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In contrast to AAV, Simian Virus 40 (rSV40) not inducing neutralizing antibodies (NAbs) allowing re-treatment seems a promising vector for neonatal treatment of inherited liver disorders. Several studies have reported efficacy of rSV40 in animal models for inherited liver diseases. In all studies the ubiquitous endogenous early promoter controlled transgene expression establishing expression in all transduced tissues. Restricting this expression to the target tissues reduces the risk of immune response to the therapeutic gene. In this study a liver specific rSV40 vector was generated by inserting a hepatocyte specific promoter. This increased the specificity of the expression of hUGT1A1 in vitro. However, in vivo the efficacy of rSV40 appeared too low to demonstrate tissue specificity while increasing the vector dose was not possible because of toxicity. In contrast to earlier studies, neutralizing antibodies were induced. Overall, the lack of a platform to produce high titered and pure rSV40 particles and the induction of NAbs, renders it a poor candidate for in vivo gene therapy., Competing Interests: The authors have declared that no competing interests exist.

The Crigler-Najjar Syndrome Type I (CNSI) is a rare genetic disorder caused by mutations in the Ugt1a1 gene. It is characterized by unconjugated hyperbilirubinemia that may result in severe neurologic damage and death if untreated. To date, liver transplantation is the only curative treatment. With the aim of generating mutant cell lines of the Ugt1 gene, we utilized the TALEN technology to introduce site-specific mutations in Ugt1 exon 4. We report a fast and efficient method to perform gene knockout in tissue culture cells, based on the use of TALEN pairs targeting restriction enzyme (RE) sites in the region of interest. This strategy overcame the presence of allele-specific single nucleotide polymorphisms (SNPs) and pseudogenes, conditions that limit INDELs' detection by Surveyor. We obtained liver-derived murine N-Muli cell clones having INDELs with efficiency close to 40%, depending on the TALEN pair and RE target site. Sequencing of the target locus and WB analysis of the isolated cell clones showed a high proportion of biallelic mutations in cells treated with the most efficient TALEN pair. Ugt glucuronidation activity was reduced basal levels in the biallelic mutant clones. These mutant liver-derived cell lines could be a very useful tool to study biochemical aspects of Ugt1 enzyme activity in a more natural context, such as substrate specificity, requirement of specific co-factors, the study of inhibitors and other pharmacological aspects, and to correlate enzyme activity to the presence of specific mutations in the gene, by adding back to the mutant cell clones specific variants of the Ugt1 gene. In addition, since genome editing has recently emerged as a potential therapeutic approach to cure genetic diseases, the definition of the most efficient TALEN pair could be an important step towards setting up a platform to perform genome editing in CNSI.

Astrocytomas and their most malignant variant glioblastoma multiforme (GBM) represent the vast majority of primary brain tumors. Despite the current progress in neurosurgery, radiation therapy and chemotherapy, most astrocytomas remain fatal disorders. Although brain tumor biology is a matter of intense research, the cell-of-origin and the complete astrocytoma-inducing signaling pathway remain unknown. To further identify the mechanisms leading to gliomagenesis, we transduced primary astrocytes on a p53(-/-) background with c-Myc, constitutively active myr-Akt or both, myr-Akt and c-Myc. Transduced astrocytes showed oncogene-specific alterations of morphology, proliferation and differentiation. Following prolonged periods of cultivation, oncogene-transduced astrocytes expressed several stem-cell markers. Furthermore, astrocytes coexpressing c-Myc and Akt were tumorigenic when implanted into the brain of immunocompetent C57BL/6 mice. Our results reveal that the loss of p53 combined with oncogene overexpression in mature astrocytes simulates pivotal features of glioma pathogenesis, providing a good model for assessing the development of secondary glioblastomas.