AAV病毒包装步骤

Hiroaki Mizukami, Takashi Okada, Takashi Matsushita, and Keiya Ozawa

Division of Genetic Therapeutics, Center for Molecular Medicine,

Jichi Medical School1. Introduction

The protocol describe at below is typical of methods that are used to

propagate and purify AAV vectors for experiments both in vitro and in vivo.2. Principles of the Triple Plasmid Transfection System

The minimum regions in helper adenovirus that mediate the replication ofthe AAV vector are E1, E2A, E4, and VA1. The 293 line of human

embryonic kidney cells encodes the E1 region of the Ad5 genome2. When ahelper plasmid that encodes the E2A, E4, and VA regions (Ad-helperplasmid) is used to transfect 293 cells, together with plasmids that encodethe genome of the AAV vector (vector plasmid), as well as the rep and capgenes (AAV-helper plasmid), the AAV vector is produced as efficiently aswhen infection by adenovirus is used as a source of helper virus. Eliminationof the heat-inactivation step directed against contaminating adenovirus canimprove the yield of the virus. Furthermore, contamination of the AAVvector stock by most adenoviral proteins can be avoided when this helpervirus-free method is used.3. Reagents

Helper plasmid DNA (pHLP, pAdeno)

Vector plasmid harboring the gene of interest flanked by inverted terminalrepeats (ITRs )

293 cells (human embryonic kidney cells)DMEM/F12 culture mediumFetal bovine serum

2 x HBS buffer, containing 290 mM NaCl, 50 mM HEPES buffer and 1.5mM Na2HPO4, pH 7.1300 mM CaCl2

Phosphate-buffered saline (PBS)

1 M HEPES buffer, pH 7.4

100 mM Tris-HCl (pH 8.0) plus 150 mM NaCl (TBS)0.5 M EDTA (pH 8.0)

40% sucrose plus 0.01% BSA in TBS

DNase buffer, containing 50 mM Hepes (pH 7.6), 0.15 M NaCl and 10 mMMgCl2

HNE buffer, containing 50 mM Hepes (pH 7.4), 0.15 M NaCl and 25 mMEDTA

A solution of CsCl in HNE (1.25 g/ml) in HNEA solution of CsCl in HNE (1.50 g/ml) in HNE

4. Plasmids

The AAV vector plasmid, pAAVlacZ, harbors a beta-galactosidase

expression cassette flanked by inverted terminal repeats (ITRs). The AAV-helper plasmid pHLP, harboring rep and cap, has been descriped previouslyas pHLP19. The Ad-helper plasmid pAdeno is identical to pVAE2AE4-5and encodes the entire E2A and E4 regions plus the VA RNA I and II genes1.5. Transfection and Extraction of Virus

This protocol is for the transfection of cells in one 225-cm2 flask. Forcultures of other sizes, multiply volumes on a linear basis.

Plate trypsinized 293 cells at 5 x 106 cells per 225-cm2 flask to generate amonolayer of 20% to 40% confluence when cells attach initially to thesurface of the flask. Use 40 ml of medium per flask and try to avoid platingclumps of cells and to make sure that cells are distributed evenly on thesubstratum. An even density of cells over the entire substratum is essentialfor high yield and it can be achieved by moving the flask of newly platedcells gently in a crosswize pattern before cells become attached. Place theplate in an incubator in 5% CO2 in air and allow cells to grow to 80%confluence (24 to 48 h.).

One hour before transfection, replace half the medium in the flask withfresh medium. Add 23 µg of vector and of each helper plasmid to 4 ml of300 mM CaCl2. Gently add this solution to 4 ml of 2x HBS and miximmediately by gentle inversion three times. Immediately pipette this

mixture into the 225-cm2 flask of 293 cells in 40 ml of DMEM/F12 medium

plus 10% FCS and swirl to produce a homogeneous solution. Immediatelyreturn the plate to the incubator and incubate at 37o for 4 to 6 hr. Do notdisturb the plate during this period. At the end of the incubation, replace themedium with pre-warmed DMEM/F12 culture that medium contains 2%FCS. Three days after transfection, add 1 ml of 0.5 M EDTA to the flask andincubate for 3 min at room temperature. Collect the suspension of cells andcentrifuge it at 300xg for 10 min. Remove the supernatant and resuspend thecells in the pellet in 2 ml of TBS.

Freeze and thaw the cells that have been suspended in TBS three times byplacing them alternately in a dry ice/ethanol bath until the suspension iscompletely frozen and in a water bath of 37oC until it is completely thawed.Return the sample to the ice bath immediately when it is completely thawed.Remove tissue debris by centrifugation at 10,000xg for 10 min and collectthe supernatant.

6. Purification of the AAV vector

Prepare supernatants, as described above from 24 flasks. Place 11 ml of asolution of 40% sucrose plus 0.01% BSA in TBS in a sterile ultracentrifugetube (Ultrabottle #3430-3870; Nalge Nunc, Rochester, NY). Carefullyoverlay the 48 ml of pooled supernatants on this solution. Pellet the crudeviral particles by centrifugation at 100,000xg for 16 hr at 4oC. Resuspend thepellet by vigorous agitation in 5 ml of DNase buffer. Add 1,000 units of

DNase I and incubate for 1 hr at 37oC. Add 250 µL of 0.5 M EDTA and thenremove debris by centrifugation at 10,000xg for 2 min. Then filter thesupernatant through a low-protein-binding 5-µm syringe filter (Sterile

Acrodisc Syringe Filter; Pall Gelman Laboratory, Ann Arbor, MI). Load thefiltered material onto a two-tier CsCl gradient (1.25 g/ml and 1.50 g/ml)prepared in HNE buffer. Spin the gradient at 35,000 rpm for 2 h at 16oC inan SW40 rotor (Beckman Instruments, Palo Alto, CA). Collect the band ofviral particles and load it on a second two-tier CsCl gradient (1.25 g/ml and1.50 g/ml) prepared in HNE buffer. Spin the gradient at 65,000 rpm for 2 hat 16oC in a VTi65.2 rotor (Beckman Instruments). Collect fractions of 0.5ml each and select the virus-rich fraction by semi-quantitative PCR analysis,Western blotting with antibodies against Cap, or quantitative DNA dot-blothybridization. Use a dialysis cassette (Slide-A-Lyzer; Pierce, Rockford, IL)

to desalt the virus-rich fraction by three cycles of dilution with 300 ml ofHNE buffer. Concentrate the material to 50 µL with Ultrafree-4 (Millipore,Bedford, MA) according to the manufacturer's instruction. The final titerusually ranges between 1 x 1013 and 5 x 1013 particles from 5 x 108 293 cells,as determined by quantitative DNA dot-blot hybridization or Southern

blotting. In the case of an AAV vector that expresses lacZ, production of thevector can be assessed functionally by titration with 293 cells in the presenceof a wild-type adenovirus type 2 at an MOI of 50. The transduced cells areincubated for 24 h at 37oC before fixation and X-gal staining. Stained cellsare counted under a light microscope. The ratio of genomes to functionalviral particles is determined from the total number of genomes divided bythe total number of functional units (as determined by X-gal staining) forverification of infectivity.7. References

1. T. Matsushita, S. Elliger, C. Elliger, G. Podsakoff, L. Villarreal, G. J.Kurtzman, Y. Iwaki, and P. Colosi, Gene Ther. 5, 938 (1998).2. F. L. Graham, J. Smiley, W. C. Russell, and R. Nairn, J. Gen. Virol. 36, 59(1977).