Targeted Delivery of siRNA for Intravenous RNAi

描述（由申请人提供）：RNA干扰（RNAi）是一种相对较新的技术，可以用短干扰RNA （sirna）敲低病理基因的表达。开发新的RNAi药物的限制因素是递送。该领域的目标是“静脉内RNAi”，即将siRNA注入外周静脉，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分子隐藏在体内的多个膜屏障后面，将血液室与mRNA分开。在目前的工作中，siRNA在产生亲和素和受体特异性单克隆抗体（MAb）融合蛋白的同时被单生物素化。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/大鼠嵌合单抗组成，可与小鼠转铁蛋白受体（TfR）结合。TfRMAb作为一个分子特洛伊木马，运送siRNA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，TfRMAb/AV融合蛋白的可用性将使脑和其他器官的体内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基因工程改造，在宿主细胞中表达，TfRMAb/AV融合蛋白将通过蛋白G亲和层析纯化，随后用小鼠TfR和生物素结合实验验证融合蛋白的双功能性。一个单一的串联载体将被设计为永久转染宿主细胞。经过筛选、扩增和稀释克隆，将转染的宿主细胞进行繁殖，并通过亲和层析纯化TfRMAb/AV融合蛋白。大规模生产的TfRMAb/AV融合蛋白将通过一系列生化试验进行验证，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RNA干扰（PUBLIC HEALTH RELEVANCE RNA interference, RNAi）是一种利用短干扰RNA （sirna）敲低病理基因表达的新技术。开发新的RNAi药物的限制因素是递送。该领域的目标是“静脉内RNAi”，即将siRNA注入外周静脉，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/大鼠嵌合单抗组成，可与小鼠转铁蛋白受体结合。

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.