Novel DNA Manufacturing Process: Cell-Free Production and Testing of a New Multiv

描述（由申请人提供）：高致病性禽流感病毒在家禽中的出现以及禽流感病毒直接传播给人类的病例数量的增加对公共卫生构成重大威胁，因为这些病毒有可能大范围传播。目前，高致病性H5N1禽流感感染的预防和治疗方案有限。许多努力都集中在疫苗的开发上。DNA的免疫原性潜力于1990年首次被描述，DNA疫苗代表了疫苗接种的一个非常有希望的未来。传统上，DNA疫苗是通过完善的发酵过程制造的。CytoGenix开发了一种替代方法，即无细胞生产技术（synDNATM），用于以最小的努力和快速的周转时间合成大量（克）DNA，用于小批量生产疫苗。由于不需要细菌来增加产量，这项技术可以应用于最少的非治疗性DNA序列。因此，这个过程比质粒发酵本质上“更清洁”，最终产品需要最少的净化。该I期提案的总体目标是验证我们的synDNATM工艺用于DNA疫苗合成的使用。作为使用我们的无细胞大规模生产方法开发基于核酸的预防性药物的第一个目标，我们选择了人类甲型流感病毒，更具体地说，是潜在的大流行株甲型流感/越南/1203/04，H5N1。包括我们自己的实验在内的几份报告表明，表达流感病毒蛋白质的DNA疫苗可以在接种疫苗的动物中单独引发强烈的免疫反应；基本上是防止他们在感染活病毒后生病。我们打算在体外合成并测试一种DNA疫苗，当注射到雪貂（人类流感动物模型的选择）体内时，能够表达这两种蛋白质，目的是引发免疫反应，以防止这种潜在的大流行性流感毒株。我们的目标是：1)利用synDNATM工艺生产DNA疫苗，并通过分析引发的免疫反应，3)优化免疫方案，在活病毒攻击的雪貂中测试其有效性。如果成功，我们的结果将为实施无细胞合成和测试使用我们的大规模生产工艺生产的DNA疫苗的标准实验方案铺平道路。这将使我们能够常规地合成和测试针对新的流感病毒株和其他病毒病原体的新DNA疫苗，并简化使用我们的生产过程制备DNA疫苗。此外，它将为以低成本快速生产有效疫苗奠定基础，这种技术可以很容易地转让给大流行病威胁最严重的发展中国家。从拟议的实验中收集的数据也将为遵循FDA“两种动物模型规则”的二期实验奠定基础，该规则适用于生物制剂的开发/测试，其中人体功效试验是不可行的或不符合伦理的。公共卫生相关性：目前，没有针对大流行性流感的强效疫苗可供公众使用。如果取得成功，本提案中所述的实验将为合成和生产新的流感DNA疫苗铺平道路，这种疫苗可以迅速适应新的病毒株，以及用常规方法制备的有效疫苗尚未生产出来的其他病毒病原体，即艾滋病毒、疟疾和结核病。

DESCRIPTION (provided by applicant): The emergence of highly pathogenic avian influenza viruses in domestic poultry and the increasing number of cases of direct transmission of avian influenza viruses to humans are a significant threat to public health because of the potential for pandemic spread of these viruses. Currently, preventive and therapeutic options for highly pathogenic avian influenza H5N1 infections are limited. Many efforts have focused on the development of vaccines. The immunogenic potential of DNA was first described in 1990 and DNA vaccines represent a very promising future for vaccination. Traditionally, DNA vaccines are manufactured through a well established fermentation process. CytoGenix has developed an alternative, i.e. a cell-free production technology (synDNATM) for the synthesis of large quantities (grams) of DNA for vaccine production in small volumes with minimal effort and rapid turn around time. Without the need for bacteria to ramp-up production, this technology can be applied with a minimum of non-therapeutic DNA sequences. This process is therefore inherently "cleaner" than plasmid fermentation and the final product requires minimal purification. The overall goal of this Phase I proposal is to validate the use of our synDNATM process for the synthesis of DNA vaccines. As a first target for the development of nucleic acid-based prophylactic made using our cell-free large scale production method, we have chosen the human influenza A virus, more specifically the potential pandemic strain influenza A/Vietnam/1203/04, H5N1. Several reports - including our own experiments - have shown that DNA vaccines expressing proteins from the flu virus can individually trigger a robust immune response in vaccinated animals; essentially preventing them from falling ill upon infection with the live virus. We intend to synthesize in vitro and test a DNA vaccine capable of expressing two of these proteins when injected into ferrets (the human influenza animal model of choice) for the purpose of eliciting an immune response that will protect against this potential pandemic flu strain. Our objectives are to 1) generate a DNA vaccine with our synDNATM process and test its efficacy in ferrets with live virus challenges by 2) analyzing the immune response triggered and 3) optimizing the immunization regimen. If successful, our results will pave the way for the implementation of standard experimental protocols for the cell-free synthesis and testing of DNA vaccines made using our large scale production process. This will enable us to routinize the synthesis and testing of new DNA vaccines against new influenza viral strains and other viral pathogens as well as simplify the preparation of DNA vaccines using our production process. Moreover, it will set the stage for the rapid production of efficacious vaccines at low cost, with a technology that can be easily transferred to developing countries where the threat of pandemics is at its highest. The data gathered from the proposed experiments will also set the stage for phase II experiments following the FDA "two animal model rule" for the development/testing of biologicals where human efficacy trials are not feasible or ethical. PUBLIC HEALTH RELEVANCE: Currently, no robust vaccines against pandemic flu are available to the general public. If successful, the experiments described in this proposal will pave the way for the synthesis and production of new DNA vaccines against influenza that can be rapidly adapted to new viral strains as well as other viral pathogens to which effective vaccines prepared by conventional methods have yet to be produced i.e. HIV, malaria and tuberculosis.