10-K

UNITED STATES

SECURITIES AND EXCHANGE COMMISSION

Washington, D.C. 20549

FORM 10-K

(Mark One)

For the fiscal year ended December 31, 2022

FOR THE TRANSITION PERIOD FROM___________TO___________

Commission File Number 001-40656

TENAYA THERAPEUTICS, INC.

(Exact name of Registrant as specified in its Charter)

171 Oyster Point Boulevard, Suite 500South San Francisco, CA 94080

(Address of principal executive offices) (Zip Code)

Registrant’s telephone number, including area code: (650) 825-6990

Securities registered pursuant to Section 12(b) of the Act:

Title of each class TradingSymbol(s) Name of each exchange on which registered

Common Stock $0.0001 par value per share TNYA Nasdaq Global Select Market

Securities registered pursuant to Section 12(g) of the Act: None

Indicate by check mark if the Registrant is a well-known seasoned issuer, as defined in Rule 405 of the Securities Act. Yes ☐ No ☒

Indicate by check mark if the Registrant is not required to file reports pursuant to Section 13 or 15(d) of the Act. Yes ☐ No ☒

Indicate by check mark whether the Registrant: (1) has filed all reports required to be filed by Section 13 or 15(d) of the Securities Exchange Act of 1934 during the preceding 12 months (or for such shorter period that the Registrant was required to file such reports), and (2) has been subject to such filing requirements for the past 90 days. Yes ☒ No ☐

Indicate by check mark whether the Registrant has submitted electronically every Interactive Data File required to be submitted pursuant to Rule 405 of Regulation S-T (§232.405 of this chapter) during the preceding 12 months (or for such shorter period that the Registrant was required to submit such files). Yes ☒ No ☐

Indicate by check mark whether the registrant is a large accelerated filer, an accelerated filer, a non-accelerated filer, smaller reporting company, or an emerging growth company. See the definitions of “large accelerated filer,” “accelerated filer,” “smaller reporting company,” and “emerging growth company” in Rule 12b-2 of the Exchange Act.

Large accelerated filer ☐ Accelerated filer ☐

Non-accelerated filer ☒ Smaller reporting company ☒

Emerging growth company ☒

If an emerging growth company, indicate by check mark if the registrant has elected not to use the extended transition period for complying with any new or revised financial accounting standards provided pursuant to Section 13(a) of the Exchange Act. ☐

Indicate by check mark whether the Registrant has filed a report on and attestation to its management’s assessment of the effectiveness of its internal control over financial reporting under Section 404(b) of the Sarbanes-Oxley Act (15 U.S.C. 7262(b)) by the registered public accounting firm that prepared or issued its audit report. ☐

If securities are registered pursuant to Section 12(b) of the Act, indicate by check mark whether the financial statements of the registrant included in the filing reflect the correction of an error to previously issued financial statements. ☐

Indicate by check mark whether any of those error corrections are restatements that required a recovery analysis of incentive-based compensation received by any of the registrant’s executive officers during the relevant recovery period pursuant to §240.10D-1(b). ☐

Indicate by check mark whether the Registrant is a shell company (as defined in Rule 12b-2 of the Exchange Act). Yes ☐ No ☒

The aggregate market value of the voting and non-voting common equity held by non-affiliates of the Registrant, based on the closing price of the shares of common stock on the Nasdaq Global Select Stock Market on June 30, 2022 was $156,445,652.

The number of shares of Registrant’s Common Stock outstanding as of March 2, 2023 was 66,865,250.

DOCUMENTS INCORPORATED BY REFERENCE

Portions of the definitive proxy statement for the Registrant’s 2022 Annual Meeting of Stockholders are incorporated by reference in Part III of this Form 10-K. Such definitive proxy statementwill be filed with the Securities and Exchange Commission within 120 days after the end of the Registrant’s 2022 fiscal year ended December 31, 2022.

Table of Contents

Page

PART I

Item 1. Business 3

Item 1A. Risk Factors 125

Item 1B. Unresolved Staff Comments 126

Item 2. Properties 126

Item 3. Legal Proceedings 126

Item 4. Mine Safety Disclosures 126

PART II

Item 7A. Quantitative and Qualitative Disclosures About Market Risk 136

Item 8. Financial Statements and Supplementary Data 137

Item 9A. Controls and Procedures 160

Item 9B. Other Information 161

Item 9C. Disclosure Regarding Foreign Jurisdictions that Prevent Inspections 161

PART III

Item 10. Directors, Executive Officers and Corporate Governance 162

Item 11. Executive Compensation 162

Item 14. Principal Accounting Fees and Services 162

PART IV

Item 15. Exhibits, Financial Statement Schedules 163

SPECIAL NOTE REGARDING FORWARD-LOOKING STATEMENTS

This Annual Report on Form 10-K, or Annual Report, contains forward-looking statements within the meaning of Section 27A of the Securities Act of 1933 and Section 21E of the Securities Exchange Act of 1934. All statements other than statements of historical facts contained in this Annual Report, including statements regarding our future results of operations and financial position, business strategy, development plans, planned preclinical studies and clinical trials, future results of clinical trials, expected research and development costs, regulatory strategy, timing and likelihood of success, as well as plans and objectives of management for future operations, are forward-looking statements. In some cases, investors can identify forward-looking statements by terms such as “may,” “will,” “should,” “would,” “expect,” “plan,” “anticipate,” “could,” “intend,” “target,” “project,” “contemplate,” “believe,” “estimate,” “predict,” “potential,” “vision,” or “continue” or the negative of these terms or other similar expressions. These forward-looking statements include, but are not limited to, statements about:

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our vision to change the treatment paradigm for heart disease;

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the ability of our ongoing preclinical studies and ongoing or planned clinical trials to demonstrate safety and efficacy of our product candidates, and other positive results;

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the timing, dosing, patient enrollment and populations, progress, and results of preclinical studies and ongoing or planned clinical trials for our current product candidates and other product candidates we may develop;

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the timing, scope and likelihood of regulatory filings and approvals, including timing of investigational new drugs (INDs), clinical trial applications (CTAs), U.S. Food and Drug Administration (FDA) approvals, and final regulatory approval of our current product candidates and any other future product candidates;

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our ability to develop and advance our current product candidates and programs into, and successfully complete, clinical trials;

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the size and the number of patients of the market opportunities we address with our product candidates;

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our manufacturing, commercialization, and marketing capabilities and strategy;

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our competitive position and the success of competing therapies that are or may become available;

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our plans relating to the further development of our product candidates, including additional indications and targets we may pursue;

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the impact of existing laws and regulations and regulatory developments in the United States, Europe and other jurisdictions;

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our intellectual property position, including the scope and length of protection we are able to establish and maintain for intellectual property rights covering our current product candidates and other product candidates we may develop, including the extensions of existing patent terms where available, the validity of intellectual property rights held by third parties, and our ability not to infringe, misappropriate or otherwise violate any third-party intellectual property rights;

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our continued reliance on third parties to conduct additional preclinical studies and clinical trials of our product candidates, and for the development and manufacture of our product candidates for preclinical studies and clinical trials;

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our ability to obtain, and negotiate favorable terms of, any collaboration, partnership, licensing or other arrangements that may be necessary or desirable to develop, manufacture or commercialize our product candidates;

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the pricing and reimbursement of our current product candidates and other product candidates we may develop, if approved, including any increase in demand as a result of the availability of reimbursement from the government and third-party payors;

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the rate and degree of market acceptance and clinical utility of our current product candidates and other product candidates we may develop;

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our estimates regarding expenses, operating losses, future revenue, cash outlays, capital requirements and needs for additional financing, including expenses arising as a result of being a public company;

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our financial performance;

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our facilities;

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the period over which we estimate our existing cash, cash equivalents and investments in marketable securities will be sufficient to fund our future operating expenses and capital expenditure requirements;

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the impact of critical accounting policies on investor’s ability to understand our financial performance; and

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our expectations regarding the period during which we will remain an emerging growth company under the JOBS Act.

We have based these forward-looking statements largely on our current expectations and projections about our business, the industry in which we operate and financial trends that we believe may affect our business, financial condition, results of operations and prospects, and these forward-looking statements are not guarantees of future performance or development. These forward-looking statements speak only as of the date of this Annual Report and are subject to a number of risks, uncertainties and assumptions described in the section titled “Risk Factors” and elsewhere in this Annual Report. Because forward-looking statements are inherently subject to risks and uncertainties, some of which cannot be predicted or quantified, investors should not rely on these forward-looking statements as predictions of future events. The events and circumstances reflected in our forward-looking statements may not be achieved or occur and actual results could differ materially from those projected in the forward-looking statements. Except as required by applicable law, we do not plan to publicly update or revise any forward-looking statements contained herein, whether as a result of any new information, future events or otherwise.

In addition, statements that “we believe” and similar statements reflect our beliefs and opinions on the relevant subject. These statements are based upon information available to us as of the date of this Annual Report, and while we believe such information forms a reasonable basis for such statements, such information may be limited or incomplete, and our statements should not be read to indicate that we have conducted an exhaustive inquiry into, or review of, all potentially available relevant information. These statements are inherently uncertain and investors are cautioned not to unduly rely upon these statements.

PART I

Item 1. Business.

Overview

We are a clinical-stage biotechnology company committed to a bold mission: to discover, develop and deliver potentially curative therapies that address the underlying drivers of heart disease. Heart disease is the leading cause of death in the world, representing an estimated 32% of all global fatalities. In the United States (U.S.), one in every five deaths is attributable to heart disease, and an estimated 40,000 infants are born each year with congenital heart disease. While there is a clear need for improved treatments, the rate of cardiovascular drug product approvals has declined in recent years and few of the approved treatments address the underlying cause of such diseases.

Leveraging an improved understanding of the genetic causes of heart disease as well as an increased recognition that precision medicine initiatives may accelerate the advancement of scientific breakthroughs, our vision is to change the treatment paradigm for heart disease and in doing so, improve and extend the lives of patients. Roughly one-third to one-half of all heart diseases are linked to genetic risks, regardless of major racial and ethnic backgrounds, and there are over 250 known genetically defined disorders where the primary source of morbidity and mortality involves the heart. Our collective understanding of the links between heart disease and genetic factors is increasing exponentially, creating new opportunities for the advancement of novel disease-modifying therapeutics that target the underlying cause of disease.

We are advancing a deep and diverse pipeline of therapeutic programs intended for both rare and highly prevalent forms of heart disease. Each of our lead product candidates, TN-201, a gene therapy for myosin binding protein C3 (MYBPC3)-associated hypertrophic cardiomyopathy (HCM), TN-301, a small molecule for heart failure with preserved ejection fraction (HFpEF), and TN-401, a gene therapy for plakophilin 2 (PKP2)-associated arrhythmogenic right ventricular cardiomyopathy (ARVC), emerged from our proprietary integrated drug discovery platforms and has progressed to the clinic or late-stage preclinical development with the support of our core internal capabilities.

In addition to our lead product candidates, we have multiple early-stage programs progressing through pre-clinical development. These programs include an adeno-associated virus (AAV)-based gene therapy designed to express the Dwarf Open Reading Frame (DWORF) gene in the heart with potentially broad utility in dilated cardiomyopathy (DCM), as well as our reprogramming program for cardiac regeneration which aims to replace heart cells lost in patients experiencing heart failure due to prior myocardial infarction (MI). While these named programs have reached candidate selection stage, we also have numerous earlier-stage programs emerging from our proprietary product platforms to address other forms of heart failure.

Our distinct, but interrelated Gene Therapy, Cellular Regeneration and Precision Medicine platforms and suite of integrated capabilities support our efforts to discover disease-modifying treatments focused on heart disease in a modality-agnostic manner. We also continue to invest in complementary new technologies and the optimization of our existing proprietary capabilities, including the use of human-induced Pluripotent Stem Cell (iPSC) disease models, machine learning and phenotypic screening, capsid engineering and novel promoter constructs to enable the discovery, design, delivery and development of therapeutics that are best suited to a given cardiovascular condition. In 2022 we also launched operations of our Genetic Medicines Manufacturing Center (GMMC) based in Union City, CA. The facility utilizes a modular, scalable design to produce AAV-based gene therapies under current Good Manufacturing Practice (cGMP) standards.

Our Product Pipeline

Our pipeline includes programs that have emerged from our internal efforts, as well as programs that are based on intellectual property licensed from academic institutions.

* US Prevalence refers to the number of patients in the U.S. with the indication based on publicly available market data

TN-201:TN-201 is our potential first-in-class gene therapy for adults and children with HCM due to MYBPC3 gene mutations, the most common cause of familial HCM. MYBPC3-associated HCM is estimated to affect more than 115,000 patients in the U.S. These mutations can cause the heart walls of affected individuals to become significantly thickened, leading to fibrosis, abnormal heart rhythms, cardiac dysfunction and heart failure. HCM is a chronic, progressive condition and those diagnosed with disease often experience significant impairment in overall heart function and quality of life. Those with sarcomeric genetic mutations, such as MYBPC3, are at increased risk of early disease onset, accelerated disease progression and disease-related mortality. TN-201 uses a differentiated approach to deliver a functional MYBPC3 gene to the heart utilizing a recombinant AAV serotype 9 (AAV9) capsid to restore expression of the cardiac myosin binding protein (MyBP-C) to halt disease progression and potentially reverse the course of genetic HCM following a single intravenous injection. TN-201 has received orphan drug designation from the FDA and orphan medicinal product designation from the European Commission (EC). In January 2023, we received notification from the FDA that clinical testing of TN-201 may proceed, and in the third quarter of 2023, we expect to begin dosing patients in a Phase 1b multi-center, open-label clinical trial, designed to assess the safety, tolerability and efficacy of a one-time intravenous infusion of TN-201. Data from the trial is anticipated in 2024.

TN-301: TN-301 is our highly specific small molecule inhibitor of histone deacetylase 6 (HDAC6). TN-301 is initially being developed for the potential treatment of HFpEF. HFpEF is characterized by a stiffening of the heart muscle resulting in an inability for the left ventricle (LV) to relax properly during normal heart rhythm, referred to as diastolic dysfunction. There are several cellular processes thought to underly the pathophysiology of HFpEF including increases in fibrosis and inflammation and defects in metabolism. Although HFpEF accounts for approximately 50% of all heart failures, there are few proven treatment options.We are currently conducting a Phase 1 clinical trial in healthy adult participants to evaluate the safety, tolerability, pharmacokinetics (PK) and pharmacodynamics (PD) of escalating oral doses of TN-301. The Phase 1 clinical trial is being conducted in two stages: a single-ascending dose (SAD) stage and a multiple-ascending dose (MAD) stage. Data from both the SAD and MAD stages of the trial are anticipated in the second half of 2023.

TN-401: We are developing a potential first-in-class AAV-based gene therapy, TN-401, designed to deliver a functional PKP2 gene in adults with ARVC due to a PKP2 genetic mutation. PKP2-associated ARVC is estimated to affect more than 70,000 patients in the U.S.PKP2 mutations can cause enlargement of the right ventricle (RV) in affected individuals, replacement of heart muscle with fibrotic tissue and fatty deposits, and severely abnormal heart

rhythms (arrhythmia) that can make it harder for the heart to function properly and result in sudden cardiac death in some adults and children. Our product candidate, TN-401, has demonstrated prevention of disease progression and survival benefit after a single dose in a mouse model of ARVC, as well as tolerability in a pilot non-Good Laboratory Practices (GLP) toxicology and biodistribution study. TN-401 has received orphan drug designation from the FDA. We have initiated investigational new drug application (IND)-enabling studies for TN-401 and expect to submit an IND to the FDA in the second half of 2023 to enable clinical development of TN-401.

Early-Stage Research Efforts: In addition to our lead product candidates, we have multiple early-stage programs progressing through preclinical development, including an AAV-based gene therapy designed to express the DWORF gene in the heart with potentially broad utility in DCM and a cellular reprogramming program for cardiac regeneration which aims to replace heart cells lost due to prior MI. Our researchers are conducting preclinical testing of these and several other genetically targeted leads emerging from our proprietary product platforms to address other forms of heart failure. In addition to our novel drug discovery efforts, we continue to invest in the further development of our gene therapy-enabling technologies and capabilities. Among the most advanced of these initiatives is an effort to identify novel AAV capsids designed to deliver cardiac gene therapy with enhanced specificity and expression compared to current AAV vectors, with the goal of optimizing gene therapy safety, dosing and efficacy.

Our Product Platforms

We have established three distinct but interrelated product platforms -- Gene Therapy, Cellular Regeneration and Precision Medicine -- to discover novel therapies for various forms of heart disease. These platforms bring together differentiated science, capabilities, and intellectual property to enable multi-modality drug discovery. We believe these three product platforms, together with our suite of proprietary capabilities, provide us greater insight into disease processes, create more opportunities for successful drug development, mitigate scientific risks, and differentiate our efforts relative to competitors.

Our Gene Therapy platform uses AAVs to deliver healthy genes to specific cells in the heart to correct or compensate for functional defects. While our lead gene therapy programs utilize AAV9 to deliver healthy genes to the heart, we have the ability to use both known AAV capsids as well as novel capsids identified through our internal capsid engineering capabilities. In the future, we may also explore other delivery options, including non-viral delivery. Depending on the nature of the disease, we may target cardiomyocytes, cardiac fibroblasts, or other cells important to the proper functioning of the heart. The product candidates arising from this platform are intended to overcome the shortcomings of traditional

therapies that are not able to address the underlying problems that contribute to heart disease. We believe this platform has potentially broad utility for both rare and prevalent forms of heart disease.

Our Cellular Regeneration platform uses viral vectors to deliver specific combinations of genes to existing cells in the heart to regenerate cardiomyocytes through two distinct in vivo approaches: One approach uses AAV vectors to deliver proprietary combinations of genes that induce the resident cardiac fibroblasts to convert to cardiomyocytes. Another approach uses non-integrating lentiviruses to deliver proprietary combinations of genes that induce the resident cardiomyocytes to undergo transient cell division. The product candidates arising from this platform are intended to overcome the shortcomings of traditional therapies that address symptoms but are not able to address the irreversible loss of cardiomyocytes. We believe this platform has potentially broad utility across a range of heart conditions that result in the loss of cardiomyocytes, including MI, chemotherapy-related toxicity, and viral infection.

Our Precision Medicine platform for target identification uses human genetic information combined with phenotypic high throughput screening in our proprietary human iPSC-CM models of human disease and machine learning algorithms for the identification and validation of novel targets for heart diseases. Targets may be further characterized using three-dimensional human engineered heart tissues. By leveraging human cells and tissues to identify and/or validate heart disease targets, this platform is intended to overcome the shortcomings of traditional drug development efforts that rely more heavily on insights from animal models. We believe this platform may also help identify promising drug targets directed to sub-populations of patients who are more likely to respond to such targeted product candidates. We believe this platform has potentially broad utility for the identification of targets and therapies in a modality-agnostic manner—including gene therapy, small molecules, and biologics—for both genetic and non-genetic forms of heart disease.

Our Core Capabilities

Foundational to our research and drug discovery efforts are our proprietary integrated core capabilities that collectively support discovery of novel targets, in vitro optimization and validation of leads, rapid product development, precise product delivery, and efficient production, which ultimately improves the probability of technical and regulatory success of our product candidates.

Our five core capabilities include:

Disease Models. We have internalized the ability to create and integrate proprietary in vitro and in vivo models within our research organization, as existing models of human heart disease may not be adequate to assess the efficacy or safety of novel therapies. Our disease modelling capabilities serve to facilitate the discovery of new leads and to characterize the activity of existing leads as we move through preclinical development. For our in vitro human iPSC-CM disease models, we use multiple methods to induce phenotypes within cell lines that simulate human diseases and then use these models for high throughput target identification and drug discovery. For our in vivo disease models, we have a dedicated onsite in vivo pharmacology group and vivarium, where we have established approximately 17 rodent heart disease models, both genetic and non-genetic, and can dose animals, perform heart surgeries, and use non-invasive imaging to assess the impact of our therapies under development.

Capsid Engineering. We have established in-house AAV capsid engineering capabilities and have successfully screened over one billion variants from more than 30 diverse, proprietary AAV libraries in multiple in vitro, in vivo, and in silico models to discover novel AAV capsids that can target the different types of cells in the heart. We have generated preclinical data to support the superiority of these capsids over parental variants in multiple species against multiple attributes to assess their potential to translate across species and into humans. Our next-generation capsids are designed to have desirable properties including the ability to more selectively target the heart versus other organs, as well as lower susceptibility to neutralizing antibodies. We believe our capsid engineering efforts will be critical in supporting the successful clinical development of future product candidates and enabling those product candidates, if approved, to reach more patients.

Promoters and Regulatory Elements. We have created novel promoters and regulatory elements that support our gene therapy and cellular regeneration programs by controlling the expression of genes

within the cells. We use these innovations to help ensure more precise and more robust expression of therapeutic payloads in the different cell types of the heart as compared to what can be achieved with currently available methods. We believe our innovations can support successful clinical development in part by improving the efficacy and safety profile of our product candidates.

Drug Delivery. We are actively exploring different routes of administration (ROAs) as well as different infusion- and injection-based methods for delivering our AAV-based therapies. We have designed a new catheter to support more targeted delivery and more efficient uptake of therapeutic payloads in the heart. We believe our discoveries in drug delivery can help widen the therapeutic index of our product candidates by reducing the dose required for a therapeutic benefit.

Manufacturing. We have internalized and integrated both cGMP and non-GMP AAV manufacturing capabilities to support our emerging portfolio of gene therapy and cellular regeneration product candidates. This includes an in-house team of approximately 45 personnel that can support process development, analytical development, quality control (QC) and GMP manufacturing. In addition, we have established a Quality Management System to oversee our GxP operations, including cGMP, GLP and Good Clinical Practices (GCP). To date, we have produced non-clinical material involving multiple parental AAV capsids at the 50L and 200L scales to support early research and IND-enabling studies in small and large animal models, as well as clinical material at the 1000L scale to support our first-in-human clinical studies. Our GMMC, a cGMP facility, is strategically located near our research labs in the San Francisco Bay Area to enable smooth scale-up of production to support our clinical studies. We have both in-licensed and internally developed manufacturing technologies to support programs emerging from our Gene Therapy and Cellular Regeneration Platforms.

Overview of Heart Disease

Heart disease is the leading cause of death in the world, representing an estimated 32% of all global fatalities. In the U.S., more than 30 million adults, or approximately 12% of the adult population, are diagnosed with heart disease. In addition, an adult dies from a cardiovascular-related health condition, such as a heart attack every 34 seconds, a gruesome statistic that translates to approximately one in five deaths in the U.S. The picture is equally bleak at the other end of the age spectrum, as approximately 40,000 infants are born in the U.S. every year with congenital heart disease, the leading cause of birth defect-related morbidity and mortality. There are over 250 known genetically defined disorders where the primary source of morbidity and mortality involves the heart, but there are few approved products that target the underlying genetic causes of such heart diseases. Recent analysis has shown that after decades of reduction in the mortality rate due to heart failure, these rates are once again rising, highlighting the need for improved treatments.

The heart is a complex organ due to its biological structure as well as its tightly regulated and coordinated electrophysiological and biomechanical properties. Heart disease comes in many forms, affects individuals at many ages, and is a result of many factors. As depicted in the below table, heart disease can be generally categorized as either directly resulting from problems associated with the heart organ, for example, heart failure, arrhythmia and heart valve disease; or indirectly resulting from problems associated with the vasculature, for example, coronary artery disease (CAD). In each case, the underlying cause could be genetic, or due to normal aging or due to environmental factors.

The table below illustrates four broad categories of heart disease:

CATEGORIES DESCRIPTION

While there is significant unmet need in the field of heart disease, historically there have been challenges in developing novel therapies for the different forms of heart disease. We are currently focused on heart failure and arrhythmia, particularly when these diseases can be traced to some underlying genetic defect.

Historic Challenges in the Development of Novel Therapies for Heart Disease

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Most development efforts have focused on treating symptoms rather than targeting the underlying causes of diseases. First-line therapies for heart failure such as generic small molecules, including angiotensin-converting enzyme (ACE) inhibitors, angiotensin II receptor blockers, beta blockers, aldosterone antagonists, and diuretics, act by reducing blood pressure or fluids to mitigate disease symptoms rather than altering the course of disease.

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Identifying relevant disease-modifying targets is challenging. There is a high reliance on animal models that are not always predictive of human heart disease. There is only an approximately 5% overall probability of successful drug development from Phase 1 through commercialization for heart disease, among the lowest of all therapeutic areas.

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Genetic diagnosis and genetic counseling are limited. Most patients presenting with heart disease do not currently obtain a genetic test as part of their diagnosis. Given there are almost no therapies that are targeted at the underlying genetic cause of the disease, physicians may believe a genetic test will not influence treatment and management decisions. Additionally, even when patients do receive a genetic diagnosis, genetic counseling and family screening are not commonly employed. As a result, family members who may be at risk of disease are not consistently identified. This lack of genetic testing also limits the availability of patients for clinical trials of genetic medicines in heart disease.

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Regenerative therapy science is still in its early stages. Historical attempts at developing cell and gene therapies for heart disease have not been successful. Much effort was devoted to regenerative medicine approaches using autologous (from self) or allogeneic (from donors) cell sources, but after more than 150 clinical studies involving thousands of patients over the last two decades, those efforts have mostly ended in failure. Factors that likely contributed to these failures include (1) an insufficient number of

new cells surviving rejection by the immune system, (2) only modest efficacy from the surviving cells, and (3) arrhythmia caused by abnormal electric activity and connections between new cells and the existing cells.

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Gene therapy science for the heart is still maturing. Early gene therapy efforts, including an AAV-based effort using AAV1 to deliver sarcoplasmic/endoplasmic reticulum Ca2+ ATPase 2a (SERCA2a) for the treatment of heart failure saw promising preclinical and early clinical results, but were stymied following unsuccessful later-stage studies. These first-generation gene therapy efforts for the heart did not have the benefit of more recent advances in capsids, promoters, delivery, and manufacturing. More recent efforts directed at rare, genetic cardiomyopathies have provided encouraging early clinical evidence of disease-modification.

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Regulatory requirements focus on survival and hospitalization outcomes. Historically, cardiovascular drug development has involved large clinical studies to demonstrate a survival benefit or reductions in hospitalizations over and above standard-of-care. This has translated into a need for very large, long, and expensive randomized and placebo-controlled clinical studies. Only more recently has the FDA indicated a willingness to consider endpoints demonstrating measurable improvements in symptoms and heart function as being potentially approvable for certain types of heart diseases, including HCM.

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Costs of development are high. In part due to the historical need for very large clinical studies, drug development for new therapies of heart disease has been very long and expensive. A recent analysis demonstrated that, on average, biopharmaceutical companies spent $1 billion in clinical development per cardiovascular drug product approval, the highest ratio among all therapeutic areas.

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Patient access barriers are challenging. In addition to being a leading cause of death, heart disease is one of the largest and most expensive categories for payers. The U.S. spends approximately $219 billion per year on cardiovascular disease. The total direct and indirect costs of heart failure are expected to increase to $70 billion by 2030. As a result, heart disease is an area of focus for cost-containment and price sensitivity for new therapies for both private and public payers.

These factors have contributed to a decline in successful heart disease drug development. Between 2000 and 2009, FDA approvals for new cardiovascular drug products declined by approximately 33% compared with the prior decade.While heart disease is a leading cause of death in the world, fewer resources have been mobilized in support of new therapies for heart disease relative to investment in other therapeutic areas, such as oncology and diseases of the central nervous system.

However, there are recent signs of improvement. There is increasing insight into the genetic causes of heart disease and a greater push for more consistent genetic testing and family counseling supported by (1) updated clinical practice guidelines such as 2020 American College of Cardiology and American Heart Association recommendations for patients with HCM, (2) the push by patient advocacy organizations for mandatory screening of young athletes, and (3) increased availability of accessible genetic testing covering more than 150 relevant genes associated with inherited arrhythmia and cardiomyopathy conditions. There are also a small but growing number of examples of clinical success with precision medicine approaches in cardiology, including in genetic cardiomyopathies. In addition, the FDA continues to issue important guidance to help streamline the development of gene therapies and mitigate safety and tolerability concerns that can delay the drug development timeline.

We believe with the evolving understanding of heart disease in the scientific community and the general public, there are significant opportunities where we can benefit from and support the evolution towards more precise diagnosis, drug development, and treatment for heart disease, as depicted in the diagram below.

Our Strategy

Our goal is to become a leading, fully integrated biotechnology company delivering next-generation therapies that address the underlying causes of heart disease identified through our multi-modality product platforms. We are taking advantage of an expanded understanding of heart biology and advances in the science of genetics and disease models to discover, develop, manufacture and ultimately commercialize a deep and diverse pipeline of novel heart disease therapies. The key components of our strategy to achieve these goals are:

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Focus exclusively on heart disease. Heart disease remains a leading cause of death globally, and the unmet medical need remains high. We see significant opportunity to address this sizable market with our dedicated strategy. The heart is a complex organ to target, in part due to the tightly regulated and coordinated electrophysiological and biomechanical properties that can complicate delivery of effective therapies and necessitates a deep understanding of heart biology. Our laser focus leads to insights that underpin our foundational and differentiated capabilities to address challenges that have historically presented barriers to the successful development of novel therapies for the heart.

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Develop disease-modifying therapies. We are focused on developing disease-modifying and potentially life-saving novel therapies that target the underlying causes of heart disease. We are particularly interested in areas where there is no current standard-of-care or where we believe the nature and the magnitude of the effect of our therapies will be significant relative to existing standards-of-care. For example, we believe our AAV-based gene therapies for genetically defined conditions have the potential to be curative after a single dose.

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Discover novel therapies using integrated product platforms. To address the wide range of issues in heart diseases, we are advancing science from three distinct product platforms that tackle different problems that have historically plagued drug development in the field of cardiology: (i) our Gene Therapy platform enables the development and delivery of a wide variety of therapeutic payloads more precisely to heart tissue, (ii) our Cellular Regeneration platform enables the replacement of heart cells lost to disease and (iii) our Precision Medicine platform enables the discovery of targeted therapies in a modality-agnostic fashion. Underpinning these platforms is a suite of bespoke internal core capabilities designed to increase the speed with which we can conduct discovery, improve the precise delivery of drug products to the tissues where they can have the desired effect while maximizing safety and productivity. These platforms represent distinct but interrelated product engines that we believe will enable a robust pipeline of promising product candidates while also mitigating overall scientific risk.

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Target defined sub-populations of patients most likely to respond to our therapies. We seek to focus on patient populations where the genetic cause of the disease is well-established, including genetic cardiomyopathies and other monogenic disorders. We also seek to use different strategies to sub-segment larger heart failure populations, such as HFpEF and heart failure with reduced ejection fraction (HFrEF), through the use of genetics or biomarkers to improve selection of patients with attributes that are more suited to the specific mechanism of action of a given therapeutic candidate. We believe this strategy can accelerate clinical development, reduce overall development costs, and improve the probability of clinical and regulatory success.

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Internalize and integrate core capabilities to support our innovation. We have five core capabilitiesthat we believe will enable us to rapidly discover, develop, and deliver heart therapies. These capabilities include: (i) Disease Models, (ii) Capsid Engineering, (iii) Promoter and Regulatory Elements, (iv) Drug Delivery and (v) Manufacturing. We believe the integration of our know-how and innovations in these areas will allow us to generate scientific insights more rapidly and improve the probability of technical and regulatory success of our product candidates. The internalization of these capabilities also reduces our reliance on third parties—be it academic labs, contract research organizations (CROs), or contract development and manufacturing organizations (CDMOs)—providing us better control of our timelines and costs.

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Advance a deep and diverse pipeline of therapies. The diversity of our programs illustrates the ambition of our vision and the versatility and depth of our scientific approach. We are currently advancing therapeutics for both rare and prevalent heart diseases across multiple treatment modalities. Our most advanced rare disease programs include two AAV-based gene therapy candidates: TN-201, our product candidate for MYBPC3-associated HCM and TN-401, our product candidate for PKP2-associated ARVC. We are also advancing TN-301, a small molecule inhibitor of HDAC6 intended to address HFpEF. Following closely at the candidate selection stage is a DWORF gene therapy for the potential treatment of DCM and a cellular reprogramming program for cardiac regeneration which aims to replace heart cells lost in patients experiencing heart failure due to prior MI. We are also working on several other early-stage programs, that we believe will add to our future pipeline opportunities.

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Seek partnerships that can expand our reach and accelerate our efforts. We believe our singular focus on heart disease and extensive platform and core capabilities make us a potential partner of choice for academics and larger companies alike who wish to access deep expertise in next-generation therapies for heart disease. We also strategically evaluate collaborations and partnerships with biopharmaceutical companies that may have more robust and complimentary capabilities and resources to accelerate the development and maximize the availability and potential of our product candidates, particularly for more prevalent indications.

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Become a fully integrated biopharmaceutical company with commercial capabilities. We aim to discover, develop, manufacture, and eventually commercialize therapies, with an initial focus on those therapies for rare disease populations that could be marketed by a relatively small salesforce.

Our Programs

TN-201: Gene Therapy for MYBPC3-associated HCM

We are developing TN-201, an investigational and potential first-in-class gene therapy for MYBPC3-associated HCM. MYBPC3 genetic mutations are the most common cause of familial HCM, estimated to affect more than 115,000 patients in the U.S. These mutations can cause the heart walls of affected individuals to become significantly thickened, leading to fibrosis, abnormal heart rhythms, cardiac dysfunction, heart failure, and increased risk of sudden cardiac death. TN-201 is an AAV-based gene therapy designed to deliver a fully functional MYBPC3 gene to restore normal levels of the cardiac MyBP-C protein and in order to halt disease progression and reverse the course of genetic HCM after a single treatment. Based on publicly available information we believe TN-201 has the potential to be the first treatment to address the underlying genetic cause of this disease.

Overview of HCM

HCM is a condition in which the heart walls become thickened (hypertrophy), resulting in a reduced ability of the LV to relax and fill (diastole) and pump (systole) blood effectively with each contraction. HCM is a chronic, progressive disease associated with significant impairment to patients’ overall quality of life, as well as an elevated risk of sudden cardiac death. Symptoms include chest pain, shortness of breath (dyspnea), fainting (syncope), fatigue and palpitations. As the disease progresses, patients may suffer premature death due to end-stage heart failure or malignant ventricular arrhythmia (VA) sometimes leading to sudden cardiac death or stroke. Disease onset can occur at any age, with HCM most frequently emerging in adults in their mid-40s. When HCM emerges in children and young adults, disease course is typically more aggressive and prognosis is worse than that observed in older patients. While a relatively rare occurrence, HCM is the leading cause of sudden cardiac death in young adults.

HCM is estimated to affect one in every 500 people, approximating more than 600,000 people in the U.S. A majority of HCM patients are currently undiagnosed, with diagnosis typically starting with the onset of symptoms, family screening, or the discovery of an abnormal electrocardiogram (ECG) pattern. A clinical diagnosis of HCM in adults is defined as a left ventricular wall thickening of greater than 15mm. Patients with HCM can present with either the obstructive form (oHCM) or the nonobstructive form (nHCM) of the disease. Both forms of the disease involve significant LV hypertrophy; however, in oHCM, the thickening of the LV wall is such that the LV outflow tract (LVOT) narrows and “obstructs” the proper flow of blood to the rest of the body. Nonobstructive HCM is more frequently characterized by diastolic dysfunction resulting in increased LV filling pressures that leads to chest pain and dyspnea.

An example of a heart from a patient who had oHCM is shown below, characterized by LV hypertrophy, high LV mass, LVOT narrowing, an overall small LV, and fibrosis.

The genetic causes of HCM may be diverse, but approximately 60% of patients with HCM have clearly identifiable familial disease with an autosomal dominant pattern of inheritance. To date, more than 2,000 mutations in eleven or more genes have been linked to HCM. Those with sarcomeric genetic mutations, including the MYBPC3 gene, are at increased risk of early disease onset, accelerated disease progression and disease-related mortality. Mutations in the MYBPC3 gene are the most common cause of HCM, estimated to represent approximately 19% of the overall HCM population and to affect approximately 115,000 patients in the U.S.

The MYBPC3 gene encodes the MyBP-C protein, which forms a key component of the cardiac sarcomere, the fundamental contractile unit of the cardiomyocyte. MyBP-C protein is central to regulation of both contraction and relaxation of the cardiac muscle. MYBPC3 gene mutations result in both oHCM and nHCM, with one study involving a series of more than 1000 patients finding that 69% of patients with truncating MYBPC3 mutations had nHCM, while 31% presented with LVOT characteristic of oHCM. The schematic below illustrates the cellular localization of MyBP-C within the heart. Cardiomyocytes contain multiple myofibrils, which are comprised of myofilaments containing many sarcomeres. The sarcomeres contain thin filaments containing actin and thick

filaments containing myosin; the myosin head binds and pulls actin like a hand on a rope and thus supports normal muscle contraction.

The reduced MyBP-C protein levels associated with heterozygous mutations in the MYBPC3 gene result in increased activity of the myosin contractile machinery, which over time leads to LV muscle thickening, known as hypertrophy, excess deposition of extracellular matrix in the cardiac muscle, known as fibrosis, and disorganized muscle cells. As a result, the LV wall stiffens, and the chamber is reduced in size, decreasing the heart’s ability to pump. The contractile strength of the muscle declines in some cases, resulting in LV systolic dysfunction, which ultimately can necessitate advanced therapies, such as an LV assist device (LVAD) or transplantation, in the most severely affected patients. Fibrosis and muscle cell disarray may also lead to arrhythmias in some patients, including life-threatening VA and atrial fibrillation, which can lead to stroke.

Infants with homozygous MYBPC3 gene mutations represent a particularly severe patient group with high risk of death within a year after birth without heart transplantation. With no ability to produce MyBP-C protein and no available treatment to address the underlying genetic mutation, the only option for this young patient population is a heart transplant.

HCM patients who are heterozygous for MYBPC3 gene mutations are typically diagnosed earlier in life, have more severe disease associated with increases in arrhythmia, sudden cardiac death and cardiovascular mortality as compared to genotype negative HCM patients.

Analysis of the hearts of patients who carry truncation mutations of the MYBPC3 gene show on average an approximately 40% reduction in the level of functional MyBP-C protein. In the most severe cases in which both copies of the gene are affected, there is a complete lack of functional MyBP-Cprotein expression. We believe these findings support the idea that mutations of the MYBPC3 gene cause human disease through haploinsufficiency, and also support the hypothesis that gene replacement may address the underlying cause of disease by increasing the levels of functional MyBP-C protein.

The current goal of HCM treatment is to relieve symptoms and prevent sudden cardiac death in people at high risk. In current guideline-directed care, patients are typically prescribed one or more symptomatic therapies, including beta-blockers, calcium channel blockers and antiarrhythmics. These therapies do not address the underlying genetic cause of HCM and do not appear to affect disease progression. No randomized clinical trials have assessed these therapies specifically in HCM. The standards of care are slightly different for patients with oHCM versus nHCM, but the unmet need is high in both forms of the disease. Cardioverter-defibrillators may be implanted for patients at high risk for malignant arrhythmias and sudden death. For a subset of oHCM patients with severe and disabling disease, invasive interventions, such as myectomy and septal ablation in which portions of the enlarged septum are removed, may be appropriate. For patients with severe nHCM implantation of an LVAD or a heart transplant may be the only options.

In recent years, a class of agents known as myosin inhibitors have emerged as potential treatments for oHCM and nHCM. One of these agents, mavacamten, was approved by the FDA in April 2022 for the treatment of oHCM. Other agents continue to be evaluated in clinical studies. Currently, there are no therapies approved or in clinical development specifically for HCM patients with MYBPC3 gene mutations.

Our Solution

We are developing TN-201, a potential first-in-class AAV-based gene therapy designed to deliver a fully functional MYBPC3 gene and to restore normal levels of MyBP-C protein, driven by our proprietary cardiac specific promoter. We believe TN-201 has the potential to address the underlying biological basis of disease in adult and pediatric HCM patients with homozygous or heterozygous MYBPC3 gene mutations.

Based on our preclinical data, we believe that gene therapy can achieve highly selective and robust expression of the MYBPC3 gene and has the potential to slow or even reverse the course of MYBPC3-associated HCM, including LV hypertrophy, outflow tract obstruction, heart failure, atrial fibrillation, and malignant arrhythmias. By improving upon these aspects of disease, TN-201 may improve heart functional capacity, stabilize or reverse disease symptoms, reduce the need for invasive treatments and improve survival. As with other AAV-based gene therapies, benefits are expected to be durable and a one-time dose may be sufficient for disease stabilization and potentially reversal.

TN-201 Clinical Development Plan

In January 2023, we received clearance of our IND from the FDA to conduct a Phase 1b clinical trial of TN-201 in symptomatic adults with the nonobstructive form of MYBPC3-associated HCM. We expect to commence patient dosing in the Phase 1b in the third quarter of 2023 and have completed all necessary manufacturing of TN-201 to supply the clinical trial.

The TN-201 Phase 1b is a multi-center, open-label clinical trial designed to assess the safety, tolerability and efficacy of a one-time intravenous infusion of TN-201. The trial will seek to enroll at least six symptomatic (New York Heart Association class II or III) adults (ages 18-65) with low tiers of AAV9 neutralizing antibodies who have been diagnosed with MYBPC3-associated nHCM and have an implantable cardioverter defibrillator (ICD). The primary endpoints for the trial include safety and tolerability, PK (as measured by transgene and mRNA expression via cardiac biopsies at 8 weeks and 52 weeks) and PD (as measured by imaging and plasma biomarkers). Additional endpoints include exercise capacity (as measured by a six-minute walk test and peak maximal oxygen consumption (VO2)) and patient-reported outcomes (as measured by a Kansas City Cardiomyopathy Questionnaire). The trial will include a preventative immunosuppressive regimen and close safety monitoring, as well as a 5-year follow-up on safety and efficacy. We expect to assess two dose levels of TN-201 in the trial, starting with 3×1013 vg/kg, a dose associated with near-maximal efficacy in preclinical studies. Three patients are expected to be enrolled in the first dose cohort and will be dosed sequentially, with a pause between patient doses to monitor for safety. An independent safety review following the initial cohort will inform plans for dose escalation to 6×1013 vg/kg, as needed, and/or enrollment of additional patients in the initial cohort. TN-201 has received orphan drug designation from the FDA and orphan medicinal product designation from the EC. Data from the trial is anticipated in 2024.

The Phase 1b clinical trial will be conducted at multiple centers in the U.S. In order to support our development efforts for TN-201, we have initiated two noninterventional studies: a study evaluating seroprevalence to AAV9 antibodies among adults with MYBPC3-associated HCM, andMyClimb, a prospective and retrospective global natural history study focused on pediatric patients with MYBPC3 mutation-associated cardiomyopathy. The objective of the natural history study is to characterize the outcomes, burden of illness, risk factors, quality of life, and biomarkers associated with disease progression in pediatric patients with cardiomyopathy due to MYBPC3 gene mutations, as well as treatments, procedures, and patient outcomes. This study complements existing disease registries focused primarily on adult patient HCM populations and may support and expedite the development of TN-201 in the pediatric patient population. We have activated more than 15 sites in the U.S. and Europe and enrolled more than 100 subjects in the MyClimb study.

Preclinical Evidence Supporting TN-201 Clinical Development Plan

In preclinical studies, we systemically administered a mouse surrogate of TN-201 (AAV:mMybpc3 or mTN-201) in two-week-old Mybpc3 KO mice. The Mybpc3 KO model develops marked LV hypertrophy, poor cardiac function, and dilation at two-weeks of age, comparable to HCM patients with truncating or null mutations. Due to the severe phenotype of the Mybpc3 KO mice and the lack of any MyBP-C protein, this is considered a demanding model to demonstrate efficacy particularly for modeling heterozygous patients, who lack only 35% to 40% of normal sarcomeric MyBP-C protein levels. As shown in the figures below, treatment with mTN-201 improved LV hypertrophy and cardiac function compared to their pre-treatment baseline levels, indicating partial reversal of the disease and dramatically extended lifespan. Treated mice exhibited an absolute improvement of EF of more than

20% versus untreated controls that eventually increases to more than 30% at 13 months, the last echocardiography measurement. EF and LV hypertrophy (LV mass normalized to body weight (BW)) improvements did not diminish over time, suggesting that a single systemic dose may be sufficient for a durable reversal of MYBPC3-associated HCM. Additionally, we observed improvements in LV diameter and ECG measurements. There is also a clear survival benefit with 100% survival in the mTN-201 arm and 100% mortality in the untreated control arm out to 18 months following dosing.

In addition, a dose-response relationship has been demonstrated with mTN-201. As shown below, 1×1013 vg/kg, 3×1013 vg/kg and 1×1014 vg/kg weight-based doses all produced significant improvements in EF, LV hypertrophy, and measures of electrophysiological function (QT interval) at eight months post-injection in the Mybpc3 KO HCM mouse model. The 1×1013 vg/kg dose had the lowest levels of efficacy, while the 3×1013 vg/kg had high improvement in the EF, similar to the 1×1014 vg/kg dose, suggesting a plateau in the dose-response curve. A similar dose response has also been observed with TN-201 in the Mybpc3 KO mouse model.

In our preclinical studies with the Mybpc3 KO model, we have not observed MyBP-C protein levels substantially above normal levels, suggesting that protein accumulation does not occur and lowers the potential concern of overexpression-related toxicities. In addition, histological assessments of mTN-201 treated Mybpc3 KO model murine hearts support the uniform and robust distribution of expression following mTN-201 infusion, suggesting gene therapy may be able to replace the missing MYBPC3 gene uniformly across the heart. This observation is consistent with heart biopsy samples from patients treated with other AAV9-based gene therapies in development.

The figure below demonstrates a restoration of MyBP-C protein levels to wildtype levels within two weeks following a single dose of mTN-201 at the 3×1013 vg/kg and 1×1014 vg/kg dose levels.

Consistent with observed therapeutic benefit, treatment of the Mybpc3 KO mice with mTN-201 is also associated with a substantial reduction of expression of genes associated with fibrosis and B-type natriuretic peptide (BNP), a circulating factor associated with cardiac wall stress.

The figures below show dose-dependent inhibition of expression of genes associated with cardiac strain (Nppa, Nppb, and Myh7) and fibrosis (Col1a1, Col4a1, and Postn) following a single dose of mTN-201 at the 1×1013 vg/kg and 3×1013 vg/kg dose levels.

Treatment with either TN-201 or mTN-201 in the Mybpc3 KO model was not associated with significant BW differences, clinical observations, or differences in histopathological assessments across dose levels. In addition, no impact on BW was observed at dose levels between 3×1013 vg/kg and 6×1014 vg/kg in pilot safety studies in wildtype neonatal mice twelve weeks after dosing.

Differentiating Characteristics for TN-201

During optimization of our MYBPC3 gene therapies, we discovered a cardiomyocyte-specific promoter, TNP-CM1, with improved performance attributes as compared to the standard cardiac troponin T (cTnT) promoter. In vitro and in vivo analyses confirmed that TNP-CM1 significantly increased expression of the MYBPC3 gene compared to what can be achieved with the standard cTnT promoter. See “Business—Our Core Capabilities—3. Promoters and Regulatory Elements.” TNP-CM1 has been tested in a human iPSC-CM disease model, in multiple murine models, and in non-human primates (NHPs). As demonstrated below, our proprietary cassette significantly improved heart function in our Mybpc3 KO mouse model in comparison to a published construct containing a standard cTnT promoter and utilizing the same AAV capsid. These data are also significant as the Mybpc3 KO models were treated at three months of age (rather than two weeks) suggesting that it is possible to reverse cardiac dysfunction even after significant onset of disease.

TN-301: HDAC6 Inhibitor Program for HFpEF

We are developing an HDAC6 small molecule inhibitor for the potential treatment of HFpEF. HFpEF is one of the greatest areas of unmet need in heart disease with more than three million patients in the U.S. and currently no approved disease-modifying therapies.A complex syndrome, the causes of HFpEF are diverse, but result in a shared pathophysiology with systemic inflammation and metabolic dysfunction leading to hypertrophy, fibrosis, and diastolic dysfunction among other characteristic consequences. The result is high morbidity and mortality in affected individuals. Our product candidate, TN-301, is a differentiated compound with unique chemical structures and high specificity for HDAC6 and based on publicly available information to date, we believe, is the first HDAC6i being developed for HFpEF.

Overview of HFpEF

HFpEF is generally defined as heart failure with an EF greater than or equal to 50%. In patients with HFpEF, the LV is stiffened and does not adequately relax, and increased pressure is needed for the ventricle to properly fill. As a result, blood begins to build up inside the left atrium of the heart and eventually swells into the lungs, veins and tissues of the body. HFpEF is a progressive disease in many patients. Symptoms initially include fatigue, shortness of breath, and tissue swelling, resulting in reduced physical activity. Over time, this results in a substantial limitation in activities and impact on quality of life, and patients are at risk of premature death.

Patients with HFpEF represent approximately half of heart failure patients, with prevalence of the disease anticipated to increase by more than 45% by 2030. The increase in HFpEF prevalence is at least in part due to the high overlap of this condition with diabetes and obesity which are also on the rise in the U.S. and globally. At least half of all hospital admissions for heart failure are related to HFpEF and approximately 24% of the HFpEF

population is considered to have New York Heart Association Class III or Class IV disease, representing a disease burden that markedly impacts quality of life and limits physical activity. Among patients hospitalized for HFpEF, readmission for heart failure and mortality rates over a five-year period are as high as 40% and 75%, respectively.

Despite limited data demonstrating efficacy in the HFpEF setting, patients generally receive therapies prescribed for HFrEF, including diuretics, beta-blockers, and ACE inhibitors. Patients with HFpEF are generally not responsive to therapies that have been shown to improve outcomes of patients with HFrEF. Without the development of more effective therapies specifically for HFpEF patients, disease management is mostly directed toward treating associated conditions and symptoms. Recently, a class of glucose lowering drugs known as sodium-glucose cotransporter-2 (SGLT2) inhibitors have demonstrated encouraging evidence of reducing hospitalizations and mortality versus placebo in HFpEF patients, with one such agent approved by the FDA for the treatment of HFpEF. In spite of this recent progress, HFpEF remains one of the greatest unmet needs in cardiovascular medicine.

Our Solution

We are developing TN-301, a highly specific small molecule inhibitor of HDAC6 for the potential treatment of HFpEF. HDAC6 is localized to the cell cytoplasm where it interacts with multiple proteins to coordinate cellular processes. In animal models intended to mimic human HFpEF, our highly selective HDAC6 inhibitors reversed preexisting cardiac hypertrophy and diastolic dysfunction, and improved lung congestion and exercise capacity, all of which are hallmarks of HFpEF.

TN-301 and our related HDAC6 inhibitors were discovered using our distinct Precision Medicine targeted drug discovery platform technologies, involving phenotypic screening and deep learning to human iPSCs. In vitro, our HDAC6 inhibitors demonstrated up to 2500-fold preferential selectivity for HDAC6, reduced sarcomeric damage and enhanced cardiac energetics. In in vivo studies in multiple mouse models of HFpEF, TN-301 and TYA-018, a structurally and functionally equivalent compound used for preclinical testing, demonstrated reductions in inflammation and metabolic dysfunction, as well as decreased fibrosis, hypertrophy and diastolic dysfunction. In a comparison study with the SGLT2 inhibitor empagliflozin, which is approved by the FDA in 2022 for the treatment of HFpEF, both TN-301 and TYA-018 improved glucose tolerance, reduced LV mass and end diastolic pressure and increased diastolic function with comparable efficacy, providing validation for our proprietary HFpEF mouse model and suggesting that preclinical results may translate to the clinic.

TN-301 Clinical Development Plan

We are conducting a randomized (3:1), double-blind, placebo-controlled Phase 1 clinical trial designed to assess the safety and tolerability of escalating oral doses of TN-301 in healthy adult participants. Secondary objectives of the clinical trial will be to assess PK and PD measures. The trial is being conducted in two stages. In the first stage, participants receive single ascending doses of either TN-301 or placebo and based on data from the SAD stage of the trial, including PD evidence of target engagement, participants in the second stage receive multiple ascending doses of TN-301 at dose levels of interest to help guide dosing in future trials. Dosing in the SAD stage of the trial began in September 2022. To date, TN-301 has been generally well tolerated. Initial target engagement (as measured by the PD biomarker of tubulin acetylation) was achieved at dose levels thought to be in therapeutic ranges, enabling the initiation of the MAD stage of the clinical trial, which commenced in February 2023. Data from both the SAD and MAD stages of the trial are anticipated in the second half of 2023.

Preclinical Evidence Supporting TN-301 Clinical Development

Treatment with TN-301 has reversed measures of HFpEF, including heart filling defects known as diastolic dysfunction, in multiple animal models. In one HFpEF model developed in-house, we surgically applied moderate aortic banding (mTAC) in wild type mice fed a high fat diet for eight weeks. These interventions induced a cardio-metabolic heart failure phenotype that simulated the systemic and cardiovascular features of HFpEF in humans. Aspects of the HFpEF phenotype included increased LV wall thickness, LV hypertrophy, increased diastolic pressure, impaired LV relaxation and filling, and glucose intolerance, while maintaining EF at or above 50%.

After the HFpEF phenotypes were established, animals were dosed orally with TN-301 or vehicle for six weeks. As illustrated below, TN-301 treatment reversed HFpEF disease phenotype across all studied parameters, including restoration of LV wall thickness, LV end diastolic pressure, LV relaxation and filling, and LV mass,

compared to control. In addition, as shown below, the treated mice exhibited a clear trend of decreased lung weight, indicative of improvement in pulmonary congestion consistent with the reduction of filling pressure.

In addition, as illustrated below, in multiple studies in HFpEF models, we have also observed an improvement in glucose tolerance suggesting that treatment with a selective HDAC6i may have a positive impact on glucose metabolism.

Consistent with the observed improvement in HFpEF phenotype, TN-301 treatment in this HFpEF model was also associated with reductions of key biomarkers of fibrosis, hypertrophy and cardiac damage, and inflammation in heart samples compared to levels observed in control animals, as shown in the figure below:

Comparison with SGLT2 Inhibitors

In order to validate our proprietary murine model of HFpEF we evaluated the efficacy of TYA-018 in comparison with empagliflozin, an SGLT2 inhibitor approved by the FDA for the treatment of HFpEF. HFpEF was induced with a combination of transaortic constriction and DIO to simulate a HFpEF phenotype. Maximally efficacious doses of TYA-018, empagliflozin or vehicle were administered for nine-weeks and compared.

In this model, empagliflozin behaved as anticipated based on the data generated from large clinical trials: fasting glucose and glucose tolerance were improved, and LV hypertrophy and diastolic dysfunction were reduced. TYA-018 demonstrated comparable benefits across multiple measures, including improvements in glucose tolerance, LV mass, LV end diastolic function and diastolic pressure.

TYA-018 also demonstrated superiority in improving markers of cardiac stress, reducing inflammatory markers and improving mitochondrial energy.

Taken together, data from our preclinical comparison studies elucidate the differentiated mechanism of action for HDAC6 inhibition and support the potential of our HDAC6 inhibition preclinical data to translate to the clinic.

PD Biomarker: Measurement of Target Engagement

HDAC6 is a cytoplasmic enzyme and one of its main substrates is tubulin. Increase in acetylated tubulin is a robust and reproducible PD marker with a high dynamic range that can be measured in both the heart and in circulating cells. We have developed an assay suitable for testing PD effect in human peripheral blood mononuclear cells that we intend to use to demonstrate proof-of-activity and target engagement in our clinical trials. The figure below illustrates dose-dependent increases in tubulin acetylation levels in the heart of a mouse model following administration of TN-301 (previously referred to as TYA-11631) (left axis), and how tubulin acetylation levels appear to correspond to levels of TN-301 as measured in plasma over time.

As part of our efforts to validate tubulin acetylation as a biomarker for target engagement, we undertook a series of experiments testing the effects of standard-of-care heart failure medication on tubulin acetylation and found that there was no impact, meaning it is a unique PD marker for TN-301.The figure below illustrates dose-dependent increases in tubulin acetylation levels in human peripheral blood mononuclear cells, illustrating how this PD biomarker can also be measured in human blood, including in healthy volunteers. This PD assay format is the same as what is being used for testing clinical samples from the TN-301 Phase 1 clinical trial.

Multi-Modal Mechanism of Action in HFpEF

The pathophysiological mechanisms underlying HFpEF is an active area of scientific research. Key aspects of HFpEF disease biology include oxidative stress and inflammation, cardiac fibrosis, cardiac hypertrophy, cardiac stiffness, which all result in diastolic dysfunction, and decreased ability of the heart to fill its chambers during contraction. Defects in glucose tolerance and insulin sensitivity and overall defective metabolism have also been proposed to play a role in HFpEF onset and progression due to high overlap in the HFpEF population with diabetes and obesity as comorbidities.

HDAC6 has been generally associated with several of these potential HFpEF mechanisms. Our preclinical data generated to date is consistent with what is known in the published literature and is suggestive of a multi-modal mechanism of action that may address multiple aspects of HFpEF disease. The schematic below shows a conceptual model of HFpEF disease biology highlighting key aspects (the yellow boxes in the figure below) for which there are external and internal data supporting the potential utility of HDAC6 inhibitors, including TN-301, in HFpEF.

Inflammation / Oxidative stress: In our preclinical studies, TN-301 has shown improvement in inflammatory markers in adipose tissue from a diet-induced obesity (DIO) model, while TYA-018 has shown improvement in inflammatory markers in a BAG3 model of DCM. Published studies have linked inhibition of HDAC6 with inflammasome biology and enhancement of regulatory T cell activity.

Defective metabolism / glucose metabolism: In our preclinical studies, TN-301 has shown improvement in glucose tolerance in a HFpEF model; dose-dependent improvements in glucose tolerance and insulin resistance in a DIO mouse model; and improvement in glucose uptake in iPSC-CMs. TYA-018 has also shown improvement in dysregulated metabolic pathways in a BAG3 model of DCM. In a published

study, HDAC6 KO mice had a significant improvement in dexamethasone-induced whole-body glucose intolerance and insulin resistance compared to wildtype mice, suggesting that HDAC6 may be an important regulator of gluconeogenesis and glucose metabolism.

Fibrosis: In our preclinical studies, TN-301 significantly improved markers of cardiac fibrosis in a HFpEF model. Published studies support our findings, having shown HDAC6 inhibition by siRNA or partially selective inhibitors attenuated myofibroblast markers and HDAC6 knockdown has been demonstrated to inhibit cardiac fibroblast proliferation.

Hypertrophy: In our preclinical studies, TN-301 has also shown improved in LV hypertrophy in multiple HFpEF models. In published studies, HDAC inhibitors prevented cardiac hypertrophy in animal models in response to various hypertrophic stimuli, and HDAC inhibition suppressed cardiac hypertrophy and fibrosis in a model of hypertension through regulation of HDAC6/HDAC8 enzyme activity.

Impaired autophagy: In our preclinical studies, TYA-018, has shown improvement in autophagy in a BAG3 model of DCM that was correlated with improvement in heart function. Published studies illustrate the role of reduced autophagy in HFpEF and in aging hearts.

Diastolic dysfunction: TN-301 has also shown improved diastolic dysfunction in multiple preclinical HFpEF models. Published studies have shown pan-HDAC inhibitors improved diastolic dysfunction in two distinct murine models of HFpEF and HDAC inhibition improved cardiopulmonary function in a feline model of diastolic dysfunction.

Potential Indications Beyond HFpEF

Data from preclinical studies evaluating TN-301 as a treatment for HFpEF suggest that there may also be a role for TN-301 in the treatment of sub-populations of HFpEF patients with obesity, diabetes or metabolic syndrome as well as potentially in sub-populations of DCM where there is strong alignment between the multi-modal mechanism of action of TN-301 with the pathophysiology of the disease.

TN-301: Preclinical Studies in Models of Metabolic Disease

In addition to improvements in glucose metabolism associated with TN-301 treatment in HFpEF mouse models, treatment with TN-301 has also led to improvements in glucose tolerance and insulin sensitivity in a DIO mouse model. As shown below, treatment with a single dose of TN-301 improves glucose tolerance in a dose-dependent manner in the DIO model. Furthermore, TN-301 treatment improves glucose tolerance in a dose-dependent manner after daily dosing for two weeks and insulin sensitivity in a dose-dependent manner after daily dosing for four weeks.

A single dose treatment of TN-301 in the DIO model is also associated with a significant reduction in inflammatory markers in adipose tissue relative to controls as shown below. Inflammatory biomarkers in adipose tissue are thought to be linked to glucose tolerance and insulin sensitivity. For example, adipose IL-6 deficiency has been associated with improvements in glucose tolerance. Loss of IL-10 has also been shown to protect mice from DIO and improve glucose tolerance and insulin sensitivity. Collectively, these data are supportive of a role for HDAC6 inhibition on glucose tolerance and insulin resistance with potential applicability to sub-populations of HFpEF patients with obesity, diabetes, or metabolic syndrome.

HDAC6 Inhibitors: Preclinical Studies in DCM

Through our target identification Precision Medicine platform, HDAC6 was initially identified as a target for a genetically defined subset of DCM, BAG3 mutant DCM. We screened a large chemical library to identify compounds able to reverse sarcomere defects in BAG3-deficient human iPSC-CMs. Sarcomere defects were rapidly and systemically assessed through our proprietary machine learning algorithms. Whereas a pan-HDAC inhibitor was identified in the initial compound screen as reversing sarcomere defects, we conducted follow-up screens using RNAi knockdowns of HDAC family members to identify HDAC6 as a potential therapeutic target in vitro.

We have validated these in vitro findings by testing our HDAC6i compounds in BAG3 mutant mice models. As shown in the figure below, treatment of a rapidly worsening mouse model of BAG3 mutant DCM with TYA-018 resulted in a greater than 20% improvement in EF after eight weeks of treatment compared to a control group treated with vehicle.

TN-401: Gene Therapy for PKP2-associated ARVC

PKP2 gene mutations are estimated to affect more than 70,000 patients in the U.S. These mutations can cause enlargement of the RV in affected individuals, replacement of heart muscle with fibrotic tissue and fatty deposits, and severely abnormal heart rhythms (arrhythmia) that can make it harder for the heart to function properly and result in sudden cardiac death in some adults and children. Based on publicly available information to date, we believe there are currently no approved treatments that address the underlying genetic cause of this disease. We are developing TN-401, a potential first-in-class AAV-based gene therapy designed to address ARVC caused by PKP2 gene mutations. We have demonstrated prevention of disease progression, reversal of RV remodeling and survival

benefit in a murine model after a single dose. We expect to submit an IND to the FDA in the second half of 2023, and those efforts are being aided by our learnings from the TN-201 IND filing experience.

Overview of ARVC

ARVC is largely an inherited disease characterized by the progressive loss of muscle cells in the heart’s RV and replacement with a composite of fibrotic tissue and fatty deposits. As a result of this structural change, the heart becomes dilated and is prone to VA and particularly ventricular tachycardia (abnormally high heart rate).

Patients with ARVC most commonly present with symptoms related to VA (such as palpitations, lightheadedness, and fainting) or cardiac arrest, with the mean age of diagnosis in patients occurring before the age of 40. ARVC is an important cause of sudden cardiac arrest in young patients, and particularly in athletes. The median age at cardiac arrest in ARVC patients is 25 years old. In an effort to reduce the risk of sudden cardiac death, patients with ARVC are typically discouraged from competitive or endurance sports activities and physical exercise may be limited.

ARVC has an estimated prevalence in the general population of approximately 1:2000. Mutations in the PKP2 gene are the most common genetic cause of ARVC, with approximately 41% to 46% of ARVC patients carrying pathogenic variants. We therefore estimate more than 70,000 patients in the U.S. are affected by PKP2 mutations. Mutations of the PKP2 gene are inherited in an autosomal dominant fashion, i.e., a mutation in one gene is sufficient to cause the disease. Over 14 mutations have been linked to the PKP2 gene. Most of these mutations are predicted to result in a truncated protein product, which suggests a disease mechanism due to loss of function, resulting in haploinsufficiency.

As illustrated below, the PKP2 protein is an integral component of cell adhesion protein complexes known as desmosomes which connect adjacent cardiomyocytes in the heart. Desmosomes are responsible for stabilizing the heart and for maintaining channels called gap junctions that allow for cellular communication among heart cells, which in turn is important to proper synchronization of cardiomyocyte contractions across the myocardium contributing to each heartbeat.

The figure below(2) analyzes heart tissue from an ARVC patient with the PKP2 mutation and compares it to the heart tissue from a normal individual. The tissue has been stained for desmosome proteins PKP2 and plakoglobin as well as other transmembrane proteins that are not part of the desmosome, but that are also present at cell-cell junctions in different body organs (e.g., N-cadherin). As illustrated, N-cadherin, PKP2, and plakoglobin are all correctly localized to the junctions between cardiomyocytes in the healthy control sample. However, when the PKP2 gene is mutated, N-cadherin continues to correctly localize, but both the PKP2 and plakoglobin proteins are no longer properly localized to the desmosome. As a result of this impairment, cardiomyocytes can become detached from each other when placed under the normal mechanical stress of the beating heart, or under the extra mechanical stress in the heart caused by athletic activity. This detachment causes cell death, which in turn causes inflammation, scar formation, and fat deposition, illustrating the crucial role the PKP2 protein plays in maintaining the structural integrity of the desmosome, and that mutations in the PKP2 gene are enough to disrupt this complex in human hearts.

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Source: Asimaki et. al. NEJM 2009.

An example of a heart from a patient who had ARVC is shown below(3). This illustrates commonly seen abnormalities in ARVC hearts as a result of the improper function of the desmosome, including dilation (enlargement) of the RV chamber and replacement of healthy heart tissue by fibrotic tissue and fatty deposits.

Following a diagnosis, ARVC patients are typically implanted with an ICD to control arrhythmias and prevent sudden cardiac death. ICD implantation is currently the only proven effective treatment for preventing sudden cardiac death in ARVC patients, but ICDs are also associated with complications, including inappropriate shocks, potential for heart perforation and need for additional surgery. Patients may progress to catheter ablation procedures which have a high rate of recurrence of VA and have not been shown to reduce risk of sudden cardiac death or improve survival. Pharmacologic options for ARVC treatment typically include beta blockers and other anti-arrhythmic or heart failure medications, intended to reduce VAs, but studies comparing the efficacy of such treatment have not been conducted. Despite the availability of these treatments, clinical heart failure has been documented in up to 40% of ARVC patients, and when heart transplantation is required, transplants occur at an average age of 40 and within seven years of the onset of heart failure symptoms. There are currently no approved therapies that address the underlying genetic causes of ARVC.

Our Solution

We are developing a potential first-in-class AAV-based gene therapy to deliver a fully functional copy of the human PKP2 gene to the hearts of ARVC patients carrying PKP2 mutations. We believe that delivery of a working PKP2 gene to cardiomyocytes represents a promising treatment that can address the underlying cause of this disease. As the disease is most often caused by haploinsufficiency, expression of a functional PKP2 gene to replace the missing PKP2 protein in cardiomyocytes is expected to restore proper structure and function of the desmosome. This in turn can help prevent adverse heart remodeling and improve heart contraction and electrical function. The PKP2

gene will be delivered using AAV9 capsid with well-established tropism for the heart and expression of the PKP2 protein will be targeted to the heart through use of a cardiomyocyte-specific promoter.

(3)

Source: Pinamonti et. al World J Cardiol 2014.

TN-401 Preclinical Studies

We developed a Pkp2 conditional knockout (Pkp2-cKO) mouse model that simulates key aspects of ARVC including dilation of the RV, decline in LV heart function, severe arrhythmia, abnormal ECG trace, and early mortality. The onset of symptoms in this model is very rapid and occurs within three weeks after induction of the gene deletion. It is worth noting that this Pkp2-cKO model is homozygous, i.e., both copies of the gene are missing and so there is no production of the PKP2 protein. As expected, the severity of disease and the rate of disease progression in this mouse model are both greater than what is normally observed in most PKP2 patients who are almost all heterozygous for PKP2 gene mutations, i.e., they have one normal, healthy copy of the gene that is producing at least some of the necessary PKP2 protein, plus one defective copy of the gene that is either producing no PKP2 protein at all or that is producing PKP2 protein that does not function properly in the desmosome. The Pkp2-cKO model is nonetheless useful as it provides important proof of concept for the potentially beneficial in vivo effect of the PKP2 protein replacement via a gene therapy approach.

In preclinical studies, we systemically administered a mouse surrogate of TN-401 (AAV:mPkp2 or mTN-401) in Pkp2-cKO mice at the 5×1013 vg/kg and 1×1014 vg/kg dose levels. Data from these studies showed that whether mTN-401 gene therapy was administered at the time of disease onset or following disease progression, several ARVC phenotypes improved compared to saline-treated controls (HBSS), including preventing RV enlargement, preventing decline of LV function, and improving survival after a single IV dose. Further, mTN-401’s beneficial effects have been shown to be durable following a single dose lasting the remainder of the Pkp2-cKO mice model’s natural life span.

We are also encouraged by the preclinical safety profile and dose-responses observed in our studies of Pkp2-cKO mice following treatment with various doses of TN-401. In these studies, we observed dose-dependent efficacy against disease attributes, including prevention of LV functional decline, with relatively low doses achieving near maximal efficacy. Single doses of TN-401 administered in Pkp2-cKO mice at the 1×1013 vg/kg, 3×1013 vg/kg and 1×1014 vg/kg dose levels were shown to achieve robust protein expression of PKP2 transgene and desmoplakin (DSP). DSP is one of the five protein components of desmosome in addition to PKP2 and its expression in response to TN-401 is an indicator of improved desmosome integrity following PKP2 protein replacement. The specificity of TN-401 for the heart and its therapeutic index have also proven to be promising in our preclinical studies with low tropism for other organs even at doses greater than ten-fold higher than those with near maximal efficacy. Taken

together, we believe these observations position TN-401 well for successful clinical development, with an encouraging safety and efficacy profile.

PD Biomarker: Preventing Ventricular Arrhythmias

PKP2 gene therapy has been shown preclinically to correct the hallmark electrophysiological defects associated with ARVC. The graphs below show nearly complete prevention of arrhythmia in Pkp2-cKO animals treated with mTN-401 versus controls, including prevention of nonsustained ventricular tachycardia (NSVT) and premature ventricular contractions (PVCs). NSVTs and PVCs were reduced to near healthy levels as evidenced by

the ECG trace and the quantification with a Ventricular Arrythmia Score measuring the incidence of spontaneous arrhythmias during 30 minutes of recording.

The graphs below show normalization of the QRS complex in Pkp2-cKO animals treated with mTN-401 versus controls, including prevention of QT elongation (as measured by QT interval) and abnormal P wave and R wave amplitudes (as measured by the P/R ratio).

Based on the preclinical evidence that PKP2 gene therapy is able to correct the arrhythmias associated with ARVC, we believe this measurement may provide a meaningful early PD biomarker for monitoring TN-401’s activity in the clinic.

TN-401 Planned Clinical Development

We have initiated IND-enabling activities and plan to submit an IND to the FDA for TN-401 in the second half of 2023. We intend to use our experience from the TN-201 IND filing and to seek feedback from multiple regulatory agencies, including the FDA, as necessary. If our IND is approved, we plan to initiate global FIH clinical trials in patients with mutations of the PKP2 gene. Additionally, in support of our development efforts for TN-401, we have initiated a global non-interventional study to collect treatment history and seroprevalence to AAV9 antibodies data among ARVC patients who carry pathogenic or likely pathogenic PKP2 gene mutations.

DWORF Program for DCM

We are developing an AAV-based gene therapy designed to deliver the DWORF gene for patients with DCM.

Dilated cardiomyopathies are estimated to affect about one million patients in the U.S. DCM is a progressive and life-threatening disease that causes enlargement and wall thinning of the LV, insufficient contraction, reduced blood flow, VA, and can result in premature morbidity and need for heart transplant in affected individuals. DWORF is a muscle-specific micro-peptide that acts on the SERCA2a pathway, widely considered to be a promising target in heart failure. We and our academic collaborators have accumulated significant preclinical proof-of-concept evidence for the therapeutic benefit and tolerability of over-expression of the DWORF gene in multiple murine models, including models of DCM. Based on publicly available information to date, we believe these are the first demonstrations of the potential benefit of AAV:DWORF. This program is currently at the candidate selection stage.

Overview of DCM

DCM is broadly defined as heart failure where the EF is below 40% and the walls of the LV are thin and over-expanded, leading to insufficient contraction, reduced blood flow pumped by the heart, and abnormal heart rhythms. DCM can be caused by a variety of mechanisms, including genetics, CAD, high blood pressure, heart attack, and viral infection.

DCM is a life-threatening and progressive disease. Once symptoms appear, a patient’s condition typically declines progressively. Typical symptoms of heart failure due to DCM include shortness of breath, fatigue, swelling in the extremities, or an irregular heartbeat. As the disease progresses, patients become increasingly debilitated and experience sustained shortness of breath, even at rest. Diastolic function, or the heart’s ability to relax and fill with blood, is also impaired because the heart is already expanded and fibrotic. The dilated LV is deprived of an adequate supply of oxygen that may contribute to further fibrosis and the risk of dangerous heart rhythm disturbances. At any stage of the disease, whether or not symptoms have appeared, DCM patients are at risk of sudden cardiac death.

It is estimated that DCM affects about one million people in the U.S., with genetic abnormalities linked to DCM estimated to be present in about 30% to 40% of DCM patients.

A subset of DCM is caused by genetic mutations in proteins involved in muscle contraction. Mutations in one such protein, phospholamban (PLN), can cause DCM. These mutations are believed to result in abnormal regulation of calcium biology instrumental in muscle contraction, leading to ventricular dilation, fibrosis and heart failure over time. Some patients with PLN mutations have a high severity of disease, including patients with R9C and R14del mutations. PLN mutations are rare with an estimated 0.5% of DCM patients carrying PLN mutations. Notwithstanding, founder effects in certain communities have led to higher concentrations of affected individuals in specific regions in the world, enabling patient identification.

DCM treatment generally utilizes medications developed and approved for HFrEF. While current pharmacologic therapies have improved prognosis and the quality of life of DCM patients, the premature morbidity and mortality rate remains unacceptably high. End-stage DCM is the leading indication for use of last line therapies, including LVADs and heart transplantation. Within five years of diagnosis, 43% of patients with advanced DCM have either died or needed a heart transplant. Thus, there is a large unmet need for targeted, disease-modifying options.

Our Solution

We have licensed intellectual property from University of Texas Southwestern Medical Center (UTSW) to develop and commercialize products relating to therapeutics overexpressing DWORF and are developing an AAV-based gene therapy to deliver the DWORF gene to cardiomyocytes for the treatment of DCM. DWORF is a recently discovered small peptide that localizes primarily to the sarcoplasmic reticulum of the cardiac muscle cell. During muscle cell activation, calcium is released from sarcoplasmic reticulum into the muscle cell’s cytosol and into the sarcomere, leading to muscle contraction. Sarcoplasmic/endoplasmic reticulum Ca2+ ATPase 2a (SERCA2a) is a major isoform of SERCA expressed in cardiomyocytes and plays an essential role in the regulation of cardiac contractility. SERCA2a transports calcium from the cytosol back into the sarcoplasmic reticulum, preserving the calcium gradient required for contraction. DWORF binds to SERCA2a and displaces the inhibitory PLN peptide, resulting in increased SERCA2a activity, increased levels of calcium pumped into the sarcoplasmic reticulum, and increased muscle contraction, ultimately leading to an improvement in heart function.

We believe DWORF is an ideal target for the treatment of particular forms of heart failure. DWORF is a small peptide that is readily expressed when delivered by AAV. The small size of the DWORF gene leaves additional room in the AAV capsid to include optimized combinations of promoters and regulatory elements to tailor DWORF gene expression levels. In addition, published studies have shown that DWORF gene expression is lower in failing human hearts compared to non-diseased hearts.

The figure below shows expression analyses in human heart failure tissue. DWORF mRNA is reduced in failing hearts whereas atrial natriuretic peptide (NPPA) mRNA, a marker of congestive heart failure, is significantly increased in failing hearts.

One therapeutic hypothesis is that restoring DWORF gene expression to normal levels, through treatment with a DWORF gene therapy, may normalize calcium flux in cardiomyocytes and increase contractile strength in DCM patients, as well as the broader HFrEF patient population. In addition, in patients with DCM caused by specific PLN mutations, the mutant PLN peptides are excessively inhibitory to SERCA2a, thereby reducing cardiac contractility. DWORF gene therapy produces DWORF peptides that directly compete with mutant PLN peptides by preferentially binding with SERCA2a, which can increase muscle contraction, potentially halting or even reversing disease progression.

Our DWORF program, illustrated below, is currently at the candidate selection stage with multiple constructs under consideration. DWORF gene expression is limited to the cardiomyocyte through use of a novel cardiomyocyte-specific promoter. Our intended product candidate will use an AAV capsid with high tropism for the heart, either AAV9 or a novel proprietary capsid developed through our capsid engineering capabilities, to deliver the DWORF gene. We are exploring different ROAs including systemic (IV) or delivery directly to the heart through an infusion catheter.

Preclinical Studies

Results in DCM (with Muscle Lim Protein (MLP) KO model): AAV:DWORF constructs have shown improvements in heart remodeling following treatment in mouse models of DCM. As shown below, we have developed multiple proprietary promoters that drive multiple different levels of expression. As shown below, AAV:DWORF constructs containing these promoters (TNP-CM2, TNP-CM4, and TNP-CM7) improved EF relative

to a saline control in the MLP KO mouse model of DCM, with improvements in EF as high as approximately 14% achieved with constructs containing the TNP-CM4 promoter. The increase in heart function was durable throughout the duration of this 24-week study:

AAV9:DWORF also improved exercise capacity, including running distance and time to exhaustion, in the MLP KO DCM mouse model 26 weeks post-treatment.

We have tested different AAV:DWORF constructs in both healthy and disease mouse models and have not observed any safety signals at clinically relevant levels of DWORF overexpression.

Planned Clinical Development and Potential Indications Beyond DCM

After selection of our product candidate, we plan to initiate IND-enabling studies. Should an IND for this program be cleared by the FDA, during clinical development, we plan to examine the role of AAV:DWORF in DCM, as well as potentially in sub-populations of HFrEF where there is alignment between AAV:DWORF with the pathophysiology of the disease. Approximately 50% of heart failure cases are HFrEF, representing a prevalence of nearly four million patients in the U.S. In addition, the incidence and prevalence of HFrEF continues to rise. This increase is driven by an aging population, improved survival from MI and other forms of heart disease, and the increasing prevalence of predisposing risk factors such as diabetes and obesity. HFrEF patients continue to have substantial unmet need despite advances in pharmacological treatments.

Reprogramming Program for Heart Failure due to Prior MI

We are developing an AAV-based approach to cellular regeneration that involves converting (or reprogramming) existing cardiac fibroblasts within the heart to turn into new cardiomyocytes and to replace cells permanently lost due to MI. There are estimated to be more than four million patients in the U.S. living with heart failure due to prior MI. The loss of cardiomyocytes in affected individuals permanently impairs heart contraction, leading to heart failure and potentially fatal arrhythmias, and the death of approximately 5% to 10% of MI survivors within the first year. There are currently no approved treatments that address the underlying loss of heart tissue. The potential utility of our unique approach to creating new cardiomyocytes was first demonstrated by our co-founder Deepak Srivastava, M.D. We have discovered a proprietary combination of three genes that can drive robust in vivo

reprogramming of cardiac fibroblasts to cardiomyocytes when delivered together in a single AAV capsid. Based on publicly available information to date, we believe our results in a pig model of heart failure due to prior MI represent the first-ever successful demonstration of the potential benefit of this approach in a human-sized heart. This program is currently at the candidate selection stage.

Overview of heart failure due to prior MI

CAD is the single most common cause of heart failure and is often associated with an MI, in which blood flow to a section of the heart, usually the LV, becomes limited, causing the cells in that section of the heart, including cardiomyocytes and cardiac fibroblasts, to die. The heart cannot replace the lost cardiomyocytes while the cardiac fibroblasts multiply significantly, resulting in scar tissue formation and stiffening of the LV walls, leading to progressive and irreversible cardiovascular remodeling. As a result, the heart continues to lose its ability to pump as strongly and may fail over time. In addition to heart failure, these patients also have a persistent risk of arrhythmias and increased likelihood of a second heart attack or sudden death.

In the U.S., greater than 800,000 people have a heart attack every year; of these approximately 200,000 already had a prior heart attack. Approximately 20% of patients age 45 and older will have another heart attack within five years of their first one. Despite advances in treatment options, mortality due to heart attack is still high; data from the U.S. National Vital Statistics Reports shows the median life expectancy among individuals aged 65 to 69 who have had a heart attack is just 8.3 years as compared to 18.7 years among those who have not.

There are no known therapies that address the loss of cardiomyocytes associated with MI and the resulting morbidity and mortality.

Our Solution: Direct In Vivo Reprogramming of Resident Cardiac Fibroblasts to Create Cardiomyocytes

Cellular reprogramming is the process of converting cells of one type into another cell type. Shinya Yamanaka and John Gurdon won the Nobel Prize for their discovery that cells in the body can be reprogrammed to become stem cells, called iPSCs, capable of developing into any other type of cell in the body using a combination of four transcriptional factors. Since then, researchers have also found other combinations of factors capable of directly converting cells from one type to another without first going through the iPSC state. Dr. Srivastava, one of our co-founders and a member of our board of directors, was the first to demonstrate direct reprogramming of cardiac fibroblasts into cardiomyocytes in both in vitro and in vivo models, creating the potential for a new approach to cardiac regeneration.

Building on this pioneering work, we have developed a novel AAV-based therapy for direct in vivo reprogramming of resident cardiac fibroblasts into cardiomyocytes to replace the cardiomyocytes lost due to an MI. Our goal is to convert the cardiac fibroblasts into new cardiomyocytes to help repair the heart after an MI, and ultimately slow down, stabilize or even potentially reverse the progression to heart failure. Our approach leverages substantial in-house advances in our reprogramming factors, capsid engineering, regulatory elements, and drug delivery to translate cardiac reprogramming science towards clinically relevant solutions.

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Reprogramming factors. Through extensive in vitro screening efforts in actual human cardiac fibroblasts, we identified a unique combination of genes encoding Myocardin and ASCL1, that together, can drive robust direct in vivo reprogramming of cardiac fibroblasts to cardiomyocytes, and that we have designed to fit into a single AAV. We use the term reprogramming factors to refer to such combination of genes and any other combinations of genes that when delivered together in a single AAV into cardiac fibroblasts, result in the direct reprogramming of the cardiac fibroblasts into cardiomyocytes.

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Capsid engineering. While AAV9 can be used to target cardiomyocytes, it does not sufficiently transduce cardiac fibroblasts. We have discovered a novel capsid, TNC-CF1, which has a higher transduction efficiency for human cardiac fibroblasts as compared to currently known AAV serotypes. Initial data suggest this novel capsid may also be less susceptible to neutralizing antibodies compared to known serotypes.

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Regulatory elements. We have pursued rigorous, iterative optimization efforts to create proprietary reprogramming products. We have further optimized Myocardin and cassette regulatory elements to both decrease cassette size and improve reprogramming efficiency. After extensive exploration of single

and double promoter strategies, we have selected the CAG promoter to drive robust expression of our reprogramming factors. We limit expression of our reprogramming factors in mature cardiomyocytes by including a miR-208 binding site that decreases reprogramming factor expression in mature cardiomyocytes after differentiation from fibroblasts.

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Drug delivery. We are developing, in conjunction with leaders in interventional cardiology, a proprietary percutaneous endomyocardial injection catheter (TND-INJ1) to inject and deliver our gene therapies around scars in the heart in a non-surgical, minimally-invasive procedure. Many potential sites for future clinical studies have experience with endomyocardial injection catheters through previous and ongoing cell therapy studies.

The schematic below summarizes the components of our intended reprogramming gene therapy product candidate and mechanism of action.

Preclinical Studies

We have conducted in vitro and in vivo experimentsto optimize our direct reprogramming approach. Ourmost advanced results have been achieved primarily with two different constructs, TN1-002 and TN1-006. A summary of certain preclinical data supporting the Reprogramming program in general and TN1-002 in particular was presented at the ASGCT conference in 2020.

Results from in vitro conversion of human cardiac fibroblasts. Our reprogramming approach has been optimized in vitro in adult human cardiac fibroblasts. We have conducted extensive iterative experiments to compare the relative efficiency of various constructs to convert cardiac fibroblasts to cardiomyocytes, whereby cardiomyocyte-specific markers like cTnT and a-Actinin are measured to determine the proportion of cells that have been converted from cardiac fibroblasts to cardiomyocytes. Results from one such an experiment demonstrate that our TN1-006 construct can convert approximately 40% of human cardiac fibroblasts to cardiomyocytes.

Results from pig disease model. We have demonstrated durable improvement in EF in a pig model of heart failure following an induced MI. In a pig model, TN1-002 was injected directly around the scar area formed 28 days after an induced MI. The figure below demonstrates approximately 10% improvement in EF compared to each animal’s own pre-dose baseline and more than 11% improvement compared to control-treated animals that remained sustained until the end of the experiment at nine weeks:

We believe these data compare favorably to published efficacy data for other cell and gene therapy interventions in large animal models. Very few previous therapeutic attempts have achieved meaningful improvement in EF compared to pre-dose baseline in large animal models, with typical improvements, when observed, of less than 5%. From an assessment of the published literature, including a meta-analysis of multiple therapeutics in HFrEF, we believe that each 5% increase in EF is expected to reduce mortality by approximately 15%.

Our preclinical findings to date provide direction to our ongoing candidate selection efforts. Further analyses of effects in individual animals revealed a clear correlation between levels of TN1-002 vector, reprogramming factor expression, and degree of functional improvement. This provides additional support that the improvements in EF seen in this experiment were a direct result of the delivery and expression of the reprogramming factors by our AAV capsid. We continue to seek ways to ensure more consistent delivery and expression of our reprogramming factors to cardiac fibroblasts, including with the use of novel capsids and novel delivery methods.

Safety. To date, no negative safety findings have been associated with either TN1-002 or TN1-006 in in vivo experiments in rat and pig models, including clinical findings, histopathology, assessment of arrhythmia, and other measures.

Planned Clinical Development

We have received feedback from the FDA through an INTERACT (INitial Targeted Engagement for Regulatory Advice on CBER producTs) review to inform the design of our future preclinical studies. After selection of our product candidate, we plan to initiate IND-enabling studies.

Our development plan is anticipated to include patients with advanced heart failure due to prior MI who meet qualifications for a heart transplant or LVAD as well as a broader patient population with severe ischemic cardiomyopathy. In the future, we also may explore potential for development in other forms of heart failure caused by a loss of cardiomyocytes, but not involving a myocardial infarction.

Pipeline Expansion Opportunities

We believe the versatility of our three product platforms and our related differentiated capabilities enables us to rapidly expand our portfolio beyond the initial areas of focus. In addition to the named programs in our current pipeline, there are several programs emerging from each of our platforms that are intended to address rare genetic cardiomyopathies as well as more prevalent forms of heart disease. We continue to research, discover and evaluate new programs arising from our three product platforms. We also continue to explore opportunities to collaborate with leading academic and biopharmaceutical organizations with complementary science and capabilities that share our bold vision for the development of next-generation therapies to benefit individuals and families fighting heart disease.

Our Product Platforms

To unlock the full potential of novel therapies across many forms of heart disease, we are advancing science from three product platforms in parallel. Each platform is intended to address different problems that have historically plagued drug development in the field of cardiology: (i) our Gene Therapy platform to deliver a wide variety of therapeutic payloads more precisely to heart tissue, (ii) our Cellular Regeneration platform to replace heart cells lost to disease, and (iii) our Precision Medicine platform to discover targeted therapies in a modality-agnostic fashion. We are advancing programs from these distinct but interrelated product platforms that combine different science, capabilities, and intellectual property.We believe these three product platforms together yield better insights into disease processes, create more opportunities for successful drug development, mitigate scientific risk, and differentiate our efforts relative to competitors.

Gene Therapy Platform

Gene therapy focuses on repairing or replacing defective or mutated genes to produce a therapeutic effect or treat a disease. AAV is a non-enveloped virus that already exists in some humans and does not cause disease. In gene therapy, the viral DNA within an AAV is replaced with new DNA to become a precisely coded vector to deliver the engineered therapeutic to specific tissues or organs within the body.

AAV vectors are the subject of significant research and development as they can be leveraged as a gene delivery vehicle for a wide range of therapeutic payloads to a wide variety of human cells. AAV-mediated gene therapy has been shown to be highly effective in targeting multiple organs, including the eye, the liver and the central nervous system. These viruses have been used to dose more than 3,300 patients in approximately 150 clinical studies around the world, and there are now several therapies that use such viruses that have been approved by the FDA and other regulatory agencies.

Third-party clinical studies have demonstrated that AAV9 can effectively transduce the hearts of infants and adults. This supports the results of several published non-clinical studies using AAV9 in murine and NHP models. Overall, data suggest that AAV9 is a highly effective parental vector for the purpose of cardiac gene therapy where the target cells are cardiomyocytes (one of the most abundant cell types in the heart responsible for contraction). In addition, we are aware of over 2,300 patients worldwide that have been treated using Novartis Pharmaceutical’s Zolgensma (developed by AveXis), a therapy utilizing IV AAV9. Based on the totality of preclinical and clinical evidence, we have also chosen to use AAV9 to support our TN-201 and TN-401 programs.

However, AAV9 has limitations. AAV9 has a well-established ability to transduce the liver and the central nervous system, in addition to the heart, which may create safety considerations. Also, some individuals have neutralizing antibodies to AAV9, making them ineligible for AAV9-based treatments. Cardiac-specific promoters like cTnT, can help limit the expression of AAV-delivered genes to cardiomyocytes, but do not enable targeted gene expression in other heart cells (e.g. cardiac fibroblasts). Additionally, the level of gene expression from these promoters may not be sufficient for therapeutic effect for some targets.

Therefore, there is significant room for improvement, and we aim to improve gene therapy for the heart in ways that expand its utility. We believe our five core internal capabilities will allow us to identify, engineer, validate, deliver and manufacture novel AAV vectors to optimize the delivery and expression of therapies more selectively to cells of interest in the heart. With our capsid engineering capabilities, we have designed and screened more than one billion AAV variants to find novel capsids with higher tropism and transduction efficiency for different types of heart cells, lower transduction efficiency for the liver and other tissues, and lower susceptibility to neutralizing antibodies. We have discovered promoters and regulatory elements that enable more precise gene expression in specific heart cells. We are developing new catheters and are exploring different ROAs to more precisely deliver vectors to heart tissue. Additionally, we have established know-how to enable more optimal manufacturing, including of novel AAV capsids.

The product candidates arising from our Gene Therapy platform are intended to overcome the shortcomings of traditional pharmacological or surgical interventions that are not able to address the underlying genetic factors contributing to heart disease. We believe our proprietary capabilities open the opportunity to deliver novel gene therapies to patients with heart disease and position us to become a leader in cardiac gene therapy. We are leveraging these capabilities to develop gene therapies for rare, genetic forms of heart disease, as well as to enable the transition to more prevalent forms as well.

Cellular Regeneration Platform

Scientists have long known that the human heart is not able to regenerate itself, unlike many other organs in the body. Acute MI—more commonly referred to as a heart attack—can kill as many as 25% of cardiomyocytes from the LV, or approximately one billion cells. The heart has no natural way to replace cells that are lost slowly with age or suddenly due to disease. Acute MI is associated with a 30% mortality rate; about 50% of the deaths occur prior to arrival at the hospital. An additional 5% to 10% of survivors die within the first year after their MI. Approximately half of all patients with an MI are re-hospitalized within one year of their first MI. The loss of healthy functional cells is a contributing factor to other forms of heart disease as well. One reason that disease is so prevalent and a leading cause of death in the world is due to the lack of regenerative potential of the heart. Finding ways to replace lost heart cells is one of the “holy grails” of regenerative medicine.

There are two abundant cell types in the heart: cardiomyocytes, which are the cells that are responsible for contraction during each heartbeat, and cardiac fibroblasts, that produce and secrete growth factors, cytokines and other signaling molecules contributing to structural, biochemical, mechanical and electrical properties of the myocardium. While cardiac fibroblasts are able to divide and proliferate, cardiomyocytes are post-mitotic, meaning they are incapable of regenerating. cardiomyocytes that are lost due to aging or disease are replaced by fibrotic scar tissue that is permanent and irreparable.

The field of cardiac regeneration has historically been dominated by ex vivo cell therapy approaches using autologous (from self) or allogeneic (from donors) cell sources to replace lost cardiomyocytes. However, there have been no successful therapies after scores of clinical studies involving thousands of patients. Any modest efficacy seen in clinical studies are now often attributed to indirect paracrine effects rather than true cardiac regeneration. Some have tried to induce regeneration by infusion or injecting cells generated from hiPSC-CMs or human embryonic stem cells, but that has been fraught with many challenges, as these cells have an embryonic phenotype and generate arrhythmias once injected into the heart; recipients need to be immunosuppressed to avoid rejection; and integration into the electric and mechanical connections of the heart is still imperfect.

We are advancing a cardiac regeneration approach based on research conducted by our founders at Gladstone Institutes and UTSW, who pioneered the idea of restoring heart function after a heart attack by in vivo regeneration of lost cardiomyocytes. Our approach is intended to achieve this by using viral vectors to deliver a proprietary combination of three genes that when delivered together in a single AAV can permanently convert—or “reprogram”—a patient’s own resident cardiac fibroblasts into new cardiomyocytes.

This approach was inspired by the Nobel-prize winning discoveries of Shinya Yamanaka. He first discovered that human cells can be “reprogrammed” with certain specific factors—which became known as the “Yamanaka factors”—to become iPSCs, and that these newly formed iPSCs were in turn capable of differentiating to become any other human cell type in the body, including heart cells. Our founders and other academic labs built on this idea and demonstrated that it is possible to directly convert cardiac fibroblasts to cardiomyocytes without first going through the iPSC stage. Dr. Srivastava, one of our co-founders and a member of our board of directors, was the first to demonstrate proof of concept of this “direct reprogramming” approach for cardiac regeneration in vivo in a mouse model and in vitro with human cells. Several independent academic labs around the world have subsequently

replicated the results with direct reprogramming for cardiac regeneration using the same factors as well as new combinations.

The figure below helps illustrate the idea of direct reprogramming of cardiac fibroblasts to cardiomyocytes using the Waddington model for cellular differentiation:

There have been several historical challenges for the field of direct reprogramming for cardiac regeneration to turn this promising scientific discovery into potentially viable therapies. Most academic efforts required anywhere from three to five factors to achieve the conversion of human cardiac fibroblasts to cardiomyocytes, and the overall conversion rate was relatively low. Some of these efforts used a combination of retroviruses and small molecules to achieve this conversion, which is not clinically applicable. The published proof-of-concept work using viral gene therapy to deliver cardiac reprogramming factors has been demonstrated in murine models of acute MI (i.e. immediately at the time of onset of heart attack), but not in models of heart failure following MI (i.e. following some period of time after the heart attack has occurred) which more accurately simulates the situation that would be adopted in the clinical setting.

We believe we are the first to potentially overcome these challenges. We have discovered a proprietary combination of three genes that can be co-packaged and co-expressed from a single proprietary AAV vector engineered for higher transduction of cardiac fibroblasts when compared to existing parental capsids. We have demonstrated higher transdifferentiation rates in vitro using human cardiac fibroblasts that are higher than rates reported in published studies using combinations of other factors intended to drive reprogramming. We have demonstrated robust and durable proof-of-concept of this approach in multiple rodent models of acute MI and heart failure post-MI. Most importantly, based on publicly available information to date, we believe our results in a pig model of heart failure due to prior MI represent the first-ever successful demonstration of the potential therapeutic benefit of this approach in a human-sized heart.

We believe our in vivo approach to cardiac regeneration may have several advantages over ex vivo cell therapies. Because the newly formed cardiomyocytes are generated from the patients’ own cells, they are not rejected by the body and no immunosuppression is needed. Since these newly formed cardiomyocytes are generated from within the patient’s heart tissue, it may be easier for them to electrically and mechanically connect with surrounding cells as they mature and to contribute to healthy heart function with lower risk for arrhythmias. In addition, it is easier to manufacture and to deliver AAV-based therapies and to offer them at commercially viable prices compared to cell-based therapies.

The initial focus of our Cellular Regeneration platform is on the development of disease-modifying treatments for heart failure due to prior MI. We believe the versatility of this product platform and related differentiated capabilities position us to expand our portfolio of therapies rapidly and pursue other indications involving loss of cardiomyocytes.

Precision Medicine Platform

The idea of “precision medicine” has been around for a number of years, with the core concept of delivering the right therapy to the right patient at the right time. Recently, the idea of precision medicine has gained traction in oncology, in particular, with the benefit of a better understanding of the genetics of different tumor types, and a growing ability to match therapies to specific mutations (e.g., Genentech’s Herceptin therapy for HER2+ breast cancer). We aim to bring this concept of precision medicine to the discovery and development of targeted therapies for heart disease.

There is an increasing understanding of the genetic basis for many cardiomyopathies, including DCM, HCM, restrictive cardiomyopathy (RCM) and arrhythmogenic cardiomyopathy (ACM). DCM provides an interesting case study. Mutations in more than 50 genes have been identified for DCM, with more than 50% of patients presenting with multiple mutations. These mutations affect different parts of the cellular apparatus of patients’ cardiomyocytes, including the sarcomere, nucleus, ion channels, and cellular membranes. Yet mutations in proteins with diverse biology present as a common disease phenotype, suggesting common nodes of disease yet to be discovered. Despite this heterogeneity of genetic background and underlying pathophysiology, the therapies used for these patients are the same as therapies used for patients with other forms of heart failure. We envision a future in which therapies are more specific to the underlying cause of disease and are used to treat patients who have been categorized based on their underlying genetic mutations.

The figure below helps illustrate our vision for “precision medicine” research and development for heart disease through the lens of DCM:

It is also necessary to have the appropriate disease models to discover new targets and to test new therapies. Unfortunately, there is still a lack of representative in vivo models; of the greater than 50 genes known to cause DCM when mutated, less than ten have relevant murine models to support drug discovery. The situation is even worse for others forms of genetic cardiomyopathy. We are committed to finding new ways to model genetic cardiomyopathies, including in vivo but also in vitro models.

There is a growing body of academic literature supporting the use of human iPSC-CMs to model human heart disease and the potential cardiotoxicity of therapeutics during drug discovery. This can be helpful where animal models for specific forms of heart disease either do not yet exist or are not yet sufficiently representative of human disease. There are also a growing number of biopharmaceutical companies that are using iPSCs for phenotypic screening and drug discovery. We are advancing a novel approach of using proprietary human iPSC-CMs disease models for target identification and drug discovery specifically for heart disease.

The figure below illustrates how we have internalized and integrated six key aspects necessary to advance the discovery of precision medicine therapeutics using human iPSC-CMs:

We have demonstrated proof of concept of this approach using a human iPSC-CM disease model representing a specific genetic DCM mutation plus machine learning algorithms to measure variations in appearance of these cells when screened with a library of several thousand small molecule compounds. We identified several biologically relevant hits and validated HDAC6 as a specific target of interest. We have since turned our findings into a product candidate in our HDAC6i program, TN-301, with in vivo activity and tolerability demonstrated in multiple heart disease models of HFpEF and DCM and evidence of target engagement during clinical evaluation.

We are currently conducting target identification screens for both gene therapy and small molecule targets in multiple human iPSC-CM disease models of DCM. We are also expanding our efforts to different genetic backgrounds including the leading genetic causes of cardiomyopathy. We believe the versatility of our Precision Medicine platform and related capabilities enables us to rapidly expand our portfolio of product candidates beyond TN-301.

Our Core Capabilities

We utilize five core internal capabilities to support our three product platforms. Our key capabilities include the creation and development of (1) disease models to more accurately simulate human heart disease phenotypes, (2) proprietary heart-tropic AAV capsids designed to enable precise tissue targeting and increase safety, (3) proprietary promoters and regulatory elements to control gene expression, (4) fit-for-purpose drug delivery methods for more optimal uptake and distribution of our product candidates and (5) scalable AAV manufacturing to better control quality, costs, timelines and supply.

We believe integration of these in-house capabilities provides us with several advantages and differentiates our efforts relative to other drug discovery companies, especially for gene therapy drug development. Through the combination of these capabilities, we are developing product candidates that can address the complicated characteristics of heart disease. For example, we believe with our capabilities in capsids and promoter design and delivery, we can overcome the limitations faced by prior cardiac gene therapy approaches by enabling more precise delivery and more robust gene expression and lowering the risk of off-target effects. We also believe that these approaches can overcome the historical challenges of drug development for heart disease, by enabling delivery of a wide range of therapeutic approaches to specific cells in the heart.

By having our capabilities in-house, we believe we are able to achieve deeper insight, shorten product development cycles, and improve the probability of technical and regulatory success for our product candidates compared to what can be achieved with a more outsourced approach. This further allows us to rapidly build a diverse pipeline of product candidates. Ultimately, we believe our differentiated capabilities can support

development of product candidates that, if approved, could address the high unmet need of patients with heart diseases.

1. Disease Models

We have internalized the ability to create and integrate in vitro and in vivo models within our research organization, which allows us to simulate human heart disease phenotypes. We believe our success will be supported by the know-how we are developing and the proprietary integration of these disease models across our programs.

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In vitro cell-based disease models: For our in vitro disease models, we have leveraged the seminal discovery of methods used to generate iPSCs to establish disease models based on human iPSC-CMs. We have implemented three primary approaches to model human heart disease in this way: (i) short interfering ribonucleic acid (siRNA) constructs to silence specific genes of interest in iPSC-CMs; (ii) CRISPR-based gene editing approaches to create isogenic iPSC-cell lines where specific genes have been altered; and (iii) iPSCs derived from patients with severe heart disease, for example, severe DCM resulting in early heart failure and transplant, sourced from commercial and academic collaborators.

In the figure below, we illustrate our primary disease model approaches based on iPSC-CMs:

These disease models can collectively help simulate the impact of human disease-causing mutations on the appearance and function of cardiomyocytes and model the impact of potentially disease-modifying treatments on such cells. In the figure below, we illustrate how, through use of gene editing and gene silencing tools, we can modify the appearance of normal iPSC-CMs to appear disorganized, and subsequently restore cell appearance with compounds from our screening library:

We initially used cells from these disease models plated in two-dimensional formats. We have since advanced our efforts to include three-dimensional engineered heart tissue disease models where the cells have a more mature phenotype and with contractility that can be measured more reliably.

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iPSC production: To conduct robust target identification and drug discovery screens using our cell-based disease models, we need to produce large volumes of these human iPSC-CMs. We have developed the necessary know-how to do so reliably and reproducibly at increasing scale.

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Imaging techniques:We use a combination of immunostaining, high-resolution imaging, and imaging algorithms to visualize and quantify phenotypic differences between our in-house iPSC-CM disease models. We can measure several details of the sarcomeres of these cell lines, including sarcomere density, disarray and Z-disc area.

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Machine learning algorithms:We have used machine learning algorithms to support high-throughput phenotypic screening of our iPSC-CM disease models. The algorithms can rapidly and reproducibly measure subtle differences in the overall appearance between wild-type iPSC-CM cells and the different disease models, as well as differences on the disease models in response to compounds in our screening libraries.

The figure below illustrates the output of a screen in a disease model of DCM, using siRNA silencing of the BAG3 gene, with a curated library of greater than 5,000 small molecule compounds. A deep learning algorithm that was trained on images of the disease model and on normal cells was used to determine which compounds caused the sarcomeres within the cells to appear more disorganized, representing more sarcomere damage (red), or more organized, representing less sarcomere damage (green), as measured by a “cardiomyocyte score”:

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In vivo models: For our in vivo disease models, we have a dedicated onsite in vivo pharmacology group and vivarium. We have established approximately 17 rodent heart disease models, both genetic and non-genetic, and continue to develop new models in-house as needed. We can dose both gene therapies as well as small molecules. We can perform heart surgeries on these rodent models and use blinded echocardiography-based imaging techniques to assess the impact of our therapies under development. The internalization of these capabilities greatly reduces our reliance on external CROs and academic organizations and significantly increases the speed and consistency with which we can iterate on product prototypes, generate data and formulate insights on our product candidates. We also work with established CROs for research efforts involving large animal models (e.g., NHPs and pigs), including for efficacy studies and evaluation of drug delivery methods. Through these efforts we have developed important insights into the advantages and limitations of specific models and have learned how to optimize the design of our experiments. This insight influences our preclinical drug development strategies and our discussions with regulatory agencies.

2. Capsid Engineering

Our goal is to discover, design, and develop novel heart-tropic AAV capsids with superior attributes in order to enable more precise cardiomyocytes targeting and to improve the safety profile of our product candidates by reducing tropism for other organs, particularly the liver. A capsid is the protective protein shell which contains the

AAV vector and AAV tropism is determined by interaction of capsid proteins and host cell surface receptors. To achieve our goals related to capsid engineering, we have established in-house AAV capsid engineering capabilities and have designed and screened over one billion variants from diverse, proprietary libraries to discover, design, and develop novel capsids to support our programs.

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Cell specificity:We are using our capsid engineering capabilities to identify novel AAV capsids with an overall higher tropism for the heart compared to other organs and selectively to target the two most abundant cell types in the heart: cardiomyocytes and cardiac fibroblasts. We already have achieved in vivo proof of concept for novel vectors for both cell types. Having capsids that more specifically target one cell type over another could help improve efficacy and safety and lower cost of goods for our future product candidates.

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Library diversity:We have screened more than one billion variants from 30 diverse libraries utilizing a range of strategies, including rational modification of surface residues as well as directed evolution efforts with peptide insertion libraries, chimeric libraries, and libraries based on systematic alteration of variable regions using different parental capsids. The diversity of approaches increases the likelihood that we will find capsids with novel properties.

The image below illustrates our efforts to achieve diverse heart-tropic AAV capsids.

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Screening models: We have performed our screens in a variety of in vitro, in vivo, and in silico libraries. Current efforts are focused on direct screening in NHPs, as well as use of machine learning algorithms. We believe our probability of finding novel variants that will translate to superior attributes in humans is highest in NHPs. We believe our in silico approaches can complement these efforts to help predict novel variants.

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Screening criteria:We have broad criteria for the selection of novel capsids, including improved tropism for the heart compared to other organs, with a particular interest in de-targeting the liver; improved transduction of specific heart cell types; lower susceptibility to neutralizing antibodies; and comparable manufacturing in both HEK293- and Sf9/rBV-based manufacturing systems. We seek capsids that can outperform the relevant parental capsids, which may vary depending on the intended use and on some or all of these criteria.

Through these efforts, we have discovered proprietary capsids with superior performance over parental variants in multiple species, including NHPs. These capsids have improved tropism for the heart compared to other organs and even for specific cells within the heart; improved transduction and expression within the heart cells; and lower susceptibility to neutralizing antibodies. We have also developed insights about the performance of novel capsids across different species including mice and NHPs. Several capsids identified have equivalent transduction in the heart and lower transduction of the liver compared to AAV9, leading to an overall better heart-to-liver transduction ratio as validated in an NHP model. Additionally, we have shown that several capsids we identified have overall better ability to evade human neutralizing antibodies compared to AAV9. We have also generated additional data that demonstrate that certain of these capsids have a greater ability to improve heart function compared to AAV9 in specific disease models.

Overall, these data provide important proof of concept of the potential utility of capsid engineering. Therefore, we have taken steps to protect the intellectual property that support the novel capsids identified from our initial capsid engineering screens, and intend to continue this practice as we generate additional data from our ongoing capsid engineering efforts.

3. Promoters and Regulatory Elements

Promoters and regulatory elements are DNA sequences whose function is to determine the level of gene expression at the RNA level and in which cells transcription will occur. These elements can therefore be essential to the success of gene therapy.Enabled by our in-house molecular biology capabilities, we have created novel heart-specific promoters, as well as regulatory elements which control gene expression within the cells to support our AAV-based programs. We are designing promoters and regulatory elements to help ensure a more precise and conditional expression of therapeutic payloads in different cell types in the heart. We believe our innovations in these elements may further support the successful clinical development of our product candidates.

Illustrative examples of our innovations in this area include:

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Heart specificity:We have developed cardiac-specific promoters that enable more selective and robust expression in the heart as compared to other organs. During optimization of TN-201, we developed a cardiomyocyte-specific promoter, TNP-CM1, with improved performance attributes as compared to the standard cTnT promoter. In vitro and in vivo analyses confirmed that TNP-CM1 significantly increased expression of the MYBPC3 gene compared to what can be achieved with the standard cTnT promoter. In addition, in a mouse model we observed 1000-fold selectivity of expression in cardiac tissue relative to other tissues, including skeletal muscle, brain and liver.

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Cell specificity:We have also developed a proprietary combination of regulatory elements that enable more optimal and selective expression in one cell type in the heart compared to others. For our Reprogramming program for cellular regeneration, we discovered ways to optimize the robust co-expression of two protein-coding genes and one micro-RNA gene delivered within a single AAV in cardiac fibroblasts, which we believe supports higher efficacy in preclinical models. We also discovered how to use specific micro-RNA binding sites to silence the translation of those same genes in both existing cardiomyocytes as well as newly created cardiomyocytes, which may provide a safety benefit and reduce the chance for off-target effects.

In the figure below, we illustrate how the use of a novel regulatory element, TNR-CF1, helped prevent the expression of a fluorescent protein in the cardiomyocytes of a mouse model and only allowed expression in the cardiac fibroblasts. We have used this regulatory element in our Reprogramming program to focus the expression of our proprietary factors in resident cardiac fibroblasts for the creation of new cardiomyocytes, but to prevent the expression of those factors both in resident cardiomyocytes and in newly created cardiomyocytes, which we believe will improve the safety profile of our future product candidates:

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Tunable gene expression:We have also demonstrated the ability to develop an entire spectrum of novel promoters to titer the expression of genes within cardiomyocytes, by combining various combinations of enhancer elements from different cardiomyocyte selective genes. Through data (not shown in the figure below) generated in our DWORF program, more than ten promoters were designed and tested in vitro in human iPSC-CMs, and in vivo in murine models to optimize the expression of the DWORF gene to be higher than what can be achieved with a standard cTnT promoter.

In the figure below, we show data for six of our promoters and cassette engineering efforts that illustrate how we have been able to create a suite of cardiac-specific constructs that are able to mediate significantly higher expression of the DWORF gene than can be achieved with a standard cTnT promoter:

4. Drug Delivery

Delivery of drugs to the heart is widely considered to be an important challenge to successful translation of cardiac gene therapy and regenerative medicines into approved products. The diversity of programs in our current pipeline necessitates the use of different delivery methods. We are actively exploring different ROAs as well as different infusion- or injection-based catheters to support more targeted delivery and more efficient uptake of therapies based on viral vectors. We believe our discoveries in drug delivery can widen the therapeutic index of our product candidates by reducing the dose required for a therapeutic benefit.

Several distinct methods of drug delivery for the heart have been explored by different groups for gene- or cell-based therapies, including infusion-based approaches, such as peripheral IV infusion, intracoronary infusion, and retrograde coronary sinus infusion, and injection-based, such as transendocardial injection and epicardial injection. These delivery methods vary significantly in terms of degree of invasiveness, distribution of therapy around the heart, degree of therapy uptake into the heart, technical difficulty of administration, and clinical

relevance and experience. For some approaches, additional methods to improve therapeutic delivery have also been tested to improve perfusion of AAV into the heart. Through these efforts, several groups have demonstrated how different delivery methods can meaningfully affect the relative uptake and biodistribution of therapies in the heart compared to peripheral organs.

Illustrative examples of various delivery methods for the heart are shown below:(1)

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(1) Source: Duan J Transl Int. Med 2020.

For the initial product candidates emerging from our Gene Therapy platform, including TN-201, we generally need broad distribution across the heart tissue that is more suited to infusion-based approaches. By contrast, for the initial product candidates emerging from our Cellular Regeneration platform, including those from our Reprogramming program, we need more precise delivery into the heart tissue directly around a scar area of the LV in a way that is more suited to injection-based approaches.

Illustrative examples of our innovations and capabilities in drug delivery include:

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Catheters: To support our Reprogramming program for cardiac regeneration, we are developing a novel transendocardial injection catheter for more precise delivery of therapeutic payloads around the scar area that is formed after heart attack, but in a way that is minimally invasive and would not require heart surgery. The prototype of our catheter was designed with the help of interventional cardiologists and is based on similar catheters that have been successfully used in clinical trials. The catheter is designed to be steered into the heart via the femoral artery in the groin area. It has a deflectable tip that can be curved to better access the different parts of the heart. This initial prototype was tested in a large animal model and was able to direct injections to all areas of the LV. We are adding mapping capabilities to the design to allow for more precise delivery during the treatment procedure.

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ROAs: We prioritize head-to-head comparison of different ROAs in large animal models to confirm the optimal method for delivery for each product candidate. For example, for TN-201, we conducted experiments in NHPs to compare the degree of drug uptake and biodistribution for peripheral IV infusion and infusions delivered directly in the heart. Based on the results, we chose systemic IV infusions as the ROA for TN-201. In addition, for our Reprogramming program, we conducted experiments in pig models to demonstrate that a less invasive catheter-based transendocardial injection to the LV inside wall can achieve a similar degree of drug uptake and biodistribution as a more invasive direct epicardial injection to the LV outside wall requiring open-heart surgery.

5. Manufacturing

We have fully integrated and internalized AAV manufacturing capabilities to support our Gene Therapy and Cellular Regeneration platforms. Our overall strategy is to have complete ownership of our PD, analytical development, MFG and QC so that we have deep insight into the attributes of our drug substance and drug product. Internalized manufacturing enables continuous process improvement, consistency (quality and productivity) and innovation that can support manufacturing requirements for clinical development and commercialization not only for rare populations, but also for more prevalent indications, and allows us to be a partner of choice in strategic drug development partnerships and with early-stage academic programs.

Overall, the internalization of these efforts provides us with know-how that yields several advantages that allow us to be in a better position to support our future capacity expansion needs or swiftly transfer technology know-how to CDMOs to achieve dual sourcing for product candidates for risk mitigation purposes.

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Vector core:We have established vector production to support early research involving both parental and novel AAV capsids at the 50L scale. We have hired key process development, analytical development and QC personnel to internalize those capabilities. We have also established the necessary process development expertise to support comparable product efficacy in both HEK293-based and Sf9/rBV-based manufacturing systems for both existing AAV serotypes as well as for novel capsids discovered from our capsid engineering efforts.

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Manufacturing Technology Development Center (MTDC): We have established in-house operations at the 200L scale to support all non-clinical studies including those involving large animal models, such as pigs and NHPs, under GLP regulations. We also rely on the MTDC for assay development and technology transfer to our dedicated cGMP facility. Our initial production at this scale has been at yields and with full/empty capsid ratios that compare favorably to industry standards.

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Genetic Medicines Manufacturing Center:Our GMMC is a dedicated cGMP facility for AAV drug product manufacturing and is located in the San Francisco Bay Area. The facility operates at the 1000L scale to support all clinical development activities from FIH clinical trials through to late stage development, as well as initial commercialization, if regulatory approval is obtained. It uses a modular design that will support scale-out and/or scale-up of manufacturing capacity in response to evolving needs.

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Intellectual property:We have in-licensed and internally developed certain manufacturing-related intellectual property to support our programs. We have filed multiple patent applications covering improvements that will support scale-up of AAV manufacturing for supply of our gene therapy product candidates intended for more prevalent heart disease populations.

Competition

The biotechnology and pharmaceutical industries are characterized by rapidly advancing technologies, intense competition and a strong emphasis on intellectual property. We believe our three product platforms, scientific know-how, five core internal capabilities, and experience provides us with competitive advantages. However, we face substantial competition from many different sources, including large and specialty pharmaceutical companies and biotechnology companies, academic research institutions and governmental agencies, and public and private research institutions. Any product candidate we develop and commercialize will have to compete with existing therapies as well as therapies currently in development and that may be developed in the future.

Due to the depth and diversity of our pipeline, we may face competition from a variety of companies, including:

TN-201: We believe the principal competition for TN-201 will be programs that address the underlying genetic cause of MYBPC3-associated HCM. Based on publicly available data, we don’t believe any such treatments have received approval from a regulatory agency or reached clinical development. Based on publicly available data, BioMarin and DiNAQOR’s BMV-293 is only program in preclinical development for treating the underlying cause of MYBPC3-associated HCM. We may also face competition from treatments for both nHCM and oHCM, including Bristol Myers Squibb’s myosin inhibitor Camzyos approved for oHCM. There are also several other programs in clinical development for HCM.

TN-301: We believe that the principal competition for TN-301 in HFpEF includes agents approved in the U.S. and/or Europe for the treatment of HFpEF, including Novartis’ Entresto and Eli Lilly and Boehringer Ingleheim’s SGLT2 inhibitor, Jardiance. While there are no approved HDAC6 inhibitors for cardiovascular indications, HFpEF clinical development is an area of robust investment and multiple additional agents for the treatment of HFpEF are in clinical development.

TN-401: We believe the principal competition for TN-401 will be programs that address the underlying genetic cause of PKP2-associated ARVC. Based on publicly available data, we don’t believe any such treatments

have received approval from a regulatory agency or reached clinical development. However, there are several programs in preclinical development for treating the underlying cause of PKP2-associated ARVC, including Rocket Pharmaceutical’s RP-A601, Lexeo Therapeutics’ LX2020 and Stridebio’s STRX-330. We may also face competition from therapies and medical devices directed to treat the symptoms of ARVC.

TNYA US Equity

TNYA · 10-K · period ended 2022-12-31

Item 1. Business.

TNYA US Equity☆

TNYA · 10-K · period ended 2022-12-31

Item 1. Business.

TNYA US Equity