Abstract
Severe combined immunodeficiency (SCID) is a group of disorders caused by mutations in genes involved in the process of lymphocyte maturation and function. CRISPR-Cas9 gene editing of the patient's own hematopoietic stem and progenitor cells (HSPCs) ex vivo could provide a therapeutic alternative to allogeneic hematopoietic stem cell transplantation, the current gold standard for treatment of SCID. To eliminate the need for scarce patient samples, we engineered genotypes in healthy donor (HD)-derived CD34+ HSPCs using CRISPR-Cas9/rAAV6 gene-editing, to model both SCID and the therapeutic outcomes of gene-editing therapies for SCID via multiplexed homology-directed repair (HDR). First, we developed a SCID disease model via biallelic knockout of genes critical to the development of lymphocytes; and second, we established a knockin/knockout strategy to develop a proof-of-concept single-allelic gene correction. Based on these results, we performed gene correction of RAG2-SCID patient-derived CD34+ HSPCs that successfully developed into CD3+ T cells with diverse TCR repertoires in an in vitro T cell differentiation platform. In summary, we present a strategy to determine the optimal configuration for CRISPR-Cas9 gene correction of SCID using HD-derived CD34+ HSPCs, and the feasibility of translating this gene correction approach in patient-derived CD34+ HSPCs.
| Original language | English |
|---|---|
| Pages (from-to) | 105-121 |
| Number of pages | 17 |
| Journal | Molecular Therapy Nucleic Acids |
| Volume | 31 |
| DOIs | |
| State | Published - 14 Mar 2023 |
Bibliographical note
Publisher Copyright:© 2022 The Author(s)
Funding
We would like to thank the members of the Somech and the Hendel Labs for reading the manuscript and providing practical advice. In addition, we want to thank Dr. Rasmus O. Bak and Dr. Andreas Reinisch for numerous important discussions and comments. Finally, we would like to thank D. Russell for providing the pDGM6 plasmid. This work was supported by the European Research Council (ERC) under the European Union Horizon 2020 research and innovation program (grant no. 755758, to A.H.), the Israel Science Foundation (grant no. 2031/19, to A.H.), and the Israel Precision Medicine Program (grant no. 3115/19, to R.S. A.H. and Y.N.L). Figure S3A was created with BioRender.com. O.I. D.A. O.K. Y.Z. D.B. A.A. and A.L. designed and conducted the experiments, evaluated, and analyzed the data. O.I. and D.B. performed the bioinformatics analyses, with the help and guidance of Y.N.L. K.B. and A.N. provided cord blood samples and R.S. provided PB samples. K.B. Y.N.L. A.N. and R.S. critically reviewed the experiments and provided important advice. A.H. supervised and conceived the research and planned the experiments and the approaches. A.H. D.A. O.I. and Y.Z. wrote the manuscript, with contributions from all authors. The authors declare no competing interests. We would like to thank the members of the Somech and the Hendel Labs for reading the manuscript and providing practical advice. In addition, we want to thank Dr. Rasmus O. Bak and Dr. Andreas Reinisch for numerous important discussions and comments. Finally, we would like to thank D. Russell for providing the pDGM6 plasmid. This work was supported by the European Research Council (ERC) under the European Union Horizon 2020 research and innovation program (grant no. 755758 , to A.H.), the Israel Science Foundation (grant no. 2031/19 , to A.H.), and the Israel Precision Medicine Program (grant no. 3115/19 , to R.S., A.H., and Y.N.L). Figure S3 A was created with BioRender.com .
| Funders | Funder number |
|---|---|
| Hendel Labs | |
| Israel Precision Medicine Program | 3115/19 |
| European Commission | |
| Israel Science Foundation | 2031/19 |
| Horizon 2020 | 755758 |
Keywords
- CRISPR-Cas9
- HSPCs
- MT: RNA/DNA Editing
- RAG2
- SCID
- gene regulation
- genome editing
- rAAV6
