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）的发病率是 虽然有治疗选择，但它们仍然主要限于手术切除，放射治疗 治疗和化疗，这往往会导致长期的发病率。RNA干扰（RNAi）似乎是一种 通过靶向治疗许多疾病（包括HPV+相关癌症）的有前途的治疗工具 病毒癌基因E6和E7与小干扰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，导致不受控制的细胞增殖。 体外敲低E6和E7已显示在HPV+宫颈细胞癌和HPV+宫颈癌中诱导细胞凋亡。 HPV+头颈部鳞状细胞癌细胞系，表明E6和E7是可行的治疗靶点 用于RNAi治疗。然而，RNAi的治疗应用需要一个递送平台， 许多挑战通常与这种形式的治疗相关。肽载体显示出巨大的前景， siRNA载体基于其理化性质和功能的多样性。最近我们 证明了我们设计的一种新型肽载体，称为RD 3AD，增强了细胞内递送， 在体外，口腔癌细胞中治疗性的化学合成siRNA的可用性达到约4倍的水平， Lipofectamine 3000，有些人认为它是体外培养的“金标准”阳性对照 核酸的递送。因此，RD 3AD可能是一种有效的运载工具， 设计用于靶向HNSCC细胞中E6和E7病毒致癌基因（siE 6和siE 7）的siRNA。RD3AD-siRNA 还观察到复合物定位于被鉴定为丝状伪足的细胞突起。这些观察结果 指示先前报道的病毒，细菌，活化受体， 脂质/复合物和外泌体，它们利用丝状伪足进行向细胞的逆行运输， 显著提高了摄入量。因此，考虑到RD 3AD的观察结果， 肽正在利用类似的机制。因此，我们的总体假设是RD 3AD-siE 6/7（siE 6或siE 7）是一种表达蛋白。 siE 7）复合物在结合细胞丝状伪足并沉默病毒后进入HPV+口腔癌细胞 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。目标1： 鉴定丝状伪足上的RD 3AD受体并利用该受体进行细胞特异性靶向。目标二： 确定RD 3AD-siRNA复合物摄取的机制。总的来说，这项建议将建立一个 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 并被肽纳米载体摄取。这项研究的结果将大大有助于解决一个问题， 关键的未满足的临床需求，并将特别影响药物输送领域，同时提供新的 对丝状伪足生物学的见解该研究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一个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.