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 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 approximately 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.
The Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford, OX3 7BN, UK.
Michele M P
MM
Lufino
Roberto
R
Manservigi
Richard
R
Wade-Martins
D000818:Animals; D016466:CHO Cells; D002784:Cholesterol / Q000494:pharmacology; D002999:Clone Cells; D006224:Cricetinae; D003412:Cricetulus; D017353:Gene Deletion; D015870:Gene Expression / Q000187:drug effects; D005786:Gene Expression Regulation; D005822:Genetic Vectors; D015894:Genome, Human; D018259:Herpesvirus 1, Human / Q000235:genetics; D006801:Humans; D045170:Matrix Attachment Regions; D010957:Plasmids / Q000235:genetics*; D011973:Receptors, LDL / Q000235:genetics*; D019076:Transgenes
D011973:Receptors, LDL; D002784:Cholesterol