HER2-targeted exosomal delivery of therapeutic mRNA for enzyme pro-drug therapy

用于酶前药治疗的 HER2 靶向外泌体递送治疗性 mRNA

• 批准号: 9333460
• 负责人: AC Matin
• 金额: $ 97.59万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-08-01 至 2020-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9333460
• 关键词: Antigens; Biochemistry; Biometry; Breast Cancer Cell; Breast Cancer cell line; Canis familiaris; Cell Communication; Cell Line; Code; Collaborations; Crystallography; DNA; Dendritic Cells; Doctor of Philosophy; Dose; Drug Kinetics; ERBB2 gene; Electroporation; Elements; Engineering; Ensure; Enzymes; Feedback; Gene Delivery; Human; Immunocompromised Host; Immunotherapeutic agent; Implant; In Vitro; Incubated; Injection of therapeutic agent; Joints; Laboratories; Letters; Ligands; Malignant Neoplasms; Mediating; Membrane Proteins; Messenger RNA; Mus; Neoplasm Metastasis; No-Observed-Adverse-Effect Level; Oncologist; Organ; Patients; Pharmacology Study; Phase; Prodrugs; Property; Rattus; Regimen; Reporter; Request for Applications; Research; Research Personnel; Resistance; Scientist; Specificity; Surface; Surgeon; System; Tail; Techniques; Testing; Therapeutic; Toxic effect; Transfection; Trastuzumab; Treatment Efficacy; United States National Institutes of Health; Veins; anticancer research; base; bioimaging; cancer cell; cell killing; cytotoxic; drug discovery; efficacy testing; exosome; improved; in vivo; killings; liquid chromatography mass spectrometry; malignant breast neoplasm; meetings; mouse model; multidisciplinary; overexpression; phenoxazine; public health relevance; receptor; response; skills; tumor; tumor xenograft

DESCRIPTION (provided by applicant): In response to NIH RFA: RM-12-014, it is proposed to engineer exosomes, the body's "natural antigen delivery system," in order to enhance their natural capacity to activate reductive prodrugs and attach to their surface ligands that specifically target HER2-positive breast cancer, many cases of which are resistant to the current therapies (e.g., trastuzumab) . This will be done for an enzyme-activated prodrug therapy newly discovered by the P.I. (A.C. Matin, Ph.D.). The prodrug is 6-chloro-9-nitro-5-oxo-5H-benzo-(a)-phenoxazine (CNOB), and the humanized enzyme improved by the PI is hChrR43. A highly helpful feature of this regimen is that the activated cytotoxic product of CNOB, 9-amino-6-chloro-5H-benzo[a]phenoxazine-5-one (MCHB), is strongly fluorescent and can be visualized in living mice. This property makes an 'observational approach' possible, and will minimize the need for mouse sacrifice and the use of more involved tests, e.g., LC/MS/MS, immunohistochemical, Westerns. Exosomes will be isolated from mouse dendritic cells, and the HER2- targeting ligand KCCYSL will be added to their surface using the exosome surface protein Lamp2b. The therapeutic regimen will consist of two types of exosomes administered by tail vein injection, those loaded with stabilized hChrR43 mRNA and those loaded with CNOB. mRNA instead of DNA will be used for gene delivery as it is proving to be better for this purpose; it will be loaded into the exosomes by electroporation and/or using the "zip code" sequence that selectively directs cellular mRNA into the exosomes. CNOB will be loaded by previously proven techniques by incubating it in PBS with the exosomes. Several types of reporter exosomes will also be constructed to optimize the conditions for specific targeting of HER2-positive breast cancer, ensuring the necessary transfection levels of cancer cells by the mRNA and its translational efficiency. The UH2 phase will concern with the construction of the directed and loaded exosomes; the optimization of their targeting specificity and HER2-positive breast cancer cell killing in vitro, followed by this optimization in vivo using implanted tumors o human HER2-positive human breast cancer cell lines in immunocompromised mice. The latter studies will also include a patient-derived breast cancer tumor xenograft mouse model that overexpress the HER2+ receptor, which is a more realistic system for testing the efficacy of the therapy. The UH3 phase will concern single dose pharmacokinetics and toxicity studies in rats and dogs, followed by repeat dose range finding toxicity studies in rats and dogs to select dose levels for the GLP subchronic studies, based on the no observed adverse effects level (NOAEL) and target organs for toxicity; and seeking pre-IND feedback from the FDA. The proposed research possesses the required mix of expertise and involves 5 Stanford scientists, a nearby exosome expert, and a premier organization, SRI for pharmacological studies.

描述(由申请人提供)：针对NIH RFA：RM-12-014，建议改造exosome，即人体的“天然抗原传递系统”，以增强它们激活还原前体药物并附着到其表面配体的自然能力，这些配体专门针对HER2阳性乳腺癌，其中许多病例对当前的治疗方法(如曲妥珠单抗)具有抗药性。这将用于P.I.新发现的一种酶激活前药物疗法(A.C.Matin，Ph.D.)。前药为6-chloro-9-nitro-5-oxo-5H-benzo-(a)-phenoxazine(CNOB)，经PI修饰的人源化酶为hchR43。该方案的一个非常有用的特点是，CNOB的激活细胞毒产物9-氨基-6-氯-5H-苯并[a]吩嗪-5-酮(MCHB)是强荧光的，可以在活体小鼠体内观察到。这一特性使“观察法”成为可能，并将最大限度地减少对小鼠的牺牲和更复杂测试的使用，例如LC/MS/MS、免疫组织化学、Western图像。将从小鼠树突状细胞中分离出外切体，并使用外切体表面蛋白Lamp2b将HER2靶向配体KCCYSL添加到它们的表面。治疗方案将包括尾静脉注射两种类型的外切体，即稳定的hchR43基因和cNOB基因。将使用信使核糖核酸而不是脱氧核糖核酸进行基因传递，因为事实证明，信使核糖核酸对此目的更好；它将通过电穿孔和/或使用选择性地将细胞信使核糖核酸定向到外切体中的“邮政编码”序列被加载到外切体中。CNOB将通过先前证明有效的技术与外切体一起在PBS中孵育而成。还将构建几种类型的报告外切体，以优化针对HER2阳性乳腺癌的特异性靶向条件，确保通过该mRNA对癌细胞进行必要的转染水平及其翻译效率。UH2阶段将涉及定向和负载外切体的构建；它们的靶向性和HER2阳性乳腺癌细胞体外杀伤的优化，然后是利用免疫低下小鼠的人HER2阳性人乳腺癌细胞株的体内移植瘤进行的优化。后一项研究还将包括一个患者来源的乳腺癌异种移植小鼠模型，该模型过度表达HER2+受体，这是一种更现实的测试治疗效果的系统。UH3阶段将涉及在大鼠和狗身上的单剂量药代动力学和毒性研究，随后将在大鼠和狗身上进行重复剂量范围寻找毒性研究，以根据未观察到的不良反应水平(NOAEL)和毒性的目标器官选择GLP亚慢性研究的剂量水平；并寻求FDA的IND前反馈。这项拟议的研究拥有所需的专业知识组合，涉及5名斯坦福大学科学家、附近的一名外显体专家和一个主要的药理学研究组织SRI。