118. In Vivo Tracking of Long Term Gene Expression of AAV1 Using Fluorescence Imaging System

Introduction:The kinetics of long term transgene expression of viral vectors is important as it directly influences the efficacy of the gene therapy. Conventional kinetic measurements require sacrificing animals at each data point and assessing the amount of transgene product in the tissue. This type of analysis is extremely laborious and contains inherent inaccuracy since it does not follow the time course of a single animal. In this study, in vivo tracking of transgene expression from adeno-aasociated virus type 1 (AAV-1) was performed using a green fluorescence protein (GFP) reporter and an specially designed optical imaging system.. Our results reveal the exact kinetics of AAV-1 expression and validate fluorescence imaging for kinetic measurements of long term expression of virally expressed genes.Materials and Method:Animals and Virus40-day-old male hairless SKH1 mice were divided into 3 groups, each containing 3 mice. The right leg of Groups 1 and 2 were injected with 1012 and 1010 plaque forming unit (pfu) of adeno-associated virus type 1 encoding green fluorescent protain (AAV-1.GFP). respectively, in 50 ul normal saline. The left legs were injected with 50 ul normal saline as an internal control. Group 3 (control group) was injected with 50 ul normal saline into the both legs. The mice were anesthetized by intraperitoneal injection of 80 mg/kg body weight ketamine at the time of each measurement, and imaged over 13 weeks.Imaging SystemSelective excitation of GFP was produced by 475 nm light emission diode through a excitation filter HQ470/40x. Emitted fluorescence was collected through an emission filter HQ525/50m (Chroma Technology Corp, Rockingham, VT) on a Hamamatsu C8484 charge-coupled device camera (Hamamatsu Photonics Systems, Bridgewater, NJ). The strength of the transgene expression was measured as the ratio of the mean of fluorescence intensity of 0.5 cm2 ROI set on the right and left leg.Results and ConclusionsThe kinetics of GFP expression was clearly measured by assessing the fluorescence intensity of each animal. The images and graphs of representative animals are shown in the Figure. In Group 1, injected with 1012 pfu AAV-1.GFP, GFP expression reached a plateau at approximately 7 weeks, which was maintained until at least 13 weeks. In Group 2, injected with 1010 pfu AAV-1.GFP, GFP expression, reached its plateau at approximately 11 weeks. These results demonstrate the relationship between injected dose and transgene expression level,