Rescue of splicing-mediated intron loss maximizes expression in lentiviral vectors containing the human ubiquitin C promoter

Abstract

Lentiviral vectors almost universally use heterologous internal promoters to express transgenes. One of the most commonly used promoter fragments is a 1.2-kb sequence from the human ubiquitin C (UBC) gene, encompassing the promoter, some enhancers, first exon, first intron and a small part of the second exon of UBC. Because splicing can occur after transcription of the vector genome during vector production, we investigated whether the intron within the UBC promoter fragment is faithfully transmitted to target cells. Genetic analysis revealed that more than 80% of proviral forms lack the intron of the UBC promoter. The human elongation factor 1 alpha (EEF1A1) promoter fragment intron was not lost during lentiviral packaging, and this difference between the UBC and EEF1A1 promoter introns was conferred by promoter exonic sequences. UBC promoter intron loss caused a 4-fold reduction in transgene expression. Movement of the expression cassette to the opposite strand prevented intron loss and restored full expression. This increase in expression was mostly due to non-classical enhancer activity within the intron, and movement of putative intronic enhancer sequences to multiple promoter-proximal sites actually repressed expression. Reversal of the UBC promoter also prevented intron loss and restored full expression in bidirectional lentiviral vectors.

A lentiviral vector bearing a reverse intron demonstrates superior expression of both proteins and microRNAs

“…oncogene dek. [2][3][4][5][6][7][8][9] We indeed observed a more significant phenotype using these 2 cDNAs. In the case of the preproinsulin gene, levels of proinsulin present in puromycin-selected cells transduced with pTREX were »11-fold higher than seen with pTRIPZ-transduced cells (Fig.…”

“…As noted above, it is well established that splicing facilitates the accumulation of mRNAs encoding proteins of interest, yet in the context of lentiviral vectors, genes are invariably expressed as unspliced cDNAs. [2][3][4][5][6][7][8][9] We previously examined the effect of splicing on a wide range of genes expressed from transfected plasmids and observed positive effects that generally ranged from 2-to 4-fold. 6 Yet, for a subset of genes, including b-globin and preproinsulin, the absence of a functional intron in the initial pre-mRNA transcript resulted in a dramatic 10-40-fold drop in the level of protein expression.…”

“…We next wished to examine whether the intron-containing pTREX vector would indeed express higher levels of inserted cDNAs, as predicted based on previous work. [2][3][4][5][6][7][8][9] As an initial test, we packaged pTREX and pTRIPZ variants bearing gfp as the transgene and then transduced na€ ıve 293T cells. Transduced cells were then selected using puromycin and the selected cells induced by addition of Dox or left untreated.…”

“…Specifically, because lentiviral vectors use RNA as their genome, any introns that are inserted into that genome are generally rapidly lost due to splicing. 2 This means that lentiviral vectors, as currently designed, are only able to express cDNA copies of the gene of interest. However, it is well established that splicing can significantly boost the expression of mRNAs by promoting mRNA polyadenylation, nuclear export and accumulation.…”

Adaptation of STARR-seq method to be used with 3rdgeneration integrase-deficient promoterless lentiviral vectors

“…Secondly, to avoid splicing out an intron -which is an element of STARR-seq plasmid -and to minimize any remaining interference from Δ5'LTR, a whole construct will be placed in a reverse orientation (Cooper et al, 2015;Poling et al, 2017). This should also help to lower risks of sub-optimal viral production due to the presence of bGH polyA sequence between LTRs ( Fig.…”