ENHANCED LIPID NANOPARTICLE DRUG DELIVERY USING A NEGATIVELY

CHARGED POLYMER

CROSS REFERENCE TO RELATED APPLICATIONS

[0001]The application claims the benefit of United States Provisional Patent Application No. 62/762,042, filed April 17, 2018, which is incorporated herein by reference in its entirety.

STATEMENT REGARDING FEDERAL FUNDING

[0002]This invention was made with government support under Grant No. D16AP00143 awarded by the Defense Advanced Research Projects Agency. The government has certain rights in this invention.

[0003] Delivery of negatively-charged drugs, such as nucleic acids including mRNA, gRNA (guide RNA), and siRNA (small interfering RNA), among other nucleic acid forms, has been researched for the treatment of diseases associated with abnormal gene expression and several other applications. A current obstacle to successful RNA drug delivery is finding an effective delivery vehicle to stabilize and deliver the nucleic acid or other polyanionic therapeutics to cells and to facilitate cellular entry and activity of the drug. One delivery vehicle for nucleic acids, e.g. RNA therapeutics, is lipid nanoparticle (LNP) technology. LNPs have been used for several decades in the delivery of small and large RNAs, including siRNA, miRNA, and mRNA. Typically, these formulations include a cationic or ionizable lipid, cholesterol or its analogues such as oxidized cholesterol or desmosterol, a“helper” lipid such as DOPC or DOPE, and a lipid conjugated to the polymer polyethylene glycol (PEG). The cationic/ionizable lipid is the active delivery agent. Cholesterol or its analogues and the helper lipid aid in the molecular packing and stability of the particle. PEG reduces the degree to which particles are cleared by immune system in vivo.

[0004]An exemplary LNP/siRNA drug product, recently approved in the United States is patisiran (ONPATTRO ^® , Anylam Pharmaceuticals), an siRNA for knocking down transthyretin, for use in treatment of hereditary transthyretin amyloidosis. A variety of other lipid-based delivery systems are undergoing clinical trials for delivery of siRNA, mRNA, and plasmid vectors, antisense reagents, among other nucleic acid or nucleic acid analog therapeutic agents.

[0005]Advances in LNP technology that, among other things, increase the specific activity of polyanionic therapeutics, such as nucleic acids and nucleic acid analogs, such as siRNA and mRNA, are desirable.

SUMMARY

[0006] In one aspect of the invention, a lipid particle, e.g., a lipid nanoparticle, is provided. The lipid particle comprises: an ionizable or cationic lipid; cholesterol or a cholesterol analogue; a helper lipid; a polyethyleneglycol-lipid (PEG-lipid) or polyethyleneglycol-cholesterol (PEG-cholesterol) comprising a polyethyleneglycol moiety and a lipid or cholestorol moiety; a polyanionic (having a plurality of negative charges) therapeutic agent; and a polyanionic helper polymer.

[0007] In a further aspect, a method of making a lipid particle, e.g., a lipid nanoparticle, is provided. The method comprises mixing in solution, e.g., an aqueous solution: an ionizable or cationic lipid; cholesterol or a cholesterol analogue; a helper lipid; a polyethyleneglycol-lipid (PEG-lipid) or polyethyleneglycol-cholesterol (PEG- cholesterol) comprising a polyethyleneglycol moiety and a lipid or cholestorol moiety; a polyanionic therapeutic agent; and a polyanionic helper polymer, in amounts effective to produce a lipid particle.

[0008] In another aspect, a method of delivering a polyanionic therapeutic agent to a cell or to a patient is provided. The method comprises, administering a lipid particle, e.g., a lipid nanoparticle, to the cell or patient comprising: an ionizable or cationic lipid; cholesterol or a cholesterol analogue; a helper lipid; a polyethyleneglycol-lipid (PEG- lipid) or polyethyleneglycol-cholesterol (PEG-cholesterol) comprising a polyethyleneglycol moiety and a lipid or cholesterol moiety; a polyanionic therapeutic agent; and a polyanionic helper polymer. In one aspect, the therapeutic agent is mRNA or an RNAi agent, such as an siRNA.

[0009]The following numbered clauses illustrate various aspects of the invention: Clause 1 . A lipid particle comprising:

an ionizable or cationic lipid;

cholesterol or a cholesterol analogue;

a helper lipid; a polyethyleneglycol-lipid (PEG-lipid) or polyethyleneglycol-cholesterol (PEG- cholesterol) comprising a polyethyleneglycol moiety and a lipid or cholesterol moiety; a polyanionic therapeutic agent; and

a polyanionic helper polymer.

Clause 2. The particle of clause 1 , wherein the polyanionic therapeutic agent is a nucleic acid or a nucleic acid analog having a negatively-charged backbone.

Clause 3. The particle of clause 1 , wherein the polyanionic therapeutic agent is RNA.

Clause 4. The particle of clause 1 , wherein the polyanionic therapeutic agent is an mRNA or an RNAi agent.

Clause 5. The particle of clause 1 , wherein the polyanionic therapeutic agent is an siRNA.

Clause 6. The particle of clause 1 , wherein the polyanionic therapeutic agent is an mRNA.

Clause 7. The particle of clause 1 , wherein the polyanionic therapeutic agent is an siRNA, and the polyanionic helper polymer is an mRNA.

Clause 8. The particle of clause 1 , wherein the polyanionic therapeutic agent is an mRNA, and the polyanionic helper polymer is an siRNA.

Clause 9. The particle of any one of clauses 1-8, wherein the anionic helper polymer has a pKa of less than 4.

Clause 10. The particle of any one of clauses 1-9, wherein the anionic helper polymer has a pKa of less than 2.

Clause 11. The particle of any one of clauses 1-10, wherein the anionic helper polymer is a polystyrene having a plurality of acidic pendant groups.

Clause 12. The particle of any one of clauses 1-11 , wherein the anionic helper polymer has a hydrocarbon backbone having pendant sulfonate, carboxyl, or phosphinic acid moieties.

Clause 13. The particle of any one of clauses 1 -12, wherein the ionizable or cationic lipid is an ionizable amine lipid.

Clause 14. The particle of clause 13, wherein the ionizable or cationic lipid is heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate (DLin-MC3- DMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1 ,3]-dioxolane (DLin-KC2-DMA), or 3060no. Clause 15. The particle of any one of clauses 1 -14, wherein the helper lipid is DSPC, DSPE, DOPC, or DOPE.

Clause 16. The particle of any one of clauses 1 -15, wherein the PEG-lipid or polyethyleneglycol-cholesterol (PEG-cholesterol) comprises a C10-C20 saturated or unsaturated fatty acid and a PEG moiety ranging from 300 g/mol to 5000 g/mol.

Clause 17. The particle of clause 16, wherein the PEG-lipid or PEG-cholesterol comprises a PEG-ceramide, a PEG-cholesterol, a PEG-phosphoethanolamine, a DMG-PEG (1 ,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000), a DSG- PEG (distearoyl-rac-glycerol-PEG2K), or a DSPE-PEG.

Clause 18. The lipid particle of any one of clauses 1 -17, wherein the polyanionic therapeutic agent or the polyanionic helper polymer is a therapeutic mRNA.

Clause 19. The lipid particle of any one of clauses 1 -17, wherein the polyanionic therapeutic or the polyanionic helper polymer is a therapeutic siRNA.

Clause 20. The lipid particle of any one of clauses 1 -19, in which:

the weight ratio of ionizable or cationic lipid to the sum of the weights of the polyanionic therapeutic agent and the helper polymer ranges from 7:1 to 10:1 ;

the weight ratio of polyanionic helper polymer to the polyanionic therapeutic agent ranges from 0.01 :1 to 1 1 :1 ;

the molecular weight of polyanionic helper polymer ranges from 4 - 100 kDa; and the molar ratio of ionizable or cationic lipid to cholesterol or cholesterol analogue to helper lipid to polyethyleneglycol-lipid (PEG-lipid) or polyethyleneglycol-cholesterol (PEG-cholesterol)is 35-55:30-50:0-20:0.5-5.

Clause 21 . The lipid particle of clause 20, comprising:

DLin-MC3-DMA, DLin-KC2-DMA, CKK-E12, or 3060iio;

cholesterol or a cholesterol analogue;

DSPC, DSPE, DOPC, or DOPE;

a PEG-lipid or PEG-cholesterol;

mRNA or siRNA; and

from 1 kDa to 200 kDa PSS.

Clause 22. The lipid particle of clause 20, comprising:

3060iio;

cholesterol or a cholesterol analogue;

DSPC or DOPE; 1 ,2-Dimyristoyl-sn-Glycero-3-Phosphoethanolamine-N [Methoxy(Polyethyle ne glycol)-2000];

mRNA or siRNA; and

from 1 kDa to 200 kDa PSS.

Clause 23. The lipid particle of clause 20, comprising:

3060iio;

cholesterol or a cholesterol analogue;

DSPC or DOPE;

1 ,2-Dimyristoyl-sn-Glycero-3-Phosphoethanolamine-N [Methoxy(Polyethylene glycol)-2000];

siRNA; and

mRNA or from 1 kDa to 200 kDa PSS.

Clause 24. The lipid particle of any one of clauses 1 -23, wherein the helper polymer has a polydispersity index of less than 2.

Clause 25. A method of making a lipid particle comprising mixing in solution:

an ionizable or cationic lipid;

cholesterol or a cholesterol analogue;

a helper lipid;

a polyethyleneglycol-lipid (PEG-lipid) or polyethyleneglycol-cholesterol (PEG- cholesterol) comprising a polyethyleneglycol moiety and a lipid or cholestorol moiety; a polyanionic therapeutic agent; and

a polyanionic helper polymer, in amounts effective to produce a lipid particle.

Clause 26. The method of clause 25, wherein the polyanionic therapeutic agent is a nucleic acid or a nucleic acid analog having a negatively-charged backbone.

Clause 27. The method of clause 25, wherein the polyanionic therapeutic agent is RNA.

Clause 28. The method of clause 25, wherein the polyanionic therapeutic agent is an mRNA or an RNAi agent.

Clause 29. The method of clause 25, wherein the polyanionic therapeutic agent is an siRNA.

Clause 30. The method of clause 25, wherein the polyanionic therapeutic agent is an mRNA. Clause 31. The method of clause 25, wherein the polyanionic therapeutic agent is an siRNA, and the polyanionic helper polymer is an mRNA.

Clause 32. The method of clause 25, wherein the polyanionic therapeutic agent is an mRNA, and the polyanionic helper polymer is an siRNA.

Clause 33. The method of any one of clauses 25-32, wherein the anionic helper polymer has a pKa of less than 4.

Clause 34. The method of any one of clauses 25-32, wherein the anionic helper polymer has a pKa of less than 2.

Clause 35. The method of any one of clauses 25-34, wherein the anionic helper polymer is a polystyrene having a plurality of acidic pendant groups.

Clause 36. The method of any one of clauses 25-35, wherein the anionic helper polymer has a hydrocarbon backbone having pendant sulfonate, carboxyl, or phosphinic acid moieties.

Clause 37. The method of any one of clauses 25-36, wherein the ionizable or cationic lipid is an ionizable amine lipid.

Clause 38. The method of clause 37, wherein the ionizable or cationic lipid is heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate (DLin-MC3- DMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1 ,3]-dioxolane (DLin-KC2-DMA), or 3060no.

Clause 39. The method of any one of clauses 25-38, wherein the helper lipid is DSPC, DSPE, DOPC, or DOPE.

Clause 40. The method of any one of clauses 25-39, wherein the cholesterol or cholesterol analogue is cholesterol, oxidized cholesterol, desmosterol, 7- dehydrocholesterol, ergosterol, lanosterol, ketosterone, cholesterol sulfate, dehydroergosterol, cholestratrienol, 5-cholestene, or pregnenolone.

Clause 41. The method of any one of clauses 25-40, wherein the PEG-lipid or PEG- cholesterol comprises a C10-C20 saturated or unsaturated fatty acid and a PEG moiety ranging from 300 g/mol to 5000 g/mol.

Clause 42. The method of clause 41 , wherein the PEG-lipid or PEG-cholesterol comprises PEG-ceramide, a PEG-cholesterol, a PEG-phosphoethanolamine, a DMG- PEG (1 ,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000), a DSG-PEG (distearoyl-rac-glycerol-PEG2K), or a DSPE-PEG. Clause 43. The method of any one of clauses 25-42, wherein the polyanionic therapeutic agent or the polyanionic helper polymer is a therapeutic mRNA.

Clause 44. The method of any one of clauses 25-42, wherein the polyanionic therapeutic or the polyanionic helper polymer is a therapeutic siRNA.

Clause 45. The method of any one of clauses 25-42, in which:

the weight ratio of ionizable or cationic lipid to the sum of the weights of the polyanionic therapeutic agent and the helper polymer ranges from 7:1 to 10:1 ;

the weight ratio of polyanionic helper polymer to the polyanionic therapeutic agent ranges from 0.01 :1 to 1 1 :1 ;

the molecular weight of polyanionic helper polymer ranges from 4 - 100 kDa; and the molar ratio of ionizable or cationic lipid to cholesterol or cholesterol analogue to helper lipid to polyethyleneglycol-lipid (PEG-lipid) or PEG-cholesterol is 35-55:30- 50:0-20:0.5-5.

Clause 46. The method of clause 45, wherein:

the ionizable or cationic lipid is DLin-MC3-DMA, DLin-KC2-DMA, CKK-E12, or 3060no; the helper lipid is DSPC, DSPE, DOPC, or DOPE;

the polyanionic therapeutic agent is mRNA or siRNA; and

the polyanionic helper polymer is from 1 kDa to 200 kDa PSS.

Clause 47. The method of clause 45, wherein:

the ionizable or cationic lipid is 3060no;

the helper lipid is DSPC;

the polyethyleneglycol-lipid (PEG-lipid) or polyethyleneglycol-cholesterol (PEG- cholesterol) is 1 ,2-Dimyristoyl-sn-Glycero-3-Phosphoethanolamine-N

[Methoxy(Polyethylene glycol)-2000];

the polyanionic therapeutic agent is mRNA or siRNA; and

the polyanionic helper polymer is from 1 kDa to 200 kDa PSS.

Clause 48. The method of clause 45, wherein:

the ionizable or cationic lipid is 3060no;

the helper lipid is DSPC;

the polyethyleneglycol-lipid (PEG-lipid) or polyethyleneglycol-cholesterol (PEG- cholesterol) is 1 ,2-Dimyristoyl-sn-Glycero-3-Phosphoethanolamine-N

[Methoxy(Polyethylene glycol)-2000];

the polyanionic therapeutic agent is siRNA; and the polyanionic helper polymer is mRNA or from 1 kDa to 200 kDa PSS.

Clause 49. The method of any one of clauses 25-48, wherein the helper polymer has a polydispersity index of less than 2.

Clause 50. A method of delivering a polyanionic therapeutic agent to a cell or to a patient, comprising, administering a lipid particle of any one of clauses 1 -24 to the cell or patient.

Clause 51 . The method of clause 50, wherein the therapeutic agent is delivered parenterally to a patient.

Clause 52. The method of clause 50, wherein the therapeutic agent is delivered orally or rectally to a patient.

Clause 53. The method of clause 50-52, wherein the therapeutic agent is an mRNA or an RNAi agent, which is administered in an amount effective to increase or decrease gene expression in the patient.

BRIEF DESCRIPTION OF THE DRAWINGS

[00010] Figure 1. Lipid nanoparticles co-delivered siRNA and mRNA in vitro. HeLa cells that stably expressed firefly luciferase were incubated with LNPs containing 10 nM of siRNA against firefly luciferase and/or 100 ng of mRNA encoding mCHERRY. Expression of both proteins was assessed 24 hours post-transfection. A) LNPs co-formulated with siRNA and mRNA resulted in greater gene silencing than LNPs formulated only with siRNA. (n = 12 - 18) B) LNP formulations 3 - 5 delivered mRNA to HeLa cells regardless of whether siRNA was included in the formulation (n = 12 - 18). The same cells generated the data in panels A and B. C) Total RNA entrapment increased at higher formulation numbers. D) The RNA cargo, but not the LNP formulation number, affected LNP size (n = 2 - 3).

[00011] Figure 2. Lipid nanoparticles synergistically co-delivered siRNA and mRNA in mice. All animals received siRNA specific for Factor VII (FVII) at a dose of 0.03 mg/kg and/or mRNA encoding firefly luciferase at a dose of 0.5 mg/kg. A) LNPs co-formulated with both RNAs (purple circles) using Formulations 3 - 5 induced higher levels of gene silencing than LNPs formulated with only siRNA (blue triangle) and only mRNA (control, red square) (n = 3) B) Co-formulated LNPs resulted in more luciferase expression than LNPs formulated with only mRNA. The same animals were used to generate the data in panels A and B. (n = 3 - 4) C) Total RNA entrapment increased with formulation number (n = 3 technical replicates) D) Co-formulation resulted in synergistic RNA delivery.“Form’d Separately” mice received an injection containing LNPs formulated with siFVII combined with LNPs formulated with mLuc. “Coformulated” mice received an injection of LNPs co-formulated with siFVII and mLuc. All LNPs were generated using Formulation 4. (n = 3).

[00012] Figure 3. A) LNP formulation 4 resulted in the highest luciferase expression in mice. The vast majority of signal was produced in the liver and spleen. IVIS images were taken 6 hours after tail vein injection of LNPs with 0.5 mg/kg mRNA encoding firefly luciferase (mLUC) and/or 0.03 mg/kg siRNA against FVII (siFVII). (n = 3) B) The weight of treated mice did not change compared to untreated mice 2 days following treatment (n = 3 - 4) C) LNP z-average diameter in formulations 1 -5 was measured using dynamic light scattering (n = 3 technical replicates) D) LNPs coformulated with siRNA and mRNA produced the highest luminescent signal in the abdomen of mice. Whole mice were imaged 6 hours after tail vein injection of LNPs in formulation 4 with 0.5 mg/kg mLUC and/or 0.03 mg/kg siFVII (n = 3). Error bars represent s.d.

[00013] Figure 4. Delivery synergy from co-formulation depended on the total RNA concentration. A) The addition of“helper” mRNA to an LNP intended to deliver siRNA improved gene silencing efficacy for intermediate total RNA concentrations. All mice were dosed with 0.03 mg/kg siRNA specific for Factor VII (FVII). (n = 3) B) The addition of“helper” siRNA to an LNP intended to deliver mRNA significantly improved luciferase protein expression at the intermediate total RNA concentration of 0.106 mg/mL. All mice were dosed with 0.5 mg/kg mRNA encoding firefly luciferase. (n = 3 - 4) C) LNP efficacy depended on the total RNA and lipidoid concentrations. LNPs containing siFVII were formulated with and without helper mRNA, and with a low or high amount of lipidoid. All mice were dosed with 0.03 mg/kg siFVII. (n = 3).

[00014] Figure 5. The addition of“helper” siRNA to LNPs loaded with luciferase encoding mRNA improved mRNA delivery for the siRNA: mRNA weight ratio of 0.006 : 0.1 . LNPs were formulated using Formulation 4 keeping the mLuc concentration constant (0.1 mg/mL) and changing the siRNA concentration (0 - 0.08 mg/mL). All mice received 0.5 mg/kg mLuc. Mice were imaged 6 hours after tail vein injection (n = 3 - 4). [00015] Figure 6. For A, C, & E LNPs were formulated using Formulation 4 with a constant siRNA concentration (0.006 mg/ml_) and varied mRNA concentrations (0.05-0.15 mg/ml_). All mice were dosed with 0.03 mg/kg siFVII. For B, D, & F LNPs were formulated using Formulation 4 with a constant mRNA concentration (0.1 mg/mL) and varied siRNA concentration (0 - 0.08 mg/mL). All mice were dosed with 0.5 mg/kg mLuc. A, B) LNPs did not induce weight change compared to untreated mice (n = 3) C, D) RNA entrapment in LNPs changed with total RNA concentration during formulation (n = 3 technical replicates) E, F) The z-average diameter of the LNPs ranged from 100 nm - 200 nm. (n = 3 technical replicates). In all panels, error bars represent s.d.

[00016] Figure 7. Neither A) siRNA-mediated Factor VII gene silencing nor B) mRNA-mediated luciferase expression in mice correlated with lipidoid concentration. All LNPs shown were dosed at 0.03 mg/kg siRNA and/or 0.5 mg/kg mLuc. C, D) A modest correlation between RNA delivery efficacy and lipidoid concentration was present in HeLa cells. The siRNA and mRNA doses were 10 nM and 100 ng, respectively (n = 9 - 10).

[00017] Figure 8. LNPs were formulated with siFVII alone, siFVII + mLuc (0.006 mg/mL) or LNPs with siFVII + mLuc (0.1 mg/mL) while keeping the lipidoid concentration constant. LNPs were delivered to mice at 0.03 mg/kg siFVII. A) LNPs did not form properly when formulated with 0.006 mg/mL siRNA, 0.1 mg/mL mLuc and a low concentration of lipidoid (0.05 mg/mL). (n = 3 technical replicates) B) The z- average diameter was measured using dynamic light scattering (n = 3 technical replicates). C) LNPs did not induce weight loss in mice compared to an untreated group (n = 3) In all panels, error bars represent s.d.

[00018] Figure 9. Inclusion of a negatively charged“helper” polymer in single RNA LNP formulations improved efficacy. A) All LNPs were dosed in mice at 0.03 mg/kg siRNA. Only the LNPs loaded with siFVII induced FVII gene silencing. LNPs formulated with helper mRNA, 70 kDa PSS, or PolyU improved knockdown compared to LNPs formulated without helper polymer (n = 3 - 4) B) All LNPs were dosed at 0.5 mg/kg mRNA, with only mLuc treatments resulting in gene expression. Compared to LNPs containing no helper material, the addition of siRNA or 6.8 kDa PSS to the formulation enhanced mLuc delivery (n = 3 - 4) C) LNPs formulated with PSS facilitated comparable FVII silencing to LNPs without PSS using three times less drug. PSS-formulated LNPs induced dose-dependent gene knockdown (n = 3).

[00019] Figure 10: The addition of siFVII or PSS to the LNP formulation significantly enhanced mLuc delivery. LNPs were formulated using Formulation 4 with mLuc and either siFVII, PSS (6.8 kDa), Poly(U), or PAA. All LNPs were dosed at 0.5 mg/kg mLuc. Mice were imaged using an IVIS 6 hours post-LNP injection (n = 3 - 4).

[00020] Figure 11. For panels A, C, & E, LNPs were formulated with siFVII and either mRNA and PSS (70 kDa), Poly(U), or PAA. For panels B, D, & F, LNPs were formulated with mLuc and either siRNA, PSS (6.8 kDa), Poly(U), or PAA. A, B) The addition of negatively charged polymers to the LNP formulation did not markedly increase total RNA entrapment (n = 3 technical replicates) C, D) LNP size ranged from 100 nm - 200 nm with or without the addition of negatively charged polymers in the formulation (n = 3 technical replicates) E, F) LNPs did not result in weight loss compared to treated groups two days post-injection (n = 3). In all panels, error bars represent s.d.

[00021] Figure 12. LNPs loaded with siFVII were formulated with or without PSS and delivered to mice at a range of siFVII doses (0.005 - 0.03 mg/kg). A) RNA entrapment decreased with decreasing siFVII dose (n = 3 technical replicates) B) The z-average diameter was measured for each LNP using dynamic light scattering (n = 3 technical replicates) C) Mice treated with LNPs did not experience weight change two days post-injection compared to untreated animals (n = 3) In all panels, error bars represent s.d.

DETAILED DESCRIPTION

[00022] The use of numerical values in the various ranges specified in this application, unless expressly indicated otherwise, are stated as approximations as though the minimum and maximum values within the stated ranges are both preceded by the word "about". In this manner, slight variations above and below the stated ranges can be used to achieve substantially the same results as values within the ranges. Also, unless indicated otherwise, the disclosure of these ranges is intended as a continuous range including every value between the minimum and maximum values. For definitions provided herein, those definitions also refer to word forms, cognates and grammatical variants of those words or phrases. [00023] As used herein, the terms“comprising,”“comprise” or“comprised,” and variations thereof, in reference to elements of an item, composition, apparatus, method, process, system, claim etc. are intended to be open-ended, meaning that the item, composition, apparatus, method, process, system, claim etc. includes those elements and other elements can be included and still fall within the scope/definition of the described item, composition, apparatus, method, process, system, claim etc. As used herein, "a" or "an" means one or more. As used herein "another" may mean at least a second or more.

[00024] As used herein, the terms "patient" or "subject" refer to members of the animal kingdom, including, but not limited to human beings.

[00025] A composition is "biocompatible" in that the composition and, where applicable, degradation products thereof, are substantially non-toxic to cells or organisms within acceptable tolerances, including substantially non-carcinogenic and substantially non- immunogenic, and are cleared or otherwise degraded in a biological system, such as an organism (patient) without substantial toxic effect. Non-limiting examples of degradation mechanisms within a biological system include chemical reactions, hydrolysis reactions, and enzymatic cleavage.

[00026] As used herein, the term "polymer composition" is a composition comprising one or more polymers. As a class, "polymers" includes, without limitation, homopolymers, heteropolymers, co-polymers, or block co-polymers and can be both natural and/or synthetic. Homopolymers contain one type of building block, or monomer, whereas copolymers contain more than one type of monomer. The term "(co)polymer" and like terms refer to either homopolymers or copolymers. Polymers can be linear, or have branched structures, such as comb, star, or dendrimeric structures. Polymers may be cross-linked. A polymer comprises a backbone, and often comprises a pendant group, such as an anionic or cationic moiety, or a reactive group.

[00027] A polymer "comprises" or is "derived from" a stated monomer if that monomer is incorporated into the polymer. Thus, the incorporated monomer (monomer residue) that the polymer comprises is not the same as the monomer prior to incorporation into a polymer, in that at the very least, certain groups are missing and/or modified when incorporated into the polymer backbone. A polymer is said to comprise a specific type of linkage if that linkage is present in the polymer. [00028] Provided herein is a dosage form, e.g., an oral dosage form, a topical dosage form, or a parenteral dosage form, e.g., an intravenous or intramuscular dosage form, for delivery of polyanions, such as nucleic acids or nucleic acid analogs to a patient. In aspects, the polyanions are single stranded or double stranded nucleic acids or nucleic acid analogs, included single-stranded DNA, single-stranded RNA, double-stranded DNA, double-stranded RNA, or modified versions of any of the preceding, or analogs of any of the preceding. With regard to overall structure and function of the nucleic acid or nucleic acid analog, the nucleic acid or nucleic acid analog may be, without limitation: mRNA, siRNA, miRNA (microRNA), gRNA (guide RNA), tRNA (transfer RNA), rRNA (ribosomal RNA), tmRNA (transfer-messenger RNA), piRNA (Piwi-interacting RNA), snRNA (small nuclear RNA), snoRNA (small nucleolar RNA), scaRNAs (small Cajal body RNA), Y RNA, eRNA (enhancer RNA), shRNA (small hairpin RNA), stRNA (small temporal RNA), DNA, chloroplast DNA, cDNA (complementary DNA), gDNA (genomic DNA), Hachimoji DNA, mitochondrial DNA, msDNA (multicopy single-stranded DNA), XNA (xeno nucleic acid), glycol nucleic acid, threose nucleic acid, hexose nucleic acid, LNA (locked nucleic acid), PNA (peptide nucleic acid), morpholino oligomers, antisense oligonucleotides, ribozymes, deoxyribozymes, aptamers, cloning vectors, phagemids, plasmids, lambda phage, cosmids, fosmids, or artificial chromosomes. The dosage form comprises a lipid particle, such as a lipid nanoparticle, comprising the polyanionic therapeutic agent and a polyanionic polymer different from the nucleic acid or nucleic acid analog, and in aspects is not a nucleic acid or nucleic acid analog.

[00029] Therapeutic/pharmaceutical compositions are prepared in accordance with acceptable pharmaceutical procedures, such as described in Remington: The Science and Practice of Pharmacy. 21 st edition, ed. Paul Beringer et ai, Lippincott, Williams & Wilkins, Baltimore, MD Easton, Pa. (2005) (see, e.g., Chapters 37, 39, 41 , 42 and 45 for examples of powder, liquid, parenteral, intravenous and oral solid formulations and methods of making such formulations). Depending on the delivery route, the dosage form may comprise additional carriers or excipients, such as water, saline (e.g., normal saline), or phosphate-buffered saline, as are broadly-known in the pharmaceutical arts. Compositions may comprise a pharmaceutically acceptable carrier, or excipient. An excipient is an inactive substance used as a carrier for the active ingredients of a medication. Although“inactive,” excipients may facilitate and aid in increasing the delivery, stability or bioavailability of an active ingredient in a drug product. Non-limiting examples of useful excipients include: anti-adherents, binders, rheology modifiers, coatings, disintegrants, emulsifiers, oils, buffers, salts, acids, bases, fillers, diluents, solvents, flavors, colorants, glidants, lubricants, preservatives, antioxidants, sorbents, vitamins, sweeteners, etc., as are available in the pharmaceutical/compounding arts.

[00030] For example, where the dosage form is an oral dosage form, it may comprise a delayed-release coating, such as an enteric coating surrounding the LNPs comprising the therapeutic agent to delay release of the therapeutic agent until it reaches to small intestine. The dosage form may be provided as a unit dosage form, e.g., with lipid particles packaged within a syringe or ampoule for single or multiple use. The dosage form also may be topical, e.g., for delivery to a body surface, such as skin, mucosa, or other topical routes, including, dermal, oral, nasal, optic, otic, or vaginal delivery routes. The dosage form also may be a suitable gastrointestinal dosage form, for example as a suppository, or through a feeding tube.

[00031] Also provided herein is a method of delivering polyanions, such as nucleic acids or nucleic acid analogs to a patient. In aspects, the polyanions include single stranded or double stranded nucleic acids or nucleic acid analogs, including single-stranded DNA, single-stranded RNA, double-stranded DNA, double-stranded RNA, or modified versions of any of the preceding, or analogs of any of the preceding. With regard to overall structure and function of the nucleic acid or nucleic acid analog, the nucleic acid or nucleic acid analog may be, without limitation: mRNA, siRNA, miRNA (microRNA), gRNA (guide RNA), tRNA (transfer RNA), rRNA (ribosomal RNA), tmRNA (transfer-messenger RNA), piRNA (Piwi-interacting RNA), snRNA (small nuclear RNA), snoRNA (small nucleolar RNA), scaRNAs (small Cajal body RNA), Y RNA, eRNA (enhancer RNA), shRNA (small hairpin RNA), stRNA (small temporal RNA), DNA, chloroplast DNA, cDNA (complementary DNA), gDNA (genomic DNA), Hachimoji DNA, mitochondrial DNA, msDNA (multicopy single-stranded DNA), XNA (xeno nucleic acid), glycol nucleic acid, threose nucleic acid, hexose nucleic acid, LNA (locked nucleic acid), PNA (peptide nucleic acid), morpholino oligomers, antisense oligonucleotides, ribozymes, deoxyribozymes, aptamers, cloning vectors, phagemids, plasmids, lambda phage, cosmids, fosmids, or artificial chromosomes. The method comprises delivering (administering) to the patient an amount of the LNP containing the polyanionic therapeutic agent effective to treat a patient. The therapeutic agent may be delivered by any suitable route, and by any suitable dosage regimen effective to treat the patient.

[00032] An “effective amount” or “amount effective” to achieve a desirable therapeutic, pharmacological, medicinal, or physiological effect is any amount that achieves the stated purpose. For example, an effective amount is a bioactive amount of a therapeutic agent delivered to the patient, or an organ or tissue of the patient. Based on the teachings provided herein, one of ordinary skill can readily ascertain effective amounts of the elements of the described dosage form and produce a safe and effective dosage form and drug product.

[00033] A therapeutic agent is any compound or composition that is delivered to a patient to achieve a desired effect, such as a beneficial, treatment, or curative effect. Therapeutic agents include proteins, such as polypeptides or proteins. In the context of the present invention, therapeutic agents are polyanions, such as single stranded or double stranded nucleic acids or nucleic acid analogs having a negatively-charged backbone, including, without limitation single-stranded DNA, single-stranded RNA, double-stranded DNA, double-stranded RNA, or modified versions of any of the preceding, or analogs of any of the preceding. With regard to overall structure and function of the nucleic acid or nucleic acid analog, the nucleic acid or nucleic acid analog may be, without limitation: mRNA, siRNA, miRNA (microRNA), gRNA (guide RNA), tRNA (transfer RNA), rRNA (ribosomal RNA), tmRNA (transfer-messenger RNA), piRNA (Piwi-interacting RNA), snRNA (small nuclear RNA), snoRNA (small nucleolar RNA), scaRNAs (small Cajal body RNA), Y RNA, eRNA (enhancer RNA), shRNA (small hairpin RNA), stRNA (small temporal RNA), DNA, chloroplast DNA, cDNA (complementary DNA), gDNA (genomic DNA), Hachimoji DNA, mitochondrial DNA, msDNA (multicopy single-stranded DNA), XNA (xeno nucleic acid), glycol nucleic acid, threose nucleic acid, hexose nucleic acid, LNA (locked nucleic acid), PNA (peptide nucleic acid), morpholino oligomers, antisense oligonucleotides, ribozymes, deoxyribozymes, aptamers, cloning vectors, phagemids, plasmids, lambda phage, cosmids, fosmids, or artificial chromosomes.

[00034] Nucleic acids and nucleic acid analogs comprise a backbone and a sequence of nucleobases. In the context of the present disclosure, the backbone monomer residues can be any suitable nucleic acid backbone monomer residues having a negative charge, such as a ribose or deoxyribose connected by a phosphodiester bond, or a backbone residue of a nucleic acid analog monomer. The backbone monomer includes both the structural“residue” component, such as the ribose in RNA, and any active groups that are modified in linking monomers together, such as the 5’ triphosphate and 3’ hydroxyl groups of a ribonucleotide, which are modified when polymerized into RNA to leave a negatively-charged phosphodiester linkage. Non-limiting examples of common nucleic acid analogs include peptide nucleic acids, such as phosphorothioate nucleic acids, locked nucleic acid (2’-0-4’-C- methylene bridge, including oxy, thio or amino versions thereof), unlocked nucleic acid (the C2’-C3’ bond is cleaved), 2’-0-methyl-substituted RNA, morpholino nucleic acid, threose nucleic acid, and glycol nucleic acid, among others, as are broadly-known.

[00035] Nucleobases are recognition moieties that bind specifically to one or more of adenine, guanine, thymine, cytosine, and uracil, e.g., by Watson-Crick or Watson-Crick-like base pairing by hydrogen bonding. A“nucleobase” includes primary nucleobases: adenine, guanine, thymine, cytosine, and uracil, as well as modified purine and pyrimidine bases, such as, without limitation, hypoxanthine, xanthene, 7- methylguanine, 5, 6, di hydrouracil, 5-methylcytosine, and 5-hydroxymethylcytosine (see also, e.g., U.S. Patent Application Publication No. 2013/0245107 A1 for additional nucleobase or nucleic acid modifications). Additional purine, purine-like, pyrimidine and pyrimidine-like nucleobases are known in the art, for example as disclosed in United States Patent Nos. 8,053,212, 8,389,703, and 8,653,254. Nucleobases may be divalent, e.g., as in International Patent Publication Nos. WO 2014/169206 and WO 2018/058091 . Suitable nucleic acids, or modified nucleic acids, such as nucleic acid analogs are broadly-known to those of skill in the art.

[00036] A“gene” is a sequence of DNA or RNA which codes for a molecule, such as a protein or a functional RNA, such as a ncRNA that has a function. Complementary refers to the ability of polynucleotides (nucleic acids) to hybridize to one another, forming inter-strand base pairs. Base pairs are formed by hydrogen bonding between nucleotide units in antiparallel polynucleotide strands. Complementary polynucleotide strands can base pair (hybridize) in the Watson-Crick manner (e.g., A to T, A to U, C to G), or in any other manner that allows for the formation of duplexes. [00037] Gene expression is the process by which information from a gene is used in the synthesis of a functional gene product, e.g., a protein or functional RNA. Gene expression involves various steps, including transcription, translation, and post- translational modification of a protein.

[00038] Transcription is the process by which the DNA gene sequence is transcribed into pre-mRNA (messenger RNA). The steps include: RNA polymerase, together with one or more general transcription factors, binds to promoter DNA. Transcription factors (TFs) are proteins that control the rate of transcription of genetic information from DNA to messenger RNA, by binding to a specific DNA sequence (i.e., the promoter region). The function of TFs is to regulate genes in order to make sure that they are expressed in the right cell at the right time and in the right amount throughout the life of the cell and the organism. The promoter region of a gene is a region of DNA that initiates transcription of that particular gene. Promoters are located near the transcription start sites of genes, on the same strand, and often, but not exclusively, are upstream (towards the 5' region of the sense strand) on the DNA. Promoters can be about 100-1000 base pairs long. Additional sequences and noncoding elements can affect transcription rates. If the cell has a nucleus (eukaryotes), the RNA is further processed. This includes polyadenylation, capping, and splicing. Polyadenylation is the addition of a poly(A) tail to a messenger RNA. The poly(A) tail consists of a chain of adenosine monophosphates. In eukaryotes, polyadenylation is part of the process that produces mature messenger RNA (mRNA) for translation. Capping refers to the process wherein the 5’ end of the pre-mRNA has a specially altered nucleotide. In eukaryotes, the 5’ cap (cap-0), found on the 5’ end of an mRNA molecule, consists of a guanine nucleotide connected to mRNA via an unusual 5’ to 5’ triphosphate linkage. During RNA splicing, pre-mRNA is edited. Specifically, during this process, introns are removed, and exons are joined together. The resultant product is known as mature mRNA. The RNA may remain in the nucleus or exit to the cytoplasm through the nuclear pore complex.

[00039] mRNA encoding a protein can be delivered to cells by the lipid particles, such as lipid nanoparticles, and methods described herein to produce the protein in the cells. The protein may be therapeutic. mRNA can be produced by in vitro transcription (IVT) as is broadly-known (see, e.g., Kwon, et al. Emergence of synthetic mRNA: In vitro synthesis of mRNA and its applications in regenerative medicine. Biomaterials 156; February 2018:172-193, describing the design and preparation of IVT mRNA, chemical modification of IVT mRNA, and therapeutic applications of IVT mRNA in regenerative medicine, cellular reprogramming, stem cell engineering, and protein replacement therapy).

[00040] RNA levels in a cell, e.g., mRNA levels, can be controlled post- transcriptionally. Native mechanisms for controlling RNA levels include endogenous gene silencing mechanisms, interference with translational mechanisms, interference with RNA splicing mechanisms, and destruction of duplexed RNA by RNAse H, or RNAse H-like activity. As is broadly-recognized by those of ordinary skill in the art, these endogenous mechanisms can be exploited to decrease or silence mRNA activity in a cell or organism in a sequence-specific, targeted manner. Antisense technology typically involves administration of a single-stranded antisense oligonucleotide (ASO) that is chemically-modified, e.g., as described herein, for bio-stability, and is administered in sufficient amounts to effectively penetrate the cell and bind in sufficient quantities to target mRNAs in cells. RNA interference (RNAi) harnesses an endogenous and catalytic gene silencing mechanism, which means that once, e.g., a microRNA, or double-stranded siRNA has been delivered into the cytosol, they are efficiently recognized and stably incorporated into the RNA-induced silencing complex (RiSC) to achieve prolonged gene silencing. Both antisense technologies and RNAi have their strengths and weaknesses, either may be used effectively to knock-down or silence expression of a gene or gene product (see, e.g., Watts, J.K., et al. Gene silencing by siRNAs and antisense oligonucleotides in the laboratory and the clinic J Pathol (2012) 226(2):365-379).

[00041] The terms "iRNA," "RNAi agent," "RNAi agent," and "RNA interference agent" as used interchangeably herein, refer to an agent that contains RNA nucleotides, and which mediates the targeted cleavage of an RNA transcript via an RNA-induced silencing complex (RISC) pathway. iRNA directs the sequence-specific degradation of mRNA through a process known as RNA interference (RNAi). The iRNA modulates, e.g., knocks down or silences, the expression of an RNA in a cell, e.g., a cell within a subject, such as a mammalian subject.

[00042] In one aspect, an RNAi agent includes a single stranded RNAi that interacts with a target RNA sequence to direct the cleavage of the target RNA. Without wishing to be bound by theory, it is believed that long double stranded RNA introduced into cells is broken down into double stranded short interfering RNAs (siRNAs) comprising a sense strand and an antisense strand by a Type III endonuclease known as Dicer. Dicer, a ribonuclease-lll-like enzyme, processes these dsRNA into 19-23 base pair short interfering RNAs with characteristic two base 3' overhangs. These siRNAs are then incorporated into an RNA-induced silencing complex (RISC) where one or more helicases unwind the siRNA duplex, enabling the complementary antisense strand to guide target recognition. Upon binding to the appropriate target mRNA, one or more endonucleases within the RISC cleave the target to induce silencing. Thus, in one aspect, the invention relates to a single stranded RNA (ssRNA) (the antisense strand of an siRNA duplex) generated within a cell and which promotes the formation of a RISC complex to effect silencing of the target gene. Accordingly, the term "siRNA" is also used herein to refer to an interfering RNA (iRNA).

[00043] In another aspect, the RNAi agent may be a single-stranded RNA that is introduced into a cell or organism to inhibit a target mRNA. Single-stranded RNAi agents bind to the RISC endonuclease, Argonaute 2, which then cleaves the target mRNA. The single-stranded siRNAs are generally 15-30 nucleotides and are chemically modified. The design and testing of single-stranded RNAs are described in U.S. Patent No. 8,101 ,348 and in Lima et al., (2012) Cell 150:883-894. Any of the RNAi agents described herein may be used as a single- stranded siRNA as described herein or as chemically modified by the methods described in Lima et al.

[00044] In another aspect, an "iRNA" or RNAi agent” for use in the compositions and methods described herein is a double stranded RNA and can be referred to herein as a "double stranded RNAi agent," "double stranded RNA (dsRNA) molecule," "dsRNA agent," or "dsRNA". The term "dsRNA", refers to a complex of ribonucleic acid molecules, having a duplex structure comprising two anti-parallel and substantially complementary nucleic acid strands, referred to as having "sense" and "antisense" orientations with respect to a target RNA. In some aspects, a double stranded RNA (dsRNA) triggers the degradation of a target RNA, e.g., an mRNA, through a post- transcriptional gene-silencing mechanism referred to herein as RNA interference or RNAi.

[00045] The majority of nucleotides of each strand of a dsRNA molecule may be ribonucleotides, but as described in detail herein, each or both strands can also include nucleotide analogs, where one or more non-ribonucleotides, e.g., a deoxyribonucleotide and/or a modified nucleotide. In addition, as used in this specification, an "RNAi agent" or“RNAi agent” may include ribonucleotides with chemical modifications; an RNAi agent may include substantial modifications at multiple nucleotides. As used herein, the term "modified nucleotide" refers to a nucleotide having, independently, a modified sugar moiety, a modified inter-nucleotide linkage, and/or modified nucleobase. Thus, the term modified nucleotide encompasses substitutions, additions or removal of, e.g., a functional group or atom, to inter-nucleoside linkages, sugar moieties, or nucleobases. The modifications suitable for use in the agents described herein include all types of modifications disclosed herein or known in the art. Any such modifications, as used in a siRNA type molecule, are encompassed by "RNAi agent" or“RNAi reagent” for the purposes of this disclosure.

[00046] The duplex region may be of any length that permits specific degradation of a desired target RNA through a RISC pathway, and may range from about 9 to 36 base pairs in length, e.g., about 15-30 base pairs in length, for example, about 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, or 36 base pairs in length, such as about 15-30, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21 , 15-20, 15- 19, 15-18, 15- 17, 18-30, 18-29, 18-28,

18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21 , 18-20, 19-30, 19-29, 19-28, 19-27,

19-26, 19-25, 19-24, 19-23, 19-22, 19-21 , 19-20, 20-30, 20-29, 20-28, 20-27, 20-26,

20-25, 20-24,20-23, 20-22, 20-21 , 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24,

21-23, or 21-22 base pairs in length. Ranges and lengths intermediate to the above recited ranges and lengths are also contemplated to be part of the invention.

[00047] The two strands forming the duplex structure may be different portions of one larger RNA molecule, or they may be separate RNA molecules. Where the two strands are part of one larger molecule, and therefore are connected by an uninterrupted chain of nucleotides between the 3'-end of one strand and the 5'-end of the respective other strand forming the duplex structure, the connecting RNA chain is referred to as a "hairpin loop." A hairpin loop can comprise at least one unpaired nucleotide. In some aspects, the hairpin loop can comprise at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 23, or more unpaired nucleotides. In some aspects, the hairpin loop can be 10 or fewer nucleotides. In some aspects, the hairpin loop can be 8 or fewer unpaired nucleotides. In some aspects, the hairpin loop can be 4-10 unpaired nucleotides. In some aspects, the hairpin loop can be 4-8 nucleotides.

[00048] Where the two substantially complementary strands of a dsRNA are comprised by separate RNA molecules, those molecules need not, but can be covalently connected. Where the two strands are connected covalently by means other than an uninterrupted chain of nucleotides between the 3'-end of one strand and the 5'-end of the respective other strand forming the duplex structure, the connecting structure is referred to as a "linker." The RNA strands may have the same or a different number of nucleotides. The maximum number of base pairs is the number of nucleotides in the shortest strand of the dsRNA minus any overhangs that are present in the duplex. In addition to the duplex structure, an RNAi agent may comprise one or more nucleotide overhangs.

[00049] In one aspect, an RNAi agent is a dsRNA, each strand of which comprises 19-23 nucleotides, that interacts with a target RNA sequence without wishing to be bound by theory, long double stranded RNA introduced into cells is broken down into siRNA by a Type III endonuclease known as Dicer. Dicer, a ribonuclease-lll-like enzyme, processes the dsRNA into 19-23 base pair short interfering RNAs with characteristic two base 3' overhangs. The siRNAs are then incorporated into an RNA-induced silencing complex (RISC) where one or more helicases unwind the siRNA duplex, enabling the complementary antisense strand to guide target recognition. Upon binding to the appropriate target RNA, one or more endonucleases within the RISC cleave the target to induce silencing. In one aspect, an RNAi agent is a dsRNA of 24-30 nucleotides that interacts with a target RNA sequence to direct the cleavage of the target RNA.

[00050] Lipid particles, and more specifically lipid nanoparticles (lipidoid nanoparticles, LNPs), as a class, have been demonstrated as being effective in delivering nucleic acids and nucleic acid analogs to cells, often in vivo (see, e.g., Kulkarni, JA, et al.,“Lipid Nanoparticles Enabling Gene Therapies: From Concepts to Clinical Utility” Nucleic Acid Ther. 2018 Jun;28(3): 146-157. doi:

10.1089/nat.2018.0721 , Hajj, KA, and KA Whitehead, Tools for translation: non-viral materials for therapeutic mRNA delivery, Nature Reviews Materials, Volume 2, Issue 10, pp. 17056 (2017), doi: 10.1038/natrevmats.2017.56, U.S. Patent Application Publication No. 20130245107 A1 , and U.S. Patent No. 8,754,062, describing exemplary lipid nanoparticles, including description of different constituents thereof, and methods of making and using lipid nanoparticles). In various embodiments, a lipid particle, e.g. a lipid nanoparticle comprises a mixture of an ionizable and/or cationic lipid, cholesterol or a cholesterol analogue, a helper lipid, such as DSPC, DOPC (dioleoyl phosphatidylcholine), DSPE (distearoyl phosphatidylethanolamine), or DOPE, a polyethylene glycol)-lipid conjugate (PEG-lipid) or PEG-cholesterol conjugate (PEG-cholesterol), and a polyanionic therapeutic agent, such as a nucleic acid or nucleic acid analog. For targeting purposes, the PEG-lipid or PEG-cholesterol may be modified with a targeting moiety, such as N-acetylgalactosamine (GalNAc) for liver targeting, or another ligand or binding reagent, such as an antibody or antibody fragment. In one non-limiting example, the lipid particle or lipid nanoparticle includes, along with the nucleic acid, e.g., siRNA, heptatriaconta-6,9,28,31 -tetraen-19-yl 4- (dimethylamino)butanoate (DLin-MC3-DMA, MC3, see, U.S. Patent Application Publication No. 2013/0245107 A1 ), 1 ,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol, and polyethylene glycol-dimyristolglycerol (PEG-DMG), e.g., at a molar ratio of 50:10:38.5:1 .5 (see, e.g., Tam, Y.Y.C. et at. “Advances in Lipid Nanoparticles for siRNA Delivery” Pharmaceutics 2013, 5, 498-507; doi:10.3390/pharmaceutics5030498). In environments where the pH is below the pKa of the ionizable lipid (e.g., pH 4.0), the amino group of MC3 is protonated and interacts with the negatively charged nucleic acids, thereby promoting the self-assembly of the formulation components into particles, such as nanoparticles, encapsulating the siRNA.

[00051] As shown herein, by adding a polyanionic helper polymer, such as a negatively-charged polymer, to the nucleic acid or nucleic acid analog to be incorporated into the lipid particle, significantly lower amounts of siRNA is required to achieve the same therapeutic effect as the same lipid nanoparticles that do not comprise the polyanionic helper polymer.

Ionizable cationic lipid

[00052] Useful ionizable or cationic lipids include a lipid moiety attached to (covalently-linked to) an ionizable or cationic group. Effective ionizable lipids tend to be positively charged at low pH (which aids in RNA complexation when it is carried out in acidic buffer) but are neutral at physiological pH (for reduced toxicity post injection). Furthermore, cellular uptake via endocytosis deposits nanoparticles into endosomal compartments, which slowly reduce their pH from approximately 6.8 to 4.5 as they morph into lysosomes. An ability for nanoparticles to ionize as the pH drops appears to be relevant to the endosomal escape process (Hajj, KA, and KA Whitehead, Nature Reviews Materials, Volume 2, Issue 10, pp. 17056 (2017)). United States Patent Nos. 8,450,298, 8,754,062, 8,969,353, 9,227,917, 9,439,968, 9,556,1 10, and 10,189,802, each of which is hereby incorporated by reference, describe various ionizable or cationic lipids useful in the formulation of LNPs. United States Patent No. 9,439,968 discloses tetrakis(8-methylnonyl) 3,3',3",3"'-(((methylazanediyl)bis(propane-3, 1 - diyl))bis(azanetriyl)) tetrapropionate (3060iio), which is described in the examples below. In one non-limiting example, the ionizable or cationic lipid is DLin-MC3-DMA, 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1 ,3]-dioxolane (DLin-KC2-DMA, KC2, see, e.g., U.S. Patent No. 8,754,062), or cKK-E12

and especially MC3 showing good promise in the delivery of siRNA reagents. In examples, the weight ratio of ionizable lipid to RNA in a typical LNP is between 5:1 and 15:1 .

Helper lipid

[00053] The helper lipid is a cationic, anionic, neutral, or zwitterionic amphiphilic lipid that, along with cholesterol or a cholesterol analog, aids in the molecular packing and stability of the particle. Helper lipids also enhance lipid nanoparticle efficacy by promoting fusion with both cell and endosomal membranes, facilitating cell uptake and endosomal release. Useful helper lipids include, for example and without limitation, DSPC, DSPE, DOPC, and DOPE. Additional useful helper lipids are known in the art, e.g. phosphatidylcholine lipids. In examples, the molar ratio of ionizable lipid to helper lipid in a typical LNP ranges from 1 : 0.001 to 1 : 0.5. In examples, the molar ratio of ionizable lipid to cholesterol or cholesterol analogue in a typical LNP ranges from 1 : 0.75 to 1 : 1.35. Non-limiting examples of cholesterol analogues include: oxidized cholesterol, desmosterol, 7-dehydrocholesterol, ergosterol, lanosterol, ketosterone, cholesterol sulfate, dehydroergosterol, cholestratrienol, 5-cholestene, or pregnenolone.

PEG -lipid and PEG-cholesterol

[00054] PEG-lipids (PEGylated lipids) comprise a polyethylene glycol) moiety attached to one or more lipid moieties, for example and without limitation, with a ceramide, succinoyl, or carbamate linkage. PEG-cholesterol (PEGylated cholesterol) comprise a polyethylene glycol) moiety attached to one or more cholesterol moieties. PEG-lipids and/or PEG-cholesterols form a protective, non-aggregating, non- immunogenic shell around the surface of the LNP. Depending on the ultimate delivery route of the LNP, the lipid group may be varied, e.g., in length, to dictate how long the PEG-lipid is associated with the LNP, with longer lipid chains tending to remain associated with the LNP for longer time periods, with shorter lipid chains being useful in providing“diffusible” PEG lipids that diffuse from the lipid nanoparticle quickly to produce an LNP with increased transfection rates in many situations, and when desirable. The PEG group or moiety of the PEG-lipid or PEG-cholesterol typically has a molecular weight ranging from 300 g/mol to 5000 g/mol, e.g., ~2000 g/mol (referred to as PEG 2000). Non-limiting examples of suitable PEG-lipids include: 1 ,2- Dimyristoyl-sn-Glycero-3-Phosphoethanolamine-N [Methoxy(Polyethylene glycol)- 2000], N-octanoyl-sphingosine-1 -{succinyl[methoxy(polyethylene glycol)2000]}, N- palmitoyl-sphingosine-1 -{succinyl[methoxy(polyethylene glycol)5000]}, 1 ,2- dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(poly ethylene glycol)- 3000] (ammonium salt), 1 ,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy(polyethylene glycol)-1000] (ammonium salt), and PEG-cholesterol, such as cholesterol-(polyethylene glycol-600). In examples, the molar ratio of ionizable lipid to PEG-lipid or PEG-cholesterol in a typical LNP ranges from 1 : 0.01 to 1 : 0.1 .

Targeting ligand

[00055] In aspects, targeting of the LNP may be achieved by including in the LNP a ligand specific to the cells to be targeted attached to a lipid moiety, such as attaching the ligand to a PEG moiety of the PEG-lipid, or attaching the ligand to a lipid moiety via any suitable linker or spacer, as are broadly-known. Suitable ligands include cell penetrating peptides (CPPs), transferrin, folate, oligosaccharides, polysaccharides, and antibodies or other binding reagents, e.g., GalNAc for liver targeting.

[00056] The term“ligand” refers to a binding moiety for a specific target, referred to as its binding partner. The molecule can be a cognate receptor, a protein a small molecule, a hapten, or any other relevant molecule. The term“antibody” refers to an immunoglobulin, derivatives thereof which maintain specific binding ability, and proteins having a binding domain which is homologous or largely homologous to an immunoglobulin binding domain. As such, the antibody operates as a ligand for its cognate antigen, which can be virtually any molecule. Natural antibodies comprise two heavy chains and two light chains and are bi-valent. The interaction between the variable regions of heavy and light chain forms a binding site capable of specifically binding an antigen (e.g., a paratope). The term“VH” refers to a heavy chain variable region of an antibody. The term “VL” refers to a light chain variable region of an antibody. Antibodies may be derived from natural sources, or partly or wholly synthetically produced. An antibody may be monoclonal or polyclonal. The antibody may be a member of any immunoglobulin class, including any of the human classes: IgG, IgM, IgA, IgD, and IgE.

[00057] Ligands, also referred to as binding reagents, having limited crossreactivity are generally preferred. In certain embodiments, suitable ligands include, for example, polypeptides, such as for example, antibodies, monoclonal antibodies, or derivatives or analogs thereof, including without limitation: Fv fragments, single chain Fv (scFv) fragments, Fabi fragments, F(ab')2 fragments, single domain antibodies, camelized antibodies and antibody fragments, humanized antibodies and antibody fragments, and multivalent versions of the foregoing; multivalent binding reagents including without limitation: monospecific or bispecific antibodies, such as disulfide stabilized Fv fragments, scFv tandems ((ScFv)2 fragments), diabodies, triabodies, tetrabodies, which typically are covalently linked or otherwise stabilized (i. e., leucine zipper or helix stabilized) scFv fragments, di-scFv (dimeric single-chain variable fragment), single-domain antibody (sdAb), or antibody binding domain fragments and other binding reagents including, for example, bi-specific T-cell engagers (BiTEs), aptamers, template imprinted materials, and organic or inorganic binding elements. In exemplary embodiments, a ligand specifically interacts with a single epitope. In other embodiments, a ligand may interact with several structurally related epitopes. [00058] The term“antibody fragment” refers to any derivative of an antibody which is less than full-length. In exemplary embodiments, the antibody fragment retains at least a significant portion of the full-length antibody's specific binding ability. Examples of binding reagents, including antibody fragments, but are not limited to, Fab, Fab', F(ab')2, Fv, Fd, dsFv, scFv, diabody, triabody, tetrabody, di-scFv (dimeric single-chain variable fragment), bi-specific T-cell engager (BiTE), single-domain antibody (sdAb), or antibody binding domain fragments. The antibody fragment may be produced by any means. For instance, the antibody fragment may be enzymatically or chemically produced by fragmentation of an intact antibody, or it may be recombinantly or partially synthetically produced. The antibody fragment may optionally be a single chain antibody fragment. Alternatively, the fragment may comprise multiple chains which are linked together, for instance, by disulfide linkages. The fragment may also optionally be a multimolecular complex. A functional antibody fragment will typically comprise at least about 50 amino acids and more typically will comprise at least about 200 amino acids. Antibody fragments also include miniaturized antibodies or other engineered binding reagents that exploit the modular nature of antibody structure, comprising, often as a single chain, one or more antigenbinding or epitope-binding sequences and at a minimum any other amino acid sequences needed to ensure appropriate specificity, delivery, and stability of the composition (see, e.g., Nelson, AL, “Antibody Fragments Flope and Flype” (2010) MAbs 2(1 ):77-83).

Polyanionic helper polymer

[00059] In the context of the present disclosure, the helper polymer is a polyanion, having an overall negative charge, e.g. a pKa of less than 4, e.g., less than 3, or 2, or approximately 1 . A polyanionic polymer may have a hydrocarbon backbone, as with polyvinyl or olefinic polymers and may have the structure -[C-C(R)]-, wherein R is a moiety comprising an acidic group or its conjugate base, such as sulfonate, carboxyl, and phosphinic acid (HOP(O)H-) groups, linked to the backbone either directly or through a C1-C6 aliphatic or aromatic hydrocarbon linker. In one example, the hydrocarbon backbone is styrene, where R includes a phenyl moiety, substituted with an acidic group or its conjugate base, such as phosphate, sulfonate, carboxylate, sulphates, alkoxydicyanoethanolates, boronates, phenolates, sulfonamides, sulfonimides, and phosphinic acid. Additional examples of polyanionic polymers useful in the present disclosure include: carboxylate and sulphonate styrene- divinylbenzene copolymer, polyglutamate, and carboxymethylcellulose.

[00060] In aspects, the helper polymer has a number average molecular weight (Mri) ranging from 1 kDa to 200 kDa, for example, from 1 kDa to 100 kDa, or from 5 kDa to 75 kDa. In aspects, the polydispersity index (PDI, where PDI = Mw/Mn, and where Mw is the weight average molecular weight of the polymer) of the helper polymer is less than ranges from 1 to 5, for example, from 1 to 2. In one aspect, the helper polymer is a polystyrene, such as a polystyrene sulfonate having an Mn ranging from 5 kDa to 75 kDa, and a PDI of less than 5. In examples, the weight ratio of helper polymer to RNA in a typical LNP is between 1 1 :1 and 0.04 : 1 .

[00061] In aspects, the therapeutic agent is an siRNA reagent, and the helper polymer is an mRNA or other low-PDI (e.g., having a PDI of less than 2) nucleic acid. In aspects, the mRNA encodes a protein. In aspects the protein is a therapeutic protein.

[00062] As indicated above, the LNP described herein comprises an ionizable or cationic lipid (ICL), cholesterol, a helper lipid, a polyethylene glycol)-lipid conjugate (PEG-lipid), a polyanionic therapeutic agent, and a helper polymer. The relative ratio of each component will vary depending on the selected constituent. Examples of specific LNP formulations are provided in the examples below. For RNA-reagents, a suitable weight ratio of total lipid (referring to the sum of the weights of ionizable or cationic lipid (ICL), cholesterol, helper lipid, PEG-lipid, and any other lipid or lipidoid constituents) to RNA, e.g., mRNA or siRNA may be between 10:1 and 30:1. In aspects, the range of ratios of various constituents of the LNPs for delivery of mRNA and/or siRNA, described herein may be as indicated in Table 1 , providing two ranges (A and B) for each example.

Table 1 - composition ranges for exemplary mRNA and/or siRNA LNPs

Examples

[00063] While mRNA and siRNA have significant therapeutic potential, their simultaneous delivery has not been previously explored. To facilitate the treatment of diseases associated with aberrant gene upregulation and downregulation, we sought to co-formulate siRNA and mRNA in a single lipidoid nanoparticle (LNP) formulation.

[00064] We accommodated the distinct molecular characteristics of mRNA and siRNA in a formulation consisting of an ionizable and biodegradable amine-containing lipidoid, cholesterol, DSPC, DOPE, and PEG-lipid. Surprisingly, the co-formulation of siRNA and mRNA in the same LNP enhanced the efficacy of both drugs in vitro and in vivo. Compared to LNPs encapsulating siRNA only, co-formulated LNPs improved Factor VII gene silencing in mice from 44 to 87% at an siRNA dose of 0.03 mg/kg. Coformulation also improved mRNA delivery, as a 0.5 mg/kg dose of mRNA coformulated with siRNA induced three times the luciferase protein expression compared to when siRNA was not included. As not all gene therapy applications require both RNA drugs, we sought to extend the synergy of co-formulated LNPs to formulations encapsulating only a single type of RNA. We accomplished this by substituting the “helper” RNA with a negatively charged polymer, polystyrene sulfonate (PSS). LNPs containing PSS mediated the same level of protein silencing or expression as standard LNPs using 2-3 fold less RNA. For example, LNPs formulated with and without PSS induced 50% Factor VII silencing at siRNA doses of 0.01 and 0.03 mg/kg, respectively. Together, these studies demonstrate potent co-delivery of siRNA and mRNA and show that inclusion of a negatively charged“helper polymer” enhances the efficacy of LNP delivery systems.

[00065] RNA drugs, including short interfering RNA (siRNA) and messenger RNA (mRNA), can theoretically treat any disease caused by gene dysregulation. Conditions associated with protein overexpression may benefit from siRNA drugs, which inhibit protein production by cleaving mRNA. On the other hand, diseases caused by insufficient protein production are candidates for mRNA therapy. Both types of RNA therapy have made significant translational progress over the past several years, often being delivered in ionizable polymer or lipid nanoparticles. The clinical translation of siRNA therapy hit a major milestone in 2017 with the completion of the first successful Phase 3 clinical trial by Alnylam Pharmaceuticals.

[00066] Delivery of both RNAs would enable simultaneous knockdown of undesirable protein(s) and expression of desirable protein(s). Such an approach would apply to many diseases, including liver cancer, which is characterized by the upregulation of oncogenes and downregulation of tumor suppressor genes. Encapsulation of siRNA and mRNA in a single particle guarantees that transfected cells receive both drugs, maximizing the intended therapeutic effect. A single formulation would also reduce production costs and regulatory hurdles compared to a therapy comprising two separate siRNA and mRNA formulations. Given the considerably different molecular characteristics of siRNA and mRNA, successful coformulation requires nanoparticle chemistry that accommodates both therapeutic molecules. For example, siRNA and mRNA have drastically different molecular weights (10 ⁴ vs. 10 ⁶ g/mol), stability, and molecular conformation.

[00067] Ionizable lipid nanoparticles (LNPs) have shown significant translational promise in the delivery of siRNA and, separately, mRNA. Typically, the LNPs used to encapsulate siRNA and mRNA consist of the same four primary components: an ionizable lipid or lipidoid compound, cholesterol, a helper lipid, and polyethylene glycol (PEG)-lipid. However, the ratio of these components, together with the ratio of the ionizable lipid to RNA can significantly alter delivery efficacy in vitro and in vivo. Previous work has demonstrated that siRNA and mRNA are most effectively delivered in distinct LNP formulations. Herein, we describe the identification of a single LNP formulation capable of simultaneous delivery of siRNA and mRNA. We chose to work with the ionizable, biodegradable, amine-containing lipidoid 3060no, which we identified as a potent RNA delivery material.

[00068] Given the vast formulation space available for testing, we began with two LNP formulations for potent siRNA or mRNA delivery. We will refer to these preparations as Formulations 1 and 5, respectively (Table 2). Compared to Formulation 1 , Formulation 5 features a higher lipidoid to RNA ratio and a lower molar percentage of lipidoid in relation to helper lipid, cholesterol, and PEG-lipid. Additionally, the helper lipid shifts from DSPC in Formulation 1 to DOPE in Formulation 5. DSPC contains two saturated aliphatic tails, while DOPE contains a c/s-double bond in each of its two aliphatic tails. This structural difference changes the packing within the lipid nanoparticle and can affect endosomal escape, which is important given the significant molecular differences between siRNA and mRNA.

Table 2. Five LNP formulations were investigated for co-delivery of siRNA and mRNA. The percentages shown are molar. DSPC = 1 ,2-distearoyl-sn-glycero- 3phosphocholine. DOPE = 1 ,2-dioleoyl-sn-glycero-3-phosphoethanolamine.

[00069] In addition to Formulations 1 and 5, which had been designed for siRNA and mRNA delivery, respectively, we evaluated three intermediate formulations. These preparations, which we refer to as Formulations 2, 3, and 4, spanned the molar and weight ratios of Formulations 1 and 5 (Table 2). Notably, each intermediate formulation contained a blend of the helper lipids DSPC and DOPE. [00070] We tested the efficacy of the five formulations for co-delivery of siRNA and mRNA in HeLa cells that stably expressed firefly and Renilla luciferase (Figure 1 ). For these experiments, siRNA targeted the firefly luciferase gene and mRNA encoded the fluorescent protein, mCHERRY. The nanoparticles were incubated with HeLa cells for 24 hours at an siRNA dose of 10 nM (27 ng) and mRNA dose of 1 .5 nM (100 ng). All five formulations were tested with cargos of siRNA only (circles), mRNA only (squares), and a mixture of siRNA with mRNA (triangles). Figure 1 (A) shows resultant luciferase gene silencing. As expected, control particles containing only mRNA induced no gene knockdown. For particles loaded with siRNA, gene silencing increased with increasing formulation number. We were surprised to note that nanoparticles co-formulated with siRNA and mRNA (purple triangles) mediated significantly higher levels of gene silencing compared to LNPs containing siRNA only (blue circles). When the same set of cells were assessed for mRNA delivery, higher formulation numbers again produced better results (Figure 1 (B)). The co-formulated LNPs did not produce statistically significant mCherry production compared to the mRNA-only nanoparticles. The improved delivery observed at higher formulation numbers was at least partially due to increases in RNA entrapment (Figure 1 (C)). LNP size corresponded to the size of the RNA cargo but not the formulation number (Figure 1 (D)).

[00071] Given the successful co-delivery of siRNA and mRNA in HeLa cells, the five formulations from Table 2 were also tested for co-delivery efficacy in mice. Because 3060no LNPs deliver RNA to the liver, we formulated LNPs with siRNA specific for the protein Factor VII (FVII). Factor VII, a blood clotting factor produced by hepatocytes, is readily measured from a blood sample using a commercially available assay. The mRNA chosen for in vivo studies encoded firefly luciferase (mLuc). 3060no has the structure:

[00072] Mice were injected by tail vein with LNPs loaded with RNA doses of 0.03 mg/kg siFVII and/or 0.5 mg/kg mLuc. Six hours post-injection, mRNA delivery- mediated luciferase expression was assessed using whole body luminescence imaging. Efficacy of siRNA delivery was measured 48 hours after injection by quantifying serum Factor VII protein levels. As shown in Figure 2A, Formulations 3-5 loaded with both RNAs produced the highest levels of Factor VII silencing (~90%, circles). This degree of knockdown was markedly better than when siRNA was delivered in Formulation 1 (~50% silencing, triangle) and the mRNA control (0% silencing, square). Co-delivery of siRNA and mRNA in Formulations 4 and 5 also improved mRNA delivery compared to LNPs containing only mRNA or only siRNA (Figures 2 (B) and 3 (A)). Data in Figure 2 (A and B) were collected from the same animals. No weight loss was observed for any of the groups (Figure 3 (B)). As with in vitro studies, RNA entrapment improved with increasing formulation number (Figure 2 (C)). Size was relatively consistent across formulations (Figure 3 (C)). Based on these efficacy data, Formulation 4 was chosen for the remainder of experiments.

[00073] Next, we confirmed that LNPs co-formulated with mRNA and siRNA were more potent than LNPs containing a single RNA species (Figure 2 (D)). We compared efficacy in two groups of mice: one group received a mixture of siRNA- formulated LNPs and mRNA-formulated LNPs (i.e., each particle contained only one type of RNA). The second group received LNPs that had been co-formulated (i.e., each particle contained both RNAs). All LNPs were made using Formulation 4. Both treated groups received an siFVII dose of 0.03 mg/kg and an mLuc dose of 0.5 mg/kg. Remarkably, co-formulated LNPs improved Factor VII silencing from 50 to 90% (open circles) and tripled luciferase expression (squares and Figure 3 (D)). The two treated animal groups received the same total amount of each LNP ingredient, including the lipidoid. These results suggest there is a substantial efficacy boost imparted through the co-formulation of siRNA and mRNA in LNPs compared to single RNA species formulations.

[00074] To better understand how co-formulation enhanced the delivery efficacy of both siRNA and mRNA, we investigated the role of total RNA concentration during the co-formulation process. The RNA concentration used for formulation does not necessarily go hand in hand with in vivo dose. In one experiment (Figure 4 (A)), a set of five LNPs were formulated in which the siRNA concentration was held constant at 0.006 mg/mL while the concentration of mRNA was gradually increased from 0 to 0.2 mg/mL. All LNPs were dosed in mice at 0.03 mg/kg of siRNA. The addition of mRNA improved FVII silencing, but only up to a total RNA concentration of 0.106 mg/mL. Beyond that, the benefit of co-formulation diminished and finally disappeared at a total RNA concentration of 0.206 mg/mL.

[00075] The influence of total RNA concentration on mRNA delivery was also investigated (Figure 4 (B)). In this case, LNPs were formulated with a constant concentration of mRNA (0.1 mg/ml_) and an increasing concentration of siRNA (0 - 0.08 mg/ml_). These formulations were dosed in mice at an mLuc dose of 0.5 mg/kg, and resultant luciferase protein expression was assessed 6 hours post-injection (Figure 5). Here, we observed that adding 0.006 mg/ml_ of siRNA to the mRNA formulation improved efficacy. Beyond a total RNA concentration of 0.12 mg/ml_, mRNA delivery efficacy decreased back to“baseline” (mRNA alone) levels. No toxicity, as determined by weight change, was observed for any animal groups (Figure 6 (A and B)). These results underscore the importance of total RNA concentration during LNP formulation on efficacy and suggest that a total RNA concentration of ~0.106 mg/ml_ is optimal.

[00076] The RNA entrapment of co-formulated LNPs was excellent (>85%) except at the highest RNA formulation concentration tested (Figure 6 (C and D)). The z-average size of LNPs remained relatively consistent across formulations (100 - 150 nm) and did not correlate with potency (Figure (E and F)). These differences in entrapment and size for the highest RNA formulation concentration may explain why it was less efficacious than lower concentrations. We also calculated the nitrogen to phosphate ratio (N:P), which stayed fairly constant at approximately 8.4 (Table 3). As such, the differences in formulation efficacy cannot be attributed to N:P.

Table 3

[00077] Table 3. The nitrogen to phosphate (N:P) ratio was constant for formulations including varied total amounts of RNA. LNPs were formulated at the concentrations shown above using formulation #4 from Table 2.

[00078] It is possible that LNPs formulated with increased RNA content and, therefore, more negative charge, were more tightly and stably formed, leading to enhanced LNP efficacy. We noticed that, when measuring RNA entrapment, an unusually high concentration of surfactant was required to disrupt any LNPs formulated at a total RNA concentration greater than 0.1 mg/mL. While some increase in particle density appears to confer efficacy, it is possible that at some increased RNA concentration, intra-particle molecular attractions are strong enough to prevent release of the RNA cargo. This may explain the trends observed in Figure 4 (A and B) in which efficacy improves only until the total RNA concentration reaches 0.106 mg/mL.

We also considered that the increase in efficacy observed with co-formulated LNPs may be due to the increasing amount of lipid. LNPs co-formulated included higher amounts of lipidoid and other lipid content, as we believed this would be necessary to provide enough material to encapsulate the higher RNA amount. Because an increase in lipidoid concentration can sometimes confer LNP efficacy, we asked if this was the reason for enhanced efficacy of both RNA species upon co-formulation.

[00079] To investigate this, we prepared four LNP formulations that all contained the same amount of siRNA. Two of the four formulations contained“helper” mRNA. We evaluated two lipidoid concentrations: the lower concentration (0.05 mg/mL) represented the standard lipidoid concentration used when formulating LNPs encapsulating only siRNA. The higher concentration (0.93 mg/mL) represented the lipidoid concentration we had been using to co-formulate both RNAs. Figure 4 (C) shows Factor VII levels following injecting of the four formulations into mice, all at a dose of 0.03 mg/kg siFVII. LNPs formulated with high lipidoid content and without “helper” mRNA only modestly improved FVII protein silencing from 37 to 49% compared to LNPs formulated at a low lipidoid concentration. We also found no correlation between lipidoid concentration and efficacy when we globally considered all the LNPs formulated and delivered in vivo (Figure 7). Together, these data confirmed that higher amounts of lipidoid were not primarily responsible for the increased efficacy observed with co-formulation. [00080] A marked increase in protein silencing to 87% was observed only when helper mRNA and increased lipidoid were included in the formulation. Efficacy disappeared if LNPs were formulated with both RNA species at a lower lipidoid concentration (rightmost bar in Figure 4 (C)). In that case, RNA entrapment was close to zero (Figure 8 (A)), suggesting that there was not enough lipid to promote LNP formation. Therefore, both a higher lipidoid concentration and increased total RNA content are needed for enhanced LNP efficacy with co-formulation. There was no relationship between LNP diameter and efficacy for the formulations tested in Figure 4 (C), and none of the formulations induced weight loss in mice (Figure 8 (B and C)). While the improvements to both mRNA and siRNA efficacy upon co-formulation are exciting, there are many instances in which treatment will require the delivery of only one type of RNA. Although the addition of a second RNA species to these LNP formulations would increase potency, it would also add cost, regulatory complexity, and the potential for unwanted biological effects. Therefore, we sought an alternative to“helper” RNA that would improve the effectiveness of single RNA formulations. We reasoned that, because RNA is a negatively charged biopolymer, it may be possible to replace it with a negatively charged synthetic polymer.

[00081] We evaluated several inexpensive options: uridine homopolymer (Poly(U)), Poly(sodium 4-sytrenesulfonate) (PSS), and Poly(acrylic acid) (PAA). Poly(U), which is a polymer of the RNA base uracil, is available at a much lower cost than sequence-specific mRNA given the increased simplicity of synthesis. PSS is a synthetic, biocompatible polymer that is used clinically to treat hyperkalemia and lithium poisoning in humans. PAA, another synthetic polymer, has also been used in biomedical applications. To assess whether these polymers could replace the efficacyenhancing effect of helper RNA, we added each of them to the LNP formulation at a concentration that provided the same total negative charge as in co-formulated LNPs. When testing for siRNA efficacy, we found that co-formulation with 70 kDa PSS provided the same degree of potency enhancement as helper mRNA (Figure 9 (A)). Poly(U) also increased protein knockdown compared to LNPs formulated without helper polymer, although not to the same degree as PSS. LNPs formulated with PAA lost siRNA delivery efficacy. LNPs formulated with control siRNA did not alter Factor VII activity regardless of PSS content (gray bars). [00082] We also tested the effect of each polymer on mRNA formulations (Figure 9 (B)). Although 70 kDa PSS did not improve efficacy (data not shown), we found that reducing the molecular weight (Mw) to a size that more closely approximated siRNA (6.8 kDa) resulted in a two-fold increase in protein expression compared to LNPs formulated without helper polymer (Figure 10). LNPs encapsulating control mRNA did not induce luciferase expression with or without PSS. Neither RNA entrapment nor LNP size correlated with efficacy (Figure 1 1 (A-D)). None of the formulations induced weight loss in mice (Figure 1 1 (E-F)).

[00083] One potential reason why PSS best mimics the effect of helper RNA is that it is negatively charged at all pH values relevant to LNP formulation and delivery. Both the PSS monomer and the phosphodiester group in the RNA backbone have pK _a values of one. LNPs are formulated under acidic conditions so that the lipidoid amines protonate and subsequently attract the negatively charged RNA. It is possible that the additional negative charge provided by the polyanion promotes the formation of a more stable and/or compact nanoparticle by increasing the electrostatic attraction inside the particle. PAA, with a higher pK _a of four, may not mediate the same electrostatic effect. Poly(U), while imparting some benefit to siRNA-loaded LNPs, may not facilitate the same intraparticle molecular interactions as mRNA because of its polydispersity (Mw 100 - 1 ,000 kDa).

[00084] Because the addition of 70 kDa PSS to siRNA-loaded LNPs was particularly successful, we wanted to understand how much siRNA drug can be saved by adding this helper polymer to the formulation. We first established that LNPs formulated without PSS and dosed at 0.03 mg/kg siRNA resulted in 49% Factor VII protein silencing (Figure 9 (C)). We then compared this result to when PSS was added to the LNP formulation and dosed from 0.005 - 0.03 mg/kg siRNA. Silencing was dose responsive, with a dose of 0.03 mg/kg now inducing 90% silencing. LNPs formulated with PSS and dosed at 0.01 mg/kg achieved the same level of silencing as those formulated without PSS and dosed at 0.03 mg/kg. In other words, the incorporation of PSS into an LNP formulation facilitated a three-fold reduction in the amount of siRNA required. Importantly, the total amount of lipidoid dosed to mice was the same for each group in Figure 9 (C). As such, the efficacy“savings” afforded by PSS will not be offset by increased toxicity due to increased lipid content. In these experiments, PSS did not improve RNA entrapment, and nanoparticle size did not correlate with efficacy (Figure 12 (A-B)). None of the treatments resulted in significant weight loss compared to the untreated mice (Figure 12 (C)).

[00085] Dual gene therapy that enables simultaneous gene expression and gene silencing through the delivery of mRNA and siRNA has the potential to benefit countless patients. Together, the data presented here show that not only is the coformulation of the two RNA drugs in the same particle possible, but that it substantially enhances efficacy compared to particles formulated with individual RNAs. This observation inspired the development of an improved LNP formulation for the delivery of single RNA species. Specifically, we propose the incorporation of the biocompatible helper polymer, PSS, into lipid nanoparticle formulations for siRNA and mRNA delivery (Table 4). Our results suggest that the additional negative charge provided by the helper polymer may promote electrostatic attraction in the particle and facilitate stability. LNPs containing PSS mediate the same degree of in vivo efficacy with only 1/2 to 1/3 the drug dose, which we anticipate will reduce the cost and potentially the off-targeting events associated with RNA treatment.

Table 4. Incorporation of the biocompatible helper polymer, PSS, into lipid nanoparticle formulations. The percentages shown are molar. DSPC = 1 ,2- distearoyl-sn-glycero-3phosphocholine. DOPE = 1 ,2-dioleoyl-sn-glycero-3- phosphoethanolamine.

Materials and Methods

Materials

[00086] Cholesterol was purchased from Sigma Aldrich (St. Louis, MO), and distearoyl-sn-glycerol-3-phosphocholine (DSPC), 1 ,2-dioleoyl-sn-glycero-3- phosphoethanolamine (DOPE), and 1 ,2-Dimyristoyl-sn-Glycero-3- Phosphoethanolamine-N [Methoxy(Polyethylene glycol)- 2000] (14:0 PEG2000-PE) were obtained from Avanti Polar Lipids (Alabaster, AL). HeLa cells were purchased from American Type Culture Collection (Manassas, VA). Dulbecco’s Modified Eagles Media (DMEM), trypsin, penicillin/streptomycin, phosphate buffered saline (PBS), and fetal bovine serum (FBS) were obtained from Thermo Fisher Scientific (Waltham, MA). Anti-firefly luciferase siRNA for Luciferase (siLuc) was purchased from Dharmacon (Lafayette, CO). Anti-Factor VII siRNA was custom ordered from Sigma Aldrich with a sense sequence of 5’-GGAucAucucAAGucuuAcT*T-3’ and an antisense sequence of 5’-GuAAGAcuuGAGAuGAuccT*T-3’, where lower case nucleotides are 2’-fluoro- modified and asterisks indicate phosphorothioate linkages. Clean Cap™ mCHERRY and firefly luciferase mRNA were purchased from TriLink Biotechnologies (San Diego, CA). Poly sodium 4-styrenesulfonate (PSS Mw 70,000 and 6,800), poly(acrylic acid) (PAA Mv -450,000), and polyuridylic acid potassium salt (Poly(U) Mw 100 - 1 ,000 kDa) were purchased from Sigma Aldrich.

Lipid nanoparticle formulation and characterization

[00087] LNPs were prepared as previously described. The lipidoid 3060no was synthesized by Michael addition of isodecyl acrylate to 3,3’-diamino-N- methyldipropylamine. The fully-substituted version, which was isolated using a Teledyne Isco chromatography system, was used for all experiments. LNPs were formulated by first dissolving the lipidoid, cholesterol, DSPC, DOPE, and PEG2000 in ethanol. A lipid solution was made by mixing these components according to the molar ratios in Table 2 in 90% ethanol and 10% 10 nM sodium citrate (by volume). The RNA (siRNA and/or mRNA) was diluted in 10 nM sodium citrate to achieve a final lipidoid:RNA weight ratio between 5:1 and 10:1 . Rapid pipet mixing was used for spontaneous formation of the LNPs. For in vivo experiments, LNPs were dialyzed for one hour in PBS to remove ethanol. Finally, LNPs were diluted in PBS to achieve the desired final concentration. [00088] For all LNP characterization studies, the nanoparticles were diluted to 1 pg/mL total RNA. LNPs were characterized for RNA entrapment using a Quant-iT™ Ribogreen® Assay (Invitrogen, Carlsbad, CA) according to the manufacture’s protocol. Dynamic light scattering (DLS) was used to characterize LNP size and polydispersity (PDI) on a Malvern Zetasizer Nano (Malvern Instruments, UK).

Transfection of co-delivered siRNA and mRNA in vitro

[00089] HeLa cells stably modified to express firefly and Renilla luciferase were grown in DMEM supplemented with 100 ml/L of FBS, 10 lU/mL of penicillin, and 10 mg/mL of streptomycin. The cells were incubated at 37 °C in a 5% CO2 environment and subcultured by partial digestion with 0.25% trypsin and ethylenediaminetetraacetic acid. Passages 10-30 were used for experiments. HeLa cells were seeded at 15,000 cells per well in a black 96 well plate. LNPs were made according to the formulations in Table 2 with either siRNA, mRNA, or siRNA + mRNA at RNA concentrations of 0.0125 mg/mL siRNA and 0.05 mg/mL mRNA. For in vitro experiments, siRNA was specific for firefly luciferase and mRNA encoded mCherry. LNPs were delivered to luciferase-expressing HeLa cells at a dose of 10 nM siRNA and/ or 100 ng (~ 1 .6 nM) mRNA for 24 hours. Resultant mCHERRY fluorescence (ex:587 nm/ em:615 nm) was measured 24 hours after transfection. Then, luciferase activity was quantified using a Dual-Glo ^® Luciferase Assay Kit (Promega, Madison, Wl) according to the manufacture’s protocol. After the first Luciferase Assay Kit reagent was added, the lysed cells were transferred to a white plate to measure luminescence. Renilla luciferase activity served as a control.

Animal studies

[00090] Animal protocols were approved by the Institutional Animal Care and Use Committee at Carnegie Mellon University (Pittsburgh, PA). C57BL/6 mice (female and male) of at least 6 weeks of age were purchased from Charles River Laboratories. Mice were housed under controlled temperature (25 °C) in 12-hour light-dark cycles. Animals were given access to standard diet and water.

[00091] LNPs for in vivo experiments were formulated with either anti-Factor VII siRNA (siFVII), mRNA encoding luciferase (mLuc), or a combination of the two and administered to mice via tail-vein injection. Six hours later, luciferase activity was assessed by administering an intraperitoneal injection of D-Luciferin substrate (130 pL at 30 mg/mL in PBS). 15 minutes later, luminescence was measured by whole mouse imaging using an IVIS (Perkin Elmer, MA) and quantified using Living Image software (Perkin Elmer). Factor VII expression was measured 48 hours after LNP injection from a submandibular blood sample. A BIOPHEN FVII assay was used according to the manufacture’s protocol (Aniara, OH). Mice were also weighed 48 hours post-injection, as significant weight loss may indicate LNP toxicity.

[00092] For the experiments in Figure 2, LNPs were formulated according to the specifications in Table 2 at concentrations of 0.006 mg/ml siFVII and/or 0.1 mg/ml mLuc. Mice were dosed with 0.03 mg/kg siRNA and/or 0.5 mg/kg mRNA. For the experiments in Figures 2 (D), 4, and 9, all LNPs were made using Formulation 4. For experiments in Figure 4 (A), the siRNA formulation concentration was kept constant at 0.006 mg/mL while mRNA concentration varied from 0 - 0.2 mg/mL. This corresponded to a final dose in mice of 0.03 mg/kg siRNA and 0 - 1 mg/kg mRNA. For Figure 4 (B), the LNP mRNA formulation concentration was kept constant at 0.1 mg/mL and the siRNA concentration varied from 0 - 0.08 mg/mL. Mice were dosed with 0.5 mg/kg mRNA and 0 - 0.4 mg/kg siRNA.

LNP formulation with negatively charged polymers

[00093] For LNP formulation with negatively charged polymers, the lipid and RNA solutions were prepared in ethanol and 10 nM sodium citrate, respectively, as described in the Lipid Nanoparticle Formulation section above. Negatively charged polymer (Poly(U), PSS, or PAA) was dissolved in PBS and added to the RNA solution. Finally, the lipid solution was added to the RNA-polymer solution and rapidly pipetted for nanoparticle formation. LNPs containing siRNA as the active drug were formulated at 0.006 mg/mL siFVII and with either mLuc (0.1 mg/mL), PSS (Mw 70 kDa, 0.064 mg/mL), Poly(U) (Mw 100 - 1 ,000 kDa, 0.1 mg/mL), or PAA (Mv 450 kDa, 0.1 mg/mL) while keeping the lipidoid concentration constant at 0.93 mg/mL. All mice were dosed with LNP formulations containing 0.03 mg/kg siFVII. LNPs containing mRNA as the active drug were formulated at 0.1 mg/mL mLuc and with either siFVII (0.006 mg/mL), PSS (Mw 6.8 kDa, 0.004 mg/mL), Poly(U) (Mw 100 - 1 ,000 kDa, 0.005 mg/mL), or PAA (Mv 450 kDa, 0.0013 mg/mL) while keeping the lipidoid concentration constant at 0.93 mg/mL. All mice were dosed with LNP formulations containing 0.5 mg/kg mLuc. The negative polymer concentrations were decided based on maintaining the same amount of total negative charge as when the LNPs are made with 0.006 mg/mL siRNA and 0.1 mg/mL mRNA. [00094] For experiments in Figure 4C, siFVII was used as the active drug and formulated at either 0.004 mg/ml_ siFVII and PSS (70 kDa, 0.066 mg/ml_), 0.002 mg/ml_ and PSS (70 kDa, 0.067 mg/ml_) or 0.001 mg/ml_ siFVII and PSS (70 kDa, 0.068 mg/ml_) and IV-injected to mice at doses of 0.005 mg/kg - 0.3 mg/kg. The negative polymer concentrations for these experiments were selected to maintain the same total negative charge as when the LNPs were formulated with 0.006 mg/ml_ siRNA and 0.1 mg/ml_ mRNA.

Statistical analysis

[00095] All statistical analysis was performed using GraphPad Prism (La Jolla, CA) software. Error bars represent standard deviation (sample sizes provided in each figure). To compare two groups, unpaired Student’s t tests were performed, assuming Gaussian distribution. Groups of three or more were compared by one-way ANOVA. Statistical significance is indicated by * p < 0.05, ** p < 0.01 and **** p < 0.0001.

[00096] The present invention has been described with reference to certain exemplary embodiments, dispersible compositions and uses thereof. However, it will be recognized by those of ordinary skill in the art that various substitutions, modifications or combinations of any of the exemplary embodiments may be made without departing from the spirit and scope of the invention. Thus, the invention is not limited by the description of the exemplary embodiments, but rather by the appended claims as originally filed.