Comparison of lentiviral vector titration methods
© Geraerts et al; licensee BioMed Central Ltd. 2006
Received: 17 March 2006
Accepted: 12 July 2006
Published: 12 July 2006
Lentiviral vectors are efficient vehicles for stable gene transfer in dividing and non-dividing cells. Several improvements in vector design to increase biosafety and transgene expression, have led to the approval of these vectors for use in clinical studies. Methods are required to analyze the quality of lentiviral vector production, the efficiency of gene transfer and the extent of therapeutic gene expression.
We compared lentiviral vector titration methods that measure pg p24/ml, RNA equivalents/ml, transducing units (TU/ml) or mRNA equivalents. The amount of genomic RNA in vector particles proves to be reliable to assess the production quality of vectors encoding non-fluorescent proteins. However, the RNA and p24 titers of concentrated vectors are rather poor in predicting transduction efficiency, due to the high variability of vector production based on transient transfection. Moreover, we demonstrate that transgenic mRNA levels correlate well with TU and can be used for functional titration of non-fluorescent transgenes.
The different titration methods have specific advantages and disadvantages. Depending on the experimental set-up one titration method should be preferred over the others.
In our laboratory we routinely produce and apply vectors derived from the human immunodeficiency virus type 1 (HIV-1). Since lentiviral vectors (LV) integrate stably into the host-cell genome of non-dividing cells such as neurons and in haematopoietic stem cells [1–3], they offer great potential for gene therapeutic applications . For biosafety reasons, the HIV-1 genome has been modified and cis and trans-acting viral sequences have been segregated over 3 to 4 different plasmids [5, 6]. Indeed, viral structural and functional proteins can be provided in trans and are encoded by 1 or 2 packaging plasmids while the envelope plasmid encodes the glycoprotein of the vesicular stomatitis virus envelope (VSV-G) and a transfer plasmid encodes the transgene of interest flanked by all cis-acting viral sequences necessary for packaging of the RNA genome (reviewed by ). Production of lentiviral vectors is routinely achieved by transient transfection of human embryonic kidney (293T) cells using high concentrations of the different plasmids, implicating the presence of residual plasmid DNA in the vector preparation, even after concentration. Transduction by lentiviral vectors matches a single-round infection and results in long-term integration into the genome of both dividing and non-dividing cells, forever linking the fate of the provirus with that of the target cell. VSV-G pseudotyping of the lentiviral vector particles not only broadens the tissue tropism of the vector, but also stabilizes the particles allowing concentration to high titers by ultracentrifugation . Since the initial development of the lentiviral vector system [2, 5, 6] the transfer plasmid was gradually optimized in order to improve biosafety as well as to increase transduction efficiency. The self-inactivating (SIN) deletion in the 3' LTR  limits vector rescue and reduces the likelihood of promoter activation after integration. The woodchuck hepatitis virus posttranscriptional regulatory element (WPRE)  stabilizes the transgene mRNA and the insertion of the central polypurine tract/central termination site (cPPT/CTS) sequence stimulates nuclear import .
Approval of lentiviral vectors for cell-marking and therapeutic studies in humans requires in-depth characterization of vector titers and expression profiles of therapeutic genes. Ample methods to evaluate lentiviral vector titers have been described (reviewed by ). These methods can roughly be divided into functional and non-functional titration methods. The latter include p24 antigen ELISA, assessment of the reverse transcriptase activity and determination of the genomic RNA concentration in vector preparations by semi-quantitative northern blotting, dot blot analysis or RT-qPCR. Generally these techniques overestimate the functional vector titer and suffer from following disadvantages: the p24 protein pool that is quantified includes a variable amount of free p24 and p24 that originates from non-functional vector particles. Similarly, RNA titers will also assess defective particles, whereas the RT-assay merely demonstrates RT activity. A more accurate, functional titer is determined by transduction of cells following limiting dilution of vector and subsequent evaluation of reporter protein activity, (e.g. beta-galactosidase positive cells) or by assessment of the number of colony forming units following antibiotic selection. The most widespread and straightforward technique to quantify functional vector titers employs eGFP fluorescence and fluorescence-activated cell sorting (FACS). However, FACS analysis of transgene expression is restricted to fluorescent reporter proteins and cannot discriminate cells with single or multiple integrations. In strict sense, the definition of a functional vector titer is the number of vector particles required to infect a cell, present in a volume. In this regard, the best measurement of the number of functional particles can be accomplished by determination of the number of integrated proviral DNA copies per cell by qPCR [13–15]. However, due to insertion in regions with different chromatin packing, the integrated proviral DNA results in varying transgene expression levels. To overcome this drawback, Lizeé et al.  described a RT-qPCR method to quantify lentiviral mRNA copies following stable transduction in cell culture. Ultimately, the method of choice will depend on the experimental set-up. Basic research and possible clinical applications are in need of a universal, functional titration method for any transgene-of-interest, for example by qPCR. When analysing different internal promoters driving transgene expression quantification of the number of integrated proviral DNA copies following titration on a reference cell line is recommended. On the other hand, to compare different lentiviral vector backbones comprising additional cis-acting elements, a non-functional titration method is preferred to normalize the number of vector particles before assessing transduction efficiency.
In this study, we developed a quantitative RT-PCR assay, for quantification of both genomic lentiviral RNA after production and of transgene transcripts following transduction. We opted for a one-step RT-qPCR to reduce both sample handling time and variability. In addition, in contrast with the published methods, samples were amplified alongside a RNA standard to correct for low reverse transcriptase efficiency. The reliability of the different titration methods (RT-qPCR, ELISA and FACS) was evaluated and the methods were subsequently applied to assess vector production quantitatively and qualitatively. Next, we analysed the correlation between transgene expression as measured by FACS analysis and RT-qPCR. Although several groups have reported on the use of TU/ml or pg p24/ml to normalize vector transduction experiments [16, 17], a careful side-by-side analysis was hitherto absent. Here, we normalized vectors for RNA and p24 values prior to transduction and evaluated the transgene expression to determine the best titration method to normalize lentiviral vectors.
Results and discussion
Validation of a one-step RT-qPCR to determine lentiviral RNA content in concentrated vector preparations
Evaluation of the different titration methods
2.68 ± 0.38 × 1010
2.23 ± 1.10 × 109
9.8 ± 5.3 × 106
5.63 ± 0.50 × 1010
5.05 ± 4.9 × 107
4.67 ± 4.5 × 106
3.83 ± 2.25 × 1010
2.92 ± 2.5 × 108
4.83 ± 4.70 × 106
2.73 ± 1.59 × 1010
1.68 ± 1.3 × 109
4.79 ± 3.45 × 106
Next, three different lentiviral vectors (H-eGFP, H-eGFP-WS and CH-eGFP-WS) were produced in parallel (Figure 1B). H-eGFP-WS contains the WPRE, known to affect mRNA stability  while the cPPT/CTS sequence in CH-eGFP-WS improves the transduction efficiency [22, 23]. For each vector the RNA equivalents, transducing units (TU/ml) and p24 concentrations were determined to compare the different titration methods. Obviously, a clear difference between the lentiviral vectors was only evidenced by measuring the transducing titer (TU/ml), whereas the RNA and p24 concentration were similar for all vector backbones, pointing out that the packaging efficiency was comparable for the different constructs. In addition, although each functional vector particle (1 transducing unit) carries two RNA copies implying a theoretical ratio of 0.5, in reality the TU/RNA ratio ranged between 0.0009 and 0.0832 (Table 1). The TU/pg value ranged between 11 and 351. Both TU/pg and TU/RNA estimate the specific activity and correlate well with improved lentiviral vector backbone design. Table 1 shows a 6 and 8-fold increase in specific activity, when comparing the H-eGFP with H-eGFP-WS vector and a 31 and 68-fold increase when comparing the H-eGFP with the CH-eGFP-WS vector for TU/pg and TU/RNA respectively. Although the specific activities correlate well with the vector backbone, the differences between TU/pg and TU/RNA demonstrate that this is not an absolute value. Indeed, variations in TU, p24 and RNA titer may also be attributed to the inherent variability of transient transfection used for vector production, which is also dependent on the number of cells plated or the state of the producer cells. The TU/pg and TU/RNA values thus give an indication of the quality of the vector production but are subjected to the variable amounts of p24 and RNA produced by the cells.
It has been shown before that RNA values overestimate functional eGFP titers (TU/ml) by 200- to 10,000-fold [13–15, 24]. In our hands, using a RNA standard, we detected an approximately 10- to 1000-fold difference between the eGFP and RNA titers depending on the vector backbone. The discrepancy between the RNA and TU titer between several groups may be dependent on the vector backbone or other factors. First, the possibility exists that incomplete, defective genomes are integrated in the vector particles . Second, during transduction, part of the functional vector particles may stay in the cell culture medium and it has been shown that changes in inoculum volume and transduction time all influence transducing titers . Third, for lentiviral vectors it was shown previously by two independent groups that only ~10% to ~18% of the initial reverse transcribed genomes actually integrate in the host-cell DNA of 293T cells after transduction, probably due to degradation in the cytoplasm [18, 26]. Fourth, not all integrated proviral genomes may result in detectable transgene expression. Several groups, except for one , demonstrated that the proviral-based qPCR overestimates eGFP titers varying from 6-to 60-fold [13, 15, 27], probably due to integration in DNA regions with reduced transcriptional activity.
Quantification of genomic lentiviral vector RNA using different primer sets
Measurement of viral RNA in concentrated lentiviral vector preparations
5.0 × 108
15.51 ± 0.01
15.65 ± 0.04
15.45 ± 0.06
5.0 × 107
17.57 ± 0.01
17.79 ± 0.10
17.51 ± 0.01
5.0 × 106
18.97 ± 0.06
19.11 ± 0.08
20.11 ± 0.05
5.0 × 105
22.06 ± 0.17
21.82 ± 0.07
22.59 ± 0.03
5.0 × 104
25.91 ± 0.05
24.06 ± 0.06
26.13 ± 0.04
22.87 ± 0.05
22.5 ± 0.02
22.04 ± 0.03
16.15 ± 0.02
16.57 ± 0.03
16.43 ± 0.05
Comparison of lentiviral vector titration methods
Most frequently used titration methods for lentiviral vectors measure the p24 antigen concentration (pg p24/ml) by ELISA or the number of transducing units (TU/ml) by FACS analysis after limiting dilution in cell culture. Whereas the p24 concentration measures both functional and non-functional vector particles, the TU strictly measures functional vector particles that result in the expression of a fluorescent reporter protein. To compare the linearity, reproducibility and variability of the different methods, a CH-eGFP-WS lentiviral vector was serially diluted (12 steps of 1/2 dilution) and subjected to RNA extraction, p24 ELISA and transduction in cell culture. All titration methods correlated well with the initial dilution series: r2 = 0.99 for RNA/ml after RT-qPCR, r2 = 0.93 for TU/ml after FACS and r2 = 0.94 for p24/ml after ELISA (Figure 2). When determining transduction titers after limiting dilution, one uses only dilutions at MOI<<1 resulting in low percentage of transduced cells, to minimize the risk for multiple integrations. Titration depends on the total volume that is covering the cells, the time of incubation with the particular vector dilution and the cell type used. Prolonged incubation or delivery at the same MOI in only half of the volume will affect titers significantly. Nevertheless, using standardized conditions, these methods allow a good estimation of vector quality and titers. As a control, we checked the correlation between vector dilution and transduction efficiency (TE, i.e. the percentage of transduced cells) as measured by FACS. The most concentrated vector dilutions resulted in near 100% transduction, whereas the most diluted samples resulted in TEs near zero (* in Figure 2) and were omitted from the linear regression. Hence, the TE correlated only over 9 dilutions with r2 = 0.95.
Moreover, to estimate the variability within each test, we calculated the coefficients of variation (CV) for the different methods. The CV for the RNA/ml, the TU/ml and the pg p24/ml were respectively 39%, 78% and 103% on average. In conclusion, the RNA and p24 concentration as well as the functional titer are reliable parameters to assess the order of magnitude of vector titers. However, absolute numbers differ between samples as shown by the CV. The highest CV was obtained for the p24 ELISA. Other disadvantages of the p24 measurement are the restricted linear range (13–200 pg/ml) and the accompanying extensive dilution of the concentrated vector sample that is required and affects reproducibility. The high CV for the TU/ml may be due to variations in the cell number upon transduction or random integration in the genome, resulting in differences in transgene expression level.
Analysis of gene expression after normalization for p24 or vector RNA concentration
Evaluation of the transduction efficiency after normalization for RNA or p24
Vector production number
median ± SD
30 ± 15
11 ± 7
Evaluation of the reliability of the titration methods to assess lentiviral vector production quality and kinetics
Reliability of the titration methods to assess lentiviral vector production quality
6.18 ± 1.71 × 109
1.07 ± 0.53 × 107
4.10 ± 2.05 × 105
below detection limit
below detection limit
2.10 ± 0.64 × 105
2.36 ± 0.63 × 1010
below detection limit
1.27 ± 0.32 × 105
8.04 ± 1.64 × 106
below detection limit
below detection limit
Our results clearly demonstrate that the p24 concentration is the least reliable for the evaluation of functional vector particles after vector production. Nevertheless, it is a fast method that can be used as quality control for routine vector production. Logan and colleagues  described how manipulation of the amounts of transfer, packaging or envelope plasmids did not alter the specific activity (TU/pg p24) but rather influenced the vector concentration in the supernatant (TU/ml and pg p24/ml). However, if non-fluorescent transgenes are encoded, a functional titer cannot be determined, and one has to rely on the RNA concentration indicating the presence of genomic RNA. How this RNA titer relates to the functional titer is dependent on the vector construct (see Table 1), the transgene and the promoter, and requires further analysis of the integrated proviral genome by qPCR or of the transgene expression level by RT-qPCR, Western blotting or immunocytochemistry after transduction.
Evaluation of transgene expression by RT-qPCR
In this paper, commonly used methods for titration of lentiviral vectors were evaluated and may serve as a guide for newcomers in the field. For basic studies and eventually clinical trials, it is imperative that the performance characteristics and the variability inherent with these titration methods are known.
Due to the transient nature of a lentiviral vector production, variations in p24, TU and RNA levels inherent to the production procedure were demonstrated by ELISA, FACS analysis and RT-qPCR, respectively. These methods can be used to measure vector titers, although absolute numbers may vary even within the same run. Nor RNA nor p24 titers, can predict a functional titer, since the functional titer is dependent on the vector construct and the cell type used for transduction. The TU/RNA and TU/pg ratios reflect the specific activity of a lentiviral vector construct and were demonstrated to correlate well with the vector backbone. Normalization of vector productions based on RNA or p24 values are hampered by variability due to the transient transfection and the titration method and should be taken into consideration. Moreover, to assess the quality of lentiviral vector productions encoding non-fluorescent genes, the RNA titer is the most reliable, since p24 titers were not affected by omission of the transfer plasmid. We also demonstrated that the quantitative analysis of transgene mRNA levels correlates very well with the eGFP fluorescence as measured by FACS and hence can be used as an alternative for titration of vectors encoding non-fluorescent transgenes or determination of the transgene expression levels in transduced cells.
Cell lines and lentiviral vector transduction
293T cells were maintained in Dulbecco's modified Eagle's medium (DMEM; Gibco BRL, Merelbeke, Belgium) supplemented with 10% heat-inactivated foetal calf serum (FCS, Harlan Sera-Lab Ltd., International Medical, Brussels, Belgium) and 100 U/mL Penicillin and 100 μg/mL Streptomycin (Gibco BRL). Chinese hamster ovary cells, CHO-K1 were maintained in Ham's F12K medium (NutMix F12, Gibco BRL) supplemented with 5% heat-inactivated FCS and 20 μg/ml gentamicin (Gibco BRL). Both cell lines were cultivated at 37°C in a humidified atmosphere containing 5% CO2. Transductions of CHO-K1 or 293T cells with HIV-1 based lentiviral vectors were carried out in 96-well plates with 10-fold serial dilutions of lentiviral vector preparations. Vector was added to the cells in their corresponding medium supplemented with 1% FCS. After 4 hours of incubation, the medium was refreshed. Three days later, cells were removed and fixed in 2% paraformaldehyde prior to determination of titers (TU/ml) by limiting dilution and FACS.
The coefficient of variation (relative standard deviation) was determined for the different titration methods, which is a measure for the biological and experimental variation and is calculated as follows: stdev/mean × 100.
Lentiviral vector production
HIV-1-derived lentiviral vector particles, pseudotyped with the VSV-G envelope, were produced by transfecting 293T cells with a second generation packaging plasmid lacking vif, vpr, vpu and nef genes (pCMVΔR8.91), a plasmid encoding the envelope of VSV (pMDG) and a pHR'-derived transfer plasmid coding for eGFP as a reporter gene. Lentiviral vectors were produced as described earlier . Lentiviral vector stocks were normalized based on p24 antigen content (HIV-1 p24 ELISA kit; PerkinElmer, Milano, Italy). Transduction titers for vectors encoding eGFP were determined by FACS analysis after limiting dilution on CHO-K1 cells.
One-step real-time RT-qPCR
RNA content of lentiviral vectors was quantified with a one-step RT-qPCR that allows reverse transcription (RT) and amplification to take place in the same reaction tube. After RNA extractions of concentrated vector preps with the RNAqueous®-Micro Kit (Ambion, Cambridgeshire, United Kingdom) a DNase treatment (RNAqueous-Micro Kit) was carried out to eliminate residual plasmid DNA from the vector production according to the manufacture's protocol. RNA was amplified using the TaqMan® One-Step RT-PCR master mix reagents kit (Applied Biosystems, Lennik, Belgium) with primers and probe located in the eGFP transgene, the LTR or the WPRE. Forward and reverse primers were developed with the Primer Express Software (Applied Biosystems) and are specific for eGFP: 5'- GGAGCGCACGATCTTCTTCA-3' and 5'-AGGGTGTCGCCCTCGAA-3'; for LTR 5'-TGTGTGCCCGTCTGTTGTGT-3' and 5'-GAGTCCTGCGTCGAGAGAGC-3' ; and for WPRE 5'-CCGTTGTCAGGCAACGTG-3' and 5'-AGCTGACAGGTGGTGGCAAT-3' . Following TaqMan probes were used : for eGFP, 5'-FAM-CTACAAGACCCGCGCCGAGGTG-TAMRA-3'; for LTR, 5'-FAM-CAGTGGCGCCCGAACAGGGA-TAMRA-3'  and for WPRE, 5'-FAM-TGCTGACGCAACCCCCACTGGT-TAMRA-3' . Amplicon sizes are for eGFP: 75 base pairs (bp), for LTR: 143 bp for WPRE: 85 bp. The kit contains the Multiscribe™ Reverse Transcriptase (MuLV) which carries out the RT-step (30 minutes at 48°C) and AmpliTaq Gold enzyme for amplification (40 cycli at 95°C for 15 seconds, followed by 1 minute at 60°C). Reactions were analyzed using the ABI Prism model 7700 sequence detection system (Applied Biosystems). During each run a 'no amplification control' (NAC) was included for each sample (i.e. sample without RT-enzyme) to detect residual DNA contaminants and this value was subtracted. To normalize the mRNA values of expressed transgenes to the total RNA content in the sample, additional primers and a VIC-TAMRA-labeled probe directed against the RNAse P housekeeping gene were included in the sample mixture (RNAse P control reagents kit, Applied Biosystems) and values were corrected for the total amount of RNA in the sample. (FAM: 6-carboxyfluorescein; TAMRA: 6-carboxytetramethylrhodamine)
Generation of RNA standards by in vitrotranscription
For quantification of the extracted vector, an RNA standard was prepared by in vitro transcription. Therefore, an LTR- (302 bp) or WPRE-fragment (640 bp) was cloned into pSPT19 or pBluescript, respectively. The plasmid was linearized by a restriction digest with an enzyme located downstream of the cloned fragment, followed by in vitro transcription according to the manufacturer's protocol (RNA labelling kit, Roche, Brussels, Belgium). The resulting RNA was quantified using a spectrophotometer and standard curves were generated by 1/10 serial dilutions. During each one-step RT-qPCR, the RNA standard was run together with the samples in duplicate and the NACs.
We thank M. Michiels, F. Coun, I. Thiry, L. Dekeyzer for outstanding technical assistance with the viral vector productions and titrations. We also thank B. Van Maele and A. Hantson for development of the eGFP primers. The HIV-1-derived lentiviral vectors used were derived from constructs kindly provided by D. Trono (University of Geneva, Switzerland), O. Danos (Généthon, Evry, France) and P. Charneau (Institut Pasteur, Paris, France). MG is funded by a grant from the Institute for Promotion of Innovation through Science and Technology in Flanders (IWT-Vlaanderen). VB is a postdoctoral fellow of the Flemish Fund for Scientific Research (FWO Vlaanderen). RG is a postdoctoral fellow funded by the EC (N)EUROPARK project (QLK3-CT-2002-02114). This work was funded by the SBO grant (IWT-30238) of the Flemish Institute supporting Scientific-Technological Research in industry (IWT), the Flemish Fund for Scientific Research (FWO Vlaanderen) G.0164.03 and G.0406.06 and the EC grant (QLK3-CT-2002-02114) (N)EUROPARK.
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