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

描述（由申请人提供）：尽管RNAi通过下调癌基因来治疗癌症具有巨大的潜力，但这种方法具有反义和基因治疗的经典递送问题：带负电荷的核酸难以运输到其组织和细胞靶点。我们实验室开发的一种低毒性的siRNA载体主要由组氨酸和赖氨酸（HK）组成。HK肽可以通过改变siRNA的氨基酸序列来将其转运到细胞中；具体来说，- hhhk -在HK末端分支中的重复模式使siRNA在体外有效运输。我们对HK聚合物的主要目标是开发一种安全有效的siRNA体内递送方法。添加Peg和肿瘤特异性配体是改善HK等载体治疗的基石。与许多载体相比，HK聚合物可以在特定位点聚乙二醇化，这可能增加稳定性并诱导更大的内体溶解。为了验证最有效的HK肽上的特定聚乙二醇化模式会显著增加siRNA向肿瘤的递送这一整体假设，我们计划了以下具体目标。目的1将比较几种未经修饰的H3K4b衍生物作为Raf-1 siRNA的全身载体的能力，以减小肿瘤移植的大小。为了将具有肿瘤抑制作用的Raf-1 siRNA系统地转运到肿瘤异种移植物中，我们选择了三种HK聚合物，它们的主要重复氨基酸序列为- hhhk -，这是siRNA运输的有效序列。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的体内载体的功效会有所不同。该目标将建立最有效的未修饰的siRNA HK载体，其特定位点的聚乙二醇化将在后续目标中进行研究。目的2将描述未修饰和聚乙二醇化的H3K4b（或衍生物）纳米颗粒的性质。将研究几种生物物理技术，包括聚乙二醇化HK:siRNA纳米复合物的形态和稳定性，以将其物理特性与siRNA递送效果联系起来。通过比较在不同位点聚乙二醇化的HK聚合物，我们的假设是，载体的关键结构特征将被确定，这将增加稳定性和增加内体溶解。根据之前的目标所获得的知识，Aim 3将比较一系列与Raf-1 siRNA复合物的聚乙二醇化H3K4b（或衍生物）的类似物对肿瘤生长的抑制能力。基于结构和机制研究，我们假设H3K4b siRNA纳米复合物上的特异性聚乙二醇化模式将显著增加Raf-1 siRNA向肿瘤异种移植物的传递。除了增加纳米复合物的肿瘤抑制活性外，在HK上的特定位置添加Peg和配体可以显著提高纳米复合物的特异性，从而降低毒性。我们期望这项研究能够加深我们对Peg和HK siRNA多聚体之间相互作用的理解，并开发出一种安全有效的HK载体，用于siRNA介导的系统性肿瘤治疗。公共卫生相关性：肿瘤抑制siRNA的有效递送对于这种核酸治疗方法的成功至关重要。为了实现这一目标，我们将利用由修饰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传递到肿瘤中。

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.