rAAV vaccine vector

描述（由申请人提供）：基于基因的递送系统作为疫苗对于传染病和癌症是非常有希望的。重组腺相关病毒载体（rAAV）在开发有效的癌症疫苗中的独特优势包括诱导持续的、高水平的转基因表达、不存在病毒结构蛋白、无致病性和无毒性。使用替代的AAV血清型衣壳来克服靶细胞难治性和在rAAV基因组中进行分子修饰以实现转基因表达的早期开始进一步增加了该载体的效用。尽管基因递送系统已被证明在癌症治疗中作为疫苗是有效的，但针对肿瘤的有效免疫活化的主要限制是抗原表达的减少，这会减少稳健的效应子活化，以及载体特异性免疫应答，这会在诱导强抗肿瘤免疫之前消除转基因表达细胞。通过本申请中概述的研究，我们提出利用rAAV作为肿瘤疫苗的优势，使用高度相关的黑色素瘤临床前小鼠模型。到目前为止，我们实验室中使用编码原型肿瘤抗原的rAAV并进行了几种修饰的研究，包括使用血清型6衣壳、含有双链基因组的rAAV、用rAAV 6多次转导随后在培养物中活化树突状细胞、以及在直接rAAV免疫后使用合成的寡核苷酸和质粒DNA佐剂，这些研究已经导致了显著的抗原特异性，C57 BL 6/J小鼠中的Th 1相关免疫应答和抗肿瘤活性。更有趣的是，这种方法成功地打破了对“自身”肿瘤抗原的免疫耐受。目前的建议将集中于开发这种载体范例，以更严格地测试使用酪氨酸酶相关蛋白（TRP）2抗原通过离体和直接体内疫苗策略在临床相关小鼠黑色素瘤模型中诱导保护性和治疗性肿瘤免疫。将在体内检查肿瘤免疫的机制。此外，将研究预先存在的抗病毒免疫对有效抗肿瘤免疫的发展的影响。 公共卫生相关性：这项转化研究的结果预计将迅速启动采用rAAV-TRP 2疫苗治疗黑色素瘤的I期临床试验，并可为以肿瘤抗原过表达为特征的其他人类癌症的新型免疫治疗策略奠定基础。基于重组AAV的独特优势，包括持续的高水平表达、非致病性和低载体免疫性，拟议的翻译研究将开发和确定rAAV疫苗与寡核苷酸和质粒DNA免疫佐剂组合在小鼠黑色素瘤模型中用于强CTL细胞应答的潜力。

DESCRIPTION (provided by applicant): Gene-based delivery systems as vaccines are highly promising for infectious diseases and cancer. Unique advantages of recombinant adeno-associated virus vector (rAAV) in the development of effective cancer vaccines include induction of sustained, high-level transgene expression, absence of viral structural proteins, non-pathogenicity, and absence of toxicity. The use of alternate AAV serotype capsids to overcome target cell refractoriness and molecular modifications in the rAAV genome to effect early onset of transgene expression further increases the utility of this vector. Although gene delivery systems have proven efficacious as vaccines in the treatment of cancer, the major limitations for effective immune activation against tumors have been diminution of antigen expression, which curtails robust effector activation, and vector-specific immune responses, which eliminate transgene-expressing cells prior to the induction of strong anti-tumor immunity. By studies outlined in this application, we propose to exploit the advantages of rAAV as a tumor vaccine using a highly relevant preclinical mouse model of melanoma. Studies so far in our lab with rAAV encoding a prototypic tumor antigen and adapting several modifications including the use of serotype 6 capsid, rAAV containing double-stranded genome, multiple transductions with rAAV6 followed by dendritic cell activation in cultures, and the use of synthetic oligonucleotide and plasmid DNA adjuvant following direct rAAV immunizations have resulted in significant antigen-specific, Th1-associated immune responses and anti-tumor activity in C57BL6/J mice. More interestingly this approach was successful in breaking immune tolerance to a "self" tumor antigen. The current proposal will focus on developing this vector paradigm to more rigorously test the induction of both protective and therapeutic tumor immunity in a clinically relevant mouse melanoma model using tyrosinase-related protein (TRP) 2 antigen by ex vivo and direct in vivo vaccine strategies. Mechanisms of tumor immunity will be examined in vivo. Further, the influence of pre-existing anti-viral immunity on the development of effective anti-tumor immunity will be studied. Public Health Relevance: Results of this translational study are anticipated to rapidly enable the initiation of phase I clinical trial employing rAAV-TRP2 vaccine for melanoma and can form the basis for a novel immunotherapy strategy for other human cancers characterized by overexpression of tumor antigens. Based on the unique advantages of recombinant AAV including sustained high-level expression, non- pathogenicity and low vector immunity, the proposed translational study will develop and determine the potential of rAAV vaccine in combination with oligonucleotide and plasmid DNA immune adjuvant for strong CTL cell response in a mouse melanoma model.