WO2015184326A1

WO2015184326A1 - Conjugates, particles, compositions, and related methods

Info

Publication number: WO2015184326A1
Application number: PCT/US2015/033284
Authority: WO
Inventors: Roy I. CASE
Original assignee: Cerulean Pharma Inc.
Priority date: 2014-05-30
Filing date: 2015-05-29
Publication date: 2015-12-03
Also published as: WO2015184326A8

Abstract

Particles and compositions containing the particles for delivering nucleic acid agents are described herein. Methods of preparing same are also described.

Description

CONJUGATES, PARTICLES, COMPOSITIONS, AND RELATED METHODS

BACKGROUND

Effective delivery of a nucleic acid agent to a therapeutic target is desirable to provide optimal use and effectiveness of that nucleic acid agent. Particle delivery systems may increase the efficacy or tolerability of the nucleic acid agent.

SUMMARY

The disclosure provides, inter alia, particles comprising nucleic acid agents, e.g., mRNA, and methods of making particles comprising nucleic acid agents, e.g., mRNA. The particles comprising nucleic acid agents, e.g., mRNA, can be used, for example, in the delivery of a nucleic acid agent, e.g., mRNA, to a therapeutic target. The particles comprising nucleic acid agents, e.g., mRNA, can be made by providing a first mixture comprising nucleic acid agent, e.g., mRNA, in a solvent, e.g., a polar aprotic solvent; contacting the first mixture with a second mixture comprising a cationic moiety, e.g., a cationic polymer, and a hydrophobic polymer in a solution comprising a solvent and a co-solvent to provide a third mixture; and contacting the third mixture with a surfactant in an aqueous solution to provide a fourth mixture, to thereby make the particle; wherein the first, second, and third mixtures each contain less than 1,000 ppm water. In some embodiments, the method further comprises a hydrophilic-hydrophobic polymer, e.g., a hydrophilic-hydrophobic polymer that is present in the second mixture. In some embodiments, the formed particles can be exposed to further processing techniques to remove the solvent(s) or to purify the particles {e.g., dialysis). In some embodiments, the particles described herein can be further processed by lyophilization to provide dried particles. The particles comprising nucleic acid agents, e.g., mRNA, can be formulated into a pharmaceutical composition or dosage form, which can be administered to a subject {e.g., a subject in need thereof), for example in the treatment of a disorder. In some embodiments, the particles are nanoparticles.

Accordingly, in a first aspect, the disclosure provides a method of making particles, the method comprising:

(a) providing a first mixture comprising nucleic acid agent, e.g., mRNA, in a solvent; (b) contacting the first mixture with a second mixture comprising a cationic moiety and a hydrophobic polymer in a solution comprising a solvent and a co- solvent to provide a third mixture; and

(c) contacting the third mixture with a surfactant in an aqueous solution to provide a fourth mixture, to thereby make the particle;

wherein the first, second, and third mixtures each contain less than 1,000 ppm water.

In some embodiments, the method further provides lyophilizing the nucleic acid agent, e.g. , mRNA, prior to incorporation into the first mixture.

In some embodiments, the cationic moiety comprises a cationic polymer (e.g. , PEI, cationic polyvinyl alcohol (cPVA), poly(histidine), poly(lysine), or poly(2-dimethylamino)ethyl methacrylate). In some embodiments, the cationic moiety comprises cPVA. In some embodiments, at least a portion of the cationic moiety is attached to at least a portion of the hydrophobic moiety, e.g. , hydrophobic polymer of (b).

In some embodiments, the hydrophobic polymer of (b) comprises poly(lactic-coglycolic acid) (PLGA). In some embodiment, the PLGA has a ratio of from about 25:75 to about 75:25 of lactic acid to glycolic acid. In some embodiments, the PLGA has a ratio of about 50:50 of lactic acid to glycolic acid. In some embodiments, the PLGA has a weight average molecular weight from about 1 kDa to about 70 kDa (e.g. , from about 4 kDa to about 66 kDa, from about 2 kDa to about 12 kDa, from about 6 kDa to about 20 kDa, from about 5 kDa to about 15 kDa, from about 6 kDa to about 13 kDa, from about 7 kDa to about 11 kDa, from about 5 kDa to about 10 kDa, from about 7 kDa to about 10 kDa, from about 5 kDa to about 7 kDa, from about 6 kDa to about 8 kDa, about 6 kDa, about 7 kDa, about 8 kDa, about 9 kDa, about 10 kDa, about 11 kDa, about 12 kDa, about 13 kDa, about 14 kDa, about 15 kDa, about 16 kDa or about 17 kDa).

In some embodiments, the cationic moiety can be covalently attached to the hydrophobic polymer, e.g. , PLGA, e.g. , PLGA- poly(histidine), PLGA-poly(lysine), PLGA-arginine, or PLGA- spermine .

In some embodiments, the surfactant is polyvinyl alcohol (PVA). In some embodiments, the particle comprises less than about 1% of PVA (e.g. , about 0.5%, about 0.4%, about 0.3%, about 0.2%, about 0.1% weight/volume). In some embodiments, the solvent comprises a polar aprotic solvent. In some embodiments, the solvent comprises a plurality of polar aprotic solvents. In some embodiments, the solvent can be selected from acetonitrile (AcN), N, N-dimethylformamide (DMF), dimethylsulfoxide (DMSO), dimethylacetamide (DMA), and propylene carbonate, acetone, cyclohexanone, methyl ethyl ketone, methyl tert-butyl ether, diethyl ether, dimethyl ether, methyl acetate, ethyl acetate and nitromethane.

In some embodiments, the solvent comprises a de- aggregating agent, e.g. , an agent that disrupts hydrogen bonding of the nucleic acid agent, e.g. , mRNA. In some embodiments, the solvent comprises a salt, e.g. , a lithium salt or a calcium salt. In some embodiments, the salt is a lithium salt, e.g. , lithium bromide. In some embodiments, the salt is a calcium salt, e.g. , calcium chloride. In some embodiments, the solvent comprises DMSO. In some embodiments, the solvent comprises lithium bromide in DMSO. In some embodiments, the solvent comprises up to about 5 mM to about 50 mM, up to about 10 mM to about 40 mM, up to about 15 mM to about 30 mM of lithium bromide in DMSO. In some embodiments, lithium bromide can be present at a concentration of 20 mM in DMSO.

In some embodiments, the co-solvent comprises a high polar index solvent, e.g. , one or more of acetonitrile, acetone, methyl ethyl ketone, methyl acetate, and ethyl acetate.

In some embodiments, the first, second, and third mixtures each contain less than 500 ppm water, less than 200 ppm water, less than 100 ppm water, or less than 50 ppm water, e.g. , anhydrous.

In some embodiments, the method further comprises a hydrophilic-hydrophobic polymer, e.g. , a hydrophilic-hydrophobic polymer that is present in the second mixture. In some embodiments, the hydrophilic-hydrophobic polymer comprises polyethylene glycol-poly(lactic- coglycolic acid) (PEG-PLGA). In some embodiments, the PEG-PLGA has a weight average molecular weight of less than 20 kDa, less than 15 kDa, e.g. , about 11 kDa.

In another aspect, the disclosure features particles comprising nucleic acid agents, e.g. , mRNA, that can be made by providing: a first mixture comprising a nucleic acid agent, e.g. , mRNA, and a cationic moiety, e.g. , a cationic polymer, in a solvent, e.g. , a polar aprotic solvent; contacting the first mixture with a second mixture comprising a hydrophobic polymer in a solution comprising a solvent and a co-solvent to provide a third mixture; and contacting the third mixture with a surfactant in an aqueous solution to provide a fourth mixture, to thereby make the particle; wherein the first, second, and third mixtures each contain less than 1,000 ppm water. In some embodiments, the method further comprises a hydrophilic-hydrophobic polymer, e.g. , a hydrophilic-hydrophobic polymer that is present in the second mixture. In some embodiments, the formed particles can be exposed to further processing techniques to remove the solvent(s) or purify the particles (e.g. , dialysis). In some embodiments, the particles described herein can be further processed by lyophilization to provide dried particles. The particles comprising nucleic acid agent, e.g. , mRNA, can be formulated into a pharmaceutical composition or dosage form, which can be administered to a subject (e.g. , a subject in need thereof), for example in the treatment of a disorder.

Accordingly, in some embodiments, the disclosure provides a method of making particles, the method comprising:

(a) providing a first mixture comprising nucleic acid agent, e.g. , mRNA, and a cationic moiety, in a solvent;

(b) contacting the first mixture with a second mixture comprising a hydrophobic polymer in a solution comprising a solvent and a co-solvent to provide a third mixture; and

In some embodiments, the cationic moiety comprises a cationic polymer (e.g. , PEI, cationic polyvinyl alcohol (cPVA), poly(histidine), poly(lysine), or poly(2-dimethylamino)ethyl methacrylate). In some embodiments, the cationic moiety comprises cPVA.

In some embodiments, the cationic moiety can be covalently attached to the hydrophobic polymer, e.g. , PLGA, e.g. , PLGA- poly(histidine), PLGA-poly(lysine), PLGA-arginine, PLGA- spermine.

In some embodiments, the surfactant is PVA. In some embodiments, the particle comprises less than about 1% of PVA (e.g. , about 0.5%, about 0.4%, about 0.3%, about 0.2%, about 0.1% weight/volume).

In some embodiments, the solvent comprises a polar aprotic solvent. In some

embodiments, the solvent comprises a plurality of polar aprotic solvents. In some embodiments, the solvent can be selected from acetonitrile, N, N-dimethylformamide, dimethylsulfoxide (DMSO), dimethylacetamide (DMA), and propylene carbonate, acetone, cyclohexanone, methyl ethyl ketone, methyl tert-butyl ether, diethyl ether, dimethyl ether, methyl acetate, ethyl acetate and nitromethane.

In some embodiments, the co-solvent comprises a high polar index solvent, e.g. , one or more of acetonitrile, acetone, methyl ethyl ketone, methyl acetate, and ethyl acetate. In some embodiments, the first, second, and third mixtures each contain less than 500 ppm water, less than 200 ppm water, less than 100 ppm water, or less than 50 ppm water, e.g. , anhydrous.

In another aspect, the disclosure provides a mixture (e.g. , a solution) comprising:

(a) a nucleic acid agent, e.g. , mRNA;

(b) a cationic moiety; and

(c) a hydrophobic polymer.

In some embodiments, the cationic moiety comprises a cationic polymer (e.g. , PEI, cationic polyvinyl alcohol (cPVA), poly(histidine), poly(lysine), or poly(2-dimethylamino)ethyl methacrylate). In some embodiments, the cationic moiety comprises cPVA. In some

embodiments, at least a portion of the cationic moiety is attached to at least a portion of the hydrophobic moiety, e.g. , hydrophobic polymer of (c).

In some embodiments, the hydrophobic polymer of (c) comprises poly(lactic-coglycolic acid) (PLGA). In some embodiment, the PLGA has a ratio of from about 25:75 to about 75:25 of lactic acid to glycolic acid. In some embodiments, the PLGA has a ratio of about 50:50 of lactic acid to glycolic acid. In some embodiments, the PLGA has a weight average molecular weight from about 1 kDa to about 70 kDa (e.g. , from about 4 kDa to about 66 kDa, from about 2 kDa to about 12 kDa, from about 6 kDa to about 20 kDa, from about 5 kDa to about 15 kDa, from about 6 kDa to about 13 kDa, from about 7 kDa to about 11 kDa, from about 5 kDa to about 10 kDa, from about 7 kDa to about 10 kDa, from about 5 kDa to about 7 kDa, from about 6 kDa to about 8 kDa, about 6 kDa, about 7 kDa, about 8 kDa, about 9 kDa, about 10 kDa, about 11 kDa, about 12 kDa, about 13 kDa, about 14 kDa, about 15 kDa, about 16 kDa or about 17 kDa). In some embodiments, the cationic moiety can be covalently attached to the hydrophobic polymer, e.g. , PLGA, e.g. , PLGA- poly(histidine), PLGA-poly(lysine), PLGA-arginine, or PLG A- spermine .

In some embodiments, the mixture further comprises a hydrophilic-hydrophobic polymer, e.g. , a hydrophilic-hydrophobic polymer that is present in the second mixture. In some embodiments, the hydrophilic-hydrophobic polymer comprises polyethylene glycol-poly(lactic- coglycolic acid) (PEG-PLGA). In some embodiments, the PEG-PLGA has a weight average molecular weight of less than 20 kDa or less than 15 kDa, e.g. , about 11 kDa.

In some embodiments, the solvent comprises a polar aprotic solvent. In some

embodiments, the solvent comprises a plurality of polar aprotic solvents. In some embodiments, the solvent can be selected from acetonitrile (AcN), N, N-dimethylformamide (DMF), dimethylsulfoxide (DMSO), dimethylacetamide (DMA), and propylene carbonate, acetone, cyclohexanone, methyl ethyl ketone, methyl tert-butyl ether, diethyl ether, dimethyl ether, methyl acetate, ethyl acetate and nitromethane.

In some embodiments, the mixture further comprises a surfactant. In some embodiments, the surfactant is PVA. In some embodiments, the particle comprises less than about 1% of PVA (e.g. , about 0.5%, about 0.4%, about 0.3%, about 0.2%, about 0.1% weight/volume).

(a) a nucleic acid agent, e.g. , mRNA;

(b) a hydrophobic polymer; and

(c) a solvent. In some embodiments, the mixture contains less than 500 ppm water, less than 200 ppm water, less than 100 ppm water, or less than 50 ppm water, e.g. , anhydrous.

In some embodiments, the solvent comprises a polar aprotic solvent. In some

In some embodiments, the solvent comprises a mixture of two solvents (e.g. , DMSO and AcN) and contains less than 1,000 ppm of water.

In another aspect, the disclosure provides particles comprising nucleic acid agent, e.g. , mRNA, which can be used, for example, in the delivery of a nucleic acid agent e.g. , mRNA, to a therapeutic target, e.g. , an mRNA vaccine. The particles include a nucleic acid agent, e.g. , mRNA, a cationic moiety, a hydrophobic polymer, and a surfactant.

Accordingly, in one aspect, the disclosure features, particles comprising:

a) a nucleic acid agent, e.g. , mRNA;

b) a hydrophobic polymer;

c) a cationic moiety; and

d) a surfactant; wherein the particles are substantially free of a hydrophilic -hydrophobic polymer.

In some embodiments, the hydrophobic polymer of (b) comprises poly(lactic-coglycolic acid) (PLGA). In some embodiment, the PLGA has a ratio of from about 25:75 to about 75:25 of lactic acid to glycolic acid. In some embodiments, the PLGA has a ratio of about 50:50 of lactic acid to glycolic acid. In some embodiments, the PLGA has a weight average molecular weight from about 1 kDa to about 70 kDa (e.g. , from about 4 kDa to about 66 kDa, from about 2 kDa to about 12 kDa, from about 6 kDa to about 20 kDa, from about 5 kDa to about 15 kDa, from about 6 kDa to about 13 kDa, from about 7 kDa to about 11 kDa, from about 5 kDa to about 10 kDa, from about 7 kDa to about 10 kDa, from about 5 kDa to about 7 kDa, from about 6 kDa to about 8 kDa, about 6 kDa, about 7 kDa, about 8 kDa, about 9 kDa, about 10 kDa, about 11 kDa, about 12 kDa, about 13 kDa, about 14 kDa, about 15 kDa, about 16 kDa or about 17 kDa). In some embodiments, the cationic moiety comprises a cationic polymer (e.g. , PEI, cationic polyvinyl alcohol (cPVA), poly(histidine), poly(lysine), or poly(2-dimethylamino)ethyl methacrylate). In some embodiments, the cationic moiety comprises cPVA.

In some embodiments, the nucleic acid agent, e.g. , mRNA, is present in an amount, e.g. , from about 0.1 to about 50% by weight of the particle (e.g. , from about 1% to about 50%, from about 1 to about 30% by weight of the particle, from about 1 to about 20% by weight of the particle, from about 4 to about 25 % by weight of the particle, or from about 5 to about 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20% by weight of the particle).

In some embodiments, the nucleic acid agent, e.g. , mRNA, is present in an amount, e.g. , from about 0.1 to about 10% by weight of the particle (e.g. , from about 0.1% to about 5%, from about 0.2% to about 4% by weight of the particle, from about 0.3% to about 3% by weight of the particle, from about 0.4% to about 2 % by weight of the particle, from about 0.5% to about 1%, e.g. , about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%, about 2%, about 3%, about 4%, about 5% by weight of the particle).

In some embodiments, the particles further comprise one or more vaccine adjuvant(s). A vaccine adjuvant is a substance that is added to the vaccine to increase the body's immune response to the vaccine. In some embodiments, the vaccine adjuvant is an aluminum gel or an aluminum salt.

In some embodiments, the formed particles can be exposed to further processing techniques to remove the solvents or purify the particles (e.g. , dialysis). In some embodiments, the particles described herein can be further processed by lyophilization to provide dried particles. In another aspect, the disclosure provides a method of eliciting an immunotherapeutic response in a subject, e.g. , a human subject, the method comprising administering to the subject a particle comprising:

a) a nucleic acid agent, e.g. , mRNA;

b) a hydrophobic polymer;

c) a cationic moiety; and

d) a surfactant; wherein the particles are substantially free of a hydrophilic -hydrophobic polymer, to thereby elicit the immunotherapeutic response in the subject e.g. , a human subject.

In some embodiments, the nucleic acid agent, e.g. , mRNA, is present in an amount, e.g. , from about 0.1 to about 50% by weight of the particle (e.g. , from about 1% to about 50%, from about 1 to about 30% by weight of the particle, from about 1 to about 20% by weight of the particle, from about 4 to about 25 % by weight of the particle, or from about 5 to about 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20% by weight of the particle). In some embodiments, the formed particles can be exposed to further processing techniques to remove the solvents or purify the particles (e.g. , dialysis). In some embodiments, the particles described herein can be further processed by lyophilization to provide dried particles.

In another aspect, the disclosure provides a method of treating a subject, e.g. , a human subject, the method comprising administering to the subject a particle comprising:

a) a nucleic acid agent, e.g. , mRNA;

b) a hydrophobic polymer;

c) a hydrophilic-hydrophobic polymer;

d) a cationic moiety; and

e) a surfactant, to thereby treat the subject, e.g. , a human subject.

In some embodiments, the hydrophilic-hydrophobic polymer comprises polyethylene glycol-poly(lactic-coglycolic acid) (PEG-PLGA). In some embodiments, the PEG-PLGA has a weight average molecular weight of less than 20 kDa, less than 15 kDa, e.g. , about 11 kDa. In some embodiments, the surfactant is PVA. In some embodiments, the particle comprises less than about 1% of PVA (e.g. , about 0.5%, about 0.4%, about 0.3%, about 0.2%, about 0.1% weight/volume).

A particle described herein can have one or more of the following properties. In one embodiment, at least a portion of the hydrophobic polymers of (b) has a carboxy terminal end. In one embodiment, a terminal end such as the carboxy terminal end is modified (e.g. , with a reactive group including a reactive group described herein). In one embodiment, at least a portion of the hydrophobic polymers of (b) has a hydroxyl terminal end. In one embodiment, the hydroxyl terminal end is modified (e.g. , with a reactive group). In one embodiment, at least a portion of the hydrophobic polymers of (b) having a hydroxyl terminal end have the hydroxyl terminal end capped (e.g. , capped with an acyl moiety). In one embodiment, at least a portion of the hydrophobic polymers of (b) have both a carboxy terminal end and a hydroxyl terminal end. In one embodiment, at least a portion of the hydrophobic polymers of (b) comprise monomers of lactic and/or glycolic acid.

In one embodiment, at least a portion of the hydrophobic polymers of (b) comprise PLA or PGA. In one embodiment, at least a portion of the hydrophobic polymers of (b) comprises copolymers of lactic and glycolic acid (i.e. , PLGA). In one embodiment, the polymer polydispersity index is less than about 2.5 (e.g. , less than about 1.5). In one embodiment, a portion of the hydrophobic polymers of (b) comprises PLGA having a ratio of from about 25:75 to about 75:25 of lactic acid to glycolic acid. In one embodiment, a portion of the hydrophobic polymers of (b) comprises PLGA having a ratio of about 50:50 of lactic acid to glycolic acid. In one embodiment, the hydrophobic polymers of (b) have a Mw of from about 1 to about 70 kDa, for example, from about 4 kDa to about 12 kDa, from about 8 kDa to about 12 kDa. In one embodiment, the hydrophobic polymers of (b) have a weight average molecular weight of from about 4 kDa to about 12 kDa (e.g. , from about 4 kDa to about 8 kDa). In one embodiment, the hydrophobic polymers of (b) comprise from about 35 to about 90% by weight in, or used as starting materials to make, the particle (e.g. , from about 35 to about 80% by weight).

In one embodiment, at least a portion of the hydrophobic polymer of (b) is covalently attached to the cationic moiety. Additional properties of the particles described herein include the following. In some embodiments, the hydrophilic-hydrophobic polymers are block copolymers. In some embodiments, the hydrophilic-hydrophobic polymers are diblock copolymers. In some embodiments, the hydrophobic portion of at least a portion of the hydrophilic-hydrophobic polymers has a hydroxyl terminal end. In some embodiments, the hydrophobic portion of at least a portion of the hydrophilic-hydrophobic polymers having a hydroxyl terminal end have the hydroxyl terminal end capped (e.g. , capped with an acyl moiety). In some embodiments, the hydrophobic portion of at least a portion of the hydrophilic-hydrophobic polymers having a hydroxyl terminal end have the hydroxyl terminal end capped with an acyl moiety.

Additional properties of the particles described herein include the following. In some embodiments, the hydrophobic portion of at least a portion of the hydrophilic-hydrophobic polymers of comprises copolymers of lactic and glycolic acid (i.e. , PLGA). In some

embodiments, the hydrophobic portion of at least a portion of the hydrophilic-hydrophobic polymers of comprises PLGA having a ratio of from about 25:75 to about 75:25 of lactic acid to glycolic acid. In some embodiments, the hydrophobic portion of at least a portion of the hydrophilic-hydrophobic polymers of comprises PLGA having a ratio of about 50:50 of lactic acid to glycolic acid.

Additional properties of the particles described herein include the following. In some embodiments, the hydrophobic portion of at least a portion of the hydrophilic-hydrophobic polymers of has a weight average molecular weight of from about 4 kDa to about 20 kDa (e.g. , from about 4 to about 12 kDa, from about 6 to about 20 kDa or from about 8 to about 15 kDa). In some embodiments, hydrophilic portion of at least a portion of the hydrophilic-hydrophobic polymers of has a weight average molecular weight of from about 1 to about 8 kDa (e.g. , from about 2 to about 6 kDa). In some embodiments, at least a portion of the plurality of hydrophilic- hydrophobic polymers of is from about 2 to about 30 by weight % in, or used as starting materials to make, the particle (e.g. , from about 4 to about 25 by weight %). In some embodiments, at least a portion of the hydrophilic portion of the hydrophilic-hydrophobic polymers of comprises PEG, polyoxazoline, polyvinylpyrrolidine, polyhydroxylpropyl- methacrylamide, or polysialic acid (e.g. , PEG). In some embodiments, at least a portion of the hydrophilic portion of the hydrophilic-hydrophobic polymers of terminates in a methoxy. Additional properties of the particles described herein include the following. In some embodiments, at least a portion of the cationic moieties comprise at least one amine (e.g. , a primary, secondary, tertiary or quaternary amine). In some embodiments, at least a portion of the cationic moieties comprise a plurality of amines (e.g. , primary, secondary, tertiary or quaternary amines). In some embodiments, at least one amine in the cationic moiety is a secondary or tertiary amine. In some embodiments, at least a portion of the cationic moiety comprises a polymer, e.g. , a polymeric cationic moiety, for example, polyethylene imine or polylysine. Polymeric cationic moieties have a variety of molecular weights (e.g. , ranging from about 500 to about 5000 Da, for example, from about 1 to about 2 kDa or about 2.5 kDa). In some embodiments, at least a portion of the cationic moieties comprise a cationic PVA (cPVA) (e.g. , as provided by Kuraray, such as CM-318 or C-506). Other exemplary cationic moieties include polyamino acids, poly(histidine) and poly(2-dimethylamino)ethyl methacrylate.

In some embodiments, the cationic moiety has a pKa of 5 or greater. In some

embodiments, the amine is positively charged at acidic pH. In some embodiments, the amine is positively charged at physiological pH. In some embodiments, at least a portion of the cationic moieties is selected from the group consisting of protamine sulfate, hexademethrine bromide, cetyl trimethylammonium bromide, spermine (e.g. , tetramethylated spermine), and spermidine. In some embodiments, at least a portion of the cationic moieties are selected from the group consisting of tetraalkyl ammonium moieties, trialkyl ammonium moieties, imidazolium moieties, aryl ammonium moieties, iminium moieties, amidinium moieties, guanadinium moieties, thiazolium moieties, pyrazolylium moieties, pyrazinium moieties, pyridinium moieties, and phosphonium moieties. In some embodiments, at least a portion of the cationic moieties are a cationic lipid. In some embodiments, at least a portion of the cationic moieties are conjugated to a non-polymeric hydrophobic moiety (e.g. , cholesterol or Vitamin E TPGS).

In some embodiments, the cationic moiety is from about 0.1 to about 60 weight by % in, or used as starting materials to make, the particle , e.g. , from about 1 to about 60 by weight % of the particle . In some embodiments, the ratio of the charge of the cationic moiety to the charge from the nucleic acid agent, e.g. , mRNA, is from about 1 : 1 to about 50: 1 (e.g. , 1 : 1 to about 10: 1 or 1 : 1 to 5: 1, about 1.5: 1 or about 1 : 1). In embodiments where the cationic moiety is a nitrogen containing moiety this ratio can be referred to as the N/P ratio. In some embodiments, at least a portion of the nucleic acid agent, e.g. , mRNA, is chemically modified (e.g. , include one or more backbone modifications, base modifications, and or modifications to the sugar) to increase the stability of the nucleic acid agent, e.g. , mRNA. In some embodiments, the nucleic acid agents, e.g. , mRNA, are from about 1 to about 50 weight % in, or used as starting materials to make, the particle (e.g. , from about 1 to about 20%, from about 2 to about 15%, from about 3 to about 12%).

Additional properties of the particles described herein include the following. In some embodiments, the particle also includes a surfactant. In some embodiments, the surfactant is a polymer such as PVA. In some embodiments, the PVA has a viscosity of from about 2 to about 27 cP. In some embodiments, the surfactant is from about 0 to about 40 weight % in, or used as starting materials to make, the particle (e.g. , from about 15 to about 35 weight %). In some embodiments, the diameter of the particle is less than about 200 nm (e.g. , from about 200 to about 20 nm, from about 150 to about 50 nm, or less than about 150 nm). In some embodiments, the surface of the particle is substantially coated with PEG, PVA, polyoxazoline,

polyvinylpyrrolidine, polyhydroxylpropylmethacrylamide, or polysialic acid (e.g. , PEG).

Additional properties of the particles described herein include the following. In some embodiments, the zeta potential of the particle is from about -20 to about 50 mV (e.g. , from about -20 to about 20 mV, from about -10 to about 10 mV, or neutral). In some embodiments, the particle is chemically stable under conditions, comprising a temperature of 23 degrees Celsius and 60% percent humidity for at least 1 day (e.g. , at least 7 days, at least 14 days, at least 21 days, at least 30 days). In some embodiments, the particle is a lyophilized particle. In some embodiments, the particle is formulated into a pharmaceutical composition. In some

embodiments, the surface of the particle is substantially free of a targeting agent.

In some embodiments, the particles described herein can deliver an effective amount of an mRNA such that translation of the protein encoded by the mRNA is increased in the subject by at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more at approximately 72 hours, 96 hours, 120 hours, 144 hours, 168 hours, 192 hours, 216 hours, 240 hours, 264 hours after administration of the particles to the subject. In one embodiment, the particles described herein can deliver an effective amount of the nucleic acid agent, e.g. , mRNA, such that expression of the targeted gene in the subject is reduced by at least 50%, 55%, 60%, 65%, 70%, 75% or 80%, approximately 120 hours after administration of the particles to the subject. In some embodiments, the level of target gene expression in a subject administered a particle or composition described herein is compared to the level of expression of the target gene seen when the nucleic acid agent, e.g. , mRNA, is administered in a formulation other than a particle (i.e. , not in a particle, e.g. , not embedded in a particle, for example, a particle described herein) or than expression of the target gene seen in the absence of the administration of the nucleic acid agent, e.g. , mRNA, or other therapeutic agent).

In another aspect, the disclosure features a composition comprising the particles described herein. In some embodiments, the composition is a pharmaceutical composition.

In some embodiments, at least 50%, 60%. 70%, 80%, 90%, 95%, 99% or all of the particles in the composition have a diameter of less than about 200 nm. In some embodiments, the particles have a D_v90 of less than 200 nm (e.g. , from about 200 to about 20 nm, from about 150 to about 50 nm, or less than about 150 nm).

In some embodiments, the composition is chemically stable under conditions, comprising a temperature of 23 degrees Celsius and 60% percent humidity for at least 1 day (e.g. , at least 7 days, at least 14 days, at least 21 days, at least 30 days). In some embodiments, the composition is a lyophilized composition.

In some embodiments, the particles described herein are formulated into a pharmaceutical composition.

In another aspect, the disclosure features a kit comprising the particles described herein or a composition described herein.

In another aspect, the disclosure features a single dosage unit comprising the particles described herein or a composition described herein.

In another aspect, the disclosure features a method of increasing target protein expression in a subject, e.g. , a subject having a disorder that can be treated by increasing expression of the targeted protein. The method comprises administering an effective amount of particles described herein or a composition described herein, wherein the mRNA delivered by the particle increases expression of the targeted protein in the subject by at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more approximately 72 hours, 96 hours, 120 hours, 144 hours, 168 hours, 192 hours, 216 hours, 240 hours, 264 hours after administration of the particles. In one embodiment, the mRNA delivered by the particles increase expression of the targeted protein in the subject by at least 50%, 55%, 60%, 65%, 70%, 75% or 80%, approximately 120 hours after administration of the particles. In some

embodiments, the level of target protein expression in a subject administered a particle or composition described herein is compared to the level of expression of the target protein seen when the mRNA is administered in a formulation other than a particle (i.e. , not in a particle, e.g. , not embedded in a particle, for example, a particle described herein), or than expression of the target protein seen in the absence of the administration of the mRNA or other therapeutic agent).

In another aspect, the disclosure features, a method of storing a particle or composition described herein, the method comprising:

providing said particle or composition disposed in a container, e.g. , an air or liquid tight container, e.g. , a container described herein, e.g. , a container having an inert gas, e.g. , argon or nitrogen, filled headspace;

storing said particle or composition, e.g. , under preselected conditions, e.g. , temperature, e.g. , a temperature described herein;

and, moving said container to a second location or removing all or an aliquot of said particle or composition, from said container.

In some embodiments, the particle or composition is evaluated, e.g. , for stability or activity of the nucleic acid agent, e.g. , mRNA, a physical property, e.g. , color, clumping, ability to flow or be poured, or particle size or charge. The evaluation can be compared to a standard, and optionally, responsive to said standard, the particle or composition, is classified.

In some embodiments, the particle or composition is stored as a re-constituted

formulation (e.g. , in a liquid as a solution or suspension).

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a bar graph depicting fluorescence as measured in the human colorectal cell line HCT-116, demonstrating that uptake of the particles comprising a nucleic acid agent, e.g. , mRNA with K-ras mutation, was higher as compared to naked mRNA. FIG. 2 shows the visualization of fluorescence as measured in HCT-116, demonstrating that uptake of the particles comprising a nucleic acid agent, e.g. , mRNA with K-ras mutation, was higher as compared to naked mRNA.

DETAILED DESCRIPTION

This disclosure is not limited in its application to the details of construction and the arrangement of components set forth in the following description or illustrated in the drawings. The disclosure is capable of other embodiments and of being practiced or of being carried out in various ways. Also, the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting. The use of "including," "comprising," or "having," "containing," "involving," and variations thereof herein, is meant to encompass the items listed thereafter and equivalents thereof as well as additional items.

Also disclosed are dosage forms containing the particles and compositions; methods of using the particles and compositions (e.g. , to treat a disorder); kits including the particles and compositions; methods of storing the particles and compositions; and methods of analyzing the particles and compositions comprising the particles.

Headings, and other identifiers, e.g. , (a), (b), (i) etc, are presented merely for ease of reading the specification and claims. The use of headings or other identifiers in the specification or claims does not require the steps or elements be performed in alphabetical or numerical order or the order in which they are presented.

Definitions

The term "about" or "approximately," as used herein refers to within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, e.g., the limitations of the measurement system, or the degree of precision required for a particular purpose. For example, "about" can mean within 1 or more than 1 standard deviations, as per the practice in the art. Alternatively, "about" can mean a range of up to 20%, up to 10%, up to 5%, and up to 1% of a given value. Where particular values are described in the application and claims, unless otherwise stated, the term "about" refers to an acceptable error range for the particular value.

The term "ambient conditions," as used herein, refers to surrounding conditions at about one atmosphere of pressure, 50% relative humidity and about 25 °C, unless specified as otherwise.

The term "attach," as used herein with respect to the relationship of a first moiety to a second moiety, e.g., the attachment of an agent to a polymer, refers to the formation of a covalent bond between a first moiety and a second moiety. In the same context, the noun "attachment" refers to a covalent bond between the first and second moiety. The attachment can be a direct attachment, e.g., through a direct bond of the first moiety to the second moiety, or can be through a linker (e.g., through a covalently linked chain of one or more atoms disposed between the first and second moiety). For example, where an attachment is through a linker, a first moiety (e.g., a cationic moiety) is covalently bonded to a linker, which in turn is covalently bonded to a second moiety (e.g., a hydrophobic polymer described herein).

The term "biodegradable" includes polymers, compositions and formulations, such as those described herein, that are intended to degrade during use. Biodegradable polymers typically differ from non-biodegradable polymers in that the former may be degraded during use. In certain embodiments, such use involves in vivo use, such as in vivo therapy, and in other certain embodiments, such use involves in vitro use. In general, degradation attributable to biodegradability involves the degradation of a biodegradable polymer into its component subunits, or digestion, e.g., by a biochemical process, of the polymer into smaller, non-polymeric subunits. In certain embodiments, two different types of biodegradation may generally be identified. For example, one type of biodegradation may involve cleavage of bonds (whether covalent or otherwise) in the polymer backbone. In such biodegradation, monomers and oligomers typically result, and even more typically, such biodegradation occurs by cleavage of a bond connecting one or more of subunits of a polymer. In contrast, another type of

biodegradation may involve cleavage of a bond (whether covalent or otherwise) internal to a side chain or that connects a side chain to the polymer backbone. In certain embodiments, one or the other or both general types of biodegradation may occur during use of a polymer.

The term "biodegradation," as used herein, encompasses both general types of biodegradation described above. The degradation rate of a biodegradable polymer often depends in part on a variety of factors, including the chemical identity of the linkage responsible for any degradation, the molecular weight, crystallinity, biostability, and degree of cross-linking of such polymer, the physical characteristics (e.g. , shape and size) of a polymer, assembly of polymers or particle, and the mode and location of administration. For example, a greater molecular weight, a higher degree of crystallinity, and/or a greater biostability, usually lead to slower biodegradation.

The term "cationic moiety" or "cationic moieties" refers to a moiety, which has a pKa 5 or greater (e.g. , a Lewis base having a pKa of 5 or greater) and/or a positive charge in at least one of the following conditions: during the production of a particle described herein, when formulated into a particle described herein, or subsequent to administration of a particle described herein to a subject, for example, while circulating in the subject and/or while in the endosome. Exemplary cationic moieties include amine containing moieties (e.g. , charged amine moieties such as a quaternary amine), guanidine containing moieties (e.g. , a charged guanidine such as a quanadinium moiety), and heterocyclic and/or heteroaromatic moieties (e.g. , charged moieties such as a pyridinium or a histidine moiety). Cationic moieties include polymeric species, such as moieties having more than one charge, e.g. , contributed by repeated presence of a moiety, (e.g. , a cationic PVA and/or a polyamine). Cationic moieties also include zwitterions, meaning a compound that has both a positive charge and a negative charge (e.g. , an amino acid such as arginine, lysine, or histidine).

The term "cationic polymer," for example, a polyamine, refers to a polymer (the term polymer is described herein below) that has a plurality of positive charges (i.e. , at least 2) when formulated into a particle described herein. In some embodiments, the cationic polymer, for example, a polyamine, has at least 3, 4, 5, 10, 15, or 20 positive charges.

The phrase "cleavable under physiological conditions" refers to a bond having a half life of less than about 50 or 100 hours, when subjected to physiological conditions. For example, enzymatic degradation can occur over a period of less than about five years, one year, six months, three months, one month, fifteen days, five days, three days, or one day upon exposure to physiological conditions (e.g. , an aqueous solution having a pH from about 4 to about 8, and a temperature from about 25 °C to about 37 °C.

An "effective amount" or "an amount effective" refers to an amount of the particle, or composition which is effective, upon single or multiple dose administrations to a subject, in treating a cell, or curing, alleviating, relieving or improving a symptom of a disorder. An effective amount of the composition may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the compound to elicit a desired response in the individual. An effective amount is also one in which any toxic or detrimental effects of the composition are outweighed by the therapeutically beneficial effects.

The term "embed" as used herein, refers to disposing a first moiety with, or within, a second moiety by the formation of a non-covalent interaction between the first moiety and a second moiety, e.g. , a nucleic acid agent e.g. , mRNA, or a cationic moiety and a polymer. In some embodiments, when referring to a moiety embedded in a particle, that moiety (e.g. , a nucleic acid agent e.g. , mRNA, or a cationic moiety) is associated with a polymer or other component of the particle through one or more non-covalent interactions such as van der Waals interactions, hydrophobic interactions, hydrogen bonding, dipole-dipole interactions, ionic interactions, and pi-stacking, and covalent bonds between the moieties and polymer or other components of the particle are absent. An embedded moiety may be completely or partially surrounded by the polymer or particle in which it is embedded.

The term "hydrophobic," as used herein, describes a moiety that can be dissolved in an aqueous solution at physiological ionic strength only to the extent of less than about 0.05 mg/mL (e.g. , about 0.01 mg/mL or less).

The term "hydrophilic," as used herein, describes a moiety that has a solubility, in aqueous solution at physiological ionic strength, of at least about 0.05 mg/mL or greater.

The term "hydrophilic -hydrophobic polymer" as used herein, describes a polymer comprising a hydrophilic portion attached to a hydrophobic portion. Exemplary hydrophilic- hydrophobic polymers include block-copolymers, e.g. , of hydrophilic and hydrophobic polymers. A "hydroxy protecting group" as used herein, is well known in the art and includes those described in detail in Protecting Groups in Organic Synthesis, T. W. Greene and P. G. M. Wuts,

3 rd edition, John Wiley & Sons, 1999, the entirety of which is incorporated herein by reference. Suitable hydroxy protecting groups include, for example, acyl {e.g., acetyl), triethylsilyl (TES), i-butyldimethylsilyl (TBDMSJ, 2,2,2-trichloroethoxycarbonyl (Troc), and carbobenzyloxy (Cbz).

The term "intact," is used herein to describe a nucleic acid agent, e.g., mRNA, which retains a sufficient amount of structure required to effectively control, e.g., protein expression. Typically, in an intact preparation of nucleic acid agent, e.g., mRNA, at least 60%, 70%, 80%, 90%, or all of the nucleic acid agent, e.g., mRNA, have the same molecular weight or length of an intact nucleic acid agent, e.g., mRNA.

"Inert atmosphere," as used herein, refers to an atmosphere composed primarily of an inert gas, which does not chemically react with the particles, compositions or mixtures described herein. Examples of inert gases are nitrogen (N₂), helium, and argon.

The term "lyoprotectant," as used herein refers to a substance present in a lyophilized preparation. Typically it is present prior to the lyophilization process and persists in the resulting lyophilized preparation. Typically a lyoprotectant is added after the formation of the particles. If a concentration step is present, e.g., between formation of the particles and lyophilization, a lyoprotectant can be added before or after the concentration step. A lyoprotectant can be used to protect particles, during lyophilization, for example to reduce or prevent aggregation, particle collapse and/or other types of damage. In some embodiments the lyoprotectant is a

cryoprotectant.

In some embodiments the lyoprotectant is a carbohydrate. The term "carbohydrate," as used herein refers to and encompasses monosaccharides, disaccharides, oligosaccharides and polysaccharides.

In some embodiments, the lyoprotectant is a monosaccharide. The term

"monosaccharide," as used herein refers to a single carbohydrate unit {e.g., a simple sugar) that cannot be hydrolyzed to simpler carbohydrate units. Exemplary monosaccharide lyoprotectants include glucose, fructose, galactose, xylose, ribose and the like.

In some embodiments, the lyoprotectant is a disaccharide. The term "disaccharide," as used herein refers to a compound or a chemical moiety formed by 2 monosaccharide units that are bonded together through a glycosidic linkage, for example through 1-4 linkages or 1-6 linkages. A disaccharide may be hydrolyzed into two monosaccharides. Exemplary

disaccharide lyoprotectants include sucrose, trehalose, lactose, maltose and the like.

In some embodiments, the lyoprotectant is an oligosaccharide. The term

"oligosaccharide," as used herein refers to a compound or a chemical moiety formed by 3 to about 15, preferably 3 to about 10 monosaccharide units that are bonded together through glycosidic linkages, for example through 1-4 linkages or 1-6 linkages, to form a linear, branched or cyclic structure. Exemplary oligosaccharide lyoprotectants include cyclodextrins, raffinose, melezitose, maltotriose, stachyose acarbose, and the like. An oligosaccharide can be oxidized or reduced.

In some embodiments, the lyoprotectant is a cyclic oligosaccharide. The term "cyclic oligosaccharide," as used herein refers to a compound or a chemical moiety formed by 3 to about 15, preferably 6, 7, 8, 9, or 10 monosaccharide units that are bonded together through glycosidic linkages, for example through 1-4 linkages or 1-6 linkages, to form a cyclic structure.

Exemplary cyclic oligosaccharide lyoprotectants include cyclic oligosaccharides that are discrete compounds, such as a cyclodextrin, β cyclodextrin, or γ cyclodextrin.

Other exemplary cyclic oligosaccharide lyoprotectants include compounds which include a cyclodextrin moiety in a larger molecular structure, such as a polymer that contains a cyclic oligosaccharide moiety. A cyclic oligosaccharide can be oxidized or reduced, for example, oxidized to dicarbonyl forms. The term "cyclodextrin moiety," as used herein refers to cyclodextrin (e.g. , an α, β, or γ cyclodextrin) radical that is incorporated into, or a part of, a larger molecular structure, such as a polymer. A cyclodextrin moiety can be bonded to one or more other moieties directly, or through an optional linker. A cyclodextrin moiety can be oxidized or reduced, for example, oxidized to dicarbonyl forms.

Carbohydrate lyoprotectants, e.g. , cyclic oligosaccharide lyoprotectants, can be derivatized carbohydrates. For example, in some embodiments, the lyoprotectant is a derivatized cyclic oligosaccharide, e.g. , a derivatized cyclodextrin, e.g. , 2 hydroxy propyl -beta cyclodextrin, e.g. , partially etherified cyclodextrins (e.g. , partially etherified β cyclodextrins) disclosed in US Patent No., 6,407,079, the contents of which are incorporated herein by this reference. Another example of a derivatized cyclodextrin is β-cyclodextrin sulfobutylether sodium. An exemplary lyoprotectant is a polysaccharide. The term "polysaccharide," as used herein refers to a compound or a chemical moiety formed by at least 16 monosaccharide units that are bonded together through glycosidic linkages, for example through 1-4 linkages or 1-6 linkages, to form a linear, branched or cyclic structure, and includes polymers that comprise polysaccharides as part of their backbone structure. In backbones, the polysaccharide can be linear or cyclic. Exemplary polysaccharide lyoprotectants include glycogen, amylase, cellulose, dextran, maltodextrin and the like.

The term "derivatized carbohydrate," refers to an entity which differs from the subject non-derivatized carbohydrate by at least one atom. For example, instead of the -OH present on a non-derivatized carbohydrate the derivatized carbohydrate can have -OX, wherein X is other than H. Derivatives may be obtained through chemical functionalization and/or substitution or through de novo synthesis— the term "derivative" implies no process-based limitation.

The term "nanoparticle" is used herein to refer to a material structure whose size in at least any one dimension (e.g. , x, y, and z Cartesian dimensions) is less than about 1 micrometer (micron), e.g. , less than about 500 nm or less than about 200 nm or less than about 100 nm, and greater than about 5 nm. In embodiments the size is less than about 70 nm but greater than about 20 nm. A nanoparticle can have a variety of geometrical shapes, e.g. , spherical, ellipsoidal, etc. The term "nanoparticles" is used as the plural of the term "nanoparticle."

The term "nucleic acid agent" refers to any synthetic or naturally occurring therapeutic agent including two or more nucleotide residues. In some embodiments the nucleic acid agent is an RNA, a DNA or a mixed polymer of RNA and DNA. In some embodiments, an RNA is an mRNA. In some embodiments a DNA is a cDNA or genomic DNA. In some embodiments, the nucleic acid agent is single stranded and in another embodiment it comprises two strands. In some embodiments, the nucleic acid agent can have a duplexed region, comprised of strands from one or two molecules. In some embodiments, the nucleic acid agent is an agent that inhibits gene expression, e.g. , an agent that promotes RNAi. In some embodiments, the nucleic acid agent is siRNA, shRNA, an antisense oligonucleotide, or a microRNA (miRNA). In some embodiments, the nucleic acid agent is an antagomir or an aptamer.

A nucleic acid agent can encode a peptide or protein, e.g. , a therapeutic peptide or protein. The nucleic acid agent can be, by way of an example, an RNA, e.g. , mRNA, or a DNA, e.g. , a nucleic acid agent that encodes a therapeutic protein. Exemplary therapeutic proteins include a tumor suppressor, an antigen, a cytotoxin, a cytostatin, a pro-drug activator an apoptotic protein and a protein having an anti- angiogenic activity. The nucleic acid agents described herein can also include one or more control regions. Exemplary control regions include, for example, an origin of replication, a promoter (e.g. , a CMV promoter, or an inducible promoter), a polyadenylation signal, a Kozak sequence, an enhancer, a localization signal sequence, an internal ribosome entry sites (IRES), and a splicing signal.

In another embodiment, a nucleic acid agent can encode antigen(s) for induction of at least one of an antibody or T cell responses, e.g. , both antibody and T cell responses. In some embodiments, the nucleic acid agent can encode antigen(s) for use as DNA or RNA vaccines (see, e.g. , Ulmer et al. Vaccine 30: 4414- 4418, 2012, which is incorporated herein by reference in its entirety).

Accordingly, in another aspect the disclosure provides particles that can be used as vaccines, e.g. , DNA or RNA vaccines.

In one embodiment, a DNA vaccine can be administered to elicit an immunotherapeutic response in patients. Examples of DNA vaccines, include without limitation: mammaglobin-A DNA vaccine for treating breast cancer patients with metastatic disease; human pro state- specific membrane antigen plasmid DNA vaccine; alpha fetoprotein plasmid DNA vaccine for treating patients with Hepatocellular Carcinoma; Heptatitis B vaccine (HBV), tyrosinase DNA vaccine for treating patients with melanoma, human papillomvirus (HPV) vaccine, lymphoma immunoglobulin derived scFV-chemokine DNA vaccines, and HIV DNA vaccines, e.g. , DNA- HlV-recombinant vaccines that can be designed to interact with CD4 (helper-inducer) and CD8 (cytotoxic) T lymphocytes (T cells) to prime CD4 and CD8 cells to respond to HIV components.

In one embodiment, a RNA vaccine, e.g. , mRNA vaccines, can be administered as active immunotherapeutic immunization in cancer therapies. For example, mRNA can be used to encode genes cloned from metastatic melanoma tumors as an autologous immunization strategy. Further embodiments include, without limitation, the administration of combinations of known tumor antigens to elicit antigen- specific immune responses. Such tumor antigens include, but are not limited to, Mucin 1 (MUC1), Carcinoembryonic antigen (CEA), telomerase, Melanoma- associated antigen 1 (MAGE-1), and tyosinase, in therapies for metastatic melanoma and renal cell carcinoma patients. In another embodiment, an RNA vaccine can be an RNA replicon vaccine, such as a bivalent vaccine including replicons encoding proteins, e.g., cytomegalovirus (CMV) gB and pp65/IEl proteins, which can generate T cell responses, e.g., polyfunctional CD4⁺ and CD8⁺ T cell responses.

In another embodiment, an RNA vaccine can be a self- amplifying RNA vaccine. For example, an RNA vaccine can be a self-amplifying RNA vaccine based on an alphavirus genome, which contains the genes encoding the alphavirus RNA replication machinery, but lacks the genes encoding the viral structural proteins required to make an infectious alphavirus particle (see, e.g., Geall et al. PNAS, 109(36): 14604-14609, 2012, which is incorporated herein by reference in its entirety).

As used herein, "particle polydispersity index (PDI)" or "particle polydispersity" refers to the width of the particle size distribution. Particle PDI can be calculated from the equation PDI =2a₂ / ai² where ai is the 1^st Cumulant or moment used to calculate the intensity weighted Z average mean size and a₂ is the 2^nd moment used to calculate a parameter defined as the polydispersity index (Pdl). A particle PDI of 1 is the theoretical maximum and would be a completely flat size distribution plot. Compositions of particles described herein may have particle PDIs of less than 0.5, less than 0.4, less than 0.3, less than 0.2, or less than 0.1.

"Pharmaceutically acceptable carrier or adjuvant," as used herein, refers to a carrier or adjuvant that may be administered to a patient, together with a particle or composition described herein, and which does not destroy the pharmacological activity thereof and is nontoxic when administered in doses sufficient to deliver a therapeutic amount of the particle. Some examples of materials which can serve as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose, mannitol and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21) other non-toxic compatible substances employed in pharmaceutical compositions.

The term "polymer," as used herein, is given its ordinary meaning as used in the art, i.e. , a molecular structure featuring one or more repeat units (monomers), connected by covalent bonds. The repeat units may all be identical, or in some cases, there may be more than one type of repeat unit present within the polymer. Polymers may be natural or unnatural (synthetic) polymers. Polymers may be homopolymers or copolymers containing two or more monomers. Polymers may be linear or branched.

If more than one type of repeat unit is present within the polymer, then the polymer is to be a "copolymer." It is to be understood that in any embodiment employing a polymer, the polymer being employed may be a copolymer. The repeat units forming the copolymer may be arranged in any fashion. For example, the repeat units may be arranged in a random order, in an alternating order, or as a "block" copolymer, i.e. , containing one or more regions each containing a first repeat unit (e.g. , a first block), and one or more regions each containing a second repeat unit (e.g. , a second block), etc. Block copolymers may have two (a diblock copolymer), three (a triblock copolymer), or more numbers of distinct blocks. In terms of sequence, copolymers may be random, block, or contain a combination of random and block sequences.

In some cases, the polymer is biologically derived, i.e. , a biopolymer. Non-limiting examples of biopolymers include peptides or proteins (i.e. , polymers of various amino acids), or nucleic acids such as DNA or RNA.

As used herein, "polymer polydispersity index (PDI)" or "polymer polydispersity" refers to the distribution of molecular mass in a given polymer sample. The polymer PDI calculated is the weight average molecular weight divided by the number average molecular weight. It indicates the distribution of individual molecular masses in a batch of polymers. The polymer PDI has a value typically greater than 1, but as the polymer chains approach uniform chain length, the PDI approaches unity (1).

As used herein, the term "prevent" or "preventing" as used in the context of the administration of an agent to a subject, refers to subjecting the subject to a regimen, e.g. , the administration of a particle or composition, such that the onset of at least one symptom of the disorder is delayed as compared to what would be seen in the absence of the regimen. As used herein, the term "subject" is intended to include human and non-human animals. Exemplary human subjects include a human patient having a disorder, e.g. , a disorder described herein, or a normal subject. The term "non-human animals" includes all vertebrates, e.g. , non- mammals (such as chickens, amphibians, reptiles) and mammals, such as non-human primates, domesticated and/or agriculturally useful animals, e.g. , sheep, dog, cat, cow, pig, etc.

As used herein, the term "treat" or "treating" a subject having a disorder refers to subjecting the subject to a regimen, e.g. , the administration of a particle or composition, such that at least one symptom of the disorder is cured, healed, alleviated, relieved, altered, remedied, ameliorated, or improved. Treating includes administering an amount effective to alleviate, relieve, alter, remedy, ameliorate, improve or affect the disorder or the symptoms of the disorder. The treatment may inhibit deterioration or worsening of a symptom of a disorder.

Particles

The particles, in general, include a nucleic acid agent, e.g. , mRNA, and at least one of a cationic moiety, a hydrophobic moiety, such as a polymer, or a hydrophilic-hydrophobic polymer. In some embodiments, the particles include a nucleic acid agent, e.g. , mRNA, and a cationic moiety, and at least one of a hydrophobic moiety, such as a polymer, or a hydrophilic- hydrophobic polymer. In some embodiments, a particle described herein includes a hydrophobic moiety such as a hydrophobic polymer or lipid (e.g. , hydrophobic polymer), a polymer containing a hydrophilic portion and a hydrophobic portion, a nucleic acid agent, e.g. , mRNA, and a cationic moiety. In some embodiments, the cationic moiety is attached to a moiety. For example, the cationic moiety can be attached to a polymer (e.g. , the hydrophobic polymer or the polymer containing a hydrophilic portion and a hydrophobic portion). In some embodiments, the cationic moiety is attached to a polymer (e.g. , a hydrophobic polymer or a polymer containing a hydrophilic and a hydrophobic portion). In some embodiments, the cationic moiety can also be attached to other moieties.

In addition to a hydrophobic moiety such as a hydrophobic polymer or lipid (e.g. , hydrophobic polymer), a polymer containing a hydrophilic portion and a hydrophobic portion, a nucleic acid agent, e.g. , mRNA, and a cationic moiety, the particles described herein may include one or more additional components such as an additional nucleic acid agent or an additional cationic moiety. A particle described herein may also include a compound having at least one acidic moiety, such as a carboxylic acid group. The compound may be a small molecule or a polymer having at least one acidic moiety. In some embodiments, the compound is a polymer such as PLGA.

In some embodiments, the particle is configured such that when administered to a subject there is preferential release of the nucleic acid agent, e.g. , mRNA, in a preselected compartment. The preselected compartment can be a target site, location, tissue type, cell type, e.g. , a disease specific cell type, e.g. , a cancer cell, or subcellular compartment, e.g. , the cytosol. In some embodiments a particle provides preferential release in a tumor, as opposed to other

compartments, e.g. , non-tumor compartments, e.g. , the peripheral blood. In some embodiments, the particle is configured such that when administered to a subject, it delivers more nucleic acid agent, e.g. , mRNA, to a compartment of the subject, e.g. , a tumor, than if the nucleic acid agent were administered free.

In some embodiments, the particle is associated with an excipient, e.g. , a carbohydrate component, or a stabilizer or lyoprotectant, e.g. , a carbohydrate component, stabilizer or lyoprotectant described herein. While not wishing to be bound be theory the carbohydrate component may act as a stabilizer or lyoprotectant. In some embodiments, the carbohydrate component, stabilizer or lyoprotectant, comprises one or more carbohydrates (e.g. , one or more carbohydrates described herein, such as, e.g. , sucrose, cyclodextrin or a derivative of

cyclodextrin (e.g. 2-hydroxypropyl- -cyclodextrin, sometimes referred to herein as ΗΡ-β-CD)), salt, PEG, PVP or crown ether. In some embodiments, the carbohydrate component, stabilizer or lyoprotectant comprises two or more carbohydrates, e.g. , two or more carbohydrates described herein. In one embodiment, the carbohydrate component, stabilizer or lyoprotectant includes a cyclic carbohydrate (e.g. , cyclodextrin or a derivative of cyclodextrin, e.g. , an α-, β-, or γ-, cyclodextrin (e.g. 2-hydroxypropyl- -cyclodextrin)) and a non-cyclic carbohydrate. Exemplary non-cyclic oligosaccharides include those of less than 10, 8, 6 or 4 monosaccharide subunits (e.g. , a monosaccharide or a disaccharide (e.g. , sucrose, trehalose, lactose, maltose) or combinations thereof).

In some embodiments, the carbohydrate component, stabilizer or lyoprotectant comprises a first and a second component, e.g. , a cyclic carbohydrate and a non-cyclic carbohydrate, e.g. , a mono-, di-, or tetra-saccharide. In some embodiments, the weight ratio of cyclic carbohydrate to non-cyclic carbohydrate associated with the particle is a weight ratio described herein, e.g. , 0.5: 1.5 to 1.5:0.5.

In some embodiments, the carbohydrate component, stabilizer or lyoprotectant comprises a first and a second component (designated here as A and B) as follows:

(A) comprises a cyclic carbohydrate and (B) comprises a disaccharide;

(A) comprises more than one cyclic carbohydrate, e.g. , a β-cyclodextrin (sometimes referred to herein as β-CD) or a β-CD derivative, e.g. , ΗΡ-β-CD, and (B) comprises a disaccharide;

(A) comprises a cyclic carbohydrate, e.g. , a β-CD or a β-CD derivative, e.g. , ΗΡ-β-CD, and

(B) comprises more than one disaccharide;

(A) comprises more than one cyclic carbohydrate, and (B) comprises more than one disaccharide;

(A) comprises a cyclodextrin, e.g. , a β-CD or a β-CD derivative, e.g. , ΗΡ-β-CD, and (B) comprises a disaccharide;

(A) comprises a β-cyclodextrin, e.g a β-CD derivative, e.g. , ΗΡ-β-CD, and (B) comprises a disaccharide;

(A) comprises a β-cyclodextrin, e.g. , a β-CD derivative, e.g. , ΗΡ-β-CD, and (B) comprises sucrose;

(A) comprises a β-CD derivative, e.g. , ΗΡ-β-CD, and (B) comprises sucrose;

(A) comprises a β-cyclodextrin, e.g. , a β-CD derivative, e.g. , ΗΡ-β-CD, and (B) comprises trehalose;

(A) comprises a β-cyclodextrin, e.g. , a β-CD derivative, e.g. , ΗΡ-β-CD, and (B) comprises sucrose and trehalose.

(A) comprises ΗΡ-β-CD, and (B) comprises sucrose and trehalose.

In some embodiments, components A and B are present in the following ratio:

0.5: 1.5 to 1.5:0.5. In some embodiments, components A and B are present in the following ratio: 3-1 : 0.4-2; 3-1 : 0.4-2.5; 3-1 : 0.4-2; 3-1 : 0.5-1.5; 3-1 : 0.5-1 ; 3-1 : 1 ; 3-1 : 0.6-0.9; and 3: 1 : 0.7. In some embodiments, components A and B are present in the following ratio: 2-1 : 0.4-2; 3-1 : 0.4-2.5; 2-1 : 0.4-2; 2-1 : 0.5-1.5; 2-1 : 0.5-1 ; 2-1 : 1 ; 2-1 : 0.6-0.9; and 2: 1 : 0.7. In some embodiments components A and B are present in the following ratio: 2-1.5 : 0.4-2; 2-1.5 : 0.4-2.5; 2-1.5 : 0.4-2; 2-1.5 : 0.5-1.5; 2-1.5 : 0.5-1 ; 2-1.5 : 1 ; 2-1.5 : 0.6-0.9; 2: 1.5 : 0.7. In some embodiments components A and B are present in the following ratio: 2.5-1.5 : 0.5-1.5;

2.2-1.6: 0.7-1.3; 2.0 -1.7: 0.8-1.2; 1.8: 1 ; 1.85: 1 and 1.9: 1.

In some embodiments, component A comprises a cyclodextrin, e.g. , a β-cyclodextrin, e.g. , a β-CD derivative, e.g. , ΗΡ-β-CD, and (B) comprises sucrose, and they are present in the following ratio: 2.5-1.5 : 0.5-1.5; 2.2-1.6: 0.7-1.3; 2.0 -1.7: 0.8-1.2; 1.8 : 1 ; 1.85 : 1 and 1.9 : 1.

In some embodiments, the particle is a nanoparticle. In some embodiments, the nanoparticle has a diameter of less than or equal to about 220 nm (e.g. , less than or equal to about 215 nm, 210 nm, 205 nm, 200 nm, 195 nm, 190 nm, 185 nm, 180 nm, 175 nm, 170 nm, 165 nm, 160 nm, 155 nm, 150 nm, 145 nm, 140 nm, 135 nm, 130 nm, 125 nm, 120 nm, 115 nm, 110 nm, 105 nm, 100 nm, 95 nm, 90 nm, 85 nm, 80 nm, 75 nm, 70 nm, 65 nm, 60 nm, 55 nm or 50 nm). In some embodiments, the nanoparticle has a diameter of at least 10 nm (e.g. , at least about 20 nm).

A particle described herein may also include a targeting agent or a lipid (e.g. , on the surface of the particle).

A composition of a plurality of particles described herein may have an average diameter of about 50 nm to about 500 nm (e.g. , from about 50 nm to about 200 nm). A composition of a plurality of particles particle may have a median particle size (Dv50 (particle size below which 50% of the volume of particles exists) of about 50 nm to about 500 nm (e.g. , about 75 nm to about 220 nm)) from about 50 nm to about 220 nm (e.g. , from about 75 nm to about 200 nm). A composition of a plurality of particles may have a Dv90 (particle size below which 90% of the volume of particles exists) of about 50 nm to about 500 nm (e.g. , about 75 nm to about 220 nm). In some embodiments, a composition of a plurality of particles has a Dv90 of less than about 150 nm. A composition of a plurality of particles may have a particle PDI of less than 0.5, less than 0.4, less than 0.3, less than 0.2, or less than 0.1.

A particle described herein may have a surface zeta potential ranging from about -20 mV to about 50 mV, when measured in water. Zeta potential is a measurement of surface potential of a particle. In some embodiments, a particle may have a surface zeta potential, when measured in water, ranging between about -20 mV to about 20 mV, about -10 mV to about 10 mV, or neutral.

In some embodiments, a particle, or a composition comprising the particles, described herein, has a sufficient amount of nucleic acid agent, e.g. , RNA, to observe an effect (e.g. , protein expression of the transcribed mRNA of the particle) when administered, for example, in an in vivo model system, (e.g., a mouse model such as any of those described herein).

In some embodiments, a particle, or a composition comprising a plurality of particles described herein, is one in which at least 30, 40, 50, 60, 70, 80, or 90% of its nucleic acid agent, e.g., mRNA, by number or weight, is intact (e.g., as measured by functionality of physical properties, e.g., molecular weight).

In some embodiments, a particle, or a composition comprising a plurality of particles, described herein, is one in which at least 30, 40, 50, 60, 70, 80, or 90% of its nucleic acid agent, e.g., mRNA, by number or weight, is inside, as opposed to exposed at the surface of, the particle.

In some embodiments, a particle, or a composition comprising a plurality of particles, described herein, when incubated in 50/50 mouse/human serum, exhibits little or no aggregation. E.g., when incubated less than 30, 20, or 10%, by number or weight, of the particles will aggregate.

In some embodiments, a particle, or a composition comprising a plurality of particles, described herein may, when stored at 25°C + 2°C/60% relative humidity + 5% relative humidity in an open, or closed, container, for 20, 30, 40, 50 or 60 days, retains at least 30, 40, 50, 60, 70, 80, 90, or 95% of its activity, e.g., as determined in an in vivo model system, (e.g., a mouse model such any of those described herein).

In some embodiments, a particle, or a composition comprising a plurality of particles, described herein may, results in at least 20, 30, 40, 50, or 60% reduction in protein and/or mRNA knockdown when administered as a single dose of 1 or 3 mg/kg in an in vivo model system, (e.g., a mouse model such as any of those described herein).

In some embodiments, a particle or a composition comprising a plurality of particles described herein results in less than 20, 10, 5%, or no knockdown for off target genes, as measured by protein or mRNA, when administered (e.g., as a single dose of 1 or 3 mg/kg) in an in vivo model system, (e.g., a mouse model such as any of those described herein).

In some embodiments, a particle or a composition comprising a plurality of particles, described herein, results in less than 2, 5, or 10 fold cytokine induction, when administered (e.g., as a single dose of 1 or 3 mg/kg) in an in vivo model system, (e.g., a mouse model such as any of those described herein). E.g., the administration results in less than 2, 5, or 10 fold induction of one, or more, e.g., two, three, four, five, six, or seven, or all, of: tumor necrosis factor-alpha, interleukin-1 alpha, interleukin-lbeta, interleukin-6, interleukin-10, interleukin-12, keratinocyte- derived cytokine and interferon-gamma.

In some embodiments, a particle, or a composition comprising a plurality of particles, described herein, results in less than 2, 5, or 10 fold increase in alanine aminotransferase (ALT) and aspartate aminotransferase (AST), when administered (e.g. , as a single dose of 1 or 3 mg/kg) in an in vivo model system (e.g. , a mouse model such as any of those described herein). In some embodiments, a particle, or a composition comprising a plurality of particles, described herein, results in no significant changes in blood count 48 hours after 2 doses of 3mg/kg in an in vivo model system, (e.g. , a mouse model such as one described herein).

In some embodiments a particle is stable in non-polar organic solvent (e.g. , any of hexane, chloroform, or dichloromethane). By way of example, the particle does not substantially invert, e.g. , if present, an outer layer does not internalize, or a substantial amount of surface components do internalize, relative to their configuration in aqueous solvent. In embodiments the distribution of components is substantially the same in a non-polar organic solvent and in an aqueous solvent.

In some embodiments a particle lacks at least one component of a micelle, e.g. , it lacks a core which is substantially free of hydrophilic components.

In some embodiments the core of the particle comprises a substantial amount of a hydrophilic component.

In some embodiments the core of the particle comprises a substantial amount e.g. , at least 10, 20, 30, 40, 50, 60 or 70% (by weight or number) of the nucleic acid agent, e.g. , mRNA, of the particle.

In some embodiments the core of the particle comprises a substantial amount e.g. , at least 10, 20, 30, 40, 50, 60 or 70% (by weight or number) of the cationic, e.g. , polycationic moiety, of the particle.

A particle described herein may include a small amount of a residual solvent, e.g. , a solvent used in preparing the particles such as acetone, ie/t-butylmethyl ether, benzyl alcohol, dioxane, heptane, dichloromethane, dimethylformamide, dimethylsulfoxide, ethyl acetate, acetonitrile, tetrahydrofuran, ethanol, methanol, isopropyl alcohol, methyl ethyl ketone, butyl acetate, or propyl acetate (e.g. , isopropylacetate). In some embodiments, the particle may include less than 5000 ppm of a solvent (e.g. , less than 4500 ppm, less than 4000 ppm, less than 3500 ppm, less than 3000 ppm, less than 2500 ppm, less than 2000 ppm, less than 1500 ppm, less than 1000 ppm, less than 500 ppm, less than 250 ppm, less than 100 ppm, less than 50 ppm, less than 25 ppm, less than 10 ppm, less than 5 ppm, less than 2 ppm, or less than 1 ppm).

In some embodiments, the particle is substantially free of a class II or class III solvent as defined by the United States Department of Health and Human Services Food and Drug

Administration "Q3c -Tables and List." In some embodiments, the particle comprises less than 5000 ppm of acetone. In some embodiments, the particle comprises less than 5000 ppm of tert- butylmethyl ether. In some embodiments, the particle comprises less than 5000 ppm of heptane. In some embodiments, the particle comprises less than 600 ppm of dichloromethane. In some embodiments, the particle comprises less than 880 ppm of dimethylformamide. In some embodiments, the particle comprises less than 5000 ppm of ethyl acetate. In some embodiments, the particle comprises less than 410 ppm of acetonitrile. In some embodiments, the particle comprises less than 720 ppm of tetrahydrofuran. In some embodiments, the particle comprises less than 5000 ppm of ethanol. In some embodiments, the particle comprises less than 3000 ppm of methanol. In some embodiments, the particle comprises less than 5000 ppm of isopropyl alcohol. In some embodiments, the particle comprises less than 5000 ppm of methyl ethyl ketone. In some embodiments, the particle comprises less than 5000 ppm of butyl acetate. In some embodiments, the particle comprises less than 5000 ppm of propyl acetate.

A particle described herein may include varying amounts of a hydrophobic moiety such as a hydrophobic polymer, e.g., from about 20% to about 90% by weight of, or used as starting materials to make, the particle {e.g., from about 20% to about 80%, from about 25% to about 75%, or from about 30% to about 70% by weight).

A particle described herein may include varying amounts of a polymer containing a hydrophilic portion and a hydrophobic portion, e.g., up to about 50% by weight of, or used as starting materials to make, the particle {e.g., from about 4 to any of about 50%, about 5%, about 8%, about 10%, about 15%, about 20%, about 23%, about 25%, about 30%, about 35%, about 40%, about 45% or about 50% by weight). For example, the percent by weight of the

hydrophobic-hydrophilic polymer of the particle is from about 3% to 30%, from about 5% to 25% or from about 8% to 23%.

In a particle described herein, the ratio of the hydrophobic polymer to the hydrophobic- hydrophilic polymer is such that the particle comprises at least 5%, 8%, 10%, 12%, 15%, 18%, 20%, 23%, 25%, or 30% by weight of a polymer of, or used as starting materials to make, the particle having a hydrophobic portion and a hydrophilic portion.

A particle described herein may include varying amounts of a cationic moiety, e.g. , from about 0.1% to about 60% by weight of, or used as starting materials to make, the particle (e.g. , from about 1% to about 60%, from about 2% to about 20%, from about 3% to about 30%, from about 5% to about 40%, from about or from about 10% to about 30%). When the cationic moiety is a nitrogen containing moiety, the ratio of nitrogen moieties in the particle to

phosphates from the nucleic acid agent, e.g. , mRNA, backbone in the particle (i.e. , N/P ratio) can be from about 1 : 1 to about 50: 1 (e.g. , from about 1 : 1 to about 25: 1, from about 1 : 1 to about 10: 1, from about 1 : 1 to about 5: 1, or from about 1 : 1 to about 1.5 to 1 : 1).

A particle described herein may include varying amounts of a nucleic acid agent, e.g. , mRNA, e.g. , from about 0.1% to about 50% by weight of, or used as starting materials to make, the particle (e.g. , from about 1% to about 50%, from about 0.5% to about 20%, from about 2% to about 20%, from about or from about 5% to about 15%).

When the particle includes a surfactant, the particle may include varying amounts of the surfactant, e.g. , up to about 40% by weight of, or used as starting materials to make, the particle, or from about 15% to about 35% or from about 3% to about 10%. In some embodiments, the surfactant is PVA and the cationic moiety is cationic PVA. In some embodiments, the particle may include about 2% to about 5% of PVA (e.g. , about 4%) and from about 0.1% to about 3% cationic PVA (e.g. , about 1%). In some embodiments, the particle may include less than about 1%, less than about 0.5%, or less than about 0.2% of cationic PVA (weight/volume).

Nucleic Acid Agents

A nucleic acid agent can be delivered using a particle, or composition described herein. Examples of suitable nucleic acid agents include, but are not limited to polynucleotides, such as mRNA, siRNA, antisense oligonucleotides, microRNAs (miRNAs), antagomirs, aptamers, genomic DNA, cDNA, and plasmids. The nucleic acid agents can target a variety of genes of interest, such as a gene whose overexpression is associated with a disease or disorder.

The nucleic acid agents, e.g. , mRNA, delivered using a particle or composition described herein, can be administered alone, or in combination, (e.g. , in the same or separate formulations). In one embodiment, multiple agents, such as, mRNAs, are administered to target different sites on the same gene for treatment of a disease or disorder. In another embodiment, multiple agents, e.g. , mRNAs, are administered to target two or more different genes for treatment of a disease or disorder.

A nucleic acid agent, e.g. , mRNA, may be present in varying amounts of a particle or composition described herein. When present in a particle, the nucleic acid agent, e.g. , mRNA, may be present in an amount, e.g. , from about 0.1 to about 50% by weight of the particle (e.g. , from about 1% to about 50%, from about 1 to about 30% by weight of the particle, from about 1 to about 20% by weight of the particle, from about 4 to about 25 % by weight of the particle, or from about 5 to about 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20% by weight of the particle).

The nucleic acid agents described herein are not attached to any of the other components of the particles described herein. However, n some embodiments, the nucleic acid agent may be associated with a polymer or other component of the particle through one or more non-covalent interactions such as van der Waals interactions, hydrophobic interactions, hydrogen bonding, dipole-dipole interactions, ionic interactions, and pi stacking. mRNA

In some embodiments, the nucleic acid agent used in the particles and compositions described herein comprises mRNA. In some embodiments, the nucleic acid agent can include modified nucleosides and modified nucleotides, which can be incorporated into a nucleic acid, e.g. , mRNA. As described herein "nucleoside" is defined as a compound comprising a five- carbon sugar molecule (a pentose or ribose) or derivative thereof, and an organic base, purine or pyrimidine, or a derivative thereof. As described herein, "nucleotide" is defined as a nucleoside comprising a phosphate group.

Modified nucleosides and nucleotides can include one or more of:

(i) alteration, e.g. , replacement, of one or both of the non-linking phosphate oxygens and/or of one or more of the linking phosphate oxygens in the phosphodiester backbone linkage;

(ii) alteration, e.g. , replacement, of a constituent of the ribose sugar, e.g. , of the 2' hydroxyl on the ribose sugar;

(iii) wholesale replacement of the phosphate moiety with "dephospho" linkers;

(iv) modification or replacement of a naturally occurring nucleobase;

(v) replacement or modification of the ribose-phosphate backbone; (vi) modification of the 3' end or 5' end of the oligonucleotide, e.g., removal, modification or replacement of a terminal phosphate group or conjugation of a moiety; and

(vii) modification of the sugar.

The modifications listed above can be combined to provide modified nucleosides and nucleotides that can have two, three, four, or more modifications. For example, a modified nucleoside and nucleotide can have a modified sugar and a modified nucleobase.

Phosphate Backbone Modifications

The Phosphate Group

In some embodiments, the phosphate group can be modified by replacing one or more of the oxygens with a different substituent. Further, the modified nucleic acids can include the wholesale replacement of an unmodified phosphate moiety with a modified phosphate as described herein. In some embodiments, the modification of the phosphate backbone can include alterations that result in either an uncharged linker or a charged linker with

unsymmetrical charge distribution.

Examples of modified phosphate groups can include phosphorothioate,

phosphoroselenates, borano phosphates, borano phosphate esters, hydrogen phosphonates, phosphoroamidates, alkyl or aryl phosphonates and phosphotriesters. In some embodiments, one of the non-bridging phosphate oxygen atoms in the phosphate backbone moiety can be replaced by any of the following groups: sulfur (S), selenium (Se), BR₃ (wherein R can be, e.g., hydrogen, alkyl, or aryl), C (e.g., an alkyl group, an aryl group, and the like), H, NR₂ (wherein R can be, e.g., hydrogen, alkyl, or aryl), or -OR (wherein R can be, e.g., alkyl or aryl). The phosphorous atom in an unmodified phosphate group is achiral. However, replacement of one of the non- bridging oxygens with one of the above atoms or groups of atoms can render the phosphorous atom chiral; that is to say that a phosphorous atom in a phosphate group modified in this way is a stereogenic center. The stereogenic phosphorous atom can possess either the "R" configuration (herein Rp) or the "S" configuration (herein Sp).

Phosphorodithioates have both non-bridging oxygens replaced by sulfur. The phosphorus center in the phosphorodithioates is achiral which precludes the formation of oligoribonucleotide diastereomers. In some embodiments, modifications to one or both non-bridging oxygens can also include the replacement of the non-bridging oxygens with a group independently selected from S, Se, B, C, H, N, and OR (R can be, e.g., alkyl or aryl).

The phosphate linker can also be modified by replacement of a bridging oxygen, (i.e., the oxygen that links the phosphate to the nucleoside), with nitrogen (bridged phosphoroamidates), sulfur (bridged phosphorothioates) and carbon (bridged methylenephosphonates). The replacement can occur at either linking oxygen or at both of the linking oxygens.

Replacement of the Phosphate Group

The phosphate group can be replaced by non-phosphorus containing connectors. In some embodiments, the charge phosphate group can be replaced by a neutral moiety.

Examples of moieties which can replace the phosphate group can include, e.g., methyl phosphonate, hydroxylamino, siloxane, carbonate, carboxymethyl, carbamate, amide, thioether, ethylene oxide linker, sulfonate, sulfonamide, thioformacetal, formacetal, oxime,

methyleneimino, methylenemethylimino, methylenehydrazo, methylenedimethylhydrazo and methyleneoxymethylimino .

Replacement of the Ribophosphate Backbone

Scaffolds that can mimick nucleic acids can also be constructed wherein the phosphate linker and ribose sugar are replaced by nuclease resistant nucleoside or nucleotide surrogates. In some embodiments, the nucleobases can be tethered by a surrogate backbone. Examples can include the morpholino, cyclobutyl, pyrrolidine and peptide nucleic acid (PNA) nucleoside surrogates.

Sugar Modifications

An oligonucleotide can include modification of all or some of the sugar groups of the nucleic acid. For example, the 2' hydroxyl group (OH) can be modified or replaced with a number of different "oxy" or "deoxy" substituents. In some embodiments, modifications to the 2' hydroxyl group can enhance the stability of the nucleic acid since the hydroxyl can no longer be deprotonated to form a 2'-alkoxide ion. The 2'-alkoxide can catalyze degradation by intramolecular nucleophilic attack on the linker phosphorus atom.

Examples of "oxy"-2' hydroxyl group modifications can include alkoxy or aryloxy (OR, wherein "R" can be, e.g., alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or a sugar); polyethyleneglycols (PEG), 0(CH₂CH₂0)_nCH₂CH₂OR wherein R can be, e.g. , H or optionally substituted alkyl, and n can be an integer from 0 to 20 (e.g. , from 0 to 4, from 0 to 8, from 0 to 10, from 0 to 16, from 1 to 4, from 1 to 8, from 1 to 10, from 1 to 16, from 1 to 20, from 2 to 4, from 2 to 8, from 2 to 10, from 2 to 16, from 2 to 20, from 4 to 8, from 4 to 10, from 4 to 16, and from 4 to 20). In some embodiments, the "oxy"-2' hydroxyl group modification can include "locked" nucleic acids (LNA) in which the 2' hydroxyl can be connected, e.g. , by a C_1-6 alkylene or Ci_6 heteroalkylene bridge, to the 4' carbon of the same ribose sugar, where exemplary bridges can include methylene, propylene, ether, or amino bridges; O-amino (wherein amino can be, e.g. , N¾; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroarylamino, ethylenediamine, or polyamino) and aminoalkoxy, 0(CH₂)_n-amino, (wherein amino can be, e.g. , NH₂; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroarylamino, ethylenediamine, or polyamino). In some embodiments, the "oxy"-2' hydroxyl group modification can include the methoxyethyl group (MOE),

(OCH₂CH₂OCH₃, e.g. , a PEG derivative).

"Deoxy" modifications can include hydrogen (i.e. deoxyribose sugars, e.g. , at the overhang portions of partially ds RNA); halo (e.g. , bromo, chloro, or fluoro); amino (wherein amino can be, e.g. , N¾; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, or amino acid); NH(CH₂CH₂NH)_nCH₂CH₂-amino (wherein amino can be, e.g. , as described herein), -NHC(0)R (wherein R can be, e.g. , alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar), cyano; mercapto; alkyl-thio-alkyl; thioalkoxy; and alkyl, cycloalkyl, aryl, alkenyl and alkynyl, which may be optionally substituted with e.g. , an amino as described herein.

The sugar group can also contain one or more carbons that possess the opposite stereochemical configuration than that of the corresponding carbon in ribose. Thus, a modified nucleic acid can include nucleotides containing e.g. , arabinose, as the sugar. The nucleotide "monomer" can have an alpha linkage at the position on the sugar, e.g. , alpha-nucleosides. The modified nucleic acids can also include "abasic" sugars, which lack a nucleobase at C- . These abasic sugars can also be further modified at one or more of the constituent sugar atoms. The modified nucleic acids can also include one or more sugars that are in the L form, e.g. L- nucleosides. Generally, RNA includes the sugar group ribose, which is a 5-membered ring having an oxygen. Exemplary, non-limiting modified nucleosides and nucleotides include replacement of the oxygen in ribose (e.g. , with sulfur (S), selenium (Se), or alkylene, such as, e.g. , methylene or ethylene); addition of a double bond (e.g. , to replace ribose with cyclopentenyl or cyclohexenyl); ring contraction of ribose (e.g. , to form a 4-membered ring of cyclobutane or oxetane); ring expansion of ribose (e.g. , to form a 6- or 7-membered ring having an additional carbon or heteroatom, such as for example, anhydrohexitol, altritol, mannitol, cyclohexanyl, cyclohexenyl, and morpholino that also has a phosphoramidate backbone). In some embodiments, the modified nucleotides can include multicyclic forms (e.g. , tricyclo; and "unlocked" forms, such as glycol nucleic acid (GNA) (e.g. , R-GNA or S-GNA, where ribose is replaced by glycol units attached to phosphodiester bonds), threose nucleic acid (TNA, where ribose is replaced with a-L- threofuranosyl-(3 '→2' )) .

Modifications on the Nucleobase

The modified nucleic acids can include a modified nucleobase. Examples of nucleobases found in RNA include, but are not limited to, adenine, guanine, cytosine, and uracil. These nucleobases can be modified or wholly replaced to provide modified nucleic acids having enhanced properties, e.g. , resistance to nucleases. The nucleobase of the nucleotide can be independently selected from a purine, a pyrimidine, a purine or pyrimidine analog. In some embodiments, the nucleobase can include, for example, naturally- occurring and synthetic derivatives of a base.

Uracil

In some embodiments, the modified nucleobase is a modified uracil. Exemplary nucleobases and nucleosides having a modified uracil include without limitation pseudouridine (ψ), pyridin-4-one ribonucleoside, 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio- uridine (s2U), 4-thio-uridine (s4U), 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy- uridine (ho⁵U), 5-aminoallyl-uridine, 5-halo-uridine (e.g. , 5 -iodo -uridine or 5-bromo-uridine), 3-

3 5 5

methyl-uridine (m U), 5-methoxy-uridine (mo U), uridine 5-oxyacetic acid (cmo U), uridine 5- oxyacetic acid methyl ester (mcmo⁵U), 5-carboxymethyl-uridine (cm⁵U), 1-carboxymethyl- pseudouridine, 5-carboxyhydroxymethyl-uridine (chm⁵U), 5-carboxyhydroxymethyl-uridine methyl ester (mchm⁵U), 5-methoxycarbonylmethyl-uridine (mcm⁵U), 5- methoxycarbonylmethyl-2-thio-uridine (mcm⁵s2U), 5-aminomethyl-2-thio-uridine (nm⁵s2U), 5- methylaminomethyl-uridine (mnm⁵U), 5-methylaminomethyl-2-thio-uridine (mnm⁵s2U), 5-

5 2 5 methylaminomethyl-2-seleno-uridine (mnm se U), 5-carbamoylmethyl-uridine (ncm U), 5- carboxymethylaminomethyl-uridine (cmnm⁵U), 5-carboxymethylaminomethyl-2-thio-uridine (cmnm ⁵s2U), 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyl-uridine (xcm⁵U),

1- taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine(xm⁵s2U), l-taurinomethyl-4- thio-pseudouridine, 5-methyl-uridine (m⁵U, i.e. , having the nucleobase deoxythymine), 1- methyl-pseudouridine (m^), 5-methyl-2-thio-uridine (m⁵s2U), l-methyl-4-thio-pseudouridine

4-thio-l -methyl -pseudouridine, 3-methyl-pseudouridine (ηι³ψ), 2-thio-l-methyl- pseudouridine, 1 -methyl- 1 -deaza-pseudouridine, 2-thio- 1 -methyl- 1 -deaza-pseudouridine, dihydrouridine (D), dihydropseudoundine, 5,6-dihydrouridine, 5-methyl-dihydrouridine (m⁵D),

2- thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxy-uridine, 2-methoxy-4-thio- uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, Nl-methyl-pseudouridine,

3- (3-amino-3-carboxypropyl)uridine (acp³U), l-methyl-3-(3-amino-3-

3 5

carboxypropyl)pseudouridine (acp ψ), 5-(isopentenylaminomethyl)uridine (inm U), 5- (isopentenylaminomethyl)-2-thio-uridine (inm⁵s2U), a-thio-uridine, 2'-0-methyl-uridine (Um), 5,2'-0-dimethyl-uridine (m⁵Um), 2'-0-methyl-pseudouridine (ψπι), 2-thio-2'-0-methyl-uridine (s2Um), 5-methoxycarbonylmethyl-2'-0-methyl -uridine (mem ⁵Um), 5-carbamoylmethyl-2'-0- methyl-uridine (ncm ⁵Um), 5-carboxymethylaminomethyl-2'-0-methyl-uridine (cmnm ⁵Um),

3 5

3,2'-0-dimethyl-uridine (m Um), 5-(isopentenylaminomethyl)-2'-0-methyl-uridine (inm Um), 1-thio-uridine, deoxythymidine, 2'-F-ara-uridine, 2'-F-uridine, 2'-OH-ara-uridine, 5-(2- carbomethoxyvinyl) uridine, 5-[3-(l-E-propenylamino)uridine, pyrazolo[3,4-d]pyrimidines, xanthine, and hypoxanthine.

Cytosine

In some embodiments, the modified nucleobase is a modified cytosine. Exemplary nucleobases and nucleosides having a modified cytosine include without limitation 5-aza- cytidine, 6-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine (m C), N4-acetyl-cytidine (act), 5- formyl-cytidine (f⁵C), N4-methyl-cytidine (m⁴C), 5-methyl-cytidine (m⁵C), 5-halo-cytidine {e.g. , 5-iodo-cytidine), 5-hydroxymethyl-cytidine (hm⁵C), 1-methyl-pseudoisocytidine, pyrrolo- cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine (s2C), 2-thio-5-methyl-cytidine, 4-thio- pseudoisocytidine, 4-thio- 1 -methyl-pseudoisocytidine, 4-thio- 1 -methyl- 1 -deaza- pseudoisocytidine, 1 -methyl- 1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine, 5-methyl- zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy-cytidine, 2-methoxy- 5 -methyl- cytidine, 4-methoxy-pseudoisocytidine, 4-methoxy-l -methyl -pseudoisocytidine, lysidine (k C), a-thio-cytidine, 2'-0-methyl-cytidine (Cm), 5,2'-0-dimethyl-cytidine (m⁵Cm), N4-acetyl-2'-0- methyl-cytidine (ac⁴Cm), N4,2'-0-dimethyl-cytidine (m⁴Cm), 5-formyl-2'-0-methyl-cytidine (f ⁵Cm), N4,N4,2'-0-trimethyl-cytidine (m⁴ ₂Cm), 1-thio-cytidine, 2'-F-ara-cytidine, 2'-F-cytidine, and 2'-OH-ara-cytidine.

Adenine

In some embodiments, the modified nucleobase is a modified adenine. Exemplary nucleobases and nucleosides having a modified adenine include without limitation 2-amino- purine, 2,6-diaminopurine, 2-amino-6-halo-purine (e.g., 2-amino-6-chloro-purine), 6-halo-purine (e.g., 6-chloro-purine), 2-amino-6-methyl -purine, 8-azido-adenosine, 7-deaza-adenine, 7-deaza- 8-aza-adenine, 7-deaza-2-amino-purine, 7-deaza-8-aza-2-amino-purine, 7-deaza-2,6- diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1 -methyl- adenosine ( r^A), 2-methyl-adenine (m²A), N6-methyl-adenosine (m⁶A), 2-methylthio-N6-methyl-adenosine (ms2m⁶A), N6- isopentenyl-adenosine (i⁶A), 2-methylthio-N6-isopentenyl-adenosine (ms²i⁶A), N6-(cis- hydroxyisopentenyl)adenosine (io⁶A), 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine (ms2io⁶A), N6-glycinylcarbamoyl-adenosine (g⁶A), N6-threonylcarbamoyl-adenosine (t⁶A), N6- methyl-N6-threonylcarbamoyl-adenosine (m⁶t⁶A), 2-methylthio-N6-threonylcarbamoyl- adenosine (ms²g⁶A), N6,N6-dimethyl-adenosine (m⁶ ₂A), N6-hydroxynorvalylcarbamoyl- adenosine (hn⁶A), 2-methylthio-N6-hydroxynorvalylcarbamoyl-adenosine (ms2hn⁶A), N6- acetyl-adenosine (ac⁶A), 7-methyl-adenine, 2-methylthio-adenine, 2-methoxy-adenine, a-thio- adenosine, 2'-0-methyl-adenosine (Am), N6,2'-0-dimethyl-adenosine (m⁶Am), N6,N6,2'-0- trimethyl-adenosine (m⁶ ₂Am), l,2'-0-dimethyl-adenosine (m'Am), 2'-0-ribosyladenosine (phosphate) (Ar(p)), 2-amino-N6-methyl-purine, 1-thio-adenosine, 8-azido-adenosine, 2'-F-ara- adenosine, 2'-F-adenosine, 2'-OH-ara- adenosine, and N6-(19-amino-pentaoxanonadecyl)- adenosine.

Guanine

In some embodiments, the modified nucleobase is a modified guanine. Exemplary nucleobases and nucleosides having a modified guanine include without limitation inosine (I), 1- methyl-inosine (m¹!), wyosine (imG), methylwyosine (mimG), 4-demethyl-wyosine (imG-14), isowyosine (imG2), wybutosine (yW), peroxywybutosine (o₂yW), hydroxywybutosine (OHyW), undermodified hydroxywybutosine (OHyW*), 7-deaza-guanosine, queuosine (Q),

epoxyqueuosine (oQ), galactosyl-queuosine (galQ), mannosyl-queuosine (manQ), 7-cyano-7- deaza-guanosine (preQ₀), 7-aminomethyl-7-deaza-guanosine (preQi), archaeosine (G⁺), 7-deaza- 8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine (m G), 6-thio-7-methyl-guanosine, 7-methyl-inosine, 6-methoxy-guanosine,

2 2 l-methyl-guanosine (m'G), N2-methyl-guanosine (m G), N2,N2-dimethyl-guanosine (m ₂G),

2 2

N2,7-dimethyl-guanosine (m ,7G), N2, N2,7-dimethyl-guanosine (m ,2,7G), 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-meth thio-guanosine, N2-methyl-6-thio-guanosine, N2,N2- dimethyl-6-thio-guanosine, a-thio-guanosine, 2'-0-methyl-guanosine (Gm), N2-methyl-2'-0-

2 2

methyl-guanosine (nTGm), N2,N2-dimethyl-2'-0-methyl- guano sine (m ₂Gm), l-methyl-2'-0- methyl-guanosine (m'Gm), N2,7-dimethyl-2'-0-methyl-guanosine (m",7Gm), 2'-0-methyl- inosine (Im), l,2'-0-dimethyl-inosine (m'lm), 2'-0-ribosylguanosine (phosphate) (Gr(p)), 1-thio- guanosine, 06-methyl-guanosine, 2'-F-ara-guanosine, and 2'-F-guanosine. siRNA

In some embodiments, the nucleic acid agent can be a "short interfering RNA" or "siRNA." As used herein, an siRNA refers to any nucleic acid molecule capable of inhibiting or down regulating gene expression or viral replication by mediating RNA interference "RNAi" or gene silencing in a sequence- specific manner. For example the siRNA can be a double- stranded nucleic acid molecule comprising self-complementary sense and antisense regions, wherein the antisense region comprises nucleotide sequence that is complementary to nucleotide sequence in a target nucleic acid molecule or a portion thereof and the sense region having nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof.

In one embodiment, the therapeutic siRNA molecule suitable for delivery with a particle or composition described herein interacts with a nucleotide sequence of a target gene in a manner that causes inhibition of expression of the target gene.

siRNA comprises a double stranded structure typically containing 15-50 base pairs, e.g. , 19-25, 19-23, 21-25, 21-23, or 24-29 base pairs, and having a nucleotide sequence identical or nearly identical to an expressed target gene or RNA within the cell. An siRNA may be composed of two annealed polynucleotides or a single polynucleotide that forms a hairpin structure. In one embodiment, the therapeutic siRNA is provided in the form of an expression vector, which is packaged in a particle or composition described herein, where the vector has a coding sequence that is transcribed to produce one or more transcriptional products that produce siRNA after administration to a subject.

The siRNA can be assembled from two separate oligonucleotides, where one strand is the sense strand and the other is the antisense strand, where the antisense and sense strands are self-complementary (i.e. , each strand comprises nucleotide sequence that is complementary to nucleotide sequence in the other strand); such as where the antisense strand and sense strand form a duplex or double stranded structure, for example where the double stranded region is about 15 to about 30 basepairs, e.g. , about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 base pairs; the antisense strand includes nucleotide sequence that is complementary to nucleotide sequence in a target nucleic acid molecule or a portion thereof and the sense strand comprises nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof (e.g. , about 15 to about 25 or more nucleotides of the siRNA molecule are

complementary to the target nucleic acid or a portion thereof). Alternatively, the siRNA is assembled from a single oligonucleotide, where the self-complementary sense and antisense regions of the siRNA are linked by means of a nucleic acid based or non-nucleic acid-based linker(s).

In certain embodiments, at least one strand of the siRNA molecule has a 3' overhang from about 1 to about 6 nucleotides in length, though may be from 2 to 4 nucleotides in length. Typically, the 3' overhangs are 1-3 nucleotides in length. In some embodiments, one strand has a 3' overhang and the other strand is blunt-ended or also has an overhang. The length of the overhangs may be the same or different for each strand. To further enhance the stability of the siRNA, the 3' overhangs can be stabilized against degradation.

The siRNAs have significant sequence similarity to a target RNA so that the siRNAs can pair to the target RNA and result in sequence- specific degradation of the target RNA through an RNA interference mechanism. Optionally, the siRNA molecules include a 3' hydroxyl group. In one embodiment, the RNA is stabilized by including purine nucleotides, such as adenosine or guanosine nucleotides. Alternatively, substitution of pyrimidine nucleotides by modified analogues, e.g. , substitution of uridine nucleotide 3' overhangs by 2'-deoxythyimidine is tolerated and does not affect the efficiency of RNAi. The absence of a 2'-hydroxyl significantly enhances the nuclease resistance of the overhang in tissue culture medium and may be beneficial in vivo.

The siRNA can be a polynucleotide with a duplex, asymmetric duplex, hairpin or asymmetric hairpin secondary structure, having self-complementary sense and antisense regions, wherein the antisense region comprises nucleotide sequence that is complementary to nucleotide sequence in a separate target nucleic acid molecule or a portion thereof and the sense region having nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof. The siRNA can be a circular single- stranded polynucleotide having two or more loop structures and a stem comprising self-complementary sense and antisense regions, where the antisense region includes nucleotide sequence that is complementary to nucleotide sequence in a target nucleic acid molecule or a portion thereof and the sense region having nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof, and where the circular polynucleotide can be processed either in vivo or in vitro to generate an active siRNA molecule capable of mediating RNAi.

The siRNA can also include a single stranded polynucleotide having nucleotide sequence complementary to nucleotide sequence in a target nucleic acid molecule or a portion thereof (for example, where such siRNA molecule does not require the presence within the siRNA molecule of nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof), where the single stranded polynucleotide can further include a terminal phosphate group, such as a 5'-phosphate (see for example Martinez et ah, Cell., 110, 563-574,2002, and Schwarz et ah, Molecular Cell, 10, 537-568, 2002), or 5',3'-diphosphate. In certain embodiments, the siRNA comprises separate sense and antisense sequences or regions, where the sense and antisense regions are covalently linked by nucleotide or non-nucleotide linkers molecules as is known in the art, or are alternately non-covalently linked by ionic interactions, hydrogen bonding, van der waals interactions, hydrophobic interactions, and/or stacking interactions.

The siRNA need only be sufficiently similar to natural RNA that it has the ability to mediate RNAi. Thus, an siRNA can tolerate sequence variations that might be expected due to genetic mutation, strain polymorphism or evolutionary divergence. The number of tolerated nucleotide mismatches between the target sequence and the RNAi construct sequence is no more than 1 in 5 basepairs, or 1 in 10 basepairs, or 1 in 20 basepairs, or 1 in 50 basepairs. In some embodiments, the agent comprises a strand that has at least about 70%, e.g., at least about 80%, 84%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% precise sequence complementarity with the target transcript over a window of evaluation between

15-29 nucleotides in length, such a sequence of at least 15 nucleotides, at least about 17 nucleotide, or at least about 18 or 19 to about 21-23 or 24-29 nucleotides in length. Alternatively worded, in an siRNA of about 19-25 nucleotides in length, siRNAs having no greater than about 4 mismatches are generally tolerated, as are siRNAs having no greater than 3 mismatches, 2 mismatches, and or 1 mismatch.

Mismatches in the center of the siRNA duplex are less tolerated, and may essentially abolish cleavage of the target RNA. In contrast, the 3' nucleotides of the siRNA (e.g. , the 3' nucleotides of the siRNA antisense strand) typically do not contribute significantly to specificity of the target recognition. In particular, 3' residues of the siRNA sequence which are

complementary to the target RNA (e.g. , the guide sequence) generally are not as critical for target RNA cleavage.

An siRNA suitable for delivery by a particle or composition described herein may be defined functionally as including a nucleotide sequence (or oligonucleotide sequence) that is capable of hybridizing with a portion of the target gene transcript (e.g. , 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50°C or 70°C hybridization for 12-16 hours; followed by washing). Additional preferred hybridization conditions include hybridization at 70°C. in IxSSC or 50°C in IxSSC, 50% formamide followed by washing at 70°C in 0.3xSSC or hybridization at 70°C in 4xSSC or 50°C in 4xSSC, 50% formamide followed by washing at 67°C in IxSSC. The hybridization temperature for hybrids anticipated to be less than 50 base pairs in length should be 5-10°C less than the melting temperature (Tm) of the hybrid, where Tm is determined according to the following equations. For hybrids less than 18 base pairs in length, Tm(°C)=2(# of A+T bases)+4(# of G+C bases). For hybrids between 18 and 49 base pairs in length,

Tm(°C)=81.5+16.6(log 10[Na+])+0.41(% G+C) (600/N), where N is the number of bases in the hybrid, and [Na+] is the concentration of sodium ions in the hybridization buffer ([Na+] for lxSSC=0.165 M). Additional examples of stringency conditions for polynucleotide hybridization are provided in Sambrook, J., et al., 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., chapters 9 and 11, and Current Protocols in Molecular Biology, 1995, F. M. Ausubel, et al., eds., John Wiley & Sons, Inc., sections 2.10 and 6.3-6.4, incorporated herein by reference. The length of the identical nucleotide sequences may be at least about 10, 12, 15, 17, 20, 22, 25, 27, 30, 32, 35, 37, 40, 42, 45, 47 or 50 bases.

As used herein, siRNA molecules need not be limited to those molecules containing only RNA, but may further encompass chemically-modified nucleotides and non-nucleotides. In certain embodiments, a therapeutic siRNA lacks 2'-hydroxy (2'-OH) containing nucleotides. In certain embodiments, a therapeutic siRNA does not require the presence of nucleotides having a 2'-hydroxy group for mediating RNAi and as such, an siRNA will not include any

ribonucleotides (e.g. , nucleotides having a 2'-OH group). Such siRNA molecules that do not require the presence of ribonucleotides to support RNAi can however have an attached linker or linkers or other attached or associated groups, moieties, or chains containing one or more nucleotides with 2'-OH groups. Optionally, an siRNA molecule can include ribonucleotides at about 5, 10, 20, 30, 40, or 50% of the nucleotide positions.

Other useful therapeutic siRNA oligonucleotides can have phosphorothioate backbones and oligonucleosides with heteroatom backbones, and in particular CH₂NHOCH₂,

CH₂N(CH₃)OCH₂, CH₂ON(CH₃)CH₂, CH₂N(CH₃)N(CH₃)CH₂, and ON(CH₃)CH₂CH₂ (wherein the native phosphodiester backbone is represented as OPOCH₂) as taught in U.S. Pat.

No. 5,489,677, and the amide backbones disclosed in U.S. Pat. No. 5,602,240.

Substituted sugar moieties also can be included in modified oligonucleotides. Therapeutic antisense oligonucleotides for delivery by particle or composition described herein can include one or more of the following at the 2' position: OH; F; O— , S— , or N-alkyl; O— , S— , or N- alkenyl; O— , S— , or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl can be substituted or unsubstituted Ci to C₁₀ alkyl or C₂ to C₁₀ alkenyl and alkynyl. Useful

modifications also can include 0[(CH₂)_nO]_mCH₃, 0(CH₂)_nOCH₃, 0(CH₂)_nNH₂, 0(CH₂)_nCH₃, 0(CH₂)_nONH₂, and 0(CH₂)_nON[(C₂)_nCH₃]₂, where n and m are from 1 to about 10. In addition, oligonucleotides can include one of the following at the 2' position: Ci to C₁₀ lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, CI, Br, CN, CF₃, OCF₃, SOCH₃, S0₂CH₃, ON0₂, N0₂, N₃, NH₂, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, groups for improving the pharmacokinetic or pharmacodynamic properties of an oligonucleotide, and other substituents having similar properties. Other useful modifications include an alkoxyalkoxy group, e.g. , 2'-methoxyethoxy (2'-OCH₂CH₂OCH₃), a dimethylaminooxyethoxy group (2'-0(CH₂)₂0N(CH₃)₂), or a dimethylamino-ethoxyethoxy group (2'-OCH₂OCH₂N(CH₂)₂). Other modifications can include 2'-methoxy (2'-OCH₃), 2'- aminopropoxy (2'-OCH₂CH₂CH₂NH₂), or 2'-fluoro (2'-F). Similar modifications also can be made at other positions within the oligonucleotide, such as the 3' position of the sugar on the 3' terminal nucleotide or in 2'-5' linked oligonucleotides, and the 5' position of the 5' terminal nucleotide. Oligonucleotides also can have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl group. References that teach the preparation of such substituted sugar moieties include U.S. Pat. Nos. 4,981,957 and 5,359,044.

An siRNA formulated with a particle or composition described herein may include naturally occurring nucleosides (e.g. , adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine), nucleoside analogs (e.g. , 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl adenosine, C5- propynylcytidine, C5-propynyluridine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5- methylcytidine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, 0(6)- methylguanine, and 2-thiocytidine), chemically modified bases, biologically modified bases (e.g. , methylated bases), intercalated bases, modified sugars (e.g. , 2'-fluororibose, ribose, 2'- deoxyribose, arabinose, and hexose). Suitable modified nucleobases include other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2- propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2- thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8- hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5- trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7- methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3- deazaguanine and 3-deazaadenine. Other useful nucleobases include those disclosed, for example, in U.S. Pat. No. 3,687,808.

A therapeutic siRNA for incorporation into a particle or composition described herein may be chemically synthesized, or derived from a longer double- stranded RNA or a hairpin RNA. The siRNA can be produced enzymatically or by partial/total organic synthesis, and any modified ribonucleotide can be introduced by in vitro enzymatic or organic synthesis. A single- stranded species comprised at least in part of RNA may function as an siRNA antisense strand or may be expressed from a plasmid vector.

By "RNA interference" or "RNAi" is meant a process of inhibiting or down regulating gene expression in a cell as is generally known in the art and which is mediated by short interfering nucleic acid molecules. In addition, as used herein, the term RNAi is meant to be equivalent to other terms used to describe sequence specific RNA interference, such as post transcriptional gene silencing, translational inhibition, transcriptional inhibition, or epigenetics. For example, therapeutic siRNA molecules suitable for delivery by a particle or composition described herein can epigenetically silence genes at both the post-transcriptional level or the pre- transcriptional level. In a non-limiting example, epigenetic modulation of gene expression by siRNA molecules can result from siRNA mediated modification of chromatin structure or methylation patterns to alter gene expression. In another non-limiting example, modulation of gene expression by an siRNA molecule can result from siRNA mediated cleavage of RNA (either coding or non-coding RNA) via RISC, or alternately, translational inhibition as is known in the art. In another embodiment, modulation of gene expression by siRNA molecules can result from transcriptional inhibition. RNAi also includes translational repression by microRNAs or siRNAs acting like microRNAs. RNAi can be initiated by introduction of small interfering RNAs (siRNAs) or production of siRNAs intracellularly (e.g. , from a plasmid or transgene), to silence the expression of one or more target genes. Alternatively, RNAi occurs in cells naturally to remove foreign RNAs (e.g. , viral RNAs). Natural RNAi proceeds via dicer-directed fragmentation of precursor dsRNA which direct the degradation mechanism to other cognate RNA sequences.

As used herein, the term siRNA is meant to be equivalent to other terms used to describe nucleic acid molecules that are capable of mediating sequence specific RNAi, and includes, for example, short interfering RNA (siRNA), double- stranded RNA (dsRNA), short hairpin RNA (shRNA), short interfering oligonucleotide, short interfering nucleic acid, short interfering modified oligonucleotide, chemically-modified siRNA, post-transcriptional gene silencing RNA (ptgsRNA), and others. miRNAs

In some embodiments, the nucleic acid agent can be a microRNA (miRNA). By

"microRNA" or "miRNA" is meant a small double stranded RNA that regulates the expression of target messenger RNAs either by mRNA cleavage, translational repression/inhibition or heterochromatic silencing (see for example Ambros, 2004, Nature, 431, 350-355; Bartel, 2004, Cell, 116, 281-297; CuUen, 2004, Virus Research., 102, 3-9; He et al., 2004, Nat. Rev. Genet., 5, 522-531 ; and Ying et al., 2004, Gene, 342, 25-28). MicroRNAs (miRNAs) are small noncoding polynucleotides, about 22 nucleotides long, which direct destruction or translational repression of their mRNA targets.

In one embodiment, the therapeutic microRNA, has partial complementarity (i.e. , less than 100% complementarity) between the sense strand or sense region and the antisense strand or antisense region of the miRNA molecule, or between the antisense strand or antisense region of the miRNA and a corresponding target nucleic acid molecule. For example, partial

complementarity can include various mismatches or non-base paired nucleotides (e.g. , 1, 2, 3, 4, 5 or more mismatches or non-based paired nucleotides, such as nucleotide bulges) within the double stranded nucleic acid molecule, structure which can result in bulges, loops, or overhangs that result between the sense strand or sense region and the antisense strand or antisense region of the miRNA or between the antisense strand or antisense region of the miRNA and a corresponding target nucleic acid molecule. Agents that act via the microRNA translational repression pathway contain at least one bulge and/or mismatch in the duplex formed with the target. In certain embodiments, a GU or UG base pair in a duplex formed by a guide strand and a target transcript is not considered a mismatch for purposes of determining whether an RNAi agent is targeted to a transcript.

In one embodiment, a therapeutic nucleic acid suitable for delivery by a particle or composition described herein is an antagomir, which is a chemically modified oligonucleotide capable of inhibition of complementary miRNA, e.g., by promoting their degradation. See, e.g. , Krutzfeldt et al., Nature, 438: 685-689, 2005, which is incorporated herein in its entirety.

Antisense oligonucleotides

In some embodiments, the nucleic acid agent can be a therapeutic "antisense

oligonucleotides" are suitable for delivery via a particle or composition described herein. The term "oligonucleotide" refers to an oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or analogs thereof. This term includes oligonucleotides composed of naturally occurring nucleobases, sugars and covalent internucleoside (backbone) linkages, as well as oligonucleotides having non-naturally occurring portions which function similarly. Such modified or substituted oligonucleotides are often preferred over native forms because of desirable properties such as, for example, enhanced cellular uptake, enhanced affinity for a nucleic acid target, and increased stability in the presence of nucleases.

A therapeutic antisense oligonucleotide is typically from about 10 to about 50 nucleotides in length (e.g. , 12 to 40, 14 to 30, or 15 to 25 nucleotides in length). Antisense oligonucleotides that are 15 to 23 nucleotides in length are particularly useful. However, an antisense

oligonucleotide containing even fewer than 10 nucleotides (for example, a portion of one of the preferred antisense oligonucleotides) is understood to be included within the disclosure so long as it demonstrates the desired activity of inhibiting expression of a target gene.

An antisense oligonucleotide may consist essentially of a nucleotide sequence that specifically hybridizes with an accessible region in the target nucleic acid. Such antisense oligonucleotides, however, may contain additional flanking sequences of 5 to 10 nucleotides at either end. Flanking sequences can include, for example, additional sequences of the target nucleic acid, sequences complementary to an amplification primer, or sequences corresponding to a restriction enzyme site.

For maximal effectiveness, further criteria can be applied to the design of antisense oligonucleotides. Such criteria are well known in the art, and are widely used, for example, in the design of oligonucleotide primers. These criteria include the lack of predicted secondary structure of a potential antisense oligonucleotide, an appropriate G and C nucleotide content (e.g. , approximately 50%), and the absence of sequence motifs such as single nucleotide repeats (e.g. , GGGG runs).

While antisense oligonucleotides are a preferred form of antisense compounds, the disclosure includes other oligomeric antisense compounds, including but not limited to, oligonucleotide analogs such as those described below. As is known in the art, a nucleoside is a base-sugar combination, wherein the base portion is normally a heterocyclic base. The two most common classes of such heterocyclic bases are the purines and the pyrimidines. Nucleotides are nucleosides that further include a phosphate group covalently linked to the sugar portion of the nucleoside. For those nucleosides that include a pentofuranosyl sugar, the phosphate group can be linked to either the 2', 3' or 5' hydroxyl moiety of the sugar. In forming oligonucleotides, the phosphate groups covalently link adjacent nucleosides to one another to form a linear polymeric molecule. The respective ends of this linear polymeric molecule can be further joined to form a circular molecule, although linear molecules are generally preferred. Within the oligonucleotide molecule, the phosphate groups are commonly referred to as forming the internucleoside backbone of the oligonucleotide. The normal linkage or backbone of RNA and DNA is a 3' to 5' phosphodiester linkage.

The therapeutic antisense oligonucleotides suitable for delivery by a particle or composition described herein include oligonucleotides containing modified backbones or non- natural internucleoside linkages. As defined herein, oligonucleotides having modified backbones include those that have a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone. For the purposes of this specification, and as sometimes referenced in the art, modified oligonucleotides that do not have a phosphorus atom in their internucleoside backbone also can be considered to be oligonucleotides.

Modified oligonucleotide backbones can include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates (e.g. , 3'-alkylene phosphonates and chiral phosphonates), phosphinates, phosphoramidates (e.g. , 3'-amino phosphoramidate and

aminoalkylphosphoramidates), thionophosphoramidates, thionoalkylphosphonates, thionoalkyl phosphotriesters, and boranophosphates having normal 3'-5' linkages, as well as 2'-5' linked analogs of these, and those having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'. Various salts, mixed salts and free acid forms are also included. References that teach the preparation of such modified backbone oligonucleotides are provided, for example, in U.S. Pat. Nos. 4,469,863 and 5,750,666.

Therapeutic antisense molecules with modified oligonucleotide backbones that do not include a phosphorus atom therein can have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a

nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH₂ component parts. References that teach the preparation of such modified backbone oligonucleotides are provided, for example, in U.S. Pat. Nos. 5,235,033 and 5,596,086.

In another embodiment, a therapeutic antisense compound is an oligonucleotide analog, in which both the sugar and the internucleoside linkage (i.e., the backbone) of the nucleotide units are replaced with novel groups, while the base units are maintained for hybridization with an appropriate nucleic acid target. One such oligonucleotide analog that has been shown to have excellent hybridization properties is referred to as a peptide nucleic acid (PNA). In PNA compounds, the sugar-backbone of an oligonucleotide is replaced with an amide containing backbone (e.g., an aminoethylglycine backbone). The nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone. References that teach the preparation of such modified backbone oligonucleotides are provided, for example, in Nielsen et al, Science 254:1497-1500 (1991), and in U.S. Pat. No. 5,539,082.

Other useful therapeutic antisense oligonucleotides can have phosphorothioate backbones and oligonucleosides with heteroatom backbones, and in particular CH₂NHOCH₂,

No. 5,489,677, and the amide backbones disclosed in U.S. Pat. No. 5,602,240.

Substituted sugar moieties also can be included in modified oligonucleotides. Therapeutic antisense oligonucleotides for delivery by a particle or composition described herein can include one or more of the following at the 2' position: OH; F; O— , S— , or N-alkyl; O— , S— , or N- alkenyl; O— , S— , or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl can be substituted or unsubstituted Ci to Cio alkyl or C₂ to Cio alkenyl and alkynyl. Useful

modifications also can include 0[(CH₂)_nO]_mCH₃, 0(CH₂)_nOCH₃, 0(CH₂)_nNH₂, 0(CH₂)_nCH₃, 0(CH₂)_nONH₂, and 0(CH₂)_nON[(C₂)_nCH₃]₂, where n and m are from 1 to about 10. In addition, oligonucleotides can include one of the following at the 2' position: Q to Cio lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, CI, Br, CN, CF₃, OCF₃, SOCH₃, S0₂CH₃, ON0₂, N0₂, N₃, NH₂, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, groups for improving the pharmacokinetic or pharmacodynamic properties of an oligonucleotide, and other substituents having similar properties. Other useful modifications include an alkoxyalkoxy group, e.g. , 2'-methoxyethoxy (2'-OCH₂CH₂OCH₃), a

dimethylaminooxyethoxy group (2'-0(CH₂)₂ON(CH₃)₂), or a dimethylamino-ethoxyethoxy group (2'-OCH₂OCH₂N(CH₂)₂). Other modifications can include 2'-methoxy (2'-OCH₃), 2'- aminopropoxy (2'-OCH₂CH₂CH₂NH₂), or 2'-fluoro (2'-F). Similar modifications also can be made at other positions within the oligonucleotide, such as the 3' position of the sugar on the 3' terminal nucleotide or in 2'-5' linked oligonucleotides, and the 5' position of the 5' terminal nucleotide. Oligonucleotides also can have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl group. References that teach the preparation of such substituted sugar moieties include U.S. Pat. Nos. 4,981,957 and 5,359,044.

Therapeutic antisense oligonucleotides can also include nucleobase modifications or substitutions. As used herein, "unmodified" or "natural" nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C), and uracil (U). Modified nucleobases can include other synthetic and natural nucleobases such as

5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine,

2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other

5- substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Other useful nucleobases include those disclosed, for example, in U.S. Pat. No. 3,687,808.

Certain nucleobase substitutions can be particularly useful for increasing the binding affinity of the antisense oligonucleotides of the disclosure. For example, 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6 to 1.2°C. (Sanghvi et al., eds., Antisense Research and Applications, pp. 276-278, CRC Press, Boca Raton, Fla. (1993)). Other useful nucleobase substitutions include 5-substituted pyrimidines,

6- azapyrimidines and N-2, N-6 and 0-6 substituted purines such as 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. It is not necessary for all nucleobase positions in a given antisense oligonucleotide be uniformly modified. More than one of the aforementioned modifications can be incorporated into a single oligonucleotide or even at a single nucleoside within an oligonucleotide. The therapeutic nucleic acids suitable for delivery by a particle or compositions described herein also include antisense oligonucleotides that are chimeric oligonucleotides. "Chimeric" antisense

oligonucleotides can contain two or more chemically distinct regions, each made up of at least one monomer unit (e.g., a nucleotide in the case of an oligonucleotide). Chimeric

oligonucleotides typically contain at least one region wherein the oligonucleotide is modified so as to confer, for example, increased resistance to nuclease degradation, increased cellular uptake, and/or increased affinity for the target nucleic acid. For example, a region of a chimeric oligonucleotide can serve as a substrate for an enzyme such as RNase H, which is capable of cleaving the RNA strand of an RNA:DNA duplex such as that formed between a target mRNA and an antisense oligonucleotide. Cleavage of such a duplex by RNase H, therefore, can greatly enhance the effectiveness of an antisense oligonucleotide.

The therapeutic antisense oligonucleotides can be synthesized in vitro. Antisense oligonucleotides used in accordance with this disclosure can be conveniently produced through known methods, e.g., by solid phase synthesis. Similar techniques also can be used to prepare modified oligonucleotides such as phosphorothioates or alkylated derivatives.

Antisense polynucleotides include sequences that are complementary to a genes or mRNA. Antisense polynucleotides include, but are not limited to: morpholinos, 2'-0-methyl polynucleotides, DNA, RNA and the like. The polynucleotide-based expression inhibitor may be polymerized in vitro, recombinant, contain chimeric sequences, or derivatives of these groups. The polynucleotide-based expression inhibitor may contain ribonucleotides,

deoxyribonucleotides, synthetic nucleotides, or any suitable combination such that the target RNA and/or gene is inhibited.

The term "hybridization," as used herein, means hydrogen bonding, which can be Watson-Crick, Hoogsteen, or reversed Hoogsteen hydrogen bonding, between complementary nucleoside or nucleotide bases. For example, adenine and thymine, and guanine and cytosine, respectively, are complementary nucleobases (often referred to in the art simply as "bases") that pair through the formation of hydrogen bonds. "Complementary," as used herein, refers to the capacity for precise pairing between two nucleotides. For example, if a nucleotide at a certain position of an oligonucleotide is capable of hydrogen bonding with a nucleotide in a target nucleic acid molecule, then the oligonucleotide and the target nucleic acid are considered to be complementary to each other at that position. The oligonucleotide and the target nucleic acid are complementary to each other when a sufficient number of corresponding positions in each molecule are occupied by nucleotides that can hydrogen bond with each other. Thus,

"specifically hybridizable" is used to indicate a sufficient degree of complementarity or precise pairing such that stable and specific binding occurs between the oligonucleotide and the target nucleic acid.

It is understood in the art that the sequence of an antisense oligonucleotide need not be 100% complementary to that of its target nucleic acid to be specifically hybridizable. An antisense oligonucleotide is specifically hybridizable when (a) binding of the oligonucleotide to the target nucleic acid interferes with the normal function of the target nucleic acid, and (b) there is sufficient complementarity to avoid non-specific binding of the antisense oligonucleotide to non-target sequences under conditions in which specific binding is desired, i.e., under conditions in which in vitro assays are performed or under physiological conditions for in vivo assays or therapeutic uses.

Stringency conditions in vitro are dependent on temperature, time, and salt concentration (see e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY (1989)). Typically, conditions of high to moderate stringency are used for specific hybridization in vitro, such that hybridization occurs between substantially similar nucleic acids, but not between dissimilar nucleic acids. Specific hybridization conditions are hybridization in 5 x SSC (0.75 M sodium chloride/0.075 M sodium citrate) for 1 hour at 40°C, followed by washing 10 times in lxSSC at 40°C and 5 x in lxSSC at room temperature.

In vivo hybridization conditions consist of intracellular conditions (e.g., physiological pH and intracellular ionic conditions) that govern the hybridization of antisense oligonucleotides with target sequences. In vivo conditions can be mimicked in vitro by relatively low stringency conditions. For example, hybridization can be carried out in vitro in 2xSSC (0.3 M sodium chloride/0.03 M sodium citrate), 0.1% SDS at 37°C. A wash solution containing 4xSSC, 0.1% SDS can be used at 37°C, with a final wash in lxSSC at 45°C.

The specific hybridization of an antisense molecule with its target nucleic acid can interfere with the normal function of the target nucleic acid. For a target DNA nucleic acid, antisense technology can disrupt replication and transcription. For a target RNA nucleic acid, antisense technology can disrupt, for example, translocation of the RNA to the site of protein translation, translation of protein from the RNA, splicing of the RNA to yield one or more mRNA species, and catalytic activity of the RNA. The overall effect of such interference with target nucleic acid function is, in the case of a nucleic acid encoding a target gene, inhibition of the expression of target gene. In the context of the disclosure, "inhibiting expression of a target gene" means to disrupt the transcription and/or translation of the target nucleic acid sequences resulting in a reduction in the level of target polypeptide or a complete absence of target polypeptide.

An antisense oligonucleotide, e.g. , an antisense strand of an siRNA may preferably be directed at specific targets within a target nucleic acid molecule. The targeting process includes the identification of a site or sites within the target nucleic acid molecule where an antisense interaction can occur such that a desired effect, e.g. , inhibition of target gene expression, will result. Traditionally, preferred target sites for antisense oligonucleotides have included the regions encompassing the translation initiation or termination codon of the open reading frame (ORF) of the gene. In addition, the ORF has been targeted effectively in antisense technology, as have the 5' and 3' untranslated regions. Furthermore, antisense oligonucleotides have been successfully directed at intron regions and intron-exon junction regions.

Simple knowledge of the sequence and domain structure (e.g. , the location of translation initiation codons, exons, or introns) of a target nucleic acid, however, is generally not sufficient to ensure that an antisense oligonucleotide directed to a specific region will effectively bind to and inhibit transcription and/or translation of the target nucleic acid. In its native state, an mRNA molecule is folded into complex secondary and tertiary structures, and sequences that are on the interior of such structures are inaccessible to antisense oligonucleotides. For maximal effectiveness, antisense oligonucleotides can be directed to regions of a target mRNA that are most accessible, i.e. , regions at or near the surface of a folded mRNA molecule. Accessible regions of an mRNA molecule can be identified by methods known in the art, including the use of RiboTAG™, or mRNA Accessible Site Tagging (MAST), technology. RiboTAG™ technology is disclosed in PCT Application Number SEO 1/02054.

Once one or more target sites have been identified, antisense oligonucleotides can be synthesized that are sufficiently complementary to the target (i.e. , that hybridize with sufficient strength and specificity to give the desired effect). The effectiveness of an antisense oligonucleotide to inhibit expression of a target nucleic acid can be evaluated by measuring levels of target mRNA or protein using, for example, Northern blotting, RT-PCR, Western blotting, ELISA, or immunohistochemical staining.

In some embodiments, it may be useful to target multiple accessible regions of a target nucleic acid. In such embodiments, multiple antisense oligonucleotides can be used that each specifically hybridize to a different accessible region. Multiple antisense oligonucleotides can be used together or sequentially. In some embodiments, it may be useful to target multiple accessible regions of multiple target nucleic acids.

Aptamers

In some embodiments, the nucleic acid agent suitable for delivery by a particle or composition described herein can be an aptamer (also called a nucleic acid ligand or nucleic acid aptamer), which is a polynucleotide that binds specifically to a target molecule where the nucleic acid molecule has a sequence that is distinct from a sequence recognized by the target molecule in its natural setting. Alternately, an aptamer can be a nucleic acid molecule that binds to a target molecule where the target molecule does not naturally bind to a nucleic acid. The target molecule can be any molecule of interest. The target molecule can be, for example, a polypeptide, a carbohydrate, a nucleic acid molecule or a cell. The target of an aptamer is a three dimensional chemical structure that binds to the aptamer. For example, an aptamer that targets a nucleic acid (e.g. , an RNA or a DNA) may include regions that bind via complementary Watson-Crick base pairing to a nucleic acid target interrupted by other structures such as hairpin loops. In another embodiment, the aptamer binds a target protein at a ligand-binding domain, thereby preventing interaction of the naturally occurring ligand with the target protein.

In one embodiment, the aptamer binds to a cell or tissue in a specific developmental stage or a specific disease state. A target is an antigen on the surface of a cell, such as a cell surface receptor, an integrin, a transmembrane protein, an ion channel or a membrane transport protein. In one embodiment, the target is a tumor-marker. A tumor-marker can be an antigen that is present in a tumor that is not present in normal tissue or an antigen that is more prevalent in a tumor than in normal tissue. The nucleic acid that forms the nucleic acid ligand may be composed of naturally occurring nucleosides, modified nucleosides, naturally occurring nucleosides with hydrocarbon linkers (e.g. , an alkylene) or a polyether linker (e.g. , a PEG linker) inserted between one or more nucleosides, modified nucleosides with hydrocarbon or PEG linkers inserted between one or more nucleosides, or a combination of thereof. In one embodiment, nucleotides or modified nucleotides of the nucleic acid ligand can be replaced with a hydrocarbon linker or a polyether linker provided that the binding affinity and selectivity of the nucleic acid ligand is not substantially reduced by the substitution (e.g. , the dissociation constant of the aptamer for the target is typically not greater than about lxl 0^"6 M).

An aptamer may be prepared by any method, such as by Systemic Evolution of Ligands by Exponential Enrichment (SELEX). The SELEX process for obtaining nucleic acid ligands is described in U.S. Pat. No. 5,567,588, the entire teachings of which are incorporated herein by reference.

Hydrophobic Polymers

A particle described herein may include a hydrophobic polymer. The hydrophobic polymer may be attached to a cationic moiety to form a conjugate (e.g. , a cationic moiety- hydrophobic polymer conjugate). In some embodiments, the hydrophobic polymer is not attached to another moiety. A particle can include a plurality of hydrophobic polymers, for example where some are attached to another moiety such as a cationic moiety and some are free.

Exemplary hydrophobic polymers include the following: acrylates including methyl acrylate, ethyl acrylate, propyl acrylate, n-butyl acrylate (BA), isobutyl acrylate, 2-ethyl acrylate, and t-butyl acrylate; methacrylates including ethyl methacrylate, n-butyl methacrylate, and isobutyl methacrylate; acrylonitriles; methacrylonitrile; vinyls including vinyl acetate, vinylversatate, vinylpropionate, vinylformamide, vinylacetamide, vinylpyridines, and

vinylimidazole; aminoalkyls including aminoalkylacrylates, aminoalkylmethacrylates, and aminoalkyl(meth)acrylamides; styrenes; cellulose acetate phthalate; cellulose acetate succinate; hydroxypropylmethylcellulose phthalate; poly(D,L-lactide); poly(D,L-lactide-co-glycolide); poly(glycolide); poly(hydroxybutyrate); poly(alkylcarbonate); poly(orthoesters); polyesters; poly(hydroxyvaleric acid); polydioxanone; poly(ethylene terephthalate); poly(malic acid);

poly(tartronic acid); polyanhydrides; polyphosphazenes; poly(amino acids) and their copolymers (see generally, Svenson, S (ed.)., Polymeric Drug Delivery: Volume I: Particulate Drug Carriers. 2006; ACS Symposium Series; Amiji, M.M (ed.)., Nanotechnology for Cancer Therapy. 2007; Taylor & Francis Group, LLP; Nair et al. Prog. Polym. Sci. (2007) 32: 762-798); hydrophobic peptide-based polymers and copolymers based on poly(L- amino acids) (Lavasanifar, A., et al., Advanced Drug Delivery Reviews (2002) 54: 169-190); poly(ethylene-vinyl acetate) ("EVA") copolymers; silicone rubber; polyethylene; polypropylene; polydienes (polybutadiene, polyisoprene and hydrogenated forms of these polymers); maleic anhydride copolymers of vinyl methylether and other vinyl ethers; polyamides (nylon 6,6); polyurethane; poly(ester urethanes); poly(ether urethanes); and poly(ester-urea).

Hydrophobic polymers useful in preparing the particles described herein also include biodegradable polymers. Examples of biodegradable polymers include polylactides,

polyglycolides, caprolactone-based polymers, poly(caprolactone), polydioxanone,

polyanhydrides, polyamines, polyesteramides, polyorthoesters, polydioxanones, polyacetals, polyketals, polycarbonates, polyphosphoesters, polyesters, polybutylene terephthalate, polyorthocarbonates, polyphosphazenes, succinates, poly(malic acid), poly(amino acids), poly(vinylpyrrolidone), polyethylene glycol, polyhydroxycellulose, polysaccharides, chitin, chitosan and hyaluronic acid, and copolymers, terpolymers and mixtures thereof. Biodegradable polymers also include copolymers, including caprolactone-based polymers, polycaprolactones and copolymers that include polybutylene terephthalate.

In some embodiments, the polymer is a polyester synthesized from monomers selected from the group consisting of D,L-lactide, D-lactide, L-lactide, D,L-lactic acid, D-lactic acid, L- lactic acid, glycolide, glycolic acid, ε-caprolactone, ε-hydroxy hexanoic acid, γ-butyrolactone, γ- hydroxy butyric acid, δ-valerolactone, δ-hydroxy valeric acid, hydroxybutyric acids, and malic acid.

A copolymer may also be used in a particle described herein. In some embodiments, a polymer may be PLGA, which is a biodegradable random copolymer of lactic acid and glycolic acid. A PLGA polymer may have varying ratios of lactic acid:glycolic acid, e.g. , ranging from about 0.1 :99.9 to about 99.9:0.1 (e.g. , from about 75:25 to about 25:75, from about 60:40 to 40:60, or about 55:45 to 45:55). In some embodiments, e.g. , in PLGA, the ratio of lactic acid monomers to glycolic acid monomers is 50:50, 60:40 or 75:25. In some embodiments, by optimizing the ratio of lactic acid to glycolic acid monomers in the PLGA polymer of the particle, parameters such as water uptake, agent release (e.g.,

"controlled release") and polymer degradation kinetics may be optimized. Furthermore, tuning the ratio will also affect the hydrophobicity of the copolymer, which may in turn affect drug loading.

In some embodiments, wherein the biodegradable polymer also has a cationic moiety attached to it, the biodegradation rate of such polymer may be characterized by a release rate of such materials. In such circumstances, the biodegradation rate may depend on not only the chemical identity and physical characteristics of the polymer, but also on the identity of material(s) attached thereto. Degradation of the subject compositions includes not only the cleavage of intramolecular bonds, e.g., by oxidation and/or hydrolysis, but also the disruption of intermolecular bonds, such as dissociation of host/guest complexes by competitive complex formation with foreign inclusion hosts. In some embodiments, the release can be affected by an additional component in the particle, e.g., a compound having at least one acidic moiety (e.g., free-acid PLGA).

In some embodiments, particles comprising one or more polymers, such as a hydrophobic polymer, biodegrade within a period that is acceptable in the desired application. In certain embodiments, such as in vivo therapy, such degradation occurs in a period usually less than about five years, one year, six months, three months, one month, fifteen days, five days, three days, or even one day on exposure to a physiological solution with a pH between 4 and 8 having a temperature of between 25 °C and 37 °C. In other embodiments, the polymer degrades in a period of between about one hour and several weeks, depending on the desired application.

When polymers are used for delivery of nucleic acid agents in vivo, it is important that the polymers themselves be nontoxic and that they degrade into non-toxic degradation products as the polymer is eroded by the body fluids. Many synthetic biodegradable polymers, however, yield oligomers and monomers upon erosion in vivo that adversely interact with the surrounding tissue (D. F. Williams, J. Mater. Sci. 1233 (1982)). To minimize the toxicity of the intact polymer carrier and its degradation products, polymers have been designed based on naturally occurring metabolites. Exemplary polymers include polyesters derived from lactic and/or glycolic acid and polyamides derived from amino acids. A number of biodegradable polymers are known and used for controlled release of pharmaceuticals. Such polymers are described in, for example, U.S. Pat. Nos. 4,291,013;

4,347,234; 4,525,495; 4,570,629; 4,572,832; 4,587,268; 4,638,045; 4,675,381 ; 4,745,160; and 5,219,980; and PCT publication WO2006/014626, each of which is hereby incorporated by reference in its entirety.

A hydrophobic polymer described herein may have a variety of end groups. In some embodiments, the end group of the polymer is not further modified, e.g. , when the end group is a carboxylic acid, a hydroxy group or an amino group. In some embodiments, the end group may be further modified. For example, a polymer with a hydroxyl end group may be derivatized with an acyl group to yield an acyl-capped polymer (e.g. , an acetyl-capped polymer or a benzoyl capped polymer), an alkyl group to yield an alkoxy-capped polymer (e.g. , a methoxy-capped polymer), or a benzyl group to yield a benzyl-capped polymer. The end group can also be further reacted with a functional group, for example to provide a linkage to another moiety such as a a cationic moiety. In some embodiments a particle comprises a functionalized hydrophobic polymer, e.g. , a hydrophobic polymer, such as PLGA (e.g. , 50:50 PLGA), functionalized with a moiety, e.g. , N-(2-aminoethyl)maleimide, 2-(2-(pyridine-2-yl)disulfanyl)ethylamino, or a succinimidyl-N-methyl ester, that has not reacted with another moiety, e.g. , a cationic moiety.

A hydrophobic polymer may have a weight average molecular weight ranging from about 1 kDa to about 70 kDa (e.g. , from about 4 kDa to about 66 kDa, from about 2 kDa to about 12 kDa, from about 6 kDa to about 20 kDa, from about 5 kDa to about 15 kDa, from about 6 kDa to about 13 kDa, from about 7 kDa to about 11 kDa, from about 5 kDa to about 10 kDa, from about 7 kDa to about 10 kDa, from about 5 kDa to about 7 kDa, from about 6 kDa to about 8 kDa, about 6 kDa, about 7 kDa, about 8 kDa, about 9 kDa, about 10 kDa, about 11 kDa, about 12 kDa, about 13 kDa, about 14 kDa, about 15 kDa, about 16 kDa or about 17 kDa).

A hydrophobic polymer described herein may have a polymer polydispersity index (PDI) of less than or equal to about 2.5 (e.g. , less than or equal to about 2.2, less than or equal to about 2.0, or less than or equal to about 1.5). In some embodiments, a hydrophobic polymer described herein may have a polymer PDI of about 1.0 to about 2.5, about 1.0 to about 2.0, about 1.0 to about 1.7, or from about 1.0 to about 1.6. A particle described herein may include varying amounts of a hydrophobic polymer, e.g., from about 10% to about 90% by weight of the particle (e.g., from about 20% to about 80%, from about 25% to about 75%, or from about 30% to about 70%).

A hydrophobic polymer described herein may be commercially available, e.g., from a commercial supplier such as BASF, Boehringer Ingelheim, Durcet Corporation, Purac America and SurModics Pharmaceuticals. A polymer described herein may also be synthesized. Methods of synthesizing polymers are known in the art (see, for example, Polymer Synthesis: Theory and Practice Fundamentals, Methods, Experiments. D. Braun et al., 4th edition, Springer, Berlin, 2005). Such methods include, for example, polycondensation, radical polymerization, ionic polymerization (e.g., cationic or anionic polymerization), or ring-opening metathesis

polymerization.

A commercially available or synthesized polymer sample may be further purified prior to incorporation into a particle or composition described herein. In some embodiments, purification may reduce the polydispersity of the polymer sample. A polymer may be purified by

precipitation from solution, or precipitation onto a solid such as Celite. A polymer may also be further purified by size exclusion chromatography (SEC).

Cationic Moieties

Exemplary cationic moieties for use in the particles described herein include amines, including for example, primary, secondary, tertiary, and quaternary amines, and polyamines (e.g., branched and linear polyethylene imine (PEI) or derivatives thereof such as

polyethyleneimine-PLGA, polyethylene imine -polyethylene glycol -N-acetylgalactosamine (PEI-PEG-GAL) or polyethylene imine - polyethylene glycol -tri-N-acetylgalactosamine (PEI- PEG-triGAL) derivatives). In some embodiments, the cationic moiety comprises a cationic lipid (e.g., l-[2-(oleoyloxy)ethyl]-2-oleyl-3-(2-hydroxyethyl)imidazolinium chloride (DOTIM), dimethyldioctadecyl ammonium bromide, 1,2 dioleyloxypropyl-3-trimethyl ammonium bromide, DOTAP, l,2-dimyristyloxypropyl-3-dimethyl-hydroxyethyl ammonium bromide, 1,2- dimyristoyl-sn-glycero-3-ethylphosphocholine (EDMPC), ethyl-PC, l,2-dioleoyl-3- dimethylammonium-propane (DODAP), DC-cholesterol, and MBOP, CLinDMA, 1,2- dilinoleyloxy-3-dimethylaminopropane (DLinDMA), pCLinDMA, eCLinDMA, DMOBA, and DMLBA). In some embodiments, for example, where the cationic moiety is a polyamine, the polyamine comprises, polyamino acids (e.g. , poly(lysine), poly(histidine), and poly(arginine)) and derivatives (e.g. poly(lysine)-PLGA, imidazole modified poly(lysine)) or polyvinyl pyrrolidone (PVP). In some embodiments, for example, where the cationic moiety is a cationic polymer comprising a plurality of amines, the amines can be positioned along the polymer such that the amines are from about 4 to about 10 angstroms apart (e.g. , from about 5 to about 8 or from about 6 to about 7).

The cationic moiety can have a pKa of 5 or greater and/or be positively charged at physiological pH.

In some embodiments, the cationic moiety is a partially hydrolyzed polyoxazoline (pOx), wherein the structure of polyoxazoline is shown below:

In some embodiments, the cationic moiety is a partially hydrolyzed pOx, e.g. , pOx45, i.e. , pOx hydrolyzed for 45 min. (about 12.5% hydrolyzed), pOx60, i.e. , pOx hydrolyzed for 60 min. (about 17.5% hydrolyzed), pOxl20, i.e. , pOx hydrolyzed for 120 min. (about 21% hydrolyzed), or pOx200, i.e. , pOx hydrolyzed for 200 min. (about 43% hydrolyzed). The ratios of x:y can be about 1 : 10, about 1 :9, about 1 :8, about 1 :7, about 1 :6, about 1 :5, about 1 :4, about 1 :3, about 1 :2, or about 1 : 1.

In some embodiments, the cationic moiety is a PVA-poly(phosphonium). In some embodiments, for example, the poly(phosphonium) comprises 20% + 5% acyl groups, 10% + 5% phosphonium groups, and 70% + 5% free hydroxyl groups, e.g. , a ratio of a/b/c of 2: 1 :7. The a:b:c ratios are about 2:0.5:7.5 for 5% density, about 2: 1 :7 for 10% charge density, about 2:3.5:3.5 for 50% density and 2:8:0 ratio for 100% charge density. The structure of the polyphosphonium is shown belo

In some embodiments, the cationic moiety is PVA-arginine (PVA-Arg), or PVA- histidine, e.g. , cationic PVA-deamino-histidine ester (PVA-His). The structure of PVA-His is shown below:

In some embodiments, the cationic moiety is PVA-dibutylammonium. In some embodiments, the cationic moiety is cationic PVA-dibutylamino-l(propylamine)-carbamate (PVA-DBA). The structure of PV -DBA is shown below:

In some embodiments, the cationic moiety is a cationic PVA that is derivatized with dimethylamino-propylamine carbamate, trimethylammonium-propyl carbonate, dibutylamino- propylamine carbamate (DBA), or arginine. In some embodiments, the cationic moiety is a cationic moiety attached to a hydrophobic polymer, e.g. , PLGA. In some embodiments, the cationic moiety is PLGA- spermine. In some embodiments, the cationic moiety is PLGA-glu-di- spermine, e.g. , bis-(Nl-spermine) glutamide-5050 PLGA- O- acetyl.

In some embodiments, the cationic moiety includes at least one amine (e.g. , a primary, secondary, tertiary or quaternary amine), or a plurality of amines, each independently a primary, secondary, tertiary or quaternary amine). In some embodiments the cationic moiety is a polymer, for example, having one or more secondary or tertiary amines, for example cationic polyvinyl alcohol (PVA) (e.g. , as provided by Kuraray, such as CM-318 or C-506), chitosan, polyamine-branched and star PEG and polyethylene imine. Cationic PVA can be made, for example, by polymerizing a vinyl acetate/N-vinaylformamide co-polymer, e.g. , as described in US 2002/0189774, the contents of which are incorporated herein by reference. Other examples of cationic PVA include those described in US 6,368,456 and Fatehi (Carbohydrate Polymers 79 (2010) 423-428), the contents of which are incorporated herein by reference.

In some embodiments, the cationic moiety includes a nitrogen containing heterocyclic or heteroaromatic moiety (e.g, pyridinium, immidazolium, morpholinium, piperizinium, etc.). In some embodiments, the cationic polymer comprises a nitrogen containing heterocyclic or heteroaromatic moiety such as polyvinyl pyrolidine or polyvinylpyrolidinone.

In some embodiments, the cationic moiety includes a guanadinium moiety (e.g. , an arginine moiety).

In some embodiments, the cationic moiety is cationic PVA, such as a cationic PVA described herein.

Additional exemplary cationic moieties include agamatine, protamine sulfate, hexademethrine bromide, cetyl trimethylammonium bromide, 1-hexyltriethyl- ammonium phosphate, 1-dodecyltriethyl-ammonium phosphate, spermine (e.g. , spermine

tetrahydrochloride), spermidine, and derivatives thereof (e.g. Nl-PLGA- spermine, Nl-PLGA- N5 ,N 10,N 14-trimethylated- spermine, (N 1 -PLGA-N5 ,N 10,N 14, N 14-tetramethylated- spermine), PLGA-glu-di-triCbz- spermine, triCbz-spermine, amiphipole, PMAL-C8, and acetyl-PLGA5050- glu-di(Nl -amino-N5,Nl 0,N 14- spermine), poly(2-dimethylamino)ethyl methacrylate), hexyldecyltrimethylammonium chloride, hexadimethrine bromide, and atelocollagen and those described for example in WO 2005/007854, U.S. Pat. No. 7,641,915, and WO 2009/055445, which are incorporated herein by reference in their entirety.

In some embodiments, a cationic moiety is one, the presence of which, in a particle described herein, is accompanied by the presence of less than 50, 40, 30, 20, orlO % (by weight or number) of the nucleic acid agent, e.g. , mRNA, on the outside of the particle.

In some embodiments, the cationic moiety is not a lipid (e.g. , not a phospholipid) or does not comprise a lipid.

In some embodiments, the cationic moiety is a cationic peptide, e.g. , protamine sulfate. In some embodiments, the cationic moiety is PLGA-glu-di- spermine, e.g. , bis- (Nl- spermine) glutamide-5050 PLGA- O- acetyl. In some embodiments, the cationic moiety is 1-hexyltriethyl- ammonium phosphate (Q6).

In some embodiments, the cationic moiety comprises O-acetyl-PLGA5050, e.g. , O- acetyl-PLGA5050 (MW: 7,000 Da). In some embodiments, the cationic moiety comprises O- acetyl-PLGA5050, e.g. , O-acetyl-PLGA5050 (MW: 7,000 Da), and spermine. In some embodiments, the cationic moiety comprises O-acetyl-PLGA5050, e.g. , O-acetyl-PLGA5050 (MW: 7,000 Da), and PVA-dibutylamino-l(propylamine)-carbamate (PVA-DBA). In some embodiments, the cationic moiety comprises O-acetyl-PLGA5050, e.g. , O-acetyl-PLGA5050 (MW: 7,000 Da), and a partially hydrolyzed polyoxazoline (pOx), e.g. , pOx45, i.e. , pOx hydrolyzed for 45 min. (about 12.5% hydrolyzed), pOx60, i.e. , pOx hydrolyzed for 60 min. (about 17.5% hydrolyzed), pOxl20, i.e. , pOx hydrolyzed for 120 min. (about 21% hydrolyzed), or pOx200, i.e. , pOx hydrolyzed for 200 min. (about 43% hydrolyzed).

Hydrophobic-Hydrophilic Polymers

A particle described herein may include a polymer containing a hydrophilic portion and a hydrophobic portion, e.g. , a hydrophobic-hydrophilic polymer. The hydrophobic-hydrophilic polymer may be attached to another moiety such as a cationic moiety. In some embodiments, the hydrophobic-hydrophilic polymer is free (i.e. , not attached to another moiety). A particle can include a plurality of hydrophobic-hydrophilic polymers, for example where some are attached to another moiety such as a cationic moiety, and some are free.

A polymer containing a hydrophilic portion and a hydrophobic portion may be a copolymer of a hydrophilic block coupled with a hydrophobic block. These copolymers may have a weight average molecular weight between about 5 kDa and about 30 kDa (e.g. , from about 5 kDa to about 25 kDa, from about 10 kDa to about 22 kDa, from about 10 kDa to about 15 kDa, from about 12 kDa to about 22 kDa, from about 7 kDa to about 15 kDa, from about 15 kDa to about 19 kDa, or from about 11 kDa to about 13 kDa, e.g. , about 9 kDa, about 10 kDa, about 11 kDa, about 12 kDa, about 13 kDa, about 14 kDa about 15 kDa, about 16 kDa, about 17 kDa, about 18 kDa or about 19 kDa). The polymer containing a hydrophilic portion and a hydrophobic portion may be attached to an agent.

Examples of suitable hydrophobic portions of the polymers include those described above. The hydrophobic portion of the copolymer may have a weight average molecular weight of from about 1 kDa to about 20 kDa (e.g. , from about 8 kDa to about 15, kDa from about 1 kDa to about 18 kDa, 17 kDa, 16 kDa, 15 kDa, 14 kDa or 13 kDa, from about 2 kDa to about 12 kDa, from about 6 kDa to about 20 kDa, from about 5 kDa to about 18 kDa, from about 7 kDa to about 17 kDa, from about 8 kDa to about 13 kDa, from about 9 kDa to about 11 kDa, from about 10 kDa to about 14 kDa, from about 6 kDa to about 8 kDa, about 6 kDa, about 7 kDa, about 8 kDa, about 9 kDa, about 10 kDa, about 11 kDa, about 12 kDa, about 13 kDa, about 14 kDa, about 15 kDa, about 16 kDa or about 17 kDa).

Examples of suitable hydrophilic portions of the polymers include the following:

carboxylic acids including acrylic acid, methacrylic acid, itaconic acid, and maleic acid;

polyoxyethylenes or polyethylene oxide (PEG); polyacrylamides (e.g. polyhydroxylpropyl- methacrylamide), and copolymers thereof with dimethylaminoethylmethacrylate, diallyl- dimethylammonium chloride, vinylbenzylthrimethylammonium chloride, acrylic acid, methacrylic acid, 2-acrylamido-2-methylpropane sulfonic acid and styrene sulfonate,

poly(vinylpyrrolidone), polyoxazoline, polysialic acid, starches and starch derivatives, dextran and dextran derivatives; polypeptides, such as polylysines, polyarginines, polyglutamic acids; polyhyaluronic acids, alginic acids, polylactides, polyethyleneimines, polyionenes, polyacrylic acids, and polyiminocarboxylates, gelatin, and unsaturated ethylenic mono or dicarboxylic acids. A listing of suitable hydrophilic polymers can be found in Handbook of Water-Soluble Gums and Resins, R. Davidson, McGraw-Hill (1980). The hydrophilic portion of the copolymer may have a weight average molecular weight of from about 1 kDa to about 21 kDa (e.g. , from about 1 kDa to about 8 kDa, from about 1 kDa to about 3 kDa, e.g. , about 2 kDa, or from about 2 kDa to about 6 kDa, e.g. , about 3.5 kDa, or from about 4 kDa to about 6 kDa, e.g. , about 5 kDa). In one embodiment, the hydrophilic portion is PEG, and the weight average molecular weight is from about 1 kDa to about 21 kDa (e.g. , from about 1 kDa to about 8 kDa, from about 1 kDa to about

3 kDa, e.g. , about 2 kDa, or from about 2 kDa to about 6 kDa, e.g. , about 3.5 kDa, or from about

4 kDa to about 6 kDa, e.g. , about 5 kDa). In one embodiment, the hydrophilic portion is PVA, and the weight average molecular weight is from about 1 kDa to about 21 kDa (e.g. , from about 1 kDa to about 8 kDa, from about 1 kDa to about 3 kDa, e.g. , about 2 kDa, or from about 2 kDa to about 6 kDa, e.g. , about 3.5 kDa, or from about 4 kDa to about 6 kDa, e.g. , about 5 kDa).

In one embodiment, the hydrophilic portion is polyoxazoline, and the weight average molecular weight is from about 1 kDa to about 21 kDa (e.g. , from about 1 kDa to about 8 kDa, from about 1 kDa to about 3 kDa, e.g. , about 2 kDa, or from about 2 kDa to about 6 kDa, e.g. , about 3.5 kDa, or from about 4 kDa to about 6 kDa, e.g. , about 5 kDa).

In one embodiment, the hydrophilic portion is polyvinylpyrrolidine, and the weight average molecular weight is from about 1 kDa to about 21 kDa (e.g. , from about 1 kDa to about 8 kDa, from about 1 kDa to about 3 kDa, e.g. , about 2 kDa, or from about 2 kDa to about 6 kDa, e.g. , about 3.5 kDa, or from about 4 kDa to about 6 kDa, e.g. , about 5 kDa).

In one embodiment, the hydrophilic portion is polyhydroxylpropylmethacrylamide, and the weight average molecular weight is from about 1 kDa to about 21 kDa (e.g. , from about 1 kDa to about 8 kDa, from about 1 kDa to about 3 kDa, e.g. , about 2 kDa, or from about 2 kDa to about 6 kDa, e.g. , about 3.5 kDa, or from about 4 kDa to about 6 kDa, e.g. , about 5 kDa).

In one embodiment, the hydrophilic portion is polysialic acid, and the weight average molecular weight is from about 1 kDa to about 21 kDa (e.g. , from about 1 kDa to about 8 kDa, from about 1 kDa to about 3 kDa, e.g. , about 2 kDa, or from about 2 kDa to about 6 kDa, e.g. , about 3.5 kDa, or from about 4 kDa to about 6 kDa, e.g. , about 5 kDa).

A polymer containing a hydrophilic portion and a hydrophobic portion may be a block copolymer, e.g. , a diblock or triblock copolymer. In some embodiments, the polymer may be a diblock copolymer containing a hydrophilic block and a hydrophobic block. In some

embodiments, the polymer may be a triblock copolymer containing a hydrophobic block, a hydrophilic block and another hydrophobic block. The two hydrophobic blocks may be the same hydrophobic polymer or different hydrophobic polymers. The block copolymers used herein may have varying ratios of the hydrophilic portion to the hydrophobic portion, e.g. , ranging from 1 : 1 to 1 :40 by weight (e.g. , about 1 : 1 to about 1 : 10 by weight, about 1 : 1 to about 1 :2 by weight, or about 1 :3 to about 1 :6 by weight).

A polymer containing a hydrophilic portion and a hydrophobic portion may have a variety of end groups. In some embodiments, the end group may be a hydroxy group or an alkoxy group (e.g., methoxy). In some embodiments, the end group of the polymer is not further modified. In some embodiments, the end group may be further modified. For example, the end group may be capped with an alkyl group, to yield an alkoxy-capped polymer (e.g., a methoxy- capped polymer), may be derivatized with a targeting agent (e.g., folate) or a dye (e.g., rhodamine), or may be reacted with a functional group.

A polymer containing a hydrophilic portion and a hydrophobic portion may include a linker between the two blocks of the copolymer. Such a linker may be an amide, ester, ether, amino, carbamate or carbonate linkage, for example.

A polymer containing a hydrophilic portion and a hydrophobic portion described herein may have a polymer polydispersity index (PDI) of less than or equal to about 2.5 (e.g., less than or equal to about 2.2, or less than or equal to about 2.0, or less than or equal to about 1.5). In some embodiments, the polymer PDI is from about 1.0 to about 2.5, e.g., from about 1.0 to about 2.0, from about 1.0 to about 1.8, from about 1.0 to about 1.7, or from about 1.0 to about 1.6.

A particle described herein may include varying amounts of a polymer containing a hydrophilic portion and a hydrophobic portion, e.g., up to about 50% by weight of the particle (e.g., from about 4 to about 50%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45% or about 50% by weight). For example, the percent by weight of the second polymer within the particle is from about 3% to 30%, from about 5% to 25% or from about 8% to 23%.

A polymer containing a hydrophilic portion and a hydrophobic portion described herein may be commercially available, or may be synthesized. Methods of synthesizing polymers are known in the art (see, for example, Polymer Synthesis: Theory and Practice Fundamentals, Methods, Experiments. D. Braun et al., 4th edition, Springer, Berlin, 2005). Such methods include, for example, polycondensation, radical polymerization, ionic polymerization (e.g., cationic or anionic polymerization), or ring-opening metathesis polymerization. A block copolymer may be prepared by synthesizing the two polymer units separately and then conjugating the two portions using established methods. For example, the blocks may be linked using a coupling agent such as EDC (l-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride). Following conjugation, the two blocks may be linked via an amide, ester, ether, amino, carbamate or carbonate linkage. A commercially available or synthesized polymer sample may be further purified prior to incorporation into a particle or composition described herein. In some embodiments, purification may remove lower molecular weight polymers that may lead to unfilterable polymer samples. A polymer may be purified by precipitation from solution, or precipitation onto a solid such as Celite. A polymer may also be further purified by size exclusion chromatography (SEC).

Additional components

In some embodiments, the particle further comprises a surfactant or a mixture of surfactants. In some embodiments, the surfactant is PEG, poly(vinyl alcohol) (PVA), poly(vinylpyrrolidone) (PVP), poloxamer, hexyldecyltrimethylammonium chloride, a polysorbate, a polyoxyethylene ester, a PEG-lipid (e.g. , PEG-ceramide, d-alpha-tocopheryl polyethylene glycol 1000 succinate), l,2-Distearoyl-sn-Glycero-3-[Phospho-rac-(l-glycerol)], lecithin, or a mixture thereof. In some embodiments, the surfactant is PVA and the PVA is from about 3 kDa to about 50 kDa (e.g. , from about 5 kDa to about 45 kDa, about 7 kDa to about 42 kDa, from about 9 kDa to about 30 kDa, or from about 11 to about 28 kDa) and up to about 98% hydrolyzed (e.g. , about 75-95%, about 80-90% hydrolyzed, or about 85% hydrolyzed) In some embodiments, the PVA has a viscosity of from about 2 to about 27 cP. In some embodiments, the PVA is a cationic PVA, for example, as described above, for example, a cationic moiety such as a cationic PVA can also serve as a surfactant. In some embodiments, the surfactant is polysorbate 80. In some embodiments, the surfactant is Solutol® HS 15. In some embodiments, the surfactant is not a lipid (e.g. , a phospholipid) or does not comprise a lipid. In some embodiments, the surfactant is present in an amount of up to about 35% by weight of the particle (e.g. , up to about 20% by weight or up to about 25% by weight, from about 15 % to about 35% by weight, from about 20% to about 30% by weight, or from about 23% to about 26% by weight).

In some embodiments, the particle is associated with an excipient, e.g. , a carbohydrate component, or a stabilizer or lyoprotectant, e.g. , a carbohydrate component, stabilizer or lyoprotectant described herein. While not wishing to be bound be theory the carbohydrate component may act as a stabilizer or lyoprotectant. In some embodiments, the carbohydrate component, stabilizer or lyoprotectant, comprises one or more sugars, sugar alcohols, carbohydrates (e.g. , sucrose, mannitol, cyclodextrin or a derivative of cyclodextrin (e.g. 2- hydroxypropyl- -cyclodextrin, sometimes referred to herein as ΗΡ-β-CD, or sulfobutyl ether of β-CD, sometimes referred to herein as CYTOSOL), salt, PEG, PVP or crown ether. In some embodiments, the carbohydrate component, stabilizer or lyoprotectant comprises two or more carbohydrates, e.g. , two or more carbohydrates described herein. In one embodiment, the carbohydrate component, stabilizer or lyoprotectant includes a cyclic carbohydrate (e.g. , cyclodextrin or a derivative of cyclodextrin, e.g. , an α-, β-, or γ-, cyclodextrin (e.g. 2- hydroxypropyl- -cyclodextrin)) and a non-cyclic carbohydrate. Exemplary non-cyclic oligosaccharides include those of less than 10, 8, 6 or 4 monosaccharide subunits (e.g. , a monosaccharide or a disaccharide (e.g. , sucrose, trehalose, lactose, maltose) or combinations thereof). In some embodiments, the lyoprotectant is a monosaccharide such as a sugar alcohol (e.g. , mannitol).

In some embodiments the carbohydrate component, stabilizer or lyoprotectant comprises a first and a second component, e.g. , a cyclic carbohydrate and a non-cyclic carbohydrate, e.g. , a mono-, di-, or tetra-saccharide.

In one embodiment, the weight ratio of cyclic carbohydrate to non-cyclic carbohydrate associated with the particle is a weight ratio described herein, e.g. , 0.5: 1.5 to 1.5:0.5.

In some embodiments the carbohydrate component, stabilizer or lyoprotectant comprises a first and a second component (designated here as A and B) as follows:

(A) comprises a cyclic carbohydrate and (B) comprises a disaccharide;

(A) comprises a cyclic carbohydrate, e.g. , a β-CD or a β-CD derivative, e.g. , ΗΡ-β-CD, and (B) comprises more than one disaccharide;

(A) comprises a β-cyclodextrin, e.g a β-CD derivative, e.g. , ΗΡ-β-CD, and (B) comprises a disaccharide; (A) comprises a β-cyclodextrin, e.g. , a β-CD derivative, e.g. , ΗΡ-β-CD, and (B) comprises sucrose;

(A) comprises a β-CD derivative, e.g. , ΗΡ-β-CD, and (B) comprises sucrose;

(A) comprises ΗΡ-β-CD, and (B) comprises sucrose and trehalose.

In some embodiments components A and B are present in the following ratio:

0.5: 1.5 to 1.5:0.5. In some embodiments, components A and B are present in the following ratio: 3-1 : 0.4-2; 3-1 : 0.4-2.5; 3-1 : 0.4-2; 3-1 : 0.5-1.5; 3-1 : 0.5-1 ; 3-1 : 1 ; 3-1 : 0.6-0.9; and 3: 1 : 0.7. In some embodiments, components A and B are present in the following ratio: 2-1 : 0.4-2; 3-1 : 0.4-2.5; 2-1 : 0.4-2; 2-1 : 0.5-1.5; 2-1 : 0.5-1 ; 2-1 : 1 ; 2-1 : 0.6-0.9; and 2: 1 : 0.7. In some embodiments components A and B are present in the following ratio: 2-1.5 : 0.4-2; 2-1.5 : 0.4-2.5; 2-1.5 : 0.4-2; 2-1.5 : 0.5-1.5; 2-1.5 : 0.5-1 ; 2-1.5 : 1 ; 2-1.5 : 0.6-0.9; 2: 1.5 : 0.7. In some embodiments components A and B are present in the following ratio: 2.5-1.5: 0.5-1.5; 2.2- 1.6: 0.7-1.3; 2.0 -1.7: 0.8-1.2; 1.8: 1 ; 1.85: 1 and 1.9: 1.

In some embodiments component A comprises a cyclodextin, e.g. , a β-cyclodextrin, e.g. , a β-CD derivative, e.g., ΗΡ-β-CD, and (B) comprises sucrose, and they are present in the following ratio: 2.5-1.5: 0.5-1.5; 2.2-1.6: 0.7-1.3; 2.0 -1.7: 0.8-1.2; 1.8 : 1 ; 1.85 : 1 and 1.9 : 1.

In some embodiments, the surface of the particle can be substantially coated with a surfactant or polymer, for example, PVA, polyoxazoline, polyvinylpyrrolidine,

polyhydroxylpropylmethacrylamide, polysialic acid, or PEG.

Cationic Moiety-Polymer Conjugates

Exemplary conjugates include cationic moiety-polymer conjugates (e.g. , a cationic moiety-hydrophobic polymer conjugate). A cationic moiety-polymer conjugate described herein includes a polymer (e.g. , a hydrophobic polymer or a polymer containing a hydrophilic portion and a hydrophobic portion) and a cationic moiety. A cationic moiety described herein may be attached to a polymer described herein, e.g. , directly (e.g. , without the presence of atoms from an intervening spacer moiety), or through a linker. A cationic moiety may be attached to a hydrophobic polymer (e.g. , PLGA). A cationic moiety may be attached to a terminal end of a polymer, to both terminal ends of a polymer, or to a point along a polymer chain. In some embodiments, multiple cationic moieties may be attached to points along a polymer chain, or multiple cationic moieties may be attached to a terminal end of a polymer via a multifunctional linker.

Modes of Attachment

A cationic moiety described herein may be directly (e.g. , without the presence of atoms from an intervening spacer moiety), attached to a polymer or hydrophobic moiety described herein (e.g. , a polymer). The attachment may be at a terminus of the polymer or along the backbone of the polymer. A cationic moiety may be attached to a polymer via a variety of linkages, e.g. , an amide, ester, sulfide (e.g. , a maleimide sulfide), disulfide, succinimide, oxime, silyl ether, carbonate or carbamate linkage. In some embodiments, a cationic moiety may be directly attached (e.g. , without the presence of atoms from an intervening spacer moiety), to a terminal end of a polymer. For example, a polymer having a carboxylic acid moiety at its terminus may be covalently attached to a hydroxy, thiol, or amino moiety of a cationic moiety, forming an ester, thioester, or amide bond. In certain embodiments, suitable protecting groups may be required on the other polymer terminus or on other reactive substituents on the cationic moiety, to facilitate formation of the specific desired cationic moiety-polymer conjugate. For example, a polymer having a hydroxy terminus may be protected, e.g. , with a silyl group (e.g. , trimethylsilyl) or an acyl group (e.g. , acetyl). A cationic moiety may be protected, e.g. , with an acetyl group or other protecting group.

In some embodiments, the process of attaching a cationic moiety to a polymer may result in a composition comprising a mixture of conjugates having the same polymer and the same cationic moiety, but which differ in the nature of the linkage between the cationic moiety and the polymer. For example, when a cationic moiety has a plurality of reactive moieties that may react with a polymer, the product of a reaction of the cationic moiety and the polymer may include a conjugate wherein the cationic moiety is attached to the polymer via one reactive moiety, and a conjugate wherein the cationic moiety is attached to the polymer via another reactive moiety. For example, where a cationic moiety has multiple reactive groups such as a plurality of amines, the product of the reaction may include a conjugate where some of cationic moiety is attached to the polymer through a first reactive group and some of the cationic moiety is attached to the polymer through a second reactive group. In some embodiments, the process of attaching a cationic moiety to a polymer may involve the use of protecting groups. For example, when a cationic moiety has a plurality of reactive moieties that may react with a polymer, the cationic moiety may be protected at certain reactive positions such that a polymer will be attached via a specified position.

In some embodiments, the cationic moieties are attached to a polymer (e.g. , 2, 3, 4, 5, 6 or more agents may be attached to a polymer). The cationic moieties may be the same or different. In some embodiments, a plurality of cationic moieties may be attached to a multifunctional linker (e.g. , a polyglutamic acid linker). In some embodiments, a plurality of cationic moieties may be attached to points along the polymer chain.

Linkers

A cationic moiety may be attached to a moiety such as a polymer or a hydrophobic moiety such as a lipid, or to each other, via a linker, such as a linker described herein. For example: a hydrophobic polymer may be attached to a cationic moiety; a hydrophilic polymer may be attached to a cationic moiety; or a hydrophobic moiety may be attached to a cationic moiety

In some embodiments, a plurality of the linker moieties is attached to a polymer, allowing attachment of a plurality of cationic moieties to the polymer through linkers, for example, where the linkers are attached at multiple places on the polymer such as along the polymer backbone. In some embodiments, a linker is configured to allow for a plurality of a first moiety to be linked to a second moiety through the linker. In some embodiments, the cationic moiety is released from the linker under biological conditions (i.e. , cleavable under physiological conditions). In another embodiment a single linker is attached to a polymer, e.g. , at a terminus of the polymer. The linker may comprise, for example, an alkylene (divalent alkyl) group. In some

embodiments, one or more carbon atoms of the alkylene linker may be replaced with one or more heteroatoms or functional groups (e.g. , thioether, amino, ether, keto, amide, silyl ether, oxime, carbamate, carbonate, disulfide, or heterocyclic or heteroaromatic moieties).

In some embodiments, a linker, in addition to the functional groups that allow for attachment of a first moiety to a second moiety, has an additional functional group. In some embodiments, the additional functional group can be cleaved under physiological conditions. Such a linker can be formed, for example, by reacting a first activated moiety such as a cationic moiety, e.g. , a cationic moiety described herein, with a second activated moiety such as a polymer, e.g. , a polymer described herein, to produce a linker that includes a functional group that is formed by joining the cationic moiety to the polymer.

Methods of Making Cationic Moiety - Polymer Conjugates

The conjugates may be prepared using a variety of methods, including those described herein. In some embodiments, to covalently link the cationic moiety to a polymer, the polymer or cationic moiety may be chemically activated using a technique known in the art. The activated polymer is then mixed with the cationic moiety, or the activated cationic moiety is mixed with the polymer, under suitable conditions to allow a covalent bond to form between the polymer and the cationic moiety. In some embodiments, a nucleophile, such as a thiol, hydroxyl group, or amino group, on the cationic moiety attacks an electrophile (e.g. , activated ester group) to create a covalent bond. A cationic moiety may be attached to a polymer via a variety of linkages, e.g. , an amide, ester, succinimide, carbonate or carbamate linkage.

In some embodiments, a cationic moiety may be attached to a polymer via a linker. In such embodiments, a linker may be first covalently attached to a polymer, and then attached to a cationic moiety. In other embodiments, a linker may be first attached to a cationic moiety, and then attached to a polymer.

Compositions Comprising Particles

In another aspect, the disclosure features compositions comprising particles comprising a nucleic acid agent, e.g. , mRNA, as described herein. Compositions comprising particles described herein may include mixtures of products. For example, the conjugation of a cationic moiety to a polymer may proceed in less than 100% yield, and the composition comprising the conjugate may thus also include unconjugated cationic moiety. In some embodiments, the compositions may also include conjugates that have the same polymer and the same cationic moiety, and differ in the nature of the linkage between the nucleic acid agent and/or cationic moiety and the polymer. For example, in some embodiments, when the conjugate is a cationic moiety-polymer conjugate and the cationic moiety includes multiple reactive groups, the composition may include polymers attached to the cationic moiety via different reactive groups present on the cationic moiety (e.g. , different reactive amines). The conjugates may be present in the composition in varying amounts. For example, when a cationic moiety having a plurality of available attachment points is reacted with a polymer, the resulting composition may include more of a product conjugated via a more reactive group (e.g. , a first hydroxyl or amino group), and less of a product attached via a less reactive group (e.g. , a second hydroxyl or amino group).

Additionally, compositions of conjugates may include cationic moieties that are attached to more than one polymer chain. For example, in the case of a cationic moiety-polymer conjugate wherein cationic moiety includes multiple reactive groups, the cationic moiety may be attached to a first polymer chain through a first reactive group (e.g. , a first amine) and a second polymer chain through a second reactive group (e.g. , a second amine).

Methods of Making Particles and Compositions

A particle described herein may be prepared using any method known in the art for preparing particles, e.g. , nanoparticles. Exemplary methods include spray drying, emulsion (e.g. , emulsion- solvent evaporation or double emulsion), precipitation (e.g. , nanoprecipitation) and phase inversion.

In some embodiments, a particle described herein can be prepared by precipitation (e.g. , nanoprecipitation). This method involves dissolving the components of the particle (i.e. , one or more polymers, an optional additional component or components, a cationic moiety and a nucleic acid agent), individually or combined, in one or more solvents to form one or more solutions. For example, a first solution containing one or more of the components may be poured into a second solution containing one or more of the components (at a suitable rate or speed). The solutions may be combined, for example, using a syringe pump, a MicroMixer, or any device that allows for vigorous, controlled mixing. In some embodiments, nanoparticles can be formed as the first solution contacts the second solution, e.g. , precipitation of the polymer upon contact causes the polymer to form nanoparticles. The control of such particle formation can be readily optimized.

In some embodiments, the particles are formed by providing one or more solutions containing the nucleic acid agent, one or more polymers, and additional components, and contacting the solutions with certain solvents to produce the particle. In a non-limiting example, mRNA can be contacted with a solvent, e.g. , a polar aprotic solvent, such as, for example, LiBr in DMSO, to provide a first mixture, e.g. , an organic solution. The first mixture can be contacted with a second mixture comprising a cationic moiety, e.g. , such as a cationic moiety described herein, e.g. , cationic PVA (cPVA), and a hydrophobic polymer, e.g. , such as a hydrophobic polymer described herein, e.g. , PLGA, in a solution comprising a solvent, e.g. , a polar aprotic solvent, and a co-solvent, e.g. , a high polar index solvent, to provide a third mixture. The third mixture can be contacted with a surfactant, e.g. , PVA, in an aqueous solution, to provide a fourth mixture, to thereby make the particles, wherein the first, second, and third mixtures each contain less than 1,000 ppm water, e.g. , less than 500 ppm, less than 200 ppm, less than 100 ppm, less than 50 ppm. In some embodiments, the first, second, and third mixtures are anhydrous. The first, second, third, and fourth mixtures may be individually sterile filtered prior to

mixing/precipitation .

In another non-limiting example, mRNA and a cationic moiety, e.g. , such as a cationic moiety described herein, e.g. , cationic PVA (cPVA), can be contacted with a solvent, e.g. , a polar aprotic solvent, such as, for example, LiBr in DMSO, to provide a first mixture, e.g. , an organic solution. The first mixture can be contacted with a second mixture comprising a hydrophobic polymer, e.g. , such as a hydrophobic polymer described herein, e.g. , PLGA, in a solution comprising a solvent, e.g. , a polar aprotic solvent, and a co-solvent, e.g. , a high polar index solvent, to provide a third mixture. The third mixture can be contacted with a surfactant, e.g. , PVA, in an aqueous solution, to provide a fourth mixture, to thereby make the particles, wherein the first, second, and third mixtures each contain less than 1,000 ppm water, e.g. , less than 500 ppm, less than 200 ppm, less than 100 ppm, less than 50 ppm. In some embodiments, the first, second, and third mixtures are anhydrous. The first, second, third, and fourth mixtures may be individually sterile filtered prior to mixing/precipitation.

In some embodiments, the co-solvent is a solvent that has a lower polar index solvent than dimethylsulf oxide (DMSO), which has a polar index of 7.2. The polar index of a solvent refers to a relative measure of the degree of interaction of the solvent with various polar test solutes. In some embodiments, the co-solvent is a solvent that has a high polar index but has a lower polar index than DMSO. In some embodiments, the co-solvent is selected from one or more of the following:

The formed nanoparticles can be exposed to further processing techniques to remove the solvents or purify the nanoparticles (e.g. , dialysis). For purposes of the aforementioned process, water miscible solvents include acetone, ethanol, methanol, and isopropyl alcohol; and partially water miscible organic solvents include acetonitrile, tetrahydrofuran, ethyl acetate, isopropyl alcohol, isopropyl acetate or dimethylformamide.

Lyophilization

A particle described herein may be prepared for dry storage via lyophilization, commonly known as freeze-drying. Lyophilization is a process which extracts water from a solution to form a granular solid or powder. The process is carried out by freezing the solution and subsequently extracting any water or moisture by sublimation under vacuum. Advantages of lyophilization include maintenance of substance quality and minimization of therapeutic compound degradation. Lyophilization may be particularly useful for developing

pharmaceutical drug products that are reconstituted and administered to a patient by injection, for example parenteral drug products. Alternatively, lyophilization is useful for developing oral drug products, especially fast melts or flash dissolve formulations.

Lyophilization may take place in the presence of a lyoprotectant, e.g. , a lyoprotectant described herein. In some embodiments, the lyoprotectant is a carbohydrate (e.g. , a carbohydrate described herein, such as, e.g. , sucrose, cyclodextrin or a derivative of cyclodextrin (e.g. 2- hydroxypropyl- -cyclodextrin)), salt, PEG, PVP or crown ether.

In some embodiments, aggregation of PEGylated particles during lyophilization may be reduced or minimized by the use of lyoprotectants comprising a cyclic oligosaccharide. Using suitable lyoprotectants provides lyophilized preparations that have extended shelf-lives.

The present disclosure features liquid formulations and lyophilized preparations that comprise a cyclic oligosaccharide. In some embodiments, the liquid formulation or lyophilized preparation can comprise at least two carbohydrates, e.g. , a cyclic oligosaccharide (e.g. , a cyclodextran or derivative thereof) and a non-cyclic oligosaccharide (e.g. , a non-cyclic oligosaccharide less than about 10, 8, 6, 4 monosaccharides in length, e.g. , a monosaccharide or disaccharide). In some embodiments, the liquid formulations also comprise a reconstitution reagent.

Examples of suitable cyclic oligosaccharides, include, but are not limited to, a- cyclodextrins, β-cyclodextrins, such as 2-hydroxypropyl-P-cyclodextrins, β-cyclodextrin sulfobutylethers sodiums, γ-cyclodextrins, any derivative thereof, and any combination thereof.

In certain embodiments, the cyclic carbohydrate, e.g. , cyclic oligosaccharide, may be included in a larger molecular structure such as a polymer. Suitable polymers are disclosed herein with respect to the polymer composition of the particle. In such embodiments, the cyclic oligosaccharide may be incorporated within a backbone of the polymer. See, e.g. , U.S. Pat. No. 7,270,808 and U.S. Pat. No. 7,091,192, which disclose exemplary polymers that contain cyclodextrin moieties in the polymer backbone that can be used in accordance with the disclosure. The entire teachings of U.S. Pat. No. 7,270,808 and U.S. Pat. No. 7,091,192 are incorporated herein by reference. In some embodiments, the cyclic oligosaccharide may contain at least one oxidized occurrence.

A lyoprotectant comprising a cyclic oligosaccharide, may inhibit the rate of

intermolecular aggregation of particles that include hydrophilic polymers such as PEG during their lyophilization and/or storage, and therefore, provide for extended shelf-life. Without wishing to be limited by theory, the mechanism for the cyclic oligosaccharide to prevent particle aggregation may be due to the cyclic oligosaccharide reducing or preventing the crystallization of the hydrophilic polymer such as PEG present in the particles during lyophilization. This may occur through the formation of an inclusion complex between a cyclic oligosaccharide and the hydrophilic polymer (e.g. , PEG). Such a complex may be formed between a cyclodextrin and, for example, the chain of polyethylene glycol. The inside cavity of cyclodextrin is lipophilic, while the outside of the cyclodextrin is hydrophilic. These properties may allow for the formation of inclusion complexes with other components of the particles described herein. For the purpose of stabilizing the formulations during lyophilization, the poly(ethyleneglycol) chain may fit into the cavity of the cyclodextrins. An additional mechanism that may allow the cyclic oligosaccharide to reduced or minimized or prevent particle degradation relates to the formation of hydrogen bonds between the cyclic oligosaccharide and the hydrophilic polymer (PEG) during lyophilization. For example, hydrogen bonding between cyclodextrin and poly(ethyleneglycol) chains may prevent ordered polyethylene glycol structures such as crystals.

The cyclic oligosaccharide may be present in varying amounts in the formulations described herein. In certain embodiments, the cyclic oligosaccharide to liquid formulation ratio is in the range of from about 0.75: 1 to about 3: 1 by weight. In preferred embodiments, the cyclic oligosaccharide to total polymer ratio is in the range of from about 0.75: 1 to about 3: 1 by weight.

In preferred aspects, the formulation contains two or more carbohydrates, e.g. , a cyclic oligosaccharide and a non-cyclic carbohydrate, e.g. , a non-cyclic oligosaccharide, e.g. , a non- cyclic oligosaccharide having 10, 8, 6, 4 or less monosaccharide units. As described herein, including a non-cyclic carbohydrate, e.g. , a non-cyclic oligosaccharide, into a liquid formulation that is to be lyophilized can promote uptake of water by the resulting lyophilized preparation, and promote disintegration of the lyophilized preparation.

In preferred aspects, the lyophilized or liquid formulation comprises a cyclic

oligosaccharide, such as an a-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, any derivative thereof, and any combination thereof, and a non-cyclic oligosaccharide, e.g. , a non-cyclic oligosaccharide described herein. In some preferred embodiments, the lyoprotectant comprises a cyclic oligosaccharide, such as an a-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, any derivative thereof, and any combination thereof, and the non-cyclic oligosaccharide is a disaccharide, such as sucrose, lactose, maltose, trehalose, and derivatives thereof, and a monosaccharide, such as glucose. In one preferred embodiment, the lyoprotectant comprises a β-cyclodextrin or derivative thereof, such as 2-hydroxypropyl-P-cyclodextrin or β-cyclodextrin sulfobutylether; and the non-cyclic oligosaccharide is a disaccharide, such as sucrose. The β-cyclodextrin or derivative thereof and the non-cyclic oligosaccharide can be present in any suitable relative amounts. Preferably, the ratio of cyclic oligosaccharide to non-cyclic oligosaccharide (w/w) is from about 0.5: 1.5 to about 1.5:0.5, and more preferably from 0.7: 1.3 to 1.3:0.7. In some examples, the ratio of cyclic oligosaccharide to non-cyclic oligosaccharide (w/w) is 0.7: 1.3, 1 :0.7, 1 : 1, 1.3: 1 or 1.3:0.7. When the liquid or lyophilized formulation comprises a particle described herein, the ratio of cyclic oligosaccharide plus non-cyclic oligosaccharide to polymer (w/w) is from about 1 : 1 to about 10: 1, and preferably, from about 1.1 to about 3: 1.

In certain embodiments, the lyophilized preparations may be reconstituted with a reconstitution reagent. In some embodiments, a suitable reconstitution reagent may be any physiologically acceptable liquid. Suitable reconstitution reagents include, but are not limited to, water, 5% Dextrose Injection, Lactated Ringer's and Dextrose Injection, or a mixture of equal parts by volume of Dehydrated Alcohol, USP and a nonionic surfactant, such as a

polyoxyethylated castor oil surfactant available from GAF Corporation, Mount Olive, N.J., under the trademark, Cremophor EL. To minimize the amount of surfactant in the reconstituted solution, only a sufficient amount of the vehicle may be provided to form a solution of the lyophilized preparation. Once dissolution of the lyophilized preparation is achieved, the resulting solution may be further diluted prior to injection with a suitable parenteral diluent. Such diluents are well known to those of ordinary skill in the art. These diluents are generally available in clinical facilities. Examples of typical diluents include, but are not limited to, Lactated Ringer's Injection, 5% Dextrose Injection, Sterile Water for Injection, and the like. However, because of its narrow pH range, pH 6.0 to 7.5, Lactated Ringer's Injection is most typical. Per 100 mL, lactated ringer's injection contains sodium chloride USP 0.6 g, sodium lactate 0.31 g, potassium chloride USP 0.03 g and calcium chloride₂H₂0 USP 0.02 g. The osmolarity is 275 mOsmol/L, which is very close to isotonicity.

Accordingly, a liquid formulation can be a resuspended or rehydrated lyophilized preparation in a suitable reconstitution reagent. Suitable reconstitution reagents include physiologically acceptable carriers, e.g. , a physiologically acceptable liquid as described herein. Preferably, resuspension or rehydration of the lyophilized preparations forms a solution or suspension of particles which have substantially the same properties (e.g. , average particle diameter (Zave), size distribution (Dvgo, Dv₅o), polydispersity, drug concentration) and morphology of the original particles in the liquid formulation of the disclosure before

lyophilization, and further maintains the therapeutic agent to polymer ratio of the original liquid formulation before lyophilization. In certain embodiments, about 50% to about 100%, preferably about 80% to about 100%, of the particles in the resuspended or rehydrated lyophilized preparation maintain the size distribution and/or drug to polymer ratio of the particles in the original liquid formulation. Preferably, the Zave, DV90, and polydispersity of the particles in the formulation produced by resuspending a lyophilized preparation do not differ from the Zave, Dvgo, and polydispersity of the particles in the original solution or suspension prior to lyophilization by more than about 5%, more than about 10%, more than about 15%, more than about 20%, more than about 15%, more than about 30%, more than about 35%, more than about 40%, more than about 45%, or more than about 50%.

Preferably liquid formulations of this aspect contain particles, and are characterized by a higher polymer concentration (the concentration of polymer(s) that form the particle) than can be lyophilized and resuspended using either a lyoprotectant that comprises one or more

carbohydrates (e.g. , a cyclic oligosaccharide and/or a non-cyclic oligosaccharide). For example, the polymer concentration can be at least about 20 mg/mL, at least about 25 mg/mL, at least about 30 mg/mL, at least about 31 mg/mL, at least about 32 mg/mL, at least about 33 mg/mL, at least about 34 mg/mL, at least about 35 mg/mL, at least about 36 mg/mL, at least about 37 mg/mL, at least about 38 mg/mL, at least about 39 mg/mL, at least about 40 mg/mL, at least about 45 mg/mL, at least about 50 mg/mL, at least about 55 mg/mL, at least about 60 mg/mL, at least about 65 mg/mL, at least about 70 mg/mL, at least about 75 mg/mL, at least about 80 mg/mL, at least about 85 mg/mL, at least about 90 mg/mL, at least about 95 mg/mL, are at least about 100 mg/mL. For example, the liquid formulation can be a reconstituted lyophilized preparation.

Methods of Storing Particles and Compositions

In another aspect, the disclosure features, a method of storing the particles or

composition, e.g. , a pharmaceutical composition, described herein.

In some embodiments, methods of storing the particles or composition described herein include, e.g. , the steps of,: (a) providing said particles or compositions disposed in a container; (b) storing said particles or composition; and, optionally, (c) moving said container to a second location or removing all or an aliquot of said particles or composition, from said container.

The particles or composition described herein can be in liquid, dry, lyophilized, or reconstituted (e.g. , in a liquid as a solution or suspension) formulation or form. The particles or composition described herein can be stored in single, or multi-dose amounts, e.g. , it can be stored in amounts sufficient for at least 2, 5, 10, or 100 dosages. In some embodiments, the method comprises dialyzing, diluting, concentrating, drying, lyophilizing, or packaging (e.g. , disposing the material in a container) the particle or composition. In some embodiments the method comprises combining the particles or composition with another component, e.g. , an excipient, lyoprotectant, or inert substance, e.g. , an insert gas. In some embodiments, the method comprises dividing a preparation of the particles or composition into aliquouts, and optionally disposing a plurality of aliquouts in a plurality of containers. In embodiments, the particles or composition, e.g. , pharmaceutical composition, described herein is stored for a period disclosed herein. In embodiments, after a period of storage, the stored particles or composition, is evaluated, e.g. , for aggregation, color, or other parameter.

In embodiments, the particles or composition described herein may be stored, e.g. , in a container, for at least about 1 hour (e.g. , at least about 2 hours, 4 hours, 8 hours, 12 hours, 24 hours, 2 days, 1 week, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 1 year, 2 years or 3 years). Accordingly, described herein are containers including the particles or composition described herein.

In embodiments, the particles or composition may be stored under a variety of conditions, including ambient conditions, or other conditions described herein. In some embodiments the particles or composition is stored at low temperature, e.g. , at a temperature less than or equal to about 5 °C (e.g. , less than or equal to about 4 °C or less than or equal to about 0 °C). The particles or composition may also be frozen and stored at a temperature of less than about 0 °C (e.g. , between -80 °C and -20 °C). The particles or composition may also be stored under an inert atmosphere, e.g. , an atmosphere containing an inert gas, such as nitrogen or argon. Such an atmosphere may be substantially free of atmospheric oxygen and/or other reactive gases, and/or substantially free of moisture.

In some embodiments, the particles or composition can be stored as a re-constituted formulation (e.g. , in a liquid as a solution or suspension).

In some embodiments, the particles or composition described herein can be stored in a variety of containers, including a light-blocking container such as an amber vial. A container can be a vial, e.g. , a sealed vial having a rubber or silicone enclosure (e.g. , an enclosure made of polybutadiene or polyisoprene). A container can be substantially free of atmospheric oxygen and/or other reactive gases, and/or substantially free of moisture.

In another aspect, the disclosure features, particles or a composition, disposed in a container, e.g. , a container described herein, e.g. , in an amount, form or formulation described herein.

Methods of Evaluating Particles and Compositions

In another aspect, the disclosure features, a method of evaluating a particle or preparation of particles, e.g. , for a property described herein. In some embodiments, the property is a physical property, e.g. , average diameter. In another embodiment, the property is a functional property, e.g. , the ability to mediate knockdown of a target gene, e.g. , as measured in an assay described herein. The method comprises:

providing a sample comprising one or a plurality of said particles, e.g. , as a composition, e.g. , a pharmaceutical composition;

evaluating, e.g. , by a physical test, a property described herein, to provide a determined value for the property,

thereby evaluating a particle or preparation of particles.

In some embodiments, the method comprises one or both of:

a) comparing the determined value with a reference or standard value, e.g. , a range of values (e.g. , value disclosed herein, or set by a regulatory agency, manufacturer, or compendia authority), or

b) responsive to said determination or comparison, classifying said particles.

In some embodiments, responsive to said determination or comparison, a decision or step is taken, e.g. , a production parameter in a process for making a particle is altered, the sample is classified, selected, accepted or discarded, released or withheld, processed into a drug product, shipped, moved to a different location, formulated, e.g. , formulated with another substance, e.g. , an excipient, labeled, packaged, released into commerce, or sold or offered for sale.

In some embodiments, the determined value for a property is compared with a reference, and responsive to said comparison said particle or preparation of particles is classified, e.g. , as suitable for use in human subjects, not suitable for use in human subjects, suitable for sale, meeting a release specification, or not meeting a release specification.

In some embodiments, a particle or preparation of particles is subjected to a measurement to determine whether an impurity or residual solvent is present (e.g. , via gas chromatography (GC)), to determine relative amounts of one or more components (e.g. , via high performance liquid chromatography (HPLC)), to measure particle size (e.g. , via dynamic light scattering and/or scanning electron microscopy), or determine the presence or absence of surface components.

In some embodiments, a particle or preparation of particles is evaluated for the average diameter of the particles in the composition. In some embodiments experiments including physical measurements are performed to determine average value. The average diameter of the composition can then be compared with a reference value. In some embodiments the average diameter for the particles is about 50 nm to about 500 nm (e.g. , from about 50 nm to about 200 nm). A composition of a plurality of particles particle may have a median particle size (Dv50 (particle size below which 50% of the volume of particles exists) of about 50 nm to about 500 nm (e.g. , about 75 nm to about 220 nm)) from about 50 nm to about 220 nm (e.g. , from about 75 nm to about 200 nm). A composition of a plurality of particles may have a Dv90 (particle size below which 90% of the volume of particles exists) of about 50 nm to about 500 nm (e.g. , about 75 nm to about 220 nm). In some embodiments, a composition of a plurality of particles has a Dv90 of less than about 150 nm. A composition of a plurality of particles may have a particle PDI of less than 0.5, less than 0.4, less than 0.3, less than 0.2, or less than 0.1. In some embodiments, the nanoparticles prepared by the methods described herein can have an average size less than 1060 nm, less than about 700 nm, less than about 500 nm, less than about 400 nm, less than about 200 nm, less than about 100 nm, less than about 40 nm. The average size is on a weight basis and is measured by light scattering, microscopy, or other appropriate methods. In some embodiments, at least 65% of the particles by weight have a particles size less than 1060 nm. In some embodiments, at least 80% of the particles are less than 1060 nm. In some embodiments, at least 95% of the particles on a weight basis have a particle size less than 1060 nm as measured by light scattering, microscopy, or other appropriate methods.

In some embodiments, a particle or preparation of particles is subjected to dynamic light scattering, e.g. , to determine size or diameter. Particles may be illuminated with a laser, and the intensity of the scattered light fluctuates at a rate that is dependent upon the size of the particles as smaller particles are "kicked" further by the solvent molecules and move more rapidly.

Analysis of these intensity fluctuations yields the velocity of the Brownian motion and hence the particle size using the Stokes-Einstein relationship. The diameter that is measured in dynamic light scattering is called the hydrodynamic diameter and refers to how a particle diffuses within a fluid. The diameter obtained by this technique is that of a sphere that has the same translational diffusion coefficient as the particle being measured.

In some embodiments, a particle or preparation of particles is evaluated using cryo scanning electron microscopy (Cryo-SEM), e.g. , to determine structure or composition. SEM is a type of electron microscopy in which the sample surface is imaged by scanning it with a high- energy beam of electrons in a raster scan pattern. The electrons interact with the atoms that make up the sample producing signals that contain information about the sample's surface topography, composition and other properties such as electrical conductivity. For Cryo-SEM, the SEM is equipped with a cold stage for cryo-microscopy. Cryofixation may be used and low-temperature scanning electron microscopy performed on the cryogenically fixed specimens. Cryo-fixed specimens may be cryo-fractured under vacuum in a special apparatus to reveal internal structure, sputter coated and transferred onto the SEM cryo-stage while still frozen.

In some embodiments, a particle or preparation of particles is evaluated using

transmission electron microscopy (TEM), e.g. , to determine structure or composition. In this technique, a beam of electrons is transmitted through an ultra thin specimen, interacting with the specimen as it passes through. An image is formed from the interaction of the electrons transmitted through the specimen; the image is magnified and focused onto an imaging device, such as a fluorescent screen, on a layer of photographic film, or to be detected by a sensor such as a charge-coupled device (CCD) camera.

In some embodiments, a particle or preparation of particles is evaluated for a surface zeta potential. In some embodiments experiments including physical measurements are performed to determine average value a surface zeta potential. The surface zeta potential can then be compared with a reference value. In some embodiments the surface zeta potential is between about -20 mV to about 50 mV, when measured in water. Zeta potential is a measurement of surface potential of a particle. In some embodiments, a particle may have a surface zeta potential, when measured in water, ranging between about -20 mV to about 20 mV, about -10 mV to about 10 mV, or neutral.

In some embodiments, a particle or preparation of particles is evaluated for the effective amount of nucleic acid agent (e.g. , mRNA) it contains. In some embodiments, particles described herein are administered, for example, in an in vivo model system, (e.g. , a mouse model), and the level of effect (e.g. , protein expression of the mRNA of the particle) observed. In embodiments, the level is compared with a reference standard.

In some embodiments, a particle or preparation of particles is evaluated for its tendency to aggregate. E.g. , aggregation can be measured in a preselected medium, e.g. , 50/50

mouse/human serum. In embodiment, when incubated 50/50 mouse human serum, the particles exhibit little or no aggregation. E.g. , less than 30, 20, or 10%, by number or weight, of the particles will aggregate. In embodiments the level is compared with a reference standard.

In some embodiments, a particle or preparation of particles is evaluated for stability, e.g. , stability at a preselected condition, e.g. , at 25°C + 2°C/60% relative humidity + 5% relative humidity, e.g. , in an open, or closed, container. In embodiments, when stored at 25°C +

2°C/60% relative humidity + 5% relative humidity in an open, or closed, container, for 20, 30, 40, 50 or 60 days, the particle retains at least 30, 40, 50, 60, 70, 80, 90, or 95% of its activity, e.g. , as determined in an in vivo model system, (e.g. , a mouse model such as one described herein). In embodiments the level of retained activity is compared with a reference standard.

In some embodiments, the particles can be evaluated for induction of cytokines, upon administration to a subject, e.g. , a human subject. A particle or preparation described herein may result in less than 2, 5, or 10 fold cytokine induction, when administered (e.g. , as a single dose of 1 or 3 mg/kg) in an in vivo model system, (e.g. , a mouse model such as any of those described herein). E.g. , the administration results in less than 2, 5, or 10 fold induction of one, or more, e.g. , two, three, four, five, six, or seven, or all, of: tumor necrosis factor-alpha, interleukin- lalpha, interleukin-lbeta, interleukin-6, interleukin-10, interleukin-12, keratinocyte-derived cytokine and interferon-gamma.

In some embodiments, the particles can be evaluated for the ability to increase in alanine aminotransferase (ALT) and or aspartate aminotransferase (AST), when administered (e.g. , as a single dose of 1 or 3 mg/kg) in an in vivo model system, (e.g. , a mouse model such as any of those described herein). In some embodiments a particle or preparation results in less than 2, 5, or 10 fold increase.

In some embodiments, the particles can be evaluated for the ability to alter blood count. In some embodiments a particle or preparation results in no changes in blood count, e.g. , no change 48 hours after 2 doses of 3 mg/kg in an in vivo model system, (e.g. , a mouse model such as any of those described herein).

A particle described herein may be subjected to a number of analytical methods. For example, a particle described herein may be subjected to a measurement to determine whether an impurity or residual solvent is present (e.g. , via gas chromatography (GC)), to determine relative amounts of one or more components (e.g. , via high performance liquid chromatography

(HPLC)), to measure particle size (e.g. , via dynamic light scattering and/or scanning electron microscopy), or determine the presence or absence of surface components.

Pharmaceutical Compositions

Provided herein are compositions, e.g. , a pharmaceutical composition, comprising a plurality of particles described herein and a pharmaceutically acceptable carrier or adjuvant.

In some embodiments, a pharmaceutical composition may include a pharmaceutically acceptable salt of a compound described herein. Pharmaceutically acceptable salts of the compounds described herein include those derived from pharmaceutically acceptable inorganic and organic acids and bases. Examples of suitable acid salts include acetate, adipate, benzoate, benzenesulfonate, butyrate, citrate, digluconate, dodecylsulfate, formate, fumarate, glycolate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, lactate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, palmoate, phosphate, picrate, pivalate, propionate, salicylate, succinate, sulfate, tartrate, tosylate and undecanoate. Salts derived from appropriate bases include alkali metal (e.g. , sodium), alkaline earth metal (e.g. , magnesium), ammonium and N-(alkyl)₄ ⁺ salts. This disclosure also envisions the quaternization of any basic nitrogen-containing groups of the compounds described herein.

Water or oil-soluble or dispersible products may be obtained by such quaternization.

Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.

Examples of pharmaceutically acceptable antioxidants include: (1) water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium

metabisulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gailate, alpha-tocopherol, and the like; and (3) metal chelating agents, such as citric acid,

ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

A composition may include a liquid used for suspending a particle or composition, which may be any liquid solution compatible with the particle or composition, which is also suitable to be used in pharmaceutical compositions, such as a pharmaceutically acceptable nontoxic liquid. Suitable suspending liquids including but are not limited to suspending liquids selected from the group consisting of water, aqueous sucrose syrups, corn syrups, sorbitol, polyethylene glycol, propylene glycol, D5W and mixtures thereof.

A composition described herein may also include another component, such as an antioxidant, antibacterial, buffer, bulking agent, chelating agent, an inert gas, a tonicity agent and/or a viscosity agent.

In one embodiment, the particle or composition is provided in lyophilized form and is reconstituted prior to administration to a subject. The lyophilized particle or composition can be reconstituted by a diluent solution, such as a salt or saline solution, e.g. , a sodium chloride solution having a pH between 6 and 9, lactated Ringer's injection solution, or a commercially available diluent, such as PLASMA-LYTE A Injection pH 7.4® (Baxter, Deerfield, IL).

In one embodiment, a lyophilized formulation includes a lyoprotectant or stabilizer to maintain physical and chemical stability by protecting the particle and active from damage from crystal formation and the fusion process during freeze-drying. The lyoprotectant or stabilizer can be one or more of polyethylene glycol (PEG), a PEG lipid conjugate (e.g. , PEG-ceramide or D- alpha-tocopheryl polyethylene glycol 1000 succinate), poly(vinyl alcohol) (PVA),

poly(vinylpyrrolidone) (PVP), polyoxyethylene esters, poloxamers, polysorbates,

polyoxyethylene esters, lecithins, saccharides, oligosaccharides, polysaccharides, carbohydrates, cyclodextrins (e.g. 2-hydroxypropyl- -cyclodextrin) and polyols (e.g. , trehalose, mannitol, sorbitol, lactose, sucrose, glucose and dextran), salts and crown ethers.

In some embodiments, the lyophilized particle or composition is reconstituted with water, 5% Dextrose Injection, Lactated Ringer's and Dextrose Injection, or a mixture of equal parts by volume of Dehydrated Alcohol, USP and a nonionic surfactant, such as a polyoxyethylated castor oil surfactant available from GAF Corporation, Mount Olive, N.J., under the trademark,

Cremophor EL. The lyophilized product and vehicle for reconstitution can be packaged separately in appropriately light-protected vials. To minimize the amount of surfactant in the reconstituted solution, only a sufficient amount of the vehicle may be provided to form a solution of the particle or composition. Once dissolution of the drug is achieved, the resulting solution is further diluted prior to injection with a suitable parenteral diluent. Such diluents are well known to those of ordinary skill in the art. These diluents are generally available in clinical facilities. It is, however, within the scope of the disclosure to package the particle or composition with a third vial containing sufficient parenteral diluent to prepare the final concentration for administration. A typical diluent is Lactated Ringer's Injection.

The final dilution of the reconstituted particle or composition may be carried out with other preparations having similar utility, for example, 5% dextrose injection, lactated ringer's and dextrose injection, sterile water for injection, and the like. However, because of its narrow pH range, pH 6.0 to 7.5, lactated ringer's injection is most typical. Per 100 mL, Lactated Ringer's Injection contains sodium chloride USP 0.6 g, Sodium Lactate 0.31 g, potassium chloride USP 0.03 g and calcium chloride USP 0.02 g. The osmolality is 275 mOsmol/L, which is very close to isotonicity.

The compositions may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. The amount of nucleic acid agent which can be combined with a pharmaceutically acceptable carrier to produce a single dosage form will vary depending upon the host being treated, the particular mode of administration. The amount of nucleic acid agent which can be combined with a pharmaceutically acceptable carrier to produce a single dosage form will generally be that amount of the compound which produces a therapeutic effect.

Routes of Administration

The pharmaceutical compositions described herein may be administered orally, parenterally (e.g. , via intravenous, subcutaneous, intracutaneous, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal, intralesional, intraocular, or intracranial injection), topically, mucosally (e.g. , rectally or vaginally), nasally, buccally, ophthalmically, via inhalation spray (e.g. , delivered via nebulzation, propellant or a dry powder device) or via an implanted reservoir.

Pharmaceutical compositions suitable for parenteral administration comprise one or more particle(s) or composition(s) in combination with one or more pharmaceutically acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents.

Examples of suitable aqueous and nonaqueous carriers which may be employed in the pharmaceutical compositions include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin. In some cases, in order to prolong the effect of a nucleic acid agent, it is desirable to slow the absorption of the agent from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material having poor water solubility. The rate of absorption of the particle or composition then depends upon its rate of dissolution which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered drug form is accomplished by dissolving or suspending the particle or composition in an oil vehicle.

Pharmaceutical compositions suitable for oral administration may be in the form of capsules, cachets, pills, tablets, gums, lozenges (using a flavored basis, usually sucrose and acacia or tragacanth), powders, granules, or as a solution or a suspension in an aqueous or nonaqueous liquid, or as an oil-in- water or water-in-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia) and/or as mouthwashes and the like, each containing a predetermined amount of an agent as an active ingredient. A composition may also be administered as a bolus, electuary or paste.

A tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared using binder (for example, gelatin or hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (for example, sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), surface-active or dispersing agent. Molded tablets may be made by molding in a suitable machine a mixture of the powdered peptide or peptidomimetic moistened with an inert liquid diluent.

Tablets, and other solid dosage forms, such as dragees, capsules, pills and granules, may optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical-formulating art. They may also be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile, other polymer matrices, liposomes and/or microspheres. They may be sterilized by, for example, filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved in sterile water, or some other sterile injectable medium immediately before use. These compositions may also optionally contain opacifying agents and may be of a composition that they release the active ingredient(s) only, or

preferentially, in a certain portion of the gastrointestinal tract, optionally, in a delayed manner. Examples of embedding compositions which can be used include polymeric substances and waxes. The active ingredient can also be in micro-encapsulated form, if appropriate, with one or more of the above-described excipients.

Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the particle or composition, the liquid dosage forms may contain inert diluents commonly used in the art, such as, for example, water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.

Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents.

Suspensions, in addition to the particle or composition, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.

Pharmaceutical compositions suitable for topical administration are useful when the desired treatment involves areas or organs readily accessible by topical application. For application topically to the skin, the pharmaceutical composition should be formulated with a suitable ointment containing the active components suspended or dissolved in a carrier. Carriers for topical administration of the a particle described herein include, but are not limited to, mineral oil, liquid petroleum, white petroleum, propylene glycol, polyoxyethylene

polyoxypropylene compound, emulsifying wax and water. Alternatively, the pharmaceutical composition can be formulated with a suitable lotion or cream containing the active particle suspended or dissolved in a carrier with suitable emulsifying agents. Suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water. The pharmaceutical compositions described herein may also be topically applied to the lower intestinal tract by rectal suppository formulation or in a suitable enema formulation. Topically-transdermal patches are also included herein.

The pharmaceutical compositions described herein may be administered by nasal aerosol or inhalation. Such compositions are prepared according to techniques well-known in the art of pharmaceutical formulation and may be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and/or other solubilizing or dispersing agents known in the art.

The pharmaceutical compositions described herein may also be administered in the form of suppositories for rectal or vaginal administration. Suppositories may be prepared by mixing one or more particle or composition described herein with one or more suitable non-irritating excipients which is solid at room temperature, but liquid at body temperature. The composition will therefore melt in the rectum or vaginal cavity and release the particle or composition. Such materials include, for example, cocoa butter, polyethylene glycol, a suppository wax or a salicylate. Compositions of the disclosure, which are suitable for vaginal administration also include pessaries, tampons, creams, gels, pastes, foams or spray formulations containing such carriers as are known in the art to be appropriate.

Ophthalmic formulations, eye ointments, powders, solutions and the like, are also contemplated as being within the scope of the disclosure. An ocular tissue (e.g. , a deep cortical region, a supranuclear region, or an aqueous humor region of an eye) may be contacted with the ophthalmic formulation, which is allowed to distribute into the lens. Any suitable method(s) of administration or application of the ophthalmic formulations of the disclosure (e.g. , topical, injection, parenteral, airborne, etc.) may be employed. For example, the contacting may occur via topical administration or via injection.

Dosages and Dosage Regimens

The particles, and compositions can be formulated into pharmaceutically acceptable dosage forms by conventional methods known to those of skill in the art.

Actual dosage levels of the active ingredients in the pharmaceutical compositions of this disclosure may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular subject, composition, and mode of administration, without being toxic to the subject. In one embodiment, the particle or composition is administered to a subject at a dosage of, e.g. , about 0.001 to 300 mg/m², about 0.002 to 200 mg/m², about 0.005 to 100 mg/m², about 0.01 to 100 mg/m², about 0.1 to 100 mg/m², about 5 to 275 mg/m², about 10 to 250 mg/m², e.g. , about 0.001, 0.002, 0.005, 0.01, 0.05, 0.1, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290 mg/m . Administration can be at regular intervals, such as every 1, 2, 3, 4, or 5 days, or weekly, or every 2, 3, 4, 5, 6, or 7 or 8 weeks. The administration can be over a period of from about 10 minutes to about 6 hours, e.g. , from about 30 minutes to about 2 hours, from about 45 minutes to 90 minutes, e.g. , about 30 minutes, 45 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours or more. In one embodiment, the particle or composition is administered as a bolus infusion or intravenous push, e.g. , over a period of 15 minutes, 10 minutes, 5 minutes or less. In one embodiment, the particle or composition is administered in an amount such the desired dose of the agent is administered. Preferably the dose of the particle or composition is a dose described herein.

In one embodiment, the subject receives 1, 2, 3, up to 10, up to 12, up to 15 treatments, or more, or until the disorder or a symptom of the disorder is cured, healed, alleviated, relieved, altered, remedied, ameliorated, palliated, improved or affected. For example, the subject receive an infusion once every 1, 2, 3 or 4 weeks until the disorder or a symptom of the disorder are cured, healed, alleviated, relieved, altered, remedied, ameliorated, palliated, improved or affected. Preferably, the dosing schedule is a dosing schedule described herein.

The particle, or composition can be administered as a first line therapy, e.g. , alone or in combination with an additional agent or agents. In other embodiments, the particle or composition is administered after a subject has developed resistance to, has failed to respond to or has relapsed after a first line therapy. The particle or composition may be administered in combination with a second agent. Preferably, the particle or composition is administered in combination with a second agent described herein. The second agent may be the same or different as the nucleic acid agent in the particle.

Kits

A particle or composition described herein may be provided in a kit. The kit includes a particle or composition described herein and, optionally, a container, a pharmaceutically acceptable carrier and/or informational material. The informational material can be descriptive, instructional, marketing or other material that relates to the methods described herein and/or the use of the particles for the methods described herein.

The informational material of the kits is not limited in its form. In one embodiment, the informational material can include information about production of the particle or composition, physical properties of the particle or composition, concentration, date of expiration, batch or production site information, and so forth. In one embodiment, the informational material relates to methods for administering the particle or composition.

In one embodiment, the informational material can include instructions to administer a particle or composition described herein in a suitable manner to perform the methods described herein, e.g. , in a suitable dose, dosage form, or mode of administration (e.g. , a dose, dosage form, or mode of administration described herein). In another embodiment, the informational material can include instructions to administer a particle or composition described herein to a suitable subject, e.g. , a human, e.g. , a human having or at risk for a disorder described herein. In another embodiment, the informational material can include instructions to reconstitute a particle described herein into a pharmaceutically acceptable composition.

In one embodiment, the kit includes instructions to use the particle or composition, such as for treatment of a subject. The instructions can include methods for reconstituting or diluting the particle or composition for use with a particular subject or in combination with a particular chemotherapeutic agent. The instructions can also include methods for reconstituting or diluting the polymer composition for use with a particular means of administration, such as by intravenous infusion.

In another embodiment, the kit includes instructions for treating a subject with a particular indication. The informational material of the kits is not limited in its form. In many cases, the informational material, e.g. , instructions, is provided in printed matter, e.g. , a printed text, drawing, and/or photograph, e.g. , a label or printed sheet. However, the informational material can also be provided in other formats, such as Braille, computer readable material, video recording, or audio recording. In another embodiment, the informational material of the kit is contact information, e.g. , a physical address, email address, website, or telephone number, where a user of the kit can obtain substantive information about a particle described herein and/or its use in the methods described herein. The informational material can also be provided in any combination of formats.

In addition to a particle or composition described herein, the composition of the kit can include other ingredients, such as a surfactant, a lyoprotectant or stabilizer, an antioxidant, an antibacterial agent, a bulking agent, a chelating agent, an inert gas, a tonicity agent and/or a viscosity agent, a solvent or buffer, a stabilizer, a preservative, a flavoring agent (e.g. , a bitter antagonist or a sweetener), a fragrance, a dye or coloring agent, for example, to tint or color one or more components in the kit, or other cosmetic ingredient, a pharmaceutically acceptable carrier and/or a second agent for treating a condition or disorder described herein. Alternatively, the other ingredients can be included in the kit, but in different compositions or containers than a particle described herein. In such embodiments, the kit can include instructions for admixing a particle or composition described herein and the other ingredients, or for using a particle or composition described herein together with the other ingredients.

In another embodiment, the kit includes a second therapeutic agent. In one embodiment, the second agent is in lyophilized or in liquid form. In one embodiment, the particle or composition and the second therapeutic agent are in separate containers, and in another embodiment, the particle or composition and the second therapeutic agent are packaged in the same container.

In some embodiments, a component of the kit is stored in a sealed vial, e.g. , with a rubber or silicone enclosure (e.g. , a polybutadiene or polyisoprene enclosure). In some embodiments, a component of the kit is stored under inert conditions (e.g. , under nitrogen or another inert gas such as argon). In some embodiments, a component of the kit is stored under anhydrous conditions (e.g. , with a desiccant). In some embodiments, a component of the kit is stored in a light blocking container such as an amber vial.

A particle or composition described herein can be provided in any form, e.g. , liquid, frozen, dried or lyophilized form. It is preferred that a particle or composition described herein be substantially pure and/or sterile. In some embodiments, the particle or composition is sterile. When a particle or composition described herein is provided in a liquid solution, the liquid solution preferably is an aqueous solution, with a sterile aqueous solution being preferred. In one embodiment, the particle or composition is provided in lyophilized form and, optionally, a diluent solution is provided for reconstituting the lyophilized agent. The diluent can include for example, a salt or saline solution, e.g. , a sodium chloride solution having a pH between 6 and 9, lactated Ringer's injection solution, D5W, or PLASMA-LYTE A Injection pH 7.4^® (Baxter, Deerfield, IL).

The kit can include one or more containers for the composition containing a particle or composition described herein. In some embodiments, the kit contains separate containers, dividers or compartments for the composition and informational material. For example, the composition can be contained in a bottle, vial, IV admixture bag, IV infusion set, piggyback set or syringe, and the informational material can be contained in a plastic sleeve or packet. In other embodiments, the separate elements of the kit are contained within a single, undivided container. For example, the composition is contained in a bottle, vial or syringe that has attached thereto the informational material in the form of a label. In some embodiments, the kit includes a plurality (e.g. , a pack) of individual containers, each containing one or more unit dosage forms (e.g. , a dosage form described herein) of a particle or composition described herein. For example, the kit includes a plurality of syringes, ampules, foil packets, or blister packs, each containing a single unit dose of a particle described herein. The containers of the kits can be air tight, waterproof (e.g. , impermeable to changes in moisture or evaporation), and/or light-tight.

The kit optionally includes a device suitable for administration of the composition, e.g. , a syringe, inhalant, pipette, forceps, measured spoon, dropper (e.g. , eye dropper), swab (e.g. , a cotton swab or wooden swab), or any such delivery device. In one embodiment, the device is a medical implant device, e.g. , packaged for surgical insertion.

Methods of Using Particles and Compositions

The particles and compositions described herein can be administered to cells in culture, e.g. in vitro or ex vivo, or to a subject, e.g. , in vivo, to treat or prevent a variety of diseases or disorders (e.g. , cancer (for example solid tumors), autoimmune disorders, cardiovascular disorders, inflammatory disorders, metabolic disorders, infectious diseases, etc.).

Thus, in another aspect, the disclosure features, a method of treating or preventing a disease or disorder in a subject wherein the disease or disorder is cancer (for example a solid tumor), an autoimmune disorder, a cardiovascular disorder, inflammatory disorder, a metabolic disorder, or an infectious disease. The method comprises administering an effective amount of a particle, or composition described herein to thereby treat the disease or disorder. In some embodiments the particles and compositions can be used as part of a first line, second line, or adjunct therapy, and can also be used alone or in combination with one or more additional treatment regimes.

In some embodiments, particles, or compositions disclosed herein can be used to treat or prevent a wide variety of diseases or disorders and can be used to deliver nucleic acid agents, for example, to a subject in need thereof, for example, mRNA; to treat diseases and disorders described herein such as cancer, inflammatory or autoimmune disease, or cardiovascular disease. In embodiments, the particles and compositions described herein can be used as part of a first line, second line, or adjunct therapy, and can also be used alone or in combination with one or more additional treatment regimes.

Vaccines

In some embodiments, the particles or compositions disclosed herein can be used to elicit an immune response in a subject.

Accordingly, in another aspect, the disclosure provides a method of eliciting an immune response to an antigen in a subject, the method comprising administering to the subject an effective amount of a particle described herein, to thereby elicit the immune response. In some embodiments, the nucleic acid agent, e.g., mRNA, can encode antigen(s) for use in eliciting an immunogenic response in a subject. In another embodiment, a nucleic acid agent, e.g., mRNA, can encode antigen(s) for induction of at least one of an antibody or T cell responses, e.g., both antibody and T cell responses. In some embodiments, the nucleic acid agent, e.g., mRNA, can encode antigen(s) for use as DNA or RNA vaccines (see, e.g., Ulmer et al. Vaccine 30: 4414- 4418, 2012, which is incorporated by reference in its entirety).

In one embodiment, the nucleic acid agent is a RNA vaccine, e.g., mRNA vaccine, which can be administered as an active immunotherapeutic immunization in cancer therapies. For example, the nucleic acid agent, e.g., mRNA, can be used to encode genes cloned from metastatic melanoma tumors as an autologous immunization strategy. Further embodiments include, without limitation, the administration of combinations of known tumor antigens to elicit antigen-specific immune responses. Such tumor antigens include, but are not limited to, Mucin 1 (MUC1), Carcinoembryonic antigen (CEA), telomerase, Melanoma-associated antigen 1 (MAGE-1), and tyosinase, in therapies for metastatic melanoma and renal cell carcinoma patients. In another embodiment, an RNA vaccine can be an RNA replicon vaccine, such as a bivalent vaccine including replicons encoding proteins, e.g. , cytomegalovirus (CMV) gB and pp65/IEl proteins, which can generate T cell responses, e.g. , polyfunctional CD4+ and CD8+ T cell responses. In another embodiment, an RNA vaccine can be a self-amplifying RNA vaccine. For example, an RNA vaccine can be a self- amplifying RNA vaccine based on an alphavirus genome, which contains the genes encoding the alphavirus RNA replication machinery, but lacks the genes encoding the viral structural proteins required to make an infectious alphavirus particle (see, e.g. , Geall et al. PNAS, 109(36): 14604-14609, 2012, which is incorporated by reference in its entirety).

Cancer

Accordingly, in another aspect, the disclosure features, a method of treating or preventing a disease or disorder in a subject wherein the disease or disorder is a proliferative disorder, e.g. , a cancer (for example a solid tumor or a liquid tumor). The method comprises administering an effective amount of a particle, or composition described herein to thereby treat the disease or disorder. In some embodiments the particles and compositions described herein can be used as part of a first line, second line, or adjunct therapy, and can also be used alone or in combination with one or more additional treatment regimes. In embodiments the particles and compositions described herein are used to treat or prevent proliferative disorders, e.g. , treating a tumor and metastases thereof wherein the tumor or metastases thereof is a cancer described herein. In some embodiments, the particles and compositions described herein can be used to evaluate or diagnose a cancer.

In embodiments, the proliferative disorder is a solid tumor, a soft tissue tumor or a liquid tumor. Exemplary solid tumors include malignancies (e.g., sarcomas and carcinomas (e.g. , adenocarcinoma or squamous cell carcinoma)) of the various organ systems, such as those of brain, lung, breast, lymphoid, gastrointestinal (e.g., colon), and genitourinary (e.g., renal, urothelial, or testicular tumors) tracts, pharynx, prostate, and ovary. Exemplary

adenocarcinomas include rectal cancer, colon cancer, colorectal cancers, renal-cell carcinoma, liver cancer, non-small cell carcinoma of the lung, and cancer of the small intestine. In embodiments the method comprises evaluating or treating soft tissue tumors such as those of the tendons, muscles or fat, and liquid tumors. In some embodiments, the cancer is a head and neck cancer.

Inflammation and Autoimmune Disease

In another aspect, the disclosure features, a method of treating or preventing a disease or disorder in a subject wherein the disease or disorder is inflammation or an autoimmune disease. The method comprises administering an effective amount of a particle or composition described herein to thereby treat the disease or disorder. In some embodiments the particles and compositions can be used as part of a first line, second line, or adjunct therapy, and can also be used alone or in combination with one or more additional treatment regimes.

Cardiovascular disease

In another aspect, the disclosure features, a method of treating or preventing a disease or disorder in a subject wherein in the disorder is a cardiovascular disease. The method comprises administering an effective amount of a particle, or composition described herein to thereby treat the disease or disorder. In some embodiments the particles and compositions can be used as part of a first line, second line, or adjunct therapy, and can also be used alone or in combination with one or more additional treatment regimes.

EXAMPLES

Example 1. Purification and characterization of 5050 PLGA.

Step A: A 3-L round-bottom flask equipped with a mechanical stirrer was charged with

5050PLGA (300 g, Mw: 7.8 kDa; Mn: 2.7 kDa) and acetone (900 mL). The mixture was stirred for 1 h at ambient temperature to form a clear yellowish solution.

Step B: A 22-L jacket reactor with a bottom-outlet valve equipped with a mechanical stirrer was charged with MTBE (9.0 L, 30 vol. to the mass of 5050 PLGA). Celite® (795 g) was added to the solution with overhead stirring at -200 rpm to produce a suspension. To this suspension was slowly added the solution from Step A over 1 hour. The mixture was agitated for an additional one hour after addition of the polymer solution and filtered through a polypropylene filter. The filter cake was washed with MTBE (3 x 300 mL), conditioned for 0.5 hour, air-dried at ambient temperature (typically 12 hours) until residual MTBE was < 5 wt% (as determined by 1H NMR analysis).

Step C: A 12-L jacket reactor with a bottom-outlet valve equipped with a mechanical stirrer was charged with acetone (2.1 L, 7 vol. to the mass of 5050 PLGA). The polymer/Celite® complex from Step B was charged into the reactor with overhead stirring at -200 rpm to produce a suspension. The suspension was stirred at ambient temperature for an additional 1 hours and filtered through a polypropylene filter. The filter cake was washed with acetone (3 x 300 mL) and the combined filtrates were clarified through a 0.45 mM in-line filter to produce a clear solution. This solution was concentrated to -1000 mL.

Step D: A 22-L jacket reactor with a bottom-outlet valve equipped with a mechanical stirrer was charged with water (9.0 L, 30 vol.) and was cooled down to 0 - 5 °C using a chiller. The solution from Step C was slowly added over 2 hours with overhead stirring at - 200 rpm. The mixture was stirred for an additional one hour after addition of the solution and filtered through a polypropylene filter. The filter cake was conditioned for 1 h, air-dried for 1 day at ambient temperature, and then vacuum-dried for 3 days to produce the purified 5050 PLGA as a white powder [258 g, 86% yield]. The 1H NMR analysis was consistent with that of the desired product and Karl Fisher analysis showed 0.52 wt% of water. The product was analyzed by HPLC (AUC, 230 nm) and GPC (AUC, 230 nm). The process produced a narrower polymer polydispersity, i.e. Mw: 8.8 kDa and Mn: 5.8 kDa.

Example 2. Synthesis, purification, and characterization of trimethylpropanaminium Polyvinyl Alcohol (PVA) (cationic PVA).

PVA (0.056 mmol, 80% hydrolyzed, viscosity 2.5-3.5 cPs, Sigma-Aldrich) was dissolved in DMSO (5 mL) at 65 °C followed by the addition of sodium hydride (12.5 mmol). The reaction mixture was stirred for an hour followed by the addition of glycidyl trimethylammonium chloride (13 mmol). (See scheme below.) The reaction mixture was stirred overnight at 65 °C. The reaction mixture was dialyzed for 5 days and lyophilized to give a light brown product. The product was analyzed by H¹ NMR.

[Cationic PVA can also be purchased from Kuraray, including for example, Cationic PVA CM- 318 (Kuraray)(CioH₂iN₂0.C₄H₆0₂.C₂H₄O.Cl)xl-Propanaminium, N, N, N-trimethyl-s-[(2- methyl-l-oxo-2-propen-l-yl)amino] -chloride (1:1), polymer with ethanol and ethenyl acetate.]

Cationic PVA CM-318 (Kuraray) Example 3. Preparation of mRNA-containing particles

Table 1

The particles described herein were prepared according to the following method and paramaters listed in Table 1. GFP mRNA (purchased from Stemgent, Inc., Cambridge, MA) was lyophilized to remove moisture. The lyophilized mRNA was then reconstituted in 20mM LiBr in DMSO to provide a solution, which was placed in a Dynamic Light Scattering (DLS) particle sizing instrument, and the observed particle size was noted. Acetonitrile (AcN) was added incrementally and the gain in particle size was attributed to aggregation, and subsequently, precipitation of the mRNA. This critical point appeared to be at about 20% AcN. Precipitation of GFP mRNA was achieved by dripping the organic solution of 10% AcN/90% LiBr in dimethyl sulfoxide (DMSO) with 20ug of GFP mRNA and 2 mg of cationic PVA, in 0.8 mL of 5050 PLGA from Example 1 in 13% AcN-DMSO, to produce a suspension of particles. The particles were washed with 10 volumes of TE buffer and concentrated using a tangential flow filtration system (300 kDa MW cutoff, membrane area = 150 cm ).

The plasma stability @ 37 °C after 12 hours was 80%.

Particle properties, evaluated by using the resulting plurality of particles made in the method above:

Z_aVg = 188 nm

Zeta = 1.52 mV

Example 4. Fluorescence Measurements in Human Colorectal Cell Line HCT-116 (HCT- 116) of Uptake of mRNA Particles HCT-116 uptake of mRNA nanoparticles was measured using fluorescence. As a positive assay control, Stemfect™ RNA transfection kit was utilized as directed by the manufacturer with 50 ng of eGFP mRNA purchased from Stemgent®. The positive control was compared to 100 ng of naked mRNA and prepared particles that were applied to the cells at concentrations of 10 ng, 25 ng, 50 ng and 100 ng and allowed to incubate at 37°C, 5% C0₂ for 24 hours. Cells were washed briefly in phosphate buffered saline (PBS) and fixed in a 4% paraformaldehyde / PBS solution. The assessment of gene expression was completed using a SpectraMax® M5 fluorescent platereader with the following settings: fluorescence (RFUs) top read, excitation 485, emission 525 and cutoff 515. As a negative control, transfection reagent was used alone. The data is presented in FIG. 1 as total measured relative fluorescent units (RFU).

HCT-116 cells were seeded onto glass coverslips. Media was removed from the cells and replaced with 4% paraformaldehyde / phosphate buffered saline (PBS) solution. Cells were briefly washed and the coverslips were mounted onto glass slides with Aqua-Poly/Mount. Slides were visualized with an AxioCam MR camera equipped with a Zeiss Epiplan-Neofluor 20x infinity corrected objected controlled by Axio Vision. Images were exported as TIF files for analysis using ImageJ software. Images for the positive control, untreated cells, naked mRNA, and prepared particles that were applied to the cells at concentrations of 50 ng and 100 ng are shown in FIG. 2.

Other embodiments are in the claims.

Claims

We claim:

1. A method of making a particle, the method comprising:

(a) providing a first mixture comprising a nucleic acid agent in a solvent;

(c) contacting the third mixture with a surfactant in an aqueous solution to provide a fourth mixture, to thereby make the particle; wherein the first, second, and third mixtures each contain less than 1,000 ppm water; and wherein the first mixture or the second mixture comprises a cationic moiety.

2. The method of claim 1, wherein the cationic moiety is present in the first mixture.

3. The method of claim 1, wherein the surfactant comprises polyvinyl alcohol (PVA).

4. The method of claim 1, wherein the solvent comprises a de-aggregating agent.

5. The method of claim 1, wherein the solvent comprises a salt.

6. The method of claim 5, wherein the salt is a lithium salt or a calcium salt.

7. The method of claim 6, wherein the salt is a lithium salt.

8. The method of claim 6, wherein the salt is a calcium salt.

9. The method of claim 1, wherein the solvent comprises dimethyl sulfoxide (DMSO).

10. The method of claim 1, wherein the solvent comprises lithium bromide in DMSO.

11. The method of claim 1, wherein the first, second, and third mixtures each contain less than 500 ppm water.

12. The method of claim 1, wherein the first, second, and third mixtures each contain less than 200 ppm water.

13. The method of claim 1, wherein the first, second, and third mixtures each contain less than 100 ppm water.

14. The method of claim 1, wherein the first, second, and third mixtures each contain less than 50 ppm water.

15. The method of claim 1, wherein the first, second, and third mixtures are anhydrous.

16. The method of claim 1, wherein the solvent comprises one or more of DMSO, dimethylacetamide (DMA), and propylene carbonate.

17. The method of claim 1, wherein the co- solvent comprises a high polar index solvent.

18. The method of claim 1, wherein the hydrophobic polymer comprises a poly(lactic-co glycolic acid) (PLGA).

19. The method of claim 18, wherein the PLGA has a ratio of from about 25:75 to about 75:25 of lactic acid to glycolic acid.

20. The method of claim 18, wherein the PLGA has a ratio of about 50:50 of lactic acid to glycolic acid.

21. The method of claim 1, wherein the hydrophobic polymer has a weight average molecular weight ranging from 1 kDa to 70 kDa.

22. The method of claim 1, wherein the cationic moiety comprises cationic PVA.

23. The method of claim 1, wherein the surfactant is present in an amount that is less than about 1%.

24. The method of claim 1, wherein the particle has a zeta potential of from about -20 mV to about +20 mV.

25. The method of claim 1, wherein the particle has an average particle diameter (Zave) of less than 500 nm.

26. The method of claim 1, further comprising a hydrophilic-hydrophobic polymer.

27. The method of claim 26, wherein the hydrophilic-hydrophobic polymer comprises polyethylene glycol-poly(lactic-coglycolic acid) (PEG-PLGA).

28. The method of claim 27, wherein the PEG-PLGA has a weight average molecular weight of less than 20 kDa.

29. The method of claim 1, further comprising lyophilizing the particle.

30. The method of claim 1, wherein the particle is a nanoparticle.

31. A mixture comprising:

(a) a nucleic acid agent;

(b) a cationic moiety; and

(c) a hydrophobic polymer.

32. The mixture of claim 31, further comprising a surfactant.

33. The mixture of claim 31, further comprising a hydrophilic-hydrophobic polymer.

34. A mixture comprising: (a) a nucleic acid agent;

(b) a hydrophobic polymer; and

(c) a solvent, wherein the solvent comprises a mixture of two solvents and contains less than 1,000 ppm of water.

35. The mixture of claim 34, further comprising a salt.

36. The mixture of claim 34, further comprising a cationic moiety.

37. The mixture of claim 34, further comprising a surfactant.

38. The mixture of claim 34, further comprising a hydrophilic-hydrophobic polymer.

I l l