The IS2 Element Improves Transcription Efficiency of Integration-Deficient Lentiviral Vector Episomes

Zhang

et al. 2019

MBoC

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.

Section: Introductionmentioning

confidence: 99%

“…, 2011a). IS2 element, which harbors SARs and HS4 sequences, can increase the percentage and expression levels of the transgene in several cell lines (Sánchez-Hernández et al. , 2018).…”

Section: Introductionmentioning

confidence: 99%

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

Zhang

et al. 2019

MBoC

“…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 ]. This is partly due to the interaction of cHS4 with the nuclear matrix though CTCF nuclear proteins [ 31 ].…”

Section: Introductionmentioning

confidence: 99%

“…For instance, our group recently designed the chimeric insulator IS2, which combines a 388 pb fragment composed of our MAR/SAR recognition signatures (MRS) pSAR2 with the genetic element cHS4_650 [ 38 ]. The insertion of this chimeric element into the 3′LTR of lentiviral vectors increases the transcriptional activity of IDLV episomes up to six-fold, but also causes a sharp reduction in the level of intracellular vector genomes, probably due to interference with the reverse transcription process which reduces particle formation [ 36 , 39 ]. The final impact of the IS2 element in IDLVs depends on the balance between these two effects on the target cells [ 36 ].…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2021

Self Cite

Integration-deficient lentiviral vectors (IDLVs) have recently generated increasing interest, not only as a tool for transient gene delivery, but also as a technique for detecting off-target cleavage in gene-editing methodologies which rely on customized endonucleases (ENs). Despite their broad potential applications, the efficacy of IDLVs has historically been limited by low transgene expression and by the reduced sensitivity to detect low-frequency off-target events. We have previously reported that the incorporation of the chimeric sequence element IS2 into the long terminal repeat (LTR) of IDLVs increases gene expression levels, while also reducing the episome yield inside transduced cells. Our study demonstrates that the effectiveness of IDLVs relies on the balance between two parameters which can be modulated by the inclusion of IS2 sequences. In the present study, we explore new IDLV configurations harboring several elements based on IS2 modifications engineered to mediate more efficient transgene expression without affecting the targeted cell load. Of all the insulators and configurations analysed, the insertion of the IS2 into the 3′LTR produced the best results. After demonstrating a DAPI-low nuclear gene repositioning of IS2-containing episomes, we determined whether, in addition to a positive effect on transcription, the IS2 could improve the capture of IDLVs on double strand breaks (DSBs). Thus, DSBs were randomly generated, using the etoposide or locus-specific CRISPR-Cas9. Our results show that the IS2 element improved the efficacy of IDLV DSB detection. Altogether, our data indicate that the insertion of IS2 into the LTR of IDLVs improved, not only their transgene expression levels, but also their ability to be inserted into existing DSBs. This could have significant implications for the development of an unbiased detection tool for off-target cleavage sites from different specific nucleases.

“…So far, several insulators or elements, such as the chicken hypersensitive site 4 (cHS4) insulator or scaffold or matrix attachment regions elements, have been found to play positive roles in enhancing transgene expression or blocking transgene silencing in previous studies [14][15][16]. The combination of HS4 and scaffold attachment region elements (chimeric insulator) was also reported to enhance transgene expression across different cell lines, such as human pluripotent stem cells, Chinese hamster ovary cells and 293T cells [17][18][19]. The most widely used is the cHS4 insulator, which possesses both barrier activity (blocking the spreading of heterochromatin from gene silencing) and enhancer blocking activity (segregating enhancer and promoter from interaction) [20,21], and was often positioned among gene expression cassettes.…”

mentioning

confidence: 99%

Characterization of the cHS4 insulator in mouse embryonic stem cells

Guo

Huang

2020

FEBS Open Bio

Synthetic biology circuits are often constructed with multiple gene expression units assembled in close proximity, and they can be used to perform complex functions in embryonic stem cells (ESCs). However, mutual interference between transcriptional units has not been well studied in mouse ESCs. To assess the efficiency of insulators at suppressing promoter interference in mouse ESCs, we used an evaluation scheme in which a tunable tetracycline response element promoter is connected to a constant Nanog promoter. The chicken hypersensitive site 4 (cHS4) insulator, widely used both for enhancer blocking and for barrier insulation in vitro and in vivo, was positioned between the two expression units for assessment. By inserting the cassette into various loci of the mouse ESC genome with PiggyBac transposon, we were able to quantitatively examine the protective effect of cHS4 by gradually increasing the transcriptional activity of the tetracycline response element promoter with doxycycline and then measuring the transcriptional activity of the Nanog promoter. Our results indicate that the cHS4 insulator has minimal insulating effects on promoter interference in mouse ESCs. Further studies show that the cHS4 insulation effect may be promoter specific and related to interaction with CCCTC‐binding factor‐mediated loop formation. In addition, we also compared DNA transposition and transgene expression with or without the cHS4 insulator using well‐established ESC reporters. The results indicate that cHS4 has no apparent effects on DNA transposition and transgene expression levels, but exerts modest protective effects on long‐term transgene silencing.