Immune Reconstitution and Preliminary Safety Analysis Of 9 Patients Treated With Somatic Gene Therapy For X-Linked Severe Combined Immunodeficiency (SCID-X1) With a Self-Inactivating Gammaretroviral Vector

X-linked severe combined immunodeficiency (SCID-X1) is a fatal genetic disease currently treated with hematopoietic stem cell transplantation. Previous gene therapy trials using a MLV-based gammaretroviral vector expressing the IL-2 receptor gamma chain (γc) controlled by the viral long-terminal repeat (LTR) induced T cell reconstitution in 18 of 20 boys with SCID-X1, but also resulted in insertional oncogenesis in 5 of 20, leading to leukemia, with 4 of the 5 demonstrating insertion at the LMO2 locus (Trial 1). We tested whether a self-inactivating (SIN) gammaretroviral vector, pSRS11.EFS.IL2RG.pre*, derived by removing the MLV LTR U3 (enhancer/promoter) region and driving expression of γc by an intronless elongation factor 1α (EF1-α) promoter, would promote T cell reconstitution in boys with X-SCID with an improved safety profile (Trial 2). Pre-clinical studies showed a lack of clonal skewing in primary and secondary transplant recipient mice in vivo and lack of insertions in either Lmo2 or Evi1 in these mice. The primary endpoints of the study are: 1. immune recovery defined as CD3+ T cell count >300/microliter and restoration of proliferation to phytohemagglutinin (PHA); and 2. Lack of insertional oncogenesis.

To date 9 boys with SCID-X1 age 3.9 to 10.5 months were enrolled with 5-32 months follow-up on the current trial. All received bone marrow derived CD34+ cells transduced with the pSRS11.EFS.IL2RG.pre* vector, infused without conditioning. Transduction was performed at each site with one of two common protocols and the same GMP-produced vector supernatant. Transduction efficiency of infused CD34+ cells ranged from 0.25-2.92 vector copy number (VCN). One patient with a low initial transduction underwent a repeat procedure 1 month after infusion of the first product. Overall survival is excellent with 8 out of 9 patients alive and well; 1 patient died of overwhelming adenoviral infection, present at study entry, at 4 months prior to full reconstitution with genetically modified T cells. Excluding 1 patient with high level maternal engraftment who is unevaluable with regard to T cell number, 5 of 7 (71%) achieved CD3+ T cell count >300/microliter. The 2 patients who did not achieve T cell count >300/microliter received CD34+ cells with the lowest vector copy number (VCN). An early rise in CD16/56+ NK cells was also associated with T cell recovery. Of 8 surviving patients, 7 of 8 (87.5%) had PHA stimulation index >15 at or before 6 months (n=8; median 85.5; range 0.2-224); the patient who had no functional recovery received mismatched allogeneic cord blood transplant. At last follow-up, the remaining 7 patients have evidence of γc transgene expression in T cells, naïve T cell generation, and normal diversity. They are free of SCID related infections and humoral immune evaluation is ongoing.

We compared the early immune recovery and peripheral blood insertion site analysis between 6 evaluable subjects on Trial 2 with the 20 subjects enrolled on the previous trials of MLV-based γc vector. The CD3+ T cell count at 6 months for Trial 2 (n=6; median 548/microliter; range 87-3960) does not appear to be lower than for Trial 1 (n=19; median 1755/microliter; range 200-4759; p=0.14 in 2-sided Wilcoxon rank sum test). However, we recognize that this test is underpowered, and results are preliminary. Because the latency time to leukemic events in the previous trial was long (33-60 months), we compared insertion sites as a surrogate measure of safety. Insertional activation of growth controlling genes has been demonstrated to be associated in some murine models with clonal skewing during in vitro expansion and in vivo reconstitution. Therefore, we hypothesized that removal of the transcription factor binding site-rich LTR U3 enhancer regions would change the insertion pattern detected in peripheral blood after transplantation away from the 5’ ends of genes important for cell proliferation or persistence. Whereas in Trial 1, LMO2, EVI1 and other lymphoid proto-oncogenes showed prominent clusters of integration sites post-transplantation, these locations were not significant clusters in the second trial (p<10^-4; comparison of n=19 Trial 1 versus n=6 Trial 2). Thus, we show promising early data that a modified gammaretroviral vector retains efficacy in treatment of SCID-X1 and is likely to have an improved safety profile.