Background

Small interfering (si)RNA mediated inhibition of oncogenes or viral genes may offer great opportunities for the treatment of several diseases such as hepatocellular carcinoma and viral hepatitis. However, the development of siRNAs as therapeutic agents strongly depends on the availability of safe and effective intracellular delivery systems. Poly(β-amino esters) (PbAEs) are, in contrast to many other cationic polymers evaluated in siRNA delivery, biodegradable into smaller, nontoxic molecules.



Methods and Results

We show for the first time that PbAE : siRNA complexes, containing 1,4-butanediol (PbAE1) or 1,6-hexanediol (PbAE2) diacrylate-based polymers, induced efficient gene silencing in both hepatoma cells and primary hepatocytes without causing significant cytotoxicity. Furthermore, carriers that slowly release the siRNA into the cytoplasm and hence induce a prolonged gene silencing are of major clinical interest, especially in fast dividing tumour cells. Therefore, we also studied the duration of gene silencing in the hepatoma cells and found that it was maintained for at least 5 days after siRNA delivery with PbAE2, the polymer with the slowest degradation kinetics.



Conclusions

From the time-dependent cellular distribution of these PbAE : siRNA complexes, we suggest that the slowly degrading PbAE2 causes a sustained endosomal release of siRNA during a much longer period than PbAE1. This may support the hypothesis that the endosomal release mechanism of PbAE : siRNA complexes is based on an increase of osmotic pressure in the endosomal vesicles after polymer hydrolysis. In conclusion, our results show that both PbAEs, and especially PbAE2, open up new perspectives for the development of efficient biodegradable siRNA carriers suitable for clinical applications. Copyright © 2008 John Wiley & Sons, Ltd.

https://users.ugent.be/~bgdgeest/Bruno/Vandenbroucke,%20R.%20Journal%20of%20Gene%20Medicine%202008.pdf

Prolonged gene silencing in hepatoma cells and primary hepatocytes after small interfering RNA delivery with biodegradable poly(beta-amino esters).

Water-soluble N-[(2-hydroxy-3-trimethylammonium)propyl]chitosan chloride as a nucleic acids vector for cell transfection.

We have investigated, for the first time, short interfering duplexes containing arabinonucleotides (ANA; the 2'-stereoisomer of RNA), as well as combinations of ANA with RNA, and their 2'-fluorinated derivatives 2F-ANA and/or 2'F-RNA. The results show that ANA is especially well accommodated in the sense strand of small interfering RNA (siRNA) duplexes, which can be extensively modified with little effect on potency. Furthermore, combining ANA with RNA and 2'F-ANA in siRNA passenger strands, particularly in patterns that bias duplex thermal stability, produces duplexes with similar (and sometimes enhanced) potency compared with native siRNA. Effective patterns of modification were identified against firefly luciferase screens in HeLa cells and then applied to knockdown of down-regulated in renal cell carcinoma (DRR), a novel and clinically tractable target for the treatment of glioblastoma.

Arabinonucleic Acids: 2′-Stereoisomeric Modulators of siRNA Activity

Hydrogel nanoparticles of poly(N,N-dimethylaminoethyl methacrylate) with hydrodynamic radii of 40–50 nm are synthesized via the copolymerization of a water-soluble monomer and N,N′-methylenebis(acrylamide) in a solution of Brij-97 reverse micelles in cyclohexane. All amino groups of nanoparticles are protonated in a weakly acidic solution; however, almost one-third of them remain inaccessible to a flexible polystyrenesulfonate polyanion, while almost two-thirds of them remain inaccessible to rigid double-helical DNA. Complexes of nanogels with plasmid DNA carrying the firefly luciferase gene transfect eukaryotic cells in a cultural medium, and the products of interaction of nanogels with small interfering RNAs suppress expression of the marker enzyme. In both systems, the replacement of linear polyamine with nanogel significantly increases the efficiency of delivery. The activity of nanogels in transfection experiments depends in an extremum pattern on the crosslink degree and achieves a maximum value at a crosslinking-agent concentration of 5 mol %. The results of this study suggest that the developed procedure offers promise for the synthesis of cationic nanogels as vectors for delivery of genetic material into living cells.

Synthesis of poly(N,N-dimethylaminoethyl methacrylate) nanogels in reverse micelles for delivery of plasmid DNA and small interfering RNAs into living cells

Development of an efficient small interfering RNA (siRNA) delivery method using non-viral carriers is necessary to determine potent therapeutic effects of RNA interference. Inflammatory responses induced by siRNA interaction with Toll-like receptors and retinoic-acid-inducible gene I protein/melanoma differentiation-associated gene 5 (RIG-I/MDA-5) are obstacles to the application of siRNAs in clinically. Here, we evaluated the effects on inflammatory responses by our siRNA delivery method using bubble lipoplexes with ultrasound (US) exposure in cultured macrophages. The effective gene suppression effects were obtained under low-toxic conditions in this siRNA transfer method. The interferon (IFN)-α after siRNA transfer using lipoplexes/bubble lipoplexes with US exposure was not detected. However, low levels of type I IFN mRNA production were induced through interaction of siRNA and cytoplasmic RIG-I/MDA-5, but not Toll-like receptors. Our findings indicate that it is possible to develop a safe and ...

Evaluation of inflammatory responses due to small interfering RNA transfer using unmodified- and mannose-modified bubble lipoplexes with ultrasound exposure in primary cultured macrophages

We determined the influence of siRNA (short interfering RNA) for expression of plasmid DNA (pDNA), when mismatched siRNA and pDNA encoding β-galactosidase (β-gal) were transfected into HeLa cells by the cotransfection method in which they were simultaneously added to the cells. Cationic liposomes (Lipofectamine2000) were used as a gene transfection reagent. The knockdown effect on β-gal was observed even when mismatched siRNA was used, and the effect depended on the amount of added mismatched siRNA. But, there was not a distinct difference of introduction of pDNA into cells between using mismatched siRNA and without using it. We considered that the cotransfection method should be avoided when we confirm RNAi efficiency. The reliable evaluation method for siRNA delivery in vitro was thus established by using NFAT reporter HeLa stable cell line or CHO (pMAM-luc) cell line that had DNA encoding luciferase. The following experimental conditions for each cell line were optimized: cell numbers seeded, total incubation times, concentrations of added inducers, and incubation times after addition of inducers. Transfection performance was compared for six commercially available reagents by this method. No commercially available transfection reagent, however, could reduce luciferase activity by less than one tenth without causing cellular cytotoxicity. Development of novel reagents providing higher transfection effects without cytotoxicity is needed.

Establishment of evaluation method for siRNA delivery using stable cell line carrying the luciferase reporter gene.

The present invention provides a composition with weak cytotoxicity for introducing a nucleic acid, such as a short oligonucleotide or a gene, into a cell and expression of the gene in the cell with improved capability for introduction of the nucleic acid, and a novel compound contained in the composition. When a compound represented by a formula (I): 
        (R 1 )n -R 2 -R 3      (I) 
wherein (R 1 )n represents a polyamino acid residue consisting of a total of n amino acid residues, which are identical to or different from one another, the n residues being selected from the group consisting of an arginine residue, a lysine residue, and a histidine residue, and n being an integer of from 4 to 16; R 2 represents a single bond or an amino acid residue; and R 3 represents a phospholipid residue with 1 or 2 identical or different unsaturated fatty acid residues having from 12 to 20 carbon atoms, and a lipid are administered or supplied to a cell together with a nucleic acid such as a gene, the nucleic acid can be efficiently introduced with weak cytotoxicity.

Composition for introduction of nucleic acid

A composition for nucleic-acid introduction which is lowly cytotoxic and is improved in the intracellular introduction of a nucleic acid to be introduced and in the intracellular expression thereof. The composition for the intracellular introduction of a nucleic acid comprises a compound represented by the following formula (I) (wherein R1 and R2 are the same or different and each means a C12-22 (un)saturated hydrocarbon group; R3 means C1-6 alkyl or C1-6 hydroxyalkyl; m means an integer of 1-10; and X means halogeno) and a phospholipid. (I)

Composition for nucleic-acid transfection

L'invention concerne une composition pour l'introduction d'un acide nucleique tel qu'un oligonucleotide court ou un gene dans une cellule, qui a une cytotoxicite faible et qui permet d'ameliorer l'efficacite de l'introduction de l'acide nucleique dans la cellule ainsi que l'expression de l'acide nucleique dans la cellule. L'invention concerne egalement un nouveau compose qui est contenu dans la composition. Un compose represente par la formule (I) et un lipide sont administres conjointement avec l'acide nucleique (par exemple, un gene) ou administres a une cellule conjointement avec l'acide nucleique, ce par quoi l'acide nucleique peut etre introduit dans la cellule avec efficacite a un taux de cytotoxicite faible. Formule (I),(R1)n-R2-R3, dans laquelle (R1)n represente un residu en acide polyamine ayant n morceaux de residus en acide amine au total (les n morceaux de residus en acide amine representant independamment un residu choisi dans le groupe constitue par un residu en arginine, un residu en lysine et un residu en histidine, et n representant un entier de 4 a 16) ; R2 represente une liaison simple ou un residu en acide amine ; R3 represente un residu phospholipide ayant 1 ou 2 residus enacide gras insature ayant chacun 12 a 20 atomes de carbone qui peuvent etre identiques ou differents les uns des autres.

Ayako Iijima

Papers

Establishment of evaluation method for siRNA delivery using stable cell line carrying the luciferase reporter gene.

Composition for introduction of nucleic acid

Composition for nucleic-acid transfection

Composition pour l'introduction d'acide nucléique

Composition for nucleic-acid introduction