- Email: [email protected]
Herpesvirus-based infectious titering of recombinant adeno-associated viral vectors
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doi:10.1016/j.ymthe.2004.08.030
Herpesvirus-Based Infectious Titering of Recombinant Adeno-Associated Viral Vectors Imran Mohiuddin,1 Scott Loiler,2,3 Irina Zolotukhin,2,3 Barry J. Byrne,1,2,3 Terence R. Flotte,1,2,3 Richard O. Snyder,1,2,* 1
Department of Molecular Genetics and Microbiology, 2Powell Gene Therapy Center, and 3Department of Pediatrics, University of Florida College of Medicine, Gainesville, FL 32610-0266, USA
*To whom correspondence and reprint requests should be addressed at the Department of Molecular Genetics and Microbiology, University of Florida, P.O. Box 100266, 1600 SW Archer Road, Gainesville, FL 32610-0266, USA. Fax: (352) 392 4290. E-mail: [email protected].
Studies in animals and human clinical trials demonstrate the safety and persistence of recombinant adeno-associated viral (rAAV) serotype 2 vectors in a variety of tissues. rAAV vectors of other serotypes are also being developed for efficient gene transfer. To date, the literature describing these vectors has relied on physical or transducing titers to determine dose, but few, if any, infectious titers have been presented. This is due in large part to the lack of reagents and methods that would facilitate the infectious titering of vectors other than serotype 2. Here, we describe reagents and methods for infectious titering of AAV2 ITR-containing vectors pseudotyped with other AAV capsid serotypes and demonstrate their utility by titering pseudotyped rAAV1 or rAAV5 vectors. Cell lines are screened for optimal transduction using a vector of a particular serotype that expresses a marker transgene. Once a cell line and vector serotype are matched, a recombinant herpes simplex virus vector expressing AAV2 rep and cap genes provides helper functions that amplify the rAAV vector genome. The vector genomes are then detected and a titer is calculated. These methods generate reliable infectious titers for AAV vectors of different serotypes, thus enhancing product characterization and reducing risk in future clinical applications. Key Words: adeno-associated virus, gene therapy, titer, herpesvirus, serotype
Gene transfer studies in animal models have shown dramatic differences in the transduction efficiency and cell specificity of recombinant adeno-associated viral (rAAV) vectors of different serotypes [1–6]. When designing these types of experiments, researchers have a choice of rAAV vectors that are bisotype,Q bpseudotype,Q or bcapsid modified.Q Isotype vectors possess inverted terminal repeats (ITRs), Rep proteins, and capsid proteins derived from the same wild-type virus, e.g., AAV2 [7–9]. Pseudotyped vectors carry ITRs and Rep proteins of a particular virus and possess capsid proteins belonging to another serotype, e.g., 2 and 1 (AAV2/1) [2,10]. Modification of the AAV capsid with epitopes has been used to target the virions to specific tissues and cell types [11–13]. AAV2-ITR vectors pseudotyped with other capsid serotypes are common because there is no need to reclone the expression cassettes used historically in AAV2 vectors. The safety profile of the AAV2 ITR sequences in animal models is better understood from the investigations of chromosomal integration efficiency and specificity [14–18], but few data have been generated with the ITRs of other AAV serotypes.
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In recent years, there have been significant enhancements in the production and purification of rAAV vectors. Along with the emergence of numerous scaleable systems [19–23], the particle output per cell has steadily increased, and the particle:infectious (P:I) ratio has decreased as purification methods have improved [24–28]. Infectious titering of rAAV vectors of different serotypes is dependent upon target cell types expressing a specific viral receptor(s) and compatible cellular enzymes [29–35]. Most titering techniques rely on the amplification of the AAV vector genome within the cell for reliable detection [24,36–40]. Since rAAV vectors are replication incompetent, this amplification is contingent upon the presence of AAV rep and cap genes, accompanied by helper virus (adenovirus (Ad)) functions. rAAV vector amplification increases the sensitivity of the titering assay and enhances the reliability of determining the infectious titer of an AAV vector stock. The replication center assay (RCA) [19,41] employs Ad and wild-type AAV (wtAAV) to supply the necessary
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components required to replicate the rAAV genome. Cells are co-infected with dilutions of rAAV together with Ad and wtAAV. When the rAAV vector infects the cell, its genome is replicated (amplified) to a level that can be detected by hybridization with DNA probes. Since the incubation period is 24–40 h, infection of neighboring cells should not occur, thus each signal represents an infection by a single infectious virion in the rAAV stock (Fig. 1A). The RCA carries a reputation for poor reproducibility that is likely due to the variability of achieving an optimal balance between the wtAAV and the Ad infections: too much wtAAV can inhibit Ad [42], too little Ad will not provide the needed help required for AAV gene expression, and too much Ad will induce cell killing too quickly. Switching to the infectious center assay (ICA; also known as the modified RCA), which incorporates rep/cap cell lines, resolves many of the problems encountered with the RCA. In the ICA, rep/cap-expressing cells are infected with dilutions of rAAV and co-infected with Ad
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[24,36,43], so the rAAV is complemented for DNA replication and the rAAV genomes are amplified. When these cells are subsequently trapped on nitrocellulose filters and probed for transgene DNA, only those cells productively infected with rAAV produce a spot (Fig. 1B). The use of rep/cap cells eliminates the need to use wtAAV to supply rep and cap and thus removes a potential source of laboratory contamination that is notoriously difficult to inactivate. However, the ICA’s dependence on a particular stable rep/cap cell line effectively limits the AAV vector serotypes and epitope-tagged capsid variants that can be assayed if they do not infect these cells efficiently. Furthermore, isolating permissive rep/capexpressing cell lines for each serotype represents a large investment of time and effort since the cytostatic and cytotoxic nature of Rep can hamper the isolation of a productive cell line [44]. To date, the literature describing different serotype vectors has relied on physical or transducing titers to FIG. 1. (A) Schematic diagram of the replication center assay (RCA). Permissive cells are infected with adenovirus, wild-type AAV, and dilutions of the rAAV vector to be titered. (B) Infectious center assay (ICA). rep/capcontaining cells are infected with adenovirus and dilutions of the rAAV vector to be titered. (C) HSV infectious center assay (HSVICA). Permissive cells are infected with a herpes vector that expresses rep and cap and dilutions of the rAAV vector to be titered. Following infection, cells are incubated for 24–48 h and analyzed for (1) transgene-specific protein expression, (2) vector genomes by PCR [38,40] or hybridization following isolation of the low-molecular-weight DNA [47], or (3) infected cells by immobilizing the cells onto a membrane, lysing the cells, denaturing the DNA, and fixing it to the membrane with NaOH and probing with vector-specific sequences.
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determine dose, but few, if any, infectious titers have been presented. This is due in large part to the lack of reagents and methods that would facilitate the infectious titering of vectors other than serotype 2. We have developed a new assay, called the herpes simplex virus ICA (HSVICA), for the infectious titering of rAAV vectors. Different cell lines are screened for their ability to be transduced with a rAAV marker vector of a
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certain serotype. This screening is achieved by comparing the expression level of a marker transgene in a variety of cell lines following rAAV-mediated transduction (Fig. 2A). The initial cell line screen is designed to screen several cell lines rapidly to determine which one(s) can be transduced, and the use of marker genes facilitates a rapid readout. If the cell line can be transduced by the rAAV serotype, then the cell is capable of vector cell
FIG. 2. (A) Permissive cell screening. 2 105 cells per well were plated into 16-well glass chamber slides (Lab-Tek, Nalge Nunc International) in 100 Al of growth medium. One day later, the cells were infected in triplicate with 1 Al of either AAV1/CB-lucEYFP-WPRE (3.0 1012 vg/ml) or AAV5/CB-lucEYFP-WPRE (1.3 1012 vg/ ml) vector per well. Six days postinfection, the cells were lysed with 100 Al of 1 CCLR lysis reagent from Promega. 10 Al of cell lysate was added to 90 Al of luciferin reagent (Promega Luciferase Assay System) and analyzed for luciferase activity. Results are FSEM. BNL CL.2 (ATCC TIB-73) cells are normal mouse liver cells grown in DMEM with 10% FBS. VA cells, a rat pulmonary artery endothelial cell line, were isolated by mechanical methods [48] and grown in medium 199 with EarleTs salts (Sigma), sodium bicarbonate (pH 7.4), 10% fetal bovine serum, 10 mM glutamine, and antibiotic/antimycotic solution at 378C in 5% CO2. (B) 293 cell transduction assay. 2 104 293 cells seeded in 96-well plates were transduced with serial dilutions of AAV1-TR-UF-11, AAV5-TR-UF-11, or AAV2-TR-UF-11 vector and co-infected with HSVr/c at an m.o.i. of 10 or 40 or with adenovirus type 5 at an m.o.i. of 2. After 30 h, GFP-positive cells were counted at different dilutions and the transducing titers (TU/ml) were calculated. Recombinant AAV vectors were produced and purified as described [25]. The CB-lucEYFP-WPRE vector is a rAAV vector with the CB promoter driving expression of a fusion protein of luciferase and enhanced yellow fluorescent protein. This vector is flanked on each end by AAV2 ITRs and also contains a WPRE for higher levels of expression. The TR-UF-11 vector is a rAAV vector with the CB promoter driving expression of GFP and is also flanked by AAV2 ITRs.
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surface binding, internalization, delivery of the vector genome to the nucleus, and second-strand synthesis. This initial screening eliminates cell lines that do not have the cell surface receptor or cannot internalize and deliver the rAAV vector genome to the nucleus. The cell line with the strongest level of marker transgene expression for the particular AAV vector serotype is then used for titering vectors of the same serotype that express nonmarker transgenes, such as secreted proteins. Infectious titering (Fig. 1C) is accomplished by coinfecting the most permissive cell line with the particular rAAV serotype vector and a herpesvirus vector that expresses AAV rep and cap (HSVr/c) [45]. HSV and AAV rep/cap gene expression supports the amplification of the AAV vector genome, which in turn increases the sensitivity of detecting vector genomes (or transgene expression). The cell line chosen in the screen above needs to be capable of being efficiently transduced by HSVr/c; thus a further screen of these candidate cell lines can be performed for best rAAV vector genome amplification. The rAAV vector genome can be detected using PCR [38,40] or DNA hybridization techniques, and transgene expression can be evaluated by determining RNA or protein levels (Fig. 1). To evaluate the transduction efficiency of the same TR-UF-11 vector pseudotyped with AAV1, AAV2, or AAV5 capsids, 293 cells were transduced in the presence of Ad type 5 or HSVr/c. GFP-positive cells were then scored at several dilutions and transducing titers calculated (Fig. 2B and Table 1). When these same transduced 293 cells were transferred to a membrane and hybridized with a vector-
specific probe, only cells co-infected with HSVr/c had detectable signals (Fig. 3 and Table 1). These results indicate that HSVr/c supports the amplification of the rAAV vector genome in permissive cells (Figs. 3B, 3D, 3E, 3G, and 3H) with vector signals that are similar to those using a rep/cap cell line (C12) infected with Ad (Fig. 3A). Adenovirus, which lacks the rep and cap genes, was unable to amplify the genome of the vectors on 293 cells (Figs. 3C, 3F, and 3I). Furthermore, when HS578T [30] cells, originally used to identify the AAV5 cellular receptor, or COS7 cells were used in the HSVICA to titer an AAV5 vector, similar titers were obtained and were comparable to 293 cells (Figs. 3G, 3H, 3J, 3K, and 3L and Table 1). The infectious titering of the rAAV1 or 5 vectors using HSVr/c on various cell lines was more sensitive than the transducing (GFP titer), and the AAV2 titers were similar. The ability to generate infectious centers is an important first step toward the quantitation of infectious titer by a variety of methods, such as Southern blotting or PCR (Fig. 1). As shown in Table 1, titers of AAV1 and AAV2 vectors using HSVr/c and the C12 cell line (a HeLabased rep/cap cell line) are 3 logs lower than titers obtained using the HSVr/c on 293 cells. This inefficiency could be due to poor HSV infection of C12 cells, inhibition of HSV by the resident Rep in the C12 cell line, or the effects of too much Rep protein produced from both the HSVr/c and the C12 cell line. In general, the HSVICA is equal to or up to 20 times more sensitive than the green cell transduction assay on the cell lines tested here (Table 1).
TABLE 1: Comparison of dot-blot (DB), green cell transduction assay (GCA), infectious titering, and particle to infectious ratio (P:I) of rAAV1, 2, or 5-UF-11 vectors using adenovirus type 5 (Ad) or HSVr/c (HSV) at the indicated m.o.i. (2, 10, 20, or 40) DB vg/ml 1.8 1.8 1.8 1.8 1.8 1.8 4.6 4.6 4.6 4.6 4.6 4.6 5.1 5.1 5.1 5.1 5.1 5.1 5.1
1014 1014 1014 1014 1014 1014 1013 1013 1013 1013 1013 1013 1013 1013 1013 1013 1013 1013 1013
Serotype
Cell line
Helper
GCA TU/ml
Infectious IU/ml
P:I ratio
AAV1 AAV1 AAV1 AAV1 AAV1 AAV1 AAV2 AAV2 AAV2 AAV2 AAV2 AAV2 AAV5 AAV5 AAV5 AAV5 AAV5 AAV5 AAV5
293 293 293 C12 C12 C12 293 293 293 C12 C12 C12 293 293 293 HS578T HS578T COS7 COS7
HSV 10 HSV 40 Ad 2 HSV 10 HSV 20 Ad 20 HSV 10 HSV 40 Ad 2 HSV 10 HSV 20 Ad 20 HSV 10 HSV 40 Ad 2 HSV 10 HSV 40 HSV 10 HSV 40
7.9 107 1.3 108 3.2 108 6.9 104 1.2 105 1.7 108 3.7 109 1.3 109 1.5 1010 1.4 106 2.8 106 1.4 1010 5.0 106 6.5 106 5.3 106 1.1 106 3.1 106 2.7 107 1.6 107
Not assayed 2.0 108 Not detectable 2.6 105 2.5 105 9.8 108 Not assayed 9.6 108 Not detectable 1.5 106 2.0 106 4.3 1010 1.2 108 7.7 107 Not detectable 4.5 107 5.1 107 1.2 108 4.1 107
Not calculated 9.0 105 Not calculated 6.9 108 7.2 108 1.8 105 Not calculated 4.8 104 Not calculated 3.1 107 2.3 107 1.1 103 4.3 105 6.6 105 Not calculated 1.1 106 1.0 106 4.3 105 1.2 106
The dot-blot assay [25] was used to determine the titer of rAAV virions that contain vector genomes.
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An additional feature of using a rep/cap-expressing herpesviral vector arises in cells used here that do not support a productive infection of the herpesviral vector. Since the herpesviral vector is replication deficient, cell
FIG. 3. ICA vs HSVICA. 96-well plates seeded with 2 104 293, HS578T, C12, or COS7 cells were infected with serial dilutions of AAV1-TR-UF-11, AAV5-TRUF-11, or AAV2-TR-UF-11 vector. These cells were co-infected with the indicated m.o.i. of either Ad or HSVr/c. After 48 h, the cells were transferred to a nylon membrane and lysed, and the nucleic acid was immobilized following treatment with NaOH. Filters were hybridized with a radiolabeled CMV immediate early enhancer probe. Cells productively infected with rAAV generated a spot following autoradiography. HSVr/c has been described and was propagated on V27 cells [22]. Adenovirus type 5 (ATCC VR-5) was propagated on 293 cells [49]. The C12 cell line [21] has been described. The 293, HS578T [50], and COS-7 (ATCC CRL-1651, transformed with SV40 T antigen) cell lines were grown in DMEM with 10% FBS.
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lysis with a resulting secondary rAAV vector infection of neighboring cells should not occur, and there is less cytopathic effect due to the defective HSVr/c vector. Thus, incubation periods can be extended, which increases assay sensitivity and results in more accurate infectious titers. The HSVICA (Figs. 1C and 3) is a versatile assay for determining titers of various rAAV vector serotypes. HSVICA offers increased flexibility because it is not dependent on the generation of unique stable rep/cap cell lines; however, utilization of the HSVr/c for titering is limited to the use of cells that are permissive for infection by the HSVr/c vector and the specific rAAV vector serotype. The proof-of-principle data presented in this article utilized the same vector genome with AAV2 ITRs packaged into three different capsid serotypes (1, 2, 5) to eliminate the variable of using different ITRs or other cassette components. In addition, the HSVr/c utilized here encodes the AAV2 Rep protein that is matched to the vector ITRs. It has been published [46] that Rep proteins from certain AAV serotypes act preferentially on their own ITRs, therefore one would predict that AAV vectors with ITRs from another serotype would be complemented by an HSVr/c harboring the Rep sequences from the same serotype. The HSVICA titering method obviates the need for wild-type AAV (used in the RCA) and wild-type adenovirus (used in the ICA), both of which can replicate and potentially contaminate laboratories producing rAAV vector stocks. Using an attenuated HSV vector to supply rep and cap provides even greater versatility and containment. Each assay for transgene expression has a unique sensitivity and the vectors may have different expression levels relating to promoter strength, so comparing infectious titers of different rAAV vectors based on transgene expression is not appropriate. Furthermore, the assays for vector genome titer do not characterize the infectious nature of rAAV preparations. Using the HSVICA or ICA assays, comparisons between different preparations of the same vector and between vectors of the same serotype that encode different transgenes are possible and provide researchers with a common unit of infectious titer when proper controls and standards are incorporated. It may not be practical to use one cell line to titer different serotypes, because the cellular receptor for each viral serotype will likely differ. As with any titering method, titers for a specific serotype obtained in vitro using the HSVICA on the best cell line will need to be correlated to the minimum effective dose, the optimal dose, and the maximum tolerated dose in a specific tissue of animals or humans. Typically, when AAV2 vectors, purified by column chromatography, are titered, the P:I ratio as determined by dot-blot hybridization and ICA on rep/cap cells is 10–1000, with a mean of 100 to 200 [24,43]. Using the HSVICA, a P:I ratio can be determined for a specific AAV serotype vector titered on a particular permissive
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cell line. This ratio can be used as a benchmark during process development to ensure that new production and purification methods do not damage the virion and to determine product lot consistency. The validation of the HSVICA will be important to support preclinical and human clinical studies. Since all validation parameters such as specificity, precision, linearity, sensitivity, and range are very much dependent on the sample matrix, the validation is dependent upon the particular combination of target cell line and AAV vector serotype. Infectious titering is an important indicator of the potency of a vector stock and is an essential method for realizing the medical and commercial potential of new therapeutic vectors. These described methods promote reliable infectious titers for AAV vectors of different serotypes. This reproducible bioassay can be utilized to characterize AAV vector products better and set product specifications so as to minimize risk in the clinic. Since several new AAV vectors based on different AAV capsid serotypes are currently being evaluated in animals (and soon in humans), it is important to establish dependable titration systems. ACKNOWLEDGMENTS We thank Kristin Good, Stefanie Shoja, Tina Phillipsburg, and Mark Potter for technical assistance and Joyce Francis for helpful comments. T.F., B.B., and R.S. are inventors on patents related to recombinant AAV technology. R.S. and B.B. own equity in a gene therapy company that is commercializing AAV for gene therapy applications. To the extent that the work in this article increases the value of these commercial holdings, T.F., B.B., and R.S. have a conflict of interest. RECEIVED FOR PUBLICATION JULY 18, 2004; ACCEPTED AUGUST 20, 2004.
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supported by a herpes simplex virus type 1 amplicon expressing Rep and Cap. J. Virol. 71: 8780 – 8789. Chiorini, J. A., Afione, S., and Kotin, R. M. (1999). Adeno-associated virus (AAV) type 5 Rep protein cleaves a unique terminal resolution site compared with other AAV serotypes. J. Virol. 73: 4293 – 4298. Hirt, B. (1967). Selective extraction of polyoma DNA from infected mouse cell cultures. J. Mol. Biol. 26: 365 – 369. Visner, G. A., Chesrown, S. E., Monnier, J., Ryan, U. S., and Nick, H. S. (1992). Regulation of manganese superoxide dismutase: IL-1 and TNF induction in pulmonary artery and microvascular endothelial cells. Biochem. Biophys. Res. Commun. 188: 453 – 462. Graham, F. L., Smiley, J., Russell, W. C., and Nairn, R. (1977). Characteristics of a human cell line transformed by DNA from human adenovirus type 5. J. Gen. Virol. 36: 59 – 74. Ross, D. T., et al. (2000). Systematic variation in gene expression patterns in human cancer cell lines. Nat. Genet. 24: 227 – 235.
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Herpesvirus-Based Infectious Titering of Recombinant Adeno-Associated Viral Vectors Imran Mohiuddin,1 Scott Loiler,2,3 Irina Zolotukhin,2,3 Barry J. Byrne,1,2,3 Terence R. Flotte,1,2,3 Richard O. Snyder,1,2,* 1
Department of Molecular Genetics and Microbiology, 2Powell Gene Therapy Center, and 3Department of Pediatrics, University of Florida College of Medicine, Gainesville, FL 32610-0266, USA
*To whom correspondence and reprint requests should be addressed at the Department of Molecular Genetics and Microbiology, University of Florida, P.O. Box 100266, 1600 SW Archer Road, Gainesville, FL 32610-0266, USA. Fax: (352) 392 4290. E-mail: [email protected].
Studies in animals and human clinical trials demonstrate the safety and persistence of recombinant adeno-associated viral (rAAV) serotype 2 vectors in a variety of tissues. rAAV vectors of other serotypes are also being developed for efficient gene transfer. To date, the literature describing these vectors has relied on physical or transducing titers to determine dose, but few, if any, infectious titers have been presented. This is due in large part to the lack of reagents and methods that would facilitate the infectious titering of vectors other than serotype 2. Here, we describe reagents and methods for infectious titering of AAV2 ITR-containing vectors pseudotyped with other AAV capsid serotypes and demonstrate their utility by titering pseudotyped rAAV1 or rAAV5 vectors. Cell lines are screened for optimal transduction using a vector of a particular serotype that expresses a marker transgene. Once a cell line and vector serotype are matched, a recombinant herpes simplex virus vector expressing AAV2 rep and cap genes provides helper functions that amplify the rAAV vector genome. The vector genomes are then detected and a titer is calculated. These methods generate reliable infectious titers for AAV vectors of different serotypes, thus enhancing product characterization and reducing risk in future clinical applications. Key Words: adeno-associated virus, gene therapy, titer, herpesvirus, serotype
Gene transfer studies in animal models have shown dramatic differences in the transduction efficiency and cell specificity of recombinant adeno-associated viral (rAAV) vectors of different serotypes [1–6]. When designing these types of experiments, researchers have a choice of rAAV vectors that are bisotype,Q bpseudotype,Q or bcapsid modified.Q Isotype vectors possess inverted terminal repeats (ITRs), Rep proteins, and capsid proteins derived from the same wild-type virus, e.g., AAV2 [7–9]. Pseudotyped vectors carry ITRs and Rep proteins of a particular virus and possess capsid proteins belonging to another serotype, e.g., 2 and 1 (AAV2/1) [2,10]. Modification of the AAV capsid with epitopes has been used to target the virions to specific tissues and cell types [11–13]. AAV2-ITR vectors pseudotyped with other capsid serotypes are common because there is no need to reclone the expression cassettes used historically in AAV2 vectors. The safety profile of the AAV2 ITR sequences in animal models is better understood from the investigations of chromosomal integration efficiency and specificity [14–18], but few data have been generated with the ITRs of other AAV serotypes.
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In recent years, there have been significant enhancements in the production and purification of rAAV vectors. Along with the emergence of numerous scaleable systems [19–23], the particle output per cell has steadily increased, and the particle:infectious (P:I) ratio has decreased as purification methods have improved [24–28]. Infectious titering of rAAV vectors of different serotypes is dependent upon target cell types expressing a specific viral receptor(s) and compatible cellular enzymes [29–35]. Most titering techniques rely on the amplification of the AAV vector genome within the cell for reliable detection [24,36–40]. Since rAAV vectors are replication incompetent, this amplification is contingent upon the presence of AAV rep and cap genes, accompanied by helper virus (adenovirus (Ad)) functions. rAAV vector amplification increases the sensitivity of the titering assay and enhances the reliability of determining the infectious titer of an AAV vector stock. The replication center assay (RCA) [19,41] employs Ad and wild-type AAV (wtAAV) to supply the necessary
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components required to replicate the rAAV genome. Cells are co-infected with dilutions of rAAV together with Ad and wtAAV. When the rAAV vector infects the cell, its genome is replicated (amplified) to a level that can be detected by hybridization with DNA probes. Since the incubation period is 24–40 h, infection of neighboring cells should not occur, thus each signal represents an infection by a single infectious virion in the rAAV stock (Fig. 1A). The RCA carries a reputation for poor reproducibility that is likely due to the variability of achieving an optimal balance between the wtAAV and the Ad infections: too much wtAAV can inhibit Ad [42], too little Ad will not provide the needed help required for AAV gene expression, and too much Ad will induce cell killing too quickly. Switching to the infectious center assay (ICA; also known as the modified RCA), which incorporates rep/cap cell lines, resolves many of the problems encountered with the RCA. In the ICA, rep/cap-expressing cells are infected with dilutions of rAAV and co-infected with Ad
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[24,36,43], so the rAAV is complemented for DNA replication and the rAAV genomes are amplified. When these cells are subsequently trapped on nitrocellulose filters and probed for transgene DNA, only those cells productively infected with rAAV produce a spot (Fig. 1B). The use of rep/cap cells eliminates the need to use wtAAV to supply rep and cap and thus removes a potential source of laboratory contamination that is notoriously difficult to inactivate. However, the ICA’s dependence on a particular stable rep/cap cell line effectively limits the AAV vector serotypes and epitope-tagged capsid variants that can be assayed if they do not infect these cells efficiently. Furthermore, isolating permissive rep/capexpressing cell lines for each serotype represents a large investment of time and effort since the cytostatic and cytotoxic nature of Rep can hamper the isolation of a productive cell line [44]. To date, the literature describing different serotype vectors has relied on physical or transducing titers to FIG. 1. (A) Schematic diagram of the replication center assay (RCA). Permissive cells are infected with adenovirus, wild-type AAV, and dilutions of the rAAV vector to be titered. (B) Infectious center assay (ICA). rep/capcontaining cells are infected with adenovirus and dilutions of the rAAV vector to be titered. (C) HSV infectious center assay (HSVICA). Permissive cells are infected with a herpes vector that expresses rep and cap and dilutions of the rAAV vector to be titered. Following infection, cells are incubated for 24–48 h and analyzed for (1) transgene-specific protein expression, (2) vector genomes by PCR [38,40] or hybridization following isolation of the low-molecular-weight DNA [47], or (3) infected cells by immobilizing the cells onto a membrane, lysing the cells, denaturing the DNA, and fixing it to the membrane with NaOH and probing with vector-specific sequences.
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determine dose, but few, if any, infectious titers have been presented. This is due in large part to the lack of reagents and methods that would facilitate the infectious titering of vectors other than serotype 2. We have developed a new assay, called the herpes simplex virus ICA (HSVICA), for the infectious titering of rAAV vectors. Different cell lines are screened for their ability to be transduced with a rAAV marker vector of a
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certain serotype. This screening is achieved by comparing the expression level of a marker transgene in a variety of cell lines following rAAV-mediated transduction (Fig. 2A). The initial cell line screen is designed to screen several cell lines rapidly to determine which one(s) can be transduced, and the use of marker genes facilitates a rapid readout. If the cell line can be transduced by the rAAV serotype, then the cell is capable of vector cell
FIG. 2. (A) Permissive cell screening. 2 105 cells per well were plated into 16-well glass chamber slides (Lab-Tek, Nalge Nunc International) in 100 Al of growth medium. One day later, the cells were infected in triplicate with 1 Al of either AAV1/CB-lucEYFP-WPRE (3.0 1012 vg/ml) or AAV5/CB-lucEYFP-WPRE (1.3 1012 vg/ ml) vector per well. Six days postinfection, the cells were lysed with 100 Al of 1 CCLR lysis reagent from Promega. 10 Al of cell lysate was added to 90 Al of luciferin reagent (Promega Luciferase Assay System) and analyzed for luciferase activity. Results are FSEM. BNL CL.2 (ATCC TIB-73) cells are normal mouse liver cells grown in DMEM with 10% FBS. VA cells, a rat pulmonary artery endothelial cell line, were isolated by mechanical methods [48] and grown in medium 199 with EarleTs salts (Sigma), sodium bicarbonate (pH 7.4), 10% fetal bovine serum, 10 mM glutamine, and antibiotic/antimycotic solution at 378C in 5% CO2. (B) 293 cell transduction assay. 2 104 293 cells seeded in 96-well plates were transduced with serial dilutions of AAV1-TR-UF-11, AAV5-TR-UF-11, or AAV2-TR-UF-11 vector and co-infected with HSVr/c at an m.o.i. of 10 or 40 or with adenovirus type 5 at an m.o.i. of 2. After 30 h, GFP-positive cells were counted at different dilutions and the transducing titers (TU/ml) were calculated. Recombinant AAV vectors were produced and purified as described [25]. The CB-lucEYFP-WPRE vector is a rAAV vector with the CB promoter driving expression of a fusion protein of luciferase and enhanced yellow fluorescent protein. This vector is flanked on each end by AAV2 ITRs and also contains a WPRE for higher levels of expression. The TR-UF-11 vector is a rAAV vector with the CB promoter driving expression of GFP and is also flanked by AAV2 ITRs.
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surface binding, internalization, delivery of the vector genome to the nucleus, and second-strand synthesis. This initial screening eliminates cell lines that do not have the cell surface receptor or cannot internalize and deliver the rAAV vector genome to the nucleus. The cell line with the strongest level of marker transgene expression for the particular AAV vector serotype is then used for titering vectors of the same serotype that express nonmarker transgenes, such as secreted proteins. Infectious titering (Fig. 1C) is accomplished by coinfecting the most permissive cell line with the particular rAAV serotype vector and a herpesvirus vector that expresses AAV rep and cap (HSVr/c) [45]. HSV and AAV rep/cap gene expression supports the amplification of the AAV vector genome, which in turn increases the sensitivity of detecting vector genomes (or transgene expression). The cell line chosen in the screen above needs to be capable of being efficiently transduced by HSVr/c; thus a further screen of these candidate cell lines can be performed for best rAAV vector genome amplification. The rAAV vector genome can be detected using PCR [38,40] or DNA hybridization techniques, and transgene expression can be evaluated by determining RNA or protein levels (Fig. 1). To evaluate the transduction efficiency of the same TR-UF-11 vector pseudotyped with AAV1, AAV2, or AAV5 capsids, 293 cells were transduced in the presence of Ad type 5 or HSVr/c. GFP-positive cells were then scored at several dilutions and transducing titers calculated (Fig. 2B and Table 1). When these same transduced 293 cells were transferred to a membrane and hybridized with a vector-
specific probe, only cells co-infected with HSVr/c had detectable signals (Fig. 3 and Table 1). These results indicate that HSVr/c supports the amplification of the rAAV vector genome in permissive cells (Figs. 3B, 3D, 3E, 3G, and 3H) with vector signals that are similar to those using a rep/cap cell line (C12) infected with Ad (Fig. 3A). Adenovirus, which lacks the rep and cap genes, was unable to amplify the genome of the vectors on 293 cells (Figs. 3C, 3F, and 3I). Furthermore, when HS578T [30] cells, originally used to identify the AAV5 cellular receptor, or COS7 cells were used in the HSVICA to titer an AAV5 vector, similar titers were obtained and were comparable to 293 cells (Figs. 3G, 3H, 3J, 3K, and 3L and Table 1). The infectious titering of the rAAV1 or 5 vectors using HSVr/c on various cell lines was more sensitive than the transducing (GFP titer), and the AAV2 titers were similar. The ability to generate infectious centers is an important first step toward the quantitation of infectious titer by a variety of methods, such as Southern blotting or PCR (Fig. 1). As shown in Table 1, titers of AAV1 and AAV2 vectors using HSVr/c and the C12 cell line (a HeLabased rep/cap cell line) are 3 logs lower than titers obtained using the HSVr/c on 293 cells. This inefficiency could be due to poor HSV infection of C12 cells, inhibition of HSV by the resident Rep in the C12 cell line, or the effects of too much Rep protein produced from both the HSVr/c and the C12 cell line. In general, the HSVICA is equal to or up to 20 times more sensitive than the green cell transduction assay on the cell lines tested here (Table 1).
TABLE 1: Comparison of dot-blot (DB), green cell transduction assay (GCA), infectious titering, and particle to infectious ratio (P:I) of rAAV1, 2, or 5-UF-11 vectors using adenovirus type 5 (Ad) or HSVr/c (HSV) at the indicated m.o.i. (2, 10, 20, or 40) DB vg/ml 1.8 1.8 1.8 1.8 1.8 1.8 4.6 4.6 4.6 4.6 4.6 4.6 5.1 5.1 5.1 5.1 5.1 5.1 5.1
1014 1014 1014 1014 1014 1014 1013 1013 1013 1013 1013 1013 1013 1013 1013 1013 1013 1013 1013
Serotype
Cell line
Helper
GCA TU/ml
Infectious IU/ml
P:I ratio
AAV1 AAV1 AAV1 AAV1 AAV1 AAV1 AAV2 AAV2 AAV2 AAV2 AAV2 AAV2 AAV5 AAV5 AAV5 AAV5 AAV5 AAV5 AAV5
293 293 293 C12 C12 C12 293 293 293 C12 C12 C12 293 293 293 HS578T HS578T COS7 COS7
HSV 10 HSV 40 Ad 2 HSV 10 HSV 20 Ad 20 HSV 10 HSV 40 Ad 2 HSV 10 HSV 20 Ad 20 HSV 10 HSV 40 Ad 2 HSV 10 HSV 40 HSV 10 HSV 40
7.9 107 1.3 108 3.2 108 6.9 104 1.2 105 1.7 108 3.7 109 1.3 109 1.5 1010 1.4 106 2.8 106 1.4 1010 5.0 106 6.5 106 5.3 106 1.1 106 3.1 106 2.7 107 1.6 107
Not assayed 2.0 108 Not detectable 2.6 105 2.5 105 9.8 108 Not assayed 9.6 108 Not detectable 1.5 106 2.0 106 4.3 1010 1.2 108 7.7 107 Not detectable 4.5 107 5.1 107 1.2 108 4.1 107
Not calculated 9.0 105 Not calculated 6.9 108 7.2 108 1.8 105 Not calculated 4.8 104 Not calculated 3.1 107 2.3 107 1.1 103 4.3 105 6.6 105 Not calculated 1.1 106 1.0 106 4.3 105 1.2 106
The dot-blot assay [25] was used to determine the titer of rAAV virions that contain vector genomes.
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An additional feature of using a rep/cap-expressing herpesviral vector arises in cells used here that do not support a productive infection of the herpesviral vector. Since the herpesviral vector is replication deficient, cell
FIG. 3. ICA vs HSVICA. 96-well plates seeded with 2 104 293, HS578T, C12, or COS7 cells were infected with serial dilutions of AAV1-TR-UF-11, AAV5-TRUF-11, or AAV2-TR-UF-11 vector. These cells were co-infected with the indicated m.o.i. of either Ad or HSVr/c. After 48 h, the cells were transferred to a nylon membrane and lysed, and the nucleic acid was immobilized following treatment with NaOH. Filters were hybridized with a radiolabeled CMV immediate early enhancer probe. Cells productively infected with rAAV generated a spot following autoradiography. HSVr/c has been described and was propagated on V27 cells [22]. Adenovirus type 5 (ATCC VR-5) was propagated on 293 cells [49]. The C12 cell line [21] has been described. The 293, HS578T [50], and COS-7 (ATCC CRL-1651, transformed with SV40 T antigen) cell lines were grown in DMEM with 10% FBS.
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lysis with a resulting secondary rAAV vector infection of neighboring cells should not occur, and there is less cytopathic effect due to the defective HSVr/c vector. Thus, incubation periods can be extended, which increases assay sensitivity and results in more accurate infectious titers. The HSVICA (Figs. 1C and 3) is a versatile assay for determining titers of various rAAV vector serotypes. HSVICA offers increased flexibility because it is not dependent on the generation of unique stable rep/cap cell lines; however, utilization of the HSVr/c for titering is limited to the use of cells that are permissive for infection by the HSVr/c vector and the specific rAAV vector serotype. The proof-of-principle data presented in this article utilized the same vector genome with AAV2 ITRs packaged into three different capsid serotypes (1, 2, 5) to eliminate the variable of using different ITRs or other cassette components. In addition, the HSVr/c utilized here encodes the AAV2 Rep protein that is matched to the vector ITRs. It has been published [46] that Rep proteins from certain AAV serotypes act preferentially on their own ITRs, therefore one would predict that AAV vectors with ITRs from another serotype would be complemented by an HSVr/c harboring the Rep sequences from the same serotype. The HSVICA titering method obviates the need for wild-type AAV (used in the RCA) and wild-type adenovirus (used in the ICA), both of which can replicate and potentially contaminate laboratories producing rAAV vector stocks. Using an attenuated HSV vector to supply rep and cap provides even greater versatility and containment. Each assay for transgene expression has a unique sensitivity and the vectors may have different expression levels relating to promoter strength, so comparing infectious titers of different rAAV vectors based on transgene expression is not appropriate. Furthermore, the assays for vector genome titer do not characterize the infectious nature of rAAV preparations. Using the HSVICA or ICA assays, comparisons between different preparations of the same vector and between vectors of the same serotype that encode different transgenes are possible and provide researchers with a common unit of infectious titer when proper controls and standards are incorporated. It may not be practical to use one cell line to titer different serotypes, because the cellular receptor for each viral serotype will likely differ. As with any titering method, titers for a specific serotype obtained in vitro using the HSVICA on the best cell line will need to be correlated to the minimum effective dose, the optimal dose, and the maximum tolerated dose in a specific tissue of animals or humans. Typically, when AAV2 vectors, purified by column chromatography, are titered, the P:I ratio as determined by dot-blot hybridization and ICA on rep/cap cells is 10–1000, with a mean of 100 to 200 [24,43]. Using the HSVICA, a P:I ratio can be determined for a specific AAV serotype vector titered on a particular permissive
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cell line. This ratio can be used as a benchmark during process development to ensure that new production and purification methods do not damage the virion and to determine product lot consistency. The validation of the HSVICA will be important to support preclinical and human clinical studies. Since all validation parameters such as specificity, precision, linearity, sensitivity, and range are very much dependent on the sample matrix, the validation is dependent upon the particular combination of target cell line and AAV vector serotype. Infectious titering is an important indicator of the potency of a vector stock and is an essential method for realizing the medical and commercial potential of new therapeutic vectors. These described methods promote reliable infectious titers for AAV vectors of different serotypes. This reproducible bioassay can be utilized to characterize AAV vector products better and set product specifications so as to minimize risk in the clinic. Since several new AAV vectors based on different AAV capsid serotypes are currently being evaluated in animals (and soon in humans), it is important to establish dependable titration systems. ACKNOWLEDGMENTS We thank Kristin Good, Stefanie Shoja, Tina Phillipsburg, and Mark Potter for technical assistance and Joyce Francis for helpful comments. T.F., B.B., and R.S. are inventors on patents related to recombinant AAV technology. R.S. and B.B. own equity in a gene therapy company that is commercializing AAV for gene therapy applications. To the extent that the work in this article increases the value of these commercial holdings, T.F., B.B., and R.S. have a conflict of interest. RECEIVED FOR PUBLICATION JULY 18, 2004; ACCEPTED AUGUST 20, 2004.
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A., and Reynolds, T. C. (1998). A highthroughput hybridization method for titer determination of viruses and gene therapy vectors. Nucleic Acids Res. 26: 2821 – 2823. 38. Clark, K. R., Liu, X., McGrath, J. P., and Johnson, P. R. (1999). Highly purified recombinant adeno-associated virus vectors are biologically active and free of detectable helper and wild-type viruses. Hum. Gene Ther. 10: 1031 – 1039. 39. Drittanti, L., Rivet, C., Manceau, P., Danos, O., and Vega, M. (2000). High throughput production, screening and analysis of adeno-associated viral vectors. Gene Ther. 7: 924 – 929.
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MOLECULAR THERAPY Vol. 11, No. 2, February 2005 Copyright C The American Society of Gene Therapy