Optimization of recombinant adeno-associated virus production using an herpes simplex virus amplicon system

Optimization of recombinant adeno-associated virus production using an herpes simplex virus amplicon system

Journal of Virological Methods 96 (2001) 97 – 105 www.elsevier.com/locate/jviromet Optimization of recombinant adeno-associated virus production usin...

206KB Sizes 0 Downloads 9 Views

Journal of Virological Methods 96 (2001) 97 – 105 www.elsevier.com/locate/jviromet

Optimization of recombinant adeno-associated virus production using an herpes simplex virus amplicon system Elisabeth Feudner a, Mahesh de Alwis b, Adrian J. Thrasher b, Robin R. Ali b,c, Sascha Fauser a,* a Uni6ersity Eye Hospital, Ro¨ntgenweg 11, 72076 Tu¨bingen, Germany Molecular Immunology Unit, Institute of Child Health, Uni6ersity College London, London, UK c Department of Molecular Genetics, Institute of Ophthalmology, Uni6ersity College London, London, UK b

Received 25 January 2001; received in revised form 2 March 2001; accepted 2 March 2001

Abstract A major limitation of adeno-associated virus (AAV) based vectors for clinical applications to date is the production of high-titer recombinant AAV vector stocks. Despite recent improvements, the amount of recombinant adeno-associated virus vectors (rAAV) particles produced per cell continues to be significantly lower than that of wild-type AAV. In this study, an HSV-based system for rAAV production was used to examine the influence of different parameters including transfection conditions (vector-to-packaging plasmid ratio, amount of total transfected DNA, cell confluency) and multiplicity of infection of herpes helper virus on the resulting titre of rAAV stocks. For herpes helper virus, time-course experiments were carried out to analyse the effect on rAAV yields up to 72 h postinfection and to determine the ideal harvesting time. Taken together, the optimized production scheme consistently yields more than 3× 103 transducing units per producer cell. © 2001 Elsevier Science B.V. All rights reserved. Keywords: Adeno-associated virus; Herpes simplex virus; Gene therapy; Viral vector

1. Introduction Vectors derived from the human parvovirus adeno-associated virus type 2 (AAV-2) are among the most promising viral vectors currently being developed for human gene therapy, because of a unique combination of attractive properties. Wild-type AAV-2 is not associated with any hu* Corresponding author. Tel: + 49-7071-2987072; fax: + 49-7071-295777. E-mail address: [email protected] (S. Fauser).

man disease (Blacklow et al., 1968) and is naturally defective, requiring co-infection with a helper virus, usually a member of the adenovirus (Atchison et al., 1965) or herpes virus family (Buller et al., 1981), for productive infection. Recombinant adeno-associated virus vectors (rAAV) have the capacity to effectively transduce both mitotic and postmitotic tissues such as lung (Flotte et al., 1993), brain (Kaplitt et al., 1994), muscle (Xiao et al., 1996), retina (Ali et al., 1996), and liver (Snyder et al., 1997). Furthermore, because rAAV vectors are deleted for all viral

0166-0934/01/$ - see front matter © 2001 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 6 - 0 9 3 4 ( 0 1 ) 0 0 2 9 8 - 1


E. Feudner et al. / Journal of Virological Methods 96 (2001) 97–105

proteins, they offer advantages over some other viral vector systems with respect to toxicity and immune response: they have been shown to be capable of long-term, high-level gene expression even in immunocompetent hosts (Kessler et al., 1996; Bennett et al., 1999) without cellular immune response or toxicity to the host. To date, a major limitation of using rAAVbased vectors for clinical applications is still the production of high-titer rAAV vector stocks. Conventional rAAV packaging systems are based on transient transfection of two plasmids into helper adenovirus infected cells. The vector plasmid carries a transgene expression cassette flanked by the AAV-2 inverted terminal repeats (ITRs) which are the only cis-elements required for rescue, replication and packaging of the recombinant genome (Samulski et al., 1987). The AAV-2 nonstructural (rep) and structural (cap) genes are supplied in trans on the second plasmid, the socalled packaging plasmid. Upon infection with helper adenovirus, the vector is excised from the plasmid backbone, amplified and finally packaged into AAV-2 capsids. Compared with wild-type AAV-2 this method of rAAV packaging is very inefficient, giving rise to only 102 recombinant viral particles per cell. Efforts to improve the packaging capacity of rAAV vectors include the construction of adenovirus helper plasmids that can be used instead of adenovirus infection (Matsushita et al., 1998; Xiao et al., 1998; Collaco et al., 1999), the generation of cell lines that contain integrated copies of some or all of the AAV genes required for packaging (Yang et al., 1994; Clark et al., 1995; Tamayose et al., 1996; Inoue and Russell, 1998), and the development of recombinant helper viruses that have been engineered to express the rAAV vector genome (Gao et al., 1998; Liu et al., 1999) or the AAV-2 rep and cap genes (Conway et al., 1999). Along with the growing understanding of the basic biology of the AAV-2 life-cycle it has become evident that two distinct factors, namely replication of the rep and cap genes and the strength of cap gene expression strongly influence the recombinant particle output. In contrast with a wild-type AAV-2 infection, rep and cap genes provided by a conventional packaging plasmid for

rAAV vector production lack the ITRs and therefore remain unamplified in the virus-producing cells, the consequence being an insufficient expression of the Rep and Cap proteins and an unbalanced genome/capsid ratio for genome encapsidation, resulting in low vector yields (Fan and Dong, 1997). Increased synthesis of AAV capsid proteins on the other hand has been shown to improve rAAV vector yields (Li et al., 1997; Vincent et al., 1997; Grimm et al., 1998). Accordingly, attempts to improve the conventional rAAV production protocol focus on the generation of new packaging plasmids that are able to replicate (Chiorini et al., 1995) or direct higher cap gene expression (Li et al., 1997). Recently both vector and packaging sequences were incorporated in herpes simplex virus type 1 (HSV-1) derived amplicon plasmids, which replicate alternatively upon infection of cells with herpes helper virus, simulating to some extent the amplification of rep and cap genes seen in a wild-type AAV infection. Facilitated production of rep and cap genes translates into a considerably improved recovery of rAAV (Zhang et al., 1999). In this study, this system was used to investigate further the influence of transfection conditions, specifically vector-to-packaging plasmid ratio, amount of total transfected DNA and cell confluency, and herpes helper virus parameters on rAAV vector yields. Whilst adenovirus is an efficient helper for rAAV production, little consideration so far has been given to other helper viruses for AAV-2 replication and packaging such as HSV-1, which is also a fully competent helper virus of AAV-2 (Buller et al., 1981; Weindler and Heilbronn, 1991). The herpes helper virus used in this study was examined specifically with regard to optimal multiplicity of infection (MOI) and ideal time of harvest for the purpose of rAAV production.

2. Materials and methods

2.1. Cells and 6iruses HeLa and 293 cells were obtained from the American Type Culture Collection (ATCC). The establishment of CR-1 cells has been described

E. Feudner et al. / Journal of Virological Methods 96 (2001) 97–105

previously (Boursnell et al., 1997; Zhang et al., 1998). These cells incorporate the genes encoding glykoprotein H (gH) of HSV-1 and therefore serve as complementing cells for growing the gH− PS1 helper virus. HeLa, 293 and CR-1 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplememted with 10% fetal bovine serum (FBS), penicillin and streptomycin. BHK cells were obtained from the European Collection of Animal Cell Cultures. BHK cells were grown in Glasgow modified Eagle’s medium supplemented with 10% FBS, 5% tryptose broth, penicillin and streptomycin. The virus PS1, which was used in this study as a helper virus for generating rAAV stocks, is a glycoprotein H and thymidinkinase deletion mutant derived from HSV-1 strain SC16 ( Hill et al., 1975) and has been described previously (Zhang et al., 1999). PS1 was grown and titrated by plaque assay on the complementing (gH+) CR-1 cell line. Adenovirus type 5 at a MOI of 50 was used in the rAAV transduction assays. Adenovirus was grown and titrated on 293 cells according to standard protocols (Graham and Prevec, 1991).


were made by gentle mixing of three components: Lipofectin (Life Technologies), peptide 6 ([K16]GACRRETAWACG), and plasmid DNA in the weight ratio 0.75:4:1. The resulting mixture was incubated at room temperature for 1.5 h. At the end of this time, the total volume was made up to 1ml with OPTIMEM, and the mixture was applied to the OPTIMEM-washed cells. Five hours later, the transfection mixture was removed and the transfected BHK cells were infected with PS1 at a MOI of 3 in DMEM 10% FBS except for experiments in which the amount of PS1 was the independent variable. Cultures were harvested usually 40 h after helper virus infection with the exception of time-course experiments by scraping the cells into the medium. Cells were collected by low-speed centrifugation in a bench-top centrifuge. The supernatant was collected and the cell pellet was resuspended in 1 ml of serum-free medium. Cells were then lysed by three freeze/ thaw cycles (dry ice with ethanol/37°C water bath) and clarified of cellular debris by centrifugation (4000g, 5 min). Contaminating HSV and amplicon particles were inactivated by incubating for 30 min at 56°C. The final vector preps were stored at − 80°C.

2.2. Plasmids 2.4. FACS analysis The packaging plasmid pHAV7.3 is a HSV-1 derived amplicon plasmid which contains the wild type AAV rep and cap genes (Zhang et al., 1999). The pHAV5 vector plasmid is a HSV-1 derived amplicon plasmid which contains the AAV ITRs flanking the cytomegalovirus early transcription promoter and the green fluorescent protein (gfp) gene cassette (Zhang et al., 1999).

2.3. Recombinant adeno-associated 6irus 6ector production All packaging experiments were carried out on 35 mm six-well dishes. 2×105 BHK cells were plated per well except otherwise stated. 24 h later, the cultures were co-transfected with pHAV5 and pHAV7.3 in the vector-to-packaging plasmid ratios described in the text using Lipofectin/Integrin targeting peptide/DNA (LID) complexes (Hart et al., 1998). Briefly, complexes

To determine transfection efficiencies, the percentage of gfp-positive cells was measured with a Becton-Dickinson flow cytometer 24 h after transfection. The cells were prepared as follows: cells were trypsinized, washed twice with PBS and were finally resuspended in 500 ml PBS with 4% paraformaldehyde. Cells transfected with packaging plasmid only were used as a negative control. A minimum of 10 000 cells were analysed for each sample. The percentage of positive cells is defined as the fraction beyond 99% of the control transfected cells.

2.5. Transduction assay To determine rAAV transduction titres, 2× 104 HeLa cells were plated in 96-well culture plates. The next day the subconfluent HeLa cells were transduced with tenfold serial dilutions of the


E. Feudner et al. / Journal of Virological Methods 96 (2001) 97–105

rAAV/gfp vector stocks and superinfected with adenovirus type 5 at a MOI of 50. Twenty four hours after transduction, before extensive cytopathic effects were observed, cells were analyzed for gfp expression using an Olympus inverted microscope IX-70 with EPI-fluorescence equipment under 20× magnification. Green fluorescent cells were counted in vector dilutions that gave well-separated fluorescent cells and the rAAV/gfp titres were determined based on the sample dilutions.

3. Results

3.1. Vector-to-packaging plasmid ratio and total DNA used for transfection In the AAV production protocol used in this study, transfection of the amplicon vector (pHAV5) and amplicon packaging plasmids (pHAV7.3) into BHK cells is followed by infection of the cells with the herpes helper virus, PS1. The herpes helper virus infection initiates the replication of the amplicon plasmids. As a result, the number of rep and cap genes available for replication and packaging of recombinant AAV genomes increases similar to the amplification seen in a wild-type AAV-2 infection. Provided that introduction of the rep and cap genes via the replicating amplicon plasmid mimicks the process in a wild-type AAV-2 infection, the optimal vector-to-(replicating) packaging plasmid DNA ratio for transfection should be 1:1. To test this hypothesis, three different vector-to-packaging plasmid ratios (1:3, 1:1, 3:1) covering a ninefold range were tested, with a total amount of transfected DNA of 4 mg. Fig. 1 shows that a 1:3 vector-topackaging plasmid ratio yielded the highest amounts of transducing rAAV vector. Given the sizes of the vector and helper plasmids, a 1:3 weight ratio is equivalent to a 1:3 molar ratio. When the vector plasmid and packaging plasmid were delivered in equal amounts (1:1), the resulting rAAV titres were already noticeably lower. An excess of vector plasmid (3:1) resulted in a drastic (tenfold) drop in transducing rAAV titres. As with non-replicating systems, the titres of rAAV

Fig. 1. Comparison of different vector/packaging plasmid ratios with regard to rAAV vector yield. 2 × 105 BHK cells were plated on the day prior to transfection in 35 mm wells. The next day, cultures were transfected with 4 mg of plasmid DNA in a vector/packaging plasmid ratio of 1:3, 1:1, or 3:1. Cells were infected with herpes helper virus PS1 5 h after transfection and harvested 40 h after PS1 infection. The results are expressed as gfp-transducing units (TU) per well 9S.D., where one unit is defined as one fluorescent cell after transduction of adenovirus-infected HeLa cells. All results are averaged from triplicate experiments.

increased proportionally to the amount of packaging plasmid used. There is, however, a limit to the amount that can be used since the rep gene products are toxic to the cell at high levels. In order to investigate whether the rAAV yield could be improved by increasing the total amount of transfected DNA, BHK cells were transfected with increasing amounts (2, 4, 6, 8, and 10 mg) of plasmid DNA in a vector-to-packaging plasmid ratio of 1:3. Interestingly, rAAV titres started to decrease significantly when more than 4 mg of DNA was used (Fig. 2). In order to confirm these results in a larger experiment, BHK cells were transfected with in-

Fig. 2. Influence of the amount of transfected DNA on rAAV yield. Cultures of BHK cells were transfected with increasing amounts of plasmid DNA in a vector/packaging plasmid ratio of 1:3. rAAV titres are expressed as gfp-TU per 35 mm well. All results are averaged from triplicate experiments.

E. Feudner et al. / Journal of Virological Methods 96 (2001) 97–105


Table 1

Fig. 3. Influence of the amount of transfected DNA and vector/packaging plasmid ratio on rAAV yield. Cultures of BHK cells in 35 mm wells were transfected with either 2, 4, 6, 8 or 10 mg plasmid DNA with vector/packaging plasmid ratios of either 1:3, 1:1 or 3:1). 5 h later, cells were infected with PS1 (MOI of 3) and harvested 40 h after infection. The results are expressed as gfp-TU per well 9 S.D. The results are averaged from two independent experiments.

rAAV production: Transfections were performed in 35 mm wells. At the time of transfection, cells were approximately 70% confluent (3×105 cells per well). Transfection efficiencies were determined by FACS analysis of gfp-positive cells 24 h after transfection. Total rAAV titer is the total rAAV yield recovered from each 35 mm well, measured in gfp-TU. RAAV titer per cell is the total rAAV titer divided by the number of cells per well (3×105). rAAV titer per gfp-positive cell is the rAAV titer per cell corrected for transfection efficiency. All results are averaged from two independent experiments.

creasing amounts (2, 4, 6, 8, and 10 mg) of plasmid DNA in various vector-to-packaging plasmid ratio (3:1, 1:1, and 1:3). Transfection efficiency was also determined by FACS analysis. Results from two independent experiments are summarized in Table 1 and Fig. 3. Irrespective of the amount of transfected DNA, a vector-to-packaging plasmid DNA ratio of 1:3 resulted in the highest rAAV yields. As before, the rAAV titres are highest when 2 or 4 mg of DNA and a vector-to-packaging plasmid ratio of 1:3 is used despite the fact that the transfection efficiencies are lower than with greater amounts of DNA.

3.2. Cell number To determine the optimal number of BHK cells per 35 mm well and to determine whether increasing the cell number per well would result

in higher rAAV yields, on the day prior to transfection, increasing cell numbers (105, 2× 105, 3×105, 4×105) were plated per 35 mm well. The next day, cells were transfected with a total of 3 mg of plasmid DNA at a vector-topackaging plasmid ratio of 1:3. Following transfection, FACS analysis showed similar overall transfection efficiencies at all cell densities (37% gfp positive cells). The highest rAAV titres however, were obtained when 2× 105 cells were plated per 35 mm well. A further increase in the number of cells per well did not result in higher rAAV titres despite constant transfection efficiencies (Fig. 4).

Fig. 4. Influence of cell numbers on rAAV yields. Cultures of BHK cells were transfected with 3 mg plasmid DNA (vector/ packaging plasmid ratio of 1:3), infected with PS1 (MOI of 3) after 5 h and harvested 42 h after infection. The results are averaged from three independent experiments.


E. Feudner et al. / Journal of Virological Methods 96 (2001) 97–105

herpes helper virus is used, time course experiments were carried out to examine the exact course of rAAV titres in pellet and supernatant after the PS1 helper virus infection. As shown in Fig. 6, the peak viral yield was obtained 20–30 h after infection of cells with PS1, with almost 90% of the rAAV particles at that time being cellassociated. Fig. 5. PS1 MOI influences rAAV yields. Cultures of BHK cells in 35 mm wells were infected with PS1 helpervirus at different MOIs 5 h after transfection of 3 mg of plasmid DNA (vector/packaging plasmid ratio of 1:3). The cultures were harvested 40 h after infection and total rAAV titer was measured in gfp-TU. All results are averaged from two independent experiments.

3.3. Herpes6irus PS1 multiplicity of infection Because traditionally most rAAV production protocols use adenovirus as the helper virus for rAAV replication and packaging, little detailed information is available about other fully competent helper viruses such as HSV-1. Therefore, the optimal amount of the herpes helper virus, PS1, was determined for the production of rAAV. Cultures of BHK cells were transfected with 3 mg of plasmid DNA (vector-to-packaging plasmid ratio of 1:3) and infected with PS1 5 h after transfection. The amount of rAAV produced was a function of the MOI of PS1 (Fig. 5). An increase in the rAAV yield was observed up to a MOI of 20. Any additional advantage gained from an MOI above 20 is outweighed by the effort required for producing the helper virus.

4. Discussion Gene therapy vectors derived from AAV are being developed for a variety of acquired and genetic diseases. One significant problem associated with rAAV vectors, that has hampered the progress, is the difficulty of consistently generating high-titer vector preparations required for in vivo applications. In an attempt to improve the yield of rAAV, several variables were analyzed in a rAAV protocol that utilises herpes simplex helper virus and amplicons (Zhang et al., 1999). Using the HSV-1 derived amplicon packaging plasmid pHAV7.3, that replicates upon herpes helper virus infection, it was found that as with a non-replicating packaging plasmid (Fan and Dong, 1997; Chirico and Trempe, 1998) the resulting rAAV vector yields increased when more rep and cap genes were transfected. This suggests that independent amplification of the rep and cap genes, mediated by the amplicon, may not be as

3.4. Har6esting of recombinant adeno-associated 6irus 6ectors following PS1 infection When using adenovirus as a helper for rAAV packaging, rAAV yields peak between 48 and 72 h after helper virus infection. Like adenovirus, herpes virus is a lytic virus that induces extensive cytopathic effects and finally leads to death of infected cells. rAAV production comes to an end when the cells die and cell lysis causes rAAV to be released into the culture medium. In order to determine the ideal time to harvest rAAV if a

Fig. 6. Time course of rAAV yields. Cultures were transfected with 3 mg of plasmid DNA (vector/packaging plasmid ratio of 1:3) and infected with PS1 helpervirus 5 h after transfection at an MOI of 20. Cultures were harvested 6, 13, 20, 30, 40, 51 and 65 h after infection, separated into cell pellet and supernatant and gfp-transducing rAAV titres were determined. All experiments were carried out in triplicate.

E. Feudner et al. / Journal of Virological Methods 96 (2001) 97–105

efficient as amplification of the wild type AAV genome. As the vector sequences are also delivered on an amplicon plasmid (pHAV5), their additional amplicon-mediated amplification may add to the imbalance between vector genomes and capsid proteins necessary for their packaging. These considerations may help to explain the comparatively lower rAAV yields if vector and packaging plasmids are transfected at equal amounts. By transfecting higher amounts of packaging plasmid, the resulting imbalance may be reduced, and higher rAAV vector yields may be achieved. Transfection is a critical step in rAAV production protocols. In general, higher transfection efficiencies result in improved yields (Chiorini et al., 1995; Mamounas et al., 1995), most likely by increasing the number of rAAV producing cells (Vincent et al., 1997). In the analysed system, increasing the amount of transfected plasmid DNA improved transfection efficiencies from 20% to over 50% but the expected increase in rAAV titres failed to materialize. rAAV titres peaked when a total of 2 mg of plasmid DNA was transfected. Although transfection efficiencies were twice as high when 4 or 6 mg of DNA were transfected, rAAV titres decreased the more DNA was transfected, possibly due to toxicity of the transfection complexes to the cell when high concentrations of DNA and lipids are used. The optimal number of BHK cells per 35 mm well was determined to be 2×105. Although transfection efficiencies remained constant up to 4×105 plated cells, a proportional increase in rAAV yields was only observed between 105 and 2× 105 cells. Increasing the number of cells further, negatively influenced the resulting rAAV titres, possibly because at higher plating densities the cells quickly became confluent. Little is known about herpes virus as a helper for rAAV packaging. The minimal set of HSV-1 genes required for rAAV replication and packaging has been identified as the early genes UL5, UL8, UL52 and UL29, genes that encode components of the DNA replication machinery but whose helper effect is not fully understood (Weindler and Heilbronn, 1991). To date, few groups have used herpes virus for the purposes of rAAV


packaging and they have used MOIs of between 0.1 (Chatterjee et al., 1999) and 3 (Zhang et al., 1999). The data suggest that rAAV titres increase with increasing herpes MOI and the highest titres were obtained at an MOI of 20. At this MOI, there appears to be the most effective balance between expression of the AAV-2 rep and cap genes, HSV-1 helper functions required for rAAV production and the cytotoxicity inherent to the herpes helper virus. rAAV released into the media following cell lysis is difficult to recover and therefore purification procedures generally only make use of the vector associated with the cell pellets. When adenovirus is used as a helper, cell-associated vector at the optimal harvesting time is reported to make up between 50% (Grimm et al., 1998) and 70– 90% (Li et al., 1997) of the total rAAV produced. In this study using herpes as a helper virus, the optimal time to harvest rAAV was as early as 20–30 h after infection and at this time almost 90% of the vector was cell-associated. Compared with adenovirus, maximal rAAV titres are reached much earlier when herpes helper virus is used. This might be connected with the observed inhibition of adenovirus DNA replication and maturation of adenovirus DNA replication centers by AAV-2 rep gene products (Weitzman et al., 1996). In contrast to adenovirus, the development of HSV-1 DNA replication centers was not affected by the presence of the rep gene (Conway et al., 1999). Under optimized conditions, this production protocol consistently yields 2× 108 transducing rAAV units (TU) / 35 mm well, which corresponds to a specific productivity of 660 TU/cell or 3300 TU/transfected cell, respectively. This represents a step towards the efficiency of wild-type AAV-2 packaging, which produces 105 –106 genomes and 104 –105 tissue culture infectious units per infected cell (Parks et al., 1967; Rose and Koczot, 1972). In combination with new chromatography-based purification procedures that allow for complete removal of helper virus products from rAAV preparations (Grimm et al., 1998; Clark et al., 1999; Zolotukhin et al., 1999), the production of rAAV from replicating amplicon plasmids and herpes helper virus should prove very useful for in vivo applications


E. Feudner et al. / Journal of Virological Methods 96 (2001) 97–105

Acknowledgements Supported by grants from the Federal Ministry of Education and Research (Fo¨ . 01KS9602) and the Interdisciplinary Center of Clinical Research Tu¨ bingen (IZKF), the British Retinitis Pigmentosa Society and Fight for Sight.

References Ali, R.R., Reichel, M.B., Thrasher, A.J., Levinsky, R.J., Kinnon, C., Kanuga, N., Hunt, D.M., Bhattacharya, S.S., 1996. Gene transfer into the mouse retina mediated by an adeno-associated viral vector. Hum. Mol. Genet. 5, 591 – 594. Atchison, R., Casto, B., Hammond, W.M., 1965. Adenovirusassociated defecitve virus particles. Science 149, 754 –756. Bennett, J., Maguire, A.M., Cideciyan, A.V., Schnell, M., Glover, E., Anand, V., Aleman, T.S., Chirmule, N., Gupta, A.R., Huang, Y., Gao, G.P., Nyberg, W.C., Tazelaar, J., Hughes, J., Wilson, J.M., Jacobson, S.G., 1999. Stable transgene expression in rod photoreceptors after recombinant adeno-associated virus-mediated gene transfer to monkey retina. Proc. Natl. Acad. Sci. USA 96, 9920 – 9925. Blacklow, N.R., Hoggan, M.D., Kapikian, A.Z., Austin, J.B., Rowe, W.P., 1968. Epidemiology of adenovirus-associated virus infection in a nursery population. Am. J. Epidemiol. 88, 368 – 378. Boursnell, M.E., Entwisle, C., Blakeley, D., Roberts, C., Duncan, I.A., Chisholm, S.E., Martin, G.M., Jennings, R., Ni, C.D., Sobek, I., Inglis, S.C., McLean, C.S., 1997. A genetically inactivated herpes simplex virus type 2 (HSV-2) vaccine provides effective protection against primary and recurrent HSV-2 disease. J. Infect. Dis. 175, 16 –25. Buller, R.M., Janik, J.E., Sebring, E.D., Rose, J.A., 1981. Herpes simplex virus types 1 and 2 completely help adenovirus- associated virus replication. J. Virol. 40, 241 – 247. Chatterjee, S., Li, W., Wong, C.A., Fisher, A.G., Lu, D., Guha, M., Macer, J.A., Forman, S.J., Wong-KK, J., 1999. Transduction of primitive human marrow and cord bloodderived hematopoietic progenitor cells with adeno-associated virus vectors. Blood 93, 1882 –1894. Chiorini, J.A., Wendtner, C.M., Urcelay, E., Safer, B., Hallek, M., Kotin, R.M., 1995. High-efficiency transfer of the T cell co-stimulatory molecule B7-2 to lymphoid cells using high-titer recombinant adeno-associated virus vectors. Hum. Gene Ther. 6, 1531 –1541. Chirico, J., Trempe, J.P., 1998. Optimization of packaging of adeno-associated virus gene therapy vectors using plasmid transfections. J. Virol. Meth. 76, 31 –41. Clark, K.R., Voulgaropoulou, F., Fraley, D.M., Johnson, P.R., 1995. Cell lines for the production of recombinant adeno-associated virus. Hum. Gene Ther. 6, 1329 – 1341.

Clark, K.R., Liu, X., McGrath, J.P., 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. Collaco, R.F., Cao, X., Trempe, J.P., 1999. A helper virus-free packaging system for recombinant adeno-associated virus vectors. Gene 238, 397 – 405. Conway, J., Rhys, C., Zolotukhin, I., Zolotukhin, S., Muzyczka, N., Hayward, G., Byrne, B., 1999. High-titer recombinant adeno-associated virus production utilizing a recombinant herpes simplex virus type I vector expressing AAV-2 Rep and Cap. Gene Ther. 6, 986 – 993. Fan, P.D., Dong, J.Y., 1997. Replication of rep-cap genes is essential for the high-efficiency production of recombinant AAV. Hum. Gene Ther. 8, 87– 98. Flotte, T., Afione, S., Conrad, C., McGrath, S., Solow, R., Oka, H., Zeitlin, P., Guggino, W., 1993. Stable in vivo expression of the cystic fibrosis transmembrane conductance regulator with an adeno-associated virus vector. Proc. Natl. Acad. Sci. USA 90, 10613 – 10617. Gao, G.P., Qu, G., Faust, L.Z., Engdahl, R.K., Xiao, W., Hughes, J.V., Zoltick, P.W., Wilson, J.M., 1998. High-titer adeno-associated viral vectors from a Rep/Cap cell line and hybrid shuttle virus. Hum. Gene Ther. 9, 2353 – 2362. Graham, F.L., Prevec, L., 1991. Manipulation of adenovirus vectors. In: Methods in Molecular Biology. The Humana Press, Clifton, NJ, pp. 109 – 127. Grimm, D., Kern, A., Rittner, K., Kleinschmidt, J.A., 1998. Novel tools for production and purification of recombinant adenoassociated virus vectors. Hum. Gene Ther. 9, 2745 – 2760. Hart, S.L., Arancibia, C.C., Wolfert, M.A., Mailhos, C., O’Reilly, N.J., Ali, R.R., Coutelle, C., George, A.J., Harbottle, R.P., Knight, A.M., Larkin, D.F., Levinsky, R.J., Seymour, L.W., Thrasher, A.J., Kinnon, C., 1998. Lipidmediated enhancement of transfection by a nonviral integrin-targeting vector. Hum. Gene Ther. 9, 575 – 585. Hill, T.J., Field, H.J., Blyth, W.A., 1975. Acute and recurrent infection with herpes simplex virus in the mouse: a model for studying latency and recurrent disease. J. Gen. Virol. 28, 341 – 353. Inoue, N., Russell, D.W., 1998. Packaging cells based on inducible gene amplification for the production of adenoassociated virus vectors. J. Virol. 72, 7024 – 7031. Kaplitt, M.G., Leone, P., Samulski, R.J., Xiao, X., Pfaff, D.W., O’Malley, K.L., During, M.J., 1994. Long-term gene expression and phenotypic correction using adeno-associated virus vectors in the mammalian brain. Nat. Genet. 8, 148 – 154. Kessler, P., Podsakoff, G., Chen, X., McQuiston, S., Colosi, P., Matelis, L., Kurtzman, G., Byrne, B., 1996. Gene delivery to skeletal muscle results in sustained expression and systemic delivery of a therapeutic protein. Proc. Natl. Acad. Sci. USA 93, 14082 – 14087. Li, J., Samulski, R.J., Xiao, X., 1997. Role for highly regulated rep gene expression in adeno-associated virus vector production. J. Virol. 71, 5236 – 5243.

E. Feudner et al. / Journal of Virological Methods 96 (2001) 97–105


Vincent, K.A., Piraino, S.T., Wadsworth, S.C., 1997. Analysis of recombinant adeno-associated virus packaging and requirements for rep and cap gene products. J. Virol. 71, 1897 – 1905. Weindler, F.W., Heilbronn, R., 1991. A subset of herpes simplex virus replication genes provides helper functions for productive adeno-associated virus replication. J. Virol. 65, 2476 – 2483. Weitzman, M.D., Fisher, K.J., Wilson, J.M., 1996. Recruitment of wild-type and recombinant adeno-associated virus into adenovirus replication centers. J. Virol. 70, 1845 – 1854. Xiao, X., Li, J., Samulski, R.J., 1996. Efficient long-term gene transfer into muscle tissue of immunocompetent mice by adeno-associated virus vector. J. Virol. 70, 8098 – 8108. Xiao, X., Li, J., Samulski, R.J., 1998. Production of high-titer recombinant adeno-associated virus vectors in the absence of helper adenovirus. J. Virol. 72, 2224 –2232. Yang, Q., Chen, F., Trempe, J.P., 1994. Characterization of cell lines that inducibly express the adeno-associated virus Rep proteins. J. Virol. 68, 4847 – 4856. Zhang, X., O’Shea, H., Entwisle, C., Boursnell, M., Efstathiou, S., Inglis, S., 1998. An efficient selection system for packaging herpes simplex virus amplicons. J. Gen. Virol. 79, 125 – 131. Zhang, X., De Alwis, M., Hart, S.L., Fitzke, F.W., Inglis, S.C., Boursnell, M.E., Levinsky, R.J., Kinnon, C., Ali, R.R., Thrasher, A.J., 1999. High-titer recombinant adenoassociated virus production from replicating amplicons and herpes vectors deleted for glycoprotein H. Hum. Gene Ther. 10, 2527 – 2537. Zolotukhin, S., Byrne, B., Mason, E., Zolotukhin, I., Potter, M., Chesnut, K., Summerford, C., Samulski, R., Muzyczka, N., 1999. Recombinant adeno-associated virus purification using novel methods improves infectious titer and yield. Gene Ther. 6, 973 – 985.

Liu, X.L., Clark, K.R., Johnson, P.R., 1999. Production of recombinant adeno-associated virus vectors using a packaging cell line and a hybrid recombinant adenovirus. Gene Ther. 6, 293 – 299. Mamounas, M., Leavitt, M., Yu, M., Wong-Staal, F., 1995. Increased titer of recombinant AAV vectors by gene transfer with adenovirus coupled to DNA-polylysine complexes. Gene Ther. 2, 429 – 432. Matsushita, T., Elliger, S., Elliger, C., Podsakoff, G., Villarreal, L., Kurtzman, G.J., Iwaki, Y., Colosi, P., 1998. Adeno-associated virus vectors can be efficiently produced without helper virus. Gene Ther. 5, 938 –945. Parks, W.P., Melnick, J.L., Rongey, R., Mayor, H.D., 1967. Physical assay and growth cycle studies of a defective adeno-satellite virus. J. Virol. 1, 171 –180. Rose, J.A., Koczot, F., 1972. Adenovirus-associated virus multiplication. VII. Helper requirement for viral deoxyribonucleic acid and ribonucleic acid synthesis. J. Virol. 10, 1–8. Samulski, R.J., Chang, L.S., Shenk, T., 1987. A recombinant plasmid from which an infectious adeno-associated virus genome can be excised in vitro and its use to study viral replication. J. Virol. 61, 3096 –3101. Snyder, R.O., Miao, C.H., Patijn, G.A., Spratt, S.K., Danos, O., Nagy, D., Gown, A.M., Winther, B., Meuse, L., Cohen, L.K., Thompson, A.R., Kay, M.A., 1997. Persistent and therapeutic concentrations of human factor IX in mice after hepatic gene transfer of recombinant AAV vectors. Nat. Genet. 16, 270 –276. Tamayose, K., Hirai, Y., Shimada, T., 1996. A new strategy for large-scale preparation of high-titer recombinant adeno-associated virus vectors by using packaging cell lines and sulfonated cellulose column chromatography. Hum. Gene Ther. 7, 507 – 513.