Targeted Delivery of siRNA for Intravenous RNAi

用于静脉 RNAi 的 siRNA 靶向递送

• 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
• 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

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”，这是将siRNA注射到外周静脉中，并发现siRNA进入远端靶器官以沉默靶mRNA分子的能力。这种靶mRNA分子隐藏在体内将血液隔室与mRNA分离的多个膜屏障后面。在本工作中，siRNA被单生物素化，同时产生抗生物素蛋白和受体特异性单克隆抗体（MAb）的融合蛋白。目前的工作将工程，表达，并验证一个新的融合蛋白组成的亲和素和小鼠/大鼠嵌合单克隆抗体的小鼠转铁蛋白受体（TfR）。TfRMAb充当分子特洛伊木马，将siRNA运送穿过表达TfR的任何靶细胞膜。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脑以及其他器官的体内RNAi成为可能。编码轻链（LC）和重链（HC）/AV融合蛋白的真核表达质粒将被遗传工程化，在宿主细胞中表达，并且TfRMAb/AV融合蛋白将通过蛋白G亲和色谱法纯化，随后用小鼠TfR和生物素结合测定验证融合蛋白的双功能性。单个串联载体将被工程化用于宿主细胞的永久转染。在选择、扩增和稀释克隆后，增殖转染的宿主细胞，并通过亲和色谱法纯化TfRMAb/AV融合蛋白。将通过一系列生化试验验证以这种大规模生产的TfRMAb/AV融合蛋白，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RNA干扰（RNAi）是一种新技术，能够用短干扰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”，这是将siRNA注射到外周静脉中，并发现siRNA进入远端靶器官以沉默靶mRNA分子的能力。目前的工作将工程，表达，并验证一个新的融合蛋白组成的亲和素和小鼠/大鼠嵌合单克隆抗体的小鼠转铁蛋白受体。