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.