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.
Lufino
Michele M P
MM
The Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford, OX3 7BN, UK.
Manservigi
Roberto
R
Wade-Martins
Richard
R
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