Mechanism of plasmid delivery by hydrodynamic tail vein injection. I. Hepatocyte uptake of various molecules.
BACKGROUND
The hydrodynamic tail vein (HTV) injection of naked plasmid DNA is a simple yet effective in vivo gene delivery method into hepatocytes. It is increasingly being used as a research tool to elucidate mechanisms of gene expression and the role of genes and their cognate proteins in the pathogenesis of disease in animal models. A greater understanding of its mechanism will aid these efforts and has relevance to macromolecular and nucleic acid delivery in general.


METHODS
In an attempt to explore how naked DNA enters hepatocytes the fate of a variety of molecules and particles was followed over a 24-h time frame using fluorescence microscopy. The uptake of some of these compounds was correlated with marker gene expression from a co-injected plasmid DNA. In addition, the uptake of the injected compounds was correlated with the histologic appearance of hepatocytes.


RESULTS
Out of the large number of nucleic acids, peptides, proteins, inert polymers and small molecules that we tested, most were efficiently delivered into hepatocytes independently of their size and charge. Even T7 phage and highly charged DNA/protein complexes of 60-100 nm in size were able to enter the cytoplasm. In animals co-injected with an enhanced yellow fluorescent protein (EYFP) expression vector and fluorescently labeled immunoglobulin (IgG), hepatocytes flooded with large amounts of IgG appeared permanently damaged and did not express EYFP-Nuc. Hepatocytes expressing EYFP had only slight IgG uptake. In contrast, when an EYFP expression vector was co-injected with a fluorescently labeled 200-bp linear DNA fragment, both were mostly (in 91% of the observed cells) co-localized to the same hepatocytes 24 h later.


CONCLUSIONS
The appearance of permanently damaged cells with increased uptake of some molecules such as endogenous IgG raised the possibility that a molecule could be present in a hepatocyte but its transport would not be indicative of the transport process that can lead to foreign gene expression. The HTV procedure enables the uptake of a variety of molecules (as previous studies also found), but the uptake process for some of these molecules may be associated with a more disruptive process to the hepatocytes that is not compatible with successful gene delivery.
Sebestyén
Magdolna G
MG
Mirus Bio Corporation, 505 S. Rosa Rd., Madison, WI 53719, USA.
Budker
Vladimir G
VG
Budker
Tatiana
T
Subbotin
Vladimir M
VM
Zhang
Guofeng
G
Monahan
Sean D
SD
Lewis
David L
DL
Wong
So C
SC
Hagstrom
James E
JE
Wolff
Jon A
JA
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000008:administration & dosage; D000952:Antigens, Polyomavirus Transforming / Q000008:administration & dosage / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics; D017123:Bacteriophage T7 / Q000235:genetics; D001483:Base Sequence; D001693:Biological Transport, Active; D005456:Fluorescent Dyes / Q000008:administration & dosage; D018014:Gene Transfer Techniques; D022781:Hepatocytes / Q000378:metabolism*; D007275:Injections, Intravenous; D008164:Luminescent Proteins / Q000235:genetics; D008562:Membrane Glycoproteins / Q000276:immunology; D051379:Mice; D008813:Mice, Inbred ICR; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D019913:Nuclear Localization Signals; D028861:Nuclear Pore Complex Proteins; D010446:Peptide Fragments / Q000008:administration & dosage / Q000235:genetics; D010957:Plasmids / Q000008:administration & dosage* / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics; D019809:Streptavidin / Q000008:administration & dosage; D013623:Tail / Q000098:blood supply
D000911:Antibodies, Monoclonal; D000952:Antigens, Polyomavirus Transforming; D001426:Bacterial Proteins; D005456:Fluorescent Dyes; D008164:Luminescent Proteins; D008562:Membrane Glycoproteins; D019913:Nuclear Localization Signals; D028861:Nuclear Pore Complex Proteins; D010446:Peptide Fragments; D011994:Recombinant Proteins; C049312:nuclear pore protein p62; C054562:yellow fluorescent protein, Bacteria; D019809:Streptavidin