Development of HIV-Resistant CAR T Cells by CRISPR/Cas-Mediated CAR Integration into the CCR5 Locus

Abstract

Adoptive immunotherapy using chimeric antigen receptor (CAR) T cells has been highly successful in treating B cell malignancies and holds great potential as a curative strategy for HIV infection. Recent advances in the use of anti-HIV broadly neutralizing antibodies (bNAbs) have provided vital information for optimal antigen targeting of CAR T cells. However, CD4+ CAR T cells are susceptible to HIV infection, limiting their therapeutic potential. In the current study, we engineered HIV-resistant CAR T cells using CRISPR/Cas9-mediated integration of a CAR cassette into the CCR5 locus. We used a single chain variable fragment (scFv) of the clinically potent bNAb 10-1074 as the antigen-targeting domain in our anti-HIV CAR T cells. Our anti-HIV CAR T cells showed specific lysis of HIV-infected cells in vitro. In a PBMC humanized mouse model of HIV infection, the anti-HIV CAR T cells expanded and transiently limited HIV infection. In conclusion, this study provides proof-of-concept for developing HIV-resistant CAR T cells using CRISPR/Cas9 targeted integration.

Keywords: CAR T cells; CRISPR/Cas9; HIV; humanized mice.

Figure 1

CRISPR-Cas9 integration of a CAR into CCR5 leads to an HIV-resistant phenotype: (A) schematic of the CAR construct; (B) comparison of CAR genes specifically integrated into CCR5 by ddPCR seven days after transduction (left axis) and tNGFR expression by flow cytometry four days after transduction (right axis); (C) CCR5 expression by flow cytometry in cells treated with or without CCR5-sgRNA/Cas9 RNP and transduced with CCR5-CAR AAV6; (D) integration rates of either anti-CD19 CAR or anti-HIV CAR into either AAVS1 or CCR5; (E) levels of eGFP expression in CD4+ CAR T cells 24 h after infection with eGFP-HIV (data presented as means ± SEM, ***: p ≤ 0.001).

Figure 2

Anti-HIV CAR T cells specifically kill HIV-infected cells in vitro: (A) cytotoxicity assay of autologous B cells at E:T 5:1; (B) cytotoxicity assay of HIV-infected autologous CD4+ T cells at E:T 1:1; (C,D) levels of p24 in supernatants following five day co-culture of CAR T cells and HIV-infected CD8-depleted autologous PBMCs at E:T 1:1. (C): CARs are integrated into AAVS1, (D) CARs are integrated into CCR5; (E) AUC of p24 curves in (C,D) (data presented as means ± SEM, *: p ≤ 0.05, **: p ≤ 0.01, ***: p ≤ 0.001).

Figure 3

Anti-CD19 CAR T cells kill B cells in vivo: (A) frequency of anti-CD19 CAR T cells based on the frequency of tNGFR+ cells per 1 × 10⁶ CD8+ T cells; (B) fold change in the frequency of CD19+ cells for each CAR T cell-treated group normalized to the frequency of CD19+ cells in mice treated with untransduced CD8+ T cells (n = 7–8 mice from 2 biological donors in each group, data presented as means ± SEM, ns: p > 0.05, ****: p ≤ 0.0001).

Figure 4

Anti-HIV CAR T cells expand and transiently limit HIV infection in vivo: (A) frequency of anti-HIV CAR T cells based on the frequency of tNGFR+ cells per 1 × 10⁶ CD8+ T cells; (B) HIV viral load as copies/mL plasma; (C) fold change in viral load for each CAR T cell-treated group normalized to the viral load in mice treated with untransduced CD8+ T cells; (D) AUC of viral load in (B) (n = 7–8 mice from 2 biological donors in each group, data presented as means ± SEM, ns: p > 0.05, ****: p ≤ 0.0001).