Elucidation of gene function using RNA interference in a cancer cell culture model.
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Date
2011
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Abstract
RNA interference (RNAi), mediated by small interfering RNA (siRNA), has emerged as a
powerful tool for elucidating gene function and also holds great potential for the treatment of
acquired and inherited diseases. The delivery of siRNAs still remains a major obstacle for their
therapeutic use. Cationic liposomes, a group of positively charged nanovesicles, represent a class
of non-viral vectors that have shown the ability to efficiently bind and deliver siRNA.
In this study, six cationic liposome formulations containing either cationic cholesterol derivative
[N-(N’,N’-dimethylaminopropane)-carbamoyl] cholesterol (Chol-T) or N,Ndimethylaminopropylaminylsuccinylcholesterylformyl-
hydrazide (MSO9) were prepared with the neutral lipid dioleoylphosphatidylethanolamine (DOPE). Varying amounts of
distearoylphosphatidylethanolamine polyethylene glycol 2000 (DSPE-PEG2000), (0, 2 and 5 mole
percent) were also included in the liposomal formulations as polyethylene glycol is known to
improve the lipoplex bioavailability in vivo.
We present evidence that siRNA may be delivered to mammalian cells, in vitro, using a novel
cationic liposome carrier system and that siRNA binding and transfection efficiency of the
cationic liposomes are affected by the degree of pegylation.
Cryoelectron microscopy revealed that the liposome vesicles were unilamellar and were in the
30 - 130 nm size range, while band shift assays confirmed the formation of complexes between
the siRNA and the liposome preparations. These siRNA lipoplexes were shown to afford
protection to their siRNA cargoes against serum nuclease degradation and were also shown to
be relatively non-toxic to the HeLa tat luc cell line which stably expresses the firefly luciferase
gene. Cryoelectron microscopy revealed that an inverse relationship exists between the lipoplex
size and the degree of pegylation. To determine the transfection efficiency of the cationic
liposome preparations in the HeLa tat luc cell line, complexes were prepared with anti-luciferase
siRNA, which is specific for the firefly luciferase gene, and knockdown of the luciferase gene
was monitored in transfected cells. The results show that liposomes containing the cytofectin
Chol-T were particularly effective, achieving up to 93.4% gene knockdown at the 30 nM siRNA
concentration. The non- pegylated and pegylated cationic liposomes that have been formulated
and examined in this study therefore warrant further development to facilitate in vivo studies.
Description
Thesis (M.Sc.)-University of KwaZulu-Natal, Westville, 2011.
Keywords
Gene therapy., RNA., Theses--Genetics.