Engineering In Vivo Chimeric Antigen Receptor Macrophages (CARMs) using mRNA-exosomes for Cancer Immunotherapy

项目总结 嵌合抗原受体(CAR)T细胞疗法使血液病的治疗发生了革命性变化 癌症。然而，对于实体瘤，CAR T细胞面临着包括瘤内异质性、动态 靶受体的表达，通常是T细胞无法运输到肿瘤以介导所需的 抗肿瘤作用。与缺乏T细胞浸润相反，许多实体瘤免疫功能丰富 抑制性髓系细胞，包括巨噬细胞。因此，将这些免疫抑制细胞转化为 杀瘤表型代表了一种基于细胞治疗的很有前途的策略。现在对……有强烈的兴趣 用病毒携带的CAR转导自体巨噬细胞生成CAR巨噬细胞 体外增强其吞噬、抗原提呈和细胞因子产生能力的载体 在重新输液后。然而，CAR巨噬细胞的体外制备是复杂的、耗时的，而且 对于巨噬细胞的非分裂性质，往往效率低下。基于信使核糖核酸的最新研究进展 治疗学，现在有可能在体内重新编程特定的免疫细胞群，从而消除 复杂的体外生产自体CAR细胞。我们目前的建议旨在提出一种创新的 用携带mRNA的外切体治疗HER2受体体内生成CAR巨噬细胞的策略 阳性乳腺癌。这将是第一个评估生产汽车巨噬细胞的可行性的研究。 利用信使核糖核酸传递平台进行体内研究及评价CAR巨噬细胞对肿瘤的抗肿瘤作用 免疫疗法。我们假设我们的战略代表着一种革命性的汽车生产方式 利用CAR信使核糖核酸的外切体体内培养巨噬细胞，为细胞治疗提供了一条新的途径 对抗实体瘤。我们之前的研究表明，我们可以有效地生产携带mRNA的外切体来 恢复实体瘤中的蛋白质表达。此外，我们的初步实验表明， 携带HER2 CAR基因的外切体能在体内产生增强效应的CAR巨噬细胞 功能。我们目前的研究将通过以下具体目标来检验我们的总体假设。在目标1中，我们 将评估使用CAR mRNA外切体在体内产生CAR巨噬细胞的动力学和毒性。在……里面 目的2，我们将评估体内产生的CAR巨噬细胞的转录和功能谱，最终， 在目标3中，我们将评估体内产生的CAR巨噬细胞对小鼠和 表达乳腺癌的人HER2。如果成功，我们提议的研究可以克服一项重大技术 这是目前细胞治疗面临的障碍。MRNAexosome平台有可能扩展到其他 CAR构建并极大地扩展了细胞治疗乳腺癌和其他实体癌症的潜在用途。

PROJECT SUMMARY Chimeric antigen receptor (CAR) T cell therapy has revolutionized the treatment of hematologic cancers. However, for solid tumors, CAR T cells face challenges including intratumor heterogeneity, dynamic expression of target receptors, and often the inability for T cells to traffic to tumors to mediate the desired antitumor effect. In contrast to the lack of T cell infiltrates, many solid tumors are abundant in immune suppressive myeloid cells including macrophages. Therefore, converting these immune suppressive cells into tumoricidal phenotype represents a promising strategy for cell-based therapy. There are now strong interest in generating CAR macrophages in which autologous macrophages are transduced with CAR delivered by viral vectors ex vivo to enhance their phagocytosis, antigen presentation and cytokine producing capabilities following re-infusion. However, ex vivo preparation of CAR macrophages is complex, time consuming, and due to the non-dividing nature of macrophages, is often inefficient. With the recent advances in mRNA-based therapeutics, it is now possible to reprogram specific immune cell populations in vivo, thus eliminating the complex ex vivo production of autologous CAR cells. Our present proposal aims to propose an innovative strategy of generating CAR macrophages in vivo using mRNA-loaded exosomes to treat HER2 receptor positive breast cancer. This will be the first study to evaluate the feasibility of producing CAR macrophages in vivo using mRNA delivery platforms and assessing the antitumor efficacy of CAR macrophages for cancer immunotherapy. We hypothesize that our strategy represents a revolutionary way to produce CAR macrophages in vivo using CAR mRNA-loaded exosome and offers a promising new approach for cell therapy against solid tumors. Our previous study showed that we can efficiently produce mRNA-loaded exosomes to restore protein expression in solid tumors. Furthermore, our preliminary experiments showed that the exosomes loaded with HER2 CAR mRNA can produce CAR macrophages in vivo with enhanced effector functions. Our current study will test our overall hypothesis by using the following specific aims. In Aim 1, we will evaluate the dynamics and toxicity of CAR macrophage production in vivo using CAR mRNA exosomes. In Aim 2, we will evaluate transcriptomic and functional profiles of in vivo generated CAR macrophages, Finally, in Aim 3, we will assess the antitumor effect of in vivo generated CAR macrophages against both murine and human HER2 expressing breast cancer. If successful, our proposed research can overcome a major technical hurdle that is currently facing cell therapy. The mRNA exosome platform could potentially be expanded to other CAR constructs and greatly expand the potential utility of cell therapy for breast and other solid cancers.