Multiplex HDR for Disease and Correction Modeling of SCID by CRISPR Genome Editing in Human HSPCs

Iancu, O.; Allen, D.; Knop, O.; Zehavi, Y.; Breier, D.; Arbiv, A.; Lev, A.; Lee, Y. N.; Beider, K.; Nagler, A.; Somech, R.; Hendel, A.

doi:10.1101/2022.10.07.511272

bioRxiv · 10.1101/2022.10.07.511272

Multiplex HDR for Disease and Correction Modeling of SCID by CRISPR Genome Editing in Human HSPCs

Iancu, O.·Allen, D.·Knop, O.·Zehavi, Y.·Breier, D.·Arbiv, A.·Lev, A.·Lee, Y. N.·Beider, K.·Nagler, A.·Somech, R.·Hendel, A.

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Abstract

Severe combined immunodeficiency (SCID) is a group of monogenic primary immunodeficiencies caused by mutations in genes involved in the process of lymphocyte maturation and function. CRISPR-Cas9 gene editing of the patients own hematopoietic stem and progenitor cells (HSPCs) ex vivo could provide a therapeutic alternative to allogeneic hematopoietic stem cell transplantation (HSCT), the current gold standard for treatment of SCID. Using CRISPR-Cas9/rAAV6 gene-editing, we engineered genotypes in healthy donor (HD)-derived CD34+ HSPCs, thus eliminating the need for rare patient samples, to model both SCID and the therapeutic outcomes of gene-editing therapies for SCID via multiplexed homology directed repair (HDR). Firstly, we developed a SCID disease model via knock-out of both alleles of genes critical to the development of lymphocytes; and secondly, we established a knock-in/knock-out (KI-KO) strategy to develop a proof-of-concept single-allelic gene correction. Since SCID is a recessive disorder, correction of only one allele is enough to cure the patient. 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 (IVTD) platform. By using CRISPR-Cas9, multiplexed HDR, HD-derived CD34+ HSPCs, and an IVTD system we outline an approach for the study of human lymphopoiesis. We present both a way for researchers to determine the optimal configuration for CRISPR-Cas9 gene correction of SCID and other recessive blood disorders, and the feasibility of translating these techniques to perform gene correction in patient-derived CD34+ HSPCs.

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Iancu, O., Allen, D., Knop, O., Zehavi, Y., Breier, D., Arbiv, A., Lev, A., Lee, Y. N., Beider, K., Nagler, A., Somech, R., Hendel, A.. 2022-10-07. Multiplex HDR for Disease and Correction Modeling of SCID by CRISPR Genome Editing in Human HSPCs. https://doi.org/10.1101/2022.10.07.511272

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