Iancu Ortal, Allen Daniel, Knop Orli, Zehavi Yonathan, Breier Dor, Arbiv Adaya, Lev Atar, Lee Yu Nee, Beider Katia, Nagler Arnon, Somech Raz, Hendel Ayal
The Institute for Advanced Materials and Nanotechnology, The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat Gan 5290002, Israel.
Pediatric Department A and the Immunology Service, Jeffrey Modell Foundation Center, Edmond and Lily Safra Children's Hospital, Sheba Medical Center, Tel-HaShomer, Ramat Gan 5266202, Israel.
Mol Ther Nucleic Acids. 2022 Dec 9;31:105-121. doi: 10.1016/j.omtn.2022.12.006. eCollection 2023 Mar 14.
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) 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 -SCID patient-derived CD34 HSPCs that successfully developed into CD3 T cells with diverse TCR repertoires in an 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.
重症联合免疫缺陷（SCID）是一组由参与淋巴细胞成熟和功能过程的基因突变引起的疾病。对患者自身的造血干细胞和祖细胞（HSPCs）进行CRISPR-Cas9基因编辑可为同种异体造血干细胞移植提供一种治疗选择，同种异体造血干细胞移植是目前治疗SCID的金标准。为了消除对稀缺患者样本的需求，我们使用CRISPR-Cas9/rAAV6基因编辑技术在健康供体（HD）来源的CD34 HSPCs中构建基因型，通过多重同源定向修复（HDR）模拟SCID以及SCID基因编辑疗法的治疗效果。首先，我们通过对淋巴细胞发育至关重要的基因进行双等位基因敲除，建立了SCID疾病模型；其次，我们建立了敲入/敲除策略，以开展概念验证性的单等位基因基因校正。基于这些结果，我们对SCID患者来源的CD34 HSPCs进行了基因校正，这些细胞在T细胞分化平台上成功发育成具有多种TCR库的CD3 T细胞。总之，我们提出了一种策略，以确定使用HD来源的CD34 HSPCs对SCID进行CRISPR-Cas9基因校正的最佳配置，以及在患者来源的CD34 HSPCs中应用这种基因校正方法的可行性。
