Gene Transfer in Skeletal and Cardiac Muscle Using Recombinant Adeno‐Associated Virus

Alisha M. Gruntman1, Lawrence T. Bish2, Christian Mueller1, H. Lee Sweeney2, Terence R. Flotte3, Guangping Gao4

1 Department of Pediatrics, University of Massachusetts Medical School, Worcester Massachusetts, 2 Department of Physiology, University of Pennsylvania School of Medicine, Philadelphia Pennsylvania, 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.3
DOI:  10.1002/9780471729259.mc14d03s28
Online Posting Date:  February, 2013
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Abstract

Adeno‐associated virus (AAV) is a DNA virus with a small (∼4.7 kb) single‐stranded genome. It is a naturally replication‐defective parvovirus of the dependovirus group. Recombinant AAV (rAAV), for use as a gene transfer vector, is created by replacing the viral rep and cap genes with the transgene of interest along with promoter and polyadenylation sequences. Only the viral inverted terminal repeats (ITRs) are required in cis for replication and packaging during production. The ITRs are also necessary and sufficient for vector genome processing and persistence during transduction. The tissue tropism of the rAAV vector is determined by the AAV capsid. In this unit we will discuss several methods to deliver rAAV in order to transduce cardiac and/or skeletal muscle, including intravenous delivery, intramuscular delivery, isolated limb infusion, intrapericardial injection in neonatal mice, and left ventricular wall injection in adult rats. Curr. Protoc. Microbiol. 28:14D.3.1‐14D.3.19. © 2013 by John Wiley & Sons, Inc.

Keywords: adeno‐associated virus; gene therapy; heart, skeletal muscle; vector delivery

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Gene Transfer to Cardiac and Skeletal Muscle Through Intravenous Delivery of rAAV in Adult Mice
  • Alternate Protocol 1: Gene Transfer to Skeletal Muscle Through Direct Intramuscular Injection in Adult Mice
  • Alternate Protocol 2: Gene Transfer to Skeletal Muscle Through Direct Intramuscular Injection with Skin Incision in Adult Mice
  • Alternate Protocol 3: Gene Transfer to Skeletal Muscle Through Isolated Limb Infusion in Adult Mice
  • Alternate Protocol 4: Gene Transfer to Cardiac Muscle Through Intrapericardial Injection in Neonatal Mice
  • Alternate Protocol 5: Gene Transfer to the Rat Myocardium via Direct Injection into the Left Ventricular Wall
  • Support Protocol 1: Preparing rAAV Vector for Delivery
  • Support Protocol 2: Ketamine/Xylazine Anesthesia Mouse
  • Support Protocol 3: Ketamine/Xylazine Anesthesia Rat
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Gene Transfer to Cardiac and Skeletal Muscle Through Intravenous Delivery of rAAV in Adult Mice

  Materials
  • rAAV prepared as described in protocol 7—diluting the vector to a volume of 200 µl in sterile PBS
  • Sterile phosphate‐buffered saline (PBS)
  • Mice (choose the strain according to planned experiments)
  • Alcohol spray or wipes
  • Heat lamp or warm water
  • Mouse restrainer
  • 1‐ml syringes equipped with 27‐G 3/8‐in. needle
  • Gauze sponges

Alternate Protocol 1: Gene Transfer to Skeletal Muscle Through Direct Intramuscular Injection in Adult Mice

  Materials
  • rAAV prepared as described in protocol 7
  • Mice (choose strain according to planned experiments)
  • Xylazine (20 mg/ml) and ketamine (100 mg/ml) or isoflurane (Webster Veterinary; http://www.webstervet.com)
  • Ophthalmic ointment (such as Puralube; Webster, cat. no. 07‐888‐2572)
  • 3/10‐ml syringe with 31‐G needle (8‐mm length needle) (e.g., BD Ultra‐Fine II short needle insulin syringes) or Hamilton Syringe
  • Nose cone or induction chamber, optional
  • Clippers with no. 40 blade or depilatory cream (e.g., Nair)

Alternate Protocol 2: Gene Transfer to Skeletal Muscle Through Direct Intramuscular Injection with Skin Incision in Adult Mice

  Materials
  • rAAV prepared as described in protocol 7
  • Mice—choose strain according to planned experiments
  • Xylazine (20 mg/ml) and ketamine (100 mg/ml)
  • Isofluorane
  • Ophthalmic ointment (e.g., Puralube; Webster, cat. no. 07‐888‐2572)
  • 2% Chlorhexidine surgical scrub and 70% isopropyl alcohol wipes
  • Rodent anesthesia workstation, including rodent ventilator and isoflurane vaporizer (available fully assembled with all necessary accessories) (Systems Specialties)
  • Clippers with no. 40 blade or depilatory cream (e.g., Nair)
  • No. 11 sterile scalpel blade with handle
  • 3/10‐ml syringe with 31‐G needle (8‐mm length needle) (e.g., BD Ultra‐Fine II short needle insulin syringes) or Hamilton syringe
  • 5‐0 monofilament suture on a cutting needle (such as polypropylene or nylon) or tissue glue (such as Dermabond)
  • Needle holder or forceps (if suturing skin)
  • Heating pad

Alternate Protocol 3: Gene Transfer to Skeletal Muscle Through Isolated Limb Infusion in Adult Mice

  Materials
  • Mice (choose strain according to planned experiments)
  • rAAV prepared as described in protocol 7
  • Xylazine (20 mg/ml) and ketamine (100 mg/ml)
  • Isoflurane
  • Ophthalmic ointment
  • 2% Chlorhexidine surgical scrub and 70% isopropyl alcohol wipes
  • 30‐G needles
  • Polyethylene PE‐10 tubing
  • Rodent anesthesia workstation, including rodent ventilator and isoflurane vaporizer (available fully assembled with all necessary accessories) (Systems Specialties)
  • Nose cone
  • Clippers with no. 40 blade or depilatory cream (e.g., Nair)
  • Small rubber bands
  • No. 11 sterile scalpel blade with handle
  • High‐pressure syringe pump (Harvard Apparatus)
  • 5‐0 monofilament suture on a cutting needle (such as polypropylene or nylon) or tissue glue (such as Dermabond)
  • Needle holder or forceps (if suturing skin)
  • Heating pad

Alternate Protocol 4: Gene Transfer to Cardiac Muscle Through Intrapericardial Injection in Neonatal Mice

  Materials
  • rAAV prepared as described in protocol 7
  • 4‐ to 5‐day‐old mouse pups
  • Ice and water mix
  • Saint Gobain Tygon microbore tubing, Formula S‐54‐HL (0.51‐mm i.d.; 0.51‐mm wall thickness; 1.53‐mm o.d.)
  • 33‐G needle (Hamilton)
  • Latex glove finger or paper‐lined tubes (for cryoanesthesia)
  • 25‐G needle, 1.5‐in. length
  • Hamilton Gastight 250‐µl glass syringe, series 1725TLL
  • Heating pad, optional

Alternate Protocol 5: Gene Transfer to the Rat Myocardium via Direct Injection into the Left Ventricular Wall

  Materials
  • Adult rats (see Troubleshooting section for information on strain selection)
  • Buprenorphine
  • Cefazolin
  • Ophthalmic eye ointment
  • 2% Chlorhexidine surgical scrub and alcohol wipes
  • rAAV prepared as described in protocol 7
  • Dobutamine (12.5 mg/ml), optional
  • Rodent work stand for intubation (Braintree Scientific, cat. no. RW A3467)
  • Rat intubation pack containing intubation speculum, endotracheal intubation tubes, an endotracheal tube guide wire, an incisor loop and a brief video tutorial on how to perform the intubations (Braintree Scientific, cat. no. RW‐A37 46)
  • Forceps
  • Otoscope for intubation—rechargeable (Braintree Scientific)
  • 10‐ml syringes
  • Sutures: 4‐0 Maxon, 4‐0 Vicryl, 5‐0 Vicryl (taper and cutting needle), and 7‐0 Prolene
  • Rodent anesthesia workstation, including rodent ventilator and isoflurane vaporizer (available fully assembled with all necessary accessories) (Systems Specialties)
  • Animal clippers
  • Standard surgical instrument pack, sterile drapes, sterile gowns, and sterile gloves
  • No. 15 scalpel blades
  • Hemostats
  • 0.5‐ml insulin syringe (U‐100) with 28‐G needle, 0.5‐in. length
  • 18‐G angiocath (1.88‐in. length; BD Angiocath Autoguard)
  • Recovery ventilator (NEMI Scientific, model 141), optional

Support Protocol 1: Preparing rAAV Vector for Delivery

  Materials
  • Recombinant adeno‐associated virus [rAAV; can be produced in‐house (see unit 14.1), through a local vector core or via a commercial manufacturer]
  • Sterile saline (normal or phosphate‐buffered saline)
  • Ice
  • 200‐µl pipet tips
  • 20‐ to 200‐µl pipets
  • Parafilm or sterile petri dish
  • Delivery syringe or pipet (see particular delivery protocol)

Support Protocol 2: Ketamine/Xylazine Anesthesia Mouse

  Materials
  • Xylazine (20 mg/ml stock concentration): 0.25 ml (10 mg/kg dose to mouse)
  • Ketamine (100 mg/ml stock concentration): 0.5 ml (100 mg/kg dose to mouse)
  • Sterile isotonic saline: 5 ml
  • Total cocktail: 5.75 ml
  • Dose of cocktail to mouse: 0.10 ml/10 g, intraperitoneally using a 25‐ to 29‐G needle with a 0.5‐ml syringe

Support Protocol 3: Ketamine/Xylazine Anesthesia Rat

  Materials
  • Xylazine (20 mg/ml stock concentration) 0.75 ml
  • Ketamine (100 mg/ml stock concentration): 1.8 ml
  • Sterile isotonic saline: 0.45 ml
  • Total cocktail: 3.0 ml
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Figures

Videos

Literature Cited

   Afione, S.A., Conrad, C.K., Kearns, W.G., Chunduru, S., Adams, R., Reynolds, T.C., Guggino, W.B., Cutting, G.R., Carter, B.J., and Flotte, T.R. 1996. In vivo model of adeno‐associated virus vector persistence and rescue. J. Virol. 70:3235‐3241.
   Bish, L.T., Morine, K., Sleeper, M.M., Sanmiguel, J., Wu, D., Gao, G., Wilson, J.M., and Sweeney, H.L. 2008. Adeno‐associated virus (AAV) serotype 9 provides global cardiac gene transfer superior to AAV1, AAV6, AAV7, and AAV8 in the mouse and rat. Hum. Gene Ther. 19:1359‐1368.
   Bish, L.T., Sweeney, H.L., Müller, O.J., and Bekeredjian, R. 2011. Adeno‐associated virus vector delivery to the heart. Methods Mol. Biol. 807:219‐237.
   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.
   Gao, G., Bish, L.T., Sleeper, M.M., Mu, X., Sun, L., Lou, Y., Duan, J., Hu, C., Wang, L., and Sweeney, H.L. 2011. Transendocardial delivery of AAV6 results in highly efficient and global cardiac gene transfer in rhesus macaques. Hum. Gene Ther. 22:979‐984.
   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.
   Hagstrom, J.E., Hegge, J., Zhang, G., Noble, M., Budker, V., Lewis, D.L., Herweijer, H., and Wolff, J.A. 2004. A facile nonviral method for delivering genes and siRNAs to skeletal muscle of mammalian limbs. Mol. Ther. 10:386‐398.
   Inagaki, K., Fuess, S., Storm, T.A., Gibson, G.A., Mctiernan, C.F., Kay, M.A., and Nakai, H. 2006. Robust systemic transduction with AAV9 vectors in mice: Efficient global cardiac gene transfer superior to that of AAV8. Mol. Ther. 14:45‐53.
   Isotani, M., Miyake, K., Miyake, N., Hirai, Y., and Shimada, T. 2011. Direct comparison of four adeno‐associated virus serotypes in mediating the production of antiangiogenic proteins in mouse muscle. Cancer Invest. 29:353‐359.
   Liu, Y.H., Yang, X.P., Nass, O., Sabbah, H.N., Peterson, E., and Carretero, O.A. 1997. Chronic heart failure induced by coronary artery ligation in Lewis inbred rats. Am. J. Physiol. 272:H722‐H727.
   Phillips, J.L., Hegge, J., Wolff, J.A., Samulski, R.J., and Asokan, A. 2011. Systemic gene transfer to skeletal muscle using reengineered AAV vectors. Methods Mol. Biol. 709:141‐151.
   Pleger, S.T., Most, P., Boucher, M., Soltys, S., Chuprun, J.K., Pleger, W., Gao, E., Dasgupta, A., Rengo, G., Remppis, A., Katus, H.A., Eckhart, A.D., Rabinowitz, J.E., and Koch, W.J. 2007. Stable myocardial‐specific AAV6‐S100A1 gene therapy results in chronic functional heart failure rescue. Circulation 115:2506‐2515.
   Prasad, K.M., Xu, Y., Yang, Z., Acton, S.T., and French, B.A. 2011. Robust cardiomyocyte‐specific gene expression following systemic injection of AAV: In vivo gene delivery follows a Poisson distribution. Gene Ther. 18:43‐52.
   Qiao, C., Koo, T., Li, J., Xiao, X., and Dickson, J.G. 2011. Gene therapy in skeletal muscle mediated by adeno‐associated virus vectors. Methods Mol. Biol. 807:119‐140.
   Rodino‐Klapac, L.R., Janssen, P.M., Montgomery, C.L., Coley, B.D., Chicoine, L.G., Clark, K.R., and Mendell, J.R. 2007. A translational approach for limb vascular delivery of the micro‐dystrophin gene without high volume or high pressure for treatment of Duchenne muscular dystrophy. J. Transl. Med. 5:45.
   Sakata, S., Lebeche, D., Sakata, N., Sakata, Y., Chemaly, E.R., Liang, L.F., Tsuji, T., Takewa, Y., del Monte, F., Peluso, R., Zsebo, K., Jeong, D., Park, W.J., Kawase, Y., and Hajjar, R.J. 2007. Restoration of mechanical and energetic function in failing aortic‐banded rat hearts by gene transfer of calcium cycling proteins. J. Mol. Cell. Cardiol. 42:852‐861.
   Shin, J.H., Nitahara‐Kasahara, Y., Hayashita‐Kinoh, H., Ohshima‐Hosoyama, S., Kinoshita, K., Chiyo, T., Okada, H., Okada, T., and Takeda, S. 2011. Improvement of cardiac fibrosis in dystrophic mice by rAAV9‐mediated microdystrophin transduction. Gene Ther. 18:910‐919.
   Sun, B., Li, S., Bird, A., and Koeberl, D.D. 2010. Hydrostatic isolated limb perfusion with adeno‐associated virus vectors enhances correction of skeletal muscle in Pompe disease. Gene Ther. 17:1500‐1505.
   Wang, B., Li, J., Fu, F.H., Chen, C., Zhu, X., Zhou, L., Jiang, X., and Xiao, X. 2008. Construction and analysis of compact muscle‐specific promoters for AAV vectors. Gene Ther. 15:1489‐1499.
   Xie, J., Xie, Q., Zhang, H., Ameres, S.L., Hung, J.H., Su, Q., He, R., Mu, X., Seher Ahmed, S., Park, S., Kato, H., Li, C., Mueller, C., Mello, C.C., Weng, Z., Flotte, T.R., Zamore, P.D., and Gao, G. 2011. MicroRNA‐regulated, systemically delivered rAAV9: A step closer to CNS‐restricted transgene expression. Mol. Ther. 19:526‐535.
   Zhang, J.C., Woo, Y.J., Chen, J.A., Swain, J.L., and Sweeney, H.L. 1999. Efficient transmural cardiac gene transfer by intrapericardial injection in neonatal mice. J. Mol. Cell. Cardiol. 31:721‐732.
   Zhang, Y. and Duan, D. 2012. Novel mini‐dystrophin gene dual adeno‐associated virus vectors restore neuronal nitric oxide synthase expression at the sarcolemma. Hum. Gene Ther. 23:98‐103.
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Key Reference
   UNIT 14D.11.
  Detailed protocols for rAAV vector production and titration/analysis.
Internet Resources
   http://www.hallowell.com/index.php?doc=2&pr=Video_Presentations
  Video demonstrating intubation using a speculum in the rat.
   http://www.theodora.com/rodent_laboratory/injections.html
  Photographs demonstrating intramuscular injection in the mouse caudal thigh.
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