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J. Biol. Chem., Vol. 269, Issue 4, 2550-2561, 01, 1994
JH Felgner, R Kumar, CN Sridhar, CJ Wheeler, YJ Tsai, R Border, P Ramsey, M Martin and PL Felgner
The application of cationic liposome reagents has advanced DNA and mRNA
transfection research in vitro, and data are accumulating which show their
utility for in vivo gene transfer. However, chemical structure- activity
data leading to a better mechanistic understanding of their biological
activity is still limited. Most of the cationic lipid reagents in use today
for this application are formulated as liposomes containing two lipid
species, a cationic amphiphile and a neutral phospholipid, typically
dioleoylphosphatidylethanolamine (DOPE). The studies reported here examine
the effects of some systematic chemical structural changes in both of these
lipid components. Cationic and neutral phospholipids were formulated
together as large multilamellar vesicles (MLV) or small sonicated
unilamellar vesicles (SUV) in water, and each formulation was assayed
quantitatively in 96-well microtiter plates under 64 different assay
conditions using COS.7 cells and an RSV- beta-galactosidase expression
plasmid. The cationic lipid molecules used for these studies were derived
from a novel series of 2,3- dialkyloxypropyl quaternary ammonium compounds
containing a hydroxyalkyl moiety on the quaternary amine. A homologous
series of dioleylalkyl (C18:1) compounds containing increasing hydroxyalkyl
chain lengths on the quaternary amine were synthesized, formulated with 50
mol % DOPE, and assayed for transfection activity. The order of efficacy
was ethyl > propyl > butyl > pentyl > 2,3-dioleyloxypropyl-1-
trimethyl ammonium bromide (DOTMA). DOTMA, which is commercially available
under the trademark Lipofectin Reagent, lacks a hydroxyalkyl moiety on the
quaternary amine. A homologous series of hydroxyethyl quaternary ammonium
derivatives with different alkyl chain substitutions were synthesized,
formulated with 50 mol % DOPE, and assayed in the transfection assay. The
order of transfection efficacy was dimyristyl (di-C14:0) > dioleyl
(di-C18:1) > dipalmityl (di-C16:0) > disteryl (di-C18:0). The
addition of 100 microM chloroquine in the transfection experiment enhanced
the activity of the dioleyl compound by 4-fold and decreased the activity
of the dimyristyl compound by 70%. For each of the compounds and
formulations examined in this report, large multilamellar vesicles (MLV;
diameter 300-700 nm) were more active than small unilamellar vesicles (SUV;
diameter 50-100 nm). The neutral phospholipid requirements for transfection
activity in COS.7 cells with these cationic lipid molecules were
examined.(ABSTRACT TRUNCATED AT 400 WORDS)
Enhanced gene delivery and mechanism studies with a novel series of cationic lipid formulations
VICAL Inc., San Diego, California 92121.
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T. Akiyama, S. Takasawa, K. Nata, S. Kobayashi, M. Abe, N. J. Shervani, T. Ikeda, K. Nakagawa, M. Unno, S. Matsuno, et al. Activation of Reg gene, a gene for insulin-producing beta -cell regeneration: Poly(ADP-ribose) polymerase binds Reg promoter and regulates the transcription by autopoly(ADP-ribosyl)ation PNAS, January 2, 2001; 98(1): 48 - 53. [Abstract] [Full Text] [PDF]
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