Long-Term Outcome of Gene Therapy for X-Linked Severe Combined Immunodeficiency (SCID-X1) Using an Enhancer-Deleted Self-Inactivating Gammaretroviral Vector

Background: X-linked severe combined immunodeficiency (SCID-X1), caused by mutations in IL2RG, is fatal unless treated with definitive cellular therapy. Autologous gene therapy (GT) using a gammaretroviral vector (γRV) showed T cell reconstitution in most subjects, but also insertional oncogenesis. We performed a multi-institutional trial of GT for SCID-X1 using an enhancer-deleted self-inactivating vector (SIN-γRV) that had been modified from the original γRV. In this SIN-γRV, the U3 enhancer in the long terminal repeat (LTR) was deleted and the elongation factor 1α short promoter (EFS) was used to drive ubiquitous expression of a non-codon optimized IL2RG transgene. A total of 9 patients were reported in 2014 (5 in Paris, 4 in US), and 5 more have since been enrolled (4 in US, 1 in London). Here we report the outcome of the 9 patients enrolled in the US and London.

Methods: Bone marrow CD34+ cells transduced with the SIN-γRV were infused fresh without chemotherapy conditioning in 8 patients, and after low dose busulfan targeted to 30 mg*h/L in 1 patient. Two patients (P4, P6) underwent 2 rounds of GT. The 11 manufactured investigational products (MIP) provided a median 7.58 x 10e6 (1.93-17.57) CD34+ cell/kg, with median vector copy number (VCN) of 0.78 copies/diploid genome (dg) (0.45-2.92) and transduction efficiency of 63.9% (27.1-91.67).

Results: Four subjects receiving MIP with lower VCN had inferior outcome with respect to T cell reconstitution. P3 (VCN 0.35) and P6 (VCN 0.77, 0.78) failed to develop T cells. P5 had persistent T cell lymphopenia with infections and was referred to allogeneic transplant 8 years after GT (VCN 0.45). P4 had T cell counts <400 cells/mcL and developed histoplasmosis pneumonia 9 years after GT (VCN 0.53, 0.51). The remaining 5 patients (VCN 0.78-2.92) have sustained gene marked T cells with median follow-up of 8.4 years (5.0-11.6), were free of opportunistic infections, and are attending school. Among the seven subjects who developed T cells (excluding P3 and P6), there was a strong correlation between T cell count at 4 years post GT and VCN in the MIP (R²=0.7752, p=0.0089).

We saw superior multilineage gene marking and immunological outcome in P9 who received low dose busulfan conditioning compared to the 5 subjects who did not receive conditioning. Gene marking in B cells and neutrophils was negligible in P1, P2, P4, P7 and P8. In contrast P9 had sustained marking in B cells (0.69-0.93 copies/dg) since 2 years post-GT and was the sole subject to show response to vaccination. Neutrophils from P9 showed sustained marking of 0.21-0.37 copies/dg since 2 years post-GT implying long-term engraftment of gene marked HSC. NK cell gene marking was low in most subjects at 4 years post-GT (0.01-0.58 copies/dg) and much higher in P9 (2.31 copies/dg).

Insertion site analysis (ISA) of 6 subjects in long-term follow-up (5.0-11.6 years) shows polyclonal engraftment and high diversity in T cells. Multilineage insertions were recovered from T, B, NK, and neutrophils from P9. To date no subject has developed insertional oncogenesis or clones representing >20% of sites recovered from whole blood.

Conclusions: GT using an enhancer-deleted SIN-γRV resulted in sustained T cell reconstitution in 6 of 9 subjects and those receiving cells with high VCN had higher T cell numbers in long-term follow-up. Conditioning with busulfan promoted engraftment with gene marked hematopoietic stem cells (HSC) leading to more functional immunologic reconstitution as demonstrated in other trials of GT. With median 9.3 years of follow-up, no subject in this trial has developed insertional oncogenesis or clonal dominance suggesting a key role of the U3 enhancer in the LTR in previously reported leukemogenesis using γRV vectors in SCID-X1.

Prockop:Jasper Therapeutics (through MSK): Research Funding; AlloVir (through MSK): Research Funding; Memorial Sloan Kettering Cancer Center (MSK): Other: Co-inventor of intellectual property transferred from Atara; Atara Biotherapeutics (through MSK): Other: Co-inventor of IP licensed to Atara. Dr Prockop transferred IP rights to MSK & has no personal financial interests in Atara. MSK has financial interests in Atara/IP interests related to this study, Research Funding. Kohn:Cimeio Therapeutics: Consultancy; Innoskel: Consultancy; MyoGene Bio: Consultancy, Other: Membership on Scientific Advisory Board; Pluto Immunotherapeutics: Consultancy, Other: Membership on Scientific Advisory Board; Allogene Therapeutics: Consultancy, Other: Member of Scientific Advisory Board; ImmunoVec: Consultancy; TransformaTx: Consultancy. Booth:SOBI: Consultancy, Honoraria; Rocket Pharmaceuticals, Inc.: Consultancy; Takeda: Honoraria; GSK: Honoraria; Orchard Therapeutics: Consultancy, Honoraria. de Oliveira:Bluebird Bio: Research Funding; Orchard Therapeutics: Research Funding. London:ArQule: Consultancy, Other: Data Safety Monitoring Board service; Jubliant Draximage: Consultancy, Other: Data Safety Monitoring Board service; Merck: Consultancy, Other: Data Safety Monitoring Board service. Walkovich:Sobi: Consultancy, Membership on an entity's Board of Directors or advisory committees; AstraZeneca: Membership on an entity's Board of Directors or advisory committees; Horizon: Membership on an entity's Board of Directors or advisory committees; Pharming: Membership on an entity's Board of Directors or advisory committees; X4 Pharma: Membership on an entity's Board of Directors or advisory committees, Research Funding; NICER Consortium: Other: Executive Chair; St. Jude: Honoraria; American Society of Hematology: Honoraria; UpToDate: Patents & Royalties. Walter:Octapharma: Research Funding; X4 Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees; CSL-Behring: Consultancy; Grifols: Consultancy; Enzyvant: Consultancy; Regeneron: Consultancy; UptoDate: Other: Medical Writer; ADMA Biologicals: Consultancy; Pharmig: Membership on an entity's Board of Directors or advisory committees; MustangBio: Research Funding; Chiesi: Research Funding; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Thrasher:Orchard Therapeutics: Consultancy; Rocket Pharmaceuticals: Consultancy; 4bio capital: Consultancy; Generation Bio: Consultancy, Current equity holder in publicly-traded company. Williams:bluebird bio: Consultancy, Other: provided GMP vector for sickle cell disease clinical trial; Orchard Therapeutics: Other: provided GMP vector for X-linked SCID lentiviral gene therapy trial; Novartis: Other: Steering Committee Novartis ETB115E2201 trial, advisory fees donated to NAAPAC; Beam Therapeutics: Membership on an entity's Board of Directors or advisory committees; Emerging Therapy Solutions: Consultancy; Skyline Therapeutics: Membership on an entity's Board of Directors or advisory committees; Biomarin: Membership on an entity's Board of Directors or advisory committees.

CD34+ autologous hematopoietic cells