Targeted Delivery of siRNA for Intravenous RNAi

描述(申请人提供)：RNA干扰(RNAi)是一种相对较新的技术，它能够用短干扰RNAs(SiRNAs)来抑制病理基因的表达。RNAi新药开发的限制因素是递送。该领域的目标是“静脉RNAi”，即将siRNA注入外周静脉的能力，并发现siRNA进入远处的目标器官，使目标mRNA分子沉默。这种靶向的信使核糖核酸分子隐藏在体内的多个膜屏障后面，这些屏障将血液隔膜与信使核糖核酸隔开。在目前的工作中，siRNA是在产生亲和素和受体特异性单抗(MAb)的融合蛋白的同时进行单生物素化的。本工作将设计、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/大鼠嵌合单抗与小鼠转铁蛋白受体(TFR)组成。TfRMAb充当分子特洛伊木马，将siRNA运送到任何表达TfR的靶细胞膜上。由于TFR在血脑屏障(BBB)上高度表达，TfRMAb/AV融合蛋白的可获得性将使大脑以及其他器官能够在体内进行RNAi。编码轻链(LC)和重链(HC)/AV融合蛋白的真核表达载体将在宿主细胞中表达，TfRMAb/AV融合蛋白将通过Protein G亲和层析纯化，随后将验证融合蛋白与小鼠TFR和生物素结合试验的双功能。一个单一的串联载体将被设计成永久地转染宿主细胞。经筛选、扩增、稀释性克隆后，对转基因宿主细胞进行增殖，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分析来验证分子的结构和功能完整性，包括验证siRNA作用和RNA干扰。与公共卫生相关的RNA干扰(RNAi)是一种利用短干扰RNA(SiRNAs)敲除病理基因表达的新技术。RNAi新药开发的限制因素是递送。该领域的目标是“静脉RNAi”，即将siRNA注入外周静脉的能力，并发现siRNA进入远处的目标器官，使目标mRNA分子沉默。本工作将设计、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/大鼠嵌合单抗与小鼠转铁蛋白受体组成。

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.