Shortened nuclear matrix attachment regions are sufficient for replication and maintenance of episomes in mammalian cells

Abstract

Matrix attachment regions (MARs) can mediate the replication of vector episomes in mammalian cells; however, the molecular mode of action remains unclear. Here, we assessed the characteristics of MARs and the mechanism that mediates episomal vector replication in mammalian cells. Five shortened subfragments of β-interferon MAR fragments were cloned and transferred into CHO cells, and transgene expression levels, presence of the gene, and the episomal maintenance mechanism were determined. Three shortened MAR derivatives (position 781–1320, 1201–1740, and 1621–2201) retained full MAR activity and mediated episomal vector replication. Moreover, the three shortened MARs showed higher transgene expression levels, greater efficiency in colony formation, and more persistent transgene expression compared with those of the original pEPI-1 plasmid, and three functional truncated MARs can bind to SAF-A MAR-binding protein. These results suggest that shortened MARs are sufficient for replication and maintenance of episomes in CHO cells.

Advances in the Development and the Applications of Nonviral, Episomal Vectors for Gene Therapy

“…Additionally, the use of a tandem array of four 155 bp modules, designed to represent the core, unwinding S/MAR portion, constitutes the minimal sequence requirements for a S/MAR to function in vivo, and this is due presumably, to the S/MAR sequence ability to recruit cellular replication factors 34 . These data complement findings that the origin of replication complex (ORC) can bind onto the episomal DNA independently of the DNA sequence, and the initiation sites of DNA replication in episomes are determined epigenetically, as is the case with mammalian chromosomes 30 2201), with these regions retaining full activity and mediating episomal vector replication 39 .…”

“…But since it appears that such chromosomal instability was not observed in other studies, including those using cosmid vectors of 38 kb 38 and later studies using the pEPI-1 36 or pEPI derived vectors 26,27 , we propose that it is not a constant feature of K562 cells but rather likely to be randomly connected to the K562 isolate used in that particular study 37 . The exact epigenetic mechanism involved in pEPI-1 as a non-integrating vector is not yet understood, but it seems that the full length S/MAR is not required for completely preventing integration, as investigated by comparing between five smaller overlapping segments of S/MAR transfected into C2C12 muscle cells 39 . Additionally, the S/MAR from the murine c-myc gene 40 could not prevent integration, suggesting this is not a property of all sequences identified as S/MAR.…”

Inexpensive and easy method for 6 fragment Golden Gate Assembly of a modular S/MARs mammalian expression vector and its variants

“…3) One module consists of the reporter gene (cGFP) with a kozack consensus sequence. [10] 4) One module consists of a shortened Nuclear Matrix Attachment Region, MARS5 [7] directly downstream of the gene. 5) One module consists of the Terminator and the poly adenylation sequence.…”

“…One module consists of a shortened Nuclear Matrix Attachment Region , MARS5 [7] directly downstream of the gene.…”

Improved Functionality of Integration-Deficient Lentiviral Vectors (IDLVs) by the Inclusion of IS2 Protein Docks

“…However, no study has focused on improving IDLVs for DSB detection The improvement in gene expression levels was achieved through the inclusion of chromatin boundary elements (CBEs) and genomic elements based on scaffold/matrix attachment regions (S/MARs) [ 25 , 26 , 27 , 28 ]. This is principally due to the capacity of SAR elements to bind transcription factors, such as special AT-rich sequence binding protein 1 (STAB1) [ 29 ], nuclear matrix protein 4 (Nmp4), and the CCCTC-binding factor (CTCF) [ 30 ]. Our team and others have also investigated other elements based on the 1.2 kb 5′chicken β-globin insulator cHS4, resulting in increased episomal efficiency [ 31 , 32 , 33 , 34 , 35 , 36 , 37 , 38 ].…”