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


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.

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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).


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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.

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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.

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Correspondence to Harry L Malech.

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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.

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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).

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