Ionizable lipid nanoparticles for the delivery of mRNA for CAR T cell engineering

项目总结 CAR T细胞免疫疗法被FDA批准用于治疗急性淋巴细胞白血病(ALL)和大 B细胞淋巴瘤，并在诱导持久缓解方面显示出成功。然而，这种疗法也与 会引起严重的、危及生命的副作用--包括细胞因子释放综合征、B细胞再生障碍性贫血和 神经毒性--70%的患者接受治疗。因此，有必要开发CAR T细胞 维持其治疗效果，同时将不良反应降至最低。目前，CAR T细胞是使用 诱导CAR永久表达的病毒，但对基于mRNA的CAR T细胞的研究-结果是 在瞬时CAR表达中-已在临床试验中使用，并显示出缓解长期 免疫疗法的副作用。为了产生这些mRNACAR T细胞，T细胞利用电穿孔 但它具有细胞毒性，没有转化为体内T细胞的潜力。因此，这项调查 目的探索可电离脂质纳米粒(LNPs)作为T细胞体外工程的载体。LNPs 在不同类型的细胞中显示出强大的信使核糖核酸递送，并且可以很容易地被修饰以改变物理化学 影响递送的属性，这将允许它们作为T细胞的递送平台进行优化。 在目标1中，将对24个新的LNPs进行筛选，以确定它们是否能够以低毒的功能传递mRNA，以及 性能最好的LNP将进一步优化，以确定输送到初级T细胞的最佳配方。在……里面 目的2，将目标1中选择的LNP制剂用于不同修饰的CAR mRNA的包囊化 确定以LNP为基础的T细胞递送的最佳信使核糖核酸载体。与顶端的LNP和CAR mRNA货物 选定后，AIM 3将验证LNPs作为CAR T细胞工程方法的有效性，并将其与电穿孔mRNA进行比较。 CAR T细胞和基于病毒的CAR T细胞通过使用全小鼠模型进行的生存研究。完成 这些目标将确定和优化T细胞的信使核糖核酸传递平台，在未来的研究中，可以 用于筛选新的汽车构造或体内递送。最终，这项工作--作为一项 生物工程系发起人与大学医学院之间的跨学科项目 这将为免疫细胞工程开发一种新型的LNP递送平台。

PROJECT SUMMARY CAR T cell immunotherapy is FDA approved for the treatment of acute lymphoblastic leukemia (ALL) and large B cell lymphoma and has shown success in inducing durable remission. However, the therapy is also associated with causing severe, life-threatening side effects—including cytokine release syndrome, B cell aplasia, and neurotoxicity—in 70% of patients receiving the treatment. Thus, there is a need to develop CAR T cells that maintain their therapeutic efficacy while minimizing adverse effects. Currently, CAR T cells are engineered using viruses that induce permanent CAR expression, but investigations into mRNA-based CAR T cells—which result in transient CAR expression—have been utilized in clinical trials and shown potential for mitigating long-term side effects of the immunotherapy. To create these mRNA CAR T cells, electroporation is utilized for T cell transfection, but it is cytotoxic and has no potential for translation to in vivo T cell delivery. Thus, this investigation aims to explore ionizable lipid nanoparticles (LNPs) as a delivery tool for the ex vivo engineering of T cells. LNPs have shown potent mRNA delivery in various cell types and can be easily modified to alter the physicochemical properties that impact delivery, which will allow for their optimization as a delivery platform for T cells specifically. In Aim 1, 24 novel LNPs will be screened for their ability to functionally deliver mRNA with low toxicity, and the top-performing LNP will be further optimized to determine the best formulation for delivery to primary T cells. In Aim 2, the LNP formulation selected in Aim 1 will be used to encapsulate CAR mRNA with different modifications to determine the best mRNA cargo for LNP-based delivery to T cells. With the top LNP and CAR mRNA cargo selected, Aim 3 will validate LNPs as a method for CAR T cell engineering as compared to electroporated mRNA- CAR T cells and virus-based CAR T cells via a survival study using an ALL mouse model. The completion of these aims will identify and optimize a mRNA delivery platform for T cells that, in future investigations, can be utilized for the screening of new CAR constructs or in vivo delivery. Ultimately, this work—conducted as an interdisciplinary project between sponsors in the Bioengineering Department and Medical School at University of Pennsylvania—will allow for the development of a novel LNP delivery platform for immune cell engineering.