Activation of silenced transgene expression in neural precursor cell lines by inhibitors of histone deacetylation.
BACKGROUND
Ex vivo gene therapy in the central nervous system (CNS) holds great promise for diseases such as the neurodegenerative disorders. However, achieving stable, long-term transgene expression in grafted cells has proven problematic. This study reports the establishment of an in vitro model of transgene down-regulation in cells grafted to the CNS using the immortalized neural progenitor cell lines HiB5 and RN33B.


METHODS
Neural cell lines were transduced at 33 degrees C with different GFP constructs, both viral and non-viral, containing either viral or non-viral promoters. Cell differentiation in vitro was obtained by culturing the cells at 37 degrees C in serum-free defined media, which halts cell division, and GFP-expression was analysed by FACS. As early as day 3 of culture at 37 degrees C, the transgene expression decreased markedly in most cell lines. To validate the assay, the same clones were grafted to the adult rat striatum and the down-regulation of GFP-expression was evaluated.


RESULTS
The temporal pattern of down-regulation was found to be similar in vitro and in vivo. Using this assay, it was shown that addition of inhibitors of histone deacetylation, but not an inhibitor of DNA methylation, reversed the silencing of GFP in quiescent neural progenitors by up to 308% of control values.


CONCLUSIONS
These results suggest that the same mechanisms controlling gene transcription of the host cell's genome are active in controlling transgene expression and that this should be taken into account when constructing vectors for gene therapy. The assay reported in this study could be used as a screening method to evaluate new vectors.
Rosenqvist
Nina
N
Wallenberg Neuroscience Center, Department of Physiological Sciences, Lund University, Sweden. nina.rosenqvist@mphy.lu.se
Hård Af Segerstad
Caroline
C
Samuelsson
Christofer
C
Johansen
Jens
J
Lundberg
Cecilia
C
D000818:Animals; D002454:Cell Differentiation; D002461:Cell Line, Transformed; D002843:Chromatin / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005786:Gene Expression Regulation / Q000187:drug effects*; D020868:Gene Silencing; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D006624:Hippocampus / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D056572:Histone Deacetylase Inhibitors; D016086:Lentivirus / Q000235:genetics; D008164:Luminescent Proteins / Q000378:metabolism; D051381:Rats; D013234:Stem Cells / Q000166:cytology / Q000378:metabolism; D019076:Transgenes
D002843:Chromatin; D004791:Enzyme Inhibitors; D056572:Histone Deacetylase Inhibitors; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins