Establishment of HIV-1 resistance in CD4+ T cells by genome editing using zinc-finger nucleases

Abstract

Homozygosity for the naturally occurring Delta32 deletion in the HIV co-receptor CCR5 confers resistance to HIV-1 infection. We generated an HIV-resistant genotype de novo using engineered zinc-finger nucleases (ZFNs) to disrupt endogenous CCR5. Transient expression of CCR5 ZFNs permanently and specifically disrupted approximately 50% of CCR5 alleles in a pool of primary human CD4(+) T cells. Genetic disruption of CCR5 provided robust, stable and heritable protection against HIV-1 infection in vitro and in vivo in a NOG model of HIV infection. HIV-1-infected mice engrafted with ZFN-modified CD4(+) T cells had lower viral loads and higher CD4(+) T-cell counts than mice engrafted with wild-type CD4(+) T cells, consistent with the potential to reconstitute immune function in individuals with HIV/AIDS by maintenance of an HIV-resistant CD4(+) T-cell population. Thus adoptive transfer of ex vivo expanded CCR5 ZFN-modified autologous CD4(+) T cells in HIV patients is an attractive approach for the treatment of HIV-1 infection.

Figure 1

ZFN-mediated disruption of CCR5 and protection from HIV-1 infection in GHOST-CCR5 cells. (a) Schematic of the CCR5 coding region showing the genomic DNA sequences targeted by CCR5 ZFNs 215/224. (b) Level of targeted gene disruption in GHOST-CCR5 cells transduced with an Ad5/35 vector encoding ZFNs targeting either CCR5 or IL-2Rγ as assessed by the Surveyor assay (Supplementary Fig. 2). Lower migrating products (arrows) are a direct measure of ZFN-mediated gene disruption. NTD, nontransduced cells. (c) Decreased CCR5 surface expression measured by flow cytometry (light blue, IL2Rγ ZFN; green CCR5 ZFN-224; orange, CCR5 ZFN-215; dark blue, nontransduced cells; red, unstained cells). (d) Protection from HIV-1_BAL measured by flow cytometry 48 h after HIV-1 challenge of CCR5 ZFN-215 and CCR5 ZFN-224-modified cells compared to IL2Rγ ZFN and control GHOST-CCR5 cells (red, IL2Rγ ZFN; green, CCR5 ZFN-224; blue, CCR5 ZFN-215). GFP fluorescence indicates HIV-1 entry and is plotted as average percent infected relative to positive control. MFI, mean fluorescence intensity. Histograms of CCR5 (c) and GFP expression (d) show one replicate for each condition; bar graphs below represent averages (±s.d.) of triplicates. Expression of CCR5 and HIV-1 infection frequency of CCR5 ZFN-treated cells is less than IL2Rγ ZFN or nontransduced cells (P < 0.001).

Figure 2

In vitro selection of CCR5-disrupted cells following HIV-1 challenge of the CD4⁺ T-cell line, PM1. (a) The level of ZFN-disrupted CCR5 alleles was determined at the indicated times post-HIV-1 challenge with R5-tropic HIV-1_BAL or after mock HIV-1 infection. Disrupted CCR5 alleles remained at stable levels in mock-infected cultures but were enriched in the presence of HIV-1. Similar results were obtained in two experiments, each extending for over 2 months. (b) The genomic CCR5 ZFN target site in ZFN-treated PM1 cells at day 52 post-HIV-1 challenge was amplified, cloned and sequenced to confirm ZFN cleavage as the molecular basis of HIV-1 resistance. Sequence alignment revealed distinct ZFN-induced insertions and deletions within the target region of CCR5.

Figure 3

Enrichment of CCR5 ZFN-modified primary CD4⁺ T cells during in vitro HIV-1 challenge. (a) Primary CD4⁺ T cells from CCR5 wild-type anonymous healthy donor were transduced with Ad5/35 vector expressing CCR5 ZFN-215, ZFN-224 or GFP at MOI of 30 and 100; percentage of total alleles are indicated below each lane. (b) Population doubling rate for CCR5 ZFN- and GFP control-transduced CD4⁺ T cells (triangle, nontransduced; square, CCR5 ZFN-224; diamond, GFP transduced). (c) Enrichment of ZFN-215-transduced CD4⁺ T cells over time following in vitro challenge with CCR5-tropic HIV-1_US1 compared to mock (square, HIV-1 infected; triangle, mock infected). An ~10% starting level of ZFN-disrupted CCR5 alleles was obtained by mixing Ad5/35-transduced CD4⁺ T cells from a 1 in 3 with unmodified CD4⁺ T cells. (d) Intranuclear 53BP1 immunostaining and epifluorescence microscopy 2 d after CD4⁺ T cells were transduced with Ad5/35 vectors expressing CCR5 ZFN pair 224, nontransduced (negative control) or 1 μM etoposide (positive control). Representative images are shown in the panels. (e) The mean (± s.d.) numbers of foci over time is shown following Ad5/35 vector transduction with CCR5 ZFN-224, GFP and nontransduced CD4⁺ T cells. Significant elevation in the number of foci was observed for CCR5 ZFN-224 treated cells on days 2 (P = 0.03) to 3 post treatment (P = 0.004, unpaired t-test, n = 4), whereas GFP-transduced cells were statistically indistinguishable from nontransduced control lymphocytes.

Figure 4

Reduction in viremia and selection for CCR5 ZFN-modified CD4⁺ T cells in the presence of HIV-1 challenge in vivo. (a) Experimental outline for adoptive transfer of modified CD4⁺ T cells and in vivo HIV-1 challenge in NOG mice. CCR5 ZFN-transduced CD4⁺ T-cell population pre-mix (day 3); CCR5 disruption level, 33%. Injected mixed samples baseline CCR5 disruption level 15% in control (mock infected) and 14% in HIV-infected group (day 7). (b) Level of ZFN-disrupted CCR5 alleles in CD4⁺ T cells isolated on day 40 from spleens of control or HIV-infected mice. Percent disruption indicated at base of each lane. One mouse from each group (HIV-infected mouse no. 8039 and control mouse no. 8022) excluded for later analysis due to inadequate CD4⁺ T-cell DNA recovery and purification. N.D., not determined. (c) Plot of in vivo disruption frequencies in spleens on day 40. Results for each group (n = 7) averaged and analyzed using an unpaired t-test with mean ± 95% confidence intervals indicated. (d–f) In an independent experiment, mice were engrafted with CCR5 ZFN-transduced CD4⁺ T cells (51% disruption) or GFP-transduced cells (mock) and followed for 50 d post HIV-1 infection. Enrichment for CCR5-disrupted CD4⁺ T cells in peripheral blood on day 50 post infection (Surveyor assay); lower migrating products (arrows) are a direct measure of ZFN-mediated gene disruption (d). Plasma viremia in mice day 10 post infection. HIV-1 viral RNA (copies/ml) is plotted for the individual mice; the mean ± 95% confidence interval is shown (e). The CCR5 ZFN-treated mice had a significantly lower viral load (P < 0.001; Mann Whitney test). Engraftment of CD4⁺ T cells in peripheral blood from days 10 to 50 post infection. The CD4⁺ T-cell counts (Trucount assay) for the mice engrafted with CCR5 ZFN-(solid symbols) and GFP-modified cells (open symbols) are plotted (f). Mice engrafted with CCR5 ZFN-treated cells had higher CD4⁺ T-cell counts on days 30–50 post infection (P = 0.04).