Liposome Vectors for In Vivo Gene Delivery

Mark Whitemore1, Song Li1, Leaf Huang1

1 University of Pittsburgh, Pittsburgh, Pennsylvanta
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Unit 12.8
DOI:  10.1002/0471142905.hg1208s20
Online Posting Date:  May, 2001
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Abstract

Gene therapy with nonviral, cationic liposomebased vectors has demonstrated promising results in a variety of in vivo models and some clinical trials. In contrast to viral vectors, cationic liposomebased vectors usually induce just mild inflammatory reactions, making them suitable for repeated injections in animals. This unit describes the preparation and delivery of two liposomebased vectorsGene therapy with nonviral, cationic liposomebased vectors has demonstrated promising results in a variety of in vivo models.

     
 
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Table of Contents

  • Basic Protocol 1: Gene Delivery to Subcutaneous Tumor Models by Direct Intratumor Injection of DNA/DC‐Cholesterol Lipoplex
  • Basic Protocol 2: Intravenous Gene Delivery to the Mouse Lung by LPD
  • Support Protocol 1: Liposome Preparation by Thin‐Film Hydration Followed by Extrusion
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Gene Delivery to Subcutaneous Tumor Models by Direct Intratumor Injection of DNA/DC‐Cholesterol Lipoplex

  Materials
  • 2 mM DC‐Chol liposome stock solution (1.07 mg/ml; see protocol 3)
  • 5.2% (w/v) dextrose (see recipe)
  • 2.0 mg/ml plasmid DNA in 5.2% dextrose (see Critical Parameters) and plasmid DNA control
  • Mouse with subcutaneous tumor
  • 0.5‐ or 1‐ml syringe with 26‐G 1/2 in. needle
NOTE: Because the stability of the lipoplex is short‐lived, the complex must be freshly prepared prior to use.NOTE: Volumes and quantities are for a single, 30‐µg dose of DNA in a volume of 50 µl. For multiple doses, volumes and quantities should be scaled up proportionately.

Basic Protocol 2: Intravenous Gene Delivery to the Mouse Lung by LPD

  Materials
  • recipe5× dextrose (26% w/v; see recipe)
  • DOTAP/cholesterol liposomes (containing 14 mM, or 10 mg/ml, DOTAP; see protocol 3)
  • 1 mg/ml plasmid DNA in sterile water (see Critical Parameters) and plasmid DNA control
  • recipe10.0 mg/ml protamine sulfate (see recipe)
  • Mice to be injected
  • 50‐ml conical polypropylene tube
  • Sterile water (autoclaved and sterile‐filtered using 0.2‐µm filter)
  • 0.5‐ or 1.0‐ml syringe with 26‐G 1/2 in. needle
NOTE: Volumes and quantities are for a single, 50‐µg dose of plasmid DNA in a volume of 300 µl. For multiple doses, volumes and quantities should be scaled up proportionately.

Support Protocol 1: Liposome Preparation by Thin‐Film Hydration Followed by Extrusion

  Materials
  • Chloroform
  • recipe2.0 mg/ml DC‐Chol stock solution (see recipe)
  • 20.0 mg/ml DOPE (1,2‐dioleoyl‐3‐phosphatidylethanolamine) in chloroform (Avanti Polar Lipids)
  • 20.0 mg/ml DOTAP (1,2‐dioleoyl‐3‐trimethylammonium propane) in chloroform (Avanti Polar Lipids)
  • recipe20.0 mg/ml cholesterol stock solution (see recipe)
  • recipe5.2% (w/v) dextrose (see recipe)
  • 30.0‐ml Corex glass centrifuge tube
  • N 2 gas tank
  • Vacuum desiccator
  • Bath sonicator
  • Water bath, 65°C
  • LiposoFast extruder (Avestin)
  • 1.0‐, 0.4‐, and 0.1‐µm polycarbonate membrane filters
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Figures

Videos

Literature Cited

Literature Cited
   Cotten, M., Baker, A., Saltik, M., Wagner, E., and Buschle, M. 1994. Lipopolysaccharide is a frequent contaminant of plasmid DNA preparations and can be toxic to primary human cells in the presence of adenovirus. Gene Ther. 1:239‐246.
   Felgner, P.L., Gadek, T.R., Holm, M., Roman, R., Chan, H.W., Wenz, M., Northrop, J.P., Ringold, G.M., and Danielsen, M. 1987. Lipofection: A highly efficient, lipid‐mediated DNA‐transfection procedure. Proc. Natl. Acad. Sci. U.S.A. 84:7413‐7417.
   Gao, X. and Huang, L. 1991. A novel cationic liposome reagent for efficient transfection of mammalian cells. Biochem. Biophys. Res. Commun. 179:280‐285.
   Gao, X. and Huang, L. 1995. Cationic liposome–mediated gene transfer. Gene Ther. 2:710‐722.
   Gao, X. and Huang, L. 1996. Potentiation of cationic liposome–mediated gene delivery by polycations. Biochemistry 35:1027‐1036.
   Goddard, C.A., Ratcliff, R., Anderson, J.R., Glenn, E., Brown, S., Gill, D.R., Hyde, S.C., MacVinish, L.J., Huang, L., Higgins, C.F., et al. 1997. A second dose of a CFTR cDNA‐liposome complex is as effective as the first dose in restoring cAMP‐dependent chloride secretion to null CF mice trachea. Gene Ther. 4:1231‐1236.
   Hofland, H. and Huang, L. 1995. Inhibition of human ovarian carcinoma cell proliferation by liposome–plasmid DNA complex. Biochem. Biophys. Res. Commun. 207:492‐496.
   Li, S. and Huang, L. 1997. In vivo gene transfer via intravenous administration of cationic lipid‐protamine‐DNA (LPD) complexes. Gene Ther. 4:981‐900.
   Li, S., Rizzo, M., Bhattacharya, S., and Huang, L. 1998. Characterization of cationic lipid‐protamine‐DNA (LPD) complexes for intravenous gene delivery. Gene Ther. 5:930‐937.
   Nabel, E., Gordon, D., Yang, Z.‐Y., Xu, L., San, H., Plautz, G., Wu, B.‐Y., Gao, X., Huang, L., and Nabel, G. 1992. Gene transfer in vivo with DNA‐liposome complexes: Lack of autoimmunity and gonadal localization. Hum. Gene Ther. 3:649‐656.
   Nabel, G., Nabel, E., Yang, Z., Fox, B., Plautz, G., Gao, X., Huang, L., Shu, S., Gordon, D., and Chang, A. 1993. Direct gene transfer with DNA‐liposome complexes in melanoma: Expression, biologic activity, and lack of toxicity in humans. Proc. Natl. Acad. Sci. U.S.A. 90:11307‐11311.
   Robbins, P.D., Tahara, H., and Ghivizzani, S.C. 1998. Viral vectors for gene therapy. Trends Biotechnol. 16:35‐40.
   Sorgi, F. and Huang, L. 1996. Large‐scale production of DC‐Chol cationic liposomes by microfluidization. Int. J. Pharm. 144:131‐139.
   Sorgi, F., Bhattacharya, S., and Huang, L. 1997. Protamine sulfate enhances lipid‐mediated gene transfer. Gene Ther. 4:961‐968.
   Sternberg, B., Sorgi, F.L., and Huang, L. 1994. New structures in complex formation between DNA and cationic liposomes visualized by freeze‐fracture electron microscopy. FEBS Lett. 356:361‐366.
   Stewart, M., Plautz, G., Del Bueno, L., Yang, Z.‐Y., Xu, L., Gao, X., Huang, L., Nabel, E., and Nabel, G. 1992. Gene transfer in vivo with DNA‐liposome complexes: Safety and acute toxicity in mice. Hum. Gene Ther. 3:267‐275.
   Wicks, I.P., Howell, M.L., Hancock, T., Kohsaka, H., Olee, T., and Carson, D.A. 1995. Bacterial lipopolysaccharide copurifies with plasmid DNA: Implications for animal models and human gene therapy. Hum. Gene Ther. 6:317‐323.
   Yang, J.‐P. and Huang, L. 1996. Direct gene transfer to mouse melanoma by intratumor injection of free DNA. Gene Ther. 3:542‐548.
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