Real-Time Quantitative Reverse Transcriptase ...

HUMAN GENE THERAPY 14:497–507 (April 10, 2003) © Mary Ann Liebert, Inc.

Real-Time Quantitative Reverse Transcriptase-Polymerase Chain Reaction as a Method for Determining Lentiviral Vector Titers and Measuring Transgene Expression GREGORY LIZÉE,1 JOERI L. AERTS,1 MONICA I. GONZALES, 1 NACHIMUTHU CHINNASAMY, 2 RICHARD A. MORGAN,1 and SUZANNE L. TOPALIAN 1

ABSTRACT The use of lentiviral vectors for basic research and potential future clinical applications requires methodologies that can accurately determine lentiviral titers and monitor viral transgene expression within target cells, beyond the context of reporter genes typically used for this purpose. Here we describe a quantitative RT-PCR (qRT-PCR) method that achieves both goals using primer sequences that are specific for the woodchuck hepatitis virus posttranscriptional regulatory element (WPRE), an enhancer contained in many retroviral vectors and that is incorporated in the 39 UTR of nascent transgene transcripts. Quantitation of titers of three recombinant lentiviruses, genetically identical except for the transgene, demonstrated consistent differences in titer that were likely due to transgene-associated toxicity in producer cells and target cells. Viruses encoding the tumor-associated antigens tyrosinase and neo-poly(A) polymerase yielded reproducibly lower titers than a virus encoding enhanced green fluorescent protein (GFP) at the viral RNA, integrated DNA, and transgene mRNA levels, as measured by WPRE qPCR. Furthermore, the magnitude of differences in expression of the three transgenes in transduced target cells could not have been predicted by measuring vector DNA integration events. Since transgene expression in target cells is the most common goal of lentiviral transduction, and since methods to quantify transgene expression on the protein level are not always readily available, qRT-PCR based on a nucleotide sequence included in the transcript provides a useful tool for titering novel recombinant lentiviruses. OVERVIEW SUMMARY A real-time quantitative polymerase chain reaction (qPCR) method was developed to assess the titers of three lentiviral vector preparations generated under identical conditions but expressing different transgenes [GFP, tyrosinase, or neo-poly(A) polymerase (neoPAP)]. The woodchuck posttranscriptional regulatory element (WPRE), present in all three vectors, was used as the template for qPCR analysis. This method allowed for rapid and accurate comparative assessments of lentiviral vector titers at three levels: vector supernatant RNA, integrated proviral DNA, and lentiviral gene (mRNA) expression in transduced cells. Significant differences in viral titers between the three lentivirus vectors were observed both at the viral RNA and integrated DNA levels, with a consistent and reproducible hierarchy of

GFP . tyrosinase . neoPAP. Furthermore, evaluation of lentiviral gene expression in transduced HeLa cells revealed a much wider discrepancy in mRNA levels among the three vectors than would have been anticipated from titer estimates based on DNA integration events.

INTRODUCTION

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are endowed with several features that make them excellent candidates for the development of novel human gene therapies (Buchschacher and Wong-Staal, 2000; Naldini and Verma, 2000; Pandya et al., 2001; Larochelle et al., 2002). Their relatively large cloning capacity, ability to stably integrate into nondividing target cells, and absence of

1Surgery 2St.

ENTIVIRAL VECTORS

Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892. Luke’s Medical Center, Milwaukee, WI 53215.

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498 vector-induced immune responses make them especially attractive tools for basic as well as clinical applications (Chinnasamy et al., 2000; Scherr and Eder, 2002). For future applications it will be essential not only to accurately determine the transducing titers of lentivirus preparations but also to assess lentiviral transgene expression in transduced target cells. Lentiviruses expressing fluorescent proteins such as green fluorescent protein (GFP), or proteins recognized by specific antibodies, can be titered with flow cytometric detection of the encoded protein in transduced cells (Dull et al., 1998; White et al., 1999). Viruses expressing reporter genes such as b-galactosidase or antibiotic resistance genes can be titered by enumerating protein-expressing cell colonies with simple chemical techniques (Chang and Zaiss, 2002; Srinivasakumar, 2002). More recently, real-time quantitative polymerase chain reaction (qPCR) approaches have been described for measuring lentivirus DNA integration events, reportedly providing a more accurate estimate of functional titers (Pan et al., 2002; Sastry et al., 2002). Such methods have the advantage of allowing titer determination for vectors encoding gene products not readily detectable by immunological methods. However, there are few reports that directly assess transgene expression by qRT-PCR (Rose et al., 2002). The identification of tumor-associated antigens (TAAs) selectively expressed in human cancer cells has heightened interest in the genes encoding these antigens as candidates for use in antitumor immunization strategies (Rosenberg, 1999; Housseau et al., 2002). Recombinant lentiviruses may provide a means for administering such immunizations. To accurately determine transducing titers of TAA-expressing lentiviral vectors as well as to assess vector transgene expression in target cells when flow cytometric or other detection methods are not feasible, we have developed a qRT-PCR assay that uses a single set of primers to achieve both ends. Our strategy utilizes primers specific for the woodchuck hepatitis virus posttranscriptional regulatory element (WPRE), a sequence known to enhance lentiviral transgene expression (Zufferey et al., 1999; Ramezani et al., 2000) that is included in the 39 untranslated region (UTR) of many lentiviral vector plasmid constructs. This system, which does not rely on reporter genes, allows the quantitation of lentiviral vector preparations at three levels: vector genomic RNA, integrated proviral DNA, and cellular lentiviral vector-mediated gene (mRNA) expression. The present study demonstrates that PCR methods for measuring vector genomic RNA and integrated proviral DNA overestimate the functional viral titers of GFP-expressing vectors as determined by flow cytometry. In addition, recombinant lentiviruses expressing the TAA tyrosinase (Topalian et al., 1994) and neo-poly(A) polymerase (neoPAP) (Topalian et al., 2001) were shown at both the genomic RNA and proviral DNA levels to have consistently lower titers than a GFP-expressing reference vector, likely due to toxic effects of the encoded TAA on 293T producer cells. Analysis of WPRE mRNA expression in transduced HeLa cells also revealed an unexpectedly wide discrepancy in lentiviral gene expression among identical vectors encoding the three different transgenes. These results underscore the limitations of using DNA integration events for performing titer estimates, and demonstrate that qRT-PCR can be used not only to obtain accurate lentiviral titers but also to

LIZÉE ET AL. precisely monitor lentiviral transgene expression in transduced target cells.

MATERIALS AND METHODS Cell lines and plasmids HeLa human cervical cancer cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA). 293T cells, transformed human embryonic kidney cells expressing simian virus 40 (SV40) T antigen, were kindly provided by G. Nolan (Stanford University, Stanford, CA). Both cell lines were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), 10 mM HEPES buffer, 2 mM L -glutamine, amphotericin B (250 ng/ml), penicillin (50 IU/ml), streptomycin (50 mg/ml), and gentamicin sulfate (50 mg/ml). The packaging constructs pCMVDR8.2 and pCMVDR8.91 (Zufferey et al., 1997), the heterologous vesicular stomatitis virus G glycoprotein (VSV-G) envelope-expressing construct pMD.G (Naldini et al., 1996), and the gene transfer vector pRRL-PGK-GFP-Wsin were gifts from D. Trono (University of Geneva, Geneva, Switzerland). pRRL-cPPT-PGK-GFPWsin (hereafter referred to as pPGK-GFP) was constructed by amplifying a 118-bp fragment of the human immunodeficiency virus type 1 (HIV-1) central polypurine tract (cPPT) from pCMVDR8.2 via PCR and inserting this product 59 to the phosphoglycerate kinase (PGK) promoter as described previously (Follenzi et al., 2000). This vector was further modified by replacing the GFP gene with a multiple cloning site (MCS) to create the vector pPGK-MCS-Wsin. cDNAs encoding fulllength human tyrosinase and neoPAP were amplified by PCR from tyrosinase- and neoPAP-containing plasmids (Topalian et al., 1994, 2001) and directionally cloned into the MluI and NheI restriction enzyme sites in pPGK-MCS-Wsin to create the plasmids pPGK-Ty-Wsin (pPGK-Ty) and pPGK-neoPAP-Wsin (pPGK-neoPAP). All new recombinant plasmids were validated by DNA sequencing.

Vector production Lentiviral vectors encoding GFP, tyrosinase, or neoPAP were prepared in parallel by transient transfection of 293T cells with the gene transfer plasmid pPGK-X-Wsin, the packaging plasmid pCMVD8.91, and the VSV-G envelope plasmid pMD.G (Follenzi and Naldini, 2002; Srinivasakumar, 2002). Briefly, 293T cells were seeded at 6 3 106 cells per flask in 175-cm2 tissue culture flasks, and then transfected 16 hr later with 8 mg of pPGK-X-Wsin, 6 mg of pCMVD8.91, and 1.5 mg of pMD.G, using the Effectine transfection reagent (Qiagen, Valencia, CA). The following day, transfected cells were washed gently with phosphate-buffered saline (PBS) and cultured in 35 ml of fresh medium for another 48–72 hr. Culture supernatants were collected, filtered through a 0.45-mm pore size filter, and concentrated approximately 30-fold by ultracentrifugation (20,000 3 g for 2 hr at 4°C) and resuspension of pellets in cold fresh medium (RPMI 1640 plus 10% FBS). Aliquots of concentrated supernatants were stored at 270°C, and were used for all subsequent HeLa cell transductions. All cal-

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LENTIVIRUS TITER AND EXPRESSION ANALYSIS culations of lentiviral genomic RNA, integrated DNA, and mRNA expression refer to concentrated supernatants.

Cell transductions and lentiviral protein expression analysis HeLa cells were seeded in 6-well tissue culture plates at 1 3 105 cells per well. The next day, cells were incubated with serial dilutions of each lentiviral supernatant preparation in a total volume of 1 ml of RPMI 1640–10% FBS plus Polybrene (8 mg/ml) (Sigma, St. Louis, MO). After 16 hr, cells were washed extensively with PBS to remove lentiviral genomic RNA and fresh medium was added. Cells were maintained in culture for another 72 hr, and then washed and harvested with trypsin–EDTA. Aliquots of cells transduced with the PGK-GFP-Wsin lentivirus were analyzed for GFP expression by flow cytometry and viral titers were calculated according to the following formula: transduction units (TU)/ml 5 (percentage of GFP-positive cells/100) 3 (number of cells infected) 3 (vector dilution factor) as described previously (Follenzi and Naldini, 2002). Only the most dilute vector samples were used to calculate GFP titer (e.g., those in the linear region of Fig. 3C), because choosing values in this range diminishes the possibility of underestimating titer due to analysis of cells containing multiple vector copies. Western blot analysis was used to assess expression of the tyrosinase and neoPAP proteins by lentiviral vectors in transduced cells. Briefly, transduced HeLa cells were lysed at 107 cells/ml in a detergent-containing buffer as described (Topalian et al., 2001). The samples were boiled for 3 min under nonreducing conditions for tyrosinase lysates or in the presence of 100 mM dithiothreitol for neoPAP lysates before loading onto 4–20% gradient Tris–glycine polyacrylamide gels (Invitrogen, Carlsbad, CA). Electrophoretically separated proteins were blotted onto nitrocellulose membranes, which were incubated for 2 hr at room temperature with either T311 (1 mg/ml), a murine monoclonal antibody specific for human tyrosinase (Vector Laboratories, Burlingame, CA), or with a polyclonal rabbit antiserum (1:2000 dilution) specific for human neoPAP (kindly provided by J. Manley and V. Vethantham, Department of Biological Sciences, Columbia University, New York, NY). Proteins were detected with horseradish peroxidase-coupled sheep anti-mouse IgG F(ab9)2 for tyrosinase or donkey anti-rabbit IgG F(ab9)2 for neoPAP. Visualization was achieved with chemiluminescence (ECL detection system; Amersham Pharmacia Biotech, Piscataway, NJ) according to the manufacturer’s instructions. Of note, neither the T311 antibody nor the polyclonal anti-neoPAP antiserum was found to be suitable for flow cytometric applications.

Real-time quantitative PCR For the quantitative analysis of genomic lentiviral RNA, proviral DNA copies, and transgene mRNA expression, the ABI Prism 7700 sequence detection system (Applied Biosystems, Foster City, CA) was used. Primers and probes were synthesized by Applied Biosystems. Probes were labeled at the 59 end with the reporter dye molecule FAM (emission wavelength, 518 nm) and at the 39 end with the quencher dye TAMRA (emission wavelength, 582 nm). The 39 end of the probe was additionally phosphorylated to prevent extension during PCR.

For detection of the WPRE sequence, primers and probe were designed using Primer Express software (version 1.0; Applied Biosystems): forward primer (1277F), 59-CCGTTGTCAGGCAACGTG-39; reverse primer (1361R), 59-AGCTGACAGGTGGTGGCAAT-39; probe (1314P), 59-FAM-TGCTGACGCAACCCCCACTGGT-TAMRA-3 9. For detection of albumin, the sequences of the primers and probe used were as follows: forward primer, 59-TGAAACATACGTTCCCAAAGAGTTT-3 9; reverse primer, 59-CTCTCCTTCTCAGAAAGTGTGCATAT-39; probe, 59-FAM-TGCTGAAACATTCACCTTCCATGCAGATAMRA-39 (Mandigers et al., 2001). For detection of b-actin, the sequences of the primers and probe used were as follows: forward primer, 59-GCGAGAAGATGACCCAGATC-3 9; reverse primer, 59-CCAGTGGTACGGCCAGAGG-3 9; probe, 59FAM-CCAGCCATGTACGTTGCTATCCAGGC-TAMRA-3 9 (Schoof et al., 2001). For the PCR, 12.5 ml of universal PCR master mix (Applied Biosystems), an 800 nM concentration of each primer, and 200 nM probe were combined and adjusted to a total volume of 20 ml with RNase-free water. Finally, either cDNA or genomic DNA was added to each reaction and the total reaction volume was adjusted to 25 ml. Standard conditions were used for the PCR (2 min at 50°C, 10 min at 95°C, and then 40 cycles of 15 sec at 95°C and 1 min at 60°C). For each PCR, a no-template reaction was included as negative control. Each DNA or cDNA sample was tested in duplicate, and the mean values are reported. Duplicate values varied by no more than 15% from the mean. Copy number quantification was based on the TaqMan principle (Heid et al., 1996). Ten-fold serial dilutions of plasmid constructs of known concentration and containing the relevant sequences (WPRE, albumin, or b-actin) were prepared so as to create standard curves for quantification of unknown samples. All dilutions were made in the presence of Escherichia coli 16S ribosomal RNA (20 ng/ml) in order to increase the stability of the plasmid dilutions.

Molecular methods for determining lentiviral titers For determination of lentiviral genomic RNA titers, RNA was isolated from 50 ml of concentrated vector supernatant or serial 10-fold dilutions of the same, using RNeasy columns (Qiagen). A DNase step was included in the column purification procedure as per the manufacturer instructions to eliminate the possibility of contamination by transfer vector plasmid DNA. Isolated RNA was eluted in 30 ml of RNase-free water and subsequently used as the template for one round of reverse transcription (RT) to generate cDNA as follows: 7.5 ml (25%) of purified RNA was added to a master mix (Applied Biosystems) containing a final concentration of 13 RT buffer, 5 mM MgCl2, 2 mM dNTPs, random hexamer primers (2.5 mM each), RNase inhibitor (0.4 U/ml), and MultiScribe reverse transcriptase (1.25 U/ml) in a 20-ml reaction. For qPCR to determine WPRE copy numbers, 2 ml (10%) of each cDNA reaction was used as the template. Lentiviral genomic RNA titers (in particles per milliliter) were calculated by taking the means of calculated titers based on WPRE copy numbers obtained from three different dilutions of viral supernatant, assuming a diploid lentiviral genome (two RNA molecules per viral particle).

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For assessment of proviral DNA copies, or lentiviral integration events, genomic DNA was isolated from HeLa cells transduced with 4-fold serial dilutions of concentrated lentiviral vector supernatants, using an Easy-DNA kit (Invitrogen). DNA from approximately 5 3 105 transduced HeLa cells was resuspended in 100 ml of distilled H2O and treated with RNase (40 mg/ml) for 30 min at 37°C. Five microliters, or 5% of the total DNA isolated, was used as the template for each qPCR. Lentiviral integration events were calculated by normalizing the number of WPRE molecules measured by qPCR to the number of HeLa cells, as quantified by the copy number of albumin molecules detected by qPCR on the same genomic DNA sample. On the basis of preliminary assessments of HeLa cell counts and corresponding albumin copy numbers, the average number of albumin genes per HeLa cell was calculated to be 3.9, consistent with the reported average HeLa ploidy of 3.66 (Hay et al., 1994). DNA titers were subsequently calculated by taking the means of titer calculations based on qPCR measurements of WPRE copy number obtained from HeLa cells transduced with four different dilutions of viral supernatant. For assessment of lentiviral gene expression at the mRNA level, total RNA was isolated from approximately 5 3 105 transduced HeLa cells, using RNeasy columns. A DNase step was included in the procedure to reduce the chance of vector plasmid or genomic DNA contamination. Purified RNA was resuspended in RNase-free water and 1 mg of total RNA was subjected to a single round of RT to generate cDNA as described above. Expression levels of WPRE message detected by qPCR were normalized, using b-actin mRNA as a control. The ratio of number of copies of WPRE to 104 copies of b-actin was used to compare relative levels of lentiviral gene expression between the three vectors. All samples analyzed demonstrated comparable b-actin mRNA copy numbers.

RESULTS Real-time qPCR strategy for detection of lentiviral vectors All of the lentiviral vectors used in this study were self-inactivating (SIN) vectors with a 400-bp deletion in the U3 region of the 39 LTR that renders integrated provirus incapable

of synthesizing vector transcripts (Zufferey et al., 1998). The WPRE, previously demonstrated to facilitate nuclear export of transcripts (Huang and Yen, 1994), was inserted immediately 39 of the transgene stop codon, resulting in transcripts that contain WPRE as part of the 39 UTR (Fig. 1A). Thus, detection of WPRE sequences could be used to quantitate viral RNA, integrated DNA, or transcribed mRNA, regardless of the nature of the recombinant transgene. In addition, the WPRE sequence has been included in a number of other retroviral vector systems, making such detection reagents widely applicable (Zufferey et al., 1999). An optimal primer and probe set for WPRE sequence amplification was designed that demonstrated the best overall amplification kinetics and lowest nonspecific amplification. Ten-fold serial dilutions of the pPGK-GFP-Wsin plasmid were used to generate standard curves for WPRE qPCR (Fig. 1B and C). Because the concentration of plasmid can be accurately measured by spectrophotometry, the standard curve can be used to calculate the number of copies of lentiviral molecules in a given sample.

Experimental overview The three lentiviral vectors used in this study were identical except for the recombinant transgene (Fig. 1A). The PGK-GFPWsin, PGK-Ty-Wsin, and PGK-neoPAP-Wsin viral vectors express the enhanced GFP, tyrosinase, and neoPAP proteins, respectively. Lentiviral supernatants containing these three vectors were generated simultaneously by transient cotransfection of 293T cells, and collection and concentration of culture supernatants were performed according to identical procedures. Analysis of lentivirus contained in concentrated supernatants was accomplished by a variety of methods (Fig. 2). Viral genomic RNA contained in supernatants was directly quantitated by real-time qRT-PCR. Transduction of HeLa cells with serial dilutions of each viral preparation was performed, followed 4 days later by harvesting of cells. An aliquot of GFP vectortransduced cells was used for flow cytometric analysis to assess GFP expression and to calculate virus titers according to conventional methods. Aliquots of cells transduced in parallel with the PGK-Ty and PGK-neoPAP vectors were also analyzed for protein expression by Western blotting. The remaining transduced HeLa cells, as well as nontransduced control cells, were split into two aliquots and were used to isolate either ge-

FIG. 1. Vector design and WPRE-based qPCR. (A) Schematic representation of recombinant HIV-1-based SIN lentiviral vectors used in this study. All vectors are identical except for the transgenes, and contain an internal PGK promoter driving expression of the GFP, tyrosinase, or neoPAP insert. Arrows depict transcription initiation sites. Transfer of the deletion in the U3 region (DU3) of the 39 LTR to the 59 LTR after reverse transcription self-inactivates the vector. A central polypurine tract (cPPT) has been added to enhance nuclear import of the proviral genome. The woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) has been inserted immediately 39 of the transgene stop codon and is included as part of the 39 UTR of lentiviral transcripts. A portion of the WPRE has been expanded to show the regions where the specific primers (1277F and 1361R) and probe (1314P) used for real-time qPCR analysis of lentiviral sequences are located. Primer/probe numerical designations refer to 59 nucleotide positions, according to GenBank accession number J04514. DGag, Deleted Gag region; RRE, Rev-responsive element. (B) Real-time qPCR amplification plots showing amplification of the pPGK-GFP-Wsin plasmid, using WPRE-specific primers and probe. Plasmid concentration was determined by spectrophotometry and standards were generated by using 10-fold serial dilutions (copy numbers of 108, 107 , 106, 105, 104, and 103 correspond to amplification curves from left to right). DRn, Change in normalized reporter signal. A standard curve (C) was generated (r2 5 0.998) by plotting cycle threshold (Ct) against the number of plasmid DNA molecules. Average Ct values obtained from experimental unknowns were compared with a standard curve amplified simultaneously in order to quantitate the WPRE copy number present in each sample.

LENTIVIRUS TITER AND EXPRESSION ANALYSIS

A

B

C

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FIG. 2. Experimental design for comparing different methods of titering concentrated lentiviral supernatants, as described in Results.

nomic DNA for direct real-time qPCR analysis of integration events, or total RNA for reverse transcription followed by qPCR to measure transgene expression.

Direct titering of viral particles contained in concentrated supernatants For determining lentiviral titers in concentrated supernatants, total RNA was extracted from serial 10-fold dilutions of supernatant. A DNase step was included in the RNA isolation procedure to eliminate plasmid DNA contamination. This lack of plasmid contamination was confirmed by PCR using primers specific for the ampicillin resistance gene, which is part of the lentiviral gene transfer plasmid but not the viral particles (data not shown). A single round of RT was performed before analyzing WPRE cDNA copy number by qPCR. The results (Fig. 3A) showed that the PGK-GFP-Wsin vector had the highest titer (5.78 3 1010 particles/ml), followed by PGK-Ty-Wsin (2.79 3 1010 particles/ml) and PGK-neoPAP-Wsin (1.40 3 1010 particles/ml) (Table 1). To ensure that these findings were not due to experimental variation, the entire lentiviral vector preparation process was repeated with similar results (data not shown). PGK-GFP lentivirus consistently had the highest titers, with PGK-Ty and PGK-neoPAP lentivirus titers being approximately 2-fold and 4-fold lower, respectively.

Determining vector titers by measuring integrated proviral DNA Several publications have described determination of lentiviral titers by measuring RNA directly from viral supernatants (Tafuro et al., 1996; Murdoch et al., 1997; Sanburn and Cornetta, 1999). However, this method is likely to severely over-

LIZÉE ET AL. estimate functional titers because of the known presence of defective contaminating lentiviral particles, which can frequently outnumber functional particles by 20-fold or more (Kirkwood and Bangham, 1994; Higashikawa and Chang, 2001). To more accurately determine transduction-competent titers, we sought to determine the number of proviral DNA copies integrated into transduced HeLa cells. Genomic DNA was isolated from HeLa cells transduced with serial dilutions of concentrated lentiviral supernatant and analyzed for WPRE copy number, using realtime qPCR. Values were normalized by quantitating albumin gene copy numbers simultaneously. The number of molecules of WPRE detected in transduced HeLa cell genomic DNA exhibited a linear relationship with vector dilution (Fig. 3B). Contamination of genomic DNA samples by plasmid carryover in transduced cells has been shown to be insignificant after extensive cell washing and 4 days in culture (Sastry et al., 2002; Srinivasakumar, 2002) and this was confirmed experimentally by our failure to amplify the ampicillin resistance gene contained in the lentiviral plasmid by conventional PCR (data not shown). Although the lentiviral titers obtained by quantitating integrated proviral DNA were approximately 200-fold lower than those obtained by directly measuring lentiviral genomic supernatant RNA, the former are likely to provide a more accurate estimate of the actual functional titers of lentiviral preparations. Importantly, the relative titers of the GFP, Ty, and neoPAP vectors when compared with each other were similar by either the RNA or DNA method (Table 1). These data suggest that although the three recombinant lentiviral vectors were produced with different efficiencies by 293T cells according to the nature of the transgene, the infectivity and integration of the three viruses into HeLa cells occurred with similar efficiencies.

Determination of vector titers by GFP expression analysis Standard methods for titering lentivirus depend on enumerating transduced cells that express the protein encoded by the transgene (Follenzi and Naldini, 2002). Thus, we assessed aliquots of HeLa cells transduced with the PGK-GFP vector by flow cytometry for green fluorescence (Fig. 3C), and a viral titer based on percentage of GFP-positive cells was calculated. This titer (3.50 3 107 TU/ml) was approximately 6-fold less than the titer obtained by measuring integrated proviral DNA (Table 1), consistent with other studies (Sastry et al., 2002). Although methods routinely employed to detect marker protein expression are considerably less sensitive than molecular methods to detect integrated DNA, they may be more appropriate when protein expression is the goal of lentiviral transduction. Measurement of integrated proviral DNA can overestimate the capacity of transduced cells to express the transgene, because a significant proportion of integrated provirus may not be transcribed.

Measuring lentiviral transgene mRNA expression in transduced cells Because transgene expression at the protein level is usually the ultimate goal of viral transduction, we sought to develop an accurate method of titering virus that would reflect protein ex-

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D

FIG. 3. Results of comparative methods for quantitating titers and transgene expression of three lentiviral vectors. (A) Lentiviral genomic RNA in concentrated supernatants was measured directly by qRT-PCR analysis, as specified in Materials and Methods. (B) Integrated proviral DNA in transduced HeLa cells as measured by qPCR. Values were normalized for total cell number, using albumin gene copy numbers as described in Materials and Methods. (C) Determination of GFP expression in PGK-GFP-Wsin vector-transduced HeLa cells, using flow cytometry. Lentiviral titer can be calculated accurately only from the most dilute samples (linear portion of the curve). (D) Analysis of transgene mRNA expression in transduced HeLa cells, using qRT-PCR. Values are normalized by measuring b-actin mRNA present in each sample. All data shown in (A–D) are representative of multiple experiments. pression and that could be applied in the absence of a transgene-specific antibody or selection marker. In addition, a generic method applicable to lentiviral vectors regardless of the identity of the transgene was desirable. Many lentiviruses currently in use contain the WPRE, which becomes part of the 39 UTR of each lentiviral transgene mRNA transcript. By measuring the number of transcripts in transduced HeLa cells by real-time qRT-PCR with WPRE-specific primers and probes, we sought to assess lentiviral gene expression for three vectors that were identical except for the identity of the transgene. Total RNA was purified from HeLa cells transduced with serial dilutions of PGK-GFP, PGK-Ty, or PGK-neoPAP, and qRTPCR was used to determine WPRE copy number. b-Actin

mRNA copy number was measured simultaneously as a control to normalize these values. As shown in Fig. 3D, the number of WPRE mRNA molecules in HeLa cells transduced with each vector showed a linear relationship with vector dilution. Surprisingly, we detected much wider variations in transgene expression by PGK-GFP, PGK-Ty, or PGK-neoPAP than would have been predicted from either the genomic RNA- or DNA-based titer estimates. At the mRNA level, GFP transgene expression was the highest among the three vectors. However, tyrosinase mRNA expression was 4.7-fold less and neoPAP mRNA expression was 56-fold less by comparison. Relative expression titers were assigned to PGK-Ty and PGK-neoPAP by reference to PGK-GFP WPRE copy number (Table 1).

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LIZÉE ET AL. TABLE 1. COMPARISON

Lentiviral vector PGK-GFP PGK-Ty PGK-neoPAP

OF

RNA, DNA, GFP,

AND

RELATIVE mRNA TITERS

OF

THREE LENTIVIRAL VECTORS a

RNA titer (particles/ml 3 10210)

DNA titer (TU/ml 3 1027)

GFP titer (TU/ml 3 1027)

Relative mRNA titer (%)

5.78 6 0.59 2.79 6 0.45 1.40 6 0.31

20.1 6 2.90 13.1 6 1.00 7.1 6 0.7

3.50 6 0.71 NA NA

100 21.0 6 2.50 1.80 6 0.37

aRNA, DNA, GFP, and relative mRNA expression titers of concentrated lentiviral supernatants. RNA titers were determined by quantitation of WPRE copy numbers in concentrated vector stocks by real-time qRT-PCR. DNA titers were determined by qPCR for integrated WPRE copy numbers and corrected for total cell numbers using albumin as a control gene, as described in Materials and Methods. GFP titer for PGK-GFP was determined by measuring GFP-positive transduced HeLa cells using flow cytometric analysis. Relative mRNA titers were determined by qRT-PCR for WPRE sequences included in the 39 UTR of transgene transcripts and normalized for b-actin mRNA expression. Viral genomic RNA titers are represented as vector particles per milliliter. DNA and GFP titers are expressed in transducing units (TU) per milliliter. mRNA titers of PGK-Ty and PGK-neoPAP are expressed relative to GFP transgene mRNA expression. All titer calculations shown in this table are means 6 standard deviations of multiple qPCR and flow cytometric measurements, as described in Materials and Methods. NA, Not applicable.

To assess whether mRNA expression detected by qRT-PCR was indicative of protein expression, Western blots of lysates from HeLa cells transduced with PGK-Ty or PGK-neoPAP vector were probed with a tyrosinase-specific antibody or neoPAPspecific antiserum, respectively. As shown in Fig. 4A and B, protein expression was detectable in lysates from both tyrosinase and neoPAP vector-transduced HeLa cells. Although the use of different antibody detection reagents cannot provide precise quantitation of relative protein levels, expression of the neoPAP protein consistently appeared to be substantially less than that of tyrosinase. This result correlated well in qualitative terms with relative mRNA levels as measured by qRT-PCR. Because neither the anti-tyrosinase antibody nor the antineoPAP antiserum was suitable for flow cytometric applications, WPRE qRT-PCR was essential for quantifying transgene expression. Taken together, these results clearly demonstrate differential transgene transcription not accounted for by differences in viral genomic RNA or proviral DNA integration.

62% growth inhibition. Similar results were observed for 293T lentiviral producer cells transiently cotransfected with each gene transfer vector in addition to the packaging and VSV-G envelope plasmids (data not shown). These results demonstrate that the nature of the transgene encoded by the lentiviral gene transfer plasmid can have a direct and substantial influence on lentivirus production efficiency in 293T cells, and suggest that these effects might also impact on target cells transduced with recombinant lentiviruses.

A

Toxicity of lentiviral transgenes Both tyrosinase (Miranda et al., 1984; Riley, 1985) and neoPAP (Topalian et al., 2002) are known to be toxic when overexpressed in certain cell types. Because high levels of lentiviral transgene expression are common in transiently transfected 293T producer cells, we were interested to know whether transgene-associated toxicity was responsible for the consistently lower viral titers measured for PGK-Ty and PGKneoPAP, compared with PGK-GFP. 293T cells were transfected with 0.5 or 1.5 mg of each gene transfer plasmid. Three days posttransfection, cell numbers and viabilities were assessed and compared with untransfected controls. Whereas no significant differences were observed in cell viabilities during the period of observation (all cultures were .90% viable), 293T cells transfected with the neoPAP plasmid showed significantly less proliferation compared with untransfected cells or cells transfected with either of the other two vectors (Fig. 5). Cells transfected with the GFP-encoding plasmid showed no signs of growth inhibition at either 0.5 or 1.5 mg compared with untransfected control cells, whereas cells transfected with 1.5 mg of the tyrosinase plasmid showed 37% growth inhibition and cells transfected with 1.5 mg of the neoPAP plasmid showed

B

FIG. 4. Transgene expression detected by Western blotting. (A) Western blot showing tyrosinase protein expression in PGK-Ty-Wsin vector-transduced HeLa cell lysates. Lanes 1–3, cells transduced with undiluted vector, vector diluted 1:4, and vector diluted 1:16, respectively; lane 4, untransduced HeLa cells; lane 5, COS-7 cells transfected with a plasmid encoding tyrosinase. Each lane was loaded with 1 3 105 cell equivalents. (B) Western blot showing neoPAP protein expression in PGKneoPAP-Wsin vector-transduced HeLa cell lysates. Lanes 1–3, cells transduced with undiluted vector, vector diluted 1:2, or vector diluted 1:4, respectively; lane 4, untransduced HeLa cells demonstrating a low level of constitutive neoPAP expression; lane 5, purified recombinant human neoPAP protein (50 ng). Each lane was loaded with 1 3 105 cell equivalents.

LENTIVIRUS TITER AND EXPRESSION ANALYSIS

FIG. 5. Toxicity of lentiviral plasmid vectors in 293T transfectants. Each of the three lentiviral gene transfer vectors (pPGK-GFP, pPGK-Ty, and pPGK-neoPAP) was used to transfect 293T cells with either 0.5 or 1.5 mg of plasmid DNA. Cells were harvested 3 days posttransfection, and cell numbers and viabilities were determined visually by trypan blue staining.

DISCUSSION Efficient delivery and expression of TAA-encoding genes presents a challenge for immunotherapy in the coming years. Lentiviruses provide an attractive vehicle for this purpose. Optimization of lentiviral vector production, gene delivery, and determination of accurate functional titers is essential not only for potential in vivo use, but also for a multitude of in vitro applications. Several methods have been described for titering lentiviral preparations, and these methods generally fall into one of three categories: quantitation of viral particles in vector supernatants, of integrated proviral DNA in target cells, or of transgene-encoded protein in target cells. Each method carries a distinct definition of viral titer. Enumeration of viral particles contained in vector supernatants is often performed indirectly by measuring concentrations of the p24 Gag protein by enzyme-linked immunosorbent assay (Naldini et al., 1996). This method can be used to obtain a rough estimate of titer, but is limited by the variable amount of free, non-particle-associated p24 that is produced by standard plasmid cotransfection methods of lentiviral production. Semiquantitative Northern blots can also be used to estimate particle number in lentiviral supernatants. However, genomic RNA titering methods are generally acknowledged to greatly overestimate functional viral titer (Kirkwood and Bangham, 1994; Higashikawa and Chang, 2001), and our results concur with these observations. Viral titers calculated by quantitation of lentiviral genomic RNA were, on average, 200-fold higher than titers obtained by measuring integrated proviral DNA (Table 1). This discrepancy is likely accounted for by the significant number of defective lentivirus particles that are unavoidably generated as part of the production process, and by functional particles that do not successfully infect target cells. Quantitation of integrated proviral DNA in transduced target cells is likely to provide a more accurate measurement of viral titer than assessing viral particles released by producer cells, because only transduction-competent viruses will be de-

505 tected by this method. Both semiquantitative Southern blotting and quantitative PCR methods have been described for detecting integrated proviral DNA (Forghani et al., 1991; Sastry et al., 2002). However, studies have demonstrated that integration events do not necessarily correlate with viral gene expression, because a significant proportion of provirus integrates into regions of the genome that are not amenable to gene transcription (Sastry et al., 2002). The most common goal of lentiviral transduction is to express transgenes of interest in target cells. Thus, methods that can detect either transgene mRNA or protein expression are likely to give the most useful assessment of functional viral titer. Traditionally, titration methods based on protein expression have relied on reporter genes. Target cell expression of transgene-encoded fluorescent or luminescent protein products such as GFP and luciferase can easily be detected by optical detection methods, and chemical methods are readily available for detecting the expression of reporter genes such as lacZ or antibiotic selection markers (Chang and Zaiss, 2002). With specific antibodies against proteins of interest, immunological methods can also be used to quantitate transgene expression. However, when no appropriate protein detection reagents are available, it is necessary to assess transgene expression at the mRNA level. While semiquantitative Northern blotting is a feasible option for this purpose, this is laborious and is limited in terms of precision. Therefore, we have developed a real-time qRT-PCR method that allows for rapid and precise quantitation of lentiviral transgene mRNA expression in transduced target cells. This method utilizes the WPRE sequence, which is included as part of the 39 UTR of lentiviral transgene transcripts in our recombinant vectors. Because the WPRE element is included in a number of retroviral systems currently in use, the method is widely applicable and can be performed independent of the nature of the recombinant transgene. By analyzing lentiviral transgene expression at the mRNA level, we observed surprising functional differences between three lentiviral vectors that could not have been predicted by measuring DNA integration events. Expression of neoPAP mRNA in transduced HeLa cells was 56-fold lower than that of GFP mRNA, despite only a 3-fold difference in proviral DNA titer. Two separate transduction experiments confirmed that neoPAP mRNA is consistently expressed at significantly lower levels than is GFP or tyrosinase mRNA in HeLa cells (data not shown). We have observed a similar phenomenon in several transduced melanoma cell lines as well as in Epstein–Barr virus-transformed B cells, demonstrating that this effect is not specific for HeLa cells (data not shown). Presumably, toxic effects resulting from overexpression of a normally tightly regulated gene product can select for overgrowth of transductants expressing relatively low levels of the transgene. In conclusion, the use of WPRE as a template for real-time qPCR allows for the rapid, sensitive, and accurate assessment of lentiviral copy number at multiple levels, including viral genomic RNA, integrated proviral DNA, and expressed mRNA in transduced cells. Viral titering and gene expression analysis by these means is widely applicable in both a basic and clinical context and, importantly, does not depend on reporter gene analysis. The ability to accurately titer lentiviruses expressing TAA such as tyrosinase and neoPAP is critical to the future development of this vector technology.

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ACKNOWLEDGMENTS The authors thank Arnold Mixon and Shawn Farid for FACS analysis, Zhili Zheng for provision of reagents, Yong Li for DNA sequencing, Patrick Hwu for helpful discussions, and Steven A. Rosenberg for advice and support.

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Address reprint requests to: Dr. Suzanne L. Topalian Surgery Branch National Cancer Institute NIH 10/2B47 Bethesda, MD 20892. E-mail: [email protected] Received for publication October 25, 2002; accepted after revision February 28, 2003. Published online: March 21, 2003.

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