Oncolytic Adenoviruses: Design, Generation, and Experimental Procedures

Julia Davydova1, Masato Yamamoto1

1 Division of Basic and Translational Research, Department of Surgery, University of Minnesota, Minneapolis, Minnesota
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Unit 12.14
DOI:  10.1002/0471142905.hg1214s78
Online Posting Date:  July, 2013
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

Oncolytic adenoviruses are designed to take advantage of the virus' native ability to replicate in cancer cells to induce oncolysis. Subsequently, the released viral progeny spread and kill the neighboring cancer cells. These characteristics, together with the ability of adenovirus to infect a broad spectrum of cells, its well understood replication machinery, and relative ease of manufacture have led to the intensive use of adenovirus as an anticancer agent. This unit describes cloning strategies, procedures to turn the intended design into virus, and quality analyses of resultant adenoviral vectors. Most of these procedures were optimized especially for oncolytic adenoviral vectors. Curr. Protoc. Hum. Genet. 78:12.14.1‐12.14.21. © 2013 by John Wiley & Sons, Inc.

Keywords: adenovirus; oncolytic; production; design; cloning; strategy; amplification; purification

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Biosafety
  • Strategic Planning
  • Basic Protocol 1: Generating Recombinant Adenovirus by Homologous Recombination in E. coli
  • Basic Protocol 2: Transient Transfection and Rescue of Oncolytic Virus
  • Basic Protocol 3: Large‐Scale Amplification of Oncolytic Adenovirus
  • Basic Protocol 4: Cesium Chloride Purification of Oncolytic Adenovirus
  • Support Protocol 1: Removal of CsCl Salts from Virus Preparation by Dialysis
  • Support Protocol 2: Virus Storage and Shipping
  • Support Protocol 3: Virus Titration by Spectrophotometry
  • Support Protocol 4: Adenovirus Titration by Plaque Forming Units (PFU) Assay
  • Support Protocol 5: Evaluation of Adenoviral Vector Quality by VP/PFU Ratio
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Generating Recombinant Adenovirus by Homologous Recombination in E. coli

  Materials
  • Shuttle plasmid: must have a design to permit the modification of the intended part of adenoviral genome as described above; in some cases, the shuttle plasmid for E1‐deleted vectors (e.g. pShuttle from Stratagene) can be used; alternatively, obtaining shuttle plasmids from a researcher reporting similar structure is the best way
  • Adenovirus backbone plasmid: use commercially available adenovirus backbone plasmid (e.g. pAd5 from O.D.260, Inc., http://od260.com/), or obtain plasmid from a researcher reporting similar structure
  • BJ5183 electroporation‐competent cells (e.g., Stratagene)
  • SOC medium or LB liquid medium (see recipes in appendix 2D)
  • LB agar plates ( appendix 2D) with appropriate antibiotic
  • LB liquid medium ( appendix 2D) containing the appropriate antibiotic
  • Restriction endonucleases (e.g., BamHI) for checking recombinants
  • DH5α competent cells (or other strain not prone to recombination)
  • 2‐mm electroporation cuvettes
  • DNase‐free microcentrifuge tubes
  • Pulser electroporator (BioRad Micro or similar apparatus)
  • 30°C orbital shaker
  • 2‐ml culture tubes
  • 30°C shaking bacterial incubator
  • Additional reagents and equipment for alkaline lysis miniprep of plasmids (unit 12.4, protocol 8) and agarose gel electrophoresis (unit 2.5)

Basic Protocol 2: Transient Transfection and Rescue of Oncolytic Virus

  Materials
  • 911 (Fallaux et al., ) or HEK‐293 (e.g., Ad‐293 from Stratagene) cells
  • Growth medium with 5% FBS (see recipe)
  • Adenovirus‐encoding recombinant plasmid (from protocol 1)
  • PacI restriction enzyme
  • Serum‐free DMEM medium
  • Transient transfection reagent (Qiagen SuperFect or equivalent)
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • 6‐well culture plate
  • Inverted tissue culture microscope
  • Cell scrapers
  • 10‐ml conical tubes
  • Additional reagents and equipment for extraction and precipitation of DNA ( appendix 3C)

Basic Protocol 3: Large‐Scale Amplification of Oncolytic Adenovirus

  Materials
  • A549 (Human Lung Carcinoma; ATCC #CCL‐185) or other adenovirus replication‐permissive cell line
  • Growth medium with 5% FBS (see recipe)
  • Virus lysate ( protocol 2, step 12)
  • 15‐cm culture dishes (or 160‐cm2 cell culture flasks)
  • Centrifuge
  • Cell scraper
  • 50‐ml conical tubes (e.g., BD Falcon)
  • 200‐ to 25‐ml conical centrifuge bottles

Basic Protocol 4: Cesium Chloride Purification of Oncolytic Adenovirus

  Materials
  • Virus lysate ( protocol 3, step 18)
  • Benzonase nuclease (Sigma‐Aldrich, 384 U/µl original concentration, or equivalent)
  • Cesium chloride (CsCl) with optical grade with purity >99.9%
  • PBS(‐): PBS without Ca or Mg (Corning cellgro, cat. no. 21‐040‐CV)
  • 50‐ml conical centrifuge tubes
  • Standard centrifuge
  • 0.22‐µm syringe filters (0.22 µm)
  • Ultra‐Clear centrifuge tubes for SW 41 Ti rotor (Beckman, ultrathin, 14 × 89 mm or equivalent)
  • Refrigerated ultracentrifuge
  • Swinging bucket rotor (SW 41 Ti rotor or equivalent)
  • 3‐ or 5‐ml syringes and 23‐G needles

Support Protocol 1: Removal of CsCl Salts from Virus Preparation by Dialysis

  Materials
  • PBS(+): Dulbecco's phosphate‐buffered saline (DPBS; with Ca and Mg; Corning cellgro, cat. no. 21‐030‐CV)
  • Sterile ultra‐pure glycerol (Invitrogene UltraPure Glycerol; Invitrogen, cat. no. 15514‐011)
  • Syringe filled with virus from protocol 4, step 10
  • 23‐G needles
  • Beakers
  • Dialysis cassettes (Thermo Slide‐A‐Lyzer, 10,000 MWCO, or equivalent)
  • Float buoys for dialysis cassettes
  • Magnetic stirrer and stir bar

Support Protocol 2: Virus Storage and Shipping

  Materials
  • Dialysis buffer: DPBS (with Ca and Mg) with 10% (v/v) glycerol (see protocol 5, step 1)
  • 10% sodium dodecyl sulfate (SDS; appendix 2D)
  • 100 mM EDTA ( appendix 2D)
  • Virus preparation (see protocols above)
  • 56°C water bath
  • Spectrophotometer
  • Spectrophotometer cuvettes

Support Protocol 3: Virus Titration by Spectrophotometry

  Materials
  • 911 (Fallaux et al., ) or HEK‐293 (e.g., Ad‐293 from Stratagene) cells
  • Growth medium with 5% FBS (see recipe)
  • Agarose (ultrapure grade)
  • Fetal bovine serum (FBS)
  • HEPES buffer (Corning cellgro, cat. no. 25‐060‐CI)
  • Adenovirus preparation (see protocols above)
  • 6‐well culture plate
  • 42°C water bath
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
   Alemany, R., Balague, C., and Curiel, D.T. 2000. Replicative adenoviruses for cancer therapy. Nat. Biotechnol. 18:723‐727.
   Altaras, N.E., Aunins, J.G., Evans, R.K., Kamen, A., Konz, J.O., and Wolf, J.J. 2005. Production and formulation of adenovirus vectors. Adv. Biochem. Eng. Biotechnol. 99:193‐260.
   Armstrong, L., Arrington, A., Han, J., Gavrikova, T., Brown, E., Yamamoto, M., Vickers, S.M., and Davydova, J. 2012a. Generation of a novel, cyclooxygenase‐2‐targeted, interferon‐expressing, conditionally replicative adenovirus for pancreatic cancer therapy. Am. J. Surg. 204:741‐750.
   Armstrong, L., Davydova, J., Brown, E., Han, J., Yamamoto, M., and Vickers, S.M. 2012b. Delivery of interferon alpha using a novel Cox2‐controlled adenovirus for pancreatic cancer therapy. Surgery 152:114‐122.
   Barber, G.N. 2004. Vesicular stomatitis virus as an oncolytic vector. Viral Immunol. 17:516‐527.
   Berk, A.J. 2007. Adenoviridae: The viruses and their replication. In Fields Virology, 5th ed. (D. Knipe and P. Howley, eds.) pp. 2355‐2394. Lippincott Williams & Wilkins, Philadelphia.
   Curiel, D.T. 1999. Strategies to adapt adenoviral vectors for targeted delivery. Ann. N.Y. Acad. Sci. 886:158‐171.
   Curiel, D.T. 2000. The development of conditionally replicative adenoviruses for cancer therapy. Clin. Cancer Res. 6:3395‐3399.
   Davydova, J., Le, L.P., Gavrikova, T., Wang, M., Krasnykh, V., and Yamamoto, M. 2004. Infectivity‐enhanced cyclooxygenase‐2‐based conditionally replicative adenoviruses for esophageal adenocarcinoma treatment. Cancer Res. 64:4319‐4327.
   Davydova, J., Gavrikova, T., Brown, E.J., Luo, X., Curiel, D.T., Vickers, S.M., and Yamamoto, M. 2010. In vivo bioimaging tracks conditionally replicative adenoviral replication and provides an early indication of viral antitumor efficacy. Cancer Sci. 101:474‐481.
   Dmitriev, I., Krasnykh, V., Miller, C.R., Wang, M., Kashentseva, E., Mikheeva, G., Belousova, N., and Curiel, D.T. 1998. An adenovirus vector with genetically modified fibers demonstrates expanded tropism via utilization of a coxsackievirus and adenovirus receptor‐independent cell entry mechanism. J. Virol. 72:9706‐9713.
   Doronin, K., Toth, K., Kuppuswamy, M., Ward, P., Tollefson, A.E., and Wold, W.S. 2000. Tumor‐specific, replication‐competent adenovirus vectors overexpressing the adenovirus death protein. J. Virol. 74:6147‐6155.
   Doronin, K., Flatt, J.W., Di Paolo, N.C., Khare, R., Kalyuzhniy, O., Acchione, M., Sumida, J.P., Ohto, U., Shimizu, T., Akashi‐Takamura, S., Miyake, K., MacDonald, J.W., Bammler, T.K., Beyer, R.P., Farin, F.M., Stewart, P.L., and Shayakhmetov, D.M. 2012. Coagulation factor X activates innate immunity to human species C adenovirus. Science 338:795‐798.
   Fallaux, F.J., Kranenburg, O., Cramer, S.J., Houweling, A., van Ormondt, H., Hoeben, R.C., and van der Eb, A.J. 1996. Characterization of 911: A new helper cell line for the titration and propagation of early region 1‐deleted adenoviral vectors. Hum. Gene Ther. 7:215‐222.
   He, T., 2004. Adenoviral vectors. Curr. Protoc. Hum. Genet. 40:12.4.1‐12.4.25.
   He, T.C., Zhou, S., da Costa, L.T., Yu, J., Kinzler, K.W., and Vogelstein, B. 1998. A simplified system for generating recombinant adenoviruses. Proc. Natl. Acad. Sci. U.S.A. 95:2509‐2514.
   Krasnykh, V., Dmitriev, I., Mikheeva, G., Miller, C.R., Belousova, N., and Curiel, D.T. 1998. Characterization of an adenovirus vector containing a heterologous peptide epitope in the HI loop of the fiber knob. J. Virol. 72:1844‐1852.
   Lichtenstein, D.L., Toth, K., Doronin, K., Tollefson, A.E., and Wold, W.S. 2004. Functions and mechanisms of action of the adenovirus E3 proteins. Int. Rev. Immunol. 23:75‐111.
   Maizel, J.V. Jr., White, D.O., and Scharff, M.D. 1968. The polypeptides of adenovirus. I. Evidence for multiple protein components in the virion and a comparison of types 2, 7A, and 12. Virology 36:115‐125.
   Mohr, I. 2005. To replicate or not to replicate: Achieving selective oncolytic virus replication in cancer cells through translational control. Oncogene 24:7697‐7709.
   Rodriguez, R., Schuur, E.R., Lim, H.Y., Henderson, G.A., Simons, J.W., and Henderson, D.R. 1997. Prostate attenuated replication competent adenovirus (ARCA) CN706: A selective cytotoxic for prostate‐specific antigen‐positive prostate cancer cells. Cancer Res. 57:2559‐2563.
   Russell, S.J. 1994. Replicating vectors for cancer therapy: A question of strategy. Semin. Cancer Biol. 5:437‐443.
   Russell, S.J. 2002. RNA viruses as virotherapy agents. Cancer Gene Ther. 9:961‐966.
   Smith, R., Huebner, R., Rowe, W., Schatten, W., and Thomas, L. 1956. Studies on the use of viruses in the treatment of carcinoma of the cervix. Cancer 9:1211‐1218.
   Toth, K., Doronin, K., Tollefson, A.E., and Wold, W.S. 2003. A multitasking oncolytic adenovirus vector. Mol. Ther. 7:435‐437.
   Toth, K., Kuppuswamy, M., Shashkova, E.V., Spencer, J.F., and Wold, W.S. 2010. A fully replication‐competent adenovirus vector with enhanced oncolytic properties. Cancer Gene Ther. 17:761‐770.
   Wold, W. and Horwitz, M. 2007. Adenoviruses. In Virology, 5 ed., vol. 2 (D. Knipe and P. Howley, eds.) pp. 2395‐2436. Lippincott Williams & Wilkins, Philadelphia.
   Yamamoto, M. 2004. Conditionally replicative adenovirus for gastrointestinal cancers. Expert Opin. Biol. Ther. 4:1241‐1250.
   Yamamoto, M. and Curiel, D.T. 2004. Nonreplicating DNA viral vectors for suicide gene therapy: The adenoviral vectors. Methods Mol. Med. 90:61‐70.
   Yamamoto, M. and Curiel, D.T. 2005. Gene Therapy and Virotherapy for Pancreatic Cancer. In Panceatic Cancer (D.D. v. Hoff, D.B. Evans, and R.H. Hurban, eds.) pp. 525‐539. Jones and Bartlett Publishers, Sudbury, Mass.
   Yamamoto, M. and Curiel, D.T. 2010. Current issues and future directions of oncolytic adenoviruses. Mol. Ther. 18:243‐250.
   Yamamoto, M., Davydova, J., Wang, M., Siegal, G.P., Krasnykh, V., Vickers, S.M., and Curiel, D.T. 2003. Infectivity enhanced, cyclooxygenase‐2 promoter‐based conditionally replicative adenovirus for pancreatic cancer. Gastroenterology 125:1203‐1218.
   Yu, W. and Fang, H. 2007. Clinical trials with oncolytic adenovirus in China. Curr. Cancer Drug Targets 7:141‐148.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library