A targeted systemic delivery platform of therapeutic modified mRNA and modRNA-derived antibodies for precision triple negative breast cancer treatment.

modified mRNA (modRNA) showed high efficacy and safety when used for COVID-19 mRNA vaccines. Upon vaccination, any cell receiving modRNA contributed to efficient expression of antigens resulting in robust immune response. However, in most disease settings it is crucial to restrict translation of therapeutic genes to clinically relevant cells. Here, we designed a breast cancer-Specific modRNA Translation system (bcSMRTs) for enriched gene expression in tumors after systemic delivery with lipid nanoparticles (LNP). Intravenous delivery of bcSMRTs led to a 114-fold increase in tumor-specific signal and a 383-fold decrease in other organs compared to regular modRNA. For therapeutic targeting, we designed modRNA-derived antibodies (modRNabs) in which host cells serve as a bioreactor to produce anti-checkpoint inhibitor antibodies such as anti-cytotoxic T lymphocyte antigen-4 (αCTLA-4). Our results show that αCTLA-4 modRNab inhibited tumor growth by 37% while bcSMRTs carrying Pip4K2c (Phosphatidylinositol-5-phosphate 4-kinase, type II, gamma) gene did so by 25%. Importantly, combining the two reduced tumor size by 75% and reorganized the immune cell landscape in a poorly immunogenic 4T1 model of breast cancer. The modular platform we created to evaluate and screen gene-based treatments in a breast cancer model can easily be adjusted to other types of cancer. The 4T1 breast cancer cell line (ATCC, #CRL-2539) was cultured in RPMI (Gibco, #72400-047) supplemented with 10% FBS and pen-strep. Cells were transfected with either Luc or Pip4K2c modRNA using Lipofectamine2000 (Invitrogen, #11668027) and collected 48h post transfection. RNA was isolated using Quick-RNA MiniPrep kit (Zymo Research, #R1055). n=2 in three independent experiments.

经修饰的信使核糖核酸（modified mRNA, modRNA）在新型冠状病毒肺炎（COVID-19）mRNA疫苗的研发中展现出优异的有效性与安全性。疫苗接种后，任何摄取modRNA的细胞均可高效表达抗原，进而引发强烈的免疫应答。然而在多数疾病场景中，将治疗性基因的翻译过程严格限制在临床相关细胞内至关重要。为此，我们设计了一款乳腺癌特异性modRNA翻译系统（breast cancer-Specific modRNA Translation system, bcSMRTs），旨在通过脂质纳米颗粒（lipid nanoparticles, LNP）进行全身递送后，在肿瘤组织中富集目的基因的表达。与常规modRNA相比，静脉递送bcSMRTs可使肿瘤特异性信号提升114倍，同时在其他器官中的信号降低383倍。针对治疗性靶向需求，我们开发了modRNA来源的抗体（modRNA-derived antibodies, modRNabs），该系统以宿主细胞作为生物反应器，生产抗免疫检查点抑制剂类抗体，如抗细胞毒性T淋巴细胞抗原-4（anti-cytotoxic T lymphocyte antigen-4, αCTLA-4）。研究结果显示，αCTLA-4 modRNabs可将肿瘤生长抑制37%，而携带Pip4K2c（磷脂酰肌醇-5-磷酸4-激酶，II型γ亚型，Phosphatidylinositol-5-phosphate 4-kinase, type II, gamma）基因的bcSMRTs可实现25%的肿瘤生长抑制。值得注意的是，二者联合使用可使肿瘤体积缩小75%，并在免疫原性较弱的4T1乳腺癌模型中重塑免疫细胞图谱。我们构建的模块化研究平台可用于在乳腺癌模型中评估与筛选基因治疗方案，且可轻松适配其他癌种。本研究使用的4T1乳腺癌细胞系（ATCC，货号#CRL-2539）于添加了10%胎牛血清与青霉素-链霉素的RPMI培养基（Gibco，货号#72400-047）中培养。使用Lipofectamine2000（Invitrogen，货号#11668027）将Luc或Pip4K2c modRNA转染至细胞中，并于转染48小时后收集细胞。采用Quick-RNA MiniPrep试剂盒（Zymo Research，货号#R1055）提取RNA。所有实验独立重复三次，每组样本量n=2。