Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Targeted gene addition in human CD34+ hematopoietic cells for correction of X-linked chronic granulomatous disease

Nature Biotechnology volume 34, pages 424–429 (2016)Cite this article

Subjects

Abstract

Gene therapy with genetically modified human CD34+ hematopoietic stem and progenitor cells (HSPCs) may be safer using targeted integration (TI) of transgenes into a genomic 'safe harbor' site rather than random viral integration. We demonstrate that temporally optimized delivery of zinc finger nuclease mRNA via electroporation and adeno-associated virus (AAV) 6 delivery of donor constructs in human HSPCs approaches clinically relevant levels of TI into the AAVS1 safe harbor locus. Up to 58% Venus+ HSPCs with 6–16% human cell marking were observed following engraftment into mice. In HSPCs from patients with X-linked chronic granulomatous disease (X-CGD), caused by mutations in the gp91phox subunit of the NADPH oxidase, TI of a gp91phox transgene into AAVS1 resulted in ∼15% gp91phox expression and increased NADPH oxidase activity in ex vivo–derived neutrophils. In mice transplanted with corrected HSPCs, 4–11% of human cells in the bone marrow expressed gp91phox. This method for TI into AAVS1 may be broadly applicable to correction of other monogenic diseases.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Learn more

Prices may be subject to local taxes which are calculated during checkout

Figure 1: AAVS1-specific ZFN and AAV6 donor-mediated targeted insertion of Venus fluorescent marker into human CD34+ HSPCs.
Figure 2: Optimization of ratios of AAVS1 ZFN mRNA to AAV6 Venus donor.
Figure 3: Optimization of AAV6 Venus donor MOI.
Figure 4: AAVS1-specific ZFN and AAV6 donor-mediated targeted insertion of gp91phox corrective gene into human CD34+ HSPCs from patients with X-linked chronic granulomatous disease (X-CGD).

References

  1. Biffi, A. et al. Lentiviral hematopoietic stem cell gene therapy benefits metachromatic leukodystrophy. Science 341, 1233158 (2013).

    Article  Google Scholar 

  2. Hacein-Bey-Abina, S. et al. Efficacy of gene therapy for X-linked severe combined immunodeficiency. N. Engl. J. Med. 363, 355–364 (2010).

    Article  CAS  Google Scholar 

  3. Cavazzana-Calvo, M. et al. Gene therapy of human severe combined immunodeficiency (SCID)-X1 disease. Science 288, 669–672 (2000).

    Article  CAS  Google Scholar 

  4. Hacein-Bey-Abina, S. et al. A serious adverse event after successful gene therapy for X-linked severe combined immunodeficiency. N. Engl. J. Med. 348, 255–256 (2003).

    Article  Google Scholar 

  5. Stein, S. et al. Genomic instability and myelodysplasia with monosomy 7 consequent to EVI1 activation after gene therapy for chronic granulomatous disease. Nat. Med. 16, 198–204 (2010).

    Article  CAS  Google Scholar 

  6. Carroll, D. Genome engineering with targetable nucleases. Annu. Rev. Biochem. 83, 409–439 (2014).

    Article  CAS  Google Scholar 

  7. Tebas, P. et al. Gene editing of CCR5 in autologous CD4 T cells of persons infected with HIV. N. Engl. J. Med. 370, 901–910 (2014).

    Article  CAS  Google Scholar 

  8. DeKelver, R.C. et al. Functional genomics, proteomics, and regulatory DNA analysis in isogenic settings using zinc finger nuclease-driven transgenesis into a safe harbor locus in the human genome. Genome Res. 20, 1133–1142 (2010).

    Article  CAS  Google Scholar 

  9. Genovese, P. et al. Targeted genome editing in human repopulating haematopoietic stem cells. Nature 510, 235–240 (2014).

    Article  CAS  Google Scholar 

  10. Carroll, D. & Beumer, K.J. Genome engineering with TALENs and ZFNs: repair pathways and donor design. Methods 69, 137–141 (2014).

    Article  CAS  Google Scholar 

  11. Urnov, F.D. et al. Highly efficient endogenous human gene correction using designed zinc-finger nucleases. Nature 435, 646–651 (2005).

    Article  CAS  Google Scholar 

  12. Moehle, E.A. et al. Targeted gene addition into a specified location in the human genome using designed zinc finger nucleases. Proc. Natl. Acad. Sci. USA 104, 3055–3060 (2007).

    Article  CAS  Google Scholar 

  13. Vierstra, J. et al. Functional footprinting of regulatory DNA. Nat. Methods 12, 927–930 (2015).

    Article  CAS  Google Scholar 

  14. Song, L. et al. Optimizing the transduction efficiency of capsid-modified AAV6 serotype vectors in primary human hematopoietic stem cells in vitro and in a xenograft mouse model in vivo. Cytotherapy 15, 986–998 (2013).

    Article  CAS  Google Scholar 

  15. Song, L. et al. High-efficiency transduction of primary human hematopoietic stem cells and erythroid lineage-restricted expression by optimized AAV6 serotype vectors in vitro and in a murine xenograft model in vivo. PLoS One 8, e58757 (2013).

    Article  CAS  Google Scholar 

  16. Orlando, S.J. et al. Zinc-finger nuclease-driven targeted integration into mammalian genomes using donors with limited chromosomal homology. Nucleic Acids Res. 38, e152 (2010).

    Article  Google Scholar 

  17. Kuhns, D.B. et al. Residual NADPH oxidase and survival in chronic granulomatous disease. N. Engl. J. Med. 363, 2600–2610 (2010).

    Article  CAS  Google Scholar 

  18. Marciano, B.E. et al. Common severe infections in chronic granulomatous disease. Clin. Infect. Dis. 60, 1176–1183 (2015).

    Article  CAS  Google Scholar 

  19. Challita, P.M. et al. Multiple modifications in cis elements of the long terminal repeat of retroviral vectors lead to increased expression and decreased DNA methylation in embryonic carcinoma cells. J. Virol. 69, 748–755 (1995).

    CAS  PubMed  PubMed Central  Google Scholar 

  20. Astrakhan, A. et al. Ubiquitous high-level gene expression in hematopoietic lineages provides effective lentiviral gene therapy of murine Wiskott-Aldrich syndrome. Blood 119, 4395–4407 (2012).

    Article  CAS  Google Scholar 

  21. Wang, 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).

    Article  CAS  Google Scholar 

  22. Langmead, B. & Salzberg, S.L. Fast gapped-read alignment with Bowtie 2. Nat. Methods 9, 357–359 (2012).

    Article  CAS  Google Scholar 

  23. Gabriel, R. et al. An unbiased genome-wide analysis of zinc-finger nuclease specificity. Nat. Biotechnol. 29, 816–823 (2011).

    Article  CAS  Google Scholar 

  24. Brenner, S. et al. Concentrated RD114-pseudotyped MFGS-gp91phox vector achieves high levels of functional correction of the chronic granulomatous disease oxidase defect in NOD/SCID/β2-microglobulin−/− repopulating mobilized human peripheral blood CD34+ cells. Blood 102, 2789–2797 (2003).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

These studies were supported in part by project 1 ZIA AI000644 of the intramural program of National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health. Human CD34+ HSPCs were obtained under NIAID IRB approved Protocol 94-I-0073 after written informed consent. Murine animal studies were performed under National Institutes of Allergy and Infectious Diseases (NIAID) Animal Care and Use Committee approved protocol Laboratory of Host Defenses (LHD) 3E.

Author information

Author notes

  1. Philip D Gregory

    Present address: Present address: blubird bio, Cambridge, Massachusetts, USA.,

  2. Suk See De Ravin and Andreas Reik: These authors contributed equally to this work.

Authors and Affiliations

  1. Laboratory of Host Defenses, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA

    Suk See De Ravin, Narda Theobald, Uimook Choi, Janet Lee, Hannah Newcombe, Sherry Koontz, Colin Sweeney, Kol A Zarember & Harry L Malech

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

    Andreas Reik, Pei-Qi Liu, Alexander H Song, Andy Chan, Jocelynn R Pearl, David E Paschon, David A Shivak, Philip D Gregory & Fyodor D Urnov

  3. MaxCyte, Inc., Gaithersburg, Maryland, USA

    Linhong Li

  4. Cancer Research Technology Program, Leidos Biomedical Research, Inc., Frederick, Maryland, USA

    Xiaolin Wu, Ling Su, Castle Raley & Madhusudan V Peshwa

Authors
  1. Suk See De Ravin
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Andreas Reik
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Pei-Qi Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Linhong Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Xiaolin Wu
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Ling Su
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Castle Raley
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Narda Theobald
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Uimook Choi
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Alexander H Song
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Andy Chan
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Jocelynn R Pearl
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. David E Paschon
    View author publications

    You can also search for this author in PubMed Google Scholar

  14. Janet Lee
    View author publications

    You can also search for this author in PubMed Google Scholar

  15. Hannah Newcombe
    View author publications

    You can also search for this author in PubMed Google Scholar

  16. Sherry Koontz
    View author publications

    You can also search for this author in PubMed Google Scholar

  17. Colin Sweeney
    View author publications

    You can also search for this author in PubMed Google Scholar

  18. David A Shivak
    View author publications

    You can also search for this author in PubMed Google Scholar

  19. Kol A Zarember
    View author publications

    You can also search for this author in PubMed Google Scholar

  20. Madhusudan V Peshwa
    View author publications

    You can also search for this author in PubMed Google Scholar

  21. Philip D Gregory
    View author publications

    You can also search for this author in PubMed Google Scholar

  22. Fyodor D Urnov
    View author publications

    You can also search for this author in PubMed Google Scholar

  23. Harry L Malech
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

This research was supported by the Intramural Research Program of the National Institute of Allergy and Infectious Diseases, National Institutes of Health under intramural project numbers Z01-AI-00644 and Z01-AI-00988. S.S.D.R., A.R., P.-Q.L., L.L. and X.W. performed most of the experiments, N.T., U.C., J.L., S.K., C.R., H.N., L.S., C.S., A.H.S., A.C., J.R.P., D.E.P. and D.A.S. developed reagent, ran assays and analyzed samples, M.V.P. and P.D.G. provided support, S.S.D.R., K.A.Z., A.R., F.D.U. and H.L.M. designed experiments and wrote manuscript.

Corresponding author

Correspondence to Harry L Malech.

Ethics declarations

Competing interests

The following authors are full-time employees of Sangamo BioSciences and might own Sangamo stock or derivatives: A.R., P.-Q.L., C.R., A.H.S., A.C., J.R.P., D.E.P., D.A.S., P.D.G. and F.D.U. L.L. and M.V.P. are full-time employees of MaxCyte Systems.

Integrated supplementary information

Supplementary Figure 1 AAVS1 Venus TI with varying AAVS1 ZFN mRNA amount and AAV6 Venus MOI

To extend conditions for transfection, experiments as described in Fig 2 were repeated using lower concentrations of AAVS1 ZFN mRNA as indicated, immediately followed by exposure to indicated AAV6 Venus donor MOIs. This figure shows cell viability (top), percent of live cells expressing Venus marker (middle) and relative viable cell numbers at each day of culture after treatment in each sample following treatment (bottom). Values were determined for each analysis at culture day 3, 4, 6 and 9 (corresponding to 1, 2, 4 and 7 days post treatment). The data demonstrated the significant impact of titrating amount of ZFN mRNA to achieve optimal outcome, which for the ZFN mRNAs used in our study, was achieved with 25gμg/mL.

Supplementary Figure 2 Optimization of culture days before treatment.

Following thaw (day 0), healthy volunteer CD34+ HSC were cultured for a period of 1, 2 or 3 days before treatment with electroporation delivery of AAVS1 ZFN mRNA followed by AAV6 Venus donor addition. This figure shows cell viability (top), and percent of live cells expressing Venus marker % Venus+ cells in the gated live cells at 2, 3, 4, and 6 days following treatment (blue, red, green and black bars respectively).

Supplementary Figure 3 Schema for molecular analysis for TI.

a. Out/Out PCR utilizes primers located outside the donor homology arm (green) with HDR-F4 and HDR-R5; Out/In PCR with one primer situated outside the left homology arm and the second primer within the construct (HDR-F4 and 2A-R (or gp91-R)), or within the construct to the right homology arm, ie the In/Out PCR (primers 2A-F (for gp91-F) and HDR-R5). The regular MiSeq is performed with primers Mi-F and Mi-R; 5’-2A-TI Miseq with primers Mi-F, 2A-R, and Mi-R; and 3’MiSeq with primers Mi-F, polyA-F, and Mi-R.

b. Schema showing the primers for extended sequencing of the AAVS1 TI junction by PacBio single molecule sequencing. PCR primer pairs with one inside the vector specific region and the other outside the vector in the non-overlapping genomic region of the AAVS1 locus results in amplification of the junctions of the integrated vector.

Supplementary Figure 4 Representative sequences of AAV6 TI junction at the AAVS1 locus.

Representative sequences showing the junctions of AAV6 Venus integration at the AAVS1 locus by HDR (i, ii) or NHEJ (iii). A representative sequence for the left arm (L) junction at the AAVS1 locus following TI by HDR shows AAVS1 sequences 5’ beyond the AAV6 vector-L arm AAVS1 junction-AAV6 vector specific sequence (i). A representative sequence of the R arm junction is shown as AAV6 vector specific sequence-R arm AAVS1 junction-AAVS1 sequence 3’ beyond the vector (ii). A representative sequence of a NHEJ-mediated junction is shown as AAVS1 sequence 5’ shared AAVS1 and AAV6 vector-AAVS1 sequence shared by L arm of AAV6 vector-ITR-L arm of AAVS1 seq shared with AAV6 vector-AAV6 vector specific sequence (iii). Since the ITR lies outside the homology region, it is not incorporated during HDR so that the junction sequence does not contain the ITR. However, during NHEJ, the ITR sequence is retained in the junction. The expected amplicon sizes range from 1.1 kb to 2.5 kb depending on whether the ITR is retained. Both AAVS1L junction and AAVS1R junction were amplified, and PacBio long single DNA molecule sequencing identified 1082 integration events without ITR sequence and 3 integration events retaining ITR sequence. The results suggest that almost all AAV integration at the AAVS1 locus we observed occurred through HDR between AAV6 vector bearing-AAVS1 sequence flanking the transgene and endogenous AAVS1 sequence.

Supplementary Figure 5 Mouse transplant study analysis at 8 weeks.

Insert figure caption here by deleting or overwriting this text; captions may run to a second page if necessary. To ensure accurate appearance in the published version, please use the Symbol font for all symbols and Greek letters.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–5 and Supplementary Table 1 (PDF 1886 kb)

Rights and permissions

Reprints and Permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

De Ravin, S., Reik, A., Liu, PQ. et al. Targeted gene addition in human CD34+ hematopoietic cells for correction of X-linked chronic granulomatous disease. Nat Biotechnol 34, 424–429 (2016). https://doi.org/10.1038/nbt.3513

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nbt.3513

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing