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

Steven J. Gray1, Vivian W. Choi1, Aravind Asokan1, Rebecca A. Haberman1, Thomas J. McCown1, Richard Jude Samulski1

1 University of North Carolina at Chapel Hill, School of Medicine, Chapel Hill, North Carolina
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 4.17
DOI:  10.1002/0471142301.ns0417s57
Online Posting Date:  October, 2011
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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. Two detailed methods are provided for viral vector delivery into the rodent brain and spinal cord, and for histological detection of transgene expression of GFP. Curr. Protoc. Neurosci. 57:4.17.1‐4.17.30. © 2011 by John Wiley & Sons, Inc.

Keywords: gene therapy; viral vectors; adeno‐associated virus; purification; serotypes; transfection; brain; spinal cord; transduction; gene delivery

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Production of Adenovirus‐Free rAAV by Transient Transfection of 293 Cells
  • Support Protocol 1: Determination of rAAV Titers by the Dot‐Blot Assay
  • Support Protocol 2: Determination of rAAV Titers by the Quantitative PCR Assay
  • Support Protocol 3: Infection of Cells In Vitro with rAAV and Determination of Titer by Transgene Expression
  • Support Protocol 4: Growing an Adenovirus Helper Stock
  • Basic Protocol 2: Stereotactic Microinjection of rAAV Into the Rat Brain
  • Basic Protocol 3: Intrathecal Administration of rAAV in Mice by Lumbar Puncture
  • Support Protocol 5: Histological Detection of Green Fluorescent Protein 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 Internet Resources for location of a map and the sequence)
  • XbaI, HindIII, Acc65I (or KpnI), and SnaBI 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 Internet Resources for location of a map and the sequence)
  • pXR series of plasmids (1‐9 and others): the AAV serotypes helper plasmid (UNC Vector Core Facility; see Internet Resources for location of a map and the sequence)
  • pXX6 plasmid: the adenoviral helper plasmid (UNC Vector Core Facility; see Internet Resources for location of a map and the sequence)
  • 293 tissue culture cell line (ATCC #CRL 1573)
  • Complete DMEM/10% FBS (see recipe)
  • 0.05% (w/v) trypsin/0.02% (w/v) EDTA
  • Serum‐free DMEM (e.g., Invitrogen)
  • 1 mg/ml polyethyleneimine (PEI; see recipe)
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • 10% (w/v) deoxycholate (DOC) solution (deoxycholic acid, Sigma)
  • Benzonase (Sigma, cat. no. E1014‐25KU)
  • 1.3 g/cm3, 1.4 g/cm3, and 1.5 g/cm3 density CsCl (see recipes)
  • 70% ethanol
  • rAAV dialysis/storage solution (see recipe)
  • 15‐cm tissue culture plates
  • 15‐ml polystyrene tubes
  • Vortex
  • Cell scrapers
  • 250‐ or 500‐ml polypropylene centrifuge bottles
  • Sorvall centrifuge with GS‐3 and SS‐34 rotors or equivalents
  • 50‐ml conical centrifuge tubes
  • Sonicator with a 3‐mm diameter probe
  • Benchtop centrifuge
  • 50‐ml high‐speed polypropylene centrifuge tubes
  • Ultracentrifuge
  • Ti70 rotor and 32 ml Beckman Ultra‐Clear tubes (or equivalent rotor and tubes)
  • 10‐ml syringe equipped with 21‐G needles
  • NVT65 rotor and corresponding 12‐ml Beckman ultraclear tubes (or equivalent rotor and tubes)
  • Pierce Slide‐A‐Lyzer dialysis cassettes (MWCO 10,000)
  • Additional reagents and equipment for restriction digestion of DNA ( appendix 1M), gel purification of DNA fragments (Moore et al., ), subcloning DNA fragments (Struhl, ), plasmid preparation and CsCl purification (Heilig et al., ), tissue culture techniques including trypsinization of cells ( appendix 3B), and determination of rAAV titers by dot‐blot assay (see protocol 2); also see appendix 1A in this manual
NOTE: All tissue culture incubations are performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified.

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

  Materials
  • Virus fractions or final virus preparation (from protocol 1)
  • DNase digestion buffer (see recipe)
  • 0.5 M EDTA ( appendix 2A)
  • Proteinase solution (see recipe)
  • Dot blot dilution buffer (see recipe)
  • 0.4 M NaOH
  • rAAV plasmid used to make recombinant virus (see protocol 1, step 1a or 1b)
  • 0.4 M Tris⋅Cl, pH 7.5 ( appendix 2A)
  • 0.5 M NaCl containing 0.5 M Tris⋅Cl, pH 7.5
  • Radiolabeled probe to transgene (made using Roche random‐primed DNA labeling kit according to manufacturer's instructions)
  • 96‐well plate
  • 1.5‐ml microcentrifuge tubes
  • 37°C incubator
  • 50°C water bath
  • 0.45‐µm nylon membrane (Hybond N+ or XL, Amersham)
  • Dot‐blot apparatus
  • Scintillation counter
  • PhosphorImager cassette, optional
  • STORM phosphoimager and ImageQuant software (GE Healthcare)
  • Additional reagents and equipment for restriction digestion of DNA ( appendix 1M), hybridization of DNA to membranes (Brown, ; Brown, or b; appendix 1A) and autoradiography (Voytas and Ke, ; appendix 1A)

Support Protocol 2: Determination of rAAV Titers by the Quantitative PCR Assay

  Materials
  • Virus fractions or final virus preparation (e.g., from protocol 1)
  • DNase digestion buffer (see recipe)
  • 0.5 M EDTA ( appendix 2A)
  • Proteinase solution (see recipe)
  • DNase/RNase‐free PCR‐grade water
  • Primers corresponding to the target sequence
  • Quantitative PCR reagents (follow the manufacturer's instructions for the reaction set up)
  • 96‐well qPCR reaction plate or individual reaction tubes (appropriate for your qPCR instrument)
  • Quantitative PCR instrument

Support Protocol 3: Infection of Cells In Vitro with rAAV and Determination of Titer by Transgene Expression

  Materials
  • HeLaRC32 cells (ACCT #CRL‐2972)
  • Complete DMEM/10% FBS (see recipe, or use tissue culture medium for other target cells using the supplier's instructions for those cells)
  • rAAV with appropriate transgene (see protocol 1)
  • Adenovirus (see protocol 5)
  • 48‐well 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 co‐infected with adenovirus (see protocol 5), 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 and should not be used in vivo. It is up to each investigator to establish a standard procedure for titering different rAAV preps.

Support Protocol 4: 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/cm3, 1.3 g/cm3, 1.4 g/cm3, and 1.5 g/cm3 density CsCl (see recipes)
  • 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
  • Autoclave
  • 50‐ml disposable polypropylene centrifuge tubes
  • Dry ice/ethanol bath
  • 37°C water bath
  • Benchtop 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)
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.

Basic Protocol 2: Stereotactic Microinjection of rAAV Into the Rat Brain

  Materials
  • 6‐ to 8‐week‐old Sprague‐Dawley rats
  • Anesthetic: pentobarbital (50 mg/kg body weight)/atropine methyl nitrate (2 mg/kg body weight) in 0.9% NaCl or ketamine (150 mg/kg body weight)
  • Betadine scrub or 70% ethanol
  • 1 mg/ml epinephrine
  • 1% procaine⋅HCl
  • rAAV preparation (see protocol 1) diluted in PBS ( appendix 2A) to desired concentration
  • Bonewax
  • Polysporin ointment
  • Rat brain atlas (e.g., Paxinos and Watson, )
  • Dental drill or Dremel with #862 Dremel engraving cutter
  • Rat stereotaxic frame (Kopf) in sterile surgical area
  • Variable speed syringe pump
  • Surgical instruments including:
    • Scalpel
    • Forceps
    • Needle holders
    • Small spatula
  • Sharp scissors or razor
  • 30‐G stainless steel for injector
  • PE‐10 intramedic tubing (Clay Adams)
  • 0 to 10‐µl syringe with Teflon‐tipped plunger (e.g., Precision Sampling glass Teflon tip plunger, gas‐tight 0 to 10‐µl syringe; Valco Instruments)
  • 1‐ml syringes equipped with 26‐ 30‐G needles
  • Animal clippers
  • Sterile gauze
  • 4‐0 silk suture
  • Heating pad, heated to 37°C
  • Clean cage without bedding
NOTE: Before beginning injections with the virus, it is important to verify the placement coordinates. This can be done by infusing dye (e.g., fast green) into one or two animals using the following procedure and then slicing the brain to verify injector placement.NOTE: A titer of at least 109 vector genomes (injected) is recommended. Using less than 108 vector genomes may not lead to a desired amount of transduced cells.

Basic Protocol 3: Intrathecal Administration of rAAV in Mice by Lumbar Puncture

  Materials
  • rAAV preparation (see protocol 1)
  • 60% (w/v) sterile lidocaine‐HCl in PBS (MPI, cat. no. 193917 or equivalent)
  • Phosphate‐buffered saline (PBS; appendix 2A) or rAAV dialysis/storage solution (see recipe)
  • Mice, at least 8 weeks old
  • 25‐ or 50‐µl Hamilton syringe, with 30‐G needle
  • Parafilm or microcentrifuge tube
  • Towel, at least 20 cm × 20 cm

Support Protocol 5: Histological Detection of Green Fluorescent Protein Expression

  Materials
  • Rodents transduced with GFP (Basic Protocols protocol 62 and protocol 73; AAV‐GFP or pAAV‐GFP virus available from UNC Vector Core Facility)
  • 4% (w/v) paraformaldehyde in PBS
  • Phosphate‐buffered saline (PBS; appendix 2A), 4°C
  • Super glue
  • 2% agar (store at 4°C in solid form, heat to 70°C to liquefy before using)
  • Distilled water
  • Fluorescent mounting medium (see recipe)
  • 1× PBS containing 3% goat serum and 0.1% Triton X‐100Anti‐GFP antibody (Millipore AB3080 rabbit anti‐GFP or equivalent)
  • VectaStain Elite Kit (rabbit; Vector Labs, cat. no. PK‐6101) containing:
    • Stock anti‐rabbit (Blue Label)
    • PBS buffer
    • Goat serum (Yellow Label)
    • Reagent A
    • Reagent B
    • ABC‐AP reagent mixing bottle
  • DAB (3,3′‐diaminobenzidine tetracholide; Polysciences, cat. no. 04008)
  • 200 mM NaHPO 4 (pH 7.4)
  • 0.5% (w/v) CoCl 2 in water
  • 1% (w/v) (NH 4) 2Ni(SO 4) 2 in water
  • 3% hydrogen peroxide
  • Standard mounting medium, e.g., Permount or Accumount
  • 50‐ml conical centrifuge tubes
  • Large forceps
  • Small forceps with hooks on the ends
  • Single‐edged razor blades
  • Vibrating microtome with blades
  • 12‐well and 24‐well tissue culture plates
  • Fine‐bristled paint brush
  • Glass petri dish, shallow
  • Dark work surface
  • Glass slides and coverslips
  • Drying rack
  • Fluorescence microscope with GFP filter
  • Shaking incubator
  • 0.22‐ or 0.45‐µm filter
  • Light microscope
  • Additional reagents and equipment for perfusion fixation and sectioning of brains (unit 1.1)
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Figures

  •   FigureFigure 4.17.1 Generation of adenovirus‐free recombinant adeno‐associated virus. 293 cells (which supply the Ad E1 gene) are transfected using three plasmids: the plasmid carrying the transgene (sub201‐gene “X,” AAV vector), the plasmid supplying the replication and capsid genes of AAV2 without terminal repeats (pXX2, AAV helper), and the plasmid supplying the adenovirus helper genes E2, E4, and VA RNA genes (pXX6, AD DNA), thereby generating Ad‐free rAAV.
  •   FigureFigure 4.17.2 (A) pSub201. The plasmid contains the terminal repeats and the replication ( rep) and capsid ( cap) genes of AAV Type 2. The rep‐cap fragment can be replaced by the gene cassette of interest, as only the terminal repeats are needed for packaging. (B) Plasmid pHpa‐trs‐SK.
  •   FigureFigure 4.17.3 CsCl step gradient before and after centrifugation. The step gradient is generated by underlying the virus‐containing clarified cell lysate with 1.3 and 1.5 g/cm3 CsCl solutions (see for instructions on forming the gradient). After centrifugation, the virus resides at the interface between the 1.3 and 1.5 g/cm3 CsCl solutions. Care should be taken when removing this layer not to remove any of the less‐dense cellular debris that remains above the 1.5 g/cm3 solution.
  •   FigureFigure 4.17.4 Green fluorescent protein (GFP) expression in the inferior colliculus and the globus pallidus 2 weeks after infusion of 1 µl of AAV‐GFP virus.

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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.
   Brown, T. 1993. Hybridization analysis of DNA blots. Curr. Protoc. Mol. Biol. 21:2.10.1‐2.10.16.
   Brown, T. 1999a. Southern blotting. Curr. Protoc. Mol. Biol. 21:2.9.1‐2.9.15.
   Brown, T. 1999b. Dot and slot blotting of DNA. Curr. Protoc. Mol. Biol. 21:2.9.15‐2.9.20.
   Burger, C., Gorbatyuk, O.S., Velardo, M.J., Peden, C.S., Williams, P., Zolotukhin, S., Reier, P.J., Mandel, R.J., and Muzyczka, N. 2004. Recombinant AAV viral vectors pseudotyped with viral capsids from serotypes 1, 2, and 5 display differential efficiency and cell tropism after delivery to different regions of the central nervous system. Mol. Ther. 10:302‐317.
   Cukor, G., Blacklow, N.R., Hoggan, D., and Berns, K.I. 1984. Biology of adeno‐associated virus. In The Parvoviruses. ( K.I. Berns, ed.) pp. 33‐66. Plenum, New York.
   Fairbanks, C.A. 2003. Spinal delivery of analgesics in experimental models of pain and analgesia. Adv. Drug Deliv. Rev. 55:1007‐1041.
   Fu, H., Muenzer, J., Samulski, R.J., Breese, G., Sifford, J., Zeng, X., and McCarty, D.M. 2003. Self‐complementary adeno‐associated virus serotype 2 vector: Global distribution and broad dispersion of AAV‐mediated transgene expression in mouse brain. Mol. Ther. 8:911‐917.
   Gray, S.J., Matagne, V, Bachaboina, L, Yadav, S, Ojeda, S, and Samulski, R.J. 2011. Preclinical differences of intravascular AAV9 delivery to neurons and glia: A comparative study of adult mice and non‐human primates. Mol. Ther. 19:1058‐1069.
   Heilig, J.S. Karen, L. Elbing, K.L., and Brent, R. 1998. Large‐scale preparation of plasmid DNA. Curr. Protoc. Mol. Biol. 41:1.7.1‐1.7.16.
   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.
   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.
   McCown, T.J., Xiao, X., Li, J, Breese, GR, and Samulski, RJ. 1996. Differential and persistent expression patterns of CNS gene transfer by an adeno‐associated virus (AAV) vector. Brain Res. 713:99‐107.
   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.
   Moore, D., Dowhan, D., Chory, J., and Ribaudo, R.K. 2002. Isolation and purification of large DNA restriction fragments from agarose gels. Curr. Protoc. Mol. Biol. 59:2.6.1‐2.6.12.
   Paxinos, G. and Watson, C. 1986. The Rat Brain in Stereotaxic Coordinates, 2nd ed. Academic Press, London.
   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., 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.
   Smith, F.E. and McCown, T.J. 1997. AAV vectors: General characteristics and potential use in the central nervous system. In Gene Transfer and Therapy for Neurological Disorders. ( E.A. Chiocca and X.O. Breakefield, eds.) pp. 79‐88. Humana Press, Totowa, N.J.
   Struhl, K. 1987. Subcloning of DNA fragments. Curr. Protoc. Mol. Biol. 13:3.16.1‐3.16.2.
   Voytas, D., and Ning Ke, N. 1999. Detection and quantitation of radiolabeled proteins and DNA in gels and blots. Curr. Protoc. Mol. Biol. 48:A.3A.1‐A.3A.10.
Key References
  McCown et al., 1996. See above.
  This paper uses the microinjection technique to demonstrate how several different brain areas can be transduced by rAAV. This is an early paper in which the virus titer is comparatively low and minimally contaminated with adenovirus. Higher titer adenovirus‐free preparations give better transduction with less cellular damage and longer duration of gene expression.
  Fairbanks et al., 2003. See above.
  This paper details the intrathecal injection protocol and its applications.
   Gray, S.J., Woodard, K.T., and Samulski, R.J. 2010. Viral vectors and delivery strategies for CNS gene delivery. Therapeutic Del. 1:517‐534.
  This review provides an overview of AAV as a gene therapy vector, discusses different routes of delivery and serotype differences, and contains information on recent accomplishments using rAAV vectors in the CNS.
   Grieger, J.C., Choi, V.W., and Samulski, R.J. 2006. Production and characterization of adeno‐associated viral vectors. Nat. Prot. 1:1412‐1428.
  This paper details the adenovirus‐free rAAV production protocol and additional protocols related to rAAV production and validation.
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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