Production and Discovery of Novel Recombinant Adeno‐Associated Viral Vectors

Christian Mueller1, Dmitry Ratner1, Li Zhong2, Miguel Esteves‐Sena3, Guangping Gao4

1 Department of Pediatrics, University of Massachusetts Medical School, Worcester, Massachusetts, 2 Department of Medicine, University of Massachusetts Medical School, Worcester, Massachusetts, 3 Department of Neurology, University of Massachusetts Medical School, Worcester, Massachusetts, 4 State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, Chengdu, People's Republic of China
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 14D.1
DOI:  10.1002/9780471729259.mc14d01s26
Online Posting Date:  August, 2012
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In this unit, we describe the detailed procedure for a three‐plasmid transfection method for rAAV production, and discuss its advantages, limitations, and troubleshooting techniques. We further discuss the rAAV purification process using CsCl gradients, as well as subsequent quality control methods using SDS‐PAGE and real‐time PCR to assess vector purity, packaging efficiency, and viral titer. Finally, we elaborate on a PCR‐based strategy that can be used to discover novel AAV capsid sequences from primate tissue, which can be used to develop newer‐generation rAAVs with a greater diversity of tissue tropism for clinical gene therapy. Curr. Protoc. Microbiol. 26:14D.1.1‐14D.1.21. © 2012 by John Wiley & Sons, Inc.

Keywords: rAAV; AAV production; transfection; AAV packaging; cap; cesium chloride

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Table of Contents

  • Introduction
  • Basic Protocol 1: Production of AAV Vector by Transient Transfection
  • Basic Protocol 2: Purification of AAV Vectors by Cesium Chloride Gradient Sedimentation
  • Basic Protocol 3: Vector Genome Copy Titration by Real‐Time PCR
  • Support Protocol 1: Preparation of Standard Set
  • Basic Protocol 4: Analysis of rAAV Purity by SDS‐PAGE and Silver Staining
  • Basic Protocol 5: PCR‐Based Method for Novel AAV Discovery
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: Production of AAV Vector by Transient Transfection

  • HEK‐293 cells (ATCC)
  • Cell growth medium (see recipe)
  • Milli‐Q‐purified water
  • 2.5 M CaCl 2 solution, filter sterilized (0.22 µm)
  • ΔF6 adeno helper plasmid (available from Penn Vector Core, )
  • pTRANS plasmids of different serotypes (Gao et al., , , ; available from Penn Vector Core, )
  • pCIS plasmid with gene of interest
  • 2× HBS solution (see recipe)
  • Resuspension buffer: 1 mM MgCl 2 in 50 mM Tris⋅Cl, pH 7.4, filter sterilized (0.22 µm)
  • 150‐mm sterile tissue culture plates
  • Humidified 37°C and 5% CO 2 cell culture incubator
  • 125‐ml sterile bottle
  • 50‐ml sterile conical tubes
  • Sterile cell scarpers
  • 500‐ml sterile centrifuge bottles
  • Refrigerated tabletop centrifuge

Basic Protocol 2: Purification of AAV Vectors by Cesium Chloride Gradient Sedimentation

  • Triple‐transfected HEK‐293 cells (see protocol 1)
  • Benzonase (EMD Chemicals)
  • 10% deoxycholic acid
  • Ultrapure cesium chloride (CsCl)
  • CsCl solutions, light and heavy (see recipe)
  • 70% (v/v) ethanol
  • Dulbecco's phosphate‐buffered saline (DPBS), calcium‐ and magnesium‐free (e.g., Invitrogen, cat. no. 14190‐250)
  • 5% (w/v) D‐sorbitol in DPBS, filter‐sterilized (0.22 µm)
  • Glycerol
  • 37°C water bath
  • Sonicator
  • Refrigerated tabletop centrifuge
  • 15‐ and 50‐ml sterile conical centrifuge tubes
  • Beckman ultracentrifuge with:
    • SW 28 or 32 Ti swinging‐bucket rotor and centrifuge tubes
    • 70.1 Ti fixed‐angle rotor and Quick‐Seal tubes
  • 10‐ml pipets
  • 1.5‐ml microcentrifuge tubes
  • Centrifuge tube holder
  • 1.5 in., 18‐G needles
  • Refractometer (Milton Roy)
  • 5‐ and 10‐ml syringes
  • Cordless centrifuge tube topper (Beckman Coulter)
  • 16‐G needle
  • 3‐ml syringe
  • Slide‐a‐Lyzer cassette, 0.5‐3 or 3‐12 ml (10,000 MWCO, Pierce)
  • 4‐liter glass beaker with stir bar, both autoclaved
  • Cryovials

Basic Protocol 3: Vector Genome Copy Titration by Real‐Time PCR

  • RNase‐free DNase I and buffer
  • Nuclease‐free water
  • Test article: purified rAAV vector (see protocol 2)
  • Control vector genome plasmid with known copy number
  • AAV validation sample with known titer
  • Vector genome standard set (see protocol 4)
  • Sample dilution buffer (see recipe)
  • 2× TaqMan Universal PCR Mix, No UNG (Applied Biosystems)
  • 9 µM forward and reverse primer stock solutions
  • 2 µM 6FAM fluorescent probe stock solution
  • 0.65‐ and 1.7‐ml microcentrifuge tubes
  • 15‐ml conical tube
  • 96‐well plates
  • Ultraclear cap strips and Cap‐It tool (or film for sealing plates)
  • PCR‐dedicated hood
  • Real‐time PCR station

Support Protocol 1: Preparation of Standard Set

  • Plasmid standard
  • Restriction enzyme with buffer
  • DNA purification kit (e.g., Qiagen, cat. no. 28104)
  • Nuclease‐free water
  • 10× PCR buffer (Applied Biosystems, cat. no. N8080189)
  • Sheared salmon sperm (SSS) DNA
  • Spectrophotometer
NOTE: Use a biosafety cabinet to prevent potential contamination from the laboratory environment. Real‐time PCR is extremely sensitive to contamination.

Basic Protocol 4: Analysis of rAAV Purity by SDS‐PAGE and Silver Staining

  • Reference standard at 1 × 1013 particles/ml
  • 2× Tris‐glycine SDS sample buffer (Invitrogen)
  • 10× NuPage reducing agent (Invitrogen)
  • Test article: purified rAAV vector (see protocol 2)
  • Protein molecular‐weight marker in the range of 20‐220 kDa
  • 10% Tris‐glycine gels (e.g., Novex 15‐well gels, Invitrogen)
  • Tris‐glycine SDS running buffer (e.g., 10× Novex, Invitrogen)
  • Silver staining kit (e.g., SilverXpress, Invitrogen)
  • 0.5‐ml microcentrifuge tubes
  • 95°C heating block
  • Gel station (e.g., BioRad)
  • Additional reagents and equipment for SDS‐PAGE of proteins ( appendix 3M)

Basic Protocol 5: PCR‐Based Method for Novel AAV Discovery

  • Tissue sample of interest
  • QIAamp Tissue DNA mini kit (Qiagen), with Buffer ATL, proteinase K, and RNase A
  • Nuclease‐free water (Qiagen)
  • 1× Platinum PCR SuperMix, High Fidelity (Invitrogen)
  • 10 µM forward and reverse PCR primer stock solutions
  • 10 ng/µl pAAVrep2/cap2 plasmid (for positive control; University of Pennsylvania, Penn Vector Core, )
  • 1% or 1.2 % agarose gel (Fisher)
  • 1× TAE buffer (Fisher)
  • TOPO TA Cloning Kit for sequencing (Invitrogen)
  • QIAprep Spin Miniprep Kit (Qiagen)
  • EcoRI restriction endonuclease and buffer
  • 5‐mm stainless steel beads (Qiagen)
  • 2‐ml microcentrifuge tubes
  • Disposable scalpels (Fisher)
  • TissueLyser (Qiagen)
  • 1.5‐ml microcentrifuge tube
  • 0.2‐ml micro‐PCR tubes
  • Thermocycler
  • Vector NTI software (Informax)
  • Additional reagents and equipment for agarose gel electrophoresis and DNA sequencing
NOTE: Prior to extraction and PCR, prepare a work space in a Biosafety Level II tissue culture hood by UV irradiating all surfaces for 15 min and then wiping all surfaces with Lysol followed by 70% ethanol.
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