CCR5 edited CD4+ T cells are resistant to HIV-1 infection
As a proof of concept, we challenged the edited cells at an early and late culture time point with GFP expressing lentiviral vectors pseudotyped with either the gp160 glycoprotein of the HIV-1 R5-tropic strain BaL or VSV-G as control. The CCR5 edited CD4+ T cells showed approximately 50% reduced susceptibility to transduction with the HIV-gp160 pseudotyped vector when compared to control cells (Fig. 2A). When challenging T cells from two Δ32 heterozygous donors, a decreased susceptibility – similar to cells from the other donors – was observed. As a CD4/CCR5-independent transduction control, we transduced the cells in parallel with VSV-G pseudotyped lentivectors. Under these conditions we did not see any alteration in transduction efficiency.
Next, we tested resistance of CCR5 edited CD4+ T cells to replication-competent HIV-1 by infecting them with either R5-tropic HIV-1JRFL or X4-tropic HIV-1NL4-3. HIV-1 p24 was quantified in the supernatant of infected cells as a measure of viral replication (Fig. S1A-B). In samples infected with HIV-1JRFL (high or low MOI), a significant protective effect (p =8.8x10-4 and p =5.7 x10-5) was observed when compared to infection of unedited cells. As expected, infections with X4-tropic HIV-1NL4-3 did not reveal significant differences (Fig S1B). Furthermore, to investigate the impact of the editing rate on the viral replication, we spiked edited CD4+ T cells with unedited cells at ratios of 1:1 (50% edited cells) and 1:3 (25% edited cells). Low MOI infection with HIV-1JRFL, as determined by p24 in the supernatant, was significantly (p =2.72x10-14) reduced in non-diluted edited cells (100%) when compared to unedited samples (Fig. 2B). The protective effect was also observed in both the 50% and 25% mix (p =8.38x10-11,p =1.13x10-4), suggesting that a cell population with ~20% of edited CCR5 alleles was able to slow down virus replication. A significant protective effect (p =3.06x10-3) was also observed when edited cells (100%) with infected with a high MOI (Fig. 2C), albeit to a lower degree as compared to the low MOI infection. After dilution (50% mix), the protective effect was lost when cells were infected at high MOI (p =0.98). In the control settings with HIV-1NL4-3infections, we did not see any impairment of virus replication in the edited cells (Fig. 2D), as expected. We conclude that editing of CD4+ T cells with CCR5 targeting TALEN leads to protection against R5-tropic HIV-1 in an editing dose-dependent manner.