Abstract
BackgroundTherapeutic gene transfer is currently being evaluated as a potential therapy for inflammatory bowel disease. This study investigates the safety and therapeutic benefit of a locally administered lentiviral vector encoding murine interleukin-10 in altering the onset and relapse of dextran sodium sulfate induced murine colitis.MethodsLentiviral vectors encoding the reporter genes firefly-luciferase and murine interleukin-10 were administered by intrarectal instillation, either once or twice following an ethanol enema to facilitate mucosal uptake, on Days 3 and 20 in Balb/c mice with acute and relapsing colitis induced with dextran sulfate sodium (DSS). DSS colitis was characterized using clinical disease activity, macroscopic, and microscopic scores. Bioluminescence optical imaging analysis was employed to examine mucosal lentiviral vector uptake and transgene expression. Levels of tumor necrosis factor-α and interleukin-6 in homogenates of rectal tissue were measured by ELISA. Biodistribution of the lentiviral vector to other organs was evaluated by real time quantitative PCR.ResultsMucosal delivery of lentiviral vector resulted in significant transduction of colorectal mucosa, as shown by bioluminescence imaging analysis. Lentiviral vector-mediated local expression of interleukin-10 resulted in significantly increased levels of this cytokine, as well as reduced levels of tumor necrosis factor-α and interleukin-6, and significantly reduced the clinical disease activity, macroscopic, and microscopic scores of DSS colitis. Systemic biodistribution of locally instilled lentiviral vector to other organs was not detected.ConclusionsTopically-delivered lentiviral vectors encoding interleukin-10 safely penetrated local mucosal tissue and had therapeutic benefit in this DSS model of murine colitis.
Highlights
Therapeutic gene transfer is currently being evaluated as a potential therapy for inflammatory bowel disease
Vector construction and preparation The pRRLsin-hCMV-fLuc vector was constructed by insertion of the firefly Luciferase gene [14], and the pRRLsin-hCMV-Murine interlukin 10 (mIL10) vector by insertion of the mIL10 coding sequence from plasmid pORF5-murine IL-10 (mIL-10) (InvivoGen, San Diego, CA, USA), respectively, into the multiple cloning site (MCS) of pRRLsin-hCMV-MCSpre, a third-generation, self-inactivating lentiviral vector (LV) construct provided by Dr Luigi Naldini (San Rafaelle Telethon Institute, Milan, Italy) [15]
Two days following topical exposure to a LV expressing fLuc, GI tract organs from each group were harvested en bloc, bathed and imaged using a cooled charge-coupled device (CCD) system (Xenogen IVIS, Caliper Life Sciences, Alameda, CA, USA) where gray-scale background photographic images of the tissues were overlaid with color images of bioluminescent signals (Living Image and IGOR-PRO image analysis software, Wave Metrics, Portland, OR, USA)
Summary
Therapeutic gene transfer is currently being evaluated as a potential therapy for inflammatory bowel disease. Inflammatory bowel disease (IBD), comprising Crohn’s disease (CD) and ulcerative disease (UC), is thought to result from abnormal interactions between gut associated lymphoid tissue and enteric microflora. This abnormal mucosal immune response is probably facilitated by defects in epithelial barrier function [1]. IL-10 has broad immunoregulatory activity and acts to suppress intestinal inflammation on several levels. There is strong evidence that IL-10 acts to promote the differentiation and augment the activity of regulatory T cells [5]
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