Helper‐Dependent Adenoviral Vectors

Kazuhiro Oka1, Lawrence Chan1

1 Baylor College of Medicine, Houston, Texas
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 16.24
DOI:  10.1002/0471142727.mb1624s69
Online Posting Date:  February, 2005
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Abstract

The helper‐dependent adenovirus (HDAd) is a recently developed adenovirus‐based vector with an improved safety profile and long‐term transgene expression. In this unit, a basic procedure for HDAd production using the Cre‐loxP system is presented. Amplification and large‐scale production of the vector can be done in both adherent and suspension cell culture systems. Included are protocols for Southern blot analysis to monitor vector amplification, slot blot assay to determine the infectious titer of the purified HDAd, and real‐time PCR to detect helper virus contamination in the preparation.

Keywords: Helper‐dependent adenoviral vector; helper virus; adherent cells; suspension cells

     
 
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Table of Contents

  • Biosafety
  • Strategic Planning
  • Basic Protocol 1: Generation of Recombinant Helper‐Dependent Adenoviral Vectors using Cre‐loxP System
  • Basic Protocol 2: Large‐Scale Helper‐Dependent Adenoviral Vector Production
  • Support Protocol 1: Monitoring Vector Amplification
  • Support Protocol 2: Determination of Infectious Titer
  • Support Protocol 3: Real‐Time PCR to Detect Helper Virus Contamination
  • Support Protocol 4: Verification of Vector Structures
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Generation of Recombinant Helper‐Dependent Adenoviral Vectors using Cre‐loxP System

  Materials
  • Expression cassette: gene of interest with promoter active in the target tissue and poly(A) signal
  • Restriction endonucleases AscI and PmeI
  • Calf intestinal phosphatase (units 3.10& 3.16)
  • Shuttle vector: pC4HSU (available from Microbix), pΔ21, pΔ28, pSTK129 or other similar shuttle vectors, with stuffer DNA of mammalian DNA origin; Sandig et al., ; Kim et al., ; Oka et al., ; Schiedner et al., )
  • Subcloning‐efficiency competent E. coli DH5α cells (Invitrogen) or other cells not prone to recombination, with transformation efficiency of ∼1 × 106 transformants/µg DNA/50 µl of competent cells in 10‐cm plate
  • 10‐cm LB/ampicillin/IPTG/Xgal plates (see recipe)
  • LB/ampicillin liquid medium: LB medium ( appendix 22) containing 100 µg/ml ampicillin
  • LB medium ( appendix 22)
  • Plasmid miniprep kit (optional; available from many major molecular biology suppliers)
  • Plasmid midiprep kit (preferably with ion‐exchange column using gravitational flow; available from many major molecular biology suppliers)
  • 1% agarose gel (unit 2.5)
  • 25:24:1 phenol/chloroform/isoamyl alcohol (unit 2.1)
  • 3 M sodium acetate, pH 5.2 ( appendix 22)
  • 70% and 100% ethanol
  • Transfection‐grade H 2O
  • E1‐function‐complementing cell lines expressing Cre recombinase, e.g., 293Cre4 (Parks et al., ; available from Microbix), 293Cre66 and suspension 293Cre66S (Schiedner et al., manuscript in preparation), or 116 (293N3S based suspension cell line; Palmer and Ng, )
  • E1‐function‐complementing cell lines (for amplification of helper virus) such as 293 (ATCC, Microbix), 293N3S (suspension cells, Microbix), PERC6, or N52.E6
  • Complete medium: α‐MEM supplemented with 10% heat‐inactivated FBS and 1× antibiotic‐antimycotic (ingredients available from Invitrogen)
  • Minimal essential medium alpha (α‐MEM; Invitrogen) without FBS or antibiotic‐antimycotic
  • 0.25% trypsin‐EDTA (Invitrogen)
  • SuperFect transfection reagent (Qiagen)
  • Helper virus (see recipe)
  • Maintenance medium for adherent cells: α‐MEM supplemented with 5% heat inactivated horse serum and 1× antibiotic‐antimycotic (ingredients available from Invitrogen)
  • 40% (w/v) sucrose (tissue culture grade) in H 2O, autoclave and filter through 0.22‐µm filter; store at room temperature
  • 15‐ml and 50‐ml conical tubes
  • 37°C orbital shaker
  • 6‐well tissue culture plates
  • 10‐cm tissue culture plates
  • Cryotubes
  • TripleFlask (TF, Nunc) or 15‐cm tissue culture dishes
  • Additional reagents and equipment for restriction endonuclease digestion (unit 3.1) and subcloning of DNA (unit 3.16), transformation (unit 1.8) and growth (units 1.2& 1.3) of E. coli, alkaline lysis miniprep of plasmid DNA (unit 1.6), agarose gel electrophoresis (unit 2.5), extraction and precipitation of DNA (unit 2.1), mammalian cell culture ( 3.NaN), and Southern blotting (unit 2.9)

Basic Protocol 2: Large‐Scale Helper‐Dependent Adenoviral Vector Production

  Materials
  • Adherent 293Cre66 or 293Cre4 cells
  • Minimal essential medium alpha (α‐MEM; Invitrogen)
  • CVL from passage 3 or additional passage (see protocol 1)
  • Helper virus (see recipe)
  • Maintenance medium for adherent cells: α‐MEM supplemented with 5% heat inactivated horse serum and 1× antibiotic‐antimycotic (ingredients available from Invitrogen)
  • 100 mM Tris⋅Cl, pH 8.0 ( appendix 22)
  • 40% (w/v) sucrose (tissue culture grade) in H 2O: autoclave and filter through 0.22‐µm filter; store at room temperature
  • Cre‐expressing, E1‐complementing cell lines adapted for suspension: e.g., 293N3SCre8, 293Cre66S (Schiedner et al., manuscript in preparation), or 116 cells
  • Growth medium for suspension cells (see recipe)
  • Maintenance medium for suspension cells (see recipe)
  • 5% sodium deoxycholate, filter‐sterilized
  • 2 M MgCl 2
  • DNase I solution (see recipe)
  • RNase A solution (see recipe)
  • CsCl density gradient solutions (density = 1.25, 1.35, and 1.41 g/ml; see recipe)
  • PBS++ : PBS ( appendix 22) containing 0.68 mM CaCl 2 and 0.5 mM MgCl 2
  • Dialysis buffer (see recipe)
  • TripleFlasks (Nunc)
  • 250‐ml centrifuge tubes (Corning)
  • 15‐cm culture dishes
  • Low‐speed centrifuge (e.g., Sorvall RC5C)
  • 250‐ and 3000‐ml spinner culture flasks and spinner culture base (Bellco or Corning)
  • Refrigerated centrifuge (e.g., Sorvall centrifuge with SS‐34 rotor)
  • NVT 65 ultracentrifuge tubes (Beckman)
  • 2‐ and 5‐ml pipets
  • Beckman LE‐80K ultracentrifuge (or equivalent ultracentrifuge with swinging‐bucket rotor)
  • 1‐ or 3‐ml syringes and 21‐G needles
  • Dialysis cassette (Pierce)
  • Additional reagents and equipment for mammalian cell culture and counting cells ( appendix 3F)

Support Protocol 1: Monitoring Vector Amplification

  • Aliquots of CVL from serial passages of the virus (see protocol 1)
  • Proteinase K reaction mixture (see recipe)
  • TE buffer, pH 8.0 ( appendix 22)
  • HindIII restriction endonuclease (or other appropriate restriction enzyme; see unit 3.1)
  • QIAamp DNA purification kit (Qiagen)
  • 6× gel loading buffer (see recipe)
  • 1.2% agarose gel (unit 2.5)
  • 1 kbPlus DNA marker (Invitrogen)
  • 400‐bp fragment containing the left ITR plus the packaging signal (contact Dr. K. Oka at ) as probe for Southern blotting, labeled with 32P using a random priming DNA labeling kit
  • Additional reagents and equipment for Southern blotting (unit 2.9) and autoradiography or phosphor imaging ( appendix 3A)

Support Protocol 2: Determination of Infectious Titer

  • 293 cells (ATCC)
  • Helper‐dependent adenoviral vector (HDAd; see protocol 1)
  • HDAd vector containing a reporter gene (e.g., lacZ, green fluorescence protein) whose infectious titer is determined independently based on the reporter gene expression on 293 cells
  • PBS++:PBS ( appendix 22) containing 0.68 mM CaCl 2 and 0.5 mM MgCl 2
  • 10× citric‐saline (see recipe)
  • 0.8 M NaOH
  • ITR or packaging signal (contact Dr. K. Oka at ) as probe for slot blotting, labeled with 32P using a random priming DNA labeling kit
  • 24‐well tissue culture plates
  • Additional reagents and equipment for slot blotting (unit 2.9) and phosphor imaging ( appendix 3A)

Support Protocol 3: Real‐Time PCR to Detect Helper Virus Contamination

  • Helper‐dependent adenoviral vectors (HDAds) to be analyzed (see protocol 1)
  • 2× SYBR Green QPCR master mix (Stratagene, Bio‐Rad, or equivalent)
  • 10 pmol/µl PCR primers (see recipe)
  • 96‐well reaction plates
  • Real time QPCR platform (Stratagene Mx3000 or equivalent)
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Figures

Videos

Literature Cited

   Donello, J.E., Loeb, J.E., and Hope, T.J. 1998. Woodchuck hepatitis virus contains a tripartite posttranscriptional regulatory element. J. Virol. 72:5085‐5092.
   Ehrhardt, A., and Kay, M.A. 2002. A new adenoviral helper‐dependent vector results in long‐term therapeutic levels of human coagulation factor IX at low doses in vivo. Blood 99:3923‐3930.
   Goncalves, M.A., Pau, M.G., de Vries, A.A., and Valerio, D. 2001. Generation of a high‐capacity hybrid vector: Packaging of recombinant adenoassociated virus replicative intermediates in adenovirus capsids overcomes the limited cloning capacity of adenoassociated virus vectors. Virology 288:236‐246.
   Kim, I.H., Jozkowicz, A., Piedra, P.A., Oka, K., and Chan, L. 2001. Lifetime correction of genetic deficiency in mice with a single injection of helper‐dependent adenoviral vector. Proc. Natl. Acad. Sci. U.S.A. 98:13282‐13287.
   Kochanek, S. 1999. High‐capacity adenoviral vectors for gene transfer and somatic gene therapy. Hum. Gene Ther. 10:2451‐2459.
   Kreppel, F., Biermann, V., Kochanek, S., and Schiedner, G. 2002. A DNA‐based method to assay total and infectious particle contents and helper virus contamination in high‐capacity adenoviral vector preparations. Hum. Gene Ther. 13:1151–1156.
   Morral, N., O'Neal, W., Rice, K., Leland, M., Kaplan, J., Piedra, P.A., Zhou, H., Parks, R.J., Velji, R., Aguilar‐Cordova, E., Wadsworth, S., Graham, F.L., Kochanek, S., Carey, K.D., and Beaudet, A.L. 1999. Administration of helper‐dependent adenoviral vectors and sequential delivery of different vector serotype for long‐term liver‐directed gene transfer in baboons. Proc. Natl. Acad. Sci. U.S.A. 96:12816‐12821.
   Ng, P., Beauchamp, C., Evelegh, C., Parks, R., and Graham, F.L. 2001. Development of a FLP/frt system for generating helper‐dependent adenoviral vectors. Mol. Ther. 3:809‐815.
   Nomura, S., Merched, A., Nour, E., Dieker, C., Oka, K., and Chan, L. 2004. Low‐density lipoprotein receptor gene therapy using helper‐dependent adenovirus produces long‐term protection against atherosclerosis in a mouse model of familial hypercholesterolemia. Gene Ther. 11:1540–1548.
   Oka, K., Pastore, L., Kim, I.H., Merched, A., Nomura, S., Lee, H.J., Merched‐Sauvage, M., Arden‐Riley, C., Lee, B., Finegold, M., Beaudet, A., and Chan, L. 2001. Long‐term stable correction of low‐density lipoprotein receptor‐deficient mice with a helper‐dependent adenoviral vector expressing the very low‐density lipoprotein receptor. Circulation 103:1274‐1281.
   Palmer, D. and Ng, P. 2003. Improved system for large‐scale production of helper‐dependent adenoviral vectors. Mol. Ther. 7:S164.
   Palmer, D. J., and Ng, P. 2004. Physical and infectious titers of helper‐dependent adenoviral vectors: A method of direct comparison to the adenovirus reference material. Mol. Ther. 10:792–798.
   Parks, R.J. and Graham, F.L. 1997. A helper‐dependent system for adenovirus vector production helps define a lower limit for efficient DNA packaging. J. Virol. 71:3293‐3298.
   Parks, R.J., Chen, L., Anton, M., Sankar, U., Rudnicki, M.A., and Graham, F.L. 1996. A helper‐dependent adenovirus vector system: Removal of helper virus by Cre‐mediated excision of the viral packaging signal. Proc. Natl. Acad. Sci. U.S.A. 93:13565‐13570.
   Parks, R., Evelegh, C., and Graham, F. 1999. Use of helper‐dependent adenoviral vectors of alternative serotypes permits repeat vector administration. Gene Ther. 6:1565‐1573.
   Recchia, A., Parks, R.J., Lamartina, S., Toniatti, C., Pieroni, L., Palombo, F., Ciliberto, G., Graham, F.L., Cortese, R., La Monica, N., and Colloca, S. 1999. Site‐specific integration mediated by a hybrid adenovirus/adeno‐associated virus vector. Proc. Natl. Acad. Sci. U.S.A. 96:2615‐2620.
   Recchia, A., Perani, L., Sartori, D., Olgiati, C., and Mavilio, F. 2004. Site‐specific integration of functional transgenes into the human genome by adeno/AAV hybrid vectors. Mol. Ther. 10:660–670.
   Sandig, V., Youil, R., Bett, A.J., Franlin, L.L., Oshima, M., Maione, D., Wang, F., Metzker, M.L., Savino, R., and Caskey, C.T. 2000. Optimization of the helper‐dependent adenovirus system for production and potency in vivo. Proc. Natl. Acad. Sci. U.S.A. 97:1002‐1007.
   Schiedner, G., Hertel, S., Johnston, M., Biermann, V., Dries, V., and Kochanek, S. 2002. Variables affecting in vivo performance of high‐capacity adenovirus vectors. J. Virol. 76:1600‐1609.
   Soifer, H., Higo, C., Kazazian, H.H. Jr., Moran, J.V., Mitani, K., and Kasahara, N. 2001. Stable integration of transgenes delivered by a retrotransposon‐adenovirus hybrid vector. Hum. Gene Ther. 12:1417‐1428.
   Soifer, H., Higo, C., Logg, C.R., Jih, L.J., Shichinohe, T., Harboe‐Schmidt, E., Mitani, K., and Kasahara, N. 2002. A novel, helper‐dependent, adenovirus‐retrovirus hybrid vector: Stable transduction by a two‐stage mechanism. Mol. Ther. 5:599‐608.
   Soudais, C., Skander, N., and Kremer, E.J. 2003. Long‐term in vivo transduction of neurons throughout the rat central nervous system using novel helper‐dependent CAV‐2 vectors. FASEB J. 18:391‐393.
   Toietta, G., Pastore, L., Cerullo, V., Finegold, M., Beaudet, A.L., and Lee, B. 2002. Generation of helper‐dependent adenoviral vectors by homologous recombination. Mol. Ther. 5:204‐210.
   Umana, P., Gerdes, C.A., Stone, D., Davis, J.R., Ward, D., Castro, M.G., and Lowenstein, P.R. 2001. Efficient FLPe recombinase enables scalable production of helper‐dependent adenoviral vectors with negligible helper‐virus contamination. Nat. Biotechnol. 19:582‐585.
   Yant, S.R., Ehrhardt, A., Mikkelsen, J.G., Meuse, L., Pham, T., and Kay, M.A. 2002. Transposition from a gutless adeno‐transposon vector stabilizes transgene expression in vivo. Nat. Biotechnol. 20:999‐1005.
Key References
   Kochanek, 1999. See above.
  An excellent comprehensive review for development of helper‐dependent adenoviral vectors.
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