Accessing the Developing CNS: Advancing AAV-Mediated Prenatal Gene Editing in the Brain

Targeted gene delivery to defined neural populations is a central challenge in neuroscience and a major barrier to gene therapy for neurodevelopmental and congenital disorders. In the developing central nervous system, this is further complicated by dynamic changes in accessibility, vector tropism, and cellular composition across developmental stages. This thesis develops new approaches for systemic prenatal CNS access, capsid engineering, and in utero genome editing in the embryonic brain. We introduce UseqFISH, a spatial transcriptomic platform that enables high-sensitivity, multiplexed detection of endogenous transcripts and barcoded viral genomes in intact tissue at single-cell resolution. This allows quantitative in situ mapping of AAV tropism and scalable evaluation of viral libraries in embryonic contexts. Using new and existing techniques, we characterize systemic AAV biodistribution across development and show that adult brain-biased serotypes do not maintain specificity in late-mid gestation embryos. We engineer modified capsids, including “null” scaffolds with reduced native tropism, which can be reprogrammed via targeting motifs. High-throughput barcoded in vivo selection identifies sequence features associated with improved CNS enrichment during embryogenesis. To expand access to midgestational delivery, we develop a surgical method enabling systemic AAV administration at embryonic day 12.5 via uterine microdissection of the vitelline circulation, achieving widespread and viable transduction through gestational development. Finally, we established an in utero genome editing platform using AAV-mediated CRISPR and homology-directed repair. We demonstrate efficient gene disruption, epitope tagging, and precise genome modification, including endogenous tagging and introduction of disease-associated alleles across cortical cell types, enabling modeling of neurodevelopmental phenotypes. Together, this work provides a framework for engineering AAV-based gene delivery and genome editing in the developing CNS, advancing tools for studying brain development, modeling disease, and enabling early therapeutic strategies.