Production of HIV vector supernatant using helper-dependent adenovirus

描述（由申请人提供）：来自柏林的一例患者的报告，该患者在接受来自a？32名CCR 5捐献者的到来再次唤起了人们对艾滋病基因治疗前景的兴奋。最有希望的载体之一是基于HIV，但目前的生产方法很费力，难以扩大规模用于可能的临床用途。在这里，我们建议使用辅助病毒依赖性腺病毒或HDAd作为一种容易地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，我们已经制备了编码所有HIV顺式和反式组分的单个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，比常规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，并在几天内产生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HIV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掺入HIV包装载体（PV）。一旦测试完成，并且1-2个最佳扩增的HDAs被证明是功能性的，将添加编码eGFP的HIV转移载体（TV），构建多达1 - 2个也具有HIV-TV的HDAs。质粒检测后，最好的3-4将被扩增，包括一个与所有三个艾滋病毒成分。为了产生HIV，将以增加的MOI用HDAs转导基于293的细胞系，收获上清液，并滴定HIV。将在pg CA/IU、基因组RNA含量/IU、辅助腺病毒污染和抑制非分裂细胞（包括巨噬细胞）的能力方面，将由HDAd制备的HIV与使用质粒转染制备的HIV进行比较。将使用悬浮293个细胞的3升旋转烧瓶来显示可扩展性。最后，将编码抗CCR 5 shRNA的TV掺入HDAd中，并显示其在转导原代T细胞和巨噬细胞以及随后保护免受M嗜性HIV攻击中的功能。在两年的资助期结束时，我们希望已经表明，有可能使用HDAd产生与常规质粒转染方法相比具有相等或更高滴度的全功能的第三代VSV G-假型HIV载体，这应该对这些载体用于广泛的先天性和获得性疾病（不仅仅是HIV）的临床用途具有影响。

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.