Lentivirus Mediated Correction of Artemis-Deficient Severe Combined Immunodeficiency

Abstract

During B and T lymphocyte maturation, V(D)J recombination is initiated by creation of DNA double-strand breaks. Artemis is an exonuclease essential for their subsequent repair by nonhomologous end-joining. Mutations in DCLRE1C, the gene encoding Artemis, cause T-B-NK+ severe combined immunodeficiency (ART-SCID) and also confer heightened sensitivity to ionizing radiation and alkylating chemotherapy. Although allogeneic hematopoietic cell transplantation can treat ART-SCID, conditioning regimens are poorly tolerated, leading to early mortality and/or late complications, including short stature, endocrinopathies, and dental aplasia. However, without alkylating chemotherapy as preconditioning, patients usually have graft rejection or limited T cell and no B cell recovery. Thus, addition of normal DCLRE1C cDNA to autologous hematopoietic stem cells is an attractive strategy to treat ART-SCID. We designed a self-inactivating lentivirus vector containing human Artemis cDNA under transcriptional regulation of the human endogenous Artemis promoter (AProArt). Fibroblasts from ART-SCID patients transduced with AProArt lentivirus showed correction of radiosensitivity. Mobilized peripheral blood CD34+ cells from an ART-SCID patient as well as hematopoietic stem cells from Artemis-deficient mice demonstrated restored T and B cell development following AProArt transduction. Murine hematopoietic cells transduced with AProArt exhibited no increase in replating potential in an in vitro immortalization assay, and analysis of AProArt lentivirus insertions showed no predilection for sites that could activate oncogenes. These efficacy and safety findings support institution of a clinical trial of gene addition therapy for ART-SCID.

Keywords: Artemis; gene therapy; lentivirus; radiation sensitivity; severe combined immunodeficiency.

Conflict of interest statement

Author Disclosure No competing financial interests exist.

Figures

Figure 1.

Schematic representations of AProArt and control cytomegalovirus–green fluorescent protein (CMV-GFP) lentivirus vectors. The human Artemis (DLCRE1C) cDNA (Hu Artemis), with its endogenous promoter (APro) was introduced into self-inactivating lentiviral vector pCS with promoter and enhancer sequences deleted (X) from the U3 region of the 3′ LTR to generate pCS-AProArt. A central polypurine tract (CPPT) was included in the vector backbone, preceded by the packaging site (ψ). The vector includes a modified woodchuck post-transcriptional regulatory element (WPRE) and a 5′ CMV promoter to generate full-length vector message for packaging. The control lentiviral vector with the same backbone, pCS-CMV-GFP, was constructed to express GFP under transcriptional regulation of the CMV promoter. LTR, long terminal repeats.

Figure 2.

In vitro assays with human skin fibroblasts. (a) Immunoprecipitation Western blot to detect Artemis protein (upper arrow) in fibroblasts from control (wild type, WT), Artemis deficient (ART-SCID) or Ligase-4 deficient (LIG4) skin fibroblasts, untransduced (left) or transduced with GFP (middle) or AProArt lentivirus (right). (b) BrdU fibroblast proliferation with WT (left), LIG4 deficient (middle), or ART-SCID (right) cells, following exposure to ionizing radiation (IR). The fibroblasts were untransduced (broken line) or transduced with control (gray line) or AProArt lentivirus (black line). Mean ± standard deviation (SD), n = 3 replicates; *p < 0.05. (c) Enumeration of γH2AX foci indicating DNA double-strand breaks, in WT (left), LIG4 deficient (middle), or ART-SCID (right) fibroblasts, following exposure to 4 Gy IR. The fibroblasts were either untransduced (broken line) or transduced with AProArt (solid line). Mean ± SD, n = 3 replicates; *p < 0.05; **p < 0.001.

Figure 3.

Relationship between vector copy number or transduction efficiency, and lentivirus concentration. Healthy control human peripheral blood mobilized CD34+ cells transduced with increasing concentrations of AProArt lentivirus (black bars) were cultured for 14 days. (a) Percentage of lentivirus transduced colonies per total hematopoietic colonies isolated from semisolid medium, determined by quantitative PCR of individual colonies. Mean ± SD, n = 3 replicates. (b) Average vector copy number per two copies of a human genomic control in cells grown in liquid bulk cultures, determined by quantitative PCR. Mean ± SD, n = 3 replicates.

Figure 4.

Analysis of Art−/− mice, transplanted with CD45.1 donor cells. (a) Percentage of donor-derived CD45.1 lymphocytes in peripheral blood at 4, 8, 12, 16, and 20 weeks post transplantation into Art−/− CD45.2 recipients pretreated with 500 μg of anti-c-kit antibody. Mice received 105 CD45.1 AProArt-transduced Art−/− (black squares, n = 5), or 104 (X's, n = 5) or 105 untransduced wild type (WT, gray circles, n = 3) hematopoietic stem cells (HSC). Horizontal lines indicate mean chimerism. (b) Proliferation of CD3+ splenocytes from untransplanted control Art−/− (clear bars, n = 5); WT (gray bars, n = 7); or Art−/− mice transplanted with 104 untransduced WT HSC (hatched bars, n = 4) or AProArt lentivirus transduced Art−/− HSC (black bars, n = 6), measured as a percentage of EdU positive cells after 4 days in medium, 4 μg/mL or 10 μg/mL Concavalin A (ConA), or 5 μg/mL anti-mouse CD3ɛ. Mean ± standard error of the mean (SEM), n = 3 replicates; *p < 0.05, **p < 0.001. (c) CD8+ (black bars) and CD4+ (gray bars) T cell numbers in control Art−/− (n = 5) and WT (n = 5) mice, and Art−/− mice transplanted with 104 WT HSC (n = 7) or AProArt lentivirus transduced Art−/− HSC (n = 6). Mean ± SEM; *p < 0.05, **p < 0.001, compared to Art−/− control. (d) B cell development in bone marrow of untransduced control Art−/− (clear bars, n = 3); WT (gray bars, n = 3); and Art−/− mice 3 months after transplant with 104 WT HSC (hatched bars, n = 5) or AProArt-transduced Art−/− HSC (black bars, n = 5). B cell progenitor subsets were: CD45+ B220+ CD43+ pro B cells; CD45+ B220+ CD43− IgM− pre B cells; and CD45+ B220+ CD43− IgM+ immature B cells, as a percentage of CD45+ B220+ B cells. Mean ± SEM; *p < 0.05. (e) Antigen-specific serum immunoglobulin (Ig)M and IgG antibody responses to immunization with NP-KLH (minus pre-immune response) in WT (black diamonds, n = 3) and Art−/− (black squares, n = 3) control mice, and Art−/− recipients of Art−/− HSC transduced with AProArt lentivirus (black triangles, n = 3) or 104 WT HSC (gray rectangles, n = 3). Mean ± SD, n = 3 replicates; *p < 0.05 compared to Art−/− controls. NP-KLH, NP24-keyhole limpet hemocyanin.

Figure 5.

Analysis of NSG mice transplanted with human CD34+ HSC. (a) Percent of human cells, identified by expression of human-specific pan-leukocyte marker CD45, detected in peripheral blood (PB), bone marrow (BM) or spleen, analyzed by flow cytometry at weeks (w) indicated post transplantation with GFP-transduced control (WT) HSC (clear bars, n = 6), AProArt-transduced ART-SCID HSC (black bars, n = 7) or GFP-transduced ART-SCID HSC (gray bars, n = 5). Mean ± SD; *p < 0.05. (b) Fraction of human CD19+ B cells (left panel) or CD3+ T cells (right panel) analyzed by flow cytometry, in total human CD45+ cells detected in the peripheral blood (PB), bone marrow (BM) and spleen of NSG mice transplanted with GFP-transduced control HSC (clear bars, n = 6), AProArt-transduced ART-SCID HSC (black bars, n = 7) or GFP-transduced ART-SCID HSC (gray bars, n = 5). Mean ± SD; *p < 0.05. (c) Quantification of human B cell Ig κ-chain rearrangement by quantitative PCR, identified as κ-chain restriction circles (KRECs) and Kde coding joints (Kde CJ), using DNA from NSG mice transplanted with GFP-transduced WT HSC (clear bars, n = 6), AProArt-transduced ART-SCID HSC (black bars, n = 7) or GFP-transduced ART-SCID HSC (gray bars, n = 5). Mean ± SD; *p < 0.05; **p < 0.001. (d) T cell receptor diversity in 24 human Vβ T cell receptor (TCR) families by spectratyping, using RNA from spleens of NSG mice transplanted with GFP-transduced control HSC (left), AProArt lentivirus transduced ART-SCID HSC (middle) or GFP-transduced ART-SCID HSC (right). Monoclonal pattern, single peak per Vβ TCR family; oligoclonal, 1–4 peaks; polyclonal, ≥5 peaks. Color images available online at www.liebertpub.com/hum

Figure 6.

In vitro replating frequency to assess immortalization potential. Replating frequency of nontransduced mock cells (n = 3), positive control samples, RSF91 (n = 8) and lv-SF (n = 3), and the test vector AProArt (n = 9). Bars, mean repopulating frequency; Q1, lower detection range between background proliferation and the first quartile of the RSF91 clonal expectation value at viral copy number 3. Negative assay points manually inserted into the graph. *p < 0.01.