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

新型 DNA 制造工艺：新型 Multiv 的无细胞生产和测试

• 批准号: 7536364
• 负责人: Frederic Kendirgi
• 金额: $ 13.67万
• 依托单位: CYTOGENIX, INC.
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-06-21 至 2009-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7536364
• 关键词: Accounting; Acquired Immunodeficiency Syndrome; Animal Model; Animals; Antibodies; Anus; Avian Influenza A Virus; Bacteria; Brain; Cells; Cessation of life; Clinical; Confidential Information; DNA; DNA Sequence; DNA Vaccines; DNA amplification; DNA biosynthesis; DNA chemical synthesis; DNA purification; Daily; Data; Developed Countries; Developing Countries; Development; Domestic Fowls; Dose; Equipment; Evaluation; Fermentation; Ferrets; Flu virus; Future; General Population; Goals; HIV; Harvest; Human; Immune response; Immunization; In Vitro; Infection; Influenza; Influenza A Virus, H5N1 Subtype; Influenza A virus; Injection of therapeutic agent; Intramuscular; Investments; Legal patent; Life; Lung; Malaria; Marketing; Methods; Monitor; Mus; Nature; Neuraminidase; Nucleic Acids; Numbers; Persons; Phase; Phase II Clinical Trials; Plasmids; Preparation; Preventive; Process; Production; Productivity; Proteins; Protocols documentation; Public Health; Purpose; Ramp; Reporting; Research; Serological; Staging; Standards of Weights and Measures; Symptoms; Technology; Testing; Therapeutic; Time; Tissue Sample; Tissues; Treatment Protocols; Tuberculosis; Tweens; United States Food and Drug Administration; Vaccinated; Vaccination; Vaccine Production; Vaccines; Vietnam; Viral; Virus; Week; base; cost; day; design; efficacy trial; falls; flu; immunogenic; manufacturing process; nonhuman primate; novel; pandemic disease; pandemic influenza; pathogen; preclinical study; prevent; prophylactic; prototype; research study; response; scale up; success; transmission process; vaccine development

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.

描述（由申请人提供）：家禽中出现高致病性禽流感病毒，以及禽流感病毒直接传播给人类的病例数量不断增加，对公众健康构成重大威胁，因为这些病毒有可能发生大流行。目前，高致病性禽流感 H5N1 感染的预防和治疗选择有限。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非常有前途的未来。传统上，DNA 疫苗是通过完善的发酵工艺生产的。 CytoGenix 开发了一种替代方案，即无细胞生产技术 (synDNATM)，用于以最少的努力和快速的周转时间合成大量（克）DNA，用于小批量疫苗生产。该技术不需要细菌来提高产量，只需最少的非治疗性 DNA 序列即可应用。因此，该过程本质上比质粒发酵“更清洁”，并且最终产品需要最少的纯化。该第一阶段提案的总体目标是验证我们的 synDNATM 工艺在 DNA 疫苗合成中的使用。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剂的第一个目标，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毒株甲型/越南/1203/04，H5N1。包括我们自己的实验在内的几份报告表明，表达流感病毒蛋白的 DNA 疫苗可以单独在接种疫苗的动物中引发强烈的免疫反应；从根本上防止他们因感染活病毒而生病。我们打算在体外合成并测试一种 DNA 疫苗，该疫苗能够在注射到雪貂（选择的人类流感动物模型）中时表达其中两种蛋白质，以引发免疫反应，从而预防这种潜在的大流行流感病毒株。我们的目标是 1) 使用 synDNATM 工艺生成 DNA 疫苗，并通过 2) 分析触发的免疫反应和 3) 优化免疫方案，在活病毒挑战的雪貂中测试其功效。如果成功，我们的结果将为实施使用我们大规模生产工艺生产的 DNA 疫苗的无细胞合成和测试标准实验方案铺平道路。这将使我们能够常规化针对新型流感病毒株和其他病毒病原体的新型 DNA 疫苗的合成和测试，并利用我们的生产流程简化 DNA 疫苗的制备。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移到流行病威胁最严重的发展中国家。从拟议实验中收集的数据还将为遵循 FDA“两种动物模型规则”的 II 期实验奠定基础，用于在人体功效试验不可行或不道德的情况下开发/测试生物制品。公共卫生相关性：目前，公众还没有针对大流行性流感的有效疫苗。如果成功，该提案中描述的实验将为合成和生产针对流感的新型 DNA 疫苗铺平道路，这种疫苗可以快速适应新的病毒株以及尚未生产出通过常规方法制备的有效疫苗的其他病毒病原体，即艾滋病毒、疟疾和结核病。