Long-Term and Therapeutic-Level Hepatic Gene Expression of Human Factor IX after Naked Plasmid Transfer in Vivo

Abstract

Naked DNA transfer of a high-expressing human factor IX (hFIX) plasmid yielded long-term (over 1 1/2 years) and therapeutic-level (0.5-2 microg/ml) gene expression of hFIX from mouse livers. The expression cassette contained a hepatic locus control region from the ApoE gene locus, an alpha1-anti-trypsin promoter, hFIX cDNA, a portion of the hFIX first intron, and a bovine growth hormone polyadenylation signal. In contrast, a hFIX plasmid containing the expression cassette without effective regulatory elements produced initially 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 indicated that at least two processes are critical for sustained gene expression: persistence of vector DNA and transcriptional/posttranscriptional activation. Liver regeneration after partial hepatectomy resulted in a significant decline in transgene expression, further suggestive of decreased episomal plasmid maintenance rather than transgene integration. Transaminase levels 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 histologically normal tissue. No significant differences were observed between rapid injection of plasmid and saline control solutions. Transient, very low level antibodies directed against hFIX did not prevent the circulation of therapeutic levels of the protein. Gene transfer of hFIX plasmid DNA into liver elicited neither transgene-specific cytotoxic effect nor long-term toxicity. These results demonstrate that long-term expression of hFIX can be achieved by nonviral plasmid transfer and suggest that this occurs independent of integration.

Persistent episomal transgene expression in liver following delivery of a scaffold/matrix attachment region containing non-viral vector

“…The rapid decline in the transgene expression in the first few days after hydrodynamic delivery may be due to a combination of elimination of transduced cells damaged in the infusion process and degradation of any unstable pDNA in the nucleus as previously suggested. 30 Thereafter, however, we observed a remarkably stable passive maintenance of the pLucA1 expression plasmid in liver hepatocytes. This plasmid vector is gradually lost during the natural turnover of liver cells with time and disappears rapidly after stimulation of hepatocyte cell division by partial hepatectomy.…”

“…The sharp decline in vector number in the immediate weeks following Persistent expression in vivo from S/MAR plasmid O Argyros et al administration is likely due to the administration procedure; transduced cells damaged in the infusion process will be eliminated and unstable pDNA in the nucleus degraded. 30 It has been suggested that, eventually, only properly established vector molecules, influenced by higher nuclear architecture in the hepatocyte nucleus, will be able to express the luciferase transgene for extended periods of time. 31 Without …”

Major role of local immune responses in antibody formation to factor IX in AAV gene transfer

“…5 Hepatic gene expression of F.IX via AAV or plasmid vector has been shown to direct sustained F.IX expression without inhibitor formation in hemophilia B mice and dogs with deletion and nonsense mutations. 3,[16][17][18][19] This route of vector administration was found to induce immune tolerance to the F.IX transgene product. 20 Tolerance induction was favored by higher levels of F.IX expression and was associated with induction of regulatory CD4 + T cells that could suppress antibody formation to F.IX after adoptive transfer.…”

Bioengineering of differentiated hepatocytes withhuman factor IX-expressing plasmidsin vitro

“…Furthermore, Non-viral vectors have the advantage of being less toxic and unable to generate harmful replication-competentby-products through recombinant events as with viral vectors. 18 Introns as cis-regulatory elements have potential to improve gene expression in a broad range of organisms. 19 Introns and their removal by spliceosomes regulate the expression of genes in different levels, including transcription, polyadenylation, nuclear mRNA export, translational efficiency and mRNA decay.…”