SIRNA MICROBUBBLE

Field of the Invention

The invention relates to a microbubble comprising siRNA and its use in therapy.

Background to the Invention

A variety of diseases and disorders are associated with an increased function of the signal transducer and transcription activator, STAT6. There is a need for new therapies for such diseases and disorders.

Summary of the Invention

The inventors have surprisingly identified a new microbubble that is capable of treating diseases or disorders associated with an increased amount or function of STAT6. The invention provides a microbubble, which comprises:

(a) at least one duplex polynucleotide comprising the sequences shown in (i) SEQ ID NOs:

1 and 2 or variants thereof having at least 90% homology to SEQ ID NOs: 1 and 2 based on sequence identity over their entire length, (ii) SEQ ID NOs: 3 and 4 or variants thereof having at least 90% homology to SEQ ID NOs: 3 and 4 based on sequence identity over their entire length, (iii) SEQ ID NOs: 5 and 6 or variants thereof having at least 90% homology to SEQ ID NOs: 5 and 6 based on sequence identity over their entire length or (iv) SEQ ID NOs: 7 and 8 or variants thereof having at least 90% homology to SEQ ID NOs: 7 and 8 based on sequence identity over their entire length; and

(b) at least one N- Acetylgalactosamine (GalNAc) molecule.

The invention also provides:

- a population of microbubbles of the invention;

a method of forming a microbubble or a population of microbubbles of the invention, comprising (a) providing an interface between a gas and a microbubble shell material and thereby forming a microbubble or population of microbubbles of the invention and (b) loading the microbubble or population of microbubbles with at least one duplex polynucleotide as defined above and at least one GalNAc molecule and thereby forming a microbubble or a population of microbubbles of the invention; a pharmaceutical composition comprising (a) a microbubble or a population of

microbubbles of the invention and (b) a pharmaceutically acceptable carrier or diluents; and

a method of treating a disease or disorder associated with an increased amount and/or function of STAT6 in a patient, comprising administering to the patient a population or a pharmaceutical composition of the invention, wherein the population or composition comprises a therapeutically effective number of microbubbles, and thereby treating the disease or disorder in the patient.

Brief Description of the Figures

Figure 1 shows milestone achievements in our techniques for ultra sound mediated micro bubble delivery of siRNA. a) shows control intact microbubbles. b) shows delivery of siRNA into cancer cells, c) shows control tagged siRNA. d) shows the viability of cells exposed to microbubbles.

Description of the Sequence Listing

SEQ ID NOs: 1 and 2 show two RNA polynucleotides which hybridise to form a duplex polynucleotide of the invention.

SEQ ID NOs: 3 and 4 show two RNA polynucleotides which hybridise to form a duplex polynucleotide of the invention.

SEQ ID NOs: 5 and 6 show two RNA polynucleotides which hybridise to form a duplex polynucleotide of the invention.

SEQ ID NOs: 7 and 8 show two RNA polynucleotides which hybridise to form a duplex polynucleotide of the invention.

Detailed Description of the Invention

It is to be understood that different applications of the disclosed products and methods may be tailored to the specific needs in the art. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments of the invention only, and is not intended to be limiting.

In addition, as used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "a microbubble" includes two or more microbubbles, reference to "a disease or disorder" includes two or more such diseases or disorders, reference to "a patient" includes two or more such patients, and the like.

All publications, patents and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety.

Microbubbles of the invention

The present invention provides a microbubble. Microbubbles, their formation and biomedical uses are known in the art (e.g. Sirsi and Borden, Bubble Sci Eng Technol. Nov 2009; 1(1 -2): 3-17).

Microbubbles are bubbles smaller than one millimetre in diameter and larger than one micrometre in diameter. The microbubble of the present invention is preferably 8μηι or less in diameter, such as 7μηι or less in diameter, 6μηι or less in diameter, 5μηι or less in diameter, 4μηι or less in diameter, 3μηι or less in diameter or 2μηι or less in diameter.

The microbubble may be formed from any substance. The general composition of a microbubble is a gas core stabilised by a shell. The gas core may comprise air or a heavy gas, such as perfluorocarbon, nitrogen or perflouropropane. Heavy gases are less water soluble and so are less likely to leak out from the microbubble leading to microbubble dissolution. Microbubbles with heavy gas cores typically last longer in circulation.

The shell may be formed from any material. The shell material preferably comprises a protein, a surfactant, a lipid, a polymer or a mixture thereof.

Suitable proteins, include but are not limited to, albumin, lysozyme and avidin. Proteins within the shell may be chemically-crosslinked, for instance by cysteine-cysteine linkage. Other crosslinkages are known in the art.

Suitable surfactants include, but are not limited to, sorbitan monopalmitate (such as SPAN-40), polysorbate detergents (such as TWEEN-40), mixtures of SPAN-40 and TWEEN-40 and sucrose stearate (mono- and di-ester).

Suitable polymers include, but are not limited to, alginate polymers, double ester polymers of ethylidene, the copolymer poly(D,L-lactide-co-glycolide) (PLGA), polyvinyl alcohol) (PVA), the copolymer polyperfluorooctyloxycaronyl-poly(lactic acid) (PLA-PFO) and other block copolymers. Block copolymers are polymeric materials in which two or more monomer sub-units that are polymerized together to create a single polymer chain. Block copolymers typically have properties that are contributed by each monomer sub-unit. However, a block copolymer may have unique properties that polymers formed from the individual sub-units do not possess. Block copolymers can be engineered such that one of the monomer sub-units is hydrophobic (i.e. lipophilic), whilst the other sub-unit(s) are hydrophilic whilst in aqueous media. In this case, the block copolymer 5 may possess amphiphilic properties and may form a structure that mimics a biological membrane.

The block copolymer may be a diblock (consisting of two monomer sub-units), but may also be constructed from more than two monomer sub-units to form more complex arrangements that behave as amphipiles. The copolymer may be a triblock, tetrablock or pentablock copolymer. Block copolymers may also be constructed from sub-units that are not classed as lipid sublet materials; for example a hydrophobic polymer may be made from siloxane or other non- hydrocarbon based monomers. The hydrophilic sub-section of block copolymer can also possess low protein binding properties, which allows the creation of a membrane that is highly resistant when exposed to raw biological samples. This head group unit may also be derived from non- classical lipid head-groups.

15 Any lipid material that forms a microbubble may be used. The lipid composition is chosen such that the microbubble has the required properties, such surface charge, packing density or mechanical properties. The lipid composition can comprise one or more different lipids. For instance, the lipid composition can contain up to 100 lipids. The lipid composition preferably contains 1 to 10 lipids. The lipid composition may comprise naturally-occurring lipids and/or 20 artificial lipids.

The lipid typically comprises a head group, an interfacial moiety and two hydrophobic tail groups which may be the same or different. Suitable head groups include, but are not limited to, neutral head groups, such as diacylglycerides (DG) and ceramides (CM); zwitterionic head groups, such as phosphatidylcholine (PC), phosphatidylethanolamine (PE) and sphingomyelin (SM);

25 negatively charged head groups, such as phosphatidylglycerol (PG); phosphatidylserine (PS), phosphatidylinositol (PI), phosphatic acid (PA) and cardiolipin (CA); and positively charged headgroups, such as trimethylammonium-Propane (TAP). Suitable interfacial moieties include, but are not limited to, naturally-occurring interfacial moieties, such as glycerol-based or ceramide- based moieties. Suitable hydrophobic tail groups include, but are not limited to, saturated

30 hydrocarbon chains, such as lauric acid (w-Dodecanolic acid), myristic acid (w-Tetradecononic acid), palmitic acid (w-Hexadecanoic acid), stearic acid (w-Octadecanoic) and arachidic (n- Eicosanoic); unsaturated hydrocarbon chains, such as oleic acid (c/s-9-Octadecanoic); and branched hydrocarbon chains, such as phytanoyl. The length of the chain and the position and number of the double bonds in the unsaturated hydrocarbon chains can vary. The length of the chains and the position and number of the branches, such as methyl groups, in the branched hydrocarbon chains can vary. The hydrophobic tail groups can be linked to the interfacial moiety as an ether or an ester.

The lipids can also be chemically-modified. The head group or the tail group of the lipids may be chemically-modified. Suitable lipids whose head groups have been chemically-modified include, but are not limited to, PEG-modified lipids, such as l,2-Diacyl-sn-Glycero-3- Phosphoethanolamine-N -[Methoxy(Poly ethylene glycol)-2000]; functionalised PEG Lipids, such as l,2-Distearoyl-sn-Glycero-3 Phosphoethanolamine-N-[Biotinyl(Polyethylene Glycol)2000]; and lipids modified for conjugation, such as l,2-Dioleoyl-sn-Glycero-3-Phosphoethanolamine-N- (succinyl) and l,2-Dipalmitoyl-sn-Glycero-3-Phosphoethanolamine-N-(Biotinyl ). Suitable lipids whose tail groups have been chemically-modified include, but are not limited to, polymerisable lipids, such as l,2-bis(10,12-tricosadiynoyl)-sn-Glycero-3-Phosphocholine; fluorinated lipids, such as l-Palmitoyl-2-(16-Fluoropalmitoyl)-sn-Glycero-3-Phosphocholi ne; deuterated lipids, such as l,2-Dipalmitoyl-D62-sn-Glycero-3-Phosphocholine; and ether linked lipids, such as 1,2-Di-O- phytanyl-sn-Glycero-3-Phosphocholine. The lipids may be chemically-modified or functionalised to facilitate coupling of the ligands, receptors ro antibodies as discussed above.

The lipid composition may comprise one or more additives that will affect the properties of the microbubble. Suitable additives include, but are not limited to, fatty acids, such as palmitic acid, myristic acid and oleic acid; fatty alcohols, such as palmitic alcohol, myristic alcohol and oleic alcohol; sterols, such as cholesterol, ergosterol, lanosterol, sitosterol and stigmasterol;

lysophospholipids, such as l-Acyl-2-Hydroxy-sn- Glycero-3-Phosphocholine; and ceramides.

The microbubble shell is preferably formed from a phospholipid. Suitable phospholipids are known in the art.

There are several commercially available lipid shell microbubble formulations such as Definity (Lantheus Medical Imaging) and Sonovue® (Bracco Diagnostics).

The microbubble may also be formed from a polymer-surfactant hybrid that involves forming poly electrolyte multilayer (PEM) shells on a preformed microbubble. The preformed microbubble is coated with a charged surfactant or protein layer, which serves as a substrate for PEM deposition. The layer-by -layer assembly technique is used to sequentially adsorb oppositely charged polyions to the microbubble shell. For instance, PEM can be deposited onto microbubbles using poly(allylamine hydrochloride) (PAH) and poly(styrene sulfonate) (PSS) for the polyion pair. PEM microbubbles with phospholipid containing the cationic headgroup trimethylammonium propane (TAP) as the underlying shell and DNA and poly(L-lysine) (PLL) as the polyion pair have also been developed.

The microbubble of the invention is typically formed by providing an interface between a gas and a microbubble shell material. Any of the materials discussed above may be used. Some materials, such as phospholipids, spontaneously form microbubbles. Phospholipids self assemble into a microbubble. Other materials require soni cation of the interface, i.e. the application of sound energy or sonic waves to the interface. Ultrasonic waves are typically used. Suitable methods are known in the art for sonication.

The microbubble of the invention comprises at least one duplex polynucleotide and at least one GalNAc molecule. These are discussed in more detail below. The at least one duplex polynucleotide is typically attached to the at least one GalNAc molecule. The at least one duplex polynucleotide is preferably covalently attached to the at least one GalNAc molecule. Methods for doing this are known in the art. The at least one duplex polynucleotide may be directly attached to the at least one GalNAc molecule. The at least one duplex polynucleotide may be attached to the at least one GalNAc molecule using one or more linkers. Preferred linkers include, but are not limited to, polymers, such as polynucleotides, polyethylene glycols (PEGs), polysaccharides and polypeptides and organic molecules. These linkers may be linear, branched or circular.

The at least one duplex polynucleotide (preferably attached to the at least one GalNAc molecule) is typically located within the gas core of the microbubble or within the microbubble shell or is attached to the microbubble shell. The at least one duplex polynucleotide (preferably attached to the at least one GalNAc molecule) may be attached to inside or the outside of the microbubble shell.

Negatively charged polynucleotides will electrostatically interact with a number of the shell materials used to form microbubbles. Polynucleotides can be attached to the inside or outside of the microbubble shell. The polynucleotide can be attached to both the inside and the outside of the microbubble shell. The polynucleotide can be attached using a variety of methods such as electrostatic attachment or covalent attachment. The at least one GalNAc molecule may also be attached to the microbubble shell using a variety of techniques. The at least one duplex polynucleotide and/or at least one GalNAc molecule may be directly attached to the microbubble shell. The at least one duplex polynucleotide and/or at least one GalNAc molecule may be attached to the microbubble shell using one or more linkers.

The microbubble may be loaded with the at least one duplex polynucleotide (preferably attached to the at least one GalNAc molecule) after formation of the microbubble or during formation of the microbubble. For instance, the microbubble may be formed by providing an interface between (i) a gas and (ii) a microbubble shell material comprising (or attached to) the at least one duplex polynucleotide and at least one GalNAc molecule. The interface is preferably sonicated to form the microbubble. Duplex polynucleotides

The microbubble of the invention comprises at least one duplex polynucleotide. The duplex polynucleotide comprises the sequences shown in (i) SEQ ID NOs: 1 and 2 or variants thereof having at least 90% homology to SEQ ID NOs: 1 and 2 based on sequence identity over their entire length, (ii) SEQ ID NOs: 3 and 4 or variants thereof having at least 90% homology to SEQ ID NOs: 3 and 4 based on sequence identity over their entire length, (iii) SEQ ID NOs: 5 and 6 or variants thereof having at least 90% homology to SEQ ID NOs: 5 and 6 based on sequence identity over their entire length or (iv) SEQ ID NOs: 7 and 8 or variants thereof having at least 90% homology to SEQ ID NOs: 7 and 8 based on sequence identity over their entire length.

A polynucleotide, such as a nucleic acid, is a polymer comprising two or more nucleotides. The nucleotides can be naturally occurring or artificial.

A nucleotide typically contains a nucleobase, a sugar and at least one linking group, such as a phosphate, 2'O-methyl, 2' methoxy-ethyl, phosphoramidate, methylphosphonate or

phosphorothioate group. The nucleobase is typically heterocyclic. Nucleobases include, but are not limited to, purines and pyrimidines and more specifically adenine (A), guanine (G), thymine (T), uracil (U) and cytosine (C). The sugar is typically a pentose sugar. Nucleotide sugars include, but are not limited to, ribose and deoxyribose. The sugar and the nucleobase together form a nucleoside. Preferred nucleosides include, but are not limited to, adenosine, guanosine, 5- methyluridine, uridine, cytidine, deoxyadenosine, deoxyguanosine, thymidine, deoxyuridine and deoxycytidine. The nucleosides are most preferably adenosine, guanosine, uridine and cytidine The nucleotide is typically a ribonucleotide or deoxyribonucleotide. The nucleotide typically contains a monophosphate, diphosphate or triphosphate. Phosphates may be attached on the 5' or 3' side of a nucleotide. Nucleotides include, but are not limited to, adenosine monophosphate (AMP), adenosine diphosphate (ADP), adenosine triphosphate (ATP), guanosine monophosphate (GMP), guanosine diphosphate (GDP), guanosine triphosphate (GTP), thymidine monophosphate (TMP), thymidine diphosphate (TDP), thymidine triphosphate (TTP), uridine monophosphate (UMP), uridine diphosphate (HDP), uridine triphosphate (UTP), cytidine monophosphate (CMP), cytidine diphosphate (CDP), cytidine triphosphate (CTP), 5-methylcytidine monophosphate, 5- methylcytidine diphosphate, 5-methylcytidine triphosphate, 5-hydroxymethylcytidine

monophosphate, 5-hydroxymethylcytidine diphosphate, 5-hydroxymethylcytidine triphosphate, cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphate (cGMP),

deoxyadenosine monophosphate (dAMP), deoxyadenosine diphosphate (dADP), deoxyadenosine triphosphate (dATP), deoxyguanosine monophosphate (dGMP), deoxyguanosine diphosphate (dGDP), deoxyguanosine triphosphate (dGTP), deoxythymidine monophosphate (dTMP), deoxythymidine diphosphate (dTDP), deoxythymidine triphosphate (dTTP), deoxyuridine monophosphate (dUMP), deoxyuridine diphosphate (dUDP), deoxyuridine triphosphate (dUTP), deoxy cytidine monophosphate (dCMP), deoxycytidine diphosphate (dCDP) and deoxy cytidine triphosphate (dCTP), 5-methyl-2'-deoxycytidine monophosphate, 5-methyl-2'-deoxycytidine diphosphate, 5-methyl-2'-deoxycytidine triphosphate, 5 -hydroxymethyl-2' -deoxycytidine monophosphate, 5-hydroxymethy 1-2' -deoxy cytidine diphosphate and 5-hydroxymethyl-2'- deoxycytidine triphosphate. The nucleotides are preferably selected from AMP, UMP, GMP, CMP, dAMP, dTMP, dGMP or dCMP. The nucleotides are most preferably selected from AMP, UMP, GMP and CMP.

The nucleotides may contain additional modifications. In particular, suitable modified nucleotides include, but are not limited to, 2'amino pyrimidines (such as 2'-amino cytidine and 2'- amino uridine), 2'-hyrdroxyl purines (such as , 2'-fluoro pyrimidines (such as 2'-fluorocytidine and 2'fluoro uridine), hydroxyl pyrimidines (such as 5'-a-P-borano uridine), 2'-0-methyl nucleotides (such as 2'-0-methyl adenosine, 2'-0-methyl guanosine, 2'-0-methyl cytidine and 2'-0-methyl uridine), 4'-thio pyrimidines (such as 4'-thio uridine and 4'-thio cytidine) and nucleotides have modifications of the nucleobase (such as 5-pentynyl-2' -deoxy uridine, 5-(3-aminopropyl)-uridine and l,6-diaminohexyl-N-5-carbamoylmethyl uridine).

One or more nucleotides in the polynucleotide may be modified, for instance with a label or a tag. The label may be any suitable label which allows the polynucleotide to be detected. Suitable labels include, but are not limited to, fluorescent molecules, radioisotopes, e.g. ¹²⁵ 1, ³⁵ S, enzymes, antibodies, antigens, other polynucleotides and ligands such as biotin.

The nucleotides in the polynucleotide may be attached to each other in any manner. The nucleotides may be linked by phosphate, 2'0-methyl, 2' methoxy-ethyl, phosphoramidate, methylphosphonate or phosphorothioate linkages. The nucleotides are typically attached by their sugar and phosphate groups as in nucleic acids. The nucleotides may be connected via their nucleobases as in pyrimidine dimers.

The polynucleotide can be nucleic acids, such as deoxyribonucleic acid (DNA) or a ribonucleic acid (RNA). The polynucleotide may be any synthetic nucleic acid known in the art, such as peptide nucleic acid (PNA), glycerol nucleic acid (GNA), threose nucleic acid (TNA), locked nucleic acid (LNA), morpholino nucleic acid or other synthetic polymers with nucleotide side chains. The polynucleotide may comprise any of the nucleotides discussed above, including the modified nucleotides.

The at least one duplex polynucleotide may comprise one strand of DNA and one strand of RNA.

The at least one duplex polynucleotide preferably comprises two strands of RNA.

A duplex polynucleotide is formed by hybridisation of two polynucleotides. For example, SEQ ID NOs: 1 and 2 or variants thereof as defined below. This is shown below for SEQ ID NOs: 1 and 2.

5 ' -GCAGGAAGAACUCAAGUUU -3 ' (SEQ ID NO: 1)

3' -TTCGUCCUUCUUGAGUUCAAA-5' (SEQ ID NO: 2)

The duplex polynucleotide may comprise any combination of two polynucleotides, such as SEQ ID NOs: 1 and 2, SEQ ID NO: 1 and a variant of SEQ ID NO: 2, a variant of SEQ ID NO: 1 and SEQ ID NO: 2 or variants of SEQ ID NOs: 1 and 2. The same applies to SEQ ID NOs: 3 and 4, SEQ ID NOs: 5 and 6 and SEQ ID NOs: 7 and 8.

Conditions that permit the hybridisation are well-known in the art (for example, Sambrook et ah, 2001, Molecular Cloning: a laboratory manual, 3rd edition, Cold Spring Harbour Laboratory Press; and Current Protocols in Molecular Biology, Chapter 2, Ausubel et al, Eds., Greene

Publishing and Wiley-lnterscience, New York (1995)). Hybridisation can be carried out under low stringency conditions, for example in the presence of a buffered solution of 30 to 35% formamide, 1 M NaCl and 1 % SDS (sodium dodecyl sulfate) at 37 °C followed by a 20 wash in from IX (0.1650 M Na+) to 2X (0.33 M Na+) SSC (standard sodium citrate) at 50 °C. Hybridisation can be carried out under moderate stringency conditions, for example in the presence of a buffer solution of 40 to 45% formamide, 1 M NaCl, and 1 % SDS at 37 °C, followed by a wash in from 0.5X (0.0825 M Na+) to IX (0.1650 M Na+) SSC at 55 °C. Hybridisation can be carried out under high stringency conditions, for example in the presence of a buffered solution of 50% formamide, 1 M NaCl, 1% SDS at 37 °C, followed by a wash in 0. IX (0.0165 M Na+) SSC at 60 °C.

RNA duplexes formed from the sequences shown in (i) SEQ ID NOs: 1 and 2, (ii) SEQ ID NOs: 3 and 4, (iii) SEQ ID NOs: 5 and 6 or (iv) SEQ ID NOs: 7 and 8 are disclosed in WO

2005/083083 and are capable of decreasing the amount and/or function of STAT6 via a small interfering RNA (siRNA) or RNA interference (RNAi) effect. Such effects are well documented in the art and are disclosed in WO 2005/083083. RNA interference or "RNAi" is a term initially coined by Fire and co-workers to describe the observation that double-stranded RNA (dsRNA) can block gene expression (Fire et al. (1998) Nature 391, 806-811; Elbashir et al. (2001) Genes Dev. 15, 188-200). Short dsRNA directs gene-specific, post-transcriptional silencing in many organisms, including vertebrates, and has provided a new tool for studying gene function. RNAi is mediated by RNA- induced silencing complex (RISC), a sequence-specific, multi-component nuclease that destroys messenger RNAs homologous to the silencing trigger. RISC is known to contain short RNAs (approximately 22 nucleotides) derived from the double stranded RNA trigger, but the protein components of this activity remained unknown. The at least one duplex polynucleotide used in the invention is capable of small interfering RNA (siRNA) or RNA interference (RNAi) effect

The at least polynucleotide duplex of the invention may comprise variant sequences based on (I) SEQ ID NOs: 1 and 2, (ii) SEQ ID NOs: 3 and 4, , (hi) SEQ ID NOs: 5 and 6 or (iv) SEQ ID NOs: 7 and 8. A variant sequence is a polynucleotide that has a nucleotide sequence which varies from that of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7 or 8 and which retains its ability to specifically hybridise to its partner (i.e. a variant of SEQ ID NO: 1 retains its ability to hybridise to SEQ ID NO: 2 or a variant thereof).

A variant "specifically hybridises" to its partner when it hybridises with preferential or high affinity to the partner but does not substantially hybridise, does not hybridise or hybridises with only low affinity to other polynucleotides. A variant "specifically hybridises" if it hybridises to its partner with a melting temperature (Tm) that is at least 2 °C, such as at least 3 °C, at least 4 °C, at least 5 °C, at least 6 °C, at least 7 °C, at least 8 °C, at least 9 °C or at least 10 °C, greater than its Tm for other polynucleotides. More preferably, the variant hybridises to its partner with a Tm that is at least 2 °C, such as at least 3 °C, at least 4 °C, at least 5 °C, at least 6 °C, at least 7 °C, at least 8 °C, at least 9 °C, at least 10 °C, at least 20 °C, at least 30 °C or at least 40 °C, greater than its Tm for other polynucleotides. Preferably, the variant hybridises to its partner with a Tm that is at least 2 °C, such as at least 3 °C, at least 4 °C, at least 5 °C, at least 6 °C, at least 7 °C, at least 8 °C, at least 9 °C, at least 10 °C, at least 20 °C, at least 30 °C or at least 40 °C, greater than its Tm for a polynucleotide which differs from its partner by one or more nucleotides, such as by 1, 2, 3, 4 or 5 or more nucleotides. The variant typically hybridises to its partner with a Tm of at least 90 °C, such as at least 92 °C or at least 95 °C. Tm can be measured experimentally using known techniques, including the use of DNA microarrays, or can be calculated using publicly available Tm calculators, such as those available over the internet.

A variant sequence is a polynucleotide that has a nucleotide sequence which varies from that of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7 or 8 and which retains its ability to decrease the amount and/or function of STAT6.

The variant sequence may comprise any of the nucleotides discussed above, including the modified nucleotides. The variant sequence is typically the same length as SEQ ID NO: 1, 2, 3, 4, 5, 6, 7 or 8, but may be longer or shorter.

Over the entire length of the sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7 or 8, a variant sequence is at least 90% homologous to that sequence based on nucleotide identity. This allows for variation, deletion or a combination thereof of two nucleotides within the sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7 or 8. Over the entire length of the sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7 or 8, a variant sequence is preferably 90.476% homologous to that sequence based on nucleotide identity.

More preferably, the variant sequence may be at least 95% homologous based on nucleotide identity to SEQ ID NO: 1, 2, 3, 4, 5, 6, 7 or 8 over its entire length. This allows for variation or deletion of one nucleotides within the sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7 or 8. More preferably, the variant sequence may be 95.238% homologous based on nucleotide identity to SEQ ID NO: 1, 2, 3, 4, 5, 6, 7 or 8 over its entire length.

Methods of measuring polynucleotide homology or identity are known in the art. For example the UWGCG Package provides the BESTFIT program which can be used to calculate homology (e.g. used on its default settings) (Devereux et al (1984) Nucleic Acids Research 12, p387-395).

The PILEUP and BLAST algorithms can also be used to calculate homology or line up sequences (typically on their default settings), for example as described in Altschul S.F. (1993) J Mol Evol 36:290-300; Altschul, S, F et al (1990) J Mol Biol 215:403-10.

Software for performing BLAST analysis is publicly available through the National Centre for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). This algorithm involves first identifying high scoring sequence pair (HSPs) by identifying short words of length W in the query sequence that either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighbourhood word score threshold (Altschul et al, supra). These initial neighbourhood word hits act as seeds for initiating searches to find HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Extensions for the word hits in each direction are halted when: the cumulative alignment score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment. The BLAST program uses as defaults a word length (W) of 11, the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1992) Proc. Natl. Acad. Sci. USA 89: 10915-10919) alignments (B) of 50, expectation (E) of 10, M=5, N=4, and a comparison of both strands.

The BLAST algorithm performs a statistical analysis of the similarity between two sequences; see e.g., Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5787. One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a sequence is considered similar to another sequence if the smallest sum probability in comparison of the first sequence to the second sequence is less than about 1, preferably less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.

A variant is typically complementary to its partner over at least 19, at least 20 or at least 21 consecutive nucleotides.

Each polynucleotide in the duplex may be any length. Each polynucleotide in the duplex is preferably 21 or 22 nucleotides in length. The microbubble may comprise any combination of (i) SEQ ID NOs: 1 and 2 or variants thereof having at least 90% homology to SEQ ID NOs: 1 and 2 based on sequence identity over their entire length, (ii) SEQ ID NOs: 3 and 4 or variants thereof having at least 90% homology to SEQ ID NOs: 3 and 4 based on sequence identity over their entire length, (iii) SEQ ID NOs: 5 and 6 or variants thereof having at least 90% homology to SEQ ID NOs: 5 and 6 based on sequence identity over their entire length or (iv) SEQ ID NOs: 7 and 8 or variants thereof having at least 90% homology to SEQ ID NOs: 7 and 8 based on sequence identity over their entire length. In particular, the microbubble may comprise {i} , {ii} , {iii} , {iv} , {i,ii}, {i,iii} , {i,iv} , {ii,iii}, {ii,iv} , {iii,iv} , {i,ii,iii} , {i,ii,iv} , {i,iii,iv} , {ii,iii,iv} or {i,ii,iii,iv} . In such combinations, each of (i) to (iv) may be the sequences shown in SEQ ID NOs: 1 to 8 or variants thereof. Any combination of duplex polynucleotides may be used.

GalNAc

The microbubble of the invention comprises at least one N-Acetylgalactosamine (GalNAc) molecule. This facilitates delivery of the at least one polynucleotide duplex so that it may have its small interfering RNA (siRNA) or RNA interference (RNAi) effect. The at least one

polynucleotide duplex and at least one GalNAc are typically in free association, i. e. are not covalently bonded together.

The microbubble may comprise any number of GalNAc molecules. The microbubble preferably comprises as many GalNAc molecules as is possible. The microbubble preferably comprises at least 1 , at least 2, at least 3, at least 4, at least 5, at least 10, at least 20, at least 30, at least 40, at least 50 or at least 100 GalNAc molecules per at least one polynucleotide duplex. If two or more GalNAc molecules are used, they may be attached, such as covalently attached, together.

The at least GalNAc molecule may be naturally occurring. The at least one GalNAc molecule may be any of the GalNAc molecules disclosed in WO 2009/073809.

The at least one GalNAc molecule may be modified to include one or more magnetic atoms or groups. This allows magnetic targeting of the microbubble as described in more detail below. The magnetic atoms or groups may be paramagnetic or superparamagnetic. Suitable atoms or groups include, but are not limited to, gold atoms, iron atoms, cobalt atoms, nickel atoms and a metal chelating groups, such as nitrilotriacetic acid, containing any of these atoms. The metal chelating group may, for instance, comprise a group selected from -C(=0)0-, -C-0-C-, -C(=0), -NH-, -C(=0)-NH, -C(=0)- CH2-I, -S(=0) ₂ - and -S-. Magnetic particles or beads

The microbubble preferably comprises one or more magnetic particles or beads. The microbubble may contain any number of magnetic particles or beads, such as 50, 100, 500, 1000 or more.

The one or more magnetic particles or beards preferably comprise a paramagnetic or a superparamagnetic material, or a paramagnetic or a superparamagnetic metal, such as iron.

Usually, the one or more magnetic particles or beads comprises a biocompatible magnetic material, such as one or more biocompatible magnetic particles or bead.

Any suitable magnetic particle or bead may be used. For instance, magnetic beads commercially available from, for instance, Clontech, Promega, Invitrogen and NEB, may be used.

In some embodiments, the one or more magnetic particles or beads comprise magnetic particles with an organic group such as a metal-chelating group, such as nitrilotriacetic acid (NTA), attached. The organic component may, for instance, comprise a group selected from -C(=0)0-, - C-O-C-, -C(=0), -NH-, -C(=0)-NH, -C(=0)-CH ₂ -I, -S(=0) ₂ - and -S-. The organic component may comprise a metal chelating group, such as NTA (nitrilotriacetic acid). Usually, a metal such as gold, iron, nickel or cobalt is also attached to the metal-chelating group. Magnetic beads of this sort are commonly used for capturing His-tagged proteins, but are also suitable for use in the invention.

Typically, the microbubble comprises a Ni-NTA or a Co-NTA magnetic bead, for instance, a Ni-NTA magnetic bead.

The presence of one or more magnetic particles or beads allows magnetic targeting of the microbubble as described in more detail below.

Targeting

The microbubble preferably comprises a ligand, a receptor or an antibody or fragment thereof on its surface. This allows the microbubble to be targeted to a cell of interest.

The ligand, receptor or antibody or fragment thereof is preferably attached to the surface of the microbubble. The ligand, receptor or antibody or fragment thereof is preferably covalently attached to the surface of the microbubble. The ligand, receptor or antibody or fragment thereof may be directly attached to the surface of the microbubble. The ligand, receptor or antibody or fragment thereof may be attached to the surface of the microbubble using one or more linkers. Suitable linkers are discussed above. For instance, the microbubble may comprise on its surface a ligand for a receptor expressed on cells which have an increased amount and/or function of STAT6. Alterantively, the microbubble may comprise on its surface a receptor for a ligand expressed on cells which have an increased amount and/or function of STAT6. The expression of the liganuVreceptor may be increased on diseased or disordered cells which have an increased amount and/or function of STAT6 compared with normal cells of the same tissue type. This can be measured using routine methods including those discussed below. A skilled person can identify and design suitable liganuVreceptor combinations for use in the invention.

The microbubble may comprise on its surface an antibody or fragment thereof which specifically binds to a protein on the surface of cells which have an increased amount and/or function of STAT6. The antibody or fragment thereof may specifically bind to a protein whose expression is increased on diseased or disordered cells which have an increased amount and/or function of STAT6 compared with normal cells of the same tissue type.

Screening methods are well known to those of skill in the art which may be used to generate and identify antibodies that are capable of specifically binding to specific proteins (e.g. Making monoclonals: A practical beginners' guide to the production and characterization of monoclonal antibodies against bacteria and viruses, Newell, Public Health Laboratory Service (1988), ISBN-13: 978-0901144232). Antibodies can be tested for specific binding by, for example, standard ELISA or Western blotting. An ELISA assay can also be used to screen for hybridomas that show positive reactivity with the protein. The binding specificity of an antibody may also be determined by monitoring binding of the antibody to the virus, for example by flow cytometry.

An antibody "specifically binds" or "specifically recognises" protein when it binds with preferential or high affinity to the protein for which it is specific but does not substantially bind, or binds with low affinity, to other proteins. The specificity of an antibody may be further studied by determining whether or not the antibody binds to other related proteins or whether it discriminates between them.

An antibody binds with preferential or high affinity if it binds with a Kd of 1 x 10 ^"7 M or less, more preferably 5 x 10 ^"8 M or less, more preferably 1 x 10 ^"8 M or less or more preferably 5 x 10 ^"9 M or less. An antibody binds with low affinity if it binds with a Kd of 1 x 10 ^"6 M or more, more preferably 1 x 10 ^"5 M or more, more preferably 1 x 10 ^"4 M or more, more preferably 1 x 10 ^"3 M or more, even more preferably 1 x 10 ^"2 M or more. A variety of protocols for competitive binding or immunoradiometric assays to determine the specific binding capability of antibodies are well known in the art (see for example Maddox et al, J. Exp. Med. 158, 1211-1226, 1993).

The microbubble may comprise whole antibodies and any antigen binding fragment (i.e., "antigen-binding portion") or single chains thereof. An antibody refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, or an antigen binding portion thereof. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. Each light chain is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). The constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.

The antibody may be a monoclonal antibody or a polyclonal antibody, and will preferably be a monoclonal antibody. The antibody may be a chimeric antibody, a CDR-grafted antibody, a nanobody, a human or humanised antibody or an antigen binding portion of any thereof. For the production of both monoclonal and polyclonal antibodies, the experimental animal is typically a nonhuman mammal such as a goat, rabbit, rat or mouse but may also be raised in other species such as camelids. Polyclonal antibodies may be produced by routine methods such as immunisation of a suitable animal, with the antigen of interest. Blood may be subsequently removed from the animal and the IgG fraction purified. Monoclonal antibodies (mAbs) of the invention can be produced by a variety of techniques, including conventional monoclonal antibody methodology e.g., the standard somatic cell hybridization technique of Kohler and Milstein. The preferred animal system for preparing hybridomas is the murine system. Hybridoma production in the mouse is a very well- established procedure and can be achieved using techniques well known in the art.

The term "antigen-binding portion" of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term "antigen-binding portion" of an antibody include a Fab fragment, a F(ab')2 fragment, a Fab' fragment, a Fd fragment, a Fv fragment, a dAb fragment and an isolated complementarity determining region (CDR). Single chain antibodies such as scFv antibodies are also intended to be encompassed within the term "antigen-binding portion" of an antibody. These antibody fragments may be obtained using conventional techniques known to those of skill in the art, and the fragments may be screened for utility in the same manner as intact antibodies.

An antibody for use in the invention may be prepared, expressed, created or isolated by recombinant means, such as (a) antibodies isolated from an animal (e.g., a mouse) that is transgenic or transchromosomal for the immunoglobulin genes of interest or a hybridoma prepared therefrom, (b) antibodies isolated from a host cell transformed to express the antibody of interest, e.g., from a transfectoma, (c) antibodies isolated from a recombinant, combinatorial antibody library, and (d) antibodies prepared, expressed, created or isolated by any other means that involve splicing of immunoglobulin gene sequences to other DNA sequences.

Once a suitable antibody has been identified and selected, the amino acid sequence of the antibody may be identified by methods known in the art. The genes encoding the antibody can be cloned using degenerate primers. The antibody may be recombinantly produced by routine methods.

Population of the invention

The invention also provides a population of microbubbles of the invention. Any number of microbubbles may be present in the population. The population of the invention preferably comprises at least about 5 x 10 ⁵ microbubbles of the invention. The population more preferably comprises at least about 1 x 10 ⁶ , at least about 2 x 10 ⁶ , at least about 5 x 10 ⁶ , at least about 1 x 10 ⁷ , at least about 2 x 10 ⁷ , at least about 5 x 10 ⁷ , at least about 1 x 10 ⁸ or at least about 2 x 10 ⁸ microbubbles of the invention. In some instances, the population may comprise at least about 1.0 x 10 ⁷ , at least about 1.0 x 10 ⁸ , at least about 1.0 x 10 ⁹ , at least about 1.0 x 10 ¹⁰ , at least about 1.0 x 10 ¹¹ or at about least 1.0 x 10 ¹² microbubbles of the invention or even more.

The population of the invention is preferably homologous. In other words, all of the microbubbles in the population are approximately the same size, contain the same type and number of duplex polynucleotides and contain the same type and number of GalNAc molecules. However, the population may be heterogeneous. In other words, the microbubbles in the population are different sizes, contain different types and/or numbers of duplex polynucleotides and/or contain different types and numbers of GalNAc molecules.

Medicaments, methods and therapeutic use

The microbubbles of the invention may be used in a method of therapy of the human or animal body. Thus the invention provides a microbubble of the invention, a population of the invention or a pharmaceutical composition of the invention for use in a method of treatment of the human or animal body by therapy. In particular, the invention concerns using the a microbubble or microbubbles of the invention to treat a disease or disorder associated with an increased amount and/or function of STAT6 in a patient.

The invention provides a method of treating a disease or disorder associated with an increased amount and/or function of STAT6 in a patient, comprising administering to the patient a population of the invention, wherein the population comprises a therapeutically effective number of microbubbles, and thereby treating the disease or disorder in the patient. The invention also provides a population of the invention for use in treating a disease or disorder associated with an increased amount and/or function of STAT6 in a patient. The invention also provides use of a population of the invention in the manufacture of a medicament for treating a disease or disorder associated with an increased amount and/or function of STAT6 in a patient.

A disease or disorder is associated with an increased amount and/or function of STAT6 if the amount and/or function of STAT6 in the diseased or disordered cells is increased compared with STAT6 amount or function in normal cells of the same tissue type. In the context of the invention, an increased function includes constitutive function.

The amount and/or function of STAT6 may be increased by any amount. For instance, the amount and/or function may be increased by at least 10%, at least 30% at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 95% compared with the amount and/or function of STAT6 in normal cells of the same tissue type. The amount and function of STAT6 may be increased to different degrees.

An increased amount of STAT6 may be an increased amount of STAT6 protein or an increased amount of STAT6 mRNA.

The amount of STAT6 protein can be measured using immunohistochemistry, western blotting, mass spectrometry or fluorescence-activated cell sorting (FACS). Suitable antibodies for use in these techniques are commercially available, for instance from Abeam® or Cell Signalling Technology®.

The amount of STAT6 mRNA can be measured using quantitative reverse transcription polymerase chain reaction (qRT-PCR), such as real time qRT-PCR, northern blotting or microarrays.

The function of STAT6 can be determined using known assays. Such assays use know techniques such as cell proliferation assay, transcription regulation assays, immunohistochemistry, western blotting, mass spectrometry and FACS.

An increased function on STAT6 may also be measured by identifying mutations in STAT6 DNA and/or mRNA which result in an increased function or constitutive function of STA6.

Various mutations which result in constitutive activation of STAT6 are known in the art (e.g. Ritz et al, Blood. Aug 2009; 114(6): 1236-1242). Mutations in the STAT6 gene may be identified using DNA sequencing including next-generation sequencing. This may also be done using Southern blotting, measuring copy-number variation and investigating STAT6 promoter methylation. Mutations in STAT6 mRNA may be identified using RNA sequencing including next-generation sequencing.

The disease or disorder associated with an increased amount and/or function of STAT6 may be selected from the an immune system disease or disorder, cancer or rheumatoid arthritis. The immune system disease or disorder is preferably selected from an allergy, non-allergic rhinitis, asthma and lymphoproliferative diseases or disorders. The cancer is preferably breast cancer, prostate cancer or lung cancer.

The microbubbles of the invention are particularly suited for treating lung diseases or disorders associated with an increased amount and/or function of STAT6, such as asthma and lung cancer. Human bronchioles have a diameter of approximately 1mm. Human alveoli have a diameter of approximately 200μηι. The smallest human lung capillaries have diameters of approximately 10 to 20μηι. The microbubbles of the invention, which are typically 8μηι or less in diameter, will enter and become trapped with the lung capillaries. This ensures the maximum dispersal of the at least one duplex polynucleotide.

The invention concerns administering to the patient a therapeutically effective number of microbubbles of the invention to the patient. A therapeutically effective number is a number which ameliorates one or more symptoms of the disease or disorder. A therapeutically effective number is preferably a number which abolishes one or symptoms of the disease or disorder. A therapeutically effective number may cure or abolish the disease or disorder. Suitable numbers are discussed in more detail below.

The microbubbles of the invention may be administered to any suitable patient. The patient is generally a human patient. The patient may be an infant, a juvenile or an adult. The patient may be known to have the disease or disorder or suspected of having the disease or disorder. The patient may be susceptible to, or at risk from, the disease or disorder. For instance, the patient may be genetically predisposed to breast cancer.

The invention may be used in combination with other means of, and substances for, treating the disease or disorder or providing pain relief. In some cases, the microbubbles of the invention may be administered simultaneously, sequentially or separately with other substances which are intended for treating the disease or disorder. The microbubbles may be used in combination with existing treatments for the disease or disorder and may, for example, be simply mixed with such treatments. Thus the invention may be used to increase the efficacy of existing treatments of the disease or disorder.

The microbubbles of the invention deliver the at least polynucleotide duplex to the site of the disease or disorder so that it can decrease STAT6 amount and/or function via a small interfering RNA (siRNA) or RNA interference (RNAi) effect. The microbubbles are preferably targeted to the disease or disorder as discussed above.

If the microbubbles are magnetic as discussed above, the method of the invention preferably comprises targeting the microbubbles to the disease or disorder using a magnetic field.

The method preferably further comprises breaking down (or destroying or bursting or dissolving) the microbubbles using ultrasonic waves. This typically releases the at least one polynucleotide duplex, especially if it is within the microbubbles, and allows it to have its effect.

Suitable methods of ultrasonic destruction of microbubbles are known in the art.

Pharmaceutical compositions and administration

The invention additionally provides a pharmaceutical composition comprising (a) a microbubble of the invention or a population of microbubbles of the invention and (b) a pharmaceutically acceptable carrier or diluent. The composition may comprise any of the microbubbles or populations mentioned herein. The invention provides a method of treating a disease or disorder associated with an increased amount and/or function of STAT6 comprising administering to the patient an effective amount of a pharmaceutical composition of the invention. Any of the therapeutic embodiments discussed above equally apply to this embodiment.

The various compositions of the invention may be formulated using any suitable method. Formulation of microbubbles with standard pharmaceutically acceptable carriers and/or excipients may be carried out using routine methods in the pharmaceutical art. The exact nature of a formulation will depend upon several factors including the microbubbles to be administered and the desired route of administration. Suitable types of formulation are fully described in

Remington's Pharmaceutical Sciences, 19 ^th Edition, Mack Publishing Company, Eastern

Pennsylvania, USA.

The microbubbles may be administered by any route. Suitable routes include, but are not limited to, enteral or parenteral routes such as via buccal, anal, pulmonary, intravenous, intraarterial, intramuscular, intraperitoneal, intraarticular, topical and other appropriate routes. If the lungs are being treated, the microbubbles may be administered by inhalation.

Compositions may be prepared together with a physiologically acceptable carrier or diluent. Typically, such compositions are prepared as liquid suspensions of microbubbles. The

microbubbles may be mixed with an excipient which is pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water, saline, dextrose, glycerol, of the like and combinations thereof.

Liquid dispersions for oral administration may be syrups, emulsions or suspensions. The syrups may contain as carriers, for example, saccharose or saccharose with glycerine and/or mannitol and/or sorbitol.

Suspensions and emulsions may contain as carrier, for example a natural gum, agar, sodium alginate, pectin, methylcellulose, carboxymethylcellulose, or polyvinyl alcohol. The suspensions or solutions for intramuscular injections may contain, together with the active substance, a pharmaceutically acceptable carrier, e.g. sterile water, olive oil, ethyl oleate, glycols, e.g. propylene glycol, and if desired, a suitable amount of lidocaine hydrochloride.

Solutions for intravenous administration or infusion may contain as carrier, for example, sterile water or preferably they may be in the form of sterile, aqueous, isotonic saline solutions. For suppositories, traditional binders and carriers may include, for example, polyalkylene glycols or triglycerides; such suppositories may be formed from mixtures containing the active ingredient in the range of 0.5% to 10%, preferably 1% to 2%.

Oral formulations include such normally employed excipients as, for example,

pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like. These compositions typically take the form of solutions or suspensions and contain 10% to 95% of active ingredient, preferably 25% to 70%. Where the pharmaceutical composition is lyophilised, the lyophilised material may be reconstituted prior to administration, e.g. a suspension. Reconstitution is preferably effected in buffer.

In addition, if desired, the pharmaceutical compositions of the invention may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, and/or adjuvants which enhance effectiveness. The composition preferably comprises human serum albumin.

One suitable carrier or diluents is Plasma-Lyte A®. This is a sterile, nonpyrogenic isotonic solution for intravenous administration. Each 100 mL contains 526 mg of Sodium Chloride, USP (NaCl); 502 mg of Sodium Gluconate (C6Hl lNa07); 368 mg of Sodium Acetate Tnhydrate, USP (C2H3Na02 ^» 3H20); 37 mg of Potassium Chloride, USP (KC1); and 30 mg of Magnesium

Chloride, USP (MgC12 ^» 6H20). It contains no antimicrobial agents. The pH is adjusted with sodium hydroxide. The pH is 7.4 (6.5 to 8.0).

The microbubbles are administered in a manner compatible with the dosage formulation and in such amount will be therapeutically effective. The quantity to be administered depends on the subject to be treated, capacity of the patient's immune system and the degree of treatment desired. Precise numbers of microbubbles required to be administered may depend on the judgement of the practitioner and may be peculiar to each patient.

Any suitable number of microbubbles may be administered to a patient. For example, at least, or about, 0.5 x 10 ⁶ , 1.5 x 10 ⁶ , 4.0 x 10 ⁶ or 5.0 x 10 ⁶ microbubbles per kg of patient may administered. For example, at least, or about, 10 ⁵ , 10 ⁶ , 10 ⁷ , 10 ⁸ , 10 ⁹ microbubbles may be administered. As a guide, the number of microbubbles of the invention to be administered may be from 10 ⁵ to 10 ⁹ , preferably from 10 ⁶ to 10 ⁸ . Typically, up to 2 x 10 ⁸ microbubbles are

administered to each patient. Any of the specific numbers discussed above with reference to the populations of the invention may be administered. The following Example illustrates the invention. Examples Example 1 - Production of microbubbles with definite volume carrying siRNA

This was evaluated using Alex Fluor 670 fluorphore tagged siRNA. Prior to exposure to ultra sound Figure 1 (a) shows intact micro bubbles carrying siRNA as showed by the red fluorescence micro bubbles.

Example 2 - Delivery of microbubble-mediated siRNA into cells

Figure 1 (b) shows not only an internalisation of siRNA delivered by microbubbles into cells but also maintain integrity of the siRNA as shown by the expression of red fluorescence from the 5' and 3' fluorescence tags. Internalisation was confirmed when compared to control cells exposed to mock tagged siRNA without any attempt to internalise with microbubble or any other transfectatnt agent. Figure 1 (c) shows that tagged siRNA was bounded to external cell membrane without any sign of internalisation when compared to Figure 1 (b).

Example 3 - Viability of transfected cells

The cells remain viable post exposure to microbubble ultra sound treatment as shown in Figure 1 (d).

SEQUENCE LISTING

SEQ ID NO: 1

GCAGGAAGAACUCAAGUUUTT

SEQ ID NO: 2

AAACUUGAGUUCUUCCUGCTT

SEQ ID NO: 3

ACAGUAC GUUACUAGCCUUT T

SEQ ID NO: 4

AAGGCUAGUAACGUACUGUTT

SEQ ID NO: 5

GAAUCAGUCAAC GUGUUGUT T

SEQ ID NO: 6

ACAACAC GUUGACUGAUUC T T

SEQ ID NO: 7

AGCACUGGAGAAAUCAUCATT

SEQ ID NO: 8

UGAUGAUUUCUCCAGUGCUTT