Genetics

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Elucidation of gene function using RNA interference in a cancer cell culture model.
(2011) Daniels, Aliscia Nicole.; Singh, Moganavelli.; Ariatti, Mario.
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.
Novel cationic lipoplexes : characterization in cell culture in vitro and in vivo.
(2010) Sewbalas, Alisha.; Ariatti, Mario.; Singh, Moganavelli.; Arbuthnot, Patrick Brian.
Amongst the more promising non-viral DNA vehicles are liposomes, with those derived from cationic lipids showing significant potential, despite moderate transfection levels in vivo. This study has investigated the effect of liposome-anchored ionophore crown ethers on lipoplex-mediated gene transfer in vitro and in vivo. Several liposomes were constructed to include the cytofectin 3β[N(N’,N’-dimethylaminopropane)-carbamoyl] cholesterol (Chol-T), the co-lipid dioleoylphosphatidylethanolamine (DOPE), and 5% (mole/mole) of the cholesteryl crown ethers RUI-128 (aza-18-crown-6) or RUI-129 (aza-15-crown-5). Liposome size and lamellarity were established by transmission electron microscopy. All liposome preparations were shown to bind, condense and protect DNA avidly in the respective band shift, ethidium displacement and nuclease protection assays. Lipoplex targeting to hepatocytes may be achieved via the asialoglycoprotein receptor (ASGP-R), which is abundantly expressed on the human hepatoblastoma cell line HepG2. Therefore six additional liposomes were formulated to include 5% (mole/mole) of the cholesteryl galactosyl RUI-90 (Gal) and cholesteryl glucosyl RUI-92 (Glu) ligands. Their hepatotropic gene delivery was examined in the HepG2 cell line using the pCMV-luc plasmid. Transfection studies in the human embryonic kidney cell line HEK293 (ASGP-R-negative) revealed an increase in transgene activity in lipoplexes displaying the RUI-129 cholesteryl derivative. No ionophore-mediated enhancement of transfection activity was observed in HepG2 cells although Chol-T:DOPE, Chol-T:DOPE:RUI-128 and Chol-T:DOPE:RUI-129 liposomes achieved very high transfection levels, exceeding those of their hepatocyte targeted counterparts. Liposome-anchored crown ethers have been shown to potentiate in vitro transfection activity of lipoplexes in the HEK293 cell line. The novel cholesteryl glycosyl derivatives were, however, unable to enhance the targeted entry of lipoplexes into HepG2 cells. The three most effective preparations from in vitro studies were taken forward for in vivo assessment in NMRI mice at the University of the Witwatersrand Molecular Medicine and Haematology unit. Three groups of mice were employed for the evaluation of Chol-T:DOPE, Chol-T:DOPE:RUI-129 and Chol-T:DOPE:RUI-129-Gal lipoplexes with the Psi-CHECK plasmid. Mice treated with hydrodynamic injection and untreated animals made up two control groups. Luciferase activity was determined on examination of the harvested liver homogenates. All liposomes showed modest, but significant transfection activity (p<0.05) and were well tolerated. The assemblies examined therefore warrant further development.
Novel siRNA lipoplexes : their targeted and untargeted delivery to mammalian cells in culture.
(2011) Dorasamy, Shantal.; Singh, Moganavelli.; Ariatti, Mario.
The high gene knockdown specificity and efficiency of RNA interference (RNAi) provides a potentially viable avenue for the development of a new class of nucleic acid therapeutics for gene-based disease conditions. However, serum instability, inefficient cellular trafficking and non-specific effects of small interfering RNAs (siRNAs), one of the functional mediators of RNAi, has necessitated the development of carriers to facilitate targeted cell delivery. The decline of viral vectors in human gene therapy as a consequence of safety issues has intensified the importance of non-viral vector development. Among the non-viral vectors available for siRNA delivery, cationic liposomes have emerged as an attractive option owing to their simplicity, versatility, relatively low toxicity and potential for cell-specific targeting. Although existing cationic lipids and liposomes traditionally used for DNA delivery have also been used for siRNAs, there still exists a need to develop cationic lipids tailored towards siRNA transfection for improved gene silencing efficiency. Among the cell specific targets available for RNAi therapeutics, hepatocytes expressing the asialoglycoprotein receptor (ASGP-R) are an ideal choice due to the large number of disease targets present for treatment. In this investigation four novel cationic liposome formulations were prepared from equi-molar quantities of either the cationic cholesterol derivative 3β [N-(N’,N’- dimethylaminopropane)-carbamoyl] cholesterol (Chol-T) or 3β [N-(N’, N’, N’- trimethylammoniumpropyl)-carbamoyl] cholesterol iodide (Chol-Q) and DOPE, with and without the hepatotropic ligand, cholesteryl-β-D-galactopyranoside. Electrophoretic gel analysis and SYBR®green displacement assays were employed to determine siRNA binding and condensation efficiencies for all cationic liposomes; while liposome and lipoplex size measurements were made by cryoTEM. SiRNAlipoplex stability in serum was determined by the nuclease protection assay. Cell studies performed on the ASGP-R+ human hepatoma cells, HepG2 and the ASGP-Rembryonic kidney cells, HEK293, to determine lipoplex toxicity and transfection efficiencies were also undertaken. We show that the cationic liposomes formulated for this investigation were able to bind synthetic siRNA optimally at a positive to negative charge ratio of ± 1 : 6. In addition, the cationic liposomes were able to afford siRNA duplexes substantial protection from ribonuclease digestion in serum. From the results obtained in this study, it appears that the cationic liposomes are well tolerated by both the HEK293 and HepG2 cells in vitro. More importantly, the results obtained demonstrated higher transfection efficiencies for the targeted lipoplexes compared with the untargeted controls, strongly supporting the notion that incorporation of the cholestryl-β-D-galactopyranoside into the liposome structure increases transfection efficiency to the targeted HepG2 cells in culture via ASGP receptor mediation. Comparative studies in the HEK293 cell line yielded low transfection effeciences in the order of 20%, with no significant difference being recorded between galactosylated and non-galactosylated lipoplexes.

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Browsing Genetics by Author "Ariatti, Mario."