WO2013032643A2 - Lipids capable of conformational change and their use in formulations to deliver therapeutic agents to cells - Google Patents

Abstract

The invention provides lipid-based composition for use in the administration of therapeutic payloads to target cells or tissues in vivo. The lipid compounds of the invention include a peptide-based moiety which is induced to change its conformation upon entering a cell or intracellular compartment. The induced conformation change in the peptide-based moiety consequently triggers the efficient release of the payload from the lipid-based composition.

Description

TITLE OF THE INVENTION

LIPIDS CAPABLE OF CONFORMATIONAL CHANGE AND THEIR USE IN FORMULATIONS TO DELIVER THERAPEUTIC AGENTS TO CELLS

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Patent Application No.

61/529,422, filed August 31, 2011, the contents of which are incorporated herein by reference in their entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention is directed to a novel class of lipid compounds capable of conformational change which are useful for the delivery of therapeutic agents or payloads to target cells.

2. Background

The beneficial biological effects of many therapeutic agents that are capable of influencing cell function at the subcellular or molecular level, including agents such as nucleic acid molecules, RNA, DNA, miRNA, siRNA, oligonucleotides, polypeptides, peptides, and small molecule compounds, can be limited by low uptake of the agents by the target cells or by intracellular breakdown of the agents after uptake. In particular regard to short RNA molecules, such agents cannot easily cross cell membranes because of their size and their polyanionic nature resulting from the negative charge of the phosphate groups. Delivery has been one of the major challenges for RNA-based therapeutics.

It is generally desired that delivery of such therapeutic agents reaches the cytoplasm or intracellular compartments of the cell for maximum therapeutic benefit or effect. This contrasts with some bioactive substances which instead exert their biological effects via the cell surface through cell receptors or interaction with extracellular components. For many agents which exert their effects only from within the intracellular environment, the cell membrane presents a selective barrier which may be impermeable.

The cell membrane, however, serves as a formidable barrier for the delivery of therapeutic agents. The barrier properties of the cell membrane are a function of the complex composition of the cell membrane which includes phospholipids, glycolipids, cholesterol, and intrinsic and extrinsic proteins, as well as a variety of cytoplasmic components. Interactions between these structural and cytoplasmic cellular components and their responses to external signals make up transport processes which are responsible for the membrane selectivity exhibited within and among cell types. Different cell types will have differing selectivities owing to their varying membrane compositions.

Several methods for improving uptake of such therapeutic agents into tissues and cells have been proposed. For example, a small pharmaceutical compound can be administered in a modified or prodrug form for transport into cells, which is designed to then undergo enzymatic conversion to an active form within the cells. However, the specificity of such uptake systems is such that the great variety of agents cannot be accommodated by this means of cellular uptake machinery.

Alternatively, the cellular processes of phagocytosis, endocytosis or pinocytosis have been exploited, for example, where a drug-containing particle becomes engulfed by a cell via phagocytic or internalization of membrane vesicles. However, this approach is limited to certain cell types.

Still another approach to enhancing the uptake of therapeutic agents by cells involves the use of fusogenic particles designed to fuse with the surface membrane of a target cell or with an internalized cellular vesicle, releasing the particle contents into the cytoplasmic compartment of the cell. Inactivated and reconstituted virus particles have been proposed for this purpose, particularly in gene therapy where large nucleic acid strands are introduced into cells. Virus-like particles composed of fusion-promoting viral proteins embedded in artificial lipid bi-layer membranes are another example. However, safety concerns and the expense associated with growing, isolating, and deactivating viral components limit these approaches.

Cellular membrane barrier function may also be overcome by delivering such therapeutic agents in lipid-based complexes that resemble the lipid composition of natural cell membranes, or at least bear appropriate characteristics that allow them to pass through the cell membrane to deliver their therapeutic agent "payload." In some cases, these lipids are able to fuse with cell membranes, and in the process, the associated therapeutic agent payload is delivered to the intracellular environment. The structure of various types of such "lipid aggregates" vary depending on a variety of factors, which include lipid composition and methods for forming the aggregates. Lipid aggregates include, for example, liposomes, unilamellar vesicles, multilamellar vesicles, micelles and the like, and may have particle sizes in the nanometer to micrometer range.

The lipids used to form the lipid aggregates can be neutral, anionic or cationic, with neutral lipids encompassing uncharged lipids as well as zwitterionic lipids that carry a net neutral charge. An important drawback to the use of anionic lipids in forming the aggregates as cell delivery vehicles is that the anionic lipid based aggregates have a negative charge that reduces the efficiency of binding to a negatively charged target cell surface. Consequently, the aggregates, e.g., liposomes, are often taken up by the cell phagocytically. Phagocytized lipid aggregates, e.g., liposomes, are delivered to the lysosomal compartment, where the cargo are subjected to the action of digestive enzymes and become degraded, which may lead to low efficiency of therapeutic benefit.

Cationic lipids have also been suggested for forming such lipid aggregate delivery vehicles. For example, it was recently discovered that a positively charged synthetic cationic lipid, N-[l-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride

(DOTMA), in the form of liposomes, or small vesicles, could interact spontaneously with DNA to form lipid-DNA complexes which are capable of fusing with the negatively charged lipids of the cell membranes of tissue culture cells, resulting in both uptake and expression of the DNA (Feigner, P. L. et al. Proc. Natl. Acad. Sci., USA 8:7413-7417, 1987 and U.S. Pat. No. 4,897,355 to Eppstein, D. et al.). However, cationic lipids also have known drawbacks. For example, no particular cationic lipid has been reported to work well with a wide variety of cell types in vivo. Since cell types differ from one another in membrane composition, different cationic lipid compositions and different types of lipid aggregates may be effective for different cell types, either due to their ability to contact and fuse with particular target cell membranes directly or due to different interactions with intracellular membranes or the intracellular environment. Additionally, no particular cationic lipid has been reported that optimally possesses sufficient efficiency in vivo in the delivery of its cargo therapeutic agent to a wide variety of cell types.

In particular regard to the delivery of nucleic acid molecule payloads, such as, for example, RNAi molecules, overcoming the cell membrane remains a major challenge (Castanotto and Rossi, Nature 2009; 457: 426-433). In addition, once the payload has crossed the cell membrane, effective release of the payload is necessary in order for the therapeutic agent to bring about its effects. For example, to trigger RNAi following systemic administration, a formulation containing short RNA molecules not only is required to (1) protect the payload from enzymatic and non-enzymatic degradation, (2) provide appropriate serum half-life and biodistribution of the formulation, but also is needed to (3) allow cellular uptake or internalization and (4) facilitate delivery to the cytoplasm of the cell and effective release therein. Many formulations that excel in criteria 1 and 2 above tend to show deficiencies in criteria 3 and 4. Thus, formulations may show excellent biodistribution but poor delivery or uptake and therefore no knockdown of a target gene. Importantly, criteria 3 and 4 are equally critical for not only systemic delivery, but local delivery as well. Indeed, even when the formulation is injected to target tissue directly (e.g., in the eye or in the tumor of interest, etc.) the payload still needs to be internalized by the cells and delivered and/or released into the cytoplasm. Moreover, it has been postulated that while endocytosis is the primary method of cellular internalization of lipid aggregate formulations, most of lipid aggregate formulations taken up by the cells fail to reach the cytoplasm and/or release their payloads and are therefore unable to trigger their intended biological effect, e.g., RNA interference with RNAi molecules.

Thus, there remains a need in the art for improved lipids and lipid aggregate formulations which are capable of delivering therapeutic agents (payloads) to a wide variety cell types with greater efficiency, and in particular, where such formulations allow effective delivery and release of the therapeutic agents or payloads (e.g., RNAi molecules) to the cell cytoplasm with improved delivery characteristics.

SUMMARY OF THE INVENTION

The invention is based, at least in part, upon the discovery of a novel means by which to modify lipids, including for example, cationic lipids, through covalent attachment of a peptide or peptide-based moiety to the lipid (1) which carries a net positive charge and thereby imparts a net positive charge on the overall molecule and (2) which undergoes a conformational change under intracellular- triggering conditions. The peptide or peptide-based moiety imparts improved performance characteristics to the overall lipid as a lipid delivery vehicle for intracellular delivery of therapeutic agents, such as nucleic acids. The improved characteristics include, but are not limited to, enhanced internalization of the lipid delivery vehicles, and thus, the payloads therein, into target cells. The improved characteristics also include enhanced payload release properties of the lipid delivery vehicles whereby the therapeutic agent payloads, e.g., nucleic acid payloads, are more effectively delivered to the cytoplasm of target cells. The intracellular conditions capable of triggering the conformational change in the peptide moiety, and thus, the resultant beneficial effects on the characteristics of the lipid delivery vehicles of the invention, can include any suitable intracellular condition that may be met by the lipid delivery vehicles upon entry of the cell. This can include, for example, changes in pH, oxidation/reduction conditions, protonation/deprotonation conditions, enzymatic cleavage (e.g., via a lysosomal enzyme) or other like conditions that may trigger a conformational change in the peptide or peptide-like moiety, and in turn, imparting the improved characteristics on the lipids of the invention.

In one aspect, the invention relates to compositions or formulations comprising lipid compounds of the invention, e.g., those defined generally herein, which comprise one or more peptide or peptide-based modifications. In another aspect, the invention relates to the modified lipid compounds themselves, i.e., the lipids of the invention having been covalently modified with the peptide or peptide-based moieties of the invention. In certain embodiments, the peptide or peptide-based moieties of the invention can be induced to undergo a conformational change, which imparts improved performance characteristics on the lipids of the invention, including enhanced internalization of the lipids of the invention by target cells and enhanced payload release characteristics. In certain other

embodiments, the peptide or peptide-based moieties of the invention have an overall net positive charge, which imparts an overall net positive charge on the modified lipids of the invention.

In still another aspect, the invention relates to the peptides or the peptide-based moieties used to modify the lipids of the invention. In certain embodiments, the peptide or peptide-based moieties have a net positive charge. In certain other embodiments, the peptides or peptide-based moieties of the invention may undergo a conformational change triggered by one or more residues of the peptide undergoing a change in pH (e.g., protonation or deprotonation state), redox state (e.g., oxidation or reduction state), chemical or biochemical cleavage or activation (e.g., enzymatic cleavage) or other change caused by or triggered by internalization of the modified lipid inside a cell or in an intracellular compartment or vesicle (e.g., endosome, lysosome, caveolae, etc.).

In yet other aspects, the invention relates to those lipids which may be modified by the peptide or peptide-based moieties of the invention. In general, any lipid may be suitable to be modified by the peptide or peptide-based moieties of the invention, but preferably the lipid has a modifiable lipid tail, such as a phospholipid. Examples of such modifiable lipids of the invention are exemplified herein. However, the present specification is not meant to be limiting as to the particular lipids which may be used in the invention, so long as the lipid can be modified in accordance with the methods of the invention. In certain embodiments, the lipids of the invention should be those lipids which are typically known and used for delivery of therapeutic payloads to cells, including, in particular, cationic lipids for the delivery of nucleic acid payloads. Preferably, the net positive peptide or peptide-based moieties of the invention should impart an overall net positive charge on the modified lipid.

In yet a further aspect, the invention relates to methods for preparing the modified lipids of the invention, as well as to methods for evaluating and testing the modified lipids of the invention.

In still other aspects, the invention relates to methods for forming or preparing lipid-based delivery vehicles which comprise at least one peptide-modified lipid of the invention and one or more payloads (e.g., nucleic acid molecule, peptide, small molecule or the like) for use in delivering to a target cell. In certain embodiments, the payload can be a nucleic acid molecule, a DNA molecule, an RNA molecule, a single-stranded nucleic acid molecule, a double- stranded nucleic acid molecule, a miRNA molecule, a siRNA molecule, a RNAi molecule, or an oligonucleotide. In a particular aspect, the therapeutic agent is a dsRNA molecule, including a siRNA or Dicer substrate ("DsiRNA) molecule. In another particular aspect, the therapeutic agent is a RNAi molecule for use in RNA interference.

In still other aspects, the present invention relates to the use of the lipid-based delivery compositions of the invention in delivering therapeutic agents, by in vitro or in vivo means to cells and/or tissues, and for treating conditions or diseases by administering therapeutically effective amounts of one or more therapeutic agents using the lipid-based delivery compositions of the invention.

The invention, in other aspects, provides kits comprising the lipid compounds, or the lipid-based compositions of the invention and instructions for use.

In still another aspect, the invention relates to a lipid delivery vehicle comprising a lipid compound of the invention.

In a particular aspect, the present invention relates to a lipid comprising a headgroup moiety and a tail moiety, wherein the tail moiety comprises a cationic peptide- based moiety that is capable of undergoing a conformational change upon contacting an intracellular condition.

In another particular aspect, the present invention relates to a cationic lipid compound having the general formula X-Y-P, wherein X is a headgroup moiety of a fatty acid, glycerolipid, glycerophospholipid, sphingolipid or saccharolipid; Y is an acyl chain; and P is a cationic moiety capable of conformational change upon internalization of the lipid into a cell or intracellular compartment and which imparts a net cationic charge to the lipid.

In certain embodiments, the lipid has an overall net positive charge. The headgroup moiety can be negatively charged, neutral, or positively charged. The headgroup moiety can also be a headgroup from a fatty acid, glycerolipid,

glycerophospholipid, sphingolipid, or saccharolipid. In certain embodiments, the glycerolipid is a triacylglyceride or glycosylglycerol; the glycerophospholipid is a phospholipid, phosphatidylcholine (PC), lecithin, phosphatidylethanolamine (PE) or phosphatidylserine (PS); the sphingolipid is a ceramide, phospho sphingolipid,

glyco sphingolipid, sphingomyelin, or ceramide phosphocholine; and the saccharolipid is Lipid A.

In certain other embodiments, the lipid is a noncationic lipid selected from the group consisting of lecithin, phosphatidylethanolamine, lysolecithin,

lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphatidic acid, cerebrosides, dicetylphosphate, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipahnitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoyl-phosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), palmitoyloleyol-phosphatidylglycerol (POPG),

dioleoylphosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane- 1 -carboxylate (DOPE-mal), dipalmitoyl-phosphatidylethanolamine (DPPE), dimyristoyl- phosphatidylethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), monomethyl-phosphatidylethanolamine, dimethyl -phosphatidylethanolamine, dielaidoyl- phosphatidylethanolamine (DEPE), DOGS, and stearoyloleoyl-phosphatidylethanolamine (SOPE).

In other embodiments, that lipid is a cationic lipid selected from the group consisting of N, N-dioleyl-N, N-dimethylammonium chloride (DODAC), N, N-distearyl- N, N-dimethylammonium bromide (DDAB), N-(l-(2, 3-dioleoyloxy)propyl)-N, N, N- trimethylammonium chloride (DOTAP), l,2-dioleoyl-3-dimethyl-ammonium-propane (DODAP), l,2-dipalmitoyl-sn-glycero-3-ethyl-phosphocholine (DpePC), N-(l-(2, 3- dioleyloxy)propyl)-N, N, N-trimethylammonium chloride (DOTMA), N, N-dimethyl-2, 3- dioleyloxypropylamine (DODMA), 1, 2-DiLinoleyloxy-N, N-dimethylaminopropane (DLinDMA), 1, 2-Dilinolenyloxy-N, N-dimethylaminopropane (DLenDMA), DSDMA, DOSPA, DC-Choi, DMRIE, l-palmitoyl-2-glutaryl-sn-glycero-3-phosphocholine (16:0- 05:0 (COOH) PC or "G1PC"), l-hexadecyl-2-azelaoyl-sn-glycero-3-phosphocholine (16- 09:0 (COOH) PC), l-palmitoyl-2-azelaoyl-sn-glycero-3-phosphocholine (16:0-09:0 (COOH) PC or "AzPC"), l-palmitoyl-2-(5'-oxo-valeroyl)-sn-glycero-3-phosphocholine (16;0-05:0 (CHO) PC) and l-palmitoyl-2-(9'-oxo-nonanoyl)-sn-glycero-3- phosphocholine. It will be appreciated that such cationic lipids may be classified as species of l,2-alkyloxy-N,N-alkylaminoalkane lipids.

In still other embodiments, the tail moiety can comprise a saturated or unsaturated fatty acid. The fatty acid can be any suitable length, and preferably at least 2 carbons but less than 50 carbons in length, or less than 40 carbons in length, or less than 30 carbons in length, or less than 20 carbons in length. More preferably, the fatty acid is between 2 and 20 carbons, or 2 and 18 carbons, or 2 and 16 carbons, or 2 and 14 carbons, or 2 and 12 carbons, or 2 and 10 carbons, or 2 and 8 carbons, or 2 and 6 carbons or 2 and 4 carbons in length. The fatty acid may also be saturated or unsaturated. If unsaturated, the fatty acid can have at least 1 double bond and up to 2, 3, 4, 5, 6, 7, 8, 9, 10 or more double bonds.

In other embodiments, the conformational change of the peptide is induced by contacting the cytosol or an intracellular body, such as an endosome, lysosome or caveolae. The conformational change may also be induced by a change in pH (e.g., protonation or deprotonation state), redox state (e.g., oxidation or reduction state), chemical or biochemical cleavage or activation (e.g., enzymatic cleavage), whereby at least one residue of the peptide moiety changes from a first state to a second state, where the second state is characterized with a conformational change in the peptide moiety. The conformational change can be reversible or irreversible.

In still other embodiments, the peptide-based moiety is a peptide having 2 to 25 amino acid residues, or 5 to 50, or 10 to 100 residues. Preferably, the peptide has between 2 to 25 amino acid residues.

In yet other embodiments, the peptide-based moiety has a high concentration or density of positively-charged residues, including arginine (R), histidine (H) and lysine (K), or any combination thereof. The peptide-based moiety, at the minimum, has at least one (1) cationic residue, e.g., arginine (R), histidine (H) and lysine (K) and the peptide-based moiety has a net positive charge. In certain embodiments, the degree of cationic residues is characterized based on a percentage of the total number of residues. Thus, if the peptide has a length of 25 amino acids, the minimum percentage of positively charged residues should preferably be 4% (1 residue out of 25 residues). Preferably, the peptide may comprise between 2% and 50%, or between 4% and 30%, or between 6% and 20%, or between 8% and 10%, so long as the total net charge is positive. The invention

contemplates that the distribution and/or location of the positively-charged residues over the length of the peptides of the invention may be at any position (or combination of positions) or region (or combination of regions) along the sequence of the peptide. For example, the positively charged residue(s) may be located towards the N-terminus of the peptide. They may also be located towards the C-terminus of the peptide. There also may be some positively charged residues near or at the N-terminus and the C-terminus, or at any residue position in between.

In certain other embodiments, the peptide-based moiety can include nonnaturally- occurring amino acid residues which include a net positive charge.

The peptide-based moiety may also comprise in certain embodiments a cleavable moiety or bond. Such cleavable moiety or bond may, when cleaved, induce a

conformational change in the peptide-based moiety. The cleavable bond may be a disulfide bridge, a bond otherwise reducible by protonation, or an enzyme cleavage site.

In certain other embodiments, the headgroup moiety and the tail moiety are co valently joined via a linker.

In other aspects, the invention relates to a pharmaceutical composition comprising a lipid of the invention and a therapeutic agent. The therapeutic agent can be a nucleic acid molecule, peptide, antibody, or small molecule, or other therapeutic agent.

In certain embodiments, the therapeutic agent is a nucleic acid molecule, such as a DNA molecule, an RNA molecule, a single-stranded nucleic acid molecule, a double- stranded nucleic acid molecule, a miRNA molecule, a siRNA molecule, a RNAi molecule, or an oligonucleotide.

In certain other aspects, the invention relates to a method of treating a disease comprising administering a therapeutically effective amount of a pharmaceutical composition of the invention.

In certain other embodiments, the peptide of the lipids of the invention and have the amino acid sequence selected from the group consisting of:

SEQ 1:— (R)_niH(R)_n2 or SEQ 2:— (K)_niH(K)_n2, wherein, nland n2 are

independently 0 to 25;

SEQ 3:— (R)_ni(HR)_n2(R)_n3 or SEQ 4:— (K)_ni(HK)_n2(K)_n3, wherein nl and n3 are independently 0 to 25 and n2 is 1 to 15;

SEQ 5:— (R)_ni(RH)_n2(R)_n3 or SEQ 6:— (K)_ni(KH)_n2(K)_n3, wherein nl and n3 are independently 0 to 25 and n2 is 1 to 15; SEQ 7:— Xi(HR)_nX₂ or SEQ 8:— Xi(HK)_nX₂, wherein XI and X2 are

independently amino acids or peptides with 2 to 25 amino acids and n is 1 to 15;

SEQ 9:— Xi(RH)_nX₂ or SEQ 10:— Xi(KH)_nX₂, wherein XI and X2 are independently amino acids or peptides with 2 to 25 amino acids and n is 1 to 15;

SEQ 11 — RHRHRHRHR ;

SEQ 12 — RHDRHDRHD ;

SEQ 13 — RHKHRQRHRPPQ ;

SEQ 14 — RHKHRQRHRPPQ;

SEQ 15 — K(RHRHR) (HRHR) ;

SEQ 16 — K(RHDRH)(DRHD); and

SEQ 17 — K(RHKHRQXRHRPPQ).

In certain other embodiments, the peptide of the lipids of the invention can have a linear amino acid sequence, or alternately, a branched amino acid sequence, or even a cyclized amino acid sequence, or a portion that is cyclized. Also, the residues of the peptides of the invention may be L-amino acids, D-amino acids, or nonnaturally-occurring amino acids or otherwise naturally-occurring residues which are derivatized, e.g., by adding a cationic group.

In yet another aspect, the cationic lipids of the invention can comprise a spacer or linker that couples or covalently links a peptide moiety with a lipid tail moiety of the invention. In certain embodiments, the spacer or linker can be a PEG (polyethylene glycol molecule), having 1-24 subunits, or preferably, 1-12 subunits, or more preferably 1-6 subunits or even 1-3 subunits. Any suitable spacer or linker molecule is contemplated.

In still another aspect, the invention relates to a method of making a lipid of the invention, comprising obtaining a peptide; and conjugating the peptide to the tail moiety.

The invention also relates to a method of making the cationic lipid of the invention, comprising: obtaining a peptide P; and conjugating the peptide P to the acyl chain Y.

The peptides used in the invention can be obtained by any suitable means, for example, purified from a biological system, purified as a fragment of a digested protein, expressed using a recombinant system, or synthesized by chemical processes.

In other embodiments, the lipid delivery vehicle compositions or formulations of the invention can be in the form of a type of lipid aggregate, including a liposome, a unilamellar liposome, a multilamellar liposome, a micelle, or an amorphous aggregate.

In certain other embodiments, the lipid delivery vehicle compositions of the invention, e.g., a lipid delivery vehicle comprising a lipid that includes a conformation- changing peptide moiety, include a therapeutic agent that can have a biological effect in a mammalian subject. The agent can be a nucleic acid molecule, a protein, a peptide, a pharmaceutical small molecule, a prodrug molecule, a vitamin, a hormone, a cytokine, a carbohydrate, or a cytotoxic agent.

In still other embodiments, the therapeutic agent is a nucleic acid molecule, that can be a DNA molecule, an RNA molecule, a single- stranded nucleic acid molecule, a double-stranded nucleic acid molecule, a miRNA molecule, a siRNA molecule, or an oligonucleotide.

In a particular embodiment, the therapeutic agent is a dsRNA, such as a siRNA or a DsiRNA, i.e., Dicer substrate.

In certain other embodiments, the lipid delivery vehicle compositions of the invention can include a second lipid compound (third, fourth, or additional lipid compounds are also contemplated). The second lipid compound can be any of those lipid compounds described herein. The second lipid compound can also be any known lipid compounds, such as any known cationic lipid compounds, anionic lipid compounds, non- cationic lipid compounds, neutral lipid compounds. In certain embodiments, the second lipid compound is N, N-dioleyl-N, N-dimethylammonium chloride (DODAC), N, N- distearyl-N, N-dimethylammonium bromide (DDAB), N-(l-(2, 3-dioleoyloxy)propyl)-N, N, N-trimethylammonium chloride (DOTAP), N-(l-(2, 3-dioleyloxy)propyl)-N, N, N- trimethylammonium chloride (DOTMA), N, N-dimethyl-2, 3-dioleyloxypropylamine (DODMA), 1, 2-DiLinoleyloxy-N, N-dimethylaminopropane (DLinDMA), 1, 2- Dilinolenyloxy-N, N-dimethylaminopropane (DLenDMA), 5-carboxyspermylglycine dioctaoleoylamide (DOGS) of dipalmitoylphophatidylethanolamine 5-carboxyspermyl- amide (DPPES).

The second lipid compound can also be, in some embodiments, a non-cationic lipid compound, that can include, for example, lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphatidic acid, cerebrosides, dicetylphosphate, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipahnitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoyl-phosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), palmitoyloleyol-phosphatidylglycerol (POPG),

dioleoylphosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane- 1 -carboxylate (DOPE-mal), dipalmitoyl-phosphatidylethanolamine (DPPE), dimyristoyl- phosphatidylethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), monomethyl-phosphatidylethanolamine, dimethyl-phosphatidylethanolamine, dielaidoyl- phosphatidylethanolamine (DEPE), stearoyloleoyl-phosphatidylethanolamine (SOPE), cholesterol, stearylamine, dodecylamine, hexadecylamine, acetyl palmitate,

glycerolricinoleate, hexadecyl stereate, isopropyl myristate, amphoteric acrylic polymers, triethanolamine-lauryl sulfate, alkyl-aryl sulfate polyethyloxylated fatty acid amides, dioctadecyldimethyl ammonium bromide, ceramide, diacylphosphatidylcholine, diacylphosphatidylethanolamine, lysophosphatidylcholine and

ly sopho sphatidylethanolamine .

In certain other embodiments, the first lipid compound is between 1 mol % and 80 mol % of the total lipid compounds of the lipid-based delivery compositions of the invention. The first lipid compound can also be between 10 mol % and 90 mol % of the total lipid compounds of said composition. In other embodiments, the first lipid compound can also be between 20 mol % and 80 mol % of the total lipid compounds of said composition. In still other embodiments, the first lipid compound can be between 30 mol % and 70 mol % of the total lipid compounds of said composition. In other embodiments, the first lipid compound can be between 40 mol % and 60 mol % of the total lipid compounds of said composition. In yet other embodiments, the first lipid compound can be between about 1 mol % and 50 mol % of the total lipid compounds of said composition.

In still other embodiments, the lipid delivery vehicle compositions of the invention can include a stabilizing component, including a PEG-lipid conjugate, ATTA-lipid conjugate, a cationic-polymer-lipid conjugate and mixtures thereof.

In yet other embodiments, the lipid delivery vehicle compositions of the invention can include a targeting moiety, including a ligand, polypeptide, nucleic acid molecule, lipid, carbohydrate, lipoprotein, hormone, cytokine, receptor or small molecule, which functions to enable the composition to interact or bind directly to a cellular or biological target more readily.

In certain embodiments, the targeting moiety can be a somatostatin (sst2), bombesin/GRP, luteinizing hormone-releasing hormone (LHRH), neuropeptide Y

(NPY/Y1), neurotensin (NT1), vasoactive intestinal polypeptide (VIP/VP AC1) or cholecystokinin (CCK/CCK2).

In certain embodiments involving dsRNA, the dsRNA is an isolated double stranded ribonucleic acid (dsRNA) comprising a first oligonucleotide strand having a 5' terminus and a 3' terminus and a second oligonucleotide strand having a 5' terminus and a 3' terminus, wherein:

said dsRNA comprises a duplex region of at least 25 base pairs;

said first strand has a length which is at least 25 nucleotides and said second strand has a length which is at least 26 nucleotides;

said second strand is 1-5 nucleotides longer at its 3' terminus than said 5' terminus of said first strand; and

said second oligonucleotide strand is sufficiently complementary to said target gene along at least 19 nucleotides of said second oligonucleotide strand length to reduce target gene expression when said dsRNA is introduced into a mammalian cell.

In still other embodiments, the first strand of the dsRNA is 25-30 nucleotides in length. In yet other embodiments, the second strand of the dsRNA is two nucleotides longer at its 3' terminus than said 5' terminus of the first strand. The 3' terminus of the first strand of the dsRNA and the 5' terminus of the second strand can form a blunt end.

In yet other embodiments, the dsRNA can include first strand and second strand lengths, respectively, selected from the group consisting of the following:

25 (first strand) and 26, 27, 28 and 29 (second strand) nucleotides, respectively;

26 (first strand) and 27, 28, 29 and 30 (second strand) nucleotides, respectively; 27 (first strand) and 28, 29 and 30 (second strand) nucleotides, respectively;

28 (first strand) and 29 and 30 (second strand) nucleotides, respectively; and

29 (first strand) and 30 (second strand) nucleotides, respectively.

In a particular embodiment, the first strand of the dsRNA is 25 nucleotides in length and the second strand is 27 nucleotides in length.

In still other embodiments, the dsRNA of the compositions of the invention is present in an amount effective to reduce target RNA levels by an amount (expressed by %) selected from the group consisting of at least 10%, at least 50% and at least 80-90% when said formulation contacts a mammalian cell in vitro.

In certain embodiments, the effective amount of dsRNA is selected from the group consisting of 1 nanomolar or less, 200 picomolar or less, 100 picomolar or less, 50 picomolar or less, 20 picomolar or less, 10 picomolar or less, 5 picomolar or less, 2, picomolar or less and 1 picomolar or less in the environment of said cell.

In yet other embodiments, the dsRNA of the compositions of the invention is present in an amount effective to reduce target RNA levels when said formulation contacts a cell of a mammalian subject by an amount (expressed by %) selected from the group consisting of at least 10%, at least 50% and at least 80-90%. In such embodiments, the effective amount of the dsRNA is a dosage selected from the group consisting of 1 microgram to 5 milligrams per kilogram of said subject per day, 100 micrograms to 0.5 milligrams per kilogram, 0.001 to 0.25 milligrams per kilogram, 0.01 to 20 micrograms per kilogram, 0.01 to 10 micrograms per kilogram, 0.10 to 5 micrograms per kilogram, and 0.1 to 2.5 micrograms per kilogram.

In yet another aspect, the present invention relates to pharmaceutical compositions comprising the lipid delivery vehicle compositions of the invention, as described above, and a pharmaceutically acceptable carrier.

In still another aspect, the present invention relates to kits comprising the lipid delivery vehicle compositions of the invention, as described above, and instructions for its use.

In still other aspects, the present invention relates to methods of using the lipid delivery vehicle compositions of the invention.

In one embodiment, the present invention provides a method for causing a biological effect in a mammalian subject comprising administering a lipid delivery vehicle composition of the invention to a mammal in an amount sufficient to cause the biological effect, wherein the conformation of the peptide-based moiety is induced to change from a first state to a second state upon internalization by a cell (i.e., entry into the cytoplasm) or upon entry into an intracellular compartment, e.g., a lysosome, thereby effectively releasing the payload. The composition can be administered in an amount sufficient to cause a biological effect in the mammal.

In another embodiment, the present invention provides a method for reducing expression of a target gene in a mammal comprising administering a lipid delivery vehicle composition of the invention to a mammal in an amount sufficient to reduce expression of a target gene in the mammal, wherein the conformation of the peptide-based moiety is induced to change from a first state to a second state upon internalization by a cell (i.e., entry into the cytoplasm) or entry into an intracellular compartment, e.g., a lysosome, thereby effectively releasing the payload. The composition can be administered in an amount sufficient to reduce expression of a target gene in the mammal.

In certain embodiments, a composition of the invention is administered at a dosage selected from the group consisting of 1 microgram to 5 milligrams per kilogram of said mammal per day, 100 micrograms to 0.5 milligrams per kilogram, 0.001 to 0.25 milligrams per kilogram, 0.01 to 20 micrograms per kilogram, 0.01 to 10 micrograms per kilogram, 0.10 to 5 micrograms per kilogram, and 0.1 to 2.5 micrograms per kilogram.

In certain other embodiments, the administering step comprises administering by intravenous injection, intramuscular injection, intraperitoneal injection, infusion, subcutaneous injection, transdermal, aerosol, rectal, vaginal, topical, oral and inhaled delivery.

BRIEF DESCRIPTION OF THE DRAWINGS

The following Detailed Description, given by way of example, but not intended to limit the invention to specific embodiments described, may be understood in conjunction with the accompanying drawings, in which:

Figure 1 depicts the results of the gel retardation assay of Example 8 which demonstrates the formation of a complex between a nucleic acid payload and a lipid composition of the invention. Figure 1A relates to the use of the lipid AzPC-PEG8-(R)9S of the invention to form a complex with a DsiRNA payload. Figures IB and 1C relate to the use of the lipid AzPC-PEG8-G(RH)₄RS of the invention to form a complex with a DsiRNA payload.

Figure 2 depicts the results of the size exclusion chromatography assay of Example 9 which demonstrates the formation of a complex between a nucleic acid payload and a lipid composition of the invention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates, in part, to the discovery that certain peptide or peptide-based modifications to lipids impart the lipids with advantageous performance characteristics in relation to their use as lipid-based delivery systems for the delivery of therapeutic payloads— including nucleic acid molecules, polypeptides, peptides, antibodies, hormones, small molecule pharmaceutical compounds, toxins, and cytokines— to a wide variety of cells and/or tissues. The lipids of the invention are modified by covalent attachment of a peptide or peptide-based moiety to the lipid, preferably at the tail moiety of the lipid molecule. The peptide or peptide-based moiety carries a net positive charge and upon contact with the intracellular environment (e.g., cytoplasm or

intracellular compartment) is induced to change its conformation or three-dimensional structure such that the release of the payload carried by the modified lipid, or by a lipid- based delivery system that comprises at least one modified lipid, is enhanced. In addition, the net positively charged peptide or peptide-based moiety imparts a net positive charge on the lipid, thereby imparting the lipid with cationic lipid

characteristics, including improved cell interaction and/or entry and/or interaction with various payloads, such as anionic payloads, such as nucleic acid molecules. The nucleic acid molecules deliverable by the compositions of the invention can include, for example, DNA molecules, RNA molecules, single-stranded nucleic acid molecules, double- stranded nucleic acid molecules, miRNA molecules, siRNA molecules, DsiRNA molecules, and oligonucleotides. In a particular aspect, the present invention is advantageous in the delivery of dsRNA, including siRNA or DsiRNA molecules, to target cells and/or tissues of interest. The present invention is based, at least in part, on the recognition that lipid compounds having been modified by the peptide and/or peptide-moieties of the invention, once introduced to intracellular conditions (e.g., changes in pH, oxidation/reduction conditions, protonation/deprotonation conditions, chemical or enzymatic conditions) can increase the efficiency of the delivery of the payload material.

Thus, the invention is based, at least in part, upon the discovery of a novel means by which to modify lipids, including for example, cationic lipids, through covalent attachment of a peptide or peptide-based moiety to the lipid (1) which carries a net positive charge and thereby imparts a net positive charge on the overall molecule and (2) which undergoes a conformational change under intracellular- triggering conditions. The peptide or peptide-based moiety imparts improved performance characteristics on the overall lipid as a lipid delivery vehicle for intracellular delivery of therapeutic agents, such as nucleic acids. The improved characteristics include, but are not limited to, enhanced internalization of the lipid delivery vehicles, and thus, the payloads therein, into target cells. The improved characteristics also include enhanced payload release properties of the lipid delivery vehicles whereby the therapeutic agent payloads, e.g., nucleic acid payloads, are more effectively delivered to the cytoplasm of target cells. The intracellular conditions capable of triggering the conformational change in the peptide moiety, and thus, the resultant beneficial effects on the characteristics of the lipid delivery vehicles of the invention, can include any suitable intracellular condition that may be met by the lipid delivery vehicles upon entry of the cell. This can include, for example, changes in pH, oxidation/reduction conditions, protonation/deprotonation conditions, enzymatic cleavage (e.g., via a lysosomal enzyme) or other like conditions that may trigger a conformational change in the peptide or peptide-like moiety, and in turn, imparting the improved characteristics on the lipids of the invention. The invention also provides methods for making or forming the lipids and lipid- based delivery compositions of the invention, which include one or more lipid compounds (e.g., the peptide-modified lipids of the invention) of the invention and a therapeutic agent, which can include nucleic acid molecules, DNA, RNA, single- stranded nucleic acids, double- stranded nucleic acids, miRNA, siRNA, oligonucleotides, polypeptides, peptides, hormones, cytokines, small molecule pharmaceuticals, toxins, and the like. In still other aspects, the present invention relates to the use of the lipid-based delivery compositions of the invention in delivering therapeutic agents, by in vitro or in vivo means to cells and/or tissues, and for treating conditions or diseases by administering therapeutically effective amounts of one or more therapeutic agents using the lipid-based delivery compositions of the invention. The invention, in other aspects, provides kits comprising the cleavable lipids, or the cleavable lipid-based compositions of the invention and instructions for use.

Thus, in one aspect, the present invention relates to a class of novel cationic lipids capable of conformational change. The formulations comprising the novel cationic lipid described in the current invention would allow improved delivery to the cytoplasm of the cell with the conformational changes to the cationic lipid in addition to providing protection of the payload from degradation and elimination, as well as allowing cellular uptake or internalization.

Conventional cationic lipids, such as DOTAP, DODAP, DC-Cholesterol, DpePC and DOTMA (defined herein), rely on the net cationic charge of their lipid head groups. The lipid tails in these molecules are considered only to provide the hydrophobic part of the molecule. These cationic lipids are extensively used in the art. While these molecules provide charge density for plasmid DNA condensation, they lack any inducible or triggered release functionality. However, no cationic lipids are described in the art that contain both net positive charge on the phospholipid tail and moieties on the phospholipid tail that are capable of conformational change. The combination of net positive charge on the phospholipid tail and moieties on the phospholipid tail that are capable of

conformational change is advantageous as the cationic charge allows condensation of the payloads, in particular, polyanionic payloads, and the moieties on the phospholipid tail that are capable of conformational change allow the payload to be released and delivered to the cell cytoplasm.

In certain aspects, the invention is broadly applicable to any anionic payload. In one embodiment of the present invention the payload is a nucleic acid. In one

embodiment of the present invention the payload is a ribonucleic acid (RNA). In one embodiment of the present invention the RNA is Dicer- substrate RNA. In one

embodiment the novel cationic lipid is utilized to fabricate a formulation that is a "vesicle- based" particle, in another embodiment the formulation is a "core-based" particle, however, the principle applies to other particulate/liposomal/micellar formulations.

Conformational change or change of structure to the cationic peptide may be induced by an intracellular condition encountered upon uptake of the lipid or a lipid-based

composition of the invention, including changes in pH, protonation/deprotonation conditions, disulphide reduction and enzymatic processing.

Definitions

Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this invention belongs. The following references provide one of skill with a general definition of many of the terms used in this invention: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, the following terms have the meanings ascribed to them below, unless specified otherwise.

As used herein, the term "cleavable moiety," as it pertains to a lipid molecule or to the peptide or peptide-based moieties of the invention, refers to a functional group, moiety or portion of a lipid or protein molecule that is susceptible to becoming cleaved by any suitable means, including, for example, by enzymatic action, hydrolysis, reduction, under certain circumstances. Cleavage can be designed to occur in vitro or in vivo, or in some combination thereof. Any suitable cleavable moiety is contemplated. In certain embodiments, the cleavable moiety is a disulfide bridge, which is cleavable upon hydrolysis or reduction. In other embodiments, the cleavable moiety is subject to cleavage via an enzyme, such as a protease, an esterase, amidase or disulfide cleaving enzyme. In one embodiment, the cleavable moiety is a disulfide bridge. In another example, if referring to the peptide or peptide-based moieties of the invention, the cleavable moiety can be a protease amino acid recognition sequence.

As used herein, the term "cationic moiety" or "positively charged moiety" refers to a functional group, moiety or portion of a compound or a lipid molecule having a net positive charge. In certain embodiments, the cationic moiety can refer to the cationic peptide or peptide-based moiety of the invention which is used to modify a lipid of the invention.

As used herein, the term "lipophilic moiety" refers to a functional group, moiety or portion of a lipid molecule of the invention having lipid characteristics, which can include where such molecules (a) tend to be insoluble in water and (b) tend to be soluble in non- polar solvents.

As used herein, the term "linker moiety" refers to any suitable functional group or moiety that joins a lipid headgroup moiety and a lipid tail moiety.

As used herein, the lipid compounds of the invention include, but are not limited to, the compounds of formula X-Y-P, as described herein, and the salts, hydrates, solvates and solvates of the salts thereof. Depending on their structures, the compounds of the invention may exist in stereoisomeric forms (enantiomers, diastereomers). The invention therefore encompasses the enantiomers or diastereomers and respective mixtures thereof of the lipid compounds of the invention. The stereoisomerically pure constituents can be isolated in a known manner from such mixtures of enantiomers and/or diastereomers.

Where the compounds of the invention can occur in tautomeric forms, the present invention encompasses all tautomeric forms.

As used herein, the term "salts", in certain instances, refers to physiologically or pharmaceutically acceptable salts of the compounds of the invention. However, salts which are themselves unsuitable for pharmaceutical applications but nevertheless can be used for isolating or purifying the compounds of the invention are also encompassed.

As used herein, "Physiologically acceptable salts" or "pharmaceutically acceptable salt(s)" of the compounds of the invention include acid addition salts of mineral acids, carboxylic acids and sulphonic acids, e.g., salts of hydrochloric acid, hydrobromic acid, sulphuric acid, phosphoric acid, methanesulphonic acid, ethanesulphonic acid, toluenesulphonic acid, benzenesulphonic acid, naphthalenedisulphonic acid, acetic acid, trifluoroacetic acid, propionic acid, lactic acid, tartaric acid, malic acid, citric acid, fumaric acid, maleic acid and benzoic acid. Physiologically acceptable salts of the compounds of the invention also include salts of conventional bases, such as, alkali metal salts (e.g. sodium and potassium salts), alkaline earth metal salts (e.g. calcium and magnesium salts) and ammonium salts derived from ammonia or organic amines having 1 to 16 carbon atoms, such as, ethylamine, diethylamine, triethylamine, ethyldiisopropylamine, monoethanolamine, diethanolamine, triethanolamine, dicyclohexylamine,

dimethylaminoethanol, procaine, dibenzylamine, N-methylmorpholine, arginine, lysine, ethylenediamine, N-methylpiperidine and choline. As used herein, the term "intracellular-triggering conditions" refers to those conditions determined by the physical and/or chemical state of the environment within a cell, which are capable of inducing a conformational change in a peptide of the invention, i.e., those peptides which are capable of undergoing a conformational change when in the presence of such conditions. Such conditions may be present in any intracellular space, including within the cytoplasm or nucleus, or any other intracellular compartment, including, for example, a lysosome, endosome, golgi, or endoplasmic reticulum.

In certain embodiments, the lipid compounds modified by the peptide or peptide- based moieties of the invention are cationic lipid compounds, and a pharmaceutically acceptable salt can be an anion which can include fluoride, chloride, bromide, iodide, sulfate, bisulfate, nitrate, phosphate, citrate, methanesulfonate, trifluoroacetate, acetate, fumarate, oleate, valerate, maleate, oxalate, isonicotinate, lactate, salicylate, tartrate, tannate, pantothenate, bitartrate, ascorbate, succinate, gentisinate, gluconate, glucaronate, saccharate, formate, benzoate, glutamate, ethanesulfonate, benzenesulfonate, p- toluensulfonate, and pamoate.

As used herein, the term "solvates" refer for the purposes of the invention to those forms of the lipid compounds of the invention which form a complex in the solid or liquid state through coordination with solvent molecules.

As used herein, the term "hydrates" refers to a specific form of solvates in which the coordination takes place with water.

The term "substituents" refers to a group "substituted" on an alkyl, cycloalkyl, alkenyl, alkynyl, heterocyclyl, heterocycloalkenyl, cycloalkenyl, aryl, or heteroaryl group, or any other functional group of atom of the lipids of the invention. Any atom of the compounds of the invention can be substituted. Suitable substituents include, without limitation, alkyl (e.g., CI, C2, C3, C4, C5, C6, C7, C8, C9, CIO, Cl l, C12, or more, straight or branched chain alkyl), cycloalkyl, haloalkyl (e.g., perfluoroalkyl such as CF₃), aryl, heteroaryl, aralkyl, heteroaralkyl, heterocyclyl, alkenyl, alkynyl, cycloalkenyl, heterocycloalkenyl, alkoxy, haloalkoxy (e.g., perfluoroalkoxy such as OCF₃), halo, hydroxy, carboxy, carboxylate, cyano, nitro, amino, alkyl amino, S0₃H, sulfate, phosphate, methylenedioxy (-0-CH₂-0- wherein oxygens are attached to same carbon (geminal substitution) atoms), ethylenedioxy, oxo, thioxo (e.g., C=S), imino (alkyl, aryl, aralkyl), S(0)_nalkyl (where n is 0-2), S(0)_n aryl (where n is 0-2), S(0)_n heteroaryl (where n is 0-2), S(0)_n heterocyclyl (where n is 0-2), amine (mono-, di-, alkyl, cycloalkyl, aralkyl, heteroaralkyl, aryl, heteroaryl, and combinations thereof), ester (alkyl, aralkyl, heteroaralkyl, aryl, heteroaryl), amide (mono-, di-, alkyl, aralkyl, heteroaralkyl, aryl, heteroaryl, and combinations thereof), sulfonamide (mono-, di-, alkyl, aralkyl,

heteroaralkyl, and combinations thereof). In one aspect, the substituents on a group are independently any one single, or any subset of the aforementioned substituents. In another aspect, a substituent may itself be substituted with any one of the above substituents.

As used herein, the term "alkyl" per se and "alk" and "alkyl" in alkoxy,

alkylamino, alkylcarbonyl, alkoxycarbonyl, alkylaminocarbonyl, alkylcarbonylamino, alkylsulphonyl, alkylsulphonylamino and alkylaminosulphonyl, refer to a linear or branched alkyl radical having 1 to 100 carbon atoms, by way of example, refers to methyl, ethyl, n-propyl, isopropyl, n-butyl and tert-butyl.

As used herein, the term "alkoxy" refers to the group -OR wherein R is alkyl, and by way of example, refers to methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy and tert- butoxy.

As used herein, the term "alkenyloxy" refers to the group -O-R wherein R is alkenyl.

As used herein, the term "alkylamino" stands for an alkylamino radical having one or two alkyl substituents (chosen independently of one another), by way of example, for methylamino, ethylamino, n-propylamino, isopropylamino, tert-butylamino, N,N- dimethylamino, N,N-diethylamino, N-ethyl-N-methylamino, N-methyl-N-n-propylamino, N-isopropyl-N-n-propylamino and N-tert-butyl-N- methylamino. Ci-C4-alkylamino stands, for example, for a mono alkylamino radical having 1 to 4 carbon atoms or for a

dialkylamino radical having 1 to 4 carbon atoms in each alkyl substituent in each case.

As used herein, the term "aryloxy" refers to the group -O-R wherein R is aryl.

As used herein, the term "aralkyloxy" refers to the group -O-R wherein R is aralkyl.

As used herein, the term "monoalkylamino" stands for an alkylamino radical having a linear or branched alkyl substituent; by way of example, it includes methylamino, ethylamino, n-propylamino, isopropylamino and tert-butylamino.

As used herein, the term "monocycloalkylamino" stands for a cycloalkylamino radical having a cycloalkyl substituent, where the other substituent at the amino radical can be hydrogen or other substituents such as alkyl. By way of example,

monocycloalkylamino groups include cyclopropylamino and cyclobutylamino.

As used herein, the term "alkylcarbonyl" stands, by way of example, for methylcarbonyl, ethylcarbonyl, n-propylcarbonyl, isopropylcarbonyl, n-butylcarbonyl and tert-butylcarbonyl.

As used herein, the term "alkoxycarbonyl" stands by way of example for methoxycarbonyl, ethoxycarbonyl, n-propoxycarbonyl, isopropoxycarbonyl, n- butoxycarbonyl and tert-butoxycarbonyl.

As used herein, the term "alkylaminocarbonyl" stands for an alkylaminocarbonyl radical having one or two alkyl substituents (chosen independently of one another), by way of example for methylaminocarbonyl, ethylaminocarbonyl, n-propylaminocarbonyl, isopropylaminocarbonyl, tert-butylaminocarbonyl, N,N-dimethylaminocarbonyl, N,N- diethylaminocarbonyl, N-ethyl-N-methylaminocarbonyl, N-methyl-N-n- propylaminocarbonyl, N-isopropyl-N-n-propylaminocarbonyl andN-tert-butyl-N- methylaminocarbonyl. C₁-C₄- Alkylaminocarbonyl stands, for example, for a

monoalkylaminocarbonyl radical having 1 to 4 carbon atoms or for a

dialkylaminocarbonyl radical having 1 to 4 carbon atoms in each alkyl substituent in each case.

As used herein, the term "alkylcarbonylamino" stands by way of example for methylcarbonylamino, ethylcarbonylamino, n-propylcarbonylamino,

isopropylcarbonylamino, n-butylcarbonylamino and tert-butylcarbonylamino.

As used herein, the term "alkylsufonyl" stands by way of example for

methylsulfonyl, ethylsulphonyl, n-propylsulfonyl, isopropylsulfonyl, n-butylsulfonyl and tert-butylsulfonyl.

As used herein, the term "alkylaminosulfonyl" stands for an alkylamino sulphonyl radical having one or two alkyl substituents (chosen independently of one another), by way of example, for methylaminosulfonyl, ethylaminosulfonyl, n-propylaminosulfonyl, isopropylaminosulfonyl, tert-butylaminosulfonyl, N,N-dimethylaminosulfonyl, N,N- diethylaminosulfonyl, N-ethyl-N-methylaminosulfonyl, N-methyl-N-n- propylaminosulfonyl, N-isopropyl-N-n-propylaminosulfonyl andN-tert-butyl-N- methylaminosulfonyl. C₁-C₄- Alkylaminosulfonyl stands for example for a

monoalkylaminosulfonyl radical having 1 to 4 carbon atoms or for a dialkylaminosulfonyl radical having 1 to 4 carbon atoms in each alkyl substituent in each case.

As used herein, the term "alkylsulfonylamino" includes, but is not limited to, methylsulfonylamino, ethyl sulfonylamino, n-propylsulfonylamino,

isopropylsulfonylamino, n-butylsulfonylamino and tert-butylsulfonylamino.

As used herein, the term "alkylene" refers to a divalent straight chain or branched chain saturated aliphatic radical. Examples of alkylene groups include methylene, ethylene, propylene, and etc..

As used herein, the term "alkylenecarboxy" refers to the group -alk-COOH where alk is alkylene.

As used herein, the term "carboxamide" or "carbamoyl" refers to the group -C(O)-

NH₂.

As used herein, the term "carbamate" refers to a moiety containing the functional

-OC(O^N^

group of \ .

As used herein, the term "cycloalkyl" stands for a monocyclic cycloalkyl group usually having 3 to 6 carbon atoms, by way of example, includes cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl.

As used herein, the term "heterocyclyl" stands for a monocyclic, heterocyclic radical having 5 or 6 ring atoms having one or more heteroatoms and/or heterogroups. Such heteroatoms and/or heterogroups include, but are not limited to, N, O, S, SO, S0₂, where a nitrogen atom may also form an N-oxide. The heterocyclyl radicals may be saturated or partly unsaturated. 5- or 6-membered, monocyclic saturated heterocyclyl groups, for example, include pyrrolidin-2-yl, pyrrolidin-3-yl, pyrrolinyl, tetrahydrofuranyl, tetrahydrothienyl, pyranyl, piperidin-l-yl, piperidin-2-yl, piperidin-3-yl, piperidin-4-yl, thiopyranyl, morpholin-l-yl, morpholin-2-yl, morpholin-3-yl, piperazin-l-yl, piperazin-2- yi-

As used herein, the term "heteroaryl" stands for an aromatic, mono- or bicyclic radical usually having 5 to 10 ring atoms having one or more heteroatoms. The

heteroatoms can be, but are not limited to, S, O and N, where a nitrogen atom may also form an N-oxide. In some instances, heteroaryl group contains 5 or 6 ring atoms. By way of example, such heteroaryl groups as used herein include thienyl, furyl, pyrrolyl, thiazolyl, oxazolyl, oxadiazolyl, pyrazolyl, imidazolyl, triazolyl, pyridyl, pyrimidyl, pyridazinyl, pyrazinyl, indolyl, indazolyl, benzofuranyl, benzothiophenyl, quinolinyl, isoquinolinyl, benzoxazolyl, benzimidazolyl.

As used herein, the term "halo" or "halogen" stands for fluorine, chlorine, bromine and iodine.

As used herein, the term "alkenyl" refers to a straight or branched hydrocarbon chain containing 2-20 carbon atoms and having one or more double bonds. Examples of alkenyl groups include, but are not limited to, allyl, propenyl, 2-butenyl, 3-hexenyl and 3- octenyl groups. One of the double bond carbons may optionally be the point of attachment of the alkenyl substituent. The term "alkynyl" refers to a straight or branched hydrocarbon chain containing 2-20 carbon atoms and characterized in having one or more triple bonds. Examples of alkynyl groups include, but are not limited to, ethynyl, propargyl, and 3- hexynyl. One of the triple bond carbons may optionally be the point of attachment of the alkynyl substituent.

As used herein, the term "steroidyl" refers to a group of lipids that contain a hydrogenated cyclopentanoperhydrophenanthrene ring system. One example of steroidyl moieties is cholesteryl. The steroidyl moieties can be substituted at any atom with any of the substituents described herein or other suitable substituents not defined herein.

As used herein, the term "structural isomer" as used herein refers to any of two or more chemical compounds, such as propyl alcohol and isopropyl alcohol, having the same molecular formula but different structural formulas.

As used herein, the term "diastereoisomer" or "stereoisomer" as used herein refers to two or more compounds which contain the same number and types of atoms, and bonds (i.e., the connectivity between atoms is the same), but which have different spatial arrangements of the atoms, for example cis and trans isomers of a double bond, enantiomers, and diastereomers.

As used herein, the term "amino acid" refers to both natural and unnatural amino acids in either their L- or D-forms. Natural amino acids include alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine. For example, unnatural amino acids include, but are not limited to

azetidinecarboxylic acid, 2-aminoadipic acid, 3-aminoadipic acid, β-alanine,

aminopropionic acid, 2-aminobutyric acid, 4-aminobutyric acid, 6-aminocaproic acid, 2- aminoheptanoic acid, 2-aminoisobutyric acid, 3-aminoisobutyric acid, 2-aminopimelic acid, 2,4-diaminoisobutyric acid, desmosine, 2,2'-diaminopimelic acid, 2,3- diaminopropionic acid, N-ethylglycine, N-ethylasparagine, hydroxylysine,

allohydroxylysine, 3-hydroxyproline, 4-hydroxyproline, isodesmosine, alloisoleucine, N- methylglycine, N-methylisoleucine, N-methylvaline, norvaline, norleucine, ornithine and pipecolic acid. In certain embodiments, the amino acids are positively charged amino acids, for example, lysine, histidine, and arginine, which provide the net positive charge to the peptide or peptide-based moieties of the invention.

As used here, the term "peptide or peptide-based moieties" refers to those peptides and peptide-based molecules used to modify a lipid molecule of the invention, wherein the peptide or peptide-based moieties (1) carry a net positive charge and thereby impart a net positive charge on the overall molecule and (2) undergo a conformational change under intracellular conditions, thereby imparting improved performance characteristics on the modified lipid molecule, including enhanced uptake of the lipids of the invention and improved release characteristics of the modified lipids of the invention. Further characteristics of the peptides or peptide-based moieties of the invention are described further herein.

As used herein, the term "modified lipids of the invention" refers to any suitable lipid that has been modified by covalent attachment of a peptide or peptide-based moiety to impart (1) a net positive charge on the lipid molecule

The term "amphipathic compound" refers to any suitable material containing both hydrophobic and hydrophilic moieties or regions. A subgroup of such compounds comprises "lipids." Hydrophilic characteristics derive from the presence of phosphate, carboxylic, sulfate, amino, sulfhydryl, nitro, carbohydrate, and other like groups.

Hydrophobicity could be conferred by the inclusion of groups that include, but are not limited to, long chain saturated and unsaturated aliphatic hydrocarbon groups and such groups substituted by one or more aromatic, cycloaliphatic or heterocyclic group(s).

Optional amphipathic compounds are phospholipids such as phosphoglycerides.

"Phospholipids" are a group of lipids having both phosphate group and one or more acyl groups. "Phosphoglycerides" are based on glycerol, wherein the three hydroxyl groups are esterified with two acyl groups and a phosphate group, which itself may be bound to one of a variety of simple organic groups. The two acyl groups can be identical, of similar length, or different. Representative examples of which include, but are not limited to, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoyl phosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine, dipalmitoylphosphatidylcholine,

dioleoylphosphatidylcholine, distearoylphosphatidylcholine or

dilinoleoylphosphatidylcholine. Other compounds, such as sphingolipids,

glycosphingolipids, triglycerides, and sterols are also amphipatic compounds.

The term "cationic lipid compound" refers to any of a number of lipid species which carry a net positive charge at physiological pH. A number of cationic moieties and related analogs, which are also useful in the present invention, have been described in U.S. Patent Publication No. 20060083780; U.S. Pat. Nos. 5,208,036; 5,264,618; 5,279,833; 5,283,185; 5,753,613; and 5,785,992; and PCT Publication No. WO 96/10390. Examples of cationic lipid moieties include certain lipid moieties discussed herein. By way of example, other cationic lipid moieties are N, N-dioleyl-N, N-dimethylammonium chloride (DODAC), dioctadecyldimethylammonium (DODMA), distearyldimethylammonium (DSDMA), N-(l-(2, 3-dioleyloxy)propyl)-N, N, N-trimethylammonium chloride

(DOTMA), N, N-distearyl-N, N-dimethylammonium bromide (DDAB), N-(l-(2, 3- dioleoyloxy)propyl)-N, N, N-trimethylammonium chloride (DOTAP), 3-(Ν-(Ν', N'- dimethylaminoethane)-carbamoyl)cholesterol (DC-Choi), N-(l, 2-dimyristyloxyprop-3- yl)-N, N-dimethyl-N-hydroxyethyl ammonium bromide (DMRIE), 1, 2-dilinoleyloxy-N, N-dimethylaminopropane (DLinDMA), 1, 2-dilinolenyloxy-N, N-dimethylaminopropane (DLenDMA), and mixtures thereof. In some cases, the cationic lipid moieties comprise a protonatable tertiary amine head group, CI 8 alkyl chains, ether linkages between the head group and alkyl chains, and 0 to 3 double bonds. Such lipid moieties include, e.g., DSDMA, DLinDMA, DLenDMA, and DODMA. The cationic lipid moieties may also comprise ether linkages and pH titratable head groups. In certain embodiments, the cationic lipids are further modifiable by the peptide or peptide-based moieties of the invention, preferably at the tail region of the cationic lipid.

The term "neutral lipid moiety" refers to any lipid species that exist either in an uncharged or neutral zwitterionic form at physiological pH. Such lipid moieties include, for example, cholesterol, DOPE, DLPE, DLPC, phosphatidylcholines,

phosphatidylethanolamines, phosphatidylserines, ceramide, sphingomyelin, cephalin, and cerebrosides. In certain embodiments, the neutral lipids are further modifiable by the peptide or peptide-based moieties of the invention, preferably at the tail region of the neutral lipid. The net charge of the modified lipid is preferably by charged.

The term "non-cationic lipid moiety" refers to any neutral lipid moiety as described above as well as anionic lipid moieties.

The term "anionic lipid moiety" refers to any lipid moiety that is negatively charged at physiological pH. These lipid moieties are, for example, phosphatidylglycerols, cardiolipins, diacylphosphatidylserines, diacylphosphatidic acids, N-dodecanoyl phosphatidylethanolamines, N-succinyl phosphatidylethanolamines, N- glutarylphosphatidylethanolamines, lysylphosphatidylglycerols,

palmitoyloleyolphosphatidylglycerol (POPG), dipalmitoylphosphatidylcholine (DPPC), and other anionic modifying groups joined to neutral lipid moieties. In certain

embodiments, the anionic lipids are further modifiable by the cationic peptide or peptide- based moieties of the invention, preferably at the tail region of the anionic lipid. The net charge of the modified lipid is preferably by charged.

As used herein, the term "lipid-based compositions or formulations" or "lipid- based delivery compositions or formulations" or "lipid-based aggregates" or "liposomes" or "lipid delivery vehicles or compositions" includes any suitable physical form of the lipid compounds of the invention, including all types of unilamellar and multilamellar liposomes, as well as micelles and more amorphous aggregates of lipid compounds or lipid mixed with amphipathic lipid compounds, such as phospholipids, or mixed with other neutral, cationic, non-cationic or anionic lipid compounds. In certain instances, the lipid-based compositions of the invention comprise at least one cleavable lipid compound of the invention.

The term "liposome" also can be known to encompass any compartment enclosed by a lipid bilayer or a lipidic particle. Some liposomes are also referred to as lipid vesicles. In order to form a liposome the lipid molecules comprise elongated non-polar (hydrophobic) portions and polar (hydrophilic) portions. The hydrophobic and

hydrophilic portions of the molecule are optionally positioned at two ends of an elongated molecular structure. When such lipid compounds are dispersed in water they may spontaneously form bilayer membranes referred to as lamellae. The lamellae are composed of two monolayer sheets of lipid compounds with their non-polar (hydrophobic) surfaces facing each other and their polar (hydrophilic) surfaces facing the aqueous medium. The membranes formed by the lipid compounds enclose a portion of the aqueous phase in a manner similar to that of a cell membrane enclosing the contents of a cell. Thus, the bilayer of a liposome has similarities to a cell membrane without the protein components present in a cell membrane. The term "liposome" includes multilamellar liposomes, which generally have a diameter in the range of 1 to 10 micrometers and are comprised of anywhere from two to hundreds of concentric lipid bilayers alternating with layers of an aqueous phase, and also includes unilamellar vesicles which are comprised of a single lipid layer and generally have a diameter in the range of about 20 to about 400 nanometers (nm), about 50 to about 300 nm, about 300 to about 400 nm, about 100 to about 200 nm, which vesicles can be produced by subjecting multilamellar liposomes to ultrasound, by extrusion under pressure through membranes having pores of defined size, or by high pressure homogenization.

The lipid-based compositions of the invention should be those that are capable of being "complexed" with a "cargo." As used herein, the concept of a cargo or payload (e.g., polypeptide, peptide, nucleic acid molecule, hormone, toxin, siRNA, DsiRNA, miRNA, and the like) being "complexed" with a lipid-based composition of the invention contemplates any suitable interaction between the lipid and the cargo, including where the cargo is contained within or inside of a compartment formed by the aggregate or liposome (e.g., a micelle form), and where the cargo is complexed or integrated (e.g., bonded via some combination of ionic, van der Waals, hydrophobic forces) directly with the lipid molecules themselves forming a type of amorphous aggregate.

As used herein, the term "delivery moiety" or "targeting moiety" is a moiety that is capable of enhancing the ability of an associated or attached lipid-based composition of the invention to associate with, bind, or enter a cell, cell of a tissue or subject, cell type, tissue or location within a subject, either in vitro or in vivo. In certain embodiments, delivery moieties are polypeptides, carbohydrates or lipids. Optionally, delivery moieties are antibodies, antibody fragments or nanobodies. Exemplary delivery moieties include tumor targeting moieties, such as somatostatin (sst2), bombesin/GRP, luteinizing hormone-releasing hormone (LHRH), neuropeptide Y (NPY/Y1), neurotensin (NT1), vasoactive intestinal polypeptide (VIP/VPAC1) and cholecystokinin (CCK/CCK2). In certain embodiments, a delivery moiety is non-covalently associated with a compound of the invention. In other embodiments, a delivery moiety is attached to a lipid compound of the invention, and is optionally covalently attached. In further embodiments, a delivery moiety is attached to a lipid compound of the invention, and is optionally covalently attached. In additional embodiments, a delivery moiety is attached to a cargo {e.g., a dsRNA, small molecule, peptide or other agent) of a formulation of the invention, optionally covalently.

As used herein, the term "cargo" or "payload" of the lipid-based compositions of the invention can be a small molecule pharmaceutical, polypeptide, protein, peptide, hormone, cytokine, antigen, compound, cytotoxic agents (e.g., camptothecin, SN-38, homo-campothothecin, paclitaxel, doxorubicin or methotrexate, and the like), or a nucleic acid molecule, such as, a DNA, RNA, dsRNA, siRNA, DsiRNA, or oligonucleotide.

Therapeutic cargoes/payloads, formulation components and functional excipients may include double-stranded RNA. Double- stranded RNA, such as the dsRNAs of the formulations of the instant invention, has different properties than single-stranded RNA, double-stranded DNA or single- stranded DNA. Each of the species of nucleic acids is bound by mostly non-overlapping sets of binding proteins in the cell and degraded by mostly non-overlapping sets of nucleases. The nuclear genome of all cells is DNA-based and as such is unlikely to produce immune responses except in autoimmune disease (Pisetsky. Clin Diagn Lab Immunol. 1998 January; 51:1-6). Single- stranded RNA

(ssRNA) is the form endogenously found in eukaryotic cells as the product of DNA transcription. Cellular ssRNA molecules include messenger RNAs (and the progenitor pre-messenger RNAs), miRNAs, small nuclear RNAs, small nucleolar RNAs, transfer RNAs and ribosomal RNAs. Single-stranded RNA can induce interferon and

inflammatory immune response via TLR7 and TLR8 receptors (Proc Natl Acad. Sci. 2004. 101:5598-603; Science. 2004. 303:1526-9; Science. 2004. 303:1529-3). Double-stranded RNA can induce interferon and inflammatory immune response via TLR3. It is noted that double-stranded RNA can induce a size-dependent immune response such that dsRNA larger than 30 bp activates the interferon response, while shorter dsRNAs tend to avoid at least the PKR-mediated pathway of immune response. MicroRNAs (miRNAs), including short temporal RNAs and small modulatory RNAs, are the only known cellular dsRNA molecules in mammals and were not discovered until 2001 (Kim. 2005. Mol. Cells. 19:1- 15). Responses to extracellular RNA in the bloodstream, double- or single- stranded of any length, include rapid excretion by the kidneys and degradation by enzymes (PLOS Biol. 2004. 2:18-20).

As used herein, the term "nucleic acid" refers to deoxyribonucleotides,

ribonucleotides, or modified nucleotides, and nucleotide analogs and polymers thereof in single- or double- stranded form. The term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, which have similar binding properties as the reference nucleic acid, and which are metabolized in a manner similar to the reference nucleotides. Examples of such analogs include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs).

As used herein, "nucleotide" is used as recognized in the art to include those with natural bases (standard), and modified bases well known in the art. Such bases are generally located at the Γ position of a nucleotide sugar moiety. Nucleotides generally comprise a base, sugar and a phosphate group. The nucleotides can be unmodified or modified at the sugar, phosphate and/or base moiety, (also referred to interchangeably as nucleotide analogs, modified nucleotides, non-natural nucleotides, non-standard nucleotides and other; see, e.g., Usman and McSwiggen, supra; Eckstein, et al., International PCT Publication No. WO 92/07065; Usman et al, International PCT

Publication No. WO 93/15187; Uhlman & Peyman, supra, all are hereby incorporated by reference herein). There are several examples of modified nucleic acid bases known in the art as summarized by Limbach, et al, Nucleic Acids Res. 22:2183, 1994. Some of the non- limiting examples of base modifications that can be introduced into nucleic acid molecules include, hypoxanthine, purine, pyridin-4-one, pyridin-2-one, phenyl, pseudouracil, 2,4,6- trimethoxy benzene, 3-methyl uracil, dihydrouridine, naphthyl, aminophenyl, 5- alkylcytidines (e.g., 5-methylcytidine), 5-alkyluridines (e.g., ribothymidine), 5-halouridine (e.g., 5-bromouridine) or 6-azapyrimidines or 6-alkylpyrimidines (e.g. 6-methyluridine), propyne, and others (Burgin, et al., Biochemistry 35:14090, 1996; Uhlman & Peyman, supra). By "modified bases" in this aspect is meant nucleotide bases other than adenine, guanine, cytosine and uracil at Γ position or their equivalents.

As used herein, "modified nucleotide" refers to a nucleotide that has one or more modifications to the nucleoside, the nucleobase, pentose ring, or phosphate group. For example, modified nucleotides exclude ribonucleotides containing adenosine

monophosphate, guanosine monophosphate, uridine monophosphate, and cytidine monophosphate and deoxyribonucleotides containing deoxyadenosine monophosphate, deoxyguanosine monophosphate, deoxythymidine monophosphate, and deoxycytidine monophosphate. Modifications of nucleotides include those naturally occurring that result from modification by enzymes that modify nucleotides, such as methyltransferases.

Modified nucleotides also include synthetic or non-naturally occurring nucleotides.

Synthetic or non-naturally occurring modifications in nucleotides include those with 2' modifications, e.g., 2'-methoxyethoxy, 2'-fluoro, 2'-allyl, 2'-0-[2-(methylamino)-2- oxoethyl], 4'-thio, 4'-CH₂-0-2'-bridge, 4'-(CH₂) ₂-0-2'-bridge, 2'-LNA, and 2'-0-(N- methylcarbamate) or those comprising base analogs. In connection with 2'-modified nucleotides as described for the present disclosure, by "amino" is meant 2'-NH₂ or 2'-0- NH₂, which can be modified or unmodified. Such modified groups are described, e.g., in Eckstein et al, U.S. Pat. No. 5,672,695 and Matulic-Adamic et al, U.S. Pat. No.

6,248,878. Additional references that describe modified nucleotides include, e.g., WO 2010/036696; US 7,666,854; US 2010/0022619; WO 2009/124295; WO 2009/117589; WO 2009/117589; WO 2009/100320; US 7,002,006; WO 2005/027962; WO

2004/113553; WO 2004/065579; WO 2003/100017; WO 2003/099840 and U.S. Patent No. 6,084,082.

In reference to the nucleic acid molecules of the present disclosure, modifications may exist upon these agents in patterns on one or both strands of the double stranded ribonucleic acid (dsRNA). As used herein, "alternating positions" refers to a pattern where every other nucleotide is a modified nucleotide or there is an unmodified nucleotide (e.g., an unmodified ribonucleotide) between every modified nucleotide over a defined length of a strand of the dsRNA (e.g., 5 ' -MNMNMN-3 ' ; 3 ' -MNMNMN-5 ' ; where M is a modified nucleotide and N is an unmodified nucleotide). The modification pattern starts from the first nucleotide position at either the 5' or 3' terminus according to a position numbering convention, e.g., as described herein (in certain embodiments, position 1 is designated in reference to the terminal residue of a strand following a projected Dicer cleavage event of a dsRNA agent of a formulation of the invention; thus, position 1 does not always constitute a 3' terminal or 5' terminal residue of a pre-processed agent of a formulation of the invention). The pattern of modified nucleotides at alternating positions may run the full length of the strand, but in certain embodiments includes at least 4, 6, 8, 10, 12, 14 nucleotides containing at least 2, 3, 4, 5, 6 or 7 modified nucleotides, respectively. As used herein, "alternating pairs of positions" refers to a pattern where two consecutive modified nucleotides are separated by two consecutive unmodified nucleotides over a defined length of a strand of the dsRNA (e.g., 5'- MMNNMMNNMMNN-3 ' ; 3 ' -MMNNMMNNMMNN-5 ' ; where M is a modified nucleotide and N is an unmodified nucleotide). The modification pattern starts from the first nucleotide position at either the 5' or 3' terminus according to a position numbering convention such as those described herein. The pattern of modified nucleotides at alternating positions may run the full length of the strand, but optionally includes at least 8, 12, 16, 20, 24, 28 nucleotides containing at least 4, 6, 8, 10, 12 or 14 modified nucleotides, respectively. It is emphasized that the above modification patterns are exemplary and are not intended as limitations on the scope of the invention.

As used herein, "base analog" refers to a heterocyclic moiety which is located at the Γ position of a nucleotide sugar moiety in a modified nucleotide that can be incorporated into a nucleic acid duplex (or the equivalent position in a nucleotide sugar moiety substitution that can be incorporated into a nucleic acid duplex). In dsRNAs of the formulations of the invention, a base analog is generally either a purine or pyrimidine base excluding the common bases guanine (G), cytosine (C), adenine (A), thymine (T), and uracil (U). Base analogs can duplex with other bases or base analogs in dsRNAs. Base analogs include those useful in the formulations, compounds and methods of the invention., e.g., those disclosed in US Pat. Nos. 5,432,272 and 6,001,983 to Benner and US Patent Publication No. 20080213891 to Manoharan, which are herein incorporated by reference. Non-limiting examples of bases include hypoxanthine (I), xanthine (X), 3β-ϋ- ribofuranosyl-(2,6-diaminopyrimidine) (K), 3-P-D-ribofuranosyl-(l-methyl-pyrazolo[4,3- d]pyrimidine-5,7(4H,6H)-dione) (P), iso-cytosine (iso-C), iso-guanine (iso-G), Ι-β-D- ribofuranosyl-(5-nitroindole), l-P-D-ribofuranosyl-(3-nitropyrrole), 5-bromouracil, 2- aminopurine, 4-thio-dT, 7-(2-thienyl)-imidazo[4,5-b]pyridine (Ds) and pyrrole-2- carbaldehyde (Pa), 2-amino-6-(2-thienyl)purine (S), 2-oxopyridine (Y), difluorotolyl, 4- fluoro-6-methylbenzimidazole, 4-methylbenzimidazole, 3-methyl isocarbostyrilyl, 5- methyl isocarbostyrilyl, and 3-methyl-7-propynyl isocarbostyrilyl, 7-azaindolyl, 6-methyl- 7-azaindolyl, imidizopyridinyl, 9-methyl-imidizopyridinyl, pyrrolopyrizinyl,

isocarbostyrilyl, 7-propynyl isocarbostyrilyl, propynyl-7-azaindolyl, 2,4,5- trimethylphenyl, 4-methylindolyl, 4,6-dimethylindolyl, phenyl, napthalenyl, anthracenyl, phenanthracenyl, pyrenyl, stilbenzyl, tetracenyl, pentacenyl, and structural derivates thereof (Schweitzer et al., J. Org. Chem., 59:7238-7242 (1994); Berger et al., Nucleic Acids Research, 28(15):2911-2914 (2000); Moran et al., J. Am. Chem. Soc, 119:2056- 2057 (1997); Morales et al., J. Am. Chem. Soc, 121 :2323-2324 (1999); Guckian et al., J. Am. Chem. Soc, 118:8182-8183 (1996); Morales et al., J. Am. Chem. Soc, 122(6): 1001- 1007 (2000); McMinn et al., J. Am. Chem. Soc, 121 : 11585-11586 (1999); Guckian et al., J. Org. Chem., 63:9652-9656 (1998); Moran et al., Proc. Natl. Acad. Sci., 94: 10506-10511 (1997); Das et al., J. Chem. Soc, Perkin Trans., 1 : 197-206 (2002); Shibata et al., J. Chem. Soc, Perkin Trans., 1 : 1605-1611 (2001); Wu et al., J. Am. Chem. Soc, 122(32):7621- 7632 (2000); O'Neill et al., J. Org. Chem., 67:5869-5875 (2002); Chaudhuri et al., J. Am. Chem. Soc, 117: 10434-10442 (1995); and U.S. Pat. No. 6,218,108.). Base analogs may also be a universal base.

As used herein, "universal base" refers to a heterocyclic moiety located at the Γ position of a nucleotide sugar moiety in a modified nucleotide, or the equivalent position in a nucleotide sugar moiety substitution, that, when present in a nucleic acid duplex, can be positioned opposite more than one type of base without altering the double helical structure (e.g., the structure of the phosphate backbone). Additionally, the universal base does not destroy the ability of the single stranded nucleic acid in which it resides to duplex to a target nucleic acid. The ability of a single stranded nucleic acid containing a universal base to duplex a target nucleic can be assayed by methods apparent to one in the art (e.g., UV absorbance, circular dichroism, gel shift, single stranded nuclease sensitivity, etc.). Additionally, conditions under which duplex formation is observed may be varied to determine duplex stability or formation, e.g., temperature, as melting temperature (Tm) correlates with the stability of nucleic acid duplexes. Compared to a reference single stranded nucleic acid that is exactly complementary to a target nucleic acid, the single stranded nucleic acid containing a universal base forms a duplex with the target nucleic acid that has a lower Tm than a duplex formed with the complementary nucleic acid. However, compared to a reference single stranded nucleic acid in which the universal base has been replaced with a base to generate a single mismatch, the single stranded nucleic acid containing the universal base forms a duplex with the target nucleic acid that has a higher Tm than a duplex formed with the nucleic acid having the mismatched base.

Some universal bases are capable of base pairing by forming hydrogen bonds between the universal base and all of the bases guanine (G), cytosine (C), adenine (A), thymine (T), and uracil (U) under base pair forming conditions. A universal base is not a base that forms a base pair with only one single complementary base. In a duplex, a universal base may form no hydrogen bonds, one hydrogen bond, or more than one hydrogen bond with each of G, C, A, T, and U opposite to it on the opposite strand of a duplex. Optionally, the universal base does not interact with the base opposite to it on the opposite strand of a duplex. In a duplex, base pairing between a universal base occurs without altering the double helical structure of the phosphate backbone. A universal base may also interact with bases in adjacent nucleotides on the same nucleic acid strand by stacking interactions. Such stacking interactions stabilize the duplex, especially in situations where the universal base does not form any hydrogen bonds with the base positioned opposite to it on the opposite strand of the duplex. Non-limiting examples of universal-binding nucleotides include inosine, l-P-D-ribofuranosyl-5-nitroindole, and/or l-P-D-ribofuranosyl-3-nitropyrrole (US Pat. Appl. Publ. No. 20070254362 to Quay et al.; Van Aerschot et al., An acyclic 5-nitroindazole nucleoside analogue as ambiguous nucleoside. Nucleic Acids Res. 1995 Nov l l;23(21):4363-70; Loakes et al., 3- Nitropyrrole and 5-nitroindole as universal bases in primers for DNA sequencing and PCR. Nucleic Acids Res. 1995 Jul l l;23(13):2361-6; Loakes and Brown, 5-Nitroindole as an universal base analogue. Nucleic Acids Res. 1994 Oct l l;22(20):4039-43).

In certain embodiments, dsRNA-mediated inhibition of a target gene is assessed. In such embodiments, target gene RNA levels can be assessed by art-recognized methods (e.g., RT-PCR, Northern blot, expression array, etc.), optionally via comparison of target gene levels in the presence of a dsRNA formulation of the invention relative to the absence of such a dsRNA formulation. In certain embodiments, target gene levels in the presence of a dsRNA formulation are compared to those observed in the presence of vehicle alone, an unformulated dsRNA, in the presence of a dsRNA and/or dsRNA formulation directed against an unrelated target RNA, or in the absence of any treatment. It is also recognized that levels of target protein can be assessed as indicative of target gene RNA levels and/or the extent to which a dsRNA formulation inhibits target gene expression, thus art-recognized methods of assessing target gene protein levels (e.g., Western blot, immunoprecipitation, other antibody-based methods, etc.) can also be employed to examine the inhibitory effect of a dsRNA formulation of the invention. A dsRNA formulation of the invention is deemed to possess "target gene inhibitory activity" if a statistically significant reduction in target gene RNA or protein levels is seen when a dsRNA formulation of the invention is administered to a system (e.g., cell-free in vitro system), cell, tissue or organism, as compared to an appropriate control. The distribution of experimental values and the number of replicate assays performed will tend to dictate the parameters of what levels of reduction in target gene RNA or protein (either as a % or in absolute terms) is deemed statistically significant (as assessed by standard methods of determining statistical significance known in the art). However, in certain embodiments, "target gene inhibitory activity" is defined based upon a % or absolute level of reduction in the level of target gene in a system, cell, tissue or organism. For example, in certain embodiments, a dsRNA formulation of the invention is deemed to possess target gene inhibitory activity if at least a 5% reduction or at least a 10% reduction in target gene RNA is observed in the presence of a dsRNA formulation of the invention relative to target gene levels seen for a suitable control. (For example, in vivo target gene levels in a tissue and/or subject can, in certain embodiments, be deemed to be inhibited by a dsRNA formulation of the invention if, e.g., a 5% or 10% reduction in target gene levels is observed relative to a control.) In certain other embodiments, a dsRNA formulation of the invention is deemed to possess target gene inhibitory activity if target gene RNA levels are observed to be reduced by at least 15% relative to an appropriate control, by at least 20% relative to an appropriate control, by at least 25% relative to an appropriate control, by at least 30% relative to an appropriate control, by at least 35% relative to an appropriate control, by at least 40% relative to an appropriate control, by at least 45% relative to an appropriate control, by at least 50% relative to an appropriate control, by at least 55% relative to an appropriate control, by at least 60% relative to an appropriate control, by at least 65% relative to an appropriate control, by at least 70% relative to an appropriate control, by at least 75% relative to an appropriate control, by at least 80% relative to an appropriate control, by at least 85% relative to an appropriate control, by at least 90% relative to an appropriate control, by at least 95% relative to an appropriate control, by at least 96% relative to an appropriate control, by at least 97% relative to an appropriate control, by at least 98% relative to an appropriate control or by at least 99% relative to an appropriate control. In some embodiments, complete inhibition of target gene is required for a dsRNA formulation to be deemed to possess target gene inhibitory activity. In certain models (e.g., cell culture), a dsRNA formulation is deemed to possess target gene inhibitory activity if at least a 50% reduction in target gene levels is observed relative to a suitable control. In certain other embodiments, a dsRNA formulation is deemed to possess target gene inhibitory activity if at least an 80% reduction in target gene levels is observed relative to a suitable control.

Target gene inhibitory activity can also be evaluated over time (duration) and over concentration ranges (potency), with assessment of what constitutes a dsRNA formulation possessing target gene inhibitory activity adjusted in accordance with concentrations administered and duration of time following administration. Thus, in certain

embodiments, a dsRNA formulation of the invention is deemed to possess target gene inhibitory activity if at least a 50% reduction in target gene activity is observed at a duration of time of 2 hours, 5 hours, 10 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days or more after administration is observed/persists. In additional embodiments, a dsRNA formulation of the invention is deemed to be a potent target gene inhibitory agent if target gene inhibitory activity (e.g., in certain embodiments, at least 50% inhibition of target gene) is observed at a concentration of 1 nM or less, 500 pM or less, 200 pM or less, 100 pM or less, 50 pM or less, 20 pM or less, 10 pM or less, 5 pM or less, 2 pM or less or even 1 pM or less in the environment of a cell.

As used herein, the term "siRNA" refers to a double stranded nucleic acid in which each strand comprises RNA, RNA analog(s) or RNA and DNA. The siRNA comprises between 19 and 23 nucleotides or comprises 21 nucleotides. The siRNA typically has 2 bp overhangs on the 3' ends of each strand such that the duplex region in the siRNA comprises 17-21 nucleotides, or 19 nucleotides. Typically, the antisense strand of the siRNA is sufficiently complementary with the target sequence of the target gene and/or RNA.

In certain embodiments, a dsRNA cargo of the formulations of the instant invention can be an siRNA, e.g., having strand lengths comprising 17-23 nucleotides in length, or even 17-25 nucleotides in length (e.g., strand lengths of 17, 18, 19, 20, 21, 22, 23, 24 and/or 25 nucleotides in length. Such siRNA cargoes may possess overhangs on one or both ends of such dsRNA structures (e.g., one or two 3' overhangs), and can also possess modifications, such as those described herein and elsewhere in the art.

In other embodiments, a dsRNA of the dsRNA formulations of the instant invention possesses strand lengths of at least 25 nucleotides. Accordingly, a dsRNA contains one oligonucleotide sequence, a first sequence, that is at least 25 nucleotides in length. In certain embodiments, strand lengths of the dsRNA cargo of the invention are no longer than about 100 nucleotides in length, no longer than about 95 nucleotides in length, no longer than about 90 nucleotides in length, no longer than about 85 nucleotides in length, no longer than about 80 nucleotides in length, no longer than about 75 nucleotides in length, no longer than about 70 nucleotides in length, no longer than about 65 nucleotides in length or no longer than about 60 nucleotides in length. In further embodiments, a dsRNA cargo of the formulations of the invention contains a first sequence that is at least 25 nucleotides in length, and that is no longer than about 55 nucleotides, about 45 nucleotides, about 40 nucleotides, about 35 nucleotides, or about 30 nucleotides. The sequence of RNA can, for example, be between about 25 and 55, 25 and 50, 25 and 45, 25 and 40, 25 and 35, 25 and 34, 25 and 33, 25 and 32, 25 and 31, 25 and 30, 25 and 29, 25 and 28, 25 and 27, 25 and 26, 26 and 55, 26 and 50, 26 and 45, 26 and 40, 26 and 35, 26 and 34, 26 and 33, 26 and 32, 26 and 31, 26 and 30, 26 and 29, 26 and 28, and 26 and 27 nucleotides in length. This sequence can be about 27 or 28 nucleotides in length or 27 nucleotides in length. The second sequence of the dsRNA agent can be a sequence that anneals to the first sequence under biological conditions, such as within the cytoplasm of a eukaryotic cell. Generally, the second oligonucleotide sequence will have at least 19 complementary base pairs with the first oligonucleotide sequence, more typically the second oligonucleotide sequence will have about 21 or more complementary base pairs, or about 25 or more complementary base pairs with the first oligonucleotide sequence. In one embodiment, the second sequence is the same length as the first sequence, and the dsRNA agent is blunt ended. In another embodiment, the ends of the dsRNA agent have one or more overhangs.

In certain embodiments, the first and second oligonucleotide sequences of the dsRNA agent exist on separate oligonucleotide strands that can be and typically are chemically synthesized. In some embodiments, both strands are between 26 and 55 nucleotides in length. In other embodiments, both strands are between 25 and 45 or 26 and 45, between 25 and 35 or 26 and 35, or between 25 and 30 or 26 and 30 nucleotides in length. In one embodiment, both strands are 27 nucleotides in length, are completely complementary and have blunt ends. In certain embodiments of the instant invention, the first and second sequences of a dsRNA exist on separate RNA oligonucleotides (strands). In one embodiment, one or both oligonucleotide strands are capable of serving as a substrate for Dicer. In other embodiments, at least one modification is present that promotes Dicer to bind to the double-stranded RNA structure in an orientation that maximizes the double-stranded RNA structure's effectiveness in inhibiting gene expression. In certain embodiments of the instant invention, the dsRNA agent is comprised of two oligonucleotide strands of differing lengths, with the dsRNA possessing a blunt end at the 3' terminus of a first strand (sense strand) and a 3' overhang at the 3' terminus of a second strand (antisense strand). The dsRNA can also contain one or more deoxyribonucleic acid (DNA) base substitutions.

Suitable dsRNA compositions that contain two separate oligonucleotides can be chemically linked outside their annealing region by chemical linking groups. Many suitable chemical linking groups are known in the art and can be used. Suitable groups will not block Dicer activity on the dsRNA and will not interfere with the directed destruction of the RNA transcribed from the target gene. Alternatively, the two separate oligonucleotides can be linked by a third oligonucleotide such that a hairpin structure is produced upon annealing of the two oligonucleotides making up the dsRNA composition. The hairpin structure will not block Dicer activity on the dsRNA and will not interfere with the directed destruction of the target RNA.

As used herein, a dsRNA, e.g., DsiRNA or siRNA, having a sequence "sufficiently complementary" to a target RNA or cDNA sequence (e.g., target gene mRNA) means that the dsRNA has a sequence sufficient to trigger the destruction of the target RNA (where a cDNA sequence is recited, the RNA sequence corresponding to the recited cDNA sequence) by the RNAi machinery (e.g., the RISC complex) or process. The dsRNA molecule can be designed such that every residue of the antisense strand is complementary to a residue in the target molecule. Alternatively, substitutions can be made within the molecule to increase stability and/or enhance processing activity of said molecule.

Substitutions can be made within the strand or can be made to residues at the ends of the strand. In certain embodiments, substitutions and/or modifications are made at specific residues within a dsRNA agent. Such substitutions and/or modifications can include, e.g., deoxy- modifications at one or more residues of positions 1, 2 and 3 when numbering from the 3' terminal position of the sense strand of a dsRNA agent; and introduction of 2'- O-alkyl (e.g., 2'-0-methyl) modifications at the 3' terminal residue of the antisense strand of dsRNA agents, with such modifications also being performed at overhang positions of the 3' portion of the antisense strand and at alternating residues of the antisense strand of the dsRNA that are included within the region of a dsRNA agent that is processed to form an active siRNA agent. The preceding modifications are offered as exemplary, and are not intended to be limiting in any manner. Further consideration of the structure of dsRNA agents, including further description of the modifications and substitutions that can be performed upon the dsRNA agents of the instant invention, can be found elsewhere herein.

The phrase "duplex region" refers to the region in two complementary or substantially complementary oligonucleotides that form base pairs with one another, either by Watson-Crick base pairing or other manner that allows for a duplex between oligonucleotide strands that are complementary or substantially complementary. For example, an oligonucleotide strand having 21 nucleotide units can base pair with another oligonucleotide of 21 nucleotide units, yet only 19 bases on each strand are

complementary or substantially complementary, such that the "duplex region" consists of 19 base pairs. The remaining base pairs may, for example, exist as 5' and 3' overhangs. Further, within the duplex region, 100% complementarity is not required; substantial complementarity is allowable within a duplex region. Substantial complementarity refers to complementarity between the strands such that they are capable of annealing under biological conditions. Techniques to empirically determine if two strands are capable of annealing under biological conditions are well know in the art. Alternatively, two strands can be synthesized and added together under biological conditions to determine if they anneal to one another.

Single- stranded nucleic acids that base pair over a number of bases are said to "hybridize." Hybridization is typically determined under physiological or biologically relevant conditions (e.g., intracellular: pH 7.2, 140 mM potassium ion; extracellular pH 7.4, 145 mM sodium ion). Hybridization conditions generally contain a monovalent cation and biologically acceptable buffer and may or may not contain a divalent cation, complex anions, e.g. gluconate from potassium gluconate, uncharged species such as sucrose, and inert polymers to reduce the activity of water in the sample, e.g. PEG. Such conditions include conditions under which base pairs can form.

Hybridization is measured by the temperature required to dissociate single stranded nucleic acids forming a duplex, i.e., (the melting temperature; Tm). Hybridization conditions are also conditions under which base pairs can form. Various conditions of stringency can be used to determine hybridization (see, e.g., Wahl, G. M. and S. L. Berger (1987) Methods Enzymol. 152:399; Kimmel, A. R. (1987) Methods Enzymol. 152:507). Stringent temperature conditions will ordinarily include temperatures of at least about 30° C, optionally of at least about 37° C, and in certain embodiments of at least about 42° C. The hybridization temperature for hybrids anticipated to be less than 50 base pairs in length should be 5-10°C less than the melting temperature (Tm) of the hybrid, where Tm is determined according to the following equations. For hybrids less than 18 base pairs in length, Tm(°C)=2(# of A+T bases)+4(# of G+C bases). For hybrids between 18 and 49 base pairs in length, Tm(°C)=81.5+16.6(log 10[Na+])+0.41 (% G+C)-(600/N), where N is the number of bases in the hybrid, and [Na+] is the concentration of sodium ions in the hybridization buffer ([Na+] for lxSSC=0.165 M). For example, a hybridization determination buffer is shown in Table 1.

Table 1.

Useful variations on hybridization conditions will be readily apparent to those skilled in the art. Hybridization techniques are well known to those skilled in the art and are described, for example, in Benton and Davis (Science 196: 180, 1977); Grunstein and Hogness (Proc. Natl. Acad. Sci., USA 72:3961, 1975); Ausubel et al. (Current Protocols in Molecular Biology, Wiley Interscience, New York, 2001); Berger and Kimmel (Antisense to Molecular Cloning Techniques, 1987, Academic Press, New York); and Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York.

As used herein, "oligonucleotide strand" is a single stranded nucleic acid molecule. An oligonucleotide may comprise ribonucleotides, deoxyribonucleotides, modified nucleotides (e.g., nucleotides with 2' modifications, synthetic base analogs, etc.) or combinations thereof. Such modified oligonucleotides can be selected over native forms because of properties such as, for example, enhanced cellular uptake and increased stability in the presence of nucleases.

As used herein, the term "ribonucleotide" encompasses natural and synthetic, unmodified and modified ribonucleotides. Modifications include changes to the sugar moiety, to the base moiety and/or to the linkages between ribonucleotides in the oligonucleotide. As used herein, the term "ribonucleotide" specifically excludes a deoxyribonucleotide, which is a nucleotide possessing a single proton group at the 2' ribose ring position. As used herein, the term "unmodified ribonucleotide" refers to a ribonucleotide containing only adenosine monophosphate, guanosine monophosphate, uridine monophosphate, or cytidine monophosphate, without further chemical

modification.

As used herein, the term "deoxyribonucleotide" encompasses natural and synthetic, unmodified and modified deoxyribonucleotides. Modifications include changes to the sugar moiety, to the base moiety and/or to the linkages between deoxyribonucleotide in the oligonucleotide.

As used herein, "Dicer" refers to an endoribonuclease in the RNase III family that cleaves a dsRNA or dsRNA-containing molecule, e.g., double- stranded RNA (dsRNA) or pre-microRNA (miRNA), into double- stranded nucleic acid fragments about 19-25 nucleotides long, usually with a two-base overhang on the 3' end. With respect to the dsRNA formulations of the invention, the duplex formed by a dsRNA region of a formulation cargo of the invention is recognized by Dicer and is a Dicer substrate on at least one strand of the duplex. Dicer catalyzes the first step in the RNA interference pathway, which consequently results in the degradation of a target RNA. The protein sequence of human Dicer is provided at the NCBI database under accession number NP_085124, hereby incorporated by reference.

Dicer "cleavage" is determined as follows (e.g., see Collingwood et ah,

Oligonucleotides 18:187-200 (2008)). In a Dicer cleavage assay, RNA duplexes (100 pmol) are incubated in 20 μΐ. of 20 mM Tris pH 8.0, 200 mM NaCl, 2.5 mM MgC12 with or without 1 unit of recombinant human Dicer (Stratagene, La Jolla, CA) at 37 °C for 18- 24 hours. Samples are desalted using a Performa SR 96-well plate (Edge Biosystems, Gaithersburg, MD). Electrospray-ionization liquid chromatography mass spectroscopy (ESI-LCMS) of duplex RNAs pre- and post-treatment with Dicer is done using an Oligo HTCS system (Novatia, Princeton, NJ; Hail et al., 2004), which consists of a ThermoFinnigan TSQ7000, Xcalibur data system, ProMass data processing software and Paradigm MS4 HPLC (Michrom BioResources, Auburn, CA). In this assay, Dicer cleavage occurs where at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or even 100% of the Dicer substrate dsRNA, (i.e., 25-30 bp, dsRNA, optionally 26- 30 bp dsRNA) is cleaved to a shorter dsRNA (e.g., 19-23 bp dsRNA, optionally, 21-23 bp dsRNA).

As used herein, "Dicer cleavage site" refers to the sites at which Dicer cleaves a dsRNA (e.g., the dsRNA region of a DsiRNA agent of a formulation of the invention). Dicer contains two RNase III domains which typically cleave both the sense and antisense strands of a dsRNA. The average distance between the RNase III domains and the PAZ domain determines the length of the short double- stranded nucleic acid fragments it produces and this distance can vary (Macrae I, et al. (2006). "Structural basis for double- stranded RNA processing by Dicer". Science 311 (5758): 195-8.). Dicer is projected to cleave certain double- stranded ribonucleic acids of the instant invention that possess an antisense strand having a 2 nucleotide 3' overhang at a site between the 21^st and 22^nd nucleotides removed from the 3' terminus of the antisense strand, and at a corresponding site between the 21^st and 22^nd nucleotides removed from the 5' terminus of the sense strand. The projected and/or prevalent Dicer cleavage site(s) for dsRNA molecules may be similarly identified via art-recognized methods, including those described in Macrae et al. While the Dicer cleavage events generate 21 nucleotide siRNAs, it is noted that Dicer cleavage of a dsRNA (e.g., DsiRNA) can result in generation of Dicer-processed siRNA lengths of 19 to 23 nucleotides in length. Indeed, in certain embodiments, a double- stranded DNA region may be included within a dsRNA for purpose of directing prevalent Dicer excision of a typically non-preferred 19mer or 20mer siRNA, rather than a 21mer.

As used herein, "overhang" refers to unpaired nucleotides, in the context of a duplex having one or more free ends at the 5' terminus or 3' terminus of a dsRNA. In certain embodiments, the overhang is a 3' or 5' overhang on the antisense strand or sense strand. In some embodiments, the overhang is a 3' overhang having a length of between one and six nucleotides, optionally one to five, one to four, one to three, one to two, two to six, two to five, two to four, two to three, three to six, three to five, three to four, four to six, four to five, five to six nucleotides, or one, two, three, four, five or six nucleotides.

As used herein, the term "RNA processing" refers to processing activities performed by components of the siRNA, miRNA or RNase H pathways (e.g., Drosha, Dicer, Argonaute2 or other RISC endoribonucleases, and RNaseH), which are described in greater detail below (see "RNA Processing" section below). The term is explicitly distinguished from the post-transcriptional processes of 5' capping of RNA and degradation of RNA via non-RISC- or non-RNase H-mediated processes. Such

"degradation" of an RNA can take several forms, e.g. deadenylation (removal of a 3' poly(A) tail), and/or nuclease digestion of part or all of the body of the RNA by one or more of several endo- or exo-nucleases (e.g., RNase III, RNase P, RNase Tl, RNase A (1, 2, 3, 4/5), oligonucleotidase, etc.).

By "homologous sequence" is meant, a nucleotide sequence that is shared by one or more polynucleotide sequences, such as genes, gene transcripts and/or non-coding polynucleotides. For example, a homologous sequence can be a nucleotide sequence that is shared by two or more genes encoding related but different proteins, such as different members of a gene family, different protein epitopes, different protein isoforms or completely divergent genes, such as a cytokine and its corresponding receptors. A homologous sequence can be a nucleotide sequence that is shared by two or more non- coding polynucleotides, such as noncoding DNA or RNA, regulatory sequences, introns, and sites of transcriptional control or regulation. Homologous sequences can also include conserved sequence regions shared by more than one polynucleotide sequence. Homology does not need to be perfect homology (e.g., 100%), as partially homologous sequences are also contemplated by the instant invention (e.g., 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80% etc.). Indeed, design and use of the dsRNA agents of the instant invention contemplates the possibility of using such dsRNA agents not only against target RNAs of target gene possessing perfect complementarity with the presently described dsRNA agents, but also against target RNAs possessing sequences that are, e.g., only 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80% etc. complementary to said dsRNA agents. Similarly, it is contemplated that the presently described dsRNA agents of the instant invention might be readily altered by the skilled artisan to enhance the extent of complementarity between said dsRNA agents and a target RNA, e.g., of a specific allelic variant of target gene (e.g., an allele of enhanced therapeutic interest). Indeed, dsRNA agent sequences with insertions, deletions, and single point mutations relative to the target sequence can also be effective for inhibition. Alternatively, dsRNA agent sequences with nucleotide analog substitutions or insertions can be effective for inhibition. Sequence identity may be determined by sequence comparison and alignment algorithms known in the art. To determine the percent identity of two nucleic acid sequences (or of two amino acid sequences), the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the first sequence or second sequence for optimal alignment). The nucleotides (or amino acid residues) at

corresponding nucleotide (or amino acid) positions are then compared. When a position in the first sequence is occupied by the same residue as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % homology=# of identical positions/total # of positions x 100), optionally penalizing the score for the number of gaps introduced and/or length of gaps introduced.

The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. In one embodiment, the alignment generated over a certain portion of the sequence aligned having sufficient identity but not over portions having low degree of identity (i.e., a local alignment). A non-limiting example of a local alignment algorithm utilized for the comparison of sequences is the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264-68, modified as in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-77. Such an algorithm is incorporated into the BLAST programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10.

In another embodiment, the alignment is optimized by introducing appropriate gaps and percent identity is determined over the length of the aligned sequences (i.e., a gapped alignment). To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Res.

25(17):3389-3402. In another embodiment, the alignment is optimized by introducing appropriate gaps and percent identity is determined over the entire length of the sequences aligned (i.e., a global alignment). A non-limiting example of a mathematical algorithm utilized for the global comparison of sequences is the algorithm of Myers and Miller, CABIOS (1989). Such an algorithm is incorporated into the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used.

Greater than 80% sequence identity, e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or even 100% sequence identity, between the dsRNA antisense strand and the portion of the target gene RNA sequence is preferred. Alternatively, the dsRNA may be defined functionally as a nucleotide sequence (or oligonucleotide sequence) that is capable of hybridizing with a portion of the target gene RNA (e.g., 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50°C or 70°C hybridization for 12-16 hours; followed by washing). Additional hybridization conditions include hybridization at 70°C in lxSSC or 50°C in lxSSC, 50% formamide followed by washing at 70°C in 0.3xSSC or hybridization at 70°C. in 4xSSC or 50°C in 4xSSC, 50% formamide followed by washing at 67°C in lxSSC. The hybridization temperature for hybrids anticipated to be less than 50 base pairs in length should be 5-10°C less than the melting temperature (Tm) of the hybrid, where Tm is determined according to the following equations. For hybrids less than 18 base pairs in length, Tm(°C)=2(# of A+T bases)+4(# of G+C bases). For hybrids between 18 and 49 base pairs in length, Tm(°C)=81.5+16.6(log 10[Na+])+0.41 (% G+C)-(600/N), where N is the number of bases in the hybrid, and [Na+] is the concentration of sodium ions in the hybridization buffer ([Na+] for lxSSC=0.165 M). Additional examples of stringency conditions for polynucleotide hybridization are provided in Sambrook, J., E. F. Fritsch, and T. Maniatis, 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., chapters 9 and 11, and Current Protocols in Molecular Biology, 1995, F. M. Ausubel et al., eds., John Wiley & Sons, Inc., sections 2.10 and 6.3-6.4. The length of the identical nucleotide sequences may be at least about 10, 12, 15, 17, 20, 22, 25, 27 or 30 bases.

By "conserved sequence region" is meant, a nucleotide sequence of one or more regions in a polynucleotide does not vary significantly between generations or from one biological system, subject, or organism to another biological system, subject, or organism. The polynucleotide can include both coding and non-coding DNA and RNA.

By "sense region" is meant a nucleotide sequence of a dsRNA molecule having complementarity to an antisense region of the dsRNA molecule. In addition, the sense region of a dsRNA molecule can comprise a nucleic acid sequence having homology with a target nucleic acid sequence.

By "antisense region" is meant a nucleotide sequence of a dsRNA molecule having complementarity to a target nucleic acid sequence. In addition, the antisense region of a dsRNA molecule comprises a nucleic acid sequence having complementarity to a sense region of the dsRNA molecule.

As used herein, "antisense strand" refers to a single stranded nucleic acid molecule which has a sequence complementary to that of a target RNA. When the antisense strand contains modified nucleotides with base analogs, it is not necessarily complementary over its entire length, but must at least hybridize with a target RNA.

As used herein, "sense strand" refers to a single stranded nucleic acid molecule which has a sequence complementary to that of an antisense strand. When the antisense strand contains modified nucleotides with base analogs, the sense strand need not be complementary over the entire length of the antisense strand, but must at least duplex with the antisense strand.

As used herein, "guide strand" refers to a single stranded nucleic acid molecule of a dsRNA or dsRNA-containing molecule, which has a sequence sufficiently

complementary to that of a target RNA to result in RNA interference. After cleavage of the dsRNA or dsRNA-containing molecule by Dicer, a fragment of the guide strand remains associated with RISC, binds a target RNA as a component of the RISC complex, and promotes cleavage of a target RNA by RISC. As used herein, the guide strand does not necessarily refer to a continuous single stranded nucleic acid and may comprise a discontinuity, optionally at a site that is cleaved by Dicer. A guide strand is an antisense strand.

As used herein, "passenger strand" refers to an oligonucleotide strand of a dsRNA or dsRNA-containing molecule, which has a sequence that is complementary to that of the guide strand. As used herein, the passenger strand does not necessarily refer to a continuous single stranded nucleic acid and may comprise a discontinuity, optionally at a site that is cleaved by Dicer. A passenger strand is a sense strand.

By "complementarity" is meant that a nucleic acid can form hydrogen bond(s) with another nucleic acid sequence by either traditional Watson-Crick or other non-traditional types. In reference to the nucleic molecules of the present invention, the binding free energy for a nucleic acid molecule with its complementary sequence is sufficient to allow the relevant function of the nucleic acid to proceed, e.g., RNAi activity. Determination of binding free energies for nucleic acid molecules is well known in the art (see, e.g., Turner et al., 1987, CSH Symp. Quant. Biol. LII pp. 123-133; Frier et al., 1986, Proc. Nat. Acad. Sci. USA 83:9373-9377; Turner et al., 1987, J. Am. Chem. Soc. 109:3783-3785). A percent complementarity indicates the percentage of contiguous residues in a nucleic acid molecule that can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, or 10 nucleotides out of a total of 10 nucleotides in the first oligonucleotide being based paired to a second nucleic acid sequence having 10 nucleotides represents 50%, 60%, 70%, 80%, 90%, and 100% complementary

respectively). "Perfectly complementary" means that all the contiguous residues of a nucleic acid sequence will hydrogen bond with the same number of contiguous residues in a second nucleic acid sequence. In one embodiment, a dsRNA molecule of a formulation of the invention comprises about 19 to about 30 (e.g., about 19, 20, 21, 22, 23, 24, 25, 26,

27, 28, 29, or 30 or more) nucleotides that are complementary to one or more target nucleic acid molecules or a portion thereof.

In one embodiment of the present invention, each sequence of a dsRNA molecule of a formulation of the invention is independently about 25 to about 55 nucleotides in length, in specific embodiments about 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, or 55 nucleotides in length. In another embodiment, the dsRNA duplexes of a formulation of the invention independently comprise about 25 to about 40 base pairs (e.g., about 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40), or in certain embodiments, about 25 to about 30 base pairs (e.g., about 25, 26, 27, 28, 29 or 30). In another embodiment, one or more strands of the dsRNA molecule of a formulation of the invention independently comprises about 19 to about 35 nucleotides (e.g., about 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35) that are complementary to a target (target gene) nucleic acid molecule. In certain embodiments, a dsRNA molecule of a formulation of the invention possesses a length of duplexed nucleotides between 25 and 55 nucleotides in length (e.g., 25, 26, 27,

28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54 or 55 nucleotides in length; optionally, all such nucleotides base pair with cognate nucleotides of the opposite strand). (Exemplary dsRNA molecules of a formulation of the invention are shown below.)

The dsRNA molecules of certain formulations of the invention are added directly, or can be complexed with cationic lipids, packaged within liposomes, or otherwise delivered to target cells or tissues. Such nucleic acid formulations can be locally administered to relevant tissues ex vivo, or in vivo through direct dermal application, transdermal application, or injection, with or without their incorporation in biopolymers, or via any other suitable route. In particular embodiments, the nucleic acid molecules of certain formulations of the invention are structures as shown below. Examples of such nucleic acid formulations consist essentially of structures defined in these exemplary structures. In another aspect, the invention provides one or more formulations containing one or more dsRNAs as described herein. The one or more formulations and/or dsRNA molecules can independently be targeted to the same or different sites.

The dsRNA molecules of certain formulations of the invention are added directly, or can be complexed with cationic lipids, packaged within liposomes, or otherwise delivered to target cells or tissues. Such nucleic acid formulations can be locally administered to relevant tissues ex vivo, or in vivo through direct dermal application, transdermal application, or injection, with or without their incorporation in biopolymers. In particular embodiments, the nucleic acid molecules of certain formulations of the invention are structures as shown below. Examples of such nucleic acid formulations consist essentially of structures defined in these exemplary structures.

In another aspect, the invention provides one or more formulations containing one or more dsRNAs as described herein. The one or more formulations and/or dsRNA molecules can independently be targeted to the same or different sites.

By "RNA" is meant a molecule comprising at least one ribonucleotide residue. By "ribonucleotide" is meant a nucleotide with a hydroxyl group at the 2' position of a β-D- ribofuranose moiety. The terms include double- stranded RNA, single-stranded RNA, isolated RNA such as partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, as well as altered RNA that differs from naturally occurring RNA by the addition, deletion, substitution and/or alteration of one or more nucleotides. Such alterations can include addition of non-nucleotide material, such as to the end(s) of the dsRNA or internally, for example at one or more nucleotides of the RNA. Nucleotides in the RNA molecules of the instant invention can also comprise non-standard nucleotides, such as non-naturally occurring nucleotides or chemically synthesized nucleotides or deoxynucleotides. These altered RNAs can be referred to as analogs or analogs of naturally-occurring RNA.

By "subject" is meant an organism, which is a donor or recipient of explanted cells or the cells themselves. "Subject" also refers to an organism to which the formulations of the invention can be administered. A subject can be a mammal or mammalian cells, including a human or human cells.

The term "mammalian subject" refers to both humans and to warm blooded animals, such as laboratory animals, e.g., rodents {e.g., mice and rats) and non-human primates, domestic animals, e.g., cats and dogs, and farm animals, e.g., cattle, pigs, goats and sheep.

As used herein "cell" is used in its usual biological sense, and does not refer to an entire multicellular organism, e.g., specifically does not refer to a human. The cell can be present in an organism, e.g., birds, plants and mammals such as humans, cows, sheep, apes, monkeys, swine, dogs, and cats. The cell can be prokaryotic (e.g., bacterial cell) or eukaryotic (e.g., mammalian or plant cell). The cell can be of somatic or germ line origin, totipotent or pluripotent, dividing or non-dividing. The cell can also be derived from or can comprise a gamete or embryo, a stem cell, or a fully differentiated cell. Within certain aspects, the term "cell" refers specifically to mammalian cells, such as human cells, that contain one or more isolated dsRNA molecules of the present disclosure. In particular aspects, a cell processes dsRNAs or dsRNA-containing molecules resulting in RNA interference of target nucleic acids, and contains proteins and protein complexes required for RNAi, e.g., Dicer and RISC.

The term "in vitro" has its art recognized meaning, e.g., involving purified reagents or extracts, e.g., cell extracts. The term "in vivo" also has its art recognized meaning, e.g., involving living cells, e.g., immortalized cells, primary cells, cell lines, and/or cells in an organism.

In certain embodiments, the phrase "consists essentially of is used in reference to the formulations of the invention. In some such embodiments, "consists essentially of refers to a composition that comprises a dsRNA formulation of the invention which possesses at least a certain level of target gene inhibitory activity (e.g., at least 50% target gene inhibitory activity) and that also comprises one or more additional components and/or modifications that do not significantly impact the target gene inhibitory activity of the dsRNA formulation. For example, in certain embodiments, a composition "consists essentially of a dsRNA formulation of the invention where modifications of the dsRNA and/or dsRNA-associated components of the formulation of the invention do not alter the target gene inhibitory activity (optionally including potency or duration of target gene inhibitory activity) by greater than 3%, greater than 5%, greater than 10%, greater than 15%, greater than 20%, greater than 25%, greater than 30%, greater than 35%, greater than 40%, greater than 45%, or greater than 50% relative to the dsRNA formulation of the invention in isolation. In certain embodiments, a composition is deemed to consist essentially of a dsRNA formulation of the invention even if more dramatic reduction of target gene inhibitory activity (e.g., 80% reduction, 90% reduction, etc. in efficacy, duration and/or potency) occurs in the presence of additional components or modifications, yet where target gene inhibitory activity is not significantly elevated (e.g., observed levels of target gene inhibitory activity are within 10% those observed for the isolated dsRNA formulations of the invention) in the presence of additional components and/or modifications.

Various methodologies of the instant invention include a step that involves comparing a value, level, feature, characteristic, property, etc. to a "suitable control", referred to interchangeably herein as an "appropriate control". A "suitable control" or "appropriate control" is a control or standard familiar to one of ordinary skill in the art useful for comparison purposes. In one embodiment, a "suitable control" or "appropriate control" is a value, level, feature, characteristic, property, etc. determined prior to performing an RNAi methodology, as described herein. For example, a transcription rate, mRNA level, translation rate, protein level, biological activity, cellular characteristic or property, genotype, phenotype, etc. can be determined prior to introducing an RNA silencing agent formulation (e.g., DsiRNA formulation) of the invention into a cell or organism. In another embodiment, a "suitable control" or "appropriate control" is a value, level, feature, characteristic, property, etc. determined in a cell or organism, e.g., a control or normal cell or organism, exhibiting, for example, normal traits. In yet another embodiment, a "suitable control" or "appropriate control" is a predefined value, level, feature, characteristic, property, etc.

"Treatment", or "treating" as used herein, is defined as the application or administration of a therapeutic agent (e.g., a dsRNA formulation, dsRNA agent or a vector or transgene encoding same) to a patient, or application or administration of a therapeutic agent to an isolated tissue or cell line from a patient, who has a disorder with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve or affect the disease or disorder, or symptoms of the disease or disorder. The term "treatment" or "treating" is also used herein in the context of administering agents prophylactically. The term

"effective dose" or "effective dosage" is defined as an amount sufficient to achieve or at least partially achieve the desired effect. The term "therapeutically effective dose" is defined as an amount sufficient to cure or at least partially arrest the disease and its complications in a patient already suffering from the disease. The term "patient" includes human and other mammalian subjects that receive either prophylactic or therapeutic treatment. The term "target gene" refers to nucleic acid sequences (e.g., genomic DNAs or mRNAs) encoding a target protein, peptide, or polypeptide, or that encode for or are regulatory nucleic acids (e.g., a "target gene" for purpose of the instant invention can also be a miRNA or miRNA-encoding gene sequence). In certain embodiments, the term "target gene" is also meant to include isoforms, mutants, polymorphisms and splice variants of target genes. In certain embodiments of the instant invention, a dsRNA of a formulation of the invention targets a sequence within the 5'-UTR, coding sequence and/or 3'-UTR of a target gene mRNA.

Lipid Compounds of the Invention

Any suitable lipid which is capable of being modified by the cationic peptide or peptide-based moiety of the invention is contemplated. The lipids to be modified may be anionic, neutral or cationic in their unmodified state. Preferably, however, once any particular lipid is modified by a cationic peptide or peptide-based moiety, the net positive charge of the modified lipid molecule is positive.

In a particular aspect, the present invention relates to a lipid comprising a headgroup moiety and a tail moiety, wherein the tail moiety comprises a cationic peptide- based moiety that is capable of undergoing a conformational change upon contacting an intracellular condition.

In another particular aspect, the present invention relates to a cationic lipid compound having the general formula X-Y-P, wherein X is a headgroup moiety of a fatty acid, glycerolipid, glycerophospholipid, sphingolipid or saccharolipid; Y is an acyl chain; and P is a cationic moiety capable of conformational change upon internalization of the lipid into a cell or intracellular compartment and which imparts a net cationic charge to the lipid.

The unmodified lipid can be any suitable lipid from any of the generally accepted categories of lipids, including a fatty acid (or more broadly, fatty acyls), glycerolipid, glycerophospholipid, sphingolipid, or saccharolipid. Preferably, the lipid has a tail region (e.g., a acyl chain of a fatty acid) since it is preferred that the peptide or peptide-based moiety is covalently attached to the tail region of a lipid. Thus, the invention may also include the use of lipids that generally do not contain a tail portion, including the broad categories of sterol lipids, prenol lipids and polyketides, so long as such molecules include a tail region is or are modified to include a tail region to allow covalent modification of the tail region to include a peptide or peptide-based moiety of the invention.

Thus, in one embodiment, the invention contemplates using fatty acyls. Fatty acyls is a generic term for describing fatty acids, their conjugates and derivatives, and are a diverse group of molecules synthesized by chain-elongation of an acetyl-CoA primer with malonyl-CoA or methylmalonyl-CoA groups in a process called fatty acid synthesis. They are made of a hydrocarbon chain that terminates with a carboxylic acid group; this arrangement confers the molecule with a polar, hydrophilic end, and a nonpolar, hydrophobic end that is insoluble in water. The fatty acid structure is one of the most fundamental categories of biological lipids, and is commonly used as a building block of more structurally complex lipids, e.g., form the "tail region" of other more complex lipids. The carbon chain, typically between four to 24 carbons long, may be saturated or unsaturated, and may be attached to functional groups containing oxygen, halogens, nitrogen and sulfur. Where a double bond exists, there is the possibility of either a cis or trans geometric isomerism, which significantly affects the molecule's molecular configuration. Czs-double bonds cause the fatty acid chain to bend, an effect that is more pronounced the more double bonds there are in a chain. This in turn plays an important role in the structure and function of cell membranes. Most naturally occurring fatty acids are of the cis configuration, although the trans form does exist in some natural and partially hydro genated fats and oils.

Examples of fatty acids are the eicosanoids, derived primarily from arachidonic acid and eicosapentaenoic acid, which include prostaglandins, leukotrienes, and thromboxanes. Other major lipid classes in the fatty acid category are the fatty esters and fatty amides. Fatty esters include important biochemical intermediates such as wax esters, fatty acid thioester coenzyme A derivatives, fatty acid thioester ACP derivatives and fatty acid carnitines. The fatty amides include N-acyl ethanolamines, such as the cannabinoid neurotransmitter anandamide.

In another embodiment, the lipids of the invention may be based on glycero lipids. Glycerolipids are composed mainly of mono-, di- and tri- substituted glycerols, the most well-known being the fatty acid esters of glycerol (triacylglycerols), also known as triglycerides. In these compounds, the three hydroxyl groups of glycerol are each esterified, usually by different fatty acids. Because they function as a food store, these lipids comprise the bulk of storage fat in animal tissues. The hydrolysis of the ester bonds of triacylglycerols and the release of glycerol and fatty acids from adipose tissue is called fat mobilization.

Additional subclasses of glycerolipids are represented by glycosylglycerols, which are characterized by the presence of one or more sugar residues attached to glycerol via a glycosidic linkage. Examples of structures in this category are the

digalactosyldiacylglycerols found in plant membranes and seminolipid from mammalian sperm cells.

In still another embodiment, the lipids of the invention may be based on glycerophospholipids. Glycerophospholipids, also referred to as phospholipids, are ubiquitous in nature and are key components of the lipid bilayer of cells, as well as being involved in metabolism and cell signaling. Neural tissue (including the brain) contains relatively high amounts of glycerophospholipids, and alterations in their composition has been implicated in various neurological disorders. Glycerophospholipids may be subdivided into distinct classes, based on the nature of the polar headgroup at the sn-3 position of the glycerol backbone in eukaryotes and eubacteria, or the sn-1 position in the case of archaebacteria.

Examples of glycerophospholipids found in biological membranes are

phosphatidylcholine (also known as PC, GPCho or lecithin), phosphatidylethanolamine (PE or GPEtn) and phosphatidylserine (PS or GPSer). In addition to serving as a primary component of cellular membranes and binding sites for intra- and intercellular proteins, some glycerophospholipids in eukaryotic cells, such as phosphatidylinositols and phosphatidic acids are either precursors of, or are themselves, membrane-derived second messengers. Typically, one or both of these hydroxyl groups are acylated with long-chain fatty acids, but there are also alkyl-linked and lZ-alkenyl-linked (plasmalogen) glycerophospholipids, as well as dialkylether variants in archaebacteria.

The lipids of the invention may also include the sphingolipids. Sphingolipids are a complex family of compounds that share a common structural feature, a sphingoid base backbone that is synthesized de novo from the amino acid serine and a long-chain fatty acyl CoA, then converted into ceramides, phospho sphingolipids, glyco sphingolipids and other compounds. The major sphingoid base of mammals is commonly referred to as sphingosine. Ceramides (N-acyl- sphingoid bases) are a major subclass of sphingoid base derivatives with an amide-linked fatty acid. The fatty acids are typically saturated or mono-unsaturated with chain lengths from 16 to 26 carbon atoms. The major

phospho sphingolipids of mammals are sphingomyelins (ceramide phosphocholines), whereas insects contain mainly ceramide phosphoethanolamines and fungi have phytoceramide phosphoinositols and mannose-containing headgroups. The

glyco sphingolipids are a diverse family of molecules composed of one or more sugar residues linked via a glycosidic bond to the sphingoid base. Examples of these are the simple and complex glycosphingolipids such as cerebrosides and gangliosides.

Saccharolipids describe compounds in which fatty acids are linked directly to a sugar backbone, forming structures that are compatible with membrane bilayers. In the saccharolipids, a monosaccharide substitutes for the glycerol backbone present in glycerolipids and glycerophospholipids. The most familiar saccharolipids are the acylated glucosamine precursors of the Lipid A component of the lipopolysaccharides in Gram- negative bacteria. Typical lipid A molecules are disaccharides of glucosamine, which are derivatized with as many as seven fatty-acyl chains. The minimal lipopoly saccharide required for growth in E. coli is Kdo₂-Lipid A, a hexa-acylated disaccharide of

glucosamine that is glycosylated with two 3-deoxy-D-manno-octulosonic acid (Kdo) residues.

It is generally the case that the broad lipid categories including fatty acid (or more broadly, fatty acyls), glycerolipids, glycerophospholipids, sphingolipids, and

saccharolipids, have lipid tail regions (e.g., fatty acid portions) that can be modified by the peptide or peptide-based moieties of the invention. However, the invention may also include the use of lipids that generally do not contain a tail portion, including the broad categories of sterol lipids, prenol lipids and polyketides, so long as such molecules include a tail region or are modified to include a tail region to allow covalent modification with the peptide or peptide-based moiety of the invention.

Thus, the invention contemplates the use of sterol lipids, such as cholesterol and its derivatives, which include a modifiable tail region. Sterols and their derivatives are an important component of membrane lipids, along with the glycerophospholipids and sphingomyelins. The steroids, all derived from the same fused four-ring core structure, have different biological roles as hormones and signaling molecules. The eighteen-carbon (CI 8) steroids include the estrogen family whereas the C19 steroids comprise the androgens such as testosterone and androsterone. The C21 subclass includes the progestogens as well as the glucocorticoids and mineralocorticoids. The secosteroids, comprising various forms of vitamin D, are characterized by cleavage of the B ring of the core structure. Other examples of sterols are the bile acids and their conjugates, which in mammals are oxidized derivatives of cholesterol and are synthesized in the liver. The plant equivalents are the phytosterols, such as β-sitosterol, stigmasterol, and brassicasterol; the latter compound is also used as a biomarker for algal growth. The predominant sterol in fungal cell membranes is ergosterol.

The invention also contemplates the use of prenol lipids which include a modifiable tail region. Prenol lipids are synthesized from the 5-carbon precursors isopentenyl diphosphate and dimethylallyl diphosphate that are produced mainly via the mevalonic acid (MVA) pathway. The simple isoprenoids (linear alcohols, diphosphates, etc.) are formed by the successive addition of C5 units, and are classified according to number of these terpene units. Structures containing greater than 40 carbons are known as polyterpenes. Carotenoids are important simple isoprenoids that function as antioxidants and as precursors of vitamin A. Another biologically important class of molecules is exemplified by the quinones and hydroquinones, which contain an isoprenoid tail attached to a quinonoid core of non-isoprenoid origin. Vitamin E and vitamin K, as well as the ubiquinones, are examples of this class. Prokaryotes synthesize polyprenols (called bactoprenols) in which the terminal isoprenoid unit attached to oxygen remains unsaturated, whereas in animal polyprenols (dolichols) the terminal isoprenoid is reduced.

The invention also contemplates the use of polyketide lipids which include a modifiable tail region. Polyketides are synthesized by polymerization of acetyl and propionyl subunits by classic enzymes as well as iterative and multimodular enzymes that share mechanistic features with the fatty acid synthases. They comprise a large number of secondary metabolites and natural products from animal, plant, bacterial, fungal and marine sources, and have great structural diversity. Many polyketides are cyclic molecules whose backbones are often further modified by glycosylation, methylation, hydroxylation, oxidation, and/or other processes. Many commonly used anti-microbial, anti-parasitic, and anti-cancer agents are polyketides or polyketide derivatives, such as erythromycins, tetracyclines, avermectins, and antitumor epothilones.

In certain embodiments, the glycerolipid is a triacylglyceride or glycosylglycerol; the glycerophospholipid is a phospholipid, phosphatidylcholine (PC), lecithin,

phosphatidylethanolamine (PE) or phosphatidylserine (PS); the sphingolipid is a ceramide, phosphosphingolipid, glycosphingolipid, sphingomyelin, or ceramide phosphocholine; and the saccharolipid is Lipid A.

In certain other embodiments, the lipid is a noncationic lipid selected from the group consisting of lecithin, phosphatidylethanolamine, lysolecithin,

lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphatidic acid, cerebrosides, dicetylphosphate, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipahnitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoyl -phosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), palmitoyloleyol-phosphatidylglycerol (POPG),

dioleoylphosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane- 1 -carboxylate (DOPE-mal), dipalmitoyl-phosphatidylethanolamine (DPPE), dimyristoyl- phosphatidylethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), monomethyl-phosphatidylethanolamine, dimethyl -phosphatidylethanolamine, dielaidoyl- phosphatidylethanolamine (DEPE), DOGS, and stearoyloleoyl-phosphatidylethanolamine (SOPE).

In other embodiments, that lipid is a cationic lipid selected from the group consisting of N, N-dioleyl-N, N-dimethylammonium chloride (DODAC), N, N-distearyl- N, N-dimethylammonium bromide (DDAB), N-(l-(2, 3-dioleoyloxy)propyl)-N, N, N- trimethylammonium chloride (DOTAP), l,2-dioleoyl-3-dimethyl-ammonium-propane (DODAP), l,2-dipalmitoyl-sn-glycero-3-ethyl-phosphocholine (DpePC), N-(l-(2, 3- dioleyloxy)propyl)-N, N, N-trimethylammonium chloride (DOTMA), N, N-dimethyl-2, 3- dioleyloxypropylamine (DODMA), 1, 2-DiLinoleyloxy-N, N-dimethylaminopropane (DLinDMA), 1, 2-Dilinolenyloxy-N, N-dimethylaminopropane (DLenDMA), DSDMA, DOSPA, DC-Choi, DMRIE, l-palmitoyl-2-glutaryl-sn-glycero-3-phosphocholine (16:0- 05:0 (COOH) PC or "G1PC"), l-hexadecyl-2-azelaoyl-sn-glycero-3-phosphocholine (16- 09:0 (COOH) PC), l-palmitoyl-2-azelaoyl-sn-glycero-3-phosphocholine (16:0-09:0 (COOH) PC or "AzPC"), l-palmitoyl-2-(5'-oxo-valeroyl)-sn-glycero-3-phosphocholine (16;0-05:0 (CHO) PC) and l-palmitoyl-2-(9'-oxo-nonanoyl)-sn-glycero-3- phosphocholine, each of which may be referred to as types or species of 1,2-alkyloxy- N,N-alkylaminoalkane.

In still other embodiments, the tail moiety can comprise a saturated or unsaturated fatty acid. The fatty acid can be any suitable length, and preferably at least 2 carbons but less than 50 carbons in length, or less than 40 carbons in length, or less than 30 carbons in length, or less than 20 carbons in length. More preferably, the fatty acid is between 2 and 20 carbons, or 2 and 18 carbons, or 2 and 16 carbons, or 2 and 14 carbons, or 2 and 12 carbons, or 2 and 10 carbons, or 2 and 8 carbons, or 2 and 6 carbons or 2 and 4 carbons in length. The fatty acid may also be saturated or unsaturated. If unsaturated, the fatty acid can have at least 1 double bond and up to 2, 3, 4, 5, 6, 7, 8, 9, 10 or more double bonds. The double bonds may be located at any position along the length of the fatty acid. The invention also contemplates the use of any pharmaceutically acceptable salt, hydrate, stereoisomer, dimer, multimer, or polymer of any of the lipids contemplated by the invention.

In certain embodiments, it may be desirable to modify a lipid to include a tail region (e.g., a fatty acid). Such lipids may include a sterol lipid, a prenol lipid or a polyketide lipid. Any known method or chemical process capable of covalently modifying any such lipid with a tail region is contemplated. General lipid chemistry and synthesis schemes will be well known to those of ordinary skill in the art and can be referenced for detailed information regarding the chemical manipulation of the lipids of the invention, for example, as means to covalently link a tail region to a sterol lipid. Such references may include, Synthesis in Lipid Chemistry, J. Tyman (author), Royal Society of Chemistry, 1^st edition (December 31, 1996); Lipid Synthesis and Manufacture (Sheffield Chemistry and Technology of Oils and Fats), Frank D. Gunstone (Ed.), Blackwell; 1^st edition (December 18, 1998); or Lipid Biotechnology, Harold W. Garnder et al. (author), CRC Press; 1^st edition (January 15, 2002), each of which are incorporated herein by reference.

The "tail region" can be any suitable lipophilic moiety which can be modifiable by the methods of the invention to covalently link a cationic peptide or peptide-based moiety thereto. The tail region can each be independently selected from the group consisting of a straight chain alkyl of 1 to 24 carbon atoms, a branched chain alkyl of 10 to 50 carbon atoms, a straight chain alkenyl of 2 to 24 carbon atoms, a branched chain alkenyl of 10 to 50 carbon atoms, a steroidyl moiety, an amine derivative, a glyceryl derivative, OP(0)(0^" )(OR₄ ), OP(0)(Ci_₃alkyl)(OR₄ ), or N(R₄ R₅ ), wherein R₄ and R₅ , for each occurrence, independently is a glyceryl derivative, a steroidyl moiety, a straight chain alkyl of 1 to 24 carbon atoms, a branched chain alkyl of 10 to 50 carbon atoms, a straight chain alkenyl of 2 to 24 carbon atoms, or a branched chain alkenyl of 10 to 50 carbon atoms. In one embodiment, the tail region is a fatty acid.

The structures of the compounds of the invention may include asymmetric carbon atoms. Accordingly, the isomers arising from such asymmetry (e.g., all enantiomers and diastereomers) are included within the scope of this invention, unless indicated otherwise. Such isomers can be obtained in substantially pure form by classical separation techniques and/or by stereochemically controlled synthesis.

Naturally occurring or synthetic isomers can be separated in several ways known in the art. Methods for separating a racemic mixture of two enantiomers include chromatography using a chiral stationary phase (see, e.g., , "Chiral Liquid Chromatography," W.J. Lough, Ed. Chapman and Hall, New York (1989)). Enantiomers can also be separated by classical resolution techniques. For example, formation of diastereomeric salts and fractional crystallization can be used to separate enantiomers. For the separation of enantiomers of carboxylic acids, the diastereomeric salts can be formed by addition of enantiomerically pure chiral bases, such as brucine, quinine, ephedrine, strychnine, and the like. Alternatively, diastereomeric esters can be formed with enantiomerically pure chiral alcohols, such as menthol, followed by separation of the diastereomeric esters and hydrolysis to yield the free, enantiomerically enriched carboxylic acid. For separation of the optical isomers of amino compounds, addition of chiral carboxylic or sulfonic acids, such as camphorsulfonic acid, tartaric acid, mandelic acid, or lactic acid can result in formation of the diastereomeric salts.

Examples of conventional noncationic lipids that are contemplated by the present invention include lecithin, phosphatidylethanolamine, lysolecithin,

lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphatidic acid, cerebrosides, dicetylphosphate, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipahnitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoyl-phosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), palmitoyloleyol-phosphatidylglycerol (POPG),

dioleoylphosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane- 1 -carboxylate (DOPE-mal), dipalmitoyl-phosphatidylethanolamine (DPPE), dimyristoyl- phosphatidylethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), monomethyl-phosphatidylethanolamine, dimethyl -phosphatidylethanolamine, dielaidoyl- phosphatidylethanolamine (DEPE), DOGS, and stearoyloleoyl-phosphatidylethanolamine (SOPE). The structure of DOGS is shown below:

Examples of conventional cationic lipids that are contemplated by the present invention include:

Table 2. A select list of conventional cationic lipids.

Table 2 cont'd.

DODMA DLinDMA Dlin-K-DMA Dlin-KC2- DDAB Dlin-MC

In addition, the present invention contemplates the use of cationic lipids that include tail regions with linkable ends, i.e., which could be covalently linked to a cationic peptide or peptide-based moiety of the invention. Examples of such conventional cationic lipids that are contemplated by the present invention include:

Table 3. A select list of conventional cationic lipids with linkable end groups on tail regions.

phosphocholine phosphocholine

Methods of Making the Compounds of the Invention

The present invention contemplates any suitable source and/or any known or attainable process available to one of ordinary skill in the art to obtain and/or prepare any of the compounds of the invention, e.g., the cationic peptide modified lipids of the invention. In certain embodiments, the compounds (or the starting materials used in the synthesis of the lipids) of the invention can be obtained from commercial sources (e.g., Sigma- Aldrich Co.). In other embodiments, the compounds of the invention can be obtained from natural sources, such as, but not limited to microorganisms, animals, plants, or other biological systems. In still other embodiments, the compounds of the invention can be prepared using enzymatic processes well known to those of ordinary skill in the art. In certain embodiments, the lipid compounds of the invention can be prepared using the synthetic schemes described herein. Other synthetic schemes that could be utilized by a person having ordinary skill in the art are also contemplated by the present invention. Essentially, the skilled artisan is required to be able to covalently modify a tail region of any suitable lipid to include a cationic peptide or peptide-based moiety of the invention. Any means which may accomplish this modification is contemplated. It will be appreciated that the particular modification scheme used will depend on the particular lipid which is being modified and the functional groups of the tail region that are present and available to be modified.

In some instances, the modified lipids of the invention can be accomplished utilizing a solid phase synthesis scheme. For example, such a scheme is illustrated with the conventional lipid AzPC (l-palmitoyl-2-azelaoyl-sn-glycero-3-phophocholine);

however, this scheme is not limited to modification of AzPC. In addition, the illustration utilizes a stable poly(arginine-histidine) peptide as the cationic peptide moiety of the invention; however, the invention is not limited to this particular peptide.

In this illustration, the poly(R-H) peptide was synthesized using Fmoc chemistry on a synthesis resin. AzPC was either purchased from Avanti (Alabaster, Alabama) or synthesized by condensing lysophosphatidyl choline and azelaic acid. A round bottom flask was charged with resin-bound peptide. A mixture of 50:50 DCM (Dichloromethane; Methylene Chloride) /THF (Tetrahydrofuran) was added to the flask in addition to carbonyldiimidazole. To this a solution of AzPC in DCM was added. Triethylamine was then added and the mixture was stirred at room temperature. The resin was then filtered to remove solvent. The reaction completion was assessed by a negative ninhydrin test.

Once the above conjugation reaction was complete, resin was cleaved from the product following standard procedures. After addition of cold diethylether to the cleavage mixture, a white powder precipitated out of solution. The solvent was decanted from the solid and the solid was washed with additional amount of cold ether. The solid was taken up in actonitrile and the solvent was removed by rotary evaporation. Finally, the residue was taken up in water and recovered by lyophilization to yield a white powder.

This scheme is outlined below:

Lysophosphatidyl choline (LPC, 16:0) Azelaic Acid

DCC, DMAP

CHCI3

Abbreviations:

DCC: Dicyclohexyl carbodiimide

DMAP: 4-Dimethylaminopyridine

CHCI3: Chloroform

In other instances, the modified lipids of the invention can be prepared utilizing a solution phase synthesis scheme. For example, two such schemes are illustrated below with the conventional lipid AzPC (l-palmitoyl-2-azelaoyl-sn-glycero-3-phophocholine); however, this scheme is not limited to modification of AzPC. In addition, the first illustration utilizes a stable poly(arginine-histidine) peptide as the cationic peptide moiety of the invention; however, the invention is not limited to this particular peptide. The second illustration utilizes a cleavable poly(arginine-histidine) peptide as the cationic peptide moiety of the invention; however, the invention is not limited to this particular peptide.

Peptide was synthesized using Fmoc chemistry with 6-Hydrazinonicotinamide (HyNic) synthon at the N-terminal of the peptide. The peptide was cleaved from the resin support following standard procedures. AzPC was reacted with Peg3-4FB (4- Formylbenzamide) in the presence of NHS (N-hydroxy succinimide), EDC (Ethylene Dichloride (1,2-Dichloroethane)) to produce AzPC-Peg3-4FB. HyNic -peptide and 4FB- AzPC (S-S-4FB, in case of disulfide cleavable conjugate) was then reacted in a mixture of 50:50 water/methanol to produce peptide- AzPC conjugate. Stable and cleavable conjugate were synthesized based on the 4-FB used in the first step of the reaction. While PEG3 moieties were employed in the below schematics, short PEG spacers or linker comprising between about 1 and 24 PEG subunits can be used between lipid tail and peptide moieties in certain embodiments.

Stable Peptide- AzPC Conjugation Scheme

Peptide-AzPC/Peg3 conjugate

Cleavable (Disulfide) Peptide- AzPC Conjugation Scheme

Peptide-AzPC/Peg3/disulfide conjugate

In certain embodiments, other chemical and/or enzymatic synthesis techniques are used to prepare and/or obtain the compounds of the invention. These synthesis techniques are well-known to those of ordinary skill in the art and thus, the description herein concerning the preparation of certain exemplary compounds is not intended to limit the invention in any way.

It will be appreciated by those of ordinary skill in the art that the general chemistry, synthesis, manufacture and sources of compounds is well known and published information and guidance regarding such is readily available in the art and which may be relied upon in the preparation of the compounds of the invention. For information and guidance as to the synthesis (e.g., enzymatic and/or chemical synthesis), manufacture, properties, and characteristics of compounds, the skilled artisan can refer to any suitable treatise, book or journal article or the like that pertains to such information, including, but not limited to: Lipid Synthesis And Manufacture (Chemistry And Technology Of Oils And Fats), Academic Press, Eds. Sheffield, 1998; Fatty Acid And Lipid Chemistry, Aspen Publishers, Eds. F. D. Gunstone, Frank Gunstone, 1996; Modifying Lipids For Use In Food, CRC Press, Eds. F. Gunstone, F. Gunstone, Frank D. Gunstone, 2006; Lipid Technologies And Applications, CRC Press, Eds. Frank D. Gunstone, Gunstone D.

Gunstone, Frank D. Gunstone, 1997; The Chemistry Of Oils & Fats Sources Composition Properties & Uses, Blackwell Science Ltd., Ed. Frank D Gunstone, 2005; and Lipid Analysis Of Oils And Fats, Springer Netherlands, Eds. R. J. Hamilton, M.d. Hamilton, Richard Hamilton, 1997, the contents of each of which are incorporated by reference.

As noted, in certain embodiments, the compounds of the invention, or starting materials that can be used to prepare the lipid compounds of the invention, can be obtained from natural sources, such as, but not limited to microorganisms, animals, plants, or other biological systems. For example, certain compounds of the invention comprise fatty acid moieties. Methods for preparing and/or isolating fatty acids from biological sources for use in the compounds of the invention can be found in U.S. Patent Nos. 7,579,174 (Enhanced production of lipids containing polyenoic/aity acid by very high density cultures of eukaryotic microbes in fermentors), 6,607,900 (Enhanced production of lipids containing polyenoic fatty acid by very high density cultures of eukaryotic microbes in fermentors), 6,582,941 (Microorganisms capable of producing highly unsaturated fatty acids and process for producing highly unsaturated fatty acids by using the

microorganisms), 6,451,567 (Fermentation process for producing long chain omega-3 fatty acids with euryhaline microorganisms), 6,255,505 (Microbial polyunsaturated fatty acid containing oil from pasteurised biomass), and 6,140,486 (Production of

polyunsaturated fatty acids by expression of polyketide-like synthesis genes in plants), each of which are incorporated herein by reference.

In other embodiments, the lipid compounds of the invention comprise sterol moieties (e.g., cholesterol). Methods for preparing and/or isolating such sterols from biological sources for use in the lipids of the invention can be found in U.S. Patent Nos. 2,729,655, 3,153,055, 3,335,154, 3,840,570, 4,148,810, 4,374,776, 4,451,564, 6,660,491, and 5,219,733, each of which are incorporated herein by reference. Sterols which may be used for the purposes of the invention may include those obtained from natural products such as, for example, soya, rapeseed, sunflower, coconut, palm kernel and palm oil.

Preferred sterols are sigmasterol, campesterol, sitosterol, brassicasterols, stigmasterol, D5 avenasterol, D7 avenasterol, ergosterol, citrostadienol, cholesterol, lanosterols, spongosterols, fungisterols, stellasterols, zymosterols and mixtures thereof and, phytosterols based on ergosterols, avenasterols (D5 and D7 avenasterol), campesterols, stigmasterols, sitosterols, brassicasterols, citrosdandiols, sigmastandiols and mixtures thereof. Any other phytosterols known to the expert may also be used. Their composition is described in "Sterinzusammensetzung und Steringehalt in 41 verschiedenen

pflanzlichen und tierischen Fetten", E. Homberg; B. Bielefeld; Fat Sci. Technol, Vol. 91, No. 1, 1989, which is incorporated herein by reference. As mentioned above, in certain embodiments, it may be desirable to modify the sterol to incorporate a tail region to which the peptide modification can be made.

The invention contemplates that the cationic peptide or peptide-based moiety used to modify the lipids of the invention be attached at any suitable location on the lipid molecule being modified, and preferable covalently attached to one or more atoms of a lipid tail region, such as fatty acid. In certain embodiments, the peptide moiety can be attached to the terminal atom or functional group of the tail region. In other embodiments, however, the peptide moiety may be attached to any position, atom or functional group on the tail region, or even to a branched moiety of the tail region.

Accordingly, it will be appreciated that the components of general formulae of the invention can be readily obtained, e.g., via synthesis using conventional methods or obtained from natural sources, such as, from plants or microorganisms, or synthesized enzymatically, or even obtained commercially, e.g., from sources including Tintagel, UK; Specs, The Netherlands; Timtec, Newark, Del.; Vitas-M Lab, Moscow, Russia.

The present invention further contemplates in certain embodiments the

modification of the lipid compounds of the invention with PEG as stabilizer components (in addition, PEG or other polymers may also be used to alter and improve biodistribution and/or reduce toxicity of a formulation). The PEG-lipid compounds can be made, for example, by reacting a glyceride moiety (e.g., a dimyristyl glyceride, dipalmityl glyceride, or distearyl glyceride) with an activating moiety under appropriate conditions, for example, to provide an activated intermediate that could be subsequently reacted with a PEG component having a reactive moiety such as an amine or a hydroxyl group to obtain a PEG-lipid. For example, a dalkylglyceride (e.g., dimyristyl glyceride) is initially reacted with Ν,Ν'-disuccinimidyl carbonate in the presence of a base (for e.g., triethylamine) and subsequent reaction of the intermediate formed with a PEG-amine (e.g., mPEG2000-NH 2) in the presence of base such as pyridine affords a PEG-lipid of interest. Under these conditions the PEG component is attached to the lipid moiety via a carbamate linkage. In another instance a PEG-lipid can be made, for example, by reacting a glyceride moiety (e.g., dimyristyl glyceride, dipalmityl glyceride, distearyl glyceride, dimyristoyl glyceride, dipalmitoyl glyceride or distearoyl glyceride) with succinic anhydride and subsequent activation of the carboxyl generated followed by reaction of the activated intermediate with a PEG component with an amine or a hydroxyl group, for instance, to obtain a PEG- lipid. In one example, dimyristyl glyceride is reacted with succinic anhydride in the presence of a base such as DMAP to obtain a hemi- succinate. The free carboxyl moiety of the hemi- succinate thus obtained is activated using standard carboxyl activating agents such as HBTU and diisopropylethylamine and subsequent reaction of the activated carboxyl with mPEH2000-NH₂, for instance, yields a PEG-lipid. In this approach the PEG component is linked to the lipid component via a succinate bridge.

Peptide Moieties of the Invention

The novel cationic lipids of the invention contain a net positive charge on their lipid tail due to the presence of one or more peptide or peptide-bases moieties which are covalently attached thereto. The cationic peptide of the invention creates a net positive charge to the overall lipid to which it modifies. The peptide or peptide-based moieties of the invention are capable of attaining a change in conformation when contacting certain conditions which are typical of the intracellular environment (including the cytosol and within intracellular compartments), such as changes in pH, protonation/deprotonation conditions, oxidation/reduction conditions, as well as changes due to chemical enzymatic modifications.

In certain embodiments, the conformation change occurs due to the reduction of a disulfide bridge, thereby cleaving the peptide. In another embodiment, the conformation change occurs due to the action of an intracellular enzyme (e.g., a protease within a lysosome) which cleaves a specific amino acid recognition sequence. In yet another embodiment, the conformation change occurs due to the action of an intracellular enzyme which modifies the structure of the peptide, e.g., adds a chemical group to one or more residues of the peptide, thereby inducing conformation change. The chemical group may carry a charge which functions to cause a conformation change in the peptide.

Exemplary peptides of the invention can include the following:

SEQ 1:— (R)_niH(R)_n2 or SEQ 2:— (K)_niH(K)_n2, wherein, nland n2 are

independently 0 to 25;

SEQ 3:— (R)_ni(HR)_n2(R)_n3 or SEQ 4:— (K)_ni(HK)_n2(K)_n3, wherein nl and n3 are independently 0 to 25 and n2 is 1 to 15;

SEQ 5:— (R)_ni(RH)_n2(R)_n3 or SEQ 6:— (K)_ni(KH)_n2(K)_n3, wherein nl and n3 are independently 0 to 25 and n2 is 1 to 15; SEQ 7:— Xi(HR)_nX₂ or SEQ 8:— Xi(HK)_nX₂, wherein XI and X2 are

independently amino acids or peptides with 2 to 25 amino acids and n is 1 to 15;

SEQ 9:— Xi(RH)_nX₂ or SEQ 10:— Xi(KH)_nX₂, wherein XI and X2 are independently amino acids or peptides with 2 to 25 amino acids and n is 1 to 15;

SEQ 11 — RHRHRHRHR ;

SEQ 12 — RHDRHDRHD ;

SEQ 13 — RHKHRQRHRPPQ ;

SEQ 14 — RHKHRQRHRPPQ;

SEQ 15 — K(RHRHR) (HRHR) ;

SEQ 16 — K(RHDRH)(DRHD); and

SEQ 17 — K(RHKHRQXRHRPPQ).

Preferably, the peptides of the invention include one or more, and preferably, a high density or a region having a high density of positively charged amino acid residues, such as, lysine, histidine and arginine. In certain other embodiments, the peptides of the invention can have a linear amino acid sequence, or alternately, a branched amino acid sequence, or even a cyclized amino acid sequence, or a portion that is cyclized. Also, the residues of the peptides of the invention may be L-amino acids, D-amino acids, or nonnaturally-occurring amino acids or otherwise naturally-occurring residues which are derivatized, e.g., by adding a cationic group.

In other embodiments, one or more peptide sequences may be combined.

Sequences may optionally contain terminal linker amino acids (e.g., Lys, Cys, Citruline- Valine (Cit-Val) or (Val-Cit) or repeat thereof) spacer amino acids (Gly, Ser, etc. or combination thereof) and spacer molecules (e.g., PEG comprising between one and about 24 PEG subunits used between lipid tail and a peptide moiety in certain embodiments).

Peptide Synthesis

There are at least four ways to obtain a peptide: (1) purification from biological system (e.g., tissue, serum, urine, etc.); (2) purification of peptide fragment after digestion of a protein; (3) genetic engineering and recombinant technologies and (4) direct chemical synthesis. The first two approaches are often impractical due to the lack of control over the peptide sequences. The first approach also suffers from low concentration of peptide in biological samples that requires significant concentrating steps prior to purification.

Typically, therefore, for shorter peptides direct chemical synthesis is an attractive option, whereas, for larger peptide recombinant technology is a better choice. Traditional synthetic approaches of organic chemistry are generally impractical for peptides with more than four or five amino acid residues due to the complexities of amino acids and peptides. The problems include multiple reactive groups in the peptide and purifying the product after each step or synthesizing a series of different peptide mixtures that are impurity to the peptide of interest.

The advent of solid phase peptide synthesis (Merrifield, 1962) in which peptide is synthesized while keeping it attached at one end to a solid support provided the major breakthrough in the direct chemical synthesis of peptides. Today, most solid phase peptide syntheses involve FMOC chemistry. Briefly, chemical synthesis proceeds from the carboxyl terminus (C terminus) to the amino terminus (N terminus). The solid phase support is an insoluble polymer or resin. The 9-fluorenyl-methoxycarbonyl (FMOC) group prevents unwanted reactions at the a-amino group of the amino acid residue. The peptide is built on a resin support one amino acid at a time using a standard set of reactions in a repeating cycle. First, the C-terminal amino acid with it a-amino group protected by FMOC group is attached to the reactive group on the resin. The protecting group on the a- amino group of the amino acid attached to the resin is removed, generally with a mild organic base. Now, the resin with the C-terminal amino acid is ready to receive the second amino acid of the peptide. Each amino acid is received protected with different chemistries at the a-amino group (FMOC) and carboxyl group (generally, Dicyclohexylcarbodiimide, DCC). The carboxyl group of the second amino acid is activated by removing DCC and reacted with the deprotected a-amino group of the first amino acid on the solid support to form the peptide bond.

At each successive step in the cycle, protective chemical groups block unwanted reactions and the sequence of (i) deprotection of the a-amino group on the nascent peptide; (ii) activation of the carboxyl group on the next amino acid and (iii) reaction to form peptide bond continues until the entire peptide sequence is synthesized. When the peptide synthesis is complete, the linkage between the resin and the peptide is cleaved off to obtain the final peptide. The state-of-the-art solid phase peptide synthesis technology is automated, and several kinds of commercial instruments are now available.

Since the solid phase synthesis is a stepwise process for longer peptides it has the important limitation of lower overall yield and therefore increased cost. For example, with a 96% stepwise yield, the overall yield for 21mer, 51mer and lOOmer peptides are 44%, 13% and 1.7%, respectively. Similarly, with a 99.8% stepwise yield, the overall yield for 21mer, 51mer and lOOmer peptides are 96%, 90% and 82%, respectively. Therefore, for longer peptides it is more cost- and time- effective to genetically engineer the sequence in an expression cassette and express them in appropriate expression system (e.g., microbial expression system such as E. coli or yeast) or mammalian expression system (cell culture). For smaller peptides, however, the cost of genetically engineer the sequence and expressing and purifying the peptides are generally not cost- and time- effective compared to the solid phase peptide synthesis.

Peptide for the current invention could be synthesized, expressed or purified using the methods described above or other methods of synthesis, expression or purification known in the art.

Peptide Charge

Positively charged amino acids are Lysine (Lys, K), Arginine (Arg, R) and

Histidine (His, H). Negatively charged amino acids are Aspartic acid or aspartate (Asp, D), Glutamic acid or glutamate (Glu, E). Overall isoelectric point (pi) value of the peptide depends on the primary sequence and especially the presence, number and location of the above mentioned charged amino acid residues.

Histidine, an essential amino acid, has a positively charged imidazole functional group. The imidazole makes it a common participant in enzyme catalyzed reactions. The unprotonated imidazole is nucleophilic and can serve as a general base, while the protonated form can serve as a general acid. The residue can also serve a role in inducible structures of peptides and proteins. The imidazole sidechain of histidine has a pKa of approximately 6, and overall, the amino acid has a pKa of 7.6. This means that at physiologically relevant pH values, relatively small shifts in pH will change its average charge. In the endocytic vesicles, as pH falls below a pH of 6, the imidazole ring becomes protonated as described by the Henderson-Hasselbalch equation. When protonated, the imidazole ring bears two NH bonds and has a positive charge. The positive charge is equally distributed between both nitrogens and can be represented with two equally important resonance structures (Lehninger Principles of Biochemistry, 3rd Ed., 2000. Edited by David L. Nelson and Michael M. Cox, Worth Publishers, New York, NY).

Conjugation Chemistry

Conjugation is performed to any amino acid residues in the peptide, e.g., the C- terminal or N-terminal amino acid residues with either terminal a-amino group, carboxyl group or to specific function group on the amino acid residue (e.g., -SH group on Cys). Any conjugation chemistry for peptide or protein known in the art maybe utilized with appropriate end group choice on the lipids. Lipid-peptide conjugates are purified and characterized for identity and purity with standard analytical methods.

Lipid Compositions of the Invention

In yet another aspect, the present invention provides lipid-based compositions for use in delivering cargo or payload materials to target cells or tissues in vitro or in vivo. In certain embodiments, the lipid compounds included in such compositions comprise a peptide or peptide-based moiety of the invention.

The lipid compounds described herein, including the peptide-modified lipid compounds of the invention, can be used as a component in a lipid-based composition or formulation, for example a liposome, micelle or aggregate comprising a desired cargo or payload, e.g., nucleic acid molecule payloads. The lipid compounds of the invention, as a component of the lipid-based compositions of the invention, upon inducing the conformational change in the peptide moiety, will advantageously release their payloads for more efficient delivery to the target cell or tissue. The conformation change of the peptide moiety in certain embodiments is induced by contacting intracellular conditions, including those of the cytoplasm or encountered in an intracellular compartment, such as an endosome or lysosome.

In one embodiment, the cargo of the inventive lipid-based compositions comprises nucleic acid molecules and the compositions can be used to administer nucleic acid based therapy, such as an DsiRNA, miRNA or siRNA, to desired target cell to affect or control gene expression. The inventive lipids are particularly advantageous in complexing polyanionic substances, such as nucleic acid molecules, because of the net positive charged imparted by the cationic peptide modification.

The lipid-based compositions of the invention can include a plurality of components.

First, the lipid-based compositions of the invention will include at least one lipid compound of the invention, which are described elsewhere as general formulae X-Y-P, wherein X is a headgroup, Y is a tail region, and P is the cationic peptide moiety of the invention.

Second, the lipid-based compositions of the invention advantageously include a desirable cargo or active ingredient, such as, a polypeptide, hormone, peptide, nucleic acid molecule (e.g., siRNA, DsiRNA, miRNA, oligonucleotides, RNA, DNA, or the like), small molecule drugs, and the like. In some embodiments, the association complex can include a plurality of therapeutic agents or active ingredients, for example two or three nucleic acid molecules that target more than one gene or different regions of the same gene. Other components can also be included in the lipid-based compositions of the invention, including lipids other than the cleavable lipids of the invention, PEG-lipids, or another structural component, such as cholesterol or a polymer component, or one or more targeting moieties.

In certain embodiments, the lipid-based compositions of the invention can include a non-cationic lipid, such as a neutral or anionic lipid which has not been modified by the peptide moieties of the invention.

Non-cationic lipid compounds used in the formulations of the present invention can be any of a variety of neutral uncharged, zwitterionic, or anionic lipids capable of producing a stable complex. Such non-cationic lipid compounds can be neutral or negatively charged. Examples of non-cationic lipid compounds include, without limitation, phospholipid-related materials such as lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphatidic acid, cerebrosides, dicetylphosphate, distearoylphosphatidylcholine (DSPC),

dioleoylphosphatidylcholine (DOPC), dipahnitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoyl-phosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), palmitoyloleyol-phosphatidylglycerol (POPG), dioleoylphosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-l- carboxylate (DOPE-mal), dipalmitoyl-phosphatidylethanolamine (DPPE), dimyristoyl- phosphatidylethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), monomethyl-phosphatidylethanolamine, dimethyl -phosphatidylethanolamine, dielaidoyl- phosphatidylethanolamine (DEPE), and stearoyloleoyl-phosphatidylethanolamine (SOPE). Non-cationic lipid compounds or sterols such as cholesterol may also be present.

Additional nonphosphorous containing lipid compounds include, e.g., stearylamine, dodecylamine, hexadecylamine, acetyl palmitate, glycerolricinoleate, hexadecyl stereate, isopropyl myristate, amphoteric acrylic polymers, triethanolamine-lauryl sulfate, alkyl- aryl sulfate polyethyloxylated fatty acid amides, dioctadecyldimethyl ammonium bromide, ceramide, diacylphosphatidylcholine, diacylphosphatidylethanolamine, and the like. Other lipid compounds such as lysophosphatidylcholine and lysophosphatidylethanolamine may be present. Non-cationic lipid compounds also include polyethylene glycol (PEG)-based polymers such as PEG 2000, PEG 5000, and polyethylene glycol conjugated to phospholipids or to ceramides (referred to as PEG-Cer), as described in U.S. patent application Ser. No. 08/316, 429.

In certain embodiments, the non-cationic lipid compounds are

diacylphosphatidylcholine (e.g., distearoylphosphatidylcholine,

dioleoylphosphatidylcholine, dipalmitoylphosphatidylcholine, and

dilinoleoylpho sphatidylcholine) , diacylpho sphatidylethanolamine (e.g.,

dioleoylphosphatidylethanolamine and palmitoyloleoyl-phosphatidylethanolamine), ceramide, or sphingomyelin. In particular embodiments, the acyl groups in these lipid compounds are acyl groups derived from fatty acids having C10-C24 carbon chains.

Optionally, the acyl groups are lauroyl, myristoyl, palmitoyl, stearoyl, or oleoyl. In certain embodiments, the non-cationic lipid compound may comprise from about 5 mol % to about 90 mol % or about 15 mol % to about 75 mol % or about 20 mol % to about 50 mol % or about 40 mol % of the total lipid compounds present in the formulation. The non- cationic lipid compound typically comprises from about 10 mol % to about 85 mol %, from about 20 mol % to about 80 mol %, or about 20 mol % of the total lipid compounds present in the formulation.

In certain other embodiments, the lipid-based components can include a cationic lipid compound or other component, such as a cholesterol. The cationic lipids may or may not be modified by the peptide of the invention.

A cationic lipid compound of a formulation of the instant invention may be, e.g., N, N-dioleyl-N, N-dimethylammonium chloride (DODAC), N, N-distearyl-N, N- dimethylammonium bromide (DDAB), N-(l-(2, 3-dioleoyloxy)propyl)-N, N, N- trimethylammonium chloride (DOTAP), N-(l-(2, 3-dioleyloxy)propyl)-N, N, N- trimethylammonium chloride (DOTMA), N, N-dimethyl-2, 3-dioleyloxypropylamine (DODMA), 1, 2-DiLinoleyloxy-N, N-dimethylaminopropane (DLinDMA), 1, 2- Dilinolenyloxy-N, N-dimethylaminopropane (DLenDMA), DSDMA, DOSPA, DC-Choi, DMRIE or mixtures thereof.

A number of these lipid compounds and related analogs have been described in U.S. Patent Publication No. 20060083780; U.S. Pat. Nos. 5,208,036; 5,264,618;

5,279,833; 5,283,185; 5,753,613; and 5,785,992; and PCT Publication No. WO 96/10390. Additionally, a number of commercial preparations of cationic lipid compounds are available and can be used in the present invention. These include, for example, LIPOFECTIN® (commercially available cationic liposomes comprising DOTMA and DOPE, from GEBCO/BRL, Grand Island, N.Y., USA); LIPOFECTAMINE®

(commercially available cationic liposomes comprising DOSPA and DOPE, from

GIBCO/BRL); and TRANSFECTAM® (commercially available liposomes comprising DOGS from Promega Corp., Madison, Wis., USA).

In certain embodiments, the modified lipid compounds of the invention may comprise from about 5 mol % to about 90 mol %, about 10 mol % to about 60 mol %, or about 40 mol % of the total lipid compounds present in the formulation. The cationic lipid compounds typically comprise from about 2 mol % to about 60 mol %, from about 5 mol % to about 50 mol %, from about 10 mol % to about 50 mol %, from about 20 mol % to about 50 mol %, from about 20 mol % to about 40 mol %, from about 30 mol % to about 40 mol %, or about 40 mol % of the total lipid compounds present in the formulation. It will be readily apparent to one of skill in the art that depending on the intended use of the formulations, the proportions of the components can be varied and the delivery efficiency of a particular formulation can be measured using, e.g., an endosomal release parameter (ERP) assay. For example, for systemic delivery, the cationic lipid compounds of the invention may comprise from about 5 mol % to about 15 mol % of the total lipid compounds present in the particle, and for local or regional delivery, the cationic lipids of the invention may comprise from about 30 mol % to about 50 mol %, or about 40 mol % of the total lipid compounds present in the formulation.

The formulations of the instant invention may further comprise cholesterol. If present, the cholesterol typically comprises from about 0 mol % to about 10 mol %, from about 2 mol % to about 10 mol %, from about 10 mol % to about 60 mol %, from about 12 mol % to about 58 mol %, from about 20 mol % to about 55 mol %, from about 30 mol % to about 50 mol %, or about 48 mol % of the total lipid compounds present in the formulation. The cholesterol may or may not be modified with a peptide moiety of the invention.

Hydrophilic polymers can also be included in the formulations of the instant invention. Hydrophilic polymers suitable for use in the formulations of the present invention are those which are readily water-soluble, can be covalently attached to a lipid of the formulations of the invention, and which are tolerated in vivo without toxic effects (i.e., are biocompatible). Exemplary suitable polymers include polyethylene glycol (PEG), polylactic (also termed polylactide), polyglycolic acid (also termed polyglycolide), a polylactic-polyglycolic acid copolymer, and polyvinyl alcohol. In certain embodiments, such polymers possess a molecular weight of from about 100 or 120 daltons up to about 5,000 or 10,000 daltons, and optionally from about 300 daltons to about 5,000 daltons. In one embodiment, the polymer is polyethyleneglycol (PEG) having a molecular weight of from about 100 to about 5,000 daltons, and optionally having a molecular weight of from about 300 to about 5,000 daltons. In a specific embodiment, the polymer is PEG of 750 daltons (PEG(750)). Polymers may also be defined by the number of monomers therein; in one embodiment, polymers of at least about three monomers, such PEG polymers consisting of three monomers (approximately 150 daltons), are used in the formulations of the invention. Additional exemplary hydrophilic polymers which may be suitable for use in the present invention include polyvinylpyrrolidone, polymethoxazoline,

polyethyloxazoline, polyhydroxypropyl methacrylamide, polymethacrylamide, polydimethylacrylamide, and derivatized celluloses such as hydroxymethylcellulose or hydroxyethylcellulose.

Conjugated lipid compounds may also be included in the formulations of the invention, including a hydrophilic polymer-lipid conjugate (e.g., a polyethyleneglycol (PEG)-lipid conjugate), a polyamide (ATTA)-lipid conjugate, a cationic-polymer-lipid conjugate (CPL), or mixtures thereof. In certain embodiments, a nucleic acid-lipid formulation of the invention comprises either a PEG-lipid conjugate or an ATTA-lipid conjugate. Optionally, a PEG-lipid conjugate or ATTA-lipid conjugate is used together with a CPL. A conjugated lipid compound of a formulation of the invention may comprise a PEG-lipid including, e.g., a PEG-diacylglycerol (DAG), a PEG

dialkyloxypropyl (DAA), a PEG-phospholipid, a PEG-ceramide (Cer), or mixtures thereof. A PEG-DAA conjugate may be a PEG-dilauryloxypropyl (C12), a PEG- dimyristyloxypropyl (C14), a PEG-dipalmityloxypropyl (C16), or a PEG- distearyloxypropyl (CI 8). Optionally, a conjugated lipid is a CPL that has the formula: A- W-Y, wherein A is a lipid moiety, W is a hydrophilic polymer, and Y is a polycationic moiety. W may be a polymer selected from the group consisting of PEG, polyamide, polylactic acid, polyglycolic acid, polylactic acid/polyglycolic acid copolymers, or combinations thereof, the polymer having a molecular weight of from about 250 to about 7000 daltons. In some embodiments, Y has at least 4 positive charges at a selected pH. In some embodiments, Y may be lysine, arginine, asparagine, glutamine, derivatives thereof, or combinations thereof. In certain embodiments, a conjugated lipid compound is present in a formulation of the instant invention from 0 mol % to about 20 mol % or about 2 mol % of the total lipid compounds present in the formulation. In addition to cationic and non-cationic lipid compounds (of which any may or may not carry the peptide modification of the invention), a formulation of the present invention can comprise a stabilizing component (SC) such as an ATTA-lipid or a PEG- lipid or other hydrophilic polymer-lipid composition as described above) such as PEG coupled to dialkyloxypropyls (PEG-DAA) as described in, e.g., PCT Publication No. WO 05/026372, PEG coupled to diacylglycerol (PEG-DAG) as described in, e.g., U.S. Patent Publication Nos. 20030077829 and 2005008689, PEG coupled to phospholipids such as phosphatidylethanolamine (PEG-PE), PEG conjugated to ceramides, or a mixture thereof (see, e.g., U.S. Pat. No. 5,885,613). In certain embodiments, the SC is a conjugated lipid compound that prevents the aggregation of formulation particles. Suitable conjugated lipid compounds include, but are not limited to, PEG-lipid conjugates, ATTA-lipid conjugates, cationic-polymer-lipid conjugates (CPLs), and mixtures thereof. In additional embodiments, formulation particles comprise either a PEG-lipid conjugate or an ATTA- lipid conjugate together with a CPL.

PEG is a linear, water-soluble polymer of ethylene PEG repeating units with two terminal hydroxyl groups. PEGs are classified by their molecular weights; for example, PEG 2000 has an average molecular weight of about 2,000 daltons, and PEG 5000 has an average molecular weight of about 5,000 daltons. PEGs are commercially available from Sigma Chemical Co. and other companies and include, for example, the following:

monomethoxy glycol (MePEG-OH), monomethoxypolyethylene glycol-succinate

(MePEG-S), monomethoxypolyethylene glycol- succinimidyl succinate (MePEG-S-NHS), monomethoxypolyethylene glycol-amine (MePEG-NH₂), monomethoxypolyethylene glycol-tresylate (MePEG-TRES), and monomethoxypolyethylene glycol-imidazolyl- carbonyl (MePEG-IM). In addition, monomethoxypolyethyleneglycol-acetic acid

(MePEG-CH₂COOH) is particularly useful for preparing PEG-lipid conjugates including, e.g., PEG-DAA conjugates.

In certain embodiments, a PEG used in a formulation of the invention has an average molecular weight of from about 550 daltons to about 10,000 daltons, optionally from about 750 daltons to about 5,000 daltons, optionally from about 1,000 daltons to about 5,000 daltons, optionally from about 1, 500 daltons to about 3,000 daltons, and optionally about 2,000 daltons or about 750 daltons. The PEG can be optionally substituted by an alkyl, alkoxy, acyl, or aryl group. The PEG can be conjugated directly to the lipid compound or may be linked to the lipid compound via a linker moiety. A linker moiety suitable for coupling the PEG to a lipid compound can be used including, e.g., non- ester containing linker moieties and ester-containing linker moieties. In certain embodiments, the linker moiety is a non-ester containing linker moiety. As used herein, the term "non-ester containing linker moiety" refers to a linker moiety that does not contain a carboxylic ester bond (-OC(O)-). Suitable non-ester containing linker moieties include, but are not limited to, amido (-C(O)NH-), amino (-NR-), carbonyl (-C(O)-), carbamate (-NHC(O)O-), urea (-NHC(O)NH-), disulfide (-S-S-), ether(-O-), succinyl (- (0)CCH₂CH₂C(0)-), succinamidyl (-NHC(0)CH₂CH₂C(0)NH-), ether, disulfide, as well as combinations thereof (such as a linker containing both a carbamate linker moiety and an amido linker moiety). Optionally, a carbamate linker is used to couple the PEG to the lipid.

In other embodiments, an ester containing linker moiety can be used to couple PEG to a lipid. Exemplary ester containing linker moieties include, e.g., carbonate (- OC(O)O-), succinoyl, phosphate esters (-O-(O)POH-O-), sulfonate esters, and

combinations thereof.

Phosphatidylethanolamines having a variety of acyl chain groups of varying chain lengths and degrees of saturation can be conjugated to PEG to form a stabilizing component. Such phosphatidylethanolamines are commercially available, or can be isolated or synthesized using conventional techniques known to those of skilled in the art. Exemplary phosphatidylethanolamines contain saturated or unsaturated fatty acids with carbon chain lengths in the range of Cio to C₂₀. Phosphatidylethanolamines with mono-or diunsaturated fatty acids and mixtures of saturated and unsaturated fatty acids can also be used. Suitable phosphatidylethanolamines include, but are not limited to, dimyristoyl- phosphatidylethanolamine (DMPE), dipalmitoyl-phosphatidylethanolamine (DPPE), dioleoylphosphatidylethanolamine (DOPE), and distearoyl-phosphatidylethanolamine (DSPE).

Additionally, a PEG can be used itself as a spacer or linker to join a peptide of the invention to the tail portion of a lipid. Preferably, the PEG in this capacity has between one and 24 PEG subunits, preferably between one and 12 PEG subunits, more preferably between one and 6 PEG subunits, and even more preferably between one and 3 PEG subunits. Any of the herein mentioned PEGS may be used in this capacity, i.e., as a spacer or linker to couple a lipid tail and peptide moiety of the invention.

In addition to the foregoing components, formulation particles of the present invention can further comprise cationic poly(ethylene glycol) (PEG) lipids or CPLs that have been designed for insertion into lipid bilayers to impart a positive charge (see, e.g., Chen et al., Bioconj. Chem., 11:433-437 (2000)). Exemplary SPLPs and SPLP-CPLs that can be used in the formulations of the instant invention, and methods of making and using SPLPs and SPLP-CPLs, are disclosed, e.g., in U.S. Pat. No. 6,852,334 and PCT

Publication No. WO 00/62813. Cationic polymer lipids (CPLs) which may also be used in the formulations of the instant invention in the present invention have the following architectural features: (1) a lipid anchor, such as a hydrophobic lipid, for incorporating the CPLs into the lipid bilayer; (2) a hydrophilic spacer, such as a polyethylene glycol, for linking the lipid anchor to a cationic head group; and (3) a polycationic moiety, such as a naturally occurring amino acid, to produce a protonizable cationic head group.

In certain instances, the formulations of the invention comprise a ligand, such as a targeting ligand or a chelating moiety for complexing calcium. In certain instances, the ligand of the formulation has a positive charge. Exemplary ligands include, but are not limited to, a compound or device with a reactive functional group and include lipids, amphipathic lipids, carrier compounds, bioaffinity compounds, biomaterials, biopolymers, biomedical devices, analytically detectable compounds, therapeutically active compounds, enzymes, peptides, proteins, antibodies, immune stimulators, radiolabels, fluorogens, biotin, drugs, haptens, DNA, RNA, polysaccharides, liposomes, virosomes, micelles, immunoglobulins, functional groups, other targeting moieties, or toxins.

The stabilizing component (e.g., PEG-lipid) can comprise from about 0 mol % to about 20 mol %, from about 0.5 mol % to about 20 mol %, from about 1.5 mol % to about 18 mol %, from about 4 mol % to about 15 mol %, from about 5 mol % to about 12 mol %, or about 2 mol % of the total lipid present in the formulation. One of ordinary skill in the art will appreciate that the concentration of a stabilizing component can be varied depending on the stabilizing component employed and the rate at which the formulation (e.g., a formulation particle) is to become fusogenic.

By controlling the composition and concentration of a stabilizing component, one can control the rate at which the stabilizing component exchanges out of a lipid formulation (where the formulation forms a particle, a formulation particle) and, in turn, the rate at which the formulation becomes fusogenic. For instance, when a

polyethyleneglycol-phosphatidylethanolamine conjugate or a polyethyleneglycol-ceramide conjugate is used as a stabilizing component, the rate at which a formulation becomes fusogenic can be varied, for example, by varying the concentration of the stabilizing component, by varying the molecular weight of the polyethyleneglycol, or by varying the chain length and degree of saturation of the acyl chain groups on the phosphatidylethanolamine or the ceramide. In addition, other variables including, for example, pH, temperature, ionic strength, etc. can be used to vary and/or control the rate at which a lipid formulation becomes fusogenic. Other methods which can be used to control the rate at which a formulation becomes fusogenic will become apparent to those of skill in the art upon reading this disclosure.

Non-limiting examples of additional lipid-based carrier systems (which may be prepared with at least one modified cationic lipid of the invention) suitable for use in the present invention include lipoplexes (see, e.g., U.S. Patent Publication No. 20030203865; and Zhang et al., J. Control Release, 100:165-180 (2004)), pH-sensitive lipoplexes (see, e.g., U.S. Patent Publication No. 2002/0192275), reversibly masked lipoplexes (see, e.g., U.S. Patent Publication Nos. 2003/0180950), cationic lipid-based compositions (see, e.g., U.S. Pat. No. 6,756,054; and U.S. Patent Publication No. 2005/0234232), cationic liposomes (see, e.g., U.S. Patent Publication Nos. 2003/0229040, 2002/0160038, and 2002/0012998; U.S. Pat. No. 5,908,635; and PCT Publication No. WO 01/72283), anionic liposomes (see, e.g., U.S. Patent Publication No. 2003/0026831), pH-sensitive liposomes (see, e.g., U.S. Patent Publication No. 2002/0192274; and AU 2003/210303), antibody- coated liposomes (see, e.g., U.S. Patent Publication No. 2003/0108597; and PCT

Publication No. WO 00/50008), cell-type specific liposomes (see, e.g., U.S. Patent Publication No. 2003/0198664), liposomes containing nucleic acid and peptides (see, e.g., U.S. Pat. No. 6,207,456), liposomes containing lipids derivatized with releasable hydrophilic polymers (see, e.g., U.S. Patent Publication No. 2003/0031704), lipid- entrapped nucleic acid (see, e.g., PCT Publication Nos. WO 03/057190 and WO

03/059322), lipid-encapsulated nucleic acid (see, e.g., U.S. Patent Publication No.

2003/0129221; and U.S. Pat. No. 5,756,122), other liposomal compositions (see, e.g., U.S. Patent Publication Nos. 2003/0035829 and 2003/0072794; and U.S. Pat. No. 6,200,599), stabilized mixtures of liposomes and emulsions (see, e.g., EP1304160), emulsion compositions (see, e.g., U.S. Pat. No. 6,747,014), and nucleic acid micro-emulsions (see, e.g., U.S. Patent Publication No. 2005/0037086).

Examples of polymer-based carrier systems (which may be prepared with at least one modified cationic lipid of the invention) suitable for use in the present invention include, but are not limited to, cationic polymer- nucleic acid complexes (i.e., polyplexes). To form a polyplex, cargo (e.g., a nucleic acid such as a DsiRNA) is typically complexed with a cationic polymer having a linear, branched, star, or dendritic polymeric structure that condenses the cargo into positively charged particles capable of interacting with anionic proteoglycans at the cell surface and entering cells by endocytosis. In some embodiments, the polyplex comprises nucleic acid (e.g., DsiRNA) complexed with a cationic polymer such as polyethylenimine (PEI) (see, e.g., U.S. Pat. No. 6,013,240;

commercially available from Qbiogene, Inc. (Carlsbad, Calif.) as In vivo jetPEI®, a linear form of PEI), polypropylenimine (PPI), polyvinylpyrrolidone (PVP), poly-L-lysine (PLL), diethylaminoethyl (DEAE)-dextran, poly(P-amino ester) (PAE) polymers (see, e.g., Lynn et al., J. Am. Chem. Soc, 123:8155-8156 (2001)), chitosan, polyamidoamine (PAMAM) dendrimers (see, e.g., Kukowska-Latallo et al., Proc. Natl. Acad. Sci. USA, 93:4897-4902 (1996)), porphyrin (see, e.g., U.S. Pat. No. 6,620,805), polyvinylether (see, e.g., U.S. Patent Publication No. 20040156909), polycyclic amidinium (see, e.g., U.S. Patent Publication No. 20030220289), other polymers comprising primary amine, imine, guanidine, and/or imidazole groups (see, e.g., U.S. Pat. No. 6,013,240; PCT Publication No. WO/9602655; PCT Publication No. W095/21931; Zhang et al., J. Control Release, 100:165-180 (2004); and Tiera et al., Curr. Gene Ther., 6:59-71 (2006)), and a mixture thereof. In other embodiments, the polyplex comprises cationic polymer-nucleic acid complexes as described in U.S. Patent Publication Nos. 2006/0211643, 2005/0222064, 2003/0125281, and 2003/0185890, and PCT Publication No. WO 03/066069;

biodegradable poly(P-amino ester) polymer-nucleic acid complexes as described in U.S. Patent Publication No. 2004/0071654; microparticles containing polymeric matrices as described in U.S. Patent Publication No. 2004/0142475; other microparticle compositions as described in U.S. Patent Publication No. 2003/0157030; condensed nucleic acid complexes as described in U.S. Patent Publication No. 2005/0123600; and nanocapsule and microcapsule compositions as described in AU 2002358514 and PCT Publication No. WO 02/096551.

In certain instances, the cargo (e.g., a nucleic acid such as a DsiRNA) may be complexed with cyclodextrin or a polymer thereof. Non-limiting examples of

cyclodextrin-based carrier systems include the cyclodextrin-modified polymer-nucleic acid complexes described in U.S. Patent Publication No. 2004/0087024; the linear cyclodextrin copolymer-nucleic acid complexes described in U.S. Pat. Nos. 6,509,323, 6,884,789, and 7,091,192; and the cyclodextrin polymer-complexing agent-nucleic acid complexes described in U.S. Pat. No. 7,018,609. In certain other instances, the cargo (e.g., a nucleic acid such as a DsiRNA) may be complexed with a peptide or polypeptide. An example of a protein-based carrier system includes, but is not limited to, the cationic oligopeptide-nucleic acid complex described in PCT Publication No. W095/21931. Cargoes of the Lipid-Based Compositions of the Invention

The instant invention is broadly applicable to formulations containing any of a number of cargoes/payloads. The invention contemplates the inclusion of any

cargo/payload that can be used as a cargo of a formulation of the instant invention.

Exemplary cargoes include nucleic acid cargoes (e.g., siRNA, DsiRNA, antisense oligonucleotide) and non-nucleic acid cargoes, including proteins, small molecules, active drugs, peptide hormones, steroid hormones, and cytotoxic agents such as camptothecin, SN-38, homo-campotothecin (BN80915), paclitaxel, doxorubicin, and methotrexate. Exemplary types of nucleic acid based cargoes are described as follows. It is

contemplated that the modified lipids of the invention which contain a cationic peptide or peptide-based moiety of the invention in the tail region of the lipid are particularly advantageous in complexing polyanionic payloads, such as nucleic acid molecules.

Single Stranded Ribonucleic Acid Cargoes

Oligonucleotide agents include microRNAs (miRNAs). MicroRNAs are small noncoding RNA molecules that are capable of causing post-transcriptional silencing of specific genes in cells such as by the inhibition of translation or through degradation of the targeted mRNA. An miRNA can be completely complementary or can have a region of noncomplementarity with a target nucleic acid, consequently resulting in a "bulge" at the region of non-complementarity. The region of noncomplementarity (the bulge) can be flanked by regions of sufficient complementarity, preferably complete complementarity to allow duplex formation. Preferably, the regions of complementarity are at least 8 to 10 nucleotides long (e.g., 8, 9, or 10 nucleotides long). A miRNA can inhibit gene expression by repressing translation, such as when the micro RNA is not completely complementary to the target nucleic acid, or by causing target RNA degradation, which is believed to occur only when the miRNA binds its target with perfect complementarity. The invention also can include double- stranded precursors of miRNAs that may or may not form a bulge when bound to their targets.

In a preferred embodiment an oligonucleotide agent featured in the invention can target an endogenous miRNA or pre-miRNA. The oligonucleotide agent featured in the invention can include naturally occurring nucleobases, sugars, and covalent

internucleoside (backbone) linkages as well as oligonucleotides having non-naturally- occurring portions that function similarly. Such modified or substituted oligonucleotides are often preferred over native forms because of desirable properties such as, for example, enhanced cellular uptake, enhanced affinity for the endogenous miRNA target, and/or increased stability in the presence of nucleases. An oligonucleotide agent designed to bind to a specific endogenous miRNA has substantial complementarity, e.g., at least 70, 80, 90, or 100% complementary, with at least 10, 20, or 25 or more bases of the target miRNA.

A miRNA or pre-miRNA can be 18-100 nucleotides in length, and more preferably from 18-80 nucleotides in length. Mature miRNAs can have a length of 19-30 nucleotides, preferably 21-25 nucleotides, particularly 21, 22, 23, 24, or 25 nucleotides. MicroRNA precursors can have a length of 70-100 nucleotides and have a hairpin conformation. MicroRNAs can be generated in vivo from pre-miRNAs by enzymes called Dicer and Drosha that specifically process long pre-miRNA into functional miRNA. The

microRNAs or precursor mi-RNAs featured in the invention can be synthesized in vivo by a cell-based system or can be chemically synthesized. MicroRNAs can be synthesized to include a modification that imparts a desired characteristic. For example, the modification can improve stability, hybridization thermodynamics with a target nucleic acid, targeting to a particular tissue or cell-type, or cell permeability, e.g., by an endocytosis-dependent or -independent mechanism. Modifications can also increase sequence specificity, and consequently decrease off-site targeting. Methods of synthesis and chemical modifications are described in greater detail below.

Given a sense strand sequence (e.g., the sequence of a sense strand of a cDNA molecule), an miRNA can be designed according to the rules of Watson and Crick base pairing. The miRNA can be complementary to a portion of an RNA, e.g., a miRNA, a pre- miRNA, a pre-mRNA or an mRNA. For example, the miRNA can be complementary to the coding region or noncoding region of an mRNA or pre-mRNA, e.g., the region surrounding the translation start site of a pre-mRNA or mRNA, such as the 5' UTR. An miRNA oligonucleotide can be, for example, from about 12 to 30 nucleotides in length, preferably about 15 to 28 nucleotides in length (e.g., 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length).

In particular, an miRNA or a pre-miRNA featured in the invention can have a chemical modification on a nucleotide in an internal (i.e., non-terminal) region having noncomplementarity with the target nucleic acid. For example, a modified nucleotide can be incorporated into the region of a miRNA that forms a bulge. The modification can include a ligand attached to the miRNA, e.g., by a linker (e.g., see diagrams OT-I through OT-IV below). The modification can, for example, improve pharmacokinetics or stability of a therapeutic miRNA, or improve hybridization properties (e.g., hybridization thermodynamics) of the miRNA to a target nucleic acid. In some embodiments, it is preferred that the orientation of a modification or ligand incorporated into or tethered to the bulge region of a miRNA is oriented to occupy the space in the bulge region. For example, the modification can include a modified base or sugar on the nucleic acid strand or a ligand that functions as an intercalator. These are preferably located in the bulge. The intercalator can be an aromatic, e.g., a polycyclic aromatic or heterocyclic aromatic compound. A polycyclic intercalator can have stacking capabilities, and can include systems with 2, 3, or 4 fused rings. The universal bases described below can be incorporated into the miRNAs. In some embodiments, it is preferred that the orientation of a modification or ligand incorporated into or tethered to the bulge region of a miRNA is oriented to occupy the space in the bulge region. This orientation facilitates the improved hybridization properties or an otherwise desired characteristic of the miRNA.

In one embodiment, an miRNA or a pre-miRNA can include an aminoglycoside ligand, which can cause the miRNA to have improved hybridization properties or improved sequence specificity. Exemplary aminoglycosides include glycosylated polylysine; galactosylated polylysine; neomycin B; tobramycin; kanamycin A; and acridine conjugates of aminoglycosides, such as Neo-N-acridine, Neo-S-acridine, Neo-C- acridine, Tobra-N-acridine, and KanaA-N-acridine. Use of an acridine analog can increase sequence specificity. For example, neomycin B has a high affinity for RNA as compared to DNA, but low sequence- specificity. An acridine analog, neo-S-acridine has an increased affinity for the HIV Rev-response element (RRE). In some embodiments the guanidine analog (the guanidinoglycoside) of an aminoglycoside ligand is tethered to an oligonucleotide agent. In a guanidinoglycoside, the amine group on the amino acid is exchanged for a guanidine group. Attachment of a guanidine analog can enhance cell permeability of an oligonucleotide agent.

In one embodiment, the ligand can include a cleaving group that contributes to target gene inhibition by cleavage of the target nucleic acid. Optionally, the cleaving group is tethered to the miRNA in a manner such that it is positioned in the bulge region, where it can access and cleave the target RNA. The cleaving group can be, for example, a bleomycin (e.g., bleomycin- A₅, bleomycin- A₂, or bleomycin-B 2), pyrene, phenanthroline (e.g., O-phenanthroline), a polyamine, a tripeptide (e.g., lys-tyr-lys tripeptide), or metal ion chelating group. The metal ion chelating group can include, e.g., an Lu(III) or EU(III) macrocyclic complex, a Zn(II) 2,9-dimethylphenanthroline derivative, a Cu(II) terpyridine, or acridine, which can promote the selective cleavage of target RNA at the site of the bulge by free metal ions, such as Lu(III). In some embodiments, a peptide ligand can be tethered to a miRNA or a pre-miRNA to promote cleavage of the target RNA, e.g., at the bulge region. For example, l,8-dimethyl-l,3,6,8,10,13-hexaazacyclotetradecane (cyclam) can be conjugated to a peptide (e.g., by an amino acid derivative) to promote target RNA cleavage. The methods and compositions featured in the invention include formulations comprising miRNAs that inhibit target gene expression by a cleavage or non- cleavage dependent mechanism.

An miRNA or a pre-miRNA can be designed and synthesized to include a region of noncomplementarity (e.g., a region that is 3, 4, 5, or 6 nucleotides long) flanked by regions of sufficient complementarity to form a duplex (e.g., regions that are 7, 8, 9, 10, or 11 nucleotides long).

For increased nuclease resistance and/or binding affinity to the target, the miRNA sequences can include 2'-0-methyl, 2'-fluorine, 2'-0-methoxyethyl, 2'-0-aminopropyl, 2'- amino, and/or phosphorothioate linkages. Inclusion of locked nucleic acids (LNA), 2- thiopyrimidines (e.g., 2-thio-U), 2-amino-A, G-clamp modifications, and ethylene nucleic acids (ENA), e.g., 2'-4'-ethylene-bridged nucleic acids, can also increase binding affinity to the target. The inclusion of furanose sugars in the oligonucleotide backbone can also decrease endonucleolytic cleavage. An miRNA or a pre-miRNA can be further modified by including a 3' cationic group, or by inverting the nucleoside at the 3'-terminus with a 3'- 3' linkage. In another alternative, the 3'-terminus can be blocked with an aminoalkyl group, e.g., a 3' C5-aminoalkyl dT. Other 3' conjugates can inhibit 3'-5' exonucleolytic cleavage. While not being bound by theory, a 3' conjugate, such as naproxen or ibuprofen, may inhibit exonucleolytic cleavage by sterically blocking the exonuclease from binding to the 3' end of oligonucleotide. Even small alkyl chains, aryl groups, or heterocyclic conjugates or modified sugars (D-ribose, deoxyribose, glucose etc.) can block 3'-5'- exonucleases.

The 5'-terminus can be blocked with an aminoalkyl group, e.g., a 5'-0-alkylamino substituent. Other 5' conjugates can inhibit 5'-3' exonucleolytic cleavage. While not being bound by theory, a 5' conjugate, such as naproxen or ibuprofen, may inhibit exonucleolytic cleavage by sterically blocking the exonuclease from binding to the 5' end of

oligonucleotide. Even small alkyl chains, aryl groups, or heterocyclic conjugates or modified sugars (D-ribose, deoxyribose, glucose etc.) can block 3'-5'-exonucleases.

In one embodiment, an miRNA or a pre-miRNA includes a modification that improves targeting, e.g. a targeting modification described herein. Examples of modifications that target miRNA molecules to particular cell types include carbohydrate sugars such as galactose, N-acetylgalactosamine, mannose; vitamins such as folates; other ligands such as RGDs and RGD mimics; and small molecules including naproxen, ibuprofen or other known protein-binding molecules.

An miRNA or a pre-miRNA can be constructed using chemical synthesis and/or enzymatic ligation reactions using procedures known in the art. For example, an miRNA or a pre-miRNA can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the miRNA or a pre-miRNA and target nucleic acids, e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used. Other appropriate nucleic acid modifications are described herein. Alternatively, the miRNA or pre-miRNA nucleic acid can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest).

Antisense-Type Oligonucleotide Cargoes

The single- stranded oligonucleotide agents featured in the invention include antisense nucleic acids. An "antisense" nucleic acid includes a nucleotide sequence that is complementary to a "sense" nucleic acid encoding a gene expression product, e.g., complementary to the coding strand of a double-stranded cDNA molecule or

complementary to an RNA sequence, e.g., a pre-mRNA, mRNA, miRNA, or pre-miRNA. Accordingly, an antisense nucleic acid can form hydrogen bonds with a sense nucleic acid target.

Given a coding strand sequence (e.g., the sequence of a sense strand of a cDNA molecule), antisense nucleic acids can be designed according to the rules of Watson and Crick base pairing. The antisense nucleic acid molecule can be complementary to a portion of the coding or noncoding region of an RNA, e.g., a pre-mRNA or mRNA. For example, the antisense oligonucleotide can be complementary to the region surrounding the translation start site of a pre-mRNA or mRNA, e.g., the 5' UTR. An antisense oligonucleotide can be, for example, about 10 to 25 nucleotides in length (e.g., 11, 12, 13, 14, 15, 16, 18, 19, 20, 21, 22, 23, or 24 nucleotides in length). An antisense

oligonucleotide can also be complementary to a miRNA or pre-miRNA.

An antisense nucleic acid can be constructed using chemical synthesis and/or enzymatic ligation reactions using procedures known in the art. For example, an antisense nucleic acid (e.g., an antisense oligonucleotide) can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and target nucleic acids, e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used. Other appropriate nucleic acid modifications are described herein. Alternatively, the antisense nucleic acid can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest).

An antisense agent can include ribonucleotides only, deoxyribonucleotides only (e.g., oligodeoxynucleotides), or both deoxyribonucleotides and ribonucleotides. For example, an antisense agent consisting only of ribonucleotides can hybridize to a complementary RNA, and prevent access of the translation machinery to the target RNA transcript, thereby preventing protein synthesis. An antisense molecule including only deoxyribonucleotides, or deoxyribonucleotides and ribonucleotides, e.g., DNA sequence flanked by RNA sequence at the 5' and 3' ends of the antisense agent, can hybridize to a complementary RNA, and the RNA target can be subsequently cleaved by an enzyme, e.g., RNAse H. Degradation of the target RNA prevents translation. The flanking RNA sequences can include 2'-0-methylated nucleotides, and phosphorothioate linkages, and the internal DNA sequence can include phosphorothioate internucleotide linkages. The internal DNA sequence is preferably at least five nucleotides in length when targeting by RNAseH activity is desired.

For increased nuclease resistance, an antisense agent can be further modified by inverting the nucleoside at the 3'-terminus with a 3'-3' linkage. In another alternative, the 3'-terminus can be blocked with an aminoalkyl group.

In one embodiment, an antisense oligonucleotide agent includes a modification that improves targeting, e.g. a targeting modification described herein.

Double-Stranded Ribonucleic Acid (dsRNA) Cargoes

In one embodiment, the invention provides a double- stranded ribonucleic acid (dsRNA) molecule packaged in an association complex, such as a liposome, for inhibiting the expression of a gene in a cell or mammal, wherein the dsRNA comprises an antisense strand comprising a region of complementarity which is complementary to at least a part of an mRNA formed in the expression of the gene, and wherein the region of

complementarity is less than 30 nucleotides in length, generally 19-24 nucleotides in length, and wherein said dsRNA, upon contact with a cell expressing said gene, inhibits the expression of said gene by at least 40%. The dsRNA comprises two RNA strands that are sufficiently complementary to hybridize to form a duplex structure. One strand of the dsRNA (the antisense strand) comprises a region of complementarity that is substantially complementary, and generally fully complementary, to a target sequence, derived from the sequence of an mRNA formed during the expression of a gene, the other strand (the sense strand) comprises a region which is complementary to the antisense strand, such that the two strands hybridize and form a duplex structure when combined under suitable conditions. Generally, the duplex structure is between 15 and 30, more generally between 18 and 25, yet more generally between 19 and 24, and most generally between 19 and 21 base pairs in length. Similarly, the region of complementarity to the target sequence is between 15 and 30, more generally between 18 and 25, yet more generally between 19 and 24, and most generally between 19 and 21 nucleotides in length. The dsRNA of the invention may further comprise one or more single- stranded nucleotide overhang(s). The dsRNA can be synthesized by standard methods known in the art as further discussed below, e.g., by use of an automated DNA synthesizer, such as are commercially available from, for example, Biosearch, Applied Biosystems, Inc.

The dsRNAs suitable for packaging in the association complexes described herein can include a duplex structure of between 18 and 25 basepairs (e.g., 21 base pairs). In some embodiments, the dsRNAs include at least one strand that is at least 21 nt long. In other embodiments, the dsRNAs include at least one strand that is at least 15, 16, 17, 18, 19, 20, or more contiguous nucleotides.

The dsRNAs suitable for packaging in the association complexes described herein can contain one or more mismatches to the target sequence. In a preferred embodiment, the dsRNA contains no more than 3 mismatches. If the antisense strand of the dsRNA contains mismatches to a target sequence, it is preferable that the area of mismatch not be located in the center of the region of complementarity. If the antisense strand of the dsRNA contains mismatches to the target sequence, it is preferable that the mismatch be restricted to 5 nucleotides from either end, for example 5, 4, 3, 2, or 1 nucleotide from either the 5' or 3' end of the region of complementarity.

In one embodiment, at least one end of the dsRNA has a single-stranded nucleotide overhang of 1 to 4, generally 1 or 2 nucleotides. Generally, the single- stranded overhang is located at the 3'-terminal end of the antisense strand or, alternatively, at the 3'-terminal end of the sense strand. The dsRNA may also have a blunt end, generally located at the 5'-end of the antisense strand. Such dsRNAs have improved stability and inhibitory activity, thus allowing administration at low dosages, i.e., less than 5 mg/kg body weight of the recipient per day. Generally, the antisense strand of the dsRNA has a nucleotide overhang at the 3'-end, and the 5'-end is blunt. In another embodiment, one or more of the nucleotides in the overhang is replaced with a nucleoside thiophosphate.

In yet another embodiment, a dsRNA packaged in an association complex, such as a liposome, is chemically modified to enhance stability. Such nucleic acids may be synthesized and/or modified by methods well established in the art, such as those described in "Current protocols in nucleic acid chemistry", Beaucage, S. L. et al. (Edrs.), John Wiley & Sons, Inc., New York, N.Y., USA, which is hereby incorporated herein by reference. Chemical modifications may include, but are not limited to 2' modifications, modifications at other sites of the sugar or base of an oligonucleotide, introduction of non- natural bases into the oligonucleotide chain, covalent attachment to a ligand or chemical moiety, and replacement of internucleotide phosphate linkages with alternate linkages such as thiophosphates. More than one such modification may be employed.

Chemical linking of the two separate dsRNA strands may be achieved by any of a variety of well-known techniques, for example by introducing covalent, ionic or hydrogen bonds; hydrophobic interactions, van der Waals or stacking interactions; by means of metal-ion coordination, or through use of purine analogues. Such chemically linked dsRNAs are suitable for packaging in the association complexes described herein.

Generally, the chemical groups that can be used to modify the dsRNA include, without limitation, methylene blue; bifunctional groups, generally bis-(2-chloroethyl)amine; N- acetyl-N'-(p-glyoxylbenzoyl)cystamine; 4-thiouracil; and psoralen. In one embodiment, the linker is a hexa-ethylene glycol linker. In this case, the dsRNA are produced by solid phase synthesis and the hexa-ethylene glycol linker is incorporated according to standard methods (e.g., Williams, D. J., and K. B. Hall, Biochem. (1996) 35:14665-14670). In a particular embodiment, the 5'-end of the antisense strand and the 3'-end of the sense strand are chemically linked via a hexaethylene glycol linker. In another embodiment, at least one nucleotide of the dsRNA comprises a phosphorothioate or phosphorodithioate groups. The chemical bond at the ends of the dsRNA is generally formed by triple-helix bonds.

In yet another embodiment, the nucleotides at one or both of the two single strands may be modified to prevent or inhibit the degradation activities of cellular enzymes, such as, for example, without limitation, certain nucleases. Techniques for inhibiting the degradation activity of cellular enzymes against nucleic acids are known in the art including, but not limited to, 2'-amino modifications, 2'-amino sugar modifications, 2'-F sugar modifications, 2'-F modifications, 2'-alkyl sugar modifications, 2'-0-alkoxyalkyl modifications like 2'-0-methoxyethyl, uncharged and charged backbone modifications, morpholino modifications, 2'-0-methyl modifications, and phosphoramidate (see, e.g., Wagner, Nat. Med. (1995) 1:1116-8). Thus, at least one 2'-hydroxyl group of the nucleotides on a dsRNA is replaced by a chemical group, generally by a 2'-F or a 2'-0- methyl group. Also, at least one nucleotide may be modified to form a locked nucleotide. Such locked nucleotide contains a methylene bridge that connects the 2'-oxygen of ribose with the 4'-carbon of ribose. Oligonucleotides containing the locked nucleotide are described in Koshkin, A. A., et al., Tetrahedron (1998), 54: 3607-3630) and Obika, S. et al., Tetrahedron Lett. (1998), 39: 5401-5404). Introduction of a locked nucleotide into an oligonucleotide improves the affinity for complementary sequences and increases the melting temperature by several degrees (Braasch, D. A. and D. R. Corey, Chem. Biol. (2001), 8:1-7).

Conjugating a ligand to a dsRNA can enhance its cellular absorption as well as targeting to a particular tissue or uptake by specific types of cells such as liver cells. In certain instances, a hydrophobic ligand is conjugated to the dsRNA to facilitate direct permeation of the cellular membrane and or uptake across the liver cells. Alternatively, the ligand conjugated to the dsRNA is a substrate for receptor-mediated endocytosis. These approaches have been used to facilitate cell permeation of antisense oligonucleotides as well as dsRNA agents. For example, cholesterol has been conjugated to various antisense oligonucleotides resulting in compounds that are substantially more active compared to their non-conjugated analogs. See M. Manoharan Antisense & Nucleic Acid Drug Development 2002, 12, 103. Other lipophilic compounds that have been conjugated to oligonucleotides include 1-pyrene butyric acid, l,3-bis-0-(hexadecyl)glycerol, and menthol. One example of a ligand for receptor-mediated endocytosis is folic acid. Folic acid enters the cell by folate-receptor- mediated endocytosis. dsRNA compounds bearing folic acid would be efficiently transported into the cell via the folate -receptor-mediated endocytosis. L.sup.l and coworkers report that attachment of folic acid to the 3'-terminus of an oligonucleotide resulted in an 8-fold increase in cellular uptake of the

oligonucleotide. L.sup.l, S.; Deshmukh, H. M.; Huang, L. Pharm. Res. 1998, 15, 1540. Other ligands that have been conjugated to oligonucleotides include polyethylene glycols, carbohydrate clusters, cross-linking agents, porphyrin conjugates, delivery peptides and lipids such as cholesterol. Other chemical modifications for siRNAs have been described in Manoharan, M. RNA interference and chemically modified small interfering RNAs. Current Opinion in Chemical Biology (2004), 8(6), 570-579.

In certain instances, conjugation of a cationic ligand to oligonucleotides results in improved resistance to nucleases. Representative examples of cationic ligands are propylammonium and dimethylpropylammonium. Interestingly, antisense oligonucleotides were reported to retain their high binding affinity to mRNA when the cationic ligand was dispersed throughout the oligonucleotide. See M. Manoharan Antisense & Nucleic Acid Drug Development 2002, 12, 103 and references therein.

The ligand-conjugated dsRNA of the invention may be synthesized by the use of a dsRNA that bears a pendant reactive functionality, such as that derived from the attachment of a linking molecule onto the dsRNA. This reactive oligonucleotide may be reacted directly with commercially-available ligands, ligands that are synthesized bearing any of a variety of protecting groups, or ligands that have a linking moiety attached thereto. The methods of the invention facilitate the synthesis of ligand-conjugated dsRNA by the use of, in some preferred embodiments, nucleoside monomers that have been appropriately conjugated with ligands and that may further be attached to a solid-support material. Such ligand-nucleoside conjugates, optionally attached to a solid-support material, are prepared according to some preferred embodiments of the methods of the invention via reaction of a selected serum-binding ligand with a linking moiety located on the 5' position of a nucleoside or oligonucleotide. In certain instances, a dsRNA bearing an aralkyl ligand attached to the 3'-terminus of the dsRNA is prepared by first covalently attaching a monomer building block to a controlled-pore-glass support via a long-chain aminoalkyl group. Then, nucleotides are bonded via standard solid-phase synthesis techniques to the monomer building-block bound to the solid support. The monomer building block may be a nucleoside or other organic compound that is compatible with solid-phase synthesis.

The dsRNA used in the conjugates of the invention may be conveniently and routinely made through the well-known technique of solid-phase synthesis. Equipment for such synthesis is sold by several vendors including, for example, Applied Biosystems (Foster City, Calif.). Any other means for such synthesis known in the art may additionally or alternatively be employed. It is also known to use similar techniques to prepare other oligonucleotides, such as the phosphorothioates and alkylated derivatives.

Teachings regarding the synthesis of particular modified oligonucleotides may be found in the following U.S. patents: U.S. Pat. Nos. 5,138,045 and 5,218,105, drawn to polyamine conjugated oligonucleotides; U.S. Pat. No. 5,212,295, drawn to monomers for the preparation of oligonucleotides having chiral phosphorus linkages; U.S. Pat. Nos. 5,378,825 and 5,541,307, drawn to oligonucleotides having modified backbones; U.S. Pat. No. 5,386,023, drawn to backbone-modified oligonucleotides and the preparation thereof through reductive coupling; U.S. Pat. No. 5,457,191, drawn to modified nucleobases based on the 3-deazapurine ring system and methods of synthesis thereof; U.S. Pat. No.

5,459,255, drawn to modified nucleobases based on N-2 substituted purines; U.S. Pat. No. 5,521,302, drawn to processes for preparing oligonucleotides having chiral phosphorus linkages; U.S. Pat. No. 5,539,082, drawn to peptide nucleic acids; U.S. Pat. No. 5,554,746, drawn to oligonucleotides having .beta.-lactam backbones; U.S. Pat. No. 5,571,902, drawn to methods and materials for the synthesis of oligonucleotides; U.S. Pat. No. 5,578,718, drawn to nucleosides having alkylthio groups, wherein such groups may be used as linkers to other moieties attached at any of a variety of positions of the nucleoside; U.S. Pat. Nos. 5,587,361 and 5,599,797, drawn to oligonucleotides having phosphorothioate linkages of high chiral purity; U.S. Pat. No. 5,506,351, drawn to processes for the preparation of 2'-0- alkyl guanosine and related compounds, including 2,6-diaminopurine compounds; U.S. Pat. No. 5,587,469, drawn to oligonucleotides having N-2 substituted purines; U.S. Pat. No. 5,587,470, drawn to oligonucleotides having 3-deazapurines; U.S. Pat. No. 5,223,168, and U.S. Pat. No. 5,608,046, both drawn to conjugated 4'-desmethyl nucleoside analogs; U.S. Pat. Nos. 5,602,240, and 5,610,289, drawn to backbone-modified oligonucleotide analogs; U.S. Pat. Nos. 6,262,241, and 5,459,255, drawn to, inter alia, methods of synthesizing 2'-fluoro-oligonucleotides.

In the ligand-conjugated dsRNA and ligand- molecule bearing sequence- specific linked nucleosides of the invention, the oligonucleotides and oligonucleosides may be assembled on a suitable DNA synthesizer utilizing standard nucleotide or nucleoside precursors, or nucleotide or nucleoside conjugate precursors that already bear the linking moiety, ligand-nucleotide or nucleoside-conjugate precursors that already bear the ligand molecule, or non-nucleoside ligand-bearing building blocks.

When using nucleotide-conjugate precursors that already bear a linking moiety, the synthesis of the sequence-specific linked nucleosides is typically completed, and the ligand molecule is then reacted with the linking moiety to form the ligand-conjugated oligonucleotide. Oligonucleotide conjugates bearing a variety of molecules such as steroids, vitamins, lipids and reporter molecules, has previously been described (see Manoharan et al., PCT Application WO 93/07883). In a preferred embodiment, the oligonucleotides or linked nucleosides of the invention are synthesized by an automated synthesizer using phosphoramidites derived from ligand-nucleoside conjugates in addition to the standard phosphoramidites and non-standard phosphoramidites that are

commercially available and routinely used in oligonucleotide synthesis.

The dsRNAs packaged in the association complexes described herein can include one or more modified nucleosides, e.g., a 2'-0-methyl, 2'-0-ethyl, 2'-0-propyl, 2'-0-allyl, 2'-0-aminoalkyl or 2'-deoxy-2'-fluoro group in the nucleosides. Such modifications confer enhanced hybridization properties to the oligonucleotide. Further, oligonucleotides containing phosphorothioate backbones have enhanced nuclease stability. Thus, functionalized, linked nucleosides can be augmented to include either or both a phosphorothioate backbone or a 2'-0-methyl, 2'-0-ethyl, 2'-0-propyl, 2'-0-aminoalkyl, 2'- O-allyl or 2'-deoxy-2'-fluoro group. A summary listing of some of the oligonucleotide modifications known in the art is found at, for example, PCT Publication WO 200370918.

In some embodiments, functionalized nucleoside sequences possessing an amino group at the 5'-terminus are prepared using a DNA synthesizer, and then reacted with an active ester derivative of a selected ligand. Active ester derivatives are well known to those skilled in the art. Representative active esters include N-hydrosuccinimide esters, tetrafluorophenolic esters, pentafluorophenolic esters and pentachlorophenolic esters.

The reaction of the amino group and the active ester produces an oligonucleotide in which the selected ligand is attached to the 5'-position through a linking group. The amino group at the 5'-terminus can be prepared utilizing a 5'-Amino-Modifier C6 reagent. In one embodiment, ligand molecules may be conjugated to oligonucleotides at the 5'-position by the use of a ligand-nucleoside phosphoramidite wherein the ligand is linked to the 5'- hydroxy group directly or indirectly via a linker. Such ligand-nucleoside phosphoramidites are typically used at the end of an automated synthesis procedure to provide a ligand- conjugated oligonucleotide bearing the ligand at the 5'-terminus.

Examples of modified internucleoside linkages or backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3'- amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3'-5' linkages, 2'-5' linked analogs of these, and those having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'.

Various salts, mixed salts and free-acid forms are also included.

Representative United States Patents relating to the preparation of the above phosphorus-atom-containing linkages include, but are not limited to, U.S. Pat. Nos.

3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799; 5,587,361; 5,625,050; and 5,697,248, each of which is herein incorporated by reference.

Examples of modified internucleoside linkages or backbones that do not include a phosphorus atom therein (i.e., oligonucleosides) have backbones that are formed by short chain alkyl or cycloalkyl intersugar linkages, mixed heteroatom and alkyl or cycloalkyl intersugar linkages, or one or more short chain heteroatomic or heterocyclic intersugar linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH 2 component parts.

Representative United States patents relating to the preparation of the above oligonucleosides include, but are not limited to, U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and 5,677,439, each of which is herein incorporated by reference.

In certain instances, an oligonucleotide included in an association complex, such as a liposome, may be modified by a non-ligand group. A number of non-ligand molecules have been conjugated to oligonucleotides in order to enhance the activity, cellular distribution or cellular uptake of the oligonucleotide, and procedures for performing such conjugations are available in the scientific literature. Such non-ligand moieties have included lipid moieties, such as cholesterol (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86:6553), cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4:1053), a thioether, e.g., hexyl-5-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660:306; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3:2765), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20:533), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10:111; Kabanov et al., FEBS Lett., 1990, 259:327; Svinarchuk et al., Biochimie, 1993, 75:49), a phospholipid, e.g., di- hexadecyl-rac-glycerol or triethylammonium l,2-di-0-hexadecyl-rac-glycero-3-H- phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651; Shea et al., Nucl. Acids Res., 1990, 18:3777), a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229), or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923). Representative United States patents that teach the preparation of such oligonucleotide conjugates have been listed above. Typical conjugation protocols involve the synthesis of oligonucleotides bearing an aminolinker at one or more positions of the sequence. The amino group is then reacted with the molecule being conjugated using appropriate coupling or activating reagents. The conjugation reaction may be performed either with the oligonucleotide still bound to the solid support or following cleavage of the oligonucleotide in solution phase. Purification of the oligonucleotide conjugate by HPLC typically affords the pure conjugate.

The modifications described above are appropriate for use with an oligonucleotide agent as described herein.

Exemplary Structures of dsRNA Cargoes

In certain aspects, the present invention provides formulations for RNA

interference (RNAi) that include a DsiRNA cargo.

Double- stranded RNA (dsRNA) agents possessing strand lengths of 25 to 35 nucleotides have been described as effective inhibitors of target gene expression in mammalian cells (Rossi et al, U.S. Patent Publication Nos. 2005/0244858 and

2005/0277610). dsRNA agents of such length are believed to be processed by the Dicer enzyme of the RNA interference (RNAi) pathway, leading such agents to be termed "Dicer substrate siRNA" ("DsiRNA") agents. Certain modified structures of DsiRNA agents were previously described (Rossi et al., U.S. Patent Publication No.

2007/0265220). Additional DsiRNA structures and specific compositions suitable for use in the formulations of the instant invention are described in U.S. patent application Ser. Nos. 12/642,371; 12/586,283; 61/257,810; 61/257,820; 12/586,281; 61/183,815;

61/183,818; 61/183,815; 61/184,735; 61/285,925; and 61/151,841. In one embodiment, a DsiRNA cargo of a formulation of the invention comprises:

5 ' -pXXXXXXXXXXXXXXXXXXXXXXXDD- 3 '

3 ' - YXXXXXXXXXXXXXXXXXXXXXXXXXp-5'

wherein "X"=RNA, "p"=a phosphate group, "Y" is an overhang domain comprised of 1-4 RNA monomers that are optionally 2'-0-methyl RNA monomers, and "D"=DNA. In one embodiment, the top strand is the sense strand, and the bottom strand is the antisense strand. Alternatively, the bottom strand is the sense strand and the top strand is the antisense strand.

In an additional embodiment, the DsiRNA comprises:

5 ' -pXXXXXXXXXXXXXXXXXXXXXXXDD- 3 '

3 ' -YXXXXXXXXXXXXXXXXXXXXXXXXXp-5'

wherein "X"=RNA, "p"=a phosphate group, "X"=2'-0-methyl RNA, "Y" is an overhang domain comprised of 1-4 RNA monomers that are optionally 2'-0-methyl RNA monomers, underlined residues are 2'-0-methyl RNA monomers, and "D"=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand.

In another embodiment, the DsiRNA comprises:

5 ' -pXXXXXXXXXXXXXXXXXXXXXXXDD- 3 '

3 ' - YXXXXXXXXXXXXXXXXXXXXXXXXXp-5'

wherein "X"=RNA, "p"=a phosphate group, "X"=2'-0-methyl RNA, "Y" is an overhang domain comprised of 1-4 RNA monomers that are optionally 2'-0-methyl RNA monomers, underlined residues are 2'-0-methyl RNA monomers, and "D"=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand.

In another embodiment, the DsiRNA comprises:

5 ' -pXXXXXXXXXXXXXXXXXXXXXXXDD- 3 '

3 ' - YXXXXXXXXXXXXXXXXXXXXXXXXXp-5'

wherein "X"=RNA, "p"=a phosphate group, "X"=2'-0-methyl RNA, "Y" is an overhang domain comprised of 1-4 RNA monomers that are optionally 2'-0-methyl RNA monomers, underlined residues are 2'-0-methyl RNA monomers, and "D"=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand.

In further embodiments, the DsiRNA comprises:

5 ' -pXXXXXXXXXXXXXXXXXXXXXXXDD- 3 '

3 ' - YXXXXXXXXXXXXXXXXXXXXXXXXXp-5' wherein "X"=RNA, "p"=a phosphate group, "X"=2'-0-methyl RNA, "Y" is an overhang domain comprised of 1-4 RNA monomers that are optionally 2'-0-methyl RNA monomers, underlined residues are 2' -O-methyl RNA monomers, and "D"=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand. In one

embodiment, the DsiRNA comprises:

5 ' -pXXXXXXXXXXXXXXXXXXXXXXXDD- 3 '

3 ' -XXXXXXXXXXXXXXXXXXXXXXXXXXXp-5'

wherein "X"=RNA, "p"=a phosphate group, "X"=2'-0-methyl RNA, and "D"=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand.

In additional embodiments, the DsiRNA comprises:

5 ' -pXXXXXXXXXXXXXXXXXXXXXXXDD- 3 '

3 ' -YXXXXXXXXXXXXXXXXXXXXXXXXXp-5'

wherein "X"=RNA, "p"=a phosphate group, "X"=2'-0-methyl RNA, "Y" is an overhang domain comprised of 1-4 RNA monomers that are optionally 2'-0-methyl RNA monomers, underlined residues are 2' -O-methyl RNA monomers, and "D"=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand. In one

embodiment, the DsiRNA comprises:

5 ' -pXXXXXXXXXXXXXXXXXXXXXXXDD- 3 '

3 ' -XXXXXXXXXXXXXXXXXXXXXXXXXXXp-5'

wherein "X"=RNA, "p"=a phosphate group, "X"=2'-0-methyl RNA, and "D"=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand.

In other embodiments, the DsiRNA comprises:

5 ' -pXXXXXXXXXXXXXXXXXXXXXXXDD- 3 '

3 ' -YXXXXXXXXXXXXXXXXXXXXXXXXXp-5'

wherein "X"=RNA, "p"=a phosphate group, "X"=2'-0-methyl RNA, "Y" is an overhang domain comprised of 1-4 RNA monomers that are optionally 2'-0-methyl RNA monomers, underlined residues are 2' -O-methyl RNA monomers, and "D"=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand. In one

embodiment, the DsiRNA comprises:

5 ' -pXXXXXXXXXXXXXXXXXXXXXXXDD- 3 '

3 ' -XXXXXXXXXXXXXXXXXXXXXXXXXXXp-5'

wherein "X"=RNA, "p"=a phosphate group, "X"=2'-0-methyl RNA, and "D"=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand. In further embodiments, the DsiRNA comprises:

5 ' -pXXXXXXXXXXXXXXXXXXXXXXXDD- 3 '

3 ' -YXXXXXXXXXXXXXXXXXXXXXXXXXp-5'

wherein "X"=RNA, "p"=a phosphate group, "X"=2'-0-methyl RNA, "Y" is an overhang domain comprised of 1-4 RNA monomers that are optionally 2'-0-methyl RNA monomers, underlined residues are 2' -O-methyl RNA monomers, and "D"=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand. In one

embodiment, the DsiRNA comprises:

5 ' -pXXXXXXXXXXXXXXXXXXXXXXXDD- 3 '

3 ' -XXXXXXXXXXXXXXXXXXXXXXXXXXXp-5'

wherein "X"=RNA, "p"=a phosphate group, "X"=2'-0-methyl RNA, and "D"=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand.

In additional embodiments, the DsiRNA comprises:

5 ' -pXXXXXXXXXXXXXXXXXXXXXXXDD- 3 '

3 ' - YXXXXXXXXXXXXXXXXXXXXXXXXXp-5'

wherein "X"=RNA, "p"=a phosphate group, "X"=2'-0-methyl RNA, "Y" is an overhang domain comprised of 1-4 RNA monomers that are optionally 2'-0-methyl RNA monomers, underlined residues are 2' -O-methyl RNA monomers, and "D"=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand. In one

embodiment, the DsiRNA comprises:

5 ' -pXXXXXXXXXXXXXXXXXXXXXXXDD- 3 '

3 ' -XXXXXXXXXXXXXXXXXXXXXXXXXXXp-5'

wherein "X"=RNA, "p"=a phosphate group, "X"=2'-0-methyl RNA, and "D"=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand.

In other embodiments, the DsiRNA comprises:

5 ' -pXXXXXXXXXXXXXXXXXXXXXXXDD- 3 '

3 ' - YXXXXXXXXXXXXXXXXXXXXXXXXXp-5'

wherein "X"=RNA, "p"=a phosphate group, "X"=2'-0-methyl RNA, "Y" is an overhang domain comprised of 1-4 RNA monomers that are optionally 2'-0-methyl RNA monomers, underlined residues are 2' -O-methyl RNA monomers, and "D"=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand. In one

embodiment, the DsiRNA comprises:

5 ' -pXXXXXXXXXXXXXXXXXXXXXXXDD- 3 ' 3 ' -XXXXXXXXXXXXXXXXXXXXXXXXXXXp-5'

wherein "X"=RNA, "p"=a phosphate group, "X"=2'-0-methyl RNA, and "D"=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand.

In certain additional embodiments, the DsiRNA comprises:

5 ' -pXXXXXXXXXXXXXXXXXXXXXXXDD- 3 '

3 ' -YXXXXXXXXXXXXXXXXXXXXXXXXXp-5'

wherein "X"=RNA, "p"=a phosphate group, "X"=2'-0-methyl RNA, "Y" is an overhang domain comprised of 1-4 RNA monomers that are optionally 2'-0-methyl RNA monomers, underlined residues are 2' -O-methyl RNA monomers, and "D"=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand. In one

embodiment, the DsiRNA comprises:

5 ' -pXXXXXXXXXXXXXXXXXXXXXXXDD- 3 '

3 ' -XXXXXXXXXXXXXXXXXXXXXXXXXXXp-5'

wherein "X"=RNA, "p"=a phosphate group, "X"=2'-0-methyl RNA, and "D"=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand.

In additional embodiments, the DsiRNA comprises:

5 ' -pXXXXXXXXXXXXXXXXXXXXXXXDD- 3 '

3 ' - YXXXXXXXXXXXXXXXXXXXXXXXXXp-5'

wherein "X"=RNA, "p"=a phosphate group, "X"=2'-0-methyl RNA, "Y" is an overhang domain comprised of 1-4 RNA monomers that are optionally 2'-0-methyl RNA monomers, underlined residues are 2' -O-methyl RNA monomers, and "D"=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand. In one

embodiment, the DsiRNA comprises:

5 ' -pXXXXXXXXXXXXXXXXXXXXXXXDD- 3 '

3 ' -XXXXXXXXXXXXXXXXXXXXXXXXXXXp-5'

wherein "X"=RNA, "p"=a phosphate group, "X"=2'-0-methyl RNA, and "D"=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand.

In further embodiments, the DsiRNA comprises:

5 ' -pXXXXXXXXXXXXXXXXXXXXXXXDD- 3 '

3 ' - YXXXXXXXXXXXXXXXXXXXXXXXXXp-5'

wherein "X"=RNA, "p"=a phosphate group, "X"=2'-0-methyl RNA, "Y" is an overhang domain comprised of 1-4 RNA monomers that are optionally 2'-0-methyl RNA monomers, underlined residues are 2'-0-methyl RNA monomers, and "D"=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand. In one

embodiment, the DsiRNA comprises:

5 ' -pXXXXXXXXXXXXXXXXXXXXXXXDD- 3 '

3 ' -XXXXXXXXXXXXXXXXXXXXXXXXXXXp-5'

wherein "X"=RNA, "p"=a phosphate group, "X"=2'-0-methyl RNA, and "D"=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand.

In another embodiment, the DsiRNA comprises strands having equal lengths possessing 1-3 mismatched residues that serve to orient Dicer cleavage (specifically, one or more of positions 1, 2 or 3 on the first strand of the DsiRNA, when numbering from the 3 '-terminal residue, are mismatched with corresponding residues of the 5 '-terminal region on the second strand when first and second strands are annealed to one another). An exemplary 27mer DsiRNA agent with two terminal mismatched residues is shown:

5 ' -pXXXXXXXXXXXXXXXXXXXXXXXXX^{1 1}

3 ' -xxxxxxxxxxxxxxxxxxxxxxxxx_M

^MMp-5

wherein "X"=RNA, "p"=a phosphate group, "M"=Nucleic acid residues (RNA, DNA or non-natural or modified nucleic acids) that do not base pair (hydrogen bond) with corresponding "M" residues of otherwise complementary strand when strands are annealed. Any of the residues of such agents can optionally be 2'-0-methyl RNA monomers - alternating positioning of 2'-0-methyl RNA monomers that commences from the 3 '-terminal residue of the bottom (second) strand, as shown for above asymmetric agents, can also be used in the above "blunt/fray" DsiRNA agent. In one embodiment, the top strand is the sense strand, and the bottom strand is the antisense strand. Alternatively, the bottom strand is the sense strand and the top strand is the antisense strand.

In certain embodiments, the present invention provides formulations for RNA interference (RNAi) where a dsNA cargo possesses one or more base paired

deoxyribonucleotides within a region of a double stranded nucleic acid (dsNA) that is positioned 3' of a projected sense strand Dicer cleavage site and correspondingly 5' of a projected antisense strand Dicer cleavage site. Such formulations of the invention comprise a dsNA which is a precursor molecule, i.e., the dsNA of a formulation of the present invention is processed in vivo to produce an active small interfering nucleic acid (siRNA). The dsNA is processed by Dicer to an active siRNA which is incorporated into RISC.

In certain embodiments, DsiRNA agents of the formulations of the invention can have any of the following exemplary structures:

In one embodiment, the DsiRNA comprises:

5 ' -XXXXXXXXXXXXXXXXXXXXXXXX_N*D_NDD-3 '

3 ' - YXXXXXXXXXXXXXXXXXXXXXXXX_N*D_NXX-5'

wherein "X"=RNA, "Y" is an optional overhang domain comprised of 0-10 RNA monomers that are optionally 2'-0-methyl RNA monomers - in certain embodiments, "Y" is an overhang domain comprised of 1-4 RNA monomers that are optionally 2'-0-methyl RNA monomers, "D"=DNA, and "N"=l to 50 or more, but is optionally 1-15 or, optionally, 1-8. "N*"=0 to 15 or more, but is optionally 0, 1, 2, 3, 4, 5 or 6. In one embodiment, the top strand is the sense strand, and the bottom strand is the antisense strand. Alternatively, the bottom strand is the sense strand and the top strand is the antisense strand.

In a related embodiment, the DsiRNA comprises:

5 ' -XXXXXXXXXXXXXXXXXXXXXXXX_N*D_NDD-3 '

3 ' - YXXXXXXXXXXXXXXXXXXXXXXXX_N*D_NDD-5'

wherein "X"=RNA, "Y" is an optional overhang domain comprised of 0-10 RNA monomers that are optionally 2'-0-methyl RNA monomers - in certain embodiments, "Y" is an overhang domain comprised of 1-4 RNA monomers that are optionally 2'-0-methyl RNA monomers, "D"=DNA, and "N"=l to 50 or more, but is optionally 1-15 or, optionally, 1-8. "N*"=0 to 15 or more, but is optionally 0, 1, 2, 3, 4, 5 or 6. In one embodiment, the top strand is the sense strand, and the bottom strand is the antisense strand. Alternatively, the bottom strand is the sense strand and the top strand is the antisense strand.

In an additional embodiment, the DsiRNA comprises:

5 ' -XXXXXXXXXXXXXXXXXXXXXXXX_N*D_NDD-3 '

3 ' -YXXXXXXXXXXXXXXXXXXXXXXXX_N*D_NZ Z-5'

wherein "X"=RNA, "X"=2'-0-methyl RNA, "Y" is an optional overhang domain comprised of 0-10 RNA monomers that are optionally 2'-0-methyl RNA monomers - in certain embodiments, "Y" is an overhang domain comprised of 1-4 RNA monomers that are optionally 2'-0-methyl RNA monomers, "D"=DNA, "Z"=DNA or RNA, and "N"=l to 50 or more, but is optionally 1-15 or, optionally, 1-8. "N*"=0 to 15 or more, but is optionally 0, 1, 2, 3, 4, 5 or 6. In one embodiment, the top strand is the sense strand, and the bottom strand is the antisense strand. Alternatively, the bottom strand is the sense strand and the top strand is the antisense strand, with 2' -O-methyl RNA monomers located at alternating residues along the top strand, rather than the bottom strand presently depicted in the above schematic.

In another such embodiment, the DsiRNA comprises:

5 ' -XXXXXXXXXXXXXXXXXXXXXXXX_N*D_NDD-3 '

3 ' -YXXXXXXXXXXXXXXXXXXXXXXXX_N*D_NZ Z-5'

wherein "X"=RNA, "X"=2'-0-methyl RNA, "Y" is an optional overhang domain comprised of 0-10 RNA monomers that are optionally 2'-0-methyl RNA monomers - in certain embodiments, "Y" is an overhang domain comprised of 1-4 RNA monomers that are optionally 2'-0-methyl RNA monomers, "D"=DNA, "Z"=DNA or RNA, and "N"=l to 50 or more, but is optionally 1-15 or, optionally, 1-8. "N*"=0 to 15 or more, but is optionally 0, 1, 2, 3, 4, 5 or 6. In one embodiment, the top strand is the sense strand, and the bottom strand is the antisense strand. Alternatively, the bottom strand is the sense strand and the top strand is the antisense strand, with 2' -O-methyl RNA monomers located at alternating residues along the top strand, rather than the bottom strand presently depicted in the above schematic.

In another such embodiment, the DsiRNA comprises:

5 ' -XXXXXXXXXXXXXXXXXXXXXXXX_N*D_NDD-3 '

3 ' -YXXXXXXXXXXXXXXXXXXXXXXXX_N*D_NZ Z-5'

wherein "X"=RNA, "X"=2'-0-methyl RNA, "Y" is an optional overhang domain comprised of 0-10 RNA monomers that are optionally 2'-0-methyl RNA monomers - in certain embodiments, "Y" is an overhang domain comprised of 1-4 RNA monomers that are optionally 2'-0-methyl RNA monomers, "D"=DNA, "Z"=DNA or RNA, and "N"=l to 50 or more, but is optionally 1-15 or, optionally, 1-8. "N*"=0 to 15 or more, but is optionally 0, 1, 2, 3, 4, 5 or 6. In one embodiment, the top strand is the sense strand, and the bottom strand is the antisense strand. Alternatively, the bottom strand is the sense strand and the top strand is the antisense strand, with 2' -O-methyl RNA monomers located at alternating residues along the top strand, rather than the bottom strand presently depicted in the above schematic.

In another embodiment, the DsiRNA comprises: 5 ' -XXXXXXXXXXXXXXXXXXXXXXXXN_* [Xl/Dl] _NDD-3 '

3' - YXXXXXXXXXXXXXXXXXXXXXXXXN* [ X2/D2]_nZ Z-5'

wherein "X"=RNA, "Y" is an optional overhang domain comprised of 0- 10 RNA monomers that are optionally 2'-0-methyl RNA monomers - in certain embodiments, "Y" is an overhang domain comprised of 1-4 RNA monomers that are optionally 2'-0-methyl RNA monomers, "D"=DNA, "Z"=DNA or RNA, and "N"=l to 50 or more, but is optionally 1- 15 or, optionally, 1-8, where at least one D1 is present in the top strand and is base paired with a corresponding D2 in the bottom strand. Optionally, D1 and D1N+I are base paired with corresponding D2_N and D2_N+i; D1_N, Dl_N+i and Dl_N+2 are base paired with corresponding D2_N, Dl_N+i and Dl_N+2, etc. "N*"=0 to 15 or more, but is optionally 0, 1 , 2, 3, 4, 5 or 6. In one embodiment, the top strand is the sense strand, and the bottom strand is the antisense strand. Alternatively, the bottom strand is the sense strand and the top strand is the antisense strand, with 2' -O-methyl RNA monomers located at alternating residues along the top strand, rather than the bottom strand presently depicted in the above schematic.

In any of the above-depicted structures, the 5' end of either the sense strand or antisense strand optionally comprises a phosphate group.

In another embodiment, a DN A :DNA-ex tended DsiRNA comprises strands having equal lengths possessing 1-3 mismatched residues that serve to orient Dicer cleavage (specifically, one or more of positions 1 , 2 or 3 on the first strand of the DsiRNA, when numbering from the 3 '-terminal residue, are mismatched with corresponding residues of the 5 '-terminal region on the second strand when first and second strands are annealed to one another). An exemplary DNA:DNA-extended DsiRNA agent with two terminal mismatched residues is shown:

5 ' -XXXXXXXXXXXXXXXXXXXXXXXXXX_N*D_N ^M

3 ' -XXXXXXXXXXXXXXXXXXXXXXXXXX_N*D_NM

uM-5'

wherein "X"=RNA, "M"=Nucleic acid residues (RNA, DNA or non-natural or modified nucleic acids) that do not base pair (hydrogen bond) with corresponding "M" residues of otherwise complementary strand when strands are annealed, "D"=DNA and "N"=l to 50 or more, but is optionally 1-15 or, optionally, 1-8. "N*"=0 to 15 or more, but is optionally 0, 1 , 2, 3, 4, 5 or 6. Any of the residues of such agents can optionally be 2'-0-methyl RNA monomers - alternating positioning of 2'-0-methyl RNA monomers that commences from the 3 '-terminal residue of the bottom (second) strand, as shown for above asymmetric agents, can also be used in the above "blunt/fray" DsiRNA agent. In one embodiment, the top strand (first strand) is the sense strand, and the bottom strand (second strand) is the antisense strand. Alternatively, the bottom strand is the sense strand and the top strand is the antisense strand. Modification and DNA:DNA extension patterns paralleling those shown above for asymmetric/overhang agents can also be incorporated into such "blunt/frayed" agents.

In one embodiment, a length-extended DsiRNA agent is provided that comprises deoxyribonucleotides positioned at sites modeled to function via specific direction of Dicer cleavage, yet which does not require the presence of a base-paired

deoxyribonucleotide in the dsNA structure. An exemplary structure for such a molecule is shown:

5 ' -XXXXXXXXXXXXXXXXXXXDDXX-3 '

3 ' -YXXXXXXXXXXXXXXXXXDDXXXX-5 '

wherein "X"=RNA, "Y" is an optional overhang domain comprised of 0-10 RNA monomers that are optionally 2'-0-methyl RNA monomers - in certain embodiments, "Y" is an overhang domain comprised of 1-4 RNA monomers that are optionally 2'-0-methyl RNA monomers, and "D"=DNA. In one embodiment, the top strand is the sense strand, and the bottom strand is the antisense strand. Alternatively, the bottom strand is the sense strand and the top strand is the antisense strand. The above structure is modeled to force Dicer to cleave a minimum of a 21mer duplex as its primary post-processing form. In embodiments where the bottom strand of the above structure is the antisense strand, the positioning of two deoxyribonucleotide residues at the ultimate and penultimate residues of the 5' end of the antisense strand is likely to reduce off-target effects (as prior studies have shown a 2'-0-methyl modification of at least the penultimate position from the 5' terminus of the antisense strand to reduce off-target effects; see, e.g., US 2007/0223427).

In one embodiment, the DsiRNA comprises:

5 ' -D_NXXXXXXXXXXXXXXXXXXXXXXXX_N*Y-3 '

3 ' -D_NXXXXXXXXXXXXXXXXXXXXXXXX_N*-5'

wherein "X"=RNA, "Y" is an optional overhang domain comprised of 0-10 RNA monomers that are optionally 2'-0-methyl RNA monomers - in certain embodiments, "Y" is an overhang domain comprised of 1-4 RNA monomers that are optionally 2'-0-methyl RNA monomers, "D"=DNA, and "N"=l to 50 or more, but is optionally 1-15 or, optionally, 1-8. "N*"=0 to 15 or more, but is optionally 0, 1, 2, 3, 4, 5 or 6. In one embodiment, the top strand is the sense strand, and the bottom strand is the antisense strand. Alternatively, the bottom strand is the sense strand and the top strand is the antisense strand.

In a related embodiment, the DsiRNA comprises:

5 ' -D_NXXXXXXXXXXXXXXXXXXXXXXXX_N*DD-3 '

3 ' -D_NXXXXXXXXXXXXXXXXXXXXXXXX_N*XX-5'

wherein "X"=RNA, optionally a 2'-0-methyl RNA monomers "D"=DNA, "N"=l to 50 or more, but is optionally 1-15 or, optionally, 1-8. "N*"=0 to 15 or more, but is optionally 0, 1, 2, 3, 4, 5 or 6. In one embodiment, the top strand is the sense strand, and the bottom strand is the antisense strand. Alternatively, the bottom strand is the sense strand and the top strand is the antisense strand.

In an additional embodiment, the DsiRNA comprises:

5 ' -D_NXXXXXXXXXXXXXXXXXXXXXXXX_N*DD-3 '

3 ' -D_NXXXXXXXXXXXXXXXXXXXXXXXX_N* Z Z-5'

wherein "X"=RNA, optionally a 2'-0-methyl RNA monomers "D"=DNA, "N"=l to 50 or more, but is optionally 1-15 or, optionally, 1-8. "N*"=0 to 15 or more, but is optionally 0, 1, 2, 3, 4, 5 or 6. "Z"=DNA or RNA. In one embodiment, the top strand is the sense strand, and the bottom strand is the antisense strand. Alternatively, the bottom strand is the sense strand and the top strand is the antisense strand, with 2'-0-methyl RNA monomers located at alternating residues along the top strand, rather than the bottom strand presently depicted in the above schematic.

In another such embodiment, the DsiRNA comprises:

5 ' -D_NXXXXXXXXXXXXXXXXXXXXXXXX_N*DD-3 '

3 ' -D_NXXXXXXXXXXXXXXXXXXXXXXXX_N* Z Z-5'

wherein "X"=RNA, optionally a 2'-0-methyl RNA monomers "D"=DNA, "N"=l to 50 or more, but is optionally 1-15 or, optionally, 1-8. "N*"=0 to 15 or more, but is optionally 0, 1, 2, 3, 4, 5 or 6. "Z"=DNA or RNA. In one embodiment, the top strand is the sense strand, and the bottom strand is the antisense strand. Alternatively, the bottom strand is the sense strand and the top strand is the antisense strand, with 2'-0-methyl RNA monomers located at alternating residues along the top strand, rather than the bottom strand presently depicted in the above schematic.

In another such embodiment, the DsiRNA comprises: 5 ' -D_NZ ZXXXXXXXXXXXXXXXXXXXXXXXX_N*DD-3 '

3 ' -D_NXXXXXXXXXXXXXXXXXXXXXXXXXX_N* Z Z-5'

wherein "X"=RNA, "X"=2'-0-methyl RNA, "D"=DNA, "Z"=DNA or RNA, and "N"=l to 50 or more, but is optionally 1-15 or, optionally, 1-8. "N*"=0 to 15 or more, but is optionally 0, 1, 2, 3, 4, 5 or 6. In one embodiment, the top strand is the sense strand, and the bottom strand is the antisense strand. Alternatively, the bottom strand is the sense strand and the top strand is the antisense strand, with 2' -O-methyl RNA monomers located at alternating residues along the top strand, rather than the bottom strand presently depicted in the above schematic.

In another such embodiment, the DsiRNA comprises:

5 ' -D_NZ ZXXXXXXXXXXXXXXXXXXXXXXXX_N*Y-3 '

3 ' -D_NXXXXXXXXXXXXXXXXXXXXXXXXXX_N*-5'

wherein "X"=RNA, "X"=2'-0-methyl RNA, "D"=DNA, "Z"=DNA or RNA, and "N"=l to 50 or more, but is optionally 1-15 or, optionally, 1-8. "N*"=0 to 15 or more, but is optionally 0, 1, 2, 3, 4, 5 or 6. "Y" is an optional overhang domain comprised of 0-10 RNA monomers that are optionally 2'-0-methyl RNA monomers - in certain

embodiments, "Y" is an overhang domain comprised of 1-4 RNA monomers that are optionally 2'-0-methyl RNA monomers. In one embodiment, the top strand is the sense strand, and the bottom strand is the antisense strand. Alternatively, the bottom strand is the sense strand and the top strand is the antisense strand, with 2' -O-methyl RNA monomers located at alternating residues along the top strand, rather than the bottom strand presently depicted in the above schematic.

In another embodiment, the DsiRNA comprises:

5 ' - [ X l / D l ] _NXXXXXXXXXXXXXXXXXXXXXXXX_N*DD-3 '

3' - [X2/D2] _NXXXXXXXXXXXXXXXXXXXXXXXX_N* Z Z-5'

wherein "X"=RNA, "D"=DNA, "Z"=DNA or RNA, and "N"=l to 50 or more, but is optionally 1-15 or, optionally, 1-8, where at least one D1_N is present in the top strand and is base paired with a corresponding D2 in the bottom strand. Optionally, D1 and D1_N+I are base paired with corresponding D2 and D2_N+I ; D1_N, D1_N+I and D1_N+2 are base paired with corresponding D2_N, Dl_N+i and Dl_N+2, etc. "N*"=0 to 15 or more, but is optionally 0, 1, 2, 3, 4, 5 or 6. In one embodiment, the top strand is the sense strand, and the bottom strand is the antisense strand. Alternatively, the bottom strand is the sense strand and the top strand is the antisense strand, with 2' -O-methyl RNA monomers located at alternating residues along the top strand, rather than the bottom strand presently depicted in the above schematic.

In a related embodiment, the DsiRNA comprises:

5 ' - [ X l / D l ] _NXXXXXXXXXXXXXXXXXXXXXXXX_N*Y- 3 '

3 ' - [ X2 /D2 ] _NXXXXXXXXXXXXXXXXXXXXXXXX_N*-5'

wherein "X"=RNA, "D"=DNA, "Y" is an optional overhang domain comprised of 0-10 RNA monomers that are optionally 2'-0-methyl RNA monomers - in certain

embodiments, "Y" is an overhang domain comprised of 1-4 RNA monomers that are optionally 2'-0-methyl RNA monomers, and "N"=l to 50 or more, but is optionally 1-15 or, optionally, 1-8, where at least one D1_N is present in the top strand and is base paired with a corresponding D2 in the bottom strand. Optionally, D1 and D1_N+I are base paired with corresponding D2 and D2_N+I ; D1_N, D1_N+I and D1_N+2 are base paired with corresponding D2_N, Dl_N+i and Dl_N+2, etc. "N*"=0 to 15 or more, but is optionally 0, 1, 2, 3, 4, 5 or 6. In one embodiment, the top strand is the sense strand, and the bottom strand is the antisense strand. Alternatively, the bottom strand is the sense strand and the top strand is the antisense strand, with 2'-0-methyl RNA monomers located at alternating residues along the top strand, rather than the bottom strand presently depicted in the above schematic.

In any of the above-depicted structures, the 5' end of either the sense strand or antisense strand optionally comprises a phosphate group.

In another embodiment, a DNA:DNA-extended DsiRNA comprises strands having equal lengths possessing 1-3 mismatched residues that serve to orient Dicer cleavage (specifically, one or more of positions 1, 2 or 3 on the first strand of the DsiRNA, when numbering from the 3 '-terminal residue, are mismatched with corresponding residues of the 5 '-terminal region on the second strand when first and second strands are annealed to one another). An exemplary DNA:DNA-extended DsiRNA agent with two terminal mismatched residues is shown:

5 ' -D_NXXXXXXXXXXXXXXXXXXXXXXXXXX_N* ^M

3 ' -D_NXXXXXXXXXXXXXXXXXXXXXXXXXX_N*M

uM- 5 '

wherein "X"=RNA, "M"=Nucleic acid residues (RNA, DNA or non-natural or modified nucleic acids) that do not base pair (hydrogen bond) with corresponding "M" residues of otherwise complementary strand when strands are annealed, "D"=DNA and "N"=l to 50 or more, but is optionally 1-15 or, optionally, 1-8. "N*"=0 to 15 or more, but is optionally 0, 1, 2, 3, 4, 5 or 6. Any of the residues of such agents can optionally be 2'-0-methyl RNA monomers - alternating positioning of 2'-0-methyl RNA monomers that commences from the 3 '-terminal residue of the bottom (second) strand, as shown for above

asymmetric agents, can also be used in the above "blunt/fray" DsiRNA agent. In one embodiment, the top strand (first strand) is the sense strand, and the bottom strand (second strand) is the antisense strand. Alternatively, the bottom strand is the sense strand and the top strand is the antisense strand. Modification and DNA:DNA extension patterns paralleling those shown above for asymmetric/overhang agents can also be incorporated into such "blunt/frayed" agents.

In another embodiment, a length-extended DsiRNA agent is provided that comprises deoxyribonucleotides positioned at sites modeled to function via specific direction of Dicer cleavage, yet which does not require the presence of a base-paired deoxyribonucleotide in the dsNA structure. Exemplary structures for such a molecule are shown:

5 ' -XXDDXXXXXXXXXXXXXXXXXXXX_N*Y-3 '

3 ' -DDXXXXXXXXXXXXXXXXXXXXXX_N*-5 '

or

5 ' -XDXDXXXXXXXXXXXXXXXXXXXX_N*Y-3 '

3 ' -DXDXXXXXXXXXXXXXXXXXXXXX_N*-5 '

wherein "X"=RNA, "Y" is an optional overhang domain comprised of 0-10 RNA monomers that are optionally 2'-0-methyl RNA monomers - in certain embodiments, "Y" is an overhang domain comprised of 1-4 RNA monomers that are optionally 2'-0-methyl RNA monomers, and "D"=DNA. "N*"=0 to 15 or more, but is optionally 0, 1, 2, 3, 4, 5 or 6. In one embodiment, the top strand is the sense strand, and the bottom strand is the antisense strand. Alternatively, the bottom strand is the sense strand and the top strand is the antisense strand. The above structures are modeled to force Dicer to cleave a minimum of a 21mer duplex as its primary post-processing form.

In any of the above embodiments where the bottom strand of the above structure is the antisense strand, the positioning of two deoxyribonucleotide residues at the ultimate and penultimate residues of the 5' end of the antisense strand is likely to reduce off-target effects (as prior studies have shown a 2'-0-methyl modification of at least the penultimate position from the 5' terminus of the antisense strand to reduce off-target effects; see, e.g., US 2007/0223427). DsiRNAs of the formulations of the invention can carry a broad range of modification patterns (e.g., 2'-0-methyl RNA patterns, e.g., within extended DsiRNA agents). Certain modification patterns of the second strand of DsiRNAs of the

formulations of the invention are presented below.

DsiRNA Cargo Design/Synthesis

It was previously shown that longer dsRNA species of from 25 to about 30 nucleotides (DsiRNAs) yield unexpectedly effective RNA inhibitory results in terms of potency and duration of action, as compared to 19-23mer siRNA agents. Without wishing to be bound by the underlying theory of the dsRNA processing mechanism, it is thought that the longer dsRNA species serve as a substrate for the Dicer enzyme in the cytoplasm of a cell. In addition to cleaving dsNA such as the DsiRNA cargoes of the invention into shorter segments, Dicer is thought to facilitate the incorporation of a single- stranded cleavage product derived from the cleaved dsNA into the RISC complex that is responsible for the destruction of the cytoplasmic RNA of or derived from the target gene. Prior studies (Rossi et al., U.S. Patent Application No. 2007/0265220) have shown that the cleavability of a dsRNA species (specifically, a DsiRNA agent) by Dicer corresponds with increased potency and duration of action of the dsRNA species. The instant invention, at least in part, provides for design of RNA inhibitory agents that direct the site of Dicer cleavage, such that certain species of Dicer cleavage products are thereby generated.

In DsiRNA processing, Dicer enzyme binds to a DsiRNA agent, resulting in cleavage of the DsiRNA at a position 19-23 nucleotides removed from a Dicer PAZ domain-associated 3' overhang sequence of the antisense strand of the DsiRNA agent. This Dicer cleavage event results in excision of those duplexed nucleic acids previously located at the 3' end of the passenger (sense) strand and 5' end of the guide (antisense) strand. (Cleavage of a DsiRNA typically yields a 19mer duplex with 2-base overhangs at each end.) This Dicer cleavage event can generate a 21-23 nucleotide guide (antisense) strand capable of directing sequence- specific inhibition of target mRNA as a RISC component.

The first and second oligonucleotides of the DsiRNA cargoes of the instant invention are not required to be completely complementary. For example, the 3'-terminus of the sense strand can contain one or more mismatches. Optionally, about two mismatches are incorporated at the 3' terminus of the sense strand. A DsiRNA cargo of the invention can also be a double stranded RNA molecule containing two RNA oligonucleotides, each of which is an identical number of nucleotides in the range of 27-35 nucleotides in length and, when annealed to each other, have blunt ends and a two nucleotide mismatch on the 3'-terminus of the sense strand (the 5'-terminus of the antisense strand). The use of mismatches or decreased thermodynamic stability

(specifically at the 3'-sense/5'-antisense position) has been proposed to facilitate or favor entry of the antisense strand into RISC (Schwarz et al., 2003; Khvorova et al., 2003), presumably by affecting some rate-limiting unwinding steps that occur with entry of the siRNA into RISC. Thus, terminal base composition has been included in design algorithms for selecting active 21mer siRNA duplexes (Ui-Tei et al., 2004; Reynolds et al., 2004). With Dicer cleavage of the dsRNA region of certain DsiRNA cargoes, a small end-terminal sequence which contains mismatches will either be left unpaired with the antisense strand (become part of a 3 '-overhang) or be cleaved entirely off the final 21-mer siRNA. Such specific forms of "mismatches", therefore, do not persist as mismatches in the final RNA component of RISC. The finding that base mismatches or destabilization of segments at the 3'-end of the sense strand of Dicer substrate improved the potency of synthetic duplexes in RNAi, presumably by facilitating processing by Dicer, was a surprising finding of past works describing the design and use of 25-30mer dsRNAs (also termed "DsiRNAs" herein; Rossi et al, U.S. Patent Application Nos. 2005/0277610, 2005/0244858 and 2007/0265220). It was also surprising to find that DsiRNAs having base-paired deoxyribonucleotides at either passenger (sense) or guide (antisense) strand positions that are predicted to be 3' of the most 3' Dicer cleavage site of the respective passenger or guide strand are at least equally effective as RNA-RNA duplex-extended DsiRNA cargoes. Such agents may also harbor mismatches, with such mismatches being formed by the antisense strand either in reference to (actual or projected hybridation with) the sequence of the sense strand of the DsiRNA cargo, or in reference to the target RNA sequence. Exemplary mismatched or wobble base pairs of cargoes possessing mismatches are G:A, C:A, C:U, G:G, A:A, C:C, U:U, I:A, I:U and I:C. Base pair strength of such cargoes can also be lessened via modification of the nucleotides of such cargoes, including, e.g., 2-amino- or 2,6-diamino modifications of guanine and adenine

nucleotides.

Modification of DsiRNA Cargoes

In certain aspects, the instant invention provides formulations comprising a dsRNA cargo. One major factor that inhibits the effect of double stranded RNAs ("dsRNAs") is the degradation of dsRNAs {e.g., siRNAs and DsiRNAs) by nucleases. A 3'-exonuclease is the primary nuclease activity present in serum and modification of the 3'-ends of antisense DNA oligonucleotides is crucial to prevent degradation (Eder et al., 1991). An RNase-T family nuclease has been identified called ERI-1 which has 3' to 5' exonuclease activity that is involved in regulation and degradation of siRNAs (Kennedy et al., 2004;

Hong et al., 2005). This gene is also known as Thexl (NM 02067) in mice or THEX1

(NM 153332) in humans and is involved in degradation of histone mRNA; it also mediates degradation of 3'-overhangs in siRNAs, but does not degrade duplex RNA (Yang et al., 2006). It is therefore reasonable to expect that 3'-end-stabilization of dsRNAs, including, e.g., DsiRNAs of certain formulations of the instant invention, will improve stability.

XRN1 (NM 019001) is a 5' to 3' exonuclease that resides in P-bodies and has been implicated in degradation of mRNA targeted by miRNA (Rehwinkel et al., 2005) and may also be responsible for completing degradation initiated by internal cleavage as directed by a siRNA. XRN2 (NM 012255) is a distinct 5' to 3' exonuclease that is involved in nuclear RNA processing. Although not currently implicated in degradation or processing of siRNAs and miRNAs, these both are known nucleases that can degrade RNAs and may also be important to consider.

RNase A is a major endonuclease activity in mammals that degrades RNAs. It is specific for ssRNA and cleaves at the 3 '-end of pyrimidine bases. siRNA degradation products consistent with RNase A cleavage can be detected by mass spectrometry after incubation in serum (Turner et al., 2007). The 3'-overhangs enhance the susceptibility of siRNAs to RNase degradation. Depletion of RNase A from serum reduces degradation of siRNAs; this degradation does show some sequence preference and is worse for sequences having poly A/U sequence on the ends (Haupenthal et al., 2006). This suggests the possibility that lower stability regions of the duplex may "breathe" and offer transient single-stranded species available for degradation by RNase A. RNase A inhibitors can be added to serum and improve siRNA longevity and potency (Haupenthal et al., 2007).

In 21mers, phosphorothioate or boranophosphate modifications directly stabilize the internucleoside phosphate linkage. Boranophosphate modified RNAs are highly nuclease resistant, potent as silencing agents, and are relatively non-toxic.

Boranophosphate modified RNAs are difficult to manufacture using standard chemical synthesis methods and instead are made by in vitro transcription (IVT) (Hall et al., 2004 and Hall et al., 2006). Phosphorothioate (PS) modifications can be readily placed in an RNA duplex at any desired position and can be made using standard chemical synthesis methods, though the ability to use such modifications within an RNA duplex that retains RNA silencing activity can be limited.

Inclusions of a multiple PS-modified deoxyribonucleotide residues in a tandem series configuration that base paired with a cognate tandem series of PS-modified deoxyribonucleotide residues abolished RNA silencing activity of an agent that was otherwise active with only unmodified deoxyribonucleotides present at these residues. Because PS moieties are likely to require greater spacing when included within an RNA duplex-containing agent in order to retain RNA inhibitory activity, extended DsiRNAs can provide a means of including more PS modifications (either PS-DNA or PS-RNA) within a single DsiRNA cargo than would otherwise be available were no such extension used. It is noted, however, that the PS modification shows dose-dependent toxicity, so most investigators have recommended limited incorporation in siRNAs, historically favoring the 3'-ends where protection from nucleases is most important (Harborth et al., 2003; Chiu and Rana, 2003; Braasch et al., 2003; Amarzguioui et al., 2003). More extensive PS modification can be compatible with potent RNAi activity; however, use of sugar modifications (such as 2'-0-methyl RNA) may be superior (Choung et al., 2006).

A variety of substitutions can be placed at the 2'-position of the ribose which generally increases duplex stability (T_m) and can greatly improve nuclease resistance. 2'- O-methyl RNA is a naturally occurring modification found in mammalian ribosomal RNAs and transfer RNAs. 2'-0-methyl modification in siRNAs is known, but the precise position of modified bases within the duplex is important to retain potency and complete substitution of 2'-0-methyl RNA for RNA will inactivate the siRNA. For example, a pattern that employs alternating 2'-0-methyl bases can have potency equivalent to unmodified RNA and is quite stable in serum (Choung et al., 2006; Czauderna et al., 2003).

The 2'-fluoro (2'-F) modification is also compatible with dsRNA (e.g., siRNA and DsiRNA) function; it is most commonly placed at pyrimidine sites (due to reagent cost and availability) and can be combined with 2'-0-methyl modification at purine positions; 2'-F purines are available and can also be used. Heavily modified duplexes of this kind can be potent triggers of RNAi in vitro (Allerson et al., 2005; Prakash et al., 2005; Kraynack and Baker, 2006) and can improve performance and extend duration of action when used in vivo (Morrissey et al., 2005a; Morrissey et al., 2005b). A highly potent, nuclease stable, blunt 19mer duplex containing alternative 2'-F and 2'-0-Me bases is taught by Allerson. In this design, alternating 2'-0-Me residues are positioned in an identical pattern to that employed by Czauderna, however the remaining RNA residues are converted to 2'-F modified bases. A highly potent, nuclease resistant siRNA employed by Morris sey employed a highly potent, nuclease resistant siRNA in vivo. In addition to 2'-0-Me RNA and 2'-F RNA, this duplex includes DNA, RNA, inverted abasic residues, and a 3'- terminal PS internucleoside linkage. While extensive modification has certain benefits, more limited modification of the duplex can also improve in vivo performance and is both simpler and less costly to manufacture. Soutschek et al. (2004) employed a duplex in vivo and was mostly RNA with two 2'-0-Me RNA bases and limited 3'-terminal PS

internucleoside linkages.

Locked nucleic acids (LNAs) are a different class of 2'-modification that can be used to stabilize dsRNA {e.g., siRNA and DsiRNA). Patterns of LNA incorporation that retain potency are more restricted than 2'-0-methyl or 2'-F bases, so limited modification is preferred (Braasch et al., 2003; Grunweller et al., 2003; Elmen et al., 2005). Even with limited incorporation, the use of LNA modifications can improve dsRNA performance in vivo and may also alter or improve off target effect profiles (Mook et al., 2007).

Synthetic nucleic acids introduced into cells or live animals can be recognized as "foreign" and trigger an immune response. Immune stimulation constitutes a major class of off-target effects which can dramatically change experimental results and even lead to cell death. The innate immune system includes a collection of receptor molecules that specifically interact with DNA and RNA that mediate these responses, some of which are located in the cytoplasm and some of which reside in endosomes (Marques and Williams, 2005; Schlee et al., 2006). Delivery of siRNAs by cationic lipids or liposomes exposes the siRNA to both cytoplasmic and endosomal compartments, maximizing the risk for triggering a type 1 interferon (IFN) response both in vitro and in vivo (Morrissey et al., 2005b; Sioud and Sorensen, 2003; Sioud, 2005; Ma et al., 2005). RNAs transcribed within the cell are less immunogenic (Robbins et al., 2006) and synthetic RNAs that are immunogenic when delivered using lipid-based methods can evade immune stimulation when introduced unto cells by mechanical means, even in vivo (Heidel et al., 2004).

However, lipid based delivery methods are convenient, effective, and widely used. A general strategy to prevent immune responses, such as the one described herein, is needed, especially for in vivo application where all cell types are present and the risk of generating an immune response is highest. Use of chemically modified RNAs may also aid in solving such immune response problems.

Although certain sequence motifs are clearly more immunogenic than others, it appears that the receptors of the innate immune system in general distinguish the presence or absence of certain base modifications which are more commonly found in mammalian RNAs than in prokaryotic RNAs. For example, pseudouridine, N6-methyl-A, and 2'-0- methyl modified bases are recognized as "self" and inclusion of these residues in a synthetic RNA can help evade immune detection (Kariko et al., 2005). Extensive 2'- modification of a sequence that is strongly immuno stimulatory as unmodified RNA can block an immune response when administered to mice intravenously (Morrissey et al., 2005b). However, extensive modification is not needed to escape immune detection and substitution of as few as two 2'-0-methyl bases in a single strand of a siRNA duplex can be sufficient to block a type 1 IFN response both in vitro and in vivo; modified U and G bases are most effective (Judge et al., 2006). As an added benefit, selective incorporation of 2'-0-methyl bases can reduce the magnitude of off-target effects (Jackson et al., 2006). Use of 2'-0-methyl bases should therefore be considered for all dsRNAs intended for in vivo applications as a means of blocking immune responses and has the added benefit of improving nuclease stability and reducing the likelihood of off-target effects.

Although cell death can result from immune stimulation, assessing cell viability is not an adequate method to monitor induction of IFN responses. IFN responses can be present without cell death, and cell death can result from target knockdown in the absence of IFN triggering (for example, if the targeted gene is essential for cell viability). Relevant cytokines can be directly measured in culture medium and a variety of commercial kits exist which make performing such assays routine. While a large number of different immune effector molecules can be measured, testing levels of IFN-0C, TNF-oc, and IL-6 at 4 and 24 hours post transfection is usually sufficient for screening purposes. It is important to include a "transfection reagent only control" as cationic lipids can trigger immune responses in certain cells in the absence of any nucleic acid cargo. Including controls for IFN pathway induction should be considered for cell culture work. It is essential to test for immune stimulation whenever administering nucleic acids in vivo, where the risk of triggering IFN responses is highest.

Modifications can be included in the DsiRNA cargoes of certain formulations of the present invention so long as the modification does not prevent the DsiRNA cargo from serving as a substrate for Dicer. It was previously found that base paired

deoxyribonucleotides can be attached to DsiRNA molecules, resulting in enhanced RNAi efficacy and duration, provided that such extension is performed in a region of the extended molecule that does not interfere with Dicer processing {e.g., 3' of the Dicer cleavage site of the sense strand/5' of the Dicer cleavage site of the antisense strand). In one embodiment, one or more modifications are made that enhance Dicer processing of the DsiRNA cargo. In a second embodiment, one or more modifications are made that result in more effective RNAi generation. In a third embodiment, one or more modifications are made that support a greater RNAi effect. In a fourth embodiment, one or more modifications are made that result in greater potency per each DsiRNA cargo molecule to be delivered to the cell. Modifications can be incorporated in the 3'-terminal region, the 5'-terminal region, in both the 3'-terminal and 5'-terminal region or in some instances in various positions within the sequence. With the restrictions noted above in mind, any number and combination of modifications can be incorporated into the DsiRNA cargo. Where multiple modifications are present, they may be the same or different. Modifications to bases, sugar moieties, the phosphate backbone, and their combinations are contemplated. Either 5'-terminus can be phosphorylated.

Examples of modifications contemplated for the phosphate backbone include phosphonates, including methylphosphonate, phosphorothioate, and phosphotriester modifications such as alkylphosphotriesters, and the like. Examples of modifications contemplated for the sugar moiety include 2'-alkyl pyrimidine, such as 2'-0-methyl, 2'- fluoro, amino, and deoxy modifications and the like (see, e.g., Amarzguioui et al., 2003). Examples of modifications contemplated for the base groups include abasic sugars, 2-0- alkyl modified pyrimidines, 4-thiouracil, 5-bromouracil, 5-iodouracil, and 5-(3- aminoallyl)-uracil and the like. Locked nucleic acids, or LNA's, could also be

incorporated. Many other modifications are known and can be used so long as the above criteria are satisfied. Examples of modifications are also disclosed in U.S. Pat. Nos.

5,684,143, 5,858,988 and 6,291,438 and in U.S. published patent application No.

2004/0203145 Al. Other modifications are disclosed in Herdewijn (2000), Eckstein (2000), Rusckowski et al. (2000), Stein et al. (2001); Vorobjev et al. (2001).

One or more modifications contemplated can be incorporated into either strand. The placement of the modifications in the DsiRNA cargo can greatly affect the characteristics of the DsiRNA cargo, including conferring greater potency and stability, reducing toxicity, enhancing Dicer processing, and minimizing an immune response. In one embodiment, the antisense strand or the sense strand or both strands have one or more 2'-0-methyl modified nucleotides. In another embodiment, the antisense strand contains 2'-0-methyl modified nucleotides. In another embodiment, the antisense stand contains a 3' overhang that is comprised of 2'-0-methyl modified nucleotides. The antisense strand could also include additional 2'-0-methyl modified nucleotides.

In certain embodiments of the formulations of the present invention, a DsiRNA cargo has one or more properties which enhance its processing by Dicer. According to these embodiments, the DsiRNA has a length sufficient such that it is processed by Dicer to produce an active siRNA and at least one of the following properties: (i) the DsiRNA is asymmetric, e.g., has a 3' overhang on the antisense strand and (ii) the DsiRNA has a modified 3' end on the sense strand to direct orientation of Dicer binding and processing of the dsRNA region to an active siRNA. In certain such embodiments, the presence of one or more base paired deoxyribonucleotides in a region of the sense strand that is 3' to the projected site of Dicer enzyme cleavage and corresponding region of the antisense strand that is 5' of the projected site of Dicer enzyme cleavage can also serve to orient such a molecule for appropriate directionality of Dicer enzyme cleavage.

The length of a dsDNA region (or length of the region comprising DNA:DNA base pairs) of an "extended" DsiRNA can be 1-50 base pairs, optionally 2-30 base pairs, optionally 2-20 base pairs, and optionally 2-15 base pairs. Thus, a DNA:DNA-extended DsiRNA of certain formulations of the instant invention may possess a dsDNA region that is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,

27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more (e.g., 51, 52, 53, 54, 55, 56, 57, 58, 59, 60 or more) base pairs in length. In some embodiments, the longest strand in the dsNA comprises 29-43 nucleotides. In one embodiment, a DsiRNA cargo is asymmetric such that the 3' end of the sense strand and 5' end of the antisense strand form a blunt end, and the 3' end of the antisense strand overhangs the 5' end of the sense strand. In certain embodiments, the 3' overhang of the antisense strand is 1-10 nucleotides, and optionally is 1-4 nucleotides, for example 2 nucleotides. Both the sense and the antisense strand may also have a 5' phosphate. In certain embodiments, the sense strand of a DsiRNA of a formulation of the invention that comprises base paired deoxyribonucleotide residues has a total length of between 26 nucleotides and 39 or more nucleotides (e.g., the sense strand possesses a length of 26, 27,

28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more (e.g., 51, 52, 53, 54, 55, 56, 57, 58, 59, 60 or more) nucleotides). In certain embodiments, the length of the sense strand is between 26 nucleotides and 39 nucleotides, optionally between 27 and 35 nucleotides, or, optionally, between 27 and 33 nucleotides in length. In related embodiments, the antisense strand has a length of between 27 and 43 or more nucleotides (e.g., the sense strand possesses a length of 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more (e.g., 51, 52, 53, 54, 55, 56, 57, 58, 59, 60 or more) nucleotides). In certain such embodiments, the antisense strand has a length of between 27 and 43 nucleotides in length, or between 27 and 39 nucleotides in length, or between 27 and 35 nucleotides in length, or between 28 and 37 nucleotides in length, or, optionally, between 29 and 35 nucleotides in length.

In certain embodiments, the sense strand of a DsiRNA cargo is modified for Dicer processing by suitable modifiers located at the 3' end of the sense strand, i.e., the DsiRNA cargo is designed to direct orientation of Dicer binding and processing via sense strand modification. Suitable modifiers include nucleotides such as deoxyribonucleotides, dideoxyribonucleotides, acyclonucleotides and the like and sterically hindered molecules, such as fluorescent molecules and the like. Acyclonucleotides substitute a 2- hydroxyethoxymethyl group for the 2'-deoxyribofuranosyl sugar normally present in dNMPs. Other nucleotide modifiers could include 3'-deoxyadenosine (cordycepin), 3'- azido-3'-deoxythymidine (AZT), 2',3'-dideoxyinosine (ddl), 2',3'-dideoxy-3'-thiacytidine (3TC), 2',3'-didehydro-2',3'-dideoxythymidine (d4T) and the monophosphate nucleotides of 3'-azido-3'-deoxythymidine (AZT), 2',3'-dideoxy-3'-thiacytidine (3TC) and 2',3'- didehydro-2',3'-dideoxythymidine (d4T). In one embodiment, deoxyribonucleotides are used as the modifiers. When nucleotide modifiers are utilized, 1-3 nucleotide modifiers, or 2 nucleotide modifiers are substituted for the ribonucleotides on the 3' end of the sense strand. When sterically hindered molecules are utilized, they are attached to the ribonucleotide at the 3' end of the antisense strand. Thus, the length of the strand does not change with the incorporation of the modifiers. For example, a DsiRNA cargo can be substituted with two DNA bases to direct the orientation of Dicer processing of the antisense strand. Optionally, two terminal DNA bases are substituted for two

ribonucleotides on the 3 '-end of the sense strand forming a blunt end of the duplex on the 3' end of the sense strand and the 5' end of the antisense strand, and a two-nucleotide RNA overhang is located on the 3'-end of the antisense strand. This is an asymmetric composition with DNA on the blunt end and RNA bases on the overhanging end.

Optionally, the modified nucleotides (e.g., deoxyribonucleotides) of the penultimate and ultimate positions of the 3' terminus of the sense strand base pair with corresponding modified nucleotides (e.g., deoxyribonucleotides) of the antisense strand (optionally, the penultimate and ultimate residues of the 5' end of the antisense strand in those DsiRNA cargoes of the instant invention possessing a blunt end at the 3' terminus of the sense strand/5' terminus of the antisense strand). The sense and antisense strands of a DsiRNA cargo of the instant invention anneal under biological conditions, such as the conditions found in the cytoplasm of a cell. In addition, a region of one of the sequences, particularly of the antisense strand, of the DsiRNA cargo has a sequence length of at least 19 nucleotides, wherein these nucleotides are in the 21-nucleotide region adjacent to the 3' end of the antisense strand and are sufficiently complementary to a nucleotide sequence of the RNA produced from the target gene to anneal with and/or decrease levels of such a target RNA.

The first and second oligonucleotides of a DsiRNA cargo of the instant invention are not required to be completely complementary. They only need to be substantially complementary to anneal under biological conditions and to provide a substrate for Dicer that produces a siRNA sufficiently complementary to the target sequence. Locked nucleic acids, or LNA's, are well known to a skilled artisan (Elman et al., 2005; Kurreck et al., 2002; Crinelli et al., 2002; Braasch and Corey, 2001; Bondensgaard et al., 2000;

Wahlestedt et al., 2000). In one embodiment, an LNA is incorporated at the 5' terminus of the sense strand. In another embodiment, an LNA is incorporated at the 5' terminus of the sense strand in duplexes designed to include a 3' overhang on the antisense strand.

Certain DsiRNA cargoes containing two separate oligonucleotides can be linked by a third structure. The third structure will not block Dicer activity on the DsiRNA and will not interfere with the directed destruction of the RNA transcribed from the target gene. In one embodiment, the third structure may be a chemical linking group. Many suitable chemical linking groups are known in the art and can be used. Alternatively, the third structure may be an oligonucleotide that links the two oligonucleotides of the DsiRNA cargo in a manner such that a hairpin structure is produced upon annealing of the two oligonucleotides making up the dsNA composition. The hairpin structure will not block Dicer activity on the DsiRNA cargo and will not interfere with the directed destruction of the target RNA.

In certain embodiments, the DsiRNA cargo of the invention has several properties which enhance its processing by Dicer. According to such embodiments, the DsiRNA cargo has a length sufficient such that it is processed by Dicer to produce an siRNA and at least one of the following properties: (i) the DsiRNA cargo is asymmetric, e.g., has a 3' overhang on the sense strand and (ii) the DsiRNA cargo has a modified 3' end on the antisense strand to direct orientation of Dicer binding and processing of the dsRNA region to an active siRNA. According to these embodiments, the longest strand in the DsiRNA cargo comprises 25-43 nucleotides. In one embodiment, the sense strand comprises 25-39 nucleotides and the antisense strand comprises 26-43 nucleotides. The resulting dsNA can have an overhang on the 3' end of the sense strand. The overhang is 1-4 nucleotides, such as 2 nucleotides. The antisense or sense strand may also have a 5' phosphate.

In certain embodiments, the sense strand of a DsiRNA cargo is modified for Dicer processing by suitable modifiers located at the 3' end of the sense strand, i.e., the DsiRNA cargo is designed to direct orientation of Dicer binding and processing. Suitable modifiers include nucleotides such as deoxyribonucleotides, dideoxyribonucleotides,

acyclonucleotides and the like and sterically hindered molecules, such as fluorescent molecules and the like. Acyclonucleotides substitute a 2-hydroxyethoxymethyl group for the 2'-deoxyribofuranosyl sugar normally present in dNMPs. Other nucleotide modifiers could include 3'-deoxyadenosine (cordycepin), 3'-azido-3'-deoxythymidine (AZT), 2',3'- dideoxyinosine (ddl), 2',3'-dideoxy-3'-thiacytidine (3TC), 2',3'-didehydro-2',3'- dideoxythymidine (d4T) and the monophosphate nucleotides of 3'-azido-3'- deoxythymidine (AZT), 2',3'-dideoxy-3'-thiacytidine (3TC) and 2',3'-didehydro-2',3'- dideoxythymidine (d4T). In one embodiment, deoxyribonucleotides are used as the modifiers. When nucleotide modifiers are utilized, 1-3 nucleotide modifiers, or 2 nucleotide modifiers are substituted for the ribonucleotides on the 3' end of the sense strand. When sterically hindered molecules are utilized, they are attached to the ribonucleotide at the 3' end of the antisense strand. Thus, the length of the strand does not change with the incorporation of the modifiers.

In certain other embodiments, the antisense strand of a DsiRNA cargo is modified for Dicer processing by suitable modifiers located at the 3' end of the antisense strand, i.e., the DsiRNA is designed to direct orientation of Dicer binding and processing. Suitable modifiers include nucleotides such as deoxyribonucleotides, dideoxyribonucleotides, acyclonucleotides and the like and sterically hindered molecules, such as fluorescent molecules and the like. Acyclonucleotides substitute a 2-hydroxyethoxymethyl group for the 2'-deoxyribofuranosyl sugar normally present in dNMPs. Other nucleotide modifiers could include 3'-deoxyadenosine (cordycepin), 3'-azido-3'-deoxythymidine (AZT), 2',3'- dideoxyinosine (ddl), 2',3'-dideoxy-3'-thiacytidine (3TC), 2',3'-didehydro-2',3'- dideoxythymidine (d4T) and the monophosphate nucleotides of 3'-azido-3'- deoxythymidine (AZT), 2',3'-dideoxy-3'-thiacytidine (3TC) and 2',3'-didehydro-2',3'- dideoxythymidine (d4T). In one embodiment, deoxyribonucleotides are used as the modifiers. When nucleotide modifiers are utilized, 1-3 nucleotide modifiers, or 2 nucleotide modifiers are substituted for the ribonucleotides on the 3' end of the antisense strand. When sterically hindered molecules are utilized, they are attached to the ribonucleotide at the 3' end of the antisense strand. Thus, the length of the strand does not change with the incorporation of the modifiers. In certain nucleic acid formulations, two DNA bases in the dsNA are substituted to direct the orientation of Dicer processing.

Optionally, two terminal DNA bases are located on the 3' end of the antisense strand in place of two ribonucleotides forming a blunt end of the duplex on the 5' end of the sense strand and the 3' end of the antisense strand, and a two-nucleotide RNA overhang is located on the 3 '-end of the sense strand. This is also an asymmetric composition with DNA on the blunt end and RNA bases on the overhanging end.

The sense and antisense strands anneal under biological conditions, such as the conditions found in the cytoplasm of a cell. In addition, a region of one of the sequences, particularly of the antisense strand, of the dsNA cargo has a sequence length of at least 19 nucleotides, wherein these nucleotides are adjacent to the 3' end of antisense strand and are sufficiently complementary to a nucleotide sequence of the target RNA to direct RNA interference.

US 2007/0265220 discloses that 27mer DsiRNAs show improved stability in serum over comparable 21mer siRNA compositions, even absent chemical modification. Modifications of DsiRNA cargoes, such as inclusion of 2'-0-methyl RNA in the antisense strand, in patterns such as detailed in US 2007/0265220, when coupled with addition of a 5' Phosphate, can improve stability of DsiRNA cargoes. Addition of 5'-phosphate to all strands in synthetic RNA duplexes may be an inexpensive and physiological method to confer some limited degree of nuclease stability.

The chemical modification patterns of the DsiRNA cargoes of the invention are designed to enhance the efficacy of such cargoes. Accordingly, such modifications are designed to avoid reducing potency of DsiRNA cargoes; to avoid interfering with Dicer processing of DsiRNA cargoes; to improve stability in biological fluids (reduce nuclease sensitivity) of DsiRNA cargoes; or to block or evade detection by the innate immune system. Such modifications are also designed to avoid being toxic and to avoid increasing the cost or impact the ease of manufacturing the instant DsiRNA cargoes of the invention.

Preparation of the Lipid-Based Compositions of the Invention

Certain formulations of the present invention, e.g., in which a desired cargo (e.g., a DsiRNA) is encapsulated in a lipid bilayer and is protected from degradation, can be formed by a method known in the art including, but not limited to, a continuous mixing method, a direct dilution process, a detergent dialysis method, or a modification of a reverse-phase method which utilizes organic solvents to provide a single phase during mixing of the components.

In certain embodiments, the cleavable lipids and any non-cationic and/or cationic lipids of the formulations of the invention are lipids as described above, or combinations thereof.

In particular embodiments, the organic solvents are methanol, chloroform, methylene chloride, ethanol, diethyl ether, or combinations thereof.

In specific embodiments, the present invention provides for cargo-lipid

formulations produced via a continuous mixing method, e.g., a process that includes providing an aqueous solution comprising a cargo such as a DsiRNA in a first reservoir, providing an organic lipid solution in a second reservoir, and mixing the aqueous solution with the organic lipid solution such that the organic lipid solution mixes with the aqueous solution so as to substantially instantaneously produce a liposome encapsulating the cargo (e.g., DsiRNA). This process and the apparatus for carrying this process are described in detail in U.S. Patent Publication No. 2004/0142025.

The action of continuously introducing lipid and buffer solutions into a mixing environment, such as in a mixing chamber, causes a continuous dilution of the lipid solution with the buffer solution, thereby producing a liposome substantially

instantaneously upon mixing. As used herein, the phrase "continuously diluting a lipid solution with a buffer solution" (and variations) generally means that the lipid solution is diluted sufficiently rapidly in a hydration process with sufficient force to effectuate vesicle generation. By mixing the aqueous solution comprising a cargo with the organic lipid solution, the organic lipid solution undergoes a continuous stepwise dilution in the presence of the buffer solution (i.e., aqueous solution) to produce a cargo-lipid formulation particle.

In some embodiments, a lipid compound of a formulation of the invention is conjugated to a cargo compound of the formulation of the invention.

In certain embodiments, the serum- stable cargo-lipid formulation particles formed using the continuous mixing method can have a size of from about 50 nm to about 150 nm, from about 60 nm to about 130 nm, from about 70 nm to about 110 nm, or from about 70 nm to about 90 nm. The formulation particles thus formed do not aggregate and are optionally sized to achieve a uniform particle size.

In yet another embodiment, the present invention provides for cargo-lipid formulation particles produced via a direct dilution process in which a third reservoir containing dilution buffer is fluidly coupled to a second mixing region. In this

embodiment, the liposome solution formed in a first mixing region is immediately and directly mixed with dilution buffer in the second mixing region. In certain aspects, the second mixing region includes a T-connector arranged so that the liposome solution and the dilution buffer flows meet as opposing 180° flows; however, connectors providing shallower angles can be used, e.g., from about 27° to about 180°. A pump mechanism delivers a controllable flow of buffer to the second mixing region. In one aspect, the flow rate of dilution buffer provided to the second mixing region is controlled to be

substantially equal to the flow rate of liposome solution introduced thereto from the first mixing region. This embodiment advantageously allows for more control of the flow of dilution buffer mixing with the liposome solution in the second mixing region, and therefore also the concentration of liposome solution in buffer throughout the second mixing process. Such control of the dilution buffer flow rate advantageously allows for small particle size formation at reduced concentrations.

These processes and the apparatuses for carrying out these direct dilution processes is described in detail in U.S. patent application Ser. No. 11/495,150. The serum-stable cargo-lipid particles formed using the direct dilution process can have a size of from about 50 nm to about 150 nm, from about 60 nm to about 130 nm, from about 70 nm to about 110 nm, or from about 70 nm to about 90 nm. The particles thus formed do not aggregate and are optionally sized to achieve a uniform particle size. In some embodiments, the particles are formed using detergent dialysis. Without intending to be bound by any particular mechanism of formation, a cargo such as a DsiRNA is contacted with a detergent solution of cationic lipids to form a coated cargo complex. These coated cargoes can aggregate and precipitate. However, the presence of a detergent can reduce this aggregation and allow the coated cargoes to react with excess lipids (e.g., non-cationic lipids) to form particles in which the cargo is encapsulated in a lipid bilayer. Thus, serum- stable cargo-lipid particles can be prepared as follows:

• (a) combining a cargo with cationic lipids in a detergent solution to form a coated cargo-lipid complex;

• (b) contacting non-cationic lipids (including, e.g., cleavable lipids as described herein) with the coated cargo-lipid complex to form a detergent solution comprising a cargo-lipid complex and non-cationic lipids; and

• (c) dialyzing the detergent solution of step (b) to provide a solution of serum- stable cargo-lipid particles, wherein the cargo is encapsulated in a lipid bilayer and the particles are serum-stable and have a size of from about 50 to about 150 nm.

An initial solution of coated cargo-lipid complexes is formed by combining the cargo with the cationic lipids in a detergent solution. In these embodiments, the detergent solution is optionally an aqueous solution of a neutral detergent having a critical micelle concentration of 15-300 mM, optionally 20-50 mM. Examples of suitable detergents include, for example, N, N'-((octanoylimino)-bis-(trimethylene))-bis-(D-gluconamide) (BIGCHAP); BRLJ 35; Deoxy-BIGCHAP; dodecylpoly(ethylene glycol) ether; Tween 20; Tween 40; Tween 60; Tween 80; Tween 85; Mega 8; Mega 9; Zwittergent® 3-08;

Zwittergent® 3-10; Triton X-405; hexyl-, heptyl-, octyl-and nonyl-P-D-glucopyranoside; and heptylthioglucopyranoside. In certain embodiments, the concentration of detergent in the detergent solution can be about 100 mM to about 2 M, optionally from about 200 mM to about 1.5 M.

In certain embodiments, cationic lipids, non-cationic lipids (e.g., cleavable cationic lipids as described herein) and cargoes may be combined to produce a charge ratio (+/-) of about 1: 1 to about 20: 1, in a ratio of about 1 : 1 to about 12: 1, or in a ratio of about 2: 1 to about 6: 1. Additionally, in certain embodiments, the overall concentration of cargo in solution can be from about 25 μg/ml to about 1 mg/ml, from about 25 μg/ml to about 200 μg/ml, or from about 50 μg/ml to about 100 μg/ml. The combination of cargoes and cationic lipids in detergent solution is kept, optionally, at room temperature, for a period of time which is sufficient for the coated complexes to form. Alternatively, the cargoes and cationic lipids can be combined in the detergent solution and warmed to temperatures of up to about 37° C, about 50° C, about 60° C, or about 70° C. For cargoes which are particularly sensitive to temperature, the coated complexes can be formed at lower temperatures, typically down to about 4° C. In some embodiments, the cargo to lipid ratios (mass/mass ratios) in a formed cargo-lipid particle will range from about 0.01 to about 0.2, from about 0.02 to about 0.1, from about 0.03 to about 0.1, or from about 0.01 to about 0.08. The ratio of the starting materials can also fall within this range. In other embodiments, the cargo-lipid particle preparation uses about 400 μg cargo per 10 mg total lipid or a cargo to lipid mass ratio of about 0.01 to about 0.08 and, optionally, about 0.04, which corresponds to 1.25 mg of total lipid per 50 μg of cargo. In certain embodiments, the particle has a cargo:lipid mass ratio of about 0.08.

A detergent solution of a coated cargo-lipid complex can then be contacted with non-cationic lipids to provide a detergent solution of cargo-lipid complexes and non- cationic lipids. Non-cationic lipids useful in this step include, diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, cephalin, cardiolipin, and cerebrosides. In certain embodiments, the non-cationic lipids are

diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, or sphingomyelin. Acyl groups in these lipids are optionally acyl groups derived from fatty acids having C₁₀- C₂4 carbon chains. In certain embodiments, the acyl groups are lauroyl, myristoyl, palmitoyl, stearoyl, or oleoyl. Optionally, the non-cationic lipids are DSPC, DOPE, POPC, egg phosphatidylcholine (EPC), cholesterol, a non-cationic lipid as described herein, or a mixture thereof. In certain embodiments, the cargo-lipid formulation particles are fusogenic particles with enhanced properties in vivo and the non-cationic lipid is DSPC or DOPE. In addition, the cargo-lipid formulation particles of the present invention may further comprise cholesterol. In other embodiments, the non-cationic lipids can further comprise polyethylene glycol-based polymers such as PEG 2,000, PEG 5,000, and PEG conjugated to a diacylglycerol, a ceramide, or a phospholipid, as described in, e.g., U.S. Pat. No. 5,820,873 and U.S. Patent Publication No. 2003/0077829. In further

embodiments, the non-cationic lipids can further comprise polyethylene glycol-based polymers such as PEG 2,000, PEG 5,000, and PEG conjugated to a dialkyloxypropyl.

The amount of non-cationic lipid which is used in the present methods can be from about 2 to about 20 mg of total lipids to 50 μg of cargo. Optionally, the amount of total lipid is from about 5 to about 10 mg per 50 μg of cargo.

After formation of a detergent solution of cargo-lipid complexes and non-cationic lipids, the detergent can be removed, e.g., by dialysis. Detergent removal can result in the formation of a lipid-bilayer which surrounds the cargo providing serum-stable cargo-lipid formulation particles which have a size of from about 50 nm to about 150 nm, from about 60 nm to about 130 nm, from about 70 nm to about 110 nm, or from about 70 nm to about 90 nm. The formulation particles thus formed do not aggregate and are optionally sized to achieve a uniform particle size.

Serum-stable cargo-lipid formulation particles can be sized by any of the methods available for sizing liposomes. The sizing may be conducted in order to achieve a desired size range and relatively narrow distribution of particle sizes.

Several techniques are available for sizing the formulation particles to a desired size. One sizing method, used for liposomes and equally applicable to the present particles, is described in U.S. Pat. No. 4,737,323. Sonicating a formulation particle suspension either by bath or probe sonication cam produce a progressive size reduction down to particles of less than about 50 nm in size. Homogenization is another method which relies on shearing energy to fragment larger particles into smaller ones. In a typical homogenization procedure, particles are re-circulated through a standard emulsion homogenizer until selected particle sizes, typically between about 60 and about 80 nm, are observed. In both methods, the particle size distribution can be monitored by conventional laser-beam particle size discrimination, or QELS.

Extrusion of the formulation particles through a small-pore polycarbonate membrane or an asymmetric ceramic membrane is also an effective method for reducing particle sizes to a relatively well-defined size distribution. Typically, the suspension is cycled through the membrane one or more times until the desired particle size distribution is achieved. The formulation particles may be extruded through successively smaller-pore membranes, to achieve a gradual reduction in size.

In certain embodiments, cargo-lipid particles of the invention can be prepared by well known conventional processes for preparing liposomes. Such liposomes can form nanocontainers, such as nanoparticles, and are commonly used for encapsulation of pharmaceutical agents. Liposomes are typically spherical in shape, and optionally have an average particle size (i.e., the average of the longest dimension, which is the diameter for spherical particles) of no greater than 1000 nanometers (nm). In certain embodiments, liposomes can be generated having an average particle size of 50 nm, e.g., in order to cross the blood brain barrier. Suitable liposomes of the instant invention may incorporate the lipids of the invention described elsewhere herein and may be prepared from, for example, phospholipids selected from the group consisting of phosphatidylserine,

pho sphatidylino sitol, pho sphatidylethanolamine, pho sphatidylcholines ,

phosphatidylglycerol, phosphatidic acid, phosphatidylmethanol, cardiolipin, ceramide, cholesterol, cerebroside, lysophosphatidylcholine, D-erythrosphingosine, sphingomyelin, dodecyl phosphocholine, N-biotinyl phosphatidylethanolamine, synthetic analogs of these molecules, derivatives of these molecules, and combinations thereof.

In some embodiments, the cargo-lipid particles of the invention are prepared, for example, by depositing a thin film of lipid on the inner wall of a flask, adding an aqueous phase (optionally including one or more cargoes, e.g., dsRNA), and shaking vigorously (e.g., by hand) in order to rehydrate the lipids as cargo-lipid particles (optionally, as liposomes). Another method may include, for example, sonication of a lipid film in an aqueous solution (optionally including one or more cargoes, e.g., dsRNA), followed by extrusion through a series of filters (e.g., of decreasing pore size). Yet another method of making cargo-lipid particles of the invention is to dialyze an aqueous solution of lipids (optionally including one or more cargoes, e.g., dsRNA) in the presence of a detergent such as sodium cholate. In certain embodiments, as the detergent is depleted, the lipids form liposomes. Still another method is based on high pressure homogenization of a lipid solution using commercially available equipment. Additional methods may include, for example, re-hydration of freeze-dried lipids and/or vesicles and reverse-phase evaporation. Descriptions and protocols for these methods are well known to those of skill in the art. See, for example, Liposomes: A Practical Approach (2nd edition, 2003), edited by

Vladimir Torchilin and Volkmar Weissig, Oxford University Press, Oxford, UK.

Materials for making such cargo-lipid particles (e.g., liposomes) are commercially available, for example, from Avestin Inc., Ottawa, Canada, Microfluidics, a division of MFIC Corp., Newton, MA, and Harvard Apparatus, a Harvard Bioscience, Inc. Company, Holliston, MA.

As noted elsewhere herein, one or more pharmaceutical agent cargoes (e.g., a dsRNA, small molecule, etc.), may be associated with a lipid formulation of the invention, such as encapsulated within a liposome, using a wide variety of mechanisms, including encapsulation within the internal compartment of a liposome, or attachment to the outer surface of a liposome through bonding or nonbonding interactions, intercalation between the double layer of lipid head groups of a liposome, and the like. For example, methods of associating one or more pharmaceutical agents with lipid formulations of the invention (e.g., liposomes) include, but are not limited to: encapsulating an agent within the aqueous core of a liposome, which can occur by preparing the liposome in the presence of the agent; causing a non-bonded interaction (e.g., van der Waals) between an agent and the hydrophilic tail of a lipid used to form a liposome, either within the core or at the outer surface of the liposome; causing an interaction between an agent and the lipid head group of a lipid used to form a liposome; intercalating an agent between the double layer of lipid head groups in a liposome; bonding (e.g., covalent, ionic, or hydrogen bonding) an agent to a molecule that makes up the structure of the lipid formulation, possibly through either a hydrophobic tail or a lipid head group of the lipid formulation; and/or causing complex formation between an agent and a cationic salt that may be a part of the structure of the lipid formulation.

In another group of embodiments, serum-stable cargo-lipid particles can be prepared as follows:

• (a) preparing a mixture comprising cationic lipids and non-cationic lipids in an organic solvent; • (b) contacting an aqueous solution of cargo with the mixture in step (a) to provide a clear single phase; and

• (c) removing the organic solvent to provide a suspension of cargo-lipid particles, wherein the cargo is encapsulated in a lipid bilayer and the particles are stable in serum and have a size of from about 50 to about 150 nm.

The cargoes (e.g., DsiRNA), cationic lipids, and non-cationic lipids which are useful in this group of embodiments are as described for the detergent dialysis methods above.

The selection of an organic solvent will typically involve consideration of solvent polarity and the ease with which the solvent can be removed at the later stages of particle formation. The organic solvent, which is also used as a solubilizing agent, is in an amount sufficient to provide a clear single phase mixture of cargo and lipids. Suitable solvents include, but are not limited to, chloroform, dichloromethane, diethylether, cyclohexane, cyclopentane, benzene, toluene, methanol, or other aliphatic alcohols such as propanol, isopropanol, butanol, tert-butanol, iso-butanol, pentanol and hexanol. Combinations of two or more solvents may also be used. Contacting the cargo with the organic solution of cationic and non-cationic lipids is accomplished by mixing together a first solution of cargo, which is typically an aqueous solution, and a second organic solution of the lipids. One of skill in the art will understand that this mixing can take place by any number of methods, for example, by mechanical means such as by using vortex mixers.

After the cargo has been contacted with the organic solution of lipids, the organic solvent is removed, thus forming an aqueous suspension of serum-stable cargo-lipid particles. The methods used to remove the organic solvent will typically involve evaporation at reduced pressures or blowing a stream of inert gas (e.g., nitrogen or argon) across the mixture.

Serum- stable cargo-lipid particles thus formed will typically be sized from about 50 nm to about 150 nm, from about 60 nm to about 130 nm, from about 70 nm to about 110 nm, or from about 70 nm to about 90 nm. To achieve further size reduction or homogeneity of size in the particles, sizing can be conducted as described above.

In other embodiments, the methods will further comprise adding non-lipid polycations which are useful to effect delivery to cells using the present compositions. Examples of suitable non-lipid polycations include, but are limited to, hexadimethrine bromide (sold under the brand name POLYBRENE®, from Aldrich Chemical Co., Milwaukee, Wis., USA) or other salts of heaxadimethrine. Other suitable polycations include, for example, salts of poly-L-ornithine, poly-L-arginine, poly-L-lysine, poly-D- lysine, polyallylamine, and polyethyleneimine.

In certain embodiments, the formation of the cargo-lipid particles can be carried out either in a mono-phase system (e.g., a Bligh and Dyer monophase or similar mixture of aqueous and organic solvents) or in a two-phase system with suitable mixing.

When formation of the complexes is carried out in a mono-phase system, the cationic lipids and cargoes are each dissolved in a volume of the mono-phase mixture. Combination of the two solutions provides a single mixture in which the complexes form. Alternatively, the complexes can form in two-phase mixtures in which the cationic lipids bind to the cargo (which is present in the aqueous phase), and "pull" it into the organic phase.

In another embodiment, serum- stable cargo-lipid particles can be prepared as follows:

• (a) contacting cargoes with a solution comprising noncationic lipids and a

detergent to form a cargo-lipid mixture;

• (b) contacting cationic lipids (e.g., the peptide-modified cationic lipids as described herein) with the cargo-lipid mixture to neutralize a portion of the negative charge of the cargoes/lipids and form a charge-neutralized mixture of cargoes and lipids; and

• (c) removing the detergent from the charge-neutralized mixture to provide the cargo-lipid particles in which the cargoes are protected from degradation.

In one group of embodiments, the solution of non-cationic lipids and detergent is an aqueous solution. Contacting the cargoes with the solution of non-cationic lipids and detergent can be accomplished by mixing together a first solution of cargoes and a second solution of the lipids and detergent. One of skill in the art will understand that this mixing can take place by any number of methods, for example, by mechanical means such as by using vortex mixers. Optionally, the cargo solution is also a detergent solution. The amount of non-cationic lipid which is used in the present method can be determined based on the amount of cationic lipid used, and is typically of from about 0.2 to about 5 times the amount of cationic lipid, optionally from about 0.5 to about 2 times the amount of cationic lipid used.

In some embodiments, the cargoes are precondensed as described in, e.g., U.S. patent application Ser. No. 09/744, 103.

A cargo-lipid mixture thus formed can be contacted with cationic lipids (e.g., the peptide-modified cationic lipids as described herein) to neutralize a portion of the negative charge which is associated with the cargoes or lipids (or other polyanionic materials) present. The amount of cationic lipids used will often be sufficient to neutralize at least 50% of the negative charge of the cargo/lipids. Optionally, the negative charge will be at least 70% neutralized, or at least 90% neutralized. Cationic lipids which are useful in the present formulations, include, for example, DLinDMA and DLenDMA. These lipids and related analogs are described in U.S. Patent Publication No. 2006/0083780.

Contacting cationic lipids with a cargo-lipid formulation mixture can be accomplished by any of a number of techniques, optionally by mixing together a solution of the cationic lipid and a solution containing the cargo-lipid mixture. Upon mixing the two solutions (or contacting in any other manner), a portion of the negative charge associated with the cargo or lipid is neutralized. Nevertheless, the cargo can remain in an uncondensed state and acquire hydrophilic characteristics.

After cationic lipids have been contacted with the cargo-lipid mixture, the detergent (or combination of detergent and organic solvent) is removed, thus forming the cargo-lipid formulation particles. The methods used to remove the detergent will typically involve dialysis. When organic solvents are present, removal is typically accomplished by evaporation at reduced pressures or by blowing a stream of inert gas (e.g., nitrogen or argon) across the mixture.

The formulation particles thus formed can be sized from about 50 nm to several microns, about 50 nm to about 150 nm, from about 60 nm to about 130 nm, from about 70 nm to about 110 nm, or from about 70 nm to about 90 nm. To achieve further size reduction or homogeneity of size in the formulation particles, the cargo-lipid formulation particles can be sonicated, filtered, or subjected to other sizing techniques which are used in liposomal formulations and are known to those of skill in the art.

In other embodiments, the methods of making formulation particles of the invention can further comprise adding non-lipid polycations which are useful to effect lipofection of cells. Examples of non-lipid polycations include, hexadimethrine bromide (sold under the brandname POLYBRENE®, from Aldrich Chemical Co., Milwaukee, Wis., USA) or other salts of hexadimethrine. Other polycations include, for example, salts of poly-L-ornithine, poly-L-arginine, poly-L-lysine, poly-D-lysine, polyallylamine, and polyethyleneimine. Addition of these salts is optionally after initial formulation particles have been formed.

In one embodiment, the cargo is a DsiRNA as described herein; a cleavable lipid as described herein is added (e.g., as a non-cationic lipid, where appropriate); optionally, a cationic lipid of the formulation is DLindMA, DLenDMA, DODAC, DDAB, DOTMA, DOSPA, DMRIE, or combinations thereof; and an optional non-cationic lipid is ESM, DOPE, PEG-DAG, DSPC, DPPC, DPPE, DMPE, DOGS, monomethyl- phosphatidylethanolamine, dimethyl-phosphatidylethanolamine, DSPE, DEPE, SOPE, POPE, cholesterol, or combinations thereof (e.g., DSPC and PEG-DAA); and the organic solvent is methanol, chloroform, methylene chloride, ethanol, diethyl ether or

combinations thereof.

In one embodiment, the cargo-lipid formulation particles prepared according to the above-described methods are either net charge neutral or carry an overall charge which provides the particles with greater gene lipofection activity. Optionally, the cargo component of the particles is a nucleic acid (e.g., a DsiRNA) which interferes with the production of an undesired protein.

In certain embodiments, the formulation cargo (e.g., a DsiRNA of the formulation) is not substantially degraded after exposure of the formulation to a nuclease at 37° C (or, in the case of a peptide or protein cargo, after exposure of the formulation to a peptidase at 37° C) for at least 20, 30, 45, or 60 minutes; or after incubation of the particle in serum at 37° C for at least 30, 45, or 60 minutes.

Administration of Lipid-Based Formulations of the Invention

Serum-stable cargo (e.g., nucleic acid)-lipid formulation particles of the present invention can be used to introduce cargoes into cells. Accordingly, the present invention also provides methods for introducing one or more cargoes into cells. The methods are carried out in vitro or in vivo by first forming the formulation particles in a manner as described above and then contacting the particles with the cells for a period of time sufficient for delivery of the one or more cargoes to occur.

The lipid-based delivery compositions of the invention preferably comprise one or more of the modified cationic-peptide-containing lipids of the invention. The invention is based, at least in part, upon the discovery of a novel means by which to modify lipids, including for example, cationic lipids, through covalent attachment of a peptide or peptide-based moiety to the lipid (1) which carries a net positive charge and thereby imparts a net positive charge on the overall molecule and (2) which undergoes a conformational change under intracellular- triggering conditions. The peptide or peptide- based moiety imparts improved performance characteristics on the overall lipid as a lipid delivery vehicle for intracellular delivery of therapeutic agents, such as nucleic acids. The improved characteristics include, but are not limited to, enhanced internalization of the lipid delivery vehicles, and thus, the payloads therein, into target cells. The improved characteristics also include enhanced payload release properties of the lipid delivery vehicles whereby the therapeutic agent payloads, e.g., nucleic acid payloads, are more effectively delivered to the cytoplasm of target cells. The intracellular conditions capable of triggering the conformational change in the peptide moiety, and thus, the resultant beneficial effects on the characteristics of the lipid delivery vehicles of the invention, can include any suitable intracellular condition that may be met by the lipid delivery vehicles upon entry of the cell. This can include, for example, changes in pH, oxidation/reduction conditions, protonation/deprotonation conditions, enzymatic cleavage (e.g., via a lysosomal enzyme) or other like conditions that may trigger a conformational change in the peptide or peptide-like moiety, and in turn, imparting the improved characteristics on the lipids of the invention.

Cargo-lipid formulation particles of the present invention can be adsorbed to almost any cell type with which they are mixed or contacted. Once adsorbed, the particles can either be endocytosed by a portion of the cells, exchange lipids with cell membranes, or fuse with the cells. Transfer or incorporation of the cargo portion of the particle can take place via any one of these pathways. In particular, when fusion takes place, the particle membrane is integrated into the cell membrane and the contents of the particle combine with the intracellular fluid.

Cargo-lipid formulation particles of the present invention can be administered either alone or in a mixture with a pharmaceutically-acceptable carrier (e.g., physiological saline or phosphate buffer) selected in accordance with the route of administration and standard pharmaceutical practice. Generally, normal buffered saline (e.g., 135-150 mM NaCl) will be employed as the pharmaceutically-acceptable carrier. Other suitable carriers include, e.g., water, buffered water, 0.4% saline, 0.3% glycine, and the like, including glycoproteins for enhanced stability, such as albumin, lipoprotein, globulin, etc.

Additional suitable carriers are described in, e.g., REMINGTON'S PHARMACEUTICAL SCIENCES, Mack Publishing Company, Philadelphia, PA, 17th ed. (1985). As used herein, "carrier" includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like. The phrase

"pharmaceutically-acceptable" refers to molecular entities and compositions that do not produce an allergic or similar untoward reaction when administered to a human. A pharmaceutically-acceptable carrier is generally added following particle formation. Thus, after a formulation particle is formed, the particle can be diluted into pharmaceutically-acceptable carriers such as normal buffered saline. The concentration of particles in the pharmaceutical formulations can vary widely, i.e., from less than about 0.05%, usually at or at least about 2 to 5%, to as much as about 10 to 90% by weight, and will be selected primarily by fluid volumes, viscosities, etc., in accordance with the particular mode of administration selected. For example, the concentration may be increased to lower the fluid load associated with treatment. This may be particularly desirable in patients having atherosclerosis-associated congestive heart failure or severe hypertension. Alternatively, particles composed of irritating lipids may be diluted to low concentrations to lessen inflammation at the site of administration.

In vivo administration

Systemic delivery for in vivo therapy, i.e., delivery of a therapeutic cargo (e.g., nucleic acid) to a distal target cell via body systems such as the circulation, has been described for nucleic acid-lipid formulation particles such as those disclosed in PCT Publication No. WO 96/40964 and U.S. Patent Nos. 5,705,385; 5,976,567; 5,981,501; and 6,410,328. Certain formats provide a fully encapsulated cargo-lipid formulation particle that protects the cargo or combination of cargoes from nuclease degradation in serum, is nonimmunogenic, is small in size, and is suitable for repeat dosing. Additional detail regarding administration of pharmaceutical compositions of the instant invention is provided below.

In vitro administration

For in vitro applications, the delivery of cargoes (e.g., nucleic acids) can be to any cell grown in culture, whether of plant or animal origin, vertebrate or invertebrate, and of any tissue or type. In certain embodiments, the cells are animal cells, e.g., mammalian cells such as human cells.

Contact between the cells and a cargo -lipid formulation particle, when carried out in vitro, takes place in a biologically compatible medium. Concentrations of particles vary widely depending on the particular application, but are generally between about 1 μπιοΐ and about 10 mmol. Treatment of the cells with the cargo-lipid formulation particles is generally carried out at physiological temperatures (about 37°C) for periods of time of from about 1 to 48 hours, e.g., from about 2 to 4 hours.

In one group of embodiments, a cargo-lipid formulation particle suspension is added to 60-80% confluent plated cells having a cell density of from about 10 to about 10⁵ cells/ml, optionally about 2 x 10⁴ cells/ml. In certain embodiments, the concentration of the suspension added to the cells can be from about 0.01 to 0.2 μg/ml, optionally about 0.1 μg/ml.

Using an Endosomal Release Parameter (ERP) assay, the delivery efficiency of a lipid-based carrier system can be optimized. An ERP assay is described in detail in U.S. Patent Publication No. 2003/0077829. More particularly, the purpose of an ERP assay is to distinguish the effect of various cationic lipids and helper lipid components of lipid- based carrier systems (e.g., SNALPs) based on their relative effect on binding/uptake or fusion with/destabilization of the endosomal membrane. This assay allows one to determine quantitatively how each component of the lipid-based carrier system affects delivery efficiency, thereby optimizing the lipid-based carrier systems. Usually, an ERP assay measures expression of a reporter protein (e.g., luciferase, β-galactosidase, green fluorescent protein (GFP), etc.), and in some instances, a SNALP formulation optimized for an expression plasmid will also be appropriate for encapsulating the cargoes (e.g., nucleic acids) described herein. In other instances, an ERP assay can be adapted to measure downregulation of transcription or translation of a target sequence in the presence or absence of the nucleic acids described herein. By comparing the ERPs for each of the various lipid-based formulations, one can readily determine the optimized system, e.g., the lipid-based formulation that has the greatest uptake in the cell.

Cells for delivery of cargoes

The compositions and methods of the present invention can be used to treat a wide variety of cell types, in vivo and in vitro. Suitable cells include, e.g., hematopoietic precursor (stem) cells, fibroblasts, keratinocytes, hepatocytes, endothelial cells, skeletal and smooth muscle cells, osteoblasts, neurons, quiescent lymphocytes, terminally differentiated cells, slow or noncycling primary cells, parenchymal cells, lymphoid cells, epithelial cells, bone cells, and the like.

In vivo delivery of cargo-lipid formulation particles of the present invention is suited for targeting cells of any cell type. The methods and compositions can be employed with cells of a wide variety of vertebrates, including mammals, such as, e.g, canines, felines, equines, bovines, ovines, caprines, rodents (e.g., mice, rats, and guinea pigs), lagomorphs, swine, and primates (e.g. monkeys, chimpanzees, and humans).

To the extent that tissue culture of cells may be required, it is well-known in the art. For example, Freshney, Culture of Animal Cells, a Manual of Basic Technique, 3rd Ed., Wiley-Liss, New York (1994), Kuchler et al, Biochemical Methods in Cell Culture and Virology, Dowden, Hutchinson and Ross, Inc. (1977), and the references cited therein provide a general guide to the culture of cells. Cultured cell systems often will be in the form of monolayers of cells, although cell suspensions are also used.

Detection ofLipidic Formulations

In some embodiments, the cargo-lipid formulation particles are detectable in the subject at about 8, 12, 24, 48, 60, 72, or 96 hours, or 6, 8, 10, 12, 14, 16, 18, 19, 22, 24, 25, or 28 days after administration of the particles. The presence of the particles can be detected in the cells, tissues, or other biological samples from the subject. The particles may be detected, e.g., by direct detection of the particles; detection of the modified cargo {e.g., nucleic acid); where the cargo is a nucleic acid, detection of a nucleic acid that silences expression of a target sequence; detection of the target and/or target sequence of interest (i.e., by detecting expression or reduced expression of the target and/or sequence of interest), or a combination thereof. A cargo-lipid formulation comprising a peptide- modified lipid of the invention, when compared to a control formulation, results in at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or even 100% increase in the detection of cargo-lipid formulation particles, as measured by a detection method, e.g., fluorescent tag or PCR.

Detection of Particles

Cargo-lipid formulation particles can be detected using any methods known in the art. For example, a label can be coupled directly or indirectly to a component of the carrier system using methods well-known in the art. A wide variety of labels can be used, with the choice of label depending on sensitivity required, ease of conjugation with the carrier system component, stability requirements, and available instrumentation and disposal provisions. Suitable labels include, but are not limited to, spectral labels such as fluorescent dyes (e.g., fluorescein and derivatives, such as fluorescein isothiocyanate (FITC) and Oregon Green™; rhodamine and derivatives such Texas red, tetrarhodimine isothiocynate (TRITC), etc. , digoxigenin, biotin, phycoerythrin, AMCA, CyDyes™, and the like; radiolabels such as 3 H, 35

^JJS, ' C, 3 ^J2"P, 3 ^J3^JP, etc .; enzymes such as horseradish peroxidase, alkaline phosphatase, etc.; spectral colorimetric labels such as colloidal gold or colored glass or plastic beads such as polystyrene, polypropylene, latex, etc. The label can be detected using any means known in the art.

Detection of Cargoes

Cargoes can be detected and quantified herein by any of a number of means well- known to those of skill in the art. The detection of peptide and/or protein cargoes can be achieved, e.g., by antibody-based methods, such as ELISA, immunoprecipitation and Western analysis. The detection of nucleic acids proceeds by well-known methods such as Southern analysis, Northern analysis, gel electrophoresis, PCR, radiolabeling, scintillation counting, and affinity chromatography. Additional analytic biochemical methods such as spectrophotometry, radiography, electrophoresis, capillary

electrophoresis, high performance liquid chromatography (HPLC), thin layer

chromatography (TLC), and hyperdiffusion chromatography may also be employed for a cargo of a formulation of the invention.

For nucleic acid cargoes, the selection of a nucleic acid hybridization format is not critical. A variety of nucleic acid hybridization formats are known to those skilled in the art. For example, common formats include sandwich assays and competition or displacement assays. Hybridization techniques are generally described in, e.g., "Nucleic Acid Hybridization, A Practical Approach," Eds. Hames and Higgins, IRL Press (1985).

Sensitivity of a hybridization assays may be enhanced through use of a nucleic acid amplification system which multiplies the target nucleic acid being detected. In vitro amplification techniques suitable for amplifying sequences for use as molecular probes or for generating nucleic acid fragments for subsequent subcloning are known. Examples of techniques sufficient to direct persons of skill through such in vitro amplification methods, including the polymerase chain reaction (PCR) the ligase chain reaction (LCR), ζ)β- replicase amplification and other RNA polymerase mediated techniques (e.g., NASBA™) are found in Sambrook et al, In Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (2000); and Ausubel et al, SHORT PROTOCOLS IN

MOLECULAR BIOLOGY, eds., Current Protocols, Greene Publishing Associates, Inc. and John Wiley & Sons, Inc. (2002); as well as U.S. Patent No. 4,683,202; PCR Protocols, A Guide to Methods and Applications (Innis et al. eds.) Academic Press Inc. San Diego, CA (1990); Arnheim & Levinson (October 1, 1990), C&EN 36; The Journal Of NIH Research, 3:81 (1991); Kwoh et al., Proc. Natl. Acad. ScL USA, 86:1173 (1989); Guatelli et al., Proc. Natl. Acad. Sci. USA, 87: 1874 (1990); Lomell et al., J. Clin. Chem., 35: 1826

(1989) ; Landegren et al, Science, 241 :1077 (1988); Van Brunt, Biotechnology, 8:291

(1990) ; Wu and Wallace, Gene, 4:560 (1989); Barringer et al, Gene, 89: 117 (1990); and Sooknanan and Malek, Biotechnology, 13:563 (1995). Improved methods of cloning in vitro amplified nucleic acids are described in U.S. Pat. No. 5,426,039. Other methods described in the art are the nucleic acid sequence based amplification (NASBA™, Cangene, Mississauga, Ontario) and QP-replicase systems. These systems can be used to directly identify mutants where the PCR or LCR primers are designed to be extended or ligated only when a select sequence is present. Alternatively, the select sequences can be generally amplified using, for example, nonspecific PCR primers and the amplified target region later probed for a specific sequence indicative of a mutation.

Nucleic acids for use as probes, e.g., in vitro amplification methods, for use as gene probes, or as inhibitor components are typically synthesized chemically according to the solid phase phosphor amidite triester method described by Beaucage et al , Tetrahedron Letts., 22: 1859 1862 (1981), e.g., using an automated synthesizer, as described in Needham VanDevanter et al, Nucleic Acids Res., 12:6159 (1984). Purification of polynucleotides, where necessary, is typically performed by either native acrylamide gel electrophoresis or by anion exchange HPLC as described in Pearson et al, J. Chrom., 255: 137 149 (1983). The sequence of the synthetic polynucleotides can be verified using the chemical degradation method of Maxam and Gilbert (1980) in Grossman and Moldave (eds.) Academic Press, New York, Methods in Enzymology, 65:499.

An alternative means for determining the level of transcription of a nucleic acid/gene (e.g., target gene) is in situ hybridization. In situ hybridization assays are well- known and are generally described in Angerer et al., Methods Enzymol, 152: 649. In an in situ hybridization assay, cells are fixed to a solid support, typically a glass slide. If DNA is to be probed, the cells are denatured with heat or alkali. The cells are then contacted with a hybridization solution at a moderate temperature to permit annealing of specific probes that are labeled. The probes are optionally labeled with radioisotopes or fluorescent reporters.

Delivery of Formulations and Con jugation of Cargoes

In certain embodiments, the present invention relates to a method for treating a subject having or at risk of developing a disease or disorder. In such embodiments, a formulation of the invention can act as a novel therapeutic agent for controlling the disease or disorder. Where the formulation comprises a DsiRNA cargo, the method comprises administering a pharmaceutical composition of the invention to the patient (e.g., human), such that the expression, level and/or activity of a target RNA is reduced. The expression, level and/or activity of a polypeptide encoded by the target RNA might also be reduced by a DsiRNA of such formulations of the instant invention.

In the treatment of a disease or disorder, a formulation can be brought into contact with the cells or tissue exhibiting or associated with a disease or disorder. For example, a formulation comprising a DsiRNA substantially identical to all or part of a target RNA sequence, may be brought into contact with or introduced into a diseased, disease- associated or infected cell, either in vivo or in vitro. Similarly, a DsiRNA cargo substantially identical to all or part of a target RNA sequence may administered directly to a subject having or at risk of developing a disease or disorder.

Therapeutic use of formulations of the instant invention can involve use of formulations comprising multiple different cargoes. For example, two or more, three or more, four or more, five or more, etc. of the presently described cargoes {e.g., DsiRNAs) can be combined to produce a formulation that, e.g., targets multiple different regions of one or more target RNA(s). For DsiRNA formulations, a DsiRNA cargo may also be constructed such that either strand of the DsiRNA independently targets two or more regions of a target RNA. Use of multifunctional DsiRNA molecules that target more then one region of a target nucleic acid molecule is expected to provide potent inhibition of RNA levels and expression. For example, a single multifunctional DsiRNA cargo can target both conserved and variable regions of a target nucleic acid molecule, thereby allowing down regulation or inhibition of, e.g., different strain variants of a virus, or splice variants encoded by a single target gene.

For certain formulations of the invention, a cargo can be conjugated {e.g., for a DsiRNA cargo, at its 5' or 3' terminus of its sense or antisense strand) or otherwise formulated with another moiety {e.g. for a nucleic acid cargo, a non-nucleic acid moiety such as a peptide can also be formulated), e.g., an organic compound {e.g., a dye, cholesterol, or the like). Modifying cargoes in this way may improve cellular uptake or enhance cellular targeting activities of the cargo and/or derivatives thereof, as compared to a corresponding unconjugated cargo, are useful for tracing cargoes and/or their derivatives in the organism/cell, and/or can improve the stability of a cargo and/or its derivative, as compared to a corresponding unconjugated cargo.

A cargo-lipid formulation comprising a lipid of the invention, when compared to a control formulation, results in at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or even 100% increase in the detection of cargo in cargo-lipid formulation particles, as measured by a detection method, e.g., fluorescent tag or PCR.

Pharmaceutical Compositions

In certain embodiments, the present invention provides for a pharmaceutical composition comprising a peptide-modified lipid-based composition of the present invention. Such compositions can be suitably formulated and introduced into the environment of the cell by any means that allows for a sufficient portion of the inventive compositions to enter the cell to deliver a cargo/payload (e.g., for DsiRNA cargoes, to induce gene silencing, if it is to occur). Many formulations are known in the art and can be used so long as the inventive formulation gains entry to the target cells so that it can act. See, e.g., U.S. published patent application Nos. 2004/0203145 Al and 2005/0054598 Al. For example, the inventive formulation of the instant invention can be further formulated in buffer solutions such as phosphate buffered saline solutions and capsids. Cationic lipids, such as lipofectin (U.S. Pat. No. 5,705,188), cationic glycerol derivatives, and polycationic molecules, such as polylysine (published PCT International Application WO 97/30731), can be used within the formulations of the instant invention. Optionally, Oligofectamine, Lipofectamine (Life Technologies), NC388 (Ribozyme Pharmaceuticals, Inc., Boulder, Colo.), or FuGene 6 (Roche) may be employed, all of which can be used according to the manufacturer's instructions.

Such compositions can include the lipidic formulation and a pharmaceutically acceptable carrier. As used herein the language "pharmaceutically acceptable carrier" includes saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Supplementary active compounds can also be incorporated into the compositions.

A pharmaceutical composition is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite;

chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

Administration can be in any manner known in the art, e.g., by injection, oral administration, inhalation (e.g., intransal or intratracheal), transdermal application, or rectal administration. Administration can be accomplished via single or divided doses. The pharmaceutical compositions can be administered parenterally, i.e., intraarticularly, intravenously, intraperitoneally, subcutaneously, or intramuscularly. In some

embodiments, the pharmaceutical compositions are administered intravenously or intraperitoneally by a bolus injection (see, e.g., U.S. Patent No. 5,286,634). Intracellular cargo delivery has also been discussed in Straubringer et al., Methods Enzymol., 101: 512; Mannino et al, Biotechniques, 6: 682; Nicolau et aJ. , Crit. Rev. Ther. Drug Carrier Syst., 6:239 (1989); and Behr, Ace. Chem. Res., 26: 274. Still other methods of administering lipid-based therapeutics are described in, for example, U.S. Patent Nos. 3,993,754;

4,145,410; 4,235,871; 4,224,179; 4,522,803; and 4,588,578. The lipid-cargo formulation particles can be administered by direct injection at the site of disease or by injection at a site distal from the site of disease (see, e.g., Culver, HUMAN GENE THERAPY,

Mary Ann Liebert, Inc., Publishers, New York. pp.70-71). The formulations of the present invention, either alone or in combination with other suitable components, can be made into aerosols (i.e., they can be "nebulized") to be administered via inhalation (e.g., intranasally or intratracheally; see, Brigham et al., Am. J. Sci., 298: 278). Aerosol formulations can be placed into pressurized acceptable propellants, such as dichlorodifluoromethane, propane, nitrogen, and the like.

In certain embodiments, the pharmaceutical compositions may be delivered by intranasal sprays, inhalation, and/or other aerosol delivery vehicles. Methods for delivering nucleic acid compositions directly to the lungs via nasal aerosol sprays have been described, e.g., in U.S. Patent Nos. 5,756,353 and 5,804,212. Likewise, the delivery of drugs using intranasal microparticle resins and lysophosphatidyl-glycerol compounds (U.S. Patent 5,725,871) is also well-known in the pharmaceutical arts. Similarly, transmucosal drug delivery in the form of a polytetrafluoroetheylene support matrix is described in U.S. Patent No. 5,780,045.

Formulations suitable for parenteral administration, such as, for example, by intraarticular (in the joints), intravenous, intramuscular, intradermal, intraperitoneal, and subcutaneous routes, include aqueous and non-aqueous, isotonic sterile injection solutions, which can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and nonaqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. In the practice of this invention, formulations can be administered, for example, by intravenous infusion, orally, topically, intraperitoneally, intravesically, or intrathecally.

Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL.TM. (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, the

composition must be sterile and should be fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle, which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, optional methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an edible carrier. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules, e.g., gelatin capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash.

Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.

Typically, oral formulations may contain at least about 0.1% of the cargo (e.g., nucleic acid)-lipid formulation particles or more, although the percentage of the particles may, of course, be varied and may conveniently be between about 1% or 2% and about 60% or 70% or more of the weight or volume of the total formulation. For formulation particles of the invention, he amount of particles in each therapeutically useful

composition may be prepared in such a way that a suitable dosage will be obtained in any given unit dose of the compound. Factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, as well as other pharmacological considerations will be contemplated by one skilled in the art of preparing such

pharmaceutical formulations, and as such, a variety of dosages and treatment regimens may be desirable.

For administration by inhalation, the formulations are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer. Such methods include those described in U.S. Pat. No. 6,468,798.

Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active formulations are formulated into ointments, salves, gels, or creams as generally known in the art.

The formulations can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

The formulations can also be administered by transfection or infection using methods known in the art, including but not limited to the methods described in

McCaffrey et al. (2002), Nature, 418(6893), 38-9 (hydrodynamic transfection); Xia et al. (2002), Nature Biotechnol., 20(10), 1006-10 (viral-mediated delivery); or Putnam (1996), Am. J. Health Syst. Pharm. 53(2), 151-160, erratum at Am. J. Health Syst. Pharm. 53(3), 325 (1996).

In certain embodiments, the formulations can also be administered by any method suitable for administration of nucleic acid agents, such as a DNA vaccine. These methods include gene guns, bio injectors, and skin patches as well as needle-free methods such as the micro-particle DNA vaccine technology disclosed in U.S. Pat. No. 6,194,389, and the mammalian transdermal needle-free vaccination with powder-form vaccine as disclosed in U.S. Pat. No. 6,168,587. Additionally, intranasal delivery is possible, as described in, inter alia, Hamajima et al. (1998), Clin. Immunol. Immunopathol., 88(2), 205-10.

Liposomes (e.g., as described in U.S. Pat. No. 6,472,375) and microencapsulation can also be used. Biodegradable targetable microparticle delivery systems can also be used (e.g., as described in U.S. Pat. No. 6,471,996).

In certain aspects, the formulations are prepared with carriers that will protect the formulations against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Such formulations can be prepared using standard techniques. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811.

Formulations suitable for oral administration can consist of, e.g.,: (a) liquid solutions, such as an effective amount of the packaged cargo (e.g., nucleic acid) suspended in diluents such as water, saline, or PEG 400; (b) capsules, sachets, or tablets, each containing a predetermined amount of the cargo, as liquids, solids, granules, or gelatin; (c) suspensions in an appropriate liquid; and (d) suitable emulsions. Tablet forms can include one or more of lactose, sucrose, mannitol, sorbitol, calcium phosphates, corn starch, potato starch, microcrystalline cellulose, gelatin, colloidal silicon dioxide, talc, magnesium stearate, stearic acid, and other excipients, colorants, fillers, binders, diluents, buffering agents, moistening agents, preservatives, flavoring agents, dyes, disintegrating agents, and pharmaceutically compatible carriers. Lozenge forms can comprise the cargo in a flavor, e.g., sucrose, as well as pastilles comprising the cargo in an inert base, such as gelatin and glycerin or sucrose and acacia emulsions, gels, and the like containing, in addition to the cargo, carriers known in the art.

In another example of their use, cargo-lipid formulation particles can be incorporated into a broad range of topical dosage forms. For instance, a suspension containing cargo-lipid formulation particles of the invention can be formulated and administered as a gel, oil, emulsion, topical cream, paste, ointment, lotion, foam, mousse, and the like.

When preparing pharmaceutical preparations of the cargo-lipid formulation particles of the invention, it can be preferred to use quantities of the particles which have been purified to reduce or eliminate empty particles or particles with cargo (e.g., nucleic acid) associated with the external surface.

The methods of the present invention may be practiced in a variety of hosts.

Exemplary hosts include mammalian species, such as primates (e.g., humans and chimpanzees as well as other nonhuman primates), canines, felines, equines, bovines, ovines, caprines, rodents (e.g., rats and mice), lagomorphs, and swine.

The amount of particles administered will depend upon the ratio of cargo (e.g., nucleic acid) to lipid, the particular cargo used, the disease state being diagnosed, the age, weight, and condition of the patient, and the judgment of the clinician, but will generally be between about 0.01 and about 50 mg per kilogram of body weight, optionally between

8 10

about 0.1 and about 5 mg/kg of body weight, or about 10 -10 particles per administration (e.g., injection).

Toxicity and therapeutic efficacy of such formulations can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD₅₀ (the dose lethal to 50% of the population) and the ED₅₀ (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD₅₀/ED₅₀. Formulations which exhibit high therapeutic indices can be preferred. While formulations that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such formulations to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such formulations optionally lies within a range of circulating concentrations that include the ED₅₀ with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any formulation used in a method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC₅₀ (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography.

As defined herein, a therapeutically effective amount of formulation (i.e., an effective dosage) depends on the formulation selected. For instance, if a DsiRNA formulation is selected, single dose amounts (of either the formulation as a whole or of a cargo component of such formulation) in the range of approximately 1 pg to 1000 mg may be administered; in some embodiments, 10, 30, 100, or 1000 pg, or 10, 30, 100, or 1000 ng, or 10, 30, 100, or 1000 μg, or 10, 30, 100, or 1000 mg may be administered. In some embodiments, 1-5 g of the formulations can be administered. The formulations can be administered from one or more times per day to one or more times per week; including once every other day. The skilled artisan will appreciate that certain factors may influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of a protein, polypeptide, nucleic acid or antibody can include a single treatment or, optionally, can include a series of treatments.

It can be appreciated that the method of introducing formulations into the environment of the cell will depend on the type of cell and the make up of its environment. For example, when the cells are found within a liquid, one optional formulation is with a lipid formulation such as in lipofectamine and the formulations can be added directly to the liquid environment of the cells. Lipid formulations can also be administered to animals such as by intravenous, intramuscular, or intraperitoneal injection, or orally or by inhalation or other methods as are known in the art. When the formulation is suitable for administration into animals such as mammals and more specifically humans, the formulation is also pharmaceutically acceptable. Pharmaceutically acceptable

formulations for administering peptides, proteins and nucleic acids (e.g., oligonucleotides) are known and can be used. For suitable methods of introducing dsNA (e.g., DsiRNA agents), see U.S. published patent application No. 2004/0203145 Al.

Suitable amounts of a formulation must be introduced and these amounts can be empirically determined using standard methods. Typically, effective concentrations of individual formulations, or of individual cargoes of a formulation, in the environment of a cell will be about 50 nanomolar or less, 10 nanomolar or less, or compositions in which concentrations of about 1 nanomolar or less can be used. In another embodiment, methods utilizing a concentration of about 200 picomolar or less, and even a concentration of about 50 picomolar or less, about 20 picomolar or less, about 10 picomolar or less, or about 5 picomolar or less can be used in many circumstances.

The method can be carried out by addition of the formulations to any extracellular matrix in which cells can live provided that the formulation is formulated so that a sufficient amount of the cargo can contact and/or enter a cell to exert its effect. For example, the method is amenable for use with cells present in a liquid such as a liquid culture or cell growth media, in tissue explants, or in whole organisms, including animals, such as mammals and especially humans.

For dsRNA formulations, the level or activity of a target RNA can be determined by any suitable method now known in the art or that is later developed. It can be appreciated that the method used to measure a target RNA and/or the expression of a target RNA can depend upon the nature of the target RNA. For example, if the target RNA encodes a protein, the term "expression" can refer to a protein or the RNA/transcript derived from the target RNA. In such instances, the expression of a target RNA can be determined by measuring the amount of RNA corresponding to the target RNA or by measuring the amount of that protein. Protein can be measured in protein assays such as by staining or immunoblotting or, if the protein catalyzes a reaction that can be measured, by measuring reaction rates. All such methods are known in the art and can be used. Where target RNA levels are to be measured, any art-recognized methods for detecting RNA levels can be used (e.g., RT-PCR, Northern Blotting, etc.). In targeting viral RNAs with the DsiRNA agents of the instant invention, it is also anticipated that measurement of the efficacy of a dsRNA (e.g., DsiRNA) cargo in reducing levels of a target virus in a subject, tissue, in cells, either in vitro or in vivo, or in cell extracts can also be used to determine the extent of reduction of target viral RNA level(s). Any of the above measurements can be made on cells, cell extracts, tissues, tissue extracts or any other suitable source material.

The determination of whether the expression of a target RNA has been reduced can be by any suitable method that can reliably detect changes in RNA levels. Typically, the determination is made by introducing into the environment of a cell undigested DsiRNA such that at least a portion of that DsiRNA agent enters the cytoplasm, and then measuring the level of the target RNA. The same measurement is made on identical untreated cells and the results obtained from each measurement are compared.

Formulations of the invention can be made as a pharmaceutical composition which comprises a pharmacologically effective amount of a cargo (e.g., peptide, protein, nucleic acid, etc.) and pharmaceutically acceptable carrier. A pharmacologically or

therapeutically effective amount refers to that amount of a cargo effective to produce the intended pharmacological, therapeutic or preventive result. The phrases

"pharmacologically effective amount" and "therapeutically effective amount" or simply "effective amount" refer to that amount of a cargo effective to produce the intended pharmacological, therapeutic or preventive result. For example, if a given clinical treatment is considered effective when there is at least a 20% reduction in a measurable parameter associated with a disease or disorder, a therapeutically effective amount of a drug for the treatment of that disease or disorder is the amount necessary to effect at least a 20% reduction in that parameter.

Suitably formulated pharmaceutical compositions of this invention can be administered by any means known in the art such as by parenteral routes, including intravenous, intramuscular, intraperitoneal, subcutaneous, transdermal, airway (aerosol), rectal, vaginal and topical (including buccal and sublingual) administration. In some embodiments, the pharmaceutical compositions are administered by intravenous or intraparenteral infusion or injection.

In general, a suitable dosage unit of formulation and/or the cargo of a formulation will be in the range of 0.001 to 0.25 milligrams per kilogram body weight of the recipient per day, or in the range of 0.01 to 20 micrograms per kilogram body weight per day, or in the range of 0.01 to 10 micrograms per kilogram body weight per day, or in the range of 0.10 to 5 micrograms per kilogram body weight per day, or in the range of 0.1 to 2.5 micrograms per kilogram body weight per day. Pharmaceutical composition comprising the cargo can be administered once daily. However, the therapeutic cargo may also be dosed in dosage units containing two, three, four, five, six or more sub-doses administered at appropriate intervals throughout the day. In that case, the cargo contained in each sub- dose must be correspondingly smaller in order to achieve the total daily dosage unit. The dosage unit can also be compounded for a single dose over several days, e.g., using a conventional sustained release formulation which provides sustained and consistent release of the cargo over a several day period. Sustained release formulations are well known in the art. In this embodiment, the dosage unit contains a corresponding multiple of the daily dose. Regardless of the formulation, the pharmaceutical composition must contain cargo in a quantity sufficient to exert its intended effect (e.g., for a dsRNA cargo, to inhibit expression of the target gene) in the animal or human being treated. The formulation can be compounded in such a way that the sum of the multiple units of cargo together contain a sufficient dose.

Data can be obtained from cell culture assays and animal studies to formulate a suitable dosage range for humans. The dosage of formulations of the invention lies within a range of circulating concentrations that include the ED₅₀ (as determined by known methods) with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any formulation used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range of the compound that includes the IC₅₀ (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels of formulation and/or cargo in plasma may be measured by standard methods, for example, by high performance liquid chromatography.

The pharmaceutical compositions can be included in a kit, container, pack, or dispenser together with instructions for administration.

Methods of Treatment

The present invention provides for both prophylactic and therapeutic methods of treating a subject at risk of (or susceptible to) a disease or disorder. In certain aspects, the disease or disorder is caused, in whole or in part, by the expression of a target RNA and/or the presence of such target RNA (e.g., in the context of a viral infection, the presence of a target RNA of the viral genome, capsid, host cell component, etc.).

"Treatment", or "treating" as used herein, is defined as the application or administration of a therapeutic agent to a patient, or application or administration of a therapeutic agent to an isolated tissue or cell line from a patient, who has the disease or disorder, a symptom of disease or disorder or a predisposition toward a disease or disorder, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve or affect the disease or disorder, the symptoms of the disease or disorder, or the predisposition toward disease.

Another aspect of the invention pertains to methods of treating subjects

therapeutically, i.e., alter onset of symptoms of the disease or disorder. These methods can be performed in vitro (e.g., by culturing the cell with the lipid-based cargo delivery formulation) or, alternatively, in vivo (e.g., by administering the lipid-based cargo delivery formulation to a subject). An effective amount of the pharmaceutical composition for treatment is one that, when compared to a control results in a delay in the symptoms or onset of symptoms by at least 5%, 10%, 25%, 50%, 70%, 80%, 90%, 95%, 99%, or 100%.

Another aspect of the invention pertains to methods of treating subjects

therapeutically, i.e., alter onset of symptoms of the disease or disorder. These methods can be performed in vitro (e.g., by culturing the cell with the immune response reducing formulation) or, alternatively, in vivo (e.g., by administering the immune response reducing formulation to a subject). An effective amount of the pharmaceutical

composition for treatment is one that, when compared to a control results in a delay in the symptoms or onset of symptoms by at least 5%, 10%, 25%, 50%, 70%, 80%, 90%, 95%, 99%, or 100%.

With regard to both prophylactic and therapeutic methods of treatment, such treatments may be specifically tailored or modified, based on knowledge obtained from the field of pharmacogenomics. "Pharmacogenomics", as used herein, refers to the application of genomics technologies such as gene sequencing, statistical genetics, and gene expression analysis to drugs in clinical development and on the market. More specifically, the term refers the study of how a patient's genes determine his or her response to a drug (e.g., a patient's "drug response phenotype", or "drug response genotype"). Thus, another aspect of the invention provides methods for tailoring an individual's prophylactic or therapeutic treatment with either the target RNA molecules of certain lipid-based cargo delivery formulations of the present invention or target RNA modulators according to that individual's drug response genotype. Pharmacogenomics allows a clinician or physician to target prophylactic or therapeutic treatments to patients who will most benefit from the treatment and to avoid treatment of patients who will experience toxic drug -related side effects.

Therapeutic agents can be tested in an appropriate animal model. For example, a lipid-based cargo delivery formulation as described herein can be used in an animal model to determine the efficacy, toxicity, or side effects of treatment with the lipid-based cargo delivery formulation. Alternatively, a therapeutic agent can be used in an animal model to determine the mechanism of action of such an agent. For example, an agent can be used in an animal model to determine the efficacy, toxicity, or side effects of treatment with such an agent. Alternatively, an agent can be used in an animal model to determine the mechanism of action of such an agent.

The practice of the present invention employs, unless otherwise indicated, conventional techniques of chemistry, molecular biology, microbiology, recombinant DNA, genetics, immunology, cell biology, cell culture and transgenic biology, which are within the skill of the art. See, e.g., Maniatis et al., 1982, Molecular Cloning (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.); Sambrook et al., 1989, Molecular Cloning, 2nd Ed. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.); Sambrook and Russell, 2001, Molecular Cloning, 3rd Ed. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.); Ausubel et al., 1992), Current Protocols in Molecular Biology (John Wiley & Sons, including periodic updates); Glover, 1985, DNA Cloning (IRL Press, Oxford); Anand, 1992; Guthrie and Fink, 1991; Harlow and Lane, 1988, Antibodies, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.); Jakoby and Pastan, 1979; Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins eds. 1984); Transcription And Translation (B. D. Hames & S. J. Higgins eds. 1984); Culture Of Animal Cells (R. I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide To Molecular Cloning (1984); the treatise, Methods In Enzymology (Academic Press, Inc., N.Y.); Gene Transfer Vectors For Mammalian Cells (J. H. Miller and M. P. Calos eds., 1987, Cold Spring Harbor

Laboratory); Methods In Enzymology, Vols. 154 and 155 (Wu et al. eds.),

Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (D. M. Weir and C. C. Blackwell, eds., 1986); Riott, Essential Immunology, 6th Edition, Blackwell Scientific Publications, Oxford, 1988; Hogan et al., Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986);

Westerfield, M., The zebrafish book. A guide for the laboratory use of zebrafish (Danio rerio), (4th Ed., Univ. of Oregon Press, Eugene, 2000).

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

EXAMPLES

The present invention is described by reference to the following Examples, which are offered by way of illustration and are not intended to limit the invention in any manner. Standard techniques well known in the art or the techniques specifically described below were utilized.

Example I: Preparing a peptide-modified cationic lipid of the invention

Synthesis schemes below are exemplified with AzPC as the lipid; however the invention is not limited to AzPC. Similarly, synthesis schemes below are exemplified with a poly(Arginine-Histidine) peptide; however the invention is not limited to this peptide. Solid Phase Synthesis

Peptide was synthesized using Fmoc chemistry on a synthesis resin. AzPC was either purchased from Avanti (Alabaster, Alabama) or synthesized by condensing lysophosphatidyl choline and azelaic acid. A round bottom flask was charged with resin- bound peptide. A mixture of 50:50 DCM (Dichloromethane; Methylene Chloride) /THF (Tetrahydrofuran) was added to the flask in addition to carbonyldiimidazole. To this a solution of AzPC in DCM was added. Triethylamine was then added and the mixture was stirred at room temperature. The resin was then filtered to remove solvent. The reaction completion was assessed by a negative ninhydrin test.

Once the above conjugation reaction was complete, resin was cleaved from the product following standard procedures. After addition of cold diethylether to the cleavage mixture, a white powder precipitated out of solution. The solvent was decanted from the solid and the solid was washed with additional amount of cold ether. The solid was taken up in actonitrile and the solvent was removed by rotary evaporation. Finally, the residue was taken up in water and recovered by lyophilization to yield a white powder. Stable Peptide-AzPC Conjugation Scheme.

Abbreviations:

DCC: Dicyclohexyl carbodiimide DMAP: 4-Dimethylaminopyridine CHC1₃: Chloroform

Solution Phase Synthesis.

Peptide was synthesized using Fmoc chemistry with 6-Hydrazinonicotinamide (HyNic) synthon at the N-terminal of the peptide. The peptide was cleaved from the resin support following standard procedures. AzPC was reacted with Peg3-4FB (4- Formylbenzamide) in the presence of NHS (N-hydroxy succinimide), EDC (Ethylene Dichloride (1,2-Dichloroethane)) to produce AzPC-Peg3-4FB. HyNic -peptide and 4FB- AzPC (S-S-4FB, in case of disulfide cleavable conjugate) was then reacted in a mixture of 50:50 water/methanol to produce peptide- AzPC conjugate. Stable and cleavable conjugate were synthesized based on the 4-FB used in the first step of the reaction.

Stable Peptide-AzPC Conjugation Scheme.

Peptide- AzPC/Peg3 conjugate Cleavable (Disulfide) Peptide-AzPC Conjugation Scheme.

Peptide-AzPC/Peg3/disulfide conjugate

These above schemes were used to form the following exemplary modified lipids of the invention:

Table 4. A Select List of Novel Cationic Lipids of the Current Invention.

linker

Example 2: Demonstration of Improved Delivery

Polyanionic payload and novel cationic lipid formulations are transfected in vitro in cell culture models to assess uptake or delivery. Appropriate cell culture models are utilized and end point measurements include, but are not limited to, one or more of the following: (i) mRNA quantification using qPCR; (ii) protein quantification using Western blot; (iii) labeled cell internalization of polyanionic payload and novel cationic lipids. Uptake or deliveries of the polyanionic payload and novel cationic lipid formulations are assessed for both the extent and duration of the above mentioned end points. In one example, transfection is performed in 24- or 48- well plates for transfecting polyanionic payload and novel cationic lipid formulations into HeLa cells. Prior to application, polyanionic payload and novel cationic lipid formulations are diluted to the cell culture media at room temperature for about 30 min. For dose-response experiments, the final concentration of polyanionic payload and novel cationic lipid formulations applied are varied within a range of 0 to 50 nM. For the time-course experiment, an optimum concentration from the dose-experiment is studied for various incubation times, e.g., 30 min to 7 days.

Functionality of polyanionic payload and novel cationic lipid formulations is also tested by differentially labeling the lipid and the polyanionic payload with fluorescent tags and performing fluorescent colocalization studies. Using this methodology and comparison with the formulations without the novel cationic lipid confirms the ability of the lipid to facilitate delivery of polyanionic payload and novel cationic lipid formulations. Novel cationic lipids' ability to deliver fluorescent label attached or polyanionic payload are measured by both measuring the total fluorescence inside the cell, measuring the fluorescent that is not stably associated with endosomal or lysosomal compartment as polyanionic payloads need to not only reach inside the cell, but also to reach cytoplasm of the cell to trigger RNAi. Conducting fluorescent localization and cellular trafficking studies are described in the art (Lu, Langer and Chen. Mol Pharm. 2009; McNaughton et al., Proc Natl Acad Sci U S A. 2009).

Example 3. Improvement Over Non-Modified Lipids

In one embodiment the achieved improvement of functionalization of the polyanionic payload and novel cationic lipid formulations compared to polyanionic payload formulation alone is about 25%. In another embodiment the achieved

improvement of functionalization of the polyanionic payload and novel cationic lipid formulations compared to polyanionic payload formulation alone is about 100%, i.e., the polyanionic payload and novel cationic lipid formulations show about 2-fold delivery compared to polyanionic payload formulation alone. In another embodiment the polyanionic payload and novel cationic lipid formulations show about 5-fold delivery compared to polyanionic payload formulation alone. In another embodiment the polyanionic payload and novel cationic lipid formulations show about 10-fold delivery compared to polyanionic payload formulation alone. In another embodiment the polyanionic payload and novel cationic lipid formulations show about 100-fold delivery compared to polyanionic payload formulation alone. In another embodiment the polyanionic payload and novel cationic lipid formulations show about 1000-fold delivery compared to polyanionic payload formulation alone.

Example 4: Preparation of a novel cationic lipid formulation of a dsRNA

Preferred target dsRNA agents are selected from a pre-screened population of dsRNAs. Design of dsRNAs can optionally involve use of predictive scoring algorithms that perform in silico assessments of the projected activity/efficacy of a number of possible dsRNAs spanning a region of sequence.

A dsRNA of the invention is formulated with novel cationic lipid formulations by any of the methods described herein above. The final formulation is characterized for particle size, polydispersity, surface charge, dsRNA entrapment and dsRNA content.

Example 5: Use of a novel cationic lipid formulation of a dsRNA to reduce expression of a target gene in a cell

Cell culture and RNA Transfection

HeLa cells are obtained from ATCC and maintained in Dulbecco's modified Eagle medium (HyClone) supplemented with 10% fetal bovine serum (HyClone) at 37 °C under 5% C0₂. For novel cationic lipid formulation of a dsRNA transfections, HeLa cells are incubated with the above mentioned formulations as indicated at a final concentration of 1 nM or 0.1 nM. Lipofectamine™ RNAiMAX (Invitrogen) dsRNAs are used as positive controls. Briefly,

of a 0.2 μΜ or 0.02 μΜ stock solution of each dsRNAs is mixed with 47.5μί of Opti-MEM I (Invitrogen). For Lipofectamine™ control, 2.5μί of a 0.2 μΜ or 0.02 μΜ stock solution of each dsRNAs is mixed with 46.5μΕ of Opti-MEM I

(Invitrogen) and

of Lipofectamine™ RNAiMAX. The resulting mix is added into individual wells of 12 well plates and incubated for 20 min at RT to allow

dsRNA:Lipofectamine™ RNAiMAX complexes to form. Meanwhile, HeLa cells are trypsinized and resuspended in medium at a final concentration of about 367 cells^L. Finally, 450μί of the cell suspension are added to each well (final volume 500μί) and plates are placed into the incubator for 24 hours. For dose response study, the

concentrations of dsRNAs are varied from initially 1 pM to 10 nM. For time course study, the incubation time of about 4 hours to about 72 hours are studied.

Assessment of Inhibition

Target gene knockdown is determined by qRT-PCR, with values normalized to HPRT expression control treatments, including Lipofectamine™ RNAiMAX alone (Vehicle control) or untreated.

RNA isolation and analysis Cells are washed once with 2mL of PBS, and total RNA was extracted using RNeasy Mini Kit™ (Qiagen) and eluted in a final volume of 30μί. ^g of total RNA is reverse-transcribed using Transcriptor 1^st Strand cDNA Kit™ (Roche) and random hexamers following manufacturer's instructions. One-thirtieth (0.66μί) of the resulting cDNA is mixed with 5μί of IQ Multiplex Powermix (Bio-Rad) together with 3.33μί of H₂0 and Ιμί of a 3μΜ mix containing primers and probes specific for human genes HPRT-1 (accession number NM_000194) and KRAS target sequences.

Quantitative RT-PCR

A CFX96 Real-time System with a CIOOO Thermal cycler (Bio-Rad) is used for the amplification reactions. PCR conditions are: 95°C for 3min; and then cycling at 95°C, lOsec; 55°C, lmin for 40 cycles. Each sample is tested in triplicate. Relative HPRT mRNA levels are normalized to target mRNA levels and compared with mRNA levels obtained in control samples treated with the transfection reagent alone, or untreated. Data are analyzed using Bio-Rad CFX Manager version 1.0 software. Expression data are presented as a comparison of the expression under the treatment of novel cationic lipid formulation of dsRNA vs the dsRNA formulation without the novel cationic lipid.

Example 6: Use of novel cationic lipid formulation of a dsRNA to reduce expression of a target gene in a subcutaneous animal tumor model

In order to assess the efficiency of delivery and subsequent functionality of the novel cationic lipid formulation of a dsRNA, we utilized subcutaneous (s.c.) tumor models (Judge et al., J Clin Invest. 2009; 119(3):661-73). Hep3B tumors are established in female SCID/beige mice by s.c. injection of 3 x 10⁶ cells in 50 μί_^ PBS into the left-hind flank. Mice are randomized into treatment groups 10-17 days after seeding as tumors became palpable. Novel cationic lipid formulation of a dsRNA or vehicle control is administered by standard intravenous (i.v.) injection via the lateral tail vein, calculated based on a mg dsRNAs/kg body weight basis according to individual animal weights. Tumors are measured in 2 dimensions (width x length) to assess tumor growth using digital calipers. Tumor volume is calculated using the equation x * y * y/2, where x = largest diameter and y = smallest diameter, and is expressed as group mean + SD. Tumor tissues are also removed from the animals of different treatment groups and gene knockdown is confirmed. Tumor volume, survival and RNA expression data are presented as a comparison between the treatments of novel cationic lipid formulation of dsRNA vs the dsRNA formulation without the novel cationic lipid. Example 7: Use of a novel cationic lipid formulation of dsRNA to reduce expression of a target gene in an orthotopic animal tumor model

In order to assess the efficiency of targeting and subsequent functionality of the novel cationic lipid formulation of dsRNA, we utilized intrahepatic tumor models (Judge et al., J Clin Invest. 2009; 119(3):661-73). Liver tumors are established in mice by direct intrahepatic injection of Hep3B or Neuro2a tumor cells. Female SCID/beige mice and male A/J mice are used as hosts for the Hep3B and Neuro2a tumors, respectively.

Maintaining the mice under gas anesthesia, a single 1.5-cm incision across the midline is made below the sternum, and the left lateral hepatic lobe is exteriorized. 1 x 10⁶ Hep3B cells or 1 x 10⁵ Neuro2a cells suspended in 25 μί_^ PBS are injected slowly into the lobe at a shallow angle using a Hamilton syringe and a 30-gauge needle. A swab is then applied to the puncture wound to stop any bleeding prior to suturing. Mice are allowed to recover from anesthesia in a sterile cage and monitored closely for 2-4 hours before being returned to conventional housing. Eight to eleven days after tumor implantation, mice are randomized into treatment groups: novel cationic lipid formulation of dsRNA, dsRNA formulation without the novel cationic lipid or vehicle control is administered by standard intravenous (i.v.) injection via the lateral tail vein, calculated based on a mg dsRNAs/kg body weight basis according to individual animal weights. Body weights are monitored throughout the duration of the study as an indicator of developing tumor burden and treatment tolerability. For efficacy studies, defined humane end points are determined as a surrogate for survival. Assessments are made based on a combination of clinical signs, weight loss, and abdominal distension to define the day of euthanization due to tumor burden. Tumor tissues are removed from the animals of different treatment groups and gene knockdown is confirmed.

Functionality of novel cationic lipid formulation of dsRNA for tumor cell uptake are also tested by labeling the lipid, dsRNA with fluorescent tags and performing fluorescent biodistribution studies using a live-animal imaging system (Xenogen or BioRad) (Eguchi et al., Nat Biotechnol. 2009; 27(6):567-71). Using this methodology, and by comparing with dsRNA formulation alone the ability of the novel cationic lipid to facilitate tumor cell internalization for novel cationic lipid formulation of dsRNA is confirmed. By contrast, dsRNA formulation alone, used as a control in this study, is unable to be taken up and delivered to the same extent to tumor surface. Efficacy end points, RNA expression and biodistribution data are presented as a comparison between the treatments of novel cationic lipid formulation of dsRNA vs the dsRNA formulation without the novel cationic lipid.

Example 8: Assay to evaluate formation of complex between a nucleic acid payload and lipid composition of the invention

This Example demonstrates an approach to evaluate the formation of nucleic acid / lipid complexes of the invention. Formation of a nucleic acid/lipid complex may be detected by the retardation of the nucleic acids on a polyacrylamide gel as compared to the noncomplexed nucleic acids.

Specifically, DsiRNA (either in nuclease-free water or in 50 mM citrate buffer, pH 3.5) was incubated with the cationic lipid AzPC-PEG8-G(R)9S (l-palmitoyl-2-azelaoyl- sn-glycero-3-phosphocholine (16:0-09:0 (COOH) PC or "AzPC" which is modified with PEG8 and the peptide moiety, G(R)₉S) or the cationic lipid AzPC-PEG8-G(RH)₄RS (1- palmitoyl-2-azelaoyl-sn-glycero-3-phosphocholine (16:0-09:0 (COOH) PC or "AzPC" which is modified with PEG8 and the peptide moiety, G(RH)₄RS), at different N/P ratios (nucleic acid / lipid-peptide ratios) at 4°C for about 2 hours. After incubation, 5 μΐ_^ of sample was mixed with 4 μL· H₂0 and 6 μΐ_^ gel loading buffer. The samples were electrophoresed in 18% TRIS-HCl pre-cast gel and stained with SYBR-Gold. The resolved siRNA bands were imaged using VersaDoc imaging system (Bio-Rad model# 4000MP). The synthesis of AzPC-PEG8-G(R)₉S or AzPC-PEG8-G(RH)₄RS may be conducted in accordance with Example 1.

Figure 1A shows the results of the gel retardation assay for complex formation between DsiRNA and AzPC-PEG8-G(R)gS at differing N/P ratios in nuclease-free water. AzPC-PEG8-G(R)9S lacking His residue demonstrated pH-independent binding as shown by gel retardation.

Figure IB shows the results of the gel retardation assay for complex formation between DsiRNA and AzPC-PEG8-G(RH)₄RS at differing N/P ratios in nuclease-free water.

Figure 1C shows the results of the gel retardation assay for complex formation between DsiRNA and AzPC-PEG8-G(RH)₄RS at differing N/P ratios in 50 mM citrate buffer at pH 3.5.

AzPC-PEG8-G(RH)₄RS contains His residues and demonstrated pH-dependent binding (i.e., no binding and gel retardation at neutral pH, but binding at low pH.

Conclusion: AzPC-PEG8-G(RH)4RS is capable of binding DsiRNA in a pH- dependent fashion. Example 9: Size exclusion chromatography (SEC) assay to evaluate formation of complex between a nucleic acid payload and lipid composition of the invention

This Example demonstrates an approach to evaluate the formation of nucleic acid / lipid complexes of the invention. Formation of a nucleic acid/lipid complex may be detected by the retardation of the encapsulated or complexed nucleic acids through a size exclusion chromatography column as compared to the noncomplexed nucleic acids or free nucleic acids.

The HPLC system used in this Example was as follows:

• Separation module: Waters 2795

• Detector: Waters 2998 - Photodiode Array Detector

• Column: SRT SEC-300 (Sepax Technologies) (4.6 x 300 mm; 5μπι) (Pore size = 300

A)

• Solvent system: PBS

The sample information was performed as follows:

• Injection volume: 50μ1

• Run time: 20 min

• Detector: UV at 260nm

• De-formulation buffer consisted of Heparin sodium (20 mg/mL) + Triton X-100 (1%)

• 20 μΐ_^ sample was mixed with either 80 μΐ_^ water or 80 μΐ_^ de-formulation buffer

• w/w ratio of Heparin sodium to AzPC-PEG8-G(RH)₄RS in de-formulation sample was

-10:1

Lipid nanoparticles were prepared using the cationic lipid, AzPC-PEG8-G(RH)₄RS (prepared in accordance with Example 1), and a nucleic acid payload, DsiRNA as described in this invention. Lipid / nucleic acid complexes or aggregates were prepared by combining about 1 mg DsiRNA formulated with about 6.8 mg of AzPC-PEG8-G(RH)₄RS cationic lipid and about 4 mg non-cationic lipids. Mole ratio of non-cationic lipids were DPPC:Cholesterol:PEG2000-DMPE = 35:62:3. The samples were prepared either in water, or in de-formulation buffer (Heparin sodium (20 mg/mL) + Triton X-100 (1% v/v)). The samples were subjected to SEC analysis. The results are shown in Figure 2.

Conclusion: AzPC-PEG8-G(RH)₄RS is capable of binding and encapsulating DsiRNA in a lipid nanoparticle formulation as is apparent from the greater retention of the complexed DsiRNA versus the free DsiRNA. EXAMPLE 10. Prophetic example to test the effectiveness of the lipid/payload compositions of the invention.

The effectiveness of therapeutic payloads that are deliverable to a subject as a complex with a lipid composition of the invention may be tested by any suitable known assay. For example, in the case of a DsiRNA payload (a Dicer substrate which will trigger the RNAi pathway for inhibition of a gene of interest), a gene knockdown experiment may be conducted under in vivo or in vitro conditions.

A DsiRNA payload is first formed into a lipid-based aggregate of the invention by combining the payload and a lipid composition of the invention. The lipid composition will comprise at least one peptide-modified cationic lipid of the invention. The lipid is designed with a peptide moiety as described herein which will undergo a conformational change upon contacting the intracellular environment of a cell. The conformational change will trigger an effective release of the payload from the aggregate. The overall effectiveness of the entire process will be a function of various factors, such as (1) whether there is enzymatic and non-enzymatic degradation of the payload, (2) the serum half-life and biodistribution of the lipid aggregate, (3) whether there is cellular uptake or internalization of the lipid aggregate into a target cell and (4) whether there is an effective release of the payload from the aggregate in the cell.

The overall effectiveness of this process can be evaluated by any number of approaches for monitoring the knockdown level in the expression of the gene targeted by the the particular DsiRNA sequence. For example, the knockdown effect may be analyzed by monitoring the mRNA level of the target gene by real-time PCT or northern-blot analysis, and by examining protein levels using western-blot analysis or by performing appropriate functional assays. Effective knockdown of a target gene will correspond with an overall effectiveness of the administration and delivery of the payload/lipid complex of the invention.

REFERENCES:

All patents, publications, and other references cited throughout this specification are incorporated herein by reference in their entirety.

1. Castanotto and Rossi, Nature 2009; 457: 426-433. 2. Lehninger Principles of Biochemistry, 3rd Ed., 2000. Edited by David L. Nelson and Michael M. Cox, Worth Publishers, New York, NY.

3. Lu, Langer and Chen. Mol Pharm. 2009; 6(3):763-71.

4. McNaughton et al., Proc Natl Acad Sci U S A. 2009 Apr 14; 106(15):6111-6116.

5. Judge et al., J. Clin. Invest. 2009; 119(3):661-673.

6. Eguchi et al., Nat Biotechnol. 2009; 27(6):567-71.

7. Yagi et al, Cancer Res. 2009; 69(16):6531-8.

8. Abrams et al., Mol Ther. 2009 Sep 8. [Epub ahead of print] .

9. Ko et al., J Control Release. 2009 Jan 19; 133(2): 132-8.

10. Noble et al., Cancer Chemother Pharmacol. 2009; 64(4):741-51.

11. Mangala et al., Methods Mol Biol. 2009; 555:29-42.

Claims

WHAT IS CLAIMED IS:

1. A lipid comprising a headgroup moiety and a tail moiety, wherein the tail moiety comprises a cationic peptide-based moiety that is capable of undergoing a conformational change upon contacting an intracellular condition.

2. The lipid of claim 1, wherein the lipid has an overall net positive charge.

3. The lipid of claim 1, wherein the headgroup moiety is negatively charged, neutral, or positively charged.

4. The lipid of claim 1, wherein the headgroup moiety is a headgroup from a fatty acid, glycerolipid, glycerophospholipid, sphingolipid, or saccharolipid.

5. The lipid of claim 4, wherein the glycerolipid is a triacylglyceride or

glyco s ylglycerol .

6. The lipid of claim 4, wherein the glycerophospholipid is a phospholipid, phosphatidylcholine (PC), lecithin, phosphatidylethanolamine (PE) or phosphatidylserine (PS).

7. The lipid of claim 4, wherein the sphingolipid is a ceramide, phospho sphingolipid, glyco sphingolipid, sphingomyelin, or ceramide phosphocholine.

8. The lipid of claim 4, wherein the saccharolipid is Lipid A.

9. The lipid of claim 1, wherein the lipid is a noncationic lipid selected from the group consisting of lecithin, phosphatidylethanolamine, lysolecithin,

lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphatidic acid, cerebrosides, dicetylphosphate, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipahnitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoyl-phosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), palmitoyloleyol-phosphatidylglycerol (POPG),

dioleoylphosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane- 1 -carboxylate (DOPE-mal), dipalmitoyl-phosphatidylethanolamine (DPPE), dimyristoyl- phosphatidylethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), monomethyl-phosphatidylethanolamine, dimethyl -phosphatidylethanolamine, dielaidoyl- phosphatidylethanolamine (DEPE), DOGS, and stearoyloleoyl-phosphatidylethanolamine (SOPE).

10. The lipid of claim 1, wherein the lipid is a cationic lipid selected from the group consisting of N, N-dioleyl-N, N-dimethylammonium chloride (DODAC), N, N-distearyl- N, N-dimethylammonium bromide (DDAB), N-(l-(2, 3-dioleoyloxy)propyl)-N, N, N- trimethylammonium chloride (DOTAP), l,2-dioleoyl-3-dimethyl-ammonium-propane (DODAP), l,2-dipalmitoyl-sn-glycero-3-ethyl-phosphocholine (DpePC), N-(l-(2, 3- dioleyloxy)propyl)-N, N, N-trimethylammonium chloride (DOTMA), N, N-dimethyl-2, 3- dioleyloxypropylamine (DODMA), 1, 2-DiLinoleyloxy-N, N-dimethylaminopropane (DLinDMA), 2,2-DiLinoleyl-4-(2-dimethylaminoethyl)-[l,3]-dioxolane (DLin-KC2- DMA), (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4- (dimethylamino)butanoate (DLin-MC3-DMA), 1, 2-Dilinolenyloxy-N, N- dimethylaminopropane (DLenDMA), DSDMA, DOSPA, DC-Chol, DMRIE, 1-palmitoyl- 2-glutaryl-sn-glycero-3-phosphocholine (16:0-05:0 (COOH) PC or "GIPC"), 1-hexadecyl- 2-azelaoyl-sn-glycero-3-phosphocholine (16-09:0 (COOH) PC), l-palmitoyl-2-azelaoyl- sn-glycero-3-phosphocholine (16:0-09:0 (COOH) PC or "AzPC"), l-palmitoyl-2-(5'-oxo- valeroyl)-sn-glycero-3-phosphocholine (16;0-05:0 (CHO) PC) and l-palmitoyl-2-(9'-oxo- nonanoyl)-sn-glycero-3-phosphocholine.

11. The lipid of claim 1, wherein the tail moiety comprises a saturated or unsaturated fatty acid.

12. The lipid of claim 11, wherein the saturated or unsaturated fatty acid is between 2 and 50 carbons in length.

13. The lipid of claim 11, wherein the saturated or unsaturated fatty acid is between 2 and 24 carbons in length.

14. The lipid of claim 11, wherein the saturated or unsaturated fatty acid is between 2 and 18 carbons in length.

15. The lipid of claim 1, wherein the conformational change is induced by contacting the cytosol or an intracellular body.

16. The lipid of claim 15, wherein the intracellular body is an endosome, lysosome or caveolae.

17. The lipid of claim 1, wherein the conformational change is induced by a change in pH (e.g., protonation or deprotonation state), redox state (e.g., oxidation or reduction state), chemical or biochemical cleavage or activation (e.g., enzymatic cleavage).

18. The lipid of claim 1, wherein the cationic peptide-based moiety is a peptide having 2 to 25 amino acid residues.

19. The lipid of claim 1, wherein the cationic peptide-based moiety is a peptide wherein at least 5-25% of the residues are positively-charged amino acids.

20. The lipid of claim 1, wherein the cationic peptide-based moiety is a peptide wherein at least 20% of the residues are positively-charged amino acids.

21. The lipid of claim 1, wherein the cationic peptide-based moiety comprises at least one arginine (R) or histidine (H) or lysine (K) residue.

22. The lipid of claim 1, wherein the cationic peptide-based moiety comprises one or more residues which are susceptible to being changed from a first state to a second state, wherein the second state induces a change in the conformation of the peptide-based moiety.

23. The lipid of claim 1, wherein the cationic peptide-based moiety comprises a cleavable bond.

24. The lipid of claim 23, wherein the cleavable bond is a disulfide bridge.

25. The lipid of claim 23, wherein the cleavable bond may be cleaved upon

protonation.

26. The lipid of claim 1, wherein the headgroup moiety and the tail moiety are covalently joined via a linker.

27. A pharmaceutical composition comprising the lipid of claim 1 and a therapeutic agent.

28. The pharmaceutical composition of claim 27, wherein the therapeutic agent is a nucleic acid molecule, peptide, antibody, or small molecule.

29. The pharmaceutical composition of claim 28, wherein the nucleic acid molecule is a DNA molecule, an RNA molecule, a single- stranded nucleic acid molecule, a double- stranded nucleic acid molecule, a miRNA molecule, a siRNA molecule, a RNAi molecule, or an oligonucleotide.

30. A method of treating a disease, comprising administering a therapeutically effective amount of the pharmaceutical composition of claim 27.

31. A cationic lipid compound having the general formula X-Y-P, wherein

X is a headgroup moiety of a fatty acid, glycerolipid, glycerophospholipid, sphingolipid or saccharolipid;

Y is an acyl chain; and

P is a cationic moiety capable of conformational change upon internalization of the lipid into a cell or intracellular compartment and which imparts a net cationic charge to the lipid.

32. The cationic lipid of claim 31, wherein the glycerolipid is a triacylglyceride or glyco sylglycerol .

33. The cationic lipid of claim 31, wherein the glycerophospholipid is a phospholipid, phosphatidylcholine (PC), lecithin, phosphatidylethanolamine (PE) or phosphatidylserine (PS).

34. The cationic lipid of claim 31, wherein the sphingolipid is a ceramide,

phosphosphingolipid, glyco sphingolipid, sphingomyelin, or ceramide phosphocholine.

35. The cationic lipid of claim 31, wherein the saccharolipid is Lipid A.

36. The cationic lipid of claim 31, wherein the lipid is a noncationic lipid selected from the group consisting of lecithin, phosphatidylethanolamine, lysolecithin,

lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphatidic acid, cerebrosides, dicetylphosphate, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipahnitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoyl-phosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), palmitoyloleyol-phosphatidylglycerol (POPG),

dioleoylphosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane- 1 -carboxylate (DOPE-mal), dipalmitoyl-phosphatidylethanolamine (DPPE), dimyristoyl- phosphatidylethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), monomethyl-phosphatidylethanolamine, dimethyl -phosphatidylethanolamine, dielaidoyl- phosphatidylethanolamine (DEPE), DOGS, and stearoyloleoyl-phosphatidylethanolamine (SOPE).

37. The cationic lipid of claim 31, wherein the lipid is a cationic lipid selected from the group consisting of N, N-dioleyl-N, N-dimethylammonium chloride (DODAC), N, N- distearyl-N, N-dimethylammonium bromide (DDAB), N-(l-(2, 3-dioleoyloxy)propyl)-N, N, N-trimethylammonium chloride (DOTAP), l,2-dioleoyl-3-dimethyl-ammonium- propane (DODAP), l,2-dipalmitoyl-sn-glycero-3-ethyl-phosphocholine (DpePC), N-(l-(2, 3-dioleyloxy)propyl)-N, N, N-trimethylammonium chloride (DOTMA), N, N-dimethyl-2, 3-dioleyloxypropylamine (DODMA), 1, 2-DiLinoleyloxy-N, N-dimethylaminopropane (DLinDMA), 2,2-DiLinoleyl-4-(2-dimethylaminoethyl)-[l,3]-dioxolane (DLin-KC2- DMA), (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4- (dimethylamino)butanoate (DLin-MC3-DMA), 1, 2-Dilinolenyloxy-N, N- dimethylaminopropane (DLenDMA), DSDMA, DOSPA, DC-Chol, DMRIE, 1-palmitoyl- 2-glutaryl-sn-glycero-3-phosphocholine (16:0-05:0 (COOH) PC or "GIPC"), 1-hexadecyl- 2-azelaoyl-sn-glycero-3-phosphocholine (16-09:0 (COOH) PC), l-palmitoyl-2-azelaoyl- sn-glycero-3-phosphocholine (16:0-09:0 (COOH) PC or "AzPC"), l-palmitoyl-2-(5'-oxo- valeroyl)-sn-glycero-3-phosphocholine (16;0-05:0 (CHO) PC) and l-palmitoyl-2-(9'-oxo- nonanoyl)-sn-glycero-3-phosphocholine.

38. The cationic lipid of claim 31, wherein the acyl chain Y comprises a saturated or unsaturated fatty acid.

39. The cationic lipid of claim 38, wherein the saturated or unsaturated fatty acid is between 2 and 50 carbons in length.

40. The cationic lipid of claim 38, wherein the saturated or unsaturated fatty acid is between 2 and 24 carbons in length.

41. The cationic lipid of claim 38, wherein the saturated or unsaturated fatty acid is between 2 and 18 carbons in length.

42. The cationic lipid of claim 31, wherein the conformational change is induced by contacting the cytosol or an intracellular body.

43. The cationic lipid of claim 42, wherein the intracellular body is an endosome, lysosome or caveolae.

44. The cationic lipid of claim 31, wherein the conformational change is induced by a change in pH (e.g., protonation or deprotonation state), redox state (e.g., oxidation or reduction state), chemical or biochemical cleavage or activation (e.g., enzymatic cleavage).

45. The cationic lipid of claim 31, wherein the cationic moiety is a peptide having 2 to 25 amino acid residues.

46. The cationic lipid of claim 31, wherein the cationic moiety is a peptide with at least 5-25% of the residues being positively-charged amino acids.

47. The cationic lipid of claim 31, wherein the cationic moiety is a peptide with at least 20% of the residues being positively-charged amino acids.

48. The cationic lipid of claim 31, wherein the cationic moiety is a peptide comprises at least one arginine (R) or histidine (H) or lysine (K) residue.

49. The cationic lipid of claim 31, wherein the cationic moiety is a peptide comprising one or more residues which are susceptible to being changed from a first state to a second state, wherein the second state induces a change in the conformation of the peptide-based moiety.

50. The cationic lipid of claim 31, wherein the cationic moiety comprises a cleavable bond.

51. The cationic lipid of claim 50, wherein the cleavable bond is a disulfide bridge.

52. The cationic lipid of claim 50, wherein the cleavable bond may be cleaved upon protonation.

53. The cationic lipid of claim 31, wherein the headgroup moiety and the acyl chain Y are covalently joined via a linker.

54. A pharmaceutical composition comprising the cationic lipid of claim 31 and a therapeutic agent.

55. The pharmaceutical composition of claim 54, wherein the therapeutic agent is a nucleic acid molecule, therapeutic amino acid, peptide, polypeptide, hormone, antibody, signalling molecule, or small molecule.

56. The pharmaceutical composition of claim 55, wherein the nucleic acid molecule is a DNA molecule, an RNA molecule, a single- stranded nucleic acid molecule, a double- stranded nucleic acid molecule, a miRNA molecule, a siRNA molecule, a RNAi molecule, or an oligonucleotide.

57. A method of treating a disease, comprising administering a therapeutically effective amount of the pharmaceutical composition of claim 54.

58. The cationic lipid of claim 31, wherein P is a peptide having the amino acid sequence selected from the group consisting of:

SEQ 1:— (R)_niH(R)_n2 or SEQ 2:— (K)_niH(K)_n2, wherein, nland n2 are

independently 0 to 25;

SEQ 3:— (R)_ni(HR)_n2(R)_n3 or SEQ 4:— (K)_ni(HK)_n2(K)_n3, wherein nl and n3 are independently 0 to 25 and n2 is 1 to 15;

SEQ 5:— (R)_ni(RH)_n2(R)_n3 or SEQ 6:— (K)_ni(KH)_n2(K)_n3, wherein nl and n3 are independently 0 to 25 and n2 is 1 to 15;

SEQ 7:— Xi(HR)_nX₂ or SEQ 8:— Xi(HK)_nX₂, wherein XI and X2 are

independently amino acids or peptides with 2 to 25 amino acids and n is 1 to 15;

SEQ 9:— Xi(RH)_nX₂ or SEQ 10:— Xi(KH)_nX₂, wherein XI and X2 are independently amino acids or peptides with 2 to 25 amino acids and n is 1 to 15;

SEQ 11: — RHRHRHRHR ;

SEQ 12: — RHDRHDRHD ;

SEQ 13: — RHKHRQRHRPPQ ;

SEQ 14: — RHKHRQRHRPPQ;

SEQ 15: — K(RHRHR) (HRHR) ;

SEQ 16: — K(RHDRH)(DRHD); and

SEQ 17: — K(RHKHRQXRHRPPQ).

59. The cationic lipid of claim 31, wherein P is a peptide having a linear amino acid sequence.

60. The cationic lipid of claim 31, wherein P is a peptide having a branched amino acid sequence.

61. The cationic lipid of claim 31, wherein P is a peptide having a cyclized amino acid sequence.

62. The cationic lipid of claim 31, wherein P is a peptide comprising L- amino acids.

63. The cationic lipid of claim 31, wherein P is a peptide comprising D-amino acids.

64. A method of making the lipid of claim 1, comprising:

obtaining a peptide; and

conjugating the peptide to the tail moiety.

65. A method of making the cationic lipid of claim 31, comprising:

obtaining a peptide P; and

conjugating the peptide P to the acyl chain Y.

66. The method of any one of claims 64 or 65, wherein the step of obtaining a peptide includes purifying the peptide from a biological system, purifying the peptide as a fragment of a digested protein, expressing the peptide using a recombinant system, or synthesizing the peptide.

67. The cationic lipid of claim 1, wherein the cationic peptide-based moiety that is capable of undergoing a conformational change upon contacting an intracellular condition is coupled to the tail moiety via a spacer.

68. The cationic lipid of claim 67, wherein the spacer is a PEG.

69. The cationic lipid of claim 68, wherein the PEG has between one and about twenty-four subunits.

70. The cationic lipid of claim 68, wherein the PEG has between one and about twelve subunits.

71. The cationic lipid of claim 68, wherein the PEG has between one and about six subunits.

72. The cationic lipid of claim 68, wherein the PEG has between one and about three subunits.