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Safe Production of High-Titer Retrovirus


Produce high-titer retroviral supernatants with safe packaging vectors

Peter Vaillancourt Coleen Miller
Stratagene

We describe a set of vectors that can be used with any MMLV-based retroviral vector to produce viral supernatants that have titers that are consistently greater than or equal to 107 cfu/ml following transient transfection. Because all of the cis and trans elements required to produce infectious virus are separated onto three plasmids, the probability of producing undesired replication-competent virus is very low. The range of target cells to be infected is determined by which one of four available envelope (env)-expressing vectors is cotransfected with the viral vector. The gag-pol and env open reading frames are all followed by an IRES linked to a downstream drug-resistance gene so that these vectors may be used to produce stable producer lines.

In recent years, tissue culture systems have been developed for the production of high-titer recombinant retrovirus that are capable of infecting a virtually limitless range of cell types. These systems have had a tremendous impact on fields for which very efficient gene delivery is essential.1 With Moloney Murine Leukemia Virus (MMLV)-based vectors, transduction efficiencies of greater than 90% are achievable for most mitotic cell types, and the copy number per cell can be easily controlled by varying the multiplicity of infection. These are clear advantages over most standard transfection methods; typically, only a small population of transfected cells is capable of the uptake and stable integration of vector DNA, and the copy number is unpredictable and often prohibitively high for many applications, such as cDNA library expression screening.2

Stratagenes pVPack retroviral system comprises a set of five vectors h exceptionally large inserts or some self-inactivating (SIN) vectors], use of the VSV-G envelope protein allows the viral supernatants to reach a concentration of several thousand-fold by simple ultacentrifugation without a substantial decrease in the stability of the virion particles.5

As an addendum, we recently used the pVPack-GP and pVPack-VSV-G vectors together with a set of ten ViraPort retroviral plasmid cDNA expression libraries7 for the large scale production of viral supernatants (see below for available supernatants). The high titers attainable with the vectors allow a consistent, high-throughput production of large numbers of frozen aliquots for each of the libraries, with each aliquot containing greater than or equal to 2 x 106 cDNA-harboring infectious virions when thawed. The ViraPort cDNA library retroviral supernatants are ready for immediate transduction and eliminate the need for plasmid library amplification, plasmid DNA preparation, and producer cell transfection for virus production.

Methods

Virus production was carried out by cotransfecting a 293-cell derivative with 3 g each of pFB-GFP, pVPack-GP, and one of the four env-expressing vectors (pVPack-VSV-G, pVPack-Ampho, pVPack-Eco or pVPack-10A1), using the Transfection MBS transfection protocol (Stratagene) modified according to Pear et al.6 Filtered viral supernatants were serially diluted in DMEM + 10% CS to a final volume of 1 ml/sample and supplemented with DEAE-Dextran (Sigma) to a final concentration of 10 g/ml. Culture media was removed from six-well tissue culture plates containing 2 x 105 NIH3T3 cells/well (plated the previous day) and replaced with 1 ml of viral dilution. Each diluted viral sample was applied to a well containing the NIH3T3 cells, and incubated for 3 hours; 1 ml of prewarmed DMEM + 10% CS was then added to each well, and the plates were then incubated for 2 days. After the 2-day incubation, cells were analyzed by fluorescence-activated cell sorting (FACS). The percent of fluorescent cells in each sample was determined using standard settings for detecting GFP. Titers were calculated using the following formula: (% fluorescent cells in gated population)(total number of cells infected)(dilution).

REFERENCES

  1. Miller, A.D. (1997). Development and Applications of Retroviral Vectors. In Retroviruses (Eds. Coffin, J.M., Hughes, S.H., and Varmus, H.E) Cold Spring Harbor Laboratory Press, Cold Spring Harbor. pp. 437-473.

  2. Onishi, M., et. al. (1996) Exp. Hematol. 24: 324-329.

  3. Soneoka, Y., et. al. (1995) Nucl. Acids Res. 23(4): 28-633.

  4. Yang, S. (1999) Hum. Gene Ther. 10: 123-132.

  5. Yee, J.-K. (1994) Generation of High Titer Pseudotyped Retroviral Vectors with Very Broad Host Range. In Meths. Cell Biol. Vol. 43. pp. 99-112.

  6. Pear, W.S., et. al. (1997) Generation of High-Titer, Helper-Free Retroviruses by Transient Transfection. In Methods in Molecular Medicine: Gene Therapy Protocols (Ed. Robbins, P.D.) Humana Press, Totawa. pp. 41-57.

  7. Felts et. al. (2000) Strategies 13: 5-18.

that can be used with any MMLV-based retroviral vector to produce viral supernatants with titers that are consistently greater than or equal to107 cfu/ml in transient triple-transfection experiments. The CMV-based### vectors include a gag-pol-expressing vector that is cotransfected with the retroviral expression vector together with one of four envelope (env)-expressing vectors. Choosing which vector to use depends on what range of cell types is desired for transduction. Because all of the cis and trans elements required to produce infectious virus are separated onto three plasmids, with minimal or no sequence overlap between the plasmids, the pVPack system is much safer than the majority of stable producer cell lines or vector-based systems, where there is a large degree of homology between the packaging vector(s) and the retroviral expression vector. In these latter systems, there is a relatively high probability of production of replication-competent retrovirus (RCR) due to homologous recombination between the vectors.1

Although the relative speed and simplicity of the transient high-titer virus production methods used here are attractive for most applications, the gag-pol and env open reading frames (ORFs) are all followed by an internal ribosome entry site (IRES) linked to a downstream drug-resistance gene so that these vectors may also be used to establish stable producer lines. The antibiotic-resistance genes used in the gag-pol and env vectors are different from each other, and from those used in most of the more popular retroviral vectors; hence, any env vector may be used with the gag-pol vector and with a wide range of antibiotic-resistant retroviral vectors to produce triple-stable viral producer lines. The position of the antibiotic-resistant gene as the second ORF in a dicistronic expression cassette, as opposed to its expression from a second cassette on the s ame plasmid, ensures that expression of the viral packaging proteins is comaintained with the antibiotic-resistant genes by including antibiotics in the media.

Replication-Defective Retroviral Gene Transfer Systems

Fig.1

Nonreplicating retroviral vectors contain all of the cis elements required for transcription of mRNA molecules encoding a gene of interest and packaging of these transcripts into infectious virus particles (Figure 1). The vectors typically comprise an E. coli plasmid backbone with the gene of interest inserted into a pair of 600-bp viral long terminal repeats (LTRs). The LTR is divided into three regions. The U3 region, containing the retroviral promoter/enhancer, is flanked in the 3 direction by the R region, which contains the viral poly-adenylation signal (pA). The U5 region follows and, along with R, contains sequences that are critical for reverse transcription. Expression of the viral RNA is initiated within the U3 region of the 5 LTR and is terminated in the R region of the 3 LTR. Between the 5 LTR and the coding sequence for the gene of interest resides an extended version of the viral packaging signal (Y+), which is required in cis for the viral RNA to be packaged into virion particles.

To generate infectious virus particles that carry the gene of interest, specialized packaging cell lines have in the past been generated that contain chromosomally integrated expression cassettes for viral gag, pol, and env proteins, all of which are required in trans to make virus. The gag gene encodes internal structural proteins, pol encodes reverse transcriptase (RT) and integrase, and the env gene encodes the viral envelope protein. The envelope protein resides on the viral surface and faci litates infection of the target cell by direct interaction with cell type-specific receptors; thus the host range of the virus is dictated not by the DNA vector but by the choice of the env gene used to construct the packaging cell.

The packaging cell line is transfected with the vector DNA and, at this point, either stable viral producer cell lines may be selected (providing the vector has an appropriate selectable marker) or mRNAs that are transiently transcribed from the vector are encapsidated and bud off into the cell supernatant. These supernatants are collected and used to infect target cells. Upon infection of the target cell, the viral RNA molecule is reverse transcribed by RT (which is present in the virion particle), and the cDNA of the gene of interestflanked by the LTRsis integrated into the host DNA. Because the vector itself carries none of the viral proteins, once a target cell is infected, the LTR expression cassette is incapable of proceeding through another round of virus production.

Due to recent advances in transfection technology high-titer viral supernatants can be produced following transient cotransfection of the viral vector together with expression vectors encoding the gag, pol, and env genes (Figure 1).3,4 This obviates the need for the production and maintenance of stable packaging cell lines.

Vector Description

Fig.2

The general structure of the packaging vectors is diagrammed in Figure 2. The vectors were designed with four aims in mind:

  • High Titer: MMLV gag-pol fusion protein and each of the envelope proteins are expressed at sufficiently high levels to allow the production of viral supernatants with titers greater than or equal to 107 cfu/ml following transient transfection.
  • Safety: Because there is minimal sequence overlap between the packaging vectors and most MMLV retroviral vectors, the probability of generating replication-competent retrovirus (RCR) by homologous recombination is greatly reduced.
  • Compatibility: Compatible selectable markers allow generation of stable producer lines.
  • Versatility: Depending on the range of cell types to be infected, there are several envelope proteins to choose from.

Traditionally, stable packaging lines and some vectors for transient virus production were constructed by preserving all or most of the MMLV genomic DNA and deleting the packaging site so the gag-pol and env-expressing genome could not be packaged itself. However, a problem inherent with these systems involves the high degree of homology between the psi-deleted packaging vector and the retroviral expression vector, which allows a high degree of RCR to be generated.1 Thus, these vectors pose a safety problem when vectors containing toxic inserts are pseudotyped with envelope proteins that permit infection of human cells. In the packaging vectors described, the only MMLV-derived sequences are the gag-pol and envelope proteins themselves (except for the VSV-G protein, which is derived from the vesicular stomatatitis virus).

Target Cell

Ecotropic

Amphotropic

10A1

VSV-G

Mouse

+

+

+

+

Rat

+

+

+

+

Hamster

+/

+

+

Rabbit

+

N.D.

+

Mink

+

+

+

Cow

+/

N.D.

+

Cat

+

+

+

Dog

+

+

+

Monkey

+

+

+

Human

+

+

+

Chicken

+/

N.D.

+

The pVPack retrovirus packaging system offers four different env-expressing vectors. Which one of the four is selected depends on the choice of host cell type (Table 1).1,5 The pVPack-Eco vector is the safest vector, providing experiments can be performed in transduced mouse or rat cells; ecotropic virus infects human cells with extremely low efficiency. The amphotropic envelope protein has historically been the env of choice for infection of human and other mammalian cell lines. The more versatile 10A1 env has been used recently as it allows the same cell-surface receptor to be recognized as the amphotropic env plus a second receptor; it can essentially infect any cell that an amphotropic virus can infect and, in some cases, with a higher efficiency. The ecotropic, amphotropic, and 10A1 proteins are all natural MMLV variants and are all relatively labile; they are considered relatively safe compared with other viral systems.

The vesicular stomatitis virus G protein (VSV-G)5 is rapidly becoming the most popular env protein. Unlike the other three MMLV-derived env proteins which recognize cell surface receptors, VSV-G recognizes a phospholipid that is present on all cell types, which theoretically allows any mitotic cell to be efficiently infected. Additionally, VSV-G confers stability, allowing viral supernatants to be concentrated to 2000-fold or more by ul tracentrifugation; this cannot be done with the other three more labile viruses.

To produce stable cells, we chose antibiotic-resistant markers to complement those used with the neomycin- and hygromycin-resistant derivatives of Stratagenes MMLV-based replication-defective vector pFB. Thus, if desired, cell lines may be first selected with histidinol to obtain gag-pol expressing lines, then a double-stable cell line may be obtained by transfecting with a puromycin-resistant env vector (it should be noted here that although stable VSV-G-expressing lines have in the past been made, in general they are very poor due to the toxicity of VSV-G to cells in which it is stably expressed.1 Therefore, we do not advise using the pVPack-VSV-G vector for stable puromycin-resistant cell line production). Finally, stable virus-producing lines may be constructed using a neo- or hyg-resistant viral vector or cotransfecting the viral vector with a pSV-Neo plasmid. The hisD and puror markers were included as downstream ORFs in IRES-containing bicistronic cassettes to ensure that selective maintenance of cells in antibiotic require expression of the gag-pol and env-encoding mRNAs.

Functional Testing of the pVPack Vectors

To test the pVPack vectors, each of the env-expressing vectors was individually transfected together with pVPack-GP and the green fluorescent protein (GFP)-expressing viral reporter vector pFB-GFP (unpublished); titers were determined by fluorescence- activated cell sorting (FACS). (Figure 3) and are calculated two ways. Titers calculated using percent-shifted values that are linear over two 10-fold dilutions (i.e., 1:103 and 1:104) are shown in bold. We previously determined titers by endpoint dilution (i.e., the smallest detectable shift that yields a percen tage above the No plasmid control; these values are also shown where applicable). All four of the vectors yielded titers greater than or equal to107 cfu/ml using the more conservative method and approached 108 cfu/ml by using the endpoint dilution method titers. These titers are higher than those reported for any other commercially available packaging system.

Fig.3

We also carried out experiments to show that the vectors can be selected and maintained in stable transfection experiments (data not shown). Stable CHO cell lines transfected with pVPack-GP were selected in histidine-free medium using a range of histidinol from 75 to 500 M and gave no significant variation in colony formation across this range. Likewise, each of the vectors (pVPack-Ampho, pVPack-Eco, and pVPack-10A1) were selected with 5 to 7 g/ml of puromycin. They showed a high level of colony formation above untransfected CHO cells, which showed no colony formation at all with the puromycin concentrations tested. The pVPack-VSV-G vector was not tested for stable cell formation due to reported toxicity of the VSV-G envelope protein to the host cell. As stated above, we do not recommend the use of this vector for stable cell production.

Conclusions

The pVPack vector system allows high-titer virus to be produced for most applications, providing that a high titer retroviral expression vector such as pFB is used, and very high transfection efficiencies are attained when producing virus. For the latter, we found that 293 cells and their derivatives work especially well when used with Stratagenes Transfection MBS transfection kit, modified according to Pear and colleagues6 (see legend to Figure 3). For certain low-titer vectors [e.g., vectors wit
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