Myology 2005 -Thursday, May 12th - Plenary lecture "Systemic delivery of genes to striated muscle using AAV "  

 

We have been exploring functional domains of dystrophin to develop a better understanding of the structural requirements for preserving muscle function. These studies have defined a variety of domains of dystrophin that are essential for functional activity, as well as several that are minimally required.  The majority of the dystrophin protein appears to play a mechanical role in muscle by linking the subsarcolemmal gamma-actin cytoskeleton to the extracellular matrix.  Actin binding is mediated by the N-terminal domain and portions of the spectrin-like repeat domain. Deletions in the N-terminal domain reduce actin binding and destabilize dystrophin, resulting in reduced mechanical protection to myofibers.  Most of the spectrin-like repeat domain can be deleted with minimal impact on mechanical protection, and a combination of spectrin-repeats that include the immediate N-terminal repeats and the terminal repeat appear capable of significant mechanical protection and flexibility.  The cysteine-rich domain encoded on exons ~62-70 binds dystroglycan and is absolutely required for function in its entirety.  Surprisingly, the C-terminal domain is redundant and can be deleted.  Since this latter region binds the syntrophins and dystrobrevins, we explored the signaling role of dystrophin by generating transgenic mice that express Dp116 in muscle.  Dp116 expression restores expression of the entire dystrophin-glycoprotein complex (DGC) except for nNOS, yet mdx muscles expressing Dp116 display a worse pathology than do mdx muscles.  Transfer of micro-dystrophins lacking 20 spectrin-like repeats and the C-terminal domain (?R4-R23/?CT) to muscles of mdx mice shows that the DGC is restored to the sarcolemmal membrane with the exception of nNOS. Interestingly, expression of larger dystrophin mini-proteins, such as?H2-R19, also fails to restore expression of nNOS, despite an apparent complete functional rescue of the muscles. These studies suggest that signaling roles for the DGC are relatively unimportant in preventing the development of dystrophic pathology and argue for a primarily mechanical role for dystrophin and the DGC.Based on these observations we have been developing methods to deliver dystrophin to muscle using AAV vectors.  Recombinant AAV vectors pseudotyped with the serotype 6 capsid (rAAV6) efficiently transduce striated muscles. These vectors have a cloning capacity of about 5 kb, necessitating the use of highly truncated “micro-dystrophin” cDNA clones.  Intravenous injection of rAAV6 vectors into mice at doses greater than 10E12 results in efficient transduction of all striated muscles of mice when the CK6 promoter is used.
Lower doses can be used with stronger promoters, such as the full CMV promoter/enhancer.  VEGF-165 increases muscle transduction at doses up to ~10E11, but has little effect when used in conjunction with higher vector doses, suggesting a role for the AAV6 capsid in enhancing muscle transduction.  Preliminary studies indicate that rAAV6 is also highly efficient at transducing canine muscles, and that the systemic delivery method is applicable in that species.  AAV6 is able to deliver a variety of expression cassettes to dystrophic muscles, opening up multiple approaches to improving muscle function.  These studies will be discussed in the context of applying gene therapy approaches for DMD using truncated micro-dystrophins.