Challenges in Gene Therapy for Somatic Reverted Mosaicism in X-Linked Combined Immunodeficiency by CRISPR/Cas9 and Prime Editing

• Published in: Genes 2022-12, Vol.13 (12), p.2348
• Main Authors: Hou, Yujuan, Ureña-Bailén, Guillermo, Mohammadian Gol, Tahereh, Gratz, Paul Gerhard, Gratz, Hans Peter, Roig-Merino, Alicia, Antony, Justin S, Lamsfus-Calle, Andrés, Daniel-Moreno, Alberto, Handgretinger, Rupert, Mezger, Markus
• Format: Article
• Language: English
• Subjects: Care and treatment; Cell cycle; Cell growth; Cell proliferation; CRISPR; CRISPR-Cas Systems - genetics; Cytokines; Gene Editing - methods; Gene therapy; Genetic aspects; Genetic Therapy - methods; Genomes; Humans; IL2RG gene; Immune system; Immunodeficiency; Interleukin 2; Lymphocytes; Lymphocytes T; Methods; Mosaicism; Mutation; Oligonucleotides; Patients; Plasmids; Point mutation; Primary immunodeficiencies; Reversion; Severe combined immunodeficiency; X-Linked Combined Immunodeficiency Diseases - genetics; X-Linked Combined Immunodeficiency Diseases - therapy
• ISSN: 2073-4425; 2073-4425
• DOI: 10.3390/genes13122348

X-linked severe combined immunodeficiency (X-SCID) is a primary immunodeficiency that is caused by mutations in the interleukin-2 receptor gamma gene. Some patients present atypical X-SCID with mild clinical symptoms due to somatic revertant mosaicism. CRISPR/Cas9 and prime editing are two advanced genome editing tools that paved the way for treating immune deficiency diseases. Prime editing overcomes the limitations of the CRISPR/Cas9 system, as it does not need to induce double-strand breaks (DSBs) or exogenous donor DNA templates to modify the genome. Here, we applied CRISPR/Cas9 with single-stranded oligodeoxynucleotides (ssODNs) and prime editing methods to generate an in vitro model of the disease in K-562 cells and healthy donors' T cells for the c. 458T>C point mutation in the gene, which also resulted in a useful way to optimize the gene correction approach for subsequent experiments in patients' cells. Both methods proved to be successful and were able to induce the mutation of up to 31% of treated K-562 cells and 26% of treated T cells. We also applied similar strategies to correct the c. 458T>C mutation in patient T cells that carry the mutation with revertant somatic mosaicism. However, both methods failed to increase the frequency of the wild-type sequence in the mosaic T cells of patients due to limited in vitro proliferation of mutant cells and the presence of somatic reversion. To the best of our knowledge, this is the first attempt to treat mosaic cells from atypical X-SCID patients employing CRISPR/Cas9 and prime editing. We showed that prime editing can be applied to the formation of specific-point mutations without inducing nonspecific on-target modifications. We hypothesize that the feasibility of the nucleotide substitution of the gene using gene therapy, especially prime editing, could provide an alternative strategy to treat X-SCID patients without revertant mutations, and further technological improvements need to be developed to correct somatic mosaicism mutations.