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同种异体干细胞移植近4年后，可能治愈了他的HIV疾病。32名CCR5供体再次激起了人们对HIV基因治疗前景的兴奋。最有希望做到这一点的载体之一是基于艾滋病毒，但目前的生产方法很费力，而且难以扩大规模以用于可能的临床应用。在这里，我们建议使用辅助腺病毒或HDAd作为一种容易制作高滴度和数量的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顺式和反式成分的单一HDAd作为第二代载体。当以高MOI导入293细胞时，HIV滴度接近108 IU/ml，比传统的多质粒转染生产方法高1-2个数量级。我们利用细胞工厂扩大了生产规模，几天后就产生了艾滋病毒载体。在这项为期两年的建议中，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上合并HIV包装载体（PV）。一旦测试完成，并且1-2个最好的扩增hdad显示出功能，将添加一个编码eGFP的HIV转移载体（TV），构建多达12个同样具有HIV-TV的hdad。在质粒检测后，最好的3-4个将被扩增，包括具有所有三种HIV成分的一个。为了产生HIV， 293细胞系将以增加MOIs的方式与hdad、收集的上清进行转导，并进行HIV滴度检测。用HDAd制成的HIV将与用质粒转染制成的HIV进行比较，包括每IU的pg CA、每IU的基因组RNA含量、辅助性腺病毒的污染以及转导非分裂细胞（包括巨噬细胞）的能力。将使用悬浮293细胞的3l旋转烧瓶显示可扩展性。最后，编码抗ccr5 shRNA的TV将被整合到HDAd中，并显示其在转导原代T细胞和巨噬细胞以及随后抵抗嗜m型HIV攻击方面的功能。在为期两年的资助期结束时，我们希望能够证明，使用HDAd生产功能齐全的第三代VSV g伪型HIV载体是可能的，与传统的质粒转染方法相比，其滴度相等或更高，这应该对这些载体在广泛的先天性和获得性疾病的临床应用产生影响，而不仅仅是HIV。