SCID-A (T-B-NK+ severe combined immunodeficiency of Athabascan-speaking Native Americans) is a primary immunodeficiency disease (PID) resulting from absence of Artemis protein, required for nonhomologous end-joining in V(D)J gene rearrangement. The only effective therapy for SCID-A is allogeneic hematopoietic stem cell (HSC) transplant, for which there is a significant risk of morbidity and mortality. We recently demonstrated that ex vivo lentiviral correction of Artemis deficiency in a mouse model of SCID-A was most effectively accomplished using the endogenous human Artemis promoter to regulate Artemis expression, forming the basis for a clinical trial of this approach to be initiated in early 2018. However, integrative gene transfer has been associated with adverse oncogenic reactions in clinical trials for X-linked SCID. As a superior genetic therapy to maintain regulated Artemis gene expression and avoid the risk of genotoxicity, we propose to develop site-directed correction of the Y192X mutation in the Artemis gene that underlies SCID-A that occurs at a higher frequency in Athabascan-speaking Native Americans in the southwest United States. To this end, we have been using the CRISPR/Cas9 system for targeted modifications and the Artemis locus and genetic correction of Artemis deficiency. In preliminary studies, we have achieved highly efficient targeting of Artemis codon 192 using the CRISPR/Cas9 nuclease system for site directed double-strand break and initiation of homology-directed recombination (HDR). In an even more elegant strategy, we have acquired the Adenosine Base Editor (ABE) system from the laboratory of Dr. David Liu for introduction of site directed single base changes without the need of making a double strand break. For correction of the Artemis gene, we will first determine effectiveness of the ABE system for targeting the Athabascan exon 8 stop codon in patient- derived fibroblasts (Aim 1). Either the base editor (if effective) or Cas9 mediated HDR will then be used for correction of the exon 8 Athascan stop codon in patient-derived human hematopoietic stem cells (Aim 2). The overall goal of this study therefore is to generate definitive proof-of-concept results supporting clinical testing of HSC genetically corrected at the Artemis locus for restoration of immunity in SCID-A. After these studies are complete, HSC from SCID-A patients will then be developed for cGMP manufacturing and clinical testing as a lead condition, with subsequent application to the treatment of other primary immune deficiencies and diseases treatable by gene editing of human hematopoietic stem cells.
Primary immune deficiencies are a rare group of inherited diseases caused by genetic deficiency in which patients suffer from the inability to fight off common infections. It is envisioned in this grant application that one way to treat these diseases would be to repair the mutated gene in the patients' blood cells, thereby restoring the ability of the patient to fight infections. The specific research proposed in this application will use cells from immunodeficient patients to test conditions for correction of the mutated gene, restoring immunity, and expanding the cells in a clean environment so that they can eventually be used to treat the disease.
