Ashley T. Martino

Capsid Mutants in Adeno-Associated Gene Therapy Viral Vectors (AAV) Have Potential to Avoid Immune Responses and Improve Gene Correction

Ashley T. Martino, College of Pharmacy and Health Sciences, Department of Pharmaceutical Sciences; Graduate Student: Enoch Bijjiga

Gene therapy clinical trials using adeno-associated virus (AAV) vectors are compromised by host immunity against corrected cells and/or vector themselves. This impedes long-term gene-correction even though it is effective short-term. This issue can be addressed by using a stealth AAV vector with capsid point mutations that will potentially increase gene-correction with lower immunity. A triple mutant on the AAV capsid replaces 3 tyrosine residues with 3 phenylalanines. This minimizes proteasomal degradation by bypassing tyrosine phosphorylation and ubiquitination. This will increase gene transfer to the nucleus and decrease the likelihood of activating cytotoxic T-cells. Triple tyrosine mutation of AAV-2 (AAV-TM) is found to increase the transgene expression in liver. In this study, AAV-1 and AAV-5 vector mutants will be developed and studied on skeletal muscle of hemophilic mice and emphysema induced lung epithelium respectively. Immunological T-cell responses against AAV-TM will be determined in a mouse model by measuring cytotoxicity of capsid specific CD8+ T cells and in vitro by a CTL assay which measures cell death of AAV-TM infected target cells when co-cultured with cap-CD8+ T-cells. Assessment of AAV-1 mutant to treat hemophilic mice by gene correction of skeletal muscle will be done by measuring the levels of corrected coagulation FIX (hFIX) in blood and determining blood clotting time. The therapeutic effect levels of AAV-5 mutant vector expressing alpha-1-antitrypsin (hA1AT) on lung epithelium of mice with induced COPD (chronic obstructive pulmonary disorder) will be determined by measuring hA1AT in blood and lung BALF (bronchial lavage fluid), assessing degree of alveolar dilation and epithelial damage, determining lung epithelial inflammation and measuring apoptosis. Following this study, using AAV capsid mutants to avoid immunity and achieve therapeutic levels of corrective protein will be established.