X-linked severe combined immunodeficiency (SCID-X1) is caused by mutations of interleukin-2 receptor γ chain (IL2RG), resulting in a lack of response to common γ-chain (γc, IL-2Rγ, or CD132)–dependent cytokines and T- B+ NK- SCID. For patients who lack matched related donors, autologous gene therapy transducing CD34+ hematopoietic progenitors with a viral vector expressing the IL2RG cDNA is a promising intervention that avoids graft-versus-host disease. Whether all IL2RG variants, particularly those that express a mutant protein, can be corrected through gene therapy is not clear.
Here, we report a novel variant in exon 8 of IL2RG (c.961_962insC, p.Leu321fsX327), an insertion at position 961-962, that causes a frameshift and premature stop codon in the cytoplasmic tail of IL2RG. This mutation disrupts the Box 2 JAK3–binding motif essential for cytokine-induced signaling. The patient (XSCID05) presented with typical SCID and underwent two infusions autologous CD34+ stem cells transduced with a self-inactivating gammaretroviral vector without chemotherapy conditioning, resulting in poor T cell reconstitution despite sustained gene marking in CD3+ T cells (Figure 1A, 1B).
Summary of abstract. A) The vector copy number (VCN) for the patient (#5) compared with other patients in the trial. B) The CD3 T cell count in patient (#5) after two rounds of gene therapy is shown compared with other patients in the trial. C) HEK-Blue (HB) reporter assay cell lines that express IL2RG, IL-2/-15, or IL-7 receptors, and a STAT5-sensitive reporter gene were transduced with lentiviral vectors expressing the patient’s IL2RG mutation (XSCID05), WT IL2RG, or empty vector control, then treated with increasing concentrations of IL-2, IL-15, or IL-7. pSTAT5-sensitive reporter gene activity was quantified using spectrophotometry. Representative data showing that overexpression of the patient mutation interfered with signaling of the endogenous IL2RG. D) Results of multiple experiments plotted as area under the curve (AUC) normalized to the empty vector control of that experiment. IL2RG mutants lacking surface expression (p.Ser94X, p.Cys62Ser) were used as additional controls. n.s. = not significant, **p = 0.01-0.001, ***p ≤ 0.001.
Summary of abstract. A) The vector copy number (VCN) for the patient (#5) compared with other patients in the trial. B) The CD3 T cell count in patient (#5) after two rounds of gene therapy is shown compared with other patients in the trial. C) HEK-Blue (HB) reporter assay cell lines that express IL2RG, IL-2/-15, or IL-7 receptors, and a STAT5-sensitive reporter gene were transduced with lentiviral vectors expressing the patient’s IL2RG mutation (XSCID05), WT IL2RG, or empty vector control, then treated with increasing concentrations of IL-2, IL-15, or IL-7. pSTAT5-sensitive reporter gene activity was quantified using spectrophotometry. Representative data showing that overexpression of the patient mutation interfered with signaling of the endogenous IL2RG. D) Results of multiple experiments plotted as area under the curve (AUC) normalized to the empty vector control of that experiment. IL2RG mutants lacking surface expression (p.Ser94X, p.Cys62Ser) were used as additional controls. n.s. = not significant, **p = 0.01-0.001, ***p ≤ 0.001.
We utilized HEK-Blue reporter assay cell lines to study the function of this mutant. HEK-Blue cells stably overexpress components of the relevant cytokine receptor and signaling pathway, including IL2RG, and a STAT5-sensitive reporter protein. We hypothesized that overexpression of WT IL2RG would render cells more sensitive to lower amounts of cytokine, shifting the curve to the left, and transduction with empty vector or mutants lacking surface expression (p.Ser94X, p.Cys62Ser) would have no effect, while the putative XSCID05 dominant negative mutant would interfere with endogenous IL2RG signaling, shifting the curve to the right. Lentiviral transduction of these constructs followed by treatment with increasing concentrations of IL-2, IL-15, or IL-7 revealed interference of mutant IL2RG with endogenous IL2RG signaling (Figure 1C, 1D). These findings suggest this mutant exerts a dominant negative effect on transgene function, providing an explanation for the poor T cell reconstitution observed in the patient and demonstrating that some IL2RG variants may not be correctable by lentiviral gene therapy. Our data highlight the need for further investigation into IL2RG variants that may mitigate otherwise successful curative use of gene therapy.
