| The liver, which is the human body’s largest internal organ, plays a key role in digestion and metabolism. It bears multiple functions such as bile secretion, sugar, fat and protein metabolism and catabolism of toxic substances. Liver has a special blood circulation system, which makes it able to accept the dual blood supply from the portal vein and hepatic artery. Lobule is the basic structural unit of the liver, hepatocytes arrange around the central vein radially to form the structure of the liver plates. Liver sinusoids exist between liver plates. Nanoparticles less than100nm can be efficiently transferred into the parenchymal cells from the vessel. The specific anatomical structures of liver, as well as its important physiological functions, making it an ideal target organ for gene therapy.With the development of molecular and cell biology, virology and immunology, the molecular genetic pathogenesis of many liver diseases have been interpreted. Besides the traditional liver cell transplantation, gene therapy has become an important and promising strategy for various genetic or acquired liver disease. The biggest problem faced by liver gene therapy is how to transfer therapeutic gene into hepatocytes safely and efficiently, with durable functional expression. Thus, researches focused on the delivery vector system used in liver gene therapy exhibit great significance. To select the delivery vector system, we should consider not only the efficiency and specificity of gene transfer, expression phase of the therapeutic transgene, but also the host immune response induced by the vector and other side effects. Idealized liver gene therapeutic vector could be able to deliver therapeutic genes to the liver tissue specifically while maintain long-time functional expression in resting hepatocytes. The vector itself must be non-toxic and non-immunogenic, simple to operate and easy for clinical applications. Currently, many researchers pay great attentions on the development and improvement of the delivery vector systems, both viral vectors (retrovirus, adenovirus, adeno-associated virus, etc.) and non-viral vector (receptor mediated vector, liposomes, Nano-particles, etc.).This study constructed a novel liver-directed gene therapeutic vector, which had long-term function with high efficiency and low toxicity, also it could manipulate multiple targets simultaneously. This vector could serve as a template system and applied to a variety of liver diseases as a multi-functional therapeutic vector by replacement of multiple target genes. The vector can be delivered into hepatocytes by the non-viral vector or a viral vector and maintain its protective effect for at least one month.Simultaneously silence CC chemokine ligand-5(CCL5) and Fractalkine (FKN) and over-expression of human interleukin-10(huIL-10) can efficiently prevent and treat NK cell-mediated acute liver injury caused by high dose adenoviruses infection or poly I:C joint D-GaIN injection. Liver diseases such as malignant tumors and viral hepatitis is usually associated with disorder of several genes, our study provides new insight into exploring the therapeutic applicability of a single vector that can manipulate multiple genes in a liver-specific manner. Our results suggest a possible extensive and practical utilization for this novel multifunctional vector.1. Construction and verification of hepatocyte-specific multifunctional VectorAfter comparing various promoters, we selected a highly efficient hepatocyte-specific promoter composed by AFP enhancer and albumin promoter. Hepatocyte-specific RNA interference was realized by microRNA-based shRNA (shRNAmir) transcribed from the hepatocyte-specific RNA polymerase Ⅱ promoter. The second generation shRNAmir showed higher interference efficiency than other shRNAs with different structures. The internal ribosome entry site sequence (IRES) and the shRNAmir was used to construct a multifunctional vector, which was able to over-express and silence different genes at the same time. A multi-reporter vector simultaneously over-expressing both P-galactosidase (LacZ) and DsRed2and knocking down both EGFP and luciferase was constructed and functionally tested, only functioned in hepatoma cell Huh7when hepatocyte-specific promoter was employed.2. Liver-directed expressing huIL-10and knocking down CCL5and FKN can effectively inhibit acute liver injury induced by adenovirus.The previous study in our laboratory demonstrated that hydrodynamic injection of adenoviral vector containing FKN small interfering RNA prevented adenovirus-induced acute liver injury significantly. Since interference of single chemokine may not be able to achieve the best potective effect, we constructed a liver-directed vector which could simultaneously manipulate a variety of functional genes. Our experimental results show that, compared with silencing FKN alone, joint interference chemokines FKN and CCL5could further suppress the lymphocyte infiltration and inflammatory cytokine production, reduce the proportion of NK cells in the liver and inhibit liver damage; Silencing FKN combined with over-expressing the inhibitory cytokine huIL-10in the liver also further reduced lymphocytes infiltration and inflammatory cytokine levels. Alleviation of liver injury was also confirmed by histological analysis. We used this hepatocyte-specific multiple function vector template to over-express both DsRed2and human IL-10(huIL10) and interfere with CCL5and FKN expression using3target sequences per chemokine; in total, these8functional fragments synergized to attenuate high dose adenovirus-induced acute hepatitis by reducing liver NK cell number/activation and serum IFN-y and TNF-a. Serum transaminase levels and pathological results also demonstrated that acute liver injury was significantly suppressed.This liver-directed multifunctional vector maintained its protection effect against acute liver injury induced by systemic infection of high dose adenovirus, as long as1months in mice. Moreover, it could be delivered both by non-viral (hydrodynamic injection) and viral (liver tropism adenovirus) approaches. When the liver-directed multifunctional vector packaged into an adenoviral vector and mixed with the vector-free replication-defective adenovirus to infect mice at a high doses, it significantly inhibited the adenovirus-induced acute liver injury with reduced liver lymphocytic infiltration and serum inflammatory cytokines.3. Liver-directed multifunctional vector suppress acute liver injury induced by Poly I:C/D-GaIN injection.D-GaIN is widely used as a liver sensitization agent in a variety of animal models, and Poly I:C mimics RNA virus to stimulate the host immune response. The results of our previous studies demonstrated that Poly I:C/D-GaIN combined injection would induce severe acute liver injury mediated by Kupffer cells and NK cells. Therefore, we employed the liver-directed multifunctional vector that could simultaneously express both huIL-10and DsRed2and silence CCL5and FKN to treat the liver damage. Mice pre-injected with multifunctional vector were found to significantly inhibit lymphocyte infiltration into the liver, also reduce the proportion of liver NK cells and serum inflammatory cytokines IFN-y and TNF-a. Histopathology, Compared with the control group, pathological results showed that there was no significant liver necrosis in mice treated with the liver-directed multifunctional vector. |
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