Human hematopoietic stem/progenitor cells modified by zinc finger nucleases targeted
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1 Supporting Online Material for: Human hematopoietic stem/progenitor cells modified by zinc finger nucleases targeted to CCR5 control HIV-1 in vivo Nathalia Holt 1, Jianbin Wang 2, Kenneth Kim 2, Geoffrey Friedman 2, Xingchao Wang 3, Vanessa Taupin 3, Gay M. Crooks, Donald B. Kohn, Philip D. Gregory 2, Michael C. Holmes 2 and Paula M. Cannon 1. 1 Keck School of Medicine of University of Southern California, Los Angeles, California, USA. 2 Sangamo BioSciences, Inc., Richmond, California, USA. 3 Childrens Hospital Los Angeles, Los Angeles, California, USA. David Geffen School of Medicine at University of California Los Angeles, Los Angeles, California, USA. This file contains: Supplementary Figures 1, 2, 3 and Supplementary Tables 1 and 2.
2 bone marrow lung IgGPerCP IgG APC IgGPerCP IgG APC spleen thymus IgGPerCP IgG FITC IgGPerCP IgG FITC small intestine blood IgGPerCP IgG APC IgG FITC IgGPerCP Supplementary Figure 1 Isotype controls for FACS analysis of human cells in mouse tissues. Staining by mouse IgG1 isotype controls is shown for each fluorochrome used: IgG1- PerCP for anti-human CD5; IgG1- APC for anti-human CD19, CD1, CD3 and CCR5; IgG1- FITC for anti-human CD. Cells were gated on FSC/ to remove debris.
3 ZFN nucleofection of CD3+ HSPC Inject neonatal NSG mice HIV 1 infection day 1 week 8 12 week Diagnostic bleeds to confirm engraftment Peripheral blood analysis Necropsy (8 12 weeks) FACS CCR5 disruption viral lload Alternate timepoints: FACS (CD5, CD, CD8) CCR5 disruption (Cel 1, pentamer) Every timepoint: viral load (qpcr) Supplementary Figure 2 Experimental outline and timeline for HSPC engraftment of NSG mice, HIV-1 infection and analyses performed.
4 blood thymus spleen small intestine FSC/ CCR CD5 FSC/ CD CC CR CD Supplementary Figure 3 Levels of CCR5 expression on human cells in HSPC engrafted NSG mice. FACS analysis of blood, thymus, spleen, and small intestine lamina propria tissues are shown from one representative mouse transplanted with untreated (neg.) HSPC and necropsied at 2 weeks post-engraftment. In an ungated analysis (top panel), a significant population of human CD5+CCR5+ cells (upper right quadrant) can be seen only for the spleen and small intestine samples. The lower panel shows the percentage of human cells (CD5+ gate) in each tissue expressing CD or CCR5, and is indicated for each quadrant. All cells were first gated on FSC/ to remove debris. No staining was observed with isotype-matched control antibodies, or for animals receiving no human graft (data not shown).
5 a CD :CD8 Ratio 8 p=.1 b HIV-1 RNA copies/ml * ZFN/NL-3 ZFN/BAL ZFN pre-inf. ZFN NL-3 ZFN BAL weeks post-infection CD5 lymphoid CD8 7 CD Supplementary Figure Effects of CXCR-tropic and CCR5-tropic HIV-1 infection. A cohort of 8 mice received ZFN-treated HSPC. At 8 weeks post-transplantation, blood was harvested (preinfection samples) and mice each were infected with either CXCR-tropic HIV-1 NL-3,orwith CCR5-tropic HIV-1 BAL. (a) Mean +/- SD ratio of human CD to CD8 T cells in peripheral blood of mice, pre-infection (n=8) or at 8 weeks post-infection (n= for each virus) and below, representative FACS plots from each group. Cells were gated on FSC/ to remove debris, on human CD5, and a lymphoid gate was applied before analysis of CD and CD8 subsets. (b) Mean +/- SD levels of HIV-1 RNA in blood of mice infected with HIV-1 NL-3 (n=)orhiv-1 BAL (n=), at indicated timepoints. Asterisk indicates statistically significant difference between the two groups (p=.3).
6 Supplementary Table 1 Human hematopoietic cells in mouse tissues tissue/cells untreated (neg.) HSPC (n=3) ZFN-treated HSPC (n=3) thymus % CD5+/total a 99 +/ /- 1 %C CD+CD8+/humanC b 95 +/ /- 16 bone marrow % CD5+/total 6 +/ /- 19 % CD19+/human 66 +/ /- 15 spleen % CD5+/total 55 +/ /- 7 % CD+/human 33 +/ /- 1 % CD8+/human 19 +/ /- 1 SE % CD5+/total 17 +/ /- 5 % CD3+/human 6 +/ /- 29 SP % CD5+/total 1 +/ /- % CD3+/human 59 +/ /- 11 LE % CD5+/total 1 +/ /- 6 % CD3+/human 68 +/ /- 18 LP % CD5+/total 18 +/ /- 3 % CD3+/human 91 +/ /- 37 a Mean +/- SEM percentage of human CD5+ cells in 5, total cells analyzed from the indicated mouse tissues. b Mean +/- SEM percentage of indicated human subsets within the human CD5+ population. Cells were harvested from adult NSG mice previously engrafted with either untreated (neg.) HSPC or ZFN-treated HSPC. Student s t-tests demonstrated no significant differences (p>.5) in the human cell populations between the neg. and ZFN groups. Abbr. S, small intestine; L, large intestine; E, intraepithelial layer; P, lamina propria.
7 Supplementary Table 2 HSPC treatment and HIV infection in mouse cohorts a ID # HSPC treatment HIV status 1 neg uninf. 2 neg uninf. 3 neg uninf. neg uninf. 5 ZFN uninf. 6* ZFN uninf. 7 ZFN uninf. 8 ZFN uninf. 9 mock uninf. 1 mock uninf. 11 mock uninf. 12 mock uninf. 13 mock uninf. 1* neg HIV+ 15* neg HIV+ 16* neg HIV+ 17* neg HIV+ 18* neg HIV+ 19* neg HIV+ 2 neg HIV+ 21 neg HIV+ 22 neg HIV+ 23* ZFN HIV+ 2 ZFN HIV+ 25* ZFN HIV+ 26* ZFN HIV+ 27 ZFN HIV+ 28 ZFN HIV+ 29 ZFN HIV+ 3 ZFN HIV+ 31 ZFN HIV+ 32 ZFN HIV+ 33 ZFN HIV+ 3 ZFN HIV+ 35 ZFN HIV+ 36* ZFN HIV+ 37* ZFN HIV+ 38 ZFN HIV+ 39 ZFN HIV+ ZFN HIV+ 1 ZFN HIV+ 2 ZFN HIV+ 3 ZFN HIV+ ZFN HIV+ 5 ZFN HIV+ 6 no graft uninf. 7 no graft uninf. Fig./Table assay mice ID #s 1b FACS 1-2, 5-6, 9-13, 1-16, c FACS 7 2a FACS 3, 7-8, 2-22, b FACS a FACS 3, 7-8, 2-22, b IHC 3-, 7-8, 2-22, 27-35, 5-6 a Cel 1/ pentamer 23-25, b Cel-1 6-7, 25, 33-3 c FACS 3, 7-8, 2-22, d FACS 3, 7-8, 2-22, CCR5 sequence 2 6a FACS/qPCR 2-22, b qpcr 1-37 Supp 1 FACS 3-, 7-8 Supp 3 FACS 3- Supp FACS/qPCR 38-5 Supp Table 1 FACS 1-3, 5, 7-8 a HSPC treatment and HIV-1 infection status of individual mice (left table), and specific animals included in each data set (right table). Animals were infected with HIV-1 at 8-12 weeks post-engraftment and were followed up to 12 weeks post-infection. Animals marked with an asterisk (*) were necropsied at earlier time points, including any found dead due to environmental contamination. Only 12 week post-infection survivors were analyzed for Figs 2a, 3, c, d and 6a. Blood analyses (FACS, Cel I and qpcr) were performed on alternating samples due to limiting sample volumes. Cel I and pentamer assays were only performed on samples that generated human CCR5 PCR fragments.
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