Production and Quality Control of High-Capacity Adenoviral Vectors
High-capacity adenovirus (HC-Ad) vectors [also called pseudoadenovirus (PAV), helper-dependent (HD-Ad), gutted, or gutless adenovirus vectors] have been developed to address capacity, toxicity, and immunogenicity problems of first- and secondgeneration adenovirus vectors. As only viral elements this vector type contains the inverted terminal repeats (ITRs), which are essential for replication of the viral DNA, and the packaging signal close to the left terminus that is required for encapsidation of the DNA into the viral capsids. Since the size of the ITRs and the packaging signal together are less than 0.6kb, up to 37kb of foreign DNA can be transported.
For practical reasons, most HC-Ad vectors will carry genes or expression cassettes that are smaller than 37kb. For stability reasons during amplification in most cases additional "stuffer" DNA has to be incorporated into the vector DNA to increase the genome size to at least around 27kb.
HC-Ad vectors cannot be produced similar to helper-independent vectors, in which most viral functions are provided from the vector. Because adenovirus is a relatively large DNA virus that expresses many different protein and RNA functions, it is unlikely that complementing cell lines can be generated to provide appropriate levels of all viral functions in trans. Therefore, a helper virus is used for production that subsequently is eliminated from the end product.
The currently preferred production system is based on excision of the packaging signal of the helper virus by a recombinase expressed in the producer cell line. Most HC-Ad vectors so far have been produced using the Cre-loxP recombination system of the bacteriophage PI. In this system the packaging signal of the helper virus is flanked by two loxP sites (Hardy et al., 1997; Parks et al., 1996). The HC-Ad vector is produced in El-complementing cells that express the recombinase constitutively. The Cre-mediated excision is surprisingly efficient and the contamination of vector by helper virus is reduced compared to the earlier production system.
The complete characterization of HC-Ad vector preparations comprises three parameters: (1) the number of infectious particles, (2) the number of total particles, and (3) the number helper virus particles remaining in the preparation after purification (Kreppel et al., 2002). Due to the fact that HC-Ad vectors do not possess any viral coding sequences, the number of infectious particles cannot be determined by plaque assay or tissue culture infectivity dose TCID50 because these methods rely on vector replication and viral protein expression in El-transformed cell lines. The number of total particles can be determined by particle lysis and subsequent measuring light absorbance at 260nm. However, the reliability of this method is usually low and strongly depends on the purity of the vector preparations. The DNA-based method described here allows for fast and reliable determination of all three parameters with standard laboratory equipment independent of viral or reporter gene expression. For quantifying the number of infectious particles, a reference cell line with defined susceptibility for Ad5 is transduced with the HC-Ad vector, cell lysates are prepared, and vector genomes that entered the cells are detected after immobilization on a nylon membran by hybridization with a radiolabeled vector-specific probe. Total particle numbers can easily be determined with the same probe on the same membrane by preparing particle lysates and immobilizing the DNA. Finally, by choosing a probe specific for the helper virus genome the number of helper virus particles can be quantified in the particle lysates.
This article is divided in two parts: in the first part the production process includes serial amplification in producer cells and ends with CsCl density centrifugation for purification. In the second part a method for titration of purified vectors is described that is based on slot blot methods and that not only allows one to determine particle and infectious vector titers, but also the contamination with helper virus.
II. PRODUCTION OF HC-Ad VECTORS
Cells and Plasmids
El-Expressing Cell Lines
Several cell lines have been described that express the adenovirus type 5 (Ad5) early region E1 (E1A and EI B) and thus support the growth of El-deleted vectors and helper viruses (Fallaux et al., 1998; Gao et al., 2000; Graham et al., 1977; Schiedner et al., 2000). In contrast to HEK293 cells, PER.C6 and N52.E6 cells have been designed not to produce replicationcompetent adenovirus (RCA) and thus are a preferred cell type for the production of El-deleted vectors and helper viruses.
Cre-Expressing Cell Lines
The production process of HC-Ad vectors builds on two viral components; vector and El-deleted helper virus with packaging signal flanked by loxP sites and serial amplifications in producer cells expressing Crerecombinase. Excision of the packaging signal from the helper virus in the producer cells results in preferential packaging of vector genomes and a reduction of helper virus contamination. Several HEK293-based Cre-expressing cell lines have been described (Hilgenberg et al., 2001; Parks et al., 1996). Similar to first-generation adenoviral vectors, the helper virus is prone to turn into RCA when HEK-293 based cells are used for HC-Ad vector production. Based on PER.C6, a Cre-expressing cell line has been described preventing the occurrence of RCA in HC-Ad vector preparations (Sakhuja et al., 2003). Similarly, a Cre-expressing cell line 73/29 has been generated (manuscript submitted) that is based on N52.E6 cells.
Helper Virus Plasmids
A preferred helper virus used for preparation of HC-Ad vectors contains loxP (Parks et al., 1996) or frt (Ng et al., 2001; Umana et al., 2001) sites flanking the packaging signal. Consequently, in Cre- or FLPerecombinase expressing producer cells the packaging signal of the helper virus is excised and vector genomes are preferentially packaged. Most published helper viruses are E1 and E3 deleted and contain a reporter gene cassette in E3 in order to allow easy quantitation of helper virus contamination (Hartigan- O'Connor et al., 2002; Hilgenberg et al., 2001; Ng et al., 2001; Palmer and Ng, 2003; Parks et al., 1996; Sandig et al., 2000; Umana et al., 2001). However, this insertion may affect helper virus yield and can provoke an immune reaction against the reporter in vivo. Recombination events between helper and vector inverted terminal repeats (ITRs) occur frequently and can result in loss of a loxP site in the helper virus genomes and thus outgrowth of mutated helper virus. Figure 1 shows the schematic structure of helper virus plasmid pGS102#21, which contains Ad5 sequences from nucleotides 1-341 with a loxP site introduced at nucleotide 192, a second loxP site introduced at nucleotide 341 followed by 4.6 kb of λ DNA, and Ad5 sequence nucleotides 3523 to 35935. The adenoviral packaging signal consists of seven functional units called A repeats with the consensus sequence [ATTTGN8GC] (Schmid and Hearing, 1998). In order to minimize sequence homologies between vector and helper virus genome, pGS102#21 contains only A repeats I to IV. In addition, sequences from phage λ were inserted in the helper virus genome in order to increase the size of the helper virus, thus improving separation of helper and vector particles in a CsCl- gradient, pGS102#21 is an infectious plasmid based on pBluescript with unique SwaI sites flanking both ITRs.
Plasmids for Cloning HC-Ad Vectors
Early observations using different sizes of HC-Ad vectors suggested that only vector genomes with sizes of at least 27kb allowed efficient and stable vector amplification (Parks and Graham, 1997). The sizes of most expression cassettes used currently for gene transfer are smaller than this 27-kb minimal size for HC-Ad vectors. Therefore, additional DNA has to be incorporated into the vector genome as stuffer DNA. Since the source of the stuffer sequences may influence transgene expression (Parks et al., 1999), the use of noncoding DNA from human origin is preferred. Figure 2 shows different plasmids constructed for the incorporation of different sized transgenes. Most plasmids contain noncoding stuffer sequences derived either from the human HPRT locus or from the human cosmid C346. In addition, all plasmids contain left (nucleotides 1-440) and right (nucleotides 35818-35935) termini of Ad5 DNA. Both left and right adenoviral termini are flanked by unique PmeI or SnaBI (pSTK142) restriction sites. The plasmid backbone in all constructs is pBluescript.
The HC-Ad vector AdGS46 has been described previously (Thomas et al., 2000) and is based on pSTK120. Plasmid pGS46 contains the Ad5 left terminus, 16 kb of HPRT stuffer, a β-Gal expression cassette containing the hCMV promoter and SV40 polyadenylation signal, a 9-kb stuffer from C346, and the Ad5 right terminus (Fig. 1).
Cell Culture Reagents
All tissue culture reagents, including (x-modified Eagle's medium (αMEM, Cat. No. 12000-063), fetal calf serum (FBS, Cat. No. 10270-106), phosphate-buffered saline (PBS, Cat. No. 14190-169), trypsin solution (Cat. No. 25300-096), and antibiotics (Cat. No. 10378-016) are from Invitrogen. G418 for selection of neomycin expression is from Sigma (geneticin, Cat. No. G5013). Tissue culture dishes (6- and 15-cm dishes) are from Renner.
Additional Reagents and Solutions
Centrifuge tubes: SW41 ultraclear centrifuge tubes (Beckmann, Cat. No. 344059)
Desalting column: PD-10 column (Amersham Bioscience, Cat. No. 17-0851-01)
Chloroform/isoamyl alcohol: Mix 10ml isoamyl alcohol with 230ml chloroform
TE: 10 mM Tris, 1 mM EDTA, pH 7.5
0.5M EDTA: Dissolve 186.1 g EDTA-2H2O in 800ml H2O, adjust pH to 8.0 using NaOH pellets (approximately 20 g), fill up to 1 liter, and autoclave
MEM agarose overlay: Dissolve 1g agarose (AppliChem, Cat. No. A2114,0500) in 100ml H2O, autoclave, and cool to 40°C. Add prewarmed (37°C) 90ml 2× MEM (Gibco, Cat. No. 61100-087), 10ml FBS, 1 ml antibiotics (Gibco, Cat. No. 10378-016), and 2 ml 5% yeast extract
Na-acetate (3M): Dissolve 24.6g Na-acetate in 100ml H2O, adjust pH to 5.2 using acetic acid, and autoclave
Phenol, buffer saturated: Gibco (Cat. No. 15513-039)
Proteinase K (Sigma, Cat. No. P6556): 5mg/ml dissolved in sterile H2O
QIAamp DNA Minikit: Qiagen (Cat. No. 51104)
RNase: DNase free (Roche, Cat. No. 1119915)
10% SDS: Dissolve 10 g SDS in 100 ml sterile H2O
Superfect transfection kit (Qiagen Cat. No. 301305)
Tris-buffered saline (TBS): 25 mM Tris, 137 mM NaCl, 2.7 mM KCl, pH 7.4, autoclave
TBS/10% glycerol: TBS containing 10% glycerol
TBS/CsCl: Dissolve 10g CsCl (Roche, Cat. No. 757306) in 20 ml sterile TBS
Ultracentrifuge, SW41 rotor: e.g., Beckmann
Yeast extract (5%): Dissolve 2g yeast extract (AppliChem, Cat. No. A1552,0100) in 40ml H2O, sterile filter
Production of Helper Virus
Preparation of DNA for Transfection
Preparation of N52.E6 Cells for Transfection
N52.E6 cells are used for the production of helper virus and are cultivated in αMEM supplemented with 10% FBS and antibiotics in 5% CO2 at 37°C. N52.E6 cells are usually passaged twice a week 1:4-5.
DNA transfections can also be performed using the calcium-phosphate coprecipitation method as described in a number of different manuals. The commercially available Effectene transfection Kit (Qiagen) also shows very high efficiency in transfection but should not be used for large-sized linearized plasmids.
Plaque Isolation and Preparation of High-Titer Helper Virus Stocks
Gradient Purification of Helper Virus
Titration of Helper Virus
DNA Preparation from CsCl-Purified Helper Virus Particles
In order to exclude helper virus rearrangement, viral DNA should be isolated from CsCl-purified particles. Take 200µl of gradient-purified and desalted vector. Isolate DNA using the QiaAmp DNA Minikit according to the manufacturer. Subsequent to ethanol precipitation, dissolve the pellet in 20µl AE buffer. Digest 10µl with the appropriate enzyme and run in an 0.8% agarose gel containing EtBr. As a positive control, double digest the corresponding helper virus plasmid with SwaI and the aforementioned enzyme.
Production of HC-Ad Vectors
Cloning of HC-Ad Vector Plasmids
General cloning reagents and equipment, as well as procedures, have been described in a number of cloning manuals. Thus, only important steps, observations, and suggestions are discussed in this section.
Preparation of Plasmid DNA for Transfection
In order to release the left and right adenoviral termini (ITR), HC-Ad vector plasmids usually contain restriction sites flanking both ITRs. ITRs in the HC-Ad vector plasmids depicted in Fig. 2 are flanked by unique PmeI sites (or SnaBI site in pSTK142). The HCAd vector plasmid is digested and further purified as described earlier for the helper virus plasmid.
Culture of 73/29 Cells
Cre-expressing 73/29 cells are used for the production of HC-Ad vectors and are cultivated in αMEM containing 10% FBS and antibiotics, supplemented with 200µg/ml G418. 73/29 cells were usually passaged twice a week and diluted 1:4-5.
Amplification of HC-Ad Vectors
Second and Third Amplification
Fourth and Fifth Amplification
After the fifth amplification it is important to titer the amount of vector present in the lysate. In case HCAd vectors express a reporter gene (e.g., β-gal, EGFP), the vector can be titered easily in a reporter gene assay. Figure 3 shows blue forming unit (bfu) titers in amplifications of AdGS46 (see Fig. 1). However, most HCAd vectors do not contain a reporter gene and thus the ratio of helper and vector genomes and possible vector genome rearrangements should be tested using DNA isolated from infected cells.
At this stage of amplification the majority of DNA within the producer cell is vector DNA with only little helper virus contamination. Therefore, if the amplification was successful, only bands corresponding to the vector should be visible with a weak background of the helper virus bands and a faint smear of cellular DNA. In addition, rearranged vector genomes can be detected. If there are no or only weak vector bands visible, one or two additional amplifications can be performed. However, it is not recommended to carry out more than seven amplifications, as the risk of outgrowth of mutated helper virus and vector rearrangements increases significantly.
Preparation of Gradient-Purified HC-Ad Vector
DNA Preparation from CsCl-Purified Vector Particles
In order to finally exclude vector rearrangement vector DNA should be isolated from CsCl-purified particles as described for extraction of helper virus DNA. The DNA should be digested with the appropriate enzyme and run on a 0.8% agarose gel containing EtBr. As a positive control, double digest the corresponding HC-Ad vector plasmid with PmeI and the aforementioned enzyme.
III. COMPLETE CHARACTERIZATION OF HC-Ad VECTOR PREPARATIONS
First Day: Seeding Cells
Cell lines: A549 (ATCC number: CCL-185) or, alternatively, HeLa cells (ATCC number: CCL-2)
Supplies: 24-well tissue culture plates
Second Day: Transduction of Cells with HC-Ad Vectors
Reagents and solutions: Tris-buffered saline (TBS, see Section II.)
Third Day: Slot Blotting of Cell and Vector Lysates; Hybridization
Shaker (e.g., Scientific Industries : Vortex Genie Model G-560E)
Vacuum pump (e.g., Biometra: Typ PM12640-026.3)
Slot-blot apparatus (e.g., Amersham Biosciences: Hoefer PR648)
Hybridisation oven (e.g., Biometra: Duo-Thermo- Oven OV5)
Positively charged nylon membrane (Pall: Biodyne B, 0.45 µm)
50µCi [32P] dCTP
RediPrime II DNA labeling kit (Amersham- Biosciences)
Reagents and Solutions
Phosphate-buffered saline (PBS): 6.46mM Na2HPO4, 1.47mM KH2PO4, 137mM NaCl, 2.7mM KCl, pH 7.4, autoclave
PBS/20mM EDTA: Add EDTA from aqueous stock solution (0.5M, pH 8, see Section II) to PBS
0.8N NaOH: Dissolve 32g NaOH pellets in 1 liter deionized H2O, always prepare fresh
(Pre-)hybridisation solution: 2 × SSC (300mM NaCl, 30mM Na-citrate, pH 7.0) containing 10% dextran sulfate, 1% SDS, 0.5% milk powder (low fat), and 0.5 mg/ml salmon sperm DNA
DNA Templates for Generation of Probes
Template for generation of HC-Ad vector-specific probe: This should be a purified PCR fragment of 600-1200bp length. Usually the PCR fragment is derived from the stuffer DNA or the transgene expression cassette of the HC-Ad vector plasmid. This probe is used for determination of both the number of infectious units and total particles. Alternatively, an Ad5-ITR probe can be used that is generated by PCR with the primers P1 (5'-CATCATCAATAATATACCTTATTTTG- 3') and P2 (5'- AACGCCAACTTTGACCCGGAACGCGG-3') from a plasmid containing at least the left Ad5 ITR.
Template for generation of helper virus-specific probe: This is a PCR fragment comprising Ad5 nucleotides 31042-32390 (Ad5 fiber gene) obtained from a plasmid containing the fiber gene of Ad5 with the primers P3 (5'-ATGAAGCGCGCAAGACCGTCTG- 3') and P4 (5'-CCAGATATTGGAGCCAAACTGCC- 3').
Plasmids for Generating Standard Curves
Comment: It is recommended to prepare a large stock of standard plasmids at appropriate concentrations (1-3E+06 copies/µl) and keep it frozen (-20°C).
The plasmid used to generate the standard curve for determining infectious and total particle contents of HC-Ad vector preparations is usually the HC-Ad vector shuttle plasmid that was used to generate the vector and/or the PCR template for the probe. The concentration of this standard plasmid should be determined as accurately as possible.
The plasmid used to generate the standard curve for determining the helper virus content of HC-Ad vector preparations is usually a shuttle plasmid for the generation of El-deleted vectors and should contain the Ad5 fiber gene. The concentration of this standard plasmid should be determined as accurately as possible.
Fourth Day: Exposition of PhosphorScreen and Signal Quantification
PhosphorScreen (e.g., Amersham-Biosciences/Molecular Dynamics: Kodak Storage Phosphor Screen SO230)
PhosphorImager (e.g., Amersham-Biosciences/Molecular Dynamics: Storm 860)
Quantification Software (e.g., Amersham- Biosciences/Molecular Dynamics: ImageQuaNT)
Reagents and Solutions
Wash buffer I: 2 × SSC (300 mM NaCl, 30 mM Na-citrate, pH 7.0) containing 0.1% SDS
Wash buffer II: 0.1 × SSC (15mM NaCl, 1.5mM Nacitrate, pH 7.0) containing 0.1% SDS
First Day: Seeding Cells
Second Day: Transduction of Cells with HC-Ad Vectors
Third Day: Slot Blotting of Cell and Particle Lysates; Hybridization
Preparation of Cell Lysates from Transduced Cells for Determining the Number of Infectious Particles
Preparation of Cell Lysates for Generation of a Standard Curve for Infectious Particles
Comment: The debris in the cell lysate that is slot blotted onto the membrane will influence the signal intensity due to partial blocking of the membrane. Therefore, to obtain reliable standard curves for determining the number of infectious HC-Ad vector particles, it is absolutely required to mix the standard plasmid with untransduced cells and prepare lysates in the same way as for transduced cells before blotting.
Preparation of Particle Lysates for Determining the Number of Total Particles
Comment: Particle lysates for determining the number of total and helper virus particles do not contain any debris that blocks the membrane. Therefore, standard plasmids are prepared in PBS/EDTA and treated the same way as particle lysates.
Preparation of a Standard for Determining the Number of Total Particles
Preparation of a Standard for Determining the Number of Helper Virus Particles
Slot Blotting of Lysates and Standard onto Positively Charged Nylon Membranes
Comment: The cell lysates and particle lysates for determining the number of infectious and total HCAd- vector particles can be blotted onto the same membrane, as the same probe is used for hybridization. Particle lysates for determining the number of helper virus particles must be blotted onto a separate membrane and will be hybridized with a different probe.
Fourth Day: Exposition of PhosphorScreen and Signal Quantification
Figure 4a shows a typical result for determination of the number of infectious particles of different HCAd vector preparations. Figure 4b shows the standard curve obtained from the signal intensities of the standard plasmid. The parameters of the standard curve (slope m and intercept b) were used to calculate the number of infectious particles of the vector preparations HC-AdV1 to HC-AdV4. These titers are given in Table I. Slot blots to determine the number of total particles and helper virus particles look similar and are omitted for brevity. The inverse bioactivity (number of total particles divided by number of infectious particles) usually is between 10 and 200, and the helper virus contamination is between 0.1 and 2%.
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