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. 2018 May 29;8(1):8214.
doi: 10.1038/s41598-018-26439-9.

Molecular Evidence of Genome Editing in a Mouse Model of Immunodeficiency

Free PMC article

Molecular Evidence of Genome Editing in a Mouse Model of Immunodeficiency

H H Abdul-Razak et al. Sci Rep. .
Free PMC article


Genome editing is the introduction of directed modifications in the genome, a process boosted to therapeutic levels by designer nucleases. Building on the experience of ex vivo gene therapy for severe combined immunodeficiencies, it is likely that genome editing of haematopoietic stem/progenitor cells (HSPC) for correction of inherited blood diseases will be an early clinical application. We show molecular evidence of gene correction in a mouse model of primary immunodeficiency. In vitro experiments in DNA-dependent protein kinase catalytic subunit severe combined immunodeficiency (Prkdc scid) fibroblasts using designed zinc finger nucleases (ZFN) and a repair template demonstrated molecular and functional correction of the defect. Following transplantation of ex vivo gene-edited Prkdc scid HSPC, some of the recipient animals carried the expected genomic signature of ZFN-driven gene correction. In some primary and secondary transplant recipients we detected double-positive CD4/CD8 T-cells in thymus and single-positive T-cells in blood, but no other evidence of immune reconstitution. However, the leakiness of this model is a confounding factor for the interpretation of the possible T-cell reconstitution. Our results provide support for the feasibility of rescuing inherited blood disease by ex vivo genome editing followed by transplantation, and highlight some of the challenges.

Conflict of interest statement

M.C.H. is an employee and P.D.G. and J.W. are former employees of Sangamo Therapeutics Inc, respectively. S.J.H. and M.E.A.-F. are employees of GSK. R.J.Y.-M. is a Trustee of the Genetic Alliance UK. The other authors declare no potential conflict of interest.


Figure 1
Figure 1
Prkdc gene editing strategy and outcomes, ZFN design and gene expression, and cutting activity in scid fibroblasts. (a) Schematic of Prkdc, including location of scid and ZFN target sites, structure of ZFN and sequences of various Prkdc alleles around scid site. (b) Production of ZFN monomers from SFFV-driven IPLV and IDLV (qPCR MOI 100). Proteins were extracted 3 d after transduction. The blotting revealed FLAG-tagged ZFN monomers (42 and 38.5 kDa for ZFN1 & ZFN2, respectively), with α-tubulin used as a loading control (49 kDa). (c) Cel-I analysis of Prkdc cutting by ZFN. Scid fibroblasts were transduced with IPLV- and IDLV-ZFN at the indicated MOI and genomic DNA was extracted 3 d later. The Cel-I-digested PCR products were separated on a polyacrylamide gel and diagnostic bands (black arrows) were used to calculate the % of indels induced by the various ZFN treatments and indicated on the gel image.
Figure 2
Figure 2
Phenotypic and molecular analyses of Prkdc gene editing in scid fibroblasts. (a) Gene editing in scid fibroblasts by plasmid transfection. Prkdc-neo template plasmid and ZFN monomer plasmids were used. All samples but the mock received repair template. G418-resistant CFUs were stained with crystal violet and counted. Statistical significance was evaluated using one-way ANOVA with Dunnett’s post hoc, comparing against template-only sample; ****p < 0.0001. (b) Prkdc gene editing in scid fibroblasts by transduction. Cells were transduced with IPLV-ZFN/IDLV-template or IDLV-ZFN/template at the indicated qPCR MOI and genomic DNA was extracted 10 d post-transduction. Scid locus was PCR-amplified with primers external to template, and ZFN-mediated gene correction was quantified from the diagnostic BsaWI band (arrow) and shown as %Prkdc correction. All samples shown were run in the same gel, from which irrelevant lanes have been cropped at places shown as thin white strips. The uncropped gel is shown in Supplementary Figure 6. (c) Restoration of DNA-PK activity after ZFN-mediated gene correction. Scid fibroblasts were transduced as above with IPLV or IDLV ZFN/template at the indicated MOI. The DNA-PK activity assay was performed using nuclear proteins extracted 10 d post transduction. Specific DNA-PK enzyme activity is plotted as a ratio between sample (gene edited cells) and mock (scid fibroblasts). Statistical significance was evaluated using a one-way ANOVA with Dunnett’s post hoc, comparing against mock sample; ***p < 0.0005. (d) Enhanced resistance of Prkdc gene-edited cells to DNA damage. Scid fibroblasts transduced with IDLV-ZFN/template at the indicated MOI were exposed to 10 μM melphalan for 1 h weekly for 3 weeks, then colonies were stained with crystal violet and CFU scored. Statistical significance was evaluated using a one-way ANOVA with Dunnett’s post hoc, comparing against mock sample; ****p < 0.00005. (e) Melphalan enrichment correlates with increase in molecular signature of Prkdc gene editing. Scid fibroblasts transduced with IDLV-ZFN/template at the indicated MOI were maintained in culture with or without weekly 10 μM melphalan treatment. Genomic DNA was extracted 3 weeks post-transduction and subjected to BsaWI digestion. The %Prkdc gene correction calculated from the intensity of the diagnostic band (arrow) is indicated on the gel.
Figure 3
Figure 3
ZFN activity in HSPC and rescue of scid mouse T-cell deficiency. (a) Cel-I analysis of ZFN-mediated Prkdc cutting. Scid HSPC were transduced with IPLV-ZFN at the indicated MOI and genomic DNA was extracted 3 d later. Diagnostic Cel-I digestion products are indicated with black arrows and the % of indels induced by ZFN treatments shown on the gel image. (b–d) T-cell populations in PBMC from primary transplant recipients, 27 weeks post-transplantation. Primary transplantation of irradiated female scid mice was done with gene-edited male scid HSPC, while secondary transplantation of female scid mice was with whole bone marrow (BM) cells. Flow cytometry analyses of (b) CD3, (c) CD4 and (d) CD8 T-cell populations from wild-type balb/c and scid mice, and balb/c, scid-IPLV-eGFP, scid-IPLV-ZFN/IDLV-template and scid-IDLV-ZFN/template transplant recipients are shown. (e) Thymic T-cell precursors in primary transplant recipients, 27–32 weeks post-transplantation. 50% of gene-edited transplant recipients were responders with double-positive CD4 CD8 cells. (f) T-cell populations in PBMC of secondary scid-IPLV-ZFN/IDLV-template transplant recipient, 35 weeks post-transplantation. (g) Proliferative response of purified spleen CD3 T-cells at 35 weeks post-secondary transplantation. CFSE-stained T-cells were cultured in the presence (black line) or absence (grey overlay) of concanavalin A and IL-2 or anti-CD3/CD28 and IL-2. After 72 h, cell expansion of the T-cell population was assessed by CFSE dilution measured by flow cytometry.

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