Gene Transfer in the Liver Using Recombinant Adeno‐Associated Virus

Seemin Seher Ahmed1, Jia Li1, Jonathan Godwin1, Guangping Gao2, Li Zhong2

1 Department of Microbiology and Physiology Systems, University of Massachusetts Medical School, Worcester, Massachusetts, 2 Co‐corresponding authors
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 14D.6
DOI:  10.1002/9780471729259.mc14d06s29
Online Posting Date:  May, 2013
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Abstract

Liver‐directed gene transfer and gene therapy are rapidly gaining attention primarily because the liver is centrally involved in a variety of metabolic functions that are affected in various inherited disorders. Recombinant adeno‐associated virus (rAAV) is a popular gene delivery vehicle for gene therapy, and intravenous delivery of some rAAV serotypes results in very efficient transduction in the liver. rAAV‐mediated gene transfer to the liver can be used to create somatic transgenic animals or disease models for studying the function of various genes and miRNAs. The liver is the target tissue for gene therapy of many inborn metabolic diseases and may also be exploited as a “biofactory” for production of coagulation factors, insulin, growth hormones, and other non‐hepatic proteins. Hence, efficient delivery of transgenes and small RNAs to the liver by rAAV vectors has been of long‐standing interest to research scientists and clinicians alike. This unit describes methods for delivery of rAAV vectors by several injection routes, followed by a range of analytical methods for assessing the expression, activity, and effects of the transgene and its product. Curr. Protoc. Microbiol. 29:14D.6.1‐14D.6.32. © 2013 by John Wiley & Sons, Inc.

Keywords: adeno‐associated virus; liver; gene transfer; gene therapy

     
 
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Table of Contents

  • Introduction
  • Vector Delivery Methods
  • Basic Protocol 1: Systemic Vector Delivery by Lateral Tail Vein Injection
  • Alternate Protocol 1: Systemic Vector Delivery by Retro‐Orbital Sinus Injection
  • Basic Protocol 2: Direct Vector Delivery by Intraportal Injection
  • Alternate Protocol 2: Vector Delivery by Intrasplenic Injection
  • Support Protocol 1: Preparation of rAAV Vectors for Injection
  • Monitoring Transduction in Live Animals
  • Basic Protocol 3: Whole‐Body Live Imaging of Transduced Firefly Luciferase Expression
  • Monitoring Transduction Using Serum Reporter Assays
  • Basic Protocol 4: A1AT Serum Reporter Assay
  • Basic Protocol 5: Gluc Serum Reporter Assay
  • Support Protocol 2: Collection and Storage of Blood and Serum
  • Tissue Collection and Storage
  • Support Protocol 3: Tissue Collection and Storage for Biodistribution Studies
  • Support Protocol 4: Tissue Collection and Storage for Transgene Expression and Histopathology Studies
  • Analytical Methods for Isolated Tissues
  • Basic Protocol 6: Quantification of Vector Genome Copy Number in rAAV‐Transduced Liver by Real‐Time PCR
  • Basic Protocol 7: Detection of Transgene Expression in rAAV‐Transduced Liver by X‐Gal Staining
  • Basic Protocol 8: Histology of Paraffin‐Embedded Liver Samples Using H&E Staining
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Systemic Vector Delivery by Lateral Tail Vein Injection

  Materials
  • Mouse
  • Ethanol
  • rAAV vector in a 1‐ml syringe with 28‐ or 30‐G needle (see protocol 5)
  • Mouse restraining device (Dan‐Kar)
  • Circulating warm water heating pad (Kent Scientific) or heat lamp (OSRAM Sylvania Products)
  • Sterile gauze

Alternate Protocol 1: Systemic Vector Delivery by Retro‐Orbital Sinus Injection

  Materials
  • Mouse
  • Isoflurane (Webster Veterinary Supply)
  • Oxygen (Airgas)
  • rAAV vector in a 1‐ml syringe with 30‐G needle (see protocol 5)
  • Alcohol wipes
  • Anesthesia machine (Vetequip, Colonial Medical Supply)
  • Sterile gauze

Basic Protocol 2: Direct Vector Delivery by Intraportal Injection

  Materials
  • Mouse
  • Isoflurane (Webster Veterinary Supply)
  • Oxygen (Airgas)
  • Ophthalmic ointment
  • Aseptic surgical scrub
  • Betadine (povidone‐iodine) or chlorhexidine antiseptic
  • 70% isopropyl alcohol
  • rAAV vector in a 1‐ml syringe with 33‐G needle (see protocol 5)
  • Buprenorphine analgesic
  • Ketoprofen or carpofen
  • Anesthesia machine (Vetequip, Colonial Medical Supply)
  • Circulating warm water heating pad (Kent Scientific)
  • Surgical drapes or towels
  • Animal hair trimmer with clipper blade size 40 or less (Oster)
  • Sterile gauze
  • Sterile sponges or cotton swabs
  • Mouse surgical package (Kent Scientific)
  • Sutures: 4‐0, 5‐0, or 6‐0 polydioxanone (PDS), Vicryl, Prolene, or nylon (Kent Scientific)
NOTE: Survival surgery should be performed using aseptic procedures with sterile surgical gloves, masks, and instruments. The surgical area should be uncluttered and disinfected. Refer to Hoogstraten‐Miller and Brown ( ) for a detailed description of setting up the surgical area. Injection material should be ready and kept on ice before the surgical procedure.

Alternate Protocol 2: Vector Delivery by Intrasplenic Injection

  Materials
  • rAAV vector(s) to be injected
  • Normal saline (0.9% NaCl) or Dulbecco's phosphate‐buffered saline (DPBS; Fisher Scientific), sterile
  • 1‐ml syringe with 28‐, 30‐, or 33‐G needle (Fisher Scientific)
NOTE: All solutions used for vector dilution should be sterile and tissue culture grade. Only sterile disposable tubes and sterile filtered pipet tips should be used. To avoid contamination, vector samples should be prepared in a culture hood dedicated for rAAVs.

Support Protocol 1: Preparation of rAAV Vectors for Injection

  Materials
  • Mice injected with rAAV‐Fluc expression vector or DPBS (see protocol 1 or 2, protocol 2 or 2)
  • Isoflurane (Webster Veterinary Supply)
  • Oxygen (Airgas)
  • 15 mg/ml beetle luciferin substrate (Promega) in Dulbecco's phosphate‐buffered saline (DPBS; Fisher Scientific)
  • ∼700 ml plastic container with lid (e.g., Tupperware)
  • Scale with 200‐g capacity
  • In vivo imaging system (IVIS, Xenogen) including light‐free warming chamber (30°C)
  • Cooled CCD camera for in vivo bioluminescence imaging (Xenogen) with image analysis software

Basic Protocol 3: Whole‐Body Live Imaging of Transduced Firefly Luciferase Expression

  Materials
  • Rabbit anti‐human‐α1‐antitrypsin (anti‐A1AT; Sigma‐Aldrich)
  • 10× coating solution (KPL; dilute to 1× in Milli‐Q water before use)
  • 20× wash solution (KPL; dilute to 1× in Milli‐Q water before use)
  • 10× blocking solution (10% bovine serum albumin, KPL; dilute to 1× in Milli‐Q water before use)
  • Sample buffer: 1:15 blocking solution concentrate and 0.05% Tween 20 in Milli‐Q water
  • Serum from mice injected with rAAV‐A1AT expression vector or DPBS (for serum collection, see protocol 9)
  • 10 mg/ml A1AT (Sigma‐Aldrich) standard stock solution
  • Horseradish peroxidase (HRP)–conjugated goat anti‐A1AT antibody (Fitzgerald)
  • ABTS peroxidase substrate (2,2′‐azino‐di(3‐ethylbenzthiazoline‐6‐sulfonate), KPL)
  • 96‐well flat‐bottom, high‐binding microtiter plates (Costar)
  • Squirt bottle, plate washer, or dunking bucket (Fisher Scientific)
  • 12‐channel multichannel pipettor (e.g., Rainin)
  • Plate sealers (adhesive sealing tape; Fisher Scientific)
  • Heat‐resistant NUNC plates (Fisher Scientific)
  • Sterile 15‐ml conical tubes (Corning)
  • Sterile 1.5‐ml microcentrifuge tubes (Fisher Scientific)
  • Plate luminometer equipped with injector (BioTek Instruments)

Basic Protocol 4: A1AT Serum Reporter Assay

  Materials
  • Concentrated hydrochloric acid (HCl, Fisher Scientific)
  • Methanol (Fisher Scientific)
  • Coelenterazine‐h or coelenterazine‐n substrate (Promega)
  • Dulbecco's phosphate‐buffered saline (DPBS, with Ca2+ and Mg2+; Fisher Scientific)
  • Serum from mice injected with rAAV‐Gluc expression vector or DPBS (for serum collection, see protocol 9)
  • Black or white opaque 96‐well microtiter plate (Costar)
  • Plate luminometer equipped with injector (BioTek Instruments)

Basic Protocol 5: Gluc Serum Reporter Assay

  Materials
  • Mice injected with rAAV vector or DPBS (see protocol 1 or 2, protocol 2 or 2)
  • Antibiotic ophthalmic ointment
  • Goldenrod animal bleeding lancet (Medipoint)
  • 19‐, 21‐, or 23‐G needle (Fisher Scientific)
  • Anti‐coagulant tubes and serum collection tubes (Fisher Scientific)
  • 50‐µl sterile Natelson capillary tube (Fisher Scientific)
  • Sterile, dry cotton pad
  • Additional reagents and equipment for injection via the lateral tail vein (see protocol 1) or retro‐orbital sinus (see protocol 2)

Support Protocol 2: Collection and Storage of Blood and Serum

  Materials
  • 10% (v/v) bleach solution (Clorox Professional Products)
  • Mice injected with rAAV vector or DPBS (see protocol 1 or 2, protocol 2 or 2)
  • Dulbecco's phosphate‐buffered saline (DPBS), cold (optional, for harvesting bone marrow)
  • Sterile absorbent surgical pads
  • Sterile, disposable surgical tools: scissors, forceps, razor blades (Kent Scientific)
  • Sterile microcentrifuge tubes (Fisher Scientific)
  • Liquid nitrogen (Middlesex Gases & Technologies)
  • Dry ice (Middlesex Gases & Technologies)
  • Sterile, disposable 3‐ml syringes with 21‐G needles (Fisher Scientific; optional, for harvesting bone marrow)

Support Protocol 3: Tissue Collection and Storage for Biodistribution Studies

  Materials
  • 10% (v/v) bleach solution (Clorox Professional Products)
  • Mice injected with rAAV vector or DPBS (see protocol 1 or 2, protocol 2 or 2)
  • Liquid nitrogen (Middlesex Gases & Technologies)
  • Dulbecco's phosphate‐buffered saline (DPBS; Fisher Scientific)
  • Optimal cutting temperature (OCT) embedding medium (Sakura Finetek)
  • Dry ice (Middlesex Gases & Technologies)
  • Ethanol (Decon Labs)
  • 4% (w/v) paraformaldehyde (Sigma‐Aldrich) or 10% neutral buffered formalin (Sigma‐Aldrich)
  • 30% (w/v) sucrose solution (Fisher Scientific)
  • Xylene (Fisher Scientific)
  • Paraffin wax (Fisher Scientific)
  • Sterile, disposable scissors and forceps (Kent Scientific)
  • 1.5‐ml microcentrifuge tubes
  • Embedding cup: made from aluminum foil using a small flat‐bottom bottle (∼1 cm diameter) or commercial vinyl embedding molds (Fisher Scientific)
  • Aluminum foil
  • Cryostat
  • Slides (Fisher Scientific)
  • Microtome (Triangle Biomedical Sciences)
  • 40°‐45°C deionized water bath
  • Small brush

Support Protocol 4: Tissue Collection and Storage for Transgene Expression and Histopathology Studies

  Materials
  • Fresh‐frozen liver tissue samples (see protocol 10)
  • QIAamp DNA Mini kit (Qiagen)
  • Proteinase K (Qiagen)
  • RNase A (Qiagen)
  • Ethanol (Decon Labs)
  • PCR standards (linearized plasmid DNA containing vector genome)
  • Mouse liver genomic DNA (gDNA, for spiking standards)
  • Master mix: GoTaq qPCR Master Mix or Perfecta SYBR Green FastMix (Applied Biosystems)
  • GoTaq Flexi DNA polymerase (Applied Biosystems)
  • Primers (Eurofins MWG Operon)
  • Probes (Applied Biosystems)
  • dNTP mix (Applied Biosystems)
  • DNase‐free water (Qiagen)
  • 2‐ml microcentrifuge tubes, sterile (Fisher Scientific)
  • 5‐mm stainless steel beads (Qiagen), autoclaved
  • TissueLyser II (Qiagen)
  • 37°, 56°, and 70°C water baths
  • 96‐well PCR plate and appropriate adhesive plate cover (Fisher Scientific)
  • 0.5‐ml microcentrifuge tubes
  • Real‐time PCR system (Applied Biosystems)
  • 96‐well plate holder (Fisher Scientific)

Basic Protocol 6: Quantification of Vector Genome Copy Number in rAAV‐Transduced Liver by Real‐Time PCR

  Materials
  • Fresh, OCT‐embedded cryostat sections (see protocol 11)
  • Dulbecco's phosphate‐buffered saline (DPBS; Fisher Scientific)
  • Fixative solution: 0.5% (w/v) glutaraldehyde (Sigma‐Aldrich) in DPBS
  • Wash solution: 2 mM MgCl 2 (Sigma‐Aldrich) in DPBS
  • 50 mg/ml 5‐bromo‐4‐chloro‐3‐indolyl‐β‐D‐galactoside (X‐gal; Sigma‐Aldrich) in dimethylformamide (DMF; Sigma‐Aldrich)
  • X‐gal staining solution (see recipe)
  • Nuclear Fast Red (Vector Laboratories)
  • Ethanol (Decon Labs)
  • Xylene (Fisher Scientific)
  • Permount (Vector Laboratories)
  • Glass or metal slide holder (Fisher Scientific)
  • Staining containers (Fisher Scientific)
  • 24 × 50–mm coverslips (Fisher Scientific)
  • Light microscope with digital camera (e.g., Olympus)
  • NIH ImageJ (NIH) or other image analysis software

Basic Protocol 7: Detection of Transgene Expression in rAAV‐Transduced Liver by X‐Gal Staining

  Materials
  • Paraffin‐embedded tissue sections (see protocol 11)
  • Xylene (Fisher Scientific)
  • Ethanol (Decon Labs)
  • Hematoxylin solution (Sigma‐Aldrich)
  • Acidic ethanol: 3 ml HCl, 92 ml ethanol, 5 ml water
  • Eosin solution (Sigma‐Aldrich)
  • Permount (Vector Laboratories)
  • 60°C drying oven
  • Glass or metal slide holder (Fisher Scientific)
  • Staining containers (Fisher Scientific)
  • Coverslips (Fisher Scientific)
  • Light microscope with digital camera (e.g., Olympus)
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Figures

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Literature Cited

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