Somatic Gene Therapy for X-Linked Severe Combined Immunodeficiency Using a Self-Inactivating Modified Gammaretroviral Vector Results in An Improved Preclinical Safety Profile and Early Clinical Efficacy in a Human Patient
Authors: Pai Sung-Yun , Notarangelo Luigi Daniele , Harris Chad , Cattaneo Federica , Wladkowski Matthew , Armant Myriam , Kao Grace S. , Wielgosz Matthew , Nienhuis Arthur W. , Modlich Ute , Brugman Martijn , Schambach Axel , Rothe Michael , Thornhill Susannah I , Gaspar H. Bobby , Parsley Kathryn , Cooray Samantha , Grassman Elke , Bushman Frederic , Shupien Sally , Oleastro Matias , Hacein-Bey-Abina Salima , Fischer Alain , Cavazzana-Calvo Marina , Malik Punam , Filipovich Alexandra , Kohn Donald B. , Baum Christopher , Williams David A. , Thrasher Adrian J 
Published: October 2019

Abstract
Abstract 164FN2
Somatic gene therapy for X-linked severe combined immunodeficiency (X-SCID) using a MLV-based gammaretroviral vector expressing the IL-2 receptor gamma chain (MFG-γc) resulted in excellent immunologic reconstitution but also in insertional oncogenesis. In 5/20 treated children, T cell leukemia developed, with insertional activation of LMO2 proto-oncogene in 4 of the 5. We reasoned that replacing the virus-derived promoter and enhancer elements with a weaker cellular promoter would result in improved safety yet retain efficacy. We therefore generated the pSRS11.EFS.IL2RG.pre* self-inactivating (SIN) gammaretroviral vector in which expression of γc is controlled by an intronless EF1-α promoter, in a MLV vector devoid of the LTR U3 (enhancer/promoter) region. In preclinical studies we determined ‘relative safety' using several surrogate assays. In a reporter assay, the pSRS11.EFS.IL2RG.pre* vector when inserted into the oncogenic LMO2 locus induced LMO2 expression 6–90-fold less than the parent MFG vector (MFG-γc). pSRS11.EFS.IL2RG.pre* had lower activity in a murine in vitro immortalization assay (0.2 clones per 10e5 cells, fitness score 0.00007), compared to MFG-γc (0.54 clones per 10e5 cells, fitness score 0.00025). Peripheral blood and bone marrow from C57BL6 mice transplanted with murine bone marrow transduced with pSRS11.EFS.IL2RG.pre* and followed in primary recipients over 4 months and in secondary recipients over 1 year in vivo showed no evidence of vector associated leukemias. Deep sequencing demonstrated 8 of 3621 insertions into the MDS-associated gene Evi1 in mice transplanted with MFG-γc-transduced cells while there were none (0 of 2690 insertions into Evi1) in mice transplanted with pSRS11.EFS.IL2RG.pre* vector transduced cells (P=0.025). In preclinical efficacy studies, circulating T and B lymphocytes were detectable in the peripheral blood of 7/7 γc-deficient mice transplanted with pSRS11.EFS.IL2RG.pre* transduced cells while 4/4 mice repopulated with SFFV-eGFP transduced cells remained alymphoid. Experimental animals were sacrificed approximately five months post-transplant for analysis of immune reconstitution. Flow cytometric analysis of the spleens and bone marrow revealed restoration of mature B220+IgM+ B cells and NK cell populations in all mice transplanted with pSRS11.EFS.IL2RG.pre* transduced cells. CD4+ and CD8+ T cells were also detected in both tissues and in thymi recovered from transplanted animals. T cells in these mice proliferated in response to mitogenic stimuli. Immunoglobulin subclasses IgG1 and IgG2a detected in the plasma from pSRS11.EFS.IL2RG.pre* reconstituted mice also indicated restored B cell function in these animals. Human preclinical studies also supported the correction of XSCID cellular defects using this vector. Based on these data, a multi-institutional phase I/II trial was initiated with the pSRS11.EFS.IL2RG.pre* vector using an identical clinical protocol as in the previous X-SCID trials, to determine efficacy and safety compared with the MFG-γc vector. The first patient was treated in December 2010. Six months post-gene therapy, he has attained CD3 T cell count of >800, normal proliferation to mitogens, and normal NK cell numbers. He has cleared medically-resistant oral ulcers, and a rotavirus infection acquired post-gene therapy. Nearly all of the circulating T cells (86%) and 41% of his NK cells express γc, albeit at modestly lower density than normal, as expected. However, the early kinetics of T cell reconstitution was comparable to several subjects treated with MFG-γc. These data suggest that the improved safety profile demonstrated with numerous surrogate preclinical studies is associated with efficacious transgene expression and functional immune recovery in the initial human patient treated.

Disclosures:
Off Label Use: CliniMACS for selection of CD34+ hematopoietic cells. Baum:Patent office: Patents & Royalties.