Laser treatment to enhance therapeutic HPV DNA vaccine potency

描述（由申请方提供）：人乳头瘤病毒E7致癌蛋白在HPV表达宫颈癌中持续表达，在诱导和维持细胞转化中具有重要作用。因此，靶向E7的免疫疗法可能为治疗HPV相关的宫颈恶性肿瘤提供机会。我们先前已经表明，皮内基因枪递送编码钙网蛋白（CRT）连接到E7（CRT/E7）的DNA疫苗可以显着增强E7特异性CD 8 + T细胞免疫应答和对E7表达肿瘤的抗肿瘤作用。这种DNA疫苗接种策略的一个主要限制是有限的体内转染效率，这可能限制DNA疫苗的效力。最近的研究描述了一种新的方法来提高体内的DNA疫苗的转染效率，使用飞秒激光束处理。因此，在目前的建议中，我们假设通过针注射或基因枪皮内注射CRT/E7 DNA疫苗，然后进行飞秒激光束处理，将显着提高DNA疫苗的转染效率，导致增强E7特异性T细胞介导的免疫应答和体内抗肿瘤作用。具体而言，我们建议：具体目标1：使用皮内针注射CRT/E7 DNA疫苗（有或无激光治疗），表征E7特异性CD 8 + T细胞免疫应答和抗E7表达肿瘤的抗肿瘤作用。具体目标二：使用基因枪给予CRT/E7 DNA疫苗（伴或不伴激光治疗），表征E7特异性CD 8 + T细胞免疫应答和抗E7表达肿瘤的抗肿瘤作用。具体目标3：对皮内针注射CRT/E7 DNA疫苗，然后进行激光治疗与基因枪注射CRT/E7 DNA疫苗，然后进行激光治疗的能力进行头对头比较，以确定其产生E7特异性CD 8 + T细胞免疫应答的能力和对E7表达肿瘤的抗肿瘤作用。具体目标4：确定激光治疗如何增强经治疗小鼠的抗原特异性CD 8 + T细胞免疫应答和抗肿瘤作用的机制。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，这项研究将可能成为未来临床翻译的重要基础。 公共卫生相关性：宫颈癌是世界范围内女性癌症死亡的第二大原因，人乳头瘤病毒（HPV）已被确定为宫颈癌的必要原因。我们的项目旨在开发一种创新的治疗性HPV DNA疫苗接种策略，采用激光技术来提高DNA疫苗的效力。这项研究的成功实施将代表一种创新的方法，用于提高靶向HPV的DNA疫苗治疗宫颈癌的效力。

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.