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）是一项相对较新的技术，可以用短的干扰RNA（siRNA）敲除病理基因的表达。新RNAi药物开发的限制因素是分娩。该领域的目的是“静脉注射RNAi”，它是将siRNA注入外周静脉的能力，并发现siRNA进入远端靶器官以使靶mRNA分子保持沉默。该靶mRNA分子隐藏在体内多个膜屏障后面，该膜屏障与mRNA分开。在目前的工作中，siRNA与产生Avidin的融合蛋白和受体特异性单克隆抗体（MAB）并联。目前的工作将设计，表达和验证由小鼠转铁蛋白受体（TFR）组成的新型融合蛋白。 TFRMAB充当分子特洛伊木马，可以将siRNA运送到表达TFR的任何靶细胞膜上。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在体内RNAi中为RNAi提供。编码轻链（LC）和重链（HC）/AV融合蛋白的真核生物表达质粒将在遗传上进行，在宿主细胞中表达，TFRMAB/AV融合蛋白将通过Protein g亲和色谱纯度纯化，然后通过对小鼠Tfr和生物素的结合融合蛋白蛋白质功能进行验证。单个串联矢量将被设计用于对宿主细胞的永久转染。在选择，扩增和稀释克隆之后，将传播转染的宿主细胞，TFRMAB/AV融合蛋白将通过亲和色谱纯化。在此大规模生产的TFRMAB/AV融合蛋白将通过一系列的生化测定验证，以验证分子的结构和功能完整性，包括验证siRNA作用和RNA干扰。公共卫生相关性RNA干扰（RNAI）是一项新技术，可以用短的干扰RNA（SIRNA）敲除病理基因的表达。新RNAi药物开发的限制因素是分娩。该领域的目的是“静脉注射RNAi”，它是将siRNA注入外周静脉的能力，并发现siRNA进入远端靶器官以使靶mRNA分子保持沉默。目前的工作将设计，表达和验证由小鼠转铁蛋白受体组成的新型融合蛋白，该融合蛋白由Avidin和小鼠/大鼠嵌合MAB组成。