Long-term and therapeutic-level hepatic gene expression of human factor IXafter naked plasmid transfer in vivo

Naked DNA transfer of a high-expressing human factor IX (hFIX) plasmid yiel ded long-term lover 1 1/2 years) and therapeutic-level (0.5-2 mug/ml) gene expression of hFIX from mouse livers. The expression cassette contained a h epatic locus control region from the ApoE gene locus, an alpha1-antitrypsin promoter, hFIX cDNA, a portion of the hFIX first intron, and a bovine grow th hormone polyadenylation signal. In contrast, a hFIX plasmid containing t he expression cassette without effective regulatory elements produced initi ally low-level gene expression that rapidly declined to undetectable levels . Southern analyses of the cellular DNA indicated that the majority of the input genome from either vector persisted as episomal forms of the original plasmids. Together with RT-PCR analyses of the transcripts, these data ind icated that at least two processes are critical for sustained gene expressi on: persistence of vector DNA and transcriptional/posttranscriptional activ ation. Liver regeneration after partial hepatectomy resulted in a significa nt decline in transgene expression, further suggestive of decreased episoma l plasmid maintenance rather than transgene integration. Transaminase level s and liver histology showed that rapid intravenous plasmid injection into mice induced transient focal acute liver damage (<5% of hepatocytes), which was rapidly repaired within 3 to 10 days and resulted thereafter in histol ogically normal tissue. No significant differences were observed between ra pid injection of plasmid and saline control solutions. Transient, very low level antibodies directed against hFIX did not prevent the circulation of t herapeutic levels of the protein. Gene transfer of hFIX plasmid DNA into li ver elicited neither transgene-specific cytotoxic effect nor long-term toxi city. These results demonstrate that long-term expression of hFIX can be ac hieved by nonviral plasmid transfer and suggest that this occurs independen t of integration.