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WO2004007721A1 - Conjugue de liberation de sequences d'arn double brin et methodes associees - Google Patents

Conjugue de liberation de sequences d'arn double brin et methodes associees Download PDF

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WO2004007721A1
WO2004007721A1 PCT/IB2003/002828 IB0302828W WO2004007721A1 WO 2004007721 A1 WO2004007721 A1 WO 2004007721A1 IB 0302828 W IB0302828 W IB 0302828W WO 2004007721 A1 WO2004007721 A1 WO 2004007721A1
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double
conjugate
stranded rna
cells
sirna
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Roger Michael Eccles
Alexandra Muratovska
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University of Otago
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/111—General methods applicable to biologically active non-coding nucleic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/65—Peptidic linkers, binders or spacers, e.g. peptidic enzyme-labile linkers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering nucleic acids [NA]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/35—Nature of the modification
    • C12N2310/351—Conjugate
    • C12N2310/3513—Protein; Peptide
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2320/00—Applications; Uses
    • C12N2320/30—Special therapeutic applications
    • C12N2320/32—Special delivery means, e.g. tissue-specific

Definitions

  • This invention relates to a conjugate for releasing a double- stranded RNA sequence, more specifically a short interfering RNA sequence and methods thereof.
  • the purpose of delivering nucleic acids into cells is to either express transgenes, or to inhibit the expression of endogenous genes in cells.
  • One approach to inhibit gene expression makes use of antisense oligomers that are designed to be complementary to a particular mRNA transcript (Ho, P.T.C. and D.R. Parkinson (1997) Seminars in Oncology 24, 187-202). This permits the antisense oligomer to undergo Watson-Crick hybridization with its mRNA target leading to inactivation of the transcript, either through digestion by RNase H or through steric blocking of the ribosome complex (Ho, P.T.C. and D.R. Parkinson (1997) Seminars in Oncology 24, 187-202).
  • oligonucleotides including double-stranded RNA and DNA, are polar which makes their delivery to the cell and intracellular distribution difficult.
  • Many methods have been used to improve the uptake of nucleic acids by cells such as fluid phase pinocytosis; receptor-mediated endocytosis; liposomes (cationic lipid encapsulation); covalent conjugation to lipid moieties or to ligands of transmembrane receptors such as the folate and transferrin receptors (Ho, P.T.C. and D.R. Parkinson (1997) Seminars in Oncology 24, 187-202).
  • all of these procedures have associated difficulties that often cause the nucleic acids to be present in too low a concentration to give a biological effect.
  • RNA interference RNA interference
  • RNAi long double stranded RNA
  • dsRNA long double stranded RNA
  • mRNA sequences that are introduced into cells either experimentally or are derived from endogenous sources such as viruses, transgenes or cellular genes (transposons) (Tuschl, T. (2001) Chembiochem 2, 239-45).
  • dsRNA molecules are processed into discrete short interfering RNA (siRNA) fragments that are 21- to 25-nucleotides in length by an enzyme known as Dicer (Hammond, S.M., et al. (2001 ) Science 293 1146-50).
  • RNA-induced silencing complex comprised of nucleases, helicases and RNA-dependent polymerases to target specific mRNAs for destruction.
  • RISC RNA- induced silencing complex
  • siRNAs While long dsRNA sequences cause non-specific mRNA degradation in cells, siRNAs induce sequence-specific degradation of only targeted mRNAs (Hammond, S.M., et al. (2001 ) Science 293 1146-50). Tusch et al. ⁇ supra) have shown that 21-nucleotide duplexes corresponding to a luciferase transgene introduced in different cell types cause specific inhibition of the luciferase gene expression (Elbashir, S.M., et al., (2001 ) Nature 411 494-8). Large dsRNAs in turn cause complete arrest of gene expression within the cells masking any sequence-specific effects.
  • siRNAs are polyanions and unassisted permeation across lipid bilayers is negligible. Double stranded RNA can be delivered to C. elegans by feeding or soaking (Bosher, J.M. and M. Labouesse (2000) Nat Cell Biol. 2, E31-6), however in mammalian cells siRNAs are typically delivered using cationic liposomes (Elbashir, S.M., et al., (2001 ) Nature 411 494-8).
  • liposomes Although the use of liposomes has shown some success the major disadvantages of this delivery method for nucleic acids are that many cell types cannot be transfected at an efficiency that yields a significant biological effect, and in each experiment several manipulations of the cells are required.
  • the development of a vector used for direct targeting of polar molecules from the extracellular environment to the cytoplasm may significantly enhance the delivery of siRNAs into cultured and somatic cells.
  • a membrane permeant conjugate used as a delivery vector for siRNAs molecules to the cytoplasm of cells where they initiate RNAi and destroy specific mRNA molecules, resulting in gene silencing.
  • One aim of the present invention is to provide a conjugate for releasing a double-stranded RNA sequence, more specifically a short interfering RNA sequence (siRNA).
  • siRNA short interfering RNA sequence
  • Another aim of the present invention is to provide a method for releasing double-stranded RNA sequence, more specifically siRNA.
  • a further aim of the present invention is to provide a method for silencing a targeted gene.
  • a still further aim of the present invention is to provide a treatment to diseases by targeted gene silencing, specifically targeting and silencing the gene causing the disease.
  • Membrane permeant peptides are suitable candidates for the delivery of polar molecules to cells. These short amphipathic peptides have been shown to translocate across lipid bilayers in an energy independent manner somewhat similar to endocytosis (Hallbrink, M., et al., (2001 ) Biochim Biophys Ada 1515, 101-9).
  • these peptides can deliver large cargo molecules across membranes including nucleic acids, peptides, proteins and even paramagnetic particles up to 200 nm in diameter (Lindgren, M., et al., (2000) Trends in Pharmacological Sciences 21 , 99-103; Schwarze, S.R. and S.F. Dowdy (2000) Trends in Pharmacol Sci. 21 , 45-8; Josephson, L, et al., (1999) Bioconjug Chem 10, 186-91 ).
  • MPPs such as, but not limited to, penetratin and transportan.
  • One advantage of this method is that the delivery of siRNAs conjugated to MPPs into the cytoplasm of cells, where the bond is reduced by the glutathione pool, allows the siRNAs to specifically target mRNA degradation by RNAi. It is reported herein the synthesis of MPP-siRNA conjugate and the ability of these compounds to influence transiently and stably expressed transgenes in cells is demonstrated.
  • X is a double-stranded RNA sequence, preferably a short interfering RNA sequence (siRNA);
  • Z is a membrane permeant peptide
  • Y is a bifunctional linker attaching X and Z together.
  • the conjugate in accordance with a preferred embodiment of the present invention wherein the membrane permeant peptide is selected from, but not limited to, the group consisting of penetratin and transportan.
  • the conjugate in accordance with a preferred embodiment of the present invention wherein Y has a labile bond with X in the reducing cytoplasmic milieu, consisting of, but not limited to, a disulfide bond or a thioether bond.
  • a method for releasing a double-stranded RNA sequence, preferably a siRNA, into the cytoplasm of a cell comprising the step of administering to the cell an effective amount of the conjugate of the present invention to the cell, wherein the double-stranded RNA sequence is released from the conjugate into the cytoplasm.
  • a method for silencing a targeted gene comprising the step of administering to a cell an effective amount of the conjugate of the present invention to the cell, wherein the double-stranded RNA sequence, preferably a siRNA, is released from the conjugate into the cytoplasm causing the silencing of the targeted gene.
  • the double-stranded RNA sequence preferably a siRNA
  • a method for treating a disease related to a gene comprising the step of administering to a patient an effective amount of the conjugate of the present invention to the patient, wherein the double-stranded RNA sequence is released from the conjugate in the cytoplasm of targeted cells causing silencing of the gene ameliorating or treating the disease caused by the gene.
  • the administration is performed by a route selected from the group consisting of in vitro incubation with cells in cell culture media, attachment to particles and transfer to cells by particle bombardment, stereotactic injection into tissues, ex vivo treatment of bone marrow and re-transplantation; somatic or stem cell line treatment in vitro and autologous or xeno-transplantation; and blood infusion.
  • kits for linking double-stranded RNA molecules, preferably siRNA, with a membrane permeant peptide, comprising a bifunctional linker, a membrane permeant peptide and instructions to link the molecules comprising a bifunctional linker, a membrane permeant peptide and instructions to link the molecules.
  • a kit for ligating double-stranded RNA molecules, preferably siRNA with a membrane permeant peptide, comprising a bifunctional linker, a membrane permeant peptide and instructions to ligate the molecules.
  • the following terms are defined below.
  • cell is intended to mean an eukaryotic cell from any species.
  • targeted gene is intended to mean a gene for which silencing is desired.
  • disease is intended to mean a disease related to a gene wherein the silencing of that gene would provide a reduction or the elimination of the symptoms of that disease.
  • diseases are, without limitation, any type of cancer or tumor in humans or other species, dominant or recessive forms of polycystic kidney disease, autoimmune diseases, degenerative diseases of the central or peripheral nervous systems, and any other disease where the silencing of gene expression could be used as a treatment.
  • membrane permeant peptide is intended to mean peptides that can translocate across cell membranes. Some of these peptides are penetratin and transportan, without limitation.
  • RNA is intended to mean a short double stranded RNA, capable of initiating RNAi or other types of RNA metabolism, and ranging from 21-25 nucleotide in length.
  • Fig. 1 illustrates the synthesis of disulfide linked MPP (SEQ ID NOS: 5 and 6) and siRNAs (SEQ ID NOS: 1 and 2);
  • Fig. 2A illustrates the effects of siRNAs on luciferase expression in COS-7 cells, where COS-7 cells were co-transfected with siRNAs and reporter plasmids at the same time point
  • Fig. 2B illustrates the effects of siRNAs on luciferase expression in COS-7 cells, where COS-7 cells were treated with siRNAs after 24 h following the reporter plasmid transfection;
  • Fig. 3 illustrates the effects of siRNAs delivered in CHO cells by either liposomes or MPP;
  • Fig. 4A is a Double fluorescence staining of cells treated with siRNAs labeled with the red fluorophore Cy3 and green fluorescence from GFP after 7 days, illustrating the silencing of GFP in mouse fibroblast cell lines stably expressing green fluorescent protein
  • Fig. 4B is a Western blot of C166-GFP and EOMA-GFP cells transfected with GFP siRNA duplex using lipofectamine 2000TM or treated with penetratin-siRNAs, transportan-siRNAs or with media only, illustrating the silencing of GFP in mouse fibroblast cell lines stably expressing green fluorescent protein
  • Fig. 4C illustrates the fluorescence of C166-GFP and EOMA-
  • GFP cells treated with or without siRNAs measured after day 1 , day 3 and day 7 by flow cytometry and illustrating the silencing of GFP in mouse fibroblast cell lines stably expressing green fluorescent protein.
  • a method for targeting siRNAs to the cytoplasm of cells was developed using the membrane permeant peptides such as penetratin or transportan that deliver them across the plasma membrane.
  • thiol containing siRNAs complementary to part of a luciferase transgene were conjugated to penetratin or transportan via a bond, preferably a disulfide bond, that is labile in the reducing cytoplasmic milieu.
  • siRNAs were then released from penetratin in the cytoplasm where they specifically targeted luciferase mRNA degradation by RNAi.
  • pen-siRNA disulfide linked penetratin-siRNA
  • transportan-siRNA transportan-siRNA
  • SiRNAs complementary short interfering ribonucleotides
  • a "r" before a nucleotide means a ribonucleotide used to synthesize RNA.
  • a pair of GL2 siRNAs and GFP siRNAs were chemically modified with a thiol group at the 5' end of one RNA strand and a 5' Cy3 on the complementary strand.
  • the thiol group allowed the reaction of the siRNAs with thiol containing membrane permeant peptides.
  • the fluorescent tag on the siRNAs enabled visualization of the siRNA uptake within cells by fluorescent microscopy.
  • the products were precipitated with diethyl ether, dissolved in 50% acetonitrile, lyophilized and redissolved in water.
  • the crude peptides were then purified by RP-HPLC using a semi preparative column (VydacTM C ⁇ 8 , 300 A, 10 x 250 mm) and the peptide purity was monitored using an analytical column (Vydac C 4 , 300 A, 4.6 x 250 mm).
  • a linear gradient starting with 0.1 % TFA in water and finishing with 90% acetonitrile in water and 0.1 % TFA was run over 30 minutes.
  • the peptide peaks were detected by absorbance at 280 nm, collected, lyophilized and dissolved in water for further use.
  • annealing buffer 100 mM potassium acetate, 30 mM HEPES-KOH at pH 7.4, 2 mM magnesium acetate
  • Annealed siRNAs containing thiol groups were desalted by incubating the hybridization mix for 7 min on ice in a preset 1 % agarose in 100 mM glucose well in an eppendorf tube (by leaving a 100 ⁇ L tip in the molten agarose mix and allowing it to set).
  • the desalted siRNAs were supplemented with 1 vol.
  • reaction buffer (10 mM HEPES, 1 mM EDTA, pH 8.0) to adjust the final concentration of the siRNAs to 17.5 ⁇ M.
  • Equivalents amount of siRNAs, membrane permeant peptides (penetratin or transportan) and the thiol oxidant diamide (Sigma) were mixed and incubated for 1 h at 40°C.
  • the MPP-siRNA conjugates were either purified by HPLC, or introduced directly into cells. Cell culture and transfections
  • COS-7 cells C166-GFP and EOMA-GFP cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% inactivated foetal calf serum (FCS) and CHO-AA8-Luc Tet-Off cells were grown in Minimum Essential Medium (Alpha Modification) with 10% FCS. All cell culture media contained 100 units. mL "1 penicillin, and 100 ⁇ g.mL "1 streptomycin.
  • C166-GFP and EOMA-GFP cells were supplemented with 200 ⁇ g.mL-1 G418 and CHO-AA8-Luc Tet-Off cells with 100 ⁇ g.mL "1 G418 and 100 ⁇ g.mL "1 hygromycin B. Cells were regularly passaged to maintain exponential growth. Twenty-four hours before transfection at -80% confluency, the cells were trypsinised, diluted 1 :3 with fresh medium (1-5 x 10 5 cells. mL "1 ) and transferred to 24-well plates (300 ⁇ L per well).
  • Co-transfection of reporter plasmids and siRNAs in COS-7 cells was carried out with LipofectamineTM 2000 (Life Technologies) as described by the manufacturer for adherent cell lines.
  • the concentrations of the plasmids per well were 1.0 ⁇ g for pGL2 (Promega) and 0.1 ⁇ g for pCB11 (Rluc expressed by the SV40 promoter).
  • the siRNA were supplemented in all cell lines at 25 nM concentrations in the presence or absence of Lipofectamine 2000.
  • Penetratin-siRNAs and transportan-siRNAs were supplemented in 25 nM concentrations without Lipofectamine 2000.
  • COS-7 cells were also treated with siRNAs 24 h after the reporter plasmid transfection.
  • Luciferase expression in COS-7 cells was assayed using a Dual luciferase kit (Promega).
  • CHO-AA8-Luc Tet- Off cells were treated with the siRNAs for up to 3 days. Luciferase expression in these cells was assayed using a BrightGloTM luciferase kit (Promega).
  • C166-GFP and EOMA-GFP cells were treated with siRNAs for up to 7 days. Gene silencing was monitored by western blotting, flow cytometry and fluorescence microscopy. SDS PAGE and Immunobloting
  • Mouse anti-GFP monoclonal antibody (Roche) diluted 1 :5000 in TBS, 0.1% fat free milk powder, 0.1 % Tween- 20TM was used for immunodetection with overnight incubation. After 3x10 min washes in TBS, 0.1% Tween-20, horseradish peroxidase conjugated goat antimouse IgG (1 :10,000, Biorad) was used as a secondary antibody. The incubation with secondary antibody was for 1 h at room temperature, followed by 3 x 10 min washes in TBS and visualized by chemiluninescence using a Pierce Super Signal R chemiluminescence substrate with Kodak X-OMATTM AR imaging film.
  • C166-GFP and EOMA-GFP cells grown in 24-well plates were treated with GFP siRNAs delivered using Lipofectamine 2000, penetratin and transportan.
  • Control cells were treated with media only as described above. These treatments were carried out for 1 , 3 and 7 days after which the cells were prepared for flow cytometry. Following each incubation period the medium was collected from the wells (to collect any detached cells) and combined with PBS used to wash the wells twice. The cells were then harvested by trypsinisation and combined with the detached cells and PBS.
  • the cells were pelleted by centrifugation at 4°C (1000 g, 5 min), washed in PBS twice and resuspended in 500 ⁇ L binding buffer [10 mM Hepes / NaOH pH 7.4, 140 mM NaCI, 5 mM CaCI 2 ]. The cells were then analyzed using a Becton Dickinson fluorescence-activated cell sorter. The fluorescence was quantitated using the Cell-QuestTM software (Becton Dickinson, San Jose, CA).
  • C166-GFP and EOMA-GFP cells grown in 35 mm dishes were treated with GFP siRNAs delivered using Lipofectamine 2000, pemetratin and transportan. Control cells were treated with media only as described above. These treatments were carried out for 1 day, 3 days and 7 days. Photographs of the cells were acquired using a ZeissTM inverted confocal microscope with a 40/1.3 Plan-Neofluar 40x/1.3 oil DIC objective, line 488 nm and Zeiss Imaging Software with equal exposure times.
  • Short interfering RNAs 21 nucleotides in length were designed to be complementary to a region of the firefly luciferase transgene.
  • One set of the siRNAs was introduced in cells by liposomes and the other set of siRNAs was chemically modified.
  • One strand of these siRNAs duplex had a free thiol group at its 5' end so that it could react with a free thiol group from a cysteine amino acid on a membrane permeant peptide (MPP).
  • the complementary strand of the duplex had a Cy3 fluorophore that enabled the siRNA localization within cells by fluorescence microscopy.
  • RNA oligonucleotides were annealed to form a duplex and their thiol groups were then reacted with thiol containing MPP to generate MPP-siRNA conjugates. This reaction was catalyzed by the thiol oxidant diamide (Fig. 1 ).
  • transgenes for luciferase enzymes were transfected, a firefly luciferase that was targeted by the GL-2siRNAs and a Renilla luciferase that was introduced as a control for the specificity of siRNA inhibition and transfection efficiency.
  • siRNAs and MPP-siRNAs were introduced in the cells together with the liposomes during transfection of the transgenes.
  • the cells were only transfected with transgenes to determine the basal (exogenous) luciferase activity within the cells.
  • the delivery of siRNAs using penetratin gave a 5-fold decrease in luciferase activity (Fig. 2A).
  • the luciferase activity was decreased 18-fold in cells treated with siRNAs delivered using liposomes (Fig. 2A).
  • SiRNAs unassisted by penetratin or liposomes could not gain entrance to the cells and the luciferase activity remained the same as cells transfected only with the transgenes.
  • the activity of the Renilla luciferase remained constant in cells treated with or without the siRNAs, suggesting that the decrease of the firefly luciferase activity was due to the sequence-specific binding of the siRNAs to its mRNA.
  • the second experiment tested whether MPP-siRNAs could specifically decrease luciferase activity when introduced into the cells 24 hours after transfection with the luciferase transgene.
  • Penetratin-siRNA and transportan-siRNA conjugates were able to enter the cells and efficiently decrease luciferase activity. Similar levels of luciferase activity were observed with siRNAs delivered by liposomes and penetratin-siRNAs delivered in conjunction with liposomes.
  • SiRNAs were incubated with cells for three days and luciferase activity was measured every 24 h.
  • Liposome delivery siRNAs decreased luciferase activity by 36% 48 h after the treatment (Fig. 3). However, the basal luciferase activity returned to normal levels 24 h later.
  • Penetratin and transportan delivered siRNAs decreased the luciferase levels by 53% and 63% respectively, within the initial 24 h after treatment and this decrease remained stable for up to 3 days (Fig. 3). Unassisted delivery of siRNAs and mock treatment of cells with media did not alter the basal luciferase activity within the cells.
  • siRNAs can be targeted directly to the cytoplasm when conjugated to MPPs, such as penetratin and transportan. Nucleic acids delivered in this way are biologically active; siRNAs are capable of silencing the expression of either transfected or endogenous genes.
  • This delivery system has some advantages over conventional delivery methods: (i) the peptide facilitates transport across the plasma membrane and the siRNA-MPPs are freely translocated into the cytoplasm, (ii) the disulfide bond is reduced in the cytoplasm, releasing the bioactive siRNA to cause sequence specific mRNA degradation and (iii) the uptake of the conjugate is rapid and occurs directly through the membrane without the need for classical receptor-mediated uptake or endo- or pinocytosis.
  • the siRNA-MPP conjugates were not cytotoxic in micromolar concentrations and were sufficiently stable within the cell to efficiently induce RNAi.
  • the penetratin-siRNA conjugate When delivered in equimolar amounts the penetratin-siRNA conjugate was able to decrease luciferase activity in cells to the same level as the liposome delivered siRNAs. However this method measures the inhibition of mRNA of a transgene that is itself introduced in cells by liposomes. Silencing of an endogenously expressed luciferase gene in cells with the penetratin-siRNA conjugate was also successful. Liposome delivery is limited to a few cell types, has variable success, and in most transfections the nucleic acids are not taken up by all cells, whereas penetratin and transportan conjugates appeared to be able to penetrate approximately 95% of cells.
  • penetratin can translocate itself and attached cargo across lipid bilayers independent of cell type, as multiple cell types were used in this study.
  • the COS-7 cell line used in this study is widely recognized as one of the most easily transfected cell lines in general use today.
  • Other cell types are generally not as efficient in liposome-mediated transfection as COS7 cells, but MPP-linked siRNAs were efficiently taken up by all cell types used to date. Therefore MPPs such as penetratin and transportan are a powerful tool for effective delivery of siRNAs to cells that are not amenable to targeting with standard transfection protocols for specific inhibition of gene expression.

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Abstract

L'invention porte sur un conjugué de formule: X-Y-Z, dans laquelle: X est un ARN double brin, de préférence une séquence d'ARN court interférant (siRNA); Y est une liaison ou une molécule de liaison liant X à Y par covalence; et Z est un peptide membranaire perméant. L'invention porte également sur un procédé de libération d'ARN double brin dans le cytoplasme d'une cellule consistant à administrer à la cellule une dose efficace dudit conjugué pour provoquer la libération de l'ARN double brin du peptide membranaire perméant dans le cytoplasme.
PCT/IB2003/002828 2002-07-17 2003-07-17 Conjugue de liberation de sequences d'arn double brin et methodes associees Ceased WO2004007721A1 (fr)

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Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1657306A1 (fr) * 2004-11-16 2006-05-17 Qiagen GmbH Silençage genique médiée au moyen d'un hybride ADN sens et ARN anisens couplé à des peptides pour faciliter leur absorption par les cellules
WO2005117991A3 (fr) * 2004-05-04 2007-01-18 Nastech Pharm Co Compositions et procedes pour l'amelioration de l'administration d'acides nucleiques dans des cellules et pour la modification de l'expression de genes cibles dans des cellules
EP1984399A4 (fr) * 2006-02-10 2010-03-03 Univ California Livraison transductrice d'arnsi par liaison arn double brin de domaines de fusion au ptd/cpps
WO2010086597A1 (fr) * 2009-01-27 2010-08-05 Trojan Technologies Ltd. Introduction d'acides nucléiques à l'aide de peptides pénétrant dans les cellules
US8299236B2 (en) 2004-05-04 2012-10-30 Marina Biotech, Inc. Compositions and methods for enhancing delivery of nucleic acids into cells and for modifying expression of target genes in cells
US9260493B2 (en) 2009-05-07 2016-02-16 The Regents Of The University Of California Transducible delivery of nucleic acids using modified dsRNA binding domains
US9950001B2 (en) 2012-08-20 2018-04-24 The Regents Of The University Of California Polynucleotides having bioreversible groups
US11597744B2 (en) 2017-06-30 2023-03-07 Sirius Therapeutics, Inc. Chiral phosphoramidite auxiliaries and methods of their use
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EP1657306A1 (fr) * 2004-11-16 2006-05-17 Qiagen GmbH Silençage genique médiée au moyen d'un hybride ADN sens et ARN anisens couplé à des peptides pour faciliter leur absorption par les cellules
WO2006053683A3 (fr) * 2004-11-16 2006-08-03 Qiagen Gmbh Silençage de genes utilisant des constructions hybrides d'adn sens et d'arn antisens couplees a des peptides facilitant l'apport dans les cellules
EP1984399A4 (fr) * 2006-02-10 2010-03-03 Univ California Livraison transductrice d'arnsi par liaison arn double brin de domaines de fusion au ptd/cpps
US8273867B2 (en) 2006-02-10 2012-09-25 The Regents Of The University Of California Transducible delivery of siRNA by dsRNA binding domain fusions to PTD/CPPS
WO2010086597A1 (fr) * 2009-01-27 2010-08-05 Trojan Technologies Ltd. Introduction d'acides nucléiques à l'aide de peptides pénétrant dans les cellules
US9260493B2 (en) 2009-05-07 2016-02-16 The Regents Of The University Of California Transducible delivery of nucleic acids using modified dsRNA binding domains
US9950001B2 (en) 2012-08-20 2018-04-24 The Regents Of The University Of California Polynucleotides having bioreversible groups
US11981703B2 (en) 2016-08-17 2024-05-14 Sirius Therapeutics, Inc. Polynucleotide constructs
US11597744B2 (en) 2017-06-30 2023-03-07 Sirius Therapeutics, Inc. Chiral phosphoramidite auxiliaries and methods of their use
US12269839B2 (en) 2017-06-30 2025-04-08 Sirius Therapeutics, Inc. Chiral phosphoramidite auxiliaries and methods of their use

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