TY - JOUR
T1 - Development of an effective gene delivery system
T2 - A study of complexes composed of a peptide-based amphiphilic DNA compaction agent and phospholipid
AU - Murphy, Eric A.
AU - Waring, Alan J.
AU - Murphy, Jason C.
AU - Willson, Richard C.
AU - Longmuir, Kenneth J.
N1 - Copyright:
Copyright 2020 Elsevier B.V., All rights reserved.
PY - 2001/9/1
Y1 - 2001/9/1
N2 - We recently described a basic technology to efficiently combine compacted DNA with phospholipids and hydrophobic peptides, to produce homogenous complexes that are completely resistant to nuclease. We have developed this technology further to form gene delivery complexes that transfect cells effectively in vitro. In addition to plasmid DNA, the complexes contained two basic components: (i) a DNA compacting peptide (-CGKKKFKLKH), either conjugated to lipid or extended to contain (WLPLPWGW-) and (ii) either phosphatidylethanolamine or phosphatidylcholine. Complexes containing a 5.5-fold charge equivalence (peptide charge/DNA charge) of WLPLPWGWCGKKKFKLKH and 5 nmol dimyristoleoylphosphatidyl-ethanolamine/μg DNA produced the highest luciferase gene expression, exceeding 1 × 109 relative light units/s/mg protein (>3 μg luciferase per mg protein). These complexes transfected OVCAR-3, COS-7 and HeLa cells at either similar or superior levels when compared to polyethylenimine or lipofectamine complexes. With green fluorescent protein reporter gene, >50% of HeLa cells were positive 30 h after addition of these complexes. Furthermore, these optimal complexes were the least sensitive to pre-treatment of cells with chloroquine, indicating efficient endosomal escape. Our results indicated that self-assembling complexes of plasmid DNA, amphiphilic peptide and phosphatidyl-ethanolamine are highly effective non-viral gene delivery systems.
AB - We recently described a basic technology to efficiently combine compacted DNA with phospholipids and hydrophobic peptides, to produce homogenous complexes that are completely resistant to nuclease. We have developed this technology further to form gene delivery complexes that transfect cells effectively in vitro. In addition to plasmid DNA, the complexes contained two basic components: (i) a DNA compacting peptide (-CGKKKFKLKH), either conjugated to lipid or extended to contain (WLPLPWGW-) and (ii) either phosphatidylethanolamine or phosphatidylcholine. Complexes containing a 5.5-fold charge equivalence (peptide charge/DNA charge) of WLPLPWGWCGKKKFKLKH and 5 nmol dimyristoleoylphosphatidyl-ethanolamine/μg DNA produced the highest luciferase gene expression, exceeding 1 × 109 relative light units/s/mg protein (>3 μg luciferase per mg protein). These complexes transfected OVCAR-3, COS-7 and HeLa cells at either similar or superior levels when compared to polyethylenimine or lipofectamine complexes. With green fluorescent protein reporter gene, >50% of HeLa cells were positive 30 h after addition of these complexes. Furthermore, these optimal complexes were the least sensitive to pre-treatment of cells with chloroquine, indicating efficient endosomal escape. Our results indicated that self-assembling complexes of plasmid DNA, amphiphilic peptide and phosphatidyl-ethanolamine are highly effective non-viral gene delivery systems.
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U2 - 10.1093/nar/29.17.3694
DO - 10.1093/nar/29.17.3694
M3 - Article
C2 - 11522841
AN - SCOPUS:0035445126
VL - 29
SP - 3694
EP - 3704
JO - Nucleic Acids Research
JF - Nucleic Acids Research
SN - 0305-1048
IS - 17
ER -