Exosomal Based micro RNA delivery for Resistant Lung Cancer

基于外泌体的 micro RNA 递送治疗耐药性肺癌

基本信息

批准号：
10629892
负责人：
Mandip Singh Sachdeva
金额：
$ 14.8万
依托单位：
FLORIDA AGRICULTURAL AND MECHANICAL UNIV
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-04-01 至 2027-03-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10629892
关键词：
Antineoplastic Agents Biological Assay Bioreactors Carboplatin Cells Clinical Research Data Dose Down-Regulation Drug Kinetics Electroporation Embryo Encapsulated Engineering Epidermal Growth Factor Receptor Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor Evaluation Exposure to Fibroblasts Formulation Future Gene Expression Goals Histological Techniques Human In Vitro Inbred BALB C Mice Induction of Apoptosis Interleukin-15 Intravenous Kidney Laboratories Lung MAP Kinase Gene Malignant Neoplasms Malignant neoplasm of lung Mesenchymal Stem Cells MicroRNAs Molecular Monoclonal Antibodies Mus Mutate Mutation NF-kappa B NOD/SCID mouse Natural Killer Cells Non-Small-Cell Lung Carcinoma Outcome Patients Pharmacodynamics Phase Prognosis Proteins Proteomics Research Personnel Resistance Resistance development Reverse Transcriptase Polymerase Chain Reaction Role Study models Techniques Testing Therapeutic Toxic effect Toxicology Tumor Burden Tyrosine Kinase Inhibitor Vimentin Western Blotting Work Xenograft procedure anti-cancer anticancer treatment cell killing comparison control cytotoxicity exosome extracellular vesicles heme oxygenase-1 in vivo kidney cell lamin B2 manufacture microRNA delivery p38 Mitogen Activated Protein Kinase particle patient derived xenograft model phase 1 study pre-clinical programmed cell death ligand 1 programmed cell death protein 1 protein expression side effect success transcriptome sequencing tumor uptake virtual

项目摘要

Abstract Non-small cell lung cancers (NSCLC) have the poorest outcome of all the cancers and after a period of responsiveness, acquired resistance (e.g. T790M mutation) occurs in virtually all NSCLC tumors exposed to Tyrosine Kinase Inhibitors (TKI). Osimertinib, an irreversible EGFR inhibitor which targets EGFR- T790M mutations is the first line treatment for mutated NSCLC but resistance develops after 12-24 months. The checkpoint proteins (PD1, PDL1) and laminB2 (LMNB2) are associated with a poor prognosis in a variety of cancers. Results from our laboratory have shown that in H1975 tumors (expressing L858R/T790M-EGFR mutations), downregulation of PDL1 and LMNB2, among other proteins, could significantly reduce tumor burden in xenotransplanted mice (proteomic analysis. Further, exosomes (EVs) derived from Natural killer cells (NK92MI, NKEVs) contain various cytolytic proteins and have shown potential as anticancer agents. In our laboratory, we observed that NKEVs (using a PBS bioreactor with IL-15), showed 40 percent cell kill at concentration of 1X1010 particles when compared to EVs derived from HEK or MSC cells which showed 15-20 percent cell kill in lung PDX cells. Further, H1975 resistant (H1975R) xenotransplanted tumors when treated with NKEVs downregulated HO1, vimentin. NF-kB, P38MAPK significantly (P<0.001) as compared to control and HEK derived EVs suggesting their anticancer role via inducing apoptosis and other possible mechanisms. Also, NKEVS were found to deliver fluorescent mir3133-TYE effectively in significant amounts (as compared to control) to H1975R tumors when given intravenously showing their targeting potential. Further we explored the micro-RNA which regulate LMNB2(mir-3133) and PDL1(mir5193) and showed that they could significantly downregulate the expression of LMNB2 and PDL1 respectively in vitro and also in PDX tumors (TM00199, Jackson labs), only when delivered as EV formulations in NSG mice. Hence based on our strong preliminary data, we hypothesize that NKEVs carrying LMNB2 and PDL1 micro RNA will deliver their payload to osimertinib resistant NSCLC and will be able to overcome resistance by using in combination with carboplatin with minimal side effects. To test this hypothesis, we propose the following independent Aims: Aim 1: Formulation of NK-EVs containing PDL1 and LMNB2 micro-RNA and evaluating them in vitro in combination with carboplatin against H1975 (R and wild type) and PDX cells Aim 2: Toxicological and Pharmacodynamic evaluation of the dual micro-RNA NKEVs in H1975 resistant and PDX models. The long-term goal of this proposal is to generate enough preclinical data with bioreactor manufactured NKEVs micro RNA formulations and to understand their role in overcoming resistance so as to apply for a R01 proposal or Phase 1 clinical studies in the future.

抽象的非小细胞肺癌（NSCLC）是所有癌症中预后最差的，经过一段时间的治疗后，反应性、获得性耐药（例如 T790M 突变）几乎发生在所有暴露的 NSCLC 肿瘤中酪氨酸激酶抑制剂（TKI）。奥西替尼，一种不可逆 EGFR 抑制剂，靶向 EGFR-T790M 突变是突变 NSCLC 的一线治疗方法，但 12-24 个月后会出现耐药性。这检查点蛋白（PD1、PDL1）和核纤层蛋白 B2（LMNB2）与多种疾病的不良预后相关癌症。我们实验室的结果表明，在H1975肿瘤（表达L858R/T790M-EGFR 突变），下调 PDL1 和 LMNB2 等蛋白质可以显着减少肿瘤异种移植小鼠的负担（蛋白质组学分析。此外，来自自然杀伤剂的外泌体（EV）细胞（NK92MI、NKEV）含有各种溶细胞蛋白，已显示出作为抗癌药物的潜力。在我们的实验室中，我们观察到 NKEV（使用含有 IL-15 的 PBS 生物反应器）显示 40% 的细胞死亡与源自 HEK 或 MSC 细胞的 EV 相比，在 1X1010 颗粒浓度下，显示肺 PDX 细胞被杀死 15-20%。此外，H1975 抗性（H1975R）异种移植肿瘤 NKEV 治疗下调 HO1、波形蛋白。 NF-kB、P38MAPK 相比显着（P<0.001）控制和 HEK 衍生的 EVs 表明它们通过诱导细胞凋亡和其他可能的抗癌作用机制。此外，NKEVS 被发现可以有效地释放大量荧光 mir3133-TYE （与对照相比）静脉注射时对 H1975R 肿瘤的影响显示出其靶向潜力。我们进一步探索了调节 LMNB2(mir-3133) 和 PDL1(mir5193) 的 micro-RNA，并表明他们可以分别在体外和体内显着下调 LMNB2 和 PDL1 的表达 PDX 肿瘤（TM00199，Jackson 实验室），仅在 NSG 小鼠中以 EV 制剂形式给药时。因此基于根据我们强有力的初步数据，我们假设携带 LMNB2 和 PDL1 micro RNA 的 NKEV 将将其有效负载传递给奥希替尼耐药的 NSCLC，并将能够通过使用来克服耐药性与卡铂联合使用，副作用最小。为了检验这个假设，我们提出以下建议独立的目标：目标 1：配制含有 PDL1 和 LMNB2 micro-RNA 的 NK-EV，并在体外对其进行评估与卡铂联合对抗 H1975（R 和野生型）和 PDX 细胞目标 2：H1975 耐药性中双 micro-RNA NKEV 的毒理学和药效学评价和 PDX 模型。该提案的长期目标是利用生物反应器生成足够的临床前数据制造 NKEV 微 RNA 制剂并了解其在克服耐药性方面的作用，以便未来申请R01提案或1期临床研究。