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

识别。

• 批准号: 7735866
• 负责人: ARCHIBALD JAMES MIXSON
• 金额: $ 32.16万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-07-01 至 2013-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7735866
• 关键词: Address; Amino Acid Repetitive Sequences; Amino Acid Sequence; Apoptosis; Asparagine; Atomic Force Microscopy; Binding; Blood Vessels; Cells; Charge; Circular Dichroism; Clinical Research; Complex; Cyclic Peptides; Cytolysis; Development; Exhibits; Fluorescence; Generic Drugs; Goals; Growth; Histidine; In Vitro; Knowledge; Laboratories; Lead; Ligands; Location; Lysine; Malignant Neoplasms; Mediating; Methods; Morphology; Names; Nuclear Magnetic Resonance; Nucleic Acids; Oncogenes; Pattern; Penetration; Peptide Synthesis; Peptides; Phase; Plasmids; Polymers; Positioning Attribute; Property; RNA Interference; S Phase; Series; Serum; Site; Small Interfering RNA; Solid; Solutions; Specificity; System; Techniques; Testing; Therapeutic; Tissues; Toxic effect; Transfection; Upper arm; Xenograft Model; Xenograft procedure; analog; base; biodegradable polymer; cancer therapy; cellular targeting; gene therapy; improved; in vivo; mouse model; nanoparticle; neoplastic cell; neovascular; physical property; prevent; public health relevance; tumor; tumor growth; tumor xenograft

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.

描述（由申请人提供）：虽然RNAi具有通过下调癌基因来治疗癌症的巨大潜力，但这种方法具有反义和基因疗法的经典递送问题：带负电荷的核酸很难运输到其组织和细胞靶点。我们实验室开发的一种有前途的低毒siRNA载体，主要由组氨酸和赖氨酸（HK）组成。 HK 肽可以通过改变其氨基酸序列来将 siRNA 转运到细胞中；具体来说，HK 末端分支中的 -HHHK- 重复模式能够实现 siRNA 的有效体外转运。我们使用 HK 聚合物的主要目标是开发一种安全有效的 siRNA 体内递送方法。添加Peg和肿瘤特异性配体是改善HK等携带者的治疗的基石。与许多载体相比，HK 聚合物可以在特定位点聚乙二醇化，这可以增强稳定性并诱导更大的内体裂解。为了检验最有效的 HK 肽上的特定聚乙二醇化模式将显着增加 siRNA 向肿瘤的递送这一总体假设，计划了以下具体目标。目标 1 将比较几种未修饰的 H3K4b 衍生物作为 Raf-1 siRNA 的系统载体减少肿瘤异种移植物大小的能力。为了将肿瘤抑制性 Raf-1 siRNA 系统性地转运至肿瘤异种移植物，我们选择了三种 HK 聚合物，其主要重复氨基酸序列为 -HHHK-，这是 siRNA 转运的有效序列。通过改变赖氨酸核心和末端分支的氨基酸序列，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，在后续目标中将研究特定位点的聚乙二醇化。目标 2 将描述未修饰和聚乙二醇化 H3K4b（或衍生物）纳米颗粒的特性。将检查几种生物物理技术，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 递送的功效相关联。通过比较在不同位点进行聚乙二醇化的 HK 聚合物，我们的假设是，将鉴定出载体的关键结构特征，这些特征将提高稳定性并增强内体裂解。根据之前目标中获得的知识，Aim 3 将比较一系列 H3K4b（或衍生物）的聚乙二醇化类似物与 Raf-1 siRNA 的复合物减少肿瘤生长的能力。基于结构和机制研究，我们假设 H3K4b siRNA 纳米复合物上的特定聚乙二醇化模式将显着增强 Raf-1 siRNA 向肿瘤异种移植物的递送。除了增加纳米复合物的肿瘤抑制活性外，将 Peg 和配体添加到 HK 上的特定位置应显着增加纳米复合物的特异性，从而降低毒性。预计该提案将增强我们对 Peg 和 HK siRNA 复合物之间相互作用的理解，并且将开发安全有效的 HK 载体用于系统性 siRNA 介导的肿瘤治疗。公共健康相关性：有效递送肿瘤抑制 siRNA 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。我们将测试这些纳米粒子是否能够将 siRNA 传递到小鼠模型的肿瘤中。