Kunjin replicons for gene therapy and protein manufacture

该赠款旨在为昆锦复制子技术用于基因治疗和蛋白质生产提供概念验证。(A)蛋白质生产。构建两个表达绿色荧光蛋白（GFP）和分泌碱性磷酸酶（SEAP）的昆津复制子构建体，分别使用FACS和SEAP生物活性报告试剂盒（Roche）监测蛋白产量。蛋白质的生产和生物活性将在瞬时转染和延长的时间内进行监测。将测试几种细胞系、培养条件和昆锦复制子载体修饰。已经安排将构建体发送给罗氏、葛兰素史克、礼来和Exelixis，以便将该系统与现有的专有蛋白质生产技术进行并排比较。(B)基因治疗。提出了两种PoC基因治疗系统用于评价昆锦复制子载体。(i)通过转染表达粒细胞巨噬细胞集落刺激因子（GMCSF）的肿瘤引起抗肿瘤CD8 T细胞的产生和随后的肿瘤排斥反应。目前的方法包括过继转移腺- gm - csf转染的肿瘤细胞，这是一个昂贵且费力的过程，只能产生短暂的表达。Imm。免疫学2001,50:373)。我们打算将昆锦复制子病毒样颗粒注射到生长中的s.c.b 16黑色素瘤中，并期望看到高感染率、持续高水平的GMCSF表达和肿瘤排斥反应。与Kunjin相反，几乎所有人都对腺病毒有抗体反应，并且需要非常高滴度的腺病毒才能获得高感染和GM-CSF表达。这两个因素限制了腺病毒在体内的使用。（ii）移植排斥反应可以通过CTLA4-Fc的表达来抑制，CTLA4-Fc是一种阻断T细胞共刺激增强同种异体移植接受的试剂（Transplantation 2000 69:1806）。高水平表达超过100天预计与最佳移植物接受相关。我们使用Kunjin在体内数月无炎症地表达β -半乳糖苷酶的能力说明了这种方法的潜力（CIB ref 15）。最初，我们打算将P815细胞注射到C57BL-6中，这些细胞通常在几天内被排斥。相比之下，具有昆津复制子介导的CTLA4-Fc表达的P815细胞应该存活较长时间。使用FACS和抗h -2d抗体很容易监测移植物存活。

This grant seeks to provide proof of concept (PoC) for the use of the Kunjin replicon technology for gene therapy and protein production. (A) Protein production. Two Kunjin replicon constructs expressing green fluorescent protein (GFP) and secreted alkaline phosphatase (SEAP) are to be constructed and protein production monitored using FACS and SEAP bioactivity reporter kit (Roche), respectively. Protein production and biological activity of the proteins will be monitored in transient transfections and over an extended time period. Several cell lines, culture conditions and Kunjin replicon vector modifications will be tested. Arrangements have also been made to send the constructs to Roche, GSK, Eli Lilly, and Exelixis for side by side comparisons of this system with existing proprietary protein production echnologies. (B) Gene therapy. Two PoC gene therapy systems are proposed to be used for evaluation of Kunjin replicon vectors. (i) Tumours expressing granulocyte macrophage colony stimulating factor (GMCSF) by transfection cause the generation of anti-tumour CD8 T cells and subsequent tumour rejection. Current approaches include adoptive transfer of adeno-GM-CSF transfected tumour cells, a costly and laborious process resulting in only transient expression (Can. Imm. Immunother 2001 50:373). We intend to inject Kunjin replicon virus like particles into growing s.c. B16 melanomas and expect to see a high infection rate, a sustained high-level expression of GMCSF, and rejection of the tumour. In contrast to Kunjin, nearly all humans have antibody responses to adenovirus, and very high titres of adenovirus are required to obtain high infection and GM-CSF expression. Both factors limit adenovirus use in vivo. (ii) Transplant rejection can be inhibited by expression in the graft of CTLA4-Fc a reagent that blocks T cell co-stimulation enhancing allo-graft acceptance (Transplantation 2000 69:1806). High-level expression for over 100 days is expected to correlate with optimal graft acceptance. Our ability to use Kunjin to express beta galactosidase for several months in vivo without inflammation illustrates the potential for this approach (CIB ref 15). Initially we intend to use P815 cells injected i.p. into C57BL-6, where they are usually rejected within a few days. In contrast, P815 cells with Kunjin replicon-mediated CTLA4-Fc expression should survive for an extended period. Graft survival is easily monitored using FACS and anti-H-2d antibodies.