Bioengineering of highly effective AAV vectors for noninvasive gene delivery to the nervous system

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Abstract: This project is designed to identify a noninvasive and highly effective gene delivery strategy to target specific neural cells in the CNS and to validate this technology by delivering regenerative molecules to mammals with CNS injury. We will determine whether systemic delivery of our newly engineered AAV9 vectors can transduce most target CNS cells and whether noninvasive delivery of the genes that target neuronal intrinsic and extrinsic factors can promote robust axon regeneration and functional recovery in rodents with spinal cord injury (SCI). A major challenge in neuroscience research is to deliver target genes to specific types of neural cells widely distributed in CNS. Engineered AAV9 vectors usually show limited efficacy after intravenous (IV) injection by transducing cells only in some CNS regions. We thus created new AAV9 vectors that include multiple features of engineered AAV9 capsids, aiming to develop highly efficient BBB-crossing AAV9 vectors (HEBC-AAV9) that can transduce most target CNS cells after IV injection. In Aim 1, we will study efficiency of our novel HEBC-AAV9-GFP vectors for selectively transducing each type of neural cells (neurons, astrocytes, oligodendrocytes, and microglia) in several strains of adult mice. To solve a crucial issue in neuroscience research with this technology, in Aim 2 we will develop a regenerative therapy for SCI by systemic delivery of genes to target neuronal let-7 miRNA. After SCI, severed axons fail to regenerate partly because of reduced intrinsic growth capacity of mature neurons. Many genes are known to control the growth ability of mature neurons, but none have been translated to clinical use. The best targets are probably those with potential to impact multiple genes. Among them, let-7 is important for regulating age-dependent decline in axon regeneration in worms. In Aim 2, we propose to use unique HEBC-AAV9-synapsin vectors to target neurons selectively for inducing expression of let-7 inhibitor, lin28, and lin41, aiming to promote robust regeneration of multiple axon tracts by enhancing growth capacity of mature neurons in SCI rodents. Chondroitin sulfate proteoglycans (CSPGs) generated by glial scars strongly suppress axon extension and are major extrinsic molecular targets for treating CNS injury. Our lab designed small peptides to block functions of CSPG receptor LAR, PTPσ, and PTPδ by targeting their critical activity domains and demonstrated their high efficiency for promoting axon regrowth. In Aim 3, we will induce astrocytic expression of secreted 3 peptides for each of LAR, PTPσ, and PTPδ with HEBC- AAV9-GFAP vectors, aiming to promote robust axon regeneration after SCI by targeting extrinsic CSPGs alone or combined with intrinsic let-7 signals. Our new viral vectors should provide a powerful tool for gene delivery in CNS and for developing effective regenerative therapies for SCI and other neurological disorders.