Lentiviral Gene Therapy with Low Dose Conditioning for X-Linked SCID Results in Complete Immune Reconstitution and No Evidence of Clonal Expansion

Introduction: Gene therapy (GT) for X-linked severe combined immunodeficiency (SCID-X1) using a gamma (γ) retroviral vector resulted in robust T cell immune reconstitution but also insertional oncogenesis. A multi-institutional trial using a self-inactivating (SIN) γ-retroviral vector resulted in equivalent T cell immune reconstitution but no insertional oncogenesis to date (Hacein-Bey-Abina et al, NEJM 2014). In both trials, conditioning was only rarely used and therefore gene marking in B cells and neutrophils was negligible and reconstitution of NK cells was limited. To improve both the immune reconstitution and safety of GT for SCID-X1, we developed a lentiviral vector (LV) in which a codon optimized IL2RG transgene is driven by the elongation factor 1 alpha short promoter (EFS) and introduced low dose busulfan conditioning to enhance hematopoietic and multilineage expression of IL2RG. An international, multicenter, Phase 1/2 clinical trial (NCT03311503, NCT03601286) is currently underway to evaluate the safety and efficacy of LV mediated gene transfer using this vector.

Methods: A total of 14 patients have been enrolled. Two patients were withdrawn due to poor stem cell mobilization and collection and manufacturing are in progress for 3 patients. Nine patients have been infused, 8 receiving mobilized peripheral blood stem cells and 1 receiving bone marrow at an average age of 5.3 months (range 3.2-7.1). Hematopoietic stem cells (CD34+ cells) were transduced with the LV vector (using transduction enhancers in 7 products) and drug products were cryopreserved. Median cell dose was 9.06 x 10e6 CD34+ cells/kg (4.55-17.87) and median vector copy number (VCN) was 0.86 copies/diploid genome(dg) (0.67-2.79). Drug products were thawed and infused after 2 days of busulfan conditioning targeted to an AUC of 30 mg*h/L. All infants tolerated busulfan exposure without any significant toxicity.

Results: As of July 2022, all treated patients are alive with robust reconstitution of gene marked T cells with median follow up of 2.2 years (0.2-4.3). T cell reconstitution was rapid with median CD3+ T cell count 552 cells/uL (132-1160) at 3 months. Seven patients evaluable at 1 year had CD3+ (2377-4900 cells/uL) and CD4+ T cell counts (1507-3080 cells/uL) normal for age at 1-year post-GT (Figure 1). Gene marking in T cells was robust averaging 1.55-5.66 copies/dg. Evidence of thymic function includes rising naïve CD4+ T cells and maintenance of a normal CD4/CD8 ratio. Multilineage engraftment of gene modified cells is evident in B cells, neutrophils, and NK cells at last follow-up with stable VCN over time (ranges 0.43-2.05, 0.24-2.11, 1.98-3.61 respectively). In contrast to limited NK cell reconstitution seen in the absence of conditioning in prior trials, NK cell counts have been sustained in 6 of 7 patients with greater than 12 months of follow-up achieving values within the normal range for age (Figure 1). Six of these 7 patients have been able to stop immunoglobulin replacement, 4 of 4 who have completed a primary vaccine series show response, with the results of 2 further patients awaited. Insertion site analysis shows uniformly polyclonal repertoire with no evidence of clonal expansion.

Conclusion: In summary, gene therapy using a lentiviral vector and low dose busulfan conditioning resulted in robust and complete immune reconstitution correcting T, NK, and B cell defects characteristic of SCID-X1 with 100% survival and no gene therapy related adverse events.

Figure 1 Immune reconstitution in 7 patients treated with >1 year follow-up A) CD3+ T cells. B) CD4+ T cells. C) NK cells.

Booth:Orchard Therapeutics: Consultancy; Revolution Medicines: Research Funding; Sobi: Honoraria. 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. Prockop: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; 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. Chandrakasan:Sobi, Inc.: Consultancy. de Oliveira:Bluebird Bio: Research Funding; Orchard Therapeutics: Research Funding. León Rico:Achilles Therapeutics: Ended employment in the past 24 months; Rocket Pharmaceuticals: Current Employment. London:Jubliant Draximage: Consultancy, Other: Data Safety Monitoring Board service; Merck: Consultancy, Other: Data Safety Monitoring Board service; ArQule: Consultancy, Other: Data Safety Monitoring Board service. Thrasher:Orchard Therapeutics: Consultancy; Rocket Pharmaceuticals: Consultancy; 4bio capital: Consultancy; Generation Bio: Consultancy, Current equity holder in publicly-traded company. Williams:Insertion Site Advisory Board, Biomarin: Consultancy; Scientific Advisory Board, Skyline Therapeutics: Consultancy; Chief Scientific Chair, Emerging Therapy Solutions: Consultancy; Scientific Advisory Board, Beam Therapeutics: Consultancy; Orchard Therapeutics: Other: Provides vector; Novartis: Consultancy, Other: Steering Committee (fees donated to NAPAAC); Insertion Site Analysis Advisory Board, Bluebird Bio: Consultancy; Bluebird: Consultancy, Other: Provides Vector; Novartis: Other: Provision of study materials, medical writing.

Autologous CD34+ selection of HSC used for gene therapy is off-label.