Targeted Delivery of siRNA for Intravenous RNAi

用于静脉 RNAi 的 siRNA 靶向递送

基本信息

批准号：
7534758
负责人：
RUBEN J. BOADO
金额：
$ 19万
依托单位：
ARMAGEN TECHNOLOGIES, INC.
依托单位国家：
美国
项目类别：
财政年份：
2008
资助国家：
美国
起止时间：
2008-09-15 至 2010-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7534758
关键词：
Affinity Affinity Chromatography Antibodies Avidin Binding Biochemical Biological Assay Biological Testing Bioreactors Biotin Blood Blood - brain barrier anatomy Brain COS Cells California Cell Density Cell Line Cell membrane Cells Chimeric Proteins Chinese Hamster Chinese Hamster Ovary Cell Chromatography Cloning Complementary DNA Conditioned Culture Media Cytomegalovirus DNA Sequence Analysis Data Development Dihydrofolate Reductase Distant Drug Delivery Systems Electroporation Elements Engineering Enzyme-Linked Immunosorbent Assay Fibroblasts G-substrate Gene Expression Genes Genetic Engineering Goals Hepatitis B Virus Hypoxanthine Hypoxanthines IgG1 Immunoglobulin G Intellectual Property Intravenous LF2000 Legal patent Licensing Light Luc Gene Luciferases Medicine Membrane Messenger RNA Methotrexate Mono-S Monoclonal Antibodies Mus Neomycin resistance gene Organ Ovary Pathologic Peripheral Phase Plasmids Polyacrylamide Gel Electrophoresis Positioning Attribute Production Public Health RNA Interference Rattus Research Robotics Seminal Series Serum Serum-Free Culture Media Simian virus 40 Small Interfering RNA Sodium Dodecyl Sulfate System Technology Thymine Transfection Transferrin Receptor Universities Validation Veins Western Blotting Work antibiotic G 418 bovine growth hormone commercialization in vivo instrument molecular trojan horse new technology plasmid DNA promoter receptor targeted delivery transcription termination vector

项目摘要

DESCRIPTION (provided by applicant): RNA interference (RNAi) is a relatively new technology that enables the knockdown of expression of pathologic genes with short interfering RNAs (siRNAs). The limiting factor in the development of new RNAi medicines is delivery. The goal of the field is "Intravenous RNAi," which is the ability to inject a siRNA into a peripheral vein, and find that the siRNA enters the distant target organ to silence the target mRNA molecule. This target mRNA molecule is hidden behind multiple membrane barriers in vivo that separate the blood compartment from the mRNA. In the present work, the siRNA is mono-biotinylated in parallel with the production of a fusion protein of avidin and a receptor specific monoclonal antibody (MAb). The present work will engineer, express, and validate a new fusion protein comprised of avidin and a mouse/rat chimeric MAb to the mouse transferrin receptor (TfR). The TfRMAb acts as a molecular Trojan horse, to ferry the siRNA across any target cell membrane that expresses the TfR. Since the TfR is highly expressed on the blood-brain barrier (BBB), the availability of the TfRMAb/AV fusion protein will enable in vivo RNAi for the brain, as well as other organs. Eukaryotic expression plasmids encoding the light chain (LC) and the heavy chain (HC)/AV fusion protein will be genetically engineered, expressed in host cells, and the TfRMAb/AV fusion protein will be purified by protein G affinity chromatography, followed by validation of the bi- functionality of the fusion protein with mouse TfR and biotin binding assay. A single tandem vector will be engineered for permanent transfection of host cells. Following selection, amplification, and dilutional cloning, the transfected host cells will be propagated, and the TfRMAb/AV fusion protein will be purified by affinity chromatography. The TfRMAb/AV fusion protein produced at this large scale will be validated with a series of biochemical assays to verify the structural and functional integrity of the molecule, including the validation of siRNA action and RNA interference. PUBLIC HEALTH RELEVANCE RNA interference (RNAi) is a new technology that enables the knockdown of expression of pathologic genes with short interfering RNAs (siRNAs). The limiting factor in the development of new RNAi medicines is delivery. The goal of the field is "Intravenous RNAi," which is the ability to inject a siRNA into a peripheral vein, and find that the siRNA enters the distant target organ to silence the target mRNA molecule. The present work will engineer, express, and validate a new fusion protein comprised of avidin and a mouse/rat chimeric MAb to the mouse transferrin receptor.

描述（由申请人提供）：RNA干扰（RNAi）是一种相对较新的技术，能够利用短干扰RNA（siRNA）敲低病理基因的表达。新 RNAi 药物开发的限制因素是递送。该领域的目标是“静脉内RNAi”，即能够将siRNA注射到外周静脉中，并发现siRNA进入远处的靶器官，从而沉默靶mRNA分子。该目标 mRNA 分子隐藏在体内多个膜屏障后面，这些膜屏障将血液隔室与 mRNA 分开。在目前的工作中，siRNA 被单生物素化，同时产生亲和素和受体特异性单克隆抗体 (MAb) 的融合蛋白。目前的工作将设计、表达和验证一种新的融合蛋白，该融合蛋白由亲和素和小鼠转铁蛋白受体 (TfR) 的小鼠/大鼠嵌合 MAb 组成。 TfRMAb 充当分子特洛伊木马，将 siRNA 运送穿过任何表达 TfR 的靶细胞膜。由于 TfR 在血脑屏障 (BBB) 上高度表达，因此 TfRMAb/AV 融合蛋白的可用性将使大脑以及其他器官的体内 RNAi 成为可能。将编码轻链（LC）和重链（HC）/AV融合蛋白的真核表达质粒进行基因工程改造，在宿主细胞中表达，并通过蛋白G亲和层析纯化TfRMAb/AV融合蛋白，然后进行验证融合蛋白的双功能与小鼠 TfR 和生物素结合测定。单个串联载体将被设计用于永久转染宿主细胞。经过选择、扩增和稀释克隆后，将增殖转染的宿主细胞，并通过亲和层析纯化TfRMAb/AV融合蛋白。如此大规模生产的 TfRMAb/AV 融合蛋白将通过一系列生化测定进行验证，以验证分子的结构和功能完整性，包括 siRNA 作用和 RNA 干扰的验证。公共卫生相关性 RNA 干扰 (RNAi) 是一种新技术，可以通过短干扰 RNA (siRNA) 敲低病理基因的表达。新 RNAi 药物开发的限制因素是递送。该领域的目标是“静脉内RNAi”，即能够将siRNA注射到外周静脉中，并发现siRNA进入远处的靶器官，从而沉默靶mRNA分子。目前的工作将设计、表达和验证一种新的融合蛋白，该融合蛋白由抗生物素蛋白和针对小鼠转铁蛋白受体的小鼠/大鼠嵌合单克隆抗体组成。