Shock wave permeabilization as a new gene transfer method.

Uptake of naked functional DNA into mammalian cells can be achieved by a number of physical methods. However, for most of these techniques possibilities for therapeutic in vivo applications--especially to solid organs--are often limited. In this report, we describe shock wave permeabilization as a new physical gene transfer method, which can be easily applied, provides great flexibility in the size and sequence of the DNA molecules to be delivered, and which should exhibit an advantageous security profile in vivo. Upon exposure to lithotripter-generated shock waves eukaryotic cells display a temporary increase in membrane permeability. This effect was shown to be caused by cavitation resulting in the transient generation of cell pores which allows the direct transfer of naked plasmid DNA. Shockwave transfection of a variety of cell lines was demonstrated. Since shock waves can be well focused within particular body regions, future applications of extracorporally generated shock waves to tissues simultaneously perfused with DNA solutions might open up the possibility of achieving a regionally enhanced in vivo gene transfer.

The HBV-producing cell line HepG2-4A5: a new in vitro system for studying the regulation of HBV replication and for screening anti-hepatitis B virus drugs.

All tissue culture systems for propagating HBV employed so far make use of tandemly arranged HBV genomes usually under the control of strong foreign promoters. Thus these systems are helpful for virus production but are of limited value in the investigation of the regulation of HBV replication or of the extent to which the expression of viral genes might be influenced by cellular signal transduction pathways. To overcome this barrier we established an HBV-producing cell line (HepG2-4A5) by stably transfecting HepG2 cells with a replication-competent, terminally redundant HBV plasmid (pSPT1.2 xHBV) that contains each of the four major HBV-ORFs only once and exclusively under the control of their own regulatory elements. HepG2-4A5 cells contain a single, nonrearranged, chromosomally integrated, replication-competent HBV genome. In the cytoplasm of HepG2-4A5 cells, all typical viral mRNAs were detectable, but no other viral transcripts were found. Furthermore, all viral gene products are synthesized in a balanced ratio, as close as possible to that found in an in vivo infection. Dane-like particles released from HepG2-4A5 cells were indistinguishable from virions synthesized in vivo, by all physical (electron microscopy, buoyant density) and biochemical (endogenous polymerase reaction, immunogenic behaviour) criteria. Because of the autologous genome organization in this system, the HepG2-4A5 cell line allows studies on the function of the HBV gene products with respect to their involvement in regulating HBV replication under conditions imitating as closely as possible the situation in vivo. Furthermore, this cell line might be a helpful tool in screening antiviral drugs and in studying their effect on regulating HBV replication.

Transactivating function and expression of the x gene of hepatitis B virus.

BACKGROUND/AIMS: The x gene of hepatitis B virus encodes a transactivating factor of 154 amino acids, termed HBx, which stimulates transcription of multiple viral and cellular genes. The transactivating function is probably associated with a tumorigenic potential of HBx, since x gene sequences, encoding functional HBx, have been repeatedly found integrated into the genome of liver carcinoma cells. METHODS: To identify the transactivating domain of HBx, we constructed x gene plasmids encoding full length HBx or HBx fragments. We determined their transactivating function after cotransfection of cells, along with a plasmid that contains a reporter gene driven by the SV40 early promoter/enhancer region. RESULTS: Our results demonstrate that a 95-amino acid fragment of HBx, encompassing amino acids 49 to 143, contains all the elements that are required for the transactivating function. Within this fragment a sequence element, encompassing amino acids 107 to 130, which contains a relatively high number of amino acids with charged side chains, appears to be crucial for the stimulation of gene expression. The influence of deletion mutations on x mRNA steady-state levels and HBx stability was examined. In essence, stable RNA and protein were produced if at least codons 1-82 or 70-154 were present in the deletion plasmids. CONCLUSION: This finding strongly suggests that the deletion of functional domains between codons 49 and 143, but not an instability of RNA and/or protein, was critical for the loss of transactivation.

Multiple simian virus 40 enhancer elements mediate the trans-activating function of the X protein of hepatitis B virus.

Transient transfections of tissue culture cells with plasmids encoding the X protein of hepatitis B virus result in a transcriptional trans-activation of certain target genes. Our experiments reveal that several individual simian virus 40 enhancer elements and a control element present in the mouse major histocompatibility class I gene H-2Kb are able to mediate the trans-activating function of the X protein. The data suggest that known cellular transcription factors that bind specifically to the multiple enhancer elements participate in trans-activation.