Fraefel C, Jacoby D R, Lage C, Hilderbrand H, Chou J Y, Alt F W, Breakefield X O, Majzoub J A
Molecular Neurogenetics Unit, Massachusetts General Hospital, Harvard Medical School, Charlestown, USA.
Mol Med. 1997 Dec;3(12):813-25.
Vectors based on herpes simplex virus type 1 (HSV-1) can efficiently transduce hepatocytes in the mouse liver, and vector genomes can persist for at least 2 months. However, 24 hr after gene transfer, the number of cells that express the transgene decreases rapidly and no transduced cells are detectable after 7 days. In this study, we examined the capability of a helper virus-free HSV/AAV hybrid amplicon vector to extend transgene expression in hepatocytes in vivo.
HSV-1 amplicon or HSV/AAV hybrid amplicon vectors that express reporter genes from different transcriptional regulatory sequences were packaged into HSV-1 virions using a helper virus-free packaging system. To determine relative transduction efficiencies, vector stocks were titered on four different cell lines, including hamster kidney (BHK21) and human lung (Hs913T) fibroblasts, and mouse (G6Pase-/-) and human (NPLC) hepatocytes. After in vivo injection of vector stocks into mouse liver, tissue sections were examined for reporter gene expression and cellular inflammatory response. Blood samples were collected to measure serum transaminase levels as a biochemical index of liver toxicity.
Expression of a reporter gene from liver-specific promoter sequences was consistently more effective in hepatic cells compared with fibroblasts, whereas the opposite was true when using an HSV-1 immediate-early promoter. Expression in hepatocytes in vivo was markedly longer from HSV/AAV hybrid vector compared with traditional HSV-1 amplicon vector: the number of transduced cells (approximately 2% of all hepatocytes) remained stable over 7 days after injection of HSV/AAV hybrid vector, whereas no transduced cells were detected 7 days after gene transfer with standard HSV-1 amplicon vector. The rapid decline in reporter gene expression from standard amplicons was not solely caused by a B or T lymphocyte-mediated immune response, as it also occurred in RAG2-/- mice. Hepatocyte toxicity and cellular inflammatory effects associated with HSV/AAV hybrid vector-mediated gene transfer were minimal, and readministration of vector stock proved equally effective in naive mice and in animals that received a first vector dose 4 weeks earlier.
HSV/AAV hybrid amplicon vectors support gene expression in vivo for considerably longer than do traditional HSV-1 amplicon vectors. Moreover, expression from these vectors does not provoke an overt inflammatory or immune response, allowing efficacious expression following repeated in vivo dosing. These characteristics suggest that such vectors may hold future promise for hepatic gene replacement therapy.
基于1型单纯疱疹病毒（HSV-1）的载体能够有效地转导小鼠肝脏中的肝细胞，并且载体基因组可以持续存在至少2个月。然而，基因转移后24小时，表达转基因的细胞数量迅速减少，7天后检测不到转导细胞。在本研究中，我们检测了一种无辅助病毒的HSV/AAV杂交扩增子载体在体内延长肝细胞中转基因表达的能力。
使用无辅助病毒包装系统将表达来自不同转录调控序列的报告基因的HSV-1扩增子或HSV/AAV杂交扩增子载体包装到HSV-1病毒粒子中。为了确定相对转导效率，在四种不同的细胞系上测定载体储备液的滴度，包括仓鼠肾（BHK21）和人肺（Hs913T）成纤维细胞，以及小鼠（G6Pase-/-）和人（NPLC）肝细胞。将载体储备液体内注射到小鼠肝脏后，检查组织切片中的报告基因表达和细胞炎症反应。采集血样以测量血清转氨酶水平作为肝毒性的生化指标。
与成纤维细胞相比，来自肝脏特异性启动子序列的报告基因在肝细胞中的表达始终更有效，而使用HSV-1立即早期启动子时情况则相反。与传统的HSV-1扩增子载体相比，HSV/AAV杂交载体在体内肝细胞中的表达明显更长：注射HSV/AAV杂交载体后7天内，转导细胞的数量（约占所有肝细胞的2%）保持稳定，而用标准HSV-1扩增子载体进行基因转移7天后未检测到转导细胞。标准扩增子报告基因表达的快速下降并非仅由B或T淋巴细胞介导的免疫反应引起，因为在RAG2-/-小鼠中也会发生这种情况。与HSV/AAV杂交载体介导的基因转移相关的肝细胞毒性和细胞炎症效应最小，并且再次给予载体储备液在未接触过的小鼠和4周前接受过首次载体剂量的动物中同样有效。
HSV/AAV杂交扩增子载体在体内支持基因表达的时间比传统HSV-1扩增子载体长得多。此外，这些载体的表达不会引发明显的炎症或免疫反应，允许在多次体内给药后有效表达。这些特性表明，此类载体可能在未来的肝脏基因替代治疗中具有前景。
