Gene therapy of liver diseases requires the development of efficient vectors for gene transfer in vivo. Retroviral and adenoviral vectors have been shown to deliver genes efficiently into hepatocytes in vitro and in vivo. However, these vectors do not allow for exclusive infection of the liver which would be highly advantageous for in vivo gene therapy strategies. We have recently demonstrated that genetically modified baculoviruses (Autographa californica nuclear polyhedrosis virus) efficiently deliver genes into cultured cells and have a strong preference for hepatocytes of different origin. Baculoviral gene transduction efficiency into human hepatocytes was determined to approach 100% and expression levels are high, provided that gene expression is controlled by mammalian promoters. In this report, we present further properties of baculoviruses regarding their use for hepatocyte gene transfer. Baculovirus-mediated gene expression declines rapidly in the hepatocellular carcinoma cell line Huh7 and more slowly in primary cultures of mouse hepatocytes. Direct application of baculoviruses for gene delivery to the liver in vivo is hampered by serum components, presumably by complement. However, we demonstrate here that baculoviral gene transfer is feasible in ex vivo perfused human liver tissue. This result suggests the development of a strategy using baculoviral vectors for liver-directed gene therapy.

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There is a discrepancy in telomere length as measured by signal intensity of telomere restriction fragments on gels and fluorescence in situ hybridization analysis. This difference has been ascribed to the X-region, a segment of subtelomeric DNA that is resistant to being cut by restriction enzymes. To explore the nature of this region, we analyzed the digestibility of an artificial seeded telomere in HeLa cells as well as the Xp/Yp autosomal telomere in human BJ fibroblasts. We found that there is a substantial fraction of subtelomeric DNA containing restriction sites that is not digested with enzymes such as EcoRI, NlaIII, and SphI. Comparison of methylation-sensitive and -resistant enzymes excluded the possibility of the X-region being maintained by DNA methylation. We show that the X-region represents a variable domain whose size changes with telomere length, and neither non-TTAGGG sequences nor cytidine methylation can adequately explain the size of the X-region.