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


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 × 107 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.


Self-inactivating (SIN) retroviral vectors lack enhancer–promoter sequences in the U3 region of their long-terminal repeats (LTRs) and use internal cis-regulatory sequences to initiate transcription of a gene of interest.1 The SIN design has several important advantages: it reduces the risk of recombination to replication-competent retroviruses (RCR), impedes the mobilization of vector RNA in case of RCR superinfection, increases the autonomy of the internal promoter1 and theoretically reduces the risk of insertional upregulation of neighboring alleles depending on the choice of the internal enhancer/promoter. These features can be achieved without compromising the potency of the integrated transgene allele. Although the deletion of enhancer sequences from the LTR impairs overall transcript levels and increases 3′ read-through,2 improved RNA processing of the internal transcript still allows the generation of SIN vectors that mediate comparable transgene expression levels as their LTR counterparts;3 SIN vectors are thus of interest for a variety of applications in human gene therapy.

On the basis of foamyvirus (FV) or lentiviruses such as the human immunodeficiency virus (HIV), SIN vectors can be produced from transiently transfected packaging cells, without substantial loss of titers compared to constructs containing intact LTR sequences.4, 5 In contrast, the first generations of gammaretroviral SIN vectors based on murine leukemia virus (MLV) suffered from strongly reduced titers.1 While MLV vectors cannot transduce non-dividing cells, they still represent important tools for human gene therapy, because they do not require the incorporation of any sequences overlapping with coding sequences of gag, pol, env or accessory genes,6 in contrast to the most common forms of vectors based on HIV or FV.4, 5 In addition, MLV SIN vectors are likely to increase the safety of human gene therapy protocols when used under conditions where MLV-based LTR vectors already show therapeutic efficiency.7, 8, 9, 10

Before the present study, the mechanisms responsible for the severe titer reduction of early generations of gammaretroviral SIN vectors were unclear. When we introduced the post-transcriptional element (PRE) from Woodchuck hepatitis virus into the 3′-untranslated region of SIN vectors, we were able to increase infectious titers above 106 transducing units per ml of unconcentrated cell-free culture supernatant.3, 11 Owing to the mode of action of the PRE,12, 13 this suggested that insufficient 3′ RNA processing of retroviral transcripts was partially responsible for reduced SIN vector yields. However, SIN titers still remained substantially reduced in comparison with LTR-driven counterparts, representing a potential limitation for clinical use. These observations suggested at least three hypotheses: (1) the gammaretroviral packaging signal is bipartite and involves sequences overlapping with the enhancer–promoter of the U3 region; (2) the interaction of the Rev-responsive element (RRE) with Rev-protein generated in producer cells is responsible for the superior titer of lentiviral SIN vectors; or (3) promoter differences between gammaretroviral and lentiviral SIN vectors lead to a superior generation of full-length transcript in the lentiviral context.

In the present study, we addressed these hypotheses by incorporating modules derived from state-of-the-art lentiviral vector plasmids14, 15 into gammaretroviral constructs. As underlined by a novel dual fluorescence reporter assay, we found that promoter competition was the major limitation for the production of gammaretroviral SIN vectors. This could be overcome by using the promoter of Rous sarcoma virus (RSV) or a tetracycline-inducible promoter to drive expression of the full-length RNA. We demonstrate the potency of this new gammaretroviral design for highly efficient transduction of rhesus monkey CD34+ cells with SIN vectors encoding a clinically relevant selection marker.


Gammaretroviral SIN vectors produce abundant internal transcripts in transfected packaging cells

In previous work, we have shown that gammaretroviral SIN vectors are as potent as their lentiviral counterparts in terms of transgene expression.16 Gammaretroviral SIN vectors, however, did not reach the same titers as their LTR-driven counterparts.3, 16 The use of the PRE from Woodchuck hepatitis virus restored SIN vector titers only partially.3

To address the underlying mechanisms, we initially compared three vector backbones, all containing the P140K mutant of methylguanine-methyltransferase (MGMT; Ragg et al.17) followed by the PREc Figure 1a). We compared our standard gammaretroviral LTR vector SF91,6, 18 the gammaretroviral SIN vector Sin11.SF16 and two third-generation lentiviral vectors (Figure 1a) containing the central polypurine tract (cPPT).14, 15 Lentiviral vectors used the internal SF promoter19 (as Sin11.SF) or the weaker phosphoglycerate kinase (PGK) promoter. Gammaretroviral supernatants were produced in 293T cell-based Phoenix-gp packaging cells,20 and lentiviral vectors in 293T cells.15 All particles contained ecotropic Env proteins21, 22 to avoid re-infection and RNA production from integrated proviruses.23

Figure 1

Gammaretroviral SIN vectors show lower titers compared to LTR-driven counterparts. (a) Vectors used for comparison of gammaretro- and lentiviral backbones (plasmid configuration). pSF91 is an LTR-driven gammaretroviral vector. pSin11.SF is the corresponding SIN gammaretroviral vector (majority of U3 deleted in the 3′-LTR), with an internal SF promoter. pRRL.PPT.SF/PGK are the lentiviral counterparts, with internal SF or PGK promoters. The U3 promoter–enhancer sequences are named after their virus origin. MP stands for myeloproliferative sarcoma virus (MPSV), SF for spleen focus-forming virus (SFFV) and RSV for Rous sarcoma virus. Within the leader regions, the primer-binding site (Θ), the packaging signal (Ψ), and splice donors and acceptors (SD, SA) are marked. In addition, the lentiviral vector RRL.PPT.SF/PGK contains 400 bp gag sequences (not shown), RRE and cPPT.15 All vectors carry the MGMT transgene and the PRE. (b) Northern blot analysis of 10 μg total RNA from Phoenix-gp (SF91, Sin11.SF) and 293T cells (RRL.PPT.SF/PGK). A PRE probe was used to detect all RNA species. The blot was reprobed with glyceraldehyde-3-phosphate dehydrogenase (GADPH) as a loading control. (c) The supernatants produced by the packaging cells were used to infect SC-1 murine fibroblasts in serial dilutions. Four days post-transduction MGMT-positive cells were analyzed by flow cytometry for intracellular staining.17 Titer is expressed as transducing units (t.u.) per ml unconcentrated supernatant. Values represent three independent transfection/transduction procedures.

The retroviral LTR vector harboring a splice-competent leader region produced both spliced and unspliced RNAs (Figure 1b, lane 1), the latter containing the packaging signal. In total RNA, a 1:1 ratio of both transcripts was reproducibly observed.3 In contrast, Sin11.SF showed a predominant signal for the internal RNA produced from the internal promoter (Figure 1b, lane 2), in expense of the amount of genomic RNA available for packaging. As this correlated with the titer determined by stable transfer of the MGMT expression cassette into SC-1 fibroblast cells (Figure 1c), promoter competition was a likely explanation for the titer reduction of gammaretroviral SIN vectors. Surprisingly, although bearing the identical internal expression cassette, lentiviral vectors showed a much better ratio of genomic vs internal RNA (Figure 1b, lane 4). However, lentiviral titers were only slightly increased, possibly because we pseudotyped the particles with the ecotropic envelope (Figure 1c).

Use of the RSV promoter improves the production of SIN genomic RNA

The 5′ region of the lentiviral backbone differs from the gammaretroviral with respect to several features: (i) R/U5, (ii) the cPPT, (iii) the RRE and (iv) the RSV promoter driving expression of the genomic RNA. We incorporated the last three modules into Sin11.SF encoding enhanced green fluorescent protein (eGFP). We found that neither the cPPT nor the RRE significantly influenced the ratio of genomic vs internal RNAs (data not shown).

We then thus focused on modifications of the 5′ promoter driving the expression of full-length RNA in the packaging cells. A new set of vectors was constructed (Figure 2a), to evaluate the extent of promoter competition with four different promoter configurations at the 5′ end, and three internal promoters (Figure 2a). The titer produced in Phoenix-gp cells is shown in Figure 2b and c provides a direct comparison of the RNA from packaging cells analyzed by Northern blot. In the Sin11.SF context, the internal SF promoter gave the highest titer in comparison to the promoters derived from PGK or cytomegalovirus (CMV). This suggests that a strong internal promoter also activates the upstream enhancer (Sin11.CMV and Sin11.SF in Figure 2c, lane 4). Although CMV was the strongest promoter in the internal position (Figure 2c), its transfer to the 5′ end (SCS vectors) did not increase titers (Figure 2b).

Figure 2

Presence of the RSV promoter in the 5′-LTR leads to a substantial titer increase. (a) Schematic drawing of the SIN vector backbone (compare Figure 1a), used to evaluate the different promoter elements. 5′ modifications included four different promoter–enhancer elements (MPSV U3, CMV, RSV, RSV+SV40 enhancer). At the internal position (iP), three different promoters were analyzed driving expression of eGFP. (b) Titer analysis of the indicated constructs performed as in Figure 1c. Note that the scale is different to Figure 1c and that titer up to 4 × 107 t.u./ml are reached. Two micrograms of transfer vector were used per transfection. Error bars indicate the standard deviations from three independent experiments. (c) Northern blot analysis of 10 μg total RNA from Phoenix-gp packaging cells. The RNA species are named on the right side. The asterisk indicates a higher molecular weight band in case of the CMV promoter. This band might represent read-through and usage of a potential polyA signal in the bacterial plasmid backbone as indicated by sequence analysis (data not shown). A PRE probe as in Figure 1 was used. The blot was reprobed with GAPDH. (d) Phosphoimager analysis. On the x–axis, the radioactive signals in relative counts for the genomic RNA (normalized according to GAPDH levels) of the Northern blot analysis shown in Figure 2c are given, and were plotted against the corresponding titers (y axis, compare Figure 2b) as determined in triplicates. The correlation coefficient is shown within the graph. (e) Titer analysis of different amounts of transfected transfer vectors.

Using the RSV promoter to drive expression of the genomic RNA (SRS vectors) resulted in a substantial increase in titer (Figure 2c). Depending on the internal promoter, titers increased up to 40-fold (Figure 2b). This correlated with an increase in the total amount of genomic RNA (Figure 2c, lanes 8–10). In the context of the internal SF promoter, the ratio of genomic vs internal RNA became similar to that observed for lentiviral vectors (compare Figure 1b, lane 4 to Figure 2c, lane 10). Thus, lentiviral and gammaretroviral SIN vectors showed equivalent results when containing the upstream RSV promoter increasing the total amount of packageable genomic RNA.

To further strengthen the 5′ promoter, we inserted the SV40 enhancer24, 25 upstream of the RSV promoter (SERS series; Figure 2a). This modification further increased the amount of genomic RNA (Figure 2c, lanes 11–13). Titers thus reached levels of 3–4 × 107 infectious units per ml unconcentrated cell-free supernatant (Figure 2b), concomitant with a further increased amount of genomic RNA (Figure 2c, lanes 11–13). To further investigate the correlation between the amount of genomic RNA and the increase in titer, we quantitated the Northern blot data by phosphoimager analysis (Figure 2d). We observed an almost linear correlation between the amount of genomic RNA and the resulting titer.

We thus reached the maximum titer that we achieved with LTR-driven vectors under our packaging conditions, indicating that even higher titers might be achievable when improving other components of the packaging systems. Of note, the vector modifications used to increase the retroviral titer leave the sequence of the integrated provirus unchanged.

We then lowered the amount of transfer vector from 2 to 0.5 μg, revealing greater differences in titer upon modification of the 5′ end (Figure 2e). The upstream RSV promoter led to 10-fold enhancement in titer when the amount of transfer vector was limiting and only to a 2.5-fold increase when the transfer vector was provided in excess (Figure 2e). Furthermore, the threshold at which the amount of genomic RNA becomes limiting was reached with the SRS vectors at 0.5 μg, in contrast to the conventional SIN vectors (5 μg of transfer vector; Figure 2e and data not shown).

In addition, the use of the RSV promoter also showed beneficial effect in the LTR context as shown by retroviral pseudotransduction26 (Supplementary Figure 1) and by integration-competent LTR vectors encoding eGFP, which showed a 3-fold titer increase (data not shown).

Performance of the new SIN vectors in primary hematopoietic cells

Using the SRS backbones, we designed efficient vectors expressing the clinically relevant selection marker MGMT transgene (Figure 3).16 The substitution of the MPSV (SIN vectors) for the RSV promoter (SRS vectors) led to a 3-fold relative increase in vector titers (determined on HT1080 cells, data now shown).

Figure 3

Performance of the new SIN vector in primary rhesus CD34+ cells. To evaluate the efficiency of the modified SIN vectors in a clinically relevant setting, the transgene was changed to MGMT (SRS11.SF.MGMT). Transduction of rhesus CD34+ cells with RD114/TR pseudotypes was shown in four independent experiments using intracellular FACS staining with a monoclonal MGMT antibody. One representative example with mock-transduced (upper left) or SRS11.SF-transduced cells using MOIs of 1, 5 and 10 (upper right, lower left and lower right, respectively) is displayed. A gate was set to separate MGMT-positive and -negative cells. The forward scatter (FSC) is given on the x axis, and MGMT fluorescence intensity on the y axis.

In order to test the performance of the vector supernatants on primary cells, we transduced Rhesus CD34+ cells with RD114/TR pseudotypes using multiplicities of infection (MOIs) of 1, 5 and 10 (Figure 3). Using an MOI of 10, productive transduction of more than 90% of Rhesus CD34+ cells was obtained (Figure 3, one representative experiment is shown). Furthermore, we transduced human CD34+ cells with MGMT encoding vectors at an MOI of 1, resulting in 43% MGMT expressing human hematopoietic cells (data not shown).

A dual fluorescent vector system suggests a Pol II occupation model for promoter competition

To address the mechanism of promoter competition, we developed a dual fluorescent vector by introducing the cDNA for the Discosoma red fluorescent protein Express (DsRed)27 upstream of the internal promoter driving eGFP (Figure 4a). The amount of DsRed should correlate with the amount of genomic RNA, and eGFP should mirror the quantity of the internal RNA. Both fluorescent proteins allow fast and quantitative analysis in single cells owing to similar maturation kinetics (Clontechniques XVII: 3, 2002) as opposed to the Northern analysis that reflects the average RNA production in a cell population.

Figure 4

Dual fluorescent vectors as a tool to analyze promoter competition. (a) Vectors encoding the DsRed Express cDNA on the genomic RNA and eGFP on the internal RNA, driven by the internal promoter (iP). The 5′ MP promoter was substituted by the RSV promoter and an additional polyA signal, derived from BGH (pA), was added upstream of the internal promoter. (b) FACS analysis of transfected Phoenix-gp cells. Cells were analyzed 48 h post-transfection. The y axis represents DsRed fluorescence correlating with the genomic RNA and the x axis shows eGFP expression from the internal promoter. The values in the upper right quadrant give the quotient of y vs x mean. The circle in the middle panel marks cells that intensify their green fluorescence after insertion of the polyA. On the right side, the transfection efficiencies of this particular experiment are given. (c) Titer analysis of the indicated constructs. Standard deviations represent three independent experiments. (d) Northern blot analysis of 10 μg total RNA from transfected Phoenix-gp cells. A PRE probe was used to detect the genomic and the internal RNA. The short RNA initiating from the 5′-LTR and terminating at the polyA cannot be detected with this probe. The asterisk marks a cryptic splice event from an efficient splice donor within DsRed to a weak acceptor site within the CMV promoter as predicted by a splice site prediction program (http://www.fruitfly.org/seq_tools/splice.html).

We started by comparing the basic retroviral SIN vectors with the RSV-modified vectors harboring two different internal promoters. Figure 4b shows a representative fluorescence-activated cell sorter (FACS) analysis of transfected Phoenix-gp cells. Standard SIN vectors showed an unfavorable ratio of green (internal RNA) vs red fluorescence (genomic transcript), as expressed by the quotient of the y vs x mean fluorescence intensity (Figure 4b, upper left panel). In contrast, the RSV modification increased this ratio sixfold; the dot-plot analyses revealed that the effect of the RSV promoter was independent of the expression level (Figure 4b, upper right panel). These data are in line with titer determinations (Figure 4c) and RNA levels (Northern blot in Figure 4d, compare lanes 2, 3 with 6, 7). We observed a direct correlation between the amount of genomic RNA and increase in titer. However, for the SRS constructs, the amount of genomic RNA increased in case of the internal CMV promoter, but it led only to minor titer increase, probably because in this setting saturating levels were already reached (Figure 4d and c, lanes 6 and 7).

We next used the dual fluorescence vectors to study the mechanisms of promoter competition. The downstream promoter might be occupied by read-through transcription of the RNA polymerase II (pol II) transcription complex originating at the upstream promoter.28, 29 Besides, epigenetic promoter modifications could occur, which are probably more important following transgene integration.30, 31, 32 If promoter occupation by RNA pol II is the relevant mechanism, inserting a transcriptional termination signal (polyA signal) 5′ of the internal promoter should rescue its activity by reducing the probability of transcriptional read-through.33 We thus cloned the bovine growth hormone (BGH) polyA in front of the internal promoter, resulting in pSRS.Red.pA.SF or pSRS.Red.pA.CMV (Figure 4a). FACS analysis of the parental vectors confirmed our previous findings (Figure 4b, lower left panel). The presence of the BGH polyA increased the eGFP signal (reflecting the internal transcript) as expressed by the ratio of mean fluorescence intensities (Figure 4b, lower right panel). The BGH polyA reduced vector titers strongly (Figure 4c and d), but not completely; this suggested that residual polymerase read-through did occur, especially when transcripts were driven by the RSV promoter (Figure 4d).

Taken together, these studies reveal promoter competition in SIN vector plasmids as a mechanism that potentially reduces the yield of genomic RNA. This can be overcome by the choice of a suitable 5′ enhancer/promoter.

Tetracycline-inducible promoters also drive high levels of genomic vector transcripts for high titer SIN vector production

Tetracycline-inducible promoters (Tet) in combination with the respective transactivators (TAs) are very potent promoter/enhancer combinations that mediate high and robust expression.34 Tet-inducible promoters have already been successfully used in the LTRs of lentiviral vectors to create all-in-one vectors with Tet-promoter and TA35, 36 and to drive the genomic RNA of lentiviral vectors in inducible packaging cells.37 Therefore, it was tempting to test whether a Tet-inducible promoter incorporated into the retroviral 5′-LTR (Figure 5a) is also capable of generating sufficient titers comparable to the SERS vector series (Figure 2). Potentially, this would allow the stable production of vectors with transgenes whose overexpression is toxic for producer cells. Furthermore, high titer virus production would be possible in cell lines where the RSV promoter is not active enough (in light of promoter competition). Figure 5b shows the results of a representative experiment. The Tet-inducible SIN vector Tet11.SF was transfected with or without the transactivator (TA) and set into comparison with the SERS11.SF vector (without TA). Titers of the Tet-inducible vector reached almost 2 × 107 transducing units per ml supernatant. Figure 5c shows the corresponding Northern blot of the packaging cell line. Interestingly, the amount of internal transcript also increased implying an interaction between the 5′ and the internal promoter. The combination of the RSV promoter and SV40 enhancer still produced more genomic RNA (Figure 5c, lane 4), but this did not translate into titer, probably because gag/pol or env were limiting (experiment conducted with saturating plasmid amounts) (Figure 2d). In summary, this indicates that Tet-inducible promoters are useful for high-titer production of gammaretroviral SIN vectors.

Figure 5

A tetracycline-inducible promoter allows high-titer gamma-retroviral SIN vector production. (a) Scheme of the vector backbone of Tet11.SF with the tet-responsive element (TRE) incorporated into the 5′-LTR U3 in comparison to to the SRS vector (Figure 2a). (b) Titers of the depicted constructs in t.u./ml as determined on SC-1 cells. For production of Tet11.SF, a transactivator plasmid (TA, 5 μg) was co-transfected. (c) Northern blot of 10 μg total RNA from Phoenix-gp packaging cells. RNA species are indicated on the right side. Loading was controlled via 28S rRNA comparison on an ethidium bromide agarose gel (data not shown).


The present study was undertaken to overcome a major limitation of gammaretroviral SIN vectors, which is the loss of titer observed upon deletion of the 3′ U3 region. Theoretically, all classes of SIN vectors are faced with the problem that a nonspecific internal promoter will be active in the packaging cells and generate RNAs that do not contribute to the titer but rather reduce the amount of genomic RNA. However, the use of a strong internal promoter is often desirable in the target cell to reach a therapeutic threshold, as for the expression of metabolic selection markers,38 genes antagonizing viral infections39 or recombinant T-cell receptors.40

Our data obtained upon transient transfection in packaging cells revealed that insufficient production of full-length transcript from the 5′ promoter is a major limitation of gammaretroviral SIN vectors. As we found that currently used lentiviral vectors produced greater amounts of genomic RNA, we screened through different modules present in the lentiviral backbone (RRE, cPPT, 5′ promoter). Interestingly, neither the RRE nor the cPPT were able to produce higher titers in the gammaretroviral background, not even in the presence of Rev (data not shown).

We rather find that high production of genomic RNA mainly depends on the choice of the 5′ promoter. Both RSV and tetracycline-inducible promoters led to high SIN vector titers. Why these promoters lead mediate production of genomic RNA might be explained by studies with lentiviral vectors. One aim during the construction of third-generation lentiviral vectors was to become Tat-independent during the production process.15 The RSV promoter was found to perform this function in the so-called third-generation lentiviral vectors and to be superior to CMV15 as in our context (Figure 2). Also, Kafri et al.35 successfully generated lentiviral SIN vectors using a tetracycline-inducible promoter to drive the genomic transcript. Our data obtained in the context of gammaretroviral vectors, which are Tat-independent a priori, implies that recruiting an elongation competent pol II complex like the Tat-dependent HIV LTR and not only the basal promoter strength is important to overrule the internal promoter. Accordingly, use of the even ‘stronger’ CMV promoter to drive genomic retroviral RNA expression did not give rise to higher titers.

If the elongation rate of RNA pol II is important, the density of transcriptionally engaged polymerases on the internal promoter suppresses its activity.28, 41, 42 Accordingly, we found that an efficient cellular termination signal placed upstream of the internal promoter activated the internal promoter, although residual read-through over the cellular polyA signal was still observed. Secondly, we enhanced transcriptional elongation by adding the 72 bp enhancer repeats from SV40 upstream of the RSV promoter,24, 43 which further increased the amount of genomic RNA and vector titers.

Competition between neighboring promoters could also occur at the level of enhancer interactions, with the stronger promoter attracting the enhancers of its neighbor. This might explain why the strong internal SFFV enhancer–promoter led to a higher SIN titer than the internal PGK promoter being the weakest of the promoters tested.17 However, the SV40 enhancer modification together with the usage of the RSV promoter makes the vectors less dependent on interactions with the internal promoter (Figure 2c).

We conclude that promoter competition is a major hindrance for the production of gammaretroviral SIN vectors and that both enhancer competition and promoter occupation need to be addressed when attempting to improve SIN vector titers. Increasing the processivity of the 5′ promoter enhanced vector titers by up to 40-fold, dependent on the cDNAs and internal promoters used. This enables the production of high titer supernatants using relatively low amounts of transfected plasmid, as required for efficient clinical-scale gene transfer. For a clinical study that explores the feasibility and safety of gene transfer into hematopoietic cells in adult patients, roughly 2 × 109 infectious particles would be required (5 × 106 CD34+ cells/kg, 70 kg body weight, two transductions with three infectious units per cell). When pseudotyping SRS and SERS vectors with human-infectious envelopes GALV and RD114, we reproducibly obtained titers of 1 × 107 infectious units per ml of unconcentrated cell culture supernatant (data not shown). Two hundred microliters supernatant would thus be sufficient for the treatment of a single patient, and 2.4 l for an entire phase I clinical trial including preclinical safety tests. Because 106 packaging cells yield about 1 ml supernatant per harvest, and at least three high titer harvests can be obtained following transient transfection, not more than 8 × 108 packaging cells would have to be transfected to obtain sufficient material for a phase I study. As 0.5 μg of SERS plasmid suffices for high titer transient production from 5 × 106 packaging cells (Figure 2e), less than 100 μg plasmid DNA would be required for clinical-grade vector production. This reduces the costs of GMP-grade plasmid production and lowers the risk of plasmid contamination of retroviral supernatants. Based on a better understanding of other limiting components of the vector production system, further improvements will likely be possible.

Materials and methods


MGMT-encoding gammaretroviral vectors (pSF91, pSin11.SF) and lentiviral vectors (pRRL.PPT.PGK and pRRL.PPT.SF) have been described previously.16 In brief, pSF91 encodes an LTR-driven vector,6, 18 and pSin11.SF a corresponding SIN vector using the same SFFVp U3 region (SF; including the enhancer; −342 to +18, relative to the transcriptional start site, GenBank no. AJ224005) as an internal promoter. pRRL.PPT.PGK and pRRL.PPT.SF are lentiviral SIN vectors with internal promoters human PGK and SF, respectively. The basic lentiviral construct pRRL.PPT.PGK.eGFP.PRE was kindly provided by Luigi Naldini (Milano, Italy).

For a functional comparison of different promoters in the 5′-LTR, we constructed a modular vector set using four different promoters in the 5′-LTR in relation to three different internal promoters. As internal promoters, we used CMV, PGK (GenBank no. M11958, nucleotides (nt) 5–516) or SF. The four versions of the 5′-LTR were as follows:

Our former SIN series uses the MPSV U3 region to transcribe the full-length RNA in transfected packaging cells.3 The SCS series represents SIN vectors containing the CMV promoter fused to the start site (+1) of the full-length RNA. SRS constructs are SIN vectors that use the RSV U3 fused to the start site of the full-length RNA. The SERS series consists of SIN vectors that use a combination of the SV40 enhancer24, 25 and the RSV U3 fused to the start site of the full-length RNA.

Modified 5′-LTRs were cloned by overlap-polymerase chain reaction (PCRs). For the amplification of the CMV promoter (GenBank no. K03104, nt −582 to −1, relative to transcriptional start site), primers 5′CMVafl (5′-IndexTermCGATCTTAAGTAGTTATTAATAGTAATCAA-3′) and 3′CMVR (5′-IndexTermGTCAATCGGAGGACTGGCGCCGGTTCACTAAACCAGCTCTG-3′), 5′CMVR (5′-IndexTermCAGAGCTGGTTTAGTGAACCGGCGCCAGTCCTCCGATTGAC-3′) and 3′Leaderbgl (5′-IndexTermCCAGATACAGATCTAGTTAGCCAA-3′) were used. PCR templates were pcDNA3 (Invitrogen, Karlsruhe, Germany) and pSF91,6 respectively. The PCR fragment was cloned into pSin11SF using AflII and BglII sites. For amplification of the RSV promoter (GenBank no. J02342, nt −233 to −1, relative to transcriptional start site) primers 5′RSVscaafl (5′-IndexTermGCTTAGTACTCTAGCTTAAGAATGTAGTCTTATGCAATACT-3′) and 3′RSVRoverlap (5′-IndexTermAGTCAATCGGAGGACTGGCGCGTTTATTGTATCGAGCTAGGC-3′), 5′RSVRoverlap (5′-IndexTermGCCTAGCTCGATACAATAAACGCGCCAGTCCTCCGATTGACT-3′) and 3′LeaderBgl (see above) were used. Templates for this overlap-PCR were pRSV-Rev (kindly provided by Tom Hope, Chicago, IL, USA) and pSF91, respectively. The PCR fragment was cloned into the pSin11.SF using AflII and BglII restriction sites. The SV40 enhancer (GenBank no. AF025845, nt 18–252), which includes two 72 bp tandem repeats was amplified using primers 5′SV40enh (5′-IndexTermCTACTTAAGACGCGTGGCCTGAAATAACCTCTGAA-3′) and 3′SV40enh (5′-IndexTermGCTACTTAAGGGACTATGGTTGCTGACTA-3′) and the pRL-SV40 plasmid (Promega, Mannheim, Germany) as a template. The PCR product was transferred into the AflII site of pSRS11.SF (upstream of the RSV promoter). To clone a tetracycline-inducible promoter into the 5′-LTR to drive the full-length vector RNA, we amplified the promoter fragment via PCR using primers 5′Tet11afl (5′-IndexTermGCTACTTAAGCTTCTTTCACTTTTCTCTGTCA-3′) and 3′Rkpn (5′-IndexTermGAGAACACGGGTACCCGGGC-3′) and plasmid ptES1-1(g)p. The tetracycline-inducible promoter consists of a tet-operator hexamer with 4C specificity fused to the Moloney murine leukemia virus minimal promoter. The resulting PCR fragment was cloned into the AflII and KpnI sites of the 5′-LTR of pSRS11.SF. All PCR fragments were confirmed by sequencing.

To insert the DsRed Express (Clontech, Mountain View, CA, USA) cDNA and the BGH polyadenylation signal (polyA) downstream of the packaging signal and upstream of the internal promoter, a new multiple cloning site (MCS) was constructed, in which successively the DsRed Express sequence and the BGH polyA were included. The phosphorylated oligonucleotides 5′leaderMCS (5′-IndexTermGCTGACGCGTACTAGCGCTGACTTCGAAGC-3′) and 3′leaderMCS (5′-IndexTermGGCCGCTTCGAAGTCAGCGCTAGTACGCGTCAGCTGCA-3′) were annealed and ligated into the PstI/NotI opened sites of the retroviral leader region of pMP71-CD34-2A-eGFP to introduce AflII, Eco47III and BstBI restriction sites. The DsRed Express cDNA (Clontech) was PCR-amplified with primers 5′DsRedmlu (5′-IndexTermGCCTACGCGTGTCGCCACCATGGCCTCCTCCGA-3′) and 3′DsRedeco47III (5′-IndexTermGTCTAGCGCTCTACAGGAACAGGTGGTGGC-3′) and cloned into the respective sites of the leader MCS. The BGH polyA (232 bp, template pcDNA3, Invitrogen) was amplified via PCR with primers 5′BGHpolyAcla (5′-IndexTermGCTAATCGATACTGTGCCTTCTAGTTGCCA-3′) and 3′BGH polyAsal (5′-IndexTermGCATGTCGACCATAGAGCCCACCGCATC-3′), digested with SalI and ClaI, treated with Klenow polymerase and ligated into the Eco47III opened leader MCS.

Cell lines, transfections and transductions

Phoenix-gp packaging cells (kindly provided by G Nolan, Stanford, CA, USA) and 293T cells were used for retroviral and lentiviral supernatant production, respectively. Phoenix-gp, 293T, HT1080 and murine fibroblast SC-1 cells were maintained in Dulbecco's modified Eagle's medium (DMEM, Gibco, Gaithersburg, MD, USA) supplemented with 10% fetal calf serum, 100 U/ml penicillin/streptomycin and 2 mM glutamine.

The day before transfection, 5 × 106 Phoenix-gp or 293T cells were plated on a 10 cm dish. The medium was exchanged and 25 μ M chloroquine (Sigma-Aldrich, Munich, Germany) was added. 0.5–5 μg transfer vector DNA, 1 μg of a eGFP reporter plasmid to determine transfection efficiencies (if eGFP was not the cDNA of the transfer vector), and 2 μg of an ecotropic envelope plasmid22 or 5 μg of an RD114/TR envelope plasmid (kindly provided by F-L Cosset, Lyon, France) were used. In addition, 10 μg of a retroviral gag/pol plasmid (M57-DAW) were transfected using the calcium phosphate precipitation method. M57 is an MLV gag/pol expression plasmid (kindly provided by Harald Wodrich, Montpellier, France) and its derivative M57-DAW is devoid of residual overlaps with the transfer vector. When producing lentiviral vectors, 5 μg of a Rev plasmid (pRSV-Rev) were co-transfected. For vector production of the tet-inducible vector, 5 μg of the expression plasmid pPGK.TP, harboring the authentic TA with 4C DNA-binding specificity, was co-transfected.

The medium was changed after 10–12 h. Equal transfection efficiency was controlled by FACS analysis. Supernatants containing the viral particles were collected 24–72 h after transfection, filtered through a 0.22 μm filter and stored at −80°C until usage.

SC-1 cells were transduced by centrifugation for 60 min at 2000 r.p.m. at 32°C in the presence of 4 μg/ml protamine sulfate (Sigma-Aldrich). After transduction, cells were grown for 4–5 days and subsequently analyzed by flow cytometry and Northern blot. Titration of the vector supernatants on SC-1 cells was performed as described previously.3

Rhesus monkey primary cells

Purpose-bred male rhesus monkeys (Macaca mulatta), each weighing 2.5–4 kg and cynomolgus monkeys (Macaca fascicularis) weighing 4–6 kg, aged 2–3 years old, were used. Housing, experiments and all other conditions were approved by an ethics committee in conformity with legal regulations in The Netherlands.

Purification of CD34+ rhesus cells was performed by positive selection using Dynalbeads (Dynal, Oslo, Norway; Neelis et al.44) Briefly, low-density cells were incubated with an IgG2A antibody against CD34 (mAb 561; from G Gaudernack and T Egeland, Rikshospitalet, Oslo, Norway) covalently linked to rat anti-mouse IgG2A beads. CD34+ cells devoid of the CD34 antibody were recovered using polyclonal antibodies against the Fab part of the CD34 antibody (Detachebead, Dynal Biotech, Hamburg, Germany). Purified CD34+ cells were analyzed by flow cytometry and prestimulated at a concentration of 105/ml for 2 days prior transduction in serum-free enriched DMEM supplemented with human recombinant growth factors fetal liver tyrosine kinase 3-ligand (Flt3-L; 50 ng/ml, kindly provided by Amgen, Thousand Oaks, CA, USA), thrombopoietin (rhTPO; 10 ng/ml, kindly provided by Genentech, South San Francisco, CA, USA) and stem cell factor (SCF; 100 ng/ml) as described previously.45

Retroviral transduction of rhesus CD34+ cells

To enhance the transduction efficiency, Falcon 1008 (35 mm) bacteriological culture dishes were coated with recombinant fibronectin fragment CH-296 (Takara Shuzo, Otsu, Japan) at a concentration of 10 μg/cm2.46 Before adding the prestimulated purified rhesus BM to the fibronectin-coated dishes, the CH-296 fragment was preincubated with virus supernatant for 1 h at 37°C.46 Subsequently, nucleated cells were resuspended in the vector-containing supernatant (MOI as indicated in Results) supplemented with hematopoietic growth factors (Flt3-L, TPO and SCF) and added to the coated and preloaded dishes in a concentration of 1–3 × 105 cells/ml. Over a period of 2 days, culture supernatant was replaced completely by resuspending non-adherent cells into fresh retrovirus supernatant and growth factors. After 2 days the cells were harvested, the transduction efficiency was analyzed by flow cytometry.

Flow cytometry

For intracellular staining of MGMT, the Cytofix/Cytoperm Kit (Becton Dickinson, Heidelberg, Germany) was used according to manufacturer's instructions. In brief, at least 3 × 105 cells were harvested and washed in phosphate-buffered saline. Cytofix/Cytoperm fixative (4% paraformaldehyde: 250 μl) was added for 20 min at 20°C. Washing with 1 ml Perm/Wash buffer was followed by incubation 30 min at 4°C with 0.25 μg of a murine anti-MGMT monoclonal antibody (Chemicon, Hampshire, UK). After two washing steps with Perm/Wash buffer, 1 μg of a goat-anti-mouse phycoerythrin-conjugated secondary antibody (Becton Dickinson) was added for 30 min at 4°C. After two additional washing steps, the samples were analyzed in a FACScalibur using CellQuest software (Becton Dickinson). A gate was set on a homogeneous cell population, as determined by scatter characteristics, and 20 000 events were monitored. A marker was set to calculate the percentage and mean fluorescence intensity of positive cells.

For the dual fluorescence assay with eGFP and DsRed Express, 2 μg transfer vector, 10 μg M57-DAW, 2 μg ecotropic MLV env were transfected into Phoenix-gp cells using the calcium phosphate technique. Three days post-transfection, the packaging cells were analyzed by FACS. Compensation of FL-1 (eGFP) and FL-2 (DsRed Express) was performed using monofluorescent constructs. A marker gate was set and the mean fluorescence intensities for eGFP- and DsRed Express-positive cells were calculated accordingly.

Northern blot

Total RNA preparation and Northern blot analysis was performed as described before.18 Specific probes (100 ng) corresponding to the PRE fragment, present in the respective retroviral and lentiviral vectors and the eGFP cDNA were radiolabeled using the DecaLabel DNA labeling kit (Fermentas, St Leon-Rot, Germany). Membranes were washed, sealed and exposed to X-ray film (Kodak X-Omat-AR, Kodak, Stuttgart, Germany) and quantified by Phosphoimager (Amersham, Freiburg, Germany) analysis.

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We thank L Naldini for providing the basic lentiviral construct, F-L Cosset for the RD114/TR envelope, E Will for providing M57-DAW, and C Klanke and M Id for technical assistance. This work was supported by the Deutsche Forschungsgemeinschaft (1837/Ba4) and by the Integrated Project CONSERT of the European Union (LSHB-CT-2004-005242).

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Correspondence to J Bohne.

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Supplementary Information accompanies the paper on Gene Therapy website (http://www.nature.com/gt)

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Schambach, A., Mueller, D., Galla, M. et al. Overcoming promoter competition in packaging cells improves production of self-inactivating retroviral vectors. Gene Ther 13, 1524–1533 (2006). https://doi.org/10.1038/sj.gt.3302807

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  • mouse leukemia virus
  • RNA processing
  • hematopoiesis

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