Optimization of AAV6 transduction enhances site-specific genome editing of primary human lymphocytes

• Published in: Molecular therapy. Methods & clinical development Vol. 23; pp. 198 - 209
• Main Authors: Rogers, Geoffrey L., Huang, Chun, Clark, Robert D.E., Seclén, Eduardo, Chen, Hsu-Yu, Cannon, Paula M.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 10.12.2021; American Society of Gene & Cell Therapy; Elsevier
• Subjects: AAV; B cell; genome editing; HSPC; Original; T cell; ZFN; genome editing; B cell; ZFN; AAV; T cell; HSPC
• ISSN: 2329-0501; 2329-0501
• DOI: 10.1016/j.omtm.2021.09.003

Adeno-associated virus serotype 6 (AAV6) is a valuable reagent for genome editing of hematopoietic cells due to its ability to serve as a homology donor template. However, a comprehensive study of AAV6 transduction of hematopoietic cells in culture, with the goal of maximizing ex vivo genome editing, has not been reported. Here, we evaluated how the presence of serum, culture volume, transduction time, and electroporation parameters could influence AAV6 transduction. Based on these results, we identified an optimized protocol for genome editing of human lymphocytes based on a short, highly concentrated AAV6 transduction in the absence of serum, followed by electroporation with a targeted nuclease. In human CD4+ T cells and B cells, this protocol improved editing rates up to 7-fold and 21-fold, respectively, when compared to standard AAV6 transduction protocols described in the literature. As a result, editing frequencies could be maintained using 50- to 100-fold less AAV6, which also reduced cellular toxicity. Our results highlight the important contribution of cell culture conditions for ex vivo genome editing with AAV6 vectors and provide a blueprint for improving AAV6-mediated homology-directed editing of human T and B cells. [Display omitted] Adeno-associated virus 6 (AAV6) vectors support site-specific genome editing in human hematopoietic cells treated with targeted nucleases by providing homology donor templates, but efficiency in lymphocytes—particularly B cells—is limited by inefficient transduction. Rogers et al. present an optimized protocol for AAV6 transduction of human B and T cells that greatly improves genome editing in these cell types.