Preclinical ex vivo IL2RG gene therapy using autologous hematopoietic stem cells as an effective and safe treatment for X-linked severe combined immunodeficiency disease

Figures

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  Figure 1. Clinical and molecular characteristics of patients with X-linked severe combined immunodeficiency disease (X-SCID). (A) Family pedigrees of the patients. The black solid symbols indicate the affected patients (P1–P9), the grey solid symbols indicate carriers of the same gene mutation, and the open symbols indicate unaffected family members. The squares indicate male subjects and the circles indicate female subjects. The crosses indicate spontaneous abortion or induced abortion because of abnormal prenatal examination and the slashes indicate death at a very young age. (B) Schematic representation of IL2RG gene map and the protein domain. The grey squares represent the exons (E1–E8). The red arrows indicate novel mutations identified in our patients and the grey arrows indicate previously reported gene mutations in P5, P6, and P8. (C) Scaled C-scores of the mutation identified in our patients. The blue dots represent missense variants and the green dots splice variants. (D) CD132 expression of total lymphocytes, NK cells, and T cells. The light grey indicates the isotype control, the dark grey shadow indicates the healthy control, and the red shadow indicates the patients. (E)…

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  Figure 2. Cell proliferation and STAT5 phosphorylation in P3 and P9. (A) A profound T cell proliferation defect in P9 but a slight proliferation defect in P3. (B) Western blot of phospho-STAT5 (pSTAT5) expression in peripheral blood mononuclear cells of P3 and HC after stimulation with IL-2. (C) The ratio of p-STAT5 to GAPDH was calculated after measuring the band densities of p-STAT5 and GAPDH using ImageJ. (D) pSTAT5 expression in lymphocytes and T cells of P9 and HC after stimulation with IL-2. (E) MFI of pSTAT5 in lymphocytes and T cells. The light grey indicates the isotype control (iso), the dark grey indicates the healthy control (HC), and the red indicates the patients (P). HC, healthy control; P, patient; MFI, mean fluorescence intensity.

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  Figure 3. Comparison of the EFS-IL2RG.wt and EFS-IL2RG.co vectors in human ED7R cells and the bone marrow stem cells. (A) Scheme of the self-inactivating (SIN) lentiviral vector containing the deleted long terminal repeats (LTRs) (Δ), the short EF1α promoter (EFS), the codon-optimized or wild type version of the IL2RG (IL2RG.co/IL2RG.wt), the HIV-1 Rev Responsive Element (RRE), and a mutated Woodchuck Post-transcriptional Response Element (WPRE). (B) Flow cytometry of CD132 expression, IL2RG-mRNA expression, and quantitative PCR analysis of VCN in EFS-IL2RG.co and EFS-IL2RG.wt vector-transduced ED7R cells. IL2RG-mRNA expression is expressed as fold change with respect to endogenous IL2RG in Jurkat cells. (C) CD132 expression and VCN in EFS-IL2RG.co and EFS-IL2RG.wt vector-transduced human bone marrow-derived CD34+ cells. VCN, vector copy number; MFI, mean fluorescence intensity; MOI, multiplicity of infection.

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  Figure 4. Small-scale transduction of EFS-IL2RG.co vector into bone marrow stem cells. (A) The purity of CD34+ cells after separation. (B, D) CD132 expression in transduced CD34+ cells at different MOIs in a small-scale transduction. (C) VCN in transduced CD34+ cells at different MOIs. (E) Hemopoietic cell colonies were observed in all samples from both the transduced and untransduced groups. (F) The total numbers of colony-forming units (CFUs) were the same in both groups (left), as were the numbers of colonies of different lineages (right). VCN, vector copy number; MFI, mean fluorescence intensity; MOI, multiplicity of infection.

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  Figure 5. Large-scale transduction of human mobilized hematopoietic stem and progenitor cells in vitro and oncogenicity analysis of transduced CD34+ cells in nude mice. (A) Cell viability of untransduced or EFS-IL2RG.co vector-transduced human CD34+ cells in the ex vivo culture; the CD132 expression in both untransduced or EFS-IL2RG.co vector-transduced CD34+ cells at different time points; VCN in the transduced cells; the numbers of colonies of different lineages formed by untransduced and vector-transduced CD34+ cells. (B) Cell viability of the final product during re-infusion monitored at different times after thawing. (C) The quality check on the frozen large-scale transduced cells at different time points (0, 1, and 6 months). From left to right: The purity of CD34+ cells being frozen at 1 and 6 months; the cell viability being frozen at 1 and 6 months; CD132 expression at 1, 3, 7, and 11 days after the transduced cells being frozen at 6 months; VCN and colony forming capacity of the final frozen cell product being frozen at 1 and 6 months. (D) Over survival of nude mice in different groups. (E) Nude mouse weights in different groups. (F) Histopathological evaluation of tumor growth…

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  Figure 6. Successful ex vivo transduction of CD34+ cells from patients with X-linked severe combined immunodeficiency disease (X-SCID). (A) CD132 expression in IL2RG defect or corrected CD34+ cells at 2 days and 7 days after transduction. (B) MFI of CD132 in CD34+ cells. (C) Mean VCNs (± standard deviation) in the bulk population of transduced CD34+ cells. (D) The total numbers of colony-forming units (CFUs) of CD34+ cells in both groups (left) and the numbers of colonies of different lineages (right). The light grey indicates the isotype control (iso), the dark grey indicates the healthy control (HC), and the red indicates patients (P). The data were presented as mean ± standard error of the mean. Statistical differences are expressed as follows: ns, non-significant; ∗∗p < 0.01. VCN, vector copy number; MFI, mean fluorescence intensity; MOI, multiplicity of infection.