Abstract
Infusion of hepatocyte-specific DNA-protein complexes into rats leads to transient recombinant gene expression in liver. The eventual deterioration of gene expression is due in part to instability of the targeted DNA. In a previous report, we noted retention of transgene sequences in liver and persistent recombinant gene expression when the animals were subjected to partial hepatectomy following in vivo gene transfer. In an attempt to define the mechanism(s) responsible for persistent gene expression following partial hepatectomy, we characterized the molecular state of the retained, liver-associated transgenes. Southern blot analysis of DNA from liver tissues harvested various times after in vivo gene transfer and partial hepatectomy (10 min to 11 weeks) demonstrated high levels of transgene DNA (100-10,000 copies/cell). The predominant form of this DNA appeared to be episomal based on analyses of uncut DNA or DNA restricted by an endonuclease with one site in the plasmid. Livers from several animals contained a small proportion of transgene sequences of unknown structure. The existence of episomal DNA in liver was confirmed in experiments in which intact plasmid was rescued from total hepatocyte DNA by transformation of bacteria. Both strands of DNA in the liver-associated plasmid retained a bacterial pattern of methylation suggesting that the plasmid had not replicated in the eukaryotic cell. These results are consistent with the hypothesis that the majority of transgene sequences are retained as stabilized plasmids. The specific form of DNA which is transcriptionally active was not identified in these studies. This represents a new mechanism for retaining foreign DNA in eukaryotic cells in vivo and has implications both for the development of somatic gene therapies and the pathogenesis of viral diseases.
Highlights
Infusion of hepatocyte-specific DNA-protein com- utility of this technology into the clinical arena
We noted retention of trans- autologous somatic cells such as lymphocytes or hepatocytes gene sequences in liver and persistent recombinant are genetically modified with recombinant viruses ex vivo and gene expression when the animals were subjected to partial hepatectomy following in vivo gene transfer
Experiments incultured cells indicate that strands ofDNA in the liver-associated plasmid re- the DNA-protein complex is internalized via the asialoglycotained a bacterial pattern of methylation suggesting protein receptor and therecombinant gene is expressed tranthat the plasmid had not replicated in the eukaryotic siently(Wuand Wu, 1987)
Summary
Partial HepatectomyFollowing in Vivo Gene TransferLeads albumin gene and a polyadenylation sequence from the bovine growth hormone gene (Wu etal., 1989,1991;Wilson et al, 1992).The vectors have been described previously and are essentially identical except for the sequences spanning an open reading frame which encodes a functional protein: cDNA for humLanDL receptor, p9-. The animal injected with p9-12alb(h)LDLR DNA-protein complex had a partial hepatectomy 10 min after injection and was sacrificed 2 weeks later. Experiments with the p912alb(h)LDLR plasmid represent a single animal in which liver tissue wasremoved at 10min (the time of partial hepatectomy) and at 2 weeks (the time a t which the animal was sacrificed)after injection. A single animal that received the p9-12albCAT vector was analyzed; liver was removedat 8weeks (the secondpartial hepatectomy) and 11weeks following DNA-protein complex injection (the time at which the animal was sacrificed). Additional samples include DNA (5 pg) isolated from liver tissue harvested 10 min (lane 3) and 2 weeks (lane 4 ) following transfection of DNA-protein complex. Total cellular DNA (10 pg) was isolated from liver harvested 8 weeks (lane 4 ) and 11 weeks ( l a n e 5) following transfection of DNA-protein complex.
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