Developing nanoplexes for RNAi-expressing plasmids

摘要 组氨酸-赖氨酸(HK)肽可以被定制为运输不同形式的核酸，如质粒或 SiRNA通过改变细胞的分支度和氨基酸序列进入细胞。香港航空公司已经展示了 在临床前模型中治疗癌症、增生性瘢痕和肝纤维化的价值。与之形成对比的是 在体外高支化的H_2K_4b多肽载体上，线性H_2K多肽在体外是一个很差的载体，但 令人惊讶的是，H_2K纳米网络以非常高的转染率靶向肿瘤。因为氢化钾和氢化钾 质粒纳米复合体在肿瘤内的积累和分布不同，显然还有其他 除EPR外，控制H_2K纳米丛肿瘤特异性的机制。因为H_2K有一个重复的 Nrp1受体(Nrp1)介导的纳米复合体的-KHHK-，转胞作用的序列模式 通过肿瘤内皮细胞为增强肿瘤靶向性和蓄积提供了理论基础。虽然 还需要关于线状肽的更多研究，这些早期发现令人鼓舞， 这个应用程序的重点。为了检验香港纳米网络的总体假设， 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确定作为载体的香港多肽在体内的构效关系。 为了将shRNA抑制质粒系统地输送到肿瘤中，我们选择了几种具有 -KHHK-(即，H_2K)的优势重复氨基酸序列，是质粒的有效序列 体内转运。通过将H_2K序列与其他氨基酸序列模式结合，并改变 分支的长度和数量，我们假设H_2K的衍生物将被识别出更多 针对不同乳腺肿瘤模型的有效体内载体。目标2将描述 转染所必需的香港纳米网络的生物物理和生物学特性。几种生物物理 将检查技术，包括形态，结合，Zeta电位，和稳定性的HK质粒 纳米网络使它们的物理性质与向肿瘤输送质粒的效果相关。通过比较 在体外和体内的生物物理方法中分支和长度不同的香港多肽，我们的 假设将确定载体的关键结构特征，这些特征将影响稳定性和分布 并增加基因的表达。基于这些结构和机制研究，目标3将 开发具有优化的HK纳米网络的靶向RNAi治疗小鼠乳腺肿瘤。我们 假设基于线性H_2K多肽和肿瘤中Nrp1水平升高的特定模式 将显著增加肿瘤抑制质粒(shRaf-1和shKras)对原位或转移瘤的输送 肿瘤。此外，在香港纳米网络中加入肿瘤靶向配体应该会增加积聚。 以及该纳米网络对肿瘤的特异性。这些改善了对肿瘤的递送的结果应该转化为 其他病理疾病，如肝纤维化/肝硬变，也有NRP-1水平升高。

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.