Episomal lentiviral vectors confer erythropoietin expression in dividing cells

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%

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

Zhang

et al. 2019

MBoC

“…This was accomplished by introducing the SV40 or EBV origins of replication in the NILV genome and the delivery of these vectors into cells expressing the SV40 large T antigen or the EBV nuclear antigen 1, respectively [ 59 , 60 ]. Alternatively, the use of a scaffold/matrix attachment region [ 61 , 62 , 63 , 64 ] or a transient induction of cell cycle arrest [ 65 ] also allows prolonged expression, even if the cells divide. Ultimately, safer gene addition therapies in dividing cells could be achieved by the use of NILVs to deliver the transgene that can then be the substrate of other integrating systems that display a safer integration profile compared to HIV-1 integrase—for example, the sleeping beauty transposase [ 66 ] or the Flp recombinase [ 67 ].…”

Section: Expression Levels Of Nilvs and Other Vector Designsmentioning

confidence: 99%

The Old and the New: Prospects for Non-Integrating Lentiviral Vector Technology

Apolonia

2020

Viruses

Lentiviral vectors have been developed and used in multiple gene and cell therapy applications. One of their main advantages over other vectors is the ability to integrate the genetic material into the genome of the host. However, this can also be a disadvantage as it may lead to insertional mutagenesis. To address this, non-integrating lentiviral vectors (NILVs) were developed. To generate NILVs, it is possible to introduce mutations in the viral enzyme integrase and/or mutations on the viral DNA recognised by integrase (the attachment sites). NILVs are able to stably express transgenes from episomal DNA in non-dividing cells or transiently if the target cells divide. It has been shown that these vectors are able to transduce multiple cell types and tissues. These characteristics make NILVs ideal vectors to use in vaccination and immunotherapies, among other applications. They also open future prospects for NILVs as tools for the delivery of CRISPR/Cas9 components, a recent revolutionary technology now widely used for gene editing and repair.

“…An in-between case is that of non-integrating viruses, which have been derived from integrating viruses engineered to be integrase-deficient, e.g. nonintegrating lentiviruses (NILVs) 3,4 . Non integrating viral vectors present an interesting development to the field and can provide stable transgene expression in quiescent as well as proliferating cells, as it has been reviewed recently 5 .…”

Section: Introductionmentioning

confidence: 99%

“…These vectors may be reinforced with scaffold/matrix attachment regions (S/MAR), yet they still harbor viral sequences in their genome and thus can induce an immune response. In addition, they have a limited packing capacity and high production cost 38,3 .…”

Section: Introductionmentioning

confidence: 99%

“…An in-between case is that of nonintegrating viruses, which have been derived from integrating viruses engineered to be integrase deficient, for example, nonintegrating lentiviruses. 3 , 4 Nonintegrating viral vectors present an interesting development to the field and can provide stable transgene expression in quiescent as well as proliferating cells, as it has been reviewed recently. 5 These vectors may be reinforced with scaffold/matrix attachment regions (S/MARs), yet they still harbor viral sequences in their genome and thus can induce an immune response.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

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

et al. 2021

Non-viral and non-integrating episomal vectors are reemerging as a valid, alternative technology to integrating viral vectors for gene therapy, due to their more favorable safety profile, significantly lower risk for insertional mutagenesis and a lesser potential for innate immune reactions, in addition to their low production cost. Over the past few years, attempts have been made to generate highly functional non-viral vectors that display long-term maintenance within cells and promote more sustained gene expression relative to conventional plasmids. Extensive research into the parameters that