215. The Use of miR-122 and Its Target Sequence in Adeno-Associated Virus-Mediated Cytotoxic Gene Therapy

Abstract

There are two challenges in adeno-associated virus (AAV)-mediated cytotoxic gene therapy: i) overexpression of the cytotoxic transgene in the producer cell lines, such as HEK293, hampers efficient AAV vector production; and ii) most, if not all, AAV serotype vectors possess hepatic tropism since liver is the major tissue for cellular metabolism. These two challenges can be partially resolved by tissue specific promoters. However, in the cases that a specific promoter is not available, constitutively active promoters, such as the chicken β-actin (CBA) promoter, have to be used. For example, there is no well-studied specific promoter for hepatic satellite cells, which are the major cell type involved in liver fibrosis. In addition, most tissue-specific are weak promoters and are often too large to be packaged into AAV vectors. To address the above problems simultaneously, we explored the use of miR122 and its target sequence for the conditional regulation of transgene expression not only in the producer cells during AAV production, but also in the liver after systemic delivery. We first established a HEK293 cell line that overexpresses miR122. The functional miR122 expression was confirmed by reverse transcriptase polymerase chain reaction (RT-PCR) as well as Western blot assays against cellular proteins that are known to be regulated by miR122, such as c-Met. The expression of miR122 showed little effect on HEK293 cell growth and AAV protein expression. Next, we generated rAAV plasmids harboring suicide genes under the control of CBA promoter and a miR122 target (miR122T) sequence in the 3’-UTR. Two strong cytotoxic genes were selected, which encodes trichosanthin (a type 1 ribosome-inactivating protein) and diphtheria toxin (an exotoxin causing diphtheria). The presence of miR122T sequence had little effect on the protein functions in miR122-free cells, but significantly reduced the protein expression in the cells overexpressing miR122. Consequently, the AAV8 vectors carrying suicide genes and miR122T sequence yielded significantly increased production, up to 75-fold, in the HEK293-miR122 cells, when compared to that in the parental HEK293 cells (Table 1). Finally, we showed that AAV8 vectors that were produced from the HEK293-miR122 cells preserved the same tropisms and full bioactivity in vivo. Most importantly, the AAV8 vectors carrying a miR122T sequence mediated little transgene expression in normal liver. Taken together, we conclude that the use of HEK293-miR122 cells and a miR122T sequence should be applied to attenuate the transgene cytotoxicity during AAV vector production and infection of normal liver tissues.HEK293-miR122 and miR122T sequence together increase yields of AAV vectors carrying suicide genes.HEK293Batch 1Batch 2Batch 3AveSDssAAV8-Fluc2.68E + 129.96E + 112.78E + 122.15E + 121.00E + 12ssAAV8-Fluc-122T8.72E + 112.28E + 121.34E + 121.50E + 127.17E + 11ssAAV8-TCS6.23E + 081.34E + 093.75E + 087.79E + 085.01E + 08ssAAV8-TCS-122T2.34E + 091.68E + 094.27E + 081.48E + 099.72E + 08HEK293-miR122Batch 1Batch 2Batch 3AveSDssAAV8-FLuc4.04E + 126.60E + 121.05E + 123.90E + 122.78E + 12ssAAV8-FLuc-122T5.61E + 122.74E + 127.80E + 117.80E + 112.43E + 12ssAAV8-TCS3.45E + 091.39E + 098.50E + 081.90E + 091.37E + 09ssAAV8-TCS-122T1.66E + 118.50E + 109.30E + 101.15E + 114.46E + 10 View Table in HTML