Production of Recombinant Adeno‐Associated Viral Vectors for In Vitro and In Vivo Use

Vivian W. Choi1, Aravind Asokan1, Rebecca A. Haberman1, Richard Jude Samulski1

1 University of North Carolina, Chapel Hill, North Carolina
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 16.25
DOI:  10.1002/0471142727.mb1625s78
Online Posting Date:  April, 2007
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Abstract

Adeno‐associated virus is a nonpathogenic human virus that has been developed into a gene‐delivery vector due to its high efficiency of infection in many different cell types and its ability to persist and lead to long‐term gene expression. The vector is also a valuable tool in molecular biology experiments. This unit describes efficient methods to generate high‐titer, research‐grade, adenovirus‐free, recombinant single‐stranded and self‐complementary adeno‐associated virus in various serotypes, along with methods to quantify the viral vectors.

Keywords: adeno‐associated virus; gene delivery; purification; serotypes; transfection; viral vectors

     
 
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Table of Contents

  • Strategic Planning
  • Basic Protocol 1: Production of Adenovirus‐Free rAAV by Transient Transfection of 293 Cells
  • Alternate Protocol 1: PEI Transfection Method
  • Alternate Protocol 2: rAAV Purification Using Heparin Sepharose Column Purification
  • Support Protocol 1: Determination of rAAV Titers by the Dot‐Blot Assay
  • Support Protocol 2: In Vitro Transduction of rAAV in Cell Culture
  • Support Protocol 3: Growing an Adenovirus Helper Stock
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Production of Adenovirus‐Free rAAV by Transient Transfection of 293 Cells

  Materials
  • pSub201 plasmid: used to clone the transgene expression cassette between AAV termini to generate single‐stranded rAAV vector (Fig. A; ATCC #68065; see for location of a map and the sequence)
  • XbaI, HindIII, Acc65I, and SalI restriction endonucleases, with appropriate buffers
  • Plasmid containing the transgene expression cassette with gene of interest
  • pHpa‐trs‐SK plasmid: used to clone the transgene expression cassette between AAV termini to generate self‐complementary rAAV vector (Fig. B; see for location of a map and the sequence)
  • pXR1, pXR2, pXR3, pXR4, pXR5 plasmid: the AAV serotypes helper plasmid (Fig. C; UNC Vector Core Facility; see for location of a map and the sequence)
  • pXX6 plasmid: the adenoviral helper plasmid (UNC Vector Core Facility; see for location of a map and the sequence)
  • 293 tissue culture cell line (ATCC #CRL 1573)
  • Complete DMEM/10% FBS (see recipe)
  • Complete IMDM/10% FBS (see recipe)
  • 0.05% (w/v) porcine trypsin/0.02% (w/v) EDTA
  • 2.5 M CaCl 2 (see recipe)
  • 2× HEPES‐buffered saline (HeBS; see recipe)
  • Complete DMEM/2% FBS (see recipe)
  • Dry ice/ethanol bath
  • Ammonium sulfate [(NH 4) 2SO 4]
  • OPTI‐MEM I (Life Technologies)
  • Saturated ammonium sulfate, pH 7.0 (see recipe), 4°C
  • 1.37 g/ml and 1.5 g/ml CsCl (see recipe)
  • 70% ethanol
  • Phosphate‐buffered saline (PBS; appendix 22)
  • 15‐cm tissue culture plates
  • 50‐ml disposable polystyrene and polypropylene centrifuge tubes
  • Cell scrapers
  • 250‐ml polypropylene centrifuge bottles
  • Sorvall centrifuge with GS‐3 and SS‐34 rotors or equivalents
  • Sonicator with a 3‐mm diameter probe
  • Tabletop centrifuge
  • 50‐ml high‐speed polypropylene centrifuge tubes
  • Beckman ultracentrifuge with SW‐41 rotor and 12.5‐ml Beckman Ultra‐Clear tubes (or equivalent ultracentrifuge, rotor, and tubes)
  • 21‐G needles
  • Pierce Slide‐A‐Lyzer dialysis cassettes (MWCO 10,000)
  • Additional reagents and equipment for restriction digestion of DNA (unit 3.1), gel purification of DNA fragments (unit 2.6), subcloning DNA fragments (unit 3.16), plasmid preparation and CsCl purification (unit 1.7), tissue culture techniques including trypsinization of cells ( appendix 3F), and determination of rAAV titers by dot‐blot assay (see protocol 4)

Alternate Protocol 1: PEI Transfection Method

  • Serum‐free DMEM (e.g., Invitrogen)
  • 1 mg/ml polyethyleneimine (PEI; see recipe)
  • 15‐ml polystyrene tubes

Alternate Protocol 2: rAAV Purification Using Heparin Sepharose Column Purification

  • PBS‐MK (see recipe)
  • 15%, 25%, 40%, and 60% iodixanol (see recipe)
  • PBS‐MK containing 1 M NaCl
  • 0.5 M NaOH
  • 20% (v/v) ethanol
  • Phenol red (Life Technologies)
  • Ethanol
  • Econo Pump peristaltic pump (Bio‐Rad)
  • 32.4‐ml Optiseal tubes (Beckman)
  • 50‐µl borosilicate glass capillary pipets (Fisher)
  • Beckman ultracentrifuge with 70Ti rotor (or equivalent)
  • 1‐ml or 5‐ml HiTrap heparin‐Sepharose columns (Amersham Pharmacia Biotech)
  • FPLC apparatus
  • Pierce Slide‐A‐Lyzer dialysis cassettes (MWCO 10,000)

Support Protocol 1: Determination of rAAV Titers by the Dot‐Blot Assay

  Materials
  • Virus fractions or final virus preparation (e.g., from protocol 1 or protocol 2 or protocol 32)
  • DNase digestion buffer (see recipe)
  • 0.5 M EDTA ( appendix 22)
  • Proteinase solution (see recipe)
  • 24:1 (v/v) buffered phenol/chloroform (unit 2.1)
  • 20 mg/ml glycogen
  • 10 M ammonium acetate
  • 70% and 100% ethanol
  • TE buffer, pH 7.5 ( appendix 22)
  • 0.5 M NaOH
  • rAAV plasmid used to make recombinant virus (see protocol 1, step 1a or b)
  • 0.5 M NaOH containing 1 M NaCl
  • 0.4 M Tris⋅Cl, pH 7.5 ( appendix 22)
  • 0.5 M NaCl containing 0.5 M Tris·Cl, pH 7.5
  • Radiolabeled probe to transgene (prepared by random primed synthesis as in unit 3.5 or using a random primed DNA labeling kit, e.g., Boehringer Mannheim)
  • 96‐well plate
  • 50°C water bath
  • Dot‐blot apparatus
  • 0.45‐µm nylon membrane (Hybond N+, Amersham)
  • STORM and ImageQuant software (GE Healthcare)
  • Additional reagents and equipment for nuclease digestion of DNA (unit 3.12), extraction and precipitation of DNA (unit 2.1), restriction digestion of DNA (unit 3.1), preparation of and hybridization to DNA dot blots (units 2.9& 2.10), and detection by autoradiography or phosphor imaging ( 3.NaN)

Support Protocol 2: In Vitro Transduction of rAAV in Cell Culture

  Materials
  • Target cells and appropriate tissue culture medium
  • rAAV with appropriate transgene (see protocol 1 or protocol 2 or protocol 32)
  • Multiwell tissue culture plates

Support Protocol 3: Growing an Adenovirus Helper Stock

  Materials
  • Adenovirus type 5 (ATCC #VR5)
  • 293 tissue culture cell line (ATCC #CRL 1573) growing in complete DMEM/10% FBS (see recipe) in 15‐cm plates and in 60‐mm plates
  • Complete DMEM/2% FBS (see recipe)
  • Tris‐buffered saline (TBS; see recipe)
  • 1.2 g/ml, 1.3 g/ml, 1.4 g/ml, and 1.5 g/ml density CsCl (see recipe)
  • 2× adenovirus storage buffer (see recipe)
  • Serum‐free DMEM
  • Plaque overlay solution (see recipe), 39°C
  • 3.3 g/liter neutral red stain (Sigma; store up to 6 months at room temperature)
  • 500‐ml polypropylene centrifuge bottle
  • Sorvall centrifuge with GSA rotor or equivalent
  • 50‐ml disposable polypropylene centrifuge tubes
  • Dry ice/ethanol bath
  • Tabletop centrifuge
  • 12.5‐ml Beckman Ultra‐Clear tubes for the SW‐41 rotor
  • Beckman ultracentrifuge with SW‐41 or equivalent ultracentrifuge and rotor
  • 5‐ml syringe
  • 21‐G needle
  • Econo Pump peristaltic pump (Bio‐Rad)
  • 50‐µl borosilicate glass capillary pipets (Fisher)
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Figures

Videos

Literature Cited

   Bi, X. and Liu, L.F. 1996. DNA rearrangement mediated by inverted repeats. Proc. Natl. Acad. Sci. U.S.A. 93:819‐823.
   Boissy, R. and Astell, C.R. 1985. An Escherichia coli recBCsbcBrecF host permits the deletion‐resistant propagation of plasmid clones containing the 5′ terminal palindrome of minute virus of mice. Gene 35:179‐185.
   Boussif, O., Lezoualc'h, F., Zanta, M.A., Mergny, M.D., Scherman, D., Demeneix, B., and Behr, J.P. 1995. A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: Polyethylenimine. Proc. Natl. Acad. Sci. U.S.A. 92:7297‐7301.
   Cukor, G., Blacklow, N.R., et al., 1984. Biology of adeno‐associated virus. In The Parvoviruses. (K.I. Berns, ed.) pp. 33‐66. Plenum, New York.
   Hermonat, P.L., Labow, M.A., Wright, R., Berns, K.I., and Muzyczka, N. 1984. Genetics of adeno‐associated virus: Isolation and preliminary characterization of adeno‐associated virus type 2 mutants. J. Virol. 51:329‐339.
   Jiang, H., Pierce, G.F., Ozelo, M.C., de Paula, E.V., Vargas, J.A., Smith, P., Sommer, J., Luk, A., Manno, C.S., High, K.A., and Arruda, V.R. 2006. Evidence of multiyear factor IX expression by AAV‐mediated gene transfer to skeletal muscle in an individual with severe hemophilia B. Mol. Ther. 14:452‐455.
   Mandel, R.J., Rendahl, K.G., Spratt, S.K., Snyder, R.O., Cohen, L.K., and Leff, S.E. 1998. Characterization of intrastriatal recombinant adeno‐associated virus‐mediated gene transfer of human tyrosine hydroxylase and human GTP‐cyclohydrolase I in a rat model of Parkinson's disease. J. Neurosci. 18:4271‐4284.
   McCarty, D.M., Fu, H., Monahan, P.E., Toulson, C.E., Naik, P., and Samulski, R.J. 2003. Adeno‐associated virus terminal repeat (TR) mutant generates self‐complementary vectors to overcome the rate‐limiting step to transduction in vivo. Gene Ther. 10:2112‐2118.
   McLaughlin, S.K., Collis, P., Hermonat, P.L., and Muzyczka, N. 1988. Adeno‐associated virus general transduction vectors: Analysis of proviral structures. J. Virol. 62:1963‐1973.
   Rabinowitz, J.E., Rolling, F., Li, C., Conrath, H., Xiao, W., Xiao, X., and Samulski, R.J. 2002. Cross‐packaging of a single‐adeno‐associated virus (AAV) type 2 vector genome into multiple AAV serotypes enables transduction with broad specificity. J. Virol. 76:791‐801.
   Rivera, V.M., Gao, G.P., Grant, R.L., Schnell, M.A., Zoltick, P.W., Rozamus, L.W., Clackson, T., and Wilson, J.M. 2005. Long‐term pharmacologically regulated expression of erythropoietin in primates following AAV‐mediated gene transfer. Blood 105:1424‐1430.
   Samulski, R.J., Chang, L.S., and Shenk, T. 1989. Helper‐free stocks of recombinant adeno‐associated viruses: Normal integration does not require viral gene expression. J. Virol. 63:3822‐3828.
   Wang, L., Calcedo, R., Nichols, T.C., Bellinger, D.A., Dillow, A., Verma, I.M., and Wilson, J.M. 2005. Sustained correction of disease in naive and AAV2‐pretreated hemophilia B dogs: AAV2/8‐mediated, liver‐directed gene therapy. Blood 105:3079‐3086.
   Xiao, X., Li, J., and Samulski, R.J. 1996. Efficient long‐term gene transfer into muscle tissue of immunocompetent mice by adeno‐associated virus vector. J. Virol. 70:8098‐8108.
   Xiao, X., Li, J., and Samulski, R.J. 1998. Production of high‐titer recombinant adeno‐associated virus vectors in the absence of helper adenovirus. J. Virol. 72:2224‐2232.
Key References
   McCarty et al., 2003. See above.
  Describes the generation of self‐complementary recombinant AAV (scAAV) vector.
   Rabinowitz et al., 2002. See above.
  Describes the generation of rAAV serotype capsids that package AAV serotype 2 ITR.
   Samulski, R.J., Sally, M., and Muzyczka, N. 1999. Adeno‐associated viral vectors. In The Development of Human Gene Therapy (T. Friedman, ed.) pp. 36:131‐172. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
  An overview of rAAV as a gene therapy vector.
   Xiao et al., 1998. See above.
  Details the adenovirus‐free rAAV production protocol.
Internet Resources
   http://www.med.unc.edu/genether/
  University of North Carolina Human Gene Therapy Center Web site. Contains map and sequence of the pSub201, pHpa‐trs‐SK, pXX2, pXR1, pXR2, pXR3, pXR4, pXR5‐Bam, and pXX6 plasmids.
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