Ident. of Structural Features of HK Polyplexes for Imprv. siRNA Deliv. to Tumors

描述(申请人提供)：尽管RNAi通过下调癌基因来治疗癌症具有巨大的潜力，但这种方法分享了反义和基因治疗的经典传递问题：带负电荷的核酸很难运输到它们的组织和细胞靶标。我们实验室开发的一种有希望的低毒siRNA载体主要由组氨酸和赖氨酸(HK)组成。HK多肽可以通过改变其氨基酸序列来量身定制将siRNA运送到细胞内；具体地说，HK末端分支中的-HHHK-的重复模式使siRNA能够在体外有效地转运。我们使用HK聚合物的主要目标是开发一种安全有效的体内siRNA递送方法。加入PEG和肿瘤特异性配体是改善如HK等携带者的治疗的基石。与许多载体不同的是，香港聚合物可以在特定的位置进行聚乙二醇化，这可能会增加稳定性并导致更大的内体溶解。为了检验这一假设，即最有效的HK多肽上的特定聚乙二醇化模式将显著增加siRNA向肿瘤的输送，计划了以下具体目标。目的1比较几种未修饰的H3K4b衍生物作为Raf-1 siRNA系统载体缩小肿瘤移植瘤体积的能力。为了系统地将抑制肿瘤的Raf-1 siRNA运送到肿瘤移植瘤中，我们选择了三种HK聚合物，它们的主要重复氨基酸序列是-HHHK-，这是一个有效的siRNA转运序列。通过改变末端分支的赖氨酸核心和氨基酸序列，我们假设H3K4b及其衍生物作为靶向Raf-1的siRNA的体内载体的有效性将有所不同。这一目标将建立最有效的未经修饰的siRNA的HK载体，在随后的AIMS中将研究特定位点的聚乙二醇化。目标2将描述未经修饰和聚乙二醇化的H3K4b(或其衍生物)纳米粒子的性质。将考察几种生物物理技术，包括聚乙二醇化的HK：siRNA纳米网络的形态和稳定性，以将其物理性质与siRNA传递的有效性相关联。通过比较已在不同位置聚乙二醇化的香港聚合物，我们的假设是，载体的关键结构特征将被识别，这将增加稳定性和增强内体裂解。根据以前AIMS获得的知识，AIM 3将比较一系列聚乙二醇化的H3K4b(或衍生物)类似物与Raf-1 siRNA的复合体，以了解它们减少肿瘤生长的能力。基于结构和机制研究，我们假设H3K4b siRNA纳米复合体上特定的聚乙二醇化模式将显著增加Raf-1 siRNA对肿瘤移植瘤的输送。除了提高纳米网络的肿瘤抑制活性外，在HK的特定位置添加聚乙二醇酯和配体应该会显著提高纳米网络的特异性，从而降低毒性。预计这项建议将加深我们对Peg和HK siRNA多聚体之间相互作用的了解，并将开发出安全有效的HK载体，用于系统的siRNA介导的肿瘤治疗。公共卫生相关性：有效传递抑制肿瘤的siRNA是这种核酸治疗方法成功的关键。为了实现这一目标，我们将利用由修饰多肽组成的纳米颗粒与这些肿瘤抑制siRNA分子相互作用。我们将在小鼠模型中测试这些纳米颗粒是否能够将siRNA运送到肿瘤中。

DESCRIPTION (provided by applicant): Although RNAi has significant potential to treat cancer by down-regulating oncogenes, this approach shares the classic delivery problem of antisense and gene therapies: negatively charged nucleic acids are poorly transported to their tissue and cellular targets. A promising carrier of siRNA with low toxicity that our lab has developed is composed primarily of histidines and lysines (HK). HK peptides can be tailored to transport siRNA into cells by altering its amino acid sequence; specifically, the repeating pattern of -HHHK- in the terminal branches of HK enables effective siRNA transport in vitro. Our primary goal with HK polymers is to develop a safe and effective delivery method for siRNA in vivo. Addition of Peg and tumor-specific ligands is a cornerstone of therapy for improvement of carriers such as HK. In contrast to many carriers, HK polymers can be pegylated at specific sites, which may augment stability and induce greater endosomal lysis. To test the overall hypothesis that specific pegylation patterns on the most effective HK peptide will markedly increase delivery of siRNA to tumors, the following specific aims are planned. Aim 1 will compare the ability of several unmodified H3K4b derivatives as systemic carriers of Raf-1 siRNA to reduce the size of tumor xenografts. To transport the tumor-inhibitory Raf-1 siRNA systemically to tumor xenografts, we have selected three HK polymers with a predominant repeating amino acid sequence of -HHHK-, an effective sequence for siRNA transport. By varying the lysine core and amino acid sequence of the terminal branches, we hypothesize that H3K4b and its derivatives will differ in their efficacy as in vivo carriers of siRNA targeting Raf-1. This Aim will establish the most effective unmodified HK carrier of siRNA to which pegylation at specific sites will be studied in subsequent aims. Aim 2 will delineate the properties of unmodified and pegylated H3K4b (or derivative) nanoparticles. Several biophysical techniques will be examined, including morphology and stability of pegylated HK:siRNA nanoplexes to correlate their physical properties with the efficacy of siRNA delivery. By comparing HK polymers that have been pegylated at different sites, our hypothesis is that key structural features of the carrier will be identified that will increase stability and augment endosomal lysis. From the knowledge gained in previous aims, Aim 3 will compare a series of pegylated analogs of H3K4b (or derivative) in complex with Raf-1 siRNA for their ability to reduce tumor growth. Based on the structural and mechanistic studies, we hypothesize that specific pegylation patterns on the H3K4b siRNA nanoplex will markedly augment delivery of Raf-1 siRNA to tumor xenografts. Besides increasing the tumor-inhibitory activity of the nanoplex, addition of Peg and a ligand to specific locations on HK should markedly increase specificity of the nanoplex, and consequently reduce toxicity. It is anticipated that this proposal will enhance our understanding of interactions between Peg and HK siRNA polyplexes and that a safe and effective HK carrier will be developed for systemic siRNA-mediated therapy against tumors. PUBLIC HEALTH RELEVANCE: Effective delivery of tumor-inhibitory siRNA is critical for this method of nucleic acid therapeutics to be successful. To achieve this goal, we will utilize nanoparticles composed of modified peptides that interact with these tumor inhibitory siRNA molecules. We will test whether these nanoparticles are able to deliver siRNA to tumors in a mouse model.