Nuclease-free Adeno-Associated Virus-Mediated Il2rg Gene Editing in X-SCID Mice

Abstract

X-linked severe combined immunodeficiency (X-SCID) has been successfully treated by hematopoietic stem cell (HSC) transduction with retroviral vectors expressing the interleukin-2 receptor subunit gamma gene (IL2RG), but several patients developed malignancies due to vector integration near cellular oncogenes. This adverse side effect could in principle be avoided by accurate IL2RG gene editing with a vector that does not contain a functional promoter or IL2RG gene. Here, we show that adeno-associated virus (AAV) gene editing vectors can insert a partial Il2rg cDNA at the endogenous Il2rg locus in X-SCID murine bone marrow cells and that these ex vivo-edited cells repopulate transplant recipients and produce CD4⁺ and CD8⁺ T cells. Circulating, edited lymphocytes increased over time and appeared in secondary transplant recipients, demonstrating successful editing in long-term repopulating cells. Random vector integration events were nearly undetectable, and malignant transformation of the transplanted cells was not observed. Similar editing frequencies were observed in human hematopoietic cells. Our results demonstrate that therapeutically relevant HSC gene editing can be achieved by AAV vectors in the absence of site-specific nucleases and suggest that this may be a safe and effective therapy for hematopoietic diseases where in vivo selection can increase edited cell numbers.

Keywords: AAV vector; genome editing; hematopoietic stem cells.

Figure 1

AAV-Mediated Gene Editing Restores T Cell Il2rg Expression (A) Wild-type and knockout Il2rg loci are shown with AAV vector maps. (B) GFP expression in vitro in AAV-scMSCV-GFP-transduced (red lines) or no vector (black lines) LSK cells 2 days after infection is shown. (C) Representative CD4⁺ and CD8⁺ populations in CD3⁺ cells from mice treated with the indicated vectors 32 weeks after transplant are shown. (D) CD4⁺ and CD8⁺ cell counts over time in the peripheral blood of mice (n = 16 for each group) treated with AAV-Il2rg3-8 (red lines), AAV-MSCV-GFP (blue lines), or no vector (black lines) are shown. Data are presented as mean ± SD. *p < 0.05; **p < 0.01 (two-way ANOVA). (E) Representative Il2rg surface expression (red lines) and isotype (black lines) in CD4⁺ and CD8⁺ cells from treated mice 20 weeks are shown. (F) Mode fluorescent intensity of Il2rg in CD4⁺ and CD8⁺ cells. Data are presented as mean ± SD. n.s., no significant difference (one-way ANOVA with Tukey’s multiple comparisons test). Each symbol represents a distinct mouse.

Figure 2

Detection of Vector Sequences in the Genomic DNA of Treated Mice (A) The qPCR products used to detect donor cells (blue bar), random integrants (green bar), and internal vector sequences (edited alleles or random integrants, red bar) are shown. (B) The frequency of donor cells, cells with random integrants, and edited cells (internal vector sequence frequency minus random integrant frequency) as determined by qPCR of DNA from peripheral blood WBCs, bone marrow cells, CD3⁺ splenocytes, and CD3^neg splenocytes of mice treated with AAV-Il2rg3-8 (black columns) or AAV-MSCV-GFP (white columns) 32 weeks after transplant (n = 4 of each) is shown. Total DNA amounts were determined by amplifying the Bcl2 gene (primers not shown) and used to calculate frequencies. Data are presented as mean ± SD. *p < 0.05 (two-tailed t test). (C) Representative GFP expression in peripheral blood WBCs at 28 weeks post-transplant is shown. (D) The frequency of GFP⁺ cells over time in the peripheral blood WBCs of mice treated with AAV-MSCV-GFP (n = 16) is shown. Data are presented as mean ± SD.

Figure 3

Edited Lymphocytes Increase in Secondary Transplant Recipients (A) Time course of CD3⁺, CD4⁺, and CD8⁺ cells in the peripheral blood of secondary transplant recipients that received bone marrow cells from mice treated with AAV-Il2rg3-8 (n = 8; red lines) or AAV-MSCV-GFP (n = 4; blue lines). Data are presented as mean ± SD. **p < 0.01 (two-way ANOVA). (B) The total number of CD3⁺, CD4⁺, and CD8⁺ cells in the spleens of secondary recipients 20 weeks after secondary transplant is shown. Data are presented as mean ± SD. **p < 0.01 (two-tailed t test). (C) Representative flow cytometry shows B and NK cell populations in the spleens. (D) The total number of B cells in these spleens is shown. Data are presented as mean ± SD. **p < 0.01 (two-tailed t test). (E) Representative flow cytometry analysis shows mature B cells (IgD⁺; B220⁺) in the splenocytes of an AAV-Il2rg3-8-treated secondary recipient.

Figure 4

Edited T Cell Characterization (A) Analysis of TCR Vβ repertoire in the CD3⁺ splenocytes of untreated wild-type mice and X-SCID mice treated with AAV-Il2rg3-8 or AAV-MSCV-GFP. (B and C) Representative flow cytometry (B) of naive CD4⁺ splenocytes (CD62L⁺; CD4⁺) and their total number (C) in mice treated with AAV-Il2rge3-8, AAV-MSCV-GFP, or no AAV (n = 16 per group) are shown. Data are presented as mean ± SD. **p < 0.01 (one-way ANOVA with Tukey’s multiple comparisons test). (D) Representative flow cytometry of naive CD8⁺ splenocytes (CD62L⁺; CD8⁺) and isotype control in AAV-Il2rg3-8-treated mice (AAV-MSCV-GFP-treated mice have no CD8⁺ cells) are shown. Spleen samples were obtained 32 weeks after transplant.

Figure 5

Immune Cells Increase after Immunization with Allogeneic Cells (A–C) Representative flow cytometry of CD3⁺ and NK1.1⁺ populations (A), CD4⁺ and CD8⁺ populations (B), and B220⁺ cells (C) in H-2Dd^neg blood cells 14 and 28 days after primary immunization with BALB/c splenocytes. (D) The number of CD4⁺ cells, CD8⁺ cells, B220⁺ cells, and NK1.1⁺ cells in peripheral blood over time is shown. Each solid or dashed line represents a different AAV-Il2rg3-8-treated or X-SCID control mouse, respectively. *p < 0.05 (two-tailed t test).

Figure 6

IL2RG Editing in Human CD34⁺ Cells (A) Map of the human IL2RG locus and AAV-IL2RGe2UNA editing vector. The IL2RG loci in CD34⁺ cell donors no. 2 and no. 3 have the indicated polymorphisms (SNP ID donor no. 2, rs11574625; donor no. 3, rs10693207). The red region indicates the qPCR amplicon. (B) GFP expression in vitro in AAV-scMSCV-GFP-transduced human CD34⁺ cells 2 days after infection is shown. (C) Editing frequencies in CD34⁺, CD133⁺ cells from donor no. 1 cultured for 7 days after infection with AAV-IL2RGe2UNA at the indicated MOIs (n = 3–5 for each condition) are shown. Data are presented as mean ± SEM. *p < 0.05; **p < 0.01 (one-way ANOVA with Tukey’s multiple comparisons test). (D) Editing frequencies in CD34⁺, CD133⁺ cells from different donors 7 days after transduction with AAV-IL2RGe2UNA (black columns) or no vector control (white columns; n = 3–5 for each condition) are shown. Data are presented as mean ± SD. *p < 0.05; **p < 0.01 (two-way ANOVA).