Gene Delivery to the Liver

Karen Kozarsky1

1 SmithKline Beecham Pharmaceuticals, Inc., King of Prussia, Pennsylvania
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Unit 13.10
DOI:  10.1002/0471142905.hg1310s22
Online Posting Date:  May, 2001
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Abstract

Viral gene transfer to the liver has proven extremely effective in animal models and is currently being evaluated in clinical trials for a variety of metabolic disorders. In rodents, a single tail vein injection of an adenoviral vector can transduce most hepatocytes in vivo. This provides a convenient model for assessing vector design as well as for evaluating the effects of specific transgenes in genetic mouse models of human disease. Protocols for optimized in vivo hepatic gene transfer are described.

     
 
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Table of Contents

  • Basic Protocol 1: Recombinant Adenovirus Delivery to Mouse Liver by Tail Vein Injection
  • Basic Protocol 2: Recombinant Adenovirus Delivery to Rabbit Liver by Peripheral Ear Vein Injection
  • Support Protocol 1: Harvesting Liver Tissue for Transgene Expression Analysis
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Tables
     
 
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Materials

Basic Protocol 1: Recombinant Adenovirus Delivery to Mouse Liver by Tail Vein Injection

  Materials
  • Adult mice, at least 6 weeks of age, 20 to 25 g
  • 70% ethanol in squirt bottle
  • 1 × 1012 particles/ml purified reciperecombinant adenovirus (see recipe) in sterile PBS
  • Mouse cage with wire lid
  • Heat lamp
  • Mouse restrainer
  • Cotton gauze pads
  • 1‐ml syringe with 27‐G (½‐in. or ∼1.25‐cm) needle

Basic Protocol 2: Recombinant Adenovirus Delivery to Rabbit Liver by Peripheral Ear Vein Injection

  Materials
  • Adult rabbits, ∼2 kg
  • 0.75–1.0 × 1013 of particles purified recombinant adenovirus (see recipe) in ∼3 ml sterile PBS
  • Restraining cage for rabbit
  • 5‐ml syringe and 21‐G butterfly needle
  • 4 × 4 gauze pads
  • Petroleum jelly

Support Protocol 1: Harvesting Liver Tissue for Transgene Expression Analysis

  Materials
  • Mouse or rabbit injected with recombinant vector (see protocol 1 or protocol 22)
  • Buffered formalin
  • OCT (optimal cutting temperature) compound
  • 2‐methylbutane
  • Sterile plastic petri dishes
  • Razor blades
  • Plastic molds
  • Dry ice
  • Cryotubes
  • Liquid nitrogen
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Figures

Videos

Literature Cited

   Bosch, A., McCray, P.B. Jr, Walters, K.S., Bodner, M., Jolly, D.J., van Es, H.H., Nakamura, T., Matsumoto, K., and Davidson, B.L. 1998. Effects of keratinocyte and hepatocyte growth factor in vivo: Implications for retrovirus‐mediated gene transfer to liver. Hum. Gene Ther. 9:1747‐1754.
   Chowdhury, J.R., Grossman, M., Gupta, S., Chowdhury, N.R., Baker, J.R., and Wilson, J.M. 1991. Long‐term improvement of hypercholesterolemia after ex vivo gene therapy in LDLR‐deficient rabbits. Science 254:1802‐1805.
   Engelhardt, J.F., Ye, X., Doranz, B., and Wilson, J.M. 1994. Ablation of E2A in recombinant adenoviruses improves transgene persistence and decreases inflammatory response in mouse liver. Proc. Natl. Acad. Sci. U.S.A. 91:6196‐6200.
   Jooss, K., Turka, L.A., and Wilson, J.M. 1998. Blunting of immune responses to adenoviral vectors in mouse liver and lung with CTLA4Ig. Gene Ther. 5:309‐319.
   Kashyap, V.S., Santamarina‐Fojo, S., Brown, D.R., Parrott, C.L., Applebaum‐Bowden, D., Meyn, S., Talley, G., Paigen, B., Maeda, N., and Brewer, H.B. Jr. 1995. Apolipoprotein E deficiency in mice: Gene replacement and prevention of atherosclerosis using adenovirus vectors. J. Clin. Invest. 96:1610‐1620.
   Kay, M.A., Graham, F., Leland, F., and Woo, S.L. 1995. Therapeutic serum concentrations of human alpha‐1‐antitrypsin after adenoviral‐mediated gene transfer into mouse hepatocytes. Hepatology 21:815‐819.
   Kozarsky, K.F., McKinley, D.R., Austin, L.L., Raper, S.E., Stratford‐Perricaudet, L.D., and Wilson, J.M. 1994. In vivo correction of LDL receptor deficiency in the Watanabe heritable hyperlipidemic rabbit with recombinant adenoviruses. J. Biol. Chem. 269:13695‐13702.
   Kozarsky, K.F., Jooss, K., Donahee, M., Strauss, J.F., and Wilson, J.M. 1996. Effective treatment of familial hypercholesterolaemia in the mouse model using adenovirus‐mediated transfer of the VLDL receptor gene. Nature Genet. 13:54‐62.
   Patijn, G.A., Lieber, A., Schowalter, D.B., Schwall, R., and Kay, M.A. 1998. Hepatocyte growth factor induces hepatocyte proliferation in vivo and allows for efficient retroviral‐mediated gene transfer in mice. Hepatology 28:707‐716.
   Rader, D.J. 1997. Gene therapy for atherosclerosis. Int. J. Clin. Lab. Res. 27:35‐43.
   Schiedner, G., Morral, N., Parks, R.J., Wu, Y., Koopmans, S.C., Langston, C., Graham, F.L., Beaudet, A.L., and Kochanek, S. 1998. Genomic DNA transfer with a high‐capacity adenovirus vector results in improved in vivo gene expression and decreased toxicity. Nat. Genet. 18:180‐183.
   Snyder, R.O., Miao, C., Meuse, L., Tubb, J., Donahue, B.A., Lin, H‐.F., Stafford, D.W., Patel, S., Thompson, A.R., Nichols, T., Read, M.S., Bellinger, D.A., Brinkhous, K.M., and Kay, M.A. 1999. Correction of hemophilia B in canine and murine models using recombinant adeno‐associated viral vectors. Nat. Med. 5:64‐70.
   Stein, C.S., Pemberton, J.L., van Rooijen, N., and Davidson, B.L. 1998. Effects of macrophage depletion and anti‐CD40 ligand on transgene expression and redosing with recombinant adenovirus. Gene Ther. 5:431‐439.
   Sullivan, D.E., Dash, S., Du, H., Hiramatsu, N., Aydin, F., Kolls, J., Blanchard, J., Baskin, G., and Gerber, M.A. 1997. Liver‐directed gene transfer in non‐human primates. Hum. Gene Ther. 8:1995‐1206.
   Walter, J. and High, K.A. 1997. Gene therapy for the hemophilias. Adv. Vet. Med. 40:119‐134.
   Wang, Q., Greenburg, G., Bunch, D., Farson, D., and Finer, M.H. 1997. Persistent transgene expression in mouse liver following in vivo gene transfer with a delta E1/delta E4 adenovirus vector. Gene Ther. 4:393‐400.
   Ye, X., Robinson, M.B., Batshaw, M.L., Furth, E.E., Smith, I., and Wilson, J.M. 1996. Prolonged metabolic correction in adult ornithine transcarbamylase‐deficient mice with adenoviral vectors. J. Biol. Chem. 271:3639‐3646.
Key References
   Ferry, N. and Heard, J.M. 1998. Liver‐directed gene transfer vectors. Hum. Gene Ther. 9:1974‐1981.
  A concise overview of different vectors available for in vivo gene transfer to the liver.
   Hitt, M.M., Addison, C.L., and Graham, F.L. 1997. Human adenovirus vectors for gene transfer into mammalian cells. Adv. Pharmacol. 40:137‐206.
  A useful review article which describes the use of different adenovirus vectors and transgenes for in vitro and in vivo use.
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