The EF‐1α promoter maintains high‐level transgene expression from episomal vectors in transfected CHO‐K1 cells

Wang, Xiaoyin; Xu, Zhen; Tian, Zheng-Wei; Zhang, Xi; Xu, Dafeng; Qin, Li; Zhang, Junhe; Wang, Tianyun

doi:10.1111/jcmm.13216

Cited by 64 publications

(50 citation statements)

References 31 publications

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“…The activity of promoters with predicted ‘ubiquitous’ expression, such as the four studied here, will still depend greatly on the lineage of the host cell (32). However, EF-1 promoter was found to be active and resistant to silencing in cells where other viral promoters may become silenced (33). Therefore, future work will be required to determine if the superior performance of EF-1 and CMV in expressing long RNA sequences can be extrapolated to other cell primary cell types.…”

Section: Discussionmentioning

confidence: 99%

Promoter Choice: Who Should Drive the CAR in T Cells?

Rad

Poudel

Tan

et al. 2020

Preprint

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Chimeric antigen receptor (CAR) T cell therapy is an effective treatment for B cell malignancies, with emerging potential for the treatment of other hematologic cancers and solid tumors. The strength of the promoter within the CAR cassette will alter CAR-polypeptide levels on the cell surface of the T cell – impacting on the kinetics of activation, survival and memory cell formation in T cells. In addition to the CAR, promoters can be used to drive other genes of interest to enhance CAR T cell function. Expressing multiple genes from a single RNA transcript can be effectively achieved by linking the genes via a ribosomal skip site. However, promoters may differ in their ability to transcribe longer RNAs, or could interfere with lentiviral production, or transduction frequencies. In this study we compared the ability of the strong well-characterized promoters CMV, EF-1, hPGK and RPBSA to drive functional expression of a single RNA encoding three products: GFP, CAR, plus an additional cell-survival gene, Mcl-1. Although the four promoters produced similarly high lentiviral titres, EF-1 gave the best transduction efficacy of primary T cells. Major differences were found in the ability of the promoters to drive expression of long RNA encoding GFP, CAR and Mcl-1, highlighting promoter choice as an important consideration for gene therapy applications requiring the expression of long and complex mRNA.

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Section: Discussionmentioning

confidence: 99%

Promoter Choice: Who Should Drive the CAR in T Cells?

Rad

Poudel

Tan

et al. 2020

Preprint

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“…The synthesized sequences replaced the CMV promoter (i.e. the CMV promoter was excised and replaced with CMV promoter mutants) in the previously described pEM vector (Figure 1 (a)) [21], and they were named as mutant-404 and mutant-542 ( Figure 1(b)). By contrast, the EF-1α and CAG promoters were generated using polymerase chain reaction (PCR).…”

Section: Vector Constructionmentioning

confidence: 99%

“…In our previous study, we investigated enhancers, various promoters and promoter variants on transgene expression in CHO-K1 cells and found that the EF-1a promoter is a potent regulatory sequence for episomal vectors and maintains high transgene expression [21]. However, only the CHO-K1 cell line, which was unfit for gene therapy owing to its differences from human cells, was tested.…”

Section: Introductionmentioning

confidence: 99%

Effect of promoter, promoter mutation and enhancer on transgene expression mediated by episomal vectors in transfected HEK293, Chang liver and primary cells

Jia

Wang

et al. 2019

Bioengineered

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The episomal vector cannot integrate into the host cell chromosome, which has no potential risk in gene therapy. However, the low level of transgene expression driven by episomal vectors needs to be solved. In this study, we investigated the effects of enhancers, promoters and promoter variants on transgene expression levels driven by episomal vectors in HEK293, Chang liver and primary cells. Results showed that all eight cis-acting elements used could increase transfection efficiency and transient eGFP expression in transfected HEK293 and Chang liver cells. In stably transfected mammalian cells, the elongation factor-1 alpha (EF-1α) promoter and mutant-404 showed high and stable transgene expression. The mechanisms might be related to the type and quantity of transcription factor regulatory elements. Moreover, quantitative reverse transcription polymerase chain reaction analysis showed that mRNA expression levels were not directly proportional to protein expression levels. Furthermore, the EF-1α promoter conferred high transgene expression levels in primary cells, and the plasmid was also present in the episomal state. Taken together, these results provided valuable information for improving transgene expression with episomal vectors in mammalian cells.

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“…More general promoters such as CMV (cytomegalovirus) 54 , 58 , 60 , 62 and EFS (elongation factor-1 alpha short) 5 , 65 can be used to express the CRISPR in multiple tissues, including muscle, retina, heart, and lung, for simultaneous gene editing of various tissues in mice. Compared with the CMV promoter, the elongation factor-1 alpha (EF1a) promoter tends to give more consistent and prolonged expression regardless of cell type or physiology 91 , 92 . EF1a is more stable in long-term culture because it maintains high transgene expression and stability and the copy number of episomal vectors 91 .…”

Section: Aav-crispr-based In Vivo Genome Editing Imentioning

confidence: 99%

In vivo genome editing in animals using AAV-CRISPR system: applications to translational research of human disease

2017

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Adeno-associated virus (AAV) has shown promising therapeutic efficacy with a good safety profile in a wide range of animal models and human clinical trials. With the advent of clustered regulatory interspaced short palindromic repeat (CRISPR)-based genome-editing technologies, AAV provides one of the most suitable viral vectors to package, deliver, and express CRISPR components for targeted gene editing. Recent discoveries of smaller Cas9 orthologues have enabled the packaging of Cas9 nuclease and its chimeric guide RNA into a single AAV delivery vehicle for robust in vivo genome editing. Here, we discuss how the combined use of small Cas9 orthologues, tissue-specific minimal promoters, AAV serotypes, and different routes of administration has advanced the development of efficient and precise in vivo genome editing and comprehensively review the various AAV-CRISPR systems that have been effectively used in animals. We then discuss the clinical implications and potential strategies to overcome off-target effects, immunogenicity, and toxicity associated with CRISPR components and AAV delivery vehicles. Finally, we discuss ongoing non-viral-based ex vivo gene therapy clinical trials to underscore the current challenges and future prospects of CRISPR/Cas9 delivery for human therapeutics.

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The EF‐1α promoter maintains high‐level transgene expression from episomal vectors in transfected CHO‐K1 cells

Cited by 64 publications

References 31 publications

Promoter Choice: Who Should Drive the CAR in T Cells?

Promoter Choice: Who Should Drive the CAR in T Cells?

Effect of promoter, promoter mutation and enhancer on transgene expression mediated by episomal vectors in transfected HEK293, Chang liver and primary cells

In vivo genome editing in animals using AAV-CRISPR system: applications to translational research of human disease

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