- Email: [email protected]
Discordance between Expression and Genome Transfer Titering of HSV Amplicon Vectors: Recommendation for Standardized Enumeration
METHOD
doi:10.1006/mthe.2000.0039, available online at http://www.idealibrary.com on IDEAL
Discordance between Expression and Genome Transfer Titering of HSV Amplicon Vectors: Recommendation for Standardized Enumeration W. J. Bowers, D. F. Howard, and H. J. Federoff1 Department of Neurology and Center for Aging and Developmental Biology, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642 Received for publication November 29, 1999, and accepted in revised form February 10, 2000
Herpes simplex virus-derived amplicon vectors are well suited to the development of gene-based therapy for neurodegenerative diseases. The plasmid-based amplicon vector system allows for facile introduction of transcription units, possesses the potential for carrying gene inserts up to approximately 130 kb in length, and can be packaged into infectious virus devoid of contaminating cytotoxic helper virus. For accurate assessments to be made regarding vector comparison and improvements in vector design, a standard for titering prepared virus stocks must be established. At present, packaged amplicon vectors are routinely titered using reporter gene expression units to quantitate numbers of infectious amplicon virions. The strength of the promoter, sensitivity of detection of the gene product, and choice of titering cell type can greatly influence the apparent numbers of infectious virus particles. This is especially evident when comparisons are made between two amplicon vectors that possess different promoters. To this end, we have developed a new titering method based on a real-time quantitative PCR technique that allows for enumeration of transducing particles. This new approach ensures that amplicon comparison experiments are initiated with equivalent transduction units, thus allowing for a fair assessment of expression and therapeutic efficacy differences. Key Words: herpes simplex virus; amplicon; quantitative PCR; gene therapy; titering.
INTRODUCTION The plasmid-based HSV-1 amplicon vector is a highly versatile gene delivery platform, owing to its facility for molecular genetic manipulation (1– 4). The backbone of the amplicon consists primarily of a eukaryotic expression plasmid modified by the addition of an HSV origin of replication (ori) and cleavage/packaging sequence (“a” sequence). The amplicon is amenable to carrying multiple heterologous transcription units of various sizes (the theoretical limit is 150 kb minus the size of the parental amplicon). Following molecular biological manipulation, the amplicon vector is packaged into virions by either the traditional helper virus-dependent system (5) or recently developed “helper-free” packaging strategies (6 – 8). The noted clinical potential of the HSV amplicon for
1 To whom correspondence should be addressed at Division of Molecular Medicine and Gene Therapy, Center for Aging and Developmental Biology, Box 645, University of Rochester School of Medicine and Dentistry, 601 Elmwood Avenue, Rochester, NY 14642. Fax: 716-442-6646. E-mail: [email protected].
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treatment of neurodegenerative diseases has spurred the efforts of numerous investigators to make safer and more efficacious iterations of this vector system. In recent years, the amplicon system has been modified in a variety of ways, including introduction of new promoters and transgenes, development of helper-free methods of virus packaging, and creation of HSV/AAV and HSV/EBV hybrid forms of the vectors (9, 10). As improvements are made to existing amplicon technologies, it becomes necessary to compare different vector iterations in an equivalent manner. Fair comparisons require the use of equal “titers” of starting virus stocks. Conventional methods of titering retrovirus, adenovirus, and HSV-based vector preparations have relied upon detection of a reporter gene product, enumeration of cells staining “positive,” and titer representation as expression units per unit volume. Recently described dot-blot techniques for titering are rapid methods for enumerating viral particles in prepared supernatants prior to infection (11). However, this approach does not assess vector transduction efficiency and as such could overestimate titer since some genomes may be within noninfectious particles. Development of a method MOLECULAR THERAPY Vol. 1, No. 3, March 2000 Copyright © The American Society of Gene Therapy
METHOD is required to determine the efficiency of viral-mediated gene transfer independent from vector-mediated promoter strength effects. In this report, we describe the development of an unbiased method for titering HSV-1 amplicon vector stocks based upon “real-time” quantitative PCR (12). This new titering assay is reproducible, amenable to high throughput, and useful for titering any amplicon vector preparation. The assay results also suggest that the transcriptional state of the packaged amplicon genome is not uniform in the tested cell types, indicating that this new method of titering may provide insight into the biological events immediately following virus infection.
MATERIALS
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METHODS
Cell culture. Baby hamster kidney (BHK) cells were maintained as described before (13). The NIH-3T3 mouse fibroblast cell line was originally obtained from American Type Culture Collection and maintained in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 units/ml penicillin, and 100 g/ml streptomycin. Vero cells were maintained as described previously (14). The HuH-7 cell line was derived from a human hepatoma and retained differentiated hepatocellular functions (15). HuH-7 cells were also maintained in DMEM plus 10% FBS, 100 units/ml penicillin, and 100 g/ml streptomycin. Amplicon plasmids. The HSVlac amplicon has been described previously (4). The glucocorticoid-regulated pHSVGRE5lac amplicon plasmid was constructed by cloning a 0.96-kb fragment from pGRE5-2 (16) into the pHSVminOriSlac amplicon vector. The cloned fragment contains five copies of tandemly repeated rat tyrosine aminotransferase (TAT) glucocorticoid-responsive elements (GRE) (17) linked to the adenovirus 2 major late promoter (Ad2-MLP), followed by a segment of the rabbit -globin exon 2–intron 2–partial exon 3 (16). pBAC-V2-mediated packaging. On the day prior to transfection, 2 ⫻ 107 BHK cells were seeded in a T-150 flask and incubated overnight at 37°C. The day of transfection, 1.8 ml Opti-MEM (Gibco-BRL, Bethesda, MD), 25 g of pBAC-V2 DNA, and 3.6 g amplicon vector DNA were combined in a sterile polypropylene tube. Seventy microliters of Lipofectamine Plus reagent (Gibco-BRL) was added over a period of 30 s to the DNA mix and allowed to incubate at 22°C for 20 min. In a separate tube, 100 l Lipofectamine (Gibco-BRL) was mixed with 1.8 ml Opti-MEM and also incubated at 22°C for 20 min. Following the incubations, the contents of the two tubes were combined over a period of 30 s and incubated for an additional 20 min at 22°C. During this second incubation, the media in the seeded T-150 flask were removed and replaced with 14 ml Opti-MEM. The transfection mix was added to the flask and allowed to incubate at 37°C for 5 h. The transfection mix was then diluted with an equal volume of DMEM plus 20% FBS, 2% penicillin/streptomycin, and 2 mM hexamethylene bisacetamide (HMBA) and incubated overnight at 34°C. The following day, media were removed and replaced with DMEM plus 10% FBS, 1% penicillin/streptomycin, and 2 mM HMBA. The packaging flask was incubated an additional 3 days before virus was harvested and stored at ⫺80°C until purification. Viral preparations were subsequently thawed, sonicated, clarified by centrifugation, and concentrated by ultracentrifugation through a 30% sucrose cushion. Viral pellets were resuspended in 100 l PBS and stored at ⫺80°C until use. Viral titering. Amplicon titers were determined either by assessing the number of cells expressing a given transduced gene using X-gal histochemistry or by assessing the number of transduced viral genomes using a Perkin–Elmer 7700 quantitative PCR-based method. For expression titers, 5 l of concentrated amplicon stock was incubated with confluent monolayers of NIH 3T3 cells. HSVGRE5lac-infected cells were cultured in media containing 100 nM dexamethasone (Sigma Chemical Co.). Following a 24-h incubation, cells were fixed with 1% glutaraldehyde for 5 min at RT and processed for X-gal histochemistry to detect the lacZ transgene product. Positively stained blue cells were counted, and expression titer was calculated and represented as blue-forming units per milliliter (bfu/ml). MOLECULAR THERAPY Vol. 1, No. 3, March 2000 Copyright © The American Society of Gene Therapy
For transduction titers, infection of confluent monolayers was carried out as described above, but following the overnight incubation, monolayers were processed for total DNA. Briefly, cells were lysed in 100 mM potassium phosphate, pH 7.8, and 0.2% Triton X-100. A 20-l aliquot of this lysate was saved for enzyme activity analysis by Galacto-Lite assay (Tropix, Inc.) and analysis performed according to manufacturer instructions. An equal volume of 2⫻ digestion buffer (0.2 M NaCl, 20 mM Tris–Cl, pH 8, 50 mM EDTA, 0.5% SDS, 0.2 mg/ml proteinase K) was added to the remainder of the lysate and the sample was incubated at 56°C for 4 h. Samples were processed further by one phenol:chloroform extraction, one chloroform extraction, and a final ethanol precipitation. Total DNA was quantitated and 50 ng of DNA was analyzed in a PE7700 quantitative PCR using a designed lacZ-specific primer/probe combination multiplexed with an 18S rRNA-specific primer/probe set. The lacZ probe sequence was 5⬘-ACCCCGTACGTCTTCCCGAGCG-3⬘, the lacZ sense primer sequence was 5⬘-GGGATCTGCCATTGTCAGACAT-3⬘, and the lacZ antisense primer sequence was 5⬘-TGGTGTGGGCCATAATTCAA-3⬘. The 18S rRNA probe sequence was 5⬘-TGCTGGCACCAGACTTGCCCTC-3⬘, the 18S sense primer sequence was 5⬘-CGGCTACCACATCCAAGGAA-3⬘, and the 18S antisense primer sequence was 5⬘-GCTGGAATTACCGCGGCT-3⬘. TaqMan quantitative PCR system. Each 25-l PCR sample contained 2.5 l of purified DNA, 900 nM of each primer, 50 nM of each probe, and 12.5 l of 2⫻ Perkin–Elmer Master Mix. Following a 2-min 50°C incubation and a 2-min 95°C denaturation step, the samples were subjected to 40 cycles of 95°C for 15 s and 60°C for 1 min. Fluorescent intensity of each sample was detected automatically during the cycles by the Perkin–Elmer Applied Biosystem Sequence Detector 7700 machine. Each PCR run included the following: no-template control samples, positive control samples consisting of either amplicon DNA (for lacZ) or cellular genomic DNA (for 18S rRNA), and two standard curve dilution series (for lacZ and 18S). Following the PCR run, real-time data were analyzed using Perkin–Elmer Sequence Detector Software version 1.6.3 and the aforementioned standard curves. Precise quantities of starting template were determined for each titering sample and results were expressed as numbers of vector genomes per milliliter of original viral stock.
RESULTS The comparison of different HSV amplicon vectors both in titer and in expression has been controversial. Conventional titering of packaged HSV-1 amplicons using expression assays precludes accurate comparison of two amplicons with different transcriptional control elements. Promoter strength and titering cell line selection greatly influence titers obtained using expression units as the measure. To circumvent this problem, we developed a method of HSV amplicon titering that is independent of promoter strength, but provides information regarding transduction efficiency of the viral vector stock. Two viruses were used in this study: HSVlac, a previously described amplicon vector that possesses the HSV IE4/5 promoter for constitutive expression of Escherichia coli -galactosidase (Fig. 1A) (4); and HSVGRE5lac, an amplicon that contains five tandem copies of the rat TAT glucocorticoid response element (GRE) and minimal promoter (Ad-MLP) to regulate the expression of -galactosidase in response to the addition of dexamethasone (Fig. 1B). We had shown previously that this repetitive transcriptional element could direct glucocorticoid-regulated expression of human growth hormone in the context of an amplicon (13). In experiments using both viruses it was difficult to ascertain the correct titer to utilize for calculation of transduction volumes as the resident promoter in each virus
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METHOD
FIG. 1. Schematic representation of HSV amplicon vectors used in this study.
vector dictated the apparent expression titer. As a result, vector-mediated transgene expression differences could not be accurately assessed. We employed the Perkin– Elmer TaqMan system to develop a high-throughput, sensitive method that we anticipated would determine numbers of vector genomes transduced into infected cells (12, 18). The TaqMan system is a real-time quantitative PCR method that measures generated PCR product accumulation using a dual-labeled fluorogenic probe, shown to possess a range of detection that encompasses at least five orders of magnitude. The two amplicon vectors (HSVlac and HSVGRE5lac) were packaged into virions according to described methods for helper virus-free packaging (6). Five microliters of virus vector stocks was used to infect confluent monolayers of four cell lines of varying origin commonly utilized for titering HSV amplicon stocks: NIH 3T3, Vero, BHK, and HuH-7 cells (13–15). Infected monolayers were analyzed 24 h later by three different assays: X-gal histochemistry for visual determination and enumeration of -galactosidase-expressing cells, quantitative PCR for assessment of amplicon-specific DNA content, and the Galacto-Lite assay for determination of -galactosidase enzyme activity. Titers derived from X-gal histochemistry were calculated as bfu/ml (expression titer), while numbers of amplicon genomes determined by quantitative
PCR were expressed as transduction units per milliliter (TU/ml; transduction titer). To determine the effect that utilizing the two different titering methods would have on experimental outcome and data interpretation, -galactosidase enzyme activity was normalized per titering unit for each amplicon on the tested cell lines. The expression titers when assayed on NIH 3T3 cells were disparate (2 ⫻ 107 bfu/ml for HSVlac and 4 ⫻ 105 bfu/ml for HSVGRE5lac; Table 1). However, when transduction titers were assessed, the titers were nearly identical, indicating that both amplicon viruses could equivalently deliver vector genome to NIH 3T3 cells. Interestingly, the transduction titers were approximately 4-fold higher (for HSVlac) and 200-fold higher (for HSVGRE5lac) than respective calculated expression titers. To ensure that potential virions bound to the cell surface would not be counted as transducing units, a subset of samples was treated with trypsin prior to cell lysis. Transduction titers were found to be unaffected by the trypsin treatment (data not shown). When -galactosidase activity for each virus-transduced sample was normalized to bfu, the HSVGRE5lac amplicon appeared to express 16fold higher activity per virion than did HSVlac. Conversely, when -galactosidase activity was normalized using transduction titering results, HSVlac exhibited a 2-fold higher enzyme activity per virion than
TABLE 1 Summary of Titering Assay Results Cell line
Amplicon
Avg bfu/ml
Avg TU/ml
-gal activity/bfua
-gal activity/TUa
NIH 3T3
HSVlac HSVGRE5lac HSVlac HSVGRE5lac HSVlac HSVGRE5lac HSVlac HSVGRE5lac
2.09 ⫻ 107 4.04 ⫻ 105 8.07 ⫻ 106 1.88 ⫻ 105 2.61 ⫻ 106 2.55 ⫻ 105 4.01 ⫻ 106 1.08 ⫻ 106
1.51 ⫻ 108 1.22 ⫻ 108 1.11 ⫻ 108 1.14 ⫻ 108 2.64 ⫻ 106 2.42 ⫻ 106 6.80 ⫻ 106 6.54 ⫻ 106
11.75 161.24 8.68 226.24 163.85 249.80 229.80 88.89
54.65 28.24 22.94 20.68 582.58 99.98 633.50 289.29
Vero BHK HuH-7
a -Galactosidase activity as measured by Galacto-Lite assay divided by either expression titer value or transduction titer value and multiplied by 104.
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MOLECULAR THERAPY Vol. 1, No. 3, March 2000 Copyright © The American Society of Gene Therapy
METHOD HSVGRE5lac. This latter result was consistent with the known transcriptional activities of both promoters, where the HSV IE4/5 promoter drives higher expression than the GRE5-AdMLP promoter unit (13). Similar results were obtained when these viruses were titered on Vero cells: the expression titer of HSVlac was 40-fold higher than that of HSVGRE5lac, whereas the transduction titers of each of the packaged amplicon vectors were nearly identical (2 ⫻ 108 TU/ml; Table 1). As in the case of the NIH 3T3 cell line, the large differences in apparent titers on Vero cells were reflected in the normalized -galactosidase enzyme activity values. Helper virus-free HSVlac and HSVGRE5lac stocks were further analyzed on BHK cells and the human hepatomaderived cell line, HuH-7, using expression- or transduction-based methods of virus quantitation. Expression titers again were different between the two viruses on each cell line, where HSVlac possessed an apparent 10- to 12fold higher titer than HSVGRE5lac (Table 1). The transduction titers of the two amplicons were nearly identical but the values were approximately 10-fold lower than those observed on either NIH 3T3 or Vero cell lines. In contrast to results obtained on NIH 3T3 and Vero cells, the expression and transduction titers on BHK and HuH-7 cells for HSVlac were nearly identical. This result suggests that there is a fundamental biological difference between BHK and HuH-7 cells and the previously studied cell lines in terms of capacity for virus uptake and support of transgene expression. This was also evident from examination of normalized -galactosidase activity per unit virion. Normalized activity values were 50- to 100-fold greater for HSVlac on BHK and HuH-7 cells than with the same virus on either NIH 3T3 or Vero cells when transduction titer was used as the method of virus quantitation.
DISCUSSION The transduction titering method, based upon TaqMan technology, allows for a better estimation of infectious HSV virion number than conventionally used techniques. We believe that transduction titering should be adopted as a standard for enumeration of vector numbers prior to comparative studies involving vectors possessing different promoters or cis elements. During the preparation of this paper, Ryncarz and colleagues published their method of using real-time quantitative PCR to detect wild-type HSV genomic DNA in clinical isolates (19). They successfully demonstrated that this sensitive and highthroughput technique can serve as a powerful tool in clinical investigations. The technique we employed in this study determined the numbers of amplicon genomes that were transferred to the tested cell lines, as well as revealed information concerning cell type-specific infectivity and amplicon transcriptional activity following infection. The relative -galactosidase activity per unit HSVlac virion in BHK and HuH-7 cells was approximately 10-fold higher than in 3T3 or Vero cells. This could reflect a more active transcriptional state of the delivered amplicon genome in BHK and MOLECULAR THERAPY Vol. 1, No. 3, March 2000 Copyright © The American Society of Gene Therapy
HuH-7 cells. Our laboratory has evidence to suggest that cellular modulators of chromatin structure play a role in dictating extent and duration of vector-mediated expression (Harvey and Federoff, unpublished observations). The relative levels of such cellular modulators in different cell types could be responsible for the disparate values of expression titers and transduction titers (i.e., NIH 3T3 and Vero). The higher transduction titer in these cell types may also reflect viral genome sequestration in subcellular compartments or may be due to higher sensitivity of the real-time quantitative PCR method as opposed to X-gal histochemistry. Different doubling rates of infected cells may also explain the disparity between expression and transduction titers. A slowly dividing cell infected with amplicon virus would likely be scored as a single X-gal-positive cell. However, a rapidly dividing cell may distribute cytoplasmic -galactosidase into two daughter cells, thus increasing the likelihood of scoring a single infection event as two X-gal-positive cells and overestimating expression titers. In fact, assessments of cell growth kinetics indicate that BHK and HuH-7 cells maintain a higher and more prolonged proliferative state than NIH 3T3 and Vero cells during the time course of titering (data not shown). These considerations likely explain why transduction and expression titers appeared different in cell types like NIH 3T3 and Vero, while appearing similar in BHK and HuH-7 cells. Transduction titer is independent of an infected cell’s proliferative state, as this method determines total virion genomes recovered from an infected cell monolayer. The IE4/5 promoter of HSV exhibits higher lacZ expression than the GRE5-AdMLP transcription unit, an observation expected because of the known transactivational properties of specific HSV nucleocapsid proteins. The strong HSV transcriptional activator protein, VP16, is released into the cell upon infection and interacts with at least two cellular proteins, HCF and Oct-1. An initial interaction occurs between VP-16 and HCF (20, 21), the latter affording stability, leading this complex to interact with Oct-1 (22). The newly formed multimeric complex binds and activates at the TAATGARAT elements found within the immediate early gene promoters of HSV. In numerous experiments performed in our laboratory, the IE4/5 promoter of HSV-1 mediates high expression levels of transgenes but its duration of expression is shorter than exhibited by cellular promoters (23). The glucocorticoid response elements cloned into the pHSVGRE5lac amplicon construct were derived from the rat TAT gene promoter. This transcription unit was previously used in the amplicon context to impart glucocorticoid-responsive expression of human growth hormone (13). The glucocorticoid receptor (GR), upon binding of the synthetic glucocorticoid, dexamethasone, undergoes a hyperphosphorylation event that results in conformational changes and binding to GRE [reviewed by (24)]. We have previously shown that the human hepatoma cell line HuH-7 supports glucocorticoid-inducible expression (13), whereas other cell types may be less capable of sup-
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METHOD TABLE 2 Proposed Standard Terminology for HSV Vector Titering Titering method
Component assayed
Titering units/vola
Expression-based (i.e., X-gal histochem, GFP fluorescence) Quantitative PCR Slot-blot hybridization ELISA Plaque assay
Transcriptional activity of transferred amplicon genome Postinfection amplicon genome content Preinfection amplicon genome content Preinfection amplicon particle number Helper virus
EU/ml TU/ml GU/ml Particles/ml pfu/ml
a EU/ml, expression units per milliliter; TU/ml, transducing units per milliliter; GU/ml, genome units per milliliter; pfu/ml, plaque-forming units per milliliter.
porting this response, presumably due to the low levels of glucocorticoid receptor expression. Promoter-mediated expression kinetics also likely play a role in the expression titer differences observed between the HSVlac and HSVGRE5lac. -Galactosidase expression from the HSVGRE5lac amplicon peaks at day 4, while expression from the IE4/5 promoter-driven HSVlac amplicon peaks at day 2 (data not shown). This may explain why expression titers analyzed 24 h following infection are so disparate between the two amplicon construct preparations. Therefore, arbitrary selection of assay time points to determine expression titers can greatly influence titer values. In contrast, transduction titering provides the number of viral genomes that gain access to the cell monolayer and is unaffected by promoter activity or expression kinetics. BHK and HuH-7 cells appeared to be less efficiently infected than either NIH 3T3 or Vero cells, as evident from lower HSVlac transduction titers on BHK and HuH-7 cells. This could reflect a reduced number of HSV receptors on the host cellular membrane. HSV particles enter host cells via interactions of their surface glycoproteins gB, gC, and gD with host-encoded receptors, including herpes entry mediators B and C (HveB and HveC) and cell-surface heparin sulfate proteoglycans (25). HveB and HveC are ubiquitously expressed, with HveC expression highest in cells of the nervous system (26). Quantitative measurement of HSV receptors on the surface of host cells will be required to definitively address this issue. Genome transfer titering, as it was adopted here for HSV amplicon vectors, can allow an investigator to rapidly determine the titer of any amplicon vector, as well as to gain insight into vector-related issues regarding promoter activity and virus infection efficiency. This method represents advancement to HSV amplicon-based experimentation. It is appreciated that some amplicon studies may not require equivalent transduction titers and therefore recommend the adoption of a conventional terminology regarding HSV vector titers (Table 2). Titers derived from a coupled infection and expression assay, what we have termed as expression titering in this paper, should be represented as expression units (EU) per unit volume. Titering data obtained via real-time quantitative PCR should be expressed as TU per unit volume. Other investigators working with HSV amplicon vectors are de-
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veloping methods for enumerating viral particles. In this instance, titering data should be represented as particles per unit volume. For conventional helper virus-based packaging, representation of helper virus titers should remain as plaque-forming units (pfu) per unit volume. Acceptance of conventional amplicon terminology will facilitate interpretation and understanding of the HSV amplicon literature. ACKNOWLEDGMENTS The authors thank Hui Huang and Brandon Harvey for their advice in method development and interpretation of experimental results. This work was supported by an AFAR Research Grant to W.J.B. and NIH Grant R01 NS364201 to H.J.F.
REFERENCES 1 Federoff, H. J., Geschwind, M. D., Geller, A. I., and Kessler, J. A. (1992). Expression of nerve growth factor in vivo, from a defective HSV-1 vector prevents effects of axotomy on sympathetic ganglia. Proc. Natl. Acad. Sci. USA 89: 1636 –1640. 2 Frenkel, N. (1983). Defective interfering herpesviruses. In The Human Herpesviruses—An Interdisciplinary Prospective (A. Nahmias, W. Dowdle, and R. Scchinazy, Eds.), pp. 91–120, Elsevier–North Holland, New York. 3 Spaete, R. R., and Frenkel, N. (1982). The herpes simplex virus amplicon: A new eucaryotic defective-virus cloning-amplifying vector. Cell 30: 305–310. 4 Geller, A. I., and Breakefield, X. O. (1988). A defective HSV-1 vector expresses Escherichia coli -galactosidase in cultured peripheral neurons. Science 241: 1667–1669. 5 Federoff, H. J. (1997). Growth of replication defective herpes virus amplicon vectors and their use for gene transfer. In Cells: A Laboratory Manual (D. L. Spector, R. D. Goldman, and L. A. Leinwand, Eds.), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 6 Stavropoulos, T. A., and Strathdee, C. A. (1998). An enhanced packaging system for helper-dependent herpes simplex virus vectors. J. Virol. 72: 7137–7143. 7 Fraefel, C., Song, S., Lim, F., Lang, P., Yu, L., Wang, Y., Wild, P., and Geller, A. I. (1996). Helper virus-free transfer of herpes simplex virus type 1 plasmid vectors into neural cells. J. Virol. 70: 7190 –7197. 8 Saeki, Y., Ichikawa, T., Saeki, A., Chiocca, E. A., Tobler, K., Ackermann, M., Breakefield, X. O., and Fraefel, C. (1998). Herpes simplex virus type 1 DNA amplified as bacterial artificial chromosome in Escherichia coli: Rescue of replication-competent virus progeny and packaging of amplicon vectors. Hum. Gene Ther. 9: 2787–2794. 9 Wang, S., and Vos, J.-M. (1996). A hybrid herpesvirus infectious vector based on Epstein–Barr virus and herpes simplex virus type 1 for gene transfer into human cells in vitro and in vivo. J. Virol. 70: 8422– 8430. 10 Johnston, K. M., Jacoby, D., Pechan, P. A., Fraefel, C., Borghesani, P., Schuback, D., Dunn, R. J., Smith, F. I., and Breakefiled, X. O. (1997). HSV/AAV hybrid amplicon vectors extend transgene expression in human glioma cells. Hum. Gene Ther. 8: 359 –370. 11 Nelson, D. M., Wahlfors, J. J., Chen, L., Onodera, M., and Morgan, R. A. (1998). Characterization of diverse viral vector preparations, using a simple and rapid whole-virion dot-blot method. Hum. Gene Ther. 9: 2401–2405. 12 Holland, P. M., Abramson, R. D., Watson, R., and Gelfand, D. H. (1991). Detection of specific polymerase chain reaction product by utilizing the 5⬘ 3 3⬘ exonuclease activity of Thermus aquaticus DNA polymerase. Proc. Natl. Acad. Sci. USA 88: 7276 –7280. 13 Lu, B., and Federoff, H. J. (1995). Herpes simplex virus type 1 amplicon vectors with glucocorticoid-inducible gene expression. Hum. Gene Ther. 6: 421– 430. 14 DeLuca, N. A., McCarthy, A. M., and Schaffer, P. A. (1985). Isolation and characterization of deletion mutants of herpes simplex virus type 1 in the gene encoding immediateearly regulatory protein ICP4. J. Virol. 56: 558 –570. 15 Nakabayashi, H., Taketa, K., Miyano, K., Yamane, T., and Sato, J. (1982). Growth of
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METHOD human hepatoma cell lines with differentiated functions in chemically defined medium. Cancer Res. 42: 3858 –3863. 16 Mader, S., and White, J. H. (1993). A steroid-inducible promoter for the controlled overexpression of cloned genes in eukaryotic cells. Proc. Natl. Acad. Sci. USA 90: 5603–5607. 17 Jantzen, H.-M., Strahle, U., Gloss, B., Stewart, F., Schmid, W., Boshart, M., Miksicek, R., and Schultz, G. (1987). Cooperativity of glucocorticoid response elements located far upstream of the tyrosine aminotransferase gene. Cell 49: 29 –38. 18 Heid, C., Stevens, J., Livak, K., and Williams, P. (1996). Real time quantitative PCR. Genome Res. 6: 986 –994. 19 Ryncarz, A., Goddard, J., Wald, A., Huang, M., Roizman, B., and Corey, L. (1999). Development of a high-throughput quantitative assay for detecting herpes simplex virus DNA in clinical samples. J. Clin. Microbiol. 37: 1941–1947. 20 O’Hare, P., and Goding, C. R. (1988). Herpes simplex virus regulatory elements and the immunoglobulin octamer domain bind a common factor and are both targets for virion transactivation. Cell 52: 435– 445.
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21 Wilson, A. C., LaMarco, K., Peterson, M. G., and Herr, W. (1993). The VP16 accessory protein HCF is a family of polypeptides processed from a large precursor protein. Cell 74: 115–125. 22 Dent, C. L., and Latchman, D. S. (1991). The overlapping octamer/TAATGARAT motif is a high-affinity binding site for the cellular transcription factors Oct-1 and Oct-2. Biochem. J. 277: 541–545. 23 Jin, B. K., Belloni, M., Conti, B., Federoff, H. J., Starr, R., Son, J. H., Baker, H., and Joh, T. H. (1996). Prolonged in vivo gene expression driven by a tyrosine hydroxylase promoter in a defective herpes simplex virus amplicon vector. Hum. Gene Ther. 7: 2015–2024. 24 Gronemeyer, H. (1992). Control of transcription activation by steroid hormone receptors. FASEB J. 6: 2524 –2529. 25 Haywood, A. M. (1994). Virus receptors: Binding, adhesion strengthening, and changes in viral structure. J. Virol. 68: 1–5. 26 Spear, P. G., Shieh, M. T., Herold, B. C., WuDunn, D., and Koshy, T. I. (1992). Heparan sulfate glycosaminoglycans as primary cell surface receptors for herpes simplex virus. Adv. Exp. Med. Biol. 313: 341–353.
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doi:10.1006/mthe.2000.0039, available online at http://www.idealibrary.com on IDEAL
Discordance between Expression and Genome Transfer Titering of HSV Amplicon Vectors: Recommendation for Standardized Enumeration W. J. Bowers, D. F. Howard, and H. J. Federoff1 Department of Neurology and Center for Aging and Developmental Biology, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642 Received for publication November 29, 1999, and accepted in revised form February 10, 2000
Herpes simplex virus-derived amplicon vectors are well suited to the development of gene-based therapy for neurodegenerative diseases. The plasmid-based amplicon vector system allows for facile introduction of transcription units, possesses the potential for carrying gene inserts up to approximately 130 kb in length, and can be packaged into infectious virus devoid of contaminating cytotoxic helper virus. For accurate assessments to be made regarding vector comparison and improvements in vector design, a standard for titering prepared virus stocks must be established. At present, packaged amplicon vectors are routinely titered using reporter gene expression units to quantitate numbers of infectious amplicon virions. The strength of the promoter, sensitivity of detection of the gene product, and choice of titering cell type can greatly influence the apparent numbers of infectious virus particles. This is especially evident when comparisons are made between two amplicon vectors that possess different promoters. To this end, we have developed a new titering method based on a real-time quantitative PCR technique that allows for enumeration of transducing particles. This new approach ensures that amplicon comparison experiments are initiated with equivalent transduction units, thus allowing for a fair assessment of expression and therapeutic efficacy differences. Key Words: herpes simplex virus; amplicon; quantitative PCR; gene therapy; titering.
INTRODUCTION The plasmid-based HSV-1 amplicon vector is a highly versatile gene delivery platform, owing to its facility for molecular genetic manipulation (1– 4). The backbone of the amplicon consists primarily of a eukaryotic expression plasmid modified by the addition of an HSV origin of replication (ori) and cleavage/packaging sequence (“a” sequence). The amplicon is amenable to carrying multiple heterologous transcription units of various sizes (the theoretical limit is 150 kb minus the size of the parental amplicon). Following molecular biological manipulation, the amplicon vector is packaged into virions by either the traditional helper virus-dependent system (5) or recently developed “helper-free” packaging strategies (6 – 8). The noted clinical potential of the HSV amplicon for
1 To whom correspondence should be addressed at Division of Molecular Medicine and Gene Therapy, Center for Aging and Developmental Biology, Box 645, University of Rochester School of Medicine and Dentistry, 601 Elmwood Avenue, Rochester, NY 14642. Fax: 716-442-6646. E-mail: [email protected].
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treatment of neurodegenerative diseases has spurred the efforts of numerous investigators to make safer and more efficacious iterations of this vector system. In recent years, the amplicon system has been modified in a variety of ways, including introduction of new promoters and transgenes, development of helper-free methods of virus packaging, and creation of HSV/AAV and HSV/EBV hybrid forms of the vectors (9, 10). As improvements are made to existing amplicon technologies, it becomes necessary to compare different vector iterations in an equivalent manner. Fair comparisons require the use of equal “titers” of starting virus stocks. Conventional methods of titering retrovirus, adenovirus, and HSV-based vector preparations have relied upon detection of a reporter gene product, enumeration of cells staining “positive,” and titer representation as expression units per unit volume. Recently described dot-blot techniques for titering are rapid methods for enumerating viral particles in prepared supernatants prior to infection (11). However, this approach does not assess vector transduction efficiency and as such could overestimate titer since some genomes may be within noninfectious particles. Development of a method MOLECULAR THERAPY Vol. 1, No. 3, March 2000 Copyright © The American Society of Gene Therapy
METHOD is required to determine the efficiency of viral-mediated gene transfer independent from vector-mediated promoter strength effects. In this report, we describe the development of an unbiased method for titering HSV-1 amplicon vector stocks based upon “real-time” quantitative PCR (12). This new titering assay is reproducible, amenable to high throughput, and useful for titering any amplicon vector preparation. The assay results also suggest that the transcriptional state of the packaged amplicon genome is not uniform in the tested cell types, indicating that this new method of titering may provide insight into the biological events immediately following virus infection.
MATERIALS
AND
METHODS
Cell culture. Baby hamster kidney (BHK) cells were maintained as described before (13). The NIH-3T3 mouse fibroblast cell line was originally obtained from American Type Culture Collection and maintained in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 units/ml penicillin, and 100 g/ml streptomycin. Vero cells were maintained as described previously (14). The HuH-7 cell line was derived from a human hepatoma and retained differentiated hepatocellular functions (15). HuH-7 cells were also maintained in DMEM plus 10% FBS, 100 units/ml penicillin, and 100 g/ml streptomycin. Amplicon plasmids. The HSVlac amplicon has been described previously (4). The glucocorticoid-regulated pHSVGRE5lac amplicon plasmid was constructed by cloning a 0.96-kb fragment from pGRE5-2 (16) into the pHSVminOriSlac amplicon vector. The cloned fragment contains five copies of tandemly repeated rat tyrosine aminotransferase (TAT) glucocorticoid-responsive elements (GRE) (17) linked to the adenovirus 2 major late promoter (Ad2-MLP), followed by a segment of the rabbit -globin exon 2–intron 2–partial exon 3 (16). pBAC-V2-mediated packaging. On the day prior to transfection, 2 ⫻ 107 BHK cells were seeded in a T-150 flask and incubated overnight at 37°C. The day of transfection, 1.8 ml Opti-MEM (Gibco-BRL, Bethesda, MD), 25 g of pBAC-V2 DNA, and 3.6 g amplicon vector DNA were combined in a sterile polypropylene tube. Seventy microliters of Lipofectamine Plus reagent (Gibco-BRL) was added over a period of 30 s to the DNA mix and allowed to incubate at 22°C for 20 min. In a separate tube, 100 l Lipofectamine (Gibco-BRL) was mixed with 1.8 ml Opti-MEM and also incubated at 22°C for 20 min. Following the incubations, the contents of the two tubes were combined over a period of 30 s and incubated for an additional 20 min at 22°C. During this second incubation, the media in the seeded T-150 flask were removed and replaced with 14 ml Opti-MEM. The transfection mix was added to the flask and allowed to incubate at 37°C for 5 h. The transfection mix was then diluted with an equal volume of DMEM plus 20% FBS, 2% penicillin/streptomycin, and 2 mM hexamethylene bisacetamide (HMBA) and incubated overnight at 34°C. The following day, media were removed and replaced with DMEM plus 10% FBS, 1% penicillin/streptomycin, and 2 mM HMBA. The packaging flask was incubated an additional 3 days before virus was harvested and stored at ⫺80°C until purification. Viral preparations were subsequently thawed, sonicated, clarified by centrifugation, and concentrated by ultracentrifugation through a 30% sucrose cushion. Viral pellets were resuspended in 100 l PBS and stored at ⫺80°C until use. Viral titering. Amplicon titers were determined either by assessing the number of cells expressing a given transduced gene using X-gal histochemistry or by assessing the number of transduced viral genomes using a Perkin–Elmer 7700 quantitative PCR-based method. For expression titers, 5 l of concentrated amplicon stock was incubated with confluent monolayers of NIH 3T3 cells. HSVGRE5lac-infected cells were cultured in media containing 100 nM dexamethasone (Sigma Chemical Co.). Following a 24-h incubation, cells were fixed with 1% glutaraldehyde for 5 min at RT and processed for X-gal histochemistry to detect the lacZ transgene product. Positively stained blue cells were counted, and expression titer was calculated and represented as blue-forming units per milliliter (bfu/ml). MOLECULAR THERAPY Vol. 1, No. 3, March 2000 Copyright © The American Society of Gene Therapy
For transduction titers, infection of confluent monolayers was carried out as described above, but following the overnight incubation, monolayers were processed for total DNA. Briefly, cells were lysed in 100 mM potassium phosphate, pH 7.8, and 0.2% Triton X-100. A 20-l aliquot of this lysate was saved for enzyme activity analysis by Galacto-Lite assay (Tropix, Inc.) and analysis performed according to manufacturer instructions. An equal volume of 2⫻ digestion buffer (0.2 M NaCl, 20 mM Tris–Cl, pH 8, 50 mM EDTA, 0.5% SDS, 0.2 mg/ml proteinase K) was added to the remainder of the lysate and the sample was incubated at 56°C for 4 h. Samples were processed further by one phenol:chloroform extraction, one chloroform extraction, and a final ethanol precipitation. Total DNA was quantitated and 50 ng of DNA was analyzed in a PE7700 quantitative PCR using a designed lacZ-specific primer/probe combination multiplexed with an 18S rRNA-specific primer/probe set. The lacZ probe sequence was 5⬘-ACCCCGTACGTCTTCCCGAGCG-3⬘, the lacZ sense primer sequence was 5⬘-GGGATCTGCCATTGTCAGACAT-3⬘, and the lacZ antisense primer sequence was 5⬘-TGGTGTGGGCCATAATTCAA-3⬘. The 18S rRNA probe sequence was 5⬘-TGCTGGCACCAGACTTGCCCTC-3⬘, the 18S sense primer sequence was 5⬘-CGGCTACCACATCCAAGGAA-3⬘, and the 18S antisense primer sequence was 5⬘-GCTGGAATTACCGCGGCT-3⬘. TaqMan quantitative PCR system. Each 25-l PCR sample contained 2.5 l of purified DNA, 900 nM of each primer, 50 nM of each probe, and 12.5 l of 2⫻ Perkin–Elmer Master Mix. Following a 2-min 50°C incubation and a 2-min 95°C denaturation step, the samples were subjected to 40 cycles of 95°C for 15 s and 60°C for 1 min. Fluorescent intensity of each sample was detected automatically during the cycles by the Perkin–Elmer Applied Biosystem Sequence Detector 7700 machine. Each PCR run included the following: no-template control samples, positive control samples consisting of either amplicon DNA (for lacZ) or cellular genomic DNA (for 18S rRNA), and two standard curve dilution series (for lacZ and 18S). Following the PCR run, real-time data were analyzed using Perkin–Elmer Sequence Detector Software version 1.6.3 and the aforementioned standard curves. Precise quantities of starting template were determined for each titering sample and results were expressed as numbers of vector genomes per milliliter of original viral stock.
RESULTS The comparison of different HSV amplicon vectors both in titer and in expression has been controversial. Conventional titering of packaged HSV-1 amplicons using expression assays precludes accurate comparison of two amplicons with different transcriptional control elements. Promoter strength and titering cell line selection greatly influence titers obtained using expression units as the measure. To circumvent this problem, we developed a method of HSV amplicon titering that is independent of promoter strength, but provides information regarding transduction efficiency of the viral vector stock. Two viruses were used in this study: HSVlac, a previously described amplicon vector that possesses the HSV IE4/5 promoter for constitutive expression of Escherichia coli -galactosidase (Fig. 1A) (4); and HSVGRE5lac, an amplicon that contains five tandem copies of the rat TAT glucocorticoid response element (GRE) and minimal promoter (Ad-MLP) to regulate the expression of -galactosidase in response to the addition of dexamethasone (Fig. 1B). We had shown previously that this repetitive transcriptional element could direct glucocorticoid-regulated expression of human growth hormone in the context of an amplicon (13). In experiments using both viruses it was difficult to ascertain the correct titer to utilize for calculation of transduction volumes as the resident promoter in each virus
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METHOD
FIG. 1. Schematic representation of HSV amplicon vectors used in this study.
vector dictated the apparent expression titer. As a result, vector-mediated transgene expression differences could not be accurately assessed. We employed the Perkin– Elmer TaqMan system to develop a high-throughput, sensitive method that we anticipated would determine numbers of vector genomes transduced into infected cells (12, 18). The TaqMan system is a real-time quantitative PCR method that measures generated PCR product accumulation using a dual-labeled fluorogenic probe, shown to possess a range of detection that encompasses at least five orders of magnitude. The two amplicon vectors (HSVlac and HSVGRE5lac) were packaged into virions according to described methods for helper virus-free packaging (6). Five microliters of virus vector stocks was used to infect confluent monolayers of four cell lines of varying origin commonly utilized for titering HSV amplicon stocks: NIH 3T3, Vero, BHK, and HuH-7 cells (13–15). Infected monolayers were analyzed 24 h later by three different assays: X-gal histochemistry for visual determination and enumeration of -galactosidase-expressing cells, quantitative PCR for assessment of amplicon-specific DNA content, and the Galacto-Lite assay for determination of -galactosidase enzyme activity. Titers derived from X-gal histochemistry were calculated as bfu/ml (expression titer), while numbers of amplicon genomes determined by quantitative
PCR were expressed as transduction units per milliliter (TU/ml; transduction titer). To determine the effect that utilizing the two different titering methods would have on experimental outcome and data interpretation, -galactosidase enzyme activity was normalized per titering unit for each amplicon on the tested cell lines. The expression titers when assayed on NIH 3T3 cells were disparate (2 ⫻ 107 bfu/ml for HSVlac and 4 ⫻ 105 bfu/ml for HSVGRE5lac; Table 1). However, when transduction titers were assessed, the titers were nearly identical, indicating that both amplicon viruses could equivalently deliver vector genome to NIH 3T3 cells. Interestingly, the transduction titers were approximately 4-fold higher (for HSVlac) and 200-fold higher (for HSVGRE5lac) than respective calculated expression titers. To ensure that potential virions bound to the cell surface would not be counted as transducing units, a subset of samples was treated with trypsin prior to cell lysis. Transduction titers were found to be unaffected by the trypsin treatment (data not shown). When -galactosidase activity for each virus-transduced sample was normalized to bfu, the HSVGRE5lac amplicon appeared to express 16fold higher activity per virion than did HSVlac. Conversely, when -galactosidase activity was normalized using transduction titering results, HSVlac exhibited a 2-fold higher enzyme activity per virion than
TABLE 1 Summary of Titering Assay Results Cell line
Amplicon
Avg bfu/ml
Avg TU/ml
-gal activity/bfua
-gal activity/TUa
NIH 3T3
HSVlac HSVGRE5lac HSVlac HSVGRE5lac HSVlac HSVGRE5lac HSVlac HSVGRE5lac
2.09 ⫻ 107 4.04 ⫻ 105 8.07 ⫻ 106 1.88 ⫻ 105 2.61 ⫻ 106 2.55 ⫻ 105 4.01 ⫻ 106 1.08 ⫻ 106
1.51 ⫻ 108 1.22 ⫻ 108 1.11 ⫻ 108 1.14 ⫻ 108 2.64 ⫻ 106 2.42 ⫻ 106 6.80 ⫻ 106 6.54 ⫻ 106
11.75 161.24 8.68 226.24 163.85 249.80 229.80 88.89
54.65 28.24 22.94 20.68 582.58 99.98 633.50 289.29
Vero BHK HuH-7
a -Galactosidase activity as measured by Galacto-Lite assay divided by either expression titer value or transduction titer value and multiplied by 104.
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MOLECULAR THERAPY Vol. 1, No. 3, March 2000 Copyright © The American Society of Gene Therapy
METHOD HSVGRE5lac. This latter result was consistent with the known transcriptional activities of both promoters, where the HSV IE4/5 promoter drives higher expression than the GRE5-AdMLP promoter unit (13). Similar results were obtained when these viruses were titered on Vero cells: the expression titer of HSVlac was 40-fold higher than that of HSVGRE5lac, whereas the transduction titers of each of the packaged amplicon vectors were nearly identical (2 ⫻ 108 TU/ml; Table 1). As in the case of the NIH 3T3 cell line, the large differences in apparent titers on Vero cells were reflected in the normalized -galactosidase enzyme activity values. Helper virus-free HSVlac and HSVGRE5lac stocks were further analyzed on BHK cells and the human hepatomaderived cell line, HuH-7, using expression- or transduction-based methods of virus quantitation. Expression titers again were different between the two viruses on each cell line, where HSVlac possessed an apparent 10- to 12fold higher titer than HSVGRE5lac (Table 1). The transduction titers of the two amplicons were nearly identical but the values were approximately 10-fold lower than those observed on either NIH 3T3 or Vero cell lines. In contrast to results obtained on NIH 3T3 and Vero cells, the expression and transduction titers on BHK and HuH-7 cells for HSVlac were nearly identical. This result suggests that there is a fundamental biological difference between BHK and HuH-7 cells and the previously studied cell lines in terms of capacity for virus uptake and support of transgene expression. This was also evident from examination of normalized -galactosidase activity per unit virion. Normalized activity values were 50- to 100-fold greater for HSVlac on BHK and HuH-7 cells than with the same virus on either NIH 3T3 or Vero cells when transduction titer was used as the method of virus quantitation.
DISCUSSION The transduction titering method, based upon TaqMan technology, allows for a better estimation of infectious HSV virion number than conventionally used techniques. We believe that transduction titering should be adopted as a standard for enumeration of vector numbers prior to comparative studies involving vectors possessing different promoters or cis elements. During the preparation of this paper, Ryncarz and colleagues published their method of using real-time quantitative PCR to detect wild-type HSV genomic DNA in clinical isolates (19). They successfully demonstrated that this sensitive and highthroughput technique can serve as a powerful tool in clinical investigations. The technique we employed in this study determined the numbers of amplicon genomes that were transferred to the tested cell lines, as well as revealed information concerning cell type-specific infectivity and amplicon transcriptional activity following infection. The relative -galactosidase activity per unit HSVlac virion in BHK and HuH-7 cells was approximately 10-fold higher than in 3T3 or Vero cells. This could reflect a more active transcriptional state of the delivered amplicon genome in BHK and MOLECULAR THERAPY Vol. 1, No. 3, March 2000 Copyright © The American Society of Gene Therapy
HuH-7 cells. Our laboratory has evidence to suggest that cellular modulators of chromatin structure play a role in dictating extent and duration of vector-mediated expression (Harvey and Federoff, unpublished observations). The relative levels of such cellular modulators in different cell types could be responsible for the disparate values of expression titers and transduction titers (i.e., NIH 3T3 and Vero). The higher transduction titer in these cell types may also reflect viral genome sequestration in subcellular compartments or may be due to higher sensitivity of the real-time quantitative PCR method as opposed to X-gal histochemistry. Different doubling rates of infected cells may also explain the disparity between expression and transduction titers. A slowly dividing cell infected with amplicon virus would likely be scored as a single X-gal-positive cell. However, a rapidly dividing cell may distribute cytoplasmic -galactosidase into two daughter cells, thus increasing the likelihood of scoring a single infection event as two X-gal-positive cells and overestimating expression titers. In fact, assessments of cell growth kinetics indicate that BHK and HuH-7 cells maintain a higher and more prolonged proliferative state than NIH 3T3 and Vero cells during the time course of titering (data not shown). These considerations likely explain why transduction and expression titers appeared different in cell types like NIH 3T3 and Vero, while appearing similar in BHK and HuH-7 cells. Transduction titer is independent of an infected cell’s proliferative state, as this method determines total virion genomes recovered from an infected cell monolayer. The IE4/5 promoter of HSV exhibits higher lacZ expression than the GRE5-AdMLP transcription unit, an observation expected because of the known transactivational properties of specific HSV nucleocapsid proteins. The strong HSV transcriptional activator protein, VP16, is released into the cell upon infection and interacts with at least two cellular proteins, HCF and Oct-1. An initial interaction occurs between VP-16 and HCF (20, 21), the latter affording stability, leading this complex to interact with Oct-1 (22). The newly formed multimeric complex binds and activates at the TAATGARAT elements found within the immediate early gene promoters of HSV. In numerous experiments performed in our laboratory, the IE4/5 promoter of HSV-1 mediates high expression levels of transgenes but its duration of expression is shorter than exhibited by cellular promoters (23). The glucocorticoid response elements cloned into the pHSVGRE5lac amplicon construct were derived from the rat TAT gene promoter. This transcription unit was previously used in the amplicon context to impart glucocorticoid-responsive expression of human growth hormone (13). The glucocorticoid receptor (GR), upon binding of the synthetic glucocorticoid, dexamethasone, undergoes a hyperphosphorylation event that results in conformational changes and binding to GRE [reviewed by (24)]. We have previously shown that the human hepatoma cell line HuH-7 supports glucocorticoid-inducible expression (13), whereas other cell types may be less capable of sup-
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METHOD TABLE 2 Proposed Standard Terminology for HSV Vector Titering Titering method
Component assayed
Titering units/vola
Expression-based (i.e., X-gal histochem, GFP fluorescence) Quantitative PCR Slot-blot hybridization ELISA Plaque assay
Transcriptional activity of transferred amplicon genome Postinfection amplicon genome content Preinfection amplicon genome content Preinfection amplicon particle number Helper virus
EU/ml TU/ml GU/ml Particles/ml pfu/ml
a EU/ml, expression units per milliliter; TU/ml, transducing units per milliliter; GU/ml, genome units per milliliter; pfu/ml, plaque-forming units per milliliter.
porting this response, presumably due to the low levels of glucocorticoid receptor expression. Promoter-mediated expression kinetics also likely play a role in the expression titer differences observed between the HSVlac and HSVGRE5lac. -Galactosidase expression from the HSVGRE5lac amplicon peaks at day 4, while expression from the IE4/5 promoter-driven HSVlac amplicon peaks at day 2 (data not shown). This may explain why expression titers analyzed 24 h following infection are so disparate between the two amplicon construct preparations. Therefore, arbitrary selection of assay time points to determine expression titers can greatly influence titer values. In contrast, transduction titering provides the number of viral genomes that gain access to the cell monolayer and is unaffected by promoter activity or expression kinetics. BHK and HuH-7 cells appeared to be less efficiently infected than either NIH 3T3 or Vero cells, as evident from lower HSVlac transduction titers on BHK and HuH-7 cells. This could reflect a reduced number of HSV receptors on the host cellular membrane. HSV particles enter host cells via interactions of their surface glycoproteins gB, gC, and gD with host-encoded receptors, including herpes entry mediators B and C (HveB and HveC) and cell-surface heparin sulfate proteoglycans (25). HveB and HveC are ubiquitously expressed, with HveC expression highest in cells of the nervous system (26). Quantitative measurement of HSV receptors on the surface of host cells will be required to definitively address this issue. Genome transfer titering, as it was adopted here for HSV amplicon vectors, can allow an investigator to rapidly determine the titer of any amplicon vector, as well as to gain insight into vector-related issues regarding promoter activity and virus infection efficiency. This method represents advancement to HSV amplicon-based experimentation. It is appreciated that some amplicon studies may not require equivalent transduction titers and therefore recommend the adoption of a conventional terminology regarding HSV vector titers (Table 2). Titers derived from a coupled infection and expression assay, what we have termed as expression titering in this paper, should be represented as expression units (EU) per unit volume. Titering data obtained via real-time quantitative PCR should be expressed as TU per unit volume. Other investigators working with HSV amplicon vectors are de-
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veloping methods for enumerating viral particles. In this instance, titering data should be represented as particles per unit volume. For conventional helper virus-based packaging, representation of helper virus titers should remain as plaque-forming units (pfu) per unit volume. Acceptance of conventional amplicon terminology will facilitate interpretation and understanding of the HSV amplicon literature. ACKNOWLEDGMENTS The authors thank Hui Huang and Brandon Harvey for their advice in method development and interpretation of experimental results. This work was supported by an AFAR Research Grant to W.J.B. and NIH Grant R01 NS364201 to H.J.F.
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