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CRISPR-engineered T cells in patients with refractory cancer
Science ( IF 56.9 ) Pub Date : 2020-02-06 , DOI: 10.1126/science.aba7365
Edward A Stadtmauer 1, 2 , Joseph A Fraietta 2, 3, 4, 5, 6 , Megan M Davis 5, 6 , Adam D Cohen 1, 2 , Kristy L Weber 2, 7 , Eric Lancaster 8 , Patricia A Mangan 1 , Irina Kulikovskaya 5 , Minnal Gupta 5 , Fang Chen 5 , Lifeng Tian 5 , Vanessa E Gonzalez 5 , Jun Xu 5 , In-Young Jung 4, 5 , J Joseph Melenhorst 3, 5, 6 , Gabriela Plesa 5 , Joanne Shea 5 , Tina Matlawski 5 , Amanda Cervini 5 , Avery L Gaymon 5 , Stephanie Desjardins 5 , Anne Lamontagne 5 , January Salas-Mckee 5 , Andrew Fesnak 5, 6 , Donald L Siegel 5, 6 , Bruce L Levine 5, 6 , Julie K Jadlowsky 5 , Regina M Young 5 , Anne Chew 5 , Wei-Ting Hwang 9 , Elizabeth O Hexner 1, 2 , Beatriz M Carreno 3, 5, 6 , Christopher L Nobles 4 , Frederic D Bushman 4 , Kevin R Parker 10 , Yanyan Qi 11 , Ansuman T Satpathy 10, 11 , Howard Y Chang 10, 12 , Yangbing Zhao 5, 6 , Simon F Lacey 5, 6 , Carl H June 2, 3, 5, 6
Affiliation  

CRISPR takes first steps in humans CRISPR-Cas9 is a revolutionary gene-editing technology that offers the potential to treat diseases such as cancer, but the effects of CRISPR in patients are currently unknown. Stadtmauer et al. report a phase 1 clinical trial to assess the safety and feasibility of CRISPR-Cas9 gene editing in three patients with advanced cancer (see the Perspective by Hamilton and Doudna). They removed immune cells called T lymphocytes from patients and used CRISPR-Cas9 to disrupt three genes (TRAC, TRBC, and PDCD1) with the goal of improving antitumor immunity. A cancer-targeting transgene, NY-ESO-1, was also introduced to recognize tumors. The engineered cells were administered to patients and were well tolerated, with durable engraftment observed for the study duration. These encouraging observations pave the way for future trials to study CRISPR-engineered cancer immunotherapies. Science, this issue p. eaba7365; see also p. 976 Multiply gene-edited human immunological T cells demonstrate safety and prolonged persistence in three patients. INTRODUCTION Most cancers are recognized and attacked by the immune system but can progress owing to tumor-mediated immunosuppression and immune evasion mechanisms. The infusion of ex vivo engineered T cells, termed adoptive T cell therapy, can increase the natural antitumor immune response of the patient. Gene therapy to redirect immune specificity combined with genome editing has the potential to improve the efficacy and increase the safety of engineered T cells. CRISPR coupled with CRISPR-associated protein 9 (Cas9) endonuclease is a powerful gene-editing technology that potentially allows the ability to target multiple genes in T cells to improve cancer immunotherapy. RATIONALE Our first-in-human, phase 1 clinical trial (clinicaltrials.gov; trial NCT03399448) was designed to test the safety and feasibility of multiplex CRISPR-Cas9 gene editing of T cells from patients with advanced, refractory cancer. A limitation of adoptively transferred T cell efficacy has been the induction of T cell dysfunction or exhaustion. We hypothesized that removing the endogenous T cell receptor (TCR) and the immune checkpoint molecule programmed cell death protein 1 (PD-1) would improve the function and persistence of engineered T cells. In addition, the removal of PD-1 has the potential to improve safety and reduce toxicity that can be caused by autoimmunity. A synthetic, cancer-specific TCR transgene (NY-ESO-1) was also introduced to recognize tumor cells. In vivo tracking and persistence of the engineered T cells were monitored to determine if the cells could persist after CRISPR-Cas9 modifications. RESULTS Four cell products were manufactured at clinical scale, and three patients (two with advanced refractory myeloma and one with metastatic sarcoma) were infused. The editing efficiency was consistent in all four products and varied as a function of the single guide RNA (sgRNA), with highest efficiency observed for the TCR α chain gene (TRAC) and lowest efficiency for the TCR β chain gene (TRBC). The mutations induced by CRISPR-Cas9 were highly specific for the targeted loci; however, rare off-target edits were observed. Single-cell RNA sequencing of the infused CRISPR-engineered T cells revealed that ~30% of cells had no detectable mutations, whereas ~40% had a single mutation and ~20 and ~10% of the engineered T cells were double mutated and triple mutated, respectively, at the target sequences. The edited T cells engrafted in all three patients at stable levels for at least 9 months. The persistence of the T cells expressing the engineered TCR was much more durable than in three previous clinical trials during which T cells were infused that retained expression of the endogenous TCR and endogenous PD-1. There were no clinical toxicities associated with the engineered T cells. Chromosomal translocations were observed in vitro during cell manufacturing, and these decreased over time after infusion into patients. Biopsies of bone marrow and tumor showed trafficking of T cells to the sites of tumor in all three patients. Although tumor biopsies revealed residual tumor, in both patients with myeloma, there was a reduction in the target antigens NY-ESO-1 and/or LAGE-1. This result is consistent with an on-target effect of the engineered T cells, resulting in tumor evasion. CONCLUSION Preliminary results from this pilot trial demonstrate that multiplex human genome engineering is safe and feasible using CRISPR-Cas9. The extended persistence of the engineered T cells indicates that preexisting immune responses to Cas9 do not appear to present a barrier to the implementation of this promising technology. CRISPR-Cas9 engineering of T cells in cancer patients. T cells (center) were isolated from the blood of a patient with cancer. CRISPR-Cas9 ribonuclear protein complexes loaded with three sgRNAs were electroporated into the normal T cells, resulting in gene editing of the TRAC, TRBC1, TRBC2, and PDCD1 (encoding PD-1) loci. The cells were then transduced with a lentiviral vector to express a TCR specific for the cancer-testis antigens NY-ESO-1 and LAGE-1 (right). The engineered T cells were then returned to the patient by intravenous infusion, and patients were monitored to determine safety and feasibility. PAM, protospacer adjacent motif. CRISPR-Cas9 gene editing provides a powerful tool to enhance the natural ability of human T cells to fight cancer. We report a first-in-human phase 1 clinical trial to test the safety and feasibility of multiplex CRISPR-Cas9 editing to engineer T cells in three patients with refractory cancer. Two genes encoding the endogenous T cell receptor (TCR) chains, TCRα (TRAC) and TCRβ (TRBC), were deleted in T cells to reduce TCR mispairing and to enhance the expression of a synthetic, cancer-specific TCR transgene (NY-ESO-1). Removal of a third gene encoding programmed cell death protein 1 (PD-1; PDCD1), was performed to improve antitumor immunity. Adoptive transfer of engineered T cells into patients resulted in durable engraftment with edits at all three genomic loci. Although chromosomal translocations were detected, the frequency decreased over time. Modified T cells persisted for up to 9 months, suggesting that immunogenicity is minimal under these conditions and demonstrating the feasibility of CRISPR gene editing for cancer immunotherapy.

中文翻译:

难治性癌症患者的 CRISPR 工程 T 细胞

CRISPR 在人类中迈出第一步 CRISPR-Cas9 是一种革命性的基因编辑技术,它提供了治疗癌症等疾病的潜力,但目前尚不清楚 CRISPR 对患者的影响。Stadtmauer 等人。报告一项 1 期临床试验,以评估 CRISPR-Cas9 基因编辑在三名晚期癌症患者中的安全性和可行性(参见 Hamilton 和 Doudna 的观点)。他们从患者身上去除了称为 T 淋巴细胞的免疫细胞,并使用 CRISPR-Cas9 破坏了三个基因(TRAC、TRBC 和 PDCD1),以提高抗肿瘤免疫力。还引入了一种癌症靶向转基因NY-ESO-1来识别肿瘤。工程细胞被施用于患者并且耐受性良好,在研究期间观察到持久的植入。这些令人鼓舞的观察结果为未来研究 CRISPR 工程癌症免疫疗法的试验铺平了道路。科学,本期第 3 页。eaba7365; 另见第 976 Multiply 基因编辑的人类免疫 T 细胞在三名患者中表现出安全性和持久性。引言 大多数癌症都被免疫系统识别和攻击,但由于肿瘤介导的免疫抑制和免疫逃避机制而可以进展。体外工程 T 细胞的输注,称为过继性 T 细胞疗法,可以增加患者的天然抗肿瘤免疫反应。重定向免疫特异性的基因疗法与基因组编辑相结合,有可能提高工程 T 细胞的功效和安全性。CRISPR 与 CRISPR 相关蛋白 9 (Cas9) 核酸内切酶相结合是一种强大的基因编辑技术,它可能允许靶向 T 细胞中的多个基因以改善癌症免疫治疗。基本原理 我们的首次人体 1 期临床试验(clinicaltrials.gov;试验 NCT03399448)旨在测试对晚期难治性癌症患者 T 细胞进行多重 CRISPR-Cas9 基因编辑的安全性和可行性。过继转移 T 细胞功效的一个限制是诱导 T 细胞功能障碍或衰竭。我们假设去除内源性 T 细胞受体 (TCR) 和免疫检查点分子程序性细胞死亡蛋白 1 (PD-1) 将改善工程 T 细胞的功能和持久性。此外,去除 PD-1 有可能提高安全性并减少自身免疫可能引起的毒性。还引入了一种合成的癌症特异性 TCR 转基因 (NY-ESO-1) 来识别肿瘤细胞。监测工程化 T 细胞的体内跟踪和持久性,以确定细胞在 CRISPR-Cas9 修饰后是否可以持续存在。结果 生产了四种临床规模的细胞产品,输注了 3 名患者(两名患有晚期难治性骨髓瘤,一名患有转移性肉瘤)。所有四种产品的编辑效率都是一致的,并且随着单向导 RNA (sgRNA) 的变化而变化,观察到 TCR α 链基因 (TRAC) 的效率最高,而 TCR β 链基因 (TRBC) 的效率最低。CRISPR-Cas9 诱导的突变对靶向位点具有高度特异性;然而,观察到罕见的脱靶编辑。注入的 CRISPR 工程 T 细胞的单细胞 RNA 测序显示,约 30% 的细胞没有可检测到的突变,而约 40% 的细胞有单突变,约 20% 和约 10% 的工程 T 细胞是双突变和三重突变分别在靶序列发生突变。编辑后的 ​​T 细胞以稳定的水平在所有三名患者中植入至少 9 个月。表达工程化 TCR 的 T 细胞的持久性比之前的三项临床试验更持久,在这三项临床试验中,T 细胞被注入保留了内源性 TCR 和内源性 PD-1 的表达。没有与工程化 T 细胞相关的临床毒性。在细胞制造过程中在体外观察到染色体易位,并且这些易位在输注到患者体内后随着时间的推移而减少。骨髓和肿瘤活检显示所有三名患者的 T 细胞都向肿瘤部位输送。尽管肿瘤活检显示残留肿瘤,但在两名骨髓瘤患者中,靶抗原 NY-ESO-1 和/或 LAGE-1 均有所减少。该结果与工程化 T 细胞的靶向效应一致,从而导致肿瘤逃避。结论 该试点试验的初步结果表明,使用 CRISPR-Cas9 进行多重人类基因组工程是安全和可行的。工程化 T 细胞的持久性表明,先前存在的对 Cas9 的免疫反应似乎不会阻碍这项有前途的技术的实施。癌症患者 T 细胞的 CRISPR-Cas9 工程。从癌症患者的血液中分离出 T 细胞(中心)。将载有三种 sgRNA 的 CRISPR-Cas9 核糖核蛋白复合物电穿孔到正常 T 细胞中,从而对 TRAC、TRBC1、TRBC2 和 PDCD1(编码 PD-1)基因座进行基因编辑。然后用慢病毒载体转导细胞以表达对癌症睾丸抗原 NY-ESO-1 和 LAGE-1 特异的 TCR(右)。然后通过静脉输注将工程化的 T 细胞返回给患者,并对患者进行监测以确定安全性和可行性。PAM,protospacer 相邻基序。CRISPR-Cas9 基因编辑提供了一个强大的工具来增强人类 T 细胞对抗癌症的自然能力。我们报告了一项首次人体 1 期临床试验,以测试多重 CRISPR-Cas9 编辑在三名难治性癌症患者中改造 T 细胞的安全性和可行性。编码内源性 T 细胞受体 (TCR) 链的两个基因 TCRα (TRAC) 和 TCRβ (TRBC) 在 T 细胞中被删除,以减少 TCR 错配并增强合成的癌症特异性 TCR 转基因 (NY-ESO) 的表达-1)。去除编码程序性细胞死亡蛋白 1 (PD-1; PDCD1) 的第三个基因以提高抗肿瘤免疫力。将工程化 T 细胞过继转移到患者体内,可以在所有三个基因组位点进行编辑,从而实现持久的植入。虽然检测到染色体易位,但频率随着时间的推移而降低。修饰后的 T 细胞持续存在长达 9 个月,这表明在这些条件下免疫原性很小,并证明了 CRISPR 基因编辑用于癌症免疫治疗的可行性。在 T 细胞中被删除以减少 TCR 错配并增强合成的癌症特异性 TCR 转基因 (NY-ESO-1) 的表达。去除编码程序性细胞死亡蛋白 1 (PD-1; PDCD1) 的第三个基因以提高抗肿瘤免疫力。将工程化 T 细胞过继转移到患者体内,可以在所有三个基因组位点进行编辑,从而实现持久的植入。虽然检测到染色体易位,但频率随着时间的推移而降低。修饰后的 T 细胞持续存在长达 9 个月,这表明在这些条件下免疫原性很小,并证明了 CRISPR 基因编辑用于癌症免疫治疗的可行性。在 T 细胞中被删除以减少 TCR 错配并增强合成的癌症特异性 TCR 转基因 (NY-ESO-1) 的表达。去除编码程序性细胞死亡蛋白 1 (PD-1; PDCD1) 的第三个基因以提高抗肿瘤免疫力。将工程化 T 细胞过继转移到患者体内,可以在所有三个基因组位点进行编辑,从而实现持久的植入。虽然检测到染色体易位,但频率随着时间的推移而降低。修饰后的 T 细胞持续存在长达 9 个月,这表明在这些条件下免疫原性很小,并证明了 CRISPR 基因编辑用于癌症免疫治疗的可行性。用于提高抗肿瘤免疫力。将工程化 T 细胞过继转移到患者体内,可以在所有三个基因组位点进行编辑,从而实现持久的植入。虽然检测到染色体易位,但频率随着时间的推移而降低。修饰后的 T 细胞持续存在长达 9 个月,这表明在这些条件下免疫原性很小,并证明了 CRISPR 基因编辑用于癌症免疫治疗的可行性。用于提高抗肿瘤免疫力。将工程化 T 细胞过继转移到患者体内,可以在所有三个基因组位点进行编辑,从而实现持久的植入。虽然检测到染色体易位,但频率随着时间的推移而降低。修饰后的 T 细胞持续存在长达 9 个月,这表明在这些条件下免疫原性很小,并证明了 CRISPR 基因编辑用于癌症免疫治疗的可行性。
更新日期:2020-02-06
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