Duchenne muscular dystrophy (DMD) is the most common form of inherited muscle disease. It usually leads to death from respiratory or cardiac failure by age 20. Currently, no effective treatment is available for this fatal disease. DMD is an X-linked genetic disease caused by dystrophin gene mutation. Gene therapy represents a very promising avenue to cure DMD. Recombinant adeno-associated virus (rAAV) mediates high-level persistent transgene expression in muscle. Recent clinical trials have further confirmed the efficiency and the safety of rAAV vectors in muscle. However, rAAVmediated DMD gene therapy has been significantly limited by the small viral packaging capacity. Only the highly truncated C-terminal-deleted versions of """"""""micro-dystrophin"""""""" genes have been attempted. Both clinical and transgenic studies show that the C-terminal-inclusive larger genes (such as the 6.0-6.3kb """"""""mini-dystrophin"""""""" genes and the approximately 4.7kb """"""""C-terminal-inclusive micro-dystrophin"""""""" genes) are therapeutically superior. Unfortunately the strong therapeutic expression cassettes derived from these genes are too large to be packaged in a single AAV virion. We have recently developed several dual vector approaches to expand AAV packaging capacity. Among these, the concatamerization-based """"""""trans-splicing"""""""" and """"""""cis-activation"""""""" strategies hold great promise for delivering the C-terminal-inclusive larger dystrophin genes. However, the expression level achieved so far is not sufficient for DMD gene therapy. In this proposal, we plan to extend our previous findings and further explore the molecular mechanisms underlying these methods, in the hope of improving the transduction efficiency for DMD gene therapy. In particular, we will try to identify and overcome the rate-limiting barriers to transgene expression. These include problems associated with dual vector co-infection, concatamerization of AAV genome inside cell, and transcription, splicing, and stability of AAV concatamers. More important, we will apply this newly obtained information to generate the most effective trans-splicing and cis-activation AAV vectors for the C-terminal-inclusive larger dystrophin genes. Therapeutic potentials of these newly developed AAV vectors will be rigorously tested in the limb muscle, diaphragm, and heart of the murine DMD model (mdx mouse). A comprehensive array of assays will be used to examine the level of gene expression and the functional improvement in muscle histology and contraction. To address safety concerns, we also plan to evaluate the potential deleterious effects from putative truncated protein production in the trans-splicing method. Taken together, our findings will lead to the eventual application of these very promising dual AAV vector strategies to the human DMD gene therapy.
Showing the most recent 10 out of 74 publications
