Gene therapy for SCID-X1 with low dose busulfan and a SIN-lentiviral vector

Project Summary Gene therapy using autologous CD34+ cells is a promising treatment for primary immunodeficiency, particularly for individuals without optimal allogeneic donors. SCID-X1 is caused by mutations in IL2 Receptor Gamma (IL2RG) which encodes the common gamma chain (γc) of multiple cytokine receptors. Boys with SCID-X1 lack T and NK cells and their B cells fail to produce antibodies due to the lack of functional IL-7, IL-15 and IL-21 signaling. This renewal application seeks to complete our currently funded Phase I/II clinical trial that addresses two major shortcomings of previous GT using gamma retrovirus (γRV) and self-inactivating (SIN)-γRV vectors, namely lack of B cell reconstitution and insertional mutagenesis. We hypothesize that this trial will improve immune reconstitution through the introduction of low dose busulfan conditioning (Aim 1) and improve safety through the change from a gammaretroviral (γRV) vector used in previous trials to the LV vector in this trial (Aim 2). Previous trials of gene therapy for SCID-X1 have infused cells without chemotherapy conditioning, which resulted in robust T cell recovery and gene marking, but negligible gene marking in B cells and failure of humoral immune reconstitution. Initial development and marking in NK cells was also not sustained. In Aim 1, we will examine the impact of low dose busulfan conditioning on 1) cell type specific engraftment and gene marking, 2) in vivo T cell reconstitution, T cell phenotype and TRB repertoire by deep sequencing, 3) in vivo humoral immune reconstitution, B cell number, phenotype, IL-21 dependent function and IGH repertoire by deep sequencing, 4) NK cell number, phenotype and function. Previous trials of gene therapy for SCID-X1 have used a γRV vector with intact viral promoters/enhancers, which resulted in 6/20 patients developing T cell leukemia due to insertional oncogenesis. Gene therapy using an enhancer deleted, self-inactivating yRV (SIN-γRV) vector funded by NIAID in which viral enhancers have been deleted shows encouraging evidence of reduced insertion sites near lymphoid oncogenes and safety, but the insertion site pattern characteristic of γRV may still be risky. The proposed trial in this application will seek to further improve safety by using a self- inactivating LV vector. In Aim 2 we will investigate the insertion site pattern in the patients’ engrafted cells, compare samples from this current trial to previous trials using γRV and SIN-γRV to delineate clustering of insertion sites in specific genes and the effect of vector backbones on clonal dynamics and expansions.