CMV-GFP AAV (Serotype Retrograde)

Directed evolution is a receptor-agnostic strategy based on the generation of AAV libraries containing cap gene mutations that confer novel tropisms on AAVs for unknown receptors on target cells. Mutated AAV capsids can be generated by error-prone PCR that introduces random point mutations, gene shuffling (DNA recombination between different AAV serotypes to generate genetic chimeric capsids), domain swapping (VR replacement between serotypes to create chimeric AAVs), or the use of degenerate oligonucleotides to generate random peptide insertions or substitutions. This highly diverse library of mutants is screened for enhanced properties through multiple rounds of in vitro or in vivo selection pressure and enrichment. Directed evolution has also been used to screen AAV variants capable of retrograde transport from neuronal axon terminals to the nucleus. The AAV2-derived rAAV2-retro was retrogradely transported to multiple regions of the mouse CNS after injection into the mouse CNS parenchyma, with a transduction capacity 133-fold greater than that of AAV2 and even higher efficiency than synthetic retrograde tracers. This novel variant has great potential in neuroscience research for probing neural circuit function and for administering vectors at small strategic locations to reach larger regions of the central nervous system. Furthermore, rAAV2-retro was administered into muscle tissue of neonatal mice, showing specific transduction of 57% of lower motor neurons projecting to muscle, and through retrograde transport, rAAV-retro achieved transduction of the spinal cord and brainstem, with some differences between subregions and DRG. Interestingly, rAAV-retro was able to diffuse through spinal nerves into the cerebrospinal fluid and was able to diffuse from blood vessels after intramuscular injection into the cerebrospinal fluid, ultimately transducing neurons in the spinal cord and reaching the central nervous system.