Large-scale GMP-compliant CRISPR-Cas9-mediated deletion of the glucocorticoid receptor in multivirus-specific T cells
Large-scale GMP-compliant CRISPR-Cas9-mediated deletion of the glucocorticoid receptor in multivirus-specific T cells
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DOI：
10.1182/bloodadvances.2020001977
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发表时间：
2020-07-01
期刊：
影响因子：
7.5
通讯作者：
Rezvani, Katayoun
中科院分区：
文献类型：
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作者：
Basar, Rafet;Daher, May;Rezvani, Katayoun
病毒特异性T细胞已被证明对治疗造血干细胞移植（HSCT）后严重和药物难治性感染非常有效。然而，这些细胞的功效受到糖皮质激素使用的阻碍，糖皮质激素通常用于治疗诸如移植物抗宿主病等并发症的患者。为了解决这一限制，我们开发了一种新的策略，用于使用聚集规则间隔短回文重复序列(CRISPR)-CRISPR相关蛋白9 （Cas9）基因编辑技术快速生成良好生产规范（GMP）级抗糖皮质激素多病毒特异性T细胞（VSTs）。我们已经证明，删除核受体亚家族3C组成员1 （NR3C1；编码糖皮质激素受体的基因）可以使VSTs抵抗糖皮质激素的淋巴细胞毒性作用。nr3c1敲除（KO） VSTs在体外和体内高剂量地塞米松存在下杀死其靶标并成功增殖。此外，我们开发了一种快速生成gmp级NR3C1 KO vst的方案，该方案具有高靶标活性和最小的脱靶编辑。这些基因工程vst有望成为一种新的方法，用于治疗在糖皮质激素治疗的hsct后危及生命的病毒感染患者。
Virus-specific T cells have proven highly effective for the treatment of severe and drug-refractory infections after hematopoietic stem cell transplant (HSCT). However, the efficacy of these cells is hindered by the use of glucocorticoids, often given to patients for the management of complications such as graft-versus-host disease. To address this limitation, we have developed a novel strategy for the rapid generation of good manufacturing practice (GMP)-grade glucocorticoid-resistant multivirus-specific T cells (VSTs) using clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) gene-editing technology. We have shown that deleting the nuclear receptor subfamily 3 group C member 1 (NR3C1; the gene encoding for the glucocorticoid receptor) renders VSTs resistant to the lymphocytotoxic effect of glucocorticoids. NR3C1-knockout (KO) VSTs kill their targets and proliferate successfully in the presence of high doses of dexamethasone both in vitro and in vivo. Moreover, we developed a protocol for the rapid generation of GMP-grade NR3C1 KO VSTs with high on-target activity and minimal off-target editing. These genetically engineered VSTs promise to be a novel approach for the treatment of patients with life-threatening viral infections post-HSCT on glucocorticoid therapy.