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

Abstract

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.

Keywords: AAV; B cell; HSPC; T cell; ZFN; genome editing.

Graphical abstract

Figure 1

Cell culture serum inhibits AAV transduction (A–C) K562 cells were nucleofected or not with CCR5 ZFN mRNA and then transduced with MOI = 10⁴ AAV6-CCR5-GFP vectors. Cells were transduced at 10⁶ cells/mL with or without 2 different batches of 10% FBS for 2 h before 10% FBS is restored for further culture. (A) Representative plots of GFP expression after 2 and 14 days by flow cytometry. (B) Quantification of GFP expression in cells treated with ZFN mRNA and AAV6 vectors after 14 days. (C) Quantification of GFP expression in cells treated with AAV6 vectors alone after 2 days. Data for (B) and (C) are mean ± SEM for n = 2 technical replicates. (D and E) Inhibition of AAV6 transduction as for (A) by MOI = 10⁴ of K562 cells was calculated over a range of serum concentrations for FBS-3 (D) or human AB serum (E), and a semi-logarithmic regression line was calculated. Data are mean ± SEM for n = 3 technical replicates. (F and G) A viral attachment assay was performed for MOI = 10⁴ AAV6-CCR5-GFP vectors on K562 cells, as diagramed (F), and GFP expression was measured after 2 days by flow cytometry (G). Bar colors correspond to treatments, and black bars are control samples transduced at 37°C without prior attachment. Data are mean ± SEM for n = 3 technical replicates.

Figure 2

Effects of culture volume and time on AAV6 transduction (A) The combined influences of AAV MOI and cell culture volume on AAV6 transduction of K562 cells were evaluated. 5 × 10⁴ K562 cells were transduced with AAV6-CCR5-GFP at MOIs of 10³–10⁶ in the indicated volumes for 2 h prior to addition of 10% FBS, and GFP expression was measured after 2 days by flow cytometry. Graph shows each individual replicate for n = 3. MOIs are as indicated on the graph: 10³ = closed triangle, 3 × 10³ = open triangle, 10⁴ = closed circle, 3 × 10⁴ = open circle, 10⁵ = closed square, 3 × 10⁵ = open square, 10⁶ = closed diamond. See also Table S1 for regression characteristics. (B and C) K562 cells were transduced with AAV6 at 10⁶ cells/mL and an MOI of 10⁴ for the indicated times. Transduction was halted by 10% FBS, and GFP expression was measured after 2 days. (B) shows that transduction over the first 2 h fits a semi-logarithmic regression, while (C) illustrates an initial jump in transduction followed by increases that fit a linear regression starting 0.5 h after transduction. (D) Transduction of K562 cells over time with AAV6 at a higher concentration of 10⁷ cells/mL and MOIs of 10⁴ or 10³ was performed as before. See also Table S2 for regression characteristics. (E) Cell-associated AAV vector copy numbers (VCNs) were measured for cells in (D) by ddPCR, and a linear regression was used to measure correlation between GFP expression and VCN. Data in (B)–(E) are shown as mean ± SEM for n = 3 technical replicates.

Figure 3

Electroporation enhances transduction by attached AAV6 particles (A and B) K562 cells were transduced with AAV6-CCR5-GFP vectors at 10⁶ cells/mL and an MOI of 10⁴, and GFP expression was measured after 2 days by flow cytometry for n = 3 technical replicates. Electroporation was performed either before (A) or after (B) AAV6 transduction. Washing was performed with PBS after transduction as indicated, and for cells not transduced in the presence of FBS, media was supplemented with 10% FBS after 2 h or after electroporation. (C) CD4⁺ T cells from n = 2 human donors were transduced with AAV6-CCR5-GFP at an MOI of 10⁴ with or without prior electroporation, and GFP expression was measured after 2 days by flow cytometry. (D) HSPCs from n = 3 donors were transduced with AAV6-CCR5-GFP at an MOI of 3 × 10³ with or without prior electroporation, and GFP expression was measured after 1 day by flow cytometry. Data are shown as mean ± SEM. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001.

Figure 4

An optimized AAV6 transduction protocol enhances genome editing in K562 cells (A) Diagram of the original and optimized protocols. In the original protocol, cells are electroporated first, then transduced with AAV6-CCR5-GFP using standard cell culture concentrations prior to addition of FBS. In the optimized protocol, cells are transduced with AAV6-CCR5-GFP at high cell concentrations prior to electroporation, then cultured under standard conditions with FBS supplementation. See Table 1 and supplemental methods for additional details of each protocol in specific cell types. (B) K562 cells were genome edited using the original protocol (far left bar) involving AAV6-CCR5-GFP transduction for 2 h after electroporation of ZFN mRNA, or the optimized protocol with varying parameters of MOI, time, and cell concentration as indicated below. Data are shown as mean ± SEM for n = 2 technical replicates.

Figure 5

Improved genome editing in CD4⁺ T cells with an optimized protocol (A–F) CD4⁺ T cells from n = 4 human donors were genome edited with either a previously published (original) or optimized protocol at the indicated AAV6-CCR5-GFP MOIs. (A) Representative plots of GFP expression measured by flow cytometry at days 2 and 10 for cells treated with the optimized protocol, with or without ZFN mRNA electroporation. (B) Stable genome editing shown by GFP expression at day 10. (C) Indel formation at CCR5 by cells electroporated with ZFN mRNA with either protocol. (D) AAV6 transduction with either protocol was measured by flow cytometry for GFP at day 2 in samples treated with AAV6 but not ZFN mRNA at the highest MOI for each protocol (original = 5 × 10⁴, optimized = 10⁴). (E and F) Cell viability (E) and total cell counts by hemocytometer (F, normalized to the original number of cells prior to genome editing) were measured 1 day after genome editing. Conditions were pooled across AAV6 MOIs. (G) CD4⁺ T cells from n = 8 human donors were genome edited with the optimized protocol at indicated MOIs, and stable genome editing was measured by GFP expression at day 10. Data are shown as mean ± SEM. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; and ns, not significant.

Figure 6

Improved genome editing in B cells with an optimized protocol CD19⁺ B cells from n = 2–6 human donors were edited with either the original or optimized protocols at the indicated AAV6-CCR5-GFP MOIs. (A) Representative plots of GFP expression measured by flow cytometry at days 2 and 8 for cells treated with the optimized protocol, with or without ZFN mRNA electroporation. (B) Stable genome editing shown by GFP expression at day 8. (C) AAV6 transduction with either protocol was measured by flow cytometry for GFP at day 2 in samples treated with AAV6 but not ZFN mRNA at the highest MOI for each protocol (original = 10⁶, optimized = 10⁵). (D and E) Cell viability (D) and total cell counts by hemocytometer (E, normalized to the original number of cells prior to genome editing) were measured 1 day after genome editing. Conditions were pooled across AAV6 MOIs. Data are shown as mean ± SEM. ∗∗p < 0.01; ∗∗∗p < 0.001.

Figure 7

Comparable efficacy of both protocols in HSPCs CD34⁺ HSPCs from n = 2–6 human donors were edited with either an original or optimized protocol at the indicated AAV6-CCR5-GFP MOIs. (A) Representative plots of GFP expression measured by flow cytometry at days 1 and 10 for cells treated with the optimized protocol, with or without ZFN mRNA electroporation. (B) Stable genome editing rates, shown by GFP expression at day 10. (C) AAV6 transduction with either protocol was measured by flow cytometry for GFP at day 1 in samples treated with AAV6 but not ZFN mRNA at the highest AAV MOI (3 × 10³). (D and E) Cell viability (D) and total cell counts by hemocytometer (E, normalized to the original number of cells prior to genome editing) were measured 1 day after genome editing. Conditions were pooled across AAV6 MOIs. Data are shown as mean ± SEM. ∗∗∗p < 0.001.