Inhibition of histone deacetylation in 293GPG packaging cell line improves the production of self-inactivating MLV-derived retroviral vectors

Abstract

Background: Self-inactivating retroviral vectors (SIN) are often associated with very low titers. Promoter elements embedded within SIN designs may suppress transcription of packageable retroviral RNA which in turn results in titer reduction. We tested whether this dominant-negative effect involves histone acetylation state. We designed an MLV-derived SIN vector using the cytomegalovirus immediate early enhancer-promoter (CMVIE) as an embedded internal promoter (SINCMV) and transfected the pantropic 293GPG packaging cell line.

Results: The SINCMV retroviral producer had uniformly very low titers (approximately 10,000 infectious retroparticles per ml). Northern blot showed low levels of expression of retroviral mRNA in producer cells in particular that of packageable RNA transcript. Treatment of the producers with the histone deacetylase (HDAC) inhibitors sodium butyrate and trichostatin A reversed transcriptional suppression and resulted in an average 106.3 +/- 4.6 - fold (P = 0.002) and 15.5 +/- 1.3 - fold increase in titer (P = 0.008), respectively. A histone gel assay confirmed increased histone acetylation in treated producer cells.

Conclusion: These results show that SIN retrovectors incorporating strong internal promoters such as CMVIE, are susceptible to transcriptional silencing and that treatment of the producer cells with HDAC inhibitors can overcome this blockade suggesting that histone deacetylation is implicated in the mechanism of transcriptional suppression.

Figure 1

SINCMV vector design and transcript expression in retroviral producer cells. A. The control SIN vector lacking an internal promoter has major deletions in the 3'LTR enhancer elements rendering its intrinsic promoter machinery transcriptionally inactive in transduced cells. B. Transgene expression in the SINCMV design is driven by the internal CMVIE promoter embedded upstream of the reporter EGFP. Three RNA transcripts are expected from SINCMV proviral DNA transcription in transfected packaging cells. The upstream CMV promoter in the 5'LTR drives the expression of a full-length ~2.6 kb transcript that can be packaged into retroparticles and a ~2.1 kb spliced form that lacks the packaging signal (Ψ). The internal CMVIE drives the expression of a shorter ~1 kb transcript. C. Hybridization with a P³²- labelled EGFP probe done on total RNA extracted from SINCMV retroviral producers treated with butyrate indicated significant increase in the level of retrovector mRNA. D. Loading control of the 3 RNA samples is shown by ribosomal RNA staining with ethidium bromide.

Figure 2

Titer of SINCMV retroviral producers treated with sodium butyrate and transduction of A549 cells with retrovirus from butyrate treated SINCMV producer cells. A. Treatment of control retroviral producer cells with the histone deacetylase inhibitor sodium butyrate for 48 hr resulted in a modest 1.6 ± 0.4-fold increase in titer (P = 0.299). B. Treatment of SINCMV retroviral producer cells with sodium butyrate for 48 hr resulted in a maximal 106.3 ± 4.6-fold increase in titer (P = 0.002) that was obtained with 20 mM butyrate. C. A549 lung carcinoma cells were transduced with same volume of retroviral supernatant that was collected from control-untreated SINCMV producers (a), producers treated with 10 mM sodium butyrate (b), and 20 mM sodium butyrate (c). % EGFP positive cells for each sample and mean EGFP reporter expression in the gated population (MnX) indicate a marked increase in gene transfer into target cells with supernatant from butyrate treated producers.

Figure 3

Histone gel assay on SINCMV producer cells treated with sodium butyrate. Treatment of SINCMV retroviral producer cells with increasing doses of sodium butyrate for 48 hr resulted in increased histone acetylation as most obvious with histone H4.

Figure 4

Titer of SINCMV retroviral producers treated with TsA and model depicting mechanism of transcriptional suppression in the SINCMV design. A. Treatment of SINCMV retroviral producer cells with the histone deacetylase inhibitor TsA for 48 hr resulted in a maximal 15.5 ± 1.3-fold increase in titer (P = 0.008) that was obtained with 3 μM TsA. B. Interferences between strong elements in the internal CMVIE enhancer-promoter and the upstream CMV promoter in the 5'LTR lead to the recruitment of histone deacetylases (HDACs) which trigger an inactive chromatin conformation at the promoter sites leading to transcriptional suppression of the retroviral RNA.