Patent Application
20100035974
The present invention relates to a composition comprising a siRNA and a transfecting compound consisting of an aminoglycoside of the class of 4,5-disubstituted 2-deoxystreptamine ring linked via a spacer molecule to a lipid moiety of formula —(R1)R2 wherein R1 and R2 represent, independently of one another, a hydrogen atom or a fatty aliphatic chain or R1 or R2 is absent, with the proviso that at least one of R1 and R2 represents a fatty aliphatic chain; or —OR3 or —NR3 wherein R3 represents a steroidal derivative. The invention also concerns in vitro and in vivo applications of these compositions.
RNA interference (RNAi) has become a powerful and widely used tool for the knockdown of target gene expression via a posttranscriptional silencing process and subsequent phenotypic analysis of gene function in cells. Therapeutic approaches involving RNA interference mechanism are also actively under investigation. To achieve efficient target gene knockdown, specific 21-25 double-stranded small interfering RNA (siRNA) molecules have to be delivered into the cytoplasmic compartment of the cell.
Different methods have been used to deliver siRNA into cells. Among them, the most widely used is based on the use of common cationic lipids which were previously developed for DNA transfection in the 90's. These cationic lipids are composed of a hydrophobic moiety linked to a cationic headgroups including a quaternary ammonium such as dioctadecyldiammonium bromide (DODAB), or dioleoyl trimethylammonium propane (DOTAP), or a polycation such as dioctadecylamidoglycylspermine (DOGS), or guanidinium residues such as bis(guanidinium)-tren-cholesterol (BGTC). Other types of cationic lipids were also synthesized for DNA transfection. Notably, compounds of formula A-Y-L, wherein A is an aminoglycoside, Y a spacer molecule and L a lipid moiety have been described in U.S. patent application Ser. No. 10/228,959 published under number US 2003-0054556 A1 (1), and which is herein incorporated by reference. The interest of these compounds for DNA transfection has further been confirmed in Sainlos et al, 2005 (2), Sainlos et al, 2004 (3) and Belmont et al, 2002 (4).
A co-lipid like dioleoyl phosphatidyl ethanolamine (DOPE) is usually combined with the cationic lipids to form liposomes. The electrostatic interactions between the plasmid DNA and cationic headgroups lead to the formation of particles whose structure has been shown to be lamellar or hexagonal depending on the co-lipid used and its proportion in the cationic liposomes.
Nevertheless, the structural characteristics of complexes formed with siRNA are still unknown, and the relevance of the structure observed with plasmid DNA remains to be elucidated for siRNA. It should also be stressed that all cationic vectors developed for DNA transfection are not necessary optimized for the delivery of nucleic acids of low molecular weight and specific chemical structure such as siRNA molecules.
Indeed, siRNA molecules display a very specific structure, very different from that of DNA generally used in DNA transfection. A siRNA is a short (usually 21-25 nucleotides) double-strand of RNA (dsRNA) with a few nucleotides (usually 2) 3′ overhangs on either end. Each strand has a 5′ phosphate group and a 3′ hydroxyl (—OH) group. This very specific structure of naturally occuring siRNAs is the result of processing by Dicer, an enzyme that converts either long dsRNAs or hairpin RNAs into siRNAs. This particular structure has been conserved for synthetic siRNAs. In contrast, transfected DNA are usually used for expressing a particular gene, which implies that the transfected DNA molecule comprises a sequence encoding said gene, as well as regulatory sequences for directing the expression of the gene into the transfected cell. Tranfected DNA molecules are thus generally long length DNA molecules, contrary to very short 21-25 nucleotides siRNAs. This very significant structure difference may result in a significant change in the transfecting efficiency of a particular tranfecting compound with one nucleic acid category or the other.
In addition, the activities of DNA expression vectors and siRNA, once transfected into a target cell, are completely different and mediated by distinct mechanisms. DNA expression vectors have to be transcribed to results in gene expression, while siRNAs assemble into endoribonuclease-containing complexes known as RNA-induced silencing complexes (RISCs), unwinding in the process, and siRNA strands subsequently guide the RISCs to complementary RNA molecules, where they cleave and destroy the cognate RNA (effecter step), thus inhibiting a particular gene expression. Cleavage of cognate RNA takes place near the middle of the region bound by the siRNA strand. The action mechanism of siRNAs is thus quite complex, and efficient transfecting compounds for siRNAs should thus allow for both the entry of siRNAs into the target cells and the release of functional siRNAs in the cell to permit an efficient interference.
Due to the particularities of siRNAs in term of structure and action mechanism, it is clear that cationic lipids which have been optimized for DNA transfection, although some of them may have some efficiency in tranfecting siRNA also, may not all be adapted to siRNAs transfection. Nevertheless, most currently known transfecting compounds have been optimized for DNA transfection there has not been many studies yet intending to optimized transfecting compounds for siRNAs transfection. There is thus a need for transfecting compounds specialized for tranfecting siRNA with a high efficiency.
The inventors have explored the efficiency of lipidic aminoglycoside derivatives to deliver siRNA molecules and knockdown gene expression in mammalian cells. They successfully synthesized new cationic lipids based upon the use of aminoglycoside as cationic headgroup linked to dioleoyl chains via a succinyl spacer. Aminoglycosides consist of oligosaccharides decorated with up to six amino groups as well as numerous hydroxyl groups thus providing a versatile polycationic frame work. The inherent flexibility of the glycoside bonds of the aminoglycosides can remodel themselves to interact with RNA molecules. This family offers the advantageous possibility to modify the cationic headgroup using different aminoglycosides as kanamycine, tobramycine, neomycine or paromomycine, known to interact with the major groove of duplex RNA or more generally with A-form nucleic acids.
The inventors found that while complexation of siRNA molecules with lipidic aminoglycosides derivatives adhere to the three stage model of colloidal stability previously proposed for plasmid DNA, siRNA complexes exhibited a smaller mean diameter and a total siRNA entrapment for lower lipidic aminoglycoside derivatives/siRNA charge ratio than that observed with siRNA complexed with guanidinium-cholesterol reagent.
They also found that lipidic aminoglycoside derivatives/siRNA complexes form specific structures consisting of self-assembled RNA-condensed particles with various morphologies depending on the aminoglycosides used as polar headgroup. Indeed, while lipidic aminoglycoside derivatives of the class of 4,6-disubstituted 2-deoxystreptamine ring formed usual “onion-like” structures made of a regular superimposition of lipid bilayer and siRNA molecules, lipidic aminoglycoside derivatives of the class of 4,5-disubstituted 2-deoxystreptamine ring surprisingly formed distinct structures with grape of small condensed structures.
In addition, comparative analysis of in vitro gene expression inhibition with lipidic aminoglycoside derivatives and cationic lipids designed for DNA transfection indicated that lipidic aminoglycoside derivatives of the class of 4,5-disubstituted 2-deoxystreptamine ring had the best efficiency to induce the interference mechanism into target cells. Indeed, although all lipidic aminoglycoside derivatives and DNA transfection reagent BGTC all allowed for a high siRNA internalization, only aminoglycoside derivatives of the class of 4,5-disubstituted 2-deoxystreptamine ring surprisingly permitted a high siRNA mediated RNAi knockdown of the target gene. Although the reasons of this phenomenon are still unknown, it may be postulated that the distinct structures of this class of that lipidic aminoglycoside derivatives/siRNA complexes may be involved in the significantly higher efficiency of these particular compounds.
Finally, comparative analysis of in vitro gene expression inhibition with lipidic aminoglycoside derivatives and commercially available reagents for siRNA delivery also demonstrated that lipidic aminoglycoside derivatives of the class of 4,5-diubstituted 2-deoxystreptamine ring displayed a higher efficiency for siRNA mediated RNAi knockdown than currently commercially available reagents for siRNA delivery.
Thus, among the general class of lipidic aminoglycoside derivatives known to mediate efficient DNA transfection, the inventors have identified a new subclass of 4,5-disubstituted 2-deoxystreptamine ring lipidic aminoglycoside derivatives particularly well adapted to the efficient and functional delivery of siRNA into mammalian cells.
The invention thus concerns a composition comprising a siRNA and a transfecting compound of general formula (I):
A-Y-L (I), wherein
or a salt, an isomer or a mixture thereof.
For the purpose of the present invention, the term “aminoglycoside” (or “aminosides”) is intended to mean natural heterosides formed by the combining of a genin of the aminocyclitol group with generally several saccharides, at least one of which is an aminosugar (osamine). Aminoglycosides may differ both in the molecular nucleus, which can be streptamine or 2-deoxystreptamine, and in the aminohexoses linked to the nucleus. Most currently used aminoglycosides have 2-deoxystreptamine as molecular nucleus and can be further segregated into two subclasses, depending on the positions of the 2-deoxystreptamine ring II to which are attached the other rings:
4,5-disubstituted 2-deoxystreptamine aminoglycosides in which the 2-deoxystreptamine nucleus is substituted in positions 4 and 5, and
4,6-disubstituted 2-deoxystreptamine aminoglycosides in which the 2-deoxystreptamine nucleus is substituted in positions 4 and 6.
The 4,5-disubstituted class includes neomycin, paromomycin, and ribostamycin, while the 4,6-disubstituted class includes tobramycin, kanamycine, amikacin and gentamicin.
The tranfecting compound of the composition according to the invention is a 4,5-disubstituted 2-deoxystreptamine aminoglycoside, as defined above. It may advantageously be selected from paromomycin, neomycin, and ribostamycin, preferably from paromomycin and neomycin. Alternatively, any synthetic or semi-synthetic 4,5-disubstituted 2-deoxystreptamine aminoglycoside, as defined above, may be used instead.
According to the present invention, the term “spacer” is intended to mean any chemical group which makes it possible both to form the bond between the aminoglycoside or its polyguanidylated derivative and the lipid component of the molecule, and to distance these two components in order to reduce any undesired interaction between them. Consequently, the spacer is a difunctional chemical group which may, for example, consist of one or more chemical functions chosen from alkyls having 1 to 6 carbon atoms, ketone, ester, ether, amine, amide, amidine, carbamate or thiocarbamate functions, glycerol, urea, thiourea or aromatic rings.
For instance, the spacer may be selected from the radicals of formula:
—C(O)—;
—NH—C(O)—CH2—CH2—;
—W—(CH2—)k—W′—;
and —(CH2—)i—W—(CH2)j—
in which i, j and k are integers chosen between 1 and 6 inclusive, and W and W′ are groups, which may be identical or different, chosen from ketone functions, ester functions, ether functions, amino functions, amide functions, amidine functions, carbamate functions, thiocarbamate functions, glycerol, urea, thiourea, and aromatic rings.
For the purpose of the present invention, the term “fatty aliphatic chains” is intended to mean saturated or unsaturated alkyl radicals containing 10 to 22 carbon atoms and optionally containing one or more hetero atoms, provided that said fatty aliphatic chains have lipid properties. Preferably, they are linear or branched alkyl radicals containing 10 to 22 carbon atoms and 1, 2 or 3 unsaturations. Preferably, said alkyl radicals comprise 10, 12, 14, 16, 18, 20 or 22 carbon atoms, more preferably 18, 20 or 22 carbon atoms. Mention may be made more particularly of aliphatic radicals selected from aliphatic radicals —(CH2)11CH3, —(CH2)13CH3, —(CH2)15CH3, —(CH2)17CH3, myristoyl, oleyl, and stearyl.
In a preferred embodiment, when L represents —(R1)R2, L is selected from dimyristoyl, dioleyl, and distearyl.
For the purpose of the present invention, the term “steroidal derivatives” is intended to mean polycyclic compounds of the cholestane type. These compounds may or may not be natural and are preferably selected from cholesterol, cholestanol, 3-α-cyclo-5-α-cholestan-6-β-ol, cholic acid, cholesteryl formiate, cholestanyl formiate, 3-α-5-cyclo-5-α-cholestan-6-β-yl formiate, cholesterylamine, 6-(1,5-dimethylhexyl)-3a,5a-dimethylhexadecahydrocyclopenta[a]cyclopropa[2,3]cyclopenta[1,2-f]naphthalen-10-ylamine, and cholestanylamine.
Preferred transfecting compounds used in compositions according to the invention are:
It is to be understood that the present invention also relates to compositions comprising the isomers of the transfecting compounds of general formula (I) when they exist, and also the mixtures thereof, or the salts thereof.
In particular, the compounds of the invention may be in the form of nontoxic and pharmaceutically acceptable salts. These nontoxic salts comprise salts with inorganic acids (for example hydrochioric, sulfuric, hydrobromic, phosphoric, nitric acid) or with organic acids (acetic, propionic, succinic, maleic, hydroxymaleic, benzoic, fumaric, methanesulfonic, trifluoroacetic or oxalic acid).
The compounds of general formula (I) according to the present invention are prepared as previously described in US 2003-0054556 A1 and in Sainlos et al, 2005 (2), Sainlos et al, 2004 (3) and Belmont et al, 2002 (4).
The compositions according to the invention may also comprise one or more adjuvants capable of associating with the transfecting compound/siRNA complexes and improving the transfecting power thereof. In another embodiment, the present invention therefore relates to compositions comprising a siRNA, a transfecting compound as described above and at least one adjuvant capable of associating with the transfecting compound/siRNA complexes and improving the transfecting power thereof.
These adjuvants which make it possible to increase the transfecting power of the compounds according to the present invention are advantageously chosen from lipids (for example neutral lipids, in particular phospholipids), peptides (for example histone, nucleolin or protamine derivatives such as those described in WO 96/25508), proteins (for example proteins of the HNG type such as those described in WO 97/12051) and/or polymers (for example polymers which make it possible to turn the transfecting compound/siRNA formulations into “stealth” formulations, such as polyethylene glycol (PEG) introduced into, the formulation on its own or in a form attached to a lipid in order to colloidally stabilize the transfecting compound/siRNA formulations, for example PEG-cholesterol).
From this point of view, the compositions of the invention may comprise, as an adjuvant, one or more neutral lipids which in particular have the property of forming lipid aggregates. The term “lipid aggregate” is a generic term which includes liposomes of all types (both unilamellar and multilamellar) and also micelles or more amorphous, aggregates.
More preferentially, the neutral lipids used in the context of the present invention are lipids containing two fatty chains. Particularly advantageously, use is made of natural or synthetic lipids which are zwitterionic or lacking ionic charge under physiological conditions. They may be chosen more particularly from dioleoylphosphatidylethanolamine (DOPE), oleoylpalmitoylphosphatidylethanolamine (POPE), distearoyl-, dipalmitoyl- and dimirystoylphosphat±dylethanolamines and also the derivatives thereof which are N-methylated 1 to 3 times, phosphatidylglycerols, diacylglycerols, glycosyldiacylglycerols, cerebrosides (such as in particular galactocerebrosides), sphingolipids (such as in particular sphingomyelins) or asialogangliosides (such as in particular asialoGM1 and asialoGM2). According to a particularly preferred alternative, the lipid adjuvants used in the context of the present invention are chosen from DOPE, DOPO, cholesterol or the lipid derivatives of nonionic surfactants, such as PEG-cholesterol.
These various lipids may be obtained either by synthesis or by extraction from organs (for example the brain) or from eggs, using conventional techniques well known to those skilled in the art. In particular, the extraction of natural lipids may be carried out using organic solvents.
Preferentially, the compositions of the invention comprise from 0.01 to 20 equivalents of adjuvant per equivalent of siRNA in mol/mol, and more preferentially from 0.5 to 5 molar equivalents, corresponding to a charge ratio (moles of positive charges)/(moles of negative charges) comprised between 0.01 and 20, preferentially between 0.5 and 5.
According to another alternative, the adjuvants cited above make it possible to improve the transfecting power of the compositions according to the present invention; in particular, the peptides, the proteins or certain polymers, such as polyethylene glycol, may be conjugated to the transfecting compounds according to the invention, and not simply mixed. In particular, the PEG may be attached covalently to the lipid derivatives of aminoglycoside according to the present invention either at a remaining amine of the aminoglycoside or at the hydroxyl radicals of the aminoglycoside.
According to a particularly advantageous embodiment, the compositions according to the present invention also comprise a targeting element which makes it possible to direct the transfer of the siRNA. This targeting element may be an extracellular targeting element which makes it possible to direct the transfer of the siRNA toward certain desired cell types or tissues (tumor cells, hepatic cells, hematopoietic cells, etc.). It may also be an intracellular targeting element which makes it possible to direct the transfer of the siRNA toward certain preferred cellular compartments (mitochondria, nucleus, etc.). The targeting element may be mixed with the transfecting compounds according to the invention and with the siRNAs and, in this case, the targeting element is preferably attached covalently to a fatty aliphatic chain (at least 10 carbon atoms) or to a polyethylene glycol. According to another alternative, the targeting element is covalently attached to the transfecting compound according to the invention, either on one of the chemical functions which make up the spacer Y, or at the end of the lipid component (for example at the end of R and/or R when they represent fatty aliphatic chains), or directly on the aminoglycoside at one of the remaining amines or at the hydroxyl radicals. Finally, the targeting element may also be attached to the siRNA, as specified previously.
Among, the targeting elements which can be used in the context of the invention, mention may be made of sugars, peptides, proteins, oligonucleotides, lipids, neuromediators, hormones, vitamins or derivatives thereof. Preferentially, they are sugars, peptides, vitamins or proteins such as, for example, antibodies or antibody fragments, cell receptor ligands or fragments thereof, receptors or receptor fragments.
For example, they may be ligands for growth factor receptors, for cytokine receptors, for receptors of the cellular lectin type or for folate receptors, or ligands containing an RGD sequence with an affinity for adhesion protein receptors such as integrins. Mention may also be made of transferrin receptors, HDL receptors and LDL receptors or the folate transporter. The targeting element may also be a sugar which makes it possible to target lectins such as the receptors for asialoglycoproteins or for sialidized species, such as sialyl Lewis X, or an Fab antibody fragment or a single-chain antibody (ScFv).
The invention is also directed to the use of the compositions according to the present invention, for transferring nucleic acids into cells in vitro, in vivo or ex vivo.
More precisely, an object of the present invention is the use of compositions according to the invention, for preparing a medicinal product intended to treat diseases, in particular diseases which result from the overrexpression of a gene product. The siRNA contained in said medicinal product specifically targets the mRNA of said gene product, thereby correcting said diseases in vivo or ex vivo.
The invention also concerns a method for inhibiting a target gene expression in a subject, comprising administering to said subject a composition according to claim 1, wherein the siRNA comprised in said composition is specifically directed to said target gene.
For uses in vivo, for example in therapy or for studying gene regulation or creating animal models of pathological conditions, the compositions according to the invention may be formulated with a view to topical, cutaneous, oral, rectal, vaginal, parenteral, intranasal, intravenous, intramuscular, subcutaneous, intraocular, transdermal, intratracheal, intraperitoneal, etc. administration.
Preferably, the compositions of the invention contain a vehicle which is pharmaceutically acceptable for an injectable formulation, in particular for direct injection into the desired organ, or for topical administration (on skin and/or mucous membrane).
They may in particular be sterile, isotonic solutions or dry compositions, in particular lyophilized compositions, which, by adding, as appropriate, sterilized water or physiological saline, allow injectable solutes to be made up. The doses of siRNAs used for the injection and also the number of administrations may be adjusted depending on various parameters, and in particular depending on the method of administration used, on the pathological condition in question, on the gene to be expressed or on the desired duration of the treatment. With regard more particular to the method of administration, it may be either a direct injection into the tissues, for example into the tumors, or an injection into the circulatory system, or it may involve treating cells in culture followed by their reimplantation in vivo, by injection or transplantation. The relevant tissues in the context of the present invention are, for example, muscles, skin, brain, lungs, trachea, liver, spleen, bone marrow, thymus, heart, lymph, blood, bones, cartilage, pancreas, kidneys, bladder, stomach, intestines, testicles, ovaries, rectum, nervous system, eyes, glands, connective tissues, etc.
Concerning in vitro applications, the invention further relates to a method for transferring a siRNA into cells in vitro, comprising:
(A) Fluorescence microscopy visualization of GFP silencing and siRNA internalization. The GFP-expressing human lung cancer H1299 cells were transfected with control siRNA (panels A,B) or 3′-rhodamine labeled anti-GFP siRNA (panels C-F). The siRNA molecules were formulated in the absence (“naked” siRNA in panels A,C,E) or in the presence of DOSP (panels B,D,F). The transfected H1299 cells were observed using a FITC filter to visualize GFP fluorescence (panels A,B, see white/light grey areas) or a rhodamine filter to visualize siRNA internalization (panels E,F, see white/light grey areas).
(B) Lamin A/C silencing efficiency using DOSP reagent as a function of siRNA amount. Anti-LaminA/C siRNA amounts ranging from 3.75 to 200 ng/well were formulated with DOSP. Results of real-time quantitative RT-PCR analysis of human Lamin A/C mRNA after transfection of HeLa cells showed that DOSP reagent is efficient with very low amount of siRNA (normalization to HPRT1). Values are relative to cells transfected under the same experimental condition with a control siRNA.
(C) Lamin A/C silencing efficiency using DOSP reagent or competitor reagent. Anti-LaminA/C siRNA at 18.75 and 37.5 ng/well were formulated with DOSP or the commercial reagents HiPerFect. Results of real-time quantitative RT-PCR analysis of human Lamin A/C mRNA after transfection of HeLa cells showed that DOSP reagent was more efficient than HiPerFect (normalization to HPRT1). Values are relative to cells transfected under the same experimental condition with a control siRNA.
1.1. Materials and Methods
1.1.1. Nucleic Acids, Cationic Lipids and Preparation of Complexes.
3′-Rhodamine labeled anti-GFP siRNA and the 5′-Rhodamine labeled control siRNA were provided by Qiagen (Chatsworth, Calif., USA). Stock. Unlabeled anti-GFP siRNA was provided by Eurogentec (Seraing, Belgium). Anti-GFP siRNAs had for sense sequence: GCAAGCUGACCCUGAAGUUCAU (SEQ ID NO:1). The Silencer® GAPDH siRNA (human, mouse, rat) targeting the GAPDH mRNA was provided by Ambion® (Cambridgeshire, United Kingdom). Human anti-Lamin A/C siRNA was provided by Santa Cruz Biotechnology (Santa Cruz, Calif., USA). The plasmid pTER, encoding anti-GFP siRNA hairpin, was obtained by inserting the modified H1 promoter between the BglII and HindII sites of pCDNA4TO (Invitrogen Life technologies, Carlsbad, Calif., USA) and was kindly provided by A. Polesskaya (Villejuif, France). Plasmids were purified from recombinant Escherichia Coli using EndoFree plasmid purification columns (Qiagen, Chatsworth, Calif., USA). Lipidic aminoglycoside derivatives and BGTC were synthesized as previously described (2-4.)
3-β-(N-(N′,N′-dimethylethane)carbamoyl)cholesterol (DC-Chol) and Dioleoyl phosphatidyl ethanolamine (DOPE) were from Aventi Polar Lipids, Inc (Alabaster, Ala., USA). Polyethyleneime (PEI) 25 kDa was from Sigma (St Louis, Mo., USA). RNAiFect, X-treme-GENE and Lipofectamine 2000 were respectively from Qiagen ( ), Roche Diagnostics ( ) and Invitrogen ( ). Cationic liposomes of lipidic aminoglycoside derivatives/DOPE and BGTC/DOPE were prepared as described in ref. With minor changes, i.e. the dispersion was sonicated for 10 minutes and stored at 4° C. Lipidic aminoglycoside derivatives/nucleic acids complexes and BGTC/nucleic acids lipoplexes were prepared by mixing equal volumes of cationic liposomes at different concentrations, with plasmid DNA or siRNA at the desired concentration in 300 mM NaCl.
1.1.2. In vitro Transfection.
d2GFP (human lung cancer) cells were cultured in flasks, at 37° C. in 5% CO2/humidificated atmosphere, in D-MEM with Glucose, 1-Glutamine and Pyruvate supplemented with 1% streptomycin/penicillin, geneticin at 0.8 mg/ml (GIBCO, Invitrogen Life technologies, Carlsbad, Calf., USA) and 10% fetal calf serum (Eurobio, Courtaboeuf, France). One day before transfection, cells were transferred onto 24-well culture plates at cell counts of 65000 resulting in approximately 70-80% confluence 24 hours later. Transfection was performed in each well by adding 50 μl of complexes in 500 μl of serum and geneticin-free D-MEM. After 2 h, the transfection medium was replaced by 1 ml of D-MEM containing 10% of fetal calf serum and 1% streptomycin/penicillin. Transfection experiments were performed in triplicate and residual GFP expression was quantified 24 hours after transfection. For the time course experiment, transfection was performed as described above with the following modifications. Transfected cells were transferred every three days onto a new 24-well culture plate with an appropriate cell counts to have 90% confluence on the day of GFP quantification. Transfection experiments were performed in triplicate and residual GFP expression was measured 1, 2, 3, 5, 6 and 9 days after transfection.
For RNA isolation, cells were placed in 6-well dishes at cell counts of 250000 cells per well. Transfection was performed in each well by adding 100 μl of complex formulation in 1 ml of serum-free and geneticin-free D-MEM. After 2 h at 37° C. the transfection medium was replaced by 1 ml of D-MEM containing 10% of fetal calf serum and 1% streptomycin/penicillin. After 24 hours, RNA was isolated.
1.1.3. Gene Expression and Inhibition.
After 24 hours, transfected cells were washed twice with 500 μl of PBS, lysed with Reporter Lysis Buffer (Promega, Madison, Wis.) supplemented with a protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany). The complete lysis was ensured by one freezing-thawing (−80° C./20° C.). Samples were then centrifuged at 10000 g for 5 min. GFP fluorescence measurements were assayed on a 180 μl aliquot of supernatant and performed on a Victor2 (Perkin Elmer, Les Ulis, France) at excitation and emission wavelengths of 485 nm and 535 nm, respectively. Fluorescence was normalized to the total protein concentration of the sample, obtained using a BCA assay kit (Pierce, Rockford, Ill., USA). Residual GFP expression was expressed as a percentage of the fluorescence of control sample.
Lamin A/C immunostaining was performed on cells which were washed with PBS and fixed 5 minutes with a PAF 2% solution, then incubated 5 minutes in a PBS-Triton 0.2% solution. First cells were incubated 1 hour with primary anti-Lamin antibody ( 1/50) (Lamin A/C (H-110), a rabbit polyclonal antibody against 231-340 mapping within an internal region of Lamin A of human origin (Santa Cruz Biotechnology, Calif., USA). Then, cells were incubated with the Rabbit IgG-Biotynilated (Sigma, St Louis, Mo., USA) ( 1/800) for 1 hour, and with the Streptavidine-Rhod ( 1/200) for 30 minutes (Molecular Probes). Cells were observed on a rhodamine excitation using the Dako Fluorescent Mounting Medium (Dako, Cytomacion, Denmark).
1.1.4. Fluorescence Microscopy.
Fluorescence microscopic examination of transfected cells was carried out using an inverted fluorescence microscopy Axiovert 200 M (CarlZeiss, Göttingen, Germany).
1.1.5. Ethidium Bromide Fluorescence Studies.
Ethidium bromide fluorescence measurements were carried out on Kontron SFM25 spectrofluorimeter at excitation and emission wavelengths of 260 nm and 590 nm, respectively. Fluorescence was monitored immediately after adding ethidium bromide (final concentration 5 μM) to samples prepared at 10 μg/ml of nucleic acid (siRNA or DNA).
1.1.6. Dynamic Light Scattering.
Dynamic light scattering measurements were made on a Zetasizer 300HSA (Malvern, Worcestershire, United Kingdom) at 20° C. Measurements were monitored on samples prepared at 10 μg/ml of nucleic acid containing various amounts of cationic lipid.
1.1.7. Cryo-TEM Micrography.
siRNA/cationic lipid complexes were prepared at 10 μg siRNA/ml with the appropriate amount of cationic lipid. A 5 μl sample was deposited onto a holey carbon coated copper grid; the excess was blotted with a filter paper, and the grid was plunged into a liquid ethane bath cooled with liquid nitrogen (Leica EM CPC). Specimens were maintained at a temperature of approximately −170° C., using a cryo holder (Gatan), and were observed with a FEI Tecnai F20 electron microscope operating at 200 kV and at a nominal magnification of 50000× under low-dose conditions. Images were recorded with a 2K×2K Gatant slow scan CCD camera.
1.1.8. Gene Expression Inhibition on a RNA Level.
Total RNA was extracted using TRIzol reagent (Invitrogen, Cergy Pontoise, France), and the Rneasy® Mini Kit (Qiagen, Chatsworth, Calif., USA). mRNA was purified using the oligotex® mRNA midi Kit (Qiagen, Chatsworth, Calif., USA). Reverse transcription was performed using the SuperScript™III (Invitrogen Life technologies, Carlsbad, Calif., USA) as described by the supplier. Real-time quantitative PCR was performed using the ABI Prism® 7900HT sequence detection system (Applied Biosystems) and the data were collected after instrument spectral compensations by the Applied Biosystems SDS 2.1 software. Sequence for GFP reverse primer was 5′-CGGGCATGGCGGACTT-3′ (SEQ ID NO:2), for the FAM-probe 5′-CAGCACGACTTCTTC-3′ (SEQ ID NO:3) and for the GFP forward primer 5′-GCTACCCCGACCACATGAAG-3′ (SEQ ID NO:4). Control DNA-free samples were run for each experiment. The cycling conditions included a hot start at 95° C. for 10 min followed by 40 cycles at 95° C. for 15 s and 60° C. for 1 min. single amplicon of the appropriate size was detected using gel electrophoresis. The HPRT gene was used for normalizing the data. Values corresponded to the percentage of normalized GFP, or GAPDH, or LaminA/C mRNA amount obtained in cells transfected with anti-GFP or GAPDH or LaminA/C siRNA versus quantity obtained with Control-siRNA.
1.2. Results
1.2.1. Gene Expression Inhibition Activities of Cationic Vectors/siRNA Complexes.
First, we assessed the potency of cationic vectors, currently used for plasmid DNA transfection, to inhibit GFP expression through the delivery of anti-GFP siRNA molecules in transformed cell line (d2GFP) expressing the cytoplasmic GFP. Anti-GFP siRNA molecules were complexes with BGTC/DOPE or DC-Chol/DOPE or PEI. One day post-transfection, the inhibition of GFP expression was monitored by fluorescence GFP measurement.
1.2.2. Chemical Structure of Lipidic Aminoglycoside Derivatives.
Lipidic aminoglycoside derivatives are amphiphiles with a self-aggregating hydrocarbon tail linked to aminoglycoside headgroup.
1.2.3. Colloidal Stability of Cationic Liposomes/Nucleic Acid Complexes.
We investigated the physicochemical properties of complexes resulting from the association of DNA or siRNA with cationic liposomes of BGTC or lipidic aminoglycoside derivatives, as a function of the cationic lipid/nucleic acids charge ratio (
Next, we investigated on the same samples, the siRNA and DNA complexation by ethidium bromide fluorescence measurements. As a general trend, fluorescence intensity decreased as the cationic lipid/nucleic acids charge ratio increased. Fluorescence intensity decreased in zone A from 100% to a value close to zero and stayed at this minimum value in zones B and C. Although, complexation of DNA with either liposomes of BGTC or the various lipidic aminoglycoside derivatives led to similar decreasing of fluorescence intensity, differences were observed with siRNA molecules. The slope of the decreasing of the fluorescence intensity as a function of the charge ratio which varied significantly between lipidic aminoglycoside derivatives was steeper with DOSN and DOSP compared to that observed with DOSK and DOST. By contrast, a lag before the fluorescence intensity decreased was observed with siRNA complexes with BGTC. Agarose gel electropheresis of cationic lipid mixed with DNA and siRNA confirmed results obtained in fluorescence experiments (data not shown).
1.2.4. Cryo Transmission Electron Micrographs of Cationic Lipsomes/siRNA Complexes.
BGTC/DOPE vesicle preparation was made of unilamellar liposomes of a diameter comprised in a range of 30-70 nm (
With DOST-DOPE and DOSK-DOPE liposomes, small complexes in a range of 100-300 nm in size were induced in the presence of siRNA molecules (
With DOSP-DOPE and DOSN-DOPE liposomes, complexes in a range of 50-300 nm in size were formed after addition of siRNA molecules (
It is important to mention that when the liposomes made with the five different lipid composition exposed to 300 mM NaCl, the small liposomes remain unilamellar vesicle while the large one became double lipid bilayer liposomes due to an osmotic effect (6 and data not shown).
1.2.5. Gene Expression Inhibition Activities of Lipidic Aminoglycoside Derivatives/siRNA Complexes.
Next, we performed RNAi experiments with siRNA targeting the endogenous Lamin A/C expression. Here, RT-PCR results indicated that d2-GFP cells transfected with DOSP/siRNA complexes had very little residual Lamin A/C mRNA when compared with cells transfected with control siRNA (
Experiments were also performed using the aminoglycoside derivatives according to the invention for transfecting siRNA, without the use of a neutral lipid adjuvant such as DOPE.
The GFP-expressing human lung cancer H1299 cells were transfected with control siRNA or 3′-rhodamine labeled anti-GFP siRNA. The siRNA molecules were formulated in the absence (“naked” siRNA) or in the presence of DOSP, but in any case without DOPE. The transfected H1299 cells were observed using a FITC filter to visualize GFP fluorescence or a rhodamine filter to visualize siRNA internalization.
Results presented in
In addition, Lamin A/C silencing efficiency using DOSP reagent complexed with anti-Lamin A/C siRNA, but without a neutral lipid adjuvant, has also been tested by RT-PCR. Anti-LaminA/C siRNA amounts ranging from 3.75 to 200 ng/well were formulated with DOSP.
Results of real-time quantitative RT-PCR analysis of human Lamin A/C mRNA after transfection of HeLa cells displayed in
Finally, the efficiency of DOSP reagent was compared with that of commercial reagent HiPerFect for transfecting anti-LaminA/C siRNA. Anti-LaminA/C siRNA at 18.75 and 37.5 ng/well were formulated with DOSP or the commercial reagents HiPerFect.
Results of real-time quantitative RT-PCR analysis of human Lamin A/C mRNA after transfection of HeLa cells displayed in
1.2.6. Gene Expression Inhibition Activities of Lipidic Paromomycine Derivatives/siRNA Complexes in Comparison with Commercial Reagents.
1.2.7. Kinetics of GFP Expression Inhibition
Next, we investigated the influence of cationic liposomes/siRNA complexes on GFP expression as a function of time (
1.2.8. In vitro Transfection of Plasmid by Amino-Glycosides Derivatives and BGTC-DOPE Liposomes
We assessed the potential of aminoglycosides derivatives to deliver not only siRNA but also plasmid DNA encoding small hairping RNA against GFP into d2GFP cells (
We also assessed the potential of aminoglycosides to co-deliver a plasmid encoding KCNE1 and the siRNA targeted against KCNE1 mRNA.
1.3. Conclusions
The colloidal stability of lipidic aminoglycoside derivatives/siRNA complexes, which depended on the charge ratio, determined three main zones-A, B and C- which corresponded to negatively, neutrally and positively charged complexes respectively. This colloidal stability behavior was previously observed with complexes resulting from association of lipopolyamine micelles (5), bisguanidinium liposomes (7, and this study) or DOTAP liposomes with DNA molecules. However, while the zone B was similar with DNA complexed with the newly synthetized lipidic aminoglycoside derivatives, it was reduced upon complexation with siRNA.
In addition, the mean diameter of lipid aminoglocoside derivatives/siRNA complexes in the three zones was smaller than that measured with lipidic aminoglycoside derivatives/DNA complexes. In Zone C, the mean diameter was about 81, 105, 58 and 57 nm for complexes resulting from the association of siRNA with DOST, DOSK, DOSP and DOSN, respectively.
Among lipidic aminoglycoside derivatives, the boundary between zone A and B was shifted toward a decreased charge ratio when siRNA was complexed with 4,5-disubstituted 2-deoxystreptamine ring aminoglycoside compared to 4,6-disubstituted 2-deoxystreptamine ring aminoglycoside and BGTC.
In addition, although regular spacing about 6.7 nm were observed in all cases, the morphology of the complexes differed with the two different classes of aminoglycosides: complexes with 4,6-disubstituted 2-deoxystreptamine ring aminoglycoside and BGTC displayed classical “onion-like” structures made of a regular superimposition of lipid bilayer and siRNA molecules, while complexes with 4,5-disubstituted 2-deoxystreptamine ring aminoglycoside surprisingly formed distinct structures with grape of small condensed structures.
The difference in morphology of complexes with the two classes of aminoglycoside may not be fully explained at the present time. However, it might be related to the geometry of the lipidic derivatives, assuming that 2-deoxystreptamine 4,6 disubstituted and 2-deoxystreptamine 4,5 disubstituted could adopt a cylindrical and conical shape, respectively. This might impact the formation and the size of the cationic liposmes. It has been shown that concentric multilamellar organization over long distance was more easily formed with large initial liposomes able to disrupt and reassemble sandwiching DNA molecules. On the other hand, small liposomes are like micelles leading to ordered lamellar microdomains.
In any case, the shift in the boundary between zones A and B and the difference in complex structure between 4,5-disubstituted 2-deoxystreptamine ring aminoglycoside and 4,6-disubstituted 2-deoxystreptamine ring aminoglycoside was correlated with the higher efficiency of complexes with ,5-disubstituted 2-deoxystreptamine ring aminoglycoside to induce functional interference in mammalian cells. Indeed, highly effective complexes for gene silencing were obtained in zone C with paromomycine headgroup characterized by their small diameter (57 nm) and high colloidal stability.
It is known that cationic lipid/nucleic acid complexes are internalized into cells through an endocytosis process mediated by electrostatic interactions between positively charged complexes and the cell membrane. Endosomal escape is recognized as one of the main limiting steps of the currently cationic reagents. Although no certain explanation for the particularly high efficiency of these complexes is available, it is possible that complexes of lipidic 4,5-disubstituted 2-deoxystreptamine ring aminoglycoside derivatives/siRNA could improve RNA releasing in the cell cytoplasm because of the specific physicochemical properties of the aminoglycoside/siRNA self assemblies such as small size and the structural features and the increase flip-flop mechanism due to the flexibility of the aminoglycoside capable to match with the negative charge density of the leaflet membrane. The particular morphology obtained with the 4,5 disubstituted 2-deoxystrepatmine ring should led to more labile supramolecular structures compared to those obtained with the 4,6 disubstituted 2-deoxystrepatmine ring conferring in this condition a more facilitated release of siRNA which is a crucial step for their incorporation into RISC complexes and the subsequent gene expression inhibition.
The obtained results also indicate that the tranfecting compounds with 4,5-disubstituted 2-deoxystreptamine ring aminoglycoside are even more efficient than commercially available transfection systems.
Globally, the inventors have thus generated and characterized transfecting compounds specially adapted to siRNA transfection.
| Number | Date | Country | Kind |
|---|---|---|---|
| 60/849000 | Oct 2006 | US | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/EP07/60571 | 10/4/2007 | WO | 00 | 4/3/2009 |
