WO2005051431A1 - Systeme d'administration colloidal pour agents therapeutiques biologiques - Google Patents

Systeme d'administration colloidal pour agents therapeutiques biologiques Download PDF

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Publication number
WO2005051431A1
WO2005051431A1 PCT/GB2004/004979 GB2004004979W WO2005051431A1 WO 2005051431 A1 WO2005051431 A1 WO 2005051431A1 GB 2004004979 W GB2004004979 W GB 2004004979W WO 2005051431 A1 WO2005051431 A1 WO 2005051431A1
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sirna
composition according
cell
cells
agarose
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PCT/GB2004/004979
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English (en)
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Anne Josephine Milner
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The University Of York
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Priority claimed from GB0327409A external-priority patent/GB0327409D0/en
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Publication of WO2005051431A1 publication Critical patent/WO2005051431A1/fr

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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
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
    • A61K48/0025—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid
    • A61K48/0041—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid the non-active part being polymeric
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1131—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against viruses
    • 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 N.A.
    • 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
    • C12N2320/00—Applications; Uses
    • C12N2320/30—Special therapeutic applications
    • C12N2320/32—Special delivery means, e.g. tissue-specific

Definitions

  • the invention relates to methods and compositions for delivery of molecules into cells.
  • the invention relates to compositions suitable for topical application to mammalian cells and tissues.
  • the first aspect has been addressed by both cell-specific and non-cell-specific approaches.
  • major delivery routes include oral, gastro-intestinal, intravenous, and topical.
  • the mode of delivery will depend upon the location, distribution and accessibility of the target tissue (s); drug toxicity; biochemical and biophysical properties of the therapeutic agent; drug formulation etc.
  • solutions to the second aspect of delivery that of enabling molecules to enter cells, include use of viral vectors such as engineered retroviruses and adenoviruses; use of liposomes to incorporate the molecule and aid its transit across the lipid bilayer of the plasma membrane; encapsulation in or conjugation to organic polymers such as dendrimers; or conjugation to delivery peptides which can pass across the plasma membrane, such as Antennapedia or the HIV TAT protein.
  • viral vectors such as engineered retroviruses and adenoviruses
  • liposomes to incorporate the molecule and aid its transit across the lipid bilayer of the plasma membrane
  • encapsulation in or conjugation to organic polymers such as dendrimers
  • conjugation to delivery peptides which can pass across the plasma membrane, such as Antennapedia or the HIV TAT protein.
  • viruses can only be used to deliver nucleic acids. It can sometimes be hard to combine cell- specific delivery methods with methods for efficiently introducing the molecule into a cell .
  • nucleic acid or proteins encased in simple liposomes cannot easily be targeted to specific cell types.
  • RNA interference RNA interference
  • RNAi RNA interference
  • RNA constructs which either assemble, or are processed, to give double-stranded siRNAs of pre-determined nucleotide sequence within cells. This approach depends upon DNA therapy to introduce the siRNA expression vectors into target cells, and carries risks associated with gene therapy in general.
  • the second approach avoids gene therapy and instead relies upon intravenous siRNA delivery.
  • High pressure injection is used to introduce siRNA into experimental animals (see Lewis et al., 2002).
  • a major problem encountered by intravenous administrat ⁇ on of siRNA is the abundance of systemic nucleases which destroy RNA. Nuclease resistance may be achieved by chemical modification of the siRNA but risks the introduction of non-specific side-effects.
  • the invention provides new methods for delivering molecules into cells.
  • the invention also provides compositions suitable for use in such methods, in particular new compositions suitable for topical application to the cells.
  • the methods may be used for delivery of, in particular, one or more siRNA(s) or other dsRNA(s), shRNAs or DNA-based. molecules encoding antisense RNAs and/or siRNAs, or other nucleic acid molecules including modified oligonucleotides .
  • the invention provides a composition
  • a composition comprising a molecule or other agent for delivery into a cell, a transfer agent to aid the entry of the molecule into the cell, and a solid or semi-solid carrier medium.
  • a semi-solid carrier medium may be, for example, a gel, sol or other colloid, or a solid matrix in a liquid support.
  • the agent for delivery may be a protein, a nucleic acid, a peptide, a drug or other therapeutic agent.
  • the composition is a composition for delivering an agent for inducing RNA interference into a cell, comprising said agent, a transfer agent and a solid or colloidal carrier medium.
  • the carrier medium may be a gel such as soft agar or agarose.
  • the carrier medium may be selected to be particularly compatible with the cells to which the molecule is to be delivered.
  • the carrier medium may be designed so that cells with certain growth characteristics can invade and grow into it, increasing the cell surface area in contact with the carrier and enhancing delivery of the molecule via its transfer agent to the cells.
  • Such cells may have the ability to solubilise, either partially or completely, the carrier medium thus enhancing delivery of the transfer agent and molecule to the cell.
  • the transfer agent may be, for example, a virus, a virus-like particle, a liposome, an organic polymer such as a dendrimer or a polylysine-transferrine-conjugate.
  • the transfer agent is a liposome-type vehicle.
  • the invention provides a method for delivering a molecule into a cell comprising contacting the cell with a composition as described herein.
  • the cell may be a cell in in vitro culture or a cell in situ in a living organism.
  • the cell may be an animal cell, or a mammalian cell.
  • the cell is a human cell.
  • the cell is a tumour cell.
  • the tumour may be, for example, a carcinoma, in particular a carcinoma of the cutaneous, squamous or cervical epithelia or an adenocarcinoma of the cervix, or a colorectal carcinoma cell.
  • the cell may be a HPV-infected cell.
  • the molecule for introduction into the cell is a siRNA, a dsRNA which may be processed into siRNA within the cell, or a nucleic acid encoding such RNA.
  • siRNA are double-stranded RNA species mediating RNA interference (RNAi) .
  • RNAi is the process of sequence-specific, post- transcriptional gene silencing in animals and plants, initiated by double-stranded RNA (dsRNA) that is homologous in sequence to the silenced gene.
  • the siRNA may be for silencing of (i.e. homologous to) a cellular gene, for example an oncogenic gene, or an exogenous gene, for example a parasitic or viral gene.
  • the gene is a gene of viral or cellular origin that promotes cellular survival, such as bcl-2.
  • the virus is preferably HPV (human papilloma virus) .
  • the gene may be, for example, HPV E6 or HPV E7.
  • Examples of siRNA sequences that may be used for silencing HPV E ⁇ and E7 and Bcl-2 are given in the sequence listing. Methods relating to the use of RNAi to silence HPV genes are described in the inventor' s previously published patent application WO 03/008573.
  • the invention provides a composition comprising an siRNA construct with a nucleotide sequence homologous to an HPV gene or portion thereof in combination with a transfer agent and a carrier medium.
  • a transfer agent is E6 or E7
  • the transfer agent is a liposome vehicle
  • the carrier medium is agarose .
  • the invention also provides provides a composition comprising an siRNA construct with a nucleotide sequence homologous to the bcl-2 gene or portion thereof in combination with a transfer agent and a carrier medium.
  • the invention also provides a method for use of a composition as described above in the preparation of a medicament for the treatment of disease, in particular cancer.
  • the cancer may be, for example, a carcinoma, in particular a carcinoma of the cutaneous, squamous or cervical epithelia, or a colorectal carcinoma.
  • the cancer may be a cancer caused by HPV infection, such as cervical cancer, labial cancer, penile cancer or squamous cell carcinoma. Other diseases caused by HPV may also be treated, including genital warts and verruca vulgaris .
  • the invention also provides a method of treatment of cancer comprising contacting the cancer cells with a composition as described herein.
  • treatment it is meant any degree of alleviation of the disease including a suppression in the rate of growth of the tumour.
  • the composition may comprise a siRNA construct with a nucleotide sequence homologous to one or more HPV genes in combination with a transfer agent and a carrier medium.
  • the HPV gene is E ⁇ and/or E7
  • the transfer agent is a liposome vehicle
  • the carrier medium is agarose
  • the cancer cervical cancer.
  • the composition comprises a siRNA construct with a nucleotide sequence homologous to a bcl-2 gene in combination with a transfer agent and a carrier medium.
  • the transfer agent is a liposome vehicle
  • the carrier medium is agarose
  • the cancer is a colorectal carcinoma.
  • the invention also provides a method for use of a composition comprising a siRNA construct with a nucleotide sequence homologous to one or more HPV genes or to a bcl-2 gene in combination with a transfer agent and a carrier medium in the preparation of a medicament for the treatment of cance .
  • Figure 1 shows detection of apoptotic cells, harvested from aqueous medium and analysed by FACS with annexin V staining (Jiang & Milner, 2003) .
  • FIG. 2 shows detection of apoptotic cells, harvested from aqueous medium and analysed by FACS with annexin V staining (Jiang & Milner, 2003).
  • Bcl2-2 refers to Bcl-2 (b) siRNA;
  • Bcl2-1 refers to Bcl-2 (a) siRNA and
  • a/s Bcl2-2 refers to antisense Bcl- 2(b) siRNA.
  • a transfer agent is any agent which can effect or aid the entry of a molecule such as a dsRNA into a cell.
  • a virus may be used as a transfer agent to introduce a desired nucleic acid molecule into a cell.
  • the transfer agent employed in a composition of the invention is a liposome. Liposomes are microscopic spherical vesicles that form when cationic lipids are mixed in aqueous medium. The lipids arrange themselves in sheets to form a bilayer membrane which encloses some of the aqueous medium in a lipid sphere.
  • the molecules for delivery into a cell are added to the aqueous medium in which the lipids are suspended, the molecules will be trapped in the aqueous centre when the liposomes form, or may associate with the lipid bilayer.
  • the lipid bilayer can merge with the lipid bilayer of a cell's plasma membrane, releasing the contents of the liposome into the cell.
  • the liposome-nucleic acid complex is prepared by sonicating the lipid mixture to be used and then mixing the sonicated mixture with the nucleic acid in an appropriate nucleic acid: lipid ratio (for example 5:3) in a physiologically acceptable diluent (for example Opti-MEMTM at 9-fold dilution) immediately prior to use.
  • the lipid carriers can be prepared from a variety of cationic lipids, including DOTAP, DOTMA, DDAB, L-PE, and the like.
  • Lipid carrier mixtures containing a cationic lipid, such as N- [1- (2, 3-dioleyloxy) propyl] -N,N,N-triethylammonium chloride (DOTMA) also known as
  • lipofectin dimethyl dioctadecyl ammonium bromide (DDAB), 1,2— dioleoyloxy-3- (trimethylammonio) propane (DOTAP) or L-lysinyl- phosphatidylethanolamine (L-PE) and a second lipid, such as dioleoylphosphatidylethanolamine (DOPE) or cholesterol (Choi) , are particularly useful for use with nucleic acids.
  • DOTMA synthesis is described in Feigner, et al . , (1987) Proc. Nat. Acad. Sciences, (USA) 84:7413-7417.
  • DOTAP synthesis is described in Stamatatos, et al., Biochemistry, (1988) 27:3917-3925.
  • Liposomes are commercially available from many sources.
  • DOPE lipid carriers can be purchased from, for example, BRL.
  • DOTAP DOPE lipid carriers can be purchased from Boehringerr Mannheim. Cholesterol and DDAB are commercially available from Sigma Corporation. DOPE is commercially available from Avanti Polar Lipids. DDAB: DOPE can be purchased from Promega. Invitrogen make liposomes under the names oligofectamineTM and lipofectamineTM .
  • liposomes In formulating with liposomes, procedures and raw materials must be considered carefully to avoid adverse effects on liposome stability.
  • liposomes In general, liposomes should be added to a formulation below 40°C using low shear mixing. Ethanol concentration should be kept below 5%, and solvents should be kept below 10%.
  • Surfactants in general should also be avoided, but low levels (up to 1%) of non-ionic high HLB surfactants are usually well tolerated. High levels of salts (>0.5%) should be avoided.
  • the recommended storage temperature of most liposome formulations is 25°C. (See New, R.R.C., Liposomes - a practical approach, IRL Press, (1990)).
  • Liposomes may designed for delivery to particular cells, tissues or organs. For example, they may incorporate an antibody which recognises a particular cell antigen, or a ligand bound by a cellular receptor.
  • Ligands may be, for example, peptides, polypeptides, lectins such as concanavalin A or sugars such as mannose, or other carbohydrates.
  • the liposome may also comprise other molecular agents for interaction with cell membranes and/or other accessible biological membranes.
  • Liposomes may also be designed for delivery of contents to the cytoplasmic or nuclear compartments of the cell through attachment of specific signal sequences such as nuclear localisation signals.
  • VLPs virus-like particles
  • HPV VLPs comprising the LI and/or L2 HPV viral protein
  • hepatitis B viral proteins e.g., viruses and virus-like particles
  • suitable VLPs may be derived from picornaviruses; togaviruses; rhabdoviruses; orthomyxoviruses; retroviruses; hepadnaviruses; papovaviruses; adenoviruses; herpesviruses; and pox viruses.
  • Delivery may employ conjugation to delivery peptides which can pass across the plasma membrane, such as Antennapedia or the HIV TAT protein.
  • the carrier medium may be a solid or a colloid.
  • a colloid is a mixture in which one substance is dispersed throughout a second substance as minute particles called colloidal particles.
  • the colloid comprises a solid phase and a liquid phase.
  • colloids include gels and liquid sols.
  • the carrier medium may exist in either a gel or a sol form, depending on external factors such as temperature and ion concentration.
  • the transition from sol form to gel form is known as gelation, and the transition from gel form to sol form is known as solation.
  • the gel/sol transition may be reversible or irreversible, depending upon the composition of the gel. For example, certain biological gels are formed by cross-linking between molecules such as polymers and undergo gelation as the degree of polymerization increases. Where the polymerization is reversible, the gel may be induced to undergo solation to form a sol.
  • the carrier medium may be simple or complex and may be formulated from natural or synthetic material. Natural materials suitable for use as carrier media include agarose and starch or cellulose derivatives. Synthetic materials suitable for use as carrier media include methacrylate derivatives or polyethylene glycol . In some embodiments, the carrier medium may comprise both natural and synthetic materials.
  • the carrier medium may include agents designed to facilitate delivery of the agent for delivery.
  • the carrier medium is biocompatible, i.e. it is not toxic to cells or tissues.
  • the carrier medium should be mucoadhesive.
  • mucoadhesive is used to define any formulation that adheres to a mucosal surface lining a body cavity or surface including the lumenal surface of the gastro-intestinal epithelium, of the colorectal epithelium and of the cervix.
  • Mucoadhesive polymers have the ability to adhere to humid or to wet mucosal tissue surfaces such as those of the colorectal epithelium or of the cervical epithelium.
  • the epithelial layer is a coherent epithelial cell sheet formed from one or more layers of cells and covering an external surface or lining a body cavity.
  • Mucus forms a protective barrier overlying the epithelial cells.
  • the mucus may form a gel or a sol, or a combination of both as observed in the airways of the respiratory tract where the mucous consists of a gel phase floating upon a sol or liquid layer.
  • the gel layer lining the airways is secreted from goblet cells and from mucus glands and floats upon the sol layer which in turn surrounds the cilia of the epithelial cells.
  • Ciliary movement operates to push the mucus up the respiratory tree; in cystic fibrosis abnormal mucous is associated with ciliary diskinesia, recurrent infections and bronchiectasis .
  • Mucosal epithelial sites are rich in a viscous secretion, mucus, which coats the epithelial cell layer and protects in from mechanical, bacterial, viral, particulate, and chemical attack.
  • Mucus consists of water, up to 95% by weight, and glycoproteins (0.5% - 5%), lipids, mineral salts (1%) and 0.5 - 1% free proteins (Woolfson et al . , 2000). The rheological and cohesive and adhesive properties of mucus are largely due to the glycoprotein component.
  • Bioadhesion is the molecular force which resists separation across the interface between a biological surface and a carrier, usually polymeric in nature.
  • Mucoadhesive polymers of natural or synthetic macromolecules, are often well accepted and used as pharmaceutical excipients for other purposes (Woolfson et al . , 2000) .
  • the carrier medium will contain a polymer to provide the mucoadhesive benefit.
  • a carrier, coating, or matrix can be, for example, a gel, such as a hydrogel, organogel or thermoreversible gel. Other useful polymer types include, but are not limited to, thermoplastics and films. Moreover, the carrier, coating, or matrix can compromise a homopolymer, copolymer or a blend of these polymer types.
  • the carrier, coating, or matrix can also include an RNAi agent-loaded microparticle dispersed within a component of the carrier, coating, or matrix, which serves as a dispersant for the microparticles .
  • Microparticles include, for example, microspheres, microcapsules and liposomes.
  • the polymer may be, for example, poly (ethylene oxide), poly (ethylene glycol) , poly (vinyl alcohol), poly (vinyl pyrrolidine) , poly (acrylic acid), poly(hydroxy ethyl methacrylate) , hydroxyethyl ethyl cellulose, hydroxy ethyl cellulose and chitosan and mixtures thereof.
  • the polymer is carboxymethylcellulose.
  • the concentration of carboxymethylcellulose in the carrier medium is preferably 0.1% to 5%, more preferably about 0.1% to about 2.5% by weight.
  • the carboxymethylcellulose is preferably in the form of sodium carboxymethylcellulose substituted to a degree that the sodium content of the sodium carboxymethylcellulose is about 1% to about 20%.
  • the carrier medium may contain other materials which provide a mucoadhesive effect.
  • materials include titanium dioxide, silicon dioxide, and clays. When formulated in colloidal dispersions, these materials are able to interact with glycoprotein, especially mucin, transforming into a viscous gel, to become effective mucoadhesive systems.
  • Useful biocompatible colloidal carrier media include polysaccharide complexes which polymerise to form gel-like matrices, such as agar.
  • Agar is a complex polysaccharide extracted from seaweeds which forms a matrix when dissolved in water or buffer, heated and allowed to set. Typical concentrations of agar solutions which set to form a soft gel are 0.5-10%.
  • the carrier, coating, or matrix can serve to immobilize the microparticles at a particular site, enhancing targeted delivery of the encapsulated RNAi agents.
  • Rapidly bioerodible polymers such as polylactide-co-glycolide, polyanhydrides, and polyorthoesters, whose carboxylic groups are exposed on the surface are useful in the coatings of use in the invention.
  • polymers containing labile bonds, such as polyesters are well known for their hydrolytic reactivity.
  • the hydrolytic degradation rates of the carrier, coating, or matrix can generally be altered by simple changes in the polymer backbone.
  • a preferred carrier medium is agarose, a highly purified form of agar. Agarose forms a gel and gelation depends upon temperature and concentration. Agarose solutions exhibit hysteresis in the liquid-gel transition, i.e. their gel point is not the same as their melting temperature.
  • Agarose gel may be prepared by dissolving powdered agarose in water or buffer such as phosphate-buffered saline) boiling and allowing to cool.
  • the agarose molecules form a matrix with pores between them. The more concentrated the agarose, the smaller the pores and the more solid the resulting gel.
  • the agarose solution should be allowed to cool to 40 °C before adding the transfer agent-siRNA.
  • the percentage agarose in the agarose solution may be, for example, 1% or less, 2%, 3%, 4% , 5% or more.
  • the final concentration of agarose in the siRNA-transfer agent-agarose composition may be 0.1% or less, 0.2%, 0.3%, 0.4%, 0.5% or more. The whole is sterile.
  • the stock agarose solution is 3%, it can be diluted to final concentration using serum-free media Opti-MEM (Invitrogen) .
  • the final concentration of the agarose aims to hold the solid phase, has no effect on cell growth, and allows the cells take up the siRNAs.
  • the optimal final concentration may vary according to different type of agarose.
  • the siRNA-transfer agent-agarose solution is applied to the cells while still liquid, and allowed to set after applying.
  • agarose powder (Sigma, type VII) is weighed out and placed in a flask. 50 ml of water or buffer (e.g. phosphate- buffered saline) is added and swirled to mix the solution. The flask containing the solution is then autoclaved in order to sterilise. The 0.1% sterile agarose gel preparation can be stored at room temperature, and melted in a water bath at 65°C or above before using. The solution is allowed to cool before incorporating the siRNA-transfer agent.
  • buffer e.g. phosphate- buffered saline
  • the carrier medium may be applied directly, for example as a gel, or it may be comprised within a pre-assembled patch structure or other device which enables apposition of the gel with the cells to be targeted.
  • a patch structure comprising a bioadhesive layer or mucoadhesive layer attached to a backing layer of suitable pliability to conform with the tissue architecture of the surface of the cervix could be employed for therapeutic delivery of drug to cervical tissues in situ.
  • the physical properties of the patch should ideally be retained over several hours to one or more days without discomfort to the patient and without displacement of the patch during normal locomotion and other body movements.
  • the bioadhesive or mucoadhesive layer will comprise a composition of the invention.
  • the backing layer may comprise, for example, poly (vinyl chloride) or hydroxypropylcellulose .
  • a bioadhesive cervical patch containing 5- fluorouracil for the treatment of cervical intraepithelial neoplasia has been successfully applied (Woolfson et al., J. Cont. Rel. 35: 49-58; 1995).
  • the patch was bilaminar in design with a drug-loaded bioadhesive film cast from a gel containg 2% w/w Carbopol 981 plasticized with 1% w/w glycerin.
  • the film was bonded directly onto a backing layer formed from thermally cured poly (vinyl chloride) emulsion.
  • the agent for delivery may be a protein, nucleic acid or other macromolecule .
  • the agent for delivery is an inducer of RNA interference or other inducer of gene silencing.
  • An inducer of RNA interference may be a siRNA, a shRNA, a longer double-stranded RNA or a DNA construct for expression of siRNA or longer RNA sequences.
  • Other inducers of gene silencing include inducers of DNA methylation, or ribozymes, or aptamers .
  • the agent may be a modulator of gene expression such as a DNA-related, RNA-related, LNA-related or PNA-related molecule.
  • PNA Peptide Nucleic Acid
  • LNA locked nucleic acid
  • LNA is a bicyclic nucleic acid where a ribonucleoside is linked between the 2 ' -oxygen and the 4 '-carbon atoms with a methylene unit. Oligonucleotides containing LNA nucleotides are very stable when bound to complementary DNA and RNA. They also have improved mismatch discrimination. The high binding affinity of LNA oligonucleotides allows for the use of short probes in e.g. SNP genotyping.
  • the agent may be a hydrophilic molecule or compound or protein derivative such as an antibody or Fab fragment, which otherwise are difficult to transport across the membrane barrier of the cell into the hydrophilic cytoplasm.
  • the agent for delivery may be a protein or polypeptide.
  • the protein or polypeptide is a therapeutic protein, such as insulin or Factor VIII.
  • Therapeutic proteins include antibodies and proteins which bind to cellular receptors such as Fab fragments, and the p53 and mdm2 proteins .
  • the agent for delivery may be a therapeutic peptide or peptide mimetic.
  • a therapeutic peptide is preferably between 5 and 100 amino acids in length, preferably less tan 50 amino acids in length.
  • a therapeutic peptide may, for example, bind to a cellular receptor, or may be a fragment of a cellular protein which retains an activity of the full length protein such as the transactivation domain of p53 or the DNA-binding domain of p53.
  • RNA interference is a process whereby the introduction of double stranded RNA (dsRNA) into a cell inhibits gene expression post-translationally, in a sequence dependent fashion. This process is also known as post-transcriptional gene silencing. Current models of RNAi indicate that it is mediated by short
  • siRNAs small interfering RNAs' (siRNA) . It appears that dsRNA is cleaved in the cell to create siRNAs. siRNAs are then incorporated into an RNA-induced silencing complex (RISC) , guiding the complex to the homologous endogenous mRNA. The activated RISC then cleaves the mRNA transcript, resulting in the destruction of the mRNA in a cell which is homologous to the siRNAs. The siRNAs are re-cycled. In this way, a relatively small number of siRNAs can selectively destroy a large excess of cellular mRNA.
  • RISC RNA-induced silencing complex
  • dsRNA may be introduced into the cell as an isolated nucleic acid fragment or via a transgene, plasmid or virus.
  • siRNA may be synthesised and introduced directly into the cell.
  • shRNA short hairpin RNA molecule
  • a shRNA consists of short inverted repeats separated by a small loop sequence. One inverted repeat is complimentary to the gene target.
  • the shRNA is then processed into an siRNA which degrades the target gene mRNA and suppresses expression.
  • shRNAs can produced within a cell by transfecting the cell with a DNA construct encoding the shRNA sequence under control of a RNA polymerase III promoter, such as the human Hi or 7SK promoter.
  • the shRNA may be synthesised exogenously and introduced directly into the cell.
  • the shRNA sequence is between 40 and 100 bases in length, more preferably between 40 and 70 bases in length.
  • the stem of the hairpin is preferably between 19 and 30 base pairs in length. The stem may contain G-U pairings to stabilise the hairpin structure.
  • siRNA sequences are selected on the basis of their homology to the gene it is desired to silence. Homology between two nucleotide sequences may be determined using a variety of programs including the BLAST program, of Altschul et al . (1990) J. Mol . Biol . 215: 403-10, or BestFit, which is part of the Wisconsin Package, Version 8, September 1994, (Genetics Computer Group, 575 Science Drive, Madison, Wisconsin, USA, Wisconsin 53711) . Sequence comparisons may be made using FASTA and FASTP (see Pearson & Lipman, 1988. Methods in Enzymology 183: 63-98).
  • Parameters are preferably set, using the default matrix, as follows: Gapopen (penalty for the first residue in a gap): -16 for nucleic acid; Gapext (penalty for additional residues in a gap) : -4 for nucleic acids; KTUP word length: 6 for nucleic acids .
  • Sequence comparison may be made over the full length of the relevant sequence, or may more preferably be over a contiguous sequence of about or 10, 15, 20, 25 or 30 bases.
  • the degree of homology between the siRNA and the target gene is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99%.
  • the degree of homology between the siRNA or dsRNA and the gene to be silenced will preferably be sufficient that the siRNA or dsRNA will hybridise to the nucleic acid of the gene sequence under stringent hybridisation conditions.
  • a sequence stated herein to be 'homologous' to a given sequence or portion thereof will be 100% homologous thereto, i.e. it will have exactly the same nucleic acid sequence.
  • Hybridisation temperature will vary depending on the GC content of the nucleic acid target sequence but will typically be between 42°C-65°C.
  • T m 81.5°C+l ⁇ .6Log[Na + ]+0.41[%G+C]-0.63 (% formamide) .
  • the siRNA may be between lObp and 30bp in length, preferably between 20bp and 25bp. Preferably, the siRNA is 20, 21 or 22bp in length.
  • the siRNA sequence may be any suitable contiguous sequence of 10-30bp from any one of the HPVl ⁇ or HPV18 E ⁇ or E7 gene sequences shown SEQ ID Nos: 15 to 18.
  • longer dsRNA fragments comprising contiguous sequences from these sequences may be used, as they will be cleaved to form siRNAs within the cell.
  • the siRNA sequences are those shown as SEQ ID Nos: 1 and 2 (for the E6 gene) or SEQ ID Nos: 3 and 4 (for the E7 gene) .
  • the siRNA sequence is preferably any suitable contiguous sequence of 10-
  • siRNA sequence is the sequence shown as SEQ ID Nos: 5 and 6.
  • a 'suitable' siRNA sequence is a sequence which can be shown to induce RNAi in cells.
  • the occurrence of RNAi can be detected by transfecting cultured cells with the siRNA, followed by RT-PCR of the mRNA of interest.
  • RNAi is induced by the siRNA
  • levels of the mRNA of interest will be reduced in transfected cells as compared to control cells.
  • a reduction in protein production can be confirmed by Western blotting of cell lysates followed by probing with an antibody reactive to the protein of interest .
  • the siRNA has the E6, E7 or Bcl-2 (b) sequences as follows:
  • siRNA TTUCCUCCUACUUUAUCUACC 5' (SEQ ID NO : 4 )
  • the siRNA has an overhang at one or both ends of one or more deoxythymidine bases.
  • the overhang is not to be interpreted as part of the siRNA sequence. Where present, it serves to increase the stability of the siRNA within cells by reducing its susceptibility to degradation by nucleases .
  • siRNA molecules may be synthesized using standard solid or solution phase synthesis techniques which are known in the art.
  • Linkages between nucleotides may be phosphodiester bonds or alternatives, for example, linking groups of the formula P(0)S, (thioate); P(S)S, (dithioate) ; P(0)NR'2; P(0)R'; P(0)0R ⁇ ; CO; or CONR'2 wherein R is H (or a salt) or alkyl (1-12C) and R ⁇ is alkyl (1-9C) is joined to adjacent nucleotides through-O-or-S- .
  • siRNA molecules or longer dsRNA molecules may be made recombinantly by transcription of a nucleic acid sequence, preferably contained within a vector as described below.
  • Modified nucleotide bases can be used in addition to the naturally occurring bases, and may confer advantageous properties on siRNA molecules containing them.
  • modified bases may increase the stability of the siRNA molecule, thereby reducing the amount required for silencing.
  • the provision of modified bases may also provide siRNA molecules which are more, or less, stable than unmodified siRNA.
  • modified nucleotide base' encompasses nucleotides with a covalently modified base and/or sugar.
  • modified nucleotides include nucleotides having sugars which are covalently attached to low molecular weight organic groups other than a hydroxyl group at the 3 'position and other than a phosphate group at the 5 'position.
  • modified nucleotides may also include 2 ' substituted sugars such as 2 '-O-methyl- ; 2-0- alkyl ; 2-0-allyl ; 2'-S-alkyl; 2'-S-allyl; 2 '-fluoro- ; 2 '-halo or 2; azido-ribose, carbocyclic sugar analogues a-anomeric sugars; epimeric sugars such as arabinose, xyloses or lyxoses, pyranose sugars, furanose sugars, and sedoheptulose .
  • 2 ' substituted sugars such as 2 '-O-methyl- ; 2-0- alkyl ; 2-0-allyl ; 2'-S-alkyl; 2'-S-allyl; 2 '-fluoro- ; 2 '-halo or 2; azido-ribose, carbocyclic sugar analogues a-anomeric sugars; epimeric sugars
  • Modified nucleotides include alkylated purines and pyrimidines, acylated purines and pyrimidines, and other heterocycles . These classes of pyrimidines and purines are known in the art and include pseudoisocyt ' osine, N4,N4- ethanocytosine, 8-hydroxy-N ⁇ -methyladenine, 4-acetylcytosine, 5- (carboxyhydroxylmethyl) uracil, 5 fluorouracil, 5-bromouracil, 5-carboxymethylaminomethyl-2-thiouracil, 5- carboxymethylaminomethyl uracil, dihydrouracil, inosine, N6- isopentyl-adenine, 1- methyladenine, 1-methylpseudouracil, 1- ethylguanine, 2, 2-dimethylguanine, 2methyladenine, 2- methylguanine, 3-methylcytosine, 5-methylcytosine,
  • the invention also provides vectors comprising a nucleotide sequence encoding an siRNA or longer RNA or DNA sequence for production of dsRNA.
  • the vector may be any RNA or DNA vector.
  • the vector is preferably an expression vector, wherein the nucleotide sequence is operably linked to a promoter compatible with the cell.
  • the vector will preferably have at least two promoters, one to direct expression of the sense strand and one to direct expression of the antisense strand of the dsRNA.
  • two vectors may be used, one for the sense strand and one for the antisense strand.
  • the vector may encode RNAs which form stem-loop structures which are subsequently cleaved by the cell to produce dsRNA.
  • the sequence to be expressed will preferably be operably linked to a promoter functional in the target cells.
  • Promoters suitable for use in various vertebrate systems are well known.
  • suitable promoters include viral promoters such as mammalian retrovirus or DNA virus promoters, e.g. MLV, CMV, RSV, SV40 IEP and adenovirus promoters and metallothionein promoter.
  • the CMV IEP may be more preferable for human use.
  • Strong mammalian promoters may also be suitable. Variants of such promoters retaining substantially similar transcriptional activities may also be used.
  • the vector may comprise a nucleic acid construct encoding a cellular, viral or other transcription factor or other regulator of gene expression.
  • the nucleic acid construct may contain a specific cellular, viral or other promoter or repressor of gene expression.
  • the promoter or repressor may be designed to reflect the context of the cell into which the construct is introduced.
  • the construct may contain a viral promoter so expression from the construct is dependent upon the presence of a viral protein, so that the construct is expressed only in viral-infected cells.
  • the construct may have a promoter or repressor specific to certain cell types or to certain developmental stages. For example, where a construct encodes a siRNA against an anti-apoptotic gene such as bcl-2 under control of a viral promoter, the siRNA will only be expressed and apoptosis induced in viral-infected cells.
  • a viral promoter which matches the disease-causing virus should be used, e.g. a HPV promoter (such as the promoter causing expression of HPV16 E6/E7) for HPV- infected cells. In this way the vector will only be expressed in the virally-infected cells.
  • picorna viral promoters for picornaviral-infected cells; toga viral promoters for togaviral-infected cells; rhabdoviral promoters for rhabdoviral-infected cells; orthomyxoviral promoters for orthomyxoviral-infected cells; retroviral promoters for retroviral-infected cells; hepadnaviral promoters for hepadnaviral-infected cells; papova viral promoters for papovaviral-infected cells; adenoviral promoters for adenoviral- infected cells; herpes viral promoters for herpesviral-infected cells; hepatitis A/B/C viral promoters for hepatitis A/B/CC- infected cells; and pox viral promoters for pox viral-infected cells .
  • Administration for hepatitis A/B/C viral promoters for hepatitis A/B/CC- in
  • compositions as described herein are to be administered to an individual, administration is preferably in a "prophylactically effective amount" or a “therapeutically effective amount” (as the case may be, although prophylaxis may be considered therapy) , this being sufficient to show benefit to the individual.
  • the concentration of the siRNA in the final composition may be, for example, 0. l ⁇ M - lOmM.
  • the final composition is administered as needed, which will depend on the disease to be treated and the size of the affected area. For example, where the composition is for topical administration, l ⁇ l-lOml may be applied. More or less composition may be applied as necessary. Administration may be, for example, daily, weekly or monthly.
  • the actual amount administered, and rate and time-course of administration, will depend on the nature and severity of what is being treated. It will also depend upon toxicity of the therapeutic agent, as determined by pre-clinical and clinical trials. Prescription of treatment, e.g. decisions on dosage etc, is within the responsibility of general practitioners and other medical doctors .
  • a composition may be administered alone or in combination with other treatments, either simultaneously or sequentially dependent upon the condition to be treated.
  • compositions of the invention may be used for a wide variety of therapeutic and cosmetic applications, both in humans and other animals. They may be used in the treatment of cancer and other diseases. For example, compositions comprising HPV or bcl-2 siRNAs may be used to treat cancer. Other applications include the promotion of tissue growth and healing, for example in the treatment of wounds, burns and scars, and following cosmetic or other surgery. Compositions for use in wound healing and treatment of burns might include cellular and tissue growth factors such as EGF, or antibiotics or agents to promote neo- vascularisation and tissue growth.
  • the invention is illustrated by the following example.
  • Cancer of the uterine cervix is the third most common cancer in women worldwide. Approximately 450,000 new cases are diagnosed each year with, worldwide, a 50% death rate. In countries such as the UK and the USA routine cervical screening has reduced the incidence of mortality. In developing countries, however, cervical cancer remains the principal cause of death by cancer in women.
  • HPV human papilloma virus
  • RNA interference was used to silence HPV gene expression with topical application of the therapeutic agent, with potential applicability as a novel anti-cancer therapy for human cervical cancer.
  • siRNA molecules per se are not cytotoxic, even at concentrations much higher than required to induce silencing of gene expression by RNA interference.
  • HPV is a human virus expressed in human cells: for reasons relating to degeneracy of the genetic code it is in human cells that trials involving RNA interference should be conducted.
  • Topical application of the therapeutic agent, E7 siRNA, prior to routine surgical resection of cervical tumour tissue may allow enhanced clinical development.
  • In vitro experiments using human cervical carcinoma cells (SiHa cell line) were thus performed.
  • siRNA-liposomes are packaged into a semi-solid gel carrier medium, e.g. low melting agarose, to produce "siRNA-liposomes- agarose". Other carrier media may substitute for agarose.
  • siRNA-liposomes-agarose is applied to the surface of the cells or tissue.
  • the semi-solid gel vehicle may be designed to favour delivery of drug to cells of a given characteristic. For example, cancer cells and normal cells grow and behave differently in soft agar (agarose) .
  • the technique has also been applied to induce apoptosis in other types of cancer cell, by preventing the expression of cellular inhibitors of apoptosis which are overexpressed in cancer cells.
  • Microscopy All specimens imaged consisted of live cells maintained in either plastic-bottomed 24-well plates or a perspex plate, with chambers formed by sealing the bottom with a coverglass using double adhesive tape. Glass-bottomed chambers were used for low-depth high resolution imaging (1.4NA oil immersion objective) and 24-well plates were used for high depth imaging (0.6NA dry objective with correction collar). Specimens were imaged in an inverted microscope. Wide-field images were acquired with a Zeiss AxioCam HR and confocal images were acquired with a Zeiss LSM 410 system using 488nm excitation for FDA fluorescence. Emission filters were selected such that channel cross-talk was undetectable . Confocal sections were typically collected at 0.8 ⁇ m intervals and axial correction factors for oil-water and air-water imaging were estimated using the built-in functions of the LSM 410 software.
  • the cells were sub-cultured as follows: cell numbers SiHa, ⁇ 4.2 x
  • 1% Agarose stock Prepare 3% agarose (low melting point agarose VII, Sigma) stock solution with PBS, autoclaved as media, and store at RT . Before the transfection , melt the 3% agarose at 65°C, then dilute it to 1% agarose using optimal media, and keep it at 38°C.
  • the transfection proceedure above was scaled up to final agarose transfection mixture volume to 300 ⁇ l (For each well, using 3 x siRNA and oligolipofectamine as above) :
  • siRNA titrations [0.3 ⁇ M; 0.59 ⁇ M; 0.71 ⁇ M; 1.43 ⁇ M; 1.18 ⁇ M; 1.78 ⁇ M; 2.37 ⁇ M; 4.29 ⁇ M; 5.71 ⁇ M] were used.
  • siRNA sequences E7 5 'AGGAGGAUGAAAUAGAUGGTT3 ' (SEQ ID NO: 3) siRNA: 3 ' TTUCCUCCUACUUUAUCUACC5 ' (SEQ ID NO: 4) BCR-ABL 5 ⁇ GAGUUCAAAAGCCCUUCAT 3' (SEQ ID NO: 5) siRNA: 3 ' TTUCUCAAGUUUUCGGGAAGU5 ' (SEQ ID NO: 6) Bcl2 (a) 5 ' GGGGCUACGAGUGGGAUGCTT3 ' (SEQ ID NO: 7) siRNA: 3 ' TTCCCCGAUGCUCACCCUACG5 ' (SEQ ID NO: 8) Bcl2(b) 5 ' GCUGCACCUGACGCCCUUCTT3 ' (SEQ ID NO: 9) siRNA: 3 ' TTCGACGUGGACUGCGGGAAG5 ' (SEQ ID NO: 10)
  • Results Agarose was employed to produce a bio-compatible gel and was applied over pre-established cultures of cells, which had been established in culture for 24 hours in normal growth medium, prior to aspiration of the growth medium and application of the agarose overlay.
  • the molten agarose 38°C was allowed to gel to 37 °C for 4 hours before further overlaying wit;h cell growth medium for a further 24 to 72 hours.
  • Cell viability and proliferation were monitored and compared with parallel cultures of cells in normal aqueous medium (i.e. without gel overlay).
  • siRNA Three experimental classes of siRNA were employed. The first controlled for active RNA interference without apparent phenotypic effect. Here we employed lamin A/C siRNA which induces RNA interference but which does not cause apoptosis or other visible effect in human cells (Jiang & Milner, 2003) . The second class of siRNA served as siRNA transfection control, ineffective for induction of RNA interference. We used BCR-ABL siRNA which has no known target in non-leukaemic human cells, and Bcl-2 (a) siRNA which does not induce RNAi (Jiang & Milner 2003) . The third class of siRNA served as positive control for induction of apoptosis by RNA interference.
  • siRNAs HPV E7 siRNA and Bcl-2 (b) siRNA.
  • HPV E7 siRNA selectively silences HPV E7 and induces apoptosis in SiHa cervical cancer cells (Jiang & Milner 2002); and that Bcl-2 (b) siRNA selectively silences human Bcl-2 and induces apoptosis in HCT116 colorectal carcinoma cells (Jiang & Milner, 2003) . All siRNA sequences are given in the table above.
  • E7 siRNA in the agarose gel overlay was examined.
  • SiHa cervical carcinoma cells were cultured for 72 hr in normal medium (aqueous) or under 0.1% or 0.3% agarose with naked E7 siRNA.
  • a range of concentrations (0.71, 1.43, 2.86, 4.29 and 5.7 ⁇ M) of the siRNA was used. No effect upon SiHa cell viability or proliferation under any of the conditions tested was observed.
  • HCT116 cells appeared to be unaffected when overlaid with agarose/Bcl-2 (b) siRNA. Failure to induce RNAi is unlikely to reflect degradation of the naked siRNA since a 4-hour incubation in cell culture medium did not cause loss of full length siRNA.
  • siRNA is not effectively delivered into cells via the agarose gel overlay. This is consistent with the cationic nature of siRNA which renders it unlikely to pass across the cell membrane or to be otherwise taken up by cells via invagination into membraneous vesicles .
  • the agarose/liposome/siRNA formulation was prepared and overlaid over SiHa cells or HCT116 cells as described above.
  • the formulation contained E7 siRNA we clearly observed the appearance of apoptotic cervical carcinoma cells by 72 hour post-treatment.
  • apoptosis was induced in HCT116 colorectal cancer cells overlaid with an agarose/liposome/Bcl-2 (b) siRNA formulation, in both 0.1% and 0.3% agarose formulations.
  • liposomal delivery confers the advantage of direct delivery into the cytoplasmic compartment of the cell through fusion with the cell membrane. This is the desired compartment for siRNAs designed to target mRNA and to induce RNA interference. Use of liposomes could thus avoid the problem of uptake and compartmentalisation into membrane-bound or other structures within cells which could reduce or to negate siRNA efficacy.
  • Muco-adhesive gel-based systems suitable for topical drug delivery have opened new avenues for treatment of tissues such as those of the oral cavity, the gastrointestinal tract, the vagina and the uterine cervix (Woolfson et al . , 2000).
  • gel-based delivery can be extended to include liposomes containing the putative therapeutic agent.
  • the gel phase functions (i) to form a hydrophilic molecular interface with a mucosal or other cell surface, and (ii) to encompass the second, liposomal phase.
  • the liposomes can be constructed to contain a therapeutic agent for delivery into mammalian cells, including macromolecules such as siRNA.
  • the basic formulation has no adverse effects on the proliferation of human cells of either normal or of cancerous origin
  • the delivery of siRNA by this formulation and consequent induction of RNA interference was evidenced by predicted phenotypic changes specific to the siRNA nucleotide sequence.
  • the structure of the liposomes appeared heterogeneous when entrapped in agarose whereas in aqueous medium the liposomes were remarkably uniform in size. Since liposome dimensions govern their fusion with mammalian cell membranes this heterogeneity may also reduce efficiency of liposomal siRNA delivery via the agarose gel matrix. Despite these limitations we show induction of apoptosis by gel-based delivery of liposomes/siRNA in two cancer cell types of major importance, namely human cervical cancer and colorectal carcinoma.
  • siRNAs include proteins, expression plasmids, other inducers of post-transcriptional gene silencing, modulators of gene expression and anti-sense RNAs already approved for clinical use.
  • the HPV E7 siRNA treatment delivered to non- infected human cells had no effect on cell growth or viability, i.e. only the HPV-infected cancer cells were killed by antiviral RNA inte ference.
  • topical application of siRNA using a novel composition can be used for selective silencing of viral gene expression in human cells. This approach permits the selective killing of cervical cancer cells without adverse side effects on normal cells. Other viral-associated tumours may similarly be targeted (over 20% of human cancers are associated with viral infection) .
  • RNA interference RNA interference

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Abstract

La présente invention se rapporte à des compositions comportant un agent de transfert et un milieu de support solide ou colloïdal pour l'introduction d'agents biologiques dans des cellules. Lesdits agents peuvent être, en particulier, des molécules d'ARN, du type siARN, et le milieu de support un polymère du type gel, par exemple agarose ou agar-agar, qui possède des propriétés de muco-adhérence telles que le gel peut être administré à des cellules corporelles à des fins thérapeutiques.
PCT/GB2004/004979 2003-11-25 2004-11-25 Systeme d'administration colloidal pour agents therapeutiques biologiques WO2005051431A1 (fr)

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9700639B2 (en) 2012-02-07 2017-07-11 Aura Biosciences, Inc. Virion-derived nanospheres for selective delivery of therapeutic and diagnostic agents to cancer cells
US9724404B2 (en) 2009-04-13 2017-08-08 INSERM (Institut National de la Santé et de la Recherche Médicale) HPV particles and uses thereof
WO2018015761A1 (fr) * 2016-07-21 2018-01-25 University Of Leeds Matrices biocompatibles pour le transfert de molécules biologiques
US10117947B2 (en) 2013-09-18 2018-11-06 Aura Biosciences, Inc. Virus-like particle conjugates for diagnosis and treatment of tumors
US11207339B2 (en) 2015-10-30 2021-12-28 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Targeted cancer therapy

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003008573A2 (fr) * 2001-07-17 2003-01-30 Anne Josephine Milner Extinction d'expression genique
US20030157030A1 (en) * 2001-11-02 2003-08-21 Insert Therapeutics, Inc. Methods and compositions for therapeutic use of rna interference

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003008573A2 (fr) * 2001-07-17 2003-01-30 Anne Josephine Milner Extinction d'expression genique
US20030157030A1 (en) * 2001-11-02 2003-08-21 Insert Therapeutics, Inc. Methods and compositions for therapeutic use of rna interference

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
JIANG MING ET AL: "Bcl-2 constitutively suppresses p53-dependent apoptosis in colorectal cancer cells", GENES AND DEVELOPMENT, COLD SPRING HARBOR LABORATORY PRESS, NEW YORK, US, vol. 17, no. 7, 1 April 2003 (2003-04-01), pages 832 - 837, XP002293683, ISSN: 0890-9369 *
JIANG MING ET AL: "Gel-based application of siRNA to human epithelial cancer cells induces RNAi-dependent apoptosis.", OLIGONUCLEOTIDES. WINTER 2004, vol. 14, no. 4, January 2004 (2004-01-01), pages 239 - 248, XP002321807, ISSN: 1545-4576 *

Cited By (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10179168B2 (en) 2009-04-13 2019-01-15 INSERM (Institut National de la Santé et de la Recherche Médicale HPV particles and uses thereof
US9724404B2 (en) 2009-04-13 2017-08-08 INSERM (Institut National de la Santé et de la Recherche Médicale) HPV particles and uses thereof
US10688172B2 (en) 2009-04-13 2020-06-23 INSERM (Institut National de la Santé et de la Recherche Médicale) HPV particles and uses thereof
EP3427755A1 (fr) 2009-04-13 2019-01-16 INSERM - Institut National de la Santé et de la Recherche Médicale Particules hpv et utilisations associées
US10596275B2 (en) 2012-02-07 2020-03-24 Aura Biosciences, Inc. Virion-derived nanospheres for selective delivery of therapeutic and diagnostic agents to cancer cells
US10300150B2 (en) 2012-02-07 2019-05-28 Aura Biosciences, Inc. Virion-derived nanospheres for selective delivery of therapeutic and diagnostic agents to cancer cells
US9700639B2 (en) 2012-02-07 2017-07-11 Aura Biosciences, Inc. Virion-derived nanospheres for selective delivery of therapeutic and diagnostic agents to cancer cells
US9855347B2 (en) 2012-02-07 2018-01-02 Aura Biosciences, Inc. Virion-derived nanospheres for selective delivery of therapeutic and diagnostic agents to cancer cells
US10117947B2 (en) 2013-09-18 2018-11-06 Aura Biosciences, Inc. Virus-like particle conjugates for diagnosis and treatment of tumors
US10588984B2 (en) 2013-09-18 2020-03-17 Aura Biosciences, Inc. Virus-like particle conjugates for diagnosis and treatment of tumors
US11110181B2 (en) 2013-09-18 2021-09-07 Aura Biosciences, Inc. Virus-like particle conjugates for diagnosis and treatment of tumors
US11806406B2 (en) 2013-09-18 2023-11-07 Aura Biosciences, Inc. Virus-like particle conjugates for diagnosis and treatment of tumors
US12029794B2 (en) 2013-09-18 2024-07-09 Biosciences, Inc. Virus-like particle conjugates for diagnosis and treatment of tumors
US11207339B2 (en) 2015-10-30 2021-12-28 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Targeted cancer therapy
WO2018015761A1 (fr) * 2016-07-21 2018-01-25 University Of Leeds Matrices biocompatibles pour le transfert de molécules biologiques

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