This is related to the unique cellular environment of the liver, which is physiologically involved in the appropriate recognition of self versus non-self molecules and pathogens [10], [11]. Reduced or absent activation of the immune system selleck kinase inhibitor after vector administration represents an advantage for the induction of transgene-specific tolerance [1]. The low immunogenicity of AAV vectors, partly due to their inefficiency in infecting dendritic cells (DC) or macrophages, favours tolerance induction [1]. Despite this, examples of transgene-directed immune responses following liver gene delivery have been reported [4], [5], [12]�C[15]. The tolerance obtained after liver gene delivery requires optimization of gene transfer strategies to eliminate transgene expression in antigen presenting cells (APC) while restricting high levels of therapeutic expression to hepatocytes [1].

The choice of regulatory elements to be included in the transgene expression cassette has a direct impact on the levels and cellular restriction of gene expression. This is crucial to improve both therapeutic efficacy and safety, and to avoid the development of transgene-directed immune responses in liver gene transfer protocols. Hepatocyte-specific promoters, such as the thyroxine-binding-globulin promoter (also called liver-specific, TBG or LSP [16]�C[25]), are often used to obtain liver-restricted transgene expression. In addition, the inclusion of microRNA (miRNA) target sequences in the vector expression cassette can help to eliminate off-target transgene expression from transduced cells that express the corresponding miRNA [26].

Moreover transgene expression levels can be improved by the inclusion, in the transgene expression cassette, of post-trascriptional regulatory elements such as the Woodchuck hepatitis virus (WHV) post-transcriptional regulatory element (WPRE, [27]), able to increase transcript levels and/or stability. Equally important is the timing of vector administration, as well as the pathological conditions, that account for alterations of the microenvironment and cell function of the liver, which could affect the efficacy and safety of liver gene transfer [4], [28]. Here we investigated the impact of the following factors on the efficacy of AAV2/8-mediated liver gene transfer in rats, commonly used as animal models for the development of gene therapy strategies: Anacetrapib i) age at vector administration, ii) presence of lysosomal storage in liver cells, and iii) regulatory elements included in the transgene expression cassette.