Production of HIV vector supernatant using helper-dependent adenovirus

描述(由申请人提供)：来自柏林的一名患者在接受32欧元CCR5捐赠者的异基因干细胞移植近4年后，可能治愈了他的艾滋病毒疾病，这一报道再次引发了人们对艾滋病毒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的，但目前的生产方法费时费力，难以扩大到可能的临床应用。在这里，我们建议使用辅助依赖腺病毒或HDAd作为一种容易地获得高滴度和高数量的HIV载体上清液的方法。在初步数据中，我们已经制作了一个编码所有HIV顺式和反式组分的HDAd作为第二代载体。当以高MOI导入293细胞时，产生的HIV滴度接近108IU/ml，比传统的多质粒转染法高1-2个数量级。我们能够使用一个细胞工厂扩大生产，在几天内产生了艾滋病毒载体。在这项为期两年的提案中，我们现在寻求将这种方法扩展到第三代艾滋病毒载体。要做到这一点，从已经编码VSV G的HDAd开始，我们首先将HIV包装载体(PV)整合到几个不同的位置和方向。一旦测试完成，并且1-2个最佳扩增的HDAD被证明是有效的，将添加编码EGFP的HIV转移载体(TV)，构建多达12个也具有HIV-TV的HDAD。经过质粒检测后，最好的3-4将被扩增，其中包括一种具有全部三种HIV成分的病毒。为了生产HIV，293细胞系将以增加的MOI与HDAd一起被转导，收获上清液，并对HIV进行滴定。由HDAd制成的HIV将在每IU PG CA、每IU基因组RNA含量、辅助腺病毒污染以及转导包括巨噬细胞在内的未分裂细胞的能力方面与使用HDAd制备的HIV进行比较。可伸缩性将使用3个L旋转烧瓶悬浮293细胞来展示。最后，编码抗CCR5 shRNA的TV将被整合到HDAd中，并被证明在转导初级T细胞和巨噬细胞以及随后保护M嗜性HIV攻击方面具有功能。在两年的资助期结束时，我们希望能够证明，使用HDAd可以生产出与传统的质粒转染法相同或更高效价的全功能第三代VSV G伪型HIV载体，这将对这些载体在广泛的先天性和获得性疾病中的临床应用产生影响，而不仅仅是HIV。

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.