Laser treatment to enhance therapeutic HPV DNA vaccine potency

描述（由申请人提供）：人乳头瘤病毒E7致癌蛋白在表达hpv的宫颈癌中一致表达，在诱导和维持细胞转化中起重要作用。因此，针对E7的免疫治疗可能为治疗hpv相关的宫颈恶性肿瘤提供了机会。我们之前已经证明，皮内基因枪递送编码钙网蛋白（CRT）与E7 （CRT/E7）的DNA疫苗可以显著增强E7特异性CD8+ T细胞免疫应答和对表达E7的肿瘤的抗肿瘤作用。这种DNA疫苗接种策略的一个主要限制是体内转染效率有限，这可能限制了DNA疫苗的效力。最近的研究描述了一种利用飞秒激光束治疗提高DNA疫苗体内转染效率的新方法。因此，在本研究中，我们假设通过针注射或基因枪皮内给药CRT/E7 DNA疫苗，然后进行飞秒激光束治疗，可以显著提高DNA疫苗的转染效率，从而增强E7特异性T细胞介导的免疫反应和体内抗肿瘤作用。具体而言，我们建议：特异性目的1：通过皮内针注射CRT/E7 DNA疫苗，在有或没有激光治疗的情况下，表征E7特异性CD8+ T细胞免疫反应和抗E7表达肿瘤的抗肿瘤作用。特异性目的2：利用基因枪给予CRT/E7 DNA疫苗伴或不伴激光治疗，表征E7特异性CD8+ T细胞免疫反应和对表达E7的肿瘤的抗肿瘤作用。特异性目的3：比较皮内针注射CRT/E7 DNA疫苗后激光治疗与基因枪给药CRT/E7 DNA疫苗后激光治疗产生E7特异性CD8+ T细胞免疫应答的能力和对表达E7的肿瘤的抗肿瘤作用。特异性目的4：确定激光治疗如何导致抗原特异性CD8+ T细胞免疫反应增强和治疗小鼠抗肿瘤作用的机制。这项研究的成功实施将代表一种提高DNA疫苗效力的创新方法。此外，本研究可能为未来临床翻译提供重要的基础。

DESCRIPTION (provided by applicant): Human papillomavirus E7 oncogenic protein is consistently expressed in HPV-expressing cervical cancers and is important in the induction and maintenance of cellular transformation. Thus, immunotherapy targeting E7 may provide an opportunity to treat HPV-associated cervical malignancies. We have previously shown that intradermal gene gun delivery of DNA vaccines encoding calreticulin (CRT) linked to E7 (CRT/E7) can significantly enhance E7-specific CD8+ T cell immune responses and antitumor effects against E7-expressing tumors. One major limitation of this DNA vaccination strategy is the limited transfection efficiency in vivo, which may limit the potency of DNA vaccines. Recent studies have described a novel approach to increase the transfection efficiency of the DNA vaccines in vivo using the femtosecond laser beam treatment. Thus, in the current proposal, we hypothesize that intradermal administration of the CRT/E7 DNA vaccine via needle injection or gene gun followed by femtosecond laser beam treatment would significantly enhance the transfection efficiency of the DNA vaccine, resulting in enhanced E7-specific T cell-mediated immune responses and antitumor effects in vivo. Specifically, we propose to: Specific Aim 1: Characterize the E7- specific CD8+ T cell immune responses and antitumor effects against an E7-expressing tumor using intradermal needle injection of the CRT/E7 DNA vaccine with or without laser treatment. Specific Aim 2: Characterize the E7-specific CD8+ T cell immune response and antitumor effects against an E7-expressing tumor using gene gun administration of the CRT/E7 DNA vaccine with or without laser treatment. Specific Aim 3: Perform a head-to-head comparison of intradermal needle injection of CRT/E7 DNA vaccine followed by laser treatment with gene gun administration of CRT/E7 DNA vaccine followed by laser treatment for their ability to generate E7-specific CD8+ T cell immune responses and antitumor effects against E7-expressing tumors. Specific Aim 4: Determine the mechanism of how laser treatment leads to enhancement in antigen- specific CD8+ T cell immune responses and antitumor effects in treated mice. The successful implementation of this study will represent an innovative approach for enhancing DNA vaccine potency. In addition, this study will potentially serve as an important foundation for future clinical translation. PUBLIC HEALTH RELEVANCE: Cervical cancer is the second leading cause of cancer deaths in women worldwide and human papillomavirus (HPV) has been identified as a necessary cause of cervical cancer. Our project aims to develop an innovative therapeutic HPV DNA vaccination strategy employing laser technology to improve DNA vaccine potency. Successful implementation of this study will represent an innovative approach for enhancing the potency of DNA vaccines targeting HPV for the treatment of cervical cancer.