Overcoming promoter competition in packaging cells improves production of self-inactivating retroviral vectors

Abstract

Retroviral vectors with self-inactivating (SIN) long-terminal repeats not only increase the autonomy of the internal promoter but may also reduce the risk of insertional upregulation of neighboring alleles. However, gammaretroviral as opposed to lentiviral packaging systems produce suboptimal SIN vector titers, a major limitation for their clinical use. Northern blot data revealed that low SIN titers were associated with abundant transcription of internal rather than full-length transcripts in transfected packaging cells. When using the promoter of Rous sarcoma virus or a tetracycline-inducible promoter to generate full-length transcripts, we obtained a strong enhancement in titer (up to 4 Â 10 7 transducing units per ml of unconcentrated supernatant). Dual fluorescence vectors and Northern blots revealed that promoter competition is a rate-limiting step of SIN vector production. SIN vector stocks pseudotyped with RD114 envelope protein had high transduction efficiency in human and non-human primate cells. This study introduces a new generation of efficient gammaretroviral SIN vectors as a platform for further optimizations of retroviral vector performance.

CTF/NF1 transcription factors act as potent genetic insulators for integrating gene transfer vectors

“…52 The insulator sequences were inserted into the NheI site of the 3¢ DU3 region, which is copied into the 5¢LTR after reverse transcription, and thus results in a design flanking the introduced expression cassette. Gamma-retroviral supernatant production was performed using 293T cells as previously described, with coexpression of ecotropic envelope proteins.…”

“…Gamma-retroviral supernatant production was performed using 293T cells as previously described, with coexpression of ecotropic envelope proteins. 42,52 Cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 100 U ml À1 penicillin/streptomycin and 2 mM glutamine. Viral titers, determined on SC-1 cells by flow cytometry, were in the range of 5Â10 6 to 2Â10 7 IU ml À1 in un-concentrated supernatants.…”

Mechanisms controlling titer and expression of bidirectional lentiviral and gammaretroviral vectors

“…Importantly, incorporating a T-cell-specific murine CD3d promoter 34 into a bidirectional GV vector did not prevent expression of the antisense message and thus did not overcome titer restrictions possibly due to interaction of the internal promoter with enhancer sequences of the 5 0 promoter as described before (Supplementary Figure S2). 35 Consequently, we focused on the screening of RNAi suppressor proteins by cotransfecting either empty vector (ctrl), HIV Tat, 36 human foamy virus Tas, 37 tomato bushy stunt virus p19, 38 betanodamuravirus B2 39 or NovB2 21,40 expression plasmid to a normal transfection mixture for the production of GV bidirectional vectors. These experiments identified NovB2 as most potent in enhancing bidirectional vector titers in a dose-dependent manner.…”

“…We thus used vectors with the standard configuration (pRBid), vectors depleted of the CTE (nC) and GV vectors with enhanced production of genomic RNA (pREBid) (pSERS backbone) 35 containing and lacking the CTE (Figure 3b). Again, in all configurations, NovB2 significantly increased vector titers up to B6 Â 10 6 t.u.…”

“…In brief, a 452 bp fragment consisting of an SV40 polyadenylation signal and a Mason-Pfizer monkey virus CTE was PCR amplified using primers 5 0 CTEpA_as MluI (5 0 -GCTACGCGTG ATCATAATCAGCCATACCACAT-3 0 ) and 3 0 CTEpA_as Not Eco47III (5 0 -GCTAGCGCTGCGGCCGCAGACCAC CTCCCCTGCGAGCTAA-3 0 ) and a pSF2-c(NMCG) template (restriction sites in bold). The PCR product was subcloned into pSRS11.PGK.eGFP.pre*, 35 additionally equipped with unique MluI and Eco47III restriction sites. Sequence identity of the subcloned PCR product was confirmed by sequencing.…”