Matskevich Alexey A, Cordelier Pierre, Strayer David S
Department of Pathology and Cell Biology, Jefferson Medical College, Philadelphia, PA 19107.
J Interferon Cytokine Res. 2003 Dec;23(12):709-21. doi: 10.1089/107999003772084824.
Chronic infection with hepatitis B virus (HBV) has potentially devastating consequences and is very difficult to treat. Therapy with recombinant interferons (IFN), especially IFN-alpha, may be effective. The blood IFN-alpha levels that are needed to maintain therapeutic IFN-alpha levels in the liver, however, often cause severe side effects. Gene delivery to the liver may provide a solution. Using a long-term expression construct could provide the desired levels of IFN locally without the need to maintain potentially problematic blood levels. Recombinant, Tag-deleted SV40-derived vectors transduce hepatocytes efficiently and provide permanent transgene expression. We designed an expression construct that was effective against HBV and whose activity was limited to HBV-infected cells. To do this, we exploited the ability of HBV X protein to activate NF-kappaB and, via NF-kappaB, to activate promoter activity of HIV long terminal repeat (LTR) in hepatocytes. Using HIVLTR as a conditional promoter upstream of human and murine IFN-alpha and IFN-gamma cDNAs, rSV40 vectors were used to test the responsiveness of IFN to HBV and the ability of these IFNs to inhibit HBV transcripts and protein production and to activate IFN signaling in neighboring untransduced cells. We found that in hepatocyte cell lines and in primary hepatocytes, HBV activated the promoter activity of the HIVLTR via NF-kappaB. When whole HBV genome was delivered to cells by transfection to simulate HBV infection, IFN expression was activated, IFNs were produced and secreted, and they protected cells from HBV. Levels of IFN proteins that were secreted in this context were comparable to targeted blood levels needed to control chronic hepatitis viral infection. Further, IFNs that were elicited and secreted in this manner were able to activate IFN-induced signaling pathways in neighboring, untransduced cells and so were likely to provide protection even to cells that the rSV40 vector did not transduce. Gene delivery using such rSV40 vectors expressing IFNs conditionally in response to HBV may be an attractive therapeutic option for the treatment of chronic hepatitis B.
慢性乙型肝炎病毒（HBV）感染可能产生毁灭性后果，且极难治疗。使用重组干扰素（IFN）进行治疗，尤其是干扰素-α，可能有效。然而，维持肝脏中治疗性干扰素-α水平所需的血液干扰素-α水平常常会引发严重的副作用。向肝脏进行基因传递或许能提供一种解决方案。使用长期表达构建体可以在局部提供所需水平的干扰素，而无需维持可能存在问题的血液水平。重组的、缺失标签的SV40衍生载体能高效转导肝细胞并实现转基因的永久表达。我们设计了一种对HBV有效的表达构建体，其活性仅限于HBV感染的细胞。为此，我们利用了HBV X蛋白激活核因子κB（NF-κB）的能力，并通过NF-κB激活肝细胞中HIV长末端重复序列（LTR）的启动子活性。将HIV LTR作为人及小鼠干扰素-α和干扰素-γ cDNA上游的条件性启动子，利用重组SV40载体来测试干扰素对HBV的反应性，以及这些干扰素抑制HBV转录本和蛋白质产生并激活邻近未转导细胞中干扰素信号传导的能力。我们发现，在肝细胞系和原代肝细胞中，HBV通过NF-κB激活HIV LTR的启动子活性。当通过转染将完整的HBV基因组导入细胞以模拟HBV感染时，干扰素表达被激活，干扰素产生并分泌，它们保护细胞免受HBV感染。在此情况下分泌的干扰素蛋白水平与控制慢性肝炎病毒感染所需的目标血液水平相当。此外，以这种方式引发和分泌的干扰素能够激活邻近未转导细胞中的干扰素诱导信号通路，因此甚至可能为重组SV40载体未转导的细胞提供保护。使用这种响应HBV条件性表达干扰素的重组SV40载体进行基因传递，可能是治疗慢性乙型肝炎的一种有吸引力的治疗选择。
