WO2005051431A1 - Colloidal delivery system for biological therapeutic agents - Google Patents

Abstract

The present invention relates to compositions comprising a transfer agent and a solid or colloidal carrier medium for delivering biological agents into cells. In particular the agents may be RNA molecules such as siRNAs and the carrier medium a polymer such as a gel, e.g. agarose or agar, which has mucoadhesive properties such that the gel may be delivered to cells of the body for treatment.

Description

COLLOIDAL DELIVERY SYSTEM FOR BIOLOGICAL THERAPEUTIC AGENTS

FIELD OF THE INVENTION

The invention relates to methods and compositions for delivery of molecules into cells. In particular, the invention relates to compositions suitable for topical application to mammalian cells and tissues.

BACKGROUND OF THE INVENTION

The delivery of molecules to and into mammalian cells is fundamental for many therapeutic treatments and also for the experimental elucidation of biological systems.

There are two aspects to delivery: delivering the molecule of interest to the cell in question, and introducing the molecule into the cell once it gets there.

The first aspect has been addressed by both cell-specific and non-cell-specific approaches.

For example, in therapeutic treatment, 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.

These methods may be combined with cell-specific approaches, which include labelling molecules with targeting moieties, such as antibodies or ligands for cellular receptors, to direct them to specific cells. 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.

Not all these methods are suitable for all types of molecules it might be desired to deliver, and all have associated disadvantages. For example, 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 . For example, nucleic acid or proteins encased in simple liposomes cannot easily be targeted to specific cell types.

For many in vitro applications, and therapeutic applications where it would be feasible, it would be useful to be able to simply apply the molecule directly to the surface of the target cells, which would solve the initial problem of delivery to the cells. For many therapeutic applications, topical delivery of specified molecules into cells and tissues would be easy, convenient and more agreeable to the patient, often being non- invasive and speedily accomplished. However, it is hard to achieve. In vitro, molecules for delivery may be brought into contact with the cells by simply adding them to the aqueous culture medium from which they may diffuse into the cells or be actively taken up by the cells. In therapeutic applications, topical application in vivo requires a formulation for delivery of the therapeutic agent to and into the cells or tissues to be treated. A therapeutic application which highlights the difficulties of achieving satisfactory delivery is RNA interference (RNAi) . In RNAi, short double-stranded RNAs are used to inhibit the translation of corresponding mRNA targets and thus prevent the production of encoded proteins. Currently there are two main approaches to inducing RNAi. The first involves expression of 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). Apart from the undesirable injection method, 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.

SUMMARY OF THE INVENTION

At its most general, 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 .

Accordingly, in a first aspect the invention provides 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. In a preferred embodiment, 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. For example, 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. Preferably, the transfer agent is a liposome-type vehicle.

In a further aspect, 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. Preferably, the cell is a human cell. In one embodiment, 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.

Additionally or alternatively, the cell may be a HPV-infected cell.

In a preferred embodiment, 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. In one embodiment, the gene is a gene of viral or cellular origin that promotes cellular survival, such as bcl-2. Where the gene is a viral gene, 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 Eβ 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.

Thus, 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. Preferably, the HPV gene is E6 or E7, the transfer agent is a liposome vehicle and 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 .

Thus, the invention also provides a method of treatment of cancer comprising contacting the cancer cells with a composition as described herein. It will be understood that by "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. Preferably, the HPV gene is Eβ and/or E7, the transfer agent is a liposome vehicle, the carrier medium is agarose and the cancer is cervical cancer. In another preferred embodiment, 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. Preferably, the transfer agent is a liposome vehicle, the carrier medium is agarose and 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 .

The invention will now be described in more detail with reference to the following figures.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows detection of apoptotic cells, harvested from aqueous medium and analysed by FACS with annexin V staining (Jiang & Milner, 2003) .

Figure 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.

Figure 3

Size (area in μm²) of liposomes in aqueous cell culture medium and in 0.1% agarose.

DETAILED DESCRIPTION

Transfer agents

A transfer agent is any agent which can effect or aid the entry of a molecule such as a dsRNA into a cell. For example, a virus may be used as a transfer agent to introduce a desired nucleic acid molecule into a cell. Preferably, 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. If 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.

To incorporate nucleic acid into liposomes, 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-MEM™ 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.

DOTMA: 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 oligofectamine™ and lipofectamine™ .

In formulating with liposomes, procedures and raw materials must be considered carefully to avoid adverse effects on liposome stability. 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.

Other vehicles suitable for use in delivering nucleic acids such as siRNAs include viruses and virus-like particles (VLPs) such as HPV VLPs comprising the LI and/or L2 HPV viral protein; or hepatitis B viral proteins. Other suitable VLPs may be derived from picornaviruses; togaviruses; rhabdoviruses; orthomyxoviruses; retroviruses; hepadnaviruses; papovaviruses; adenoviruses; herpesviruses; and pox viruses. Otherwise delivery may employ conjugation to delivery peptides which can pass across the plasma membrane, such as Antennapedia or the HIV TAT protein.

Carrier media

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. Preferably, the colloid comprises a solid phase and a liquid phase. Such colloids include gels and liquid sols.

In some embodiments, 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.

Preferably, the carrier medium is biocompatible, i.e. it is not toxic to cells or tissues. Where the composition is for use on epithelial tissues with mucosal surfaces, for example when it is to be used to treat cervical or colorectal carcinomas, the carrier medium should be mucoadhesive.

The term 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) .

Preferably, the carrier medium will contain a polymer to provide the mucoadhesive benefit.

Many polymers can be utilized to form the carrier, coating, matrix or graft. 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. In one embodiment, 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%.

In alternative embodiments, the carrier medium may contain other materials which provide a mucoadhesive effect. Such 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. In addition, 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. Ideally, 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.

Optimally, the siRNA-transfer agent-agarose solution is applied to the cells while still liquid, and allowed to set after applying.

A typical protocol for the preparation of an agarose gel is given below. PREPARATION OF 0.1% AGAROSE GEL

0.5 grams of 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.

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. For example, 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 .

For example, 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. Other examples include polymeric systems composed of bioadhesive copolymers PMVE/MA and PVP for delivery to the oral cavity (Jones et al . , J. Pharm Sci. 92: 995-1007; 2003) , and tetracaine-based self-adhesive patches formulated from hydroxypropylcellulose discs (Long et al . , Br J Anaesthesia 91: 514-518; 2003), and other systems as described in Woolfson et al (2000) .

Agent for delivery

The agent for delivery may be a protein, nucleic acid or other macromolecule .

In a preferred embodiment, 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 .

In other embodiments, 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) is an analogue of DNA in which the backbone is a pseudopeptide rather than a sugar. PNA mimics the behaviour of DNA and binds complementary nucleic acid strands. The neutral backbone of PNA results in stronger binding and greater specificity than normally achieved. LNA (locked nucleic acid) . is a high-affinity nucleic acid analogue. 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.

In another embodiment, the agent for delivery may be a protein or polypeptide. Preferably, 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 .

In an alternative embodiment, 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

RNA interference (RNAi) 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

(typically 20-25 nucleotides) dsRNAs known as 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.

To induce RNA interference in a cell, dsRNA may be introduced into the cell as an isolated nucleic acid fragment or via a transgene, plasmid or virus. Alternatively, siRNA may be synthesised and introduced directly into the cell.

Another alternative is the expression of a short hairpin RNA molecule (shRNA) in the cell. 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. Alternatively, the shRNA may be synthesised exogenously and introduced directly into the cell. Preferably, 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.

Preferably 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.

Preferably, 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.

Typical hybridisation conditions use 4-6 x SSPE; 5-lOx Denhardts solution, 5g polyvinylpyrrolidone and 5g bovine serum albumin; lOOμg-l g/ml sonicated salmon sperm DNA; 0.1-1% sodium dodecyl sulphate; optionally 40-60% deionised formamide. Hybridisation temperature will vary depending on the GC content of the nucleic acid target sequence but will typically be between 42°C-65°C. Sambrook et al (2001) Molecular Cloning: A Laboratory Approach (3^rd Edn, Cold Spring Harbor Laboratory Press) . A common formula for calculating the stringency conditions required to achieve hybridisation between nucleic acid molecules of a specified homology is:

T_m = 81.5°C+lβ.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.

Where the gene to be silenced is an HPV Eβ or E7 gene, the siRNA sequence may be any suitable contiguous sequence of 10-30bp from any one of the HPVlβ or HPV18 Eβ or E7 gene sequences shown SEQ ID Nos: 15 to 18. Alternatively, longer dsRNA fragments comprising contiguous sequences from these sequences may be used, as they will be cleaved to form siRNAs within the cell.

Preferably, 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) .

Where the gene to be silenced is a bcl-2 gene, the siRNA sequence is preferably any suitable contiguous sequence of 10-

30bp from the sequence (accession no. NM 00657.1) shown as SEQ ID NO: 19. Alternatively, longer dsRNA fragments comprising contiguous sequences from the sequences of SEQ ID NO: 19 may be used, as they will be cleaved to form siRNAs within the cell. Preferably, the 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. Where 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 .

In preferred embodiments, the siRNA has the E6, E7 or Bcl-2 (b) sequences as follows:

E6 5' GAGGUAUAUGACUUUGCUUTT (SEQ ID NO : 1 )

SiRNA TTCUCCAUAUACUGAAACGAA 5' (SEQ ID NO: 2)

E7 5'AGGAGGAUGAAAUAGAUGGTT (SEQ ID NO: 3) siRNA: TTUCCUCCUACUUUAUCUACC 5' (SEQ ID NO : 4 )

BCR-ABL 5' AGAGUUCAAAAGCCCUUCATT (SEQ ID NO: 5) siRNA: 3 ' TTUCUCAAGUUUUCGGGAAGU 5' (SEQ ID NO: 6)

In some embodiments, 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)0Rβ; CO; or CONR'2 wherein R is H (or a salt) or alkyl (1-12C) and Rβ is alkyl (1-9C) is joined to adjacent nucleotides through-O-or-S- .

Alternatively, 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.

For example, 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.

The term ^modified nucleotide base' encompasses nucleotides with a covalently modified base and/or sugar. For example, 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. Thus 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 .

Modified nucleotides are known in the art and 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-Nβ-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, Nβ- ethyladenine, 7-methylguanine, 5-methylaminomethyl uracil, 5- methoxy amino methyl-2-thiouracil, -D-mannosylqueosine, 5- methoxycarbonylmethyluracil, 5methoxyuracil, 2 methylthio-Nβ- isopentenyladenine, uracil-5-oxyacetic acid methyl ester, psueouracil, 2-thiocytosine, 5-methyl-2 thiouracil, 2- thiouracil, 4-thiouracil, 5methyluracil, N-uracil-5-oxyacetic acid methylester, uracil 5-oxyacetic acid, queosine, 2- thiocytosine, 5-propyluracil, 5-propylcytosine, 5-ethyluracil, 5ethylcytosine, 5-butyluracil, 5-pentyluracil, 5-pentylcytosine, and 2, β, diaminopurine, methylpsuedouracil, 1-methylguanine, 1- methylcytosine .

Vectors

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.

Alternatively, two vectors may be used, one for the sense strand and one for the antisense strand. Alternatively the vector may encode RNAs which form stem-loop structures which are subsequently cleaved by the cell to produce dsRNA.

Where the vector is an expression vector, 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. For example, 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. For example, 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. Similarly, 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.

Where the vector is for use in virally infected cell such as cells infected with HPV, 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. Other examples are 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

Where a composition as described herein is 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. Where the molecule for delivery in to a cell is a siRNA, the concentration of the siRNA in the final composition may be, for example, 0. lμM - 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, lμl-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.

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.

There is no consistently effective therapy for those who progress to malignancy. In developed countries patients may be treated with surgery, radiotherapy and/or chemotherapy. In less well-developed countries the availability of such treatments is limited or is simply not available. Metastatic cancer cells invade and proliferate in adjacent healthy organs and eventually death ensues.

Cancer of the cervix is caused by infection with the human papilloma virus (HPV) . It is a sexually transmitted disease and it is estimated that between 50% to 70% of the at-risk population are positive for HPV infection. Progression from infection to malignancy is unpredictable and is variable in terms of time. In some cases it takes decades, in other patients the development of malignancy is much earlier. This aspect is poorly understood. The balance between tolerance and immunosurveillance by the host immune system is a likely factor. Another factor concerns the virus itself: progression to malignancy correlates with integration of the viral genome into the host cell genome. This leads to elevated expression of two viral genes, namely HPV Eβ and HPV E7. The HPV E6 and E7 proteins are the driving forces of malignancy.

During the development of human cervical cancer the HPV-positive tumour cells become accessible at the surface of the uterine cervix. Indeed the tumour cells can be scraped from the surface of the uterine cervix and are detected by the PAP smear test. This forms the foundation of screening for cellular abnormalities of the uterine cervix. Here, 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. In vitro studies have demonstrated that siRNA molecules per se are not cytotoxic, even at concentrations much higher than required to induce silencing of gene expression by RNA interference.

For this new technology extensive pre-clinical trials with experimental animals are of dubious value and may be dangerously misleading. 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.

To summarise the procedure: 1. E7 siRNA is packaged into liposome-type transfer agent to produce "siRNA-liposomes".

2. 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. 3. 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.

Experimental procedures

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 0.8μm intervals and axial correction factors for oil-water and air-water imaging were estimated using the built-in functions of the LSM 410 software.

Cell lines and cell culture

The cells were sub-cultured as follows: cell numbers SiHa, ~4.2 x

10³; HCTllβ, -5.6 x 10³; normal human diploid fibroblasts (NDF) , ~7 x 10² per well, into 96-well (~0.28cm²) plates with 200 μl antibiotic-free media for 24 hours before the transfection. For 15mm glass cover-slip culture chambers, the cell numbers were SiHa, 2.6 x 10⁴; HCT116, 4.4 x 10⁴; NDF, 4.4 x 10³; with 0.5 ml antibiotic-free media. Cells were cultured at 37 °C in 5% C0₂ in air .

Agarose overlay For 9β-well plates:

Final concentration 0.1% agarose, lμM/2μM siRNA

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.

Dilute oligolipofectamine with lOμl optimal media, mix and leave it at RT for 5-10mins. Dilute siRNA with 30μl optimal media. Mix the oligolipofectamine and siRNA together, and leave at RT for 15-20mins. Adjust the mixture to 90μl (0.1% agarose final con.) or 70μl (0.3% agarose final con.), and pre-warm at 38°C, followed by adding the appropriate 1% agarose to final volume of lOOμl. Wash the cells with optimal media once, and apply the lOOμl transfection mix onto the cells, leave the agarose to set, then incubate the cells in 37°C, 5% C0₂ for 4 hours. Finally, add 50μl antibiotic-free media with 3 x FCS, and culture the cells for further 24 to 72 hours.

For 15mm glass cover slip chambers, the transfection proceedure above was scaled up to final agarose transfection mixture volume to 300μl (For each well, using 3 x siRNA and oligolipofectamine as above) :

Final concentration 0.1% agarose, lμM /2μM siRNA

For siHa cells, siRNA titrations [0.3μM; 0.59μM; 0.71μM; 1.43μM; 1.18μM; 1.78μM; 2.37μM; 4.29μM; 5.71μM] were used.

SiRNA sequences : E7 5 'AGGAGGAUGAAAUAGAUGGTT3 ' (SEQ ID NO: 3) siRNA: 3 ' TTUCCUCCUACUUUAUCUACC5 ' (SEQ ID NO: 4) BCR-ABL 5ΑGAGUUCAAAAGCCCUUCAT 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)

LaminA/C 5 ' CUGGACUUCCAGAAGAACAT 3 ' (SEQ ID NO: 11) siRNA: 3 ' TTGACCUGAAGGUCUUCUUGU3 ' (SEQ ID NO: 12)

Cy3Luciferase 5 ' CGUACGCGGAAUACUUCGATT3 ' (SEQ ID NO: 13) GL2siRNA: 3 ' TTGCAUGCGCCUUAUGAAGCU5 ' (SEQ ID NO: 14)

FDA (Fluorescein Diacetate) staining live cells

Dissolve FDA in acetone (lmg/ml) and store at 4°C. Add FDA stock solution into cell culture at 1:2000 dilution, and incubate for 30mins at 37°C, 5% C0₂.

Soft agar clonogenic assay

Mix the cells (NDF, 2 x 10⁴; HCT, 2 x 10⁵; SiHa, 1.5 x 10^s) in 1ml normal culture media with 0.3% agarose at 37°C. Place the cell/agarose mix in 35mm petri dish, a 6-well plate. Leave the agarose to set, incubate them at 37°C, 5% C0₂. Monitor and count the colonies after 1, 2 and 3 weeks.

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).

Human cervical carcinoma cells (SiHa) and colorectal carcinoma cells (HCT116) cell viability was determined at 0, 24, 48 and 72 hours. For both cell lines, proliferation appeared unaffected by either 0.3% or 0.1% agarose overlay and was indistinguishable from cells cultured in normal aqueous growth medium. The proliferation of normal, non-cancerous cells (human diploid fibroblasts) was also tested. It is well established that, in contrast to cancer cells, normal adherent cells are unable to proliferate and to form colonies when suspended in soft agar or agarose. Indeed, in the present study we confirmed that both SiHa and HCT116 cells readily form colonies in an agarose-based clonogenic assay, whereas normal human diploid fibroblasts fail to divide under identical conditions. However, for adherent fibroblasts overlaid with agarose gel, we demonstrate that normal cell viability and proliferation are unaffected by the gel overlay.

In order to screen for gel-based delivery of siRNA to cultured cells we required a phenotypic marker of RNAi, detectable by visualisation through the agarose gel overlay. Induction of apoptosis was chosen since this effect is evident by microscopy. The process of apoptosis may be confirmed by cell harvesting and further analysis, but in the present experiments the recovery of cells from beneath the agarose gel was not an option. However, apoptosis was confirmed in parallel controls, cultured in aqueous medium without agarose, by annexin V staining and FACS analysis (Figs 1 and 2) .

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. Here we employed two siRNAs: HPV E7 siRNA and Bcl-2 (b) siRNA. We have already established that 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.

The simple inclusion of 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 μM) of the siRNA was used. No effect upon SiHa cell viability or proliferation under any of the conditions tested was observed. Similarly 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. We therefore conclude that naked 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 .

(a) Effects of agarose-siRNA and of agarose-liposome-siRNA formulations on the viability and proliferation of as indicated. Top panel = untreated control cells; second panel = cells overlaid with agarose-E7 siRNA; third panel = cells overlaid with agarose-liposomes-E7 siRNA; and the bottom panel = cells overlaid with agarose-liposomes-lamin A/C siRNA (as indicated) . Inserts show confocal images with dual labelling for live cells (green) and apoptotic cells (red; see methods) . Note apoptotic cells in 0.1% agarose but not in 0.3% agarose-liposome-E7 siRNA formulation.

(b) SiHa cells treated -agarose. siRNA concentrations are as indicated. The cells appear normal up to 5.71 μM E7 siRNA.

We next explored the possibility that the polymeric agarose matrix might support liposomes and enable the delivery of liposomal contents to the cells, to determine whether a formulation of agarose-liposomes-siRNA can enable effective delivery of siRNA to cells. The agarose/liposome/siRNA formulation was prepared and overlaid over SiHa cells or HCT116 cells as described above. When the formulation contained E7 siRNA we clearly observed the appearance of apoptotic cervical carcinoma cells by 72 hour post-treatment. Similarly 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. In the case of the HCT116 cells, apoptosis was evident at 48 hr post-treatment, consistent with the kinetics previously observed following induction of RNAi targeted against Bcl-2 mRNA in these cells (Jiang & Milner; 2003) .

In both cases the induction of apoptosis was specific to the siRNA sequence since identical treatment with lamin A/C siRNA, Bcl-2 (a) siRNA or BCR-ABL siRNA had no observable effect on the viability or proliferation of either of the two cell lines. Thus we demonstrate that the chosen formulation of agarose-liposome- siRNA by itself has no adverse effects on cell proliferation. In contrast, agarose formulations with liposomes containing pro- apoptotic E7-siRNA or Bcl-2 (b) siRNA induce apoptosis of SiHa cells and HCT116 cells respectively. Apoptotic cell killing is thus specific to the siRNA nucleotide sequence and is attributable to the induction of RNA interference. Effective delivery of siRNA into human cells causes RNAi with degradation of the target mRNA and this process is believed to occur in the cytoplasmic compartment of the cell. It is noteworthy that 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.

In order to visualise the liposomes-siRNA we used Cy3-labelled siRNA. The agarose-liposome-siRNA formulation was analysed by confocal scanning microscopy, as detailed above. Figure 3 shows the size of liposomes in aqueous cell culture medium and in 0.1% agarose .

Discussion

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). Here we have demonstrated that gel-based delivery can be extended to include liposomes containing the putative therapeutic agent. Thus a two-phase formulation is envisaged in which 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. For this purpose we used induction of apoptosis as a phenotypic marker of RNA interference in cervical cancer cells (treated with E7 siRNA) and in colorectal carcinoma cells (treated with Bcl-2 siRNA) .

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.

Thus we have established the feasibility of gel-based topical delivery of siRNA to human cells. This holds promise for further refinement and development of gel-based delivery of siRNA as a novel therapeutic approach to treat human disease. Moreover, this novel delivery method is not restricted to siRNAs, and additional agents suitable for delivery via gel/liposome formulations include proteins, expression plasmids, other inducers of post-transcriptional gene silencing, modulators of gene expression and anti-sense RNAs already approved for clinical use.

It is thus demonstrated that selective silencing of expression of HPV E6 and E7 in human cervical cancer cells and of bcl-2 in colorectal carcinoma cells can be achieved by RNA interference via topical application of siRNA using a novel compositions of the invention. This was demonstrated on human cancer cell lines derived from actual tumours and grown in cell culture. A single dose of treatment was sufficient to silence gene expression and cause the cancer cells to die.

Importantly, 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. Thus we have established that 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) .

References

Dykxhoorn DM, Novina CD, Sharp PA. Killing the messenger: short RNAs that silence gene expression. Mol Cell Biol, 4; 457-467 (2003)

Jiang M, Milner J. Selective silencing of viral gene expression in HPV-positive human cervical carcinoma cells treated with siRNA, a primer of RNA interference. Oncogene, 21; 6041-6048 (2002)

Brummelkamp TR, Bernards R, Agami R. A system for stable expression of short interfering RNAs in mammalian cells. Science, 296; 550-53 (2002)

Lewis DL, Hagstrom JE, Loomis AG, Wolff JA, Herweijer H. Efficient delivery of siRNA for inhibition of gene expression in postnatal mice. Nature Genet, 32; 107-108 (2002) McCaffrey AP, Meuse L, Pham TT, Conklin DS, Hannon GJ, Kay MA. RNA interference in adult mice. Nature, 418; 38-39 (2002)

Novina CD, Murray MF, Dykxhoorn DM, Beresford PJ, Riess J, Lee SK, Collman RG, Lieberman J, Shankar P, Sharp PA. siRNA-directed inhibition of HIV-1 infection. Nature Med, 8; 681-686 (2002)

Capodici J, Kariko K, Weissman D. Inhibition of HIV-1 infection by small interfering RNA-mediated RNA interference. J Immunol, 169; 5196-5201 (2002)

Coburn GA, Cullen BR. Potent and specific inhibition of human immunodeficiency virus type 1 replication by RNA interference. J Virol, 76; 9225-9231 (2002)

Jacque JM, Triques K, Stephenson M. Modulation of HIV-1 replication by RNA interference. Nature, 418; 435-438 (2002)

Woolfson AD, Malcolm RK, MCCarron PA, Jones DS . Bioadhesive Drug Delivery Systems. Polymeric Biomaterials 2^nd Edn (Ed S Dumitriu) . Marcel Dekker New York; 1063-1082 (2000)

Sanchez-Velar N, Udofia EB, Yu Z, Zapp ML. hRIP, a cellular cofactor for Rev function, promotes release of HIV RNA from the perinuclear region. Genes Dev, 18; 23-34 (2004)

Oshima , Kawasaki H, Soda Y, Tani K, Asano S, Taira K. Maxizymes and small hairpin-type RNAs that are driven by a tRNA promoter specifically cleave a chimeric gene associated with leukemia in vitro and in vivo. Cancer Res, 63; 6809-14 (2003)

Wilda M, Fuchs U, Wossman W, Borkhardt A. Killing of leukemic cells with a BCR/ABL fusion gene by RNA interference (RNAi) . Oncogene, 21; 5716-24 (2002) SEQUENCE LISTING

<110> Milner, Anne J

<120> Colloidal Delivery System For Biological Therapeutic Agents

<130> AHB/CP6247712

<140> PCT/GB <141> 2004-11-25

<150> US 60/549,919

<151> 2004-03-05

<150> GB 0327409.9

<151> 2003-11-25

<160> 19

<170> Patentln version 3.1

<400> 1

5 ' GAGGUAUAUGACUUUGCUUTT <400> 2

TTCUCCAUAUACUGAAACGAA 5'

<400> 3

5 ' AGGAGGAUGAAAUAGAUGGTT3 '

<400> 4

3 ' TTUCCUCCUACUUUAUCUACC5 '

<400> 5 5ΑGAGUUCAAAAGCCCUUCATT3'

<400> 6

3 ' TTUCUCAAGUUUUCGGGAAGU5 ' <400> 7

5 ' GGGGCUACGAGUGGGAUGCTT3 '

<400> 8

3 ' TTCCCCGAUGCUCACCCUACG5 '

<400> 9 5 'GCUGCACCUGACGCCCUUCTT3'

<400> 10

3 ' TTCGACGUGGACUGCGGGAAG5 '

<400> 11

5 ' CUGGACUUCCAGAAGAACATT3 '

<400> 12 3 ' TTGACCUGAAGGUCUUCUUGU3 *

<400> 13

5 ' CGUACGCGGAAUACUUCGATT3 ' <400> 14

3 ' TGCAUGCGCCUUAUGAAGCU5 '

<210> 15 <211> 477

<212> DNA

<213> Human papillomavirus type 18

<400> 15 atggcgcgct ttgaggatcc aacacggcga ccctacaagc tacctgatct gtgcacggaa 60 ctgaacactt cactgcaaga catagaaata acctgtgtat attgcaagac agtattggaa 120 cttacagagg tatttgaatt tgcatttaaa gatttatttg tggtgtatag agacagtata 180 ccgcatgctg catgccataa atgtatagat ttttattcta gaattagaga attaagacat 240 tattcagact ctgtgtatgg agacacattg gaaaaactaa ctaacactgg gttatacaat 300 ttattaataa ggtgcctgcg gtgccagaaa ccgttgaatc cagcagaaaa acttagacac 360 cttaatgaaa aacgacgatt tcacaacata gctgggcact atagaggcca gtgccattcg 420 tgctgcaacc gagcacgaca ggaacgactc caacgacgca gagaaacaca agtataa 477

<210> 16

<211> 318 <212> DNA

<213> Human papillomavirus type 18

<400> 16 atgcatggac ctaaggcaac attgcaagac attgtattgc atttagagcc ccaaaatgaa 60 attccggttg accttctatg tcacgagcaa ttaagcgact cagaggaaga aaacgatgaa 120 atagatggag ttaatcatca acatttacca gcccgacgag ccgaaccaca acgtcacaca 180 atgttgtgta tgtgttgtaa gtgtgaagcc agaattgagc tagtagtaga aagctcagca 240 gacgaccttc gagcattcca gcagctgttt ctgaacaccc tgtcctttgt gtgtccgtgg 300 tgtgcatccc agcagtaa 318

<210> 17

<211> 477

<212> DNA <213> Human papillomavirus type 16

<400> 17 atgcaccaaa agagaactgc aatgtttcag gacccacagg agcgacccag aaagttacca 60 cagttatgca cagagctgca aacaactata catgatataa tattagaatg tgtgtactgc 120 aagcaacagt tactgcgacg tgaggtatat gactttgctt ttcgggattt atgtatagta 180 tatagagatg ggaatccata tgctgtatgt gataaatgtt taaagtttta ttctaaaatt 240 agtgagtata gacattattg ttatagtgtg tatggaacaa cattagaaca gcaatacaac 300 aaaccgttgt gtgatttgtt aattaggtgt attaactgtc aaaagccact gtgtcctgaa 360 gaaaagcaaa gacatctgga caaaaagcaa agattccata atataagggg tcggtggacc 420 ggtcgatgta tgtcttgttg cagatcatca agaacacgta gagaaaccca gctgtaa 477

<210> 18

<211> 297

<212> DNA

<213> Human papillomavirus type 16

<400> 18 atgcatggag atacacctac attgcatgaa tatatgttag atttgcaacc agagacaact 60 gatctctact gttatgagca attaaatgac agctcagagg aggaggatga aatagatggt 120 ccagctggac aagcagaacc ggacagagcc cattacaata ttgtaacctt ttgttgcaag 180 tgtgactcta cgcttcggtt gtgcgtacaa agcacacacg tagacattcg tactttggaa 240 gacctgttaa tgggcacact aggaattgtg tgccccatct gttctcagaa accataa 297

<210> 1! <211> 618 <212> DNA

<213> Human Bcl-2 cDNA

<400> 19 atggcgcacg ctgggagaac ggggtacgac aaccgggaga tagtgatgaa gtacatccat 60 tataagctgt cgcagagggg ctacgagtgg gatgcgggag atgtgggcgc cgcgcccccg 120 ggggccgccc ccgcaccggg catcttctcc tcccagcccg ggcacacgcc ccatccagcc 180 gcatcccgcg acccggtcgc caggacctcg ccgctgcaga ccccggctgc ccccggcgcc 240 gccgcggggc ctgcgctcag cccggtgcca cctgtggtcc acctggccct ccgccaagcc 300 ggcgacgact tctcccgccg ctaccgcggc gacttcgccg agatgtccag ccagctgcac 360 ctgacgccct tcaccgcgcg gggacgcttt gccacggtgg tggaggagct cttcagggac 420 ggggtgaact gggggaggat tgtggccttc tttgagttcg gtggggtcat gtgtgtggag 480 agcgtcaacc gggagatgtc gcccctggtg gacaacatcg ccctgtggat gactgagtac 540 ctgaaccggc acctgcacac ctggatccag gataacggag gctgggtagg tgcatctggt 600 gatgtgagtc tgggctga 618

Claims

1. A composition for delivering an agent into a cell, comprising said agent, a transfer agent and a solid or colloidal carrier medium.

2. A composition according to claim 1 wherein the agent for delivery is a protein, a nucleic acid, a peptide, a drug or other therapeutic agent.

3. A composition according to claim 1 wherein the agent for delivery is an agent for inducing RNA interference in a cell.

4. A composition according to claim 3 wherein the agent for inducing RNA interference is a siRNA, a dsRNA, an shRNA, or DNA encoding such RNA.

5. A composition according to claim 4 wherein the agent for inducing RNA interference is a siRNA.

6. A composition according to claim 5 wherein the siRNA is homologous to a portion of an HPV gene.

7. A composition according to claim 6 wherein the HPV gene is E6.

8. A composition according to claim 6 wherein the HPV gene is E7.

9. A composition according to claim 7 wherein the siRNA has the sequence shown as SEQ ID Nos: 1 and 2.

10. A composition according to claim 8 wherein the siRNA has the sequence shown as SEQ ID Nos: 3 and 4.

11. A composition according to claim 5 wherein the siRNA is homologous to a portion of the bcl-2 gene.

12. A composition according to claim 11 wherein the siRNA has the sequence shown as SEQ ID Nos: 5 and 6.

13. A composition according to any one of the above claims wherein the carrier medium is a gel.

14. A composition according to claim 13 wherein the gel comprises a synthetic gel.

15. A composition according to claim 13 or claim 14 wherein the gel comprises a natural gel.

16. A composition according to claim 15 wherein the gel comprises a synthetic and a natural gel.

17. A composition according to any one of claims 13 to 16 which is a mucoadhesive gel.

18. A composition according to any one of claims 13 to 17 wherein the gel comprises agar or agarose.

19. A composition according to any one of claims 1 to 18 wherein the transfer agent is a virus, a liposome, a dendrimer or a polylysine-transferrine-conjugate .

20. A composition according to any one of claims 7 to 10 wherein the transfer agent is a liposome and the carrier medium is agarose.

21. A composition according to any one of claims 11 to 12 wherein the transfer agent is a liposome and the carrier medium is agarose.

22. A composition according to claim 13 wherein the transfer agent is a liposome and the carrier medium is agarose.

23. A method of delivering a molecule into a cell comprising contacting the cell with the composition of any one of claims 1 to 22.

24. The method of claim 23 wherein the composition is a composition according to claim 20.

25. The method of claim 23 wherein the composition is a composition according to claim 21.

26. The method of claim 23 wherein the cell is an HPV-infected cell.

27. The method of any one of claims 23 to 26 wherein the cell is a tumour cell.

28. The method of claim 27 wherein the tumour is a carcinoma of the cutaneous, squamous or cervical epithelia, or a colorectal carcinoma .

29. The method of claim 28 wherein the tumour is a cervical carcinoma.

30. The method of claim 28 wherein the tumour is a colorectal carcinoma .

31. A method of treating cancer comprising administering a composition according to any one of claims 1 to 22 to an individual in need thereof.

32. A method according to claim 31 wherein the cancer is a carcinoma of the cutaneous, squamous or cervical epithelia, or a colorectal carcinoma.

33. A method according to claim 32 wherein the cancer is a cervical carcinoma.

34. A method according to claim 32 wherein the cancer is a colorectal carcinoma.

35. A method according to claim 33 wherein the composition is a composition of claim 20.

36. A method according to claim 34 wherein the composition is a composition of claim 21.

37. A composition according to any one of claims 1 to 22 for use in a method of treatment.