Tolerogenic lipid NANO-particles

Abstract

Disclosed are lipid nano-particle pharmaceutical formulations comprising retinoic acid, and methods of using and making the formulations.

Description

Leydig 770805 University 06252 1 TOLEROGENIC LIPID NANO-PARTICLES CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This patent application claims the benefit of U.S. Provisional Patent Application No.63 / 529,249, filed July 27, 2023, which is incorporated by reference in its entirety herein. BACKGROUND OF THE INVENTION

[0002] Delivery of therapeutic active agents is an important tool in managing diseases. However, some subjects experience increased inflammatory cytokines and / or an undesirable immune response following administration of therapeutic active agents, e.g., mRNA vaccines. Accordingly, new formulations for safe administration of therapeutic active agents are needed. BRIEF SUMMARY OF THE INVENTION

[0003] An aspect of the invention provides lipid nano-particle pharmaceutical formulations comprising: a. from about 5 Mol % to about 50 Mol % lipidoid, b. from about 5 Mol % to about 50 Mol % phospholipid, c. from about 0.5 Mol % to about 5 Mol % poly(ethylene glycol), d. from about 0.5 Mol % to about 30 Mol % cholesterol, or a derivative thereof, and e. from about 0.5 Mol % to about 20 Mol % retinoic acid.

[0004] Another aspect of the invention provides methods of mitigating formation of antibodies against at least one active agent in a subject, the methods comprising administering a formulation of an aspect of the invention to a subject.

[0005] A further aspect of the invention provides methods of immune-silencing at least one active agent in a subject, the methods comprising administering a formulation of an aspect of the invention to a subject.

[0006] Another aspect of the invention provides methods of making lipid nano-particle pharmaceutical formulations of an aspect of the invention.Leydig 770805 University 06252 2 BRIEF DESCRIPTION OF THE VIEWS OF THE DRAWINGS

[0007] Figure 1 is a graph showing MYD88 levels following quantitative PCR (qPCR) 36 hours after transfection of RAW cells using a formulation without retinoic acid and methods of an aspect of the invention.

[0008] Figure 2 is a graph showing IFNα levels following quantitative PCR 36 hours after transfection of RAW cells using a formulation without retinoic acid and methods of an aspect of the invention.

[0009] Figure 3 is a graph showing MYD88 levels following quantitative PCR 36 hours after transfection of RAW cells using a 2.5% or 5% retinoic acid formulation and methods of an aspect of the invention.

[0010] Figure 4 is a graph showing IFNα levels following quantitative PCR 36 hours after transfection of RAW cells using a 2.5% or 5% retinoic acid formulation and methods of an aspect of the invention.

[0011] Figure 5 is a graph showing MYD88 levels following quantitative PCR 4 hours after transfection of RAW cells using a formulation and methods of an aspect of the invention.

[0012] Figure 6 is a graph showing IFNα levels following quantitative PCR 4 hours after transfection of RAW cells using a formulation and methods of an aspect of the invention.

[0013] Figure 7 is a graph showing RIG1 levels following quantitative PCR 4 hours after transfection of RAW cells using a formulation and methods of an aspect of the invention.

[0014] Figure 8 is a graph showing MDA5 levels following quantitative PCR 4 hours after transfection of RAW cells using a formulation and methods of an aspect of the invention.

[0015] Figure 9 is a graph showing IFNβ levels following quantitative PCR 4 hours after transfection of RAW cells using a formulation and methods of an aspect of the invention.

[0016] Figure 10A is a graph showing the results of qRT-analysis of MYD88 mRNA expression in different LNP transfected culture conditions: untransfected cells (N=2), mCherry-ZFR (zinc finger) mRNA-306O10 with 0% RA transfected cells (N=2), mCherry- ZFR mRNA-306O10with 2.5% RA transfected cells (N=2), mCherry-ZFR mRNA-306O10with 7.5% RA transfected cells (N=2), mCherry-ZFR mRNA-306O10 with 15% RA transfected cells (N=2), mCherry mRNA-306O10 with 0% RA transfected cells (N=2), mCherry mRNA-306O10 with 2.5% RA transfected cells (N=2), mCherry mRNA-306O10 with 7.5% RA transfected cells (N=2), and mCherry mRNA-306O10 with 15% RALeydig 770805 University 06252 3 transfected cells (N=2). Fold changes were relative to the untransfected control (N=2). Data are presented as mean + s.e.m.

[0017] Figure 10B is a graph showing the results of qRT-analysis of TGFβ1 mRNA expression in different LNP transfected culture conditions: untransfected cells (N=2), mCherry-ZFR mRNA-306O10with 0% RA transfected cells (N=2), mCherry-ZFR mRNA- 306O10 with 2.5% RA transfected cells (N=2), mCherry-ZFR mRNA-306O10 with 7.5% RA transfected cells (N=2), mCherry-ZFR mRNA-306O10with 15% RA transfected cells (N=2), mCherry mRNA-306O10 with 0% RA transfected cells (N=2), mCherry mRNA-306O10 with 2.5% RA transfected cells (N=2), mCherry mRNA-306O10 with 7.5% RA transfected cells (N=2), and mCherry mRNA-306O10with 15% RA transfected cells (N=2). Fold changes were quantified relative to the untransfected control (N=2). Data are presented as mean + s.e.m.

[0018] Figure 10C is a graph showing qRT-analysis of TGFβ2 mRNA expression in different LNP transfected culture conditions: untransfected cells (N=2), mCherry-ZFR mRNA-306O10 with 0% RA transfected cells (N=2), mCherry-ZFR mRNA-306O10 with 2.5% RA transfected cells (N=2), mCherry-ZFR mRNA-306O10with 7.5% RA transfected cells (N=2), mCherry-ZFR mRNA-306O10 with 15% RA transfected cells (N=2), mCherry mRNA-306O10with 0% RA transfected cells (N=2), mCherry mRNA-306O10with 2.5% RA transfected cells (N=2), mCherry mRNA-306O10 with 7.5% RA transfected cells (N=2), and mCherry mRNA-306O10with 15% RA transfected cells (N=2). Fold changes were quantified relative to the untransfected control (N=2). Data are presented as mean + s.e.m.

[0019] Figure 10D is a graph showing the results of qRT-analysis of TGFβ3 mRNA expression in different LNP transfected culture conditions: untransfected cells (N=2), mCherry-ZFR mRNA-306O10with 0% RA transfected cells (N=2), mCherry-ZFR mRNA- 306O10 with 2.5% RA transfected cells (N=2), mCherry-ZFR mRNA-306O10 with 7.5% RA transfected cells (N=2), mCherry-ZFR mRNA-306O10 with 15% RA transfected cells (N=2), mCherry mRNA-306O10with 0% RA transfected cells (N=2), mCherry mRNA-306O10with 2.5% RA transfected cells (N=2), mCherry mRNA-306O10 with 7.5% RA transfected cells (N=2), and mCherry mRNA-306O10with 15% RA transfected cells (N=2). Fold changes were quantified relative to the untransfected control (N=2). Data are presented as mean + s.e.m.

[0020] Figure 10E is a graph showing the results of qRT-analysis of IL10 mRNA expression in different LNP transfected culture conditions: untransfected cells (N=2),Leydig 770805 University 06252 4 mCherry-ZFR mRNA-306O10with 0% RA cells (N=2), mCherry-ZFR mRNA- 306O10 with 2.5% RA transfected cells (N=2), mCherry-ZFR mRNA-306O10 with 7.5% RA transfected cells (N=2), mCherry-ZFR mRNA-306O10with 15% RA transfected cells (N=2), mCherry mRNA-306O10 with 0% RA transfected cells (N=2), mCherry mRNA-306O10 with 2.5% RA transfected cells (N=2), mCherry mRNA-306O10with 7.5% RA transfected cells (N=2), and mCherry mRNA-306O10 with 15% RA transfected cells (N=2). Fold changes were quantified relative to the untransfected control (N=2). Data are presented as mean + s.e.m.

[0021] Figure 10F is a graph showing the results of qRT-analysis of TNFα mRNA expression in different LNP transfected culture conditions: untransfected cells (N=2), mCherry-ZFR mRNA-306O10 with 0% RA transfected cells (N=2), mCherry-ZFR mRNA- 306O10with 2.5% RA transfected cells (N=2), mCherry-ZFR mRNA-306O10with 7.5% RA transfected cells (N=2), mCherry-ZFR mRNA-306O10 with 15% RA transfected cells (N=2), mCherry mRNA-306O10with 0% RA transfected cells (N=2), mCherry mRNA-306O10with 2.5% RA transfected cells (N=2), mCherry mRNA-306O10 with 7.5% RA transfected cells (N=2), and mCherry mRNA-306O10with 15% RA transfected cells (N=2). Fold changes were quantified relative to the untransfected control (N=2). Data are presented as mean + s.e.m. DETAILED DESCRIPTION OF THE INVENTION

[0022] Improved lipid nano-particle formulations have been developed that allow targeted delivery of an active agent to immune related cells, while also becoming immune- silenced. The formulations modulate immunity in two ways - humoral immunity and cellular immunity (e.g., antibody and CD8+ T cell responses). The formulations described herein as aspects of the invention contain retinoic acid at concentrations that provide the desirable features. The tolerogenic formulations of an aspect of the invention lower inflammatory cytokines. Further, the formulations of an aspect of the invention are useful with different types of active agents, including vaccines that use mRNA.

[0023] In one aspect, the invention provides lipid nano-particle pharmaceutical formulations comprising: a. from about 5 Mol % to about 50 Mol % lipidoid, b. from about 5 Mol % to about 50 Mol % phospholipid, c. from about 0.5 Mol % to about 5 Mol % poly(ethylene glycol), d. from about 0.5 Mol % to about 30 Mol % cholesterol, or a derivative thereof, and e. from about 0.5 Mol % to about 20 Mol % retinoic acid.Leydig 770805 University 06252 5

[0024] In one aspect, the invention lipid nano-particle pharmaceutical formulations comprising: a. from about 30 Mol % to about 40 Mol % lipidoid, b. from about 35 Mol % to about 45 Mol % phospholipid, c. from about 2 Mol % to about 3 Mol % poly(ethylene glycol), d. from about 5 Mol % to about 25 Mol % cholesterol, or a derivative thereof, and e. from about 2 Mol % to about 17 Mol % retinoic acid.

[0025] The formulation of an aspect of the invention can further comprise at least one active agent. In an aspect of the invention, the active agent is from about 0.1 Mol % to about 15 Mol % of the formulation (e.g., from about 1 Mol % to about 14 Mol % of the formulation, from about 2 Mol % to about 13 Mol % of the formulation, from about 3 Mol % to about 12 Mol % of the formulation, from about 4 Mol % to about 11 Mol % of the formulation, or from about 5 Mol % to about 10 Mol % of the formulation).

[0026] The active agent can be any suitable active agent. For example, the active agent can be a small molecule or biologic, protein, peptide, or polynucleotide that can be formulated with lipid nano-particles. In an aspect of the invention, the at least one active agent is DNA. In another aspect of the invention, the at least one active agent is RNA. In another aspect of the invention, the at least one active agent is a vaccine. In another aspect of the invention, the at least one active agent is an mRNA vaccine.

[0027] The at least one active agent can also be an mRNA. In another aspect of the invention, the at least one active agent is an anti-inflammatory mRNA. The anti- inflammatory mRNA may increase the tolerogenic impact of the lipid nano-particle pharmaceutical formulation.

[0028] In an aspect of the invention, the active agent can be a MYD88 suppressor, a MYD88 inhibitor, a MYD88 transcriptional repressor,, a transforming growth factor beta (TGFβ, e.g., TGFβ1, TGFβ2, or TGFβ3) suppressor, an interleukin-10 (IL10) suppressor, or tumor necrosis factor alpha (TNFα) suppressor.

[0029] In an aspect of the invention, the formulation is administered with designer epigenetic editing therapeutic tools (e.g., CRISPR) that target the gene of interest, e.g., the Myd88 gene. The designer epigenetic editing therapeutic tools and the formulation of an aspect of the invention can be administered simultaneously or sequentially in any order. When administered sequentially, the time frame of administration is not particularly limited, but could be administered within several minutes (e.g., within 10, 20, 30, 40, or 50 minutes) or within several hours (e.g., within 1, 2, 4, 8, 12, or 24 hours), or within several days (e.g., 1, 2, 5, or 7), or within several weeks (e.g., 1, 2, 3, or 4) of one another.Leydig 770805 University 06252 6

[0030] In a further aspect of the the formulation is co-administered with zinc fingers. For example, the formulation can be administered with zinc fingers that target the Myd88 gene. The zinc fingers and formulations can be administered as separate formulations or as a single formulation, or the two agents can be administered sequentially in any order. When administered sequentially, the timeframe of administration is not particularly limited, but could be administered within several minutes (e.g., within 10, 20, 30, 40, or 50 minutes) or within several hours (e.g., within 1, 2, 4, 8, 12, or 24 hours), or within several days (e.g., 1, 2, 5, or 7), or within several weeks (e.g., 1, 2, 3, or 4) of one another.

[0031] The active agent can also be a reporter protein, e.g., a fluorescent reporter protein. For example, the active agent in an aspect of the invention can be a mFruit, e.g., mCherry fluorescent reporter protein.

[0032] In an aspect of the invention, the active agent can be an antigen associated with a gene therapy virus (e.g., an adeno-associated virus (AAV) or adenovirus).

[0033] In another aspect of the invention, the active agent can be a gene therapy cargo such as Factor VIII or IX in hemophilia.

[0034] In one aspect, the invention provides lipid nano-particle pharmaceutical formulations comprising from about 5 Mol % to about 50 Mol % lipidoid (e.g., from about 5 Mol % to about 50 Mol % lipidoid, from about 10 Mol % to about 50 Mol % lipidoid, from about 15 Mol % to about 50 Mol % lipidoid, from about 20 Mol % to about 50 Mol % lipidoid, from about 25 Mol % to about 45 Mol % lipidoid, from about 30 Mol % to about 40 Mol % lipidoid, from about 33 Mol % to about 37 Mol % lipidoid, or about 35 Mol % lipidoid).

[0035] The lipidoid can be any suitable lipidoid. In an aspect of the invention, lipidoids are prepared from the conjugate addition of alkylamines to acrylates as described in U.S. Patent Nos.9,439,968 and 9,227,917 (incorporated herein by reference). In an aspect of the invention, the lipidoids are prepared from the conjugate addition of alkylamines to acrylates as described in Hajj et al., Small, 15: 1805097 (2019). In one aspect of the invention, the lipidoid is an amine-containing branched chain lipidoid. In an aspect of the invention, the lipidoid has 3 or more (e.g., 3, 4, 5, or 6) tails containing from about 6 to about 18 carbons (i.e., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 carbons, or from about 6 to about 14 carbons, from about 7 to about 13 carbons, from about 8 to about 12 carbons, from about 9 to about 11 carbons).Leydig 770805 University 06252 7

[0036] In another aspect of the the lipidoid is prepared from the conjugate addition of amine 306 (3,3′-diamino-N-methyldipropylamine) with an acrylate tail as shown below in Schematic 1, wherein each x in the lipidoid and acrylate tail is independently an integer from 6 to 18. Schematic 1 O6,O8, O9, O10, Oi10, O11, O12, O13, O14, O16, or O18, as shown below. In an aspect of the invention, the acrylate tail is branched (e.g., Oi10).

[0038] In another aspect of the invention, the lipidoid is 306O10 .Leydig 770805 University 06252 8

[0039] In another aspect of the the lipidoid is prepared from the conjugate addition of amine 304with an acrylate tail as shown in Schematic 1, wherein each x in the lipidoid is independently an integer from 6 to 18. In a further aspect of the invention, each acrylate tail of the lipidoid is independently O6, O8, O9, O10, Oi10, O11, O12, O13, O14, O16, or O18, as shown above.

[0040] In an aspect of the invention, the lipidoid is 304Oi10. In a further aspect of the invention, the lipidoid contains three Oi10acrylate tails.

[0041] In one aspect, the invention provides lipid nano-particle pharmaceutical formulations comprising from about 5 Mol % to about 50 Mol % phospholipid (e.g., from about 10 Mol % to about 50 Mol % phospholipid, from about 15 Mol % to about 50 Mol % phospholipid, from about 20 Mol % to about 50 Mol % phospholipid, from about 25 Mol % to about 50 Mol % phospholipid, from about 30 Mol % to about 50 Mol % phospholipid, from about 35 Mol % to about 45 Mol % phospholipid, from about 37 Mol % to about 42 Mol % phospholipid, or about 40 Mol % phospholipid).

[0042] The phospholipid can be any suitable phospholipid. In an aspect of the invention, the phospholipid is selected from the group consisting of DOPS (1,2-dioleoyl-sn-glycero-3- phospho-L-serine), DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine), DOPC (1,2- dioleoyl-sn-glycero-3-phosphocholine), SOPC (1-stearoyl-2-oleoyl-sn-glycero-3- phosphocholine), (1,2-distearoyl-sn-glycero-3-phosphocholine) DSPC, or a combination thereof. In an aspect of the invention, the phospholipid is DOPS.

[0043] In one aspect, the invention provides lipid nano-particle pharmaceutical formulations comprising from about 0.5 Mol % to about 20 Mol % retinoic acid (e.g., from about 1 Mol % to about 18 Mol % retinoic acid, 1.5 Mol % to about 17 Mol % retinoic acid, or from about 2 Mol % to about 16 Mol % retinoic acid).

[0044] In one aspect, the invention provides lipid nano-particle pharmaceutical formulations comprising from about 1 Mol % to about 5 Mol % retinoic acid (e.g., from about 1 Mol % to about 4 Mol % retinoic acid, from about 2 Mol % to about 3 Mol % retinoic acid, or about 2.5 Mol % retinoic acid).Leydig 770805 University 06252 9

[0045] In one aspect, the invention lipid nano-particle pharmaceutical formulations comprising from about 5 Mol % to about 10 Mol % retinoic acid (e.g., from about 6 Mol % to about 9 Mol % retinoic acid, from about 7 Mol % to about 8 Mol % retinoic acid, or about 7.5 Mol % retinoic acid).

[0046] In one aspect, the invention provides lipid nano-particle pharmaceutical formulations comprising from about 10 Mol % to about 20 Mol % retinoic acid (e.g., 11 Mol % to about 19 Mol % retinoic acid, 12 Mol % to about 18 Mol % retinoic acid, from about 13 Mol % to about 17 Mol % retinoic acid, from about 14 Mol % to about 16 Mol % retinoic acid, or about 15 Mol % retinoic acid).

[0047] The retinoic acid can be any suitable retinoic acid. In an aspect of the invention, the retinoic acid is all-trans retinoic acid.

[0048] In one aspect, the invention provides lipid nano-particle pharmaceutical formulations comprising from about 0.5 Mol % to about 5 Mol % poly(ethylene glycol) (e.g., 1 Mol % to about 4.5 Mol % poly(ethylene glycol), from about 1.5 Mol % to about 3.5 Mol % poly(ethylene glycol), from about 2 Mol % to about 3 Mol % retinoic acid, or about 2.5 Mol % poly(ethylene glycol)).

[0049] The poly(ethylene glycol) can be any suitable poly(ethylene glycol). In an aspect of the invention, the poly(ethylene glycol) has a 14 to 18 carbon backbone. In another aspect of the invention, the poly(ethylene glycol) has an average molecular weight of from about 1,500 to about 2,500 Da (e.g., from about 1,750 to about 2,250 Da, or about 2000 Da). In an aspect of the invention, the poly(ethylene glycol) is PEG 2000.

[0050] In one aspect, the invention provides lipid nano-particle pharmaceutical formulations comprising from about 0.5 Mol % to about 30 Mol % cholesterol, or a derivative thereof (e.g., from about 2 Mol % to about 28 Mol % cholesterol, or a derivative thereof, from about 4 Mol % to about 26 Mol % cholesterol, or a derivative thereof, or from about 6 Mol % to about 24 Mol % cholesterol, or a derivative thereof).

[0051] In one aspect, the invention provides lipid nano-particle pharmaceutical formulations comprising from about 15 Mol % to about 25 Mol % cholesterol, or a derivative thereof (e.g., from about 18 Mol % to about 22 Mol % cholesterol, or a derivative thereof, from about 19 Mol % to about 21 Mol % cholesterol, or a derivative thereof, or about 20 Mol % cholesterol, or a derivative thereof).

[0052] In one aspect, the invention provides lipid nano-particle pharmaceutical formulations comprising from about 15 Mol % to about 25 Mol % cholesterol, or a derivativeLeydig 770805 University 06252 10 thereof (e.g., from about 18 Mol % to about Mol % cholesterol, or a derivative thereof, from about 19 Mol % to about 21 Mol % cholesterol, or a derivative thereof, or about 15 Mol % cholesterol, or a derivative thereof).

[0053] In one aspect, the invention provides lipid nano-particle pharmaceutical formulations comprising from about 5 Mol % to about 10 Mol % cholesterol, or a derivative thereof (e.g., from about 6 Mol % to about 9 Mol % cholesterol, or a derivative thereof, from about 7 Mol % to about 8 Mol % cholesterol, or a derivative thereof, or about 7.5 Mol % cholesterol, or a derivative thereof).

[0054] Any suitable cholesterol (cholest-5-en-3β-ol), or a derivative thereof, can be used in the formulations of an aspect of the invention. For example, a cholesterol derivative such as 3β[N—(N′,N′-dimethylaminoethane)-carbamoyl]cholesterol (DC-cholesterol) can be used. In an aspect of the invention, the amount of cholesterol in the formulations can be adjusted to accommodate the desired amount of retinoic acid.

[0055] In an aspect of the invention, the formulations comprise a. from about 30 Mol % to about 40 Mol % lipidoid, b. from about 35 Mol % to about 45 Mol % phospholipid, c. from about 2 Mol % to about 3 Mol % poly(ethylene glycol), d. from about 15 Mol % to about 25 Mol % cholesterol, or a derivative thereof, and e. from about 2 Mol % to about 3 Mol % all-trans retinoic acid.

[0056] In an aspect of the invention, the formulations comprise a. from about 30 Mol % to about 40 Mol % lipidoid, b. from about 35 Mol % to about 45 Mol % phospholipid, c. from about 2 Mol % to about 3 Mol % poly(ethylene glycol), d. from about 10 Mol % to about 20 Mol % cholesterol, or a derivative thereof, and e. from about 7 Mol % to about 8 Mol % all-trans retinoic acid.

[0057] In an aspect of the invention, the formulations comprise a. from about 30 Mol % to about 40 Mol % lipidoid, b. from about 35 Mol % to about 45 Mol % phospholipid, c. from about 2 Mol % to about 3 Mol % poly(ethylene glycol), d. from about 7 Mol % to about 8 Mol % cholesterol, or a derivative thereof, and e. from about 14 Mol % to about 16 Mol % all-trans retinoic acid.

[0058] In an aspect of the invention, the formulations comprise about 35 Mol % lipidoid, about 40 Mol % phospholipid, about 2.5 Mol % poly(ethylene glycol), about 20 Mol % cholesterol, and about 2.5 Mol % retinoic acid.Leydig 770805 University 06252 11

[0059] In an aspect of the invention, the comprise about 35 Mol % lipidoid, about 40 Mol % phospholipid, about 2.5 Mol % % poly(ethylene glycol), about 17.5 Mol % cholesterol, and about 5 Mol % retinoic acid.

[0060] In an aspect of the invention, the formulations comprise about 35 Mol % lipidoid, about 40 Mol % phospholipid, about 2.5 Mol % % poly(ethylene glycol), about 15 Mol % cholesterol, and about 7.5 Mol % retinoic acid.

[0061] In an aspect of the invention, the formulations comprise about 35 Mol % lipidoid, about 40 Mol % phospholipid, about 2.5 Mol % % poly(ethylene glycol), about 7.5 Mol % cholesterol, and about 15 Mol % retinoic acid.

[0062] In an aspect of the invention, the formulations comprise about 35 Mol % 306O10lipidoid, about 40 Mol % DOPS, about 2.5 Mol % PEG 2000, about 20 Mol % cholesterol, and about 2.5 Mol % all-trans retinoic acid.

[0063] In an aspect of the invention, the formulations comprise about 35 Mol % 306O10 lipidoid, about 40 Mol % DOPS, about 2.5 Mol % PEG 2000, about 17.5 Mol % cholesterol, and about 5 Mol % all-trans retinoic acid.

[0064] In an aspect of the invention, the formulations comprise about 35 Mol % 306O10lipidoid, about 40 Mol % DOPS, about 2.5 Mol % PEG 2000, about 15 Mol % cholesterol, and about 7.5 Mol % all-trans retinoic acid.

[0065] In an aspect of the invention, the formulations comprise about 35 Mol % 306O10 lipidoid, about 40 Mol % DOPS, about 2.5 Mol % PEG 2000, about 7.5 Mol % cholesterol, and about 15 Mol % all-trans retinoic acid.

[0066] In an aspect of the invention, the formulations comprise about 35 Mol % 304Oi10lipidoid, about 40 Mol % DOPS, about 2.5 Mol % PEG 2000, about 20 Mol % cholesterol, and about 2.5 Mol % all-trans retinoic acid.

[0067] In an aspect of the invention, the formulations comprise about 35 Mol % 304Oi10 lipidoid, about 40 Mol % DOPS, about 2.5 Mol % PEG 2000, about 17.5 Mol % cholesterol, and about 5 Mol % all-trans retinoic acid.

[0068] In an aspect of the invention, the formulations comprise about 35 Mol % 304Oi10 lipidoid, about 40 Mol % DOPS, about 2.5 Mol % PEG 2000, about 15 Mol % cholesterol, and about 7.5 Mol % all-trans retinoic acid.

[0069] In an aspect of the invention, the formulations comprise about 35 Mol % 304Oi10lipidoid, about 40 Mol % DOPS, about 2.5 Mol % PEG 2000, about 7.5 Mol % cholesterol, and about 15 Mol % all-trans retinoic acid.Leydig 770805 University 06252 12

[0070] In one aspect, the invention methods of mitigating formation of antibodies against at least one active agent in a subject, the method comprising administering the formulation of an aspect of the invention to a subject.

[0071] In another aspect, the invention provides methods of immune-silencing at least one active agent in a subject, the method comprising administering the formulation of an aspect of the invention to a subject.

[0072] In a further aspect of the invention, the administration of the formulation reduces the level of inflammatory cytokines released by the subject, as compared to the level of inflammatory cytokines released when the at least one active agent is not administered as part of the formulation to the subject. Inflammatory cytokines include interferons.

[0073] In another aspect of the invention, the administration of the formulation increases the level of regulatory T cells specific to the at least one active agent in the subject, as compared to the level of regulatory T cells specific to the at least one active agent when the at least one active agent is not administered as part of the formulation to the subject.

[0074] In another aspect of the invention, the administration of the formulation modulates humoral immunity (e.g., modulation of antibody response). In further aspect of the invention, the administration of the formulation modulates cellular immunity (e.g., CD8+ T cell responses).

[0075] The formulations can be administered in any suitable means. For example, the formulations can be administered to a subject by via parental (subcutaneous, intramuscular, intradermal, intraperitoneal, intrathecal, intravenous, and intratumoral), topical, oral, or local administration. In an aspect of the invention, the formulations are administered one time to the subject. In another aspect, the formulations are administered more than once to the subject (i.e., multiple times, two, three, four, or more times to the subject).

[0076] The formulations of an aspect of the invention can be made by any suitable means. In one aspect of the invention, the method of making the lipid nano-particle pharmaceutical formulations comprises a. mixing together: i. from about 5 Mol % to about 50 Mol % lipidoid, ii. from about 5 Mol % to about 50 Mol % phospholipid, iii. from about 0.5 Mol % to about 5 Mol % poly(ethylene glycol), iv. from about 0.5 Mol % to about 30 Mol % cholesterol, or a derivative thereof, and v. from about 0.5 Mol % to about 20 Mol % retinoic acid to form a lipid nano-particle formulation, and b. mixing the lipid nano-particle formulation with at least one active agent. The mixing can be done using any suitable means, e.g., with a pipette.Leydig 770805 University 06252 13

[0077] Aspects, including the subject matter described herein may be beneficial alone or in combination, with one or more other aspects or embodiments. Without limiting the foregoing description, certain non-limiting aspects of the disclosure numbered 1- 23 are provided below. As will be apparent to those of skill in the art upon reading this disclosure, each of the individually numbered aspects may be used or combined with any of the preceding or following individually numbered aspects. This is intended to provide support for all such combinations of aspects and is not limited to combinations of aspects explicitly provided below:

[0078] (1) A lipid nano-particle pharmaceutical formulation comprising: a. from about 5 Mol % to about 50 Mol % lipidoid; b. from about 5 Mol % to about 50 Mol % phospholipid; c. from about 0.5 Mol % to about 5 Mol % poly(ethylene glycol); d. from about 0.5 Mol % to about 30 Mol % cholesterol, or a derivative thereof; and e. from about 0.5 Mol % to about 20 Mol % retinoic acid.

[0079] (2) The formulation of aspect 1, further comprising: f. at least one active agent.

[0080] (3) The formulation of aspect 2, wherein the active agent is from about 0.1 Mol % to about 15 Mol % of the formulation.

[0081] (4) The formulation of aspect 2 or 3, wherein the at least one active agent is DNA or RNA.

[0082] (5) The formulation of any one of aspects 2-4, wherein the at least one active agent is a vaccine.

[0083] (6) The formulation of any one of aspects 2-5, wherein the at least one active agent is an mRNA vaccine.

[0084] (7) The formulation of any one of aspects 2-4, wherein the at least one active agent is anti-inflammatory mRNA.

[0085] (8) The formulation of aspect 7, wherein the anti-inflammatory mRNA is an MYD88 suppressor.

[0086] (9) The formulation of any one of aspects 1-8, wherein the retinoic acid is all- trans retinoic acid.

[0087] (10) The formulation of any one of aspects 1-9, wherein the lipidoid is an amine-containing branched chain lipidoid.Leydig 770805 University 06252 14

[0088] (11) The formulation of one of aspects 1-10, wherein the lipidoid is 306O10.

[0089] (12) The formulation of any one of aspects 1-10, wherein the lipidoid is 304Oi10.

[0090] (13) The formulation of any one of aspects 1-12, wherein the phospholipid is selected from the group consisting of DOPS, DOPE, DOPC, SOPC, DSPC, or a combination thereof.

[0091] (14) The formulation of any one of aspects 1-13, wherein the phospholipid is DOPS.

[0092] (15) The formulation of any one of aspects 1-14, wherein the poly(ethylene glycol) is PEG 2000.

[0093] (16) The formulation of any one of aspects 1-15, comprising: a. from about 30 Mol % to about 40 Mol % lipidoid; b. from about 35 Mol % to about 45 Mol % phospholipid; c. from about 2 Mol % to about 3 Mol % poly(ethylene glycol); d. from about 15 Mol % to about 25 Mol % cholesterol, or a derivative thereof; and e. from about 2 Mol % to about 3 Mol % all-trans retinoic acid.

[0094] (17) The formulation of any one of aspects 1-15, comprising: a. from about 30 Mol % to about 40 Mol % lipidoid; b. from about 35 Mol % to about 45 Mol % phospholipid; c. from about 2 Mol % to about 3 Mol % poly(ethylene glycol); d. from about 10 Mol % to about 20 Mol % cholesterol, or a derivative thereof; and e. from about 7 Mol % to about 8 Mol % all-trans retinoic acid.

[0095] (18) The formulation of any one of aspects 1-15, comprising: a. from about 30 Mol % to about 40 Mol % lipidoid; b. from about 35 Mol % to about 45 Mol % phospholipid; c. from about 2 Mol % to about 3 Mol % poly(ethylene glycol); d. from about 7 Mol % to about 8 Mol % cholesterol, or a derivative thereof; and e. from about 14 Mol % to about 16 Mol % all-trans retinoic acid.

[0096] (19) A method of mitigating formation of antibodies against at least one active agent in a subject, the method comprising:Leydig 770805 University 06252 15 administering the formulation of of aspects 2-18 to a subject.

[0097] (20) A method of immune-silencing at least one active agent in a subject, the method comprising: administering the formulation of any one of aspects 2-18 to a subject.

[0098] (21) The method of aspect 19 or 20, wherein administration of the formulation reduces the level of inflammatory cytokines released by the subject, as compared to the level of inflammatory cytokines released when the at least one active agent is not administered as part of the formulation to the subject.

[0099] (22) The method of any one of aspects 19-21, wherein administration increases the level of regulatory T cells specific to the at least one active agent in the subject, as compared to the level of regulatory T cells specific to the at least one active agent when the at least one active agent is not administered as part of the formulation to the subject.

[0100] (23) A method of making a lipid nano-particle pharmaceutical formulation, the method comprising a. mixing together: i. from about 5 Mol % to about 50 Mol % lipidoid; ii. from about 5 Mol % to about 50 Mol % phospholipid; iii. from about 0.5 Mol % to about 5 Mol % poly(ethylene glycol); iv. from about 0.5 Mol % to about 30 Mol % cholesterol, or a derivative thereof; and v. from about 0.5 Mol % to about 20 Mol % retinoic acid to form a lipid nano-particle formulation; and b. mixing the lipid nano-particle formulation with at least one active agent.

[0101] The following example further illustrates aspects of the invention but, of course, should not be construed as in any way limiting its scope. EXAMPLES

[0102] Example 1

[0103] This example demonstrates that formulations of an aspect of the invention in in vitro studies are effective at reducing inflammation.

[0104] The following formulations of an aspect of the present invention were prepared: (a) 35 Mol % 306O10lipidoid, 40 Mol % DOPS, 2.5 Mol % PEG 2000, and 22.5 Mol % cholesterol (0 % all-trans retinoic acid formulation);Leydig 770805 University 06252 16 (b) 35 Mol % 306O10lipidoid, 40 % DOPS, 2.5 Mol % PEG 2000, 20 Mol % cholesterol, and 2.5 Mol % all-trans retinoic acid (2.5 % all-trans retinoic acid formulation); (c) 35 Mol % 306O10lipidoid, 40 Mol % DOPS, 2.5 Mol % PEG 2000, 17.5 Mol % cholesterol, and 5 Mol % all-trans retinoic acid (5 Mol % all-trans retinoic acid formulation); and (d) 35 Mol % 306O10 lipidoid, 40 Mol % DOPS, 2.5 Mol % PEG 2000, 15 Mol % cholesterol, and 7.5 Mol % all-trans retinoic acid (7.5 Mol % all-trans retinoic acid formulation).

[0105] To evaluate expression levels, RAW 264.7 cells were transfected with the lipid nano-particles carrying the mCherry or MYD88 regulator mRNAs.

[0106] Twenty-four hours after transfection, the expression levels of mCherry were evaluated using a microscope. Specifically, 10X images showing expression levels with non- purified mCherry-306O10 as part of the 2.5 % all-trans retinoic acid formulation (final concentration was 0.005 mg / ml) and 10X images showing expression level with non-purified mCherry-306O10 as part of the 5 % all-trans retinoic acid formulation (final concentration was 0.005 mg / ml) were evaluated. Abundant expression was present within the transfected cells while the untransfected control cells exhibited no expression.

[0107] The cells were harvested 36 hours after transfection. MYD88 (see Figures 1 and 5) and IFNα (see Figures 2 and 4) levels were detected using quantitative PCR. The MYD88 gene provides instructions for making a protein involved in signaling within immune cells.

[0108] The cells were harvested 4 hours after transfection. MYD88 (see Figure 5), IFNα (see Figure 6), RIG1 (see Figure 7), MDA5 (see Figure 8), and IFNβ (see Figure 9) levels were detected using quantitative PCR.

[0109] These results demonstrate that formulations of an aspect of the invention can reduce inflammation (cytokines) as compared to similar formulations that do not contain retinoic acid. The formulations of an aspect of the invention can also successfully deliver an active agent to a target cell. This may be especially useful for creating tolerogenic agents. For example, it may be possible to use retinoic acid in synergy with the cargo of the lipid nano-particles such as an MYD88 suppressor, IL-10 or TGF-β to further enhance the tolerogenic capacity of the lipid nano-particles.

[0110] Example 2

[0111] This example demonstrates that formulations of an aspect of the invention in in vitro studies are effective at reducing inflammation.Leydig 770805 University 06252 17

[0112] To evaluate expression levels, 264.7 cells were transfected with the lipid nano-particles carrying the mCherry or MYD88 regulator mRNAs, with and without a MYD88 transcriptional repressor (a zinc finger, ZFR).

[0113] qRT-PCR was utilized to quantify gene expression levels of several key anti- and pro-inflammatory markers: MYD88, TGFβ1, TGFβ2, TGFβ3, IL10, and TNFα. Based on qPCR results, MYD88 mRNA expression levels exhibit an elevation in response to retinoic acid treatment. Further, co-administration of a MYD88 transcriptional repressor ZFR with retinoic acid leads to attenuated upregulation of MYD88 transcript levels, as compared to retinoic acid treatment alone (Fig.10A). In addition, qPCR results suggest a dose-dependent trend indicating that increased levels of all-trans retinoic acid can lead to elevated mRNA levels of anti-inflammatory markers such as TGFβ2 and TGFβ3, and that this increase is enhanced in an immunosuppressed environment under MYD88 repression (Figs.10B-D). Co-administration of retinoic acid and a MYD88 transcriptional repressor ZFR leads to a synergistic, dose-dependent upregulation of IL10 mRNA expression. The results in Figure 10E indicate the synergism between a MYD88 repression and retinoic acid specifically enhances IL10 expression in a dose-responsive manner.

[0114] In summary, these results indicate that formulations of an aspect of the invention can mitigate inflammation, as evidenced by reduced cytokine levels, compared to analogous formulations without retinoic acid. Additionally, the retinoic acid-containing formulations retained the capacity to successfully deliver cargo molecules to target cells.

[0115] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.

[0116] The use of the terms “a” and “an” and “the” and “at least one” and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The use of the term “at least one” followed by a list of one or more items (for example, “at least one of A and B”) is to be construed to mean one item selected from the listed items (A or B) or any combination of two or more of the listed items (A and B), unless otherwise indicated herein or clearly contradicted by context. The terms “comprising,” “having,” “including,” and “containing”Leydig 770805 University 06252 18 are to be construed as open-ended terms “including, but not limited to,”) unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0117] Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

Claims

Leydig 770805 University 06252 19 CLAIMS:

1. A lipid nano-particle pharmaceutical formulation comprising: a. from about 5 Mol % to about 50 Mol % lipidoid; b. from about 5 Mol % to about 50 Mol % phospholipid; c. from about 0.5 Mol % to about 5 Mol % poly(ethylene glycol); d. from about 0.5 Mol % to about 30 Mol % cholesterol, or a derivative thereof; and e. from about 0.5 Mol % to about 20 Mol % retinoic acid.

2. The formulation of claim 1, further comprising: f. at least one active agent.

3. The formulation of claim 2, wherein the active agent is from about 0.1 Mol % to about 15 Mol % of the formulation.

4. The formulation of claim 2 or 3, wherein the at least one active agent is DNA or RNA.

5. The formulation of claim 2 or 3, wherein the at least one active agent is a vaccine.

6. The formulation of claim 2 or 3, wherein the at least one active agent is an mRNA vaccine.

7. The formulation of claim 2 or 3, wherein the at least one active agent is anti- inflammatory mRNA.

8. The formulation of claim 7, wherein the anti-inflammatory mRNA is an MYD88 suppressor.Leydig 770805 University 06252 20 9. The formulation of any one 1-3, wherein the retinoic acid is all-trans retinoic acid.

10. The formulation of any one of claims 1-3, wherein the lipidoid is an amine- containing branched chain lipidoid.

11. The formulation of any one of claims 1-3, wherein the lipidoid is 306O10.

12. The formulation of any one of claims 1-3, wherein the lipidoid is 304Oi10.

13. The formulation of any one of claims 1-3, wherein the phospholipid is selected from the group consisting of DOPS, DOPE, DOPC, SOPC, DSPC, or a combination thereof.

14. The formulation of any one of claims 1-3, wherein the phospholipid is DOPS.

15. The formulation of any one of claims 1-3, wherein the poly(ethylene glycol) is PEG 2000.

16. The formulation of any one of claims 1-3, comprising: a. from about 30 Mol % to about 40 Mol % lipidoid; b. from about 35 Mol % to about 45 Mol % phospholipid; c. from about 2 Mol % to about 3 Mol % poly(ethylene glycol); d. from about 15 Mol % to about 25 Mol % cholesterol, or a derivative thereof; and e. from about 2 Mol % to about 3 Mol % all-trans retinoic acid.

17. The formulation of any one of claims 1-3, comprising: a. from about 30 Mol % to about 40 Mol % lipidoid; b. from about 35 Mol % to about 45 Mol % phospholipid; c. from about 2 Mol % to about 3 Mol % poly(ethylene glycol);Leydig 770805 University 06252 21 d. from about 10 Mol about 20 Mol % cholesterol, or a derivative thereof; and e. from about 7 Mol % to about 8 Mol % all-trans retinoic acid.

18. The formulation of any one of claims 1-3, comprising: a. from about 30 Mol % to about 40 Mol % lipidoid; b. from about 35 Mol % to about 45 Mol % phospholipid; c. from about 2 Mol % to about 3 Mol % poly(ethylene glycol); d. from about 7 Mol % to about 8 Mol % cholesterol, or a derivative thereof; and e. from about 14 Mol % to about 16 Mol % all-trans retinoic acid.

19. A method of mitigating formation of antibodies against at least one active agent in a subject, the method comprising: administering the formulation of claim 2 or 3 to a subject.

20. A method of immune-silencing at least one active agent in a subject, the method comprising: administering the formulation of claim 2 or 3 to a subject.

21. The method of claim 19, wherein administration of the formulation reduces the level of inflammatory cytokines released by the subject, as compared to the level of inflammatory cytokines released when the at least one active agent is not administered as part of the formulation to the subject.

22. The method of claim 19, wherein administration increases the level of regulatory T cells specific to the at least one active agent in the subject, as compared to the level of regulatory T cells specific to the at least one active agent when the at least one active agent is not administered as part of the formulation to the subject.Leydig 770805 University 06252 22 23. A method of making a lipid particle pharmaceutical formulation, the method comprising a. mixing together: i. from about 5 Mol % to about 50 Mol % lipidoid; ii. from about 5 Mol % to about 50 Mol % phospholipid; iii. from about 0.5 Mol % to about 5 Mol % poly(ethylene glycol); iv. from about 0.5 Mol % to about 30 Mol % cholesterol, or a derivative thereof; and v. from about 0.5 Mol % to about 20 Mol % retinoic acid to form a lipid nano-particle formulation; and b. mixing the lipid nano-particle formulation with at least one active agent.

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