Cross-Species Evolution of New AAV Variants

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2023

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Abstract

Therapeutic gene transfer and genome editing require effective delivery of genetic cargo to target cells and tissues. Recombinant adeno-associated viral (AAV) vectors are a promising delivery platform, but ongoing clinical trials continue to highlight a relatively narrow therapeutic window. Efforts to optimize vector dosing or engineer improved vectors are confounded, at least in part, by differences in AAV biology across animal species. Here, we present a broadly applicable, cross-species evolution approach to tackle this challenge. Specifically, I iteratively cycled AAV libraries administered intravenously and amplified isolates from CNS tissue in pigs, mice, and non-human primates to generate cross-species compatible AAVs (ccAAVs). By sequentially evolving AAV libraries in three different species, we discover a highly potent variant (AAV.cc47) that demonstrates improved attributes benchmarked against AAV serotype 9 (AAV9). Increased potency of AAV.cc47 is evidenced through robust reporter gene expression as well as Cre-mediated recombination and CRISPR/Cas9-mediated genome editing in a fluorescent reporter mouse model. Enhanced transduction efficiency of AAV.cc47 vectors is further corroborated in macaques and pigs, providing a strong rationale for potential clinical translation into human gene therapies. Lastly, we report increased expression of a therapeutic acid alpha-glucosidase (GAA) transgene in a mouse model of Pompe disease and enhanced restoration of dystrophin through CRISPR/Cas9 gene editing in the mdx mouse model of Duchenne Muscular Dystrophy using AAV.cc47 vectors. We discovered another cross-species compatible variant (AAV.cc84) with enhanced CNS transduction and de-targeted from the liver in vivo compared to AAV9. We demonstrate improved targeting of neurons in the brain following both systemic and intracerebroventricular (ICV) injection of reporter vectors in mice. Reporter gene expression and vector genome biodistribution reveal a liver detargeted phenotype with AAV.cc84 compared to AAV9. Lastly, enhanced neuronal transduction was confirmed in the pig CNS following intrathecal infusion of AAV.cc84 reporter vectors. Taken together, we envision that ccAAV vectors can potentially improve predictive modeling in preclinical studies as well as clinical translatability by broadening the therapeutic window of AAV based gene therapies.

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Gonzalez, Trevor John (2023). Cross-Species Evolution of New AAV Variants. Dissertation, Duke University. Retrieved from https://hdl.handle.net/10161/27589.

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