Adeno-Associated Viruses (AAV) have emerged as the vector platform of choice for therapeutic gene transfer towards multiple genetic disorders with neurological manifestations. My doctoral thesis was focused on a) evaluating CNS spread, transduction profiles, receptor interactions and clearance; and b) understanding the physiological and biochemical checkpoints governing AAV biologics in the CNS. In our first study, we engineered an AAV4 variant (AAV4.18) that shows expanded tropism from ependymal cells to migrating progenitors in the developing brain. AAV4.18 revealed a striking shift in glycan engagement from 2,3-linked Sialic acid (SA) to 2,8-linked Polysialic acid (PSA). PSA is an important biomarker of neurogenesis. We also report opposing roles of SA and PSA on CNS transduction of AAV4. Overall, carbohydrate content can be exploited to regulate viral tropism in the brain. We then evaluated a lab-derived chimeric AAV2g9, developed through rational vector design. Direct brain injections of AAV2g9 resulted in widespread neuronal transduction with reduced glial tropism. As compared to AAV9, AAV2g9 displayed minimal systemic leakage and off-target gene expression within peripheral organs. We utilized AAV2g9 for brain-specific deletion of ubiquitously expressed MIR137 schizophrenia risk gene using CRISPR/Cas9 technology. Our approach exemplifies control over AAV tropism at both cellular and organ levels to potentially improve vector safety. Lastly, AAV mediated CNS gene therapy requires a deeper understanding of factors affecting transduction efficiency and clearance. Dysfunction in aquaporin-4 (AQP4) mediated CSF flux during aging or disease is correlated with ineffective solute clearance from the brain. Aged mice showed mislocalized AQP4 expression and increased AAV9 deposition following intraCSF administration. We further compared wildtype (WT) and AQP4 knockout (AQP4-/-) mouse CNS. Minutes after intraCSF administration, AQP4-/- mice exhibited highly restricted spread of fluorophore labeled AAV9. Transgene expression was markedly increased 2 weeks post AAV9 administrations in AQP4-/- mice. Further, AQP4-/- mice showed markedly reduced AAV biodistribution and transgene expression in peripheral organs. This suggests that AQP4-deregulation affects CNS spread, transduction efficiency and systemic leakage of AAV vectors. We hypothesize that altered CSF flux under conditions of aging, CNS disease or injury can impact AAV residence time and gene transfer efficiency.