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Journal of Virology, February 2004, p. 1384-1392, Vol. 78, No. 3
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.3.1384-1392.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Multiple Modifications Allow High-Titer Production of Retroviral Vectors Carrying Heterologous Regulatory Elements

Juraj Hlavaty,^1,2 Anika Stracke,¹ Dieter Klein,¹ Brian Salmons,³ Walter H. Günzburg,^1* and Matthias Renner³

Institute of Virology, University of Veterinary Medicine,¹ Austrianova, A-1210 Vienna, Austria,³ Cancer Research Institute, Slovak Academy of Sciences, Bratislava, Slovak Republic²

Received 17 June 2003/ Accepted 16 October 2003

Top Abstract Introduction Materials and Methods Results Discussion References

 
Tumor-specific expression of therapeutic genes is a prerequisite in many approaches to retrovirus-mediated cancer gene therapy. However, tissue specificity is often associated with a reduction in viral titer. To overcome this problem, we constructed a series of murine leukemia virus (MLV)-based retroviral promoter conversion (ProCon) vectors carrying either the simian virus 40 poly(A) signal trimer (3pA) inserted in the 3' long terminal repeat (LTR) of these vectors or the human cytomegalovirus enhancer region (CMVe) inserted 5' and 3' of the retroviral LTRs. Furthermore, an extended AT stretch/attachment site (AT/att) of wild-type MLV was introduced into the vector. In the vector-producing cells, insertion of the CMVe and/or the 3pA resulted in a three- to fourfold-enhanced marker gene expression compared to the parental vector, whereas insertion of the AT/att gave a slight decrease in expression. The combination of all three modifications had no additional effects. In contrast, however, neomycin selection of infected cells revealed only a slight increase in virus titer with vectors carrying the 3pA modification; the titer was increased by 1 with vectors containing the extended AT/att, although the viral DNA copy numbers in infected cells were similar with both types of vectors. Thus, insufficient integration rather than insufficient reverse transcription and/or production of virus RNA is the major cause for the low titer obtained with the ProCon vectors. The combination of all three modifications resulted in a 2- to 3-log increase in the virus titer. These modifications result in expression targeted ProCon vectors with titers similar to those of nonmodified MLV-based vectors.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Moloney murine leukemia virus (MoMLV)-derived retroviral vectors are widely used to transfer genes of interest into a variety of mammalian cells, both in vitro and in vivo (reviewed in reference 34). Despite their ability to integrate into the host genome permitting long-term gene expression, the use of MoMLV-derived vectors as a universal gene transfer vehicle is still far from a reality. Both the generally low transduction efficiencies and the difficulties in targeting have proven to be stumbling blocks for the use of retroviral vectors in in vivo gene therapy protocols. Since proteins that are therapeutic in the context of one tissue or cell type may be harmful in another (34), such targeting is especially important to in vivo approaches because, unlike in ex vivo approaches, the physical isolation of the cells to be transduced, and their testing prior to reimplantation is not possible (30). However, most efforts to modify retroviral vectors to ensure tissue-specific targeting are associated with additional reductions in their already low transduction efficiency. Thus, it is imperative to find methods to achieve targeting without concomitant loss of infectivity (13).

Targeting can be achieved either at the level of the infection event or, later, at the level of expression of the transduced therapeutic genes. Targeting at the level of gene expression offers the opportunity to maintain titer, which is often reduced as soon as the surface protein of MoMLV is modified (34, 36), while achieving the necessary limited expression of therapeutic gene product. Previous attempts have thus been made to replace the almost ubiquitously active 72-bp MoMLV enhancer with a heterologous tissue-specific enhancer (4, 7, 19). However, a recent study has shown that the 72-bp enhancer is not the only enhancer in the MLV-U3 region (11) and that viruses deleted in the 72-bp enhancer are still able to replicate (25). Since it would be advantageous to delete all constitutive MLV enhancers in a tissue-specific vector we have previously reported construction of the ProCon system in which almost all of the U3-region of the retroviral 3' long terminal repeat (3'LTR) is replaced by an inducible or tissue-specific promoter, whereas the 5'LTR remains intact (22, 23, 29). After infection of target cells with such retroviral ProCon vectors, the heterologous promoter is duplicated and one copy translocated to the U3-region of the 5'LTR so that it is the only promoter regulating the expression of the therapeutic gene. However, although cell and tissue specificity could be demonstrated by using these vectors (23, 29), the virus titer was generally found to be reduced by 10- to 100-fold. This result was not unexpected since major modifications in the LTRs are known to often result in decrease of viral titer.

In the present study, we have comprehensively analyzed the potential causes for the observed reduction in titer and have identified three responsible factors. Modification of ProCon vectors containing mouse mammary tumor virus (MMTV) or whey acidic protein (WAP) promoters by insertion of (i) a CMV enhancer to increase viral RNA production in virus-producing cells, (ii) a strong polyadenylation signal in the modified 3'LTR to prevent readthrough of viral RNA and to stabilize mRNA, and (iii) an elongated attachment site, together with an untranslated region upstream of the 3'LTR to increase the integration efficiency of the provirus, result in ProCon vectors with titers similar to those of nonmodified MoMLV-based (standard) vectors.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Plasmid construction. Plasmid pVL (Fig. 1) was constructed by inserting the luciferase (luc) gene released from plasmid pGL3-Basic (Promega) into the BamHI-linearized plasmid pLXSN (21). The human cytomegalovirus (CMV) enhancer was excised from plasmid pCI-neo (Promega) by BglII-PvuI digestion; the resulting 659-bp fragment was cloned in either a sense or an antisense orientation into vector pVL. The presence of a single copy of the enhancer and its orientation was shown by restriction and sequencing analysis. The resulting vectors pVL5Cs and pVL5Ca carry the CMV enhancer upstream of the viral 5'LTR and pVL3Cs and pVL3Ca containing it downstream of the 3'LTR, respectively (Fig. 1). Plasmid pVE (Fig. 1) was created by digesting vector pLXSNEGFP (14) with EcoRI and AgeI and religation of the vector backbone. Vectors pVEA, pVEmA, and pVEwA containing a triple polyadenylation signal 27 bp downstream of the wild-type poly(A) signal (Fig. 1) were obtained by inserting the 3083-bp KpnI fragment of pRC2Ubipac-3pAsense into the KpnI sites of vectors pVE, pVEm, and pVEw (identical to pLXPCWapEGFP) (23), respectively. Plasmid pRC2Ubipac-3pAsense is identical to pRC2/3pA (31) except for the orientation of the polyadenylation signal trimer (3pA).

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FIG. 1. Retroviral vector constructs. VL, MLV-based vector (LTR consisting of unique 3' (U3), repeated (R), and unique 5' (U5) regions) containing luciferase gene (Luc), as well as an internal SV40 promoter (SV40) controlling expression of a neomycin resistance gene (neo); VL5Cs or VL5Ca, VL with CMV enhancer (checked box) cloned in a sense or an antisense orientation upstream of the 5'LTR; VL3Cs or VL3Ca, VL with CMV enhancer (checked box) cloned in a sense or an antisense orientation downstream of the 3'LTR; VE, MLV-based vector containing the EGFP in place of the Luc gene; VEA, VE with poly(A)-trimer (3pA, gray checked box) cloned 3' of the 3'LTR R region; VEm or VEw, ProCon vector with the MMTV or WAP promoter (black box) in the 3' U3 region; VEmA or VEwA, MMTV or WAP ProCon vector with poly(A)-trimer cloned 3' of the 3'LTR R region; VETm or VETw, MMTV or WAP ProCon vector containing the extended AT/att cloned 5' of the 3'LTR and in the 5' end of the 3'LTR U3 region (AT/att, dotted boxes); VEmA5Cs or VEmA5Ca and VEwA5Cs or VEwA5Ca, MMTV and WAP ProCon vector with CMV enhancer cloned in a sense or an antisense orientation upstream of the 5'LTR and poly(A)-trimer cloned 3' of the 3'LTR R region; VETmA5Cs or VETmA5Ca and VETwA5Cs or VETwA5Ca, MMTV and WAP ProCon vector with CMV enhancer cloned in a sense or an antisense orientation upstream of the 5'LTR, extended AT/att and poly(A)-trimer. Abbreviations used in vector nomenclature: V, vector; E, EGFP; L, LUC; m, MMTV promoter; w, WAP promoter; A, poly(A)-trimer; T, extended AT/att; 5C, CMV enhancer cloned upstream of the 5'LTR; 3C, CMV enhancer cloned downstream of the 3'LTR; s, sense; a, antisense.

To clone pVEm (Fig. 1), plasmid pLXSN was digested with AflIII and BamHI; the fragment containing 5'LTR and packaging signal was ligated to an AflII-BamHI fragment from p125.6 (30) carrying the simian virus 40 promoter-neomycingene (SV40neo) cassette, the pBRori sequence, and the hybrid mtv2/MLV 3'LTR. The resulting plasmid was named pLX125 and, after removal of an unwanted SacII site, was renamed pLXPCMTV. Finally, the enhanced green fluorescent protein (EGFP) gene from plasmid pEGFP-1 (Clontech) was ligated to the linearized plasmid pLXPCMTV to generate pVEm (Fig. 1). A DNA fragment containing the WAP negative regulatory element (NRE) promoter was amplified by PCR with the plasmid pWAP2hGH (9) as the template and ligated to the 7,547-bp SacII-MluI fragment of pVEm. The resulting plasmid was named pVEw (Fig. 1). Again, the human CMV enhancer was inserted in sense and antisense orientations upstream of the 5'LTR into these vectors by using the 1,906-bp PvuI-AscI fragment from pVL5Cs or pVL5Ca, respectively. The resulting constructs were named pVEmA5Cs, pVEmA5Ca, pVEwA5Cs, and pVEwA5Ca (Fig. 1). A 92-bp DNA fragment containing the elongated attachment site was PCR amplified from plasmid pLESN1aM by using the primers JH22 (5'-GGA TCG ATG GGC CCG ATA AAA TAA AAG ATT TTA TTT AG-3') and JH24 (5'-CAT CCA AAC CGT TCG ATC GGG CGC CAC-3') and inserted into 8,725-, 8,015-, 9,644-, and 8,973-bp vector fragments of pVEm, pVEw, pVEmA5Cs, pVEmA5Ca, pVEwA5Cs, and pVEwA5Ca, resulting in vectors pVETm, pVETw, pVETmA5Cs, pVETmA5Ca, pVETwA5Cs, and pVETw5Ca, respectively (Fig. 1).

Cell lines. Human 2GP19Talf amphotropic retroviral packaging cells (24) and murine PA-317 packaging cells (20) were grown in Dulbecco’s modified Eagle’s medium (DMEM)/Glutamax (Life Technologies) supplemented with 10% fetal calf serum (Life Technologies). NIH 3T3 cells (ATCC CRL-1658) were maintained in DMEM/Glutamax supplemented with 5% fetal calf serum.

Transfection. Transfections were performed by calcium phosphate coprecipitation (6) as recommended by the supplier (Amersham Biosciences). For transient-transfection experiments, cells were lysed 2 days later and subjected to luciferase assay as described elsewhere (33). For stable transfections the transfected cells were treated with trypsin and selected in DMEM containing 0.4 mg of Geneticin (G418; Life Technologies)/ml until colonies were formed.

Infection. Culture supernatant from 2 x 10⁶ virus-producing cells was used to infect 4 x 10⁵ of NIH 3T3 target cells as described elsewhere (24). For titer estimation, infected cells were split 24 h after infection and selected in medium containing 0.4 mg of G418/ml. After 10 to 14 days of culture, drug-resistant colonies were counted, and the CFU per milliliter of vector supernatant were calculated.

FACS analysis. Stably transfected cells were analyzed by fluorescence-activated cell sorting (FACS) immediately after the medium supernatant was used for infection experiments. For FACS analysis, cells were trypsinized, washed twice with phosphate-buffered saline, and then 10,000 cells per sample were analyzed for fluorescence with a FACS analyzer (FACSCalibur; Becton Dickinson). The mean fluorescence intensity (MFI) of positive cells was determined.

Nucleic acid extraction, real-time PCR, and real-time RT-PCR. Viral RNA from cell culture supernatant was extracted by using QIAamp viral RNA minikit (Qiagen) as recommended by the manufacturer. The amount of viral RNA in the supernatant of vector producing cell lines was estimated by real-time reverse transcription-PCR (RT-PCR). The primer and probe sequences for the EGFP real-time RT-PCR assay have been published previously (15). The neomycin-specific real-time RT-PCR assay consists of primers Neo364f (5'-CGGCTGCATACGCTTGATC-3') and Neo529r (5'-ATCGACAAGACCGGCTTCC-3') and the probe Neo423p (5'-FAM-AACATCGCATCGAGCGAGCACGT-TAMRA-3'). The conditions of the real-time RT-PCR assays have been already described (15, 16). The detected fluorescence signals were analyzed by using sequence detection software (version 1.6.3; Applied Biosystems). The amount of viral RNA per 5-µl sample was calculated by using a serial dilution of either in vitro transcribed RNA or viral RNA as a standard. The reaction efficiency of each assay was calculated by the method described previously (16) and revealed only minimal differences (<5%). Viral DNA of infected cells was extracted by using the DNeasy tissue kit (Qiagen) as recommended by the manufacturer. The cell number per sample was estimated by using a real-time PCR-assay targeting the 18S ribosomal DNA (15). The amount of total viral DNA was estimated by using the EGFP-specific real-time PCR-assay described above (15).

Top Abstract Introduction Materials and Methods Results Discussion References

 
The in vivo use of retrovirus vectors in clinical trials is often limited by the relatively low production of infectious virus. This limitation is even more pronounced in vectors with modifications in the LTR sequences. We speculated that one possibility to raise the titer might be to increase the amount of viral RNA production in the packaging cells. Thus, we analyzed whether introduction of a transcriptional enhancer sequence might increase viral vector production. The retroviral vector VL, based on the MLV LXSN-vector family, encodes the firefly luciferase (luc) reporter gene driven by the MLV promoter in the 5'LTR, and the neomycin resistance gene is under the transcriptional control of the internal SV40 promoter (LTR-luc-SV40neo-LTR). A 659-bp sequence of the human CMV immediate-early enhancer (CMVe) was inserted upstream of the 5'LTR or downstream of the 3'LTR, respectively, in this plasmid. The CMVe was inserted as a single copy in sense and antisense orientation with respect to the proviral sequences.

Equimolar amounts of plasmid DNA of the four thus-generated constructs (4 x 10¹¹ molecules) were transiently transfected into mouse PA-317 and human 2GP19Talf packaging cell lines, and the luciferase activity was measured. In both PA-317 cells (Fig. 2A) and 2GP19Talf cells (Fig. 2B) constructs containing the CMV enhancer (pVL3Cs, pVL3Ca, pVL5Cs, and pVL5Ca; Fig. 1) yielded higher levels of luciferase activity than the enhancerless construct (pVL; Fig. 1). With the exception of the construct pVL3Ca, luciferase levels were increased between two- and fourfold. A stronger and more consistent enhancement was observed in 2GP19Talf cells (Fig. 2B), and it has been shown by others that the CMV enhancer works especially well in cells of primate or human origin (1).

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FIG. 2. Effect of the human CMV enhancer on marker gene expression in the context of a retrovirus vector. Relative Luc activity in PA-317 (A) and 2GP19Talf (B) packaging cells transiently transfected with constructs containing the hCMV enhancer (pVL3Cs; pVL3Ca, pVL5Cs, and pVL5Ca) and with the parental plasmid pVL. A total of 2.5 x 105 PA-317 or 5 x 105 2GP19Talf packaging cells were seeded per well of a six-well tissue culture plate, allowed to adhere overnight, and transfected with 4 x 1011 molecules of plasmid DNA (3.0 µg for pVL and 3.26 µg for CMVe-containing vectors). Cells were lysed 2 days later and subjected to luciferase assay. Insertion of the enhancer upstream of the 5'LTR resulted in stronger transgene expression than did insertion downstream of the 3'LTR. Moreover, insertion of the enhancer sequence in the sense orientation led to higher transgene expression than did insertion in the antisense orientation. A stronger expression enhancement was observed with human 2GP19Talf cells than with mouse PA-317 cells. A significant difference from parental, unmodified vector as determined by using a Student t test is indicated (, P = 0.05; , P = 0.1).

In further experiments, the effect of the CMVe on virus titer was studied by infecting NIH 3T3 cells with the vector viruses VL, VL5Cs, and VL5Ca derived from the stably transfected 2GP19Talf producer cells, and G418-resistant colonies were scored (G418 titer). In parallel, the supernatants used for infections were subjected to real-time RT-PCR with primers specific for the neomycin gene to measure the amount of genome containing virus particles. Although not statistically significant in a Student t test, the number of genome containing virions (real-time titer) in the supernatant, as well as the number of infection events (G418 titer), was doubled (Table 1) with CMVe containing vectors (VL5Cs and VL5Ca) compared to the vector without the CMVe (VL), suggesting that the activity of the enhancer in the packaging cells results in increased transcription and a concomitant increase in genome carrying viral particles and titer.

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TABLE 1. Effect of the human CMV enhancer on vector productiona

Sequence elements within the 3'LTR provide signals for correct RNA polyadenylation. Most of the sequences that have been identified to date are located within the R region, however, a number of viruses (avian sarcoma virus, MMTV, and human T-cell leukemia virus type 1) carry these signals within their U3 region (8). Additional sequences have also been identified and suggest that modifications of the 3'LTR U3 region might affect transcriptional termination, thereby influencing virus production. Thus, we analyzed whether introduction of a strong poly(A) signal after the R region in the 3'LTR might lead to an increase in production of recombinant viruses. A DNA fragment consisting of three copies of the SV40 early mRNA polyadenylation signal [poly(A)-trimer, 3pA] was inserted either into the basic MLV-based vector pVE or into the MLV-based ProCon vectors pVEm and pVEw harboring modifications in the U3-region of the 3'LTR. In pVEm the MLV 3'LTR U3-region is replaced by the MMTV U3-region and in pVEw by the WAP promoter (Fig. 1). The constructs pVEA, pVEmA, and pVEwA containing the poly(A)-trimer, as well as parental vectors without this modification, were used to generate stable mass populations of virus-producing cells based on the 2GP19Talf packaging cell line. The production of recombinant viruses and level of transgene expression in transfected and infected cells was examined.

Estimation of MFI values was used to monitor EGFP expression levels. In the transfected packaging cells, MFI values obtained for pVE, pVEm, and pVEw [parental vectors without the extra poly(A)-trimer] were very similar (MFIs of 130 to 140, Fig. 3A). Since pVE carries wild-type MLV/MSV U3 sequences at both ends, whereas the other two constructs carry heterologous U3 sequences in the 3'LTR (MMTV in VEm and WAP in VEw), these results suggest that promoter interference between the heterologous promoter in the 3'LTR and the MLV promoter of the 5'LTR is negligible (Fig. 3A). However, a two- to threefold increase in MFI was observed in cells transfected with constructs carrying the additional triple poly(A) signal (pVEA, pVEmA, and pVEwA) compared to cells carrying the respective parental vectors (Fig. 3A).

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FIG. 3. Effect of an additional strong heterologous polyadenylation signal on virus production and transgene expression in virus-producing cells. 2GP19Talf-based cells were transfected with (i) the parental construct pVE; (ii) two ProCon derivatives, pVEm and pVEw, containing the MMTV and WAP promoters in their 3'LTR U3 regions, respectively; and (iii) the same constructs harboring additional triple poly(A) signals in the 3'LTR U5 region (3pA). Populations of virus-producing cells were tested for transgene expression levels (EGFP; MFI values, black bars) (A) and for virus production as measured by real-time RT-PCR (open bars) and by the number of infected NIH 3T3 cells (G418 titer, shaded bars) (B). The introduction of a heterologous polyadenylation signal in the U5 region of the 3'LTR had a slight positive effect on the level of transgene expression (two- to threefold increase) and on the amount of infectious virus as measured by G418 selection (two- to threefold increase). At least three independent infection experiments were performed. Shown are data from a representative experiment.

The effect of poly(A)-trimer insertion on virus production and virus titer was studied by measuring the amount of virus particles in the supernatant of virus-producing cells via real-time RT-PCR, as well as by counting of G418-resistant colonies after infection of NIH 3T3 cells. These experiments revealed that insertion of 3pA has only a slight effect (two- to threefold increase) on G418 virus titer of the MMTV and WAP ProCon vectors (Fig. 3B, shaded bars, compare pVEm versus pVEmA and pVEw versus pVEwA). Surprisingly, a similar effect was observed with parental vector transfected cells in which the vector 3'LTR does not contain any modification compared to wild-type MoMLV and therefore should serve as a strong transcriptional termination signal (Fig. 3B, shaded bars, compare pVE and pVEA). In addition, a >100-fold difference in G418 titer was still observed between the parental pVE vectors with or without 3pA and the ProCon vectors pVEm and pVEw with or without 3pA (Fig. 3B, shaded bars). In contrast, determination of the amount of viral RNA packaged into virus particles from pVE, pVEm, pVEw, pVEA, pVEmA, and pVEwA transfected producer cells by using an EGFP-specific real-time RT-PCR revealed that about the same amount of genome-containing virions are produced by the parental and ProCon vector transfected cells (Fig. 3B, open bars, compare pVE, pVEm, and pVEw) and that only a minimal increase of up to twofold in RNA-containing virions is detectable when constructs are analyzed that contain the additional poly(A)-trimer (Fig. 3B, open bars, compare constructs with or without 3pA). Based on these data, we can assume that neither promoter interference nor lack or instability of proviral RNA as a result of insufficient transcription termination is responsible for the low infection potential of ProCon vectors.

The clear discrepancy between the similar amounts of RNA-containing virions detected in parental and ProCon transfected packaging cell supernatants (Fig. 3B, open bars) and the number of infection events later measured by G418 titer (Fig. 3B, shaded bars) suggests that events between virus production and the expression of the integrated provirus in the target cell must be responsible for the differences in titer observed.

The +primer binding site (+PBS), which consists of a stretch of purines located immediately 5' of the 5' boundary of the 3'LTR, is essential for the initiation of second strand DNA synthesis during RT. The +PBS has been defined as a 13-bp region with the sequence 5'-AGAAAAAGGGGGG-3' in MLV and is present in the ProCon vector immediately 3' of the inserted heterologous gene and 5' of the 3'LTR (Fig. 4A). However, a number of recent studies have suggested that an additional AT stretch immediately 5' of the polypurine tract (PPT) may also be critical for efficient positive-strand priming (2, 26, 27, 35). The retroviral attachment site consisting of a 13-bp inverted repeat at the 5' end of the U3 region and at the 3' end of the U5 region of MLV is essential, for instance, for integration of viral DNA in the infected cells (5, 28). ProCon vectors carry this 13-bp inverted repeat, followed directly by the heterologous promoter. However, recently it has been suggested that a longer attachment site improves the efficiency of integration (3, 32, 37). Thus, the sequence containing an A-stretch and a T-stretch upstream of the PPT, as well as 23 bases naturally occurring in the MLV-LTR U3 region 3' of the inverted repeats, was introduced in the 3'LTR of the ProCon vectors (Fig. 4A).

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FIG. 4. (A) Structure of the noncoding region with extended attachment sites. By using standard PCR techniques, the noncoding sequence between the 3' end of the env gene and 5' end of the 3'LTR, as well as the first 36 bases of the 3'LTR U3 region of wild-type MLV, were introduced into ProCon vectors. The inserted AT/att sequence contains the A-stretch, the T-stretch, and PPT upstream of 3'LTR and 13 bases of inverted repeats, followed by 26 bases of the sequence naturally occurring in the MLV-LTR U3 region 3' of the inverted repeats. Shown are also the ClaI and SacII restriction sites (in italics) used for exchange of the original sequence with the modified sequence. (B) Effect of the noncoding region with AT-stretch/extended attachment site on the virus production and transgene expression in virus-producing cells. 2GP19Talf-based cells were transfected with the parental ProCon vector plasmids pVEm and pVEw, as well as with the same constructs harboring the extended AT/att sequence. Populations of virus-producing cells were tested for transgene expression levels (EGFP; MFI values, black bars) (B) and for virus production as measured by real-time RT-PCR (open bars) and by the number of infected NIH 3T3 cells (G418 titer, shaded bars) (C). Introduction of the extended AT/att sequence has a slight negative effect on the level of transgene expression (B). However, an increased number of infected cells were observed after infection with viruses containing the extended AT/att sequence (see panel C). At least three independent infection experiments were performed. Data from a representative experiment are shown.

2GP19Talf packaging cells were stably transfected with plasmids encoding ProCon vectors either with (pVETm and pVETw) or without (pVEm and pVEw) the extended AT stretch/attachment site (AT/att; Fig. 1). Subsequently, the level of transgene (EGFP) expression was analyzed by measuring the MFI in these cells. Surprisingly, insertion of the AT/att sequence led to a decrease in transgene expression (Fig. 4B, black bars, MFI 134 versus 100 for pVEm versus pVETm and MFI 138 versus 69 for pVEw versus pVETw). EGFP-specific real-time RT-PCR of virus supernatant, however, revealed no significant differences in amount of viral particles produced from modified and parental vector-producing cells (Fig. 4C, open bars). In contrast, the number of infection events as measured by number of G418-resistant colonies after infection of NIH 3T3 cells was increased 29-fold after infection with vector VETm compared to vector VEm carrying no extended attachment site (Fig. 4C, shaded bars) and 7-fold increased with vector VETw compared to vector VEw (Fig. 4C, shaded bars). Thus, insufficient integration and/or RT as a consequence of changes in sequence elements upstream and/or within the 3'LTR seem to be responsible for much of the reduction in titers observed with the original ProCon vectors (VEm and VEw). To distinguish between insufficient integration or insufficient RT as the cause of low infection titers, the numbers of EGFP gene copies detected by an EGFP-specific real-time PCR on total DNA from NIH 3T3 cells infected with VEm, VETm, VEmA5Ca, and VETmA5Ca (Fig. 1) were compared to corresponding G418 titers (see Table 3). No significant differences in EGFP copy numbers normalized to 10⁶ cells per 10⁸ virions per ml of supernatant have been observed between NIH 3T3 cells infected with VEm versus VETm and with VEmA5Ca versus VETmA5Ca, respectively. These data reveal that RT of virus RNA is not significantly increased by inserting the elongated attachment site (see Table 3, compare data for VEm versus VETm and data for VEmA5Ca versus VETmA5Ca, respectively). In contrast, G418 titers are increased 30-fold (Table 3, VETm versus VEm) and 20-fold (see Table 3, VETmA5Ca versus VEmA5Ca). Thus, comparison of the ratios of the G418 titers of VEm versus VETm and VEmA5Ca versus VETmA5Ca, respectively, with the ratios of the respective EGFP copy numbers clearly suggests that the increase in virus titer with vectors carrying the AT/att is due to enhanced integration rather than to enhanced RT.

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TABLE 3. Insertion of the extended AT/att site influences integration of the proviral DNA rather than RT

Finally, to optimize virus production and transgene expression in ProCon vectors, the AT/att modification was combined with the addition of the poly(A) sequence trimer and the CMV enhancer element (pVETmA5Cs, pVETmA5Ca, pVETwA5Cs, and pVETwA5Ca; Fig. 1). 2GP19Talf packaging cells were transfected with vectors carrying one, two, or all three modifications. Vectors carrying both the CMVe and the 3pA showed a three- to fourfold increase in EGFP MFI compared to cells transfected with the parental vector (Table 2, line 3 versus lines 6 and 7 and line 10 versus lines 13 and 14). Combination of all three modifications had no additional positive or negative effect on EGFP expression levels (Table 2, lines 6 and 7 versus lines 8 and 9 and lines 13 and 14 versus lines 15 and 16). The combination of 3pA and CMVe led to a five- to sixfold increase in infectious virus titer (Table 2, line 3 [4.4 x 10³ CFU/ml] versus lines 6 and 7 [2.1 x 10⁴ and 2.6 x 10⁴ CFU/ml]) for MMTV-containing ProCon vectors and a >1-log increase (Table 2, line 10 [26-fold, 4.7 x 10² CFU/ml] versus lines 13 and 14 [1.2 x 10⁴ CFU/ml]) in virus titer for WAP-containing ProCon vectors as measured by G418 selection of infected cells. The combination of all three modifications led to an overall titer increase for MMTV-containing ProCon vectors of >2 logs (Table 2, line 3 [4.4 x 10³ CFU/ml for VEm] versus lines 8 and 9 [6.3 x 10⁵ and 1.3 x 10⁶ CFU/ml for VETmA5Cs and VETmA5Ca, respectively]) and for WAP-containing ProCon vectors of >3 logs (Table 2, line 10 [4.7 x 10² CFU/ml for VEw] versus lines 15 and 16 [4.6 x 10⁵ and 9.8 x 10⁵ CFU/ml for VETwA5Cs and VETwA5Ca, respectively]), bringing the absolute values into the range of parental MLV-based vectors without modifications in the 3'LTR (Table 2, line 1 [1.83 x 10⁶ CFU/ml for VE]). Again, comparison of the data obtained by real-time RT-PCR, the real-time PCR data of EGFP copy numbers from total DNA of infected cells, and the infection data (titer) obtained after G418 selection clearly suggests that insufficient integration rather than insufficient RT and/or production of virus particles and/or promoter interference are the major cause for the low titer obtained with MMTV and WAP ProCon vectors used up to now. However, the effect of the CMV enhancer on titer increase is also substantial.

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TABLE 2. Characterization of 293alf producer cells stably transfected with ProCon vectors showing the effects of different modifications

Top Abstract Introduction Materials and Methods Results Discussion References

 
The use of retroviruses as a delivery tool for human in vivo gene therapy is often limited by relatively low levels of virus production (34). Vectors containing modifications in the LTR sequences, either for safety reasons (self-inactivating vectors) or as an attempt to ensure targeted expression of transduced genes, often show even lower titers. In the present study we explored three different strategies aiming to achieve high-titer ProCon vectors containing the promoters of either MMTV or the WAP gene.

We speculated that one way to increase the titer might be to increase viral RNA production in the packaging cells. Thus, we analyzed whether the introduction of a strong transcriptional enhancer sequence outside of the vector cassette could increase virus expression levels in the packaging cells and thus virus vector production. Indeed, insertion of CMVe into a plasmid encoding the retroviral vector led to an increase in vector transgene expression in transiently transfected cells. The effect was stronger when the enhancer was localized upstream of the 5'LTR compared to its insertion downstream of the 3'LTR. This effect was reproducibly observed in two independent packaging cell lines tested, and therefore in further experiments we focused only on constructs with CMVe inserted upstream of the 5'LTR. Measurement of the number of packaged vector genomes from virus containing cell supernatants by real-time RT-PCR revealed an 2-fold increase in the number of genome containing virions in the supernatant and a corresponding 2-fold increase in the number of subsequently infected cells, as measured by G418 resistance.

Efficient polyadenylation has been suggested to play a crucial role in the fate of mRNA. Its proposed functions include conferring mRNA stability, promoting efficiency of mRNA translation, and playing a role in the processing of the primary transcript, as well as nucleocytoplasmic transport of the mRNA (10). The retroviral LTR contains elements that control the correct initiation and termination of viral RNA synthesis, providing signals necessary for viral polyadenylation at the R/U5 junction. Like other eukaryotes, polyadenylation is dependent on the presence of a canonical hexanucleotide sequence AAUAAA located 16 to 25 bp upstream of the actual site of polyadenylation and a GU-rich sequence located 20 to 30 bp downstream. In some retroviruses, additional upstream signals located in the U3 region have also been identified (8). We speculated that modification within the U3 region in ProCon vectors might result in defective polyadenylation and thereby the stability of the viral mRNA could be influenced. Moreover, inefficient polyadenylation might lead to the generation of readthrough transcripts, which impairs packaging of virus RNA and thereby decreases the amount of infective virus particles. To counteract this, we inserted a sequence consisting of three copies of the strong SV40 early mRNA polyadenylation signal at the 5' end of the U5 region in the 3'LTR. Analysis of packaging cells stably transfected with these constructs showed an improved vector function, resulting in an increase in transgene expression, as well as an increase in infectious virus titer from both ProCon vectors (VEmA and VEwA), as well as from the MLV-based vector VEA. These data are consistent with those obtained from a lentiviral vector system, in which the U3 region of the 3'LTR had been deleted except for the first 24 nucleotides forming the left integrase attachment site, and the U5 region substituted with an additional heterologous polyadenylation sequence from bovine growth hormone gene gave a vector titer that was nearly twice as high as that from an unmodified vector (12). When the polyadenylation signal from the viral R region was the only polyadenylation signal present in the 3'LTR without any substitution of U5, virus production dropped to 1/10 of the virus production of the unmodified vector. These results are also consistent with recently published data (18), suggesting that sequences at the 3' end of the U3 region of MLV may also play a role in the polyadenylation of the viral transcript.

Despite introduction of a strong heterologous polyadenylation signal into ProCon vectors, the infection titer, measured by G418 selection of infected NIH 3T3 cells, was still low compared to the unmodified MLV-based vector. Nevertheless, a relatively high virus load in the supernatant from virus-producing cells was detected by real-time RT-PCR. Based on this discrepancy, we speculated that the rate of RT and viral DNA synthesis and/or integration in the ProCon vectors is influenced in a negative way. Our vectors, like all other retroviral vectors, carry the +PBS or PPT located immediately 5' of the 3'LTR. Similarly, like other retroviral vectors, we have maintained the attachment site (att site), also known as the inverted repeat, a region of 11 nucleotides immediately downstream of the PPT (13 nucleotides when located terminally at the 5' end of the proviral genome prior to integration). However, a number of researchers (2, 17, 26, 27, 35) have recently speculated that an additional 23 nucleotides located 3' of the previously described att site may be important for efficient integration. Similarly, it has been speculated that an additional AT stretch, located 5' of the previously described PPT, may also play a role in efficient RT.

The introduction of the extended attachment sequence and the AT stretch into our vectors (VETm and VETw; Fig. 1) resulted in an up to 20-fold increase in infectious titer from 4.4 x 10³ G418 CFU/ml for VEm and 4.7 x 10² G418 CFU/ml VEw to 1.1 x 10⁵ G418 CFU/ml for VETm and 3.2 x 10³ G418 CFU/ml for VETw (Table 2). However, consistent with the observations of others (2, 17, 26, 27, 35), these two regions do not affect the level of expression of virus-encoded proteins: the MFIs of VEm and VEw virus-producing cells is similar to those observed in VETm and VETw virus-producing cells (MFIs of 134 ± 5 versus 100 ± 2 and of 138 ± 21 versus 70 ± 2; Fig. 4B), respectively. Further, in contrast to previously reported data showing an up to 30-fold drop in the amount of virion RNA in the supernatant of MLV virus after deletion of 17 nucleotides downstream from the env stop codon (35), viral RNAs from VEm and VEw were packaged with the same efficiency as RNA from VETm and VETw, as determined by estimation of the genome-containing viral particles by real-time RT-PCR (Fig. 4C). The copy numbers of EGFP in total DNA of cells infected with vectors with or without the AT/att sequence compared to their respective G418 titers (Table 3) revealed that integration rather than RT is impaired in vectors lacking the AT-stretch/extended attachment site.

The neomycin phosphotransferase gene is expressed independently from the SV40 promoter in all of the vectors analyzed; however, modifications in the LTR or in other parts of the vector may directly or indirectly influence the SV40 promoter activity and/or transcript stability. Although we cannot rigorously rule out this possibility, it seems unlikely since, if this was true, the number of clones obtained after stable transfection of packaging cells with the various vector constructs would vary according to the vector used. However, in our experiments this effect was not observed. Similar numbers of clones were obtained during the generation of mass populations producing different viral vectors, indicating that the modifications had a negligible effect on the levels of expression and/or transcript stability. Further, when vector series carrying the MMTV promoter and one or more of the various modifications were compared, there was no significant difference in the number of G418 colonies obtained with virus-producing cells and, likewise, when comparisons were made within and among the various WAP promoter carrying vectors, again suggesting that the modifications were having little effect on expression levels and/or stability of the neomycin phosphotransferase-containing transcripts. In summary, we describe here an improvement for ProCon vectors containing the MMTV or WAP promoter via modifications that result in potentially expression targeted ProCon vectors with titers similar to those of nonmodified MLV-based vectors. Our results are generally applicable to the improvement of retroviral vectors and suggest that the inclusion of a powerful enhancer outside the body of the vector, the introduction of a strong polyadenylation signal, and the elongation of both the nucleotide stretch involved in priming second-strand DNA synthesis (+PBS) and the attachment site required for efficient integration can increase the titer of the retroviral vector by >2 logs.

 
We thank Elisabeth Knapp for excellent technical support in performing real-time PCR and RT-PCR. We thank Martina Urabl for providing plasmid pVL, Birgit Leichsenring for plasmid pLESN1aM, and Feride Öztürk-Winder for plasmids pVEm and pVEw.

 
* Corresponding author. Mailing address: Research Institute for Virology and Biomedicine, University of Veterinary Medicine, Veterinaerplatz 1, A-1210 Vienna, Austria. Phone: 43-1-25077-2301. Fax: 43-1-25077-2390. E-mail: walter.guenzburg{at}vu-wien.ac.at.

Top Abstract Introduction Materials and Methods Results Discussion References

 

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Journal of Virology, February 2004, p. 1384-1392, Vol. 78, No. 3
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.3.1384-1392.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

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