Recombinant adeno-associated viral (AAV) vectors are a promising gene delivery platform, but ongoing clinical trials continue to highlight a relatively narrow therapeutic window. Effective clinical translation is confounded, at least in part, by differences in AAV biology across animal species. Here, we tackle this challenge by sequentially evolving AAV capsid libraries in mice, pigs and macaques. We discover a highly potent, cross-species compatible variant (AAV.cc47) that shows improved attributes benchmarked against AAV ser- otype 9 as evidenced by robust reporter and therapeutic gene expression, Cre recombination and CRISPR genome editing in normal and diseased mouse models. Enhanced transduction efficiency of AAV.cc47 vectors is further cor- roborated in macaques and pigs, providing a strong rationale for potential clinical translation into human gene therapies. We envision that ccAAV vectors may not only improve predictive modeling in preclinical studies, but also clinical translatability by broadening the therapeutic window of AAV based gene therapies.Genetic engineering of T cells in mouse models is essential for investigating immune mechanisms. We aimed to develop an approach to manipulate T cells in vivo using an evolved adeno-associated virus (AAV) capsid named Ark313. Delivery of a transient transgene expression cassette was feasible using Ark313, and this serotype outperformed natural serotypes. A single intravenous injection of a Cre recombinase-expressing Ark313 in the Ai9 fluorescent reporter mouse model achieved permanent genetic modifications of T cells. Ark313 facilitated in vivo gene editing in both tissue-resident and splenic T cells and validation of immunotherapy targets in solid tumor models. Ark313 delivered large DNA donor templates to T cells in vivo and integrated transgenes in primary CD4+ and CD8+ T cells, including naive T cells. Ark313-mediated transgene delivery presents an efficient approach to target mouse T cells in vivo and a resource for the interrogation of T cell biology and for immunotherapy applications.
The difficulty of delivering genes to the kidney has limited the translation of genetic medicines, particularly for the more than 10% of the global population with chronic kidney disease. Here we show that new variants of adeno-associated viruses (AAVs) displaying robust and widespread transduction in the kidneys of mice, pigs and non-human-primates can be obtained by evolving capsid libraries via cross-species cycling in different kidney models. Specifically, the new variants, AAV.k13 and AAV.k20, were enriched from the libraries following sequential intravenous cycling through mouse and pig kidneys, ex vivo cycling in human organoid cultures, and ex vivo machine perfusion in isolated kidneys from rhesus macaques. The two variants transduced murine kidneys following intravenous administration, with selective tropism for proximal tubules, and led to markedly higher transgene expression than parental AAV9 vectors in proximal tubule epithelial cells within human organoid cultures and in autotransplanted pig kidneys. Following ureteral delivery, AAV.k20 efficiently transduced kidneys in pigs and macaques. The AAV.k13 and AAV.k20 variants are promising vectors for therapeutic gene-transfer applications in kidney diseases and transplantation.Gene therapy for neurological disorders requires effective viral transduction within the human central nervous system (CNS). To date, CNS-targeting adeno-associated viral (AAV) capsids have been developed exclusively in preclinical animal models, in turn yielding vectors with potentially limited utility/efficacy in human clinical settings. Here, to devise novel capsid variants optimized for human CNS, we used organotypic human brain tissue explants to perform structure-guided evolution of AAVs. Capsid libraries based on AAV8 or AAV9, with mutations in two distinct surface epitopes, were cycled on human neocortical tissue explants, which were then collected for genetic and histochemical analyses of viral transduction. Barcode analysis of pooled capsid candidates revealed the enrichment of distinct serotype and epitope-dependent phenotypes: specifically, while AAV8 variants exhibited only moderately increased viral uptake, they were robustly more effective at yielding tissue expression of payload RNA in both neuronal and non-neuronal cell types. AAV9 variants, on the other hand, particularly those harboring the integrin-binding (RGD) motif, showed a strong correlation between tissue uptake and payload expression. AAV9 variants also exhibited increased tropism for non- neuronal cell types in human gray and white matter. By affording increased efficacy and tropism for human CNS populations, these human-optimized) AAV capsids may hold increased utility for research and clinical applications. More broadly, our ex vivo approach to human-tailored capsid generation represents a powerful strategy for optimizing gene therapy vectors.
Grants and other efforts supported by GTRC
The Duke Gene Therapy Resource Core has supported multiple federal funded grants: 3x RO1s, 1 UH3 and a UO1
Two Duke spinoff companies in the form of SRAs and the Danaher Beacon project
