Improving gene replacement by intracellular formation of linear homologous DNA.
BACKGROUND
Gene targeting is a potential tool for gene therapy but is limited by the low rate of homologous recombination. Using highly homologous linear DNA improves gene targeting frequency but requires microinjection into nuclear cells to be effective. Because transfection of circular DNA is more efficient than transfection of linear DNA and adaptable to viral vectors, we developed a system for the intracellular release of linear fragments from circular plasmids.


METHODS
Only one cutting site inside the "donor" DNA was not convenient because it led to integration of exogenous sequences into the target. So we constructed several "donor" plasmids containing the homologous sequences flanked by two I-Sce I recognition sites. Expression of I-Sce I allowed intracellular delivery of "ends-out" (replacement) vectors. We compared the efficiency of different constructions to correct a mutated gfp target.


RESULTS
Co-transfection of "donor" plasmids and an I-Sce I expression vector into CHO cells enhanced the correction of an extrachromosomal mutated gfp target by at least 10 times. Maximum correction was observed with the greatest homology size and maximum effect of I-Sce I was obtained when the long hemi-sites of the duplicated I-Sce I sites were contiguous to the homologous sequence. Unexpectedly, the reverse orientation of I-Sce I sites provided little or no effect, probably due to the asymmetrical activity of the I-Sce I meganuclease.


CONCLUSIONS
Releasing homologous DNA fragments with I-Sce I enhances gene replacement. This work provides the basis for the future design of viral vectors for gene replacement.
de Piédoue
G
G
INSERM emi 0111, laboratoire de Thérapie Génique Hématopoïétique, Institut Universitaire d'Hématologie, Hôpital Saint-Louis, 1 Av. C. Vellefaux, 75010 Paris, France.
Maurisse
R
R
Kuzniak
I
I
Lopez
B
B
Perrin
A
A
Nègre
O
O
Leboulch
P
P
Feugeas
J-P
JP
D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D004270:DNA, Circular / Q000235:genetics*; D015252:Deoxyribonucleases, Type II Site-Specific / Q000494:pharmacology*; D018390:Gene Targeting / Q000379:methods; D005822:Genetic Vectors / Q000032:analysis* / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000235:genetics*; D011995:Recombination, Genetic; D029701:Saccharomyces cerevisiae Proteins; D014162:Transfection
D004270:DNA, Circular; D029701:Saccharomyces cerevisiae Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; C037725:SCEI protein, S cerevisiae; D015252:Deoxyribonucleases, Type II Site-Specific