Liver-specific promoters are often used to restrict viral vector-mediated expression to hepatocytes. As aberrant transgene expression has been reported with both synthetic and native hepatocyte-specific http://www.selleckchem.com/products/lapatinib.html promoters [35], [62], we tested if this is also the case with the widely used TBG promoter. Although we found that AAV2/8-TBG-mediated transduction is restricted to hepatocytes in both wild-type and MPS VI liver, we observed detectable eGFP expression in the spleen and kidney of wild-type rats injected systemically with AAV2/8-TBG-eGFP at P4, suggesting that the TBG promoter is not hepatocyte-specific, at least in newborn rats. This partially agrees with data reported from Bell et al. and Wang et al., showing that low levels of eGFP mRNA are detected in the spleen of dogs [25] and non-human primates [22] injected sistemically with AAV2/8-TBG-eGFP.

However, we can not exclude that the eGFP expression we observed in non-hepatic rat tissues may derive from the promoter activity associated with the AAV inverted terminal repeats (ITR) [63]. Interestingly, AAV vector genomes were higher in the spleen from rats injected at P30 compared to those injected at P4; thus, proliferation-related dilution of AAV vector genomes may occur in newborn spleen as observed for the liver. Alternatively, efficiency of adult and newborn spleen cells transduction after systemic AAV2/8 administration might be different. In addition, it is possible that different cell types are transduced in newborn and adult spleen after systemic AAV2/8-injection, and this could explain the absence of detectable eGFP expression in the spleen from rats injected at P30, despite the high number of gc/mdg.

Off-target transgene expression in splenic APC cells after systemic injection of lentiviral vectors has been described to induce transgene-directed immune responses in the context of liver-directed gene transfer [35]. This was avoided by the inclusion of target sites for miR142-3p in the transgene expression cassette. Even though AAV vectors are considered overall inefficient at APC transduction [1], [64], transgene expression in DC or macrophages has been reported after AAV delivery [65], [66]. We hypothesized that this may contribute to the anti-hARSB immune responses we observed in MPS VI rats after liver transduction with AAV2/8-TBG-hARSB [14], [15] and that this could be prevented by the inclusion of the miR142-Tx4 element in the AAV2/8-TBG-hARSB expression cassette.

However, the low ARSB serum levels and the high anti-ARSB antibody titers we found despite the use of the miR142-3p target sequence argue against this. This is not surprising since ARSB, like other lysosomal enzymes, is secreted from producing cells and can be efficiently Anacetrapib uptaken via the mannose-6-phosphate receptor pathway from non-transduced cells [32], possibly including APC, thus resulting in immune system activation independently of APC transduction.