Research Projects

Rationale: Spinal muscular atrophy (SMA) is an autosomal recessive neurological disease caused by mutations in SMN1 gene, causing muscle atrophy, weakness and paralysis. Current gene therapies, such as FDA approved, Adeno-associated virus (AAV) serotype 9 based gene therapy (Zolgensma), does not provide a lasting cure. They are further associated with severe adverse events such as hepatotoxicity. Open FDA database has reported 100 deaths worldwide after therapy. Given the high cost of treatment (USD 2 million per dose) and the significant drawbacks associated with current treatment, we propose multiple overlapping strategies to establish optimal vectors that can be utilised for SMA treatment. We seek to understand and overcome (i) the viral vector specific rate limiting post translational modifications (PTM) on vector capsid that prevent successful central nervous system (CNS)/muscle cell transduction, (ii) modify viral capsid epitopes that lead to T cell or B cell activation (iii) establish SMA patient’s genotype and immune status, so that they could be lead candidates in future, for receiving the novel gene therapy products developed in this project. Objectives: 1. To generate bioengineered AAV8 or AAV9 vectors carrying an optimized sequence of human SMN1 gene and validate them in vitro and in vivo. 2. Demonstrate the safety of engineered vectors for clinical translation by comprehensive evaluation in murine models of SMA. 3. Perform genotype-phenotype correlation and immune profiling in SMA patients (n=100) in India for disease prognosis and selection of candidates for clinical trials. Hypothesis We hypothesize that a combination of engineered muscle-specific and CNS-specific vectors (AAV8 /AAV9) developed in this project, and a comprehensive knowledge of underlying genetic/ immune status of the SMA patients in India will significantly contribute to the long-term safety and efficacy of SMA gene therapy. Methods: Objective 1: To maximize the yield of SMN protein particularly in central nervous system and muscle, we will codon-optimize the sequence of human SMN1 gene and package it in modified AAV8 and AAV9 capsids. These vectors will be tested for their infectivity in vitro in neuronal and muscle cell lines. The in vivo validation of these vectors will be performed in a murine model of SMA. For assessment of motor and functional outcomes a series of in vivo assays will be performed. Objective 2: To determine the pre-clinical safety profile we will perform several dosage escalation studies and evaluate different parameters like systemic biodistribution of vectors, off-target effects, immunogenicity, toxicology and vector shedding. The goal will be to find the optimum dosage which results in maximum survival and minimal side effects. Objective 3: We will perform molecular profiling of various genetic and immune markers in SMA patients (n=100). This will improve disease prognosis and may uncover novel therapeutic biomarkers. Further, it will be helpful in patients stratification for future gene therapy clinical trials. Expected outcome: • Phenotypic response in animal models: We expect increased survival of treated mice, compared to untreated mice. The untreated mice has an average life span of only 15 days. We also expect improved motor function and functional outcomes in treated mice. Finally, with comprehensive biodistribution studies, a proof-of-concept will be established demonstrating the efficacy and safety of bioengineered vectors in murine models of SMA.