Interest in recombinant adeno-associated virus vectors (rAAV) has steadily increased because of the ability of this vector to mediate long-term, robust in vivo gene expression in numerous nondividing cell types (i.e., hepatocytes, neurons, and skeletal myocytes). Efficient in vivo gene transfer via rAAV transduction requires reasonably high multiplicities of infection, estimated between 103 to 105 DNA-containing particles per cell, depending on the cell type targeted. Moreover, based on large animal studies, a clinical dose in humans will require 1012 to 1014 rAAV vector particles, depending on the level of therapeutic protein expression needed for treatment efficacy1. Therefore, the ability to produce high-titer rAAV is critical for clinical applications and has historically been the primary impediment to the widespread use of this vector system. This review discusses the recent advances in vector production technology that have resulted in the routine isolation openface>104 recombinant viral particles per cell. The vast majority of rAAV is produced in one of two ways. The first method, referred to as transient transfection production, relies on the use of plasmid DNA transfection to introduce required AAV production components into human cell lines. The second approach focuses on the use of stable rAAV producer cell lines for the large-scale production of rAAV. Transient transfection is amenable to higher throughput, allowing the investigator more flexibility to generate and test multiple rAAV vector constructs. It is hampered by the requirement for the transfection of large numbers of mammalian cells in culture. Conversely, although stable cell line approaches are readily scaleable, they are less flexible with regards to the number of vectors that can be rapidly generated and assayed. Both production methods require three essential components for rAAV production. The first element is the rAAV vector, which is a transgene expression cassette flanked by AAV inverted terminal repeats (ITRs). The AAV ITRs are located at the ends of the AAV genome and contain all of the cis sequence information necessary for vector DNA replication and encapsidation2,3. Thus, rAAV vectors contain only a very small proportion (6%) of the wild-type AAV genome and lack all viral coding sequences. Second, the two AAV genes (rep and cap), which encode replication and AAV structural proteins, are expressed in trans. The physical separation of these genes and the ITR packaging signal is critical for avoiding the formation of wild-type AAV. The rep gene possesses two promoters (p5 and p19) from which 4 Rep proteins are expressed (Rep78, Rep68, Rep52, and Rep40). The p5 derived Rep proteins (Rep78 or Rep68) are essential for rAAV replication. Three capsid proteins (VP1, VP2, and VP3) are synthesized following transcription from the rep-inducible p40 cap gene promoter. Finally, because AAV and recombinant derivatives are replication defective, these viruses require helper virus gene products from adenovirus or herpesvirus for efficient viral production. The helper virus (or specific helper virus genes) provides a favorable intracellular milieu for robust rAAV replication and viral synthesis. A schematic of both production strategies is shown in Figure 1.
Figure 1.
rAAV production schematic. The top half of the figure depicts rAAV production via transient transfection. The process involves the cotransfection of a rep-cap–containing plasmid and an rAAV vector plasmid containing the AAV ITRs (arrowheads) flanking a gene expression cassette [promoter (pr), desired transgene (tg), and polyadenylation signal (pA)]. Helper functions for efficient rAAV synthesis are supplied by adenovirus infection or an adenovirus helper plasmid containing the VA, E2A, E4 genes. The lower portion of the figure depicts rAAV production using stable cell line approaches. Generally, HeLa cells containing integrated rep-cap and/or rAAV vector sequences are infected with either adenovirus or recombinant HSV to provide the needed helper functions. rAAV is then rapidly purified from crude cellular lysates using affinity chromatography (antibody or heparin sulfate).
Full figure and legend (80K)ADVANCES IN TRANSIENT TRANSFECTION PRODUCTION
rAAV is most commonly generated in cell culture by co-transfecting 293 or 293T cells with a rAAV vector plasmid and an AAV rep-cap plasmid followed by helper adenovirus infection. Yields of between 102 to 103 viral particles per cell are typical using an optimized calcium phosphate coprecipitation method. For purposes of clarity, all references to rAAV particles, unless otherwise indicated, assume that these are DNA-containing particles. Unfortunately, this method of production is inherently inefficient because of the requirement for three separate "hits" on a cell to produce rAAV (cotransfection of two plasmids plus infection with adenovirus) and is difficult to perform reproducibly on a large scale. However, three recent observations have resulted in 
10-fold increases in vector yields. The first was the recognition that below a threshold level, AAV-2 capsid protein levels can limit rAAV production4. Second, downmodulation of Rep78 protein expression results in increased capsid protein synthesis and rAAV production4,5,6,7. Finally, by introducing the necessary adenovirus helper genes into cells via plasmid DNA transfection rather than by standard adenovirus infection, moderate increases in rAAV yields are possible7,8,9,10,11,12. Importantly, the resulting rAAV preparations were free of contaminating infectious adenovirus and most adenovirus structural proteins. Vincent et al observed that transient transfection vector yields could be increased approximately 10-fold if AAV capsid expression levels were increased using a strong constitutive promoter [human immediate early CMV promoter/enhancer (CMV IE)] in place of the endogenous p40 cap gene promoter4. They also observed that overexpression of the Rep78 protein (using the RSV promoter) resulted in reduced capsid protein production. The authors suggested that Rep78 exerts its negative effect on cap expression by translational repression of capsid mRNA. Moreover, the finding that a minimum threshold level of capsid protein is necessary for efficient intranuclear AAV-2 virion formation added further support to the conclusion that capsid protein levels can be a limiting element in rAAV production12,13,14. Li et al also observed that overexpression of Rep78 protein correlated with decreased capsid protein levels, reduced rAAV genome replication, and rAAV titers6. Conversely, when the authors downmodulated Rep78/68 expression (by using a nonconsensus ACG start codon), they observed increased capsid protein production and rAAV yields by approximately 10-fold. Using a similar strategy, Grimm et al achieved low level Rep expression by using the mouse mammary tumor virus LTR promoter (under non-induced conditions) in place of the native p5 rep promoter7. These authors also observed increased capsid and rAAV production (by a factor of 10-fold). How high-level Rep78 expression acts to reduce capsid expression and vector yields in the transient production schema is not completely understood, but because the AAV Rep proteins have pleiotropic effects on cellular and AAV gene expression, tightly regulated Rep expression appears necessary for optimal rAAV titers. For example, Rep78/68 has been shown to inhibit cap mRNA translation, but these same proteins are required for efficient cap mRNA transcription from the native p40 promoter. Additionally, elevated Rep78/68 expression can repress adenovirus replication and inhibit adenovirus helper functions, resulting in reduced rAAV production capacity. Thus, although clearly needed for AAV replication and transcriptional activation, excess Rep78 levels appear deleterious to efficient rAAV formation. Regardless of the molecular mechanism, the use of these downmodulated rep expression plasmids has resulted in increased rAAV production. The third significant advancement in transient rAAV vector production was the development of helper virus-free packaging systems that use a DNA plasmid to supply all helper adenovirus functions but is incapable of generating infectious adenovirus. Earlier work defined five adenovirus gene regions that were required for complete helper functions for wild-type and rAAV synthesis (E1, E2, VA I/II RNA, and E4). Subsequent research refined the minimal set to E1a, E1b, VA I/II RNA, E2A, and E4 ORF611. Although this approach added a third plasmid to the transfection mixture, rAAV yields were equal to or higher than vector stocks produced by standard adenovirus infection. Among the numerous variations, Xiao et al reported the generation of a miniadenovirus genome plasmid termed pXX6, which deleted several adenovirus structural genes and the MLP promoter9. When cotransfected into 293T cells, the authors reported an approximately threefold increase in vector yield over adenovirus infection. Similarly, Matsushita et al also found that adenovirus gene transfection resulted in vector particle yields that were four times higher than adenovirus infection11. Two groups have combined the rep-cap and adenovirus helper genes on a single plasmid and reported 10-fold increases in rAAV yields7,10. Elevated rAAV yields may be due to the absence of competition between two concurrent viral infections (rAAV and adenovirus) for the host cell macromolecular synthesis machinery. Clearly, the major benefit of this approach is the complete removal of contaminating adenovirus (and most structural proteins) that could deleteriously affect rAAV-mediated transgene expression through host immune activation. An important question is whether all five gene products are necessary for high-level rAAV production. When using the native rep-cap promoters for rAAV production, it appears that all five genes contribute to optimal rAAV yields11. Interestingly, when the rep and cap gene products were expressed by constitutive promoters (the mouse metallothionein gene 1 and CMV promoters, respectively), only the E4 ORF6 adenovirus protein appeared necessary for high-level rAAV production in 293 cells15. Thus, under the appropriate rep and cap conditions, it appears that only the E1 region and the E4 ORF6 protein are required for rAAV production. A summary of the various rAAV production methods is shown in Table 1.
Table 1 - Estimated rAAV DNase resistant particle per cell yields of several production methods.
Full table STABLE CELL LINE PRODUCTION
In addition to transient transfection strategies, there are several methods currently in use for the large-scale production of rAAV using packaging cell lines16,17,18,19,20,21. In 1990, Vincent et al demonstrated that a cell line expressing rep and cap could be used to package rAAV; however, the method still relied on transfection of the rAAV genome into the cell line, and reported titers were low22. Recently, we and others have developed a simplified method for generating rAAV based on stable HeLa cell lines containing integrated copies of the AAV rep-cap genes and a rAAV vector16,17. To generate rAAV, the cells are simply infected with wild-type adenovirus, which results in the induction of the p5 rep promoter. rAAV producer cell lines are initially isolated by first cloning the desired cDNA sequence into a rAAV vector plasmid. Also present on the same plasmid (or a separate plasmid) is a selectable marker gene and the native rep-cap helper genes16,17. Following HeLa cell transfection, stable cell lines are screened for viral production by adenovirus infection and yields quantitated by polymerase chain reaction (PCR) or dot blot hybridization23. Depending on the producer cell line, yields between 4 
103 to 2 104 particles/cell are typical. Importantly, recombinant virus generated in this manner appears free of replication competent wild-type-like AAV (<1 IU of wild-type-like AAV/1011 rAAV particles). A variation on this basic approach is to infect a stable rep-cap expressing HeLa cell line (C12 or B50) with a recombinant adenovirus harboring a rAAV vector genome in the adenovirus E1 region18,20,24,25. Coinfection with wild-type adenovirus is also required to supply the missing E1 helper gene products. Upon coinfection, the rAAV vector is excised from the adenovirus genome, replicated, and packaged into infectious virions. The primary advantage of this strategy is that repeated stable cell line selection is not necessary and reported rAAV yields were between 104 to 105 rAAV particles/cell20. Another potential strategy would be to generate a second recombinant adenovirus containing the AAV rep-cap genes; fortunately, such a vector has been isolated recently (Note added in proof). Interestingly, human herpes virus type 6 contains a functional rep gene homologue26, and HSV amplicon vectors that carry and express the AAV Rep proteins in a stable manner have been reported27,28. This has led to the development of a novel stable cell line production system based on a recombinant herpes simplex virus type I vector (rHSV-1) carrying the AAV rep-cap genes (d27.1-rc)21. In this production scheme, rHSV d27.1-rc provides complete helper functions for rAAV production. Moreover, d27.1-rc is itself replication defective because it does not produce a required herpes protein (ICP27) and, therefore, can only be grown in the appropriate complementing cell line. Infection of a stable cell line containing an integrated rAAV genome with d27.1-rc resulted in vector yields openface>104 particles per cell. It is notable that to date all stable cell line production methods rely on viral infection to supply helper gene products, and therefore, the potential for contamination of rAAV vector stocks with helper virus or associated viral proteins remains a cause for concern. Clearly, improved purification methods (discussed later here) have alleviated many of these concerns, but the development of stable packaging cells that supply all rAAV helper functions remains an important goal for future production strategies. As with all stable cell line methods, the process is scaleable. Currently, several groups are using microcarrier beads, spinner cells, or adherent cell bioreactors (Corning Cell Cube, Acton, MA, USA; or Nunc Cell Factory, Rochester, PA, USA) for the growth openface>1010 to 1011 producer cells. Assuming rAAV particle per cell yields of 104, stable cell line approaches can yield between 1014 to 1015 rAAV particles per large-scale preparation.
RAAV PRODUCTION USING REP-CAP GENE AMPLIFICATION
The wild-type AAV genome (rep-cap genes) is replicated to 106 copies per cell, of which a significant proportion is in a double-stranded, transcriptionally active conformation. Thus, a logical goal for rAAV production systems is to mimic as closely as possible wild-type AAV-2 rep-cap gene amplification as it occurs during a productive infection. Accordingly, it has been documented that the transfection of additional rep-cap plasmid copies relative to the rAAV vector plasmid augments rAAV yields proportionally28. Several production systems have been reported that allow for varying levels of rep-cap gene amplification, although no system to date approaches wild-type levels. The initial report placed a simian virus 40 (SV40) origin of replication on an AAV rep-cap plasmid. This helper plasmid was electroporated into COS cells (SV40 T-antigen expressing African green monkey cells) in an attempt to achieve episomal rep-cap gene amplification via SV40 ori/T-antigen interaction. However, low viral yields were reported (1000 particles per cell), and it was unclear whether rep-cap amplification was actually achieved in this system29. Limited repcap gene amplification was also reported using an HSV amplicon system30. In this production system, the AAV rep-cap genes and rAAV vector sequences were incorporated into HSV amplicon plasmids that contained the HSV origin of replication and packaging signal (oriS-pac). Upon transfection, the plasmids were amplified by infection with a replication defective HSV helper virus (glycoprotein H deleted). This virus supplied both helper rAAV functions and replicative ability to the HSV amplicon plasmids. Vector yields openface>104 rAAV particles per cell were reported. A rep-cap amplification strategy was also developed using a stable HeLa cell line system, which permitted inducible amplification (4-fold) of integrated rep-cap sequences from a nearby SV40 origin of replication using a doxycycline activated SV40 T antigen gene cassette31. Viral yields of 104 particles per cell were also reported. Recently, adenovirus inducible rep-cap gene amplification was also documented in stable, HeLa rAAV producer cell lines by two groups32,33. Quantitative PCR documented episomal rep-cap gene amplification of approximately 100-fold in response to adenovirus infection, which corresponded to rep-cap copy numbers of approximately 400 to 500 per cell. This observation provides a plausible mechanism by which stable HeLa producer cell lines produce high levels of rAAV even though the initial AAV rep-cap copy number is low (typically 5 to 10 copies per cell). Curiously, adenovirus-inducible rep-cap gene amplification was not observed when the rep-cap–containing plasmid was transiently transfected into HeLa cells, indicating that a dramatic difference exists in the amplification potential of transfected rep-cap sequences versus stably integrated sequences. In conclusion, there are multiple, viable methods for the production of rAAV in excess of 104 particles per cell. These production levels should permit the widespread use of this technology for clinical applications Table 1.
VECTOR PURIFICATION
Another significant advance in rAAV vector technology has been the recent development of several refined chromatography-based purification methods that render traditional isopycnic CsCl gradient purification methods obsolete7,23,34. Importantly, vector purity, biological potency, and process throughput are all increased using these chromatographic methods. All three methods allow for one-day vector purification from clarified cell lysates in volumes in excess of 1 L. One method uses an immunoaffinity approach, whereby a monoclonal antibody (A20) that recognizes a conformational epitope on the intact viral particle is coupled to a sepharose resin. Viral purification is achieved by a single pass of a crude cell lysate containing rAAV, followed by washing and viral elution with 2.5 mol/L MgCl2. Percent recoveries of 70% were reported, and vector preparations contained low levels (
20%) of contaminating cellular proteins7. The other two approaches use heparin sulfate (a recently identified co-receptor for AAV-2) as an affinity ligand for viral purification. Clark et al reported a single-pass, crude lysate purification procedure using a commercial heparin sulfate resin (HE POROS; Applied Biosystems, Foster City, CA, USA) and column elution with a linear salt gradient23. Recoveries were on average 70% with purity in excess of 95%. Column capacity (1.4 mL bed volume) was>1014 DNA-containing particles. A second method also used the HE POROS resin after partial rAAV purification using the iso-osmotic gradient reagent Iodixanol34 and reported viral recoveries and purity similar to Clark et al. All three methods appear to produce lower rAAV particle:infectious particle ratios (100:1) than that observed using CsCl purification. Importantly, all three methods greatly decrease the level of contaminating cellular and helper virus proteins and result in a product that is of sufficient purity for clinical testing.
VECTOR TITRATION
The lack of a single method for determining the titer of rAAV stocks has led to some confusion in the field. Essentially, rAAV can be measured in four ways: (1) A physical particle titer enumerates the total number of viral particles (whether containing DNA or not) present in a rAAV preparation. A particle titer can be determined by EM, a commercial A20 antibody-based enzyme-linked immunosorbent assay (ELISA)35 or can be calculated using the known molecular mass of AAV-2 and the protein concentration of a highly purified vector stock (1.5 1011 particles per 1 
g of AAV-2 protein). Grimm et al and we have independently estimated that the majority (80%) of vector particles in a rAAV preparation are devoid of DNA23,35. Accordingly, this may imply that the efficiency of rAAV genome encapsidation may be a rate-limiting step in infectious rAAV formation. (2) The second method determines the number of Dnase-resistant vector genomes (encapsidated viral genomes) present in the rAAV vector stock. Briefly, to derive this value, a rAAV vector preparation is treated with DNase to degrade residual nonencapsidated vector genomes. The DNase is inactivated by proteinase K treatment, and the remaining viral nucleic acid quantitated by PCR or dot blot hybridization and compared with a known plasmid DNA standard curve. The advantage to this particle titer determination is that using quantitative PCR, titers can be rapidly generated in four hours23. Moreover, comparable Dnase-resistant genome titers can be derived for all vector preparations, regardless of the transgene or promoter used in the construct. (3) A third method, also applicable to all vector stocks, determines the proportion of viral particles that can infect and replicate in a target cell line. This method, commonly referred to as a replication center assay, determines the number of infectious units in a rAAV preparation by intracellular amplification of the rAAV genome in the presence of wild-type AAV and adenovirus36. Detection of rAAV replication foci is accomplished by filter hybridization of infected cell monolayers. Recently, this approach has been refined using stable rep expressing cell lines and real time PCR detection of the amplified vector genomes12,23,37. (4) Finally, a transduction (expression) titer measures the number of virus particles competent to infect and express the transgene in the target cell. Although a common method, widely varying titers (over three orders of magnitude) can be generated from the same vector stock by altering the target cell line or the intracellular environment (e.g., concurrent helper adenovirus infection or cell confluency). Therefore, comparisons of rAAV efficacy in different target cells or between vectors with different promoters become highly suspect. How do these various titers relate to each other? Although each vector preparation will possess a specific particle to infectivity ratio, an approximate ratio for heparin purified particles can be roughly given as 2500 total particles: 500 Dnase-resistant vector genome containing particles: 10 replication units: 1 transduction unit. Ideally, for each rAAV production method, the viral yields should be expressed in the form of infectious units per cell and Dnase-resistant vector genome particles per cell.
VECTOR QUALITY CONTROL
Prior to in vivo use, all rAAV vector stocks should be characterized with respect to four basic properties: (1) sterility, (2) replication competent AAV, (3) replication competent helper virus, and (4) purity. The effect that stock impurities have on rAAV transgene expression has been well documented38,39. For example, replication competent adenovirus can dramatically increase rAAV-mediated transgene expression in vitro and can activate host immune responses in vivo. Furthermore, crude vector preparations containing transgene protein can lead to artifactual gene expression through a process termed pseudotransduction38. Therefore, each rAAV vector preparation should be characterized with respect to these four properties prior to use. Purified rAAV vector stocks can be sterile filtered through 0.45 m membranes with little loss in vector titer (typically 20%) and subsequently assayed for sterility using thioglycolate media. The presence of replication competent adenovirus can be readily assayed using 1% of the rAAV preparation and a 293-plaque assay over a five-day period. Purity can be qualitatively monitored by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and a more thorough estimate can be calculated by comparing the preparation's total protein content and the total particle yield (using the A20 particle ELISA)35. Contamination of vector stocks by replication competent AAV has been a persistent problem when using the transient transfection production method to produce rAAV. Wild-type-like AAV can be formed as a result of nonhomologous recombination between the rep-cap helper plasmid and the rAAV vector plasmid and can result in contamination levels of 0.1 to 10%40,41. To address this problem, split packaging rep-cap plasmids that do not contain any sequence homology between the rAAV vector and the AAV helper plasmid were created40,41, and this has greatly reduced or eliminated contaminating wild-type AAV from rAAV vector stocks. Fortunately, stable cell line production approaches do not appear to produce detectable wild-type-like AAV16,20,21. To test vector stocks for replication competent AAV, 1% of the rAAV stock should be used in a serial passage assay (typically on adenovirus-infected 293 or HeLa cells) to confirm the absence of wild-type AAV replication intermediates (monomeric and dimeric forms) in DNA isolated from the P2 cell population16.
Notes
ZANG X, LI CY: Generation of recombinant adeno-associated virus vectors by a complete adenovirus-mediated approach. Mol Ther 3:787–792, 2001
References
| 1. | Grimm D & Kleinschmidt JA. Progress in adeno-associated virus type 2 vector production: Promises and prospects for clinical use. Hum Gene Ther 1999; 10: 2445−2450. | Article | PubMed | ISI | ChemPort | |
| 2. | McLaughlin SK, Collis P, Hermonat PL & Muzyczka N. Adeno-associated virus general transduction vectors: Analysis of proviral structures. J Virol 1989; 62: 1963−1973. | ISI | |
| 3. | Samulski RJ, Chang LS & Shenk T. Helper-free stocks of recombinant adeno-associated viruses: Normal integration does not require viral gene expression. J Virol 1989; 63: 3822−3828. | PubMed | ISI | ChemPort | |
| 4. | Vincent KA, Piraino ST & Wadsworth SC. Analysis of recombinant adeno-associated virus packaging and requirements for rep and cap gene products. J Virol 1997; 71: 1897−1905. | PubMed | ISI | ChemPort | |
| 5. | Ogasawara Y, Urabe M & Ozawa K. The use of heterologous promoters for adeno-associated virus (AAV) protein expression in AAV vector production. Microbiol Immunol 1998; 42: 177−185. | PubMed | ISI | ChemPort | |
| 6. | Li J, Samulski RJ & Xiao X. Role for highly regulated rep gene expression in adeno-associated virus vector production. J Virol 1997; 71: 5236−5243. | PubMed | ISI | ChemPort | |
| 7. | Grimm D, Kern A, Rittner K & Kleinschmidt JA. Novel tools for production and purification of recombinant adeno-associated virus vectors. Hum Gene Ther 1998; 9: 2745−2760. | PubMed | ISI | ChemPort | |
| 8. | Ferrari FK, Samulski T, Shenk T & Samulski RJ. Second-strand synthesis is a rate-limiting step for efficient transduction by recombinant adeno-associated virus vectors. J Virol 1996; 70: 3227−3234. | PubMed | ISI | ChemPort | |
| 9. | Xiao X, Li J & Samulski RJ. Production of high-titer recombinant adeno-associated virus vectors in the absence of helper adenovirus. J Virol 1998; 72: 2224−2232. | PubMed | ISI | ChemPort | |
| 10. | Collaco RF, Cao X & Trempe JP. A helper virus-free packaging system for recombinant adeno-associated virus vectors. Gene 1999; 238: 397−405. | Article | PubMed | ISI | ChemPort | |
| 11. | Matsushita T, Elliger S & Elliger C et al. Adeno-associated virus vectors can be efficiently produced without helper virus. Gene Ther 1998; 5: 938−945. | Article | PubMed | ISI | ChemPort | |
| 12. | Salvetti A, Oreve S & Chadeuf G et al. Factors influencing recombinant adeno-associated virus production. Hum Gene Ther 1998; 9: 695−706. | PubMed | ISI | ChemPort | |
| 13. | Weger S, Wistuba A, Grimm D & Kleinschmidt JA. Control of adeno-associated virus type 2 cap gene expression: Relative influence of helper virus, terminal repeats, and Rep proteins. J Virol 1997; 71: 8437−8447. | PubMed | ISI | ChemPort | |
| 14. | Wistuba A, Kern A & Weger S et al. Subcellular compartmentalization of adeno-associated virus type 2 assembly. J Virol 1997; 71: 1341−1352. | PubMed | ISI | ChemPort | |
| 15. | Allen JM, Halbert CL & Miller AD. Improved adeno-associated virus vector production with transfection of a single helper adenovirus gene, E4orf6. Mol Ther 2000; 1: 88−95. | Article | PubMed | ISI | ChemPort | |
| 16. | Clark KR, Voulgaropoulou F, Fraley DM & Johnson PR. Cell lines for the production of recombinant adeno-associated virus. Hum Gene Ther 1995; 6: 1329−1341. | PubMed | ISI | ChemPort | |
| 17. | Tamayose K, Hirai Y & Shimada T. 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 1996; 7: 507−513. | PubMed | ISI | ChemPort | |
| 18. | Liu XL, Clark KR & Johnson PR. Production of recombinant adeno-associated virus vectors using a packaging cell line and a hybrid recombinant adenovirus. Gene Ther 1999; 6: 293−299. | Article | PubMed | ISI | ChemPort | |
| 19. | Inoue N & Russell DW. Packaging cells based on inducible gene amplification for the production of adeno-associated virus vectors. J Virol 1998; 72: 7024−7031. | PubMed | ISI | ChemPort | |
| 20. | Gao GP, Qu G & Faust LZ et al. High-titer adeno-associated viral vectors from a Rep/Cap cell line and hybrid shuttle virus. Hum Gene Ther 1998; 9: 2353−2362. | PubMed | ISI | ChemPort | |
| 21. | Conway JE, Rhys CM & Zolotukhin I et al. High-titer recombinant adeno-associated virus production utilizing a recombinant herpes simplex virus type I vector expressing AAV-2 Rep and Cap. Gene Ther 1999; 6: 986−993. | Article | PubMed | ISI | ChemPort | |
| 22. | Vincent KA, Moore GK & Haigwood NL. Replication and packaging of HIV envelope genes in a novel adeno-associated virus vector system. Vaccine 1990; 90: 353−359. |
| 23. | Clark KR, Liu X, McGrath JP & Johnson PR. Highly purified recombinant adeno-associated virus vectors are biologically active and free of detectable helper and wild-type viruses. Hum Gene Ther 1999; 10: 1031−1039 10.1089/10430349950018427. | Article | PubMed | ISI | ChemPort | |
| 24. | Thrasher AJ, de Alwis M & Casimir CM et al. Generation of recombinant adeno-associated virus (rAAV) from an adenoviral vector and functional reconstitution of the NADPH-oxidase. Gene Ther 1995; 2: 481−485. | PubMed | ISI | ChemPort | |
| 25. | Fisher KJ, Kelley WM, Burda JF & Wilson JM. A novel adenovirus-adeno-associated virus hybrid vector that displays efficient rescue and delivery of the AAV genome. Hum Gene Ther 1996; 7: 2079−2087. | PubMed | ISI | ChemPort | |
| 26. | Thomson BJ, Efstathiou S & Honess RW. Acquisition of the human adeno-associated virus type-2 rep gene by human herpesvirus type-6. Nature 1991; 351: 78−80. | Article | PubMed | ISI | ChemPort | |
| 27. | Conway JE, Zolotukhin S & Muzyczka N et al. Recombinant adeno-associated virus type 2 replication and packaging is entirely supported by a herpes simplex virus type 1 amplicon expressing Rep and Cap. J Virol 1997; 71: 8780−8789. | PubMed | ISI | ChemPort | |
| 28. | Fan PD & Dong JY. Replication of rep-cap genes is essential for the high-efficiency production of recombinant AAV. Hum Gene Ther 1997; 8: 87−98. | PubMed | ISI | ChemPort | |
| 29. | Chiorini JA, Wendtner CM & Urcelay E et al. 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 1995; 6: 1531−1541. | PubMed | ISI | ChemPort | |
| 30. | Zhang X, De Alwis M & Hart SL et al. High-titer recombinant adeno-associated virus production from replicating amplicons and herpes vectors deleted for glycoprotein H. Hum Gene Ther 1999; 10: 2527−2537. | Article | PubMed | ISI | ChemPort | |
| 31. | Inoue N & Russell DW. Packaging cells based on inducible gene amplification for the production of adeno-associated virus vectors. J Virol 1998; 72: 7024−7031. | PubMed | ISI | ChemPort | |
| 32. | Liu X, Voulgaropoulou F & Chen R et al. Selective rep-cap gene amplification as a mechanism for high-titer recombinant AAV production from stable cell lines. Mol Ther 2000; 2: 394−403. | Article | PubMed | ISI | ChemPort | |
| 33. | Chadeuf G, Favre D & Tessier J et al. Efficient recombinant adeno-associated virus production by a stable rep-cap HeLa cell line correlates with adenovirus-induced amplification of the integrated rep-cap genome. Gene Med 2000; 2: 260−268. | ChemPort | |
| 34. | Zolotukhin S, Byrne BJ & Mason E et al. Recombinant adeno-associated virus purification using novel methods improves infectious titer and yield. Gene Ther 1999; 6: 973−985. | Article | PubMed | ISI | ChemPort | |
| 35. | Grimm D, Kern A & Pawlita M et al. Titration of AAV-2 particles via a novel capsid ELISA: packaging of genomes can limit production of recombinant AAV-2. Gene Ther 1999; 6: 1322−1330. | Article | PubMed | ISI | ChemPort | |
| 36. | Yakobson B, Koch T & Winocour E. Replication of adeno-associated virus in synchronized cells without the addition of a helper virus. J Virol 1987; 61: 972−981. | PubMed | ISI | ChemPort | |
| 37. | Clark KR, Voulgaropoulou F & Johnson PR. A stable cell line carrying adenovirus-inducible rep and cap genes allows for infectivity titration of adeno-associated virus vectors. Gene Ther 1996; 3: 1124−1132. | PubMed | ISI | ChemPort | |
| 38. | Alexander IE, Russell DW & Miller AD. Transfer of contaminants in adeno-associated virus vector stocks can mimic transduction and lead to artifactual results. Hum Gene Ther 1997; 8: 1911−1920. | PubMed | ISI | ChemPort | |
| 39. | Tenenbaum L, Hamdane M & Pouzet M et al. Cellular contaminants of adeno-associated virus vector stocks can enhance transduction. Gene Ther 1999; 6: 1045−1053. | Article | PubMed | ISI | ChemPort | |
| 40. | Wang XS, Khuntirat B & Qing K et al. Characterization of wild-type adeno-associated virus type 2-like particles generated during recombinant viral vector production and strategies for their elimination. J Virol 1998; 72: 5472−5480. | PubMed | ISI | ChemPort | |
| 41. | Allen JM, Debelak DJ, Reynolds TC & Miller AD. Identification and elimination of replication-competent adeno-associated virus (AAV) that arise by nonhomologous recombination during AAV vector production. J Virol 1997; 71: 6816−6822. | PubMed | ISI | ChemPort | |
