Functional genetic correction of the X-linked Severe Combined Immunodeficiency using engineered nucleases (TALENs or CRISPR/Cas)

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Severe combined immunodeficiencies (SCID) are devastating diseases of the immune system and for most of them disease causing genes have been identified. The most common form is SCID-X1 that is caused by mutations of the IL2RG gene. Allogeneic hematopoietic stem cell transplantation can cure the disease but the outcomes are impaired when no matched donor is available or the child has acquired infections. An ideal therapy for a genetic disease would be the correction of the genetic defect in autologous hematopoietic cells. Previous trials of gene therapy have shown general feasibility and efficacy but also uncovered the risks associated with the semi-random integration of the viral vectors that were used which lead to insertional mutagenesis. Recently, new methods have been developed that enable targeted correction of the mutated gene at its original site. They rely on the creation of a site-specific DNA break and introduction of a non-mutated version of the gene. The cell can introduce this template into the site of the break by homologous recombination. This replaces the mutated version of the gene by its correct version while the natural mechanisms of gene regulation are preserved. Tools for this gene editing are zinc finger nucleases, TAL effector nucleases (TALENs) and the CRISPR/Cas system. In preliminary work both TALEN based and CRISPR/Cas based functional gene editing approaches targeting the gene from exon 1 downstream have been identified as the most promising platforms for correction of the IL2RG gene. A series of assays has been used to compare this approach against the most viable gene editing competitors (exon 5 based approach, zinc finger nucleases) and determined that the exon 1 TALENs and CRISPR/Cas nucleases would be a treatment option for a much larger proportion of patients with SCID-X1 and that the reagents have better activity and toxicity profiles than ZFN-based approaches. In this project the defects of the IL2RG gene locus will be corrected by TALENs or the CRISPR/Cas system in human hematopoietic stem and progenitor cells to establish a multilineage hemato- and lymphopoiesis with functional signal transduction and the methods will be advanced to make translation into a clinical trial possible. It aims to refine this strategy by optimizing the functional gene correction methods in order to increase on-target activity while using advanced techniques for extensive safety testing. Its application will be increased to large-scale GMP conditions in order to prepare a first trial in humans. SCID-X1 is an ideal candidate disease for the application of the new gene editing methods because relatively small number of corrected cells are sufficient, but, if successful, the impact of this project will be broad because it will establish that gene editing of tissue specific stem cells can cure devastating diseases and thus will prompt the development of similar programs for other genetic diseases of hematopoiesis.