Developing nanoplexes for RNAi-expressing plasmids

开发 RNAi 表达质粒的纳米复合物

• 批准号: 10017994
• 负责人: ARCHIBALD JAMES MIXSON
• 金额: $ 33.9万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-15 至 2024-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10017994
• 关键词: Abbreviations; Affect; Amino Acid Repetitive Sequences; Amino Acid Sequence; Binding; Biodistribution; Biological; Biological Assay; Biophysics; Branched-Chain Amino Acids; Breast Cancer Model; Breast Cancer Treatment; Buffers; Calorimetry; Cells; Charge; Cirrhosis; Complex; DNA; Development; Disease; Disulfides; Endosomes; Endothelium; Equilibrium; Exhibits; Gel; Gene Expression; Goals; Histidine; Hypertrophic Cicatrix; In Vitro; Lead; Length; Ligands; Liver Fibrosis; Luciferases; Lysine; Malignant Neoplasms; Mammary Neoplasms; Mediating; Modeling; Morphology; Mus; Neoplasm Metastasis; Neuropilin-1; Neuropilins; Nucleic Acids; Oncogenes; Pathologic; Pathology; Pathway interactions; Pattern; Peptides; Periodicity; Plasmids; Polymers; Pre-Clinical Model; Property; RNA Interference; RNA Interference Therapy; Research Personnel; Safety; Small Interfering RNA; Specificity; Structure; Structure-Activity Relationship; Surface; System; Testing; Time; Toxic effect; Transfection; Translating; Transmission Electron Microscopy; Tumor Tissue; Variant; base; biophysical techniques; gene therapy; improved; in vivo; malignant breast neoplasm; mouse model; nanoparticle; nucleic acid delivery; nucleic acid-based therapeutics; physical property; plasmid DNA; receptor; small hairpin RNA; transcytosis; tumor; tumor specificity; zeta potential

Abstract Histidine-lysine (HK) peptides can be tailored to transport different forms of nucleic acids such as plasmids or siRNA into cells by altering their degree of branching and amino acid sequence. The HK carrier has shown value in treating cancer, hypertrophic scars, and liver fibrosis in pre-clinical models. In contrast to the effective in vitro highly-branched H2K4b peptide carrier of plasmids, the linear H2K peptide is a poor carrier in vitro, yet surprisingly, H2K nanoplexes target tumors with very high transfection efficiency. Because H2K4b and H2K plasmid nanoplexes differ in their accumulation and distribution within the tumor, there are clearly other mechanisms besides EPR that govern the tumor specificity of H2K nanoplexes. Because H2K has a repeating sequence pattern of –KHHK-, transcytosis of the nanoplex mediated by the neuropilin-1 receptor (NRP1) through the tumor endothelium provides a rationale for enhanced tumor targeting and accumulation. Although additional development concerning the linear peptide is required, these early findings are encouraging and are the focus of this application. To test the overall hypothesis that characterization of HK nanoplexes containing a shRNA plasmid targeting Kras/Raf-1 will facilitate improved antitumor activity, the following aims are planned. Aim 1 will determine the structure-activity relationship of HK peptides as carriers of plasmids in vivo. To transport the shRNA-inhibitory plasmids systemically to tumors, we have selected several HK polymers with a predominant repeating amino acid sequence of –KHHK- (i.e., H2K), an effective sequence for plasmid transport in vivo. By combining the H2K sequence with other amino acid sequence patterns, and varying the length and number of branches, we hypothesize that derivatives of H2K will be identified that are more effective in vivo carriers of plasmids targeting different breast tumor models. Aim 2 will delineate the biophysical and biological properties of HK nanoplexes essential for transfection. Several biophysical techniques will be examined, including morphology, binding, zeta potential, and stability of HK plasmid nanoplexes to correlate their physical properties with the efficacy of plasmid delivery to tumors. By comparing HK peptides that differ in their branching and length with in vitro and in vivo biophysical methods, our hypothesis is that key structural features of the carrier will be identified that will affect stability and distribution within the tumor and increase gene expression. Based on these structural and mechanistic studies, Aim 3 will develop a targeted RNAi therapeutic with an optimized HK nanoplex toward breast tumors in mice. We hypothesize that the specific patterns based on the linear H2K peptide and elevated levels of NRP1 in tumors will markedly augment delivery of tumor-inhibitory plasmids (shRaf-1 and shKras) to orthotopic or metastatic tumors. Moreover, addition of a tumor-targeting ligand to the HK nanoplex should increase the accumulation and specificity of the nanoplex to the tumor. These results of improved delivery to the tumor should translate to other pathological disease such as liver fibrosis/cirrhosis, which also have elevated levels of NRP-1.

摘要 组氨酸-赖氨酸（HK）肽可以被定制以转运不同形式的核酸，例如质粒或质粒。 siRNA通过改变其分支程度和氨基酸序列进入细胞。香港航空公司已经表明， 在临床前模型中治疗癌症、增生性瘢痕和肝纤维化的价值。与有效的 在质粒的体外高度支化的H2 K4 b肽载体中，线性H2K肽是体外差的载体，但 令人惊讶地，H2K纳米复合物以非常高的转染效率靶向肿瘤。因为H2 K4 b和H2K 质粒纳米复合物在肿瘤内的积累和分布不同，但显然存在其他差异。 除了EPR之外，还存在控制H2K纳米复合物的肿瘤特异性的机制。因为H2K有一个重复 -KHHK-的序列模式，由神经纤毛蛋白-1受体（NRP 1）介导的纳米复合物的转胞吞作用 通过肿瘤内皮细胞提供了增强肿瘤靶向和积聚的基本原理。虽然 关于线性肽的进一步发展是需要的，这些早期的发现是令人鼓舞的， 这个应用程序的重点。为了检验总体假设，表征含有 靶向Kras/Raf-1的shRNA质粒将有助于提高抗肿瘤活性，计划实现以下目标。 目的1研究HK肽作为质粒载体的体内构效关系。 为了将shRNA抑制性质粒系统地转运至肿瘤，我们选择了几种HK聚合物， -KHHK-的主要重复氨基酸序列（即，H2K），质粒的有效序列 体内运输通过将H2K序列与其他氨基酸序列模式组合，并改变H2K序列， 分支的长度和数量，我们假设H2K的衍生物将被鉴定为更多 靶向不同乳腺肿瘤模型的质粒的有效体内载体。目标2将描述 转染所必需的HK纳米复合物的生物物理和生物学性质。几种生物物理 将检查技术，包括HK质粒的形态、结合、zeta电位和稳定性 纳米复合物，以将它们的物理性质与质粒递送至肿瘤的功效相关联。通过比较 HK肽在其分支和长度上与体外和体内生物物理方法不同， 假设是将鉴定影响稳定性和分布的载体的关键结构特征 并增加基因表达。基于这些结构和机制研究，目标3将 开发具有针对小鼠乳腺肿瘤的优化HK nanoplex的靶向RNAi治疗剂。我们 假设基于线性H2K肽和肿瘤中NRP 1水平升高的特定模式 将显著增加肿瘤抑制质粒（shRaf-1和shKras）向原位或转移性肿瘤的递送， 肿瘤的此外，向HK纳米复合物中添加肿瘤靶向配体应增加肿瘤靶向配体的积累。 和对肿瘤的特异性。这些改善肿瘤递送的结果应该转化为 其他病理性疾病如肝纤维化/肝硬化，其也具有升高的NRP-1水平。