WO2016079197A1 - Delivery enhancers for conjugated sirna and lipid nanoparticles - Google Patents

Abstract

The present invention relates to lipid nanoparticles and pharmaceutical formulations containing them that exhibit enhanced cellular delivery of active pharmaceutical ingredients such as siRNA. The present invention also relates to a process for preparing such lipid nanoparticles and pharmaceutical formulations and their use.

Description

DELIVERY ENHANCERS FOR CONJUGATED SIRNA

AND LIPID NANOP ARTICLES

FIELD OF THE INVENTION

[01] The present invention relates to delivery enhancers for facilitating the cellular delivery of (i) an active pharmaceutical ingredient (e.g., an siRNA) in a lipid nanoparticle or (ii) a conjugated siRNA. The present invention also relates to pharmaceutical compositions including (a) the delivery enhancer an (b) (i) a lipid nanoparticle containing an active pharmaceutical ingredient or (ii) a conjugated siRNA. The invention further relates to processes for preparing such pharmaceutical compositions and their use.

BACKGROUND OF THE INVENTION

[02] Interfering with gene expression has long been proposed as a potential therapeutic strategy. The combination of potent RNAi therapeutics and innovative delivery strategies has opened new opportunities to efficiently silence disease-associated genes at therapeutically relevant doses. Numerous delivery platforms, such as viruses (Silva et al, Virology Journal, 7, 248, 2010, liposomes (Buyens et al, J. Controlled Release, 158(3), 362-370, 2012), polycationic polymers (Castillo et al, J. Drug Delivery, 218940, 2012), conjugates (Lopez et al., Curr. Opin. in Pharmacol., 12(4), 414-419, 2012); and lipid nanoparticles (LNPs) (Zimmermann et al, Nature, 441(7089), 1 11-114, 2006; Akinc et al, Mo., Ther., 17(5), 872-879, 2009; Akinc et al, Mol. Ther. J. Am. Gene Soc, 18(7), 1357-1364, 2010; Love et al, Nat. Acad. Sci. USA, 107(5), 1864-1869, 2010; Jayarman et al, Angew. Chem. Int. Ed. Engl, 51(34), 8429-8533, 2012; Gilleron et al, Nature Biotech., 31(7), 638-646, 2013), are now being used to deliver siRNAs in vivo. Advances in the development of these delivery technologies have enabled the entry of numerous systemic RNAi products into the clinic (Coelho et al, N. Eng. J. Med., 369(9), 819- 829, 2013; Kanasty et al, Nature Mat., 12(1 1), 967-977, 2013).

[03] The development of short interfering RNA sequences (siRNAs) as therapeutics has been hindered by problems in delivering the siRNA to its target. siRNA rapidly undergoes enzymatic degradation resulting in a short half-life in the blood, and has poor cellular update and tissue bioavailability. As a result, there has been significant research on delivering siRNA in lipid nanoparticles (LNPs).

[04] Nevertheless, existing platforms for siRNA delivery may still be further improved and particularly efficient systemic delivery to extra-hepatic cells and tissues remains a challenge (Pei et al, KNA, 16(12), 2553-2563, 2010; Gilleron et al, Nature Biotech., 31(7), 638-646, 2013; Xu et al, Mol. Pharmaceutics, 11(5), 1424- 1434, 2014). Delivery is a multistep process consisting of targeting to the appropriate tissue and cell types, cellular uptake and escape of the siRNAs from the endosomes into the cytosol for loading on the RNA-induced silencing complex (RISC) (Akinc et al, Cold Spring Harbor Perspectives in Biology, 5(11), a016980, 2013). Recently, significant emphasis has been placed on the targeting step and some solutions have emerged (Lee et al, Methods in Enzymology, 502, 91-122, 2012; Nielson et al, Exp. Opin. Drug Deliv., 11(5), 791-822, 2014). Most notably, efficient systemic delivery to hepatocytes has been achieved by combining multivalent GalNAc ligands with advanced siRNA chemistry resulting in a potent platform suitable for subcutaneous administration. However, improving uptake and especially release from unproductive intracellular compartments remains a challenge for many other tissues and cell types (Gilleron et al, Nature Biotech., 31(7), 638-646, 2013; Xu et al, Mol. Pharmaceutics, 11(5),1424-1434, 2014).

[05] There is, therefore, a need for improved LNPs that exhibit enhanced delivery of active pharmaceutical ingredients, such as siRNA.

SUMMARY OF THE INVENTION

[06] The present inventors have discovered compounds ("delivery enhancers") that enhance cellular delivery of (i) a siRNA conjugated to a ligand (such as a targeting ligand) (hereinafter referred to as a "conjugated siRNA") and (ii) an active pharmaceutical ingredient (such as siRNA) when combined with a lipid nanoparticle (LNP). Some of these compounds enhanced the uptake of the conjugated siRNA or the LNP, while others enhanced the intracellular release of the siRNA from endosomes. [07] In one aspect, the invention relates to a composition (such as a pharmaceutical formulation) comprising (i) a conjugated siRNA and (ii) a delivery enhancer compound which enhances the cellular uptake of the conjugate siRNA and/or the intracellular release of the siRNA (for example, from endosomes). In one preferred embodiment, the composition is suitable for parenteral administration.

[08] In one embodiment, the delivery enhancer compound which enhances the cellular uptake of the conjugate siRNA and/or the intracellular release of the siRNA (for example, from endosomes) is selected from compounds of Formula (I):

wherein each occurrence of Ri is, independently, alkyl (e.g., C]-C₈ alkyl, Ci-C₆ alkyl or C]-C₄ alkyl, such as methyl);

R₂ is H and R₃ is OH; or R₂ and R₃, together with the carbon atom to which they are attached, form a carbonyl (C=0) group;

R4 is hydrogen or OH;

R₅ is hydrogen, alkyl (e.g., Cj-C₈ alkyl, Ci-C₆ alkyl or Ci-C₄ alkyl, such as methyl); or alkenyl (e.g., C₂-C₈ alkenyl, C₂-C₆ alkenyl or C₂-C₄ alkenyl, such as -C(=CH₂)CH₃));

R⁶ is hydrogen or alkyl (e.g., Ci-C₈ alkyl, Ci-C₆ alkyl or C₁-C4 alkyl, such as methyl); R⁷ is hydrogen, hydroxy or alkyl (e.g., Q-Q alkyl, Q-C6 alkyl or Q-C4 alkyl, such as methyl); n is 1 or 2; each occurrence of is an optional carbon carbon double bond; and

Z is OH or -N(Ri)₂. The compounds of Formula (I) are preferably used with conjugated siRNA.

[09] In one embodiment, the compound of formula (I) does not contain two carbon-carbon double bonds. In one embodiment, the compound of formula (I) contains zero or one carbon- carbon double bond.

[10] A preferred compound of formula (I) is a compound of formula (IA):

wherein Ri-R₇ and Z are as defined above for the compound of formula (I).

[11] In one embodiment of the compound of formula (IA): each occurrence of Ri is C1-C4 alkyl, such as methyl; hydrogen and R₃ is hydroxyl; and

Z is OH. [12] In another preferred embodiment of the compound of formula (IA): each occurrence of R] is Q-C4 alkyl, such as methyl; R₂ is hydrogen and R₃ is hydroxyl; R5 is hydrogen or C1-C4 alkyl, such as methyl; R₆ is hydroxyl or Q-C4 alkyl, such as methyl; R₇ is hydrogen or C₁-C₄ alkyl, such as methyl; and Z is OH.

[13] In another embodiment, the compound of formula (I) is a compound of formula (IB):

wherein R R₇ and Z are as defined above for the compound of formula (I). [14] In a preferred embodiment of the compound of formula (IB): each occurrence of R] is Q-C₄ alkyl, such as methyl;

R₂ and R₃, together with the carbon atom to which they are attached, form a carbonyl (C=0) group; and Z is OH.

[15] In another preferred embodiment of the compound of formula (IB) : each occurrence of Ri is C1-C4 alkyl, such as methyl;

R₂ and R₃, together with the carbon atom to which they are attached, form a carbonyl (C=0) group;

R5 is hydrogen;

R₆ and R₇ are both C1-C4 alkyl, such as methyl; and

Z is OH.

In yet another embodiment, the compound of formula (I) is a compound of formula (IC):

wherein R]-R₇ and Z are as defined above for the compound of formula (I). [17] In a preferred embodiment of the compound of formula (IC): each occurrence of Ri is C1-C4 alkyl, such as methyl;

R₂ is hydrogen and R₃ is hydroxyl; and

Z is OH. [18] In another preferred embodiment of the compound of formula (IC) : each occurrence of Ri is Q-C4 alkyl, such as methyl; R₂ is hydrogen and R₃ is hydroxyl;

R₅ is alkenyl (e.g., C₂-C₈ alkenyl, C₂-C₆ alkenyl or C₂-C₄ alkenyl, such as -

C(=CH₂)CH₃));

R and R₇ are hydrogen; and Z is OH.

[19] In another preferred embodiment, the compound of formula (I) has the following stereochemistry:

[20] In a further embodiment, delivery enhancer compounds which enhance the cellular uptake of the conjugate siRNA and/or the intracellular release of the siRNA (for example, from endosomes) include, but are not limited to, pentacyclic triterpenes and pentacyclic steroids, such as ursolic acid, oleanolic acid, moronic acid, betulinic acid, corosolic acid, and combinations thereof. [21] In a further embodiment, delivery enhancer compounds which enhance the cellular uptake of the conjugate siRNA and/or the intracellular release of the siRNA (for example, from endosomes) include, but are not limited to, those listed in Table A below.

Table A Compound

ADD029 G19

Compound

Tegaserod maleate

CBN039 O20

[22] The pharmaceutical formulation may include one or more pharmaceutically acceptable excipients, such as isotonicity agents.

[23] In one embodiment, the concentration of delivery enhancer compound in the pharmaceutical formulation ranges from about 0.1 μΜ to about 100 μΜ, such as from about 0.5 to about 50 μΜ, from about 1 μΜ to about 16 μΜ, or from about 5 μΜ to about 15 μΜ (e.g., about 5 μΜ or about 10 μΜ).

[24] According to another aspect, the invention relates to a process for preparing a composition by mixing conjugated siRNA with one or more delivery enhancer compounds selected from those described above, such as a delivery enhancer compound selected from formula I, IA, IB, IC or Table A. [25] Another embodiment is a pharmaceutical formulation prepared by the process described above.

[26] Yet another embodiment is a method of administering (e.g., parenterally such as intravenously) an siRNA by administering a pharmaceutical formulation containing a conjugated siRNA of the present invention.

[27] In yet another aspect, the invention relates to lipid nanoparticles comprising an active pharmaceutical ingredient where the lipid nanoparticles are treated with one or more delivery enhancer compounds (e.g., those selected from Table B or of Formula II, IIA, III, IIIA, or IIIB) that enhance the cellular uptake of the active pharmaceutical ingredient and/or the intracellular release of the active pharmaceutical ingredient (for example, from endosomes). The active pharmaceutical ingredient can be a nucleic acid, such as a siRNA.

[28] In one embodiment, the delivery enhancer compound which enhances cellular uptake of LNPs and/or the intracellular release of the siRNA (for example, from endosomes) is of formula

II:

wherein

Ring A and Ring B are each, independently, selected from substituted and unsubstituted aryl (e.g., phenyl or naphthyl) and substituted and unsubstituted heteroaryl (e.g., pyridinyl or pyrimidinyl); each occurrence of Ri is, independently, selected from alkyl (e.g., C₁-C₄ alkyl), aryl, heteroaryl, arylalkyl and heteroarylalkyl; each occurrence of L is, independently, selected from -0-, -S-, -NRg-, -C(0)0-, -OC(O)-, -NRg(CO)- and -C(0)NR₈-; each occurrence of R₈ is hydrogen or alkyl (e.g., C1-C4 alkyl); each occurrence of X is, independently, selected from -O- and -S-; and each occurrence of n is independently 1, 2, 3, 4, 5 or 6.

In one preferred embodiment, each of rings A and B is bound to group L at the para position relative to the C(R ₂ group.

In a preferred embodiment, both Ring A and Ring B are, independently, selected from substituted and unsubstituted aryl (e.g., phenyl).

[29] In one embodiment, the compound of formula (II) is a compound of formula (IIA)

wherein R¹, L, X and n are as defined above for formula (II).

[30] Preferred embodiments of the compounds of formula (II) and (IIA) include one or more of the following:

(i) each occurrence of Ri is, independently, alkyl (e.g., -Q alkyl, C_\-C alkyl or C1-C4 alkyl);

(ii) each occurrence of Ri is methyl;

(iii) each occurrence of L is, independently, selected from -O- and -S-; (iv) each occurrence of L is -0-;

(v) each occurrence of X is -0-; and

(vi) each occurrence of n is 1.

[31] In another embodiment, the delivery enhancer compound which enhances cellular uptake of LNPs and/or the intracellular release of the siR A (for example, from endosomes) is of formula III:

wherein each occurrence of Xi is, independently, CH or N; Y is -0-, -NH-, or -S-;

R" is -CH₃, -CH(CH₃)₂, -(CH₂)₂C0₂H, -CH₂C₆H4 or

each occurrence of R] is, independently, H, alkyl (e.g., C alkyl) or aryl (e.g., phenyl);

R₂ is H, alkyl (e.g., C\A alkyl) or aryl (e.g., phenyl); and each bond identified by * can independently be in the R or S configuration.

[32] For example, the compound of formula (III) may be a compound of formula (IIIA) or (IIIB):

[33] Preferred embodiments of the compounds of formula (III), (IIIA) and (IIIB) include one or more of the following:

(i) each occurrence of X] is CH;

(ii) Y is -0-;

(iv) R" is -CH₂C₆H₄;

(v) Z is -OH; and

(vi) R₂ is H.

[34] In a further embodiment, delivery enhancer compounds which enhance cellular uptake of LNPs and/or the intracellular release of the siRNA (for example, from endosomes) include, but are not limited to, those in Table B below.

Table B

Compound

CBN040I13

Pterostilbene

APIOLE

[35] In another aspect, the invention relates to lipid nanoparticles comprising an active pharmaceutical ingredient where the lipid nanoparticles are treated with one or more delivery enhancer compounds selected from a compound of Formula II, IIA, III, IIIA, or IIIB, or Table B that enhances delivery of the active pharmaceutical ingredient. The active pharmaceutical ingredient can be a nucleic acid, such as a siRNA.

[36] Another embodiment is compacted lipid nanoparticles prepared by treating lipid nanoparticles with one or more delivery enhancer compounds selected from a compound of Formula II, IIA, III, IIIA, or IIIB, or Table B, such as BADGE. The lipid nanoparticles comprise an active pharmaceutical ingredient. The compacted lipid nanoparticles may be incorporated in a pharmaceutical formulation as described herein.

[37] In another aspect, the invention relates to lipid nanoparticles comprising (a) an active pharmaceutical ingredient, such as a nucleic acid (e.g., a siRNA), and (b) one or more delivery enhancer compounds selected from a compound of formula II, IIA, III, IIIA, or IIIB, or Table B.

[38] Another embodiment is a pharmaceutical formulation comprising lipid nanoparticles, where the lipid nanoparticles comprise an active pharmaceutical ingredient, and one or more delivery enhancer compounds that enhance delivery of an active pharmaceutical ingredient such as siRNA. The delivery enhancer compounds can be selected from a compound of Formula II, IIA, HI, IIIA, or IIIB, and Table B. In one embodiment, each nanoparticle comprises a cationic lipid and an active pharmaceutical ingredient (such as a nucleic acid). In one embodiment, the lipid nanoparticles have a d₅o ranging from about 5 to about 500 nm, such as from about 5 to about 200 nm, from about 10 to about 50 nm, from about 25 to about 50 nm or from about 35 to about 45 nm. Preferably, the pharmaceutical formulation is suitable for parenteral administration.

[39] In one embodiment, the delivery enhancer compound is selected from BADGE and CP WOOl J18 and mixtures thereof:

[40] Another embodiment is a pharmaceutical formulation comprising lipid nanoparticles and one or more delivery enhancer compounds selected from a compound of formula II, IIA, III, IIIA, or IIIB, and Table B, where each lipid nanoparticle comprises a cationic lipid and an active pharmaceutical ingredient (such as a nucleic acid), and the lipid nanoparticles have a d₅₀ ranging from about 10 to about 50 nm, such as from about 25 to about 50 nm or from about 35 to about 45 nm.

[41] In one embodiment, the concentration of delivery enhancer compound in the pharmaceutical formulation ranges from about 0.1 μΜ to about 100 μΜ, such as from about 0.5 to about 50 μΜ, from about 1 μΜ to about 16 μΜ, or from about 5 μΜ to about 15 μΜ (e.g., about 5 μΜ or about 10 μΜ).

[42] In one preferred embodiment, the lipid nanoparticles comprise:

(a) an active pharmaceutical ingredient (e.g., a nucleic acid),

(b) a cationic lipid, (c) a non-cationic lipid (such as a neutral lipid),

(d) an aggregation reducing agent (such as polyethylene glycol (PEG) or PEG-modified lipid), and

(e) optionally, a sterol.

[43] In one embodiment, the cationic lipid has a pKa ranging from about 4 to about 11, and preferably from about 5 to about 7.

[44] In one embodiment, the formulation includes less than about 3, about 2, about 1.5, about 1, or about 0.5 mole percent of the aggregation reducing agent (such as PEG or PEG-modified lipid), based upon the total moles of lipid (e.g., total moles of cationic lipid, non-cationic lipid, sterol, and aggregation reducing agent) in the lipid nanoparticle.

[45] In one embodiment, the formulation further comprises one or more isotonicity agents. Preferably, the formulation includes a sufficient amount of the isotonicity agent(s) to render the formulation physiologically isotonic (i.e., have a pharmaceutically acceptable osmolality) in order to avoid cell distortion or lysis.

[46] In a preferred embodiment, the active pharmaceutical ingredient in the lipid nanoparticles is a nucleic acid, such as a siRNA. The nucleic acid-lipid particle preferably has an encapsulation efficiency of greater than about 90, 92, 95, or 98%, after storage of the formulation for 1 month at about 4°C.

[47] The formulations containing conjugated siR As or LNPs described herein may be solutions or suspensions.

[48] According to another aspect, the present invention relates to a process for preparing lipid nanoparticles. The process includes the step of incubating lipid nanoparticles in the presence of one or more delivery enhancer compounds selected from a compound of formula II, IIA, III, IIIA, or IIIB, and Table B. The lipid nanoparticles comprise an active pharmaceutical ingredient. [49] Another embodiment is a process for preparing a pharmaceutical formulation containing LNPs according to any of the embodiments described herein. The process includes adding one or more delivery enhancer compounds to one or more lipid nanoparticles. The process may include, for example, mixing and/or incubating the delivery enhancer compound with the lipid nanoparticles.

[50] In one embodiment, the process comprises incubating the lipid nanoparticles and the delivery enhancer compound. The incubation can be for, for example, from about 1 to about 48 hours. In one embodiment, the process involves incubating overnight (e.g., for about 12 or 16 hours). In another embodiment, the process involves incubating at a temperature between about 0 ° C and about 10° C, such as between about 2° C to about 5° C, e.g., at about 4° C. In one embodiment, the process involves incubating overnight (e.g., for about 12 or 16 hours) at about 4° C. In one embodiment, the incubation is conducted in an Eppendorf tube.

[51] Yet another embodiment is a method of preparing a pharmaceutical formulation of the present invention comprising:

(i) preparing lipid nanoparticles comprising a cationic lipid and an active pharmaceutical ingredient (such as a nucleic acid);

(ii) adding a delivery enhancer of the present invention (such as a compound of formula II, IIA, III, IIIA, or IIIB, or one from Table B) and mixtures thereof; and

(iii) optionally incubating (e.g., incubating overnight at about 4° C, e.g., in Eppendorf tubes) the mixture of step (ii).

[52] Yet another embodiment is a method of reducing the size of lipid nanoparticles comprising incubating the lipid nanoparticles in the presence of one or more delivery enhancer compounds selected from a compound of Formula II, IIA, III, IIIA, or IIIB or Table B. The incubation can be for, for example, from about 1 to about 48 hours. In one embodiment, the process involves incubating overnight (e.g., for about 12 or 16 hours). In one embodiment, the process involves incubating at a temperature between about 0 ° C and about 10° C, such as between about 2° C to about 5° C, e.g., at about 4° C. In one embodiment, the process involves incubating overnight (e.g., for about 12 or 16 hours) at about 4° C. In one embodiment, the incubation is conducted in an Eppendorf tube.

[53] Yet another embodiment is a pharmaceutical formulation prepared by any of the processes described herein.

[54] Yet another embodiment is a method of administering (e.g., parenterally such as intravenously) an active pharmaceutical ingredient, such as an siRNA, by administering a pharmaceutical formulation of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[55] Figure la shows images of LNP-siRNA-gold detected in HeLa cells in vitro by electron microscopy after treatment with DMSO, Compound 1, or Compound 2. siRNA-gold were detected in the cytosol (arrows) or within several endocytic compartments. Magnified images (insets) permit appreciation of the cytosolic localization of siRNA-gold.

[56] Figure lb is a bar graph of the total number of siRNA-gold (representing the uptake) found per μπι² of cells with LNP-siRNA-gold treated with with DMSO, Compound 1 or Compound 2. The values are reported as mean values ± standard error (n=3).

[57] Figure lc is a bar graph of the ratio between the number of cytosolic siRNA-gold found and the total number of siRNA-gold internalized (representing the percentage of siRNA escape). The values are reported as mean values ± standard error (n=3).

[58] Figure Id is a bar graph of the number of cytosolic siRNA-gold found per um² of cells. The values are reported as mean values ± standard error (n=3).

[59] Figure 2a shows images of LNP-siRNA-gold by electron microscopy after incubation with DMSO or Compound 1. A bar graph in the right panel shows LNPs mean size diameter. The values are reported as mean values ± standard error (n=3). [60] Figure 2b is a graph showing the fluorescence intensity (uptake kinetics) of LNP-siR A- alexa647 (40nM) treated with DMSO (black curve) or Compound 1 (red curve, ΙΟμΜ) over time.

[61] Figure 2c is a bar graph of percentage uptake of LNP-siR A-alexa647 treated with DMSO (black columns) or Compound 1 (white columns) in HeLa cells after silencing of key regulators of CME (CLTC) and Macropinocytosis (ARF-1, RAC1). The values are reported as mean values ± standard error (n=3).

[62] Figure 3a is a bar graph of the number of siRNA-alexa647 positive vesicles per area in mouse primary endothelial cells for LNPs treated with DMSO or Compound 1. The values are reported as mean values ± standard error (n=3) (** P-value < 0.01).

[63] Figure 3b is a bar graph of the percentage of GFP intensity in primary endothelial cells isolated from GFP-lifeact mice after 72 hours of transfection with LNP-siRNA treated with DMSO or Compound 1. The values are reported as mean values ± standard error (n=3) (* P- value < 0.05).

[64] Figure 3c is a bar graph of the number of LNP-siRNA-alexa647 per view field in vivo after treatment with DMSO or Compound 1. The values are reported as mean values ± standard error (n=3) (*** P-value < 0.001).

DETAILED DESCRIPTION OF THE INVENTION

Definitions

[65] The term "subject" or "patient" refers to a mammal, such as a human, domestic animal, such as a feline or canine subject, farm animal (e.g., bovine, equine, caprine, ovine, and porcine subject), wild animal (whether in the wild or in a zoological garden), research animal, such as mouse, rat, rabbit, goat, sheep, pig, dog, and cat, avian species, such as chicken, turkey, and songbird. The "subject" or "patient" can also be a plant.

[66] The terms "treat" and "treatment" refer to (a) relief from or alleviation of at least one symptom of a disorder in a subject, (b) relieving or alleviating the intensity and/or duration of a manifestation of a disorder experienced by a subject, (c) slowing or reversing the progression of such condition, and (d) arresting, delaying the onset (i.e., the period prior to clinical manifestation of a disorder) and/or reducing the risk of developing or worsening a disorder.

[67] As used herein, the term "intravenous infusion" or "IV infusion" refers to a method of administration of a composition directly into the vein of a patient. IV infusion allows for direct administration of a pharmaceutical formulation to the bloodstream of a patient. This can be performed, for example, via subcutaneous or intradermal infusion. IV infusion can be performed in many ways, including through the use of an injection needle, or with an infusion pump. It can be provided as, for example, a continuous infusion, an intermittent infusion, a patient-controlled infusion, or a circadian infusion.

[68] An "isotonicity agent" generally refers to a compound that is physiologically tolerated and imparts a suitable tonicity to a formulation to prevent the net flow of water across cell membranes that are in contact with the formulation.

[69] The term "alkyl" refers to a straight or branched chain saturated hydrocarbon moiety. In one embodiment, the alkyl group is a straight chain saturated hydrocarbon. Unless otherwise specified, the "alkyl" group contains from 1 to 24 carbon atoms, such as from 1 to 12 carbon atoms, from 1 to 8 carbon atoms, from 1 to 6 carbon atoms or from 1 to 4 carbon atoms. Representative saturated straight chain alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, n-butyl, n-pentyl, and n-hexyl; while saturated branched alkyl groups include isopropyl, sec-butyl, isobutyl, tert-butyl, and isopentyl.

[70] The term "alkenyl" refers to a straight or branched chain hydrocarbon moiety having one or more carbon-carbon double bonds. In one embodiment, the alkenyl group contains 1, 2, or 3 double bonds and is otherwise saturated. Unless otherwise specified, the "alkenyl" group contains from 2 to 24 carbon atoms, such as from 2 to 12 carbon atoms, from 2 to 8 carbon atoms, from 2 to 6 carbon atoms or from 2 to 4 carbon atoms. Alkenyl groups include both cis and trans isomers. Representative straight chain and branched alkenyl groups include ethylenyl, propylenyl, 1-butenyl, 2-butenyl, isobutylenyl, 1-pentenyl, 2-pentenyl, 3-methyl-l-butenyl, 2-methyl-2-butenyl, and 2,3-dimethyl-2-butenyl.

[71] The term "aryl" refers to an aromatic monocyclic, bicyclic, or tricyclic hydrocarbon ring system. Examples of aryl moieties include, but are not limited to, phenyl, naphthyl, anthracenyl, and pyrenyl.

[72] The term "heteroaryl" refers to an aromatic 5-8 membered monocyclic, 7-12 membered bicyclic, or 1 1-14 membered tricyclic ring system having 1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9 heteroatoms if tricyclic, where the heteroatoms are selected from O, N, or S (e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of N, O, or S if monocyclic, bicyclic, or tricyclic, respectively). The heteroaryl groups herein described may also contain fused rings that share a common carbon-carbon bond.

[73] The term "halogen" or "halo" refers to fluoro, chloro, bromo and iodo.

[74] The term "substituted", unless otherwise indicated, refers to the replacement of one or more hydrogen radicals in a given structure with the radical of a specified substituent including, but not limited to: halo, alkyl, alkenyl, aryl, heteroaryl, and any combination thereof.

Conjugated siR A [75] The term "conjugated siRNA" refers to an siRNA molecule conjugated to at least one ligand (preferably, at least one targeting ligand). The siRNA may have a duplex region that is 17-21 or 19-21 nucleotides in length. Suitable conjugated siRNA are described, for example, in U.S. Patent Publication Nos. 2013/0184328, 2013/0203836, 2013/0202652, 2013/0317080, 2013/0211063, and 2013/0184324, which are hereby incorporated by reference. Other RNA conjugates are described in U.S. Patent Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465;

5,541,313; 5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,591,584; 5,109,124; 5,118,802

5, 138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735

4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013

5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469

5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203

5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481

5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599.928 and 5,688,941; 6,294,664; 6,320,017

6,576,752; 6,783,931; 6,900,297; 7,037,646; each of which is herein incorporated by reference.

[76] SiRNAs are generally RNA duplexes normally 16-30 nucleotides long that can associate with a cytoplasmic multi-protein complex known as RNAi-induced silencing complex (RISC). RISC loaded with siRNA mediates the degradation of homologous mRN A transcripts, therefore siRNA can be designed to knock down protein expression with high specificity.

[77] While the first described siRNA molecules were RNA:RNA hybrids comprising both an RNA sense and an RNA antisense strand, it has now been demonstrated that DNA sense:RNA antisense hybrids and RNA sense'.DNA antisense hybrids are capable of mediating RNA interference (Lamberton, J. S. and Christian, A. T., (2003) Molecular Biotechnology 24: H il l 9). Thus, the term "siRNA", unless otherwise specified, includes the use of small interfering RNA molecules comprising any of these different types of double-stranded molecules. In addition, it is understood that RNAi molecules may be used and introduced to cells in a variety of forms. Accordingly, as used herein, siRNA molecules encompasses any and all molecules capable of inducing an RNA interference response in cells, including, but not limited to, (i) a double-stranded oligonucleotide comprising two separate strands, i.e. a sense strand and an antisense strand, (ii) a double-stranded oligonucleotide comprising two separate strands that are linked together by a non-nucleotidyl linker, (iii) a oligonucleotide comprising a hairpin loop of complementary sequences, which forms a double-stranded region, e.g., shKNAi molecules, and (iv) expression vectors that express one or more polynucleotides capable of forming a double- stranded polynucleotide alone or in combination with another polynucleotide.

[78] The siRNA may be a single strand siRNA compound or a double stranded siRNA compound. A "single strand siRNA compound" as used herein, is an siRNA compound which is made up of a single molecule. It may include a duplexed region, formed by intra-strand pairing, e.g., it may be, or include, a hairpin or pan-handle structure. Single strand siRNA compounds may be antisense with regard to the target molecule.

[79] A single strand siRNA compound may be sufficiently long that it can enter the RISC and participate in RISC mediated cleavage of a target mRNA. A single strand siRNA compound is at least 14, and in other embodiments at least 15, 20, 25, 29, 35, 40, or 50 nucleotides in length. In certain embodiments, it is less than 80 or 60 nucleotides in length.

[80] Hairpin siRNA compounds will have a duplex region equal to or at least 17, 18, 19, 29, 21, 22, 23, 24, or 25 nucleotide pairs. The duplex region will may be equal to or less than 30, 25, 23, or 22 nucleotide pairs in length. In certain embodiments, ranges for the duplex region are 15- 30, 17 to 23, 19 to 23, and 19 to 21 nucleotide pairs in length. The hairpin may have a single strand overhang or terminal unpaired region. In certain embodiments, the overhangs are 2-3 nucleotides in length. In some embodiments, the overhang is at the sense side of the hairpin and in some embodiments on the antisense side of the hairpin.

[81] A "double stranded siRNA compound" as used herein, is an siRNA compound which includes more than one, and typically two, strands in which interchain hybridization can form a region of duplex structure. The antisense strand of a double stranded siRNA compound may be equal to or at least, 14, 15, 16, 17, 18, 19, 25, or 29 nucleotides in length. It may be equal to or less than 30, 25, 23, or 21 nucleotides in length. Ranges may be 17 to 25, 19 to 23, and 19 to 21 nucleotides in length. As used herein, term "antisense strand" means the strand of an siRNA compound that is sufficiently complementary to a target molecule, e.g. a target RNA.

[82] The sense strand of a double stranded siRNA compound may be equal to or at least 14, 15, 16, 17, 18, 19, 25, or 29 nucleotides in length. It may be equal to or less than 30, 25, 23, or 21 nucleotides in length. Ranges may be 17 to 25, 19 to 23, and 19 to 21 nucleotides in length.

[83] The double strand portion of a double stranded siRNA compound may be equal to or at least 14, 15, 16 17, 18, 19, 20, 21, 22, 23, 24, 25, 29, or 30 nucleotide pairs in length. It may be equal to or less than 30, 25, 23, or 21 nucleotides pairs in length. Ranges may be 15 to 30, 17 to 23, 19 to 23, and 19 to 21 nucleotide pairs in length.

[84] In many embodiments, the siRNA compound is sufficiently large that it can be cleaved by an endogenous molecule, e.g., by Dicer, to produce smaller siRNA compounds, e.g., siRNAs agents.

[85] The sense and antisense strands may be chosen such that the double-stranded siRNA compound includes a single strand or unpaired region at one or both ends of the molecule. Thus, a double- stranded siRNA compound may contain sense and antisense strands, paired to contain an overhang, e.g., one or two 5' or 3' overhangs, or a 3' overhang of 1-3 nucleotides. The overhangs can be the result of one strand being longer than the other, or the result of two strands of the same length being staggered. Some embodiments will have at least one 3' overhang. In one embodiment, both ends of an siRNA molecule will have a 3' overhang. In some embodiments, the overhang is 2 nucleotides.

[86] The siRNA compounds described herein, including double-stranded siRNA compounds and single-stranded siRNA compounds can mediate silencing of a target RNA, e.g., mRNA, e.g. a transcript of a gene that encodes a protein (a target gene). In general, the RNA to be silenced is an endogenous gene or a pathogen gene. In addition, RNAs other than mRNA, e.g., tRNAs, and viral RNAs, can also be targeted. [87] In one embodiment, an siRNA compound is "sufficiently complementary" to a target RNA, e.g., a target mRNA, such that the siRNA compound silences production of protein encoded by the target mRNA. In another embodiment, the siRNA compound is "exactly complementary" to a target RNA, e.g., the target RNA and the siRNA compound anneal, for example to form a hybrid made exclusively of Watson-Crick base pairs in the region of exact complementarity. A "sufficiently complementary" target RNA can include an internal region (e.g., of at least 10 nucleotides) that is exactly complementary to a target RNA. Moreover, in certain embodiments, the siRNA compound specifically discriminates a single-nucleotide difference. In this case, the siRNA compound only mediates RNA interference if exact complementary is found in the region (e.g., within 7 nucleotides of) the single-nucleotide difference.

[88] The ligand may be conjugated to any monomer in the siRNA, either directly or indirectly (such as through an intervening tether). The ligand may be conjugated to a monomer at the 5' or 3' terminus of the siRNA molecule or may be attached to an internal monomer. For example, the ligand can be attached to the 3' end of the sense strand.

[89] The ligand can facilitate targeting and/or delivery of the siRNA. The ligand cholesterol, for example, promotes entry into a cell. In one preferred embodiment, the ligand includes a lipophilic moiety. While not wishing to be bound by theory, it is believed the attachment of a lipohilic agent increases the lipophilicity of the siRNA.

[90] In preferred embodiments, a ligand alters the distribution, targeting or lifetime of an siRNA into which it is incorporated. In one preferred embodiment, a ligand provides an enhanced affinity for a selected target, e.g., molecule, cell or cell type, compartment, e.g., a cellular or organ compartment, tissue, organ or region of the body, as, e.g., compared to a species absent such a ligand. For example, the ligand may be a liver targeting ligand. Preferred ligands can improve transport, hybridization, and specificity properties and may also improve nuclease resistance of the resultant natural or modified siRNA. [91] Ligands in general can include therapeutic modifiers, e.g., for enhancing uptake; cross- linking agents; and nuclease-resistance conferring moieties. General examples include lipophiles, lipids, steroids (e.g., uvaol, hecigenin, and diosgenin), terpenes (e.g., triterpenes, e.g., sarsasapogenin, Friedelin, and epifriedelanol derivatized lithocholic acid), vitamins (e.g., folic acid, vitamin A, biotin, and pyridoxal), carbohydrates, proteins, protein binding agents, integrin targeting molecules, polycationics, peptides, polyamines, and peptide mimics.

[92] The ligand can have endosomolytic properties. The endosomolytic ligands promote the lysis of the endosome and/or transport of the siRNA from the endosome to the cytoplasm of the cell. The endosomolytic ligand may be a polyanionic peptide or peptidomimetic which shows pH-dependent membrane activity and fusogenicity. In one embodiment, the endosomolytic ligand assumes its active conformation at endosomal pH. The "active" conformation is that conformation in which the endosomolytic ligand promotes lysis of the endosome and/or transport of the siRNA from the endosome to the cytoplasm of the cell. Exemplary endosomolytic ligands include the GALA peptide (Subbarao et al, Biochemistry, 1987, 26: 2964-2972), the EALA peptide (Vogel et al, J. Am. Chem. Soc, 1996, 118: 1581-1586), and their derivatives (Turk et al., Biochem. Biophys. Acta, 2002, 1559: 56-68). In one embodiment, the endosomolytic component may contain a chemical group (e.g., an amino acid) which will undergo a change in charge or protonation in response to a change in pH. The endosomolytic component may be linear or branched. Exemplary primary sequences of peptide based endosomolytic ligands are shown in Table 1 of US 2013/0203836, which is hereby incorporated by reference. Endosomal release agents include, but are not limited to, imidazoles, poly- or oligoimidazoles, PEIs, peptides, fusogenic peptides, polycarboxylates, polyacations, masked oligo or poly cations or anions, acetals, polyacetals, ketals/polyketyals, orthoesters, polymers with masked or unmasked cationic or anionic charges, and dendrimers with masked or unmasked cationic or anionic charges.

[93] Ligands can include a naturally occurring substance, (e.g., human serum albumin (HSA), low-density lipoprotein (LDL), or globulin); carbohydrate (e.g., a dextran, pullulan, chitin, chitosan, inulin, cyclodextrin or hyaluronic acid); amino acid, or a lipid. The ligand may also be a recombinant or synthetic molecule, such as a synthetic polymer, e.g., a synthetic polyamino acid. Examples of polyamino acids include, but are not limited to, a polylysine (PLL), poly L- aspartic acid, poly L-glutamic acid, styrene-maleic acid anhydride copolymer, poly(L-lactide-co- glycolied) copolymer, divinyl ether-maleic anhydride copolymer, N-(2- hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacryllic acid), N-isopropylacrylamide polymers, or polyphosphazine. Examples of polyamines include, but are not limited to, polyethylenimine, polylysine (PLL), spermine, spermidine, polyamine, pseudopeptide-polyamine, peptidomimetic polyamine, dendrimer polyamine, arginine, amidine, protamine, and cationic moieties, e.g., cationic lipid, cationic porphyrin, quaternary salt of a polyamine, or an alpha helical peptide.

[94] Ligands can also include targeting groups, e.g., a cell or tissue targeting agent, e.g., a lectin, glycoprotein, lipid or protein, e.g., an antibody, that binds to a specified cell type such as a kidney cell. A targeting group can be a thyrotropin, melanotropin, lectin, glycoprotein, surfactant protein A, Mucin carbohydrate, multivalent lactose, multivalent galactose, N-acetyl- galactosamine, N-acetyl-gulucosamine multivalent mannose, multivalent fucose, glycosylated polyaminoacids, multivalent galactose, transferrin, bisphosphonate, polyglutamate, polyaspartate, a lipid, cholesterol, a steroid, bile acid, folate, vitamin B12, biotin, or an RGD peptide or RGD peptide mimetic. A preferred targeting ligand is GalNAc. Exemplary targeting ligands are described in Table 2 of US 2013/0203836, which is hereby incorporated by reference.

[95] Other examples of ligands include dyes, intercalating agents (e.g. acridines), cross-linkers (e.g. psoralene, mitomycin C), porphyrins (TPPC4, texaphyrin, and Sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine and dihydrophenazine), artificial endonucleases (e.g. EDTA), lipophilic molecules (e.g., cholesterol, cholic acid, adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone, glycerol (e.g., esters and ethers thereof, e.g., Ci₀-C₂o alkyl, e.g., l,3-bis-0(hexadecyl)glycerol, l ,3-bis-0(octaadecyl)glycerol), geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3 -propanediol, heptadecyl group, palmitic acid, myristic acid, 03-(oleoyl)lithocholic acid, 03-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine), peptide conjugates (e.g., antennapedia peptide and Tat peptide), alkylating agents, phosphate, amino, mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]₂, polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes, haptens (e.g. biotin), transport/absorption facilitators (e.g., aspirin, vitamin E, and folic acid), synthetic ribonucleases (e.g., imidazole, bisimidazole, histamine, imidazole clusters, acridine-imidazole conjugates, and Eu3+ complexes of tetraazamacrocycles), dinitrophenyl, HRP, or AP.

[96] Ligands can be proteins, e.g., glycoproteins, or peptides, e.g., molecules having a specific affinity for a co-ligand, or antibodies e.g., an antibody, that binds to a specified cell type such as a cancer cell, endothelial cell, or bone cell. Ligands may also include hormones and hormone receptors. They can also include non-peptidic species, such as lipids, lectins, carbohydrates, vitamins, cofactors, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl- gulucosamine multivalent mannose, or multivalent fucose. The ligand can be, for example, a lipopolysaccharide, an activator of p38 MAP kinase, or an activator of NF-KB.

[97] The ligand can be a substance, e.g., a drug, which can increase the uptake of the siRNA into the cell, for example, by disrupting the cell's cytoskeleton, such as by disrupting the cell's microtubules, microfilaments, and/or intermediate filaments. The drug can be, for example, taxon, vincristine, vinblastine, cytochalasin, nocodazole, japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, or myoservin.

[98] The ligand can increase the uptake of the siRNA into the cell, for example, by activating an inflammatory response. Exemplary ligands that have such an effect include tumor necrosis factor alpha (TNF-ot), interleukin-1 beta, or gamma interferon.

[99] In one embodiment, the ligand is a lipid or lipid-based molecule. Such a lipid or lipid- based molecule may bind a serum protein, e.g., human serum albumin (HSA). An HSA binding ligand allows for distribution of the conjugate to a target tissue, e.g., a non-kidney target tissue of the body. For example, the target tissue can be the liver, including parenchymal cells of the liver. Other molecules that can bind HSA can also be used as ligands. For example, neproxin or aspirin can be used. A lipid or lipid-based ligand can (a) increase resistance to degradation of the conjugate, (b) increase targeting or transport into a target cell or cell membrane, and/or (c) can be used to adjust binding to a serum protein, e.g., HSA.

[100] A lipid based ligand can be used to modulate, e.g., control the binding of the conjugate to a target tissue. For example, a lipid or lipid-based ligand that binds to HSA more strongly will be less likely to be targeted to the kidney and therefore less likely to be cleared from the body. A lipid or lipid-based ligand that binds to HSA less strongly can be used to target the conjugate to the kidney.

[101] In one embodiment, the lipid based ligand binds HSA. For example, it may bind HSA with a sufficient affinity such that the conjugate will be preferably distributed to a non-kidney tissue. However, it is preferred that the affinity not be so strong that the HSA-ligand binding cannot be reversed.

[102] In another preferred embodiment, the lipid based ligand binds HSA weakly or not at all, such that the conjugate will be preferably distributed to the kidney. Other moieties that target to kidney cells can also be used in place of or in addition to the lipid based ligand.

[103] In another aspect, the ligand is a moiety, e.g., a vitamin, which is taken up by a target cell, e.g., a proliferating cell. These are particularly useful for treating disorders characterized by unwanted cell proliferation, e.g., of the malignant or non-malignant type, e.g., cancer cells. Exemplary vitamins include vitamins A, E, and K. Other exemplary vitamins include B vitamins, e.g., folic acid, vitamin B₁₂, riboflavin, biotin, pyridoxal or other vitamins or nutrients taken up by cancer cells. Also included are HSA and low density lipoprotein (LDL).

[104] In another embodiment, the ligand is a cell-permeation agent, such as a helical cell- permeation agent. The agent may be amphipathic. An exemplary agent is a peptide such as tat or antennopedia. The helical agent may be an alpha-helical agent, for example, which has a lipophilic and a lipophobic phase.

[105] The ligand can be a peptide or peptidomimetic. A peptidomimetic (also referred to as an oligopeptidomimetic) is a molecule capable of folding into a defined three-dimensional structure similar to a natural peptide. The peptide or peptidomimetic moiety can be about 5-50 amino acids long, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids long (see, for example, Table 3 of US 2013/0203836, which is hereby incorporated by reference).

[106] A peptide or peptidomimetic can be, for example, a cell permeation peptide, cationic peptide, amphipathic peptide, or hydrophobic peptide (e.g., consisting primarily of Tyr, Trp or Phe). The peptide moiety can be a dendrimer peptide, constrained peptide or crosslinked peptide. In another alternative, the peptide moiety can include a hydrophobic membrane translocation sequence (MTS). An exemplary hydrophobic MTS-containing peptide is RFGF having the amino acid sequence AAVALLPAVLLALLAP. An RFGF analogue (e.g., amino acid sequence AALLPVLLAAP) containing a hydrophobic MTS can also be a targeting moiety. The peptide moiety can be a "delivery" peptide, which can carry large polar molecules including peptides, oligonucleotides, and protein across cell membranes. For example, sequences from the HIV Tat protein (GRKKRRQRRRPPQ) and the Drosophila Antennapedia protein (RQIKIWFQNRRMKWKK) have been found to be capable of functioning as delivery peptides. A peptide or peptidomimetic can be encoded by a random sequence of DNA, such as a peptide identified from a phage-display library, or one-bead-one-compound (OBOC) combinatorial library (Lam et al., Nature, 354:82-84, 1991). In one embodiment, the peptide or peptidomimetic tethered to an siRNA via an incorporated monomer unit is a cell targeting peptide such as an arginine-glycine-aspartic acid (RGD)-peptide, or RGD mimic. A peptide moiety can range in length from about 5 amino acids to about 40 amino acids. The peptide moieties can have a structural modification, such as to increase stability or direct conformational properties.

[107] An RGD peptide moiety can be used to target a tumor cell, such as an endothelial tumor cell or a breast cancer tumor cell (Zitzmann et al., Cancer Res., 62:5139-43, 2002). An RGD peptide can facilitate targeting of an siRNA to tumors of a variety of other tissues, including the lung, kidney, spleen, or liver (Aoki et al., Cancer Gene Therapy 8:783-787, 2001). In one embodiment, the RGD peptide will facilitate targeting of an siRNA to the kidney. The RGD peptide can be linear or cyclic, and can be modified, e.g., glycosylated or methylated to facilitate targeting to specific tissues.

[108] Peptides that target markers enriched in proliferating cells can be used. For example, RGD containing peptides and petomimetics can target cancer cells, in particular cells that exhibit an integrin. In addition to RGD, one can use other moieties that target the integrin ligand. Generally, such ligands can be used to control proliferating cells and angiogeneis. The ligands can target PECAM-1, VEGF, or other cancer gene, e.g., a cancer gene described herein.

[109] The ligand can target the liver. For example, a liver-targeting agent can be a lipophilic moiety. Examples of such lipophilic moieties include, but are not limited to, lipids, cholesterols, oleyl, retinyl, and cholesteryl residues. Other lipophilic moieties that can function as liver- targeting agents include cholic acid, adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone, l,3-bis-0(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, 03- (oleoyl)lithocholic acid, 03-(oleoyl)cholenic acid, dimethoxytrityl, and phenoxazine.

[110] An siRNA can also be targeted to the liver by association with a low-density lipoprotein (LDL), such as lactosylated LDL. Polymeric carriers complexed with sugar residues can also function to target siRNA to the liver.

[I l l] A targeting agent that incorporates a sugar, e.g., galactose and/or analogues thereof, is particularly useful. These agents target, in particular, the parenchymal cells of the liver. For example, a targeting moiety can include more than one or preferably two or three galactose moieties, spaced about 15 angstroms from each other. The targeting moiety can alternatively be lactose (e.g., three lactose moieties), which is glucose coupled to a galactose. The targeting moiety can also be N-acetyl-galactosamine, N-Ac-glucosamine. A mannose or mannose-6- phosphate targeting moiety can be used for macrophage targeting.

[112] A "cell permeation peptide" is capable of permeating a cell, e.g., a microbial cell, such as a bacterial or fungal cell, or a mammalian cell, such as a human cell. A microbial cell- permeating peptide can be, for example, an alpha-helical linear peptide (e.g., LL-37 or Ceropin PI), a disulfide bond-containing peptide (e.g., alpha-defensin, beta-defensin or bactenecin), or a peptide containing only one or two dominating amino acids (e.g., PR-39 or indolicidin). A cell permeation peptide can also include a nuclear localization signal (NLS). For example, a cell permeation peptide can be a bipartite amphipathic peptide, such as MPG, which is derived from the fusion peptide domain of HIV- 1 gp41 and the NLS of SV40 large T antigen (Simeoni et ah, Nucl. Acids Res. 31:2717-2724, 2003).

[113] In one embodiment, a targeting peptide can be an amphipathic alpha-helical peptide. Exemplary amphipathic alpha-helical peptides include, but are not limited to, cecropins, lycotoxins, paradaxins, buforin, CPF, bombinin-like peptide (BLP), cathelicidins, ceratotoxins, S. clava peptides, hagfish intestinal antimicrobial peptides (HFIAPs), magainines, brevinins-2, dermaseptins, melittins, pleurocidin, H_2A peptides, Xenopus peptides, esculentinis-1, and caerins. A number of factors may be considered to maintain the integrity of helix stability. For example, a maximum number of helix stabilization residues may be utilized (e.g., leu, ala, or lys), and a minimum number of helix destabilization residues nay be utilized (e.g., proline or cyclic monomeric units). The capping residue may be considered (for example Gly is an exemplary recapping residue and/or C-terminal amidation can be used to provide an extra H-bond to stabilize the helix). Formation of salt bridges between residues with opposite charges, separated by i±3, or i±4 positions can provide stability. For example, cationic residues such as lysine, arginine, homo-arginine, ornithine or histidine can form salt bridges with the anionic residues glutamate or aspartate.

[1 14] Peptide and peptidomimetic ligands include those having naturally occurring or modified peptides, e.g., D or L peptides; alpha, beta, or gamma peptides; N-methyl peptides; azapeptides; peptides having one or more amide, i.e., peptide, linkages replaced with one or more urea, thiourea, carbamate, or sulfonyl urea linkages; or cyclic peptides.

[115] The targeting ligand can be any ligand that is capable of targeting a specific receptor. Examples include, but are not limited to, folate, GalNAc, galactose, mannose, mannose-6P, clusters of sugars such as a GalNAc cluster, mannose cluster, galactose cluster, or an apatamer. A cluster is a combination of two or more sugar units. The targeting ligand can also be an integrin receptor ligand, Chemokine receptor ligand, transferrin, biotin, serotonin receptor ligand, PSMA, endothelin, GCPII, somatostatin, or LDL or HDL ligand. The ligand can also be based on a nucleic acid, e.g., an aptamer. The aptamer can be unmodified or have any combination of modifications disclosed herein.

Active Pharmaceutical Ingredients

[1 16] The active pharmaceutical ingredient can be any compound suitable for incorporation into a lipid nanoparticle. In one embodiment, the active pharmaceutical ingredient is encapsulated within an aqueous interior of the lipid nanoparticle. In another embodiment, the active pharmaceutical ingredient is present within one or more lipid layers of the lipid nanoparticle. In yet another embodiment, the active pharmaceutical ingredient is bound to the exterior or interior of the lipid surface of the lipid nanoparticle.

[1 17] The active pharmaceutical ingredient can be any compound capable of exerting a desired effect on a cell, tissue, organ, or subject. Such effects may be biological, physiological, or cosmetic, for example. The active pharmaceutical ingredient can be a nucleic acid, peptide, polypeptide (e.g., an antibody), cytokine, growth factor, apoptotic factor, differentiation-inducing factor, cell surface receptor or a corresponding ligand, or hormone. Suitable active pharmaceutical ingredient include, but are not limited to, anti-inflammatory compounds, anti-depressants, stimulants, analgesics, antibiotics, birth control medication, antipyretics, vasodilators, anti-angiogenics, cytovascular agents, signal transduction inhibitors, cardiovascular drugs (e.g., anti-arrhythmic agents), vasoconstrictors, hormones, steroids, and oncology drugs (e.g., an anti-tumor agent, an anti-cancer drug, or anti-neoplatic agent).

[118] In a preferred embodiment, the active pharmaceutical ingredient is a nucleic acid. The nucleic acid can be an interfering RNA (such as a siRNA, including conjugated siRNA as described herein), an antisense oligonucleotide, a DNAi oligonucleotide, a ribozyme, an aptamer, a plasmid, or any combination of any of the foregoing. For example, the nucleic acid can be encoded with a product of interest including, but not limited to, RNA, antisense oligonucleotide, an antagomir, a DNA, a plasmid, a ribosomal RNA (rRNA), a micro RNA (miRNA) (e.g., a miR A which is single stranded and 17-25 nucleotides in length), transfer RNA (tRNA), a small interfering RNA (siRNA), small nuclear RNA (snRNA), antigens, fragments thereof, proteins, peptides, and vaccines or mixtures thereof. In one embodiment, the nucleic acid is an oligonucleotide (e.g., 15-50 nucleotides in length (or 15-30 or 20-30 nucleotides in length)). An siRNA can have, for instance, a duplex region that is 16-30 nucleotides long (e.g., 17-21 or 19- 21 nucleotides long). In another embodiment, the nucleic acid is an immunostimulatory oligonucleotide, decoy oligonucleotide, supermir, miRNA mimic, or miRNA inhibitor. A supermir refers to a single stranded, double stranded or partially double stranded oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or both or modifications thereof, which has a nucleotide sequence that is substantially identical to an miRNA and that is antisense with respect to its target. miRNA mimics represent a class of molecules that can be used to imitate the gene silencing ability of one or more miRNAs. The term "microRNA mimic" refers to synthetic non-coding RNAs (i.e. the miRNA is not obtained by purification from a source of the endogenous miRNA) that are capable of entering the RNAi pathway and regulating gene expression.

[1 19] The nucleic acid that is present in a lipid nanoparticle can be in any form. The nucleic acid can, for example, be single- stranded DNA or RNA, or double-stranded DNA or RNA, or DNA-RNA hybrids. Non-limiting examples of double-stranded RNA include siRNA. Single-stranded nucleic acids include, e.g., antisense oligonucleotides, ribozymes, microRNA, and triplex-forming oligonucleotides. The nucleic acid can be conjugated to one or more ligands (e.g., a targeting ligand).

[120] In further embodiments, the nucleic acid is selected from an interfering RNA, an antisense oligonucleotide, a DNAi oligonucleotide, a ribozyme, an aptamer, a plasmid, and any combination of any of the foregoing. In one embodiment, the RNA is selected from siRNA, aiRNA, miRNA, Dicer-substrate dsRNA, shRNA, ssRNAi oligonucleotides, and any combination of any of the foregoing.

[121] In a more preferred embodiment, the active pharmaceutical ingredient is an siRNA (e.g., an siRNA having a duplex region that is 17-21 or 19-21 nucleotides long). Formulations containing siRNA are useful in down-regulating the protein levels and/or mRNA levels of target proteins. The siRNA may be unmodified oligonucleotides or modified, and may be conjugated with lipophilic moieties such as cholesterol.

[122] In another embodiment, the active pharmaceutical ingredient is a micro RNA.

[123] In one preferred embodiment, the active pharmaceutical ingredient (e.g., a nucleic acid) is fully encapsulated in the lipid nanoparticle.

Cationic Lipids

[124] The lipid nanoparticle may include any cationic lipid suitable for forming a lipid nanoparticle. Preferably, the cationic lipid carries a net positive charge at about physiological pH.

[125] The cationic lipid may be an amino lipid. As used herein, the term "amino lipid" is meant to include those lipids having one or two fatty acid or fatty alkyl chains and an amino head group (including an alkylamino or dialkylamino group) that may be protonated to form a cationic lipid at physiological pH.

[126] The cationic lipid may be, for example, N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), 1,2- dioleoyltrimethyl ammonium propane chloride (DOTAP) (also known as N-(2,3-dioleoyloxy)propyl)-N,N,N- trimethylammonium chloride and l,2-Dioleyloxy-3-trimethylaminopropane chloride salt), N-(l- (2,3- dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), N,N-dimethyl-2,3- dioleyloxy)propylamine (DODMA), 1 ,2-DiLinoleyloxy-N,N-dimethylaminopropane (DLinDMA), l,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), l,2-di-y- linolenyloxy-N,N-dimethylaminopropane (γ-DLenDMA), 1 ,2-Dilinoleylcarbamoyloxy-3- dimethylaminopropane (DLin-C-DAP), 1 ,2-Dilinoleyoxy-3-(dimethylamino)acetoxypropane (DLin-DAC), l,2-Dilinoleyoxy-3-morpholinopropane (DLin-MA), l,2-Dilinoleoyl-3- dimethylaminopropane (DLinDAP), l,2-Dilinoleylthio-3-dimethylaminopropane (DLin-S- DMA), l-Linoleoyl-2-linoleyloxy-3 -dimethyl aminopropane (DLin-2-DMAP), 1,2- Dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl), l,2-Dilinoleoyl-3- trimethylaminopropane chloride salt (DLin-TAP.Cl), l,2-Dilinoleyloxy-3-(N- methylpiperazino)propane (DLin-MPZ), or 3-( ,N-Dilinoleylamino)-l,2-propanediol (DLinAP), 3-(N,N-Dioleylamino)- 1 ,2-propanedio (DOAP), 1 ,2-Dilinoleyloxo-3-(2-N,N- dimethylamino)ethoxypropane (DLin-EG-DMA), 2,2-Dilinoleyl-4-dimethylaminomethyl-[l,3]- dioxolane (DLin-K-DMA) or analogs thereof, (3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)- octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][l,3]dioxol-5-amine, (6Z,9Z,28Z,31Z)- heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate (MC3), l,l'-(2-(4-(2-((2- (bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-l- yl)ethylazanediyl)didodecan-2-ol (C12-200), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[l,3]- dioxolane (DLin-K-C2-DMA), 2,2-dilinoleyl-4-dimethylaminomethyl-[l,3]-dioxolane (DLin-K- DMA), (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino) butanoate (DLin-M-C3-DMA), 3-((6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,3 l-tetraen-19-yloxy)-N,N- dimethylpropan-1 -amine (MC3 Ether), 4-((6Z,9Z,28Z,31 Z)-heptatriaconta-6,9,28,31-tetraen-19- yloxy)-N,N-dimethylbutan-l -amine (MC4 Ether), or any combination of any of the foregoing.

[127] Other cationic lipids include, but are not limited to, N,N-distearyl-N,N- dimethylammonium bromide (DDAB), 3P-(N-(N',N'-dimethylaminoethane)- carbamoyl)cholesterol (DC-Choi), N-(l-(2,3-dioleyloxy)propyl)-N-2-

(sperminecarboxamido)ethyl)-N,N-dimethylammonium trifluoracetate (DOSPA), dioctadecylamidoglycyl carboxyspermine (DOGS), l,2-dileoyl-sn-3-phosphoethanolamine (DOPE), l,2-dioleoyl-3-dimethylammonium propane (DODAP), N-(l,2-dimyristyloxyprop-3- yl)-N,N-dimethyl-N-hydroxyethyl ammonium bromide (DMRIE), and 2,2-Dilinoleyl-4- dimethylaminoethyl-[l,3]-dioxolane (XTC). Additionally, commercial preparations of cationic lipids can be used, such as, e.g., LIPOFECTIN (including DOTMA and DOPE, available from GIBCO/BRL), and LIPOFECTAMINE (comprising DOSPA and DOPE, available from GIBCO/BRL).

[128] Other suitable cationic lipids are disclosed in International Publication Nos. WO 09/086558, WO 09/127060, WO 10/048536, WO 10/054406, WO 10/088537, WO 10/129709, and WO 2011/153493; U.S. Patent Publication Nos. 2011/0256175, 2012/0128760, and 2012/0027803; U.S. Patent Nos. 8,158,601; and Love et al, PNAS, 107(5), 1864-69, 2010.

[129] Other suitable amino lipids include those having alternative fatty acid groups and other dialkylamino groups, including those in which the alkyl substituents are different {e.g., N-ethyl- N-methylamino-, and N-propyl-N-ethylamino-). In general, amino lipids having less saturated acyl chains are more easily sized, particularly when the complexes must be sized below about 0.3 microns, for purposes of filter sterilization. Amino lipids containing unsaturated fatty acids with carbon chain lengths in the range of C₁₄ to C₂₂ may be used. Other scaffolds can also be used to separate the amino group and the fatty acid or fatty alkyl portion of the amino lipid.

[130] In certain embodiments, amino or cationic lipids of the invention have at least one protonatable or deprotonatable group, such that the lipid is positively charged at a pH at or below physiological pH (e.g. pH 7.4), and neutral at a second pH, preferably at or above physiological pH. It will, of course, be understood that the addition or removal of protons as a function of pH is an equilibrium process, and that the reference to a charged or a neutral lipid refers to the nature of the predominant species and does not require that all of the lipid be present in the charged or neutral form. Lipids that have more than one protonatable or deprotonatable group, or which are zwiterrionic, are not excluded from use in the invention.

[131] In certain embodiments, the protonatable lipids have a pKa of the protonatable group in the range of about 4 to about 11 , e.g., a pKa of about 5 to about 7.

[132] Lipid particles can include two or more cationic lipids. The cationic lipids can be selected to contribute different advantageous properties. For example, cationic lipids that differ in properties such as amine pK_a, chemical stability, half-life in circulation, half-life in tissue, net accumulation in tissue, or toxicity can be used in the lipid nanoparticle. In particular, the cationic lipids can be chosen so that the properties of the mixed-lipid particle are more desireable than the properties of a single-lipid particle of individual lipids.

[133] Net tissue accumulation and long term toxicity (if any) from the cationic lipids can be modulated in a favorable way by choosing mixtures of cationic lipids instead of selecting a single cationic lipid in a given formulation. Such mixtures can also provide better encapsulation and/or release of the active pharmaceutical ingredient.

[134] In one example, a series of structurally similar compounds can have varying pK_a values that span a range, e.g. of less than 1 pKg unit, from 1 to 2 pKa units, or a range of more than 2 pK_a units. Within the series, it may be found that a pK_a in the middle of the range is associated with an enhancement of advantageous properties (greater effectiveness) or a decrease in disadvantageous properties (e.g., reduced toxicity), compared to compounds having pK_a values toward the ends of the range. In such a case, two (or more) different compounds having pK_a values toward opposing ends of the range can be selected for use together in a lipid nanoparticle. In this way, the net properties of the lipid nanoparticle (for instance, charge as a function of local pH) can be closer to that of a particle including a single lipid from the middle of the range. Cationic lipids that are structurally dissimilar (for example, not part of the series of structurally similar compounds mentioned above) can also be used in a mixed-lipid nanoparticle.

[135] In some cases, two or more different cationic lipids may have widely differing pK_a values, e.g., differing by 3 or more pKa units. In this case, the net behavior of a mixed lipid nanoparticle will not necessarily mimic that of a single-lipid particle having an intermediate pK_a. Rather, the net behavior may be that of a particle having two distinct protonatable (or deprotonatable, as the case may be) site with different pKa values. In the case of a single lipid, the fraction of protonatable sites that are in fact protonated varies sharply as the pH moves from below the pKa to above the pKa (when the pH is equal to the pKa value, 50% of the sites are protonated). When two or more different cationic lipids may have widely differing pK_a values (e.g., differing by 3 or more pKa units) are combined in a lipid nanoparticle, the lipid nanoparticle can show a more gradual transition from non-protonated to protonated as the pH is varied.

[136] The cationic lipid can comprise from about 20 mol % to about 70 or 75 mol % or from about 45 to about 65 mol % or about 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, or about 70 mol % of the total lipid present in the particle. In another embodiment, the lipid nanoparticles include from about 25% to about 75% on a molar basis of cationic lipid, e.g., from about 20 to about 70%, from about 35 to about 65%, from about 45 to about 65%, about 60%, about 57.5%, about 57.1%, about 50% or about 40% on a molar basis (based upon 100% total moles of lipid in the lipid nanoparticle).

[137] In one embodiment, the ratio of cationic lipid to nucleic acid is from about 3 to about 15, such as from about 5 to about 13 or from about 7 to about 11.

Non-Cationic Lipids

[138] The non-cationic lipid incorporated in the LNP can be a neutral lipid, an anionic lipid, or an amphipathic lipid. Neutral lipids, when present, can be any of a number of lipid species which exist either in an uncharged or neutral zwitterionic form at physiological pH. Such lipids include, for example, diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, dihydrosphingomyelin, cephalin, and cerebrosides. The selection of neutral lipids for use in the particles described herein is generally guided by consideration of, e.g., lipid particle size and stability of the lipid particle in the bloodstream. Preferably, the neutral lipid is a lipid having two acyl groups (e.g., diacylphosphatidylcholine and diacylphosphatidylethanolamine). In one embodiment, the neutral lipids contain saturated fatty acids with carbon chain lengths in the range of Cio to C₂₀. In another embodiment, neutral lipids with mono or diunsaturated fatty acids with carbon chain lengths in the range of Cio to C₂o are used. Additionally, neutral lipids having mixtures of saturated and unsaturated fatty acid chains can be used.

[139] Suitable neutral lipids include, but are not limited to, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl- phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoyl- phosphatidylethanolamine 4-(N- maleimidomethyl)-cyclohexane-l- carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), dimyristoyl phosphatidylcholine (DMPC), distearoyl-phosphatidyl-ethanolamine (DSPE), SM, 16-0- monomethyl PE, 16-O-dimethyl PE, 18-1 -trans PE, l-stearoyl-2-oleoyl- phosphatidyethanolamine (SOPE), cholesterol, or a mixture thereof.

[140] Anionic lipids suitable for use in lipid particles of the invention include, but are not limited to, phosphatidylglycerol, cardiolipin, diacylphosphatidylserine, diacylphosphatidic acid, N-dodecanoyl phosphatidylethanoloamine, N-succinyl phosphatidylethanolamine, N-glutaryl phosphatidylethanolamine, lysylphosphatidylglycerol, and other anionic modifying groups joined to neutral lipids.

[141] Amphipathic lipids refer to any suitable material, wherein the hydrophobic portion of the lipid material orients into a hydrophobic phase, while the hydrophilic portion orients toward the aqueous phase. Such compounds include, but are not limited to, phospholipids, aminolipids, and sphingolipids. Representative phospholipids include sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoyl phosphatdylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine, dipalmitoylphosphatidylcholine, dioleoylphosphatidylcholine, distearoylphosphatidylcholine, or dilinoleoylphosphatidylcholine. Other phosphorus-lacking compounds, such as sphingolipids, glycosphingolipid families, diacylglycerols, and β-acyloxyacids, can also be used.

[142] The non-cationic lipid can be from about 5 mol % to about 90 mol %, about 5 mol % to about 10 mol %, about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, or about 90 mol % of the total lipid present in the particle. In one embodiment, the lipid nanoparticles include from about 0% to about 15 or 45% on a molar basis of neutral lipid, e.g., from about 3 to about 12%) or from about 5 to about 10%. For instance, the lipid nanoparticles may include about 15%, about 10%, about 7.5%, or about 7.1% of neutral lipid on a molar basis (based upon 100% total moles of lipid in the lipid nanoparticle).

Sterols

[143] A preferred sterol for incorporation in the LNP is cholesterol. [144] The sterol can be about 10 to about 60 mol % or about 25 to about 40 mol % of the lipid particle. In one embodiment, the sterol is about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or about 60 mol % of the total lipid present in the lipid particle. In another embodiment, the lipid nanoparticles include from about 5% to about 50% on a molar basis of the sterol, e.g., about 15 to about 45%, about 20 to about 40%, about 48%, about 40%, about 38.5%, about 35%, about 34.4%, about 31.5% or about 31% on a molar basis (based upon 100% total moles of lipid in the lipid nanoparticle).

Aggregation Reducing Agent

[145] The aggregation reducing agent in the LNP can be a lipid capable of reducing aggregation. Examples of such lipids include, but are not limited to, polyethylene glycol (PEG)-modified lipids, monosialoganglioside Gml, and polyamide oligomers (PAO) such as those described in U.S. Patent No. 6,320,017, which is incorporated by reference in its entirety. Other compounds with uncharged, hydrophilic, steric-barrier moieties, which prevent aggregation during formulation, like PEG, Gml or ATTA, can also be coupled to lipids. ATTA-lipids are described, e.g., in U.S. Patent No. 6,320,017, and PEG-lipid conjugates are described, e.g., in U.S. Patent Nos. 5,820,873, 5,534,499 and 5,885,613, each of which is incorporated by reference in its entirety.

[146] The aggregation reducing agent may be, for example, a polyethyleneglycol (PEG)-lipid including, without limitation, a PEG-diacylglycerol (DAG), a PEG-dialkylglycerol, a PEG- dialkyloxypropyl (DAA), a PEG-phospholipid, a PEG-ceramide (Cer), or a mixture thereof (such as PEG-Cerl4 or PEG-Cer20). The PEG-DAA conjugate may be, for example, a PEG- dilauryloxypropyl (Ci₂), a PEG-dimyristyloxypropyl (C₁₄), a PEG-dipalmityloxypropyl (Ci₆), or a PEG- distearyloxypropyl (C]₈). Other pegylated-lipids include, but are not limited to, polyethylene glycol-didimyristoyl glycerol (C14-PEG or PEG-C14, where PEG has an average molecular weight of 2000 Da) (PEG-DMG); (R)-2,3-bis(octadecyloxy)propyl-l-(methoxy polyethylene glycol)2000)propylcarbamate) (PEG-DSG); PEG-carbamoyl-1,2- dimyristyloxypropylamine, in which PEG has an average molecular weight of 2000 Da (PEG- cDMA); N-Acetylgalactosamine-((R)-2,3-bis(octadecyloxy)propyl- 1 -(methoxy poly(ethylene glycol)2000)propylcarbamate)) (GalNAc-PEG-DSG); mPEG

(mw2000)-diastearoylphosphatidyl-ethanolamine (PEG-DSPE); and polyethylene glycol - dipalmitoylglycerol (PEG-DPG). In one embodiment, the aggregation reducing agent is PEG- DMG. In another embodiment, the aggregation reducing agent is PEG-c-DMA.

[147] The average molecular weight of the PEG moiety in the PEG-modified lipids can range from about 500 to about 8,000 Daltons (e.g., from about 1,000 to about 4,000 Daltons). In one preferred embodiment, the average molecular weight of the PEG moiety is about 2,000 Daltons.

[148] The concentration of the aggregation reducing agent may range from about 0.1 to about 15 mol %, based upon the 100% total moles of lipid in the lipid particle. In one embodiment, the formulation includes less than about 3, 2, or 1 mole percent of PEG or PEG-modified lipid, based upon the total moles of lipid in the lipid particle.

[149] In another embodiment, the lipid nanoparticles include from about 0.1% to about 20% on a molar basis of the PEG-modified lipid, e.g., about 0.5 to about 10%, about 0.5 to about 5%, about 10%, about 5%, about 3.5%, about 1.5%, about 0.5%, or about 0.3% on a molar basis (based on 100% total moles of lipids in the lipid nanoparticle).

Lipid Nanoparticles (LNPs)

[150] The lipid nanoparticles may have the structure of a liposome. A liposome is a structure having lipid-containing membranes enclosing an aqueous interior. Liposomes may have one or more lipid membranes. Liposomes can be single-layered, referred to as unilamellar, or multi-layered, referred to as multilamellar. When complexed with nucleic acids, lipid particles may also be lipoplexes, which are composed of cationic lipid bilayers sandwiched between DNA layers.

[151] The formulation is preferably substantially free of aggregates of lipid nanoparticles. For instance, the formulation may have a deo (i.e., 90% of the particles have a particle size) less than about 1, about 0.9, about 0.8, about 0.7, or about 0.6 μηι. In one preferred embodiment, the formulation includes less than about 5, about 4, about 3, about 2, or about 1% by volume of aggregates greater than about 2, about 1.5, about 1 , or about 0.8 μνα..

[152] In certain embodiments, the lipid nanoparticles in the formulation have a d9₈ of less than 1 micron, such as less than about 500 nm, less than about 400 nm, less than about 300 nm, less than about 250 nm, less than about 200 nm, less than about 150 nm or less than about 100 nm. For example, the lipid nanoparticles have a d₉₉ of less than 1 micron, such as less than about 500 nm, less than about 400 nm, less than about 300 nm, less than about 250 nm, less than about 200 nm, less than about 150 nm or less than about 100 nm. In additional embodiments, the particle has a d₅₀ of less than about 100 nm, such as less than about 75nm, less than about 50 nm, less than about 40 nm, less than about 30 nm, less than about 20 nm or less than about 10 nm. For instance, the lipid nanoparticles may have a d₉₉ ranging from about 50 to about 200 nm, or from about 75 to about 150 nm. The lipid nanoparticles may have a d₅₀ ranging from about 5 to about 50 nm, such as from about 10 to about 50 nm, from about 25 to about 50 nm or from about 35 to about 45 nm. In another embodiment, the lipid nanoparticles have a d₅₀ ranging from about 10 to about 40 nm or from about 20 to about 30 nm.

[153] In general, lipid nanoparticles treated with Compound 1 have a reduced particle size. The lipid nanoparticles may have the previously mentioned particle sizes pre- or post-treatment with Compound 1.

[154] In another embodiment, the lipid nanoparticles have a median diameter size of from about 50 nm to about 300 nm, such as from about 50 nm to about 250 nm, for example, from about 50 nm to about 200 nm.

[155] In one preferred embodiment, the d₅₀, d₉₈ or d₉ of the lipid nanoparticles in the formulation does not vary by more than 40, 30, 20, 10, or 5% after 1, 3, 6, 9, 12, and 24 months of storage at 4° C. In one embodiment, after 1 month of storage at 4° C, the lipid nanoparticles in the formulation have d₅o, d₉₈ and/or d₉₉ values as set forth above. For instance, after 1 month storage at 4° C, the lipid nanoparticles in the formulation have d₉₈ or d₉₉ of less than 1 micron, such as less than about 500 nm, less than about 400 nm, less than about 300 nm, less than about 250 nm, less than about 200 nm, less than about 150 nm or less than about 100 nm.

[156] In yet another embodiment, the lipid nanoparticles in the formulation of the present invention have a single mode particle size distribution (i.e., they are not bi- or poly-modal).

[157] The lipid nanoparticles may further comprise one or more lipids and/or other components in addition to those mentioned above. Other lipids may be included in the liposome compositions for a variety of purposes, such as to prevent lipid oxidation or to attach ligands onto the liposome surface. Any of a number of lipids may be present in lipid particles, including amphipathic, neutral, cationic, and anionic lipids. Such lipids can be used alone or in combination.

[158] Additional components that may be present in a lipid particle include bilayer stabilizing components such as polyamide oligomers {see, e.g., U.S. Patent No. 6,320,017, which is incorporated by reference in its entirety), peptides, proteins, and detergents.

[159] Different lipid nanoparticles having varying molar ratios of cationic lipid, non-cationic (or neutral) lipid, sterol (e.g., cholesterol), and aggregation reducing agent (such as a PEG- modified lipid) on a molar basis (based upon the total moles of lipid in the lipid nanoparticles) are provided in Table 1 below.

Table 1

[160] In one embodiment, the weight ratio of lipid to siRNA is at least about 0.5: 1, at least about 1 :1, at least about 2:1, at least about 3:1, at least about 4:1, at least about 5:1, at least about 6: 1, at least about 7: 1, at least about 11: 1 or at least about 33:1. In one embodiment, the weight ratio of lipid to siRNA is from about 1:1 to about 35:1, about 3: 1 to about 15: 1, about 4:1 to about 15:1, or about 5:1 to about 13:1. In one embodiment, the weight ratio of lipid to siRNA is from about 0.5:1 to about 12:1.

[161] In one embodiment, the lipid nanoparticles have an in vivo half life (ti_/2) (e.g., in the liver, spleen or plasma) of less than about 3 hours, such as less than about 2.5 hours, less than about 2 hours, less than about 1.5 hours, less than about 1 hour, less than about 0.5 hour or less than about 0.25 hours.

The Medium

[162] In one embodiment, the medium containing the lipid nanoparticles is substantially free of negative counter-ions (i.e., anions).

[163] In another embodiment, the medium comprises a non-ionic or substantially non-ionic diluent, and preferably includes a non-ionic or substantially non-ionic diluent that does not destabilize the formulation. In one embodiment, the non-ionic or substantially non-ionic diluent increases the stability of the lipid nanoparticles, such as against mechanical disturbances, and/or inhibits the aggregation of the lipid nanoparticles. The medium may comprise water. In a preferred embodiment, the medium is deionized (e.g., deionized water). The water in the medium may have been purified, for example, by reverse osmosis. In a preferred embodiment, the medium (such as water) contains less than about 50 ppm of mineral acid(s), such as less than about 40 ppm, less than about 30 ppm, less than about 20 ppm, less than about 10 ppm, less than about 5 ppm or less than about 1 ppm of mineral acid(s).

[164] The medium may include an acid so long as it is not predominantly in its dissociated form. In one embodiment, the formulation further comprises an acid, wherein the ratio of (a) the concentration of the anions formed from the acid to (b) the concentration of the acid is less than about 0.5, such as less than about 0.4, less than about 0.3, less than about 0.2 or less than about 0.1. In a particular embodiment, the ratio of anion concentration to acid concentration is less than about 0.2 to 0.5. The anions present in the formulation may be derived from the acid in the medium. In one embodiment, the anion is a monovalent anion (such as an anion derived from acetic acid).

Isotonicity Agents

[165] The isotonicity agent(s) included in the formulation are preferably substantially free of anions (e.g., substantially non-ionic), and more preferably are non-ionic. Suitable non-ionic isotonicity agents include, but are not limited to, polyols (e.g., a sugar alcohol such as a C₃-C₆ sugar alcohol), sugars (such as sucrose, fructose, dextrose, trehalose, or glucose), amino acids (such as glycine), and albumin. Suitable sugar alcohols include, but are not limited to, glycerol, erythritol, threitol, arabitol, xylitol, ribitol, sorbitol, mannitol, dulcitol and iditol. In one embodiment, the isotonicity agent is a sugar such as a glucose.

[166] In one embodiment, the concentration of sugar (e.g., glucose) in the medium is at most about 300 mM, such as at most about 200, 100, 75, or 50 mM.

[167] The amount of the isotonicity agent is preferably sufficient for the formulation to obtain an isotonic level.

[168] In another embodiment, the formulation is free or substantially free of isotonicity agents. Pharmaceutical Formulation

[169] For formulations containing conjugated siRNA, the concentration of conjugated siRNA in the formulation may range from about 0.01 to about 50 mg/mL. In one embodiment, the concentration of conjugated siRNA in the formulation ranges from about 0.1 to about 10 mg mL, such as 0.5 to about 5 mg/mL. In another embodiment, the concentration of conjugated siRNA in the formulation is about 0.5, about 0.75, about 1, about 1.5, about 2, about 2.5, about 3, about 4, or about 5 mg/mL.

[170] For LNPs, the concentration of lipid nanoparticles in the formulation may range from about 0.01 to about 50 mg/mL. In one embodiment, the concentration of lipid nanoparticles in the formulation ranges from about 0.1 to about 10 mg/mL, such as 0.5 to about 5 mg/mL. In another embodiment, the concentration of lipid nanoparticles in the formulation is about 0.5, about 0.75, about 1, about 1.5, about 2, about 2.5, about 3, about 4, or about 5 mg/mL.

[171] These formulations may be administered parenterally, for example, intradermally, subcutaneously, intramuscularly, intravenously, or intraperitoneally. In one embodiment, the formulation is directly injected into a subject (e.g., by intravenous infusion). In another embodiment, the formulation is added to an intravenous fluid which is intravenously administered. Because many intravenous fluids contain significant quantities of anions which may over time cause aggregation of LNPs, the LNP-containing formulation of the invention is preferably added to the intravenous fluid shortly before (e.g., within 5, 10 or 15 minutes of) or simultaneously with the intravenous administration to the subject.

[172] These formulations may further include additional pharmaceutically acceptable diluents, excipients, and/or carriers. Example of excipients include, but are not limited to, isotonicity agents, pH adjusting and buffering agents. The formulations may also include lipid-protective agents which protect lipids against free-radical and lipid-peroxidative damages on storage. Such agents include, but are not limited to, lipophilic free-radical quenchers, such as a-tocopherol and water-soluble iron-specific chelators, such as ferrioxamine.

[173] The formulation can be sterilized by known sterilization techniques. The aqueous solutions can then be packaged for use or filtered under aseptic conditions and lyophilized, the lyophilized preparation being combined with a sterile aqueous solution prior to administration.

[174] The concentration of conjugated siRNA in the formulation can range, for example, from less than about 0.01% (e.g., at or at least about 0.05-5%) to as much as 10 to 30% by weight. The dose of conjugated siRNA is dependent on many factors, including the disorder and active pharmaceutical ingredient. In one embodiment, the dose of conjugated siRNA administered may range from about 0.01 and about 50 mg per kilogram of body weight (e.g., from about 0.1 and about 5 mg/kg of body weight).

[175] The concentration of lipid nanoparticles in the formulation can range, for example, from less than about 0.01% (e.g., at or at least about 0.05-5%) to as much as 10 to 30% by weight. The dose of lipid nanoparticles is dependent on many factors, including the disorder and active pharmaceutical ingredient. In one embodiment, the dose of lipid nanoparticles administered may range from about 0.01 and about 50 mg per kilogram of body weight (e.g., from about 0.1 and about 5 mg/kg of body weight). [176] The formulation can be provided in kit form. The kit will typically be comprised of a container that is compartmentalized for holding the various elements of the kit. The kit may contain the lipid nanoparticles, the LNP-containing formulation (such as in dehydrated or concentrated form), or the conjugated siR A formulation with instructions for their rehydration or dilution and administration.

Conjugated siR A - Methods of Manufacture

[177] The conjugated siRNA can be prepared by methods known in the art, such as those described in U.S. Patent Publication Nos. 2013/0184328, 2013/0203836, 2013/0202652, 2013/0317080, 2013/0211063, and 2013/0184324, which are hereby incorporated by reference.

Lipid Nanoparticles - Methods of Manufacture

[178] The lipid nanoparticles may be prepared by an in-line mixing method as follows. In this method, both the lipids (e.g., the cationic lipid, non-cationic lipid, sterol, and aggregation reducing agent) and the nucleic acid are added in parallel into a mixing chamber. The mixing chamber can be a simple T-connector. This method is disclosed, for example, in International Publication No. WO 2010/088537, U.S. Patent Nos. 6,534,018 and US 6,855,277, U.S. Patent Publication No. 2007/0042031 and Pharmaceuticals Research, Vol. 22, No. 3, Mar. 2005, p. 362-372, which are hereby incorporated by reference in their entirety.

[179] In one embodiment, individual and separate stock solutions are prepared - one containing lipid (e.g., the cationic lipid, non-cationic lipid, sterol, and aggregation reducing agent) and the other an active pharmaceutical ingredient, such as a nucleic acid (e.g., siRNA). A lipid stock solution containing a cationic lipid, non-cationic lipid, sterol, and an aggregation reducing agent (e.g., a PEG-modified lipid) is prepared by solubilizing the lipids in a solution of an alcohol (e.g., ethanol) at, for example, a lipid concentration of 25 mg/mL. The nucleic acid (e.g., siRNA) is solubilized in acetate buffer, for example, at a concentration of 0.8 mg/mL. For small scale, 5 mL of each stock solution may be prepared. [180] Preferably, the stock solutions are completely clear, and the lipids are completely solubilized before combining them with the nucleic acid. The stock solutions may be heated to completely solubilize the lipids.

[181] The individual stock solutions (i.e., the lipid stock solution and the nucleic acid stock solution) may be combined by pumping each solution to a T-junction (i.e., by in-line mixing). This results in the formation of the lipid nanoparticles.

[182] Once the lipid nanoparticles have been formed, they may be contacted with the delivery enhancer (e.g., Compound 1, Compound 2, or a mixture thereof) by any method known in the art. For instance, the delivery enhancer may be added to and/or mixed and/or incubated with the lipid nanoparticles.

[183] In another embodiment, the delivery enhancer compound is added during the formation of the lipid nanoparticles. For instance, the delivery enhancer compound may be added to and/or mixed and/or incubated with the lipid stock solution and the nucleic acid stock solution.

[184] In one embodiment, the method includes incubating the lipid nanoparticle with the delivery enhancer. The incubation can be for from about 1 to about 48 hours. In one embodiment, the process involves incubating overnight (e.g., for about 12 or 16 hours). In one embodiment, the process involves incubating at a temperature between about 0 ° C and about 10° C, such as between about 2° C to about 5° C, e.g., at about 4° C. In one embodiment, the process involves incubating overnight (e.g., for about 12 or 16 hours) at about 4° C. In one embodiment, the incubation is conducted in an Eppendorf tube.

[185] Following the formation of the lipid nanoparticles, the medium of the lipid nanoparticles may be exchanged to one which is (a) non-ionic or substantially non-ionic and/or (b) free of or substantially free of anions. This exchange can be performed by dialysis or tangential flow filtration.

[186] For example, the lipid nanoparticles may be dialyzed into reverse osmosis / deionized (RO/DI) water, and then concentrated (e.g., using centrifuge tubes). The dispersion medium can then be changed to, for example, 300 mM glucose by adding an appropriate stock solution, for example, to give final lipid nanoparticles at ~lmg/n L (based on siRNA).

[187] Alternatively, the medium may be exchanged as follows. The lipid nanoparticles are diluted into RO/DI water. The diluted lipid nanoparticles are then concentrated using tangential flow filtration. The concentration step includes washing with lOx larger volume-compared to concentrated formulation volume-of RO/DI water. The dispersion medium can then be changed to, for example, 300 mM glucose by adding an appropriate stock solution, for example, to give final lipid nanoparticles at ~lmg mL (based on siRNA).

Examples

[188] The examples below are provided to describe specific embodiments of the present invention. By providing these specific examples, the applicants do not limit the scope and spirit of the present invention.

Materials siRNAs and Formulation into Lipid Nanoparticles (LNP) or Cholesterol-Conjugated siRNA

[189] In the examples below, the siRNAs used target GFP (eGFP plasmid, Clonetech, Mountain Via, CA). The siRNAs were labelled with Alexa Fluor 647 (alexa637) and formulated into LNPs or conjugated to cholesterol. The procedure used to produce LNP-siRNA, LNP- siRNA-alexa647 and LNP-siRNA-gold are as described in Gilleron et al, Nature Biotech., 31(7), 638-646, 2013.

Cell Culture and Cell Lines

[190] GFP-HeLa cells (Bramsen et al, Nucleic Acid Res., 37(9), 2867-2881, 2009) were cultured in DMEM media complemented with 10% FBS and 1% penicillin-streptomycin at 37° C and 5% C0₂. Primary human fibroblasts (GM00041, Coriell Institute, Camden, NJ) were cultured and infected with Rab5-GFP as previously described (Pal et al, J. Cell Biol, 172(4), 605-618). Primary mouse hepatocytes and endothelial cells were obtained from GFP-lifeact transgenic mice (Reidl et al, Nature Methods, 7(3), 168-169, 2010), following isolation and culture protocols described in Zeigerer et al, Nature, 485(7399), 465-470, 2012 and Limmer et al., Nature Methods, 6, 1348-1354, 2000). When required the cells were seeded on 24 (for electron microscopy analysis) or 96 (for fluorescence microscopy analysis) well plates.

Example 1

Identification of Compounds as Delivery Enhancers

[191] A high throughput screen was performed to identify chemical compounds that improve delivery of (i) cholesterol-conjugated siRNA and (ii) lipid nanoparticles containing siRNA. The primary screen was performed in HeLa cells, a cell line which exhibits the key general features of the mammalian endocytic pathway. The results were validated by performing a secondary screen in primary cells, fibroblasts and hepatocytes. The assay consisted of GFP-expressing HeLa cells transfected with suboptimal doses of LNPs containing an anti-GFP siRNA. The assay was optimized to obtain 20% GFP expression reduction as measured by quantitative microscopy. As a pre-requisite for the screen, it was verified that under the same conditions silencing could be boosted to higher than 80% by adding a known transfection reagent. This demonstrated that the amount of siRNA per se was not limiting, but rather cellular uptake and/or escape from the endolysosomal system was suboptimal, allowing us to carry out a screen to improve these steps.

[192] To optimize the protocol, a pilot screen was initially performed on a small set of compounds, with or without overnight pre-incubation of the compounds with the delivery system prior to adding the mixtures to the cells. This pilot screen revealed that an overnight preincubation increased the number of hits for LNPs. Therefore, an overnight compound preincubation step was incorporated for the LNP screen. After 5 hours of incubating the cells with the compound and siRNA mixtures, the cells were washed, and further incubated with fresh media for 72 hours. The cells were fixed, nuclei were stained with Hoechst and the GFP expression was quantified on an automated fluorescence microscope. All transfections were performed in serum containing media to mimic blood flow conditions. More than 50,000 compounds were tested. 25 and 27 compounds improved the silencing activity of LNPs and cholesterol-conjugated siRNAs, respectively.

Example 2

Human Primary Fibroblasts

[193] The compounds identified from the high throughput screen in Example 1 were tested in human primary fibroblasts expressing Rab5-GFP. These cells were selected because they generally internalize LNPs inefficient and consequently are also poorly transfected. Gilleron, supra.

[194] Rab5-GFP human primary fibroblasts were transfected with LNP-siRNA formulation preincubated or not with the compounds (following a similar procedure as that in Example 1). After 72 hours, the cells were fixed with PFA 4% (pH 7.2 in phosphate buffer) for 20 minutes at room temperature. After washing, cells nuclei were labelled with Dapi and cytosol with SytoBlue. Acquisition and analysis of images (at least 25 fields per conditions) were done on an ArrayscanVTI with Twisterll automated wide field microscope (TDS, MPI-CBG, Dresden).

[195] The results for the lipid nanoparticles and cholesterol-conjugated siRNA are provided in Tables 2 and 3 below, respectively.

Table 2

Compound GFP Intensity

Untreated 100

LNP with no enhancer 80.16

Compound GFP Intensity

119.18

CBN040 D12

87.63

CBN040 K7

Table 3

Compound GFP Intensity

ChC + DMSO (Chol-siRNA) 100

Compound GFP Intensity

101.74

CBN052 E20

98.27

ADD054 05

Cell death

Tegaserod maleate

Compound GFP Intensity

72.35

HO

Indirubin-3 '-monoxime

HF13-4.10.2-1 93.09

Example 3

Primary Mouse Hepatocytes

[196] The procedure in Example 2 was repeated with primary mouse hepatocytes expressing Lifeact-GFP instead of human primary fibroblasts. The results for the lipid nanoparticles and cholesterol-conjugated siRNA are provided in Tables 4 and 5 below, respectively.

Table 4

Compound GFP Intensity

DMSO (vehicle) 100

Compound GFP Intensity

72.90

CBN007 F7

103.96

<xxVxx

CBN040 D12

82.73

CBN040 H10 Compound GFP Intensity

51.03

CBN040I13

93.31

CP WOOl J18

99.03

ADD029F15

Table 5

Compound Concentration GFP

(μηι) Intensity

MOCK - 100 r^~ 10 104.42

Ursolic acid

Example 4

Uptake Assay

[197] LNPs treated with BADGE or CP WOO 1 J18 or DMSO (control) were analyzed by electron microscopy. Morphological experiments were analysed in a blind fashion using a code that was not broken until the quantitation was completed. For electron microscopy analysis on HeLa cells, cells were transfected with LNP-siRNA-gold and fixed with 2.5% glutaraldehyde (in phosphate buffer) overnight. Then, cells were post-fixed in ferrocyanide reduced osmium as described in Karnovsky, Proceedings of the Eleventh Annual Meeting of the American Society or Cell Biology, 284, 186, 1971. Cells were dehydrated in an increasing bath of ethanol for 10 minutes, and infiltrated with a mixture of ethanol and Epon (3:1 and 1:3) and pure Epon for 1 hour. After Epon polymerization overnight at 60° C, the 24 well plates were broken and pieces of Epon were glued on Epon sticks. 70 to 50 nm sections were then cut and stained with uranyl acetate and lead citrate following a standard procedure (see Karnovsky, Proceedings of the Eleventh Annual Meeting of the American Society for Cell Biology, 284, 186, 1971). Supermontaging of 100 images were randomly collected at 1 lOOOx magnification on a Tecnai 12 TEM microscope (FEI) (electron microscopy facility, mpi-cbg, Dresden) and the images stitching was achieved using the open access software Blendmont (Boulder Laboratory, University of Colorado, USA).

[198] To quantify the total uptake as well as the ratio of structures labelled versus unlabelled in a reliable manner, a stereological approach based on randomly distributed crosses was applied allowing a relative loading index calculation and normalization of the number of structure counted (Lucocq, Transgenic Research, 16(2), 133-145, 2007. To quantify the ratio of siR A escape from endosome, a plug-in was developed that automatically counts the total number of gold particles per montage. Images were processed by performing morphological bottom-hat filtering on the grayscale input image (Soille, Morphological Image Analysis, Principles and Applications, Springer-Verlag Telos, 1999). The structuring element used for this was a circle of a radius bigger than the object of interest (radius 4). Following this, image equalization was performed to the interval [0;1] and thresholding with a threshold set at 0.3. The binarized images were then analyzed by the watershed transform to split contiguous gold particles. A last postprocessing step was performed to remove uncertain gold particles (particles having the average intensity value less than 5 standard deviations of the median intensity value in the whole image). Then, for a set of images, the number of particles were automatically counted and manually counted and an error rate (< 1%) was determined, confirming that the procedure succeeded in correctly identifying gold particles. Finally, the procedure to determine the total number of gold particles in the images was applied. Then, the number of gold particles within the cytoplasm were counted manually based on morphological recognition. For this analysis, three pieces of Epon were selected containing cells incubated for 6 hours with LNP-siRNA-gold. For each piece, several sections were cut that were collected on eight grids. Among these eight grids covering a large portion of the cells, three grids were randomly selected. Five super-montaging, at random places but in areas containing cells, were made for each grid. The super-montaging covered approximately two cells. Altogether, in each experiment, approximately 45 super- montaging were analyzed corresponding roughly to 90 cells per condition. Three independent experiments were analyzed, amounting to -270 cells per condition. For each condition, at least 100,000 siR A-gold particles were automatically counted. [199] BADGE increased the uptake of LNPs as indicated by the -15 fold increase in the amount of siRNA-gold internalized by the cells shown in Figure lb. The ratio of siRNA-gold in the cytosol versus the total amount internalized was not increased (Figure lc). Without wishing to be bound by theory, these results suggest that BADGE acts exclusively on the uptake, rather than on the release of siR As from endosomes. In contrast, CPWOOl J18 was found to slightly reduce the total amount of siRNAs-gold internalized (Figure lb) while increasing the ratio of siRNA-gold in the cytosol versus the total amount internalized by approximately 5 fold as shown in Figure lc. Again, without being bound by theory, these results suggest that CPWOOl J18 improves the release of siRNAs from endosomes. Overall, the total amount of cytosolic siRNA- gold was significantly more elevated with treatment of BADGE or CPWOOl J18 compared to DMSO (Figure Id). This is also evidenced by electron micrograph images. See the arrows in Figure la. Thus, BADGE and CPWOOl J18 act via different mechanisms, improving not only uptake but also endosomal escape.

[200] To investigate whether the compounds acted upon the delivery systems or the cells, the silencing enhancers were either pre-incubated with the cells or were added to the cells concomitantly with the delivery systems. If a compound were acting primarily on the cell, its silencing enhancer effect would be expected to occur also when added to the cells prior to the siRNAs. By this criterion, we found that 28% of the enhancer compounds were active only when incubated with the LNPs whereas the remaining 72% were most probably acting on the cells. Interestingly, almost all compounds that improved silencing activity by facilitating endo- lysosomal escape were active when pre-incubated with the cells, suggesting that they act upon the cells. The results are provided for the lipid nanoparticles and cholesterol conjugated siRNAs in tables 6 and 7, respectively.

Table 6 - LNPs

Mechanism of Action Compounds Uptake value

BADGE 16.73

Acts on LNPs Uptake

CPW 001 N14 7.44 ADD031 Oi l 7.16

Estradiol valerate 4.35

CBN047 D17 1.77

Release CBN007 F7 0.61

CBN002 B19 4.47

ADD025 H20 4.32

ADD042 P18 2.6

CBN40:B8 2.48

Uptake

CBN040 113 2.35

CBN040 J8 1.75

Pterostilbene 1.6

APIOLE 1.45

Acts on Cells

CPWOOl F10 1.19

CBN040 H10 0.92

CPWOOl J18 0.76

ADD041 D14 0.73

Release

ADD029 F15 0.66

CBN035 C21 0.63

CBN040 D12 0.5

CBN040 K7 0.45

Table 7 - Cholesterol Conjugated siRNAs

Mechanism of Action Compounds Uptake value

CBN052 E20 21.15

ADD051 E7 22.67

Uptake

Tetrandrine 24.28

ADD054 05 30.24 CBN026 N14 5.54

CBN039 O20 4.87

ADD029 G19 1.58

Ursolic acid 1.81

Indirubin (also referred to as 1.34

indirubin-3 ' -monoxime)

Lomatin 0.92

CBN007 E18 1.02

Release CBN053 M19 0.91

Methoxychlor 0.99

HF13-4 0.75

Example 5

Size Analysis of Lipid Nanoparticles Treated with BADGE

[201] Electron micrograph negative staining was performed on LNPs treated with BADGE and DMSO (control) to determine the differences in morphological properties, such as size, of the LNPs.

[202] An in vitro uptake assay was performed with these LNPs. HeLa cells were transfected with LNP-siRNA-alexa647 treated with BADGE or DMSO. After 72 hours, the cells were fixed with PFA 4% (pH 7.2 in phosphate buffer) for 20 minutes at room temperature. After washing, cells nuclei were labelled with Dapi and cytosol with SytoBlue. Images were acquired on an Opera automated confocal microscope (TDS, MPI-CBG, Dresden) and analyzed on MotionTracking software as previously described in Gilleron et at, Nature Biotech., 31(7), 638- 646, 2013). [203] To determine the endocytic pathway used by LNPs to enter the cell, a depletion of key endocytic machinery (namely, clathrin (CLTC), ARF-1, and RAC-1) was performed as previously described in Gilleron et al, Nature Biotech., 31(7), 638-646, 2013.

By electron micrograph negative staining analysis, it was found that BADGE had an effect on the nanoparticles themselves. BADGE reduced the size of LNPs by ~2 fold as shown in Figure 2a. This reduction in size was associated with a dramatic acceleration of uptake kinetics as shown in Figure 2b. The uptake of LNPs exposed to BADGE is much less sensitive to the knockdown of clathrin, ARF-1 and RAC-1 compared to the control (Figure 2c), suggesting that the smaller LNPs are captured through a broader set of endocytic mechanisms. Without wishing to be bound by theory, the present inventors theorize that the reduction in size of the LNPs is due to particle compaction, not fission.

Example 6

Enhanced Delivery to Endothelial Cells

[204] The uptake of LNPs treated with BADGE or DMSO was measured in primary endothelial cells by the same method as in Example 5. Endothelial cells are important in a number of diseases and generally difficult to transfect with siRNAs. BADGE treated LNPs were delivered twice as efficiently as control LNPs in primary endothelial cells as shown in Figure 3 a. Consistently, this increase in uptake was accompanied by a similar (~2-fold) increase in silencing activity, as determined by analyzing the GFP intensity in primary endothelial cells isolated from GFP-lifeact transgenic mice (Figure 3b).

[205] To test whether BADGE could improve delivery to the endothelium in a tissue, BADGE treated LNPs were injected into the heart of mice. LNPs pre-treated with BADGE were strongly captured by the endocardium cell layer as evidenced by numerous fluorescent vesicles. The quantification of the number of vesicles loaded with LNP-siRNA-alexa647 revealed that the treatment with BADGE increased the uptake in the endothelial cell in vivo by approximately 14 fold. See Figure 3c. [206] These and other changes can be made to the embodiments in light of the above-detailed description. In general, in the following claims, the terms used should not be construed to limit the claims to the specific embodiments disclosed in the specification and the claims, but should be construed to include all possible embodiments along with the full scope of equivalents to which such claims are entitled. Accordingly, the claims are not limited by the disclosure.

The invention furthermore comprises the following items:

1. Lipid nanoparticles treated with a delivery enhancer described herein and mixtures thereof, wherein the lipid nanonparticles comprise an active pharmaceutical ingredient.

2. The lipid nanoparticles of item 1, wherein the lipid nanoparticles are treated with a sufficient amount of the delivery enhancer to reduce the size of the lipid nanoparticles.

3. Lipid nanoparticles comprising (a) an active pharmaceutical ingredient and (b) a delivery enhancer described herein and mixtures thereof.

4. The lipid nanoparticles of any one of items 1-3, wherein the lipid nanoparticles further comprise a cationic lipid, a non-cationic lipid, an aggregation reducing agent, and optionally a sterol.

5. The lipid nanoparticles of any one of items 1-4, wherein the active pharmaceutical ingredient is a nucleic acid.

6. The lipid nanoparticles of item 5, wherein the active pharmaceutical ingredient is an siR A.

7. A pharmaceutical formulation comprising the lipid nanoparticles of any one of items 1-6.

8. A pharmaceutical formulation comprising:

(a) lipid nanoparticles comprising an active pharmaceutical ingredient, and

(b) a delivery enhancer described herein and mixtures thereof.

9. The pharmaceutical formulation of item 8, wherein the active pharmaceutical ingredient is a nucleic acid.

10. The formulation of item 9, wherein the nucleic acid is selected from an interfering RNA, an antisense oligonucleotide, a DNAi oligonucleotide, a ribozyme, an aptamer, a plasmid, and any combination of any of the foregoing. 11. The formulation of item 10, wherein the interfering RNA is selected from siRNA, aiRNA, miRNA, Dicer-substrate dsRNA, shR A, ssRNAi oligonucleotides, and any combination of any of the foregoing.

12. The pharmaceutical formulation of item 8, wherein the active pharmaceutical ingredient is a siRNA.

13. The pharmaceutical formulation of any one of items 9-12, wherein the nucleic acid is fully encapsulated in the lipid nanoparticle.

14. The pharmaceutical formulation of any one of items 9-13, wherein the lipid nanoparticles have a d₅₀ of about 5 to about 500 nm.

15. The pharmaceutical formulation of any one of items 9-14, wherein the lipid nanoparticles further comprise a cationic lipid, a non-cationic lipid (such as a neutral lipid), an aggregation reducing agent (such as polyethylene glycol (PEG) or PEG-modified lipid), and optionally, a sterol.

16. The pharmaceutical formulation of item 15, wherein the cationic lipid has a pKa ranging from about 5 to about 7.

17. A process for preparing lipid nanoparticles comprising the step of incubating lipid nanoparticles in the presence of a delivery enhancer described herein and mixtures thereof, wherein the lipid nanoparticles comprise an active pharmaceutical ingredient.

18. A process for preparing a pharmaceutical formulation comprising adding a delivery enhancer described herein and mixtures thereof to lipid nanoparticles, wherein the lipid nanoparticles comprise an active pharmaceutical ingredient.

Claims

Claims

1. Lipid nanoparticles treated with a delivery enhancer described herein and mixtures thereof, wherein the lipid nanonparticles comprise an active pharmaceutical ingredient.

2. The lipid nanoparticles of claim 1, wherein the lipid nanoparticles are treated with a sufficient amount of the delivery enhancer to reduce the size of the lipid nanoparticles.

3. The lipid nanoparticles of claim 1 or 2, wherein the lipid nanoparticles further comprise a cationic lipid, a non-cationic lipid, an aggregation reducing agent, and optionally a sterol.

4. The lipid nanoparticles of any one of claims 1-3, wherein the active pharmaceutical ingredient is a nucleic acid, preferably an siRNA.

5. A pharmaceutical formulation comprising the lipid nanoparticles of any one of claims 1- 4.

6. The pharmaceutical formulation of claim 5, wherein the active pharmaceutical ingredient is a nucleic acid.

7. The formulation of claim 6, wherein the nucleic acid is selected from an interfering RNA, an antisense oligonucleotide, a DNAi oligonucleotide, a ribozyme, an aptamer, a plasmid, and any combination of any of the foregoing, wherein the interfering RNA is preferably selected from siRNA, aiRNA, miRNA, Dicer-substrate dsRNA, shRNA, ssRNAi oligonucleotides, and any combination of any of the foregoing.

8. The pharmaceutical formulation of claim 6 or 7, wherein the nucleic acid is fully encapsulated in the lipid nanoparticle.

9. The pharmaceutical formulation of any one of claims 6-8, wherein the lipid nanoparticles have a d₅₀ of about 5 to about 500 nm.

10. The pharmaceutical formulation of any one of claims 6-9, wherein the lipid nanoparticles further comprise a cationic lipid, a non-cationic lipid (such as a neutral lipid), an aggregation reducing agent (such as polyethylene glycol (PEG) or PEG-modified lipid), and optionally, a sterol.

11. The pharmaceutical formulation of claim 10, wherein the cationic lipid has a pKa ranging from about 5 to about 7.

12. A process for preparing lipid nanoparticles comprising the step of incubating lipid nanoparticles in the presence of a delivery enhancer described herein and mixtures thereof, wherein the lipid nanoparticles comprise an active pharmaceutical ingredient.

13. A process for preparing a pharmaceutical formulation comprising adding a delivery enhancer described herein and mixtures thereof to lipid nanoparticles, wherein the lipid nanoparticles comprise an active pharmaceutical ingredient.