Production of Recombinant Adeno‐Associated Viral Vectors

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 Human Genetics
Unit Number:  Unit 12.9
DOI:  10.1002/0471142905.hg1209s53
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 for many different cell types and its ability to persist and lead to long‐term gene expression. 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; AAV; gene therapy; gene delivery

     
 
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Table of Contents

  • 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: Infection of Cells In Vitro with rAAV and Determination of Titer by Transgene Expression
  • 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 (and pXX2), 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 density CsCl (see recipe)
  • 70% ethanol
  • Phosphate‐buffered saline (PBS; appendix 2D)
  • 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 for the SW‐41 (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 (e.g., CPMB UNIT ), gel purification of DNA fragments (CPMB UNIT ), subcloning DNA fragments (CPMB UNIT ), plasmid preparation and CsCl purification (unit NaN.NaN in this manual), tissue culture techniques including trypsinization of cells ( appendix 3G in this manual), and determination of rAAV titers by dot‐blot assay (see protocol 4)
NOTE: All tissue culture incubations are performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified.

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 2D)
  • Proteinase solution (see recipe)
  • 24:1 phenol/chloroform
  • 20 mg/ml glycogen
  • 10 M ammonium acetate
  • 70% and 100% ethanol
  • TE buffer, pH 7.5 ( appendix 2D)
  • 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 2D)
  • 0.5 M NaCl containing 0.5 M Tris·Cl, pH 7.5
  • Radiolabeled probe to transgene (made using Boehringer Mannheim random‐primed DNA labeling kit according to manufacturer's instructions)
  • 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 restriction digestion of DNA ( appendix 1M), hybridization of DNA to membranes (e.g., unit 2.7) and autoradiography (CPMB APPENDIX )

Support Protocol 2: Infection of Cells In Vitro with rAAV and Determination of Titer by Transgene Expression

  Materials
  • Target cells
  • Tissue culture medium for target cells (see supplier's instructions for cells)
  • rAAV with appropriate transgene (see protocol 1 or protocol 2 or protocol 32)
  • Tissue culture plates (multiwell plates recommended for assaying transducing titer)
NOTE: All tissue culture incubations are performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified. Any manipulations using virus should be carried out in a tissue culture hood dedicated for virus work.NOTE: To assay transducing titer, cells can be coinfected with adenovirus (see unit 12.4), which acts as a helper virus and increases the transduction efficiency. The addition of adenovirus gives a better indication of the number of particles that are competent to transduce a cell by inducing an optimal environment for AAV infection. However, the adenovirus has a cytopathic effect because of its antigenicity and should not be used in vivo. Thus, a titer derived with use of adenovirus in vitro may not accurately reflect an in vivo competency. It is up to each investigator to establish a standard procedure for titering different rAAV preps.
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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., et al., 1984. Genetics of adeno‐associated virus: Isolation and preliminary characterization of adeno‐associated virus type 2 mutants. J. Virol. 51:329‐339.
   Mandel, R.J., Rendahl, K.G., et al., 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(11):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., et al., 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.
   Samulski, R.J., Chang, L.S., et al., 1989. Helper‐free stocks of recombinant adeno‐associated viruses: Normal integration does not require viral gene expression. J. Virol. 63(9):3822‐3828.
Key References
   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.
  This paper 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, pXR5Bam, and pXX6 plasmids.
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