Rescue of Murine IL-7 Receptor Deficiency with Human IL-7 Receptor Gene Therapy

In humans, interleukin 7 (IL-7) receptor (IL-7R) deficiency causes approximately 10% of cases of severe combined immunodeficiency (SCID). IL-7R deficient SCID is a T-B+NK+ SCID and is caused by autosomal recessive deficiency of the IL-7R alpha chain gene (IL7R). IL-7R is a heterodimeric receptor comprised of the alpha chain and the IL-2 receptor common gamma chain (IL2RG). In both mouse and human, IL-7R is a marker of the common lymphoid progenitor cell, and IL-7 signaling leads to STAT5 phosphorylation and proliferation of developing T and B cells. Mice lacking IL7R, Il7r ^-/-, lack both T and B cells (Peschon, JJ, et al. J Exp Med. 1994). T cells do not progress to TCR beta chain rearrangement and B cell development is halted at the pre-pro-B cell stage. Similar to the mouse, IL-7 signaling in humans is required for T cell receptor beta gene rearrangement and T cell maintenance, however humans lacking IL-7R can develop B cells.

A prior attempt to rescue murine IL-7R deficiency utilized a retroviral vector (mouse stem cell virus, MSCV), the MSCV retroviral promoter, and the murine Il7r gene (Jiang, Q, et al. Gene Therapy. 2005). This strategy did restore T cells and had variable restoration of B cells. However, retroviral-based gene addition of Il7r led to a myeloproliferative condition with significant neutrophilia and splenomegaly. Transduced bone marrow cells formed myeloid progenitors in response to IL-7 in vitro. We evaluated a novel gene therapy for IL-7R deficient SCID that utilizes the human IL7R gene. To prevent lineage skewing, we sought to limit ectopic expression of IL7R in non-lymphoid cells by utilizing the endogenous enhancers and promoters of IL7R. These sequences were identified as sites of high sequence conservation across species and DNA accessibility/hypersensitivity (DHS) in human lymphocytes. We are testing these sequences alone or in combination with the constitutive phosphoglycerate kinase promoter (PGK) in VSV-G pseudotyped lentiviral vectors (LV): vPGK_DHS_hIL7R and vDHS_hIL7R. Here we present the first data evaluating the ability of the human IL-7R protein to functionally replace the murine IL-7R protein and the ability of IL7R gene addition to rescue the murine Il7r ^-/- immunodeficient phenotype in vivo.

Transduction of Il7r ^-/- bone marrow cells with IL7R encoding LV rescued the formation of lymphocyte precursors from murine bone marrow cells in colony forming unit (CFU) assays (pre-B CFU with human IL-7), with the most robust response seen with vPGK_DHS_hIL7R. Mouse bone marrow from Il7r ^-/- animals transduced ex vivo engrafted in lethally irradiated (8 Gy) Il7r ^-/-oppositegender recipients and there were no significant aberrations in absolute neutrophil count, hemoglobin or platelet count. Absolute lymphocyte counts in mice receiving transduced Il7r ^-/-bone marrow cells was higher (mean 2555/μL) than in mice receiving untransduced bone marrow (mean 1410/μL). The proportion of leukocytes that were T cells was 4.2-fold and 9.8-fold higher at 1 and 2 months post-transplant, respectively. B cells were only seen in mice receiving vPGK_DHS_hIL7R: 7.4% of leukocytes versus 1.5% in controls. A reciprocal decrease in the fraction of Gr1+ cells (neutrophils and monocytes) was seen at two months post-transplant in transduced marrow recipients compared to untransduced controls: 36.5% versus 63% Gr1+, respectively. Lymphocyte subsets are being further analyzed, bone marrow and thymic lymphoid precursors assessed, and T and B cell function in response to immunizations are in progress. Further evaluation in human derived IL7R deficient human cells is warranted.

For individuals with IL-7R deficient SCID, but no HLA matched hematopoietic stem cell (HSC) donor, there is a difficult choice between the risks of GVHD with a mismatched HSC donor and supportive care with the hope of identifying a matched HSC donor in the future. In immunodeficiencies however age and serious infection are both associated with increased mortality (Pai, SY, et al. NJEM. 2014). This novel approach to IL7R gene replacement has the potential to be a therapeutic and expedient option for those without a matched donor. Additionally, this would be an ideal disorder for HSC conditioning with less toxic, HSC-targeted strategies given gene corrected lymphocytes and progenitors will preferentially expand post-transplant.