嵌合抗原受体 (CAR) T 细胞疗法已被证明是过去十年中癌症治疗的突破，在对抗血液恶性肿瘤方面取得了前所未有的成果。所有批准的 CAR T 细胞产品以及许多正在临床试验中评估的产品都是使用病毒载体将外源遗传物质部署到 T 细胞中而产生的。病毒载体在基因传递方面具有悠久的临床历史，因此经过迭代优化以提高其效率和安全性。尽管如此，它们半随机整合到宿主基因组中的能力使它们通过插入突变和关键细胞基因失调而具有潜在的致癌性。 CAR T 细胞给药后继发性癌症似乎是一种罕见的不良事件。然而，过去几年记录的几起案例使这个问题成为人们关注的焦点，鉴于 CAR T 细胞疗法的部署相对较新，到目前为止，这个问题可能被低估了。此外，在血液恶性肿瘤中取得的初步成功尚未在实体瘤中复制。现在很清楚，需要进一步增强，以使 CAR T 细胞能够增加长期持久性、克服疲劳并应对免疫抑制的肿瘤微环境。为了实现这一目标，多种基因组工程策略正在评估中，其中大多数依赖于 CRISPR/Cas9 或其他基因编辑技术。这些方法很容易在产物细胞中引入意想不到的、不可逆的基因组改变。在本综述的第一部分中，我们将讨论用于生成 CAR T 细胞的病毒和非病毒方法，而在第二部分中，我们将重点关注基因编辑和非基因编辑 T 细胞工程，其中特别关注优点、限制和安全性。最后，我们将批判性地分析不同的基因部署和基因组工程组合，为下一代 CAR T 细胞的生产制定具有卓越安全性的策略。版权所有 © 2024 Rossi 和 Breman。

Chimeric antigen receptor (CAR) T-cell therapy has proven a breakthrough in cancer treatment in the last decade, giving unprecedented results against hematological malignancies. All approved CAR T-cell products, as well as many being assessed in clinical trials, are generated using viral vectors to deploy the exogenous genetic material into T-cells. Viral vectors have a long-standing clinical history in gene delivery, and thus underwent iterations of optimization to improve their efficiency and safety. Nonetheless, their capacity to integrate semi-randomly into the host genome makes them potentially oncogenic via insertional mutagenesis and dysregulation of key cellular genes. Secondary cancers following CAR T-cell administration appear to be a rare adverse event. However several cases documented in the last few years put the spotlight on this issue, which might have been underestimated so far, given the relatively recent deployment of CAR T-cell therapies. Furthermore, the initial successes obtained in hematological malignancies have not yet been replicated in solid tumors. It is now clear that further enhancements are needed to allow CAR T-cells to increase long-term persistence, overcome exhaustion and cope with the immunosuppressive tumor microenvironment. To this aim, a variety of genomic engineering strategies are under evaluation, most relying on CRISPR/Cas9 or other gene editing technologies. These approaches are liable to introduce unintended, irreversible genomic alterations in the product cells. In the first part of this review, we will discuss the viral and non-viral approaches used for the generation of CAR T-cells, whereas in the second part we will focus on gene editing and non-gene editing T-cell engineering, with particular regard to advantages, limitations, and safety. Finally, we will critically analyze the different gene deployment and genomic engineering combinations, delineating strategies with a superior safety profile for the production of next-generation CAR T-cell.Copyright © 2024 Rossi and Breman.