Lentiviral Hematopoietic Stem Cell Gene Therapy for Older Patients with X-Linked Severe Combined Immunodeficiency

Background: X-linked severe combined immunodeficiency (SCID-X1) is caused by mutations in the IL2RG gene that encodes the common gamma chain (gc). Hematopoietic stem cell (HSC) transplant can be curative, but for infants without a matched sibling donor, haploidentical parental hematopoietic stem cell transplant (HSCT) without myeloconditioning is life-saving but restores only T-cell immunity. In infants with SCID-X1, autologous HSC transduced with murine gamma retrovirus (gRV) carrying gc infused without conditioning also only effectively restores T-cell immunity. Similar gene therapy of older children with SCID-X1 who have persistent immune defects despite haploidentical transplant in infancy failed to achieve significant gene marking or sustained improvement of T-cell immunity. Furthermore, the gRV gene therapy in the infants also resulted in vector-associated leukemia in 25% of patients. Here, we use a lentiviral vector (CL20-i4-EF1a hgcOPT) containing a 400bp insulator fragment from the chicken beta-globin locus within the self-inactivating long-terminal repeat (LTR), driven by the eukaryotic elongation factor alpha (EF1alpha) promoter to express a codon-optimized gc cDNA. Herein, we provide the initial data on subjects enrolled and treated in this study (NIAID protocol 11-I-0007).

Subjects and Methods: SCID-X1 Subjects >2years of age with persistent immune deficiency and medical problems undergo G-CSF and plerixafor mobilized peripheral blood apheresis and CD34 isolation. For transduction, CD34+ cells are prestimulated for one day in SCF, FLT-3L, and TPO (100ng/ml), followed by daily exposure to vector for 6-8 hours daily on 2 consecutive days. On day 3, transduced cells were harvested and infused. Prior to cell infusion, subjects receive i. v. busulfan 6mg/kg total. Subjects are monitored for hematologic engraftment, immunological phenotype and functional profile, as well as gene marking in sorted immune cell lineages. Vector integration site analysis by modified linear amplification-mediated PCR and high-throughput deep sequencing, as well as replication-competent lentivirus (RCL) assays are performed for safety monitoring.

Results: Five (23, 24,7, 16 and 10 year old) subjects (P1-5, respectively) with worsening immune dysfunction and complex medical problems, with IgG supplementation dependence despite one or more prior haploidentical HSC transplants have been treated with a follow up period of 30-, 27, 4, 1 and <1 months, respectively (as of abstract submission). Busulfan conditioning was well tolerated with no serious adverse events. In the 2 older patients with significantly longer follow-up, we observe stable engraftment of gc-expressing cells with expansion of gene corrected T-, B-, and NK-cells. Gene marking in the myeloid lineages stabilize by a year following treatment to 8-10% (=0.1 vector genome (vg)/cell), while continuing to increase in B- (38%/0.38 vg/cell), T- (13-55%/0.13-0.55vg/cell) and NK-(56-76%/0.56-0.76vg/cell) cells. Early gene marking in P3 is comparable at 6% or 0.06vg/cell in myeloid lineages with increasing IgM production. Chimerism studies of their T-cells show increasing host cell contribution that reflects gradual replacement of the donor T-cell graft over time. Increase in NK-cells in P2 corresponds to an improvement in chronic warts. Both P1 and P2 produce IgG and antigen-specific responses with protective titer response to immunization, and clearance of chronic norovirus, and resolution of protein-losing enteropathy. Vector tracking confirms diverse repertoire of gene insertion sites and absence of oncogene-targeted clones observed in earlier gRV gene therapy.

Conclusion: Lentiviral-gene therapy with reduced-intensity conditioning appears safe and achieves unprecendented pan-immunologic correction of T, B, and NK cell compartments due to engraftment of relatively high levels of transduced HSCs in the bone marrow. This is the first demonstration of the use of gene therapy to salvage failed allogeneic HSCT in older SCID-X1 patients.