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

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• 批准号: 8071231
• 负责人: ARCHIBALD JAMES MIXSON
• 金额: $ 29.39万
• 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/8071231
• 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; Health; 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; Xenograft Model; Xenograft procedure; analog; arm; base; biodegradable polymer; cancer therapy; cellular targeting; gene therapy; improved; in vivo; mouse model; nanoparticle; neoplastic cell; neovascular; physical property; prevent; 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）：尽管RNAi具有通过下调癌基因来治疗癌症的显著潜力，但该方法具有反义和基因疗法的经典递送问题：带负电荷的核酸难以转运至其组织和细胞靶标。本实验室开发的一种有前途的低毒性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定制以将siRNA转运到细胞中;具体地，HK末端分支中的-HHHK-的重复模式使得能够在体外有效转运siRNA。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用于改善载体如HK的治疗的基石。与许多载体相反，HK聚合物可以在特定位点聚乙二醇化，这可以增加稳定性并诱导更大的内体裂解。为了测试最有效的HK肽上的特定聚乙二醇化模式将显著增加siRNA向肿瘤的递送的总体假设，计划了以下具体目标。目的1将比较几种未修饰的H3 K4 b衍生物作为Raf-1 siRNA的全身载体以减小肿瘤异种移植物的大小的能力。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，我们选择了三种具有主要重复氨基酸序列-HHHK-（siRNA转运的有效序列）的HK聚合物。通过改变末端分支的赖氨酸核心和氨基酸序列，我们假设H3 K4 b及其衍生物作为靶向Raf-1的siRNA的体内载体的功效不同。该目的将建立最有效的siRNA的未修饰HK载体，在随后的目的中将研究其在特定位点的聚乙二醇化。目的2将描述未修饰和聚乙二醇化的H3 K4 b（或衍生物）纳米颗粒的性质。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，以将其物理性质与siRNA递送的功效相关联。通过比较HK聚合物，已在不同的网站聚乙二醇化，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强内体裂解。根据先前目标中获得的知识，目标3将比较与Raf-1 siRNA复合的一系列H3 K4 b聚乙二醇化类似物（或衍生物）减少肿瘤生长的能力。基于结构和机制研究，我们假设H3 K4 b siRNA纳米复合物上的特定聚乙二醇化模式将显著增加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之外，将PEG和配体添加到HK上的特定位置应显著增加纳米复合物的特异性，并因此降低毒性。预期该提议将增强我们对Peg和HK siRNA聚合复合物之间相互作用的理解，并且将开发安全有效的HK载体用于针对肿瘤的系统性siRNA介导的治疗。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，我们将利用由修饰的肽组成的纳米颗粒，这些肽与这些肿瘤抑制siRNA分子相互作用。我们将测试这些纳米颗粒是否能够在小鼠模型中将siRNA递送到肿瘤。