Gene therapy has the potential to treat or cure a variety of monogenetic diseases. Adeno-associated virus (AAV), a human parvovirus, is a particularly promising vector for several reasons. AAV is non-pathogenic, capable of site-specific integration into the host cell genome, and long-term gene expression. However, a relatively small genetic payload, roughly 4.7kb or 2.3kb for self-complimentary vectors, limits its potential as a gene therapy vector. AAV is classified as a dependovirus, because its gene expression is dependent on gene products of a helper virus, further recombinant vectors do not integrate predictably into the host cell genome and are maintained as episomes, like the parental virus. The very broad tropism of the wild type virus is both beneficial and detrimental to its development as a therapeutic, because it can transduce a large number of cells however is limited in its ability to specifically transduce single cell types. We will examine the potential to narrow the vector's tropism to motor neurons for the purpose of treating neurodegenerative disease. An ideal vector will exhibit a high specificity for motor neurons as well as a high level of retrograde transport, a characteristic AAV already exhibits although inefficiently.;The ability to modify the natural tropism of AAV could greatly enhance its potential as a gene therapy vector. Previous studies have shown that AAV will tolerate genetic insertion of short linear peptides at specific locations in the capsid proteins. A recently identified 12 amino acid peptide, tet1, exhibits a binding capacity similar to that of tetanus toxin c-terminal fragment, for the Gt1b receptor located at the neuro-muscular junction of motor neurons. We have shown that genetic insertion of this peptide increases AAV's specificity for motor neurons, while simultaneously reducing its specificity for non-neuronal cells. However, modifications can result in poor capsid protein folding or assembly. Recent molecular dynamics models predict targeting ligand and "linker sequences" least likely to negatively effect capsid folding and assembly. Our tet1-modified vector does indeed exhibit markedly decreased vector yield relative to unmodified vector. We will examine the potential of alternate insertion sites to alleviate the decrease in vector yield while maintaining motor neuron specificity. Another approach to achieve effective transduction of spinal and peripheral motor neurons may involve intravenous injection of recombinant AAV9 vector. Previous studies suggest that rAAV9 transduces motor neurons both after crossing the blood brain barrier (BBB) and peripheral vascular endothelium. However, other studies suggest that rAAV9 preferentially transduces astrocytes in vivo. We hypothesize that incorporation of our tet1 peptide in the capsid monomers of AAV9, guided by our understanding of sites that tolerate short linear peptide inserts in rAAV2 and rAAV1, will increase the specificity of rAAV9 for both spinal and peripheral motor neurons.
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