Challenges with Gene Therapy Based on CRISPR/Cas9 and Prime Editing for Somatic Reverted Mosaicism of X-linked Combined Immunodeficiency

Abstract

X-linked severe combined immunodeficiency (X-SCID) is a rare primary immunodeficiency disorder with X-chromosome-linked recessive inheritance, that is caused by the mutations of the interleukin-2 receptor gamma (IL2RG) gene. The IL2RG gene encodes the interleukin-2 receptor common gamma chain (IL-2Rγ, also known as γc). The IL-2Rγ is essential not only for lymphocyte development and immune function but also for maintaining the structure of the IL-2R complex and the link of intracellular signaling molecules. The mutations of IL2RG can result in impaired lymphocytes and deficiency immunity. Some patients of atypical X-SCID present mild syndrome due to hypomorphic or revertant IL2RG mutations. This study describes a novel IL2RG variant (c.458T>C, p.Ile153Thr) in three 20-year-old brothers with atypical X-SCID. They had a regular frequency of lymphocytes in peripheral blood but an inverted CD4+/CD8+ ratio. IL-2Rγ expression was detected commonly in lymphocyte subsets, while their CD3+ T cell proliferation in vitro was impacted. They have occasionally suffered from respiratory infections and skin warts. The mutant and wild-type (the mutation reverted to the wild type) nucleotides at the same site of IL2RG gene (c.458T>C/T) were co- exist in their T-cell populations. Despite the observation of somatic revertant mosaicism as a naturally occurring mechanism of gene correction, the patients still exhibit symptoms. Therefore, gene editing offers a potential therapy to increase the frequency of corrected T cells. Here, advanced genome editing tools, CRISPR/Cas9-ssODN and prime editing, were applied to modify the IL2RG gene at the specific locus 458 in exon 4. These two methods were successfully applied to establish an in vitro model of X-SCID in K562 cells and healthy donors’ T cells to introduce the c. 458T>C mutation in IL2RG gene. However, the similar strategies were not able to correct the IL2RG c. 458T>C mutation in patients’ T cells due to the limited in vitro proliferation of mutant cells and the presence of somatic reversion. This study describes that gene editing in a mixed population of mutated and reverted cells might hinder gene therapy application, and summarizes the main challenges and limitations confronted with. Nevertheless, results of this project implicate the feasibility of precise nucleotide substitution of IL2RG gene locus, especially by prime editing. Gene therapy based on CRISPR/Cas9-ssODN and prime editing could provide an alternative treatment for X-SCID patients. Further technology improvement needs to be developed to correct the mutations in somatic mosaicisms.