Targeted genome editing in Recombination activating gene 1 (RAG): a precise correction of the genetic defect in human SCID

Severe combined immunodeficiency (SCID) caused by defect in Recombination Activating Gene 1 (RAG1) are an heterogenous group of disease including forms characterized by  a block in T and B cells or in the case of hypomorphic defects by severe inflammation and autoimmunity (Omenn syndrome), atypical SCID and combined immunodeficiency with granuloma (CID_AG). Bone marrow transplantation is the treatment of choice, however limited by the availability of compatible donors and risk to develop severe side effects due to the conditioning regimes used to obtain efficient engraftment. Several groups have attempted to develop alternative therapeutic strategies exploiting gene therapy, however because of the tight regulation of RAG1 gene during lymphocyte differentiation and cell cycle, results obtained so far are poor in terms of immune reconstitution and genotoxicity. Furthermore, poor overall survival due to very low levels of T cell engraftment and absence of donor B cells has been reported after haploidentical HSC transplantation with no or limited conditioning, clearly indicating that myeloablative conditioning is required to achieve long-term T and B cell recovery. All these data indicate the clinical need to develop novel strategies of treatment available for all patients. In our project, we plan to establish a novel therapeutic platform based on the use of nucleases (CRISP-CAS9) able to cut the double strand DNA in a preselected DNA portion and introduce the corrective RAG1 sequence under the genomic regulatory regions control. This process, called Homology driven repair, allows to correct and maintain the gene in the same genomic region and under the same regulatory regions. We will develop this platform in hematopoietic stem cell precursors (HSCP) that will be tested by in vitro and in vivo studies with the final aim to demonstrate the efficacy and safety of our approach. In parallel, we will test novel conditioning regimen that will allow to deplete and “make space “ in hematopoietic organs to host corrected gene edited HSCP. These compounds named “non genotoxic” drugs are based on the use of monoclonal antibodies that should be less toxic and preserve tissue organs. Overall, we will provide a novel platform to cure RAG1 deficiencies.