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

Yujuan Hou; Guillermo Ureña-Bailén; Tahereh Mohammadian Gol; Paul Gerhard Gratz; Hans Peter Gratz; Alicia Roig-Merino; Justin S Antony; Andrés Lamsfus-Calle; Alberto Daniel-Moreno; Rupert Handgretinger; Markus Mezger

doi:10.3390/genes13122348

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

Genes (Basel). 2022 Dec 13;13(12):2348. doi: 10.3390/genes13122348.

Affiliations

¹ Department of Pediatrics I, Hematology and Oncology, University Children's Hospital, University of Tübingen, 72076 Tuebingen, Germany.
² MaxCyte Inc., Rockville, MD 20850, USA.

Abstract

X-linked severe combined immunodeficiency (X-SCID) is a primary immunodeficiency that is caused by mutations in the interleukin-2 receptor gamma (IL2RG) 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 IL2RG 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 IL2RG 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 IL2RG mutations without inducing nonspecific on-target modifications. We hypothesize that the feasibility of the nucleotide substitution of the IL2RG 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.

Keywords: CRISPR/Cas9; IL2RG; SCID; prime editing; somatic mosaicism; ssODN.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

CRISPR-Cas Systems / genetics
Gene Editing / methods
Genetic Therapy / methods
Humans
Mosaicism
X-Linked Combined Immunodeficiency Diseases* / genetics
X-Linked Combined Immunodeficiency Diseases* / therapy

Supplementary concepts

Severe combined immunodeficiency, atypical

Grants and funding

This research was funded by the Clinician Scientist Program (N°. 440-0-0); Stefan Morsch Stiftung, MaxCyte Inc.; and the University Children’s Hospital of Tübingen. We acknowledge support from the Open Access Publishing Fund of the University of Tübingen.