Development of a CRISPR/Cas9 gene editing platform to correct Severe Combined Immunodeficiency caused by mutations in the IL7RA gene

Severe combined immunodeficiency (SCID) is a rare genetic disorder caused by numerous genetic mutations and characterized by the disturbed development of the immune system. Mutations in the IL7RA gene cause the third most common form of SCID. IL7RA SCID patients do not develop T-cells and are usually affected by severe bacterial, viral, or fungal infections early in life. These babies, if untreated, usually die within one year due to severe infections unless they have undergone successful hematopoietic stem cell transplantation. More recently, gene therapy for different forms of SCID has been successfully attempted as an alternative to bone marrow transplant. For the gene therapy procedure, hematopoietic stem cells are corrected in a laboratory and then infused back to the patient where they start producing healthy immune cells. However, gene therapy attempts to introduce a correct copy of IL7RA in hematopoietic stem cells showed that constitutive and unregulated expression of the gene pre-disposes to leukemia. An alternative approach is to correct the hematopoietic stem cells by introducing a normal copy of the gene directly in its endogenous locus, thus maintaining its physiological regulation, through the use of gene editing. Our preliminary data demonstrates the feasibility of such application for IL7RA SCID. We have indeed developed gene editing reagents and protocols to efficiently knock-in a reporter gene into the IL7RA locus in human hematopoietic stem cells. We now aim to move forward and knock-in a correct copy of IL7RA, in order to restore IL7RA expression in IL7RA-deficient hematopoietic stem cells from SCID patients. We also aim to demonstrate that transplantation of corrected stem cells allows the development of all blood cells, and in particular of T-cells, and that the process causes minimal to no toxicity. The development of a gene editing-based therapeutic approach to treat IL7R protein deficiency will provide a valuable alternative for children with this disease. Thanks to our expertise in gene editing and manipulation of hematopoietic stem cells we feel we are in the best position to achieve this goal.

The severe combined immunodeficiencies (SCID) are a diverse set of diseases caused by numerous genetic mutations that share a common pathophysiological path of a quantitative deficiency in T-cell numbers. IL7RA deficiency causes the third most common form of SCID. Absence of T lymphocytes results in profound failure of the immune response, with severe and opportunistic infections leading to fatal outcome. While allogeneic hematopoietic stem cell transplantation is curative for all forms of SCID, incomplete immune reconstitution and toxicities cause significant morbidities, meaning alternative cures should be developed. Gene therapy has been successfully developed for ADA SCID and SCID X1, however its application to IL7RA SCID showed that constitutive and unregulated expression of the gene predisposes to leukemia. An alternative to gene therapy is to utilize genome editing, which could correct the endogenous IL7RA locus while avoiding issues of unregulated transgene expression. The goal of this proposal is to develop a CRISPR/Cas9-based genome editing platform to functionally correct mutations in the IL7RA gene in hematopoietic stem and progenitor cells (HSPCs) derived from SCID patients. Our preliminary results showed that by delivering our optimized CRISPR/Cas9 reagents together with an AAV6 donor template we are able to efficiently knock-in a reporter cassette into the IL7RA locus in up to 47% of HSPCs, while maintaining high cell viability and colony forming ability. We now aim to move forward and restore IL7R expression in IL7RA-deficient cells from SCID patients, to establish a viable gene editing-based therapeutic approach to treat IL7RA deficiency and potentially other blood disorders that require correction at the stem cell level.