An S/MAR-based infectious episomal genomic DNA expression vector provides long-term regulated functional complementation of LDLR deficiency

Episomal gene expression vectors offer a safe and attractive alternative to integrating vectors. Here we describe the development of a high capacity episomal vector system exploiting human episomal retention sequences to provide efficient vector maintenance and regulated gene expression through the...

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Bibliographic Details
Published in:Nucleic acids research 2007-08, Vol.35 (15), p.e98-e98
Main Authors: Lufino, Michele M.P., Manservigi, Roberto, Wade-Martins, Richard
Format: Article
Language:English
Subjects:
Animals
CHO Cells
Cholesterol - pharmacology
Clone Cells
Cricetinae
Cricetulus
Gene Deletion
Gene Expression - drug effects
Gene Expression Regulation
Genetic Vectors
Genome, Human
Herpes simplex virus 1
Herpesvirus 1, Human - genetics
Humans
Matrix Attachment Regions
Methods Online
Plasmids - genetics
Receptors, LDL - genetics
Transgenes
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Summary:Episomal gene expression vectors offer a safe and attractive alternative to integrating vectors. Here we describe the development of a high capacity episomal vector system exploiting human episomal retention sequences to provide efficient vector maintenance and regulated gene expression through the delivery of a genomic DNA locus. The iBAC-S/MAR vector is capable of the infectious delivery and retention of large genomic DNA transgenes by exploiting the high transgene capacity of herpes simplex virus type 1 (HSV-1) and the episomal retention properties of the scaffold/matrix attachment region (S/MAR). The iBAC-S/MAR vector was used to deliver and maintain a 135 kb genomic DNA insert carrying the human low density lipoprotein receptor (LDLR) genomic DNA locus at high efficiency in CHO ldlr−/− a7 cells. Long-term studies on CHO ldlr−/− a7 clonal cell lines carrying iBAC-S/MAR-LDLR demonstrated low copy episomal stability of the vector for >100 cell generations without selection. Expression studies demonstrated that iBAC-S/MAR-LDLR completely restored LDLR function in CHO ldlr−/− a7 cells to physiological levels and that this expression can be repressed by ∼70% by high sterol levels, recapitulating the same feedback regulation seen at the endogenous LDLR locus. This vector overcomes the major problems of vector integration and unregulated transgene expression.
ISSN:0305-1048
1362-4962
DOI:10.1093/nar/gkm570