Development of optimized AAVrh74 vectors for gene therapy of muscular dystrophies

点击翻译按钮获取中文摘要

PROJECT SUMMARY/ABSTRACT: Recombinant adeno-associated virus (AAV) vectors are currently being used in three separate clinical trials for gene therapy of Duchenne muscular dystrophy (DMD), sponsored by Solid Biosciences, Pfizer, Inc., and Sarepta Therapeutics, respectively. The use of the first generation of AAV9 vectors in both Solid Biosciences and Pfizer trials have led to a number of serious adverse events, including the death of a patient in the Pfizer trial. Although the first generation of AAVrh74 vectors in the Sarepta trial were shown to be tolerated well, a recent Phase II trial in patients with DMD failed to meet its primary functional endpoint. We have argued, based on the fact that AAV evolved as a virus, and not as a vector for the purposes of delivery of therapeutic genes, and thus, the full potential of the first generation of recombinant AAV vectors is unlikely to be realized. By modifying the AAV capsid protein, we have developed the next generation (‘NextGen’) of AAV vectors, which are up to 30-fold more efficacious at reduced doses. These vectors are also less immunogenic. We have also modified the AAV vector genome to develop the generation X (‘GenX’) AAV vectors. These vectors mediate up to 8-fold enhanced transgene expression. We have combined both these strategies to generate the optimized (‘Opt’) AAV serotype vectors. These vectors are 20-30-fold more efficient at further reduced doses. We have also documented that inclusion of specific regulatory elements within the AAV inverted terminal repeats can significantly increase transgene expression levels. In this application, we propose to develop the Opt AAVrh74 vectors, and test the following hypotheses: (i) Liver de-targeted Opt AAVrh74 vectors containing novel muscle- specific enhancer elements will lead to further increased levels of transgene expression at significantly lower doses; and (ii) Opt AAVrh74 vectors can be delivered to target muscle cells at significantly reduced doses following systemic administration in a mouse model of DMD in vivo. The following three specific aims will be pursued: Specific Aim 1: Development of capsid+genome-modified, liver de-targeted Opt AAVrh74 vectors for high-efficiency transduction of primary human skeletal muscle cells in vitro, and in a mouse model in vivo. Specific Aim 2: Development of novel Opt AAVrh74 vectors with muscle-specific enhancer elements to augment transgene expression at further reduced doses in a mouse model in vivo. Specific Aim 3: Development of a novel Opt AAVrh74-micro-dystrophin vector (Opt74-µDys) for systemic gene therapy of DMD in a mouse model in vivo. The development of optimized AAVrh74 vectors that are effective at lower doses, are likely to reduce the probability of inducing the host immune responses, the vector production costs, as well as the economic cost per patient, for the potential gene therapy of muscular dystrophies in humans.