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Item 1. Business

2024-03-01 compared with 2023-03-09 · 680 added, 1528 removed, 676 unchanged (77% of the section changed)

We are a biotechnology company committed to delivering a new class of differentiated one-time curative genetic therapies, Prime Editors, to address the widest spectrum of diseases by deploying Prime Editing technology, which we believe is a versatile, precise, efficient and broad gene editing technology.

Genetic mutations implicated in disease are diverse and can range from errors of a single base, known as point mutations, to errors that extend beyond a single base, such as insertions, deletions, duplications, or combinations thereof.

Other mutations can affect regulatory sequences that control the function of genes and can affect the function of larger biochemical and genetic pathways.

Furthermore, natural genetic variations, revealed by population-level genomic studies, are known to protect against or to increase risk of disease.

To maximize the impact of these genetic insights, we believe the ability to alter the human genome at the foundational level may confer the greatest therapeutic impact on human disease.

Gene editing, including platforms such as Prime Editing, is a novel technology that is not yet clinically validated for human therapeutic use.

Over the last decade, the field of genetic medicine has evolved tremendously, with groundbreaking advances in gene therapy, cell therapy, RNA, or ribonucleic acid, therapy, and, more recently, gene editing.

These technologies represent dramatic advancements for genetic therapies, but lack the versatility to precisely and efficiently correct the diverse range of mutations or deoxyribonucleic acid, or DNA, alterations implicated in disease.

Non-targeted gene therapy, which involves introducing a new copy of a gene into a patient’s cell, lacks the ability to target a specific, desired genetic location, resulting in the risk of random genomic integration, potentially waning durability and lack of native physiological gene regulation.

Nuclease gene editing technologies, such as CRISPR, zinc finger nucleases, or ZFNs, engineered meganucleases and transcription activator-like effector nucleases, or TALENs, create a targeted double-stranded break in the DNA, and then rely on cellular mechanisms to complete the editing process, thereby limiting their use.

While such approaches can be efficient in the disruption of gene expression, they lack control of the editing outcome, have low efficiency of precise gene correction, and can result in unwanted DNA modifications with potentially deleterious implications.

The recent emergence of base editing technology has made it possible for more precise gene editing at the single base level without making a double-stranded break in the DNA.

Despite this promise, base editing can only edit four out of the twelve types of single point mutations, cannot address errors that extend beyond those single base changes and has the potential to make certain unwanted on-target by-products known as bystander edits.

We believe Prime Editing technology has transformative potential that could change the course of how disease is treated and overcome the challenges associated with current genetic therapies.

Although Prime Editing technology is a developing field and is not yet validated in clinical studies, it has been extensively validated in vitro and in animal studies, as first described in a Nature publication in December 2019 and replicated in over 50 papers published in the primary scientific literature since then.

Our in-licensed Prime Editing technology was described in the Nature publication and further validated in other published papers, although we believe publications have not disclosed or used any of the specific pegRNA, ngRNA or Prime Editor protein sequences that are being used in our current programs.

In addition, in response to the Nature publication, more than 1,500 academic laboratories requested the substances, compounds, or sequences used to carry out the laboratory experiments, or reagents, from Dr.

Liu’s laboratory to replicate the experiments described in Nature and to perform Prime Editing in their laboratories, demonstrating the impact this new technology has had on the gene editing academic community.

We believe that the number of requests for reagents demonstrates the excitement in the academic community about the potential of Prime Editing as most scientific publications tend to generate a much smaller number of requests for reagents.

Prime Editing technology, as developed by Dr.

Anzalone, has broad theoretical potential therapeutic applications.

We are a biotechnology company committed to delivering a new class of differentiated one-time curative genetic therapies to address the widest spectrum of diseases.

We are deploying Prime Editing technology, which we believe is a versatile, precise, and efficient gene editing technology.

In the past forty years, the genetic disorders causing many diseases have become more clear.

Genetic mutations implicated in disease are diverse and can range from a single base error, which are known as point mutations, to errors that extend from two bases to several to thousands of bases, including multi-base insertions, deletions, duplications, or combinations thereof.

Some mutations affect the coding regions of genes while others affect regulatory sequences that control the function of genes and can affect the function of larger biochemical and genetic pathways.

Furthermore, as revealed by population-level genomic studies, natural genetic variations are known to protect against or to increase risk of disease.

Given these insights, we believe that gene editing has the potential to treat and even cure many human diseases.

The field of genetic medicine has evolved tremendously over the last decade, with groundbreaking advances in gene therapy, cell therapy, ribonucleic acid, or RNA, therapy, and, more recently, gene editing.

This past year saw the first CRISPR/Cas9-based gene editing therapy (CASGEVY™) approval by the U.S.

Food and Drug Administration, or the FDA, for the treatment of sickle cell disease.

These technologies represent significant advancements for genetic therapies but we believe Prime Editing is the only gene editing technology that, by itself, can edit, correct, insert and delete deoxyribonucleic acid, or DNA, sequences in any target tissue.

We believe Prime Editing technology has transformative potential that could change the course of how many diseases is treated and overcome the challenges associated with current genetic therapies.

Prime Editing, originally developed by Dr.

Anzalone and first described in a Nature publication in 2019, has potentially broad therapeutic applications.

Prime Editing is the only gene editing technology that can edit, correct, insert and delete DNA sequences in any target tissue.

It can correct mutations across many tissues, organs, and cell types, in dividing and non-dividing human cells.

For example, Prime Editing technology has the ability to repair diverse mutations, including all types of point mutations, deletion mutations, insertion and duplication mutations and insertion-deletion mutations.

Our analysis of more than 75,000 pathological, or disease-causing, mutations found in the National Center for Biotechnology Information ClinVar Database shows that those addressable by Prime Editing technology account for approximately 90 percent of genetic variants associated with disease.

As such, we believe Prime Editing technology has the theoretical potential for repairing approximately 90 percent of known disease-causing mutations across many organisms, organs and cell types.

We have chosen to strategically focus on disease settings where we believe that Prime Editing technology could offer compelling advantages over both current standard-of-care and novel therapeutic modalities in development.

Currently, at Prime Medicine, we are leveraging the breadth of our in-licensed Prime Editing technology to focus on our current portfolio of 18 investigational therapeutic programs.

As such, we believe Prime Editing technology has the theoretical potential for repairing approximately 90 percent of known disease-causing mutations across many tissues, organs and cell types.

In addition, we believe our Prime Assisted Site-Specific Integrase Gene Editing, or PASSIGE TM technology, may enable Prime Editing to insert gene-sized sequences precisely, potentially addressing large patient markets.

PASSIGE uses Prime Editing to insert one or more recombinase recognition sequences at precisely chosen locations in the genome.

In our preclinical studies, we have shown that a site-specific recombinase can locate the recombinase recognition sequence and carries out DNA recombination, resulting in the desired large DNA sequence insertion at the desired location in the genome.

We believe that such a technology has the potential to precisely insert “gene-sized” pieces of DNA, at a predetermined and specific site in the genome.

Taken together, Prime Editing’s versatile gene editing capabilities have the potential to unlock opportunities across thousands of potential indications.

Prime Editors also have the ability to create permanent modifications at their natural genomic location, resulting in durable edits that are passed on to daughter cells, and retain their native physiological control.

Our next generation gene editing technology is capable of producing a wide variety of precise, predictable and efficient genetic outcomes at the targeted sequence, while minimizing unwanted bystander edits and off-target edits and avoiding double-stranded DNA breaks.

Our next generation gene editing technology is designed to produce a wide variety of precise, predictable and efficient genetic outcomes at the targeted sequence, while minimizing unwanted bystander edits and off-target edits and avoiding double-stranded DNA breaks.

Our Prime Editors are designed to make only the right edit at the right position within a gene.

If nuclease gene editing approaches are “scissors” for the genome, and base editors are “pencils,” erasing and rewriting a subset of single letters in the gene, then Prime Editing is a “word processor,” searching for the correct location and replacing or repairing a wide variety of target DNA.

Our novel Prime Editors have two main components that act together to edit DNA:

(i) a Prime Editor protein, having a Cas protein and a reverse transcriptase enzyme that may be fused together, and (ii) a pegRNA, that targets the Prime Editor to a specific genomic location and provides a template for making the desired edit to the target DNA sequence.

Prime Editing leverages the established DNA-targeting capabilities of CRISPR-Cas proteins modified to nick, but not cause double-stranded DNA breaks, and combines these with the DNA synthesis capabilities of reverse transcriptase enzymes, which have been engineered to efficiently and precisely copy a pegRNA-encoded edited sequence into target DNA.

This proprietary combination enables the precise and targeted editing of any single base pair of DNA to any other desired base pair, the precise insertion or deletion of DNA, and combinations of these edits, which has not been previously possible.

To maximize the potential of our Prime Editing technology to provide one-time curative genetic therapies to the broadest set of diseases possible, we have purposefully built a diversified portfolio organized around four strategic indication categories, each set of indications chosen to deliver a different strategic goal:

• Immediate target indications:

Deliberately chosen as potentially the fastest, most direct path to demonstrate technological success of Prime Editing in patients.

We are initially focusing on diseases of the blood via ex vivo delivery to hematopoietic stem cells and on diseases of the liver, the eye and the ear.

• Differentiation target indications:

Aimed to create therapeutics to address the underlying cause of severe genetic diseases with therapeutics that we believe could not have been created before, especially using other gene-

editing approaches.

These include repeat expansion diseases, or diseases where expansion of pathological DNA repeats results in serious disease.

• “Blue sky” indications:

Intended to push new and innovative technological developments in Prime Editing and extend its application beyond rare genetic diseases and towards our goal of more broadly addressing human disease.

These programs remain in the early stages of conception and will become an increasing focus over the next few years.

• “March up the chromosome” approaches:

Represents opportunities to deliver upon our overarching vision to ultimately treat all patients with a disease and correct the full set of mutations in a particular gene.

This category overlaps with other strategic indication categories, where most of our disclosed indications across other categories have a plan that can accommodate expansion opportunities to address additional mutations in that disease.

To maximize the potential of our Prime Editing technology to provide one-time curative genetic therapies to the broadest set of diseases possible, we have built a diversified portfolio of investigational therapeutic programs organized around core areas of focus:

hematology and immunology, liver, lung, ocular, and neuromuscular.

We are advancing additional programs as potential partnership opportunities.

Recent highlights of the programs in our portfolio include the following:

• PM359, our first product candidate within our hematology area of focus, targets the p47phox variant of chronic granulomatous disease, or CGD, a serious life-threatening disease that presents in childhood.

PM359 is comprised of autologous hematopoietic stem cells, or HSC, modified ex vivo using Prime Editors that have been designed to correct a high percentage of cells containing the disease-causing mutation.

We plan to submit an investigational new drug, or IND, application with the FDA and/or clinical trial application, or CTA, in the first half of 2024.

We believe Prime Editing is uniquely well-suited to address this form of CGD.

We have completed our preliminary clinical trial design and selected global trial sites to maximize access to patients and expedite enrollment for PM359 clinical trials.

In August 2023, we received rare pediatric drug designation, or RPDD, from the FDA for PM359.

In addition, in January 2024, we received orphan drug designation, or ODD, from the FDA for PM359.

• Also in our hematology and immunology area of focus, in June 2023, we entered into a research collaboration with Cimeio Therapeutics, Inc., or Cimeio, to combine our Prime Editing platform and Cimeio’s Shielded Cell and Immunotherapy Pairs, or SCIP, platform to develop Prime Edited SCIP for genetic diseases, acute myeloid leukemia, and myelodysplastic syndrome.

The overall goal of the research is to reduce the toxicity of conditioning regimens and introduce new therapeutic options to meaningfully expand the utility of HSC transplant and enable the in vivo selection of edited HSCs to potentially remove the need for transplantation entirely.

• We have demonstrated Prime Editing of cells preclinically at predicted therapeutically relevant levels for all of our leading programs, including Wilson’s Disease and Glycogen Storage Disease 1b, or GSD1b, in our liver area of focus, Retinitis Pigmentosa/Rhodopsin, or RHO, in our ocular area of focus, and Friedreich’s Ataxia in our neuromuscular area of focus.

In 2023, we presented preclinical research across multiple programs, including proof-of-concept data from in vivo rodent and large animal studies.

Specifically:

▪ In October 2023, we reported preclinical data demonstrating the ability of liver-targeted Prime Editors to precisely correct with high efficiency one of the most prevalent disease-causing mutations of GSD1b in non-human primates, or NHPs, and mouse models.

These data are the first Prime Editing data in NHPs, which we believe provide further proof-of-concept for our Prime Editing approach to potentially address a wide range of diseases.

▪ We presented additional in vivo data in October 2023, demonstrating that Prime Editors can efficiently and precisely correct the predominant mutations that cause RHO associated autosomal dominant retinitis pigmentosa.

These data suggest that our proprietary dual adeno-associated virus, or AAV, platform can effectively deliver Prime Editors to the eye, with the potential to treat a range of retinal diseases.

• In our lung area of focus, we have expanded our efforts to develop Prime Editors for the treatment of Cystic Fibrosis, or CF, and in January 2024, we entered into a therapeutic development agreement with the Cystic Fibrosis Foundation, or CFF, in which CFF agreed to provide Prime Medicine with up to $15 million to support development of hotspot editing and PASSIGE in CF, as well as our ongoing efforts to develop lipid nanoparticles, or LNPs, for delivery to the lung.

Through hotspot editing, we aim to address multiple mutations at mutational hotspots using a small number of Prime Editors, potentially addressing a large percentage of individuals with CF with only a few Prime Editors.

In parallel, using PASSIGE, we aim to address nearly all people with CF using a single superexon insertion strategy.

• In our chimeric antigen receptor T cell, or CAR-T, program, we presented preclinical data in December 2023, demonstrating that PASSIGE was greater than 80 percent efficient for non-viral, site-specific delivery of chimeric antigen receptor to primary human T-cells to generate CAR-T cells, and can be

multiplexed with Prime Editing at other target sites by non-viral one-step delivery with no loss of efficiency.

• Lastly, our comprehensive suite of assays used to identify potential off-target events has been expanded to include new, unbiased genome-wide tools.

These analyses have continued to demonstrate minimal to no detectable off-target edits, chromosomal rearrangements or translocations.

No off-target activity has been detected in any of our leading programs, including CGD, Wilson’s Disease, GSD1b, and RHO.

We believe these preliminary analyses, across multiple editing programs, suggest a potentially best-in-class safety profile.

• We believe our Prime Editing programs are well-positioned to leverage the clinical, regulatory, and manufacturing advancements made to date across gene therapy, gene editing, and delivery modalities to accelerate progression to clinical trials and potential approval.

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For a given tissue type, we intend to use the delivery modality with the most compelling biodistribution.

Our initial, immediate programs rely on three distinct delivery methodologies:

Our initial programs rely on three distinct delivery methodologies:

(a) electroporation for efficient delivery to blood cells and immune cells ex vivo ;

(b) lipid nanoparticles, or LNPs, for non-viral in vivo delivery to the liver and potentially other organs in the future;

and (c) adeno-associated virus, or AAV, for viral delivery in vivo to the eye, ear, and potentially the central nervous system, or CNS, and muscle.

We have constructed our portfolio of 18 investigational therapeutic programs, including one partnered program, across our first two strategic indication categories in disease settings where we believe the unique characteristics of Prime Editing could offer compelling advantages over current standard-of-care and novel therapeutic modalities in development.

Our current portfolio includes the following 18 programs:

We have established preclinical proof-of-concept in vivo with long term engraftment of ex vivo Prime Edited human CD34 cells in mice in our partnered sickle cell disease program, where we have precisely corrected the disease-causing mutation.

This program is closely followed by Prime Editing for patients with chronic granulomatous disease where we have designed Prime Editors with high levels of correction of the disease-causing mutation in the cells that must be targeted.

We have selected a development candidate, designated PM359, for this program and will initiate IND-enabling studies with PM359.

We have demonstrated preclinical Prime Editing of cells in vitro at predicted therapeutically relevant levels for all of our remaining named programs.

We have designed proprietary high throughput methods to identify highly efficient Prime Editors and have advanced the reach and efficiency of the Prime Editing technology.

We have incorporated dual-flap Prime Editing technology enabling us to establish

Prime Editors with greater than 75 percent precise removal of pathological expansion repeats in five different repeat expansion diseases.

We expect that key upcoming events will continue to drive the Prime Medicine platform forward.

The following outlines a summary of select ongoing activities and next steps for Prime Medicine.

All our in vivo studies are preliminary to date.

We will continue to expand preclinical proof-of-concept in vivo, including data from in vivo rodent studies and non-human primate studies in several programs in 2023.

If successful, we expect to next initiate investigational new drug, or IND, enabling studies for several of our lead programs, with the first IND filing potentially as early as 2024, and with the potential for additional IND filings as early as 2025.

We also anticipate continuing to name additional programs as they advance over the next few years.

In addition, we are continuing to optimize non-viral and viral systems for delivery and are demonstrating meaningful delivery of our Prime Editors to various target tissues in animal models;

to demonstrate a superior “off target” profile for Prime Editing programs;

and to expand Prime Editing using proprietary recombinase and/or retrotransposon technologies for new and existing programs.

We continue to build key capabilities and infrastructure as we build an organization, culture, and expertise to meet our ambitious goals.

This includes increasing research and development, or R&D, and Chemistry, Manufacturing and Controls, or CMC, resources and building out translational medicine and clinical development capabilities to support rapid entry of a broad portfolio of programs to the clinic.

We began operations in the summer of 2020, after being co-founded by a world-renowned leader in the field of gene editing, David Liu, Ph.D.

Liu was joined as co-founder by Andrew Anzalone, M.D., Ph.D., who conceived of and developed Prime Editing along with Dr.

(b) lipid nanoparticle, or LNP, for non-viral in vivo delivery to the liver and potentially other organs in the future;

and (c) AAV for viral delivery in vivo to the eye, ear, and the central nervous system, or CNS, and muscle.

• We believe the modularity of our platform means that we will be able to accelerate our ongoing efforts and enable rapid generation of new product candidates.

We believe the core components, such as Prime Editors, delivery, manufacturing, off-target assays, clinical, and regulatory can be leveraged to drive acceleration, efficiency and execution of our pipeline.

We began operations in the summer of 2020, after being co-founded by David Liu, Ph.D., a world-renowned leader in the field of gene editing, along with co-founder Andrew Anzalone, M.D., Ph.D., who conceived of and developed Prime Editing along with Dr.

Liu and others.

Anzalone joined as our Head of Platform Development with years of experience in Prime Editing.

This has helped us to rapidly and effectively extend our Prime Editing technology beyond the academic research laboratory and into the company for drug discovery and development.

Drawn by the promise of Prime Editing’s ability to transform the field of gene editing, we have assembled a diverse and growing team that has grown to 175 as of December 31, 2022.

Our research and development team consists of experts in gene editing and Prime Editing, computational biology, automation, data sciences, off-target biology, structural biology, RNA chemistry, protein engineering and molecular evolution, genetics, pharmacology, translational medicine and the manufacturing and delivery of genetic medicines.

Keith Gottesdiener, M.D., joined in 2020 as our President and Chief Executive Officer.

Drawn by the promise of Prime Editing’s ability to transform the field of gene editing, we have since assembled a diverse and growing team that has grown to approximately 230 as of December 31, 2023, with all key functional leadership and employees in place.

Our research, technical, and clinical development teams consist of experts in gene editing and Prime Editing, computational biology, automation, data sciences, off-target biology, structural biology, RNA chemistry, protein engineering and molecular evolution, genetics, pharmacology, translational medicine, the manufacturing and delivery of genetic medicines, and clinical medicine and regulatory affairs.

Relationship with David Liu, Ph.D.

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In addition to being a co-founder, Dr.

Liu is the chair of our Scientific Advisory Board and a Board observer, meets regularly with Company representatives, and provides consulting services to us pursuant to a consulting agreement, or the Liu Consulting Agreement, related to any and all gene editing and related technology for any and all human therapeutic or prophylactic uses.

Liu is the chair of our Scientific Advisory Board and a Board observer, meets regularly with Company representatives, and provides consulting services to us pursuant to a consulting agreement, or the Liu Consulting Agreement, related to gene editing and related technology for human therapeutic or prophylactic uses.

We have also licensed certain improvements to Prime Editing from Dr.

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Liu has entered into an agreement with us pursuant to which he is obligated to assign to us any inventions with respect to the services he performs for us.

However, such obligations are subject to limitations and do not extend to his work in other fields or to the intellectual property arising from his employment with Harvard University, or Harvard, Howard Hughes Medical Institute, or HHMI, and Broad Institute.

To obtain such intellectual property rights, we would need to enter into license agreements with such institutions, and such license agreements may not be available on commercially reasonable terms or at all.

For more information, see the risk factors entitled “The gene editing field is relatively new and is evolving rapidly, making us subject to additional development challenges and risks.

We are focusing our research and development efforts on gene editing using Prime Editing technology, but other gene editing technologies may be discovered that provide significant advantages over Prime Editing, which could materially harm our business.” and “Our rights to develop and commercialize our Prime Editing platform technology and product candidates are subject to the terms and conditions of licenses granted to us by others.

If we fail to comply with our obligations in the agreements under which we license intellectual property rights from third

parties or otherwise experience disruptions to our business relationships with our licensors, we could lose license rights that are important to our business.”

Our goal is to transform the lives of patients with debilitating diseases through the application of our ground-breaking Prime Editing platform and technology.

We are committed to developing safe and efficient therapeutics using Prime Editing approaches to address high unmet need across a broad spectrum of diseases, from rare genetic diseases to severe, chronic and acute diseases, and ultimately to prevent disease before it occurs.

We are committed to developing safe and efficient therapeutics using Prime Editing approaches to address high unmet need across a broad spectrum of diseases, from rare genetic

diseases to severe, chronic and acute diseases, and ultimately to prevent disease before it occurs.

Key components of our strategy are as follows:

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• Deploy our technology to extend the application of one time potentially curative therapeutics to areas that we believe were not addressable before.

To unlock the full potential of our Prime Editing technology across a wide range of therapeutic applications, we intend to advance multiple therapeutic targets into clinical programs grouped into a series of four strategic indication categories:

(1) immediate target indications, (2) differentiation target indications, (3) “blue sky” indications and (4) “march up the chromosome” approaches.

We deliberately chose our immediate target indications based on our belief that they are the most direct path to demonstrate technological success of Prime Editing in patients.

Our differentiation target indication programs aim to create therapeutics that we believe could not have been created before, especially using other gene-editing approaches.

Our “blue sky” target indications are intended to push new and innovative technological developments in Prime Editing and extend the range of diseases we can treat.

Finally, our “march up the chromosome” category represents opportunities to deliver upon our overarching vision to ultimately treat all patients with a disease and correct the full set of mutations in a particular gene.

• Advance our pipeline while simultaneously enhancing, validating and enabling our Prime Editing platform.

We have established a diverse pipeline of 18 Prime Editing programs, including one partnered program, with a primary initial focus on the first two of our strategic indication categories.

In progressing our current immediate target indication pipeline using validated delivery methods, we believe we will enhance the probability of clinical success for future programs as we achieve early human proof-of-concept with our technology.

We intend to advance these immediate programs carefully and quickly into the clinic and through development.

We also believe we have the ability to move quickly into similar follow-on programs in each target organ as we achieve therapeutic success.

In advancing our current differentiation target indications, we aim to develop programs that build on the advantages of our technology to widen the possibilities for gene editing.

Our initial focus on repeat expansion diseases is one of many potential areas of differentiation from other gene therapy and editing approaches, and was chosen to demonstrate where Prime Editing has a unique genetic approach that could be applied to a large set of related diseases with high unmet need:

To unlock the full potential of our Prime Editing technology across a wide range of therapeutic applications, we intend to advance multiple therapeutic programs into the clinic, initially focused on genetic diseases that we believe have a fast, direct path to treating patients, and those with high unmet need not currently addressable using other gene-editing approaches.

Within our neuromuscular programs, for example, our initial focus on repeat expansion diseases is one of many potential areas of differentiation from other gene therapy and editing approaches, and was chosen to demonstrate where we believe Prime Editing has a unique genetic approach that could be applied to a large set of related diseases with high unmet need:

the precise removal of pathogenic repeats at the natural gene location, returning the patient’s genome to wild-type genetics.

Over time, we intend to push new and innovative technological developments to maximize Prime Editing’s versatile therapeutic potential, and unlock broad opportunities beyond the genetic diseases in our initial pipeline, potentially including immunological diseases, cancers, infectious diseases, and targeting genetic risk factors in common diseases.

• Advance our pipeline while simultaneously enhancing, validating and enabling our modular platform.

We have established a diverse pipeline of investigational therapeutic programs organized around core areas of focus:

hematology and immunology, liver, lung, ocular, and neuromuscular.

In addition, we are advancing additional programs, such as CAR-T, as potential partnership opportunities.

We have designed a modular platform within each core area, which we believe will accelerate our ongoing efforts and enable rapid generation of new product candidates.

We believe the core components, such as Prime Editors, off-target assays, delivery, manufacturing, clinical, and regulatory can be leveraged to accelerate our pipeline to clinical trials and potential approval.

To unlock the full potential of our Prime Editing technology across our areas of focus, we are pursuing numerous clinically validated delivery modalities in parallel.

For a given tissue type, we intend to use the delivery modality with the most compelling biodistribution and Prime Editing efficiency.

We are currently focusing on three delivery modalities:

(a) electroporation for delivery to blood cells and immune cells ex vivo ;

(b) LNP for non-viral in vivo delivery to the liver, lung and potentially other organs in the future;

and (c) AAV for viral in vivo delivery to the eye, ear, and potentially the central nervous system, lung and muscle.

Our goal is to develop highly modular delivery systems that allow us to rapidly develop new products targeting the same cells/tissues/organs by leveraging the approaches and data that precedes them.

• Continue to push the frontier of innovation in gene editing by optimizing and expanding our Prime Editing technology and capabilities.

We plan to continue investing in our technology, team and intellectual property with a focus on reinforcing our leadership position and making fundamental progress towards better therapies for patients.

We are also leveraging and investing in a full range of validated, as well as novel delivery modalities in order to retain optionality for our portfolio and select the most appropriate delivery method for each program.

• Opportunistically evaluate synergistic and value-creating partnerships to maximize the broad potential of our platform.

Our pipeline programs have been internally generated, and we retain worldwide development and commercialization rights to all but one of our programs.

Given the broad potential of our technology, we may enter into complementary collaborations with external parties in order to maximize the potential applications of our platform.

Our pipeline programs have been internally generated, and we retain worldwide development and commercialization rights to all of our programs.

Given the broad potential of our technology, we intend business development to play an important role in building Prime Medicine, with the goal of accelerating our pipeline, bolstering our financial resources, and maximizing the potential of Prime Editing.

Our overall partnership strategy includes:

1) partnering within our core areas to accelerate and globalize our current pipeline programs at the “right” stage of development;

2) outside our core areas,

entering into collaboration or license agreements for programs now that we would not otherwise pursue in the near term;

and 3) accessing enabling innovations, such as delivery and manufacturing capabilities.

• Lead with our culture of integrity, ethics, innovation and respect in everything we do.

We believe the potential of Prime Editing can only be achieved through the coordinated effort of our team and the support of our partners across academia and industry.

To push the boundaries of where gene editing can go, we are committed to jointly defining and maintaining a culture that is transparent, develops trust, values integrity and ethics, puts patients first, is science data driven, and encourages innovation.

Prime Editors:

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To maximize the impact of these genetic insights, we believe the ability to alter the human genome at the foundational level in a versatile, precise, efficient and broad manner may confer the greatest therapeutic impact on human disease.

Over the last decade, groundbreaking advances in gene therapy, cell therapy and RNA therapeutics have resulted in several approvals for genetic medicines that have transformed the treatment of certain severe genetic diseases and cancers as well as the prevention of infectious diseases, such as COVID-19.

More recently, the first generation of CRISPR-Cas based gene editing approaches for gene knockout have demonstrated initial evidence of the ability to correct pathogenic genetic mutations, via either in vivo or ex vivo delivery to humans.

Finally, the first base editing investigational medicine, that enables targeted editing of certain point mutations, has received IND clearance by the U.S.

Food and Drug Administration, or the FDA, and clinical trials have begun.

Despite this progress, there remain considerable limitations to current genetic medicine approaches that impede their ability to truly deliver on the promise of a curative, one-time therapy to the broadest set of patients.

While each gene editing technology differs, the barriers that prohibit one or more of the existing technologies from addressing genetic diseases widely include:

• Limits in the types of edits they can make

• Limits in the types of cells in which they can make edits

• Limits in the precision of gene correction

• Reliance on double-stranded breaks

• Inability to correct the mutated gene at its physiological site

Due to these limitations, we believe that it is critical that new approaches be developed that can edit genes across most therapeutically relevant mutations, precisely at the edited site with minimal off-target, or unwanted, activity elsewhere in the genome, in clinically relevant organs, and at the physiological location to keep an edited gene under native gene control.

We believe our in-licensed and company-owned Prime Editing technology has the potential to address approximately 90 percent of known disease-causing mutations.

By overcoming challenges associated with current methods in gene therapy and gene editing, we believe Prime Editing technology has the potential to provide life-long cures after a single treatment.

Furthermore, we believe Prime Editing could accelerate progression of product candidates into clinical trials by leveraging the clinical, regulatory, and manufacturing advancements made to date in the field of genetic medicine.

More recently, the first generation of CRISPR-Cas based gene editing approaches for gene disruption have demonstrated evidence of the ability to address diseases caused by genetic mutations, via either in vivo or ex vivo delivery to humans.

In 2023, the first CRISPR/Cas9-based therapy (CASGEVY™) was approved by the FDA for the treatment of sickle cell disease, followed soon after by its approval for use in beta-thalassemia.

In addition to first generation CRISPR approaches, several base editing investigational medicines, which enable targeted introduction of certain point mutations, have received IND clearance by the FDA, and clinical trials have begun.

Current Challenges for the Field of Genetic Medicines

Despite significant progress within gene therapy, cell therapy, and RNA therapeutics, there remain considerable limitations to current genetic medicine approaches that impede their ability to truly deliver on the promise of a curative, one-time therapy to the broadest set of patients.

Non-Targeted Gene Therapy

Non-targeted gene therapy includes using viral vectors, such as Adeno-Associated Virus, or AAV, or retroviruses such as lentiviruses, to deliver new copies of genes, or transgenes, to cells.

It also includes the broad field of mobile

gene elements, such as retrotransposons and transposons.

These approaches generally do not correct genes but insert new whole genes into cells in a non-targeted manner.

While having some important benefits, non-targeted gene therapy approaches can have many of the following key limitations:

• Lack of programmability, or the ability to target the gene therapy approach to a specific, desired genetic location.

• For transposons and retrotransposons, integration may occur randomly at hundreds or thousands of sites in the human genome.

• Variable gene expression due to inability to fine tune the vector copy number per cell.

• Lack of normal endogenous regulation of gene expression.

• Limited durability for non-integrating viral vectors, such as AAV.

• Pre-existing immunity to AAV vectors that could limit their use.

• Inability to re-dose in the context of lack of persistence due to certain immune responses to AAV.

• Inability to correct the mutated gene which may lead to diminished efficiency of a transgene due to competition with mutated protein.

• Risk of random genomic integration of the vector, or insertional mutagenesis, for permanent integrating viral vectors, such as lentiviral vectors.

• Potentially curative only for loss-of-function mutations.

Nuclease Gene Editing

Non-targeted gene therapy includes using viral vectors, such as AAV, or retroviruses such as lentiviruses, to deliver new copies of genes, or transgenes, to cells.

It also includes the broad field of mobile gene elements, such as retrotransposons and transposons.

These approaches generally do not correct genes but insert new copies of genes or parts of genes into cells in a non-targeted manner.

While having some important benefits, non-targeted gene therapy approaches have many key limitations.

Certain non-integrating viral vectors, such as AAV, may have limited durability, and pre-existing immunity to the vectors could limit their use and ability to be re-dosed.

For approaches that integrate genes, including transposons, retrotransposons, and retroviral vectors, gene integration may occur randomly at hundreds or thousands of sites in the human genome because it is not currently possible to direct their integration to a specific, desired genetic location.

Randomly integrating approaches also carry the risk of insertional mutagenesis.

In addition, non-targeted gene therapy approaches do not take advantage of normal endogenous regulation of gene expression, and instead lead to variable gene expression due to an inability to fine tune the vector copy number per cell.

Nuclease Gene Editing and Base Editing

First generation gene editing methods rely on a class of enzymes called nucleases, such as CRISPR, ZFNs, engineered meganucleases and TALENs, to create double-stranded breaks in DNA at a targeted location.

The DNA can then be repaired by one of two naturally occurring DNA repair pathways:

(1) non-homologous end joining, or NHEJ, which patches the broken ends of the chromosomes back together but can randomly insert indels, or unwanted insertions and deletions;

can then be repaired by one of two naturally occurring DNA repair pathways:

(1) non-homologous end joining, or NHEJ, which patches the broken ends of the chromosomes back together and randomly creates indels, or insertions and deletions;

or (2) homologous directed repair, or HDR, which can more precisely replace DNA at the target cut site with the delivery of a template of corrected DNA.

However, given NHEJ is typically the dominant repair pathway in cells and due to the low efficiency of repair and complexity associated with HDR, most nuclease-based editing programs in the clinic have focused on an NHEJ-directed knock out approach to alter or silence gene expression.

Nuclease based gene editing approaches can have the following key limitations:

• Lack of predictability in genetic outcomes at the target site in NHEJ, such as randomly inserting indels (efficient if the goal is to disrupt or knock out a gene).

• Low percentage editing and efficiency with HDR to make correction, replacement or insertions.

• Inability to correct genes in non-dividing cells since currently, HDR DNA repair machinery is only expressed in dividing cells.

• Requirement for DNA template with desired, corrected gene sequence needs to be delivered simultaneously which increases complexity.

• Unwanted DNA modifications associated with double-stranded breaks, including cell death response, genomic instability, off-target editing and the potential for oncogenesis.

• Inability to multiplex edit due to potential for large scale translocations and rearrangements from multiple double-stranded breaks.

Nuclease based gene editing approaches have several key limitations.

First, there is a lack of predictability in genetic outcomes at the target site in NHEJ, such as randomly creating indels (efficient if the goal is to disrupt or knock out a gene).

Using HDR to make corrections, replacements, or insertions has low percentage editing efficiency, does not have the ability to correct genes in non-dividing cells because HDR DNA repair machinery is only expressed in dividing cells, and requires a DNA template with the desired, corrected gene sequence to be delivered simultaneously, which increases complexity.

Nuclease editing also leads to unwanted DNA modifications associated with double-stranded breaks, including cell death response, genomic instability, off-target editing and the potential for oncogenesis.

Finally, making multiple edits with nucleases that generate double-stranded breaks at multiple genomic locations has the potential to lead to unwanted large scale translocations and rearrangements, potentially limiting applicability to multiplex editing.

Base editing is an emerging gene editing technology that harnesses CRISPR-Cas9 to deliver a deaminase to a target DNA site, which can edit a single base efficiently.

Base editing avoids double-stranded breaks and the deleterious effects associated with first generation nuclease editing.

Base editing can have the following key limitations:

• Edits can reliably correct only four out of 12 possible single base mutations, and base editing has no ability to perform or correct insertion or deletions, which limits the number of diseases base editing can address.

• Ability for each base editor to correct or introduce only a single point mutation at a time.

• Potential to make certain types of unwanted on-target by-products, called bystander edits, near the targeted site, e.g.

modifying nearby bases which are not being targeted.

• Potential for limited optionality due to its smaller editing window.

Base editing avoids double-stranded breaks and the deleterious effects associated with first generation nuclease editing, while enabling C-to-T or A-to-G base substitutions edits using either a Cytosine Base Editor, or CBE, or Adenine Base Editor, or ABE, respectively.

Base editing has several key limitations.

Currently, base editing can reliably correct only four out of 12 possible single base mutations, and base editing cannot make or correct insertion or deletions, which limits the number of diseases base editing can address.

Further, each base editor (CBE or ABE) has the ability to correct or introduce only a single point mutation at a specific location.

Base editing also has been shown to make certain types of unwanted on-target by-products, called bystander edits, near the targeted site, e.g., modifying nearby bases which are not being targeted but fall within the editing window.

Finally, base editing may have limited optionality for targeting mutations due to its smaller editing window.

Prime Editing:

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Although Prime Editing is a developing technology and is not yet validated in clinical studies, it was first described in a Nature publication in December 2019 and has since been extensively validated in vitro and in animal studies, both by our company and in over 150 papers published in the primary scientific literature to date.

In addition, in response to the Nature publication, more than 1,500 academic laboratories requested the reagents, from Dr.

Prime Editors are designed to produce edits across many organisms, organs and types of cells and to work broadly across most types of gene mutations at the natural genomic location, while minimizing unwanted DNA modifications.

This approach uses a process designed to produce a wide variety of precise, predictable and efficient genetic outcomes at the targeted sequence, which we believe will dramatically increase the impact of gene editing for a broad range of therapeutic applications.

The below image illustrates the potential of Prime Editing relative to some of the current genetic medicine approaches, using the example of correcting misspellings in a sentence from the Preamble to the U.S.

Constitution.

In this example, where the sentence represents a target of genetic code, gene therapy is unable to make a precise correction of the misspellings and instead inserts a new corrected sentence either randomly into the paragraph or outside of the paragraph (which is not shown below).

In most cases, nuclease editing inserts or deletes letters (indels) within the existing sentence, which results in a sentence that has completely lost its initial meaning.

Base editing enables the precise correction of specific letters within the existing sentence, but can only make specific changes like a G to an A, but cannot correct a C to an A.

We believe Prime Editing allows for a much broader scope of corrections to the sentence, by either correcting all the misspellings or even modifying the meaning of the sentence by inserting and deleting whole words or groups of words.

Our Prime Editing Platform

Summary of Gene Editing Technologies

The below table describes features of different gene therapy and gene editing methodologies, including Prime Editing, based on our assessment of publicly available data and our own data.

It does not represent the results of head-to-head comparison studies and is not intended to represent superiority of any one methodology over any other in any of the displayed categories.

This table is based on our assessment of publicly available data, including representative citations listed below, as well as our own data.

For Prime Editing references, see:

Anzalone, et al.

Genome editing with CRISPR–Cas nucleases, base editors, transposases and prime editors.

Nat Biotechnol 38, 824–844 (2020);

and Anzalone, A.V.

et al, Programmable deletion, replacement, integration and inversion of large DNA sequences with twin Prime Editing.

Nat Biotechnol (2021).

For Non-Targeted Gene Delivery, see:

at al, Viral vector platforms within the gene therapy landscape.

Sig Transduct Target Ther 6, 53 (2021);

and Tipanee, J.

et al, Transposons:

moving forward from preclinical studies to clinical trials.

Hum Gene Ther 28, 1087-1104 (2017).

For Nuclease-Based Gene Editing, see:

Anzalone, A.V., et al, Nat Biotechnol Ibid (2020);

et al, Therapeutic genome editing:

prospects and challenges.

Nat Med 21, 121–131 (2015);

and Li, H.at al, Applications of genome editing technology in the targeted therapy of human diseases:

mechanisms, advances and prospects.

Sig Transduct Target Ther 5, 1 (2020).

For Base Editing, see:

Rees, H.A.et al, Base editing:

precision chemistry on the genome and transcriptome of living cells.

Nat Rev Genet 19, 770–788 (2018);

and Anzalone, A.V., et al, Nat Biotechnol Ibid (2020).

Advantages of our Platform

Advantages of our Prime Editing Platform

We believe Prime Editing is a versatile, precise, efficient and broad gene editing technology with the following key advantages:

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• Programmable, which means that both the specified target location in the genome and the directed type of edit can be easily modified by replacing the Prime Editing guide RNA, or pegRNA, element of a Prime Editor.

• Modular for targeting a broad set of mutations, meaning that by redesigning the pegRNA a new mutation can be targeted for correction.

• Multiple potential therapeutic applications, including but not limited to targeted gene correction, gene silencing or activation such as by altering the regulatory regions of genes, inserting or creating premature stop codons, or by modifying splicing sequences, hotspot region replacement, multiplex editing of several genes simultaneously, and wild-type variant modification to protect against or modify risk for a disease.

• Capable of inserting, deleting or inverting kilobase amounts of genomic DNA by combining Prime Editing with proprietary recombinase technology.

• Capable of inserting, deleting or inverting kilobase amounts of genomic DNA by combining Prime Editing with proprietary recombinase technology in an approach we call PASSIGE.

Highly specific and predictable gene editing

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• Potential ability to repair approximately 90 percent of all types of mutations known to cause genetically driven disease.

• Broad therapeutic potential, including rare, genetic diseases as well as severe, chronic, and acute diseases.

Beyond correcting disease-causing mutations, potential for gene modification to edit naturally occurring variations within genes known to protect against or modify risk for a disease.

Our novel Prime Editors have two main components that are designed to act together to edit DNA:

target DNA sequence.

Prime Editing leverages the established DNA-targeting capabilities of CRISPR-Cas proteins, which have been modified so that they do not cause double-stranded DNA breaks, and combines these with the DNA synthesis capabilities of reverse transcriptase enzymes, which have been engineered to efficiently and precisely copy a pegRNA-encoded edited sequence into target DNA.

Illustration of Prime Editor and Two Main Components (Cas domain and RT domain)

Mechanism in Detail

• Broad therapeutic potential extending beyond rare genetic diseases to also potentially include severe, chronic, and acute diseases.

In addition to correcting disease-causing mutations, potential for gene modification to edit naturally occurring variations within genes known to protect against or modify risk for a disease.

Prime Editors have at least two major components, a Prime Editor protein and a pegRNA.

Our Prime Editor proteins contain two protein domains.

The first domain is a programmable DNA binding domain, often a CRISPR-Cas domain, or Cas domain.

Cas proteins enable targeting of specific DNA sequences, and they have been adapted and engineered to target desired genomic locations in human cells with high specificity, yet modified such that they do not cause a double-stranded break in the DNA.

Our Prime Editors most often use Cas9 proteins, though other Cas proteins can also be used to target DNA and we have ongoing efforts to expand our selection of Cas proteins.

Cas proteins enable targeting of specific DNA sequences, and they have been adapted and engineered to target desired genomic locations in human cells with high specificity.

In Prime Editors, programmable DNA binding domains, such as Cas domains, for example Cas9 proteins, are modified such that they do not cause a double-stranded break in the DNA.

The second protein domain of Prime Editors is a reverse transcriptase enzyme domain, or RT domain.

Reverse transcriptases are DNA polymerase enzymes that write new DNA sequences by copying from an RNA template, provided by the pegRNA.

In Prime Editing, the RT copies the edited DNA sequence directly into the target genomic site where the edit is made.

Reverse transcriptases are DNA polymerase enzymes that write new DNA sequences by

copying from an RNA template.

In Prime Editing, the RT domain copies the edited DNA sequence directly into the target genomic site where the edit is made.

The other main component in Prime Editing is the pegRNA.

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The pegRNA also contains a second sequence unique to Prime Editing, a replace sequence, or edit template, which provides a blueprint for the edit that will be made to the target DNA sequence.

As shown in the second panel in the figure below, our Prime Editor and the pegRNA locate the DNA target site using the pegRNA’s search sequence.

As shown in the second panel in the figure below, our Prime Editor protein, exemplified using a Cas protein, and the pegRNA locate the DNA target site using the pegRNA’s search sequence.

When the correct DNA target is found (referred to as “edit check 1,” as described below), the Prime Editor’s Cas domain cleaves, or nicks, one of the two DNA strands, creating a single-stranded 3’ flap.

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Next, the 3’ flap binds to a region of the replace sequence in the pegRNA (“edit check 2”) and “primes” the DNA synthesis, which is shown in the third panel below.

The Prime Editor’s reverse transcriptase, or RT, domain copies the pegRNA’s replace sequence, directly writing the corrected DNA sequence into the gene, as shown in the fourth panel.

The Prime Editor’s RT domain copies the pegRNA’s replace sequence, directly writing the corrected DNA sequence into the gene, as shown in the fourth panel.

After the corrected sequence is fully copied, cellular DNA repair preferentially incorporates the corrective 3’ flap (“edit check 3”) while removing the excess original DNA sequence.

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Illustration of Editing Mechanism by Prime Editor – No Double-Stranded DNA Breaks

A key feature of Prime Editing is that it is fully programmable, meaning that both (1) the location in the genome and the edit can be chosen specifically, and (2) the location targeted and the edit directed, can both be changed easily, based on simple design rules.

By changing the search sequence of a pegRNA, we can quickly and precisely program our Prime Editors to different genomic locations based on their gene sequences.

By changing the replace sequence of a pegRNA, we can control which edit is made.

Therefore, to make a different correction edit in a new location in the genome, we can readily reprogram the Prime Editor to specifically target a new DNA sequence and to make the precise edit that is required, simply by changing the pegRNA sequence.

Most often this will be performed by swapping out one pegRNA and replacing it with another, keeping other parts of the Prime Editor unchanged.

Characteristics of Prime Editing

We believe Prime Editing’s unique and differentiated mechanism makes it an extremely versatile, precise, efficient and broad gene editing technology.

We believe these features, along with its programmable nature, allow Prime Editors to be rapidly customized for specific diseases, creating a broad array of potential new therapeutic programs.

We believe Prime Editing can make diverse sequence edits at nearly any desired location in the human genome, enabling multiple therapeutic applications.

Prime Editors are able to change any base pair to any other base pair to correct all twelve types of single base pair point mutations, delete DNA sequences to correct insertion mutations, or insert DNA sequences to correct deletion mutations.

We can also make combinations of these types of edits with the same Prime Editor.

Notably, Prime Editors also have the ability to make direct corrections of DNA, alter the regulatory regions of genes, insert or create premature stop codons, and modify splicing sequences, differentiating the Prime Editing approach to addressing genetic disease.

Illustration of Versatility of Prime Editors’ Ability to Correct All Twelve Potential Base Pair Mutations as well as to Address Sequence Insertions and Deletions

For example, Prime Editing can be used to convert an A to either a C, G or T, to change a C to an A, G or T, to convert a G to an A, C or T, or to change a T to an A, C or G, as shown in the figure below, left.

Prime Editing can also make larger insertions or deletions, such as the precise 18-base insertion or 80-base deletion shown in the figure below, right.

By combining Prime Editors with proprietary recombinase technology, kilobase amounts of genomic DNA can be inserted, deleted, or inverted.

All of these changes can be made with highly efficient and potentially therapeutically relevant levels of precise gene correction, which we believe are generally unachievable by other gene editing approaches.

Prime Editors’ Have Shown Ability to Address All Twelve Potential Base Pair Mutations (left) as well as Larger Insertions and Deletions (right)

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The data displayed represent early demonstrative experiments that established the feasibility of performing each edit type.

Prime Editing components were not individually optimized for each edit, and therefore the efficiencies shown are not intended to reflect general or optimal editing efficiencies for the corresponding edit type.

Prime Editing can also target a wide range of mutations throughout the genome that may not be accessible to other technologies.

The targeting abilities of Cas proteins, like those used in Prime Editing and other gene editing technologies, to successfully dock onto the target DNA region, requires the presence of an approximately 3-base sequence, known as a PAM, which is adjacent to the target DNA sequence.

Other CRISPR gene editing technologies, such as base editing and Cas9-HDR, can generally edit only within a limited sequence window around the location of the PAM.

Importantly, Prime Editing is less constrained by PAM availability and can make edits near or far from the PAM sequence.

For example, based on in vitro experiments, Prime Editing can make gene edits up to about 60 bases and potentially more from the PAM sequence;

one supporting in vitro experiment is shown below.

Because Prime Editing has a larger editing window, the likelihood that a PAM exists in a suitable location nearby a

targeted mutation is higher than for other gene editing technologies.

The larger Prime Editing window also offers greater flexibility and opportunity for optimization, since there may be multiple PAM options that have Prime Editing windows that cover the location of the targeted mutation.

This offers the potential for greater flexibility and optionality for correcting a given target mutation and could broaden the number of mutations that Prime Editing can reach within a gene.

Prime Editing Less Constrained by PAM Availability and Can Correctly Perform Edits Far Removed from PAM Sequence

__________________

The blue triangles in the left graphic indicate base positions that are within the Prime Editing window, and corresponding examples of Prime Editing at those positions are shown in the bar graph on the right.

This experiment was designed to provide initial proof-of-concept of Prime Editing capabilities and was not optimized.

Specifically, we believe that Prime Editing could be extended to additional therapeutic applications, including gene correction, gene modification, gene silencing and activation, multiplex editing, and hotspot editing.

While we anticipate pursuing many of these applications as future programs, our current programs do not yet address all of these applications.

Gene Correction:

Prime Editing technology has the ability to repair diverse mutations, including all types of point mutations, deletion mutations, insertion and duplication mutations and insertion-deletion mutations.

As such, we believe Prime Editing technology has the theoretical potential for repairing approximately 90 percent of known disease-causing mutations across many organisms, organs and cell types.

Currently, at Prime Medicine, we are leveraging the breadth of our in-licensed Prime Editing technology to focus on our current portfolio of 18 investigational therapeutic programs.

Percentage Distribution of Pathological Genetic Variants Associated with Disease

Gene Modification:

We believe that our Prime Editors are also capable of making precise modifications to genes and their controlling elements to mimic natural genetic variations that are known to protect against or modify risk for a disease.

For example, the apolipoprotein E4 genotype, or APOE4, is known to confer a higher risk of Alzheimer’s Disease, whereas the “Icelandic” variant of the amyloid precursor protein gene significantly lowers the risk.

By converting such variants from the high risk form to the low risk form, we believe Prime Editing could reduce risk of disease in high risk individuals.

Gene Silencing and Activation:

We believe the precision of our Prime Editors is ideally suited for modulation of biochemical pathways that require upregulation, activation, downregulation, or silencing, to prevent or treat disease.

Precise editing of regulatory regions of genes at specific bases to achieve the desired effect avoids causing broader disruptions to adjacent regions that may still have important regulatory functions.

Our Prime Editors can also be used to silence the expression of genes, without requiring a double-stranded break, either by the conversion of certain short gene sequences, called codons, into STOP codons, by the insertion or deletion of nucleotides that create a STOP codon, or by the disruption of splice donor-acceptor sites.

Multiplex Editing:

We believe that our Prime Editors may be particularly advantageous for situations in which multiple sequences in the genome must be simultaneously targeted because they avoid creating double-stranded breaks.

The simultaneous creation of multiple double-stranded breaks by nucleases can cause unwanted large-scale genomic rearrangements, such as translocations and deletions.

These genomic rearrangements appear to occur more frequently as the number of double-stranded breaks increases.

Conversely, Prime Editors do not create double-stranded breaks.

The utility of cell therapies is currently limited by the immune recognition of donor cells by the recipient’s immune system.

Multiplex editing has the potential to be used to create cell therapies that can evade recipient’s immune system and be given to multiple different individuals.

Similarly, xenotransplantation or porcine organs for human disease is currently limited by immune recognition by the recipient’s immune system.

We believe that multiplex editing can be used to limit immune detection of porcine organs.

Hotspot Editing:

Mutational hotspots are regions within genes where clusters of distinct mutations associated with disease have been found in the human population.

By designing the replace sequence of a pegRNA so that it corrects an entire hotspot region, Prime Editing has the potential to correct many mutations within a hotspot using a single pegRNA, making it applicable for correcting multiple distinct but neighboring mutations, each found in different patients.

Currently, the replace sequence of a pegRNA is able to target regions approximately 100 bases in length.

In the hypothetical graphic shown below, three patients have distinct but neighboring mutations within a gene, representing a hotspot.

Each mutation could be corrected by the single pegRNA edit template.

By expanding on this approach to target multiple hotspot regions throughout a gene, a larger proportion of mutations could be addressed by a single Prime Editor, enabling one of our broader goals to treat all patients’ mutations in a given disease.

Illustration of How Prime Editors Can Address Hotspots Using a Single pegRNA

Prime Editing is designed to make only the right edit at the right position within a gene, which greatly minimizes on-target by-products at the site of editing, and results in low, or minimal off-target editing in other places in the genome.

Importantly, our Prime Editors do not create double-stranded DNA breaks, which supports the precision of our technology.

Prime Editing requires three “edit checks” or places where there must be a match between the editor and the target DNA in order to complete an edit.

We believe that these “edit checks” also lead to highly specific and precise edits, as described above for our mechanism.

Precision at the Target Site

Prime Editing is precise in making corrections and edits at the target site in the genome.

For example, as shown in the top panel of the graphic below, a specific pegRNA can be designed to edit only the first intended G to an A, and the bases before and after the target G are not edited, even if neighboring G bases are present.

In the bottom panel, a different, specific pegRNA can be designed to edit only the second G to an A, again without affecting neighboring bases.

With precise editing at the target site, Prime Editing minimizes bystander edits to nearby base sequences.

Using the same graphic below, we see that only the intended G is edited, and the nearby G is not edited.

This precision contrasts with base editing where it is challenging to selectively edit a single base pair when additional, similar target bases are present in the target window, such as GG in the graphic below, thus leading to bystander edits.

We have demonstrated that, following Prime Editing, more than 99 percent of the time, either a precise edit occurs in a cell or the uncorrected target DNA sequences remain unmodified and fully intact without production of unwanted by-products.

Therefore, much of our approach to the optimization of Prime Editing at a target site is focused on increasing the relative percentage of edited cells to unedited, intact cells.

We identify that information with “percent precise edits” on our graphs.

In addition, we can also readily assess and optimize our choices, by screening and optimizing many pegRNA sequences, to reduce both on-target and off-target edits.

Prime Editing Is Designed to Make the Right Edit While Minimizing Bystander Edits at Neighboring Bases

Precision at Off-Target Sites

Prime Editing shows low to no evidence of off-target editing at other locations in the genome.

As mentioned above, we believe this is also due to the lack of double- stranded breaks in DNA, as well as our three “edit checks” that are integral parts of our editing mechanism.

An example of the potential for off-target activity is shown in the figure below, which compares Prime Editing to CRISPR-Cas9 editing in a head-to-head comparison.

In this figure, the top panel reports percent of off-target editing (from 0 to 100 percent of cells) by CRISPR-Cas9 nuclease editing, and the bottom panel by Prime Editing.

The experiment involved four well-known genes that were selected for gene editing (labeled at the bottom of the figure).

A single location was edited in each of these genes.

For each edit, four different pegRNAs were designed, along with a matched single guide, or sgRNA, for CRISPR-Cas9.

CRISPR-Cas9 makes unwanted edits at well-characterized sites elsewhere in the genome, known as “off-target sites.” For each gene that was edited, there are four off-target sites (#1, #2, #3, #4) where off-target gene editing activity was quantified.

The sites are well-established sentinel sites, or sites where off-target editing has been demonstrated previously with CRISPR-Cas9 nuclease editing in preclinical studies.

The graphic shows the expected, and in some cases quite extensive, resulting off-target edits caused by CRISPR-Cas9.

In contrast, the results from Prime Editing using identical conditions generally was very low, minimal or at undetectable levels at all genes and sites.

The only exception was off-target site #3 of the HEK4 gene locus, where Prime Editing resulted in some off-target edits.

However, as described below, we believe Prime Editing has the ability to optimize guides and other parameters to improve editing.

As is shown for pegRNA#1 at the same site and gene, an “optimized” pegRNA was selected with markedly less off target activity.

The actual significance of off-target editing activity is not fully understood, but we believe that the less it occurs, the more likely this will result in a long-term safety advantage to patients.

Recent publications have shown that CRISPR-Cas9 editing can be optimized for limited off-target activity, but the ability to do that widely across programs is not clear.

We believe that markedly lower levels of off-target activity, along with the greater opportunity to optimize pegRNAs to attain even lower levels, is a major advantage of the Prime Editing technology.

Cas9 Editing Can Result in Significant Off-Target Indels (Top);

Prime Editing Has Minimal Off-Target Indels and Use of Different pegRNAs Can Further Mitigate Off-Target Effects (Bottom)

Our off-target validation approaches are described in more detail in Specificity in Prime Editing:

On-and-off target unwanted edits below, where we also describe our unbiased genome-wide analysis of off-target editing for our chronic granulomatous disease program.

As of the date of the filing of this Annual Report on Form 10-K, no off-target editing has been identified in this program.

See "—Specificity in Prime Editing:

On-and-off target unwanted edits."

The Importance of Avoiding Double-Stranded DNA Breaks

Unlike first-generation nuclease-based technologies, Prime Editors do not generate double-stranded DNA breaks.

Emerging literature supports that double-stranded DNA breaks can result in many disadvantages, including:

• Lack of editing precision at the target site, leading to many indels.

• Increased likelihood of off-target edits elsewhere in the genome.

• The possibility of large deletions, structural rearrangements, and chromosomal translocations.

• Activation of p53, a gene that makes a protein that is found inside the nucleus of cells and plays a key role in controlling cell division and cell death, leading to cell death, and possible selection for somatic cells with p53 inactivation.

• Possible reduction of cell viability in edited cells.

As shown below in a preclinical experiment, cell viability was observed to be similar with or without Prime Editing, which we believe means that Prime Editing does not affect cell viability.

In this experiment to test cell viability, cells underwent identical procedures with full Prime Editing (left), then with inactive Prime Editing components that prevented Prime Editing correction to occur (#1:

inactive reverse transcriptase;

inactive Cas9 nuclease;

#3 inactive Cas9).

The results show similar cell viability in the presence and absence of Prime Editing.

In conclusion, Prime Editing is highly precise and specific, and we believe that these advantageous features of Prime Editing will potentially contribute to better patient outcomes and improved overall safety.

We believe that with a single treatment, Prime Editing could create permanent, positive corrections of disease- causing mutations, resulting in restoration of the gene back to its wild-type healthy sequence.

A corrected gene would persist in an edited cell, working naturally and being passed along to daughter cells, resulting in a potentially durable cure or therapeutic outcome.

Unlike some other gene editing approaches, Prime Editing occurs in situ, or at the gene’s naturally occurring site, which preserves a normal number of copies of the gene in the cell, allows for normal physiology, or activity, and gene regulation, normal splice variants and protein isoforms.

All of these benefits have the potential for optimal gene regulation, which we believe could result in long-lasting benefits to patients.

Although Prime Editing is a developing technology and is not yet validated in clinical studies, it has been validated in extensive preclinical testing, both by our company and in over 50 papers published in primary scientific literature to date.

Our Prime Editors have demonstrated in preclinical studies the ability to repair mutations with comparable or superior editing efficiency relative to nuclease-based approaches such as Cas9- initiated homology-directed-repair.

Continued modifications and optimization aims to further increase the editing efficiency of Prime Editors.

Prime Editing is a compelling approach for a wide range of therapeutic applications at the genomic level, and can make precise, targeted edits in an array of cell types, tissues and organs.

We believe this breadth in applications and ability to target multiple cell types will enable Prime Editing to bring potentially curative gene editing approaches to a broader set of diseases, beyond genetic disease and towards severe, chronic, and acute diseases.

Further Enhancing the Prime Editing Platform

Over the last two years since Prime Editing was first described, an increase in efficiency as well as an expansion in the scope of applications have been demonstrated and reported in multiple publications and abstracts as well as contributions from our team.

The figure below summarizes some of these key advances.

Over the last four years since Prime Editing was first described, an increase in efficiency as well as an expansion in the scope of applications have been demonstrated and reported in multiple publications and abstracts as well as contributions from our team.

The versatile nature of Prime Editing allows for the selection of the right tools for a specific gene edit from up to ten thousand potential choices to optimize for desired effects with high efficiency and precision at the targeted site, while minimizing off-target edits at more distant chromosomal sites.

An important element of our capability is leveraging high-throughput screening and machine learning, coupled with automation of workflow, to build a data-driven model for designing optimized Prime Editing systems that can potentially accelerate our therapeutic candidate development and enhance efficiency.

We have also optimized individual subcomponents of our Prime Editors to enhance their capability beyond the first generation of Prime Editors.

Some notable developments include engineered pegRNAs and DNA mismatch repair modulation to further enhance efficiency of our Prime Editors where appropriate and expanding the array of gene edits by incorporating recent innovations in Prime Editing, including dual-flap Prime Editing and targeted integration, deletion and inversion of gene-sized DNA, using Prime Assisted Site Specific Integrase Gene Editing, or PASSIGE TM , which is described in further detail below, all of which are highlighted in the figure above.

We believe that we have built a leading position in Prime Editing by consolidating technology and intellectual property in the field as well as by establishing extensive internal capabilities to deliver on the promise of this next generation gene editing technology.

It is our belief that performing Prime Editing with high efficiency and precision, unlocking its broad applications in different genetic settings, and rapidly progressing towards clinical therapeutics requires great skill, know-how, and knowledge of the intricacies of Prime Editing.

We think this expertise differentiates us from the other gene editing platforms, and could allow us to rapidly and efficiently deliver on the promise of Prime Editing.

Enhancements to Improve Efficiency

Automated Screening and Know-How

We are building a high-throughput automated screening engine to rapidly test up to thousands of pegRNAs and hundreds of ngRNAs, for every target edit of interest.

Because pegRNA and ngRNA sequences can be chosen from a very large number of possible sequence designs, and since the choice of these designs can meaningfully influence Prime Editing efficiency, identifying the best performing molecules requires both expertise in pegRNA and ngRNA design as well as high- throughput screening capabilities for testing their activities.

The figure below depicts the current screening and know-how acquisition engine, which continues to evolve.

This process enables identification of optimized Prime Editing systems for a desired target edit, and it provides data that can be used to develop proprietary machine learning algorithms for pegRNA and ngRNA activity prediction, as described below.

We Employ Proprietary High-Throughput Screening and Design Algorithms to Identify Optimal pegRNA and ngRNA Sequences

Machine Learning

PegRNAs contain multiple sequence elements that can be optimized in isolation or together to improve Prime Editing efficiency and specificity.

For each target edit, there may be as many as tens of search sequence options, and for each of those, there exist tens to hundreds of possible replace sequences, tens of possible 3’ RNA motifs, and tens of additional options for varying other sequence elements.

As a result, the combinatorial design space for pegRNAs that target a particular mutation could reach the tens of thousands.

One arm of our approach to improving pegRNA design is to assemble our large collection of data derived from our high-throughput screening platform, then use those data to train machine learning algorithms that can accurately predict highly active pegRNA molecules.

This algorithm, known as PEGASUS™, allows us to more quickly screen and identify highly active pegRNAs in silico starting from a vast sequence space containing tens of thousands of pegRNA designs.

PEGASUS has already achieved a 76% reduction in testing of pegRNA during screening, and efforts to achieve further improvement are ongoing.

This capability is greatly enhancing our ability to efficiently and rapidly identify pegRNA sequences with the highest activity and specificity.

Novel and Improved Prime Editor Proteins

We have developed several generalizable proprietary enhancements to our first-generation Prime Editor proteins, that on average have provided more than double the level of activity.

We have developed a curated database of more than 30,000 reverse transcriptases, or RTs, which we have screened to identify novel and differentiated Prime Editing ability.

The figure below shows 80 novel RTs that efficiently perform Prime Editing in conjunction with a pegRNA and Cas domain.

Many of these have properties that differentiate from Moloney Murine Leukemia Virus, or MMLV-RT, and are as small as one-third the size of the MMLV-RT used in our first generation Prime Editor

We are improving these novel RTs along with several different Cas domains using powerful protein engineering and evolution methods to work with high efficiency in Prime Editing.

Engineered pegRNAs

Professor David Liu’s laboratory at Broad Institute recently developed epegRNAs that can improve Prime Editing efficiency by 3-fold or more in multiple human cell types, an enhancement that is now being incorporated into our research activities.

In addition to the elements found in standard pegRNAs, epegRNAs have an extra element called a 3’ RNA motif.

We believe that 3’ RNA motifs stabilize the replace sequence of pegRNA in cells, extending the duration of Prime Editing, and thereby leading to higher editing efficiency.

Multiple classes and sequences of 3’ RNA motifs can be used in epegRNAs, and therefore represent another lever that we can apply to optimize Prime Editing efficiency.

We have exclusively in-licensed and adopted the use of these epegRNAs and are actively developing our own classes of epegRNAs to enhance Prime Editing.

Other Improvements to pegRNA Design

We have developed several other generalizable proprietary enhancements to our first-generation Prime Editing systems through optimization of our Prime Editor pegRNAs.

By using powerful RNA engineering methods, we have established different generalized optimizations that can be applied to pegRNAs to improve their activity.

in the figure below, these optimization processes can yield marked increases in activity, and have been observed to lead to a more than 10-fold increase in pegRNA activity.

We believe combining these enhancements and others will enable us to build next-generation Prime Editors that are optimized for efficiency, breadth, precision, and therapeutic delivery.

We outline what we believe to be other, important additional improvements below.

DNA Mismatch Repair Modulation

Recently, it has been shown in in vitro experiments that a DNA surveillance system, which is variably active across cells of the body, called mismatch repair pathway, or MMR, can influence Prime Editing outcomes.

Transient suppression of MMR, specifically a part of the system called MLH1, in the tissue where Prime Editing is occurring, can moderately boost efficiency, as shown in the figures below.

As a result, although many of these approaches require further validation, we are developing approaches to transiently modify MMR so that desired Prime Edits are favored, and any undesired by-products are minimized.

We are evaluating several different approaches to modulating the MMR response including active pharmaceutical ingredients, such as siRNA, or small interfering RNA, or other approaches that transiently modulate MMR activity which could be co-administered with a Prime Editor.

While inherited mutations in genes encoding the protein factors in the MMR pathway can increase the risk of neoplasms of epithelial tissues including colon and skin appearing in adulthood, we believe short-term (days) suppression of MMR is likely to be generally well-tolerated.

Safety studies will be required to establish a safety profile of transient MMR suppression.

Suppressing the MMR Pathway, For Example by Inhibiting MLH1, Can Boost Editing Efficiency and Minimize By-Products

Co-Administration of siRNA with our Prime Editors was Observed to Increase Editing Efficiency

Enhancements to Broaden the Universe of Edits for Prime Editing

Dual-flap Prime Editing

Multiple enhancements to our Prime Editing platform, including engineered pegRNAs, enhanced Prime Editors, and DNA mismatch repair modulation, provide us with a versatile toolbox for applying Prime Editing to a wide range of diseases.

In addition, our focus on high-throughput screening and machine learning are allowing us to grow our internal technical expertise for Prime Editing optimization and are being used to develop Prime Editors that are both

more efficient and more precise.

Finally, we are broadening the types of edits that we can make by incorporating recent innovations in Prime Editing, including dual-flap Prime Editing, long-flap Prime Editing, and PASSIGE.

Dual-flap Prime Editing and long-flap Prime Editing

We have in-licensed certain dual-flap Prime Editing technology developed by David Liu’s laboratory at Broad Institute, and expanded and improved on its uses.

Compared to traditional Prime Editing, dual-flap Prime Editing uses two Prime Editors instead of one.

In different places, each of the Prime Editors creates a nick in the DNA and creates a flap;

the two flaps are designed to bind tightly to each other.

This results in the looping out of the DNA between the Prime Editors, with replacement of new DNA.

In different places on a target gene, each of the Prime Editors creates a nick in the DNA and creates a flap;

the two flaps are often designed to bind tightly to each other.

This results in the looping out of the DNA between the Prime Editors, with replacement by new DNA.

Dual-flap Prime Editing is designed to achieve efficient editing of a broader range of edit types, including the precise replacement or insertion of DNA sequences that are a hundred bases or more in length with potentially higher efficiency than standard Prime Editing.

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In addition to its high efficiency, it achieves the same level of precision, and we believe it results in minimal off-target editing, as shown in preclinical studies, similar to the more standard forms of Prime Editing.

As illustrated in the figure below, dual-flap Prime Editing could be used to delete expanded repeat sequences like those that occur in repeat expansion diseases, to replace mutation hotspots with corrected sequences, or to insert sequences at safe harbor or other locations in the genome.

The figure below depicts examples that use dual-flap Prime Editing to:

replace a 64-base mutation hotspot in the gene that causes the metabolic disorder, Phenylketonuria, delete approximately 3,000 bases, or insert 38 bases at a targeted location in the genome.

Dual-Flap Prime Editing Uses Two pegRNAs to Potentially Expand Applications

Dual-Flap Prime Editing Precisely Corrects a Gene with a Human Mutational Hotspot, Precisely Removes Large Sequence of Pathogenic DNA and Precisely Inserts a 38 Base Pair Sequence into the Human Genome

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Error bars represent standard error of N=5 experiments.

PASSIGE TM – Precise introduction of gene-sized pieces of DNA into the genome

Dual-flap Prime Editing could be used to delete expanded repeat sequences like those that occur in repeat expansion diseases, to replace mutation hotspots with corrected sequences, or to insert sequences at safe harbor or other locations in the genome, as can be applied to our PASSIGE approach.

Additionally, we have developed a long-flap Prime Editing approach that, compared to standard Prime Editing, is designed to more efficiently insert or replace larger stretches of DNA that are a hundred bases or more in length, while also enabling precise deletions of up to thousands of base pairs.

Long-flap Prime Editing can be applied for similar applications as dual-flap Prime Editing, including editing of hotspot regions in DNA, insertions of recombinase sites for PASSIGE, and the excision of expanded repeats.

Together, dual-flap Prime Editing and long-flap Prime Editing broaden the capabilities of our Prime Editing platform.

PASSIGE – Precise introduction of gene-sized pieces of DNA into the genome

We have in-licensed from the Broad Institute and are developing a technology that allows us to expand our gene editing toolbox to include programmable insertion, deletion, or inversion of thousands of bases of DNA.

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PASSIGE™ – Extending Prime Editing to insert gene sized sequences precisely in the genome

We have delivered Prime Editing components and DNA recombination components in a single step to human primary T cells.

The short recombinase DNA target sequence, used by the site specific recombinase enzyme known as Bxb1, was inserted into human primary T cells with greater than 90% efficiency, and a 3.5 kilobase gene of interest was precisely inserted into that recombinase site location in more than 60% of the T cells, resulting in positive expression of the gene product by those T cells.

As a result, the T cells acquired a new cell function dependent on cell dose in a cell assay, indicating that the 3.5 kilobase gene was functional.

In addition, our focus on high-throughput screening and machine learning are allowing us to grow our internal technical expertise for Prime Editing optimization, and are being used to develop Prime Editors that are both more efficient and more precise.

Finally, we are broadening the types of edits that we can make by incorporating recent innovations in Prime Editing, including dual-flap Prime Editing and PASSIGE™.

Translating Prime Editors into Product Candidates

Multi-modality Delivery of Prime Editors

The efficient delivery of our Prime Editors is critical for the development of our therapeutic pipeline indications.

We are investing in Prime Editor protein and mRNA discovery and development, pegRNA design from high throughput screening (HTS) through to process chemistry, and a broad, multi-modal delivery approach.

We have established in-house capabilities, tools, and partnerships to pursue a comprehensive suite of validated delivery technologies, and we continue to evaluate novel experimental delivery approaches.

For each program in our pipeline, we evaluate the best options for delivery, and select the delivery technology with the most compelling biodistribution for a given tissue type.

Our initial programs rely on three distinct delivery methodologies:

(i) LNPs for non-viral in vivo delivery to the liver, lung, and potentially other organs, as well as ex vivo cells;

(ii) electroporation for efficient delivery to blood and immune cells ex vivo;

and (iii) AAV for viral delivery in vivo to the eye, ear, CNS and muscle.

By leveraging these diverse delivery technologies in parallel, we believe we could avoid overreliance on any single delivery method and create optionality by advancing a broad portfolio.

We believe these delivery technologies are foundational to successfully bringing our pipeline programs to the clinic and we are actively building capabilities and investing in development and optimization of the delivery technologies to accelerate our pipeline progress.

Moreover, we also continue to evaluate and leverage the many advancements in novel and experimental delivery approaches that are being made in the cell and gene therapy field, and intend to license complementary delivery technologies, as appropriate.

Our multi-pronged delivery strategy to enable our portfolio includes the following:

• Non-Viral Delivery.

We are designing Prime Editing product candidates to provide a “once and done” treatment and we see a non-viral future for delivery.

We have developed internal protein engineering capabilities to optimize the Prime Editor proteins and recombinase proteins for human therapeutic use, and we have developed internal mRNA discovery, chemistry, and process development capabilities to optimize drug candidate properties and characterize the Prime Editor mRNA for efficient, tolerable, and consistent delivery and translation of the protein.

Beyond our automated pegRNA high throughput screening capabilities, we are building internal pegRNA synthesis capacity, pegRNA modifications with structure-activity-relationship to improve drug candidate properties, and pegRNA process chemistry.

• Initially, we are utilizing existing LNP formulations and technologies for in vivo delivery to the liver.

We have also established end-to-end capabilities across our R&D organization consisting of lipid design, chemistry, and synthesis, high throughput LNP screening using bar coding technology, LNP formulation process development for tissue targeted delivery, and production to support our preclinical studies.

Translating Prime Editors into Product Candidates - Multiple Modalities for Prime Editors

The optimal design and efficient generation of our Prime Editors are fundamental for the development of our pipeline.

We have established capabilities to design and optimize our Prime Editors, and to design and develop the components needed for LNPs, vector genomes, Prime Editing of ex vivo cells, as well as to develop the manufacturing processes and analytical assays to ensure robust, scalable production of quality intermediates and products to support our programs.

Many of these workflows are automated to allow rapid machine learning and/or artificial intelligence-based data analysis, correlation, visualization, and iterative optimization and innovation.

For each program in our pipeline, we determine the best option for delivering the Prime Editor and select the delivery technology with the most compelling biodistribution for a given tissue type.

Our initial programs rely on three distinct delivery modalities:

(a) electroporation for delivery to blood cells and immune cells ex vivo ;

(b) LNPs, for non-viral in vivo delivery to the liver, lung and potentially other organs in the future;

and (c) AAVs for viral in vivo delivery to the eye and ear, and potentially the central nervous system, lung and muscle.

A key feature of Prime Editing and associated delivery methods is the modularity of the technology platforms.

Once the first program for each delivery platform is established, the design algorithms, workflows, non-clinical and CMC data, as well as the manufacturing process and majority of assays can be leveraged and applied to the next program which differs only in the pegRNA.

We believe these delivery technologies are foundational to successfully advancing our pipeline programs to the clinic and we are strategically developing our delivery platforms and generating data to accelerate our pipeline progress.

Moreover, we continue to assess the many advancements in novel and experimental delivery approaches that are being made in the cell and gene therapy field and intend to license innovative delivery technologies that prove to provide a breakthrough.

We are designing Prime Editing product candidates to provide a “once and done” treatment.

Our multi-pronged approach to enable our portfolio includes the following:

• pegRNA Design, High Throughput Screening and Synthesis:

An important element of our capability is leveraging high throughput automated screening and design algorithms to identify optimal pegRNA sequences.

The data is also used to develop proprietary machine learning algorithms for pegRNA activity prediction.

Internal chemistry capabilities facilitate high-throughput optimization and manufacture of oligonucleotides for in vivo studies.

We have established internal high-throughput pegRNA synthesis, pegRNA modifications with structure-activity-relationship to improve drug candidate properties, and pegRNA process chemistry.

• Optimization of Prime Editing proteins and recombinase proteins:

We have developed internal protein engineering capabilities to optimize the Prime Editor proteins and recombinase proteins (for PASSIGE) for human therapeutic use, and have developed internal mRNA design and optimization, enzymatic chemistry, and process development capabilities to enhance drug candidate properties and characterize the mRNA for efficient, tolerable, and consistent delivery and translation of the Prime Editor protein.

• Prime Editing Specificity and Assays:

A robust and unbiased evaluation of all potential off-target activities is a critical element of our efforts.

Our approach to minimizing off-target editing is to start by screening for Prime Editor candidates with very low off-target activity.

We then use comprehensive, sensitive, and state-of-the-art methods to identify all putative off-target sites by identifying places where a Prime Editor has a possibility (no matter how small) to nick the DNA.

We have developed multiple, complementary, but distinct, methods to measure such possible events.

Our approach includes evaluation of:

(a) off-target activity in the genome that is specific to the sequence of a particular pegRNA or the ngRNA;

(b) similar activity that is independent of the pegRNA or ngRNA sequences;

and (c) genomic rearrangements.

• Electroporation:

A second delivery approach is electroporation for transfecting ex vivo cells.

Electroporation is being used for our chronic granulomatous disease program with ex vivo CD34+ cells.

We are building internal cell process development capabilities, initially for our CD34+ cell programs.

Electroporation is a clinically and commercially validated technology for ex vivo delivery to CD34+ cells which utilizes electrical pulses to increase the cell membrane permeability to deliver the Prime Editing components.

Electroporation is being used in our CGD program with ex vivo CD34+ cells.

We have established a modular cell processing manufacturing platform process that can be used for autologous CD34+ cells, and it can be leveraged for the next ex vivo HSC programs, as well as for allogeneic T-cells with multiplexing.

In the future, we plan to transition to in vivo editing of stem cells and other lymphocytes.

LNP delivery has initially focused on in vivo delivery of Prime Editing to the liver.

We have established end-to-end capabilities across our R&D organization consisting of lipid design, lipid synthesis, high throughput LNP discovery from our proprietary lipid library using screening with bar coding technology, LNP formulation process development for tissue targeted delivery, and manufacturing to support our preclinical and IND enabling studies.

We are developing a universal liver targeting LNP comprised of 5 components and plan to leverage its modularity for our various programs aimed at Prime Editing in the liver, as well as to address additional mutations within the same indication.

Similar approaches are being taken for developing modular LNPs to lung, and HSCs and T cells.

• Viral Delivery:

Finally, we are using viral delivery to tissues and locations that can only currently be reached with Adeno-associated Virus, or AAV.

We consider this a stop gap measure until we can identify a highly specific, non-persisting delivery approach.

To enable this delivery approach, we have developed internal capabilities to design and optimize each of the components of the AAV genome at scale to efficiently deliver Prime Editors to the target tissue.

We have developed internal capabilities to generate, purify, and quality control with release testing, AAV within a dedicated AAV Reagent Production Core.

In parallel, we are working with several partners to facilitate production and quality at scale.

• Critical Assays.

Prime has established comprehensive analytical, and assay development functions to enable careful, rapid, and comprehensive development and optimization of functional and cell-based potency assays, product characterization assays, quality control release assays, stability testing and, compendial assays.

We are using viral delivery to tissues and locations that can currently only be reached with AAV.

To enable this delivery approach, we have developed capabilities to design and optimize the vector genome to efficiently deliver Prime Editors to the target tissue.

We use our internal AAV Reagent Production Core, analytical development team, as well as outsourced resources and partners to generate AAV Prime Editors, quality control test as well as characterize them.

• Strategic Manufacturing Partnerships:

Our overall strategy is to design manufacturing platforms to make the Prime Editing components and associated delivery systems with high throughput, high quality, high purity, modularity, and scalability.

We are developing manufacturing processes and analytical methods both internally and partnering with suppliers to ensure the quality and consistency of the Prime Editor components and Prime Edited drug products needed for preclinical studies, IND application submission, and future clinical studies.

To maximize the potential of our Prime Editing technology, we have built a diversified portfolio of investigational therapeutic programs organized around core areas of focus:

hematology and immunology, liver, lung, ocular, and

neuromuscular.

We are advancing additional programs as potential partnership opportunities.

The following table summarizes the status of certain of our programs:

Our Blood Programs

Chronic Granulomatous Disease

Chronic granulomatous disease, or CGD, is a rare inherited hematologic disorder characterized by susceptibility to severe, difficult-to-treat infections, and inflammatory/autoimmune complications.

CGD is caused by mutations in any one of the subunits comprising the NADPH oxidase complex, which is required for phagocytic cells, in particular neutrophils, to destroy many invasive microorganisms.

CGD causative mutations are estimated to occur between one in 100,000 and one in 200,000 births in the United States, and most children are diagnosed within the first three years of life.

Beginning in childhood, patients with CGD develop infections from a range of both typical and unsual bacteria, fungi and mycobacteria.

These infections may present in various organ systems, and protracted infections can lead to long-term organ damage and failure.

In addition, patients have non-infectious inflammatory disease, most commonly presenting as inflammatory bowel disease, soft tissue granulomas, and strictures of the urinary or digestive tract.

Undiagnosed or untreated, the infectious manifestations of CGD are rapidly fatal.

Approximately 60 percent of patients with CGD reach age 30 and refractory or antimicrobial resistant infection is the leading cause of mortality.

The NADPH oxidase complex has five domains encoded by five separate genes.

Loss-of-function mutations in any of these genes can present as CGD.

The second most common form, which represents approximately 25 percent of cases, is caused by biallelic loss-of-function mutations, in both copies of the NCF1 gene encoding the p47phox protein.

More than 78 percent of p47phox CGD patients have a specific, 2-nucleotide deletion, or ΔGT, in the NCF1 gene.

The NCF1 gene location is complex, and also contains pseudogenes, or copies of the NCF1 gene that in most healthy individuals, and in individuals with CGD, are inactivated by the ΔGT mutation.

Preclinical studies have demonstrated that correcting just one copy of the ΔGT mutation in either the NCF1 gene or any pseudogene restores protein expression and full NADPH oxidase activity.

Our Approach and Results:

Direct correction of prevalent CGD mutations or hotspots

PM359, our first product candidate within our hematology and immunology area of focus, targets the p47phox variant of CGD.

We have been able to demonstrate that Prime Editing precisely corrects the ΔGT mutation in the NCF1 gene to restore p47phox protein expression and NADPH oxidase activity.

PM359 is comprised of autologous HSCs modified ex vivo using Prime Editors that have been designed to correct a high percentage of cells containing

the disease-causing mutation.

PM359 is delivered as an autologous HSC transplant, and is designed to restore normal immunologic function in individuals with p47phox CGD, without the risks of graft-versus-host disease, or GVHD, graft rejection and post-transplant immunosuppression associated with allogeneic transplantation.

In order to develop PM359, a therapeutic product candidate to treat patients with p47phox CGD, we screened pegRNA and ngRNA to identify Prime Editing guides and guide pairs that have high activity and perform precise editing at the NCF1 locus.

Because healthy donors have NCF1 pseudogenes bearing the same ΔGT mutation, we have been able to utilize healthy donor CD34+ HSCs to demonstrate precise editing of the ΔGT mutation.

Using healthy donor CD34+ HSCs we also have been able to demonstrate a very low off-target potential, and robust engraftment and hematopoietic functionality of Prime Edited HSCs in mouse models.

We have confirmed these findings using p47phox CGD patient-derived HSCs, and in addition have demonstrated that Prime Editing is able to restore NADPH oxidase activity in patient-derived cells.

The overall process for generating PM359 follows a similar paradigm to that employed by other investigational ex vivo HSC CRISPR-Cas9 therapeutics that have been validated in the clinic, with a few notable modifications.

The Prime Editor complex is delivered to CD34+ HSCs using electroporation.

The Prime Editor mRNA is generated by in vitro transcription, and the pegRNA and ngRNA are generated by solid phase RNA synthesis.

The Prime Editor complex is delivered by simultaneously electroporating mRNA encoding the Prime Editor protein along with pegRNA and ngRNA.

We believe the mRNA is translated into the Prime Editor protein during a period of incubation, then the Prime Editor protein assembles with pegRNA or ngRNA, and the complex enters the nucleus with Prime Editing commencing at the target site in the genome.

We have identified a Prime Editor complex that is able to correct over 80% of healthy donor CD34+ cells or CGD patient CD34+ cells at at least one ΔGT locus (pseudogene in healthy donor;

pseudogene or NCF1 in patient).

In order to be able to understand the effects of Prime Editing on the functionality of modified CD34+ cells and to determine the ability of Prime Edited cells to persist in vivo , we administered Prime Edited CD34+ cells to a specialized immunocompromised mouse model, the NBSGW mouse, which is capable of supporting engraftment of human HSCs as shown in the figure below.

Mice are typically followed for 16 weeks in these experiments to allow for engraftment of the long-term HSCs, repopulation of the immune system by long-term HSCs, and evaluate durability of effect.

When Prime Edited healthy donor or CGD patient CD34+ cells are administered to NBSGW mice, they were able to engraft, and edited cells proliferated to repopulate the hematopoietic system.

The left and middle panels in the figure below demonstrate results from a representative experiment in which healthy donor CD34+ cells, either Prime Edited or mock treated, were administered to NBSGW mice.

In the left panel of the figure below, human hematopoietic cells, distinguished by the human CD45+ surface marker, were able to successfully engraft at high efficiency, with no differences between mock treated and Prime Edited cells.

In the middle panel, after 16 weeks in the mice, long-term engrafted human CD34+ HSCs retained a very high degree of editing:

over 80% of cells exhibited correction of the ΔGT mutation at at least one locus, compared to 0% of mock edited cells.

Based on natural history studies of carriers of CGD-causative mutations, restoration of p47phox function in a minimum of 20% of neutrophils is believed to be sufficient to confer protection from serious infection with CGD-associated pathogens.

Together these data suggest that Prime Editing is able to correct the ΔGT mutation at an efficiency many-fold above the lower bound of the projected threshold for therapeutic benefit, and that Prime Edited cells may effectively home to and stably engraft in the marrow after infusion.

CD34+ HSCs from an individual with p47phox CGD were Prime Edited ex vivo , and these cells were differentiated ex vivo to allow assessment of NADPH oxidase activity in myeloid progeny.

The panel on the right in the figure below demonstrates the results of one commonly used measure of NADPH oxidase activity, the dihyrorhodamine (DHR) assay.

Mock edited CGD patient-derived and differentiated cells produced negligible amounts of NADPH oxidase, reflected in the absence of any DHR positive cells.

In contrast, NADPH oxidase activity was restored in

approximately 80 percent of Prime Edited CGD patient cells, very closely corresponding to the editing rate observed in healthy donor CD34+ cells in the middle panel.

This level significantly exceeds the projected therapeutic minimum threshold of DHR activity in 20 percent of neutrophils thought to be sufficient to prevent serious infection based on natural history studies.

Additionally, these results confirm that editing rates in CGD patient derived CD34+ cells are similar to those in healthy donor cells, and that healthy donor cells are a useful proxy for understanding the Prime Editing reagents used to generate PM359.

HSC = hematopoietic stem cell;

LT-HSC = long term HSC;

DHR = dihydrorhodamine;

normalized to healthy donor control.

Data presented at ASGCT and ESGCT 2023.

Prime Editing has exhibited a very low level of undesirable genetic changes.

Extensive in silico, ex vivo and in vivo analyses have been performed.

The figure below demonstrates representative analyses.

In the left panel, 550 of the most likely candidate off-target sites were nominated based on in silico assessment, and interrogated for off-targeting editing in Prime Edited healthy donor CD34+ cells;

no significant off-target editing was detected.

In the middle panel, the marrow of NBSGW mice receiving either mock or Prime Edited human CD34+ cells was assessed for large deletions or translocations 16 weeks after engraftment;

there was no difference between mock treated and Prime Edited cells, and neither had any detectable evidence of deletions or translocation above the level of significance.

In the right panel, a human cell line was transfected with spCas9 targeting NCF1 without the Prime Editing machinery.

Unlike Prime Editing, spCas9 introduces double strand breaks in DNA as part of its expected mechanism of action.

In contrast to the Prime Editor, spCas9 by itself introduced a high rate of translocations.

1 Analysis of edited CD34+ cells from CGD program:

Targeted in vitro Analysis of 550 potential off-target sites of off-target editing.

2 Data from in vivo analysis from mouse bone marrow harvested 16 weeks after engraftment was complete.

3 Positive control.

Overall, the data presented in the two figures above strongly support the clinical evaluation of PM359 in patients with p47phox CGD.

We believe these data suggest that PM359 has the potential to meet and exceed the projected mutation correction rate sufficient to achieve disease amelioration, and that PM359 has a low probability of safety events potentially associated with ex vivo gene-edited HSC products.

Based on these results, we have selected a development candidate, designated PM359 and are in the process of completing IND-enabling studies with this candidate.

We plan to submit an IND and/or CTA with the the FDA, in the first half of 2024.

We have completed our preliminary clinical trial design and selected leading CGD transplant centers across the United States and other countries as clinical trial sites to maximize access to patients and expedite enrollment for PM359 clinical studies.

Study Prime-0101 is a planned, multinational, first-in-human trial designed to assess the safety, biological activity and preliminary efficacy of PM359 in adult and pediatric study participants who have p47phox CGD due to the ΔGT mutation in NCF1, and are medically suitable to undergo autologous HSCT.

Autologous CD34+ cells will be collected by mobilization and apheresis and transferred to a centralized manufacturing facility, where they will be electroporated with Prime Editing reagents, cryopreserved and quality control tested, to generate PM359.

PM359 will be infused after myeloablative conditioning, and study participants will be followed for three years in the primary study, and an additional 12 years as part of the long-term follow-up period.

Initial study participants will be adults with stable disease.

Once safety and biological activity has been demonstrated in initial participants, the study will enroll participants with active infection or inflammation, as well as adolescent and pediatric participants, under the supervision of a Data Monitoring Committee.

Participants will be followed for safety, including engraftment and reconstitution of the hematopoietic system, as well as early biological markers of restored immune function including the DHR assay, and for the long-term resolution and prevention of infectious and inflammatory complications of CGD.

We believe PM359 is uniquely well-suited to restore immune function and resolve infectious and inflammatory disease in patients with p47phox CGD.

In August 2023, we received RPDD from the FDA for PM359 for the treatment of CGD.

Companies that receive approval for a New Drug Application, or NDA, or Biologics License Application, or BLA, for a rare pediatric disease may be eligible to receive a voucher for priority review of a subsequent marketing application for a different product.

If we receive a priority review voucher, it may be used by us or sold to a third party.

In addition, in January 2024, we received Orphan Drug designation from the FDA for PM359 for the treatment of CGD.

Other Programs in Discovery

We continue to make progress in other programs in our hematology and immunology area of focus.

We are exploring cell shielding to expand our HSC area of focus beyond rare diseases.

In June 2023, we entered into a research collaboration with Cimeio to combine our Prime Editing platform and Cimeio’s SCIP platform to develop Prime Edited SCIP for genetic diseases, acute myeloid leukemia, and myelodysplastic syndrome.

Additionally, we are also researching Fanconi anemia, a rare and life-threatening DNA repair disorder that arises from loss-of-function mutations in any of 23 genes.

Our Liver Programs

Non-Viral Delivery In Vivo with Lipid Nanoparticles

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We believe that building an iterative and integrated system will increase efficiencies in identifying potent and safe LNPs capable of delivering Prime Editors to extra-hepatic tissues.

For our first in vivo Prime Editor program, we are leveraging existing LNP technology that we believe will allow us to move the program in to the clinic quickly and establish proof of concept.

We have developed a model system in mice to iteratively study and optimize the properties of our LNP formulations and the Prime Editor cargo.

For our first in vivo Prime Editor programs, we are building upon learnings from existing LNP technologies to develop a Universal Targeted LNP delivery system that is targeted to the liver, and specifically targets the LNP to the hepatocytes.

This approach, we believe, will improve biodistribution to the target cell type.

The LNP system will be modular in that simply swapping out the pegRNA (and ngRNA where required) will result in a new product.

This approach will allow us to move the existing liver programs in to the clinic quickly and establish proof of concept, and potentially bring forward additional liver programs, quickly thereafter.

To develop and optimize the LNP platform, Prime has developed a model system in mice to iteratively study and optimize the properties of our LNP formulations and the Prime Editor cargo.

In this system, we inactivate the PCSK9 gene by precisely introducing a stop codon into the gene.

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This system enables us to look at levels of PCSK9 protein in the blood in response to editing.

Prime Editors containing three different mRNA lots were formulated with one of our LNP formulations.

One of our optimized mRNA lots showed more than 40% editing in whole liver, resulting in more than 90% reduction in circulating PCSK9 protein levels.

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Following optimizations in this system, simply swapping the guide RNAs for PCSK9 Prime Editing for program specific guide RNAs will result in test articles that can be evaluated in humanized mice or in NHPs for the liver programs.

Optimization of Prime Editors containing three different mRNA lots were formulated with one of our LNP formulations.

One of our optimized mRNA lots showed more than 40 percent editing in whole liver, resulting in more than 90 percent reduction in circulating PCSK9 protein levels.

Panel shows an experiment delivering LNPs to the liver containing a Prime Editor precisely introducing a stop codon into the mouse PCSK9 gene.

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Therefore, maximum editing possible is predicted to be no more than 60%.

Our LNP-based delivery system encapsulates mRNA, pegRNA, ngRNA, and additional potential Prime Editor components into nanoparticles for in vivo delivery of the Prime Editor as a single dose.

We are systematically optimizing the Prime Editor components and LNP formulation to further improve in vivo Prime Editing efficiency, and to build a LNP delivery platform.

By changing the pegRNA and ngRNA pairs, we are initially applying this LNP formulation to our Wilson’s Disease and Glycogen Storage Disease liver programs.

Electroporation

Electroporation is a clinically validated technology for ex vivo delivery of a therapeutic payload such as mRNA into cells, which are then reinfused back into the patient(s).

Electroporation utilizes electrical pulses to increase the cell membrane permeability.

We are using electroporation to deliver Prime Editor components (for example, pegRNA/ngRNA guide pairs, and mRNA encoding a Prime Editor protein, or guide pairs with purified Prime Editor protein as a ribonucleoprotein complex) into blood cells and immune cells.

We have established electroporation delivery capabilities, and the analytical characterization for the transfected cells to support of our leading cell therapy program(s).

We are also monitoring and evaluating novel technologies that can be a viable alternative to electroporation that will improve process efficiencies and product quality.

Viral Delivery In Vivo with AAV

AAV is a validated viral vector that is non-pathogenic to humans and does not integrate into the genome.

For AAV delivery, we are optimizing AAV with respect to serotype, capsids, and other aspects of the Prime Editing cassette in the AAV genome.

We are employing internal and external process development and analytical QC to progress our preclinical development of our dual AAV mediated PE programs.

In the figure below, successful Prime Editing is demonstrated from initial proof of concept experiments using different Prime Editor AAVs to edit the PCSK9 gene as a control site by in vivo delivery to the CNS via a cerebrospinal fluid infusion or local infusion in a mouse model.

By changing the pegRNA sequences, a similar AAV delivery platform is being applied to our CNS indications, and work on Friedreich’s Ataxia is currently underway.

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Panel shows an experiment delivering AAVs to the CNS containing a Prime Editor to precisely introduce a stop codon into the mouse PCSK9 gene.

The experiment in the left graph shows dual AAV delivered to the cerebrospinal fluid by ICV infusion.

Three weeks later the cortex was sampled.

Approximately 50% of cortical cells were transduced by AAV.

Of those, approximately 90% were precisely edited.

On the right, AAVs were delivered locally to the CNS by infusion.

This approach resulted in transduction of 95% of neurons and, of those, approximately 80% were precisely edited.

Overall, these preliminary experiments with two key delivery technologies planned for our clinical programs, demonstrate our expanding capabilities for delivery of our Prime Editors.

Specificity in Prime Editing:

On- and off-target unwanted edits

A key element of evaluating safety in gene-editing relates to the specificity of the edits:

is the edit precise at the targeted site, and/or are there off-target edits, or unwanted edits, at more distant locations in the genome, that are the result of gene editing?

In particular, the ability to demonstrate the absence of low, even minimal, levels of off-target editing is a major differentiator for Prime Editing compared to most other gene editing technologies, and we believe this may result in a more benign safety profile for Prime Editing.

A robust and unbiased evaluation of all potential off-target activities is a critical element of our efforts.

Our approach to minimizing off-target editing is to start by screening for Prime Editor candidates with very low off-target activity.

We are developing multiple, complementary, but distinct, methods to measure such possible events.

Our approach includes evaluation, among other methods, of:

(a) off-target activity in the genome that is specific to the sequence of a particular pegRNA or the ngRNA;

(b) similar activity that is independent of the pegRNA or ngRNA sequences;

and (c) genomic rearrangements.

To establish a preliminary set of potential off-target sites, we initially evaluate our promising pegRNAs with computer algorithms that identify other sites in the genome with some degree of sequence similarity.

In parallel, we identify a comprehensive set of putative sites where the Prime Editor could nick the DNA, as determined by several orthogonal experiments performed in cell-free conditions with DNA.

Combining these sets of sites, we then evaluate each of those locations extensively using sequencing methods to identify the potential for very low frequency off-target edits and perform the evaluation in cells that are relevant to the disease for which the Prime Editor is intended.

From these parallel methods, there may be up to thousands of potential sites evaluated for off-target activity for each potential Prime Editor and we are able to quantify the absence or presence of Prime Editing at these sites.

As an example, from our chronic granulomatous disease program, we have performed preliminary evaluation of a Prime Editor from the program using one of these key methods.

We identified 550 potential off-target sites in the genome to evaluate for guide-dependent off-target activity, where the Prime Editor might be predicted to cause a nick.

We evaluated these 550 sites in healthy donor CD34+ hematopoietic stem cells, or HSCs, following Prime Editing and did not detect any Prime Editing at these potential sites with a sensitivity threshold of <0.5%.

We have performed similar analyses of edited hepatocytes using a Prime Editor from the Wilson’s disease program described in further detail below, where we identified 170 potential off-target sites.

Targeted sequencing and analysis of these 170 potential off-target sites did not detect any off-target sites, with a sensitivity threshold of <0.5%.

To evaluate off-target effects independent of the pegRNA or ngRNA sequences, we Prime Edit stem cells, and then expand populations of single cell clones, or cells that are genetically identical.

From this large pool, we have the ability to evaluate very low frequency events, such as rare off-target edits, using unbiased methods.

Each clone undergoes whole genome sequencing and is compared to appropriate controls, which allows evaluation of locations that are not predicted from the pegRNA sequence or genomic location.

Using this same type of approach, and additional approaches, we also are employing a combination of methods to look for genomic rearrangements, including targeted or random rearrangements, using whole genome sequencing of clonally expanded Prime Edited stem cells.

Manufacturing Prime Editor Product Candidates

Due to the breadth of potential therapeutic indications that can be served by Prime Editing, we are developing broad manufacturing capabilities and know-how needed to support the rapid advancement of parallel programs into clinical studies.

We are investing in building a strong technical development and operations team with extensive CMC experience providing a good line of sight to biologics license application, or BLA, and commercialization.

This gives us the ability to develop the manufacturing processes and analytical controls needed to produce reliable and high-quality Prime Editing drug products focusing on the most critical CMC activities early.

Early and strategic CMC investment is critical for cell and gene therapy success.

We have three key strategies that guide our early CMC investments described in the figure below.

Early CMC investment in areas such as identifying critical manufacturing process parameters and developing functional potency assays help to provide deep process and product knowledge that is crucial for facilitating tech transfer, troubleshooting manufacturing and supporting future regulatory comparability strategies.

New manufacturing technologies may be incorporated to improve scalability, reliability, and cost of goods of the manufacturing process in the future.

We are also employing automation, data management, and machine learning that will be important for gaining the insights needed to optimize and ensure reliable control of our manufacturing processes, as well as for supporting justification of specifications needed for product regulatory approvals.

Collaboration and relationship building with external contract manufacturers and partners are underway.

As explained above, one of our preferred configurations for the Prime Editor complex consists of two main components.

The first component is the Prime Editor protein consisting of a Cas nickase domain fused to a reverse transcriptase domain.

The Prime Editor protein is generated either by (i) recombinant DNA technology or (ii) in vivo expression from mRNA that is made via in vitro transcription.

The second component is synthetic guide RNA, which is referred to as Prime Editing guide RNA or pegRNA, and nick guide RNA, or ngRNA.

We have established internal capabilities and external partnerships to synthetically produce guide RNA by solid phase synthesis.

We are also designing new chemistry routes along with purification steps to improve scalability, purity, throughput, and modularity.

Prime Editor proteins may be produced using an optimized microbial system.

The proteins are purified and quality controlled, and activity is tested using various biophysical measurements.

The Prime Editor ribonucleoprotein, or RNP, is formulated by mixing the protein with the pegRNA and ngRNA pair.

Prime Editing has been achieved by electroporation of HSCs ex vivo, as well as demonstrated by LNP delivery in primary hepatocyte cells, and other targeted tissues in vitro.

Prime Editor protein may be delivered as an mRNA, which is translated into the Prime Editor protein following delivery to the target cells.

For mRNA production, our efforts are focused on designing mRNA modifications to improve stability, half-life and expression, developing robust purification steps, and evaluating new technologies aimed at speed, purity, and reduced cost.

We have been focused on developing the manufacturing processes both internally and partnering with suppliers to ensure the quality of the Prime Editor components needed for preclinical studies, and IND submission.

In the figure below, the high purity of our pegRNA is shown by high performance liquid chromatography, or HPLC.

On the right panel, the electropherogram similarly shows the high purity of the mRNA that has been produced.

We believe that

the quality of these materials demonstrates our ability to meet our internal Prime Editing requirements, and the regulatory expectations for IND submission.

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The high purity of pegRNA produced in partnership with our preferred external supplier and internally analyzed by HPLC is shown on the left, and a capillary electrophoresis electropherogram shows the high purity of a lot of mRNA produced internally.

We will continue to leverage the significant advances and progress that are being made in the field of manufacturing sciences and analytical controls of genetic medicines and therapies, while focusing specifically on the application and optimization of those technologies for Prime Editing.

In addition, our collaboration with Beam Therapeutics Inc., or Beam, allows us access to specified know-how, methods, and intellectual property in certain fields that we believe will be useful to these activities.

Our overall strategy is to design manufacturing platforms to make the Prime Editing components and associated delivery systems with high throughput, high quality and purity, modularity, and scalability.

Manufacturing platforms will provide the framework for rapidly developing Prime Editors for subsequent new target indications.

Modularity refers to a collection of components that can be rapidly recombined for the construction of a new product candidate.

For example, we believe that once a Prime Editor is established, a new drug product candidate may only require that the relevant pegRNA and ngRNA to be replaced.

Similarly, we believe that the pegRNA and ngRNA will be able to be produced with modularity.

We intend to collaborate seamlessly with various strategic partners to license their technologies and leverage their capabilities and expertise.

We intend to establish strategic partnerships with contract manufacturing organizations with established good manufacturing practice, or GMP, manufacturing capabilities and relevant manufacturing experience in genetic medicines, and where we will bring our Prime Editing process and product knowledge and technical expertise.

Over the longer term, we may decide to build our own manufacturing facilities, especially for critical Prime Editing components where we may decide to leverage our core capabilities in process and product characterization.

Our Portfolio

We believe that Prime Editing has transformative potential that could change the course of how disease is treated.

To maximize the potential of Prime Editing to provide one-time curative therapies to the broadest set of diseases possible, we have purposefully built a diversified portfolio organized around four strategic indication categories, each set of indications chosen to deliver a different strategic goal.

We have constructed our portfolio of 18 programs, including one partnered program, across our strategic indication categories in disease settings where the unique characteristics of Prime Editing could offer compelling advantages over current standard-of-care and novel therapeutic modalities in development.

Our Four Strategic Indication Categories

Immediate Target Indications

Our immediate target indications were chosen as potentially the fastest, most direct paths to demonstrate technological success of Prime Editing in humans.

We prioritized and advanced programs in this strategic category based on a number of criteria including high unmet medical need where the underlying genetic pathogenesis, or

cause of the disease, is well-understood, and where there were well-characterized delivery methods targeting specific organs or tissues.

We also considered the availability of strong clinical and preclinical biomarkers, well-established animal models, a readily accessible patient population, and the regulatory path.

In most cases, the correction of the target gene is initially focused on a predominant mutation or set of mutations, and we intend to expand to additional mutations within each indication.

We also intend on moving quickly into similar follow-on programs in each target organ as we achieve therapeutic success.

For our initial immediate target indications, we have focused on diseases of the blood via ex vivo delivery to hematopoietic stem cells, the liver and the eye.

We have initiated six preclinical programs across these organs/tissues, in addition to one program partnered with Beam and other programs in earlier stages of development.

In addition, we have initiated two preclinical programs for undisclosed indications in the ear.

We believe each of these programs has the potential to deliver rapid preclinical and clinical proof-of-concept for Prime Editing in patients.

Differentiation Target Indications

Our differentiation target indications are focused on areas where Prime Editing can potentially overcome limitations of other gene therapies and editing approaches, with the ability to do precise and much more diverse, targeted edits, in a broader array of organs, tissues and types of cells.

We also focus on areas where our technology has a special impact on a category of genetic diseases, such as the ability to loop out unwanted repeat sequences.

While several potential indications in this category also rely on validated delivery methods, some of our longer-term targets may require novel delivery development.

Programs in this category include:

• repeat expansion diseases, most of which are CNS diseases, or neuro-muscular diseases with pathological, or disease-causing expanded numbers of DNA repeat sequences.

These diseases are particularly tailored for Prime Editing approaches, in that Prime Editing can loop out large numbers of unwanted repeats, so pathologic repeats of different lengths can, in principle, be contracted to a single, healthy repeat sequence

• diseases characterized by mutational hotspots

• diseases caused by mutations in extremely large genes, which we refer to as “big gene” diseases

• diseases in difficult to edit cell types

• multiplex editing without the introduction of double-stranded breaks

• edits to regulatory sequences modulating physiological pathways

• diseases requiring precise physiologic control, where too much or too little activity would be a concern

• extremely high-fidelity locations

We have initiated four preclinical programs for repeat expansion diseases, including Friedreich’s Ataxia, Myotonic Dystrophy type 1, Amyotrophic Lateral Sclerosis and Fuch’s Endothelial Corneal Dystrophy.

In addition we have programs in three undisclosed repeat expansion disease indications, and two additional undisclosed differentiation target indication programs in earlier stages of development.

We believe these programs have the potential to address difficult and complex diseases of great unmet medical need, which are often not accessible to other forms of gene editing approaches.

“Blue Sky” Target Indications

Our “blue sky” target indications category pushes new and innovative technological developments in Prime Editing to extend its application outside of rare genetic diseases and towards our goal of broadly addressing human disease.

We have already conducted a process of enhancements, some of which are being implemented in potential new programs, and we are committed to continue to push the frontier of innovation in genomic medicines by optimizing and expanding our Prime Editing technology and capabilities further.

We believe these advancements to our technology should allow us to proceed more rapidly into opportunities beyond rare genetic diseases, including:

• transforming chronic therapies into a single-dose, one-time permanent therapeutic correction

• preventing serious diseases by targeting the causes before the onset

• treating the genetic basis of common diseases

• inserting or replacing whole exons or genes

• treating infectious diseases

• treating cancers by correcting underlying germline or other mutations or by broadening the reach of immunological approaches to cancers

• using multiplex editing to treat immunological diseases

• treating diseases that require insertion, replacement or inversion of large sequences, enabling novel cell therapies

• enabling other important technologies such as xenotransplantation

While these programs remain in the early stages of conception, we expect this category to become an increasing focus for our company over the next few years.

“March Up the Chromosome” Approaches

As part of our commitment to patients, we envision a truly personalized medicine approach in which we can treat every individual patient with a given disease by "marching up a chromosome," correcting each individual mutation in a gene.

Because Prime Editing can search and replace, by simply swapping out the pegRNA while keeping other elements of a program the same, such as clinical trial design and manufacturing, we believe we will be able to march from mutation to mutation, or from hotspot to hotspot, along a single gene, and eventually treat every individual patient with a specific disease, not just the few with the most prevalent mutations.

This effort will require a multi-year, multi-step strategic approach to identify a limited set of data to support registration across the set of mutations within a gene.

This category can overlap with other strategic indication categories, where we are designing each of our previously-described strategic indications with this approach in mind.

As such, most of our disclosed indications have a plan that can accommodate expansion opportunities to address additional mutations in that disease.

Our current portfolio is focused primarily on the first two strategic indication categories, and includes the following 18 programs, including the Sickle Cell Disease program which is partnered with Beam.

Prime Milestones

We expect that key upcoming events will continue to drive the Prime Medicine platform forward.

The following outlines a summary of select ongoing activities and next steps for Prime Medicine.

All our in vivo studies are preliminary to date.

We will continue to expand preclinical proof-of-concept in vivo , including data from in vivo rodent studies and non-human primate studies in several programs in 2023.

If successful, we expect to next initiate IND-enabling studies for several of our lead programs, with the first IND filing potentially as early as 2024, and

with the potential for additional IND filings as early as 2025.

Since we are in early stages of product candidate development, we will provide an update on our timelines moving forward.

We also anticipate continuing to name additional programs as they advance over the next few years.

In the near-term, we plan to define the early-stage manufacturing processes and controls to produce representative drug product using our multi-modal delivery approaches consisting of electroporation for our ex vivo programs, LNP, as well as AAV.

We are also investing in a dedicated chemistry facility for medicinal chemistry, process development, and analytical chemistry groups, including a non-GMP piloting lab for making guide RNA, mRNA and synthesizing lipids to support our research activities.

Immediate Target Indications

OUR BLOOD PROGRAMS

Chronic Granulomatous Disease – Our program using ex vivo electroporation of hematopoietic stem cells

Chronic granulomatous disease, or CGD, is a rare inherited hematologic disorder that results in a failure of immune defense against extracellular pathogens.

In CGD patients, myeloid cells lack a functional NADPH oxidase, or NOX2, complex, which renders patients susceptible to prolonged and recurrent bacterial and fungal infections and inflammatory complications.

NOX2 is only produced by certain types of bone marrow-derived myeloid cells.

CGD causative mutations occur in approximately one in 200,000 births in the United States, and most children are diagnosed within the first three years of life.

Approximately 60 percent of patients with CGD reach age 30 and Aspergillus infection is the leading cause of mortality.

The NOX2 protein complex has five domains encoded by five separate genes.

Loss-of-function mutations in any of these genes can present as CGD.

The most common form, which represents approximately 65 percent of cases, is caused by mutations in the CYBB gene encoding the gp91 phox protein.

We have identified hotspots in exons 7 and 9 that are amenable to Prime Editing.

The second most common form, which represents approximately 25 percent of cases, is caused by biallelic loss-of-function mutations, in both copies of the NCF1 gene encoding the p47 phox protein.

More than 78 percent of p47 phox CGD patients have a specific, 2-nucleotide deletion, or ΔGT, in the NCF1 gene.

The NCF1 gene location is complex, and also contains pseudogenes, or non-functioning copies of the NCF1 gene.

Preclinical studies have demonstrated that correcting just one copy of the ΔGT mutation restores protein expression and full NOX2 activity.

Limitations of Current Approaches

For individuals with an HLA-matched donor, an allogenic CD34+ hematopoietic stem cell transplant, or HSCT, may provide a possible cure, but the three-year event-free survival rate for patients that receive HSCT may be as low as 70 percent and patients often experience many frequent, debilitating complications, such as graft versus host disease.

Many patients are not able to find a suitable donor for the HSCs and, without transplantation, 50 percent of patients will die by the fourth decade of life.

Antibiotics also provide important supportive care.

Our Approach and Results:

Direct correction of prevalent CGD mutations or hotspots

We are using Prime Editing in in vitro studies to precisely correct the ΔGT mutation in one copy of the NCF1 gene to restore p47 phox protein expression and NOX2 activity.

Our approach is to mobilize a patient’s CD34+ cells into the blood stream followed by apheresis, and separation of these cells by ex vivo enrichment.

Prime Editing components will then be delivered to these CD34+ cells ex vivo via electroporation.

These Prime Edited cells are cryopreserved, quality control tested, and the thawed cells are injected intravenously back to the patient for engraftment.

A critical element of assessing this approach is the presence of long-term engraftment, which is discussed below under Sickle Cell Disease.

Using the programmable features of our Prime Editing technology and ability to address hotspots, our plan is to expand our targets to include key mutations of the more common CYBB gene.

We have screened pegRNA and ngRNA to identify Prime Editing guides and guide pairs that have high activity and perform precise editing at the NCF1 locus.

Initial experiments have utilized healthy donor mobilized CD34+ HSCs which are readily available, and we measure precise editing of ΔGT as a surrogate for editing in patient CD34+ HSCs.

We have performed a series of optimizations to tailor activity for editing at this locus, and several different, high activity Prime Editor proteins remain under evaluation.

The Prime Editor complex is delivered to CD34+ HSCs using electroporation by an established method.

The Prime Editor mRNA is generated by in vitro transcription, and the pegRNA and ngRNA are generated by solid phase RNA synthesis.

The Prime Editor complex is delivered by simultaneously electroporating mRNA encoding the Prime Editor protein along with pegRNA and ngRNA.

The mRNA is translated into the Prime Editor protein during a period of incubation, then the Prime Editor protein assembles with pegRNA or ngRNA, and the complex enters the nucleus with Prime Editing commencing at the target site in the genome.

We have identified a series of Prime Editor complexes that demonstrate approximately 55 percent precise correction at the NCF gene copies in the target cells, or human primary HSCs, as shown in the figure on the left side.

Following cloning of myeloid-differentiated clones after 14 days, in this study, clonal analysis showed that nearly 90 percent of clones had received at least one precise corrective edit to ΔGT, as shown in the figure on the right side below.

This greatly exceeds the approximately 15 percent precise editing target threshold that is predicted to provide a clinical benefit.

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Note that each data point represents the result from a different, individual Prime Editor, with the average percent precise correction of cells shown in the bar graph.

In a second study, we have edited HSCs from patients with the ΔGT mutation, and again demonstrated approximately 80% correction of one or more copies of NCF1.

The figure below (left panel) shows that when control (CGD patient, unedited) patient HSCs are differentiated into myeloid cells, they do not produce p47 phox protein, while cells from healthy donors show 100% of cells expressing p47phox, as expected (healthy donors, unedited).

In contrast, following Prime Editing, approximately 80% of the patient cells demonstrated normal expression of the p47 phox protein (CGD patient, Prime Edited).

These results demonstrate that precise correction by Prime Editing restores the missing protein.

The same Prime Edited myeloid cells were tested for a normally functioning NOX2 protein complex by the ability of NOX2 to produce oxygen radicals (oxidase activity), the key functional activity that is missing in patients.

As shown in the figure below, approximately 80% of the Prime Edited patient cells had fully restored normal NOX2 oxidase activity (right panel).

This NOX2 assay measures directly the functional defect that causes the disease is used to diagnose patients with CGD, and we anticipate using this assay in the clinical trial for diagnostic as well as

clinical efficacy evaluation.

The results support that the genetic correction of the gene has the desired effect of restoring production of the missing protein and restoring the function of the missing protein complex.

The next step is to transition to evaluating engraftment of Prime Edited long-term HSCs, or LT-HSCs in vivo .

We edit CD34 HSCs with Prime Editor, cryopreserve, or freeze, the cells, transfer, thaw cells and infuse.

In these experiments we infuse into immunodeficient mice.

LT-HSCs take up permanent residence in the bone marrow and produce blood lineages (e.g.

red blood cells, white blood cells, platelets) if they engraft and remain healthy.

HSCs Prime Edited with the Prime Editor to correct the NCF1 gene demonstrated long-term and high-level engraftment of the edited HSCs in bone marrow in mice.

The figure below outlines an experiment in rodents that demonstrates, we believe, the potential feasibility of a similar approach for CGD patients in clinical trials.

• In this experiment shown below, HSCs are Prime Edited via ex vivo electroporation to correct the ΔGT mutation in NCF1 genes, and then cryopreserved.

• After thawing, the edited cells are introduced in mice, and allowed to engraft in the bone marrow.

• Mice are studied for 16 weeks.

After 16 weeks, only LT-HSCs remain in the bone marrow, and produce blood cells.

• The proportion of precisely edited cells is evaluated at the beginning of the experiment, and after 16 weeks, looking for evidence that edited cells have long-term durability.

• Prime Edited HSCs showed similar level of engraftment to unedited healthy donor HSCs (bottom panel left), showing the robustness of the engraftment.

• Prime Edited HSCs produced all blood lineages at 16 weeks, similarly to unedited healthy donor CD34+ HSCs.

• At 16 weeks, 84 percent of the LT-HSC population were Prime Edited, similar to the level in the CD34+ HSCs delivered to mice at 0 weeks, indicating LT-HSCs are efficiently Prime Edited, and that the Prime Edited LT-HSCs remain healthy.

In more recent in vivo studies, following further optimizations to the Prime Editor, 92 percent of the LT-HSC population were Prime Edited.

• Finally, the LT-HSCs at 16 weeks generated all blood lineages in the normal proportions, consistent with Prime Edited LT-HSCs retaining fully multipotency.

• Animals were healthy without any evidence of toxicity.

As shown in the figure, edited cells were shown to have long-term duration with no decrease in the percent of precisely edited cells.

This is a critical proof-of-concept that Prime Edited HSCs successfully can engraft, and once engrafted, permanently populate the bone marrow.

We believe these results greatly increase the probability of success of any HSC-based Prime Editing clinical indication.

Based on these results, we have selected a development candidate, designated PM359, from leading Prime Editors for this program and will initiate IND-enabling studies with this development candidate.

In addition, we will also evaluate alternative approaches to delivery, and have begun developing Prime Editors targeted to CYBB mutational hotspots or replace the whole CYBB gene using Prime Editing with recombinase approach known as PASSIGETM.

Sickle Cell Disease (partnered with Beam)

We are partnering with Beam on the preclinical efforts related to the Prime-Edited Sickle Cell Disease, a program which they have licensed from us.

Some of the results from these efforts provide important proof-of-concept for key aspects of our Prime Editing technology, which is the focus of the description below.

Sickle cell disease, or SCD, is a serious inherited autosomal recessive blood disorder caused by a single point mutation in the beta globin gene HBB at the sixth amino acid.

The mutant protein is also known as hemoglobin S, or HbS.

The mutation causes the beta globin protein to aggregate in long rigid biomolecules that bend red blood cells into a sickle shape when oxygen concentrations are low.

The sickled red blood cells obstruct small blood vessels and have a shortened life span.

This results in anemia, severe pain crises, tissue infarctions, local and systemic infections, stroke, and premature death.

SCD is the most common inherited blood disorder in the United States affecting approximately 100,000 individuals.

Current therapeutics are very limited.

The Prime Editing Approach and Results:

Direct correction of the actual disease-causing point mutation in Sickle Cell Disease

We and our partner, Beam, which has conducted all studies described in this section, are using Prime Editing in in vitro studies to precisely correct the disease-causing HbS mutation back to the normal genomic sequence, resulting in wild type hemoglobin.

The approach is similar to that described above for CGD.

Published studies suggest that a 20 percent correction may be sufficient to cure the disease.

Prime Editing is differentiated from other genetic approaches in that it can precisely correct the HbS mutation, restoring normal hemoglobin and directly addressing the underlying cause of SCD, without causing double-stranded breaks.

Double-stranded breaks may result in detrimental insertions or deletions of sequence at this gene location.

In addition, correction of the HBB gene at its natural site leads to permanent, physiological production of normal hemoglobin directly.

To achieve proof-of-concept and demonstrate precision editing and safety in primary HSCs, we have screened pegRNAs and ngRNAs to identify guides and guide pairs that have high activity and perform precise editing at the HBB locus.

We have created an SCD model using readily available healthy donor mobilized CD34+ HSCs to install the HBB E6V mutation, thereby creating the SCD mutation and phenotype and acting as a surrogate for Prime Editing at that unique location in the gene.

As noted in the below, these Prime Editors have demonstrated approximately 60 percent precise edits at the HBB locus (left), and when differentiated into red blood cells, under various conditions, they retain the approximately 60 percent editing (middle), and contain more than 50 percent of hemoglobin as HbS, showing we have created the phenotype of SCD.

Active Prime Editors That Correct E6V HBB Mutation Identified.

Prime Editing to Install the E6V Mutation in Erythroid Cells Resulted in HbS Production

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001 and 002 represent the two Prime Editors that were screened and install the E6V mutation.

Importantly, the Prime-Edited SCD program demonstrated long-term and high-level engraftment of the edited HSCs in bone marrow.

The figure below outlines an experiment in rodents that represents the approach planned for humans:

• In this experiment, HSCs are Prime Edited via ex vivo electroporation to introduce the disease-causing sickle mutation, and then cryopreserved.

• After thawing, the edited cells are introduced back in mice, and allowed to engraft in the bone marrow.

• The proportion of precisely edited cells is evaluated at the beginning of the experiment, after eight weeks, and then again after 16 weeks, looking for evidence that edited cells have long-term durability.

Ex Vivo Prime Editing of Mobilized Peripheral Blood Human CD34+ Cells to Install the E6V HBB Resulted in Long Term (16 Week) Engraftment of Precisely Edited Hematopoietic Stem Cells In Vivo

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In this experiment, each data point represents the results from a single mouse.

001 and 002 represent the two Prime Editors that were screened.

As shown in the figure, edited cells were shown to have long-term duration with no decrease in the percent of precisely edited cells.

This is a critical proof-of-concept that Prime Edited HSCs can successfully engraft, and once engrafted, permanently populate the bone marrow.

We believe these results greatly increase the probability of success of any HSC-based Prime Editing clinical indication, such as CGD described above.

Fanconi Anemia:

Another indication using ex vivo electroporation of HSCs

Fanconi anemia, or FA, is a rare and life-threatening DNA repair disorder that arises from loss-of-function mutations in any of 23 genes whose protein products are involved in the Fanconi Anemia/Breast Cancer DNA repair pathway.

The FA core complex consists of 10 individual proteins.

Almost all cases of FA result from inactivation of FA genes on both chromosomes.

The protein products of FA genes form the Fanconi complex, which responds to and repairs DNA breaks that occur naturally during cellular replication or in response to radiation or DNA crosslinking agents.

Clinical presentation includes congenital anomalies, loss of many types of blood cells and progressive bone marrow failure, as well as a predisposition to cancers such as leukemia and head and neck cancers.

The majority of FA patients show serious disease within the first decade of life.

Limitations of Current Approaches

Allogeneic hematopoietic stem cell transplant, or allo-HSCT, is currently considered the standard of care for FA and can result in hematologic correction of the disorder.

However, HSCT is associated with both acute and long-term risks, including transplant-related mortality, graft versus host disease, as well as increased risk of subsequent cancers.

Additionally, the sensitivity of FA patient cells to DNA damage complicates allo-HSCT because of the reliance on alkylating agents and radiation for pre-transplant conditioning.

Median survival for all FA patients, despite standard of care, is 24 years.

Our Approach and Results

Inherited pathogenic variants in FANCA, FANCC or FANCG genes, all members of the FA core complex, account for approximately 90 percent of FA cases.

Among these, mutations in FANCA account for more than 60 percent of patients, FANCC for 15 percent of patients and FANCG for 10 percent of patients.

We are initially focusing on two

predominant FANCC mutations and two predominant FANCA mutations with our initial approach to design Prime Editors to correct each predominant FANCC or FANCA mutation independently.

We are targeting 50 percent correction as heterozygotes have no disease, but evidence suggests moderately lower rates of correction have the potential to be therapeutic due to a survival advantage of corrected cells.

Unlike most ex vivo HSC indications, there is strong clinical evidence that engraftment of HSCs can occur without the need for strong bone marrow conditioning.

Our preliminary screening process has identified Prime Editors that achieve approximately 40 percent precise editing in preclinical studies.

Our initial Prime Editors have not yet been optimized for FA, nor have they been tested with enhancements such as ngRNAs.

We intend to continue to optimize these Prime Editors to improve the efficiency of the edits.

In addition, Prime Editors targeted at the second predominant mutation within the FANCC and the FANCA genes are currently under evaluation.

Ultimately, we intend to develop Prime Editors to address all known mutations in FA across the three genes.

Expansion Opportunities in Hematology Pipeline

We plan to add additional hematology-related indications to our pipeline.

In addition to establishing the indications above, the experience and methods developed with Prime Editing should enable our ability to advance other hematology programs.

This highlights the versatility and modularity of our platform that potentially enables the rapid creation of new product candidates by merely replacing the pegRNA and ngRNA components.

OUR LIVER PROGRAMS

Panel shows a schematic of the Universal targeted LNP encapsulating the Prime Editor drug components (left) and dose responsive whole liver in vivo Prime Editing of the mouse PCSK9 gene using 2 different leading LNP formulations.

on experiment delivering LNPs to the liver containing a Prime Editor precisely introducing a stop codon into the mouse PCSK9 gene.

Note that the targeted LNPs deliver to hepatocytes in liver.

Therefore, maximum editing possible is predicted to be no more than 60%.

Leading Prime Editor LNP formulations resulted in dose responsive Prime Editing of whole liver mouse PCSK9.

Following optimizations of the components and their formulations in this system, by swapping only the guide RNAs (pegRNA and ngRNA) for PCSK9 Prime Editor for program specific guide RNAs test articles that can be evaluated in humanized mice or in NHPs for the liver programs, Wilson’s Disease and Glycogen Storage Disease.

Wilson’s Disease

Our lead Prime Editing liver program using LNP delivery technology

Wilson’s disease, or WD, is a devastating rare disease of the liver, with manifestations throughout the body, that is caused by copper accumulation.

Most people are diagnosed with WD between ages five and 35 years and with reported prevalence rates ranging between 1/10,000 and 1/30,000, it is expected to affect upwards of 35,000 to 100,000 patients in the United States and Europe.

Most people are diagnosed with WD between ages five and 35 years and with reported prevalence rates ranging between 1 in 10,000 and 1 in 30,000, it is expected to affect upwards of 35,000 to 100,000 patients in the United States and Europe.

It is also understood that there may be significant under-diagnosis of WD.

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Genotyping of ATP7B is not routinely performed during diagnosis and is used to confirm the symptomatic diagnosis when necessary.

Limitations of Current Approaches

There are no therapies that target the underlying cause of WD.

Current therapy includes removal of copper from the body using copper chelators d-penicillamine and Trientine and prevention of dietary absorption of copper in the intestine using zinc.

In five to 10 percent of the patients that present with severe or sudden onset, or fulminant, WD, liver transplantation is the primary treatment option.

The disease is fatal if undiagnosed and/or untreated.

Most patients improve on chelator therapies;

nevertheless, a lack of compliance is associated with rapid progression to death, and treatments can include significant and intolerable side effects.

Patients are eligible for liver transplant if they have fulminant liver failure or severe progressive liver cirrhosis.

Successful transplant has a good response with many patients but requires life-long immunosuppression, and five-year patient survival after transplant has been reported to be 65 percent.

Our Approach and Results:

Direct correction of prevalent ATP7B mutations

Our initial approach to Wilson’s disease is to correct the prevalent mutations ATP7B H1069Q and R778L in hepatocytes of the liver at their genomic location.

Our initial approach to WD is to correct the prevalent mutations ATP7B H1069Q and R778L in hepatocytes of the liver at their genomic location.

A Prime Editor that corrects R778L will also correct R778W and R778G mutations, rarer mutations that are seen in the U.S.

We are also evaluating hotspot editors in the ATP7B gene region around R778L that could address additional patients.

We have performed pegRNA and ngRNA screens and identified guide combinations that correct the disease-causing point mutations.

Correction of the gene in the liver should address all aspects of the disease by normalizing the process in which the body removes copper in the liver.

In a hepatocyte cell line with the human WD mutation, we have identified Prime Editors that demonstrate precise correction of H1069Q ATP7B in 77 percent of cells as shown in the figure below on the left.

We have observed similar results in primary human hepatocytes with the R778L mutation, which is shown in the figure below on the right.

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Note that each data point represents the result from a different, individual Prime Editor, with the average percent precise correction of cells shown in the bar graph and the red circle highlights the best performing Prime Editor from the 5 demonstrated.

This high level of precise editing in primary hepatocytes meets our threshold of 25-50 percent for predicted clinically relevant effects.

In a hepatocyte cell line with the human WD mutation, we have identified Prime Editors that demonstrate precise correction of H1069Q ATP7B in approximately 80 percent of cells as shown in the figure below on the left.

We have further demonstrated precise correction of approximately 40 percent in primary humanized mouse hepatocytes bearing the human ATP7B gene with the H1069Q mutation, which is shown in the figure below in the middle, and we have also observed approximately 50 percent precise correction using a surrogate Prime Editor in primary NHP hepatocytes, which is shown in the figure below on the right.

Note that each data point in the figure on the left represents the result from a different, individual Prime Editor, and the red circle highlights the best performing Prime Editor, with the average percent precise correction of cells shown in the bar graphs.

This high level of precise editing in primary hepatocytes meets our threshold of 25 to 50 percent for predicted clinically relevant effects.

To support this, we performed a copper toxicity challenge in liver cells that are normal and liver cells with a pathogenic H1069Q mutation with varying degrees of precise editing correction.

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left bar) and liver cells with a pathogenic mutation that are unedited (0 percent;

The third and fourth bars show that with different degrees of precise correction, such as 14 percent and 55 percent, the

ability of Prime Edited cells to survive copper toxicity returns towards normal levels the greater the level of correction.

We are currently conducting preclinical studies to confirm the ability to correct the human R778L sequence and the human H1069Q sequence in humanized mouse models, using LNP delivery technology that we have demonstrated to efficiently deliver Prime Editing to the liver in vivo as described above in “—Translating Prime Editors into Product Candidates”.

We are also currently optimizing LNP formulations for validation of Prime Editing experiments in non-human primates.

The third and fourth bars show that with different degrees of precise correction, such as 14 percent and 55 percent, the ability of Prime Edited cells to survive copper toxicity returns towards normal levels the greater the level of correction.

Initial genome-wide studies using patient cells and human hepatocytes have not identified any detectable off-target editing.

Leveraging our modular universal targeted LNP delivery platform, we are currently conducting preclinical studies to optimize our H1069Q and R778L Prime Editors in humanized mouse models and NHP.

Glycogen Storage Disease 1b

Another Prime Edited liver indication using LNP delivery technology

Glycogen Storage Disease 1b, or GSD1b, is a rare, serious progressive disease affecting approximately 1,500 patients and caused by impaired glycogen metabolism.

In October 2023, we reported new preclinical data demonstrating the ability of liver-targeted Prime Editors to efficiently and precisely correct one of the most prevalent disease-causing mutations of GSD1b in NHPs and mouse models.

These data are the first Prime Editing data in NHPs and we believe provide further proof-of-concept for our Prime Editing approach to potentially address a wide range of diseases.

GSD1b is a rare, serious progressive and fatal disease affecting approximately 1,500 patients and caused by impaired glycogen metabolism.

This autosomal recessive disease is caused by mutations in the glucose-6-phosphate transporter, G6PT also known as SLC37A4.

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Multiple other serious manifestations can occur.

Limitations of Current Approaches

Current care focuses on nutritional therapy to avoid fasting hypoglycemia with small, frequent feedings high in complex carbohydrates, and limitation of fructose, sucrose, and lactose.

There are no disease modifying therapies for patients with GSD1b.

Others are developing genetic therapies for patients with a similar disease, GSD1a.

Our Approach and Results:

Direct correction of prevalent mutations in SLC37A4

Our initial approach to treating patients with GSD1b is to apply Prime Editing via LNP delivery to hepatocytes in the liver to correct the two most prevalent mutations that cause the disease, which are located very close to each other in the gene.

Our initial approach to treating patients with GSD1b is to use our Universal Targeted LNP platform to deliver Prime Editor complex to hepatocytes in the liver to correct the two most prevalent mutations that cause the disease, which are located very close to each other in the gene:

L348fs, and G339C.

In Caucasian populations, these two predominant mutations together are found in 45 percent of patients.

Based on prevalence data we estimate there are approximately 650 patients in the United States and 1,450 patients in Europe with GSD1b, and we estimate there are approximately 950 patients with these mutations.

Heterozygote carriers have no disease and animal studies of GSD1b suggest that little as 11 percent of normal activity has the potential to restore normoglycemia.

As shown in the figure below, in our initial screening studies, we have identified Prime Editors that demonstrate editing of the first mutation with approximately 80 percent efficiency (left), before any optimization.

initial screening studies have identified Prime Editors that demonstrate editing of the second mutation with approximately 50 percent efficiency (right), also prior to optimization.

We are currently evaluating whether a single Prime Editor could correct both prevalent mutations (hotspot editing) since the mutations are only 26 base pairs separated in the gene.

We are establishing patient-derived hepatocyte cultures to establish a genotype-phenotype biomarker response, and have established novel mouse models harboring the human gene.

We will use LNP delivery technology that we have demonstrated to efficiently deliver Prime Editing to the liver in vivo (see Delivery section).

We are also capitalizing on the learnings from the Wilson’s disease program to formulate Prime Editors within LNPs for delivery to the liver.

In addition, we are evaluating whether Prime Editing could address additional patients with GSD1b.

Based on prevalence data we estimate there are approximately 650 patients in the United States and 1,450 patients in Europe with GSD1b, and we estimate there are

approximately 950 patients with these mutations.

While heterozygote carriers have no disease and animal studies of GSD1b suggest that as little as 11 percent of normal activity has the potential to restore normoglycemia, we use an estimate that 20 percent of activity normalizes fasting glucose.

As shown in the left figure below, we have identified Prime Editors that demonstrate precise correction of the first mutation with approximately 80 percent efficiency in primary hepatocytes harboring the L348fs mutation.

We formulated Prime Editors in our Universal Targeted LNP platform and, as shown in the right figure below, also demonstrated in vivo editing of the first mutation with approximately 80 percent efficiency in the livers of humanized SLC37A4 mice where the mouse gene has been replaced with the human gene harboring the L347fs mutation.

The NHP Prime Editor is directed to the same location in the SLC37A4 gene as the human Prime Editor, but the NHP genomic sequence is slightly different from the human sequence, hence the use of a surrogate.

To further evaluate our Universal Targeted LNP platform, we were also able demonstrate in vivo editing with approximately 70 percent efficiency in NHPs using surrogate SLC37A4 Prime Editor, as shown in the figure below.

In our preliminary safety studies, the Universal Targeted LNPs were well tolerated in both rats and NHPs.

Initial genomewide studies using patient cells and human hepatocytes have not identified any detectable off-target editing.

We are currently performing lead optimization of our GSD1b Prime Editors, establishing an efficacy and safety data package including genotype-phenotype biomarker response, and off-target, safety.

In addition, we have developed a series of Prime Editors that precisely correct the G339C mutation with high levels of editing efficiency and are evaluating them in in vivo humanized mouse studies.

Expansion Opportunities in the Liver Pipeline

Now that we have established the ability to deliver Prime Editors via LNPs to hepatocytes, we could potentially advance other Prime Editing liver programs to the clinic quickly.

This highlights the versatility and modularity of our platform, which potentially enables the rapid creation of new product candidates by merely changing pegRNAs.

In addition, in each of our liver indications, our “march up the chromosome” personalized medicine approach allows expansion opportunities into the larger set of pathological mutations that exist in patients with these debilitating diseases.

OUR EYE PROGRAMS

To accelerate our liver programs, we are continuing to develop our proprietary universal targeted LNP platform in liver.

Now that we have established the ability to deliver Prime Editors via LNPs to hepatocytes in vivo , we could potentially advance other Prime Editing liver programs more quickly, reflecting the versatility and modularity of our platform, which potentially enables the rapid creation of new product candidates by merely swapping out pegRNAs.

Our Lung Programs

Cystic Fibrosis

CF is a progressive lung disease characterized by production of thick mucus lung secretions which lead to blockage of airways, inflammation, and lung infection, progressing ultimately to lung failure.

It also affects the pancreas gland and biliary system of the liver in a similar way, leading to exocrine pancreatic failure and mild to moderate cholestatic liver disease in some patients.

Most patients are diagnosed before two years of age through newborn screening or because of symptoms of lung disease, combined by salty skin which can be confirmed using a sweat test.

Through supportive care and antibiotic therapies patient median survival has increased to early thirties before lung failure necessitates lung transplantation, if available.

Overall CF prevalence in the United States and Europe is

approximately 70,000 to 90,000 people (~40,000 in the United States), and while significant progress in the last decade has created therapeutic options for many patients (e.g.

Trikaftor), there is no cure and existing treatments are ineffective or not tolerated for approximately 15 percent of patients.

The disease is inherited recessively and caused by loss-of-function mutations in a chloride protein transporter called CF transmembrane conductance regulator, or CFTR.

Approximately 65 to 75 percent of CF patients have a three-nucleotide deletion in the CFTR gene known as F508del.

The vast majority of remaining patients have one of several prevalent mutations in a small number of genetic hotspots in the CFTR gene, including mutations such as N1303K, W1282X, G542X, or G553X / G551D and I507del.

F508del and several other mutations result in misfolding of the CFTR protein which fails to reach the plasma membrane, whereas other mutations lead to complete absence of protein or a protein which does not function even though it is localized at the correct site in the cell.

The failure of CFTR to function at the cell surface leads to cell secretions that lack sufficient salt and water, resulting in high viscosity and inability to clear secretions from lung and pancreas.

Our Approach:

Correct prevalent mutations and mutational hotspots in the CFTR gene

We intend to progress two distinct strategies for applying Prime Editing to treat CF:

hotspot editing and PASSIGE.

Through hotspot editing, we aim to address multiple mutations at CFTR mutational hotspots with a small number of Prime Editors.

This strategy has the potential to address a large percentage of individuals having CF with only a few Prime Editors, with a particular focus on the 15 percent of patients who cannot be treated with current therapy.

Preclinical data generated by us suggest that using only eight hotspot Prime Editors could benefit more than 93 percent of all people with CF, including those living with nonsense and rare mutations whose disease is not amenable to treatment with currently approved therapies, as well as those who do not tolerate existing therapies.

In parallel, with PASSIGE, we aim to address nearly all people with CF with a single CFTR superexon insertion strategy.

Our preliminary screens have identified hotspot Prime Editors that achieve high levels of precise correction.

As shown in the figure below, we observed approximately 70 percent precise editing of the G542X mutational hotspot (left bar), and approximately 50 percent precise editing of the hotspot encompassing I507del and prevalent F508del mutations (right bar) in patient cells.

1 G542X and I507del are “high unmet need” mutations;

F508del is one of the most prevalent CF mutations;

2 data show correction in patient induced pluripotent stem cells.

Each dot shows a different Prime Editor.

In initial proof of concept studies, we developed an intestinal organoid swelling assay that enabled us to test the impact of our Prime Editors on CFTR protein function.

As shown in the figure below, healthy donor intestinal organoids swell when stimulated as a result of the CFTR channel pumping salt and water into the organoid (top left image).

Organoids from CF patients do not swell (top right image).

We then edited the organoids from patients with

the G542X mutation and demonstrated that our Prime Editors restore swelling to levels seen in healthy donor organoids (bottom right image).

With CFF’s support, pursuant to the therapeutic development agreement with the Cystic Fibrosis Foundation, or CFF, we are aiming to deliver one-time, non-viral therapy that offers first cure to all patients living with CF.

We will perform optimizations of the hotspot Prime Editors from the early screens to increase efficiency and optimize Prime Editors for additional mutations.

In addition, and in parallel we are developing a superexon insertion approach using Prime’s PASSIGE technology.

Screening for Prime Editors and superexon templates for PASSIGE is underway and we are building a series of assays to evaluate our Prime Editors on restoration of CFTR protein function.

We are testing Prime Editors in patient-derived cells, including iPSCs, intestinal organoids, and human bronchial epithelium.

Humanized mice with part of the mouse CFTR gene replaced with human CFTR gene containing the human mutation, have been developed for us to deliver Prime Editors initially to the lung epithelial basal cells which contain a population of lung stem cells.

We are developing and optimizing LNP formulations to efficiently deliver our Prime Editors to human bronchial epithelial cells in vitro and to lung basal cells in vivo .

Our Ocular Programs

Retinitis Pigmentosa Caused by Rhodopsin Mutations:

Our lead eye indication using AAV delivery technology

Retinitis pigmentosa, or RP, is a subset of related inherited retinal diseases, or IRDs, where disease progression is characterized by loss of night vision in childhood or early adulthood, followed by loss of peripheral vision in adulthood characterized by constricting visual field and eventual loss of central vision leading to blindness later in life.

Our first eye indication using AAV delivery technology

Retinitis pigmentosa, or RP, is a subset of related inherited retinal diseases, or IRDs, where disease progression is characterized by loss of night vision in childhood or early adulthood, followed by loss of peripheral vision and eventual loss of central vision leading to blindness later in life.

One of the most common IRDs is autosomal dominant RP, or adRP, caused by mutations in the RHO gene which encodes the light sensitive Rhodopsin protein, or RhoP, expressed by rod photoreceptors of the retina.

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Approximately 6,000-7,000 patients have adRP in the United States caused by RHO mutations.

We are initially focused on one predominant mutation, P23H, which is highly prevalent in the United States and has been identified as causing disease in approximately 30 percent of all patients (approximately 2,000-2,500 patients).

As we advance our portfolio, we believe that hotspots and other frequent mutations may also be suitable targets for Prime Editing.

Limitations of Current Approaches

There are currently no disease modifying therapies for the P23H form of RHO, and patients are currently managed by supportive care.

We are initially focused on the predominant mutations of P23H and two mutations V345L and P347L which occur in a mutational hotspot.

These three mutations are highly prevalent in the United States and have been identified as causing disease in approximately 60 percent of all patients (approximately 3,000-4,000 patients).

As we advance our portfolio, we believe that other frequent mutations may also be suitable targets for Prime Editing.

Our Approach and Results:

Directly correct prevalent mutations in the RHO gene in photoreceptors of the retina

Our initial approach to adRP is to correct the RHO P23H mutation in rod photoreceptors of the retina at their natural genomic location.

We believe a Prime Editor that corrects P23H will also correct rarer, nearby P23L and P23A mutations.

We have performed pegRNA and ngRNA screens and identified guide combinations that correct the disease-causing P23H point mutation.

Our initial approach to adRP is to develop two Prime Editors to correct the RHO P23H point mutation and a mutational hotspot in RHO in rod photoreceptors of the retina at their natural genomic location.

We believe a Prime Editor that corrects the P23H mutation will also correct rarer, nearby P23L and P23A mutations, while a single hotspot editor could correct 18 different pathogenic mutations in that genomic location, including the most prevalent mutations V345L and P347L.

Natural history studies suggest that correction of only 25 percent of rod photoreceptors would have an important clinical impact, because when 25 percent or more of rods are preserved, there is full preservation of cone photoreceptors that are critical to central vision.

We have identified Prime Editors that demonstrate approximately 65 percent precise correction of the RHO P23H mutant locus.

To deliver our Prime Editors to the eye, we are initially leveraging the tropism of AAV capsids that efficiently transfect the retina to deliver our Prime Editor as a transgene along with our pegRNA (and ngRNA, if necessary) to rod photoreceptors.

Given our lead Prime Editor is larger than AAV packaging capacity, we are using a split AAV system that delivers the Prime Editor with two AAV vectors, similar to the approach for delivery to the CNS, as described above “—Translating Prime Editors into Product Candidates”.

Once inside the rod photoreceptor, the two halves of the Prime Editor protein are recombined to create a functional Prime Editor protein.

We are currently conducting preclinical studies designed to test and optimize RHO P23H Prime Editors in donor human retinal explants.

We also plan to evaluate our approach in non-human primate studies where the Prime Editors will be delivered by subretinal injections to mimic the anticipated route of administration in the clinic.

In parallel, we are generating patient-stem cell derived human retinal organoids to evaluate Prime Editor potency in addressing the phenotypic changes in Rhodopsin localization and rod photoreceptor function.

Finally, we have also generated new mouse models that have introduced the human RHO gene, replacing the mouse RHO gene.

While we are advancing the RHO P23H program, we are also identifying Prime Editors that can correct other prevalent mutations and hotspots in the RHO adRP gene as part of our “march up the chromosome” approach.

Retinitis Pigmentosa and Usher Syndrome:

Our second eye indication using AAV delivery technology

RP can also be caused by mutations in other genes different from RHO.

Mutations in USH2A encoding the very large usherin protein account for approximately 20 percent of autosomal recessive RP cases and can result in Usher syndrome.

Approximately 14,300 patients in the United States have RP due to mutations in USH2A.

While patients with various forms of RP present generally as described for RHO above, each type of RP has differences.

In the case of Usher syndrome, patients present with ocular disease given normal usherin is involved in the regulation of protein transport in photoreceptors.

Patients also present with severe hearing loss and potential vestibular defects, because normal usherin is involved in the maintenance of hair bundle formation during inner ear development in the inner ear.

The USH2A 2299delG mutation is the most prevalent mutation in the USH2A gene, accounting for approximately 16 to 44 percent of all USH2A mutant alleles.

A second predominant mutation USH2A C759F accounts for approximately 15 percent of all pathogenic USH2A alleles.

These two mutations occur within a mutation hotspot.

The precise number of rod and cone photoreceptors that need to be corrected is not known.

Individuals with a single mutant copy of USH2A have no disease, therefore correcting 50 percent of all gene copies would restore patients to healthy gene function.

Based on natural history studies from other genetic forms of RP and macular degeneration, correction of one gene copy in 10 percent and 25 percent of cone and rod photoreceptors respectively is sufficient to preserve vision.

Therefore, correcting mutations in 25 percent of rods and cones may be sufficient to halt disease.

Limitations of Current Approaches

There are currently no treatments available for USH2A RP, or Usher syndrome.

However, hearing aids or cochlear impanation can be used to support hearing loss.

Our Approach and Results:

Directly correct prevalent mutations in the USH2A gene in photoreceptors of the retina.

Our initial approach to USH2A RP is to correct the USH2A 2299delG mutation in rod and cone photoreceptors of the retina at their genomic location.

We have performed pegRNA and ngRNA screens and identified guide combinations that correct the disease-causing nucleotide deletion.

As shown in the figure below, through our initial screening approach, we have identified Prime Editors that demonstrate approximately 54 percent precise correction of the USH2A 2299delG mutation.

Our approach for USH2A is similar to our approach for adRP.

While we are advancing the USH2A 2299delG project, as part of our “march up the chromosome” effort, we are identifying Prime Editors designed to correct other prevalent mutations and hotspots in the USH2A gene.

Expansion Opportunities in the Ophthalmic Pipeline

We plan to add additional eye-related indications to our pipeline.

Once we have established delivery to the retina of a Prime Editor through AAV delivery, there are numerous other retinal diseases where our editing technologies could be applied.

By merely changing the pegRNA and ngRNA sequences, we may be able to rapidly create new product candidates using the same AAV production and delivery approaches pioneered in these retinitis pigmentosa programs.

OUR EAR PROGRAMS

Our Approach to Genetic Hearing Loss

Genetic hearing loss consists of a group of diseases with high unmet need, despite the availability of current therapies.

These diseases result in learning difficulties, behavioral problems and social isolation.

We estimate that there are approximately 6,000 new cases each year in the United States alone, and approximately 470,000 individuals in the United States with genetic deafness.

These diseases are all caused by mutations in proteins that are expressed by specialized cells of the inner ear, or cochlea.

Although mutations in many different genes have been identified, mutations in a small number of genes cause hearing loss in the majority of patients.

Many of these mutated genes have prevalent or closely-clustered mutations.

Similar to our other immediate indications, well-characterized delivery methods are established for delivery of genetic therapies to the inner ear and can be applied to Prime Editing.

Methods to objectively measure changes in appreciation of sound may facilitate early detection of benefit for patients.

Our initial approach is to correct prevalent mutations in genes causing progressive hearing loss, targeting delivery to the cells of the inner ear where those proteins are expressed.

We are currently performing screens to identify Prime Editors that precisely correct prevalent mutations in several genes that cause hearing loss.

We have two initial hearing loss programs.

Usher’s Syndrome Type III:

Our First Hearing Loss Program

Usher’s syndrome type III, or USH3, is characterized by progressive post-lingual hearing loss, variable vestibular dysfunction, as well as adolescent-onset progressive vision loss due to retinitis pigmentosa.

The hearing loss is progressive during childhood years, resulting in complete deafness in adolescents or early adults, learning difficulties, behavioral difficulties, and social isolation.

Usher syndrome is found in 4 to 17 per 100,000 children.

USH3 is inherited recessively and caused by mutations in the Clarin 1 protein encoded by the CLRN1 gene.

Clarin 1 is produced in inner and outer hair cells of the inner ear and, to a lesser extent, by the spiral ganglion cells, where it plays an important function in the maintenance of structures of the hair cells.

In certain populations USH3 accounts for 40 to 50 percent of all Usher patients.

N48K is a mutation commonly found in patients of Ashkenazi Jewish descent, whereas S50fs is commonly found in those descended from Northern Europeans and Y176X is commonly found in individuals of Finnish descent.

Limitations of Current Approaches

There are no disease modifying therapies for these patients.

Hearing aids and speech therapy provide support.

Cochlear implants have offered limited benefit to patients, likely because the disease usually manifests post-lingually.

Our Approach and Results:

Correct prevalent mutations in the CLRN1 gene to restore normal Clarin 1 and hair cell function in the cochlea

Our initial approach to treating patients with USH3 is to apply Prime Editing via dual AAV delivery to hair cells in the organ of Corti in the cochlea to correct the most prevalent mutations that cause the disease, N48K and S50fs.

As shown in the figure below, following optimization from our initial screening studies, we have identified Prime Editors that demonstrate precise correction of the N48K mutation with more than 90% efficiency.

We are currently exploring similar Prime Editors to correct the neighboring S50fs mutation.

To deliver our Prime Editors directly to the cochlea by local injection, we are initially leveraging the tropism of AAV capsids that efficiently transduce the supporting cells and hair cells to deliver our Prime Editor as a transgene along with our pegRNA.

Similar to the retinal programs, we will initially use a dual AAV approach to deliver both the Prime Editor protein and pegRNA.

We are currently building preclinical assays using patient stem cells differentiated into hair cells to confirm that Prime Editing of N48K will correct Clarin 1 expression and hair cell function.

In parallel we are developing assays to optimize delivery of Prime Editors to the cochlea.

Non-Syndromic Hearing Loss – GJB2:

A Follow-On Hearing Loss Program

Two thirds of genetic hearing loss is non-syndromic and is inherited recessively.

The GJB2 gene, which encodes the connexin 26 protein, is expressed by supporting cells of the cochlea and contributes to the normal production of endolymph in the cochlea.

GJB2 is the most commonly-mutated gene in non-syndromic hearing loss, found in 1:2,000 live births and accounting for approximately 1,875 newly diagnosed patients each year in the United States and similar number in Europe, with a very common mutation, c.35delG, found on average in 57% of these patients.

Additional mutations are found in patients of Asian (c.235delC, V37I), Indian/European (W24X) and Ashkenazi Jewish descent (c.167delG).

Since the widespread adoption of newborn and pre-school hearing screening for early diagnosis, many patients are diagnosed with abnormal but preserved hearing and show progressive disease.

Because the disease can lead to profound hearing loss (complete deafness) in the first few years of life, patients suffer learning difficulties, speech difficulties, social isolation and behavioral problems.

Limitations of Current Approaches

There are no disease modifying therapies.

Current therapies include hearing aids, cochlear implants and speech and language therapies.

These approaches do not treat the cause of disease and result in limited benefits.

In particular, cochlear implants provide a partial restoration of sound but require extensive speech and language retraining with variable outcomes.

Moreover, implants require permanent wearing of large external devices.

Our Approach:

Correct prevalent mutations in the GJB2 gene to restore normal connexin 26 function in the cochlea and prevent hearing loss progression

Our initial screens are to find Prime Editors to precisely correct c.35delG.

After identifying preliminary Prime Editors that show installation of the c.35delG mutation with good levels of efficiency, we are currently screening for Prime Editors to precisely correct this mutation.

We will leverage the work from our USH3 program to deliver Prime Editors to the cochlea with a focus on targeting support cells.

We are currently building preclinical assays using patient stem cells differentiated into inner ear cells to confirm that Prime Editing of c.35delG will correct GJB2 expression and connexin 26 function.

Expansion Opportunities in the Hearing Loss Pipeline

We plan to add additional hearing loss-related indications to our pipeline.

Once we have established delivery to the cochlea of a Prime Editor through AAV delivery, we believe there are many other hearing loss diseases where our editing technologies could be applied.

Differentiation Target Indications

OUR REPEAT EXPANSION DISEASE PROGRAMS

Our Approach to Repeat Expansion Diseases

Repeat Expansion Diseases, or REDs, are a collection of more than 50 progressive diseases, mainly affecting the CNS and musculoskeletal system.

These diseases are all caused by mutations that are an expansion of repeat DNA sequences, often triplets of nucleotides, found in the normal genome.

We believe these repeats can potentially be exponentially expanded from typically approximately five to 15 repeats in normal tissues to up to many thousands of repeats in diseased tissues.

The repeats are found within parts of the coding region of genes, called exons;

in non-coding regions of a gene between exons, called introns;

and in non-coding regions of the gene, either at the beginning or end of a gene, that are not translated in RNA, called untranslated regions, or UTRs.

In addition, sometimes REDs are described by the size of the expansion, ranging from short and medium repeats (often less than 200 extra repeats) to medium to long repeats (often more than 200 extra repeats to several thousand extra repeats), as described in the figure below.

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The above figure exemplifies our initial approach to repeat expansion diseases, showing disease names, primary organs affected by disease, approximate estimates of prevalence or incidence, type of expansion mutation, gene where expansion mutation occurs, location and approximate length of expansion mutation within the gene.

In certain repeat expansion diseases, the repeats often cause a pathological gain-of-function, or a new deleterious impact, and are inherited dominantly.

In other settings, the disease is inherited recessively.

Prime Editing enables us to fix the underlying and fundamental causes of these diseases by precisely and completely removing the repeat expansion copies from the DNA, potentially replacing them with the normal, usually small number of healthy repeats, or possibly removing any repeats from the gene.

Our initial approach is to establish the utility and breadth of Prime Editing technology by precisely removing repeat sequences in the pathological target tissues.

In settings where the repeats are in an exon, Prime Editing may be applied to remove the repeat and replace the repeat with the healthy coding sequence precisely.

In other settings, such as repeats in introns, we have more flexibility to our approach.

To build the foundation for the potential correction of all 50 or more repeat expansion diseases, which would address a huge unmet medical need, we are evaluating many different types of these diseases to understand where Prime Editing can be efficient.

For our initial studies, we broadly selected diseases of high unmet need with a path to deliver the Prime Editor to target tissues.

We are studying repeats within exons, introns and the UTR regions, as well as diseases with small-to-medium and medium-to-very large numbers of expansion repeats.

In each individual indication, beyond exploring the potential for Prime Editing to perform these precise corrections or completely remove the pathological repeats, we are also evaluating whether genetic correction will result in phenotypic or biochemical results consistent with a clinical improvement.

We have focused on REDs since inception of our initial preclinical program efforts.

Independently published proof-of-concept of this idea was demonstrated in a repeat expansion disease called Fragile X syndrome, providing corroboration of the potential of this approach.

More than 50 repeat expansion diseases are known to be pathogenic, and we believe our broad approach supports that most, if not all, of the diseases may be amenable to Prime Editing corrections of the target DNA, thereby potentially halting, preventing, or even curing such diseases.

In summary we have achieved predicted therapeutically relevant Prime Editing in almost all repeat expansion disease programs.

The figure below shows the levels of editing achieved for each of the programs and current activity stage of these programs in our drug discovery work-flows.

Similarly to the liver programs, we have developed a modular delivery platform for delivery of Prime Editors to the retina.

In the case of retina, our modular delivery platform is a dual AAV system.

We have optimized the AAV genomes to precisely and efficiently deliver the Prime Editor protein and the Prime Editor guide RNAs, and have optimized the AAV capsid for delivery to photoreceptors.

As shown in the figure below, we have identified Prime Editors that demonstrate up to 65 to 70 percent percent precise correction of photoreceptors in vivo and that were generally well-tolerated with no detectable immune response.

In regions of the retina delivered to, Prime Editors prevented retinal degeneration in vivo .

There were also no off-target edits detected in human photoreceptors and no detectable evidence of viral vector integration into retinal cells.

For our ocular programs, we are optimizing the modular dual-AAV system comprised of an optimized AAV capsid to improve transduction of photoreceptors, optimized AAV genome to increase expression of Prime Editor components, and novel AAV production processes that we believe will enable us to efficiently support multiple products.

We are evaluating the modular dual-AAV in humanized RHO adRP mice including studies to evaluate preservation of the retina following Prime Editor delivery, and we plan to evaluate our approach in NHP studies where the Prime Editors will be delivered by subretinal injections or suprachoroidal injections to mimic the anticipated route of administration in the clinic.

While we are advancing the RHO program with two Prime Editors for P23H and V345L-P347L, we are also identifying additional Prime Editors that can correct other prevalent mutations in the RHO adRP gene.

Other Programs in Discovery

Additionally, in our ocular area of focus, building on the modularity of our ocular platform and learnings from Rhodopsin program, we are also researching RP caused by mutations in USH2A resulting in Usher syndrome.

simply swapping out the pegRNA and ngRNA in our retinal AAV delivery system we have the potential to accelerate the Usher syndrome program as well as advance additional retinal degeneration programs in the future.

In addition, we are researching Fuch’s endothelial corneal dystrophy, a common repeat expansion disease affecting the cornea leading to progressive corneal opacification and blindness.

We continue to make progress in both of these programs.

Our Neuromuscular Programs

Friedreich’s Ataxia

Our lead repeat expansion disease indication

Friedreich’s Ataxia, or FRDA, is a multisystem, autosomal recessive neurodegenerative disorder affecting the central and peripheral nervous system as well as the heart and other organs.

FRDA significantly reduces survival for patients, with the mean age of death being 39 years.

FRDA is characterized by progressive ataxia, or lack of muscle

control or coordination of voluntary movements, with mean age at onset of approximately five to 16 years.

FRDA is characterized by progressive ataxia, or lack of muscle control or coordination of voluntary movements, with mean age at onset of approximately five to 16 years.

A vast majority of patients progress to loss of unsupported sitting within two years and loss of ambulation on average 10 to 15 years from diagnosis.

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In the United States, it is estimated that around 4,000 individuals are affected by FRDA, while there are estimated to be 15,000 to 19,000 patients globally.

FRDA is caused by GAA-repeat nucleotide sequence expansions in the 1st intron of the FXN gene encoding the frataxin protein, which plays important roles in mitochondria.

FRDA is a repeat expansion disease caused by GAA-repeat nucleotide sequence expansions in the 1st intron of the FXN gene encoding the frataxin protein, which plays important roles in mitochondria.

The expanded repeats occur early in the gene, and cause disruptions in transcribing the FXN gene into RNA resulting in low levels of the frataxin protein, the pathogenesis of the clinical disease.

Published literature shows that removal of expanded repeats can restore frataxin expression in vitro .

Limitations of Current Approaches

There is no approved disease-modifying therapy, and current clinical management guidelines mainly focus on symptom management.

Our Approach and Results:

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Our Prime Editing technology enables us to precisely remove the expanded repeat sequences that cause FRDA.

Using the dual-flap technology with a Prime Editor targeted both upstream, or before the GAA pathological repeats, and downstream, or after the repeats, our goal is to precisely remove the repeat sequence from intron 1 of the FXN gene to restore normal FXN regulation and normal expression of frataxin;

the approach is shown in the figure below.

Removal of repeats from FXN in the myocardium is also highly desirable to prevent cardiomyopathy and reduce mortality.

The primary target tissues are for areas of the brain and spinal cord, but we plan to address cardiomyocytes as well.

Our Approach to Restore Normal FXN Regulation and Normal Expression of Frataxin

Our goal is to precisely remove the pathological expanded GAA repeat sequence from intron 1 of the FXN gene to restore normal FXN regulation and normal expression of frataxin using dual-flap and long-flap Prime Editing technologies.

The primary target tissues are for areas of the brain and spinal cord, but removal of repeats from FXN in the myocardium is also highly desirable to prevent cardiomyopathy and reduce mortality, and we plan to address cardiomyocytes as well.

We have performed screens to identify the pegRNA pairs that achieve highly efficient and precise removal of the expanded repeats.

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In addition, the figure on the right shows up to 66 percent precise editing in FRDA patient-derived induced Pluripotent Stem Cells, or iPSCs, which contain larger numbers of pathological repeats, numbering from 420 to 541 nucleotide triplet repeats.

In later, preliminary experiments, some of our Prime Editors have achieved greater than 80 percent precise editing out of the pathogenic repeat region.

Remarkably, the total length of sequence precisely removed can be more than 7,000 nucleotides, or seven kilobase, using dual-flap Prime Editing.

In addition, preliminary in vitro experiments have shown that Prime Editing-mediated removal of pathological repeats resulted in correction of hypermethylation of the FXN gene, restoring gene expression back to wild-type levels.

Our initial experiments are encouraging.

In our models in donor- and patient-derived iPSCs we measure the amount of frataxin expression in both FRDA patient cells, as well as cells derived from healthy donors.

As expected, and as shown in first panel of the figure below, we show that patient cells have low levels of frataxin (control;

left bar), and healthy donor cells (both control and edited;

right bars) have high levels of frataxin.

When we Prime Edit the FRDA patient cells in this model, as indicated by the arrow, each of five individual Prime Editors, in association with high levels of precise correction, restore levels of frataxin towards that seen in healthy donor cells.

As each data point, or dot, represents a different Prime Editor, our best candidate from this experiment can restore frataxin levels to approximately 80 percent of normal levels, which exceeds the 30 percent-of-normal threshold predicted to provide a potential clinical benefit to patients.

The objective of precisely removing expanded repeats from the FXN gene with Prime Editing is to restore normal expression of frataxin mRNA and protein.

To test the effect of Prime Editors on frataxin expression, we edited patient cells with different Prime Editors and, as shown in the figure below, left panel.

In addition, we have established a tight correlation between the efficiency of editing among different Prime Editors and the restoration of frataxin levels with Prime Editors that demonstrated high editing activity showing restoration of frataxin to within 80-90 percent of normal levels, as shown in the left panel below.

The results below, right panel, demonstrate that when we isolated and cloned cells, so that the whole cell population had precise correction of the unwanted repeats, frataxin levels returned completely to normal without over expression.

Since we can correct the gene defect at the physiological site in the genome, an important element is to evaluate all different forms, or isoforms, of frataxin to determine if all potentially important mRNA messages from this gene are restored.

We were able to demonstrate the key frataxin isoforms were restored in this experiment, providing

confidence that we believe supports that this approach may potentially be able to provide a genetic cure by Prime Editing to patients with FRDA.

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*Quantitative polymerase chain reaction data for FXN transcripts normalized to housekeeping control.

**Patient iPSCs contain 541 and 420 GAA repeats.

Consistent with the high levels of precise correction observed in patient iPSCs, we have also observed a restoration of frataxin expression to levels that approach those observed in healthy donor iPSCs.

This is shown in the figure below which illustrates restoration of frataxin protein expression after delivery of the Prime Editor to patient iPSCs.

FRDA = Friedreich’s Ataxia and GAPDH = a control housekeeping protein (glyceraldehyde-3-phosphate dehydrogenase).

One of the hallmarks of Friedreich’s Ataxia is the degeneration of the dorsal root ganglia, or DRGs.

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When we correct 100 percent of the copies of FXN gene there is complete restoration of the sensory axon growth from the patient DRGs (Patient 100% Corrected).

Even, when we correct 50 percent of the copies of the FXN gene there is also complete restoration of the

sensory axon growth from the DRGs.

Even, when we correct 50 percent of the copies of the FXN gene there is also complete restoration of the sensory axon growth from the DRGs.

We believe these in vitro results indicate that Prime Editors may have the potential to restore normal function of patient sensory neurons.

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Fluorescence microscopy images at low magnification of dorsal root ganglia, or DRG, from healthy donor or patient, showing cell nuclei (blue) and axons (green).

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Following Prime Editing to remove the expanded repeats and precisely correct the FXN gene, patient DRGs show normal axon growth.

The heart is a second organ affected by Friedreich’s Ataxia.

We have established preliminary methods to deliver Prime Editors to terminally differentiated patient cardiomyocytes and plan to test editing and recovery of cardiomyocyte function and frataxin expression in this cell system.

Then, to ultimately deliver Prime Editors to heart and other tissues, we expect initially to rely on the tropism of AAV capsids, each optimized to deliver our Prime Editors to the central nervous system or heart.

We have established an AAV delivery system for efficient delivery of Prime Editing to neurons and glial cells in vivo, as demonstrated above in “—Translating Prime Editors into Product Candidates”.

We are now optimizing this system and planning to evaluate our Prime Editors in a disease model in mice which contain the human frataxin gene with pathological repeats.

While AAV delivery is our primary route of delivery for early programs such as this, we are actively determining whether a non-viral delivery system could be used to efficiently deliver the Prime Editor to one or more of our key target organs.

We have established a preliminary dual-AAV delivery system for efficient delivery of Prime Editing to neurons and glial cells in viv o, as demonstrated in the figure below where we have observed robust transduction of over 95 percent of neurons within the injection site (left bar), and where we observed precise editing in approximately 80 percent of the transduced neurons (right bar);

this high editing efficiency supports the robustness of the dual -AAV system.

This system allows us to interrogate promising Prime Editors while we continue to develop an optimized dual-AAV delivery system as described in “Next Steps” below.

Tool Prime Editors were packaged into our preliminary dual-AAV system and together with a reporter AAV were injected into the brains of healthy adult mice.

Four weeks later, brain tissues were collected and fluorescent regions indicating successful AAV transduction were isolated and evaluated for editing of target site.

For our neuromuscular programs, we are establishing a modular dual-AAV system comprised of an optimized AAV capsid to improve transduction across key brain regions using minimally-invasive route of administration, optimized AAV genome to increase safety, specificity and expression of Prime Editor components, and novel AAV production that we believe will enable us to efficiently support multiple products.

In addition to the dual-flap technology used in our preliminary experiments, we are also evaluating a long flap approach to precisely correct the expanded GAA repeats in FXN, and the most promising Prime Editors of either approach will be incorporated into our modular dual-AAV system to be further evaluated in humanized FXN mice containing expanded GAA repeats to confirm editing efficiency in vivo .

We will also evaluate the modular dual-AAV approach using tool or surrogate Prime Editors in NHPs.

Myotonic Dystrophy Type 1

Another repeat expansion disease indication

Myotonic Dystrophy type I, or DM1, is a common autosomal dominant muscular dystrophy among people of European ancestry and is principally a muscle disease affecting skeletal and cardiac muscle with multisystem manifestations.

Patients often initially present with muscle weakness.

Recent newborn screening studies indicate that the true prevalence of DM1 is 1 in 2,300 (approximately 140,000 patients in the United States).

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childhood/juvenile DM1, and adult-onset DM1.

Congenital DM1, where patients typically have more than 800 repeats, often occurs from asymptomatic parents and presents at birth with severe weakness, hyporeflexia, or lack of reflexes, and respiratory insufficiency, and has a 40 percent mortality, with cardiac conduction abnormalities accounting for approximately 70 percent of that mortality.

Congenital DM1, where patients typically have more than 800 repeats, presents at birth with severe weakness, hyporeflexia, or lack of reflexes, and respiratory insufficiency, and has a 40 percent mortality, with cardiac conduction abnormalities accounting for approximately 70 percent of that mortality.

Survivors have distal weakness, cognitive impairment, and neuropsychological disorders.

Childhood/juvenile DM1 is more similar to adult disease presenting at ages of five to 15 years with developmental delays and speech and

learning difficulties.

Childhood/juvenile DM1 is more similar to adult disease presenting at ages of five to 15 years with developmental delays and speech and learning difficulties.

In adolescent patients, muscle weakness, myotonia, or the inability for muscles to relax, and gastrointestinal symptoms are most prominent.

DM1 is caused by expanded CTG repeats in the 3’ UTR of one copy of the DMPK gene.

When transcribed into RNA, the expanded repeat nucleotides in the RNA, in the case of DM1, form toxic RNA foci in the nucleus that, sequester critical nuclear splicing factors, thereby preventing the correct function of many genes that regulate cell function.

Limitations of Current Approaches

Current therapy includes supportive care to manage symptoms, though many experimental approaches are under consideration.

DM1 is a repeat expansion disease caused by expanded CTG repeats in the 3’ UTR of one copy of the DMPK gene.

When transcribed into RNA, the expanded repeat nucleotides form toxic RNA foci in the nucleus that, sequester critical nuclear splicing factors, thereby preventing the correct function of many genes that regulate cell function.

Our Approach and Results:

Directly and precisely remove the pathological repeats in the DMPK gene

Our dual-flap Prime Editing technology enables the removal of the expanded repeat sequences that cause disease.

Our goal in DM1 is to leverage our Prime Editing technology to precisely remove the repeat sequence from the UTR region of the DMPK gene, to restore DMPK regulation and expression of DMPK protein back to normal levels.

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We have performed screens to identify pegRNA pairs that achieve highly efficient and precise removal of the expanded repeats and have demonstrated precise removal of pathological repeats from the DMPK gene.

In the example provided in the figure on the left below, we have established precise removal of the smaller number of repeats in healthy cell lines with more than 80 percent efficiency.

In a second set of experiments as shown in the figure on the right, in patient-derived iPSCs, which contain approximately 1,600 pathological repeats, we have demonstrated precise removal of repeats, with our best Prime Editors achieving more than 90 percent precise editing and removal of the pathological repeats.

These data are also shown in the figure on the right, with each dot representing the data of a different individual Prime Editor.

DM1 is primarily a disease of muscle.

A key target tissue is the cardiac muscle, or myocardium of the heart consisting of cardiomyocytes, a terminally differentiated cell-type.

We developed systems to generate terminally differentiated and functioning cardiomyocytes from patient cells that rhythmically contract and relax, or beat.

This patient tissue can be used to test our ability to achieve high efficiency of Prime Editing in these cells and to test the

ability of Prime Editors to correct cardiomyocyte function.

The figure below shows dose dependent Prime Editing in cardiomyocytes, demonstrating high efficiency can be achieved.

In patient-derived iPSCs, which contain approximately 1,600 pathological repeats, we have demonstrated precise removal of repeats, with our best Prime Editors achieving more than 90 percent precise editing and removal of the pathological repeats.

These data are shown in the figure below, with each dot representing the data of a different individual Prime Editor.

As mentioned above, a hallmark of the disease is toxic RNA foci , formed from the repeats, that sequester key splicing factors in the cells with this disease.

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We edited patient cardiomyocytes to remove the pathological repeats with one of our Prime Editors and evaluated the impact on the formation of toxic RNA repeats.

As is also shown in the left side of the figure below, cardiomyocytes that have 100 percent of pathological DMPK gene corrected, RNA foci are no longer detectable (right column, top);

As is also shown in the right side of the figure below, cardiomyocytes that have 100 percent of pathological DMPK gene corrected, RNA foci are no longer detectable (right column, top);

nor is MBNL1 staining detectable (right column, bottom) with only background staining detectable.

The right side shows a quantification and analysis of the results, with 100 percent correct patient cells showing levels of RNA foci similar to those in healthy donor cells.

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Left hand panel shows fluorescence microscopy images at high magnification of patient cardiomyocyte nuclei.

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Columns showing patient cardiomyocytes 100% corrected or 0% corrected (unedited) and healthy donors.

We have established a preliminary dual-AAV delivery system for efficient delivery of Prime Editing to muscle, as demonstrated in the figures below, left panel, where using tool Prime Editors we have observed precise editing of up to 90 percent in cardiomyocytes, and right panel, where we observed precise editing of up to 50 percent in skeletal myotubes.

This system allows us to investigate promising DM1 Prime Editors while we continue to develop an optimized muscle delivery system as described in “Next Steps" below.

Human iPSC-derived cardiomyoctes (above left) and mouse skeletal myotubes differentiated from myoblasts (above right) were treated with tool Prime Editors packaged in dual-AAV system at various titers and evaluated for precise editing of target site.

We are evaluating the ability of Prime Editing to correct the mis-splicing of a panel of genes that are known to be mis-spliced as a result of the toxic RNA foci.

In parallel, we will perform similar experiments in patient-derived skeletal muscle cells.

Then, to ultimately deliver Prime Editors to heart and skeletal muscle, we expect initially to rely on the tropism of AAV capsids, optimized to deliver our Prime Editors to the heart and skeletal muscle.

We have established an AAV system for efficient delivery of Prime Editing in neurons and glial cells in vivo, as demonstrated above in “—Translating Prime Editors into Product Candidates”.

We are now optimizing this system for the DM1 program and planning to evaluate our Prime Editors in a disease model in mice which contain the human DMPK gene with pathological repeats.

In parallel, we plan to perform similar experiments in patient-derived skeletal muscle cells.

To ultimately deliver Prime Editors to heart and skeletal muscle, we expect initially to rely on the tropism of AAV capsids, optimized to deliver our Prime Editors to the heart and skeletal muscle.

We have established an AAV system for efficient delivery of Prime Editing in neurons and glial cells in vivo , as demonstrated

above in Friedreich’s Ataxia.

We are optimizing this system for the DM1 program and planning to evaluate our Prime Editors in a disease model in mice which contain the human DMPK gene with pathological repeats.

Amyotrophic Lateral Sclerosis:

Another repeat expansion disease indication

Amyotrophic lateral sclerosis, or ALS, is a rapidly progressive neurodegenerative disease characterized by progressive motor neuron loss.

Mean age of onset for ALS is 58 to 60 years, with mean survival of three to four years after onset.

The disease selectively results in dysfunction of upper and lower motor neurons, which later degenerate and die.

Degeneration of these cells primarily causes impairment of motor function, and leads to muscle weakness, changes in speech, and difficulty breathing and swallowing, with death caused by paralysis and respiratory failure.

Overall ALS prevalence in the United States and Europe is approximately 40,000 patients.

Approximately 11 percent of ALS cases have pathological expansions of the hexa repeat GGGGCC in intron 1 of the ALS C9orf72 gene.

The same repeat expansions have been found in a study to cause as high as 53 percent of frontal temporal dementia and an overlap syndrome known as ALS/FTD;

in addition, C9orf72 is thought to be causative in a proportion of Parkinson’s disease, Huntington’s disease, Corticobulbar syndrome and Olivopontocerebellar degeneration.

Eleven or fewer repeats are normal, but the disease is associated with expansion to hundreds or even thousands of repeats.

Longer repeats are associated with earlier onset and more severe disease.

The primary pathology is thought

to be a toxic RNA gain-of-function by which the mRNA sequesters nuclear factors in a different but analogous way to the repeats in DM1.

Limitations of Current Approaches

Currently resveratrol, a type of naturally occurring polyphenol, is the only approved therapy for ALS which modestly slows disease and may function as an anti-oxidant.

All other therapies are supportive.

Our Approach and Results:

Directly and precisely remove the pathological repeats in the C9orf72 gene

Our initial approach is the correction of the C9orf72 mutation by removing the GGGGCC repeat expansion tract using dual-flap Prime Editing, thereby restoring normal C9orf72 regulation and expression.

We have performed screens to identify pegRNA pairs that achieve highly efficient and precise removal of the repeats in the first intron of C9orf72.

As shown in the example provided in the figure below, left panel, we have established precise removal of repeats in healthy cell lines with more than 90 percent efficiency.

The panel on the right shows that Prime Editors are highly efficient in removing repeats from patient iPSCs with more than 160 pathological repeats of GGGGCC sequence.

We have developed ALS patient-derived motor neuron cultures and are building assays to establish the impact of Prime Editing on patient-derived motor neuron function.

Our approach to delivery is similar to that of other repeat expansion diseases, such as FRDA described above.

Similarly, we will assess editing efficiency in mouse models, eventually progressing to non-human primate studies.

Fuch’s Endothelial Corneal Dystrophy:

Another repeat expansion disease indication

Fuch’s Endothelial Corneal Dystrophy, or FECD, is a common disease of the cornea of the eye leading to progressive corneal opacification and blindness.

FECD patients present with blurred vision, visual acuity loss, bright light sensitivity and presence of extracellular matrix excrescences called guttae, which can cause pain.

The disease starts with degeneration of the corneal endothelial cells which supply nutrients to the inner layer of the cornea and maintain transparency.

FECD is estimated to affect over 600,000 patients in the United States, usually presenting in patients over 40 years of age.

We estimate that between 14,000 and 14,500 cases annually progress to visual deterioration in the United States sufficient to require corneal transplantation.

Approximately 79 percent of FECD patients have a mutation in the TCF4 gene, with expanded nucleotide triplet repeats;

the repeats result in aggregates that sequester cell splicing factors, similar to what occurs in patients with Myotonic Dystrophy.

Limitations of Current Approaches

Early treatment is localized, but as the disease progresses, surgical management becomes necessary.

Current approaches are surgical and require corneal grafts, which are sourced from cadavers and are in scarce supply.

Surgery is also complicated by graft loss;

there is only approximately 64 percent graft survival at 10 years following surgery.

The procedure requires general anesthesia, may require immunosuppression, may require long periods off work, and may be complicated by infections, glaucoma, cataract, and astigmatism, and it can still take up to one year following the procedure for full sight restoration.

Our Approach and Results:

Correct the TCF4 mutation in corneal endothelial cells

Our initial approach is to deliver Prime Editors to the anterior chamber of the eye to correct the TCF4 mutation in corneal endothelial cells by looping out the pathological repeats.

The advantages include avoiding surgery and the lack of requirement for cadaveric tissue, among others, as well as the potential to achieve a permanent correction.

We have performed screens to identify dual-flap Prime Editors that can achieve highly efficient and precise removal of the repeats in the TCF4 gene, achieving precise removal of repeats from healthy cell lines with more than 70 percent efficiency, as shown in the figure below.

Our next steps include validating the activity of these Prime Editors in patient-derived endothelial cells.

Our goal is to deliver Prime Editors to the corneal endothelial cells before degeneration of the capillary network.

Our initial approach will be to encapsulate Prime Editors, to be delivered as either ribonucleoprotein or RNA, and deliver them to the endothelium by anterior chamber microinjection.

Oculopharyngeal Muscular Dystrophy:

Another Repeat Expansion Disease Indication

Oculopharyngeal muscular dystrophy, or OPMD, is a rare autosomal dominant disease, characterized by progressive weakness in the muscles around the eyelids as well as in the tongue and pharynx.

This weakness manifests as

difficulties with vision, swallowing and speaking.

As the disease progresses it affects the neck, shoulders and the limbs, resulting in difficulty walking.

The disease is often first diagnosed in adults in their 40’s, with a small minority ultimately requiring a wheelchair.

Major manifestations include eyelid drooping, dysphagia (difficulty swallowing) and facial muscle weakness.

Dysphagia can result in malnutrition, aspiration and pneumonia.

OPMD is often identified in individuals of French Canadian descent, Ashkenazi Jewish, and Latino populations from central and northern America where prevalence may be high (e.g.

in French Canadian populations it is 1 in 1,000).

Overall prevalence estimates in Europe are approximately 1 in 100,000.

OPMD is caused by expansion of a GCG repeat sequence in the first exon of the PABPN1 gene which encodes the poly-A binding nuclear protein-1.

The GCG repeat encodes a short track of alanine residues with 7 CGC repeats.

Short expansions of 11 to 18 repeats are sufficient to cause disease.

OPMD affects all voluntary muscles but appears to spare smooth and cardiac muscle.

The disease is characterized histologically by intranuclear inclusions in muscle of aggregated PABPN1 protein.

Aggregation of the mutant PABPN1 protein is thought to be an important part of the pathological mechanism, due to sequestration of normal PABPN1 or a toxic gain-of-function to the formation of poly-Alanine protein.

Limitations of Current Approaches

Current therapies are supportive and may involve surgical procedures to the eyelids and esophagus, including local botulinum toxin injections.

There are currently no disease modifying agents.

Our Approach:

Precisely remove the repeats from the mutant PABPN1 gene and replace with healthy sequence to restore normal PABPN1 function in the eyelid and pharyngeal muscles.

Our initial approach is the correction of the PABPN1 mutation by removing the GCG repeat expansion tract using Prime Editing and replacing with a sequence encoding 7 alanine amino acids, thereby restoring normal function to the PABPN1 gene.

In our initial screening studies, we have identified Prime Editors that demonstrate editing of the first mutation with approximately 25 percent efficiency.

We plan to perform additional screens and to optimize currently identified Prime Editors to increase efficiency, then validate Prime Editors in patient-derived cells and demonstrate correction of the PABMP1 localization protein function.

We are currently evaluating whether a local therapeutic approach to inject muscles of the eyelid, pharynx and tongue using a LNP encapsulating Prime Editor cargo of an mRNA and pegRNAs would provide a strong therapeutic benefit to patients.

Fragile X Syndrome:

Another Repeat Expansion Disease Indication

Fragile X syndrome, or FXS, is an X chromosome-linked dominant rare disease, which is the most common monogenic cause of childhood intellectual disability and autism.

Patients have cognitive, behavioral, and sensory deficits, and may have cardiac and skeletal manifestations.

FXS is found globally, and is estimated to affect 40,000 to 60,000 in the United States, with a similar number in Europe.

FXS is caused by expansion of a CGG repeat tract in the 5’ untranslated region of the FMR1 gene, leading to aberrant gene silencing and loss of the Fragile X mental retardation protein (FMRP), a protein important for brain development.

Loss of FMRP leads to abnormal protein synthesis from many genes involved in synaptic function leading to abnormal synaptic signaling and abnormal morphology of neurons.

CGG is repeated more than 200 times in FXS patients, whereas 5-40 repeats regularly occur in healthy people.

More than 200 repeats usually leads to aberrant hypermethylation of both the promoter region and the expanded repeat itself, and this results in transcriptional silencing of FMR1.

Some FXS patients with a full mutation but unmethylated FMR1 gene may only have anxiety and/or behavioral issues without intellectual disability.

Individuals carrying a premutation (between 55 to 200 repeats) are associated with increased risk for Fragile X associated tremor/ataxia syndrome which has later onset.

Limitations of Current Approaches

Current approaches are aimed at reducing symptoms.

Several therapies are in development including small molecules targeting symptoms and gene therapies, but the level of expression is FMR1 protein is tightly regulated and over-expression can be toxic.

Therefore there a few if any therapies in development to restore gene function.

Our Approach and Results:

Correct the FMR1 mutation in neurons

Our initial approach is to deliver Prime Editors to neurons of the CNS to correct the mutation in the FMR1 gene, restoring normal expression of the FMRP protein and synaptic function.

The Prime Editor will be designed to loop out the pathological repeats in the FMR1 gene.

This approach aims to correct the underlying cause of disease and has the potential to achieve a permanent correction.

We are focusing on particular regions to deliver Prime Editors including caudate nucleus, hippocampus and various areas of the cortex.

Guided by human genetics, we believe restoration of FMRP to 15-20 percent of normal FMRP may be sufficient to restore normal function to patients.

We have performed screens to identify dual-flap Prime Editors that can achieve highly efficient and precise removal of repeats in the FMR1 gene, achieving precise removal of repeats from healthy cell lines with more than 80 percent efficiency, as shown in the figure below.

In addition, we have demonstrated Prime Editors can remove pathological repeats from patient cells containing more than 450 CGG repeats with high efficiency.

More importantly, early experiments have shown that precise removal of pathological repeats from patient cells restores the expression of the FMR1 mRNA.

Previous approaches to restore FMR1 expression have been elusive;

therefore we believe that these important new results provide impetus for Prime Medicine to pursue Prime Editing approaches for patients with Fragile-X syndrome.

We are validating the activity of these Prime Editors in patient-derived neurons to demonstrate restoration of normal FMRP protein expression and normal synaptic function.

Our goal is to deliver Prime Editors to the cortical and deep brain structures.

Similar to our delivery approach for Friedreich’s ataxia, we will initially evaluate a dual AAV system to deliver the Prime Editor, but we are actively evaluating non-viral delivery approaches as an alternative.

Huntington’s Disease:

Another Repeat Expansion Disease Indication

Huntington’s disease, or HD, is an autosomal dominant progressive neurodegenerative disease affecting teenagers through middle aged adults.

Most individuals are diagnosed by age 45, with approximately 15 percent of patients diagnosed as teenagers.

Prevalence estimates are 1 in 10,000, suggesting between 20,000-40,000 individuals in the United States have the disease.

It is characterized by progressive loss of motor and cognitive function often with involuntary limb movements and fine motor impairments which progress steadily.

Natural history studies indicate substantial preservation of brain parenchyma (functional tissue) at the time of diagnosis, and in those with prodromal disease suggest cellular dysfunction precedes loss of neurons.

The first tissues to degenerate in the CNS are the striatum, followed by the cortex.

More than 95 percent of neurons in the striatum are medium spiny neurons which control muscle movement of limbs, body, eyes, as well as reward reinforcement and aversion responses.

These specialized neurons are the first to degenerate.

Typical progression to end-stage disease occurs over 10 years.

HD is caused by an expansion of a CAG repeat sequence in the first exon of the HTT gene which encodes the huntingtin protein.

The repeat encodes a run of glutamate amino acids.

Whereas 5-35 repeats are found in healthy individuals, having more than 40 repeats is considered pathological, with repeats often expanding well beyond 120 and with as many as 700 repeats observed in individual disease-affected cells.

Many distinct toxic or pathological consequences of the expanded repeat sequence occurring in the HTT mRNA or huntingtin protein have been documented.

These include toxic protein fragments and protein aggregation, transcriptional dysregulation, disrupted protein homeostasis, mitochondrial dysfunction, altered synaptic plasticity and defects in axonal transport.

These findings suggest mutant huntingtin has widespread pathological effects on neurons.

Limitations of Current Approaches

There are supportive measures and therapies to modify symptoms, but there are currently no disease modifying agents.

Our Approach:

Precisely remove the repeats from the mutant HTT gene and replace with healthy sequence to restore normal huntingtin function in the CNS

Our initial approach is the correction of the HTT mutation by removing the CAG repeat expansion tract using dual-flap Prime Editing or single long flap Prime Editing, and replacing with a sequence encoding 5 glutamate amino acids, thereby restoring normal function to the HTT gene.

We are evaluating the best method to deliver Prime Editing to deep brain structures including the striatum (caudate nucleus, putamen, nucleus accumbens), as well as the hippocampus, thalamus and cerebral cortex.

This is an early-stage program, where we have performed initial screens and identified Prime Editors that achieve efficient and precise removal of the repeats in the first exonic sequence while simultaneously recoding the normal 5 glutamate repeat or 10 glutamate repeats with some Prime Editors achieving greater than 40% precise correction.

We are currently performing optimizations to the Prime Editors to increase efficiency above 50%.

Similar to our other programs, we will evaluate promising Prime Editors in patient cells and differentiate patient-derived stem cells into medium spiny neurons in order to evaluate the impact of correction of HTT on normal huntingtin protein function.

Such assays include the restoration of normal huntingtin protein, as well as detection and quantification of inclusion bodies.

Expansion Opportunities in Repeat Expansion Diseases

In five of our repeat expansion diseases, including the four described in detail above, we have identified in our preliminary experiments at least one candidate Prime Editor that can achieve greater than 75 percent precise editing at each gene locus.

In particular for repeat expansion diseases, our approach has been to pick important surrogate examples, so that progress in the pipeline would considerably increase the probability of success for others of the approximately 50

repeat expansion diseases.

We plan to use our knowledge from existing programs to expand our pipeline of preclinical candidates, based on rapid progress in looping out pathological repeats.

OUR OTHER INITIAL DIFFERENTIATION TARGETS

Duchenne Muscular Dystrophy:

“Differentiation” Disease Indication

Duchenne muscular dystrophy, or DMD, is an X chromosome-linked recessive disease affecting boys that is characterized by early onset progressive muscle weakness affecting limbs.

It is frequently diagnosed between 2 to 3 years of age, and progresses to most patients being wheelchair bound by age 10.

In teenage years patients develop progressive cardiomyopathy and respiratory weakness, and patients die in their early 20’s from related complications.

It is estimated to affect 1 in 3,500 live births with diagnosed prevalent cases in the United States of 16,000 to 17,000.

The disease is caused by loss-of-function mutations in the DMD gene, which is one of the largest genes in humans.

The DMD gene encodes the dystrophin protein which is a critical protein component in the membrane of muscle fibers.

Dystrophin stabilizes the membrane of muscle fibers and connects the actin-myosin muscle machinery to the cell membrane.

In the absence of dystrophin, myofibers are susceptible to use injury.

Use injury causes damage and inflammation of the myofibers resulting in myofiber degeneration, inflammation, fibrosis and fatty replacement.

Many of the mutations causing DMD are large deletions in the DMD gene resulting in out-of-frame gene sequence, complete loss-of-function, and the absence of any dystrophin protein.

A smaller number of mutations are large duplications, and a small number of mutations are substitutions or small insertions/deletions.

A milder form of DMD, called Becker muscular dystrophy is caused by in-frame mutations in the DMD gene which produce shortened forms of the dystrophin protein.

The Becker disease ranges from a milder form of DMD with teenage or adult onset, through to asymptomatic individuals with a normal lifespan.

Asymptomatic individuals often have large deletions in the central region of the dystrophin protein, indicating that production of a truncated protein is compatible with normal or near normal muscle function.

Because muscle is a syncytium (a single muscle fiber may be several feet in length containing several hundred nuclei), correction of the gene defect in a limited number of nuclei may be sufficient to completely restore protein expression in the muscle fiber.

Studies of DMD in dogs suggest gene correction in as few as 11 percent of nuclei restores dystrophin to within 90 percent of normal levels.

Limitations of Current Approaches

In addition to supportive care, patients benefit from steroid therapy which reduces inflammation and has a modest impact slowing progression of disease.

An oligonucleotide delivered by regular IV infusions, which promotes exon skipping of the 51 st exon during mRNA splicing and can result in reframing of the DMD gene to result in a functional DMD protein, has offered minimal to modest benefits to some patients.

Gene therapies in development that are delivering an engineered “microdystrophin” protein to patients have demonstrated effective protein delivery to muscle, but to date have resulted in limited or no functional benefit.

Our Approach:

Precisely reframe the DMD gene to restore functional dystrophin protein in muscle

Prime Editing can precisely introduce or remove nucleotides from the DMD gene at pre-specified positions to reframe the gene so that a shortened but highly functioning protein is produced instead of no protein.

Guided by human biology of patients with Becker muscular dystrophy, we have initially designed Prime Editors to reframe the DMD gene in positions such that many individuals with different mutations, particularly large deletions in the central region of the DMD gene, will achieve functional dystrophin protein restoration from a single Prime Editor.

This is an early program, where we are performing screening to identify Prime Editors that demonstrate efficient and precise nucleotide insertion or deletion to reframe the gene.

Based on known DMD mutations, a small number of different Prime Editors could reframe the DMD gene and restore functional protein for more than half of the DMD patients.

Following optimization of the Prime Editors we will use patient-derived cardiomyocytes to evaluate the efficacy of selected Prime Editors to restore dystrophin protein and function.

Using similar delivery methods to those we are establishing for delivery of Prime Editors to skeletal and cardiac muscle for Myotonic Dystrophy type I, such as a dual AAV delivery approach, we will deliver lead Prime Editors to a humanized animal model and establish their pharmacokinetic and pharmacodynamic properties.

Cystic Fibrosis:

“Differentiation” Disease Indication

Cystic fibrosis, or CF, is a progressive lung disease characterized by production of thick mucus lung secretions which lead to blockage of airways, inflammation, and lung infection, progressing ultimately to lung failure.

It also affects the pancreas gland and biliary system of the liver in a similar way, leading to exocrine pancreatic failure and mild to moderate cholestatic liver disease in some patients.

Most patients are diagnosed before 2 years of age through newborn screening or because of symptoms of lung disease, combined by salty skin which can be confirmed using a sweat test.

Through supportive care and antibiotic therapies patient median survival has increased to early 30’s before lung failure necessitates lung transplantation, if available.

Overall CF prevalence in the United States and Europe is approximately 70,000 to 90,000 patients.

The disease is inherited recessively and caused by loss-of-function mutations in a chloride protein transporter called cystic fibrosis transmembrane conductance regulator, or CFTR.

Approximately 65 to 75 percent of CF patients have a three-nucleotide deletion in the CFTR gene known as F508del.

The vast majority of remaining patients have one of several prevalent mutations in a small number of genetic hotspots in the CFTR gene, including mutations such as N1303K, W1282X, G542X, or G551D.

The failure of CFTR to function at the cell surface leads to cell secretions that lack sufficient salt and water, resulting in high viscosity and inability to clear secretions from lung and pancreas.

Limitations of Current Approaches

In addition to supportive care, antibiotics and lung transplantation, patients also receive pancreatic enzyme supplements daily.

Recently, a combination medicine containing three small molecules, known as Trikaftor has proven highly effective at correcting the folding of the CFTR protein that harbors either the F508del mutation or one of a few other mutations.

Trikaftor has improved lung function and survival for those patients.

For 15 to 30 percent of patients (those with CF caused by other mutations) there is currently no disease modifying therapy.

In addition, adverse events and discontinuations reported for individuals taking Trikaftor (or one or more of the constituent active pharmaceutical ingredients), indicates patients with F508del also continue to have unmet need.

Our Approach:

Correct prevalent mutations and mutational hotspots in the CFTR gene

We are using either classical Prime Editing or dual-flap Prime Editing to correct different CFTR mutations to restore CFTR protein regulation, expression and function.

This is an early program, where we are performing screens to identify pegRNAs or pegRNA pairs that achieve efficient and precise correction of CFTR at multiple sites in the gene, including hotspots.

Our goal is to identify Prime Editors to target mutations initially in seven hotspots or at prevalent mutations in the CFTR gene.

From these early screens we have identified Prime Editors with high activity at each of these seven hotspots or prevalent mutations, with some Prime Editors achieving more than 70 percent precise correction.

For example, preclinical proof-of-concept data demonstrated greater than 70 percent precise editing of the G542X mutational hotspot in vitro .

We are also exploring whether the PASSIGETM approach could be applied to the mutant CFTR gene to develop a therapy for additional patients.

In initial proof of concept studies, we developed an intestinal organoid swelling assay that enabled us to test the impact of our Prime Editors on CFTR protein function.

Healthy donor intestinal organoids swell when stimulated as a result of the CFTR channel pumping salt and water into the organoid.

Organoids from CF patients do not swell.

preliminary studies, we edited organoids from patients with the G542X mutation and demonstrated that our Prime Editors restore swelling to levels seen in healthy donor organoids.

Initially we will perform optimizations of the Prime Editors from the early screens to increase efficiency and optimize Prime Editors for additional mutations.

We are building a series of assays to evaluate our Prime Editors on restoration of CFTR protein function.

We will test Prime Editors in patient-derived cells, including iPSCs, intestinal organoids, and human bronchial epithelium.

Humanized mice with CFTR mutations have been developed for us to deliver Prime Editors initially to the lung epithelial basal cells which contain a population of lung stem cells.

Our initial approach to delivery of Prime Editors to the lung epithelium includes the use of lipid nanoparticles with tropism to basal cells of the lung airways.

Our Future Opportunities in Expanding the Prime Editing Pipeline

To maximize the potential of Prime Editing, we have purposefully built a diversified portfolio organized around four strategic indication categories:

(1) immediate target indications, (2) differentiation target indications, (3) “blue sky” indications and (4) “march up the chromosome” approaches, with each set of indications chosen to deliver a different strategic goal.

We constructed our current portfolio of 18 programs, including one partnered program, across our first two strategic indication categories in disease settings where the unique characteristics of Prime Editing could offer compelling advantages over current standard-of-care and novel therapeutic modalities in development.

We expect to achieve preclinical proof-of-concept in vivo , which would include data from in vivo rodent studies in several programs in 2023.

If successful, we expect to next initiate IND-enabling studies for several of our lead programs, with the first IND filing potentially as early as 2024, and with the potential for additional IND filings as early as 2025.

Since we are in early stages of product candidate development, we will provide an update on our timelines moving forward.

We also anticipate continuing to name additional programs as they advance over the next few years .

Beyond these current 18 programs, we believe we have the ability to advance quickly into similar follow-on programs in blood, liver, eye and ear as we achieve therapeutic success.

We also will continue to invest in research around our “blue sky” target indications, which are intended to push new and innovative technological developments in Prime Editing and delivery and extend its application beyond rare genetic diseases and towards our goal of more broadly addressing human disease.

These programs remain in the early stages of conception and will become an increasing focus over the next few years.

Finally, our “march up the chromosome” approaches represent opportunities to deliver upon our overarching vision to ultimately treat all patients with a disease and correct the full set of mutations in a particular gene.

Many of our disclosed indications across our other strategic categories have a plan that can accommodate expansion opportunities to address additional mutations in that disease.

Complementary retrotransposon mediated gene-insertion technology to enable an all-RNA approach to insert gene-sized DNA into the genome

We are continuing to evaluate opportunities that we see as potentially uniquely-positioned to advance our gene editing toolbox, including new technologies to help us to fulfil our strategic goals across both our “blue sky indications” and “march up the chromosome” approaches.

For these efforts to be successful, we believe it is important to pursue and to develop technologies that enable the targeted insertion of gene-sized pieces of DNA into the genome.

In December 2021, we entered into the Myeloid Agreement with Myeloid, a company primarily focused on harnessing and reprogramming myeloid cells for treating cancers.

Through this research collaboration, we received an exclusive option to obtain ownership of certain patent rights and know-how that relate to a new retrotransposon-based technology to enable the insertion of gene-sized DNA sequences into the genome that, if successful, we believe could open new opportunities for programmable gene editing.

See “Our License and Collaboration Agreements—Research Collaboration, Option and License Agreement with Myeloid.”

We believe this retrotransposon-based approach is complementary to Prime Editing and, if successfully deployed alongside Prime Editing, could expand the applicability of our Prime Editing technologies towards our goal of more broadly addressing human diseases.

The Myeloid team includes experts in retrotransposon biology, complementing

our deep in-house gene editing expertise.

In addition, Myeloid maintains an intellectual property position in retrotransposon-based editing, which, if we exercise our option, can be transferred to us.

This emerging approach uses human LINE-1 retrotransposase, which has been engineered by Myeloid to insert transgenes into the genome when delivered as an RNA, without the need for co-administration of DNA.

We believe this approach may be complementary to the combined Prime Editor with the site-specific recombinase approach outlined above.

Genomic Integration of Transgenes (>3kb) Using LINE-1 mRNA (No Donor DNA Required)

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An mRNA carrying the LINE-1 genes (ORF1 and ORF2) and transgene cargo (teal) is delivered to the cell.

Inside the cell, the LINE-1 mRNA is translated, producing ORF1p (green) and ORF2p (purple and pink) proteins.

The ORF2p protein contains an endonuclease domain (En, purple) and a reverse transcriptase domain (RT, pink).

The ORF1p and ORF2p proteins bind to the LINE-1 mRNA to form a ribonucleoprotein complex (LINE-1 RNP).

At permissive sites in the genome, the LINE-1 RNP binds and nicks the DNA using the En domain, and then reverse transcribes the mRNA into the genome using the RT domain, thereby integrating the transgene sequence into the genome.

En = endonuclease domain;

RT = reverse transcriptase domain;

RNP = ribonucleoprotein complex.

This retrotransposase technology is still at an early-stage of development and has not yet been optimized to operate with the same efficiency or programmability as Prime Editing (See Figure below).

Using our protein engineering capabilities, we plan to adapt the LINE-1 retrotransposase technology to increase its programmability so that it can be directed to pre-specified, targeted site(s) in the genome, similar to a Prime Editor.

Protein engineering and other methods, like those employed to increase the efficiency of Prime Editing, will also be used to increase the efficiency of transgene integration by the LINE-1 retrotransposon.

If this research collaboration with Myeloid is successful, we intend to utilize this technology initially in ex vivo cell therapies, with the potential to expand and deliver in vivo to enable precise gene insertion.

Insertion of a Functioning Transgene using human LINE-1 Retrotransposon Technology

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Example of human LINE-1 retrotransposon inserting a green fluorescent protein transgene into primary human monocytes following electroporation of LINE-1 mRNA carrying the sequence for the transgene (Source:

Other Programs in Discovery

Within our neuromuscular area of focus, we are also exploring other repeat expansion diseases, including amyotrophic lateral sclerosis, a rapidly progressive neurodegenerative disease characterized by progressive motor neuron loss;

Huntington’s disease, an autosomal dominant progressive neurodegenerative disease affecting teenagers through middle aged adults;

Fragile X syndrome, an X chromosome-linked dominant rare disease that is the most common monogenic cause of childhood intellectual disability and autism;

and oculopharyngeal muscular dystrophy, a rare autosomal dominant disease, characterized by progressive weakness in the muscles around the eyelids as well as in the tongue and pharynx.

In addition to the repeat expansion diseases, we are also exploring Duchenne muscular dystrophy, an X chromosome-linked recessive disease affecting boys that is characterized by early onset progressive muscle weakness affecting limbs.

Additional Programs

As one part of our overall partnering strategy, we are advancing the following programs as partnering opportunities:

Chimeric Antigen Receptor T-cell program (CAR-T)

We are developing investigational CAR-T cell therapies for autoimmune and oncology indications.

In combination with multiplex Prime Editing, we believe PASSIGE may be able to overcome existing challenges in developing CAR-T cells for human therapeutic use — such as manufacturing time, cost, yield for autologous cell therapy, cell quality issues, and safety risks associated with semi-random integration and double strand breaks at multiple genomic loci.

Multiplex Prime Editing with PASSIGE has the potential to create a best-in-class allogeneic CAR-T cell product.

In December 2023, we presented preclinical data suggesting PASSIGE is up to 80 percent efficient for non-viral, site-specific delivery of chimeric antigen receptor to primary human T-cells to generate CAR-T cells, and can be multiplexed with Prime Editing at other target sites by non-viral one-step delivery with no loss of efficiency.

PASSIGE-generated CAR-T cells showed potent antigen-specific function and cytotoxicity ex vivo and in vivo against an established human B-cell tumor growing in immunodeficient mice.

The potential advantages, we believe, of our CAR-T approach using Prime-Edited are shown in the table below.

As shown in the figure below, we have achieved multiplex Prime Editing in T cells to knock out the B2M gene and the endogenous T cell receptor, or TRAC, without loss in efficiency compared to single site editing (left panel).

We have also delivered Prime Editing components and DNA recombinase components (PASSIGE components) in a single step to human primary T cells, without the use of viruses.

The short recombinase DNA target sequence, used by the site-specific recombinase enzyme known as Bxb1, was inserted into human primary T cells at the TRAC

locus with greater than 95 percent efficiency, and a 3.5 kilobase anti-CD19 CAR gene cassette was precisely inserted into that recombinase site location in up to 80 percent of the T cells, resulting in positive expression of the CAR by those T cells (middle panel).

As a result, the T cells acquired CD19+ tumor cell killing activity that was dependent on cell dose in a cell assay, indicating that the integrated CAR was functional (right panel).

PASSIGE and Multiplex Prime Editing Could Create Potentially Best-in-Class Allogenic CAR-T Cell Product

This exploratory program is approaching lead optimization.

Additional optimization of the Prime Editor and PASSIGE components is ongoing, and the scalable cell therapy process is being developed.

A suite of assays to evaluate cell potency and cell fitness are currently being developed and we plan to evaluate CAR-T cell leads in vivo in established tumor killing studies.

Other Programs in Discovery

In addition to CAR-T, we are also exploring genetic hearing loss, including Usher’s syndrome type III, which is characterized by progressive post-lingual hearing loss, variable vestibular dysfunction, as well as adolescent-onset progressive vision loss due to retinitis pigmentosa caused by mutations in the Clarin 1 protein encoded by the CLRN1 gene;

and non-syndromic hearing loss due to mutations in GJB2, the most commonly mutated gene in non-syndromic hearing loss, which accounts for two thirds of genetic hearing loss.

Our License and Collaboration Agreements

Strategic relationship with Beam Therapeutics

In September 2019, we established a strategic, collaborative relationship with Beam Therapeutics Inc., or Beam, a biotechnology company developing precision genetic medicines.

One of our founders, David Liu, is also a founder of Beam.

Through our relationship, we collaborate with Beam on the research, development, manufacture and commercialization of certain Prime Editing products within a specified field and provide each other with access and licenses to certain proprietary technology to advance the other’s progress.

Mutual access and licenses under the Beam Collaboration Agreement

We entered into a collaboration and license agreement with Beam, which we refer to as the Beam Collaboration Agreement, under which we agree to provide each other with access to, and licenses under, certain technology, know-how and patent rights controlled by each of us for a limited number of years after the effective date, known as the initial term, and certain improvements thereto.

Certain licenses we grant to Beam are limited to exploiting licensed products in the Beam field, as further described below, and certain licenses Beam grants to us are limited to exploiting products in the Prime field, as further described below.

Under the Beam Collaboration Agreement, we grant to Beam an exclusive (even as to us and our affiliates), worldwide license under (i) certain Prime Editing technology, know-how and patent rights that we control during the initial term, and improvements thereto that we control for a specified number of years following the initial term, and (ii) our interest in certain jointly-owned collaboration technology, in each case, solely to develop, make, have made, use, offer for sale, sell, import and commercialize licensed products only in the Beam field, which is limited to (a) the prevention, modification, improvement, amelioration or treatment of human disease, including cell-based therapies and the creation of one or more protective mutations, through administration of a licensed product that incorporates or contains a qualifying Prime Editing agent, which is a macromolecule or macromolecular complex that uses Prime Editing to make one or more transition point mutations (that is, C to T, T to C, A to G or G to A) in the sequence of one or more DNA targets, without intentionally making any non-transition mutations or other changes, including insertions, deletions, duplications, indels, transversions or combinations thereof, and does not incorporate or contain any other Prime Editing agent or other gene editing approach that is not a qualifying Prime

Editing agent or (b) the prevention, modification, improvement, amelioration or treatment of sickle cell disease through administration of a licensed product that incorporates or contains a more broadly defined Prime Editing agent.

We refer to each of clause (a) and clause (b) of the Beam field as subfields.

We also grant to Beam a non-exclusive, worldwide license under certain CRISPR or delivery-related technology, know-how and patent rights that we control during the initial term, and improvements thereto that we control for a specified number of years following the initial term, solely to develop, make, have made, use, offer for sale, sell, import and commercialize licensed products only in the Beam field.

Under the Beam Collaboration Agreement, Beam grants to us certain non-exclusive, worldwide licenses under certain technology, know-how and patent rights, including under certain CRISPR or delivery-related technology, know-how and patent rights, that it controls during the initial term, and improvements thereto that Beam controls for a specified number of years following the initial term, solely to develop, make, have made, use, offer for sale, sell, import and commercialize products only in the Prime field, which is limited to the prevention, modification, improvement, amelioration or treatment of human disease (excluding sickle cell disease), including cell-based therapies and the creation of one or more protective mutations, through administration of a product or service containing or incorporating a Prime Editing agent that is not a qualifying Prime Editing agent, but excluding (a) the Beam field, (b) the administration of any product or service containing or incorporating a base editor and (c) a field related to microbial cells in the human flora in certain Asia territories and the development of products targeting four named gene targets.

For clarity, the Prime field includes products or services that contain or incorporate (x) at least one Prime Editing agent that is not a qualifying Prime Editing agent and (y) any other gene-editing approach, including other Prime Editing agents, which may include one or more qualifying Prime Editing agents, subject to the aforementioned exclusions.

The licenses granted to us by Beam under the Beam Collaboration Agreement are subject to the terms of certain third-party agreements and certain rights retained by third parties.

In addition to the ongoing licenses, under the Beam Collaboration Agreement, we are both obligated to adhere to a technology transfer plan, under which each of us agrees to disclose or otherwise share the technology, know-how and patent rights licensed to the other and to provide the other party with reasonable assistance in the exercise of its licenses.

The licenses granted to each party under the Beam Collaboration Agreement are sublicensable to affiliates and third parties, subject to certain requirements, including providing the other party a copy of each executed sublicense agreement, and ensuring any sublicensee comply with the terms of the Beam Collaboration Agreement.

Beam’s development and commercialization of licensed products in the Beam field

Unless we exercise our profit sharing option for a licensed product, as described below, Beam is solely responsible for the development and commercialization of licensed products in the Beam field under the Beam Collaboration Agreement.

Beam is required to use commercially reasonable efforts to develop and seek marketing approval for at least one licensed product in each subfield of the Beam field in each of (a) the United States and (b) one other specified major market country, and to commercialize any such licensed product that achieves marketing approval.

As described further below, we are entitled to receive ongoing milestone and royalty payments from Beam based on Beam’s development and commercialization of each licensed product.

Our profit sharing option

Subject to the provisions in the next paragraph, on a licensed product-by-licensed product basis, we have the right to elect to share equally with Beam in the profits and losses in the United States for Beam’s licensed products.

We may exercise such right for each licensed product within a specified period of time.

Any such licensed product for which we exercise such right we refer to as a collaboration product.

If we exercise such right, we agree to share equally in the costs, profits and losses of each such collaboration product in the United States, rather than receiving milestones and royalties based on development and sales thereof by Beam in the United States.

For clarity, we are still entitled to receive milestones and royalties on the development and sales of each such collaboration product outside of the United States.

We also have the right to elect, within a specified time period, to co-promote with Beam each collaboration product in the United States, in addition to sharing in the profits and losses.

To the extent we exercise our co-promote option with respect to a given collaboration product, we and Beam must use commercially reasonable efforts to commercialize such collaboration product, in each case, in the Beam field in the major markets

in which marketing authorization has been obtained.

After we have exercised our right to profit share on a collaboration product, we are able to, at any time during the term of the Beam Collaboration Agreement, on a collaboration product-by-collaboration product basis, opt-out of the profit and loss share and co-promotion activities with respect to any collaboration product with prior written notice to Beam within a certain time period.

Notwithstanding the rights described above, at any time prior to or within 30 days of the filing of an IND for a licensed product, Beam may designate up to a mid-single digit number of licensed products for which (i) we are not permitted to exercise our profit sharing right, and (ii) Beam assumes sole control and decision-making authority and bears all costs and expenses, with respect to the development and commercialization of such products.

Under the Beam Collaboration Agreement, a “protected product” is a licensed product for which either (a) we have not exercised our profit share option or (b) Beam has designated as a protected product pursuant to the foregoing sentence.

For clarity, we are entitled to ongoing milestones and royalties from Beam based on its development and commercialization of protected products worldwide.

Upon Beam’s designation of a licensed product as a protected product , Beam is required to pay us $5.0 million if the product is developed for non-sickle cell disease or $10.0 million if the product is developed for sickle cell disease.

Consideration

As partial consideration for the licenses and rights granted to each other under the Beam Collaboration Agreement, Beam issued to us $5.0 million in shares of its common stock and we issued to Beam an aggregate of 1,608,337 shares of our common stock.

Beam was also entitled to appoint a representative to our board of directors, which right expired.

Beam initially appointed its CEO, John Evans, to our board of directors.

Evans resigned from our board of directors effective September 13, 2022.

We are entitled to receive development milestone payments from Beam on Beam’s development of protected products (which, for clarity, includes any licensed product for which we have not exercised our profit share option) and collaboration products.

For protected products, we are entitled to receive up to a total of $35.5 million on a protected product-by-protected product basis based on Beam’s development of such protected product and, for collaboration products, up to a total of approximately $17.8 million on a collaboration product-by-collaboration product basis based on Beam’s development of such collaboration product outside of the United States, in each case, with such amounts lowered if such licensed product achieves a given milestone for use in treating an orphan disease.

We are also entitled to receive sales-based milestone payments from Beam based on net sales of licensed products.

For protected products, we are entitled to receive up to a total of $84.5 million on a protected product-by-protected product basis based on net sales of such protected product worldwide, and, for collaboration products, up to a total of approximately $42.3 million on a collaboration product-by-collaboration product basis based on net sales of collaboration products outside of the United States.

The sickle cell disease product partnered with Beam is a licensed product under the Beam Collaboration Agreement.

Beam has not designated this product as a protected product and we have not received any development or sales-based milestones with respect to Beam’s exploitation thereof.

Beam is obligated to pay to us tiered royalties ranging from a high-single digit percentage to a low double-digit percentage, but less than teens on net sales of protected products worldwide on a protected product-by-protected product basis and net sales of collaboration products outside of the United States on a collaboration product-by-collaboration product basis.

Our royalties are subject to customary offsets and reductions, to a floor that takes into account any royalties we are obligated to pay to our third-party licensors, including Broad Institute.

In addition, certain of the rights licensed under the Beam Collaboration Agreement are sublicensed from third parties, and Beam agrees to reimburse us for certain payments we are required to make to our third-party licensors attributable to Beam’s exercise of any sublicense we grant to Beam, including payments we make to Broad Institute under the Broad License Agreement.

If we develop a product that is covered by the technology, know-how or patent rights that Beam licenses to us under the Beam Collaboration Agreement, which we refer to as a Prime product, we are obligated to pay to Beam a low single digit percentage royalty on our worldwide net sales of any such product on a Prime product-by-Prime product and country-by-country basis, subject to certain customary reductions, to a floor.

Each party’s obligation to pay the other royalties expires on a country-by-country and product-by-product basis on the latest of (a) the expiration of the last to expire valid claim of an issued patent or pending patent application within the applicable licensed patent rights that cover such product in such country, (b) the expiration of regulatory exclusivity for such product in such country or (c) ten (10) years after the first commercial sale of such product in such country.

If we exercise our option to profit share on collaboration products, we share equally in the profits and losses of any such collaboration product in the United States and share in a lower portion of any development or commercialization costs attributable to such collaboration product outside of the United States.

Intellectual property ownership and patent prosecution

Under the Beam Collaboration Agreement, Beam assigns ownership to us of certain improvements Beam makes, itself or jointly with us or others, to certain technology, know-how and patent rights we license to Beam, and we assign to Beam ownership of all improvements we make, ourselves or jointly with Beam or others, certain technology, know-how and patent rights Beam licenses to us.

Each party grants back to the other certain exclusive and non-exclusive licenses to such improvements.

Except for any such improvements, each party owns any other inventions that it developed under the Beam Collaboration Agreement and an equal, undivided interest with the other party in any inventions jointly developed.

We are responsible for prosecution and maintenance of the patent rights we license to Beam, while keeping Beam reasonably informed and providing Beam the opportunity to provide comments and make requests of us, in each case regarding the patent rights that we exclusively license to Beam in the field of the exclusive license.

Beam has a step-in right to the extent we decline or fail to prosecute any patent rights that are exclusively licensed to Beam and applicable to the Beam field.

Beam is responsible for prosecution and maintenance of the patent rights it licenses to us, while keeping us reasonably informed and providing us the opportunity to provide comments and make requests of us, in each case with respect to any patent rights that Beam exclusively licenses to us in the field of the exclusive license.

Beam has the first right to enforce any patent rights we exclusively license to Beam in the Beam field against any third party developing a product in the Beam field that is competitive with a licensed product Beam is developing under the Beam Collaboration Agreement.

We have a step-in right on any such enforcement to the extent Beam declines or fails to initiate such enforcement action.

Term and termination

Unless earlier terminated in accordance with its terms, the Beam Collaboration Agreement will expire on the later of (a) expiration of the last royalty term for a product on which a party is obligated to pay royalties to the other party or (b) with respect to any collaboration product, the date on which neither party is developing or commercializing any such collaboration product in the United States.

After expiration of the initial term, Beam can terminate the Beam Collaboration Agreement for convenience in its entirety, or on a licensed product-by-licensed product or subfield-by-subfield basis, with ninety (90) days’ prior written notice to Prime.

Each party may terminate the Beam Collaboration Agreement for (a) the other party’s uncured material breach within ninety (90) days of notice of such breach, (b) upon the insolvency or bankruptcy of the other party if such proceeding is not dismissed within ninety (90) days after the filing thereof or (c) immediately to the extent the other party (or its affiliates or sublicensees) challenges a patent right licensed to such party.

License agreements with Broad Institute

2 unchanged sentences

Under the Broad License Agreement, Broad Institute grants to us certain rights and licenses under certain patent rights it owns or controls related to editing of DNA sequences using a Prime Editor.

Certain of the licensed patent rights are co-owned by Broad Institute with MIT and Harvard.

Certain of the licensed patent rights are co-owned by Broad Institute with MIT and Harvard and certain are co-owned by Broad Institute with Harvard.

In December 2022, following the timely exercise of an option under an existing option agreement with Broad Institute we entered into a second license agreement with Broad Institute, which we refer to as the 2022 Broad License Agreement.

2022 Broad License Agreement, Broad Institute grants to us certain rights and licenses under certain patent rights it owns or controls related to MMR inhibition and prime editing improvements.

Under the 2022 Broad License Agreement, Broad Institute grants to us certain rights and licenses under certain patent rights it owns or controls related to MMR inhibition and prime editing improvements.

The licensed patent rights are co-owned by Broad Institute with Harvard, The Trustees of Princeton University, or Princeton, and The Regents of the University of California, or University of California.

License rights under the Broad License Agreement

Broad License Agreement

The licenses Broad Institute grants to us under the Broad License Agreement are limited to the field of prevention or treatment of human disease, and most licenses granted to us under the Broad License are further limited to the prevention or treatment of human disease by editing (including modifying or converting) or targeting DNA ex vivo , in vivo , or through xeno-transplantation methods.

We refer to this field as the Prime Broad Field.

The Prime Broad Field specifically excludes the prevention or treatment of human disease using small or large molecules that are not otherwise “prime editor products” and other specified agricultural and livestock applications of the technology covered by the licensed patent rights.

Under the Broad License Agreement, “prime editors” are macromolecules or macromolecule complexes intended to insert DNA sequence into, delete DNA sequence from, or replace one or more bases of a target DNA sequence, using a combination of (i) natural or engineered reverse transcriptase(s) or any other nucleic acid polymerase enzyme and (ii) a nucleic acid binding protein that can be programmed to bind to a DNA sequence to be so changed.

“Prime editor products” are products that combine Prime Editors and nucleic acid molecules that bind to and direct the Prime Editors to specified DNA sequences and that contain template sequences for introducing the intended alteration into the specified DNA sequences.

Under the Broad License Agreement, Broad Institute grants to us (i) an exclusive, worldwide license under the licensed patent rights solely to offer for sale, sell, have sold and import products covered by such licensed patent rights, or licensed products, solely for use within the Prime Broad Field (subject to certain specified limitations and exclusions with respect to certain applications), (ii) a non-exclusive, worldwide license under the licensed patent rights solely to make, have made, offer for sale, sell, have sold, and import licensed products solely for use in the Prime Broad Field, (iii) a non-exclusive, worldwide license under the licensed patent rights solely to make, have made, offer for sale, sell, have sold and import other products that are enabled by (a) the licensed patent rights or (b) the use of certain materials transferred to us by Broad Institute, solely for the prevention or treatment of human diseases, which we refer to as enabled products, and (iv) a non-exclusive, worldwide license solely for internal research.

Further, with respect to DNA delivery or targeting applications covered by the licensed patent rights, the exclusive license granted to us by Broad Institute is limited only to “prime editor” products and specifically excludes applications relating to the production or processing of small or large molecules, including for the prevention or treatment of human disease.

All of the above license grants (i) specifically exclude (a) human germline modification, (b) the stimulation of biased inheritance of particular genes or traits within a plant or animal population and (c) certain modifications of the tobacco plant, and (ii) are subject to certain retained rights of Broad Institute, MIT and Harvard and the U.S.

All of the above license grants specifically exclude human germline modification, the stimulation of biased inheritance of particular genes or traits within a plant or animal population, and certain modifications of the tobacco plant, and are subject to certain retained rights of Broad Institute, MIT and Harvard and the U.S.

federal government.

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Our licenses are subject to Broad Institute’s inclusive innovation model, pursuant to which Broad Institute retains the right, under specified circumstances, to grant to third parties (other than specified competitors of ours) licenses under the licensed patent rights that would otherwise fall within the scope of the exclusive license granted to us.

If a third party provides Broad Institute with a bona fide proposal to develop a product covered by the licensed patents and directed to a particular gene target, Broad Institute may notify us of the proposal, including the identity of such

gene target and the proposing third party.

Broad Institute is not required to share any other information provided by the requester with us in connection with the inclusive innovation model.

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If we notify Broad Institute that we have a good faith interest in sublicensing our rights to such third party or another third party, we have a specified period of time to negotiate and enter into a sublicense agreement with a third party.

If we (i) notify Broad Institute that we are not interested in developing such product (internally or with another third party) or do not respond to the proposed product notice, or (ii) notify Broad Institute of our interest as outlined above and do not complete or, for an internal program, commence, those activities within the specified time periods, Broad Institute has the right, subject to certain conditions, to terminate our rights to such gene target and may grant to such proposing third party an exclusive or non-exclusive license under the patent rights to exploit products covered by the licensed patent rights and directed to such gene target, which we refer to as a march-in license.

proposing third party an exclusive or non-exclusive license under the patent rights to exploit products covered by the licensed patent rights and directed to such gene target, which we refer to as a march-in license.

Broad has not yet granted any march-in license to a third party.

In addition to the inclusive innovation model, our licenses are also subject to Broad Institute’s right to designate a single-digit number of gene targets per year in which it has a good faith interest in reserving for its own development of products covered by the patent rights directed to such gene targets.

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Broad Institute has not yet exercised its right to designate any reserved gene targets.

This includes several developmental milestones that we are required to meet with respect to licensed products within a specified number of years.

Under the Broad License Agreement, we are required to use commercially reasonable efforts to develop licensed products in the Prime Broad Field in accordance with a development plan that we prepared and submitted to Broad Institute, which includes several developmental milestones for licensed products that we are required to meet within a specified number of years.

We may update the development plan from time to time if we believe, in our good faith judgment, that such update is needed to improve our ability to meet such development milestones.

Broad Institute has the right to terminate the Broad License Agreement if we fail to use commercially reasonable efforts or to achieve a development milestone, subject to our right to extend or amend such milestone in accordance with certain procedures.

If, despite using commercially reasonable efforts, we will not achieve a development milestone, we may request an extension of the development milestone timelines by providing a reasonable explanation for the extension and a reasonable, detailed, written plan for promptly achieving such reasonable extended or amended milestone to Broad Institute, and following Broad Institute’s approval of the request to delay, the applicable milestone deadline will be automatically amended (to the extent we request an extension of less than a specified number of years).

We may request an extension of the development milestone timelines by providing a reasonable explanation and plan to Broad Institute, and following Broad Institute’s approval of the request to delay, the applicable milestone deadline will be automatically amended (to the extent we request an extension of less than a specified number of years).

We have not yet requested any such extension and have met the deadlines for diligence milestones that have already occurred.

In addition to the diligence obligations to achieve the milestones in the development plan, for any products that attain regulatory approval, we are required to use commercially reasonable efforts to introduce any such licensed product into the commercial market and to commercialize and make such licensed products reasonably available to the public.

Consideration under the Broad License Agreement

If we are successfully able to gain regulatory approval for any licensed product, we are required to use commercially reasonable efforts to introduce any such licensed product into the commercial market and to commercialize and make such licensed products reasonably available to the public.

As partial consideration for the rights granted to us under the Broad License Agreement, we paid Broad Institute an upfront fee of $0.5 million, and issued Broad Institute an aggregate of 623,529 shares of our common stock.

We also are obligated to pay to Broad Institute an annual license maintenance fee ranging from the low- to mid-five figures to the low six-figures, depending on the particular calendar year, for the term of the Agreement.

Under the February 2021 and December 2022 amendments, as partial consideration for the addition of licensed patent rights relating to prime editing improvements, we paid Broad Institute amendment fees of approximately $0.1 million and $0.1 million, respectively.

We also are obligated to pay to Broad Institute an annual license maintenance fee in the low six-figures for the term of the Agreement.

Broad Institute is also entitled to receive clinical and regulatory milestone payments up to a total of $20.0 million per licensed product, depending on the patient population to be treated by the licensed product achieving the applicable milestone.

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(i) the expiration of the last to expire valid claim of an issued patent or pending patent application within the licensed patent rights covering such product in such country, (ii) the period of regulatory exclusivity for such product in such country or (iii) ten (10) years after the first commercial sale of such product in such country.

We estimate the last patent right licensed under the Broad License Agreement will expire in 2043, without giving effect to any potential patent term extensions or patent term adjustments.

Broad Institute is also entitled to a percentage of consideration that we receive from our sublicensees, with such percentage at low double-digits and decreasing to high single digits, dependent on the development stage of products under the Broad License Agreement at the time of sublicense execution.

If we (or one of our affiliates or sublicensees) initiate a patent challenge of the licensed patent rights, among other things, our payment obligations could be doubled and we could lose exclusivity of our license.

Prosecution and enforcement of licensed patent rights under the Broad License Agreement

Broad Institute is also entitled to a percentage of consideration that we receive from our sublicensees, with such percentage at low double-digits and

decreasing to high single digits, dependent on the development stage of products under the Broad License Agreement at the time of sublicense execution.

Broad Institute is responsible for the prosecution and maintenance of all licensed patent rights, although we are entitled to certain consultation, comment and review rights with respect to such prosecution and maintenance activities of the exclusively licensed patent rights.

We are obligated to reimburse Broad Institute for its documented, out-of-pocket costs incurred while prosecuting and maintaining such licensed patent rights.

So long as we remain the exclusive licensee of licensed patent rights in the Prime Broad Field, we have the first right to enforce the licensed patent rights in the Prime Broad Field, where we reasonably determine that a third party is marketing or has specific plans and is preparing to market an infringing product in any country that competes with one of our licensed products in the Prime Broad Field.

Broad Institute has a step-in right to the extent we decline to exercise such first right to enforce.

Term and termination of the Broad License Agreement

So long as we remain the exclusive licensee of licensed patent rights in the Prime Broad Field, we have the first right to enforce the licensed patent rights in the Prime Broad Field.

Unless earlier terminated, the Broad License Agreement will remain in effect until the later of (i) the last to expire valid claim of an issued patent or pending patent application within the licensed patent rights covering our licensed products or (ii) the expiration of the last royalty term for a licensed product in a country.

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Broad Institute may also immediately terminate the Broad License Agreement (i) to the extent we (or our affiliates or sublicensees) challenge a licensed patent right, (ii) upon our bankruptcy or insolvency or (iii) if we fail to procure and maintain insurance.

Amendments to the Broad License Agreement

We amended the Broad License Agreement in May 2020 to correct an exhibit to the Broad License Agreement and amended the Broad License Agreement in February 2021 and December 2022 to include additional licensed patent rights.

2022 License Agreement with Broad Institute

Following our timely exercise of an option under an existing option agreement with the Broad Institute, in December 2022, we entered into a second license agreement with Broad Institute, which we refer to as the 2022 Broad License Agreement.

Under the 2022 Broad License Agreement, Broad Institute grants to us certain rights and licenses under the patent rights it owns or controls related to MMR inhibition and prime editing improvements.

Other than as summarized below, the general terms of the 2022 Broad License Agreement, including the scope and field of the license grants, are the same in all material respects as the terms of the Broad License Agreement, as summarized above.

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The royalty term for a royalty-bearing product under the 2022 Broad License Agreement is determined in the same way as in the Broad License Agreement.

As of the date of the issuance of the Annual Report, we estimate the last patent right licensed under the Broad Second License Agreement will expire in 2042, without giving effect to any potential patent term extensions or patent term adjustments.

Pledge to Broad Institute and Harvard

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The Pledge is intended to be used for research and development related to new genome editing technologies, for example Prime Editing, improve on existing genome-editing technologies, identify delivery mechanisms for these technologies and apply these technologies to the understanding and treatment of rare genetic diseases.

We can terminate the Pledge at our discretion, subject to providing one year of funding from the date of termination.

We can terminate the Pledge at our discretion, subject to providing one year of funding from the

date of termination.

In August 2022, we amended and restated the Pledge to clarify that the funds may be used by the laboratory of David Liu, who is a member of Broad Institute and a faculty member at Harvard.

Research Collaboration, Option and License Agreement with Myeloid

In December 2021, we entered into a research collaboration and exclusive option agreement with Myeloid Therapeutics Inc., or Myeloid, and such agreement, the Myeloid Agreement.

Under the Myeloid Agreement, we collaborate with Myeloid, a related party, on the research and development of LINE-1 retrotransposon technology.

This retrotransposon-based approach is complementary to Prime Editing and, if successfully deployed alongside Prime Editing, could expand the applicability of our technologies towards our goal of more broadly addressing

human diseases.

In connection with the Myeloid Agreement, we also entered into a subscription agreement with Myeloid under which we were obligated to issue an aggregate of 1,101,525 shares of our common stock as additional consideration for the license.

Myeloid grants to us an exclusive option, exercisable during the research term and for 60 days thereafter, to obtain ownership of certain patent rights and know-how owned by Myeloid that relate to LINE-1 retrotransposon technology.

If we exercise our option, in addition to assigning us ownership of the applicable patent rights and know-how, Myeloid also agrees to grant us certain exclusive and non-exclusive licenses, including to certain improvements and other enabling technology.

Following the exercise of our option, we agree to grant Myeloid, in addition to certain other licenses, an exclusive, worldwide license under the assigned patent rights and know-how to develop and commercialize products in the field of myeloid cells and myeloid cell engineering, or the Myeloid Field.

As of December 31, 2022, we have not exercised our option.

Upon entering into the Myeloid Agreement, Myeloid was entitled to receive an upfront payment of $30.0 million in cash and an aggregate of 1,101,525 shares of our common stock, with a then fair value of $12.0 million, both of which Myeloid received in January 2022.

If the research agreement meets its goals, then (i) during the research term, Myeloid is entitled to cash payments of up to $35.0 million in the aggregate upon the achievement of certain milestones reflecting the technology’s development;

and (ii) if we exercise our option, we agree to pay to Myeloid an option exercise fee of $80.0 million in cash, and shares of our common stock with a then fair value of $30.0 million.

Additionally, if the research collaboration meets its goal and we exercise our option, and we are able to proceed with the development and commercialization of a product that is covered by (a) the patent rights or know-how subject to our option or (b) the patent rights or know-how developed by one or both of the parties during the research term related to LINE-1 retrotransposon technology, or, collectively, a Prime Product, Myeloid would be eligible to receive, for the first five Prime Products, development and regulatory milestone payments of up to $120.0 million on a Prime Product-by-Prime Product basis and sales-based milestone payments of up to $210.0 million on a Prime Product-by-Prime Product basis.

Myeloid is also eligible to receive tiered low to mid single-digit percentage royalties on our annual aggregate global net sales of Prime Products on a Prime Product-by-Prime Product and country-by-country basis, subject to customary offsets and reductions to a floor.

On a country-by-country and Prime Product-by-Prime Product basis, the period during which royalties will be paid will continue until the latest of (i) the expiration date of the last to expire valid claim of an issued patent or pending patent application within the patent rights subject to our option or the patent rights developed by one or both of the parties during the research term related to LINE-1 retrotransposon technology, in each case, covering the applicable Prime Product, (ii) loss of regulatory exclusivity for such Prime Product in such country, or (iii) ten (10) years after the first commercial sale of such Prime Product in such country.

Following the exercise of our option and for a period of two years thereafter, Myeloid will have the right to select up to three targets, subject to certain exclusions, for the development and commercialization of products directed at such targets in all fields and we will be eligible to receive the development, regulatory and sales-based milestone payments and royalty payments as set forth above from Myeloid with respect to such products.

Unless earlier terminated based on customary termination rights, the Myeloid Agreement will continue on a Prime Product-by-Prime Product and country-by-country basis until the expiration of the royalty term for such Prime Product in such country.

If we exercise our option, neither party will have the right to terminate the Myeloid Agreement for any reason.

Collaboration and License Agreement with Beam Therapeutics

In September 2019, we entered into a collaboration and license agreement, which we refer to as the Beam Collaboration Agreement, with Beam Therapeutics Inc., or Beam.

One of our founders, David Liu, is also a founder of Beam.

Under the Beam Collaboration Agreement, we grant to Beam an exclusive (even as to us and our affiliates), worldwide license under (i) certain Prime Editing know-how that we control during the initial term, and improvements thereto that we control for a specified number of years following the initial term, and patent rights that cover such Prime Editing know-how during the term of the Agreement, and (ii) our interest in certain jointly-owned collaboration technology, in each case, solely to develop, make, have made, use, offer for sale, sell, import and commercialize licensed products only in the Beam field.

The Beam field is limited to (a) the prevention, modification, improvement, amelioration or treatment of human disease, including cell-based therapies and the creation of one or more protective mutations, through administration of a licensed product that incorporates or contains a qualifying Prime Editing agent, which is a macromolecule or macromolecular complex that uses Prime Editing to make one or more transition point mutations (that is, C to T, T to C, A to G or G to A) in the sequence of one or more DNA targets, without intentionally making any non-transition mutations or other changes, including insertions, deletions, duplications, indels, transversions or combinations thereof, and does not incorporate or contain any other Prime Editing agent or other gene editing approach that is not a qualifying Prime Editing agent or (b) the prevention, modification, improvement, amelioration or treatment of sickle cell disease through administration of a licensed product that incorporates or contains a more broadly defined Prime Editing agent.