Improved in vivo delivery of siRNA

改善 siRNA 的体内递送

• 批准号: 7404770
• 负责人: LANCE P FORD
• 金额: $ 17.24万
• 依托单位: BIOO SCIENTIFIC CORPORATION
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-06-01 至 2010-11-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7404770
• 关键词: Animal Disease Models; Animals; Antibodies; Antigen Targeting; Binding; Biotechnology; Cathepsins; Cell Culture System; Cell physiology; Cell surface; Cells; Clinical Research; Complex; Condition; Crosslinker; Cultured Cells; Development; Disease; Disulfides; Double-Stranded RNA; Down-Regulation; Drug Delivery Systems; Drug Metabolic Detoxication; Eukaryota; Eukaryotic Cell; Fluorescence; Fluorescence Microscopy; Fluorescence Spectrometry; Future; Gene Expression; Gene Silencing; Gene Targeting; Genes; Genetic; Goals; Human; Human Cell Line; Hydrazones; Immunoconjugates; Injection of therapeutic agent; Life; Malignant Neoplasms; Mammalian Cell; Mammals; Measurement; Measures; Methods; Mus; Nucleotides; Nude Mice; Numbers; Peptides; Pharmaceutical Preparations; Pharmacologic Substance; Phase; Plasma; Protein Overexpression; Proteins; RNA Interference; RNA Interference Pathway; RNA-Binding Proteins; Reagent; Recombinants; Research; Serum; Small Interfering RNA; Structure; Surface Antigens; Techniques; Testing; Therapeutic; Therapeutic Agents; Tissues; Validation; Work; Xenograft procedure; base; cancer cell; cell type; crosslink; design; drug metabolism; fighting; gene function; health organization; improved; in vivo; interest; loss of function; neoplastic cell; novel; pre-clinical; research study; success; therapeutic gene; therapeutic target; tool

DESCRIPTION (provided by applicant): The overall goal of this proposal is to develop novel reagents to efficiently deliver functional siRNA into animals for gene silencing experiments. Pharmaceutical and biotechnology companies are actively testing siRNAs as therapeutic agents and to validate drug targets. Unfortunately siRNA delivery in animals is usually not efficient and it has proven difficult to deliver siRNA to some therapeutically important cell types and tissues. The commercial success of in vivo applications of siRNA hinges on the development of new delivery methods. The proposed research will result in the development of improved antibody-based conjugates to target siRNAs to cells overexpressing the appropriate cell surface antigen. Two different types of immunoconjugates will be developed. The first immunoconjugate type is produced by chemically crosslinking large recombinant RNA binding proteins to antibodies. The siRNA noncovalently associates with the targeting antibody via the RNA binding protein. The second type is produced by directly attaching functional modified siRNAs to antibodies using heterobifunctional crosslinking agents containing internal cell labile structures to promote release of the siRNA after internalization. Specific aims for Phase I: 1. Develop novel immunoconjugates to significantly increase the amount of siRNA loaded per antibody molecule in the antibody/siRNA complex. 2: Covalently conjugate siRNA to antibodies using cleavable linkers to produce immunoconjugates which are stable in plasma but efficiently release functional siRNA in cells. 3. Use the novel immunoconjugates produced in Aims 1 and 2 to specifically deliver functional siRNA into cultured cells overexpressing the appropriate cell surface antigen and into tumor cells in mouse xenografts. The siRNA binding capacity and stability of each of immunoconjugates will be directly compared using fluorescence measurements. The ability of the immunoconjugates to deliver functional siRNA into targeted cells to silence specific genes will also be tested using qRT-PCR. In Phase II, we will expand and adapt the technique to work with chemically modified siRNAs designed for in vivo applications. Narrative: The versatility and potency of siRNA in downregulating target gene expression has made it a preferred tool to study gene function in mammalian cell culture systems. As a result of this success, the therapeutic potential of siRNA is being actively investigated in animal disease models and in humans. Unfortunately, the current methods to deliver siRNA in animals are very inefficient and difficult to control. The promise of therapeutic siRNA will not be realized until more efficient modes of delivery are developed. Therefore, we outline an efficient research plan to develop improved methods of in vivo siRNA delivery. Our strategy involves the use of antibody-based delivery agents to target and deliver siRNA into specific cell types and if successful will greatly advance the delivery of siRNA in vivo for understanding and treating cancer.

描述（由申请人提供）：本提案的总体目标是开发新型试剂，以有效地将功能性siRNA传递到动物体内进行基因沉默实验。制药和生物技术公司正在积极地测试sirna作为治疗药物和验证药物靶点。不幸的是，siRNA在动物体内的传递通常效率不高，而且已经证明很难将siRNA传递到一些治疗上重要的细胞类型和组织中。siRNA在体内应用的商业成功取决于新的递送方法的发展。拟议的研究将导致改进的基于抗体的偶联物的发展，将sirna靶向过度表达适当细胞表面抗原的细胞。将开发两种不同类型的免疫偶联物。第一种免疫偶联物是通过化学交联大型重组RNA结合蛋白与抗体产生的。siRNA通过RNA结合蛋白与靶向抗体非共价结合。第二类是使用含有细胞内部不稳定结构的异双功能交联剂将功能性修饰的siRNA直接附着在抗体上，以促进内化后siRNA的释放。第一阶段的具体目标：开发新的免疫偶联物，显著增加抗体/siRNA复合物中每个抗体分子的siRNA负载量。2：利用可切割的连接物将siRNA与抗体共价结合，产生在血浆中稳定但在细胞中有效释放功能性siRNA的免疫偶联物。3. 使用Aims 1和Aims 2中产生的新型免疫偶联物特异性地将功能性siRNA传递到过表达适当细胞表面抗原的培养细胞和小鼠异种移植物的肿瘤细胞中。每个免疫偶联物的siRNA结合能力和稳定性将使用荧光测量直接比较。免疫偶联物将功能性siRNA传递到靶细胞以沉默特定基因的能力也将使用qRT-PCR进行测试。在第二阶段，我们将扩展和调整该技术，使其与用于体内应用的化学修饰sirna一起工作。