AAV Gene Therapy in Cardiovascular Disease

AAV Gene Therapy in Cardiovascular Disease

Cardiovascular disease (CVD) remains the leading cause of morbidity and mortality worldwide. Despite advancements in pharmacotherapy and medical devices, many conditions like heart failure (HF), inherited cardiomyopathies, and ischemic heart disease still lack curative therapies. Gene therapy using recombinant adeno-associated viruses (rAAVs) has emerged as a promising platform to correct underlying molecular pathologies in CVD by enabling long-term, tissue-specific expression of therapeutic genes.

AAV vectors are non-pathogenic, have low immunogenicity, and can drive persistent expression in non-dividing cells such as cardiomyocytes. Over the past decade, significant progress has been made in optimizing AAV serotypes for cardiac delivery, identifying therapeutic targets, and advancing clinical trials. This review synthesizes current knowledge and highlights the translational potential of AAV gene therapy for CVD.

AAV Serotypes for Cardiac Gene Delivery

The efficacy of AAV gene therapy hinges significantly on the serotype’s tropism for cardiac tissue, which dictates its ability to efficiently deliver therapeutic genes to myocardial cells. Several AAV serotypes have been extensively investigated for their potential in treating cardiovascular diseases, each possessing distinct characteristics regarding their transduction efficiency and tissue tropism (Figure 1):

1. AAV1: This serotype has demonstrated strong and reliable transduction in both skeletal and cardiac muscle tissues. Its ability to effectively deliver genes to these muscle types has made it a candidate for clinical translation, notably being utilized in the CUPID trial for heart failure (HF), aiming to restore proper calcium handling in cardiac cells.
2. AAV6: Known for its efficient transduction capabilities in cardiomyocytes and vascular cells, AAV6 is a frequently employed serotype in preclinical models of heart failure. Its efficacy in targeting both heart muscle and the surrounding vascular structures makes it valuable for investigating various aspects of cardiovascular disease pathology and potential therapeutic interventions.
3. AAV8: While primarily recognized for its liver tropism, AAV8 also exhibits the capacity to transduce the heart. The inherent liver tropism can lead to some off-target delivery and potential immune responses. Consequently, significant research efforts have focused on developing engineered variants of AAV8 with modifications designed to improve its cardiac specificity and reduce liver uptake, thereby enhancing its therapeutic index for heart-related conditions.
4. AAV9: This serotype stands out for its highly cardiotropic nature. AAV9 provide superior global cardiac gene transfer compared to other serotypes, allowing for systemic administration and efficient delivery of genes directly to the heart muscle. Its robust and widespread transduction of cardiac tissue has led to its widespread use in both preclinical research and ongoing clinical studies for a variety of cardiovascular disorders, including Duchenne muscular dystrophy-associated cardiomyopathy and other genetic heart diseases.
5. Other Serotypes and Engineered Variants: Recent advancements have focused on identifying and engineering novel AAV capsids to further enhance cardiac specificity and reduce resistance from pre-existing neutralizing antibodies.

• AAVrh.74 and AAVrh.10: These rhesus macaque-derived serotypes have shown particular propensity for heart muscle cells, making them promising candidates for cardiac gene delivery.
• AAVM41, AAV2-THGTPAD, AAV2-NLPGSGD, and AAVHSC15: These serotypes and engineered variants have also demonstrated tropism for heart muscle cells.
• Engineered Variants (e.g., AAV2i8 and Anc80L65): Efforts are actively underway to design and select AAV variants with enhanced cardiac tropism. AAV2i8., for example, a variant that has the AAV2 hexapeptide (RGNRQA) and the corresponding peptide from AAV8 (QQNTAP), has been used in clinical trials for non-ischemic cardiomyopathy. Similarly, Anc80L65 is an engineered variant that has shown promising results in pre-clinical settings for improving cardiac transduction efficiency. These modifications aim to overcome challenges such as insufficient gene transduction in the human heart and the high cost of producing sufficient AAV vectors. PackGene is actively contributing to this advancement with its π-Icosa AAV Serotype Screening Platform. This platform is designed to identify and optimize AAV capsids with enhanced tropism and transduction efficiency for specific tissues, especially the heart. The ongoing development of such rationally designed and novel capsids, alongside high-throughput screening and directed evolution strategies, is crucial for advancing cardiac gene therapy.

Figure 1. AAV serotypes are tested in heart tissue

The continuous development and characterization of these diverse AAV serotypes and engineered variants are crucial for advancing the field of AAV gene therapy, paving the way for more effective and safer treatments for a wide spectrum of cardiovascular diseases.

PackGene produces over thousands of AAV lots annually for the above-mentioned serotypes with titers ranging from 2e12 GC to 5e14 GC. Our consistently high yields across most serotypes ensure reliable support for your research, offering both research-grade and GMP-grade AAV products to seamlessly meet your project needs.

AAV Targets in Cardiovascular Disease

Gene therapy for CVD aims to rectify pathogenic genetic variants that lead to debilitating conditions such as hypertrophic, dilated, and arrhythmogenic cardiomyopathies, as well as severe forms of hypercholesterolemia. The strategies primarily involve two main avenues: delivering exogenous (external) genes to supplement insufficient or missing protein levels, or employing advanced genome editing technologies like CRISPR/Cas9 to precisely correct, delete, or modify mutant gene sequences, thereby restoring normal protein function.

Preclinical studies, particularly those conducted in large animal models (like pigs, sheep, and dogs), are crucial for evaluating the safety and efficacy of these gene therapies before human trials. These models offer a more physiologically relevant system, with heart sizes and physiological responses closer to humans than rodent models, allowing for better assessment of optimal dosing, distribution, and potential side effects of AAV gene therapy.

Here are some of the key targets and approaches being investigated in AAV gene therapy for CVD:

• Calcium Handling Proteins: The precise regulation of calcium (Ca2+) within cardiac muscle cells is fundamental for proper heart contraction and relaxation. Disruptions in calcium handling are a hallmark of many forms of heart failure.

  • Sarco/endo-plasmic reticulum Ca2+-ATPase (SERCA2a): This protein is crucial for pumping calcium ions back into the sarcoplasmic reticulum after muscle contraction, enabling relaxation and efficient subsequent contractions. Downregulation of SERCA2a is a significant factor in various forms of heart failure, including heart failure with preserved ejection fraction (HFpEF). Gene therapies delivering a functional SERCA2a gene using AAV vectors have been extensively studied. Notably, AAV1-based delivery of SERCA2a was among the first gene therapies for heart failure to advance into human clinical trials. Preclinical evidence for SERCA2a includes studies in:

    • Pigs using AAV1
    • Sheep using AAV6, AAV1, and AAV9
    • Dogs using AAV6 and AAV1

  • Other Calcium Handling Modulators: Researchers are also exploring other proteins that influence calcium dynamics, such as SUMO1 (tested with AAV1 in Pigs), S100A1 (AAV9 in Pigs and AAV6 in Pigs), and I1c (AAV9 in Pigs and AAV2i8 (BNP116) in Pigs). Additionally, silencing of Phospholamban (PLB) via shRNA-PLB delivered by AAV6 in Dogs aims to enhance SERCA2a activity indirectly.

• Structural Proteins: Mutations in genes encoding the structural proteins that form the heart muscle (myocardium) are direct causes of many cardiomyopathies. Gene therapies in this area aim to replace the faulty gene with a healthy copy.

  • Plakophilin-2 (PKP2): Mutations in the PKP2 gene are a primary cause of arrhythmogenic right ventricular cardiomyopathy (ARVC), a serious genetic heart disease characterized by progressive replacement of heart muscle with fatty and fibrous tissue, leading to arrhythmias and sudden cardiac death. Companies like Tenaya Therapeutics are developing AAV9-based gene therapies (e.g., TN-401) to deliver a functional PKP2 gene. Rocket Pharmaceuticals is also pursuing this target with RP-A601 for PKP2-ACM.
  • Myosin Binding Protein C3 (MYBPC3): Mutations in the MYBPC3 gene are a common cause of hypertrophic cardiomyopathy (HCM), a condition where the heart muscle becomes abnormally thick. Tenaya’s TN-201 is designed to deliver a working MYBPC3 gene using AAV to correct this underlying genetic defect.

• Lysosome-Associated Membrane Protein 2 (LAMP2B):

  • Danon Disease: This is a rare, severe X-linked genetic disorder primarily affecting the heart muscle (cardiomyopathy), skeletal muscles, and intellect. It is caused by mutations in the LAMP2 gene, specifically often affecting the LAMP2B isoform, which is critical for autophagy – the cellular process of recycling waste. The deficiency of functional LAMP2B leads to the damaging accumulation of waste products within heart cells. Rocket Pharmaceuticals’ RP-A501 utilizes an AAV9 vector to deliver the wildtype human LAMP2B gene, aiming to restore proper lysosomal function and ameliorate the disease.

• Other Targets and Mechanisms: Beyond directly addressing protein deficiencies or structural defects, gene therapy research in CVD also explores broader therapeutic mechanisms:

  • Gene Editing Technologies: Advanced tools such as CRISPR/Cas9, base editors, and prime editors offer the potential for highly precise and potentially permanent corrections to disease-causing genes at the DNA level. These technologies can introduce specific genetic changes to restore normal protein sequences, delete harmful mutations, or even introduce premature stop codons to degrade abnormal protein products. This approach holds significant promise for a wide range of genetic cardiovascular diseases.
  • Beta-Adrenergic Pathway Modulation: The beta-adrenergic signaling pathway plays a crucial role in regulating heart rate and contractility. Dysregulation of this pathway is implicated in heart failure. Gene therapies targeting components like βARKct have been explored (e.g., AAV6 in Pigs) to restore proper signaling and improve cardiac function.
  • Therapeutic Angiogenesis: This strategy focuses on stimulating the growth of new blood vessels to improve blood supply to ischemic (oxygen-deprived) heart tissue, which can be critical after events like a heart attack or in chronic coronary artery disease. Growth factors like Vascular Endothelial Growth Factor (VEGF), Fibroblast Growth Factor (FGF), and Hepatocyte Growth Factor (HGF) have shown promise in preclinical studies. Specific examples from large animal models include:

    • VEGF/Ang1 (AAV1 in Pigs)
    • VEGF-A/PDGF-B (AAV9 in Pigs)
    • VEGF-B (AAV9 in Dogs)

  • Inflammation Modulation: Chronic inflammation contributes to the progression of various CVDs. Gene therapies targeting inflammatory pathways, such as delivering Heme Oxygenase-1 (HMOX1) via AAV9 in Pigs, are being investigated to reduce damaging inflammatory responses in the heart.

The diversity of these targets and the increasing sophistication of AAV vectors highlight the significant progress and potential for gene therapy to revolutionize the treatment of cardiovascular diseases by addressing their fundamental genetic underpinnings.

Current Companies and AAV Programs in Heart Disease

Several biotechnology companies are actively pursuing AAV-based gene therapies for various cardiovascular indications (Table 1):

• Tenaya Therapeutics: A clinical-stage company focused on curative therapies for heart disease.

  • TN-201: AAV9-based gene therapy for MYBPC3-associated hypertrophic cardiomyopathy (HCM). It’s designed to deliver a working MYBPC3 gene to cardiomyocytes. The MyPEAKTM-1 Phase 1b clinical trial started dosing in October 2023.
  • TN-401: AAV9-based gene therapy for PKP2-associated arrhythmogenic right ventricular cardiomyopathy (ARVC). It aims to deliver a functional PKP2 gene. The RIDGE-1 Phase 1b clinical trial is on track to begin dosing in the second half of 2024.
  • Tenaya also has early-stage preclinical programs and is involved in capsid engineering efforts to enhance AAV delivery.

• Affinia Therapeutics: An innovative gene therapy company developing AAV gene therapies for cardiovascular and neurological diseases.

  • AFTX-201: Their lead program for BAG3 Dilated Cardiomyopathy. Preclinical data for this program has been presented, and the company has rationally designed novel capsids with increased tropism for cardiac muscle.

• Rocket Pharmaceuticals: Utilizes AAV platforms for disorders affecting the heart.

  • RP-A501: An AAV9-based gene therapy for Danon Disease, delivering the wildtype human LAMP2B gene intravenously. It has entered pivotal trials.
  • RP-A601: An AAV vector-based gene therapy for PKP2-related Arrhythmogenic Cardiomyopathy (PKP2-ACM). It has shown the ability to stabilize or improve heart function in a Phase 1 clinical trial.
  • Also has a preclinical program targeting BAG3-associated dilated cardiomyopathy (DCM).

• Medera / Sardocor Corp.: Medera is a clinical-stage company with a broad pipeline of cardiovascular indications based on AAV-based treatments. Sardocor Corp. is solely owned by Medera.

  • SRD-001: An AAV-mediated first-in-human gene therapy for HFpEF. It is directly delivered to cardiac ventricular muscle cells via a proprietary intracoronary delivery technique. Patients have been dosed in a Phase 1/2A clinical trial.

• Asklepios Biopharmaceutical, Inc. (AskBio):

  • AB-1002 (NAN-101): A chimeric AAV (AAV2i8) based gene delivery of constitutively active inhibitor-1 for patients with congestive heart failure. This is a Phase 1 open-label, dose-escalation study using intracoronary delivery.

Table 1. Key companies work on cardiovascular AAV gene therapy

Experimental Considerations to deliver AAVs to the Heart

For researchers embarking on AAV gene therapy studies in preclinical models of heart disease, a comprehensive thought process for experimental design is paramount. Recognizing the ongoing need for deeper insights into mechanisms of AAV uptake and efficient gene delivery, and drawing upon extensive experience in successful AAV delivery to the heart, the following considerations have been meticulously compiled (Figure 2). These points are designed to guide investigators through critical decisions, ensuring robust study design and meaningful outcomes for AAV-based therapeutic development in this challenging area.

1. AAV Serotype Selection: Choose an appropriate AAV serotype for cardiac targeting. As mentioned, AAV1, AAV6, and AAV9 are commonly used for heart-specific gene delivery. Newer variants or engineered AAV capsid may also offer superior transduction efficiency.
2. AAV Delivery Method: Consider the most appropriate method of AAV delivery for effective cardiac transduction. The route significantly impacts cardiac and off-target transduction and must align with the species and disease model.
3. In Vitro Gene Functionality Test: Confirm the desired biological activity of the delivered DNA cargo in vitro.
4. Dose Optimization and Controls: Before disease model studies, optimize the AAV dose in a wildtype preclinical model (same animal strain, sex, species, age) under basal conditions. This helps mitigate issues related to vector toxicity, immunogenicity, and insufficient target gene expression in cardiac tissue. Always include appropriate controls.
5. Target Gene Expression Confirmation in Heart: Verify target gene expression in cardiac tissue using quantitative polymerase chain reaction (mRNA) or western blot (protein). If specific antibodies are unavailable, consider tagging the target protein for detection. Indirect readouts (e.g., altered downstream target activity) may also be appropriate.
6. Cardiomyocyte Transduction Efficiency Determination: Assess the proportion of cardiomyocytes expressing the AAV-delivered gene. This can involve including a known epitope tag (e.g., haemagglutinin, FLAG, c-myc, V5) fused to the recombinant protein, allowing detection via immunofluorescence. Confirm the tag does not impair protein functionality.
7. AAV Genome Quantification in Cardiac Tissue: Quantify absolute vector genome copy number per microgram of gDNA in experimental cardiac samples. This is achieved using real-time PCR with SYBR Green dye, AAV promoter-specific primers, and a standard curve generated from known quantities of plasmid AAV DNA.
8. Assess Therapeutic Efficacy: Evaluate the therapeutic impact on cardiac function (e.g., echocardiography, hemodynamic measurements), structural remodeling (e.g., hypertrophy, fibrosis, cellular changes via histology), and relevant metabolic parameters of heart disease. Consider longitudinal assessments to capture long-term effects and disease progression.
9. Off-Target AAV Genome Detection: Check for AAV genomes in biopsies of non-cardiac tissues (e.g., liver, lung, spleen, skeletal muscle, adipose tissue, kidney, brain) at the study endpoint using the method detailed in consideration #7. Additionally, investigate target gene or AAV expression in these tissues to assess the tissue-specificity of the gene therapy.

Figure 2. Experimental considerations for preclinical AAV Gene therapy studies in cardiovascular disease

Conclusion:

AAV gene therapy stands at the forefront of cardiovascular innovation, holding transformative potential for a wide range of debilitating heart conditions, including various forms of heart failure and inherited cardiomyopathies. While initial efforts in this field revealed significant hurdles, particularly concerning optimal AAV delivery efficiency and achieving precise AAV vector tropism, the landscape has evolved dramatically.

Through dedicated research and development, a new era of precision genetic medicine is rapidly emerging. This progress is largely driven by the design of advanced second-generation AAV capsids that offer improved targeting and transduction profiles, alongside the development of smarter, more effective AAV delivery methods (like retrograde infusion techniques) that enhance vector delivery to the myocardium while minimizing off-target effects.

Despite these significant advancements, the successful and widespread adoption of AAV gene therapy in human cardiology will depend on overcoming several critical challenges:

• Development of Cardiomyocyte-specific, high-efficiency vectors: Ensuring that therapeutic genes are delivered precisely and effectively to the intended heart muscle cells, with minimal impact on other tissues. PackGene has the full capability to design, construct, and produce high-quality AAV vectors for research and clinical use.
• Creation of minimally invasive yet effective delivery systems: Refining administration techniques to be less burdensome for patients while maximizing therapeutic gene transfer.
• Rigorous long-term safety monitoring: Establishing robust surveillance mechanisms to track patient outcomes and identify any unforeseen long-term effects.

With numerous promising programs currently advancing through various clinical stages, the next five years are poised to be pivotal. We anticipate that these ongoing efforts will finally see the realization of AAV gene therapy as a critical and routine clinical mainstay in the management and treatment of complex cardiovascular diseases.

Reference:

1. Rode L, et al. AAV capsid engineering identified two novel variants with improved in vivo tropism for cardiomyocytes. Mol Ther. 2022;30(8):2769-2782.
2. Chan C, et al. Rational design of AAV-rh74, AAV3B, and AAV8 with limited liver targeting. Viruses. 2023;15(9):2168.
3. Katz MG, et al. Efficient cardiac gene transfer and early-onset expression of a synthetic adeno-associated viral vector, Anc80L65, after intramyocardial administration. J Thorac Cardiovasc Surg. 2022;163(6):e417-e429.
4. Zsebo K, et al. Long-term effects of AAV1/SERCA2a gene transfer in patients with severe heart failure: analysis of recurrent cardiovascular events and mortality. Circ Res. 2014;114(1):101-108.
5. Pleger ST, et al. Cardiac AAV9-S100A1 gene therapy rescues post-ischemic heart failure in a preclinical large animal model. Sci Transl Med. 2011;3(92):92ra64.
6. Ishikawa K, et al. Cardiac I-1c overexpression with reengineered AAV improves cardiac function in swine ischemic heart failure. Mol Ther. 2014;22(12):2038-2045.
7. Woitek F, et al. Intracoronary cytoprotective gene therapy: a study of VEGF-B167 in a pre-clinical animal model of dilated cardiomyopathy. J Am Coll Cardiol. 2015;66(2):139-153.
8. Zhang H, et al. AAV-mediated gene therapy: advancing cardiovascular disease treatment. Front Cardiovasc Med. 2022;9:952755.
9. Chamberlain K, et al. Cardiac gene therapy with adeno-associated virus-based vectors. Curr Opin Cardiol. 2017;32(3):275-282.
10. Yamada KP, et al. Consideration of clinical translation of cardiac AAV gene therapy. Cell Gene Ther Insights. 2020;6:785-798.
11. Kim Y, et al. Gene therapy in cardiovascular disease: recent advances and future directions in science: a science advisory from the American Heart Association. Circulation. 2024;150(23):e471-e480.
12. Ishikawa K, et al. Human cardiac gene therapy. Circ Res. 2018;123(5):601-613.
13. Katz MG, et al. Use of adeno-associated virus vector for cardiac gene delivery in large-animal surgical models of heart failure. Hum Gene Ther Clin Dev. 2017;28(3):157-164.
14. Ishikawa K, et al. Human gene therapy clinical development. Circ Res. 2018;123(5):599-600.
15. Weeks KL. Adeno-associated viruses as gene delivery tools for diabetic heart disease and failure: key considerations for clinicians and preclinical researchers. Heart Lung Circ. 2025;34(2):118-124.