Functional gene delivery to and across brain vasculature of systemic AAVs with endothelial-specific tropism in rodents and broad tropism in primates

Abstract

Delivering genes to and across the brain vasculature efficiently and specifically across species remains a critical challenge for addressing neurological diseases. We have evolved adeno-associated virus (AAV9) capsids into vectors that transduce brain endothelial cells specifically and efficiently following systemic administration in wild-type mice with diverse genetic backgrounds, and in rats. These AAVs also exhibit superior transduction of the CNS across non-human primates (marmosets and rhesus macaques), and in ex vivo human brain slices, although the endothelial tropism is not conserved across species. The capsid modifications translate from AAV9 to other serotypes such as AAV1 and AAV-DJ, enabling serotype switching for sequential AAV administration in mice. We demonstrate that the endothelial-specific mouse capsids can be used to genetically engineer the blood-brain barrier by transforming the mouse brain vasculature into a functional biofactory. We apply this approach to Hevin knockout mice, where AAV-X1-mediated ectopic expression of the synaptogenic protein Sparcl1/Hevin in brain endothelial cells rescued synaptic deficits.

Adeno-Associated Viral Tools to Trace Neural Development and Connectivity Across Amphibians

“…Other differences in the performance of serotypes between species may be explained by highly evolvable residues on these proteins, or their recruitment of entirely different combinations of glycan and protein receptors. Our results extend observations in mammals, where comparisons across mouse strains and species of non-human primates have revealed substantial differences of AAV tropism 103 .…”

“…Despite having significant potential for research on lesser-studied species not amenable to transgenesis, broader use of AAVs has been limited. These viral vectors have only recently been employed in neuroscience applications in non-human primates 103,104 , birds 105–108 , and reptiles 109 . Previous experiments in zebrafish 110 and frogs 42 either did not find, or found negligible, AAV transduction in the brain.…”

Spatial genomics of AAVs reveals mechanism of transcriptional crosstalk that enables targeted delivery of large genetic cargo

“…Diversification of the AAV capsid through directed evolution has yielded a toolkit of AAVs with varied tissue tropism [11][12][13][14][15][16][17][18][19][20][21][22][23] . Further refinement of expression can be achieved through inclusion of regulatory elements, including enhancer sequences [24][25][26][27][28][29][30][31][32][33] .…”

“…Importantly, this paradigm does not require transgenic organisms, and so may be easily applied to a variety of models, including disease models where crossing of transgenics would not be feasible, as well as models where Cas9-expressing transgenics are not readily available. This approach is particularly exciting given the recent development of AAV capsids that provide genetic access to the nervous systems of non-human primates following systemic administration [15][16][17]23,65 and the identification of gene regulatory elements for cell type-specific expression across species 24,[27][28][29]33 . The small size of minimal promoters and terminator sequences allows easy integration of even large CRISPR effectors, including fusion proteins for AAV-based gene activation 66 , base editing 67 , or prime editing 68 .…”

“…Maintenance of the AAV genome as circular monomeric or concatemeric episomes provides long-term expression [6][7][8][9][10] . The tropism of AAVs can be altered by modifying residues on the AAV capsid surface and directed evolution has yielded a toolkit of capsids with diverse tropisms, including variants that can efficiently and broadly transduce target organs following systemic administration [11][12][13][14][15][16][17][18][19][20][21][22][23] .…”