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Homology-driven genome editing in hematopoietic stem and progenitor cells using ZFN mRNA and AAV6 donors

Nature Biotechnology volume 33, pages 1256–1263 (2015)Cite this article

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Abstract

Genome editing with targeted nucleases and DNA donor templates homologous to the break site has proven challenging in human hematopoietic stem and progenitor cells (HSPCs), and particularly in the most primitive, long-term repopulating cell population. Here we report that combining electroporation of zinc finger nuclease (ZFN) mRNA with donor template delivery by adeno-associated virus (AAV) serotype 6 vectors directs efficient genome editing in HSPCs, achieving site-specific insertion of a GFP cassette at the CCR5 and AAVS1 loci in mobilized peripheral blood CD34+ HSPCs at mean frequencies of 17% and 26%, respectively, and in fetal liver HSPCs at 19% and 43%, respectively. Notably, this approach modified the CD34+CD133+CD90+ cell population, a minor component of CD34+ cells that contains long-term repopulating hematopoietic stem cells (HSCs). Genome-edited HSPCs also engrafted in immune-deficient mice long-term, confirming that HSCs are targeted by this approach. Our results provide a strategy for more robust application of genome-editing technologies in HSPCs.

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Figure 1: HSPCs are efficiently transduced by AAV6.
Figure 2: Combination of ZFN mRNA and AAV6 vectors promotes high levels of site-specific genome editing at the CCR5 locus in HSPCs.
Figure 3: Site-specific genome editing by AAV6 vectors uses HDR.
Figure 4: Rates of bulk culture cell growth and genome modification in erythroid and myeloid lineages.
Figure 5: Genome editing in different HSPC subsets.
Figure 6: Engraftment of NSG mice with gene-edited HSPCs.

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Acknowledgements

This work was supported by funding from the California HIV/AIDS Research Program ID12-USC-245 and F10-USC-207, US National Institutes of Health grants P01 HL73104, R01 DE025167, and U19 HL129902, the California Institute for Regenerative Medicine grant RT3-07848, and the James B. Pendleton Charitable Trust. We thank L. Truong, G. Lee and Y. Lee for CD34+ cells, E. Lopez, E. Seclen and J. Rathbun for assistance with humanized mice, E. Killingbeck and P. Cheung for MiSeq analysis, A. Goodwin, A. Kang, and H. Tran for AAV production, A. Reik and F. Urnov for AAVS1 ZFNs, and C. Wang for helpful discussions.

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  1. Jianbin Wang and Colin M Exline: These authors contributed equally to this work.

  2. Michael C Holmes and Paula M Cannon: These authors jointly directed this work.

Authors and Affiliations

  1. Sangamo BioSciences, Inc., Richmond, California, USA

    Jianbin Wang, Joshua J DeClercq, Samuel B Hayward, Patrick Wai-Lun Li, David A Shivak, Richard T Surosky, Philip D Gregory & Michael C Holmes

  2. Department of Molecular Microbiology and Immunology, Keck School of Medicine, University of Southern California, Los Angeles, California, USA

    Colin M Exline, G Nicholas Llewellyn & Paula M Cannon

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Contributions

J.W., C.M.E. and J.J.D. performed most of the experiments; S.B.H., P.W.-L.L., D.A.S., R.T.S. and G.N.L. developed assays and analyzed samples; J.W., C.M.E., P.D.G., M.C.H. and P.M.C. designed the experiments and analyzed data; J.W., C.M.E., M.C.H. and P.M.C. wrote the manuscript.

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Correspondence to Michael C Holmes or Paula M Cannon.

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Competing interests

The following authors are full-time employees of Sangamo BioSciences, Inc.; J.W., J.J.D., S.B.H., P.W.L., D.A.S., R.T.S., P.D.G. and M.C.H.

Integrated supplementary information

Supplementary Figure 1 Timing of AAV6 vector addition relative to ZFN mRNA electroporation.

Mobilized blood CD34+ HSPCs were transduced with AAV6 vectors containing a CCR5-XhoI donor (2,000 vg/cell) at the indicated times, pre- or post- electroporation with CCR5 ZFN mRNA (60 μg/ml). Cells were collected 5 days later and genomic DNA analyzed by Illumina deep sequencing to measure the extent of genome modification by (a) RFLP insertions and (b) indels. Data were combined from two independent experiments.

Supplementary Figure 2 AAV6 vectors and ZFN mRNA promote high levels of genome editing at the CCR5 locus in mobilized blood HSPC.

Mobilized blood CD34+ HSPCs were transduced with AAV6 vectors carrying the CCR5-RFLP donor at indicated doses (vg/cell) for 16 hours and/or electroporated with 40-60 μg/ml CCR5 ZFN mRNA. Cells were analyzed 4-5 days post-electroporation by Illumina deep sequencing to measure the efficiency of genome modification (% indels and RFLP). Results (mean ± SD) are combined from 19 separate experiments using 7 different HSPC donors. ns, no significant difference (p>0.05, unpaired t-test) in the % RFLP modification obtained at the 3,000 and 10,000 vg/cell doses of CCR5-RFLP, in combination with CCR5 ZFN mRNA.

Supplementary Figure 3 AAV6 vectors and ZFN mRNA promote high levels of genome editing at the CCR5 locus in fetal liver CD34+ HSPCs.

Fetal liver CD34+ HSPCs were transduced with AAV6-CCR5-GFP donor (1,000 vg/cell) for 24 hours then electroporated with CCR5 ZFN mRNA. Cells were analyzed at days 1 and 10 post electroporation by flow cytometry (a, c) and by In-Out PCR at day 10 (b). (a) is one representative experiment while (c) is the mean +/- SD for flow cytometry data from 4 independent experiments. **** p<0.0001, one-way ANOVA, Newman-Keuls post-test to compare all columns.

Supplementary Figure 4 AAV6 vectors and ZFN mRNA promote high levels of genome editing at the AAVS1 locus in mobilized blood HSPCs.

(a) Schematics of AAV vectors used to deliver AAVS1 homology donors. R and L refer to AAVS1 genomic sequences, comprising 801 and 840 bp respectively. (b) Mobilized blood CD34+ HSPCs were transduced with AAV6 vectors carrying the AAVS1-RFLP donor at indicated doses (vg/cell) for 16 hours and/or electroporated with AAVS1 ZFN mRNA. Cells were analyzed 3-5 days post-electroporation by Illumina deep sequencing to measure the efficiency of genome modification (% indels and RFLP). Results are combined from 6 experiments using 5 HSPC donors, and show mean +/- SD. No significant difference (p>0.05, unpaired t-test) on % RFLP was detected between cells treated with 1,000 and 3,000 vg/cell AAVS1-GFP in combination with ZFN mRNA treatment. (c) Confirmation of insertion of HindIII site at the AAVS1 locus by RFLP assay. One representative experiment is shown. (d) Mobilized blood HSPCs were treated as described in (b), but using the AAVS1-GFP donor. Cells were collected 3-6 days post-transduction and analyzed by flow cytometry for % GFP+, and by deep sequencing to measure % indels. Results were combined from 6 experiments using 4 HSPC donors and show mean +/- SD. * p<0.05, unpaired t-test between GFP+. (e) Flow cytometry plots from a representative experiment using 10,000 vg/cell AAVS1-GFP donor at 5 days post-electroporation.

Supplementary Figure 5 AAV6 vectors and ZFN mRNA promote high levels of genome editing at the AAVS1 locus in fetal liver HSPCs.

Fetal liver CD34+ HSPCs were transduced with AAVS1-GFP vectors (1,000 vg/cell) for 24 hours, then electroporated with/without AAVS1 ZFN mRNA. Cells were analyzed at day 10 post electroporation. Representative flow cytometry plots (a), or a specific In-Out PCR (b), are shown as measures of GFP addition, with the mean +/- SD for flow cytometry data from 3 independent experiments shown in (c). **** p<0.0001, unpaired t-test.

Supplementary Figure 6 Possible outcomes for AAV donor genomes.

(a) If an AAV genome containing homology arms flanking a transgene (eg promoter-GFP cassette) is introduced into a cell with matched ZFN, the possible outcomes include on-target insertion at the ZFN target site mediated by (ii) HDR, or (iii) NHEJ. In addition, off-target insertions by end-capture can occur at other DSB, which can arise from off-target activity of the nuclease (iv), or from random cellular events (v). (b) Expanded data set from Fig. 3f, showing frequency of different events. Data is mean ± SD from n=3 samples except the AAVS1 ZFN, no donor and CCR5 ZFN, no donor treatments, n=1. * p < 0.05, one-way ANOVA. (c) Relative contribution of different events to stable GFP expression at each locus, extrapolated from data in part (b), suggesting that 95.7% and 96.5% of events at the CCR5 and AAIVS1 loci, respectively, are HDR-mediated on-target GFP addition.

Supplementary Figure 7 Lineage analysis of human cells in NSG mice engrafted with genome edited HSPCs.

Neonatal NSG mice were engrafted with fetal liver HSPCs, either mock or treated with AAV6 donors (CCR5-GFP or CCR5-RFLP) and CCR5 ZFN mRNAs. Peripheral blood was harvested at 8, 12, and 16 weeks (a), and bone marrow and spleen harvested at 16 weeks (b), and analyzed for the indicated lineages of human cells. There were no significant differences in levels of each lineage between mice receiving mock and treated HSPCs in the blood (two-way ANOVA) or either tissue (one-way ANOVA).

Supplementary Figure 8 Genome edited cells are present in different lineages.

Four neonatal NSG mice were engrafted with fetal liver HSPCs, treated with CCR5-GFP vectors and CCR5 ZFN mRNA. Peripheral blood was analyzed by flow cytometry at 8, 12 and 16 weeks for GFP+ levels in each indicated human cell lineage. At the 16 week time point, bone marrow was similarly analyzed for GFP expression. No significant differences were noted in levels of GFP+ cells between lineages except in the bone marrow, where levels of GFP + cells in the CD4 + T cell fraction were significantly higher than in the other cell types (p <0.05, one-way ANOVA, Newman-Keuls post test to compare all cell types). (a) Combined data from 4 animals at each time point. (b) Representative plots showing GFP + cells in CD19 + B cells or CD4 + T cells, from blood of two different mice.

Supplementary Figure 9 Comparison of engraftment of NSG mice with fetal liver versus mobilized blood CD34+ HSPCs.

Neonatal NSG mice were transplanted with 1 million fetal liver (FL) HSPCs, and adult NSG mice were transplanted with 1 million mobilized blood (Mob) CD34+ HSPCs. The rates of engraftment with human cells achieved with mobilized blood CD34+ cells were significantly lower in blood and tissues (bone marrow and spleen) at later time points (**** = p<0.0001).

Supplementary Figure 10 Supporting data for Figure 2.

Uncropped images of gels shown in (a) Figure 2d and (b) Figure 2g. * indicates structures that can form during the last re-annealing step of the PCR, combining different types of CCR5 sequences (wild-type, indel-, or XhoI-containing sequences), and thereby resulting in heteroduplexes that are resistant to XhoI digestion. Alternatively, more than two single stranded amplicons can anneal to form complex structures.

Supplementary Figure 11 Supporting data for Figure 3.

Uncropped image of gel shown in Figure 3d.

Supplementary Figure 12 Supporting data for Figure 5.

(a) Gating strategy used to isolate primitive (P), early (E) and committed (C) progenitors from mobilized blood CD34+ HSPCs. (b) Uncropped image of gel shown in Figure 5d. (c) Uncropped image of gel shown in Figure 5g. (d) Uncropped image of gel shown in Figure 5h.

Supplementary Figure 13 Supporting data for Figure 6.

Uncropped image of gels shown in (a) Figure 6c (* = non-specific band; # = duplicate samples) and (b) Figure 6f.

Supplementary Figure 14 Amino acid sequences of AAVS1 ZFN pair used in this study.

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Supplementary Figures 1–14 and Supplementary Tables 1–4 (PDF 1824 kb)

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Wang, J., Exline, C., DeClercq, J. et al. Homology-driven genome editing in hematopoietic stem and progenitor cells using ZFN mRNA and AAV6 donors. Nat Biotechnol 33, 1256–1263 (2015). https://doi.org/10.1038/nbt.3408

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