447. Lentiviral Gene Therapy in a Preclinical Model of Omenn Syndrome

Patients with Omenn syndrome (OS), carrying hypomorphic recombination activating genes (RAG) mutations, develop a peculiar severe combined immunodeficiency associated with autoimmune-like manifestations. RAG1 and RAG2 genes play an essential role in the generation of antigen receptors during lymphocyte development. HLA-matched bone marrow transplantation is curative, however, hematopoietic stem cell gene therapy (GT) could represent an alternative treatment for patients lacking a suitable donor. The aim of the present study was to investigate the efficacy of GT in a knock-in murine model carrying the Rag2 R229Q mutation that mimics most of the symptoms of human OS. To this aim, we are testing a lentiviral vector encoding for codon optimized human RAG2 (hRAG2co-LV vector) under the control of the ubiquitous chromatin opening element (UCOE).We initially measured the efficiency of transduction of Rag2R229Q/R229Q lineage negative (lin-) cells by testing the presence of the integrated viral vector by real-time PCR in lin- cells transduced at a multiplicity of infection (MOI) 5 and 10 and in vitro cultured for 8 days. We observed that the hRAG2co-LV vector transduced Rag2R229Q/R229Q lin- cells very efficiently at MOI 5. Then, we tested the in vivo efficacy of hRAG2co-LV-mediated GT infusing transduced lin- BM cells isolated from male Rag2R229Q/R229Q mice in lethally irradiated females. Control Rag2R229Q/R229Q mice received wild-type (BMT-WT) or untransduced (BMT-OS) lin- BM cells. Analysis of immune cells in peripheral blood was evaluated overtime starting from six weeks after the treatment. GT-treated mice showed the appearance and progressive increase of B cells and amelioration of T cell frequency and absolute count as compared to BMT-OS mice. In parallel, the frequency of myeloid cells started to decrease 13 weeks after GT indicating a normalization of peripheral immune cell distribution. Our preliminary data demonstrate the feasibility of GT in the preclinical model of OS. Additional experiments aimed at evaluating long-term efficacy and safety are ongoing.