Protection of human CD4+ T cells in peripheral blood of HIV-infected mice transplanted with ZFN-modified HSC (a) FACS plots showing human CD4+ and CD8+ T cells in peripheral blood of representative animals from each of three cohorts: uninfected mice previously engrafted with either untreated or ZFN-treated HSC (uninf.), and HIV-1 infected animals previously engrafted with either untreated HSC (neg.) or ZFN-treated (ZFN) HSC, at 4 weeks post-infection. The total number of animals analyzed in each cohort is indicated. Cells were gated on FSC/SSC to remove debris, on human CD45, and a lymphoid gate applied. Percentage of cells in indicated compartments is shown. (b) Ratio of human CD4+ to CD8+ lymphocytes in peripheral blood of individual mice transplanted with untreated (neg.) or ZFN-modified HSC, measured pre-infection, and at 6–8 weeks post-infection. Statistical analysis comparing neg. and ZFN cohorts at each time-point is shown.

Control of HIV-1 replication in mice receiving ZFN-treated HSC. (a) Mean +/− SD levels of HIV-1 RNA (left) and percent CD4+ human T cells (right) in peripheral blood of mice transplanted with untreated (neg.) or ZFN-treated HSC, at indicated times post-infection. Dashed line is limit of detection of assay. Asterisk indicates a statistically significant difference between two groups (p<0.05). (b) Mean +/− SD HIV-1 RNA levels in small and large intestine lamina propria from neg. or ZFN mice, from animals necropsied between 8 and 12 weeks post-infection. Numbers of mice analyzed at each time point are shown above the appropriate bar. Dashed line indicates limits of detection of assay. Asterisk indicates undetectable levels.

Effects of HIV-1 infection on human cells in transplanted NSG mice. (a) FACS analysis of human cells in tissues of representative NSG mice from three cohorts: uninfected mice previously engrafted with either untreated or ZFN-treated HSC (uninf.), and HIV-1 infected animals previously engrafted with either untreated HSC (neg.) or ZFN-treated (ZFN) HSC. Mice were necropsied at 12 weeks post-infection, or at the equivalent time-point for uninfected animals. The total number of animals analyzed in each cohort is indicated. FACS analysis was performed as described in Figure 1. Small intestine sample is lamina propria, and similar results were obtained when samples from the large intestine were analyzed. Percentage of cells in indicated compartments is shown. (b) Immunohistochemical analysis of human CD3 expression in small intestine (SI), and CD4 expression in spleen of representative NSG mice, transplanted with untreated (neg.) or ZFN-treated (ZFN) HSC, with and without HIV-1 infection. Animals were necropsied at 12 weeks post-infection, or at the same time-point for uninfected animals. Control animals receiving no human HSC (no graft) were also analyzed. The number of animals analyzed in each cohort is shown. Bars represent 50 μM.

ZFN activity produces heterogeneous mutations in CCR5. Sequence analysis was performed on 60 cloned human CCR5 alleles, PCR amplified from intraepithelial cells from the large intestine of an HIV-infected mouse previously transplanted with ZFN-treated HSC, and at 12 weeks post-infection. The number of nucleotides deleted or inserted at the ZFN target site in each clone is indicated on the right of each sequence, together with the number of times the sequence was found. Dashes (-) indicate deleted bases compared to the wildtype sequence, uppercase letters are point mutations, underlined upper case letters are inserted bases. Some specific mutations of CCR5 occurred more frequently, in particular a 5bp duplication at the ZFN target site that was identified 13 times (bottom sequence). No mutations in CCR5 were observed in a similar analysis performed on control samples from a mouse receiving non-modified HSC (data not shown).

ZFN-mediated disruption of CCR5 gene in HSC. (a) Representative gel showing extent of CCR5 disruption in CD34+ HSC, 24 hours after cells were nucleofected with ZFN-expressing plasmids (ZFN) or mock nucleofected (mock). Neg. is untreated HSC. CCR5 disruption was measured by PCR amplification across the ZFN target site, followed by Cel I nuclease digestion and quantitation of products by PAGE. (b) Graph showing mean +/− SD percentage of human CD45+ cells in peripheral blood of mice at 8 weeks post-transplantation with either untreated, mock nucleofected, or ZFN nucleofected HSC, (n=5 each group). (c) FACS profiles of human cells from various organs of one representative mouse transplanted with ZFN-treated HSC. Cells were gated on FSC/SSC to remove debris. Staining for human CD45, a pan leukocyte marker, was used to reveal the level of human cell engraftment in each organ. CD45+ gated populations were further analyzed for subsets, as indicated: CD19 (B cells) in bone marrow, CD14 (monocytes/macrophages) in lung, CD4 and CD8 (T cells) in thymus and spleen, and CD3 (T cells) in the small intestine (lamina propria). The CD45+ population from the small intestine was further analyzed for CD4 and CCR5 expression. Peripheral blood cells from CD45+ and lymphoid gates were analyzed for CD4 and CD8 expression. The percentage of cells in each indicated area is shown. No staining was observed with isotype-matched control antibodies (Supplementary Fig. 1 online), or in animals receiving no human graft (data not shown).

HIV-1 infection selects for disrupted CCR5 alleles. (a) Mean +/- SD levels of CCR5 disruption (Cel 1 assay, black bars) in sequential peripheral blood samples taken from mice transplanted with ZFN-treated HSC and infected with HIV-1. Upper limit of linearity of Cel I assay is 44% (19) and is indicated by the dotted line; upper limit of sensitivity of assay is 70–80%. White bars show the frequency of a common 5bp duplication at the ZFN target site that typically comprises 10–30% of total CCR5 mutations (19). Numbers of mice analyzed at each time point, and in each assay, are shown above the appropriate bar. (b) Mean +/− SD levels of CCR5 disruption (Cel I assay) in indicated tissues from mice transplanted with ZFN-treated HSC and necropsied at 12 weeks post-infection (black bars), or at an equivalent time-point for uninfected ZFN-treated animals (white bars). Numbers analyzed in each group are shown above the appropriate bar. One representative Cel I analysis from the large intestine (lamina propria) of uninfected and infected mice is shown. Animals receiving untreated HSC gave no Cel I digestion products at any time-point analyzed (data not shown). Asterisk indicates levels too low to quantify. (c) Contour FACS analyses of human CD4+ cells in the small intestine (lamina propria) and spleen of one representative animal from each indicated cohort are shown. Cells were gated on FSC/SSC to remove debris and gated on human CD45 and CD4. Numbers indicate the percentage of cells that are CCR5+. (d) Mean +/− SD numbers of human CD4+ cells (grey bars) and CD4+CCR5+ cells (white bars) per 5,000 human CD45+ cells analyzed from different sections of the intestine, and from the indicated cohorts. Asterisk indicates levels too low to quantify. Number of animals analyzed in each cohort is indicated. Abbr. S, small intestine; L, large intestine; E, intraepithelial lymphocytes; P lamina propria lymphocytes; BM, bone marrow.