rAAV vaccine vector

描述（由申请人提供）：基于基因的递送系统作为疫苗在传染病和癌症方面非常有前途。重组腺相关病毒载体（rAAV）在开发有效的癌症疫苗方面的独特优势包括诱导持续、高水平的转基因表达、不含病毒结构蛋白、无致病性和无毒性。利用替代的AAV血清型衣壳来克服靶细胞的难治性和rAAV基因组中的分子修饰，从而影响转基因表达的早期开始，进一步增加了该载体的实用性。虽然基因传递系统已被证明是治疗癌症的有效疫苗，但有效的肿瘤免疫激活的主要限制是抗原表达的减少，这削弱了强大的效应激活，以及载体特异性免疫反应，在诱导强大的抗肿瘤免疫之前消除表达转基因的细胞。通过本申请中概述的研究，我们建议利用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，在我们实验室的研究中，rAAV编码一个原型肿瘤抗原，并适应几种修饰，包括使用血清型6衣壳，rAAV含有双链基因组，rAAV6的多次转导，然后在培养中激活树突状细胞，以及在直接rAAV免疫后使用合成寡核苷酸和质粒DNA佐剂，在C57BL6/J小鼠中产生了显著的抗原特异性，th1相关的免疫反应和抗肿瘤活性。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。目前的建议将侧重于开发这种载体范式，以更严格地测试在临床相关的小鼠黑色素瘤模型中使用酪氨酸酶相关蛋白（TRP） 2抗原通过体外和直接体内疫苗策略诱导保护性和治疗性肿瘤免疫。肿瘤免疫机制将在体内进行研究。进一步，我们将研究预先存在的抗病毒免疫对形成有效的抗肿瘤免疫的影响。

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.