Production of HIV vector supernatant using helper-dependent adenovirus

使用辅助依赖性腺病毒生产 HIV 载体上清液

• 批准号: 8340244
• 负责人: Richard Sutton
• 金额: $ 22.13万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-02-01 至 2014-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8340244
• 关键词: Adenoviruses; Allogenic; Berlin; CCR5 gene; Capsid; Capsid Proteins; Cell Line; Cells; Clinical; Data; Disease; Funding; Future; Generations; Genes; Genomics; HIV; HIV Infections; Harvest; Hematopoietic stem cells; Immune Sera; Individual; Interphase Cell; Lentivirus Vector; Life; Methods; Patients; Plasmids; Production; RNA; Reagent; Reporting; Resistance; Site; Stem cell transplant; Suspension substance; Suspensions; System; T-Lymphocyte; Testing; Time; Transfection; Ultracentrifugation; Vesicular stomatitis Indiana virus; Virus; base; flasks; gene therapy; interest; macrophage; neuronal cell body; particle; scale up; small hairpin RNA; therapeutic gene; tissue culture; vector; viral RNA

DESCRIPTION (provided by applicant): The report of a single patient from Berlin who may be cured of his HIV disease almost 4 years after allogeneic stem cell transplant from a ?32 CCR5 donor has raised renewed excitement of the prospects for HIV gene therapy. One of the most promising vectors for doing so is based upon HIV, but current production methods are laborious and difficult to scale-up for possible clinical use. Here we propose the use of helper-dependent adenovirus or HDAd as a way to easily make HIV vector supernatants of high titer and quantity. In preliminary data we have made a single HDAd encoding all the HIV cis and trans components as a second generation vector. When introduced into 293 cells at high MOI resultant HIV titers were close to 108 IU/ml, 1-2 orders of magnitude greater than conventional multiplasmid transfection production methods. We were able to scale up production using a cell factory, and HIV vector was generated over several days. In this two-year proposal we now seek to extend this method to a third generation HIV vector. To do so, starting with an HDAd that already encodes VSV G, we will first incorporate an HIV packaging vector (PV) in several different sites and orientations. Once that testing is complete and the 1-2 best amplified HDAds are shown to be functional, an HIV transfer vector (TV) that encodes eGFP will be added, constructing up to a dozen HDAds that also have HIV-TV. After plasmid testing, the best 3-4 will be amplified, including one with all three HIV components. To produce HIV, 293-based cell lines will be transduced at increasing MOIs with the HDAds, supernatant harvested, and titered for HIV. HIV made from HDAd will be compared to that made using plasmid transfection in terms of pg CA per IU, genomic RNA content per IU, contamination with helper adenovirus, and ability to transduce non-dividing cells, including macrophages. Scalability will be shown using 3 l spinner flasks of suspension 293 cells. Finally, a TV encoding an anti-CCR5 shRNA will be incorporated into the HDAd and shown to be functional in transducing primary T cells and macrophages and protecting against M-tropic HIV challenge subsequently. At the end of the two year funding period we hope to have shown that it is possible to produce a fully functional, third generation VSV G-pseudotyped HIV vector using HDAd, of equal or greater titer compared to conventional plasmid transfection methods, which should have implications for the clinical use of these vectors for a wide range of congenital and acquired diseases, not merely HIV. PUBLIC HEALTH RELEVANCE: Although there is no cure for HIV, it may be possible to modify the body's cells to make them resistant to the virus. This proposal attempts a new way to be able to make vectors in order to introduce genes into cells. If successful, it would have implications not just for HIV but for many different diseases in which defective genes need to be replaced or new genes inserted.

描述（由申请人提供）：来自柏林的一名患者在接受 ?32 CCR5 捐赠者的同种异体干细胞移植近 4 年后，其 HIV 疾病可能被治愈，该报告使人们对 HIV 基因治疗的前景重新感到兴奋。最有前途的载体之一是基于 HIV 的载体，但目前的生产方法很费力，并且难以扩大规模以用于可能的临床应用。在这里，我们建议使用辅助依赖性腺病毒或 HDAd 作为一种轻松制备高滴度和高数量的 HIV 载体上清液的方法。 在初步数据中，我们制作了一个编码所有 HIV 顺式和反式成分的 HDAd 作为第二代载体。当以高 MOI 引入 293 细胞时，所得 HIV 滴度接近 108 IU/ml，比传统多质粒转染生产方法高 1-2 个数量级。我们能够使用细胞工厂扩大生产规模，并在几天内生成了 HIV 载体。 在这个为期两年的提案中，我们现在寻求将这种方法扩展到第三代艾滋病毒载体。为此，从已经编码 VSV G 的 HDAd 开始，我们将首先在几个不同的位点和方向合并 HIV 包装载体 (PV)。一旦测试完成并且 1-2 个最佳扩增 HDAd 被证明具有功能，将添加编码 eGFP 的 HIV 转移载体 (TV)，从而构建多达 12 个也具有 HIV-TV 的 HDAd。质粒测试后，最好的 3-4 个将被扩增，其中一个包含所有三种 HIV 成分。为了产生HIV，将以增加的MOI与HDAd转导基于293的细胞系，收获上清液并针对HIV进行滴度测定。由 HDAd 制备的 HIV 与使用质粒转染制备的 HIV 将在每 IU 的 pg CA、每 IU 的基因组 RNA 含量、辅助腺病毒污染以及转导非分裂细胞（包括巨噬细胞）的能力方面进行比较。将使用 3 L 转瓶悬浮 293 细胞来显示可扩展性。最后，编码抗 CCR5 shRNA 的 TV 将被整合到 HDAd 中，并显示出在转导原代 T 细胞和巨噬细胞以及随后防止 M 向性 HIV 攻击方面具有功能。在两年资助期结束时，我们希望能够证明，使用 HDAd 生产功能齐全的第三代 VSV G 假型 HIV 载体是可能的，与传统的质粒转染方法相比，其滴度等于或更高，这应该具有这些载体的临床应用对广泛的先天性和获得性疾病（而不仅仅是艾滋病毒）的影响。 公共卫生相关性：虽然艾滋病毒无法治愈，但有可能改变人体细胞，使其对病毒具有抵抗力。该提案尝试了一种能够制造载体以将基因引入细胞的新方法。如果成功，它不仅会对艾滋病毒产生影响，还会对许多需要替换缺陷基因或插入新基因的不同疾病产生影响。