An investigation of how filopodia can be exploited by peptide carriers for enhanced uptake of RNAi cargo for the treatment of HPV+ oral cancers.

研究肽载体如何利用丝状伪足来增强 RNAi 货物的摄取，从而治疗 HPV 口腔癌。

• 批准号: 10678166
• 负责人: Charles Everett Holjencin
• 金额: $ 5.6万
• 依托单位: MEDICAL UNIVERSITY OF SOUTH CAROLINA
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-07-01 至 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10678166
• 关键词: Address; Amino Acids; Apoptosis; Bacteria; Binding; Biological Assay; Biology; Cancer Biology; Cancer Etiology; Cancerous; Cell Line; Cell Nucleus; Cell Proliferation; Cell Survival; Cells; Cervical; Characteristics; Clinical; Co-Immunoprecipitations; Complex; Data; Development; Disease; Disseminated Malignant Neoplasm; Drug Delivery Systems; Endocytosis; Excision; Fellowship; Filopodia; Fluorescence Microscopy; Fostering; Future; Gene Silencing; Generations; Genome; Head and Neck Squamous Cell Carcinoma; Heparan Sulfate Proteoglycan; Human; Human Papillomavirus; Impairment; In Vitro; Incidence; Induction of Apoptosis; Investigation; Knock-out; Knowledge; Label; Laboratories; Lipofectamine; Malignant Neoplasms; Mass Spectrum Analysis; Mediating; Messenger RNA; Methods; MicroRNAs; Mitochondrial DNA; Modification; Molecular; Morbidity - disease rate; Nucleic Acids; Oncogenes; Oncogenic; Oncolytic; Oncoproteins; Operative Surgical Procedures; Oral health; Outcome; Pattern; Peptides; Production; Property; Quality of life; RNA Interference; RNA Interference Therapy; RNA-targeting therapy; Radiation therapy; Receptor Cell; Reporting; Research; Retinoblastoma Protein; Role; Scientist; Series; Small Interfering RNA; Specificity; Stains; System; TP53 gene; Technology; Testing; Therapeutic; Toxic effect; Training; Treatment Efficacy; Tumor Suppressor Proteins; University of Pittsburgh Cancer Institute; Variant; Viral; Viral Oncogene; Virus; Work; cancer cell; cancer therapy; chemical synthesis; chemotherapy; delivery vehicle; design; exosome; improved; inhibitor; insight; knock-down; malignant mouth neoplasm; microscopic imaging; nanocarrier; novel; nucleic acid delivery; nucleic acid-based therapeutics; oral HPV-positive head and neck cancers; overexpression; receptor; retrograde transport; standard of care; therapeutic RNA; therapeutic gene; tool; uptake

Abstract The incidence of human papillomavirus-positive (HPV+) head and neck squamous cell carcinoma (HNSCC) is rising and while treatment options are available, they remain limited primarily to surgical excision, radiation therapy, and chemotherapy which often result in long term morbidity. RNA interference (RNAi) appears to be a promising therapeutic tool for the treatment of many diseases, including HPV+ related cancers, through targeting viral oncogenes, E6 and E7, with small interfering RNAs (siRNA). The expression of E6 and E7 viral oncoproteins inhibits the p53 and pRb tumor suppressors, respectively, resulting in uncontrolled cellular proliferation. Knockdown of E6 and E7 in vitro has been shown to induce apoptosis in both HPV+ cervical cell carcinoma and HPV+ head and neck squamous cell carcinoma cell lines, indicating that E6 and E7 are viable therapeutic targets for RNAi therapy. However, the therapeutic application of RNAi requires a delivery platform that can overcome numerous challenges typically associated with this form of therapy. Peptide carriers show great promise as siRNA carriers based on the diversity of their physiochemical properties and functions. Recently, we demonstrated that a novel peptide carrier we designed, termed RD3AD, enhanced the intracellular delivery and availability of therapeutic, chemically synthesized siRNAs in oral cancer cells, in vitro, to levels that were ~4x that of Lipofectamine 3000, which some would consider to be the “gold standard” positive control for in vitro delivery of nucleic acids. It therefore stands to reason that RD3AD could be an effective delivery vehicle for siRNAs designed to target the E6 and E7 viral oncogenes (siE6 and siE7) in HNSCC cells. RD3AD-siRNA complexes were also observed to localize to cellular projections identified as filopodia. These observations were indicative of patterns previously reported for cellular uptake of viruses, bacteria, activated receptors, lipo/polyplexes, and exosomes, which utilized filopodia to undergo retrograde transport toward the cell, resulting in significant enhancements of uptake. It is therefore possible, given the observations with RD3AD, that this peptide is exploiting similar mechanisms. Thus, our overarching hypothesis is that RD3AD-siE6/7 (siE6 or siE7) complexes enter HPV+ oral cancer cells after binding to cellular filopodia and silence viral oncogenic E6 or E7 mRNA, in vitro. We will test our hypothesis through the following specific aims. Aim 1: Identify the RD3AD receptor on filopodia and exploit this receptor for cell specific targeting. Aim 2: Determine the mechanism(s) of RD3AD-siRNA complex uptake. In aggregate, this proposal will establish a molecular therapeutic option for HPV+ HNSCCs, while describing a novel mechanism of cell-specific delivery and uptake by a peptide nanocarrier. The results from this study will contribute significantly to a solution for a critical unmet clinical need and will be particularly impactful on the field of drug delivery while providing new insights into filopodia biology. This fellowship will also provide training in drug delivery and cancer biology and will foster the development of the trainee into a unique oral health academic clinician/scientist.

抽象的 人乳头瘤病毒阳性（HPV+）头颈鳞状细胞癌（HNSCC）的发病率是 上升，虽然有可用的治疗选择，但它们仍然主要限于手术切除、放射治疗 治疗和化疗通常会导致长期发病。 通过靶向治疗有前途的疾病，包括 HPV+ 相关癌症的治疗工具 病毒癌基因 E6 和 E7，以及小干扰 RNA (siRNA) E6 和 E7 病毒癌蛋白的表达。 分别抑制 p53 和 pRb 肿瘤抑制因子，导致细胞增殖失控。 体外敲低 E6 和 E7 已被证明可诱导 HPV+ 宫颈细胞癌和 HPV+头颈鳞状细胞癌细胞系，表明E6和E7是可行的治疗靶点 然而，RNAi 的治疗应用需要一个能够克服这些问题的递送平台。 与这种形式的治疗相关的许多挑战显示出巨大的前景。 siRNA载体基于其理化性质和功能的多样性。 证明我们设计的一种新型肽载体，称为 RD3AD，增强了细胞内递送并 口腔癌细胞中治疗性化学合成 siRNA 的体外可用性达到约 4 倍的水平 Lipofectamine 3000，有些人认为它是体外阳性对照的“金标准” 因此，RD3AD 可能是一种有效的核酸递送载体。 设计用于靶向 HNSCC 细胞中的 E6 和 E7 病毒癌基因（siE6 和 siE7）的 siRNA。 还观察到复合物定位于被识别为丝状伪足的细胞投影。 先前报道的细胞摄取病毒、细菌、激活受体的模式指标， 脂质/多聚复合物和外泌体，利用丝状伪足逆行运输至细胞，产生 因此，根据 RD3AD 的观察结果，这可能会显着增强吸收。 因此，我们的总体假设是 RD3AD-siE6/7（siE6 或 siE7) 复合物在与细胞丝状伪足结合后进入 HPV+ 口腔癌细胞并沉默病毒 体外致癌 E6 或 E7 mRNA 我们将通过以下具体目标来检验我们的假设： 识别丝状伪足上的 RD3AD 受体并利用该受体进行细胞特异性靶向目标 2： 确定 RD3AD-siRNA 复合物摄取的机制 总的来说，该提案将建立一个机制。 HPV+ HNSCC 的分子治疗选择，同时描述了细胞特异性递送的新机制 和肽纳米载体的摄取本研究的结果将为解决方案做出重大贡献。 未满足的关键临床需求将对药物输送领域产生特别影响，同时提供新的 该奖学金还将提供药物输送和癌症生物学方面的培训。 将促进学员发展成为独特的口腔健康学术临床医生/科学家。