Enhanced gene expression from retroviral vectors
© Blø et al; licensee BioMed Central Ltd. 2008
Received: 05 November 2007
Accepted: 25 February 2008
Published: 25 February 2008
Retroviruses are widely used to transfer genes to mammalian cells efficiently and stably. However, genetic elements required for high-level gene expression are incompatible with standard systems. The retroviral RNA genome is produced by cellular transcription and post-transcriptional processing within packaging cells: Introns present in the retroviral genomic transcript are removed by splicing, while polyadenylation signals lead to the production of ineffective truncated genomes. Furthermore strong enhancer/promoters within the retroviral payload lead to detrimental competition with the retroviral enhancer/promoter.
By exploiting a new method of producing the retroviral genome in vitro it is possible to produce infectious retroviral particles carrying a high-level expression cassette that completely prohibits production of infectious retroviral particles by conventional methods.
We produced an expression cassette comprising a strong enhancer/promoter, an optimised intron, the GFP open reading frame and a strong polyadenylation signal. This cassette was cloned into both a conventional MMLV retroviral vector and a vector designed to allow in vitro transcription of the retroviral genome by T7 RNA polymerase.
When the conventional retroviral vector was transfected into packaging cells, the expression cassette drove strong GFP expression, but no infectious retrovirus was produced. Introduction of the in vitro produced uncapped retroviral genomic transcript into the packaging cells did not lead to any detectable GFP expression. However, infectious retrovirus was easily recovered, and when used to infect target primary human cells led to very high GFP expression – up to 3.5 times greater than conventional retroviral LTR-driven expression.
Retroviral vectors carrying an optimized high-level expression cassette do not produce infectious virions when introduced into packaging cells by transfection of DNA. Infectious retrovirus carrying the same cassette is readily produced when packaging cells are transfected with in vitro transcribed retroviral genomic RNA. The applications of this technique are not limited to producing the higher levels of transgene expression demonstrated here. For example, novel reporters with alternatively spliced exon-intron configurations could readily be transduced into virtually any cell. Furthermore, because the in vitro transcripts are not translated within the packaging cells, retroviruses carrying genes lethal to the packaging cells can also be produced.
Results and Discussion
In spite of the popularity of retroviral vectors, little has been done to address these limitations. We recently developed a simple approach that dramatically expands the range of genetic elements transferable by retroviral vectors . The system employs in vitro generated RNA to nucleate retroviral virions, completely circumventing packaging cell genomic transcription and translation (Figure 1D).
To demonstrate the utility of this innovation, we endeavoured to create a high expression retroviral vector using a known optimal constellation of genetic elements predicted to be incompatible with current systems. Indeed we show that a retroviral vector with substantially higher transgene expression in primary human cells can be constructed using the in vitro transcribed retroviral genomic RNA approach, while the same gene cassette prohibited the production of virus by the conventional system.
In contrast, in vitro transcription system based vectors (pT) carrying the same CMV expression cassette readily generated infectious virus following transfection of in vitro transcribed RNA genomes into packaging cells. The pT vectors produced no detectable GFP expression in the packaging cells, due to the lack of requisite translation signals (e.g. 5' cap) on the RNA transcripts, a notable advantage of the system (not shown;). Primary human umbilical cord endothelial cells (HUVEC) were readily infected with viral supernatants generated by the pT-vectors carrying the strong GFP expression cassette (pT-cGreen and pT-neerGc) and expressed markedly higher (up to 3.5-fold) levels than obtained with the conventional LTR-driven (pC-GFP, pT-GFP) vectors (Figure 3C). The reverse orientation cassette (pC-neerGc, pT-neerGc) consistently gave higher expression than the forward orientation cassette (pC-cGreen, pT-cGreen) (Figure 3A, C). The reason for this is unknown but presumably reflects a more favorable context for CMV-driven expression proximal the 3' LTR compared to near the packaging sequence (Ψ).
In this report we demonstrate how in vitro-transcribed retroviral RNA genomes can be utilized to transfer a high-level expression cassette which includes genetic elements not compatible with commonly used retroviral packaging systems. Using this cassette we observe an increase in expression level of up to 3.5 times in primary human cells, reflecting the provision of an intron and the use of strong enhancer/promoter and polyadenylation signals. The ability to circumvent packaging cell transcription, post-transcriptional editing and translation of the vector genome, while maintaining assembly into virions, effectively addresses the shortcomings of current retroviral systems. Thus, this approach is based on standard molecular biology techniques, yet allows virtually any genetic sequence constellation to be manifested by the integrated retroviral provirus: Strong enhancers/promoters, introns and polyadenylation signals for boosting gene expression; gene reporters that encompass multiple exon-introns; post-transcriptional signal sequences to improve gene trapping; cytotoxic gene expression or shRNA expression cassettes that target essential genes.
Vectors pT-cGreen/neerGc (L311/L312) were based on vector L139 pT7RU5mcsSIN, a derivative of LB-G  in which the GFP has been removed and the 3'LTR replaced with a self-inactivating 3'LTR (SIN) from the vector pTra . L139 thus carries in the following order: A T7 site fused to the transcriptional start site of the MMLV RU5 region of the LTR, an extended packaging sequence (Ψ), a multiple cloning site and a self-inactivating 3'LTR followed by forty A residues. Vectors pC-cGreen/neerGc (L309/L310) were based on vector L060 pCRU5 Puro2Amcs, a derivative of p96.7 . This vector carries a composite CMV promoter fused to the transcriptional start site of the MMLV RU5 region of the LTR, an extended packaging sequence (Ψ), multiple cloning site and 3'LTR. Vector pCI (Promega) contains a CMV promoter, an optimized chimeric intron and an SV40 polyadenylation site selected to drive strong constitutive expression . A T7 polymerase initiation site within the expression cassette of pCI was removed by deleting the sequence between the unique NheI site (filled) and the ScaI site 22 bp upstream (pCIdeltaT7). An EcoRI/NotI fragment from pCGFP  encompassing the GFP coding sequence was then cloned into the corresponding sites of pCIdeltaT7. The complete CMV-intron-GFP-SV40pA cassette was excised with BglII and ClaI, blunt ended and cloned into L060 (XhoI, EcoRI, blunted) and L139 (PmeI). Both orientations of insert were obtained, producing pT-cGreen/neerGc and pC-cGreen/neerGc.
Genomic RNA production
To generate the uncapped retroviral genomic transcripts, MluI-linearized pT-vector DNA was transcribed with T7 polymerase (MEGAscript, Ambion, Austin, TX). RNA was quantified (ND-1000 spectrophotometer, NanoDrop Technologies, Wilmington, DE) and the quality checked by denaturing agarose gel electrophoresis  (data not shown).
Transfection and retroviral transduction
Standard retroviral vectors (pC) were transfected into 293-based Phoenix A packaging cells by the standard protocol [12, 15] which for pC-GFP typically produces titers of around 1 × 107 gfu/ml for infection of primary human endothelial cells.
The in vitro transcribed genomic RNA was introduced into the packaging cells using the cationic lipid DMRIE-C (Invitrogen, San Diego, CA). The RNA was mixed with lipid at a ratio of 1 μg RNA to 3.5 μl DMRIE-C in a final volume of 250 μl of serum-free OptiMEM (Invitrogen, San Diego, CA) and incubated at room temperature for 20 minutes. Subconfluent (80%) Phoenix-A packaging cells in poly-lysine coated 12-well plates were prepared by rinsing with serum-free medium and 360 μl OptiMEM was added per well. The RNA-lipid mixture was added dropwise to the cells and incubated for 2 hours before the cells were washed and incubated in complete medium. pT virus-containing medium (1 ml) was harvested 18 hours later. Titers were typically approximately 1 × 105 gfu/ml for infection of primary human endothelial cells .
Primary human umbilical vein endothelial cells (HUVEC, passage 1–3; Cambrex, Rockland, ME) were cultured according to the supplier's recommendations. Cells were infected as described (e.g. plated at 40,000/12-well; 5 μg/ml protamine sulfate) . Viral supernatants were diluted in medium to obtain a multiplicity-of-infection yielding 10–20% GFP-positive cells corresponding to single viral infections. Typical dilutions used were 1/100 (pC-GFP) or 1/3–1/9 (pT vectors). GFP expression measurements were performed on a FACSCalibur cytometer (Becton Dickinson, San Jose, CA) and analysis was carried out using Flowjo .
The authors wish to thank Sissel Vik Berge and Paulina Ruurs for expert technical assistance. The study was supported by funding from the Norwegian Research Council and University of Bergen pre-doctoral (MB) and postdoctoral (DM) fellowships.
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