In both humans and canines, X-linked severe combined immunodeficiency disease (XSCID) is caused by mutations in the interleukin-2 receptor gamma chain gene (IL2RG) which results in a lack of response to common gamma-chain (gammaC) dependent cytokines and abnormal development of T and B lymphocytes, and natural killer (NK) cells. Death from infections usually occurs before 1 year of age unless allogeneic hematopoietic cell transplantation (HCT) is performed. While HCT is successful if an HLA-matched sibling donor is available, transplants from mismatched and unrelated donors are associated with greater morbidity and overall survival can be as low as 50%. To circumvent these complications, several clinical trials are testing the possibility of utilizing blood and marrow stem cells from the patient for ex vivo gene therapy to treat X-SCID. Although these trials show promising results, they require expensive GMP cell manufacturing that are not accessible to many patients, and may also necessitate low level of conditioning to improve engraftment of gene-corrected cells. With these limitations in mind, we have explored in vivo gene therapy as a treatment for X-SCID. We previously showed that foamy virus vectors (FVs), exhibit a potentially more favorable integration profile compared to lenti- and gamma-retroviral vectors. In vivo delivery of a gammaC-FV in dogs resulted in immune reconstitution with gene-corrected T cells in dogs but the treated animals still developed infections and had low levels of immunoglobulin levels. We hypothesized that an increased transduction of hematopoietic stem/progenitor cells in vivo might result in more rapid and sustained immune reconstitution. Thus, in the current study, we used cG-CSF and AMD3100 to mobilize hematopoietic stem/progenitor cells into the peripheral blood prior to in vivo injection with a FV expressing the gammaC gene driven by a PGK promoter (PGK-gammaC-FV). We mobilized two X-SCID dogs at ~3 weeks of age with 5ug/kg of cG-CSF bi-daily from day -4 to -1 prior to FV injection, and with 4mg/kg of AMD3100 on the morning of the injection with 4x10e8 IU of PGK-gammaC-FV. Our mobilization protocol resulted in a 10-fold increase in CD34+ cells in the peripheral blood of mobilized X-SCID dogs as compared to a unmobilized normal littermate control (Figure 1 A). Lymphocyte recovery and gene marking in the mobilized animals was significantly improved as compared to animals that were previously injected with similar doses of either PGK-gammaC-FV or EF1a-gammaC-FV but without mobilization. As illustrated in Figure 1B-C, lymphocyte counts expanded to ~3000 cells/uL with ~75% gene marking in the mobilized animals treated with PGK-gammC-FV within 30 days, as compared to <1500 cells/uL with <5% gene marking in unmobilized dogs treated with EF1a-gammaC-FV and to <1000 cells/uL with <50% gene marking in unmobilized dogs treated with PGK-gammaC-FV at all time points post-therapy. The expansion of CD3+ T-cells at 6 weeks post injection for the mobilized dogs was about 2700 cells/uL, as compared to <380 cells/uL in the PGK-gammaC-FV and <210 cells/uL in the EF1a-gammaC-FV unmobilized dogs. Notably, in human clinical trials, CD3 T cell counts were <250 cells/uL following transplantation with autologous CD34+ cells modified with EF1a-gammaC-SIN gamma-retrovirus (Hacein-Bey-Abina, NEJM, 2014). In conclusion, mobilization with cG-CSF and AMD3100 prior to in vivo injection of PGK-gammaC-FV substantially improved the lymphocyte expansion and immune reconstitution in X-SCID dogs and resulted in a higher level of gene marking in myeloid cells (about 1%) at one-month post injection than seen in our previous studies in unmobilized dogs. These results suggest remarkable potential for an accessible and portable approach for treatment of human X-SCID clinical trials using combination of hematopoietic stem/progenitor cells mobilization and in vivo foamy viral vector delivery.


Adair:Rocket Pharmaceuticals: Consultancy, Equity Ownership. Scharenberg:bluebird bio: Consultancy, Equity Ownership, Research Funding; Alpine Immune Sciences: Consultancy. Kiem:Rocket Pharmaceuticals: Consultancy, Equity Ownership, Research Funding.

Author notes


Asterisk with author names denotes non-ASH members.