WO2010059829A2

WO2010059829A2 - Compositions and methods for triggered release rna therapeutics

Info

Publication number: WO2010059829A2
Application number: PCT/US2009/065153
Authority: WO
Inventors: Roger C. Adami; Rachel E. Johns
Original assignee: Mdrna, Inc.
Priority date: 2008-11-19
Filing date: 2009-11-19
Publication date: 2010-05-27
Also published as: WO2010059829A3

Abstract

Provided are compositions for delivery of an active agent including a ribonucleic acid, an RNAi-inducing agent, or an antisense agent, comprising a DILA2 amino acid compound and an intracellularly-cleavable peptide of from six to 100 amino acid residues in length, the intracellularly-cleavable peptide comprising two or more nucleic acid-binding region amino acid sequences connected by one or more intracellularly-cleavable linker amino acid sequences, wherein the nucleic acid-binding regions each contain two or more positively-charged amino acid residues. Further provided are methods of using the compositions to efficiently deliver an active agent such as a nucleic acid.

Description

Compositions and Methods for Triggered Release RNA Therapeutics

FIELD OF THE INVENTION

This disclosure relates generally to processes, compositions and uses for delivery of biologically active agents and drug agents. The processes and compositions of this disclosure are useful for delivery of therapeutic agents to selected cells, tissues, organs or subjects. Embodiments of this invention may provide for delivery of pharmaceuticals and therapeutic agents, including nucleic acid agents, and methods for making and using materials to effect drug delivery. In particular, this invention relates to processes and compositions containing liposomes or lamellar vesicles, and other forms of delivery - enhancing compositions and formulations, as well as therapeutic methods and uses for these delivery materials.

SEQUENCE LISTING

This application includes a sequence listing submitted herewith via EFS-WEB as an ASCII file created on November 17, 2009, named MD-08-17PCT.txt, which is 99,909 bytes in size, and is hereby incorporated by reference in its entirety.

BACKGROUND

The delivery of a therapeutic compound to a subject can be impeded by limited ability of the compound to reach a target cell or tissue, or by restricted entry or trafficking of the compound within cells. Delivery of a therapeutic material is in general restricted by membranes of cells. These barriers and restrictions to delivery can result in the need to use much higher concentrations of a compound than is desirable to achieve a result, which brings the risk of toxic effects and side effects.

One strategy for delivery is to improve transport of a compound into a cell is to carry the compound within a nanoscale particle in which the compound is bound and can be released within the cell. The nanoscale particles or materials can take advantage of mechanisms that exist for selective entry into a cell, while still excluding exogenous molecules such as nucleic acids and proteins.

For example, a peptide or other biological molecule may interact with a drug agent and provide contact with a cell membrane. The peptide may aggregate or condense the agent into particles as carriers. The carrier particles can protect the agent from degradation while improving its uptake by cells. Also, positively charged carrier particles may interact with negatively charged cell membranes to initiate transport across a membrane.

In general, a drawback of carrier particles is that the agent can be bound in the particle and unavailable to cause a biological effect in the cell. The agent should be released from the carrier particle after entry into the cell.

The understanding of regulatory RNA and the development of RNA interference (RNAi), RNAi therapy, RNA drugs, antisense therapy, and gene therapy, among others, has increased the need for effective means of introducing active nucleic acid agents into cells. In general, nucleic acids are stable for only limited times in cells or plasma. However, nucleic acid-based agents can be stabilized in compositions and formulations which may then be dispersed for cellular delivery.

This disclosure provides compositions, methods and uses for improving systemic and local delivery of drugs and biologically active molecules. Among other things, this application provides novel compositions and methods for making and using delivery structures and carriers which can release the agent within a cell and increase the efficiency of delivery of biologically active molecules.

BRIEF SUMMARY

This invention overcomes these and other drawbacks by providing a range of carrier compositions for delivering a biological agent to a cell. More particularly, this disclosure provides carrier structures that are nanoscale in size, can reduce toxicity, and can function to condense a nucleic acid agent into small particles. The carrier particles can have increased stability in delivery, and can efficiently deliver a drug agent to modulate gene expression or activity.

This disclosure includes compositions comprising a DILA2 amino acid compound and an intracellularly-cleavable peptide of from six to 100 amino acid residues in length, the intracellularly-cleavable peptide comprising two or more nucleic acid-binding region amino acid sequences connected by one or more intracellularly-cleavable linker amino acid sequences, wherein the nucleic acid-binding regions each contain two or more positively-charged amino acid residues, and further comprising a ribonucleic acid, an RNAi-inducing agent, or an antisense agent.

The intracellularly-cleavable linker may be a portion of a Cathepsin B, D, or L substrate, or Val-Cit. The peptide may contain a binding region sequence. The peptide may bind to a ribonucleic acid, an RNAi-inducing agent, or an antisense agent. In some embodiments, the composition may contain a ribonucleic acid, an RNAi- inducing agent, or an antisense agent. The peptide may be complexed with a ribonucleic acid, an RNAi-inducing agent, or an antisense agent. In some aspects, the composition may contain a nanoparticle formed from the peptide and a ribonucleic acid, an RNAi- inducing agent, or an antisense agent. The composition may contain a nanoparticle formed from the intracellularly-cleavable peptide and a ribonucleic acid, an RNAi- inducing agent, or an antisense agent, or a liposomal particle, or an endosomolytic agent.

In some embodiments, this disclosure encompasses a method for delivering an active agent to a cell comprising preparing a composition containing a cleavable or crosslinkable peptide and treating the cell with the composition.

In certain embodiments, this disclosure encompasses a method for delivering an active agent to a cell comprising preparing a composition containing a cleavable or crosslinkable peptide and treating the cell with the composition.

In some embodiments, this disclosure encompasses a method for inhibiting expression of a gene in a cell comprising preparing a composition containing a cleavable or crosslinkable peptide and treating the cell with the composition.

In certain embodiments, this disclosure encompasses a method for inhibiting expression of a gene in a mammal comprising preparing a composition containing a cleavable or crosslinkable peptide and administering the composition to the mammal. In certain embodiments, this disclosure encompasses a method for treating a disease in a human, the disease being selected from inflammatory diseases including rheumatoid arthritis, metabolic diseases including hypercholesterolemia, liver disease, encephalitis, bone fracture, heart disease, viral disease including hepatitis and influenza, and cancer, comprising preparing a composition containing a cleavable or crosslinkable peptide and administering the composition to the human.

This summary, taken along with the detailed description of the invention, as well as the figures, if any, the appended examples and claims, as a whole, encompass the disclosure of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 : Figure 1 shows that the binding of a polyarginine binding region to dsRNA increased with the length of the polyarginine binding region. In Figure 1, the strongest binding (best ability to displace SYBR-GoId dye) was observed with PN3499, peptide (SEQ ID NO: 159) RRRRRCCRRRRR, which was a dimer peptide containing at total of 10 arginines. DETAILED DESCRIPTION

This disclosure relates generally to novel compounds and compositions, as well as methods and uses thereof, for delivery of biologically active agents and drug agents. The compounds and compositions of this disclosure are useful for delivery of therapeutic agents to selected cells, tissues, organs or subjects. More particularly, this disclosure relates to the delivery of therapeutic agents, including nucleic acid agents, and methods for making and using materials containing peptides to effect delivery of biologically active agents and drug agents.

This invention relates generally to the fields of carriers for nucleic acids. Carriers for nucleic acids include compounds and compositions formed with peptide components including crosslinkable and cleavable peptide structures. More particularly, this invention provides crosslinkable peptide structures and cleavable peptide structures which bind with a nucleic acid to form complexes or condensate compositions.

In some aspects, this disclosure provides peptide-nucleic acid complexes and compositions which include peptide-nucleic acid core structures, and core structures having various layers of peptides.

The compounds and compositions of this disclosure can provide stable delivery systems for biologically active agents and drug agents.

In some respects, the compounds and compositions of this disclosure can provide biological activity with reduced toxicity.

Methods of using the carriers, peptides, peptide-nucleic acid constructs, and compositions of this disclosure in altering gene expression or activity are also provided, optionally in combination with targeting components, liposomal formulations, and other pharmaceutical formulation components. This invention provides a range of carrier compositions for delivering a biologically active agent to a cell. More particularly, this disclosure provides carrier structures that are nanometer scale in size, can reduce toxicity, and can function to condense a nucleic acid agent into small particles. The carrier particles can have increased stability in delivery, and can efficiently deliver an active agent. In some aspects, the carrier can efficiently deliver an active agent to modulate gene expression or activity.

The compositions and methods of this disclosure are useful for delivery of therapeutic, prophylactic, and diagnostic agents such as nucleic acids, polynucleotides, peptides, proteins, and small molecule compounds and drugs. These compositions may include nanoparticles of various diameters.

This disclosure provides novel compounds, compositions and formulations for intracellular and in vivo delivery of an active agent for use, ultimately, as a therapeutic, which in general maintain cytoprotection and relatively low toxicity. The compounds and compositions of this disclosure are useful for delivery of active agents to selected cells, tissues, organs or compartments in order to alter a disease state or a phenotype.

In some aspects, this disclosure provides compounds, compositions and methods to deliver RNA structures to cells to produce the response of RNA interference, antisense effects, or the regulation or modulation of genomic expression.

In some variations, this disclosure provides compounds, compositions and methods to deliver DNA structures or DNA-containing materials to cells.

As used herein, the term peptide-nucleic acid refers to a peptide bound or complexed to a nuclei acid. The term peptide-nucleic acid does not refer to a "PNA" or "peptide nucleic acid" which is a synthetic polymer.

Carriers and peptide binding regions

In some aspects, carrier compounds and compositions of this disclosure may be formed with peptide components that condense with a biologically active nucleic acid component by binding to the nucleic acid to form particles of nanometer dimensions.

A carrier may be formed when one peptide having one or more binding regions binds to a nucleic acid.

In certain variations, more than one cationic binding region of a peptide may bind to the same or different nucleic acid molecules. The crosslinkable and cleavable peptide structures of this disclosure may advantageously have a plurality of cationic residues which are distributed along the peptide chain in one or more binding regions. Variation of the number and distribution of cationic residues can be used vary the strength of binding of the peptide to an active agent. Peptides of this invention include a cationic peptide having a binding region with sufficient positive charge to bind to a nucleic acid and one or more linker groups. A binding region of a peptide of this invention may have sufficient positive charge to bind to a nucleic acid. Linker groups can link to each other to crosslink two or more peptides into a single molecule. Peptides capable of condensing with an active nucleic acid agent to form a carrier particle of this disclosure may have sufficient positive charge to bind to a nucleic acid and sufficient linker groups to form a self-crosslinked construct that includes a bound nucleic acid. This disclosure provides peptides having sufficient positively-charged residues to bind to a nucleic acid, and being capable of forming a crosslinked peptide.

In some embodiments, the biologically active agent is a nucleic acid agent which can bind with a cationic peptide. A nucleic acid agent may bind one, two, three, four, five, or six peptides, or more, to form a complex. A condensate particle may be formed by aggregation and binding of nucleic acid-pep tide complexes.

In some embodiments, a nucleic acid agent may bind portions of more than one peptide such that the peptide attaches to more than one nucleic acid agent.

Carrier structures or constructs can be formed by admixing a crosslinkable or cleavable peptide of this invention with a biologically active agent to which the peptide binds. Binding of the peptide to the agent can be performed at the same time as crosslinking of the peptide occurs, or before or after the peptides are crosslinked.

In some aspects, the carrier is a crosslinked peptide construct which may be a peptide-nucleic acid condensate. The condensate may form a carrier particle of nanometer dimension which can incorporate a biologically active agent such as a nucleic acid.

Crosslinkable peptides

In some embodiments, the crosslinkable peptides of this invention may contain a crosslinkable terminal residue or group. For example, a crosslinkable peptide may have a single terminal cysteine residue which may crosslink by forming an interpeptide disulfide bond resulting in dimers of the peptide.

In some variations, the peptides may contain one or more sulfhydryl groups which can crosslink to form a multimeric peptide construct which binds to, and may be a carrier for a biologically active agent.

In some embodiments, a crosslinkable group may form a cleavable crosslink that may be cleaved at low pH or may be cleaved by the action of a protein or enzyme. Examples of cleavable crosslinks include chemically-cleavable acid labile crosslinks and enzyme-cleavable crosslinks. Examples of crosslinkable groups include organic groups having up to 1000 atoms, a bifunctional linker, a bifunctional crosslinker, and a heterobifunctional linker. The crosslinkable groups may be substituents of a peptide residue, or may be attached at the terminus of the peptide. Crosslinkable groups include In certain embodiments, crosslinkable peptide structures include peptides having crosslinkable groups at each terminus. In some variations, crosslinkable peptide structures include dimers, trimers, and multimers of peptides having crosslinkable groups at each terminus.

Cleavable peptides

In some aspects, this disclosure provides cleavable peptides containing an internal cleavable linker group located between portions of a peptide sequence.

In some embodiments, a cleavable peptide may have two cationic binding regions linked together by a cleavable group. The cleavable group may be cleaved to detach various binding regions of the peptide from each other.

The cationic binding regions may bind to a biologically active agent such as a nucleic acid.

In some variations, cleavage of the linker group of the peptide to detach the binding regions can allow more rapid dissociation of a peptide from a biologically active agent compared to a peptide that would not be cleaved.

An intracellularly-cleavable linker may be cleaved by chemical reduction, or by the action of various proteins or enzymes in the intracellular environment.

Condensate particles and releasable forms Compounds and compositions of this disclosure include condensate particles or carriers composed of one or more peptide components and one or more active agents.

In general, condensate particles formed with a peptide and an active agent may be anionic, neutral, or cationic. For delivery of the carrier particles in vivo, a neutral or cationic form may be preferred. A condensate particle may be referred to as a core particle.

In some embodiments, a condensate particle may be formed with a first portion of a crosslinkable peptide and an active agent. One or more additional layers of the same or different crosslinkable peptide may be added to the particle. In some variations, a condensate particle may be formed with a first portion of a cleavable peptide and an active agent. One or more additional layers of the same or different cleavable peptide may be added to the particle.

In certain embodiments, a condensate particle may be formed with a first portion of a cleavable peptide and an active agent. One or more additional layers of a crosslinkable peptide may be added to the particle.

In some variations, a condensate particle may be formed with a first portion of a crosslinkable peptide and an active agent. One or more additional layers of a cleavable peptide may be added to the particle. In some embodiments, a condensate particle that is anionic may be formed with a first portion of a crosslinkable or cleavable peptide and an active nucleic acid agent. An additional layer or layers of a cationic crosslinkable or cleavable peptide may be added to the anionic particle to form a neutral or cationic carrier particle.

In certain variations, a condensate particle that is anionic may be formed with a first portion of a crosslinkable or cleavable peptide and an active nucleic acid agent. An additional layer or layers of a cationic crosslinkable or cleavable peptide may be added to the anionic particle to form a neutral or cationic carrier particle. An additional layer or layers of an anionic endosomolytic compound may be added to the neutral or cationic carrier particle to form a layered neutral or cationic carrier particle. In some aspects, the active agents may be one or more drug compounds, one or more antisense agents, one or more RNAi-inducing agents, or one or more DNA- containing agents.

In optional embodiments, a composition or formulation of this disclosure may be prepared by loading condensate particles or layered carrier particles into cationic liposomes.

In some embodiments, the compositions and methods of this disclosure may provide delivery of therapeutic agents in releasable forms or compositions. Releasable forms and compositions include molecules that bind and release an active agent, molecules that bind an active agent and discharge a moiety that assists in release of the agent, molecules that bind an active agent and are subsequently modulated in form within a biological compartment to assist in release of the agent, and compositions containing molecules that bind an active agent admixed with a release mediator compound.

As used herein, releasable forms include those containing a crosslinkable or cleavable peptide of this disclosure, or a form containing an endosomolytic compound or material. A condensate or carrier particle may contain a cleavable peptide structure or matrix. Cleavage of a peptide structure can be triggered by certain events such as entry of the carrier into a biological environment or compartment containing a compound which can cleave the peptide crosslinks. Cleavage of peptide linker groups can occur intracellular^ in the cytosol or in various cellular or extracellular compartments.

Cleavage of disulfide peptide linker groups can be done chemically, for example, by reduction of the disulfide with tris(2-carboxyethyl) phosphine hydrochloride (TCEP), dithiothreitol (DTT), or mercaptoethanol.

In certain embodiments, a disulfide reductase may be used to cleave peptide disulfide bonds .

Once within a cell, disulfide crosslinks may be reduced, thereby releasing an active agent for efficient delivery. The environment of the endosome is believed to be reducing and to mediate disulfide reduction and release of the active agent.

Release within a cell can occur by disruption or cleavage of the peptide crosslinks, as well as by dissociation of the biologically active agent from the peptide.

The peptides and peptide constructs of this invention may advantageously contain one, two, or more binding regions having one or more positively-charged amino acid residues. The binding regions can be attached in a chain where one positively-charged binding region is cleavably-linked to the next binding region by a cleavable crosslink. The cationic regions may serve as binding regions for an active agent, such as a nucleic acid agent, and several cationic regions may bind to the same active agent to cooperatively attach the peptide to the active agent.

In certain embodiments, a releasable form of this disclosure includes a peptide- nucleic acid condensate particle, where the peptide component includes crosslinks that can be cleaved to effect release of the nucleic acid. Cleavage of linker groups of the peptides may be triggered by a change in the environment of the peptide such as would occur in transport from extracellular to intracellular domains, or during endocytosis or uptake and delivery of endosomes by cells.

Examples of cleavable linkers for peptides include acid-cleavable groups such as hydrazone which may be cleaved during endocytosis or through intracellular interaction with lysosomes.

In some embodiments, release of the active agent may be provided by an acid- labile linker.

Examples of acid-labile linkers include linkers containing an orthoester group, a hydrazone, a cis-acetonyl, an acetal, a ketal, a silyl ether, a silazane, an imine, a citriconic anhydride, a maleic anhydride, a crown ether, an azacrown ether, a thiacrown ether, a dithiobenzyl group, a cis-aconitic acid, a cis-carboxylic alkatriene, methacrylic acid, and mixtures thereof.

Examples of acid-labile groups and linkers are given in U.S. Patent Nos. 7,098,032; 6,897,196; 6,426,086; 7,138,382; 5,563,250; and 5,505,931.

Examples of cleavable linkers for peptides include Cathepsin-cleavable linkers such as Val-Cit which may be cleaved by intracellular Cathepsins. Examples of substrate sequences for Cathepsin B, D, and L are shown in Tables 1, 2, and 3, respectively. Cleavable linkers include di-, tri-, and tetrapeptide subunits of Cathepsin B, D, and L substrates (P2-P2').

In some variations, a releasable form of this disclosure includes a peptide-nucleic acid condensate particle and an endosomolytic compound. In these variations, an endosomolytic compound can assist in release of the core particle and active agent into the cell from an endosome, while the peptide component can include crosslinks that may be cleaved to effect release and dissociation of the nucleic acid from the core condensate particle within the cell.

Examples of endosomolytic compounds include Chloroquin, 4-aminoquinoline, aminoquinoline, Amodiaquine, cell penetrating peptides, Transportan, Penetratin, a hemagglutinin fusion peptide from influenza virus (see for example Han et al., Nat. Struct. Biol. Vol. 8, 715-720, 2001), and influenza-based peptide diINF7.

In some embodiments, condensates of this invention may optionally be delivered to a cell using a liposome composition. Methods for preparing optional liposomal formulations of this disclosure include those discussed in U.S. Provisional Pat. Application No. 61/106,062.

In certain embodiments, carrier particles or constructs can be formulated with a targeting agent for cellular or sub-cellular delivery. In some variations, a carrier particle may be combined with a synthetic polymer stealthing agent such as polyethylene glycol (PEG) to reduce non-specific effects or interaction with blood components. A suitable synthetic polymer includes a polyethylene glycol chain (PEG), or a PEG copolymer such as PEG-polyurethane or PEG-polypropylene. See, e.g., J. Milton Harris, Poly(ethylene glycol) chemistry: biotechnical and biomedical applications (1992). Methods of use

This disclosure includes a method for delivering a therapeutic nucleic acid to a cell comprising preparing a composition containing a carrier particle containing a nucleic acid agent and treating a cell with the composition. This disclosure includes a method for inhibiting expression of a gene in a cell comprising preparing a composition containing a carrier particle containing a nucleic acid agent and treating a cell with the composition.

This disclosure includes a method for inhibiting expression of a gene in a mammal comprising preparing a composition containing a carrier particle containing a nucleic acid agent and administering the composition to the mammal.

This disclosure includes a method for treating a disease in a human, the disease being selected from inflammatory diseases including rheumatoid arthritis, metabolic diseases including hypercholesterolemia, liver disease, encephalitis, bone fracture, heart disease, viral disease including hepatitis and influenza, and cancer, comprising preparing a liposomal composition and administering the composition to the human.

Active agents

In some aspects, this disclosure provides methods for making compositions suitable for delivery of therapeutic agents. The methods of this disclosure may provide compositions of nucleic acid agents, such as condensed RNA nanoparticles, two- or three- stranded RNA structures, RNA peptide conjugates, dicer substrate RNAs, dsRNAs, siRNAs, microRNAs, hairpin RNAs, other active and regulatory RNA forms, antisense therapeutic forms including antisense RNA and DNA, and DNA and DNA-containing forms. The active agent of this disclosure may be a single-stranded or double- stranded nucleic acid. The active agent of this disclosure may be an antigenic or immunogenic protein or polypeptide.

The active agent of this disclosure may be a peptide condensate of an active agent. For example, an active agent may be composed of nanoparticles formed by condensing an active agent with a peptide or other biomolecule, or a condensate or complex of an active agent with a peptide, biomolecule, or polymeric molecule. Nanoparticles or condensates may be crosslinked. Nanoparticles or condensates can optionally be loaded as cargo into a liposomal composition.

The active agent of this disclosure may be an antisense or sense, DNA or RNA oligonucleotide, or a modified DNA or RNA oligonucleotide which binds to target nucleic acid sequence to block transcription or translation of the target sequence by various interactions. An antisense or sense agent may form a triple helix with a nucleotide double helix, or may be a ribozyme, or may encode transcriptional or translational regulatory sequences including promoter sequences or enhancer sequences. An antisense or sense oligonucleotide may be used to block expression of a protein and may have modified nucleobases or sugar groups, or other groups, or may be a conjugate with a biomolecule, peptide, or protein, for enhanced stability or activity. An antisense or sense oligonucleotide may be delivered into a cell containing its target nucleic acid by the compositions and methods described herein. An antisense or sense oligonucleotide may be delivered into a cell containing its target nucleic acid using an oligonucleotide-carrier complex, or optionally a liposomal formulation, as described herein.

Crosslinkable and cleavable peptides

Crosslinkable peptides of this invention include those having the structure shown in Formula I:

A-B Formula I

where A is a peptide of from two to about 16 amino acid residues which may contain a cationic binding region, and B is a crosslinkable group, wherein A contains one or more positively charged residues at pH 7.

Examples of B include cysteine.

Other examples of B include organic groups having up to 1000 atoms, a bifunctional linker, a bifunctional crosslinker, a heterobifunctional linker, a carbamate, and an ester.

Examples of A include cationic peptides.

Examples of A include cationic peptides having the structure shown in Formula II:

Formula II

where Xaa is an amino acid residue, each of Xaa¹, Xaa², Xaa³, and Xaa⁴ are independently selected amino acid residues which are the same or different, each of m, n, o, and p is from zero to four provided that the sum of m, n, o, and p is two or more, wherein one or more of Xaa¹, Xaa², Xaa³, and Xaa⁴ is a positively charged residue at pH 7. Cationic peptides can be prepared where, for example, a residue of A has a basic side chain. Examples of amino acids having a basic side chain include arginine (Arg), homoarginine (homoArg) (side chain -(CH₂)₄NH(C=NH)NH₂), norarginine (norArg) (side chain -(CH₂)₂NH(C=NH)NH₂), nor-norarginine (nornorArg) (side chain -(CH₂)NH(C=NH)NH₂), ornithine, lysine, homolysine, histidine, 1-methylhistidine, pyridylalanine (Pal), asparagine, N-ethylasparagine, glutamine, and 4-aminophenylalanine, and side chain modified derivatives thereof.

As used herein, the term "homo," when referring to an amino acid, means that an additional carbon is added to the side chain, while the term "nor," when referring to an amino acid, means that a carbon is subtracted from the side chain. Thus, homolysine refers to side chain-(CH₂)₅NH₂.

Cationic peptides can also be prepared where the side chain of a residue contains an ionizable group or substituent.

In some embodiments, the cationic residue is N^G-methylarginine, symmetric or asymmetric N^G,N^G-dimethylarginine, N^G-methyl -homoarginine, symmetric or asymmetric N^G,N^G-dimethyl-homoarginine, N^G-methyl-norarginine, symmetric or asymmetric N^G,N^G-dimethyl-norarginine, or N^G-methyl-nor-norarginine, symmetric or asymmetric N^G,N^G-dimethyl-nor-norarginine.

In some embodiments, the cationic residue is N^G-ethylarginine, symmetric or asymmetric N^G,N^G-diethylarginine, N^G-ethyl-homoarginine, symmetric or asymmetric N^G,N^G-diethyl-homoarginine, N^G-ethyl-norarginine, symmetric or asymmetric N^G,N^G- diethyl-norarginine, or N^G-ethyl-nor-norarginine, symmetric or asymmetric N^G,N^G- diethyl-nor-norarginine.

In certain embodiments, the cationic residue is N^G-alkylarginine, symmetric or asymmetric N^G,N^G-dialkylarginine, N^G-alkyl-homoarginine, symmetric or asymmetric N^G,N^G-dialkyl-homoarginine, N^G-alkyl-norarginine, symmetric or asymmetric N^G,N^G- dialkyl-norarginine, or N^G-alkyl-nor-norarginine, symmetric or asymmetric N^G,N^G- dialkyl-nor-norarginine.

In some embodiments, the cationic residue is an amino acid having a guanidine- or amidine-containing side chain. For example, the side chain of the Xaa residue may contain a group such as guanido, amidino, dihydroimidazole, 4-guanido-phenyl, 4-amidino-phenyl, N-amidino-piperidine, N-amidino-piperazine, 4,5-dihydroimidazole, 2-(N-amidino)-pyrrolidinyl, or 4-[(2-aminopyrimidinyl)]ethyl.

Examples of cationic residues may have side chains that include the following structures, as well as their salt forms:

Cleavable peptides of this invention include those which are dimers of the structure shown in Formula I, for example, the dimer A-B-B-A, wherein the linker groups B are capable of linking to each other, and where the linkage -B-B- can be cleaved.

For example, the dimer A-B-B-A may be A-B-(S-S)-B-A where (S-S) is a disulfide linkage. Other examples of linkage -B-B- include organic groups having up to 1000 atoms, a linkage formed with a bifunctional linker, a linkage formed with a bifunctional crosslinker, a linkage formed with a heterobifunctional linker, a hydrzone linker, a carbamate linkage, and an ester linkage.

Crosslinkable peptides of this invention include those having the structure shown in Formula II:

B-A-B Formula II

where A is a peptide of from about two to about 16 amino acid residues, and B is a crosslinkable group as defined above, wherein A contains one or more positively charged residues at pH 7. Examples of B include cysteine.

Examples of A include cationic peptides.

Cleavable peptides of this invention include those which are dimers, trimers, or multimers of the structure shown in Formula II, for example, the dimer B-A-B-B-A-B, and the multimer -(B-A-B)_n-, wherein the linker groups B are capable of linking to each other, and where the linkage -B-B- can be cleaved. Some of these cleavable peptides remain crosslinkable because they retain a crosslinkable group at each terminus.

Examples of cationic binding regions suitable for preparation of peptides of this disclosure are shown in Table 4. A crosslinkable peptide of this disclosure may have a binding region shown in Table 4 with a cysteine attached at either the N-terminus or the C-terminus of the peptide shown in Table 4. A crosslinkable peptide of this disclosure may form a dimer.

Table 4: Binding regions for preparation of peptides

Examples of cleavable peptides of this disclosure are shown in Table 5.

Table 5: Cleavable Peptides

As used herein, amino acid names and designations refer to any stereoisomer of the corresponding amino acid.

In Table 5, a group which is internal to the peptide sequence may provide a cleavage site. For example, an internal cleavage site can be a disulfide bond or a Val-Cit linkage. Examples of cleavable linkages include Phe-Lys, Val-Cit, Ala-Leu, Leu- Ala-Leu, and Ala-Leu-Ala-Leu (SEQ ID NO: 375), as described in U.S. Pat. Publ. No. 20080166363.

Carrier nanoparticles

The carrier particles of this disclosure are generally of uniform particle size. The carrier particle size may be about 300 nm in diameter or less, or about 250 nm or less, or about 200 nm or less, or about 180 nm or less, or about 160 nm or less, or about 150 nm or less, or about 140 nm or less, or about 130 nm or less, or about 120 nm or less, or about 110 nm or less, or about 100 nm or less, or about 90 nm or less, or about 80 nm or less, or about 70 nm or less.

The active agent carrier particles of this disclosure may have a range of particle sizes, for example, from about 50 nm to about 500 nm, or from about 60 nm to about 400 nm, or from about 70 nm to about 300 nm, or from about 70 nm to about 200 nm, or from about 70 nm to about 160 nm, or from about 80 nm to about 160 nm.

The active agent carrier particles of this disclosure may be used in pharmaceutical compositions. Administration of the active agent liposomal compositions of this disclosure to a subject may be parenteral, oral, by inhalation, topical, mucosal, rectal, or buccal. Parenteral use includes subcutaneous, intracutaneous, intravenous, intramuscular, intraarticular, intrasynovial, intrastemal, intrathecal, intralesional, and intracranial injection or infusion techniques.

Effective amount

An effective amount of an active agent composition of this disclosure for treating a particular disease is generally an amount sufficient to ameliorate or reduce a symptom of the disease. An effective amount of an active agent composition of this disclosure may be an amount sufficient to cause any biological effect attributed to the agent. The composition may be administered as a single dosage, or may be administered by repeated dosing.

Efficient delivery of RNAi and antisense agents

In some aspects, the compositions and methods of this invention provide efficient delivery of an active agent by providing carrier particles having a high concentration or density of carrier molecules to active agent molecules. For example, in certain embodiments, based on the charges of the cationic peptides combined with a nucleic acid agent such as an RNAi-inducing agent or an antisense agent, the carrier particle can include structures in which up to six or more peptide binding regions may bind to an active RNA agent. In some variations, based on the mass of the cationic peptides combined with a nucleic acid agent such as an RNAi-inducing agent or an antisense agent, the carrier particle can have over 5,000, or over 6,000, or over 7,000, or over 8,000, or over 9,000, or over 10,000 molecules of the RNA agent per particle.

For example, in some aspects, for spherical particles having an N: P of 2, a density of 1 g/cc, a particle volume of 1.26x10 nm³, composed of peptide having MW 3781.2

(net 7 cationic charges) and duplex RNA having MW 13,500 (net 40 anionic charges), the mass of a particle is 1.26xlO^"9 ug, and the particle has 11.4 peptides per duplex RNA. In this example, the ratio of the mass of peptide to RNA is 4.32 x 10⁴, where the fraction of the mass of the particle represented by RNA is 0.24, the number of duplex RNA molecules in the particle is 13,369, and the number of peptide molecules per particle is 1.53 xlO⁵.

Optional liposomal variations

In some aspects, the carrier particles of this invention may optionally be delivered as encapsulated in a liposomal formulation. For example, carrier particles may optionally be delivered as encapsulated in a liposomal formulation such as the liposomal compositions disclosed in U.S. Pat. Application No. 12/114,284.

For example, in some embodiments, a pharmaceutical formulation of carrier particles of this invention delivered as encapsulated in a liposomal formulation may increase the payload of a duplex RNA by 20-fold compared to a liposomal formulation of the RNA without the peptide carrier particle composition of this invention.

For example, in some embodiments, a pharmaceutical formulation of carrier particles of this invention delivered as encapsulated in a liposomal formulation may decrease the amount of carrier mass by 45% compared to a liposomal formulation of the RNA without the peptide carrier particle composition of this invention.

For example, in some embodiments, a pharmaceutical formulation of carrier particles for an RNA agent includes a peptide-based delivery system which uses peptides in delivering nucleic acids. This system can increase the payload of RNA agent which can be incorporated into a liposomal formulation. Using a peptide-based nanoparticle, the efficiency of delivery may be enhanced, as well as the tissue distribution pattern of the delivery system. In some embodiments, the delivery system may demonstrate an increase in RNA payload up to 20-fold per liposomal particle, while reducing the total amount of carrier excipients by approximately 45 percent. In some variations, the system may achieve a 30% reduction in RNA agent dose as compared to a liposomal formulation without peptides, for example, as measured by in vivo knockdown of ApoB, while maintaining 85% knockdown in mouse liver and knockdown in mouse jejunum. Thus, a pharmaceutical formulation of carrier particles of this invention may significantly improve the delivery efficiency of an RNA agent, such as an siRNA, mdRNA, or an antisense agent.

DILA2 amino acid compounds

Liposomal compositions of this disclosure may include DILA2 amino acid compounds as disclosed in U.S. Pat. Application No. 12/114,284.

In some aspects, DILA2 amino acid compounds of this disclosure may provide delivery of a therapeutic agent in a releasable form. Releasable forms and compositions are designed to provide sufficient uptake of an agent by a cell to provide a therapeutic effect.

Releasable forms include DILA2 amino acid compounds that bind and release an active agent. In some embodiments, release of the active agent may be provided by an acid-labile linker. Examples of hydrolysable and modulatable groups are given in U.S. Patent Nos.

6,849,272; 6,200,599; as well as Z. H. Huang and F. C. Szoka, "Bioresponsive liposomes and their use for macromolecular delivery," in: G. Gregoriadis (ed.), Liposome Technology, 3rd ed. (CRC Press 2006).

Examples of lipids which are modulatable from anionic to neutral forms include cholesteryl hemisuccinate (CHEMS) as described in U.S. Patent Nos. 6,897,196; 6,426,086; and 7,108,863.

Examples of pH-sensitive polymeric materials are given in U.S. Patent No. 6,835,393.

In some embodiments, release of the active agent may be provided by an enzyme- cleavable peptide.

In some embodiments, a range of DILA2 amino acid compounds corresponding to Formula I are represented by the structures

Structure IA

and

Structure IB

where R¹, R², R^N, R³, and R⁴ are defined as above. In some embodiments, R³ and R⁴ are independently selected lipid-like tails which impart sufficient lipophilic character or lipophilicity, such as defined by water/octanol partitioning, to provide delivery across a membrane or uptake by a cell. These tails provide, when used in a DILA2 amino acid compound structure, an amphipathic molecule. Lipid-like tails may be derived from phospholipids, glycolipids, triacylglycerols, glycerophospholipids, sphingolipids, ceramides, sphingomyelins, cerebrosides, or gangliosides, among others, and may contain a steroid.

In certain embodiments, R³ and R⁴ may independently be a lipid-like tail having a glycerol backbone.

In some embodiments, R³ and R⁴ may independently be ClOalkyl, Cl lalkyl, C12alkyl, C13alkyl, C14alkyl, C15alkyl, Clδalkyl, C17alkyl, Clδalkyl, C19alkyl, C20alkyl, C21alkyl, or C22alkyl.

In some embodiments, R³ and R⁴ may independently be lipophilic tails having one of the following structures:

In the figure above, X represents the atom of the tail that is directly attached to the amino acid residue terminus, and is counted as one of the atoms in the numerical designation, for example, "18:3." In some embodiments, X may be a carbon, nitrogen, or oxygen atom.

Phytanoyl

where X is as defined above.

In some embodiments, R³ and R⁴ are independently selected lipid-like tails which may contain a cholesterol, a sterol, or a steroid such as gonanes, estranes, androstanes, pregnanes, cholanes, cholestanes, ergostanes, campestanes, poriferastanes, stigmastanes, gorgostanes, lanostanes, cycloartanes, as well as sterol or zoosterol derivatives of any of the foregoing, and their biological intermediates and precursors, which may include, for example, cholesterol, lanosterol, stigmastanol, dihydrolanosterol, zymosterol, zymostenol, desmosterol, 7-dehydrocholesterol, and mixtures and derivatives thereof. In certain embodiments, R³ and R⁴ may independently be derived from fatty acid- like tails such as tails from myristic acid (C14:0)alkenyl, palmitic acid (C16:0)alkenyl, stearic acid (C18:0)alkenyl, oleic acid (C18: l, double bond at carbon 9)alkenyl, linoleic acid (C18:2, double bond at carbon 9 or 12)alkenyl, linonenic acid (C18:3, double bond at carbon 9, 12, or 15)alkenyl, arachidonic acid (C20:4, double bond at carbon 5, 8, 11, or 14)alkenyl, and eicosapentaenoic acid (C20:5, double bond at carbon 5, 8, 11, 14, or 17)alkenyl. Other examples of fatty acid- like tails are found at Donald Voet and Judith Voet, Biochemistry, 3rd Edition (2005), p. 383.

In some embodiments, R³ and R⁴ may independently be derived from an isoprenoid. As used herein, the term "amino acid" includes naturally-occurring and non- naturally occurring amino acids. Thus, a DILA2 amino acid compound of this invention can be made from a genetically encoded amino acid, a naturally occurring non-genetically encoded amino acid, or a synthetic amino acid.

Examples of amino acids include Ala, Arg, Asn, Asp, Cys, GIn, GIu, GIy, His, He, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and VaI.

Examples of amino acids include azetidine, 2-aminooctadecanoic acid, 2- aminoadipic acid, 3-aminoadipic acid, 2,3-diaminopropionic acid, 2-aminobutyric acid, A- aminobutyric acid, 2,3-diaminobutyric acid, 2,4-diaminobutyric acid, 2-aminoisobutyric acid, 4-aminoisobutyric acid, 2-aminopimelic acid, 2,2'-diaminopimelic acid, 6-aminohexanoic acid, 6-aminocaproic acid, 2-aminoheptanoic acid, desmosine, ornithine, citrulline, N-methylisoleucine, norleucine, tert-leucine, phenylglycine, t-butylglycine, N-methylglycine, sacrosine, N-ethylglycine, cyclohexylglycine, 4-oxo- cyclohexylglycine, N-ethylasparagine, cyclohexylalanine, t-butylalanine, naphthylalanine, pyridylalanine, 3-chloroalanine, 3-benzothienylalanine, A- halophenylalanine, 4-chlorophenylalanine, 2-fluorophenylalanine, 3-fluorophenylalanine, 4-fluorophenylalanine, penicillamine, 2-thienylalanine, methionine, methionine sulfoxide, homoarginine, norarginine, nor-norarginine, N-acetyllysine, 4-aminophenylalanine, N-methylvaline, homocysteine, homoserine, hydroxylysine, allo-hydroxylysine, 3- hydroxyproline, 4-hydroxyproline, isodesmosine, allo-isoleucine, 6-N-methyllysine, norvaline, O-allyl-serine, O-allyl-threonine, alpha-aminohexanoic acid, alpha- aminovaleric acid, and pyroglutamic acid.

As used herein, the term "amino acid" includes alpha- and beta- amino acids.

Other amino acid residues can be found in Fasman, CRC Practical Handbook of Biochemistry and Molecular Biology, CRC Press, Inc. (1989).

In general, a compound may contain one or more chiral centers. Compounds containing one or more chiral centers may include those described as an "isomer," a "stereoisomer," a "diastereomer," an "enantiomer," an "optical isomer," or as a "racemic mixture." Conventions for stereochemical nomenclature, for example the stereoisomer naming rules of Cahn, Ingold and Prelog, as well as methods for the determination of stereochemistry and the separation of stereoisomers are known in the art. See, for example, Michael B. Smith and Jerry March, March's Advanced Organic Chemistry, 5th edition, 2001. The compounds and structures of this disclosure are meant to encompass all possible isomers, stereoisomers, diastereomers, enantiomers, and/or optical isomers that would be understood to exist for the specified compound or structure, including any mixture, racemic or otherwise, thereof.

Examples of DILA2 amino acid compounds include R³-(C=O)-Arg-NH-R⁴ wherein Arg is D- or L-arginine, and R³ and R⁴ are independently alkyl or alkenyl.

Examples of DILA2 amino acid compounds include the following structures:

Examples of DILA2 amino acid compounds include the following structures:

Examples of DILA2 amino acid compounds include the following structures:

Examples of DILA2 amino acid compounds include R³-(C=O)-norArg-NH-R⁴ n norArg is D- or L-norarginine, and R³ and R⁴ are independently alkyl or alkei Examples of DILA2 amino acid compounds include the following structures:

10

Examples of DILA2 amino acid compounds include R³-(C=O)-nornorArg-NH-R⁴ wherein nornorArg is D- or L-nor-norarginine, and R³ and R⁴ are independently alkyl such as heptyl, octyl, nonyl, decyl, and undecyl.

Examples of DILA2 amino acid compounds include the following structures:

Examples of DILA2 amino acid compounds include R³-(C=O)-homoArg-NH-R⁴ wherein homoArg is D- or L-homoarginine, and R³ and R⁴ are independently alkyl such as heptyl, octyl, nonyl, decyl, and undecyl.

Examples of DILA2 amino acid compounds include

R³-(C=O)-4-pyridylalanine-NH-R⁴ wherein the pyridylalanine is D- or L-pyridylalanine, and R³ and R⁴ are independently alkyl such as heptyl, octyl, nonyl, decyl, and undecyl. Examples of R³-(C=O)-pyridylalanine-NH-R⁴ DILA2 amino acid compounds include pharmaceutically-acceptable pyridyl salts, such as 4- [N-methylpyridyl] alanine chloride. Examples of pyridylalanine DILA2 amino acid compounds include the following structures:

Examples of DILA2 amino acid compounds include R³-(C=O)-Lys-NH-R⁴ wherein R³ and R⁴ are independently alkyl or alkenyl.

Examples of DILA2 amino acid compounds include the following structures:

Examples of DILA2 amino acid compounds include R³-(C=O)-His-NH-R⁴ wherein R³ and R⁴ are independently alkyl or alkenyl. Examples of His DILA2 amino acid compounds include the following structures:

Examples of DILA2 amino acid compounds include R³-(C=O)-Xaa-O-R⁴ wherein

R³ is alkyl and R⁴ is a sphingoid.

Examples of DILA2 amino acid compounds include the following structures:

Examples of DILA2 amino acid compounds include R³-(C=O)-Xaa-NH-R⁴ wherein R³ and R⁴ are alkyl or alkenyl. Examples of DILA2 amino acid compounds include the following structure:

Examples of DILA2 amino acid compounds include

(ClOacyl)-Arg-NH-(ClOalkyl) (SEQ ID NO: 11), (C12acyl)-Arg-NH-(C12alkyl) (SEQ ID NO: 11), (C14acyl)-Arg-NH-(C14alkyl) (SEQ ID NO: 11),

(C16acyl)-Arg-NH-(C16alkyl) (SEQ ID NO: 11), (C18acyl)-Arg-NH-(C18alkyl) (SEQ ID NO: 11), (C14acyl)-homoArg-NH-(C14alkyl), (C16acyl)-homoArg-NH-(C16alkyl), (C18acyl)-homoArg-NH-(C18alkyl), (ClOacyl)-norArg-NH-(ClOalkyl), (C12acyl)-norArg-NH-(C12alkyl), (C14acyl)-norArg-NH-(C14alkyl), (C16acyl)-norArg-NH-(C16alkyl), (C18acyl)-norArg-NH-(C18alkyl), (C 1 Oacyl)-nornorArg-NH-(C 1 Oalkyl), (C 12acyl)-nornorArg-NH-(C 12alkyl), (C 14acyl)-nornorArg-NH-(C 14alkyl), (C 16acyl)-nornorArg-NH-(C 1 δalkyl), (C 18acyl)-nornor Arg-NH-(C 18alkyl) , (C 10acyl)-4-Pal-NH-(C 1 Oalkyl) , (C12acyl)-4-Pal-NH-(C12alkyl), (C14acyl)-4-Pal-NH-(C14alkyl), (C 16acyl)-4-Pal-NH-(C 1 δalkyl), (C 18acyl)-4-Pal-NH-(C 18alkyl), (C 10acyl)-4-Pal(Me)-NH-(C 1 Oalkyl) , (C 12acyl)-4-Pal(Me)-NH-(C 12alkyl) , (C 14acyl)-4-Pal(Me)-NH-(C 14alkyl) , (C 16acyl)-4-Pal(Me)-NH-(C 1 δalkyl) , and (C18acyl)-4-Pal(Me)-NH-(C18alkyl).

In general, the designation "C14-norArg-C14," for example, refers to (C13alkyl)-(C=O)-norArg-NH-(C14alkyl) which is the same as (C 14acyl)-norArg-NH-(C 14alkyl).

Examples of DILA2 amino acid compounds include (Clθacyl)-D-Arg-L- Arg-NH-(C1 Oalkyl), (C12acyl)-D-Arg-L-Arg-NH-(C12alkyl), (C14acyl)-D-Arg-L- Arg-NH-(C 14alkyl) , (C 16acyl)-D- Arg-L- Arg-NH-(C 1 δalkyl) , (C 18acyl)-D- Arg-L- Arg-NH-(C18alkyl), (ClOacyl)-D-homoArg-L-homoArg-NH-(ClOalkyl), (C12acyl)-D- homoArg-L-homoArg-NH-(C 12alkyl), (C 14acyl)-D-homoArg-L- homoArg-NH-(C 14alkyl), (C 16acyl)-D-homoArg-L-homoArg-NH-(C 1 δalkyl), (Cl 8acyl)-D-homoArg-L-homoArg-NH-(Cl 8alkyl), (ClOacyl)-D-norArg-L- norArg-NH-(ClOalkyl), (C12acyl)-D-norArg-L-norArg-NH-(C12alkyl), (C14acyl)-D- norArg-L-norArg-NH-(C 14alkyl), (C 16acyl)-D-norArg-L-norArg-NH-(C 1 δalkyl), (C18acyl)-D-norArg-L-norArg-NH-(C18alkyl), (ClOacyl)-D-nornorArg-L- nornorArg-NH-(ClOalkyl), (C^acy^-D-nornorArg-L-nornorArg-NH^C^alkyl), (C 14acyl)-D-nornor Arg-L-nornor Arg-NH-(C 14alkyl) , (C 16acyl)-D-nornor Arg-L- nornorArg-NH-(C 1 δalkyl), (C 18acyl)-D-nornorArg-L-nornorArg-NH-(C 18alkyl).

Examples of DILA2 amino acid compounds include (Clθacyl)-His- Arg-NH-(C1 Oalkyl), (C12acyl)-His-Arg-NH-(C12alkyl), (C14acyl)-His- Arg-NH-(C14alkyl), (C 16acyl)-His-Arg-NH-(Cl δalkyl), (C18acyl)-His- Arg-NH-(C18alkyl), (ClOacyl)-His-Arg-NH-(ClOalkyl), (C12acyl)-His- Arg-NH-(C12alkyl), (C14acyl)-His-Arg-NH-(C14alkyl), (Clδacyl)-His- Arg-NH-(C1 δalkyl), (C18acyl)-His-Arg-NH-(C18alkyl), (ClOacyl)-His-Arg-(ClOalkyl), (C 12acyl)-His- Arg-NH-(C 12alkyl) , (C 14acyl)-His- Arg-NH-(C 14alkyl) , (C 16acyl)-His- Arg-NH-(C1 δalkyl), (C18acyl)-His-Arg-NH-(C18alkyl), (Clθacyl)-His- Arg-NH-(C1 Oalkyl), (C12acyl)-His-Arg-NH-(C12alkyl), (C14acyl)-His- Arg-NH-(C 14alkyl), (C 1 OaCyI)-HiS-ATg-NH-(C 1 δalkyl), (C 18acyl)-His- Arg-NH-(C18alkyl).

Examples of DILA2 amino acid compounds include (Clθacyl)-His- Asp-NH-(ClOalkyl), (C12acyl)-His-Asp-NH-(C12alkyl), (C14acyl)-His- Asp-NH-(C14alkyl), (C 16acyl)-His-Asp-NH-(Cl δalkyl), (C18acyl)-His- Asp-NH-(C18alkyl), (ClOacyl)-His-Asp-NH-(ClOalkyl), (C12acyl)-His- Asp-NH-(C12alkyl), (C14acyl)-His-Asp-NH-(C14alkyl), (C16acyl)-His- Asp-NH-(C1 δalkyl), (C18acyl)-His-Asp-NH-(C18alkyl), (ClOacyl)-His-Asp-(ClOalkyl), (C12acyl)-His-Asp-NH-(C12alkyl), (C14acyl)-His-Asp-NH-(C14alkyl), (C16acyl)-His- Asp-NH-(C1 δalkyl), (C18acyl)-His-Asp-NH-(C18alkyl), (Clθacyl)-His- Asp-NH-(ClOalkyl), (C12acyl)-His-Asp-NH-(C12alkyl), (C14acyl)-His- Asp-NH-(C 14alkyl), (C 16acyl)-His- Asp-NH-(C 1 δalkyl), (C 18acyl)-His- Asp-NH-(C18alkyl).

Examples of DILA2 amino acid compounds include (Clθacyl)-Pal- Arg-NH-(ClOalkyl), (C12acyl)-Pal-Arg-NH-(C12alkyl), (C14acyl)-Pal- Arg-NH-(C14alkyl), (C 16acyl)-Pal-Arg-NH-(Cl δalkyl), (C18acyl)-Pal- Arg-NH-(C18alkyl), (ClOacyl)-Pal-Arg-NH-(ClOalkyl), (C12acyl)-Pal- Arg-NH-(C12alkyl), (C14acyl)-Pal-Arg-NH-(C14alkyl), (Clδacyl)-Pal- Arg-NH-(Clδalkyl), (C18acyl)-Pal-Arg-NH-(C18alkyl), (ClOacyl)-Pal-Arg-(ClOalkyl), (C12acyl)-Pal-Arg-NH-(C12alkyl), (C14acyl)-Pal-Arg-NH-(C14alkyl), (Clδacyl)-Pal- Arg-NH-(C1 δalkyl), (C18acyl)-Pal-Arg-NH-(C18alkyl), (Clθacyl)-Pal- Arg-NH-(ClOalkyl), (C12acyl)-Pal-Arg-NH-(C12alkyl), (C14acyl)-Pal- Arg-NH-(C14alkyl), (C 16acyl)-Pal-Arg-NH-(Cl δalkyl), (C18acyl)-Pal- Arg-NH-(C18alkyl). DILA2 amino acid compounds can be prepared as poly-mer or multi-mer species, such as dimers, trimers, or tetramers. The poly-mer or multi-mer species can be prepared from a single DIL A2 amino acid compound, or from more than one species. Poly-mer or multi-mer DILA2 amino acid species can be prepared in some embodiments by providing a sulfhydryl group or other cross-linkable group on a side chain of the amino acid, or with linked or tethered amino acid structures such as desmosine or citrulline. In other embodiments, a poly-mer or multi-mer DILA2 amino acid species can be prepared with bioconjugate linker chemistries.

Examples of DILA2 amino acid compounds include the following structures:

A DILA2 amino acid compound can be prepared as a conjugate having a peptide or polymer chain covalently attached to the amino acid side chain. The peptide or polymer chain can be attached using a reactive group of the amino acid side chain, for example, using the thiol or methylmercaptan group of cysteine or methionine, respectively, or the alcohol group of serine, or the amino group of lysine. The peptide or polymer chain can be attached using any reactive group of a substituted or modified amino acid side chain. Various linker groups such as NHS, maleimido, and bioconjugate techniques and linkers can be used. DILA2 amino acid compounds can be prepared as constructs attached to an oligomeric or polymeric framework. For example, a DILA2 amino acid compound can be attached to polyethylene glycol, polypropylene glycol, an oligonucleotide network or lattice, a poly(amino acid), a carbohydrate, a dextran, a hydrogel, or a starch.

DILA2 amino acid compounds can be prepared as constructs attached to a pharmaceutical drug compound or composition, or a biologically active agent. For example, a DILA2 amino acid compound can be conjugated to a nucleic acid drug such as a regulatory or interfering RNA.

Examples of DILA2 amino acid compounds include the following structures:

where R is any amino acid side chain.

The compounds and compositions of this disclosure may incorporate solubilizing or functionalizing groups or structures including polymeric structures. See, e.g., R. L. Dunn and R. M. Ottenbrite, Polymeric Drugs and Drug Delivery Systems, ACS Symp. Ser. 469 (1991). DILA2 amino acid compounds can be derivatized to enhance solubility such as, for example, to attach a diol, to prepare a quaternary ammonium or charged group, to attach hydroxyl or amine groups such as alcohols, polyols, or polyethers, or to attach a polyethyleneimine, a polyethyleneglycol or a polypropyleneglycol. The molecular mass of an attached polymeric component such as a polyethyleneglycol can be any value, for example, 200, 300, 400, 500, 750, 1000, 1250, 1500, 2000, 3000, 4000, 5000, 7500, 10,000, 15,000, 20,000, 25,000, or 30,000 Da, or greater. For example, a polyethyleneglycol chain can be attached through an amino group or other reactive group of an amino acid side chain.

In general, as used herein, general chemical terms refer to all groups of a specified type, including groups having any number and type of atoms, unless otherwise specified. For example "alkenyl" refers broadly to alkyls having 2 to 22 carbon atoms, as defined below, while (C18:l)alkenyl refers to alkenyls having 18 carbon atoms and one double bond.

The term "alkyl" as used herein refers to a saturated, branched or unbranched, substituted or unsubstituted aliphatic group containing from 1-22 carbon atoms. This definition applies to the alkyl portion of other groups such as, for example, alkoxy, alkanoyl, aralkyl, and other groups defined below. The term "cycloalkyl" as used herein refers to a saturated, substituted or unsubstituted cyclic alkyl ring containing from 3 to 12 carbon atoms.

The term "alkenyl" as used herein refers to an unsaturated, branched or unbranched, substituted or unsubstituted alkyl or cycloalkyl having 2 to 22 carbon atoms and at least one carbon-carbon double bond. The term "alkynyl" as used herein refers to an unsaturated, branched or unbranched, substituted or unsubstituted alkyl or cycloalkyl having 2 to 22 carbon atoms and at least one carbon-carbon triple bond.

The term "alkoxy" as used herein refers to an alkyl, cycloalkyl, alkenyl, or alkynyl group covalently bonded to an oxygen atom. The term "alkanoyl" as used herein refers to -C(=O)-alkyl, which may alternatively be referred to as "acyl." The term "alkanoyloxy" as used herein refers to -O-C(=O)-alkyl groups. The term "alkylamino" as used herein refers to the group -NRR', where R and R' are each either hydrogen or alkyl, and at least one of R and R' is alkyl. Alkylamino includes groups such as piperidino wherein R and R' form a ring. The term "alkylaminoalkyl" refers to -alkyl-NRR'.

The term "aryl" as used herein refers to any stable monocyclic, bicyclic, or polycyclic carbon ring system of from 4 to 12 atoms in each ring, wherein at least one ring is aromatic. Some examples of an aryl include phenyl, naphthyl, tetrahydro- naphthyl, indanyl, and biphenyl. Where an aryl substituent is bicyclic and one ring is non- aromatic, it is understood that attachment is to the aromatic ring. An aryl may be substituted or unsubstituted.

The term "heteroaryl" as used herein refers to any stable monocyclic, bicyclic, or polycyclic carbon ring system of from 4 to 12 atoms in each ring, wherein at least one ring is aromatic and contains from 1 to 4 heteroatoms selected from oxygen, nitrogen and sulfur. Some examples of a heteroaryl include acridinyl, quinoxalinyl, pyrazolyl, indolyl, benzotriazolyl, furanyl, thienyl, benzothienyl, benzofuranyl, quinolinyl, isoquinolinyl, oxazolyl, isoxazolyl, pyrazinyl, pyridazinyl, pyridinyl, pyrimidinyl, pyrrolyl, and tetrahydroquinolinyl. A heteroaryl includes the N-oxide derivative of a nitrogen- containing heteroaryl. The term "heterocycle" or "heterocyclyl" as used herein refers to an aromatic or nonaromatic ring system of from five to twenty-two atoms, wherein from 1 to 4 of the ring atoms are heteroatoms selected from oxygen, nitrogen, and sulfur. Thus, a heterocycle may be a heteroaryl or a dihydro or tetrathydro version thereof. The term "aroyl" as used herein refers to an aryl radical derived from an aromatic carboxylic acid, such as a substituted benzoic acid. The term "aralkyl" as used herein refers to an aryl group bonded to an alkyl group, for example, a benzyl group.

The term "carboxyl" as used herein represents a group of the formula -C(=0)0H or -C(=O)O^~. The terms "carbonyl" and "acyl" as used herein refer to a group in which an oxygen atom is double-bonded to a carbon atom >C=O. The term "hydroxyl" as used herein refers to -OH or -O^~. The term "nitrile" or "cyano" as used herein refers to -CN. The term "halogen" or "halo" refers to fluoro (-F), chloro (-Cl), bromo (-Br), and iodo

(-1).

The term "substituted" as used herein refers to an atom having one or more substitutions or substituents which can be the same or different and may include a hydrogen substituent. Thus, the terms alkyl, cycloalkyl, alkenyl, alkynyl, alkoxy, alkanoyl, alkanoyloxy, alkylamino, alkylaminoalkyl, aryl, heteroaryl, heterocycle, aroyl, and aralkyl as used herein refer to groups which include substituted variations. Substituted variations include linear, branched, and cyclic variations, and groups having a substituent or substituents replacing one or more hydrogens attached to any carbon atom of the group. Substituents that may be attached to a carbon atom of the group include alkyl, cycloalkyl, alkenyl, alkynyl, alkoxy, alkanoyl, alkanoyloxy, alkylamino, alkylaminoalkyl, aryl, heteroaryl, heterocycle, aroyl, aralkyl, acyl, hydroxyl, cyano, halo, haloalkyl, amino, aminoacyl, alkylaminoacyl, acyloxy, aryloxy, aryloxyalkyl, mercapto, nitro, carbamyl, carbamoyl, and heterocycle. For example, the term ethyl includes without limitation -CH₂CH₃, -CHFCH₃, -CF₂CH₃, -CHFCH₂F, -CHFCHF₂, -CHFCF₃, -CF₂CH₂F, -CF₂CHF₂, -CF₂CF₃, and other variations as described above. In general, substituents may be further substituted with any atom or group of atoms.

DILA2 amino acid compounds of this invention or variants thereof can be synthesized by methods known in the art.

Methods to prepare various organic groups and protective groups are known in the art and their use and modification is generally within the ability of one of skill in the art. See, e.g., Stanley R. Sandler and Wolf Karo, Organic Functional Group Preparations (1989); Greg T. Hermanson, Bioconjugate Techniques (1996); Leroy G. Wade, Compendium Of Organic Synthetic Methods (1980); examples of protective groups are found in T. W. Greene and P. G. M. Wuts, Protective Groups In Organic Synthesis (3rd ed. 1991).

A pharmaceutically acceptable salt of a peptide or protein composition of this invention which is sufficiently basic may be an acid-addition salt with, for example, an inorganic or organic acid such as hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, chlorosulfonic, trifluoroacetic, citric, maleic, acetic, propionic, oxalic, malic, maleic, malonic, fumaric, or tartaric acids, and alkane- or arenesulfonic acids such as methanesulfonic, ethanesulfonic, benzenesulfonic, chlorobenzenesulfonic, toluenesulfonic, naphthalenesulfonic, naphthalenedisulfonic, and camphorsulfonic acids. A pharmaceutically acceptable salt of a peptide or protein composition of this invention which is sufficiently acidic may be an alkali metal salt, for example, a sodium or potassium salt, or an alkaline earth metal salt, for example, a calcium or magnesium salt, or a zinc or manganese salt, or an ammonium salt or a salt with an organic base which provides a physiologic ally- acceptable cation, for example, a salt with methylamine, dimethylamine, trimethylamine, triethylamine, ethanolamine, diethanolamine, triethanolamine, ethylenediamine, tromethamine, N-methylglucamine, piperidine, morpholine or tris-(2-hydroxyethyl)amine, and including salts of amino acids such as arginate, and salts of organic acids such as glucuronic or galactunoric acids. See, for example, Berge et ah, J. Pharm. ScL (5(5:1-19, 1977. A salt or pharmaceutically-acceptable salt of a composition of this disclosure which contains an interfering-RNA agent and a lipid, peptide, or protein, among other components, may contain a salt complex of the interfering-RNA agent and the lipid, peptide, or protein. A salt complex of the interfering-RNA agent and the lipid, peptide, or protein may be formed from a pharmaceutically-acceptable salt of an interfering-RNA agent, or from a pharmaceutically-acceptable salt of the lipid, peptide, or protein.

Some compounds of this disclosure may contain both basic and acidic functionalities that may allow the compounds to be made into either a base or acid addition salt.

Some compounds, peptides and/or protein compositions of this invention may have one or more chiral centers and/or geometric isomeric centers (E- and Z-isomers), and it is to be understood that the invention encompasses all such optical isomers, diastereoisomers, geometric isomers, and mixtures thereof.

This disclosure encompasses any and all tautomeric, solvated or unsolvated, hydrated or unhydrated forms, as well as any atom isotope forms of the compounds, peptides and/or protein compositions disclosed herein. In some aspects of this invention, DILA2 amino acid compounds in combination with lipids may be employed for delivery and administration of regulatory RNA components, RNA antagonists, interfering RNA, or nucleic acids. More particularly, a composition of this invention may include one or more DILA2 amino acid compounds along with a lipid or non-cationic lipid.

Some examples of lipids are described in U.S. Patent Nos. 4,897,355; 5,279,833; 6,733,777; 6,376,248; 5,736,392; 5,334,761 ; 5,459,127; 2005/0064595; 5,208,036; 5,264,618; 5,279,833; 5,283,185; 5,753,613; and 5,785,992.

Uses for regulatory RNA and RNA interference

In some aspects, this disclosure relates generally to the fields of regulatory RNA and RNA interference, antisense therapeutics, and delivery of RNA therapeutics. More particularly, this invention relates to compositions and formulations for ribonucleic acids, and their uses for medicaments and for delivery as therapeutics. This invention relates generally to methods of using ribonucleic acids in RNA interference for gene- specific inhibition of gene expression in cells, or in mammals to alter a disease state or a phenotype.

RNA interference refers to methods of sequence- specific post-transcriptional gene silencing which is mediated by a double- stranded RNA (dsRNA) called a short interfering RNA (siRNA). See Fire, et al, Nature J9i:806, 1998, and Hamilton, et al, Science

286:950-951, 1999. RNAi is shared by diverse flora and phyla and is believed to be an evolutionarily-conserved cellular defense mechanism against the expression of foreign genes. See Fire, et al, Trends Genet. i5:358, 1999.

RNAi is therefore a ubiquitous, endogenous mechanism that uses small noncoding RNAs to silence gene expression. See Dykxhoorn, D. M. and J. Lieberman, Annu. Rev. Biomed. Eng. S:377-402, 2006. RNAi can regulate important genes involved in cell death, differentiation, and development. RNAi may also protect the genome from invading genetic elements, encoded by transposons and viruses. When a siRNA is introduced into a cell, it binds to the endogenous RNAi machinery to disrupt the expression of mRNA containing complementary sequences with high specificity. Any disease-causing gene and any cell type or tissue can potentially be targeted. This technique has been rapidly utilized for gene-function analysis and drug-target discovery and validation. Harnessing RNAi also holds great promise for therapy, although introducing siRNAs into cells in vivo remains an important obstacle. The mechanism of RNAi, although not yet fully characterized, is through cleavage of a target mRNA. The RNAi response involves an endonuclease complex known as the

RNA-induced silencing complex (RISC), which mediates cleavage of a single-stranded

RNA complementary to the antisense strand of the siRNA duplex. Cleavage of the target RNA takes place in the middle of the region complementary to the antisense strand of the siRNA duplex (Elbashir, et al, Genes Dev. 75:188, 2001).

One way to carry out RNAi is to introduce or express a siRNA in cells. Another way is to make use of an endogenous ribonuclease III enzyme called dicer. One activity of dicer is to process a long dsRNA into siRNAs. See Hamilton, et al, Science 286:950- 951, 1999; Berstein, et al, Nature 409:363, 2001. A siRNA derived from dicer is typically about 21-23 nucleotides in overall length with about 19 base pairs duplexed.

See Hamilton, et al, supra; Elbashir, et al, Genes Dev. 75:188, 2001. In essence, a long dsRNA can be introduced in a cell as a precursor of a siRNA.

This invention provides a range of compositions, formulations and methods which include a regulatory RNA, an interfering nucleic acid or a precursor thereof in combination with various components including lipids, DILA2 amino acid compounds, and natural or synthetic polymers.

The term "dsRNA" as used herein refers to any nucleic acid molecule comprising at least one ribonucleotide molecule and capable of inhibiting or down regulating gene expression, for example, by promoting RNA interference ("RNAi") or gene silencing in a sequence- specific manner. The dsRNAs of this disclosure may be suitable substrates for

Dicer or for association with RISC to mediate gene silencing by RNAi. One or both strands of the dsRNA can further comprise a terminal phosphate group, such as a

5'-phosphate or 5', 3 '-diphosphate. As used herein, dsRNA molecules, in addition to at least one ribonucleotide, can further include substitutions, chemically-modified nucleotides, and non-nucleo tides. In certain embodiments, dsRNA molecules comprise ribonucleotides up to about 100% of the nucleotide positions.

Examples of dsRNA molecules can be found in, for example, U.S. Patent

Application No. 11/681,725, U.S. Patent Nos. 7,022,828 and 7,034,009, and PCT International Application Publication No. WO/2003/070897.

Examples of modified nucleosides are found in U.S. Patent Nos. 6,403,566, 6,509,320,

6,479,463, 6,191,266, 6,083,482, 5,712,378, and 5,681,940.

In addition, as used herein, the terms "dsRNA," "RNAi-inducing agent, "and

"RNAi-agent" are meant to be synonymous with other terms used to describe nucleic acid molecules that are capable of mediating sequence specific RNAi including meroduplex RNA (mdRNA), nicked dsRNA (ndsRNA), gapped dsRNA (gdsRNA), short interfering nucleic acid (siRNA), siRNA, microRNA (miRNA), single strand RNA, short hairpin RNA (shRNA), short interfering oligonucleotide, short interfering substituted oligonucleotide, short interfering modified oligonucleotide, chemically-modified dsRNA, and post-transcriptional gene silencing RNA (ptgsRNA), as well as precursors of any of the above.

The term "large double-stranded (ds) RNA" refers to any double-stranded RNA longer than about 40 base pairs (bp) to about 100 bp or more, particularly up to about 300 bp to about 500 bp. The sequence of a large dsRNA may represent a segment of an mRNA or an entire mRNA. A double-stranded structure may be formed by self-complementary nucleic acid molecule or by annealing of two or more distinct complementary nucleic acid molecule strands.

In some aspects, a dsRNA comprises two separate oligonucleotides, comprising a first strand (antisense) and a second strand (sense), wherein the antisense and sense strands are self-complementary (i.e., each strand comprises a nucleotide sequence that is complementary to a nucleotide sequence in the other strand and the two separate strands form a duplex or double-stranded structure, for example, wherein the double-stranded region is about 15 to about 24 base pairs or about 26 to about 40 base pairs); the antisense strand comprises a nucleotide sequence that is complementary to a nucleotide sequence in a target nucleic acid molecule or a portion thereof (e.g., a human mRNA); and the sense strand comprises a nucleotide sequence corresponding (i.e., homologous) to the target nucleic acid sequence or a portion thereof (e.g., a sense strand of about 15 to about 25 nucleotides or about 26 to about 40 nucleotides corresponds to the target nucleic acid or a portion thereof). In some embodiments, the dsRNA may be assembled from a single oligonucleotide in which the self-complementary sense and antisense strands of the dsRNA are linked by together by a nucleic acid based- linker or a non-nucleic acid-based linker. In some embodiments, the first (antisense) and second (sense) strands of the dsRNA molecule are covalently linked by a nucleotide or non-nucleotide linker as described herein and known in the art. In some embodiments, a first dsRNA molecule is covalently linked to at least one second dsRNA molecule by a nucleotide or non-nucleotide linker known in the art, wherein the first dsRNA molecule can be linked to a plurality of other dsRNA molecules that can be the same or different, or any combination thereof. In some embodiments, the linked dsRNA may include a third strand that forms a meroduplex with the linked dsRNA. In some respects, dsRNA molecules described herein form a meroduplex RNA (mdRNA) having three or more strands, for example, an 'A' (first or antisense) strand, 'Sl' (second) strand, and 'S2' (third) strand in which the 'Sl' and 'S2' strands are complementary to and form base pairs (bp) with non-overlapping regions of the 'A' strand (e.g., an mdRNA can have the form of A:S1S2). The Sl, S2, or more strands together essentially comprise a sense strand to the 'A' strand. The double- stranded region formed by the annealing of the 'Sl' and 'A' strands is distinct from and non-overlapping with the double- stranded region formed by the annealing of the 'S2' and 'A' strands. An mdRNA molecule is a "gapped" molecule, meaning a "gap" ranging from 0 nucleotides up to about 10 nucleotides. In some embodiments, the A:S1 duplex is separated from the A:S2 duplex by a gap resulting from at least one unpaired nucleotide (up to about 10 unpaired nucleotides) in the 'A' strand that is positioned between the A:S1 duplex and the A:S2 duplex and that is distinct from any one or more unpaired nucleotide at the 3'-end of one or more of the 'A', 'Sl', or 'S2' strands. In some embodiments, the A:S1 duplex is separated from the A:B2 duplex by a gap of zero nucleotides (i.e., a nick in which only a phosphodiester bond between two nucleotides is broken or missing in the polynucleotide molecule) between the A: Sl duplex and the A:S2 duplex - which can also be referred to as nicked dsRNA (ndsRNA). For example, A:S1S2 may be comprised of a dsRNA having at least two double-stranded regions that combined total about 14 base pairs to about 40 base pairs and the double- stranded regions are separated by a gap of about 0 to about 10 nucleotides, optionally having blunt ends, or A:S1S2 may comprise a dsRNA having at least two double- stranded regions separated by a gap of up to 10 nucleotides wherein at least one of the double-stranded regions comprises between about 5 base pairs and 13 base pairs. As described herein, a dsRNA molecule which contains three or more strands may be referred to as a "meroduplex" RNA (mdRNA). Examples of mdRNA molecules can be found in U.S. Provisional Patent Application Nos. 60/934,930 and 60/973,398.

A dsRNA or large dsRNA may include a substitution or modification in which the substitution or modification may be in a phosphate backbone bond, a sugar, a base, or a nucleoside. Such nucleoside substitutions can include natural non-standard nucleosides (e.g., 5-methyluridine or 5-methylcytidine or a 2-thioribothymidine), and such backbone, sugar, or nucleoside modifications can include an alkyl or heteroatom substitution or addition, such as a methyl, alkoxyalkyl, halogen, nitrogen or sulfur, or other modifications known in the art. In addition, as used herein, the term "RNAi" is meant to be equivalent to other terms used to describe sequence specific RNA interference, such as post transcriptional gene silencing, translational inhibition, or epigenetics. For example, dsRNA molecules of this disclosure can be used to epigenetically silence genes at the post-transcriptional level or the pre-transcriptional level or any combination thereof.

In some aspects, this invention provides compositions containing one or more RNAi-inducing agents which are targeted to one or more genes or target transcripts, along with one or more delivery components. Examples of delivery components include lipids, peptides, polymers, polymeric lipids, and conjugates thereof. The compositions and formulations of this disclosure may be used for delivery of

RNAi-inducing entities such as dsRNA, siRNA, mdRNA, miRNA, shRNA, or RNAi- inducing vectors to cells in intact mammalian subjects, and may also be used for delivery of these agents to cells in culture.

This disclosure also provides methods for the delivery of one or more RNAi- inducing agents or entities to cells, organs and tissues within the body of a mammal. In some respects, compositions containing an RNAi-inducing entity may be introduced by various routes to be transported within the body and taken up by cells in one or more organs or tissues, where expression of a target transcript is modulated.

In general, this disclosure encompasses RNAi-inducing agents that are useful therapeutics to prevent and treat diseases or disorders characterized by various aberrant processes. For instance, viruses that infect mammals can replicate by taking control of cellular machinery of the host cell. See, e.g., Fields Virology (2001). Thus, dsRNAs are useful to disrupt viral pathways which control virus production or replication.

This disclosure includes methods for treating or preventing a viral infection in a subject by use of one or more therapeutic RNAi-inducing agents having a broad spectrum of efficacy against strains of a target virus. An RNAi-inducing agent of this invention can be targeted to a sequence of a viral gene in a known variant strain or variants of a virus, and exhibit sequence-specific gene silencing of the targeted viral gene in those variants. For example, an RNAi-inducing agent may be targeted to, and exhibit efficacy against a seasonal strain of influenza virus, as well as variant strains of influenza.

Compositions and formulations of this disclosure may be used for delivery of drug agents or biologically active agents to a variety of cells in vitro. Examples of cells for which in vitro delivery is encompassed include epithelial cells such as A549, immortal cell lines such as HeLa, hepatoma cells such as HepG2, rat gliosarcoma cells such as 9L/LacZ, human monocyte cells such as THP-I, Madin-Darby canine kidney cells (MDCK), various fibroblast cell lines, and primary cells in culture in the presence or absence of various sera, among others.

Compositions and formulations of this disclosure may be used for delivery of drug agents or biologically active agents to a variety of cells, tissues or organs in vivo. Modalities for delivering an agent in vivo include topical, enteral, and parenteral routes. Examples of modalities for delivering an agent in vivo include inhalation of particles or droplets, delivery of nasal or nasal-pharngyl drops, particles, or suspensions, transdermal and transmucosal routes, as well as injection or infusion by intramuscular, subcutaneous, intravenous, intraarterial, intracardiac, intrathecal, intraosseus, intraperitoneal, and epidural routes.

In some embodiments, an agent can be administered ex vivo by direct exposure to cells, tissues or organs originating from a mammalian subject.

A drug agent or biologically active agent to be delivered using a composition or formulation of this disclosure may be found in any form including, for example, a pure form, a crystalline form, a solid form, a nanoparticle, a condensed form, a complexed form, or a conjugated form.

This invention also provides methods for the delivery of one or more RNAi- inducing entities to organs and tissues within the body of a mammal. In some embodiments, compositions containing an RNAi-inducing entity, one or more DILA2 amino acid compounds, and one or more additional lipid components are introduced by various routes to be transported within the body and taken up by cells in one or more organs or tissues, where expression of a target transcript is modulated.

This disclosure provides pharmaceutically acceptable nucleic acid compositions with various lipids useful for therapeutic delivery of nucleic acids and gene-silencing RNAs. In particular, this invention provides compositions and methods for in vitro and in vivo delivery of dsRNAs for decreasing, downregulating, or silencing the translation of a target nucleic acid sequence or expression of a gene. These compositions and methods may be used for prevention and/or treatment of diseases in a mammal. In exemplary methods of this invention, a ribonucleic acid molecule such as an siRNA or shRNA is contacted with a DILA2 amino acid compound to formulate a composition which can be administered to cells or subjects such as mammals. In some embodiments, this invention provides methods for delivering an siRNA or shRNA intracellularly by contacting a nucleic acid-containing composition with a cell.

In exemplary embodiments, this invention includes compositions containing a nucleic acid molecule, such as a double-stranded RNA (dsRNA), a short interfering RNA (siRNA), or a short hairpin RNA (shRNA), admixed or complexed with a DILA2 amino acid compound, and a polymeric lipid to form a composition that enhances intracellular delivery of the nucleic acid molecule. In some embodiments, a delivery composition of this invention may contain a dsRNA and one, two, or more DILA2 amino acid compounds, which may be cationic or non-cationic. In some variations, a delivery composition may contain a dsRNA, DIL A2 amino acid compounds, and one or more polymeric lipids. In some embodiments, a delivery composition may contain a dsRNA, DILA2 amino acid compounds, one or more additional lipids, and one or more polymeric lipids. The compositions of this invention can form stable particles which may incorporate a dsRNA as an interfering RNA agent. Compositions and formulations of this invention may include further delivery-enhancing components or excipients.

In some embodiments, compositions of this invention contain stable RNA-lipid particles having diameters from about 5 nm to about 400 nm. In some embodiments, the particles may have a uniform diameter of from about 10 nm to about 300 nm. In some embodiments, the particles may have a uniform diameter of from about 50 nm to about 150 nm.

Within exemplary compositions of this invention, a double- stranded RNA may be admixed or complexed with DILA2 amino acid compounds to form a composition that enhances intracellular delivery of the dsRNA as compared to contacting target cells with naked dsRNA.

In some embodiments, a composition of this invention may contain one or more DILA2 amino acid compounds which are from about 0.5% to about 70% (mol%) of the total amount of lipid and delivery-enhancing components, including any polymeric component, but not including the RNA component. In some embodiments, a composition of this invention may contain one or more DILA2 amino acid compounds from about 10% to about 55%. In some embodiments, a composition of this invention may contain one or more DILA2 amino acid compounds from about 15% to about 35%.

In certain embodiments, a composition of this invention may contain one or more non-amino acid non-cationic lipids, where the non-amino acid non-cationic lipids are from about 2% to about 95% (mol%) of the total amount of lipid and delivery-enhancing components, including any polymeric component, but not including the RNA component. In some embodiments, a composition of this invention may contain one or more non-cationic lipids from about 20% to about 75%, or from about 45% to about 75%, or from about 45% to about 55%. In some embodiments, a composition of this invention may contain one or more non-cationic lipids from about 10% to about 50%. In some embodiments, a composition of this invention may contain one or more polymeric lipids, where the polymeric lipids are from about 0.2% to about 20% (mol%) of the total amount of lipid and delivery-enhancing components, including any polymeric component, but not including the RNA component. In some embodiments, a composition of this invention may contain one or more polymeric lipids from about 0.5% to about 10%. In some embodiments, a composition of this invention may contain one or more polymeric lipids from about 1% to about 5% of the composition.

Compositions and uses for nucleic acid therapeutics

In some embodiments, this invention provides a method of treating a disease or disorder in a mammalian subject. A therapeutically effective amount of a composition of this invention containing an interfering RNA, an DILA2 amino acid compounds, a non- amino acid non-cationic lipid, a polymeric lipid, and one or more delivery-enhancing components or excipients may be administered to a subject having a disease or disorder associated with expression or overexpression of a gene that can be reduced, decreased, downregulated, or silenced by the composition.

This invention encompasses methods for treating a disease of the lung such as respiratory distress, asthma, cystic fibrosis, pulmonary fibrosis, chronic obstructive pulmonary disease, bronchitis, or emphysema, by administering to the subject a therapeutically effective amount of a composition. This invention encompasses methods for treating inflammatory disease, rheumatoid arthritis, liver disease, encephalitis, bone fracture, heart disease, viral disease including hepatitis and influenza, or cancer.

Methods for making liposomes are given in, for example, G. Gregoriadis, Liposome Technology (CRC Press 1984), and M. J. Ostro, Liposomes (Marcel Dekker 1987).

The nucleic acid component, DILA2 amino acid compounds, and any additional components may be mixed together first in a suitable medium such as a cell culture medium, after which one or more additional lipids or compounds may be added to the mixture. Alternatively, the DILA2 amino acid compounds can be mixed together first in a suitable medium such as a cell culture medium, after which the nucleic acid component can be added.

Within certain embodiments of the invention, a dsRNA is admixed with one or more DILA2 amino acid compounds, or a combination of one or more DIL A2 amino acid compounds and non-amino acid non-cationic lipids. The interfering RNA agent may also be complexed with, or conjugated to a DILA2 amino acid compound or polymeric lipid, and admixed with one or more non- amino acid non-cationic lipids, or a combination of one or more non-amino acid non- cationic and non-amino acid cationic lipids. An interfering RNA agent and a DILA2 amino acid compound may be mixed together first, followed by the addition of one or more non-amino acid non-cationic lipids, or a combination of non-amino acid non-cationic and non-amino acid cationic lipids added in a suitable medium such as a cell culture medium. Alternatively, the DILA2 amino acid compounds and lipid components may be mixed first, followed by the addition of the RNA agent in a suitable medium.

This invention provides compositions and methods for modulating gene expression using regulatory RNA such as by RNA interference. A composition of this invention can deliver a ribonucleic acid agent to a cell which can produce the response of RNAi. Examples of nucleic acid agents useful for this invention include double-stranded nucleic acids, modified or degradation-resistant nucleic acids, RNA, siRNA, siRNA, shRNA, miRNA, piRNA, RNA antagonists, single-stranded nucleic acids, DNA-RNA chimeras, antisense nucleic acids, and ribozymes. As used herein, the terms siRNA, siRNA, and shRNA include precursors of siRNA, siRNA, and shRNA, respectively. For example, the term siRNA includes an RNA or double-stranded RNA that is suitable as a substrate of dicer enzyme.

Ribonucleic acid agents useful for this invention may be targeted to various genes. Examples of human genes suitable as targets include TNF, FLTl, the VEGF family, the ERBB family, the PDGFR family, BCR-ABL, and the MAPK family, among others. An RNA of this disclosure to be delivered may have a sequence that is complementary to a region of a viral gene. For example, some compositions and methods of this invention are useful to regulate expression of the viral genome of an influenza virus. In some embodiments, this invention provides compositions and methods for modulating expression and infectious activity of an influenza by RNA interference. Expression and/or activity of an influenza can be modulated by delivering to a cell, for example, a short interfering RNA molecule having a sequence that is complementary to a region of a RNA polymerase subunit of an influenza. Examples of RNAs targeted to an influenza virus are given in U.S. Patent Publication No. 20070213293 Al. Uses for delivery of active agents

The compounds and compositions of this invention may be used for delivery of any physiologically active agent, as well as any combination of active agents, as described above or known in the art. The active agent may be present in the compositions and uses of this invention in an amount sufficient to provide the desired physiological or ameliorative effect.

The compounds and compositions of this invention are directed toward enhancing delivery of a range of drug agents and biologically active agents in mammalian subjects including small molecule compounds and drugs, peptides, proteins, and vaccine agents. Examples of active agents include a peptide, a protein, a nucleic acid, a double- stranded RNA, a hematopoietic, an antiinfective; an antidementia; an antiviral, an antitumoral, an antipyretic, an analgesic, an anti-inflammatory, an antiulcerative, an antiallergenic, an antidepressant, a psychotropic, a cardiotonic, an antiarrythmic, a vasodilator, an antihypertensive, a hypotensive diuretic, an antidiabetic, an anticoagulant, a cholesterol-lowering agent, a therapeutic for osteoporosis, a hormone, an antibiotic, a vaccine, a cytokine, a hormone, a growth factor, a cardiovascular factor, a cell adhesion factor, a central or peripheral nervous system factor, a humoral electrolyte factor, a hemal organic substance, a bone growth factor, a gastrointestinal factor, a kidney factor, a connective tissue factor, a sense organ factor, an immune system factor, a respiratory system factor, a genital organ factor, an androgen, an estrogen, a prostaglandin, a somatotropin, a gonadotropin, an interleukin, a steroid, a bacterial toxoid, an antibody, a monoclonal antibody, a polyclonal antibody, a humanized antibody, an antibody fragment, and an immunoglobin.

Compositions and Formulations for Administration As used herein, the terms "administering" and "administration" encompass all means for directly and indirectly delivering a compound or composition to a site of action. The compounds and compositions of this disclosure may be administered alone, or in combination with other compounds, compositions, or therapeutic agents which are not disclosed herein. The compositions and methods of the invention may be administered to subjects by a variety of mucosal administration modes, including by oral, rectal, vaginal, intranasal, intrapulmonary, or transdermal delivery, or by topical delivery to the eyes, ears, skin or other mucosal surfaces. In some aspects of this invention, the mucosal tissue layer includes an epithelial cell layer. The epithelial cell can be pulmonary, tracheal, bronchial, alveolar, nasal, buccal, epidermal, or gastrointestinal. Compositions of this invention can be administered using conventional actuators such as mechanical spray devices, as well as pressurized, electrically activated, or other types of actuators.

Compositions of this invention may be administered in an aqueous solution as a nasal or pulmonary spray and may be dispensed in spray form by a variety of methods known to those skilled in the art. Pulmonary delivery of a composition of this invention may be achieved by administering the composition in the form of drops, particles, or spray, which can be, for example, aerosolized, atomized, or nebulized. Pulmonary delivery may be performed by administering the composition in the form of drops, particles, or spray, via the nasal or bronchial passages. Particles of the composition, spray, or aerosol can be in a either liquid or solid form. Preferred systems for dispensing liquids as a nasal spray are disclosed in U.S. Patent No. 4,511,069. Such formulations may be conveniently prepared by dissolving compositions according to the present invention in water to produce an aqueous solution, and rendering said solution sterile. The formulations may be presented in multi-dose containers, for example in the sealed dispensing system disclosed in U.S. Patent No. 4,511,069. Other suitable nasal spray delivery systems have been described in Transdermal Systemic Medication, Y. W. Chien ed., Elsevier Publishers, New York, 1985; and in U.S. Patent No. 4,778,810. Additional aerosol delivery forms may include, for example, compressed air-, jet-, ultrasonic-, and piezoelectric nebulizers, which deliver the biologically active agent dissolved or suspended in a pharmaceutical solvent, for example, water, ethanol, or mixtures thereof. Nasal and pulmonary spray solutions of the present invention typically comprise the drug or drug to be delivered, optionally formulated with a surface active agent, such as a nonionic surfactant (e.g., polysorbate-80), and one or more buffers. In some embodiments of the present invention, the nasal spray solution further comprises a propellant. The pH of the nasal spray solution may be from about pH 6.8 to 7.2. The pharmaceutical solvents employed can also be a slightly acidic aqueous buffer of pH 4-6. Other components may be added to enhance or maintain chemical stability, including preservatives, surfactants, dispersants, or gases. In some embodiments, this invention is a pharmaceutical product which includes a solution containing a composition of this invention and an actuator for a pulmonary, mucosal, or intranasal spray or aerosol.

A dosage form of the composition of this invention can be liquid, in the form of droplets or an emulsion, or in the form of an aerosol. A dosage form of the composition of this invention can be solid, which can be reconstituted in a liquid prior to administration. The solid can be administered as a powder. The solid can be in the form of a capsule, tablet or gel.

Methods of making lipid compositions include ethanol injection methods and extrusion methods using a Northern Lipids Lipex Extruder system with stacked polycarbonate membrane filters of defined pore size. Sonication using probe tip and bath sonicators can be employed to produce lipid particles of uniform size. Homogenous and monodisperse particle sizes can be obtained without the addition of the nucleic acid component. For in vitro transfection compositions, the nucleic acid component can be added after the transfection agent is made and stabilized by additional buffer components. For in vivo delivery compositions, the nucleic acid component is part of the formulation.

The compositions and formulations of this invention may be administered by various routes, for example, to effect systemic delivery via intravenous, parenteral, or intraperitoneal routes. In some embodiments, an agent may be delivered intracellularly, for example, in cells of a target tissue such as lung or liver, or in inflamed tissues.

Included within this disclosure are compositions and methods for delivery of an agent by removing cells of a subject, delivering an agent to the removed cells, and reintroducing the cells into a subject. In some embodiments, this invention provides a method for delivery of an agent in vivo. A composition may be administered intravenously, subcutaneously, or intraperitoneal^ to a subject. In some embodiments, the invention provides methods for in vivo delivery of an agent to the lung of a mammalian subject.

Additional Embodiments

All publications, references, patents, patent publications and patent applications cited herein are each hereby specifically incorporated by reference in entirety.

While this invention has been described in relation to certain embodiments, and many details have been set forth for purposes of illustration, it will be apparent to those skilled in the art that this invention includes additional embodiments, and that some of the details described herein may be varied considerably without departing from this invention. This invention includes such additional embodiments, modifications and equivalents. In particular, this invention includes any combination of the features, terms, or elements of the various illustrative components and examples.

The use herein of the terms "a," "an," "the" and similar terms in describing the invention, and in the claims, are to be construed to include both the singular and the plural. The terms "comprising," "having," "including" and "containing" are to be construed as open-ended terms which mean, for example, "including, but not limited to." Thus, terms such as "comprising," "having," "including" and "containing" are to be construed as being inclusive, not exclusive. Recitation of a range of values herein refers individually to each and any separate value falling within the range as if it were individually recited herein, whether or not some of the values within the range are expressly recited. For example, the range "4 to 12" includes without limitation any whole number, integer, fractional, or rational value that is greater than or equal to 4 and less than or equal to 12. Specific values employed herein will be understood as exemplary and not to limit the scope of the invention.

Recitation of a range of number of carbon atoms herein refers individually to each and any separate value falling within the range as if it were individually recited herein, whether or not some of the values within the range are expressly recited. For example, the term "Cl-22" includes without limitation the species Cl, C2, C3, C4, C5, C6, C7, C8, C9, ClO, CI l, C12, C13, C14, C15, C16, C17, C18, C19, C20, C21, and C22.

Definitions of technical terms provided herein should be construed to include without recitation those meanings associated with these terms known to those skilled in the art, and are not intended to limit the scope of the invention. Definitions of technical terms provided herein shall be construed to dominate over alternative definitions in the art or definitions which become incorporated herein by reference to the extent that the alternative definitions conflict with the definition provided herein.

The examples given herein, and the exemplary language used herein are solely for the purpose of illustration, and are not intended to limit the scope of the invention. When a list of examples is given, such as a list of compounds or molecules suitable for this invention, it will be apparent to those skilled in the art that mixtures of the listed compounds or molecules are also suitable.

EXAMPLES

EXAMPLE 1 Peptide Binding Regions The relative strength of the binding of a cationic peptide to an RNAi-inducing agent was measured with a dye binding assay. The RNAi-inducing agent was prepared at 7.8 μl in 10ml to make a 20 μg/ml stock, then 75 μl/well. A SYBR gold dilution was prepared at 3.75 μl in 15 ml for 1 :4000 dilution for a 2.5X stock.

Peptides were dissolved in Hepes buffer with 5% dextrose and diluted. Peptides were further diluted so that 75 μl could be added to each well resulting in the desired N: P (ranging from 0-4). Peptides were assumed to have a purity of 50%, but actual peptide amount was unknown.

A SYBR-GOLD Dye Binding Assay was performed. A 96-well plate assay with sample volume of 150 μl per well. Final dsRNA concentration was lOμg/ml in 1OmM hepes/5%dextrose at pH 7.4. Peptides were diluted into different working solutions such that equal volumes were added to reach different N/P ratios. For the addition procedure, dsRNA was first added (75 μl of 20 μg/ml), followed by 150μl of 2.5X SYBR Gold. Peptide (75μl) was then added to compete off the SYBR dye. Total volume was 300μl. Fluorescence was corrected from background of dye alone in buffer. SYBR-GoId ex/em was 495nm/537nm, read on Molecular Devices plate reader.

Formulation particle sizes were determined by transferring to 384-well plate to be tested using the Wyatt particle sizer. Each well of the 96 well plate was transferred in duplicate. Volume remaining in plate was 200 μl.

Peptide release was triggered by disulfide reduction or enzymatic cleavage where appropriate. Cysteine-terminated peptides were cleavable by glutathione reduction. V- Cit containing peptides were cleavable by enzymatic cleavage by Cathepsin B. Glutathione was present at 0.1-10 mM intracellularly; Cathepsin B was 1 mM in lysosomes. (For Cathepsin B at 0.14ng/μl, see Teich et al BMC Gastroenterology 2002, 2:16). For release, the appropriate molecule was added to a final concentration of ImM in one of the duplicated wells followed by measurement of SYBR GOLD fluorescence with time.

As shown in Figure 1, the binding of a polyarginine binding region to dsRNA increased with the length of the polyarginine binding region. In Figure 1, the strongest binding (best ability to displace SYBR-GoId dye) was observed with PN3499, peptide (SEQ ID NO: 159) RRRRRCCRRRRR, which was a dimer peptide containing at total of 10 arginines. EXAMPLE 2 In Vitro Assay for PPIB Gene Expression Knockdown in A549 cells

Cyclophilin B (PPIB) gene knockdown measurements can be used as a primary activity-based in vitro assay for interfering RNA delivery formulations. Typically, the measurements were made as described below, with minor variations.

Cyclophilin B (PPIB) gene expression knockdown was measured in A549 human alveolar basal epithelial cells. For PPIB gene knockdown measurements, A549 cells were transfected with an interfering RNA formulation, total RNA prepared 24 hours after transfection, and PPIB mRNA assayed by RT-PCR. QRT-PCR of 36B4 (acidic ribosomal phosphoprotein PO) mRNA expression was performed for normalization.

A549 cells were seeded at 7,500 cells/well (96-well) and incubated overnight in medium. Confluency was about 50% at the time of transfection. Transfection complex was prepared by adding an interfering RNA to medium (OptiMEM™) and vortexing, separately adding a delivery formulation to medium (OptiMEM™) and vortexing, and finally mixing the interfering RNA in medium with the delivery formulation in medium and incubating 20 minutes at room temperature to make the transfection complex. The medium for incubated cells was replaced with fresh OptiMEM™ and transfection complex was added to each well. Cells were incubated for 5 hrs at 37°C and 5% CO_2, then complete medium was added (to a final fetal bovine serum concentration 10%) and incubation continued until 24 hours post-transfection.

For PPIB gene knockdown cells were lysed and RNA prepared (Invisorb RNA Cell HTS 96-Kit/C, Invitek, Berlin, or RNeasy 96 Kit, Qiagen). Quantitative RT-PCR was performed using One-Step qRT-PCR kit (Invitrogen) on a DNA Engine Opticon2 thermal cycler (BioRad). Primers used for PPIB were:

(SEQ ID NO: 160)

5' -GGCTCCCAGTTCTTCATCAC-S' (forward) and (SEQ ID NO:161)

5' -CCTTCCGCACCACCTC-S' (reverse) with (SEQ ID NO: 162)

5' -FAM-CTAGATGGCAAGCATGTGGTGTTTGG-TAMRA-S ' for the probe.

For 36B4, primers were: (SEQ ID NO: 163) 5' -TCTATCATCAACGGGTACAAACGA-S' (forward) and (SEQ ID NO: 164)

5' -CTTTTCAGCAAGTGGGAAGGTG-S ' (reverse) with (SEQ ID NO: 165) 5 '-FAM-CCTGGCCTTGTCTGTGGAGACGGATTA-TAMRA-S ' for the probe.

The structures of some double- stranded RNAs (dsRNA) of this disclosure are shown in Table 6.

Table 6: Double-stranded RNAs

In Table 6, "mU" represents 2'-O-methyl uridine, "mC" represents 2'-O-methyl cytidine, and "s" represents a phosphorothioate linkage. EXAMPLE 3 PPIB gene expression knockdown using a layered carrier and triggered release peptide

Nanoparticle carriers for an RNAi-inducing agent were tested for PPIB gene knockdown activity in A549 cells. A binary complex of a dsRNA RNAi-inducing agent with a triggered release peptide was initially formed at a particular N/P ratio. An endosomolytic agent was added, which adjusted the N/P ratio to a final value.

Formulations of layered carriers were in general prepared by first vortexing a dsRNA into HEPES/Dextrose buffer. Triggered release peptide was added with vortexing to complex the dsRNA. The complex was incubated for 15 minutes. Glutaraldehyde was added and the core allowed to crosslink for 1.5 h. The reaction was quenched by addition of 1 M Tris buffer pH 7.4 Endosomolytic agent was added, and the carrier mixture incubated for 15 minutes before adding to cells.

PPIB gene expression knockdown measurements using a layered carrier comprising a triggered release peptide are shown in Table 7. The results in Table 7 indicate that the carrier comprising a triggered release peptide was effective in the presence of an endosomolytic agent to deliver an active dsRNA agent to cells to produce a significant gene silencing effect.

Table 7: PPIB gene expression knockdown using a triggered release peptide

Materials used in this example were the following:

PN4110

SEQ ID NO: 179 WWHHKKRRCCRRKKHHWW

PN3033 (diINF7)

SEQ ID NO: 180

NH₂-GLFEAIEGFIENGWEGMIDGWYGC-CO₂H

The effect of the final N/P ratio on PPIB gene expression knockdown measurements using a layered carrier comprising a triggered release peptide was determined, and the results are shown in Table 8. The results in Table 8 indicate that the carrier comprising a triggered release peptide was effective in the presence of an endosomolytic agent to deliver an active dsRNA agent to cells to produce a significant gene silencing effects. Further, the results in Table 8 indicate that in vitro knockdown for a layered carrier comprising a triggered release peptide is enhanced at a lower final N/P ratio of 2.5-3.5.

Table 8: Effect of final N/P ratio on gen knockdown

Claims

WHAT IS CLAIMED IS:

1. A composition comprising a DILA2 amino acid compound and an intracellularly-cleavable peptide of from six to 100 amino acid residues in length, the intracellularly-cleavable peptide comprising two or more nucleic acid-binding region amino acid sequences connected by one or more intracellularly-cleavable linker amino acid sequences, wherein the nucleic acid-binding regions each contain two or more positively-charged amino acid residues, and further comprising a ribonucleic acid, an RNAi-inducing agent, or an antisense agent.

2. The composition of claim 1, wherein the intracellularly-cleavable linker is a portion of a Cathepsin B, D, or L substrate, or Val-Cit.

3. The composition of claim 1, wherein the intracellularly-cleavable peptide contains a nucleic acid-binding region sequence selected from SEQ ID NOS: 1-82.

4. The composition of claim 1 , wherein the intracellularly-cleavable peptide has the sequence of any one of SEQ ID NOS: 83- 158.

5. The composition of claim 1, wherein the intracellularly-cleavable peptide binds to a ribonucleic acid, an RNAi-inducing agent, or an antisense agent.

6. The composition of claim 1, wherein the intracellularly-cleavable peptide is complexed with a ribonucleic acid, an RNAi-inducing agent, or an antisense agent.

7. The composition of claim 1, further comprising a nanoparticle formed from the intracellularly-cleavable peptide and a ribonucleic acid, an RNAi-inducing agent, or an antisense agent.

8. The composition of claim 1, wherein the composition contains a liposomal particle.

9. The composition of claim 1, further comprising an endosomolytic agent.

10. A method for delivering an active agent to a cell comprising preparing a composition according to any one of claims 1 to 9 and treating the cell with the composition.

11. A method for inhibiting expression of a gene in a cell comprising preparing a composition according to any one of claims 1 to 9 and treating the cell with the composition.

12. A method for inhibiting expression of a gene in a mammal comprising preparing a composition according to any one of claims 1 to 9 and administering the composition to the mammal.

13. A method for treating a disease in a human, the disease being selected from inflammatory diseases including rheumatoid arthritis, metabolic diseases including hypercholesterolemia, liver disease, encephalitis, bone fracture, heart disease, viral disease including hepatitis and influenza, and cancer, comprising preparing a composition according to any one of claims 1 to 9 and administering the composition to the human.

14. A use of a composition according to any one of claims 1 to 9 in the preparation of a medicament for treating a disease including inflammatory diseases including rheumatoid arthritis, metabolic diseases including hypercholesterolemia, liver disease, encephalitis, bone fracture, heart disease, viral disease including hepatitis and influenza, and cancer.

15. A composition according to any one of claims 1 to 9 for use in treating a disease selected from inflammatory diseases including rheumatoid arthritis, metabolic diseases including hypercholesterolemia, liver disease, encephalitis, bone fracture, heart disease, viral disease including hepatitis and influenza, and cancer.