Figure 1.
AAV6 Is Superior to AAVHSCs in Facilitating HDR in CD34+ Cells and Requires a Double-Strand Break
(A) Table of tested AAV capsid variants and corresponding AAVHSC variant name from Smith et al.2 (B) Diagram of donor template packaged into the tested AAV capsids, containing GFP driven from UBC promoter with flanking human beta-globin homology arms and AAV ITRs. Episomal expression from the AAV genome facilitates low-intensity GFP fluorescence (top), while expression after HDR facilitates long-lived high-intensity GFP fluorescence (bottom).27 (C) Molecular modeling of location of AAVHSC variants in relation of parental capsid AAV9. Outer (top) and inner (bottom) surface representation of AAV9 capsid crystal structure: individual viral protein (VP) monomers are highlighted in white, black, or gray and HSC point mutations are highlighted magenta. (D) Gating strategy used for quantification of episomal or HDR-mediated GFP expression in CD34+ HSPCs. Samples from one representative experiment are shown. (E) Quantification of episomal or HDR-mediated GFP expression with standard error measurements from three individual donors, quantified 12–14 days post-transduction. Episomal expression was calculated by subtracting GFP high (HDR) cells from total GFP-positive cells after using AAV6+RNP as gating control for cells that underwent HDR.