title: Adenovirus or HA-2 fusogenic peptide-assisted lipofection increases cytoplasmic levels of plasmid in nondividing endothelium with little enhancement of transgene expression.
abstract: BACKGROUND
  Adenovirus-assisted lipofection has been reported to increase transfection efficiency through mechanisms potentially involving endosome escape and/or nuclear targeting activity. Similarly, transfection with the viral fusogenic peptide HA-2 of the influenza virus hemagglutinin can increase transfection efficiency. However, there are few studies examining the mechanism and intracellular trafficking of these viral and/or viral fusogenic peptide-assisted lipofections.
  
  
  RESULTS
  Endosome escape was directly assayed with T7 RNA polymerase bound to plasmid (pTM beta gal) expressing beta-galactosidase under a T7 promoter to detect transcribable plasmid that escapes the endosomal compartment. Lipofection of pTM beta gal with replication-deficient adenovirus (Ad5-null) at a multiplicity of infection (MOI) of 100 and 1000 increased cytoplasmic levels of transcribable plasmid by 24- and 117-fold, respectively, over lipofection alone, without an effect on total plasmid uptake. However, lipofection of pCMV beta gal with Ad5-null at a MOI of 100 and 1000 increased transgene expression only seven- and eight-fold, respectively, over lipofection alone. Thus, a 24-fold increase in endosome escape saturated expression from pCMV beta gal and provided only a seven-fold benefit in nondividing cells, which was not significantly increased with further increases in endosome escape. A cationic form of HA-2 (HA-K(4)) also caused significant enhancements in endosome escape, as detected with the cytoplasmic transcription assay. However, HA-K(4) enhancement of endosome escape did not correlate with transgene expression from pCMV beta gal, consistent with the detection of HA-K(4)-mediated partitioning of plasmid to the insoluble fraction of the cell lysate.
  
  
  CONCLUSIONS
  These results indicate that enhancement of endosome escape in nondividing cells does not fully alleviate rate limits related to nuclear import of the plasmid.
authors.affiliation: Institute for Medicine and Engineering, Department of Chemical Engineering, 1024 Vagelos Research Laboratories, Philadelphia, PA 19104, USA.
authors.forename: Ajit
authors.initials: A
authors.lastname: Subramanian
authors.forename: Haiching
authors.initials: H
authors.lastname: Ma
authors.forename: Kris N
authors.initials: KN
authors.lastname: Dahl
authors.forename: Jingya
authors.initials: J
authors.lastname: Zhu
authors.forename: Scott L
authors.initials: SL
authors.lastname: Diamond
mesh_terms: D000256:Adenoviridae; D000818:Animals; D001011:Aorta; D002417:Cattle; D011992:Endosomes; D004727:Endothelium; D015870:Gene Expression; D018014:Gene Transfer Techniques; D006389:Hemagglutinins, Viral; D008081:Liposomes / Q000737:chemistry*; D010957:Plasmids; D014161:Transduction, Genetic; D014162:Transfection; D019076:Transgenes
chemical_list: D006389:Hemagglutinins, Viral; D008081:Liposomes; C452559:hemagglutinin HA-2 fusogenic peptide, Influenza virus