Laser treatment to enhance therapeutic HPV DNA vaccine potency

描述(申请人提供)：人乳头瘤病毒E7癌基因蛋白在表达HPV的宫颈癌中持续表达，并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机会。我们之前已经证明，皮内基因枪注射编码E7连接的钙网蛋白(CRT)的DNA疫苗(CRT/E7)可以显著增强E7特异性CD8 T细胞免疫应答和对E7表达的肿瘤的抗肿瘤效应。这种DNA疫苗策略的一个主要限制是体内有限的转染率，这可能会限制DNA疫苗的效力。最近的研究描述了一种利用飞秒激光处理提高DNA疫苗体内转染率的新方法。因此，在目前的方案中，我们假设，通过针头注射或基因枪皮内注射CRT/E7 DNA疫苗，然后再进行飞秒激光治疗，将显著提高DNA疫苗的转染率，从而增强体内E7特异性T细胞介导的免疫反应和抗肿瘤效应。具体地说，我们建议：具体目标1：通过皮内注射CRT/E7 DNA疫苗，无论是否进行激光治疗，表征E7特异性CD8 T细胞免疫反应和抗E7表达肿瘤的效果。特定目的2：用基因枪注射CRT/E7 DNA疫苗，并结合激光治疗，研究E7特异性CD8 T细胞免疫应答和抗E7表达肿瘤的作用。具体目的3：比较皮内注射CRT/E7 DNA疫苗、基因枪注射CRT/E7 DNA疫苗和激光治疗后产生E7特异性CD8 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：确定激光治疗如何导致抗原特异性CD8 T细胞免疫反应增强和抗肿瘤作用的机制。这项研究的成功实施将代表着一种提高DNA疫苗效力的创新方法。此外，这项研究也可能成为未来临床翻译的重要基础。 公共卫生相关性：宫颈癌是全球女性癌症死亡的第二大原因，人类乳头瘤病毒(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，利用激光技术来提高DNA疫苗的效力。这项研究的成功实施将代表着一种创新的方法，以提高针对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.