Gene Transfer in the Lung Using Recombinant Adeno‐Associated Virus

Alisha M. Gruntman1, Christian Mueller2, Terence R. Flotte3, Guangping Gao4

1 Gene Therapy Center, University of Massachusetts Medical School, Worcester, Massachusetts, 2 Department of Pediatrics, University of Massachusetts Medical School, Worcester, Massachusetts, 3 Department of Microbiology and Physiological Systems, University of Massachusetts Medical School, Worcester, Massachusetts, 4 State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, Chengdu, Sichuan, People's Republic of China
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 14D.2
DOI:  10.1002/9780471729259.mc14d02s26
Online Posting Date:  August, 2012
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Abstract

Adeno‐associated virus (AAV) is a small replication‐deficient DNA virus belonging to the Parvovirinae family. It has a single‐stranded ∼4.7‐kb genome. Recombinant AAV (rAAV) is created by replacing the viral rep and cap genes with the transgene of interest along with promoter and polyadenylation sequences. The short viral inverted terminal repeats must remain intact for replication and packaging in production, as well as vector genome processing and persistence in the transduction process. The AAV capsid (serotype) determines the tissue tropism of the rAAV vector. In this unit we will discuss serotype selection for lung targeting along with the factors effecting efficient delivery of rAAV vectors to the murine lung. Detailed procedures for lung delivery (intranasal, orotracheal, and surgical tracheal injection), sample collection, and post‐mortem tissue processing will be described. Curr. Protoc. Microbiol. 26:14D.2.1‐14D.2.17. © 2012 by John Wiley & Sons, Inc.

Keywords: adeno‐associated virus; vector delivery; lung; gene therapy

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Intranasal Recombinant Adeno‐Associated Virus Vector Delivery
  • Alternate Protocol 1: Speculum/Otoscope‐Assisted Orotracheal Recombinant Adeno‐Associated Virus Vector Delivery
  • Alternate Protocol 2: Surgical Tracheal Recombinant Adeno‐Associated Virus Vector Delivery
  • Alternate Protocol 3: BioLITE‐Assisted Orotracheal Recombinant Adeno‐Associated Virus Vector Delivery
  • Support Protocol 1: Preparing rAAV Vector for Delivery
  • Support Protocol 2: Post‐Mortem Bronchoalveolar Lavage
  • Support Protocol 3: Collecting Lung Tissue for Histology and/or Immunohistochemistry or Immunofluorescence
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Intranasal Recombinant Adeno‐Associated Virus Vector Delivery

  Materials
  • Animal: 6‐week‐old mice or older (18 g minimum)
  • Xylazine (20 mg/ml; see recipe)
  • Ketamine (100 mg/ml; see recipe)
  • 3/10 ml syringes equipped with 31‐G needles (8‐mm length needle; e.g., BD Ultra‐Fine II short needle insulin syringes)
  • Ophthalmic ointment (such as Puralube; Webster, cat. no. 07‐888‐2572; http://www.webstervet.com)
  • Vector solution (see protocol 5)
  • Rodent Intubation Stand (Braintree Scientific, cat. no. RIS‐100) or Rodent Work Stand (Braintree Scientific, cat. no. RW‐A3467)
  • Incisor loop (Hallowell, cat. no. 210A3490A)
  • Animal cages

Alternate Protocol 1: Speculum/Otoscope‐Assisted Orotracheal Recombinant Adeno‐Associated Virus Vector Delivery

  • 2% lidocaine HCl jelly (Webster, cat. no. 07‐835‐7610)
  • Mouse Intubation Pack (Braintree Scientific, cat. no. RW‐A3746), including: incisor loop, intubation speculum (to use with the otoscope), pointed cotton‐tipped applicators, mirror for verification of placement, stylet, lidocaine applicator, and tutorial video
  • Welch Allyn otoscope [NICAD (Braintree Scientific, cat. no. RW‐A3749) or LI Ion (Braintree Scientific, cat. no. RW‐A3754)]
  • 20‐G, 1.25‐in. catheter (BD Angiocath, Becton Dickinson)
  • Chilled mirror
  • Lung inflation bulbs (Braintree Scientific, cat. no. LIB‐03)
  • 0.35‐ml syringes
  • 1‐ml tuberculin slip tip syringe

Alternate Protocol 2: Surgical Tracheal Recombinant Adeno‐Associated Virus Vector Delivery

  • Depilatory cream (such as Nair), scissors, or clippers to remove hair from surgical sight
  • Chlorhexadine or betadine scrub and 70% alcohol
  • Gauze sponge
  • No. 15 scalpel blade and handle or 41/2‐in. curved tissue scissors
  • Adson tissue forceps (or other delicate tissue forceps)
  • IV butterfly catheter with 23‐ or 25‐G needle, optional
  • 4‐0 monofilament absorbable suture on a cutting needle (such as PDSII) or tissue glue (such as Dermabond)
  • Needle holder (for suturing skin)

Alternate Protocol 3: BioLITE‐Assisted Orotracheal Recombinant Adeno‐Associated Virus Vector Delivery

  • BioLITE Mouse Kit (MI‐Kit; Braintree Scientific) including:
    • Fiber optic illuminator
    • Fiber optic stylet
    • Lung inflation bulbs
    • Catheters (20‐G, 1‐in.)
  • Rodent Intubation Stand (Braintree Scientific, RIS‐100) or Rodent Work Stand (Braintree Scientific, RW‐A3467)

Support Protocol 1: Preparing rAAV Vector for Delivery

  Materials
  • rAAV vector
  • Ice
  • 200‐µl pipet tips
  • 20‐ to 200‐µl pipets
  • Parafilm or sterile petri dish

Support Protocol 2: Post‐Mortem Bronchoalveolar Lavage

  • Sterile saline
  • Iris scissors
  • 3‐ml syringes
  • 1.5‐ml microcentrifuge tubes
  • Additional reagents and equipment for euthanizing the animal (Donovan and Brown, )

Support Protocol 3: Collecting Lung Tissue for Histology and/or Immunohistochemistry or Immunofluorescence

  • Sterile saline
  • Neutral buffered formalin
  • 5‐ml syringes
  • 27‐G needles
  • 10‐ml conical tubes
  • Additional reagents and equipment for euthanizing the animal (Donovan and Brown, )
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Figures

Videos

Literature Cited

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   Boutin, S., Monteilhet, V., Veron, P., Leborgne, C., Benveniste, O., Montus, M.F., and Masurier, C. 2010. Prevalence of serum IgG and neutralizing factors against adeno‐associated virus (AAV) types 1, 2, 5, 6, 8, and 9 in the healthy population: Implications for gene therapy using AAV vectors. Hum. Gene Ther. 21:704‐712.
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   Donovan, J. and Brown, P. 2006. Euthanasia. Curr. Protoc. Immunol. 73:1.8.1‐1.8.4.
   Flotte, T.R., Afione, S.A., and Zeitlin, P.L. 1994. Adeno‐associated virus vector gene expression occurs in nondividing cells in the absence of vector DNA integration. Am. J. Resp. Cell Mol. Biol. 11:517‐521.
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   Gao, G., Vandenberghe, L.H., Alvira, M.R., Lu, Y., Calcedo, R., Zhou, X., and Wilson, J.M. 2004. Clades of adeno‐associated viruses are widely disseminated in human tissues. J. Virol. 78:6381‐6388.
   Grimm, D., Pandey, K., Nakai, H., Storm, T.A., and Kay, M.A. 2006. Liver transduction with recombinant adeno‐associated virus is primarily restricted by capsid serotype not vector genotype. J. Virol. 80:426‐439.
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   Halbert, C.L., Miller, A.D., McNamara, S., Emerson, J., Gibson, R.L., Ramsey, B., and Aitken, M.L. 2006. Prevalence of neutralizing antibodies against adeno‐associated virus (AAV) types 2, 5, and 6 in cystic fibrosis and normal populations: Implications for gene therapy using AAV vectors. Hum. Gene Ther. 17:440‐447.
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Internet Resources
   http://www.hallowell.com/index.php?doc=1&pr=Video_Presentations
  Detailed procedural video demonstrating the orotracheal intubation technique. Note minor modification from the technique described above. We do recommend the 20‐G catheters listed in the materials list over those presented in the video because they are disposable and a new catheter can be used for each mouse. They are also made of a somewhat softer material and, therefore, less likely to induce trauma on placement.
   http://www.kentscientific.com/products/productView.asp?productID=6354&Mouse_Rat=Respiratory&Products=Small+Animal+Intubation+Kit
  Detailed procedural video demonstrating the BioLite intubation technique.
   http://www.jove.com/video/1941/direct‐tracheal‐instillation‐of‐solutes‐into‐mouse‐lung
  Detailed procedural video demonstrating the surgical tracheal injection technique.
   http://www.medipoint.com/html/directions_for_use1.html
  Video demonstrating blood collection from facial vein/artery using lancet.
  https://www.aalaslearninglibrary.org/swfs/course2451/stabpst1.swf
  Video demonstrating blood collection from facial vein/artery using needle.
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