WO2004007721A1

WO2004007721A1 - Conjugate for double-stranded rna sequence releasing and methods thereof

Info

Publication number: WO2004007721A1
Application number: PCT/IB2003/002828
Authority: WO
Inventors: Roger Michael Eccles; Alexandra Muratovska
Original assignee: University of Otago
Current assignee: University of Otago
Priority date: 2002-07-17
Filing date: 2003-07-17
Publication date: 2004-01-22
Anticipated expiration: 2005-01-17
Also published as: AU2003281078A1

Abstract

The present invention relates to a conjugate having the following formula: X-Y-Z, wherein X is a double-stranded RNA, preferably short interfering RNA sequence (siRNA); Y is a bound or a binding molecule covalently linking X and Z together; and Z is a membrane permeant peptide. The present invention also relates to a method for releasing double-stranded RNA into 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 is released from the membrane permeant peptide into the cytoplasm.

Description

Conjugate for double-stranded RNA sequence releasing and methods thereof

BACKGROUND OF THE INVENTION (a) Field of the Invention

This invention relates to a conjugate for releasing a double- stranded RNA sequence, more specifically a short interfering RNA sequence and methods thereof.

(b) Description of Prior Art Genetic information now available from the human genome sequence may be exploited for the design of specific agents to modulate the function of genes or their protein products for the treatment of disease. A number of strategies have been used to manipulate gene expression such as gene therapy, ribozymes and DNA hypermethylating agents. However, despite these strategies the relatively inefficient delivery of genetic material into either somatic or cultured cells remains a major obstacle hindering progress in this field. Methods to deliver nucleic acids into cells presently include microinjection, transfection, electroporation, and particle bombardment. These techniques all have limitations generally with regard to efficiency of delivery and/or potential size-limit of the nucleic acid being transported.

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). The final consequence of this inactivation is the specific inhibition of synthesis of a particular protein product. Most 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). However, 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.

Recently, a naturally occurring process was discovered in fungi and plants that involves sequence-specific post-transcriptional silencing of gene expression (Fire, A. et al. (1998) Nature 391 , 806-11 ). Subsequently this process was shown to occur in bacterial and animal cells and the term RNA interference (RNAi) was coined for this phenomenon (Fire, A. et al. (1998) Nature 391 , 806-11 , Tuschl, T. (2001) Chembiochem 2, 239-45). The silencing "triggers" for the RNAi are long double stranded RNA (dsRNA) molecules complementary to 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). These 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). The siRNA are subsequently presented to a 500 kDA ribonuclear protein complex known as the RNA- induced silencing complex (RISC) comprised of nucleases, helicases and RNA-dependent polymerases to target specific mRNAs for destruction. The exact identity of the RISC proteins and its mechanism of action are under investigation, but the process of RNAi has spurred interest in the field of biotechnology.

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.

The efficient delivery of polar molecules such as siRNAs across lipid bilayers of cells presents something of a technological challenge because of their impermeance. 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). 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.

It would be highly desirable to be provided with 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.

SUMMARY OF THE INVENTION

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).

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 (MPPs) 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). In addition, 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 ). Here is provided a conjugate for directly targeting siRNAs to the cytoplasm of cells, with delivery across the plasma membrane using MPPs, such as, but not limited to, penetratin and transportan. Thiol containing siRNAs complementary to part of a luciferase transgene, or to part of a GFP transgene, were synthesized and conjugated to MPP via a bond that is labile in the reducing cytoplasmic milieu. 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.

In accordance with the present invention, there is provided a conjugate having the following formula: X-Y-Z wherein

X is a double-stranded RNA sequence, preferably a short interfering RNA sequence (siRNA);

Z is a membrane permeant peptide; and 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. In accordance with the present invention, there is provided 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.

In accordance with the present invention, there is provided 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.

In accordance with the present invention, there is provided 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 method in accordance with a preferred embodiment of the present invention, wherein 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.

In accordance with the present invention, there is provided a kit 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. ln accordance with the present invention, there is provided 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. For the purpose of the present invention the following terms are defined below.

The term "cell" is intended to mean an eukaryotic cell from any species.

The term "targeted gene" is intended to mean a gene for which silencing is desired.

The term "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. Such 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.

The term "membrane permeant peptide" is intended to mean peptides that can translocate across cell membranes. Some of these peptides are penetratin and transportan, without limitation.

The term "siRNA" 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. BRIEF DESCRIPTION OF THE DRAWINGS

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 2000™ 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; and 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.

DETAILED DESCRIPTION OF THE INVENTION In accordance with the present invention, there is provided a conjugate for releasing a short interfering RNA sequence and methods thereof.

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. To do this, 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. Once these penetratin-siRNA or transportan-siRNA conjugates had translocated across the lipid bilayer the disulfide bond was reduced by the cytoplasmic glutathione pool. The siRNAs were then released from penetratin in the cytoplasm where they specifically targeted luciferase mRNA degradation by RNAi. Herein it is reported the synthesis and characterization of the disulfide linked penetratin-siRNA (pen-siRNA) and transportan-siRNA, and their ability to decrease transgene expression in cells is demonstrated.

MATERIALS AND METHODS

Four sets of complementary short interfering ribonucleotides (siRNAs) were purchased from Dharmacon Research (Lafayette, CO). SiRNAs were designed to target the GL2 luciferase mRNA (Ace. NO. X65324), corresponding to the coding region 153-173 relative to the first nucleotide of the start codon (5'-rCrGrUrArCrGrCrGrGrArArUrArCrUrUrC rGrATT-3' (SEQ ID NO:1 ) and 5'-rUrCrGrArArGrUrArUrU rCCrGrCrGrUrArCrGTT-3' (SEQ ID NO:2) and the GFP mRNA (Ace. No. U55762), corresponding to the coding region 540-565 (5¹- rArCrUrArCrCrArGrCrArGrArArCrArCrCrCrCTT-3' (SEQ ID NO:3) and 5'- rGrGrGrGrUrGrUrU rCrUrGrCrUrGrGrUrArGrUTT (SEQ ID NO:4)). In the previous sequences, 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.

Solid phase peptide synthesis

Peptide synthesis for penetratin (KKWKMRRNQFWIKIQR (SEQ ID NO:5)) and transportan (GWTLNSAGYLLKINLKALAALAKKIL (SEQ ID NO:6)) was carried out on a 0.25 mmol scale (manual synthesis) starting from a Fmoc-lys WANG resin (substitution value 0.75 mmol/g resin). All Fmoc (9-fluorenylmethoxycarbonyl) amino acids in 4-fold excess were assembled using 2-(1-H-benzotriazol-1-yl)-1 ,1 ,3,3-tetramethyl-uronium (HBTU) and diisopropylethylamine (DIPEA) as coupling reagents. The Fmoc groups protecting the amine groups on amino acids were removed using 50% piperidine in dimethylformamide (DMF) (Sole N.A. and G. Baranay (1992) Journal of Organic Chemistry 57, 5399-5403). Both penetratin and transportan were synthesized with a cystein residue at the N terminus. Both peptides were cleaved from their resins by TFA and scavengers (TFA : H₂O : phenol : triisopropylsilane, 88 : 5 : 5 : 2) under nitrogen at 0°C for 1 h (Sole N.A. and G. Baranay (1992) Journal of Organic Chemistry 57, 5399-5403). 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 (Vydac™ Cι₈, 300 A, 10 x 250 mm) and the peptide purity was monitored using an analytical column (Vydac C₄, 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.

Synthesis

For annealing of siRNAs, 35 μM of single strands were incubated in annealing buffer (100 mM potassium acetate, 30 mM HEPES-KOH at pH 7.4, 2 mM magnesium acetate) for 1 min at 90°C followed by 1 h incubation at 37°C. 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 μL 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 μM. 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

Cells were grown at 37°C and 5% CO₂ in humidified atmosphere. 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 μg.mL^"1 streptomycin. In addition, C166-GFP and EOMA-GFP cells were supplemented with 200 μg.mL-1 G418 and CHO-AA8-Luc Tet-Off cells with 100 μg.mL^"1 G418 and 100 μg.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 10⁵ cells. mL^"1) and transferred to 24-well plates (300 μL per well). Co-transfection of reporter plasmids and siRNAs in COS-7 cells was carried out with Lipofectamine™ 2000 (Life Technologies) as described by the manufacturer for adherent cell lines. The concentrations of the plasmids per well were 1.0 μg for pGL2 (Promega) and 0.1 μg 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 BrightGlo™ 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

Cell samples were lysed in 20 μL loading buffer (50 mM Tris, 4% SDS, 12% glycerol, 2% 2-mercaptoethanol, 0.01 % eoomassie brilliant blue) and were separated on a 12.5% Tris-glycine gel. Gels were then either fixed and stained with eoomassie brilliant blue (0.1 % (w/v) eoomassie brilliant blue R-250, 45% (v/v) methanol and 10% (v/v) acetic acid], or electrotransfered onto 0.2 μm nitrocellulose (100 v, 1 h) in transfer buffer (25 mM Tris-HCI, pH 8.3, 192 mM glycine, 20% methanol) and then blocked with 2% (w/v) fat-free milk powder in TBS (5 mM Tris.HCI, pH 7.4, 20 mM NaCI), 0.1% Tween-20. Mouse anti-GFP monoclonal antibody (Roche) diluted 1 :5000 in TBS, 0.1% fat free milk powder, 0.1 % Tween- 20™ 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-OMAT™ AR imaging film.

Flow cytometry

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 μL binding buffer [10 mM Hepes / NaOH pH 7.4, 140 mM NaCI, 5 mM CaCI₂]. The cells were then analyzed using a Becton Dickinson fluorescence-activated cell sorter. The fluorescence was quantitated using the Cell-Quest™ software (Becton Dickinson, San Jose, CA).

Fluorescence microscopy

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 Zeiss™ 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.

RESULTS

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. The 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 ).

Two experiments were carried out to test the efficiency of delivery of the MPP-siRNA on its own, or MPP-siRNA in the presence of liposomes. In addition, experiments were carried out to test the delivery efficiency of siRNA that had no penetratin moiety conjugated to it, with and without liposomes. An assay was used to measure the delivery of the siRNAs, which relied on targeted degradation of the firefly luciferase mRNA. This was determined by assaying for luciferase activity in cell transfected with a transgene for the luciferase enzyme. Two 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.

In the first experiment the siRNAs and MPP-siRNAs were introduced in the cells together with the liposomes during transfection of the transgenes. In control experiments 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. Unmodified siRNAs did not decrease luciferase activity, as they could not enter the cells. These data indicate that penetratin and transportan are effective vectors for the delivery of siRNAs to the cytoplasm of cells. The translocation of penetratin leaves the plasma membrane structurally and functionally intact and once inside the cytoplasm penetratin is reduced from the siRNAs allowing them to induce sequence-specific mRNA degradation. The MPP-siRNA conjugate did not appear to be toxic to cells. The efficiency of the MPP conjugated sirRNAs at silencing endogenous gene expression in cells was then investigated in Chinese hamster ovary (CHO) cells that stably expressed luciferase. 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. These results lend support to the idea that conjugation of MPPs to siRNA is an effective delivery method for siRNA duplexes, which can efficiently silence gene expression in cells. Fluorescent microscopy showed that liposomes generally targeted ~50% of cells whereas penetratin and transportan targeted more than 90% of cells. This explain the higher luciferase activity in cells where the siRNAs were delivered using liposomes.

To further test the specificity and efficiency of the silencing effects of MPP-linked siRNAs we used two mouse fibroblast cell lines that stably expressed GFP. The selection was based on the availability of techniques able to quantitate the silencing of GFP. C166-GFP and EOMA- GFP cells were transfected with GFP siRNAs using liposomes or treated with penetratin-siRNAs and transportan-siRNAs. Silencing of GFP was monitored for one, three and seven days after the treatment, to allow turnover of the protein of the targeted mRNA. Fluorescence microscopy was used initially to show the general decrease in fluorescence of cells treated with siRNAs delivered by liposomes and MPP (Fig. 4A). The fluorescence of cells treated with media only was unchanged over seven days. The intensity of the cell fluorescence decreased dramatically with prolonged exposure to siRNAs. This was most evident after seven days of siRNA treatment where the fluorescence was at its lowest.

Western blots were then used to show qualitative decrease of GFP following treatment of C166-GFP and EOMA-GFP with siRNA delivered by liposomes, penetratin and transportan. Cells treated with media only showed abundant GFP in the absence of RNAi (Fig. 4B). The silencing of GFP expression was more pronounced in the EOMA-GFP cell line, where treatment with MPP-siRNAs resulted in -90% decrease in protein expression. In the C166-GFP cell line the amount of GFP was silenced, but only by -50%, presumably due to the presence of multiple GFP transgene in the stably transfected cell line.

As before C166-GFP and EOMA-GFP cells were treated with siRNAs delivered by liposomes and MPP and the delivery efficiencies were compared by quantifying GFP expression using flow cytometry. Consistent with the previous data, the liposome delivered siRNAs decreased the cell fluorescence by 65% after 7 days (Fig. 4C), penetratin and transportan siRNA delivery resulted in 73% and 80% decrease in cell fluorescence, respectively (Fig. 4C). These data further support the effective delivery of siRNAs using MPP, eliminating the need for transfections using liposomes that can be both labor intensive and target only about 50% of the cell population with highly variable outcomes.

DISCUSSION

Here is shown that 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. In addition, the siRNA-MPP conjugates were not cytotoxic in micromolar concentrations and were sufficiently stable within the cell to efficiently induce RNAi.

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. In addition, 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.

While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth, and as follows in the scope of the appended claims.

Claims

WHAT IS CLAIMED IS:

1. A conjugate having the following formula:

X-Y-Z wherein

X is a double-stranded RNA; Z is a membrane permeant peptide; and Y is a bifunctional linker attaching X and Z together.

2. The conjugate of claim 1 , wherein said double-stranded RNA is a short interfering RNA sequence (siRNA).

3. The conjugate of claim 1 , wherein said membrane permeant peptide is selected from the group consisting of penetratin and transportan.

4. The conjugate of claim 1 , wherein Y has a labile bond with X in reducing cytoplasmic milieu.

5. The conjugate of claim 1 , wherein Y is selected from the group consisting of a disulfide bond and a thioether bond.

6. A method for releasing double-stranded RNA into the cytoplasm of a cell comprising the step of administering to said cell an effective amount of the conjugate of claim 1 to said cell, wherein said siRNA is released from said conjugate into said cytoplasm.

7. The method of claim 6, wherein said double-stranded RNA is a siRNA.

8. The method of claim 6, wherein said membrane permeant peptide is selected from the group consisting of penetratin and transportan.

9. The method of claim 6, wherein Y has a labile bond with X in reducing cytoplasmic milieu.

10. The method of claim 6, wherein Y is selected from the group consisting of a disulfide bond and a thioether bond.

11. A method for silencing a targeted gene comprising the step of administering to a cell an effective amount of the conjugate of claim 1 to said cell, wherein said double-stranded RNA is released from said conjugate into the cytoplasm causing the silencing of said targeted gene.

12. The method of claim 11 , wherein said double-stranded RNA is a siRNA.

13. The method of claim 11 , wherein said membrane permeant peptide is selected from the group consisting of penetratin and transportan.

14. The method of claim 11 , wherein Y has a labile bond with X in reducing cytoplasmic milieu.

15. The method of claim 11 , wherein Y is selected from the group consisting of a disulfide bond and a thioether bond.

16. 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 claim 1 to said patient, wherein said double-stranded RNA is released from said conjugate in the cytoplasm of targeted cells causing silencing of said gene ameliorating or treating the disease caused by said gene.

17. The method of claim 16, wherein said double-stranded RNA is siRNA.

18. The method of claim 16, wherein said membrane permeant peptide is selected from the group consisting of penetratin and transportan.

19. The method of claim 16, wherein Y has a labile bond with X in reducing cytoplasmic milieu.

20. The method of claim 16, wherein Y is selected from the group consisting of a disulfide bond and a thioether bond.

21. The method of claim 16, wherein said disease is selected from the group consisting of cancer, tumor, dominant forms of polycystic kidney disease, recessive forms of polycystic kidney disease, autoimmune diseases, degenerative diseases of the central nervous systems and degenerative diseases of the peripheral nervous systems.

22. The method of any one of claims 6 to 21 , wherein said administering 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.

23. A kit for linking double-stranded RNA molecules with a membrane permeant peptide, comprising a bifunctional linker, a membrane permeant peptide and instructions to link said molecules.

24. The kit of claim 23, wherein said double-stranded RNA is siRNA.

25. A kit for ligating double-stranded RNA molecules with a membrane permeant peptide, comprising a bifunctional linker, a membrane permeant peptide and instructions to ligate said molecules.

26. The kit of claim 25, wherein said double-stranded RNA is siRNA.