Persistence of Transgene Expression in Synovium

描述(由申请人提供)： 由于无法维持关节内转基因表达，关节炎基因治疗的临床应用进展缓慢。普遍的观察是，转基因表达只持续两到三周。目前的建议旨在检验以下假设：1)外源(非自身)转基因产物的免疫不相容导致转基因表达的提前终止；2)滑膜内的特定细胞类型能够支持稳定、持久的转基因表达；3)在正常的、具有免疫能力的动物中，编码同源(自身)或免疫相容的基因产物的cDNA将在滑膜内长期持续表达。为了确定各种体外和体内基因传递系统的相对益处和局限性，将对每种系统在裸鼠膝部的转基因持久性进行评估。因为在这种动物中，表达外源蛋白的转导细胞可以在自然寿命内存活，所以它将作为一个“假同源”系统，在这个系统中，我们可以使用诊断标记基因来模拟将编码“自身”蛋白的转基因运送到正常关节。使用绿色荧光蛋白的cDNA来表型标记滑膜中的转基因细胞，我们将使用流式细胞术来表征转导细胞的数量。通过研究它们如何随时间变化，我们将确定滑膜衬里中可能允许长期表达的特定细胞群。然而，这项研究背后的真正动机是确定外源转基因在免疫活性动物的正常关节和关节炎关节中表达的时间有多长。为此，将在正常大鼠身上研究两种不同的基因系统，它们不会激活免疫清除。第一种将是可溶形式的大鼠肿瘤坏死因子受体II型；第二种将是转录但不翻译的“基因”结构，完全避免了外来蛋白质产品的免疫问题。1)比较评价滑膜成纤维细胞、真皮成纤维细胞和间充质干细胞作为体外关节内基因载体向裸鼠膝关节内持续转基因表达的能力及其生物分布；2)比较评估重组慢病毒、AAV和高容量腺病毒作为载体在裸鼠膝关节内实现持续转基因表达及其生物分布的能力；3)比较滑膜中转基因细胞的特征并确定它们在体内随时间的变化；以及4)在免疫活性大鼠体内，评估免疫兼容转基因系统在正常膝关节和关节炎膝关节中的表达在体外和体内分娩后的持久性。

DESCRIPTION (provided by applicant): Progress toward the clinical application of gene therapy for arthritis has been slowed by the inability to sustain intraarticular transgene expression. The prevailing observation is that transgene expression persists for only two to three weeks. The current proposal is designed to test the following hypotheses: 1) The immunologic incompatibility of foreign (non-self) transgene products leads to this premature termination of transgene expression, 2) Specific cell types within the synovium are capable of supporting stable, persistent transgene expression, and 3) In normal, immunocompetent animals, expression of cDNAs encoding homologous (self) or immunologically compatible gene products will persist long-term within the synovium. To determine the relative benefits and limitations of various ex vivo and in vivo gene delivery systems, each will be evaluated for transgenic persistence in the knees of athymic, nude rats. Because transduced cells that express foreign proteins survive for their natural life-span in this animal, it will serve as a "pseudohomologous" system within which we can use diagnostic marker genes to simulate the delivery of transgenes encoding "self" proteins to normal joints. Using the cDNA for Green Fluorescent Protein to phenotypically tag genetically modified cells in the synovium, we will use flow cytometry to characterize the populations of transduced cells. By studying how they change with time, we will identify the specific cell populations within the synovial lining that may permit long-term expression. The true motivation behind the study is however to determine for how long exogenous transgenes may be expressed in normal and arthritic joints in immunocompetent animals. For this, two different gene systems will be studied in normal rats that will not activate immune clearance. The first will be a soluble form of the rat TNF receptor type II; the second will be a "gene" construct that is transcribed, but not translated, avoiding altogether the immune issues of foreign protein products. The following specific aims will be addressed: 1) To comparatively evaluate synovial fibroblasts, dermal fibroblasts and mesenchymal stem cells for their capacity to enable persistent transgenic expression, and their biodistribution when used as vehicles for ex vivo intra-articular gene delivery to the knees of athymic nude rats, 2) To comparatively evaluate recombinant lentivirus, AAV and high capacity adenovirus for their capacity to enable persistent transgenic expression and their biodistribution when used as vehicles for direct intraarticular gene delivery to the knees of athymic nude rats, 3) To characterize genetically modified cell populations in the synovium and to determine how they change with time in vivo, and 4) Within immunocompetent rats, evaluate persistence of expression using immunocompatible transgene systems in normal and arthritic knees following ex vivo and in vivo delivery.