TN-201

The AAV9 serotype has been extensively studied in gene therapy, particularly for its ability to target tissues like heart, central nervous system (CNS), liver, and skeletal muscles. While it is difficult to provide an exact number of patients who have been treated with AAV9-based gene therapies across the world, either in clinical trials or with the approved gene therapy products, more than 5500 patients have been dosed in 50 countries with Zolgensma alone based on the latest figures released from Novartis.

Zolgensma, an AAV9-mediated gene therapy, was approved by the FDA in 2017 to treat pediatric patients less than two years of age with spinal muscular atrophy with bi-allelic mutations in the SMN1 gene. Zogensma is now approved in over 47 countries, and thousands of children have been treated. Data from a long-term follow-up study of Zolgensma demonstrate durability up to 7.5 years and 100% achievement of all assessed milestones in children treated prior to SMA symptom onset.

There have also been several individuals affected by Duchenne muscular dystrophy who have been treated with an AAV9-based gene therapy by Pfizer and Solid Biosciences (Manini 2022). Preliminary data demonstrate encouraging safety and efficacy, with robust expression of micro-dystrophin and improvements in functional scores. (Manini A et al. Adeno-Associated Virus (AAV)-Mediated Gene Therapy for Duchenne Muscular Dystrophy: The Issue of Transgene Persistence. Front. Neurol., 05 January 2022).

Finally, AAV9-based gene therapy is being studied in individuals with Danon disease, an X-linked cardiomyopathy. Results from the ongoing Phase 1 Danon Disease trial represent one of the most comprehensive investigational gene therapy datasets for any cardiac condition. RP-A501 was generally well tolerated with evidence of durable treatment activity and improvement of Danon disease for both pediatric patients with up to nine months of follow-up and four adult patients with up to 36 months of follow-up. All adult and pediatric patients who received a closely monitored immunomodulatory regimen showed improvements across tissue, laboratory, and imaging-based biomarkers, as well as in New York Heart Association (NYHA) class (from II to I) and Kansas City Cardiomyopathy Questionnaire (KCCQ) scores with follow-up of six to 36 months. (https://ir.rocketpharma.com/news-releases/news-release-details/rocket-pharmaceuticals-reaches-fda-alignment-pivotal-phase-2)

Gene replacement therapy and gene editing use genetic material to treat or prevent disease. Most gene therapy approaches work by delivering a functional gene into a cell to replace the genetic variant underlying a given condition and restore healthy protein levels necessary for proper function. Gene editing has a similar goal but differs by delivering genetic material that can directly edit pieces of DNA within a cell. This changes the instructions encoded by the DNA to correct the protein produced by the DNA and restore proper cell function. CRISPR-Cas9 is a common gene editing treatment approach for gene editing. (www.asgct.org)

AAV gene therapy is typically used for the treatment of genetic disorders. COVID vaccines are designed to protect against SARS-CoV-2 infection and COVID-19 disease in the general population (Hakroush & Tampe, 2021).

AAV gene therapy and COVID vaccines differ in their mechanisms of action and targets. AAV gene therapy involves the use of adeno-associated viruses (AAVs) to deliver therapeutic genes into cells, aiming to correct genetic disorders and uses a DNA-based approach (Speer et al., 2022). The COVID vaccine payload doesn’t enter the cell’s nucleus; instead, it directs the cells to produce spike protein at the ribosome in the cytoplasm, and is designed to stimulate an immune response against the SARS-CoV-2 virus, which causes COVID-19 (Hakroush & Tampe, 2021).

AAV gene therapy focuses on specific genetic mutations that cause diseases, aiming to provide a long-term or permanent correction at the genetic level (Speer et al., 2022). In contrast, COVID vaccines target the spike protein of the SARS-CoV-2 virus, which is responsible for viral entry into human cells and prompts an immune response without causing disease. (Hakroush & Tampe, 2021).

The AAV capsid is recognized by receptors at the cell surface of the host and taken into the cell via endosomes. Following endosomal escape, AAV enters the nucleus, where it undergoes capsid uncoating to release the transgene or gene of interest. The transgene undergoes circularization, creating a stable genome called episomal DNA, leading to gene expression that persists in postmitotic cells. Importantly, the episomal DNA does not integrate into the host DNA. (Wang D et al. Adeno-associated virus vector as a platform for gene therapy delivery. Nature Reviews. May 2019.) The episomal DNA then undergoes transcription to messenger RNA, which travels to the ribosomes for production of the intended protein (e.g., TN-201 will restore functional myosin binding protein C). Because the episome is stable and adult cardiomyocytes do not replicate, a single dose of TN-201 has the potential to produce functional myosin-binding protein indefinitely.

Immunosuppression is required in AAV-mediated gene therapy clinical trials to maximize safety and efficacy for treated individuals. The immune system may recognize AAV vectors as foreign invaders and mount an immune response against them. Immunosuppression helps reduce this immune reaction, allowing the therapy to be more effective.

In Tenaya’s clinical trials of AAV9-based gene therapies, we utilize two components of the immunosuppressive regimen: Sirolimus and Prednisone. These are administered pre-dose on Day -7. Patients will be given a loading dose of sirolimus and then continue daily dosing based on sirolimus trough levels. Sirolimus will be continued for approximately 15 weeks, or until 1 week after the corticosteroids have been tapered off, or until the patient’s biomarkers and LFTs indicate that it can be stopped, whichever is longest. Following the administration of gene therapy, patients’ liver enzymes are closely monitored. Close monitoring continues throughout the tapering process. The duration and intensity of immunosuppression depend on the specific gene therapy being used and the patient’s immune status, requiring a balance between reducing immune rejection and minimizing risks.

Intact rAAV particles in endosomes undergo a series of pH-dependent structural changes necessary for transduction and traffic through the cytosol via the cytoskeletal network. After endosomal escape, rAAV enters the nucleus through the nuclear pore complex, where it undergoes capsid uncoating to release the genome. Following uncoating, a small fraction of viral peptides is targeted to the lysosome and degraded. (Wang D et al. 2019; Siddhartha Mukherjee, 2022)

Because AAV and associated parvoviruses naturally infect mammals, it is important that patients be tested for pre-existing neutralizing antibodies (NAbs), which could block AAV-mediated gene transfer and interfere with successful transduction. Global estimates of the general population suggest 70% of adults have no or low pre-existing AAV9 NAbs, but rates vary based on geography, demographics, and age (Verma, S., et al., Hum Gene Ther, 2023;34(9-10):430-438.). In our RIDGE^TM Natural History study of 144 patients with PKP2-associated ARVC, 93% of patients show AAV9 neutralizing antibody (NAb) titers ≤1:40, meeting eligibility criteria for TN-401 gene therapy.

AAV vectors, including AAV9, are generally considered to have a low risk of viral shedding and transmission due to their non-pathogenic nature and inability to replicate in human cells. Therefore, they do not pose a risk of transmission to others. (Suckau et al., 2009). However, it is always important to exercise caution and follow appropriate safety measures when working with viral vectors to minimize any potential risks.

In general, most health care facilities recommend universal/standard precautions with patient material between 14 and 30 days after administration for both health care staff and direct family members, dependent on viral shedding analysis. Additionally, instructions are provided to family members and caregivers to practice good hand hygiene for a minimum of 2 weeks after the injection. This requires washing hands with soap regularly and using appropriate protective gloves if coming into direct contact with patients’ bodily fluids and waste. (Ghosh S et al., 2020.)

There are two types of immune responses: innate and adaptive. Most adverse events in gene therapy studies have been linked to adaptive immune responses. Adaptative/Cellular Immune Response (Hepatotoxicity) happens at week 1-12 after the vector administration.

Immune-mediated liver toxicities can be encountered when an AAV vector is administered systemically. The presentation of the symptoms is normally: transaminase elevations (ALT most pronounced), decline in transgene-protein expression, IFN- ELISPOT to AAV capsid peptides. It occurs 1 week post vector administration, with a second peak (higher) at 1 month, concurrent with steroid wean or discontinuation. In the Zolgensma clinical trial participants, the vast majority (90%) had elevation in AST and ALT, but importantly, none had elevations of bilirubin more than 2x ULN, though all these recipients were on prophylactic steroids. Notably, only a small minority had very high elevations in liver enzymes.

The risk of myocarditis with AAV gene therapy appears to be very low but is a noted concern, particularly in specific patient populations, such as those receiving treatment for conditions like Duchenne muscular dystrophy (DMD) in which cases of myocarditis have been reported during clinical trials, including the death of one patient.  Based on the investigation by an external data monitoring committee, all cases of myocarditis that occurred appeared to be associated with a specific pattern of genetic mutation in the DMD gene.  Enrollment criteria was subsequently adjusted and no further incidents have been reported to date. (C.G. Bonnemann, 2023)

Although the etiology of AAV vector-induced myocarditis is not yet fully understood it seems feasible that the inflammatory milieu of the damaged muscle tissues in DMD patients favors induction of an immune response to the AAV vectors’ transgene product or alternatively that the added inflammatory reaction induced by the AAV vectors promotes auto-reactive T cell responses to muscle cells. (Ertl,  2022)

Continuous monitoring and safety evaluations are crucial in ongoing trials to assess the incidence and severity of this potential side effect.

To date, there have been no reports of AAV-induced tumor formations in humans.

AAVs are generally non-pathogenic and do not integrate into the host genome in a manner that leads to significant mutagenesis. However, there are concerns about potential insertional mutagenesis, where the therapeutic gene could inadvertently disrupt important genes or regulatory elements in the host DNA. Ongoing research is focused on better understanding these risks and enhancing the safety profiles of AAV vectors.

Recombinant AAV integrations leading to hepatocellular carcinoma have been reported in mice. In a large study of 695 mice evaluating serotypes 1, 2, 5, 7, 8, and 9, only one liver tumor was observed resulting in an incidence of 0.14% (Bell P, Wang L, Lebherz C, et al. 2005). In total, AAV2 posed the greatest risk of insertional mutagenesis (Bell et al. 2005).

Interim data from the first three patients enrolled in the company’s MyPEAK^TM-1 Phase 1b/2 clinical trial of TN-201 were highlighted in a Late-Breaker presentation at the 2025 American College of Cardiology Scientific Sessions (ACC.25) by Milind Desai, M.D., MBA, Haslam Family Endowed Chair in Cardiovascular Medicine, Vice Chair of Education in the Heart Vascular Thoracic Institute, Director of the Hypertrophic Cardiomyopathy Center at Cleveland Clinic, and an investigator for the MyPEAK^TM-1 Phase 1b/2 clinical trial.

Interim data include results from serial biopsies and assessments of Patients 1 and 2 at Week 52 and Patient 3 at Week 26, analyzing changes over time in the first three patients to receive a one-time infusion of TN-201 gene therapy (Cohort 1).

• TN-201 was generally well tolerated at 3E13 vg/kg, and treatment-emergent adverse events (AEs) were primarily mild, manageable and/or reversible.
• Serial biopsies taken at two timepoints for all three patients demonstrated sustained presence of TN-201 DNA in the heart (0.8 to 1.4 vg/dg) and robust TN-201 RNA expression (>1.25×105 copies per microgram of RNA) that increased as much as 13-fold from Week 8 to Week 52 post-dose. MyBP-C protein levels increased from 56 to 59% and from 62 to 64% of normal between Week 8 and Week 52 for Patients 1 and 2, respectively. This increase, combined with the increase observed in TN-201 mRNA expression, suggests that TN-201 gene therapy is successfully being transcribed and expressed after reaching target cells. Patient 3 was the first patient on study to receive a baseline biopsy, which is expected to offer insight into the total change in protein levels following TN-201 treatment. The post-dose biopsy sample from Patient 3 was not evaluable; a second post-dose biopsy is planned later this year and will be reported in a future data readout.
• Cardiac troponin, a biomarker of myocardial injury, was elevated in Cohort 1 patients at baseline and decreased by more than 60% in two patients, whose levels are now normal or near normal. NT-proBNP, a biomarker of cardiac strain, increased and remained elevated while patients were on immunosuppression, but returned to baseline as immunosuppressive drugs were discontinued. Key measures of hypertrophy, or enlargement of the heart, improved in two patients while other assessments remained stable.
• Left ventricular posterior wall thickness, which was elevated at baseline, decreased in two patients by up to 40% into the normal range for healthy individuals. In one patient, left ventricular mass (LVMI) improved by 10%. Additional measures of hypertrophy and diastolic function remained stable.
• All three patients in Cohort 1 had objectively severe disease at the time of enrollment with mild-to-moderate heart failure symptoms (New York Heart Association (NYHA) classification II or III) that interfered with activities of daily living. All three have now achieved NYHA Class I, defined as having no limitations on physical activity.

The transgene is cloned into a plasmid containing the AAV “inverted terminal repeat” (ITR) sequences and a cardiac-specific promoter, in this order: 5’-ITR —promoter-human MYBPC3– ITR-3’. The cardiac specific promoter facilitates expression of the transgene in cardiomyocytes and limits the off-target expression of the transgene. The ITR sequences are derived from AAV and are necessary “messages” for the DNA construct to be subsequently packaged into an AAV vector during cell culture production. Also, the ITR sequences are the only sequences of viral origin remaining in the construct.

Tenaya manufactures gene therapy investigational products using production processes that are very similar to those used by other gene therapy companies. The process uses recombinant cell culture technology in Sf9 cells , production of the TN-201 gene therapy vector in a large-scale bioreactor, and a multistep purification process followed by sterile vial filling.

For the purposes of cardiac gene transfer, AAV1, AAV6, and AAV9 have emerged as the most promising AAVs. But in particular, AAV9 has proven to be the most powerful AAV serotype to efficiently transduce cardiomyocytes in mice, rats, and swine. (Zincarelli C, et al., 2008; Chamberlain K et al. 2017; Li J et al., 2022). Furthermore, AAV9 is the only serotype that has now demonstrated efficient transduction and evidence of restored expression of deficient LAMP2 protein in cardiomyocytes of Danon disease patients. (https://ir.rocketpharma.com/news-releases/news-release-details/rocket-pharmaceuticals-receives-fda-regenerative-medicine-1)

Preclinical treatment in non-human primates was performed to determine the optimal route of administration for TN-201. Intravenous dosing performed comparably to intra-coronary delivery in non-human primates based on transduction analyses, greatly simplifying the development of TN-201.

AAV gene therapy does not make changes to genes in a person’s reproductive cells. The working gene cannot be passed from a parent to a child, so receiving AAV gene therapy will not change the risk of potentially passing on a genetic condition to children. (Goswami R, et al. Gene therapy leaves a vicious cycle. Front. Oncol. 2019;9:297.)

Currently, a person can receive an AAV-based gene therapy only once. The viral makeup of the gene therapy construct primes the immune system to potentially attack the viral components the next time it sees the same or similar (other AAV serotypes) therapy. Redosing of AAV gene therapies is an ongoing area of research.

While gene therapy for inherited heart conditions like MYBPC3-associated HCM is still under investigation, one of the potential benefits is to enable patients to stop or decrease other medications and devices.

The patients will continue to make dysfunctional protein alongside WT protein after TN-201 has been administered. Truncated mutant peptides, if expressed at all in cardiomyocytes with heterozygous MYBPC3 mutations, are rapidly degraded such that any scarce residual amount is unlikely to play a role in disease pathogenesis. https://doi.org/10.1172/jci.insight.133782

Only patients with truncating MYBPC3 mutations (loss-of-function variants) are eligible for TN-201 therapy. Mutations classified as truncating mutations include frameshift, nonsense, splicing, and large rearrangements. Mutations in MYBPC3, the gene encoding MyBP-C, are the most common cause of familial HCM, an autosomal dominant condition with incomplete penetrance. In 70% of cases, the disease is caused by loss-of-function variants in the MYBPC3 gene which lead to the protein deficiency due to premature termination codons that cause a reduction of MyBP-C protein levels in HCM hearts. (O’Leary, et al, J Mol Cell Cardiol, 2020) TN-201 produces the MyBP-C proteins needed to power normal heart function.

It is not yet known how long it will take for transduction, transgene expression, protein expression, or benefit to take place in humans. Based on the experience of other sponsors, we know that gene therapy can take several weeks to months to show efficacy (as opposed to the near-immediate effects possible with other therapeutic modalities), and that relatively low levels of protein expression appear to have growing benefit over time, and that results can be durable, lasting for several years.

TN-201 has the potential to be durable. Cardiomyocytes are considered the most compatible for use of AAV gene therapy, as cardiomyocyte turnover is negligible in adults.

There are several examples of the duration of gene therapy: a study by Pasi et al. (2020) reported on the multiyear follow-up of AAV5-hFVIII-SQ gene therapy for hemophilia A. The results showed durable efficacy, long-term safety, and clinical and biologic outcomes in adults with severe hemophilia A who received a single infusion of AAV5-hFVIII-SQ (Pasi et al., 2020). There are now 15-year follow-up data reported in the literature for hemophilia patients dosed with AAV-based gene therapy (George LA, Ragni MV, Rasko JEJ, et al. 2020. Long-term follow-up of the first in human intravascular delivery of AAV for gene transfer: AAV2-hFIX16 for severe hemophilia B. Mol. Ther)

Another study by Pasi et al. (2020) described 2-year and 3-year safety and efficacy data after the administration of AAV5-hFVIII-SQ in men with severe hemophilia A (Pasi et al., 2020). The study not only provided clinical safety and efficacy data but also contributed to a mechanistic understanding of the variability observed in gene therapy studies, shedding light on mechanisms of DNA persistence and durable expression (Pasi et al., 2020).

Latest data from two Long-Term Follow-Up (LTFU) studies, LT-001 and LT-002, show the continued efficacy and durability of Zolgensma across a range of patient populations, with an overall benefit-risk profile that remains favorable. (Mendell J. et al. Long-Term Follow-Up of Onasemnogene Abeparvovec Gene Therapy in Symptomatic Patients with Spinal Muscular Atrophy Type 1. / Connolly A. et al. Intravenous and Intrathecal Onasemnogene Abeparvovec Gene Therapy in Symptomatic and Presymptomatic Spinal Muscular Atrophy: Long-Term Follow-Up Study). Highlighting the remarkable durability of Zolgensma, data from LT-001, an ongoing 15-year LTFU study of patients who completed the Phase 1 START study, showed that up to 7.5 years post-dosing, children who were treated after presenting symptoms of SMA maintained all previously achieved motor milestones.

Fibrotic cells are not expected to transduce gene therapy and produce proteins. MyPEAK^TM-1 is focused on patients with preserved systolic function, where most of the cardiomyocytes are functional. As part of the clinical trial eligibility criteria, participants who are most likely to respond to gene therapy will be required to have an LVEF > 50%.

TN-201 DNA transduction and RNA expression in heart tissue are quantified using RV septal biopsy samples performed at baseline, post-dose, and one year. Since the MyBP-C protein produced by TN-201 gene therapy is identical to the endogenous protein, we look for transgene delivery into target cells (vector genome copy number), expression of TN-201 mRNA, and differences in protein levels over time based on serial biopsies.

Interim data include results from serial biopsies and assessments of Patients 1 and 2 at Week 52 and Patient 3 at Week 26, analyzing changes over time in the first three patients to receive a one-time infusion of TN-201 gene therapy (Cohort 1).

Serial biopsies taken at two timepoints for all three patients demonstrated sustained presence of TN-201 DNA in the heart (0.8 to 1.4 vg/dg) and robust TN-201 RNA expression (>1.25×105 copies per microgram of RNA) that increased as much as 13-fold from Week 8 to Week 52 post-dose. MyBP-C protein levels increased modestly between Week 8 and Week 52 for Patients 1 and 2, respectively. This increase, combined with the increase observed in TN-201 mRNA expression, suggests that TN-201 gene therapy is successfully being transcribed and expressed after reaching target cells.

Following IV infusion, TN-201 has been designed to deliver the MYBPC3 transgene to the cardiomyocyte, increase MyBP-C protein production, and restore normal function in the sarcomere. In the mouse model, a single dose of TN-201 provided durable expression of MyBP-C in transduced cardiomyocytes. TN-201 gene therapy is designed to directly restore the MyBP-C content in the cardiomyocyte, whose deficit is the biological basis of this genetic cardiomyopathy, which would be irreversible.

Thrombotic Microangiopathy (TMA) is the most significant acute safety concern following AAV gene therapy infusion. It has been observed with systemic AAV9 (and non-AAV9 LK03) vectors with the dose >6E13 vg/kg across at least 5 diseases. TMA occurrence is uncommon with AAV9 gene therapy. In the MyPEAK^TM study, we do not use the dose >6E13 vg/kg. Hospitalization with daily safety laboratories from Day -1 to Day 7 will increase the safety of TN-201 gene therapy treatment by enabling intensive monitoring and early treatment for hepatoxicity and TMA. Preliminary data from three patients in the first dose cohort of 3E13 vg/kg (Cohort 1) showed that TN-201 was generally well tolerated without any signs of TMA or thrombocytopenia.

Preliminary data from three patients in the first dose cohort of 3E13 vg/kg (Cohort 1) showed that TN-201 was generally well tolerated. All patients (n=3) experienced transient, reversible elevations in liver enzymes following dosing. Elevations normalized after additional steroid treatment. One mild adverse event classified as a Serious Adverse Event (SAE) with inpatient steroids. The majority of treatment-emergent adverse events were mild, transient, or reversible. Two other SAEs occurred, both unrelated to TN-201. No signs of cardiotoxicity, TMA, or thrombocytopenia were detected. All patients have completed every visit and remain in the study.

MYBPC3 protein levels in the heart are regulated to normal levels even if mRNA is overexpressed. Preliminary data from three patients in the first dose cohort of 3E13 vg/kg (Cohort 1) demonstrated a modest increase in MyBP-C Protein Levels.

AAV9:mMybpc3 (mouse homolog) given at 3E14 vg/kg, which was 10-fold higher than the dose that rescued cardiac function in double knock out (Mybpc3-/-) mice, exhibited a 6.5X increase in Mybpc3 RNA (normalized to GAPDH) above vehicle-treated animals and a concomitant 1.5-fold increase in MYBPC3 protein. This substantial increase in protein is consistent with published findings, using transgenic mouse lines over-expressing Mybpc3 RNA, that expression of MYBPC3 protein beyond wild-type levels has not been achieved (Van Dijk et al. 2016; Yang et al. 1998). In adult naïve mice, a greater than 6-fold increase in MYBPC3 RNA overexpression, with a concomitant 50% increase in MYBPC3 protein (G20-04), 10 weeks post-injection, has been seen. This disconnect between RNA and protein levels highlights the tight homeostatic control of MYBPC3 protein that is carefully maintained in cardiomyocytes.

Interim results from three patients in the first dose cohort of 3E13 vg/kg (Cohort 1) demonstrated no alterations of ejection fraction.

In a knockout (KO) mouse model, animals received the gene therapy dose when their EFs were about 35%. The earliest clinical benefit for any AAV-based gene therapy typically begins around 2-3 weeks post-injection, and the TN-201 preclinical data support this: at 2 weeks post-injection, EF increased +6% vs. Vehicle; at 4 weeks +14% vs. Vehicle; at 6 weeks +20% vs. Vehicle. The EF did not decrease further post-injection in any of the animals. The EF increased within 6 weeks after the administration and stabilized at about 50-55% for the duration of the study at 13 months.

Preliminary data from three patients in the first dose cohort of 3E13 vg/kg (Cohort 1) demonstrated that TN-201 expression, as measured by MYBPC3 RNA levels, was highly cardiac-selective.

None of the three patients in the MyPEAK^TM study had myocarditis.

The length of the short-term immunosuppression will be determined by individual responses to TN-201 but is expected to last 3-4 months. The immunosuppression regimen for MyPEAK^TM-1 aims to diminish the immediate effects of the innate immune reaction, the subsequent adaptive T-cell responses to the AAV9 capsid, which occur within the first 4 weeks following gene therapy. Increases in circulating AAV capsid-specific CD8+ T cells coincide with hepatotoxicity and elimination of AAV-transduced cells. Of note, because MYBPC3-associated HCM patients in this study are heterozygous and express MyBP-C protein, they likely have immune tolerance to this protein, and so adaptive responses to the TN-201 transgene product are not expected.