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

摘要 人乳头瘤病毒阳性(HPV+)头颈部鳞状细胞癌(HNSCC)发病率为 虽然有治疗选择，但仍主要局限于手术切除、放射治疗。 治疗和化疗往往会导致长期的发病率。RNA干扰(RNAi)似乎是一种 通过靶向治疗包括HPV+相关癌症在内的许多疾病的有前景的治疗工具 病毒癌基因E6和E7，带有小干扰RNA(SiRNA)。E6和E7病毒癌蛋白的表达 分别抑制P53和pRb肿瘤抑制基因，导致细胞不受控制的增殖。 体外敲除E6和E7基因可诱导HPV阳性宫颈癌细胞和 HPV+头颈部鳞状细胞癌细胞系，提示E6和E7是可行的治疗靶点 接受RNAi治疗。然而，RNAi的治疗应用需要一个能够克服 与这种治疗形式相关的许多挑战通常都存在。多肽载体显示出巨大的前景 SiRNA载体基于其物理化学性质和功能的多样性。最近，我们 证明了我们设计的一种名为RD3AD的新型多肽载体，增强了细胞内的递送和 口腔癌细胞中治疗性的、化学合成的siRNA在体外的可用性达到~4倍的水平 脂质体3000，有人会认为它是体外阳性对照的“金标准” 运送核酸。因此理所当然，RD3AD可能是一种有效的交付工具 针对HNSCC细胞中的E6和E7病毒癌基因(siE6和siE7)的siRNAs。RD3AD-siRNA 还观察到复合体定位于被鉴定为丝状足基的细胞突起。这些观察结果是 表明以前报道的细胞摄取病毒、细菌、激活的受体、 脂/多复合体和外体，它们利用丝状足细胞进行逆行运输到细胞，结果 在显著提高摄取能力方面。因此，根据RD3AD的观察，这是可能的 多肽也在利用类似的机制。因此，我们的总体假设是RD3AD-siE6/7(siE6或 SiE7)复合体与细胞丝状伪足结合后进入HPV+口腔癌细胞并沉默病毒 在体外，致癌的E6或E7mRNA。我们将通过以下具体目标来检验我们的假设。目标1： 鉴定丝状伪足上的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复合体摄取的机制(S)。总的来说，这项提议将建立一个 HPV+HNSCCs的分子治疗选择，同时描述了一种细胞特异性递送的新机制 并被多肽纳米载体摄取。这项研究的结果将对解决 关键的未得到满足的临床需求，将在药物输送领域产生特别大的影响，同时提供新的 对丝状足纲生物学的洞察。该研究金还将提供药物输送和癌症生物学方面的培训，以及 将培养学员成为一名独特的口腔健康学术临床医生/科学家。

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.