Semi-mechanistic pharmacodynamic modelling of gene expression and silencing processes.
BACKGROUND
Suppressed expression of transgene is a major obstacle in gene therapy. Understanding of the mechanisms involved in expression and silencing of exogenous genes is required to overcome gene therapy hurdles.


OBJECTIVE
To develop a semi-mechanistic model describing the effects of transgenes over the activity of an expression cassette.


METHODS
Twelve Balb/c mice received 40microg of plasmid DNA. Animals were assigned to one of the following treatments: (I) 20microg of the plasmid expressing luciferase (pEF-Luc) and 20microg of "empty" plasmid; (II) pEF-Luc (20microg) and 20microg of plasmid expressing murine interferon alpha (IFNalpha); and (III) pEF-Luc (20microg), and 20microg of plasmid expressing beta-galactosidase (pCMVbeta). The expression of luciferase over time, quantified by a noninvasive method, was used as a measured of pEF-Luc activity and modelled using NONMEM.


RESULTS
The selected model suggests the co-existence of two forms of active DNA differing in their transcription efficiencies. The core model was expanded to describe reversible and irreversible silencing processes, induced by the coexpression of IFNalpha or beta-galactosidase, respectively.


CONCLUSIONS
Coupling noninvasive in vivo imaging and mathematical modelling allows quantitative description of gene transfer, providing a tool to select the best regulatory elements to construct a therapeutic expression cassette.
Berraondo
Pedro
P
Division of Hepatology and Gene Therapy, Center for Investigation in Applied Medicine (CIMA), Avda. Pío XII 55, 31008 Pamplona, Spain.
González-Aseguinolaza
Gloria
G
Trocóniz
Iñaki F
IF
D000465:Algorithms; D000818:Animals; D002855:Chromatography, Thin Layer; D004247:DNA / Q000737:chemistry / Q000235:genetics; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D015870:Gene Expression / Q000187:drug effects*; D020868:Gene Silencing / Q000187:drug effects*; D015316:Genetic Therapy; D016898:Interferon-alpha / Q000096:biosynthesis / Q000235:genetics; D008156:Luciferases / Q000235:genetics; D049449:Luminescence; D051379:Mice; D008807:Mice, Inbred BALB C; D008957:Models, Genetic; D010957:Plasmids / Q000737:chemistry / Q000235:genetics; D001616:beta-Galactosidase / Q000096:biosynthesis / Q000235:genetics
D016898:Interferon-alpha; D004247:DNA; D008156:Luciferases; D001616:beta-Galactosidase