Compact Bidirectional Promoters for Dual-Gene Expression in a Sleeping Beauty Transposon.

Kevin He,Aarati Poudel,S. M. Ali Hosseini Rad,Alexander Donald McLellan

doi:10.3390/ijms21239256

Kevin He, Aarati Poudel + Show 2 more

Open Access

https://doi.org/10.3390/ijms21239256

Copy DOI

Abstract

Promoter choice is an essential consideration for transgene expression in gene therapy. The expression of multiple genes requires ribosomal entry or skip sites, or the use of multiple promoters. Promoter systems comprised of two separate, divergent promoters may significantly increase the size of genetic cassettes intended for use in gene therapy. However, an alternative approach is to use a single, compact, bidirectional promoter. We identified strong and stable bidirectional activity of the RPBSA synthetic promoter comprised of a fragment of the human Rpl13a promoter, together with additional intron/exon structures. The Rpl13a-based promoter drove long-term bidirectional activity of fluorescent proteins. Similar results were obtained for the EF1-α and LMP2/TAP1 promoters. However, in a lentiviral vector, the divergent bidirectional systems failed to produce sufficient titres to translate into an expression system for dual chimeric antigen receptor (CAR) expression. Although bidirectional promoters show excellent applicability to drive short RNA in Sleeping Beauty transposon systems, their possible use in the lentiviral applications requiring longer and more complex RNA, such as dual-CAR cassettes, is limited.

Highlights

Bidirectional promoters allow transcription from both the sense and antisense direction within a region defined as
We investigated the utility of bidirectional transgene expression for driving stable gene expression within a genome-integrated Sleeping Beauty system
As the majority of transcription binding sites are clustered in the RPL13 proximal promoter region of RPBSA [13], we further examined whether deletion of the intron and exon would affect the strength of bidirectional activity