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Long-term and therapeutic-level hepatic gene expression of human factor IX after naked plasmid transfer in vivo.
- Source :
-
Molecular therapy : the journal of the American Society of Gene Therapy [Mol Ther] 2001 Jun; Vol. 3 (6), pp. 947-57. - Publication Year :
- 2001
-
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.
- Subjects :
- Animals
Cell Line
DNA Primers chemistry
Factor IX metabolism
Genetic Therapy methods
Genetic Vectors
Hemophilia B blood
Hemophilia B genetics
Hemophilia B therapy
Humans
Immunoglobulin G immunology
In Vitro Techniques
Injections, Intravenous
Locus Control Region
Mice
Mice, Inbred C57BL
Plasmids administration & dosage
Reverse Transcriptase Polymerase Chain Reaction
Factor IX genetics
Gene Expression
Liver metabolism
Plasmids genetics
Transfection
Subjects
Details
- Language :
- English
- ISSN :
- 1525-0016
- Volume :
- 3
- Issue :
- 6
- Database :
- MEDLINE
- Journal :
- Molecular therapy : the journal of the American Society of Gene Therapy
- Publication Type :
- Academic Journal
- Accession number :
- 11407909
- Full Text :
- https://doi.org/10.1006/mthe.2001.0333