New Methods to Titrate EIAV-Based Lentiviral Vectors
Authors: Enca Martin-Rendon, Linda J. White, Anna Olsen, Kyriacos A. Mitrophanous, Nicholas D. Mazarakis
Journal: Molecular Therapy (2002)

Ideally, gene transfer vectors used in clinical protocols should only express the gene of interest. So far most vectors have contained marker genes to aid their titration. We have used quantitative real-time PCR to titrate equine infectious anemia virus (EIAV) vectors for gene therapy applications. Viral RNA was isolated from vector preparations and analyzed in a one-step RT-PCR reaction in which reverse transcription and amplification were combined in one tube. The PCR assay of vector stocks was quantitative and linear over four orders of magnitude. In tandem, the integration efficiency of these vectors has also been determined by real-time PCR, measuring the number of vector genomes in the target cells. We have found that these methods permit reliable and sensitive titration of lentiviral vectors independent from the expression of a transgene. They also allow us to determine the integration efficiency of different vector genomes. This technology has proved very useful, especially in the absence of marker genes and where vectors express multiple genes. Ideally, gene transfer vectors used in clinical protocols should only express the gene of interest. So far most vectors have contained marker genes to aid their titration. We have used quantitative real-time PCR to titrate equine infectious anemia virus (EIAV) vectors for gene therapy applications. Viral RNA was isolated from vector preparations and analyzed in a one-step RT-PCR reaction in which reverse transcription and amplification were combined in one tube. The PCR assay of vector stocks was quantitative and linear over four orders of magnitude. In tandem, the integration efficiency of these vectors has also been determined by real-time PCR, measuring the number of vector genomes in the target cells. We have found that these methods permit reliable and sensitive titration of lentiviral vectors independent from the expression of a transgene. They also allow us to determine the integration efficiency of different vector genomes. This technology has proved very useful, especially in the absence of marker genes and where vectors express multiple genes. IntroductionThere have been several reports on gene transfer to clinically relevant tissues and cell types, such as the nervous system and CD34+ cells, using lentiviral vectors [1Trono D. Lentiviral vectors: turning a deadly foe into a therapeutic agent.Gene Ther. 2000; 7: 20-23Crossref PubMed Scopus (240) Google Scholar, 2Kordower J.H. Neurodegeneration prevented by lentiviral vector delivery of GDNF in primate models of Parkinsons' disease.Science. 2000; 290: 767-773Crossref PubMed Scopus (1139) Google Scholar, 3Mitrophanous K. Stable gene transfer to the nervous system using a nonprimate lentiviral vector.Gene Ther. 1999; 6: 1808-1818Crossref PubMed Scopus (248) Google Scholar]. The success of gene therapy protocols is highly dependent on high-titer vector preparations. Traditionally, transduction efficiency on target cells has helped to estimate viral titers. Titers correspond to the ability of the vector particles to infect the target cells, integrate into the host genome, and express the transgene. In most cases the transgene is an antibiotic resistance gene that takes 2 weeks of drug exposure to select and to determine the vector titer [4Onodera M. A simple and reliable method for screening retroviral producer clones without selectable markers.Hum. Gene Ther. 1997; 8: 1189-1194Crossref PubMed Scopus (55) Google Scholar, 5Sanburn N. Cornetta K. Rapid titer determination using quantitative realtime PCR.Gene Ther. 1999; 6: 1340-1345Crossref PubMed Scopus (46) Google Scholar]. There is concern about the immunogenic potential of these genes in animal models and clinical trials. Accordingly, scientists have preferred to remove drug resistance genes from viral vector genomes. In other cases the transgene is either LacZ or GFP, whose expression is high in mammalian cells due to the stability of the proteins. Transgene expression may vary between cell types depending on the strength and specificity of the promoter used—making a “titer” cell specific. Finally, the capacity of the retroviral vectors to infect the target cells might be impaired at stages before the integration of the vector genome into the host DNA, therefore making gene expression inappropriate as the inherent measure of titer. There is an urgent need to devise an objective measure of titer that is independent of the target cell type and does not require a marker gene.In the absence of marker genes, the titers of viral vectors expressing therapeutic genes have to be determined by alternative methods. RNA dot-blot assays have been used to estimate vector RNA from viral supernatants [6Huszar D. Ellis J. Bernstein A. A rapid and accurate method for determining the titre of retrovirus vectors lacking a selectable marker.Technique. 1989; 1: 28-32Google Scholar, 7Turfuro S. Zentilin L. Falaschi A. Giacca M. Rapid retrovirus titration using competitive polymerase chain reaction.Gene Ther. 1996; 3: 679-684PubMed Google Scholar, 8Murdoch B. Pereira D.S. Wu X. Dick J.E. Ellis J. A rapid screening procedure for the identification of high-titer retrovirus packaging clones.Gene Ther. 1997; 4: 744-749Crossref PubMed Scopus (26) Google Scholar]. These methods, although helpful, are semiquantitative. Real-time RT-PCR technology is a more sensitive and accurate method [5Sanburn N. Cornetta K. Rapid titer determination using quantitative realtime PCR.Gene Ther. 1999; 6: 1340-1345Crossref PubMed Scopus (46) Google Scholar, 8Murdoch B. Pereira D.S. Wu X. Dick J.E. Ellis J. A rapid screening procedure for the identification of high-titer retrovirus packaging clones.Gene Ther. 1997; 4: 744-749Crossref PubMed Scopus (26) Google Scholar]. It allows the processing of large numbers of samples, combines reverse transcription and PCR in one tube, and provides real-time read-out.We have previously developed a vector system based on the equine infectious anemia virus (EIAV) that is able to transduce dividing and nondividing cells [3Mitrophanous K. Stable gene transfer to the nervous system using a nonprimate lentiviral vector.Gene Ther. 1999; 6: 1808-1818Crossref PubMed Scopus (248) Google Scholar] and sustains long-term expression of a transgene. Our minimal EIAV vector system has a high safety profile and the potential to be used in gene therapy protocols in the clinic. Vector preparations pseudotyped with the vesicular stomatitis virus (VSV-G) and rabies virus (rabies-G) glycoproteins are conventionally produced by three-plasmid cotransfection in a transient system [9Soneoka Y. Transient three-plasmid expression system for the production of high titer retroviral vectors.Nucleic Acids Res. 1995; 23: 628-633Crossref PubMed Scopus (608) Google Scholar] and can be concentrated successfully [10Rohll J.B. The design, production, safety, evaluation and clinical applications of non-primate lentiviral vectors.Methods Enzymol. 2002; 346: 466-500Crossref PubMed Scopus (64) Google Scholar]. Here we have developed assays that use quantitative real-time PCR to determine the performance of EIAV vector preparations that only express therapeutic genes. This technology is broadly applicable to all lentiviral-based strategies for gene therapy.Results and DiscussionValidation of the Product-Enhanced Reverse Transcriptase and Viral RNA AssaysWe prepared EIAV viral vectors expressing GFP (pONY8G) and pseudotyped with VSV-G using the transient system. The vector particles were disrupted and the EIAV reverse transcriptase present in the particles determined by product-enhanced reverse transcriptase (PERT) assay. Figure 1 represents the mean Ct values (threshold cycle at which there is significant increase in signal generated by any given set of PCR conditions) and standard deviation of 16 independent PERT assays carried out on the same vector stock on different occasions. The assay is linear over the range tested (4 orders of magnitude) with a correlation coefficient of 0.99.Similarly, we isolated the genome RNA from the viral preparations and used it as template in a one-step RT-PCR reaction. DNA contamination due to plasmid carryover in the transfection was eliminated by treating the isolated RNA with DNase I. Serial dilutions (10–2 to 10–5) of the DNA-free viral RNA were carried out. To validate the viral RNA assays, we carried out three independent RNA extractions from the same vector preparation. We performed quantitative real-time RT-PCR once or twice for each extraction on separate occasions. Three samples of the RT-PCR reaction were assayed each time. Figure 2 shows the mean Ct values and standard deviation of pONY8G viral RNA. This assay is also linear over the range of dilutions tested (4 orders of magnitude) with a correlation coefficient of 0.99. The Ct values obtained with parallel control reactions in which the reverse transcriptase was omitted (–RT) were approximately 10–13 cycles (0.08–0.1%) lower, indicating that the RT-PCR products represent the amplification of the viral RNA template and not contaminating DNA (data not shown). The Ct values of +RT and –RT reactions differ by only three cycles (eightfold) when the samples were not treated with DNase I suggesting that there is some contaminating DNA, most probably carried over from the production of vector (data not shown).FIG. 2Ct values (mean and standard deviation) obtained from five independent viral RNA assays. The viral RNA was extracted three times from the same viral vector preparation, pONY8G. Each lot of viral RNA was assayed once or twice in an ABI Prism 7700 Sequence Detection System using a set of primers and probe specific for the CMV promoter present in the vector genome.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The small standard deviation obtained in both PERT and viral RNA assays demonstrates that these methods are consistent.Correlation between Viral RNA Content and Viral Titers on a Target Cell TypePERT assays determine the relative total number of particles, whereas the RNA assays estimate the content of viral RNA in a vector preparation and therefore the potential of that vector to transduce target cells. We have analyzed three pONY8G vector preparations with different biological titers on D17 cells by PERT and RNA assays to determine whether there is any correlation between the three measurements. The titers were 1.6 × 109 t.u./ml (vector 1), 7 × 108 t.u./ml (vector 2), and 1.0 × 108 t.u./ml (vector 3). Figure 3 shows the mean Ct values obtained from three experiments. The three vector stocks contain a similar number of particles, as determined by PERT (Fig. 3A), suggesting that there is no correlation between PERT and the titers obtained by transduction of D17 cells. The results are consistent over the range of dilutions performed on the particles. However, there is a positive correlation between the viral RNA content in the vector stocks and the biological titers on D17 cells (Fig. 3B). The gradients are essentially the same (y = –3.3x + 11.74) for all three viral vector stocks, with a correlation coefficient of 0.99. These results indicate that the RNA assay is more accurate in determining the potential of a vector preparation to transduce target cells. The PERT/RNA assays carried out on any given vector stock provide an indication of the particles/infectivity (P/I) ratio of that vector preparation. The differences in titers observed with the three vector stocks described above are likely to represent different P/I ratios, indicating that these ratios could be optimized and used to improve vector production.FIG. 3PERT and viral RNA assays carried out on three independent pONY8G vector preparations with different biological titers on D17 cells. The assays were performed over three 10-fold dilutions of the particles or viral RNA template, respectively. The Ct values represent the mean of three independent experiments. (A) PERT assays. (B) Viral RNA assays.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Transduction Efficiency of EIAV Vectors on Different Target CellsEIAV vectors can transduce a variety of cell types. However, the transduction efficiency of these vectors varies in different cell lines. We used a pONY8G vector preparation to transduce D17, HEK293T, and HeLa cells and found different biological titers on the different cell lines (Table 1). Titers on HeLa cells seem to be 0.77–0.83% of those on D17 and HEK293T cells. The transduction efficiency of EIAV vectors on HeLa cells might be compromised either at the integration level or at the transgene expression level. If the former is true, the number of EIAV genomes in the host chromosomes will be different between the different cell lines. In contrast, if the latter is true, the integration efficiency will be equivalent in the different cell lines.TABLE 1:Titers of pONY8G on three different target cell linesCell lineMean titer (t.u./ml)aThe results represent the average of three independent experiments.Standard deviationaThe results represent the average of three independent experiments.D173.16 × 1090.64 × 109HEK293T4.22 × 1090.15 × 109HeLa2.63 × 1070.21 × 107a The results represent the average of three independent experiments. Open table in a new tab HEK293T and HeLa cells transduced with pONY8G were passaged at least three times before total DNA was extracted (approximately 8 days post-transduction). We used TaqMan assays to determine the efficiency of integration of EIAV vectors. The relative integration efficiency of EIAV genomes/β-actin (dCt values) is equivalent in the cell types used in this study. Figure 4 shows the average result of three experiments. Clearly, the difference in titers observed on HEK293T and HeLa cells is not reflected in the amount of reverse-transcribed and integrated genomes. We cannot rule out that transgene inactivation might be a consequence of proviral DNA rearrangements. Genomic rearrangements and mutations have been previously reported to be the source of genetic variation in other retroviral vectors such as murine leukemia virus (MLV) and human immunodeficiency virus (HIV) [11Parthasarathi S. Varela-Echavarria A. Ron Y. Preston B.D. Dougherty J.P. Genetic rearrangements occurring during a single cycle of murine leukaemia virus vector replication: characterization and implications.J. Virol. 1995; 69: 7991-8000PubMed Google Scholar, 12Mansky L.M. Temin H.M. Lower in vivo mutation rate of human immunodeficiency virus type 1 than that predicted from the fidelity of purified reverse transcriptase.J. Virol. 1995; 69: 5087-5094Crossref PubMed Google Scholar]. Considering that EIAV vectors would show similar rates of recombination and mutation as MLV or HIV, the genetic variation will be vector-dependent and not cell-typedependent. Therefore it may not account for the drop in transduction efficiency of EIAV vectors in HeLa cells compared with HEK293T cells. Thus, this difference in titers is likely to be due to either different transgene expression levels (that is, promoter activity, mRNA stability) or genesilencing mechanisms. This assay might be useful in quantifying the expression levels of a transgene driven by different promoters.FIG. 4Relative integration efficiency of EIAV genomes/p-actin. HeLa and HEK293T cells were transduced with pONY8G at an MOI of 30 or 3, according to the titers on D17 cells. Total DNA (100 µg) from the transduced cells was used as template. Parallel real-time PCR reactions were set up in which primers and probes specific to either the CMV promoter in the vector genomes or the β-actin gene were added. The dCt values are the mean from three experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Titration of Viral Vectors That Lack Marker GenesLentiviral vectors are good candidates to use in the clinic as gene transfer vectors. In this context, reporter genes will need to be removed from the vector genomes to make them safer. Therefore it seems appropriate to develop new methods to assess the potential of these vectors to transduce target cells. We have developed a pONY8Tric vector (Fig. 5A) as a gene transfer vector for the treatment of Parkinson's disease. This vector genome expresses three therapeutic genes involved in the dopamine biosynthetic pathway in a single transcription unit. The three genes are linked by two internal ribosome entry sites (IRESes) and their expression is driven by the cytomegalovirus (CMV) promoter. There was a need to maximize the cloning capacity of the EIAV vectors to carry these three genes and no markers. We compared pONY8Tric with pONY8G. Figure 5B shows the PERT and viral RNA Ct values of these vector preparations. The total number of vector particles in these preparations is very similar, whereas the viral RNA content differs slightly, suggesting that the P/I ratios are different. D17 target cells were transduced with both vector stocks at a multiplicity of infection (MOI) of 10, determined by transduction of D17 with pONY8G and matching the vector stocks for RNA content. We have also used an integrase minus mutant EIAV-LacZ (pONY8Z(Int–)) viral preparation as a control. The PCR results are indicative of equivalent transduction of D17 cells using the EIAV vectors pONY8G, pONY8Tric, and pONY8Z (Fig. 5C). The dCt values obtained when cells were transduced with integrase-deficient particles were similar to those obtained with the untransduced cells control (dCt ~ 1.98 ± 0.35) and much lower than those of pONY8G, pONY8Tric, and pONY8Z genomes (< 0.005%), demonstrating that the assay is measuring integrated copies of EIAV genomes. The transduction efficiency of the vectors, measured as a direct correlate of transgene expression, varies according to factors such as expression levels, gene silencing, and/or genomic variation [11Parthasarathi S. Varela-Echavarria A. Ron Y. Preston B.D. Dougherty J.P. Genetic rearrangements occurring during a single cycle of murine leukaemia virus vector replication: characterization and implications.J. Virol. 1995; 69: 7991-8000PubMed Google Scholar, 12Mansky L.M. Temin H.M. Lower in vivo mutation rate of human immunodeficiency virus type 1 than that predicted from the fidelity of purified reverse transcriptase.J. Virol. 1995; 69: 5087-5094Crossref PubMed Google Scholar]. The PCR assay only measures integration and does not take into account gene inactivation by mutations or rearrangements. pONY8Tric is ~ 110% in size relative to the EIAV wild-type genome. Thus our results indicate that EIAV capsids can efficiently package genomes that are larger than the wild-type viral RNA in size. We have also demonstrated that the methods described in this paper can be systematically used to assess the performance of vector preparations intended for use in the clinic.FIG. 5Performance of EIAV vectors that express therapeutic genes. (A) Configuration of an EIAV-TRIC genome used in this study. Tric, tricistronic; CMVp, cytomegalovirus promoter; ϕ, EIAV packaging signal; LTR, long terminal repeats; HA, c-myc and FLAG; epitope tags; AADC, aromatic L-amino acid dopa decarboxylase; TH, tyrosine hydroxylase; CH, GTP-cyclohydrolase; IRES, internal ribosome entry site. (B) PERT and viral RNA assays carried out on pONY8G and pONY8-TRIC vector preparations. The titer of pONY8G on D17 cells was ~ 1 × 109 t.u./ml. (C) Relative integration efficiency of EIAV genomes/β-actin on D17 cells. The dCt values represent the mean of three experiments. The dCt value obtained from untransduced cells was ~ 1.98.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Materials and MethodsMinimal EIAV vector systemPlasmid pRV67 [9Soneoka Y. Transient three-plasmid expression system for the production of high titer retroviral vectors.Nucleic Acids Res. 1995; 23: 628-633Crossref PubMed Scopus (608) Google Scholar] expressing the VSV-G protein from the CMV promoter was used to pseudotype EIAV viral particles. pONY3.1 [10Rohll J.B. The design, production, safety, evaluation and clinical applications of non-primate lentiviral vectors.Methods Enzymol. 2002; 346: 466-500Crossref PubMed Scopus (64) Google Scholar] expresses the EIAV gag/pol. An integrase minus (pONY3.1 Int–) mutant derived from pONY3.1 was used as control to assess gene transfer in the absence of integration [10Rohll J.B. The design, production, safety, evaluation and clinical applications of non-primate lentiviral vectors.Methods Enzymol. 2002; 346: 466-500Crossref PubMed Scopus (64) Google Scholar]. pONY8G (GFP) and pONY8Z (LacZ) were used as vector genomes. These genomes do not express any of the EIAV accessory genes and have the first two ATG codons of gag mutated to prevent expression of Gag proteins [13Mazarakis N.D. Rabies virus glycoprotein pseudotyping of lentiviral vectors enables retrograde axonal transport and access to the nervous system after peripheral delivery.Hum. Mol. Genet. 2001; 10: 2109-2121Crossref PubMed Scopus (372) Google Scholar]. pONY8Tric is a minimal EIAV vector genome based on pONY8 that carries three genes linked by two IRESes driven by the CMV promoter (Fig. 5A). Details of the construction of this vector genome are given elsewhere (E.M.-R. et al., manuscript in preparation).Viral vector productionEIAV viral vector stocks pseudotyped with VSV-G were prepared using the HEK 293T transient system previously described [9Soneoka Y. Transient three-plasmid expression system for the production of high titer retroviral vectors.Nucleic Acids Res. 1995; 23: 628-633Crossref PubMed Scopus (608) Google Scholar, 10Rohll J.B. The design, production, safety, evaluation and clinical applications of non-primate lentiviral vectors.Methods Enzymol. 2002; 346: 466-500Crossref PubMed Scopus (64) Google Scholar]. Briefly, 16 µg vector plasmid, 16 µg gag/pol plasmid (pONY3.1), and 8 µg envelope plasmid were used to transfect HEK 293T cells plated on 10-cm dishes using the calcium-phosphate method. Approximately 36–48 hours post-transfection, viral supernatants were filtered (0.45 µm) and concentrated in two steps. First, by low-speed centrifugation of 6000g (JLA-10.500) for 16 hours at 4°C, followed by ultracentrifugation at 50,000g (SW40Ti rotor) for 90 minutes at 4°C. The viral vector stocks were resuspended in phosphate buffered saline (PBS) for 3–4 hours, aliquotted, and stored at –70°C. Vector stocks expressing a marker gene, such as LacZ or GFP, were titrated by the serial dilution method on D17, canine osteosarcoma cells. The transduction of target cells was carried out in 12-well plates (seeded at 8 × 104 cells/well) in triplicate in the presence of 8 µg/ml Polybrene (Sigma) for 2–5 hours. The Polybrene-containing medium was removed and fresh medium was added to the cells. β-Galactosidase expressing colonies were counted 48 hours post-transfection. GFP expressing colonies were counted after 48 hours at 37°C and 72 hours at 32°C. Titers were expressed as transducing units per ml (t.u./ml).PERT assaysEIAV reverse transcriptase associated with vector particles was released by mild detergent treatment and used to synthesize cDNA using MS2 bacteriophage RNA as template. The MS2 RNA template and primer were present in excess in the reaction, hence the amount of cDNA synthesized is proportional to the amount of RT released from the particles and therefore proportional to the number of particles. MS2 cDNA was then quantitated using TaqMan technology [14Lie Y.S. Petropolous C.J. Advances in quantitative PCR technology: 5′ nuclease assays.Curr. Opin. Biotechnol. 1998; 9: 43-48Crossref PubMed Scopus (227) Google Scholar]. Serial dilutions of the viral vector stocks were carried out in PBS. Typically 10–4, 10–5, 10–6, and 10–7 dilutions were used for concentrated viral stocks. The particles were disrupted by adding 25 µl diluted vector to 25 µl particle disruption buffer [15Arnold B.A. Hepler R.W. Keller P.M. One-step fluorescent probe product-enhanced reverse transcriptase assay.Biotechniques. 1998; 25: 98-106PubMed Google Scholar] (40 mM Tris-HCl, pH 7.5 (20°C), 50 mM KCl, 20 mM DTT, 0.2% NP-40). Disrupted particles (10 pl) were added to 90 µl one-step RT-PCR reaction mix. The reaction consisted of 300 µM dATP, dCTP, and dGTP, 600 µM dUTP, 5.5 mM MgCl2, 300 nM PERT forward and reverse primers, 150 nM PERT probe, 0.025 U/µl AmpliTaq Gold, 0.4 U/µl RNase Inhibitor, 1× TaqMan Buffer A, MS2 RNA (Roche), and nuclease-free water. Aliquots of 25 µl were transferred to the 96-well optical plate. The plate was capped and spun at 1100 rpm for 3 minutes. Amplification was carried out using the ABI Prism 7700 Sequence Detection System: one cycle, 48°C for 30 minutes; one cycle, 95°C for 10 minutes; 40 cycles, 95°C for 15 seconds, 60°C for 1 minute. The primers and probe used in the PERT assay recognize the MS2 RNA. Their sequences are as follows: forward primer, 5'-TCCTGCTCAACTTCCTGTCGA-3'; reverse primer, 5'-CACAGGTCAAACCTCCTAGGAATG-3'; probe, 5'-CGAGACGCTACCATGGCTATCGCTGTAG-(TAMRA)-3'.Viral RNA assaysVector RNA was isolated in a two-stage process. In the first step, the RNA was extracted from concentrated vector stocks, and in the second, the isolated RNA was treated with DNase I to remove contaminating DNA, in particular plasmid DNA carried over from the production of vector. Briefly, the viral RNA was extracted using a viral RNA extraction kit (Qiagen) from a 5 µl aliquot diluted in 70 µl PBS. Initially, the reproducibility of the extraction procedure was tested by carrying out several RNA extractions in parallel. The RNA was resuspended in a final volume of 60 µl and stored at –80°C in aliquots. To remove any contaminating DNA, 5 µl viral RNA was treated with 3 µl DNase I (2 U/µl; Ambion, DNA-free kit) in a final volume of 50 µl at 37°C for 30 minutes. Serial 10-fold dilutions of the template RNA in nuclease-free water were carried out before setting up the TaqMan reactions. Each dilution of the template RNA (10 pl) was added to 90 µl one-step RT-PCR reaction mix. The RT-PCR reaction mix consisted of 300 µM dATP, dCTP and dGTP, 600 µM dUTP, 5.5 mM MgCl2, 0.25 U/µl Multiscribe reverse transcriptase, 0.025 U/µl AmpliTaq Gold, 0.4 U/µl RNase inhibitor, 1× TaqMan buffer A, template, 300 nM each forward and reverse primers, 125 nM probe, and RNase-free water to volume. Aliquots of 25 µl were transferred to the 96-well optical plate. The plate was capped and spun at 1100 rpm for 3 minutes. Amplification was carried out using the ABI Prism 7700 Sequence Detection System as described in the PERT assays. To determine which proportion of the amplified product was the result of DNA contamination, parallel control reactions were set up in which the reverse transcriptase was omitted (-RT). The sequence of primers and probe used in the viral RNA assay correspond to the CMV promoter. These are as follows: forward primer, 5'-CATATATGGAGTTCCGCGTTACAT-3'; reverse primer, 5'-GTATGTTCCCATAGTAACGCCAATAG-3’ and probe, 5'-TGGCTGACCGCCCAACGACC-(TAMRA)-3'.Integration assaysTransduction experiments were carried out in duplicate in the presence of Polybrene (Sigma) diluting the vector stocks according to titers obtained on D17 cells. Generally two different MOIs were used. In order to reach the steady state of integrated DNA, the transduced cells were subjected to at least three passages (after a period of 7–10 days post-transduction) before the isolation of the DNA. Total cellular DNA was isolated from approximately 5 × 105 cells using the QIAamp DNA Mini Kit (Qiagen). The concentration of DNA was determined by absorbance measurement at 260 nm. Reactions were carried out in triplicate with 100 ng of total DNA per reaction in a final volume of 25 µl. The reaction mix was composed of 12.5 µl (2X) Master Mix (Applied Biosystems), 300 nM forward and reverse primers, 125 nM probe to detect the internal CMV promoter in the vector, and nuclease-free water. Reactions were carried out separately to measure β-actin as an endogenous reference (900 nM forward and 300 nM reverse primers and 100 nM probe). The sequences of the β-actin primers and probe are as follows: forward primer, 5'-AGCGCG-GCTACAGCTTCA-3'; reverse primer, 5'-GGCGACGTAGCACAGCTTCT-3'; and probe, 5'-TCACGCACGATTTCCCGCTCG (TAMRA)-3'.As described above, 25 ml aliquots were added to the 96-well optical plate. The plate was capped and spun at 1100 rpm for 3 minutes. The amplification was performed on ABI Prism 7700 Sequence Detection System: one cycle, 50°C for 2 minutes; one cycle, 95°C for 10 minutes; 40 cycles, 95°C for 15 seconds and 60°C for 1 minute.Data interpretationThe instructions are described in the user bulletins (Bulletin#2) available at http://www.appliedbiosystems.com/ (Services and Support/Documents on Demand/). The threshold cycle or Ct value is the cycle at which there is significant increase in the signal generated by any given set of PCR conditions. This parameter reflects the amount of template or target. The lower the Ct value, the higher the amount of target in the PCR reaction. The dCt values are the differences in threshold cycles (Ct) for target and reference, that is, CtEIAV - Ctβactin. IntroductionThere have been several reports on gene transfer to clinically relevant tissues and cell types, such as the nervous system and CD34+ cells, using lentiviral vectors [1Trono D. Lentiviral vectors: turning a deadly foe into a therapeutic agent.Gene Ther. 2000; 7: 20-23Crossref PubMed Scopus (240) Google Scholar, 2Kordower J.H. Neurodegeneration prevented by lentiviral vector delivery of GDNF in primate models of Parkinsons' disease.Science. 2000; 290: 767-773Crossref PubMed Scopus (1139) Google Scholar, 3Mitrophanous K. Stable gene transfer to the nervous system using a nonprimate lentiviral vector.Gene Ther. 1999; 6: 1808-1818Crossref PubMed Scopus (248) Google Scholar]. The success of gene therapy protocols is highly dependent on high-titer vector preparations. Traditionally, transduction efficiency on target cells has helped to estimate viral titers. Titers correspond to the ability of the vector particles to infect the target cells, integrate into the host genome, and express the transgene. In most cases the transgene is an antibiotic resistance gene that takes 2 weeks of drug exposure to select and to determine the vector titer [4Onodera M. A simple and reliable method for screening retroviral producer clones without selectable markers.Hum. Gene Ther. 1997; 8: 1189-1194Crossref PubMed Scopus (55) Google Scholar, 5Sanburn N. Cornetta K. Rapid titer determination using quantitative realtime PCR.Gene Ther. 1999; 6: 1340-1345Crossref PubMed Scopus (46) Google Scholar]. There is concern about the immunogenic potential of these genes in animal models and clinical trials. Accordingly, scientists have preferred to remove drug resistance genes from viral vector genomes. In other cases the transgene is either LacZ or GFP, whose expression is high in mammalian cells due to the stability of the proteins. Transgene expression may vary between cell types depending on the strength and specificity of the promoter used—making a “titer” cell specific. Finally, the capacity of the retroviral vectors to infect the target cells might be impaired at stages before the integration of the vector genome into the host DNA, therefore making gene expression inappropriate as the inherent measure of titer. There is an urgent need to devise an objective measure of titer that is independent of the target cell type and does not require a marker gene.In the absence of marker genes, the titers of viral vectors expressing therapeutic genes have to be determined by alternative methods. RNA dot-blot assays have been used to estimate vector RNA from viral supernatants [6Huszar D. Ellis J. Bernstein A. A rapid and accurate method for determining the titre of retrovirus vectors lacking a selectable marker.Technique. 1989; 1: 28-32Google Scholar, 7Turfuro S. Zentilin L. Falaschi A. Giacca M. Rapid retrovirus titration using competitive polymerase chain reaction.Gene Ther. 1996; 3: 679-684PubMed Google Scholar, 8Murdoch B. Pereira D.S. Wu X. Dick J.E. Ellis J. A rapid screening procedure for the identification of high-titer retrovirus packaging clones.Gene Ther. 1997; 4: 744-749Crossref PubMed Scopus (26) Google Scholar]. These methods, although helpful, are semiquantitative. Real-time RT-PCR technology is a more sensitive and accurate method [5Sanburn N. Cornetta K. Rapid titer determination using quantitative realtime PCR.Gene Ther. 1999; 6: 1340-1345Crossref PubMed Scopus (46) Google Scholar, 8Murdoch B. Pereira D.S. Wu X. Dick J.E. Ellis J. A rapid screening procedure for the identification of high-titer retrovirus packaging clones.Gene Ther. 1997; 4: 744-749Crossref PubMed Scopus (26) Google Scholar]. It allows the processing of large numbers of samples, combines reverse transcription and PCR in one tube, and provides real-time read-out.We have previously developed a vector system based on the equine infectious anemia virus (EIAV) that is able to transduce dividing and nondividing cells [3Mitrophanous K. Stable gene transfer to the nervous system using a nonprimate lentiviral vector.Gene Ther. 1999; 6: 1808-1818Crossref PubMed Scopus (248) Google Scholar] and sustains long-term expression of a transgene. Our minimal EIAV vector system has a high safety profile and the potential to be used in gene therapy protocols in the clinic. Vector preparations pseudotyped with the vesicular stomatitis virus (VSV-G) and rabies virus (rabies-G) glycoproteins are conventionally produced by three-plasmid cotransfection in a transient system [9Soneoka Y. Transient three-plasmid expression system for the production of high titer retroviral vectors.Nucleic Acids Res. 1995; 23: 628-633Crossref PubMed Scopus (608) Google Scholar] and can be concentrated successfully [10Rohll J.B. The design, production, safety, evaluation and clinical applications of non-primate lentiviral vectors.Methods Enzymol. 2002; 346: 466-500Crossref PubMed Scopus (64) Google Scholar]. Here we have developed assays that use quantitative real-time PCR to determine the performance of EIAV vector preparations that only express therapeutic genes. This technology is broadly applicable to all lentiviral-based strategies for gene therapy.