A protocol for generation of high-capacity adenoviral vectors lacking all viral coding sequences is presented. Cloning of transgenes contained in the vector genome is based on homing endonucleases. Virus amplification in producer cells grown as adherent cells and in suspension relies on a helper virus providing viral genes in trans.
High-capacity adenoviral vectors (HCAdV) devoid of all viral coding sequences represent one of the most advanced gene delivery vectors due to their high packaging capacity (up to 35 kb), low immunogenicity, and low toxicity. However, for many laboratories the use of HCAdV is hampered by the complicated procedure for vector genome construction and virus production. Here, a detailed protocol for efficient cloning and production of HCAdV based on the plasmid pAdFTC containing the HCAdV genome is described. The construction of HCAdV genomes is based on a cloning vector system utilizing homing endonucleases (I-CeuI and PI-SceI). Any gene of interest of up to 14 kb can be subcloned into the shuttle vector pHM5, which contains a multiple cloning site flanked by I-CeuI and PI-SceI. After I-CeuI and PI-SceI-mediated release of the transgene from the shuttle vector the transgene can be inserted into the HCAdV cloning vector pAdFTC. Because of the large size of the pAdFTC plasmid and the long recognition sites of the used enzymes associated with strong DNA binding, careful handling of the cloning fragments is needed. For virus production, the HCAdV genome is released by NotI digest and transfected into a HEK293 based producer cell line stably expressing Cre recombinase. To provide all adenoviral genes for adenovirus amplification, co-infection with a helper virus containing a packing signal flanked by loxP sites is required. Pre-amplification of the vector is performed in producer cells grown on surfaces and large-scale amplification of the vector is conducted in spinner flasks with producer cells grown in suspension. For virus purification, two ultracentrifugation steps based on cesium chloride gradients are performed followed by dialysis. Here tips, tricks, and shortcuts developed over the past years working with this HCAdV vector system are presented.
For gene therapeutic applications it is of great importance to avoid cytotoxic and immunogenic side effects caused by expression of viral proteins, the transgene itself, or by incoming viral proteins. Adenovirus vectors (AdV) are widely used to introduce foreign DNA into a wide variety of cells to investigate the impact of transgene expression 1,2. The most advanced version of AdV is represented by high-capacity adenovirus vectors (HCAdV) lacking all viral coding sequences 3,4 and thereby offering a packaging capacity up to 35 kb combined with low immunogenicity and low toxicity 5-8. Due to their high packaging capacity they allow delivery of large or multiple transgenes using a single vector dose. Therefore, they represent a valuable tool for the research community.
In contrast to first- or second-generation AdV lacking the early genes E1 and/or E3 that can be easily produced using commercial kits, vector genome construction and virus production of HCAdV is more complex. The system for the construction of HCAdV genomes is based on the plasmid pAdFTC carrying a HCAdV genome devoid of all viral coding sequences and the shuttle plasmid pHM5 9-12. Any gene of interest of up to 14 kilobases (kb) can be cloned into the shuttle vector pHM5 in which the multiple cloning site is flanked by recognition/cleaving sites of the homing endonucleases PI-SceI and I-CeuI. Therefore, a cloned gene of interest can be released by consecutive PI-SceI and I-CeuI digests for subsequent directed insertion into the same restriction sites present in the HCAdV genome contained in the plasmid pAdFTC. In pAdFTC the transgene insertion site located between the PI-SceI and I-CeuI cleavage sites is flanked by stuffer DNA and the noncoding adenoviral sequences required for genome packaging such as the 5' and 3' inverted terminal repeats (ITRs) at both ends and the packaging signal downstream of the 5'ITR. The additional stuffer DNA provides optimal size of the final HCAdV genome ranging from 27 to 36 kb to ensure efficient packaging during virus production. Since pAdFTC is a large plasmid with up to 45 kb (dependent on the size of the inserted transgene) and the usage of homing endonucleases with comparably long DNA recognition sites exhibits strong DNA binding, several cleanup steps are necessary during transfer of the transgene from pHM5 to pAdFTC. Careful handling avoiding shearing forces is recommended.
The ITRs of the HCAdV genome are flanked by NotI restriction enzyme recognition sites located directly upstream of the 5'ITR and downstream of the 3'ITR 12. Therefore, HCAdV can be released by NotI digest for subsequent transfection of the viral genome into the HCAdV producer cell line. Note that the usage of the restriction enzyme NotI for release of the viral genome from the plasmid pAdFTC implies that the inserted transgene is devoid of NotI DNA recognition sites. The HEK293 cell based producer cells (116 cells) stably express Cre recombinase. For virus amplification 116 cells are co-infected with a helper virus (HV) providing all AdV genes needed for replication and packaging in trans 3,4. The HV is a first-generation AdV with a floxed packing signal which is removed during virus amplification by Cre recombinase expressed in 116 cells 4. This ensures that predominantly HCAdV genomes containing an intact packaging signal are encapsidated.
Pre-amplification of the HCAdV is performed by conducting serial passaging steps in 116 cells grown on surfaces in tissue culture dishes. After each passage viral particles are released from infected cells by conducting three consecutive freeze-thaw steps. With every passage increasing numbers of cells are infected with 1/3 of cell lysate from the preceding passage. Finally lysate from the last pre-amplification step is used to infect producer cells grown in suspension in a spinner flask for large scale amplification. Virions are purified from the suspension cells by performing ultracentrifugation in a cesium chloride density gradient 4,12. With this procedure empty particles and fully assembled particles are separated into two distinct bands. To further concentrate the HCAdV particles a second non-gradual ultracentrifugation step is performed. Subsequently the resulting band containing the HCAdV is collected and dialyzed against a physiological buffer. Final vector preparations are characterized with respect to numbers of absolute viral particles, infectious particles and HV contamination levels. Absolute viral particles can be determined by lysing viral particles and measuring the absorbance at 260 nm or by performing quantitative real-time PCR (qPCR) 12. Infectivity of the purified virus particles can be determined by qPCR measuring HCAdV genomes present within infected cells 3 hr post-infection.
1. Construction of Recombinant HCAdV Genomes based on the Plasmid pAdFTC
Note: All plasmids have been described previously 11,12 and are available upon request. The cloning procedure is schematically shown in Figure 1.
2. Release of HCAdV-genomes from pAdFTC Plasmid and Preamplification of HCAdV Vectors in the Producer Cell Line 116
3. Monitoring of the Amplification Process using Quantitative-Real Time PCR (qPCR) (see also Figure 4A).
4. Large Scale Amplification of HCAdV Vectors in 116 cells Growing in Suspension
5. Purification and Dialysis of HCAdV
6. Measuring the Physical Titer of Final HCAdV Preparations by Optical Density (OD)
7. Measuring Total Particles, Infectious Units of the HCAdV and HV Contamination Levels in the Final Vector Preparation by qPCR. A Scheme of the Titration Procedure is Shown in Figure 6
Here representative examples for cloning, amplification and purification of HCAdV preparations are shown. An overview of the cloning strategy (Figure 1) and representative examples for cloning and release of the HCAdV genome by restriction enzyme digest are provided (Figure 2). A typical restriction pattern after release of the GOI-expression cassette from pHM5 by PI-SceI and I-CeuI digest and subsequent phenol-chloroform extraction and EtOH precipitation (see also steps 1.2-1.5) is shown (Figure 2A). Typically a ~3 kb band corresponding to the pHM5 plasmid backbone and a second band corresponding to the expression cassette of the GOI can be observed. The band containing the GOI-expression cassette is then purified from the agarose gel. The purified GOI-expression cassette is analyzed on an agarose gel together with the purified, dephosphorylated pAdVFTC that was digested with PI-SceI and I-CeuI respectively (step 1.7) to verify the correct size and to estimate the relative amount of molecules for subsequent ligation (Figure 2B). After ligation (see step 1.8) followed by phenol-chloroform extraction and EtOH precipitation and SwaI digest to eliminate uncut pAdFTC and subsequent phenol-chloroform extraction and EtOH precipitation (step 1.9), ligation products are transformed into bacteria and clones are selected on ampicillin containing LB- agar plates. Usually dozens to hundreds of clones are obtained, from which 5-10 clones are prepared for plasmid DNA mini-preparations. A restriction pattern of an analytical HincII digest of positive and negative clones together with an empty pAdFTC as negative control (see also step 1.10) is shown (Figure 2C). Here positive clones do not show a 5 kb band, that is present in empty pAdFTC and negative clones and a 1.7 kb fragment appears that is not present in the empty pAdFTC and negative clones. For this respective GOI this indicates tha cloning was successful. A positive clone that has been purified via midiprep was then digested with NotI to release the HCAdV-genome carrying the GOI from pAdVFTC-GOI (step 2.2). Finally NotI digested DNA ist purified twice using phenol-chloroform extraction and EtOH precipitation to ensure high purity of the DNA for subsequent transfection into 116 cells. Analysis of a small fraction of the NotI digested pAdVFTC-GOI on an agarose gel shows a ~9 kb band corresponding to the pAdFTC plasmid backbone and a large band of up to ~36 kb (depending on the size of the GOI expression cassette) corresponding to the HCAdV genome (Figure 2D). An overview of the amplification and purification pipeline is schematically shown (Figure 3). Representative examples of the monitoring of the vector pre-amplification process are provided (Figure 4). A typical amplification curve of viral genomes during the pre-amplification process by serial passaging of viral lysates, each time coinfecting with HV (section 2), that was monitored by qPCR (section 3) is shown (Figure 4A). Note that the number of vector genomes usually descreases during the first passages and strongly increases in later passages. A vector copy number of 106-107 per 15,000 cells is sufficient to infect 116 cells for large scale production in a 3-L spinner flask. If the GOI within the HCAdV vector genome contains a GFP expression cassette, the pre-amplification process (section 2) can also be monitored by fluorescent microscopy. Representative pictures of each passage are shown (Figure 4B). Note that the number of GFP positive cells usually decreases during first passages and strongly increases in late passages. If the number of GFP positive cells reaches ~100%, the vector was amplified sufficiently to infect 116 cells for large scale production in a 3-L spinner flask. The large scale amplification process of a HCAdV containing GFP expression cassette was monitored by microscopic analysis of culture medium and cells that was transfered from the spinner flask to a tissue culture dish. Microscopic pictures of cells producing HCAdV in suspension culture (see step 4.4 and 4.11) are provided (Figure 4C). Note that cells are growing in clumps indicating that the preceeding cell growth was sufficient and cells are healthy. When using fluorescent microscopy ~100% of these cells were GFP positive, indicating efficient transduction of suspension cells in the bioreactor and efficient vector production. Vector particles were purified from the medium and cells of the 3-L suspension culture by CsCl ultracentrifugation. Representative pictures of the HCAdV vector purification using CsCl density gradient ultracentrifugation are shown (Figure 5). After an initial centrifugation using a CsCl step gradient usually two bands are observed. The upper band contains empty of partial capsids whereas the lower band is comprised of DNA containing vector particles (step 5.4-5.5) (Figure 5A). DNA containing vector particles are subsequently harvested (step 5.6) and pooled for a second ultracentrifugation step to concentrate the vector particles. This step usually results in a single band of DNA containing vector particles that is finally harvested for subsequent dialysis (step 5.7-5.8) (Figure 5B). Dialyzed vector preparations were characterized by measuring the number of absolute particles, infectious particles and HV contamination. A schematic outline of the titration procedure (section 6 and 7) is provided (Figure 6). Representative results for characterization of the final vector preparation are shown (Figure 7). The bar chart shows typical vector particle numbers that are obtained by the procedure that is described in this protocol. Absolute particles numbers of vector particles were determined by optical density (section 6), infectious vector particles and HV contamination were measured by qPCR (section 7) (Figure 7A). Experience shows, that the OD titer overestimates the number of virus particles. Absolute viral particles measured by OD can be 20-500x higher than infectious viral particles measured by q-PCR. Infectious vector particles usally range between 5-10% of absolute particles when both were measured by q-PCR (section 7). If the vector preparation has a very good quality, more than 20% of absolute particles are infectious. The typical ratio between HV and infectious HCAdV particles (step 7.14) usually ranges between 1-5% of infectious particles (Figure 7B). This is acceptable as the HV is replication deficient. Nevertheless as less HV contamination as possible (< 1% of infectious particles) would be preferred.
Figure 1. Flowchart of cloning the GOI into pAdFTC. The GOI is cloned into the MCS of the pHM5 shuttle plasmid. Plasmids pAdFTC and pHM5-GOI are digested with I-CeuI and PI-SceI and purified by phenol-chloroform extraction and EtOH precipitation. The digested pHM5 is loaded on a preparative agarose gel to purify the gene of interest (see also step 1.5), whereas the digested pAdFTC plasmid is dephosphorylated. Subsequently the ligation reaction with CIP-treated pAdFTC and the transgene expression cassette is set up (see step 1.8) and after completion purified by phenol-chloroform extraction and EtOH precipitation. The subsequent SwaI digest of the ligation product followed by phenol-chloroform extraction and EtOH precipitation prevents growth of clones carrying pAdFTC without insert. Black triangles indicate restriction enzyme recognition sites; grey boxes indicate AdV5 inverted terminal repeats (ITR), white boxes marked with Ψ indicate AdV5 packaging signal, red arrow indicates promoter, green boxes indicate transgene (gene of interest, GOI) to be expressed, blue boxes indicate polyadenylation signal, orange or blue arrows indicate antibiotic resistance genes of the plasmid backbone, respectively. Please click here to view a larger version of this figure.
Figure 2. Representative results for the cloning procedure and release of the HCAdV genome from pAdVFTC. (A) Release of GOI-expression cassette from pHM5 by PI-SceI and I-CeuI digest (see also steps 1.2-1.5). (B) purified GOI-expression cassette and purified, dephosphorylated pAdVFTC digested with PI-SceI and I-CeuI respectively (step 1.7). (C) Analytical HincII digest of pAdVFTC clones with and without GOI (see also step 1.10). (D) Release of the HCAdV-genome carrying the GOI from pAdVFTC-GOI by NotI digest (step 2.2) Please click here to view a larger version of this figure.
Figure 3. Schematic diagram for high-capacity adenoviral vector amplification and purification. (A) HCAdV DNA transfection and helper virus (HV) infection: Transfection of linearized HCAdV genome carrying the GOI into the HCAdV producer cell line (116 cells [4]) and subsequent infection with the HV AdNG163R-2 [4]. (B) Amplification of HCAdV: After pre-amplification steps by serially transferring cell lysate to a new tissue culture dish and co-infecting with HV, large-scale production is performed in a 3-L suspension culture. (C) HCAdV purification: For purification, virus is isolated by ultracentrifugation using cesium chloride gradients. (D) Dialysis: Purified HCAdV particles are dialyzed against 2 L storage buffer. HCAdV, high-capacity adenoviral vector; ITR, adenovirus serotype 5 inverted terminal repeat; Ψ, packaging signal; HV, helper virus; MOI, multiplicity of infection. Please click here to view a larger version of this figure.
Figure 4. Representative results of the amplification process. (A) Monitoring of the pre-amplification process by amplification of viral genomes using qPCR (see also section 3); (B) If the GOI contains GFP as a transgene, the pre-amplification process can be monitored by fluorescent microscopy. (C) Representative results of the large scale amplification process in a spinner flask examplified for HCAdV encoding GFP. The left panel shows a microscopic picture of infected 116 cells from the spinner flask. A sample of culture medium and cells were transferred to a 60-mm tissue culture dish. Note that clumps of amplified cells are visible. The right panel shows the same picture using fluorescent microscopy. Almost 100% of cells are transduced with HCAdV carrying GFP. Please click here to view a larger version of this figure.
Figure 5. Representative results after CsCl gradient centrifugation. (A) After performing a step gradient (see also step 5.5); (B) After continuous gradient (see also step 5.7). Please click here to view a larger version of this figure.
Figure 6. Schematic outline of the titration procedure. (A) Measurement of infectious particles: Cells in a multi-well plate are infected with different volumes of purified virus and collected as cell pellets. (B) Measurement of absolute particles: Unifected cells form a multi-well are collected by trypsinization and centrifugation. After resuspension purified virus is added and genomic DNA is isolated. (C) Isolation of genomic DNA. (D) To quantify viral genome copy numbers a quantitative PCR (q-PCR) is performed. Please click here to view a larger version of this figure.
Figure 7. Representative results for characterization of the final vector preparation. (A) Absolute numbers of particles of a vector preparation according to the presented protocol determined with different methods: OD titer measured by optical density (section 6), infectious titer and HV contamination measured by qPCR (section 7). (B) Ratio between total infectious HCAdV particles and HV contamination levels measured by qPCRs (section 7). Please click here to view a larger version of this figure.
The protocol presented here allows purification of HCAdV vectors based on human adenovirus type 5 based on previously described procedures 4,12. The HCAdV genome within the pAdFTC plasmid is devoid of all adenovirus genes and only carries the 5'- and 3'- ITRs and the packaging signal. In this strategy the HV AdNG163R-2 4 provides all necessary genes for efficient virus production in trans. This offers a packaging capacity of up to 35 kb, which clearly outcompetes first and second generation AdVs or the widely used lentivirus (LV) – or adeno associated virus (AAV) based vectors. A second advantage of HCAdV especially for in vivo applications is the fact that in contrast to first- or second-generation AdVs they display reduced levels of cytotoxic and immunogenic side effects caused by the expression of viral proteins 5-8.
Nevertheless the protocol described here is more time and work intensive than for other viral vectors such as LV- or AAV-vectors as well as first- or second-generation AdVs. A major obstacle is the cloning of large transgenes and the subsequent transfer to the virus production plasmid pAdV-FTC. The use of homing endonucleases PI-SceI and I-CeuI offer precise and directed insertion of the transgene from the shuttle plasmid pHM5, but have the disadvantage of strongly sticking to the cleaved DNA. For that reason many phenol-chloroform cleanup steps are necessary when preparing plasmid- and insert-DNA during the cloning procedure. Therefore, rigorously following the provided cloning protocol is strictly recommended. After cloning the HCAdV genome is released from the plasmid pAdFTC by NotI restriction enzyme digest and subsequently transfected into the producer cell line. Note that initial transfection efficiencies are crucial for achieving sufficient virus amplification. Importantly, the protocol offers several steps from which the procedure can be restarted in case subsequent steps fail. During pre-amplification one third of the lysates can be stored as backup to start the procedure from that point again or to shorten a repetition in case more virus needs to be produced.
HCAdV carrying large, complex or several transgenes or certain stuffer DNA may be amplifying slower than expected. Therefore, it is crucial to carefully follow the pre-amplification process before suspension culture is grown in a spinner flask. If pre-amplification is not successful, the procedure should be stopped and started from the beginning again. If pre-amplification is inefficient, additional passaging may be necessary until the amount of virus is high enough to infect the cells grown in a 3-L spinner flask. As an alternative to suspension culture in a spinner flask, 20-30 150-mm tissue culture dishes can be used for the final passage. However, this may be more time and work intensive.
During CsCl gradient centrifugation it is be beneficial to split the lysate from the spinner flask into two fractions and spin them separately as this will not dramatically reduce virus titers. As soon as infective HCAdV are successfully produced it is relatively easy to re-amplify the viral stock by co-infecting a suspension culture from a spinner flask with purified viral particles and HV.
For qPCR measurements gDNA can either be purified via the protocol mentioned here or any commercially available kit for the isolation of gDNA from cultured cells. By using commercially available kit the protocol can be shortened one day but a varying portion of the HCAdV genome copies that are present in infected cells may be lost during purification, depending on the kit used. Therefore HCAdV genomes will be slightly underrepresented in following q-PCR measurements when compared to the method presented in step 7.4).
Obtained total infectious titers in one final vector preparation derived from one spinner flask range from 1 x 1010 to 5 x 1011 infectious particles. This amount is sufficient to perform numerous in vitro experiments. Dependent on the target cells, also several in vivo experiments can be conducted. For instance, for achieving sufficient liver transduction after systemic administration 1 x 109 infectious particles are required.
In summary, the wide cell tropism and the possibility to produce capsid-modified adenovirus together with the improved safety profile of HCAdV vectors devoid of all viral genes render these HCAdV vector system a highly valuable tool for gene therapeutic applications.
The authors have nothing to disclose.
This work was supported by DFG grant EH 192/5-1 (A.E.), the EU (E-rare-2) project Transposmart (A.E.), the UWH Forschungsförderung (E.S. and W.Z), and the PhD programme of the University Witten/Herdecke (P.B.). J.L. was supported by a stipend of the Chinese Scholarship council and T.B. and M.G by the Else Kröner-Fresenius foundation (EKFS).
I-CeuI | New England Biolabs | R0699S | restriction digest |
PI-SceI | New England Biolabs | R0696S | restriction digest |
T4 Ligase | New Engand Biolabs | M0202S | ligation |
SwaI | New England Biolabs | R0604S | restriction digest |
NotI | New England Biolabs | R0189S | restriction digest |
Calf Intestinal Alkaline Phosphatase (CIP) | New England Biolabs | M0290S | dephosphorylation of digested plasmids |
Hygromycin B | PAN Biotech | P02-015 | selection of CRE expressing 116 cells |
DMEM | PAN Biotech | P04-03590 | Hek293T cell culture medium |
Minimal Essential Medium (MEM) Eagle | PAN Biotech | P04-08500 | 116 cell culture medium |
Dulbecco’s phosphate buffer saline (DPBS) | PAN Biotech | P04-36500 | washing of cells, resuspension of cells |
250 ml storage bottle | Sigma | CLS430281-24EA | infection of 116 cells grown in suspension |
500mL PP CentrifugeTubes | Sigma | CLS431123-36EA | sedimentation of cells from suspension culture |
Spinner flask | Bellco | 1965-61030 | growth of 116 cells in suspension |
Ultra Clear Ultracentrifuge tubes | Beckmann Coulter | 344059 | density gradient centrifugation |
Ultracentrifuge | Beckmann Coulter | – | density gradient centrifugation |
SW-41 rotor | Beckmann Coulter | – | density gradient centrifugation |
Spectrum Laboratories Spectrapor Membrane | VWR | 132129 | dialysis tubing |
ready-to-use dialysis cassettes | Thermo | 66383 | dialysis |
one shot DH10B electrocompetent E. coli | invitrogen | C4040-52 | transformation of ligation reactions |
PureYield™ Plasmid Midiprep System | Promega | A2495 | midiprep |
peqGOLD Tissue DNA Mini Kit | Peqlab | 12-3396-02 | isolation of genomic DNA |
SuperFect Transfection Reagent | Qiagen | 301305 | tranfection of 116 cells |
opti MEM (10 % FBS) | Gibco | 31985-062 | transfection of 116 cells |
iQ SYBR Green Supermix | BioRad | 170-8882 | q-PCR |
CFX 96 C1000 touch | Biorad | qPCR machine | |
Phenol/Chloroform/Isoamyl alcohol | Carl Roth | A156.1 | purification of DNA |
Cesium chloride | Carl Roth | 8627.1 | density gradient centrifugation |
sodium acetate 99% | Carl Roth | 6773.2 | DNA precipitation |
LB medium | Carl Roth | X968.3 | bakterial growth medium |
ethanol 99,8% pure | Carl Roth | 9065.5 | DNA precipitation and washing |
SDS 99,5% | Carl Roth | 2326.2 | lysis buffer |
EDTA | Carl Roth | 8043.2 | lysis buffer |
Tris-HCl 99% | Carl Roth | 9090.3 | dialysis buffer/ lysis buffer |
glycerol 99,5% | Carl Roth | 3783.1 | dialysis buffer |
MgCl2 98,5% | Carl Roth | KK36.2 | dialysis buffer |
NaCl | Carl Roth | 3957.1 | optional dialysis buffer |
KH2PO4 | Carl Roth | 3904.2 | optional dialysis buffer |
sucrose | Carl Roth | 9286.1 | optional dialysis buffer |
Na2HPO4x2H2O | Carl Roth | 4984.2 | optional dialysis buffer |
1,5ml tubes | sarstedt | 72,730,005 | storage of virus preparations at -80°C |