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

Diana E Jaalouk,Jacques Galipeau,Milena Crosato,Pnina Brodt

doi:10.1186/1743-422x-3-27

Abstract

BackgroundSelf-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.ResultsThe SINCMV retroviral producer had uniformly very low titers (~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.ConclusionThese 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.

Highlights

Self-inactivating retroviral vectors (SIN) are often associated with very low titers
We further demonstrate that treatment of the SINCMV retroviral producers with the histone deacetylase (HDAC) inhibitors sodium butyrate and Trichostatin A (TsA) reversed the transcriptional suppression and resulted in a significant increase in the SIN retroviral titer
SINCMV retrovector design and increased retroviral RNA transcription with sodium butyrate treatment in transfected 293GPG producer cells A SIN vector was derived from a Moloney murine leukemia virus (Mo-murine leukemia virus (MLV))-based vector, pLTRGFP, by creating a 311-bp NheI-SacI deletion to the 3'long terminal repeats (LTRs), removing all the retroviral enhancers and the CAAT box (Figure 1A)

Summary

Introduction

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. Retroviral vectors derived from C-type mammalian retroviruses are characterized by their ability to integrate into the chromosomal DNA of their target cells. The transcriptional activity of an embedded promoter can be compromised by interferences from the strong enhancer and promoter machinery in the flanking retroviral LTRs [3,4,5] To bypass this problem, self-inactivating retroviral vectors (SIN) have been designed whereby the viral enhancer and/or promoter sequences are deleted from the U3 region of the 3'LTR. Following reverse transcription in transduced cells, the 3' LTR deletions will be copied to the 5'LTR by template switch rendering the vector transcriptionally inactive [68]

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Conclusion