Lipid nanoparticles with RNA for cosmetic treatment

Abstract

A method for directing a change in cell state of white adipose tissue (WAT) which involve treating WAT cells with mRNA encapsulated inside of a lipid nanoparticle (LNP). The mRNA encodes a sequence for synthesizing UCP1 or other proteins that may transition adipose tissue from WAT to brown adipose tissue (BAT). The LNP may contain (C14-4: NCL: Cholesterol: PEG / Lipid Conjugate) at the molar ratio of (35: 16: 46.5: 2.5), (50: 10: 38.5: 1.5) or (56.5: 10:31.8: 1.7).

Description

[0001] LIPID NANOPARTICLES WITH RNA FOR COSMETIC TREATMENT

[0002] Sequence Listing

[0003] The application contains a sequence listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on May 29, 2025, is named 12447_012652-WO0.xml and is 239,261 bytes.

[0004] Background

[0005] Traditionally, adipose tissue is categorized into two main types with distinct functions: white adipose tissue (WAT) and brown adipose tissue (BAT). WAT, composed primarily of large, unilocular adipocytes, serves as the body's principal site for storing energy in the form of triglycerides. Conversely, BAT is specialized for energy expenditure through non-shivering thermogenesis, a process driven by the unique mitochondrial protein, Uncoupling Protein 1 (UCP1). Dysfunction within these adipose depots, particularly the excessive expansion and inflammation of WAT characteristic of overweight / obesity, is intimately linked to the development of metabolic disorders and diseases, including impaired glucose tolerance, metabolic syndrome, type 2 diabetes, cardiovascular disease, non-alcoholic fatty liver disease, and at least thirteen different types of cancer. Therefore, understanding and manipulating adipose tissue function represents a significant frontier in treating human disease.

[0006] A paradigm shift in adipose tissue biology occurred with the confirmation of metabolically active BAT in adult humans. That paradigm shift was further expanded by the discovery of inducible “browning” or “beigeing” of WAT. Beige adipose tissue (BeAT) is defined by the emergence or development of cells, in WAT, that express UCP1. The “beigeing” process can be activated by specific stimuli such as chronic cold exposure or treatment with B- adrenergic agonists. Like classical brown adipocytes, beige cells possess multilocular lipid droplets, abundant mitochondria, and express UCP1, enabling them to consume caloric fuel via heat production. This process increases the body's thermogenic capacity.

[0007] UCP1, also known historically as thermogenin, is an inner mitochondrial membrane protein, primarily found in brown and beige adipocytes. UCP1 provides an alternative pathway for proton re-entry into the mitochondrial matrix, effectively bypassing ATP synthase. This regulated "proton leak" dissipates the energy stored in the PMF directly as heat, rather than converting it into chemical energy in the form of ATP. This conversion of the PMF to heat is the core mechanism of non- shivering thermogenesis mediated by brown and beige fat.

[0008] There is an unmet need in the art for an agent to induce UCP1 expression in WAT and other tissues, thereby bypassing natural upstream controls. This strategy offers the potential advantage of inducing thermogenesis independent of external stimuli like cold, while also circumventing the body's inherent negative controls on upregulating heat production. An ideal agent would thus increase local energy expenditure by generating heat and thereby reduce the size of adipose tissue deposits.

[0009] Summary

[0010] To address the above-mentioned unmet needs, the present technology presents methods, compositions and systems for directing a change in the cell state of human WAT to BeAT, agents to achieve that change and systems / protocols for taking advantage of the thermogenic and calorie-expending properties of BeAT in order to achieve local reductions of adipose tissue and systemic change in the properties of adipose tissue.

[0011] The present invention provides methods and compositions for increasing the quantities and / or the ratios of brown / beige adipocytes in WAT. In various aspects, the cellular manipulations described herein are guided and / or mediated by the expression of sequences encoded in in vitro transcribed (“IVT” or “synthetic”) messenger RNA (mRNA) which provides cellular instructions to synthesize the protein structures encoded thereby transitioning of a cell from one state to a different cellular state. Specifically, cells in WAT are transfected by lipid nanoparticle (LNP) -packaged synthetic mRNA.

[0012] An aspect of the present invention relates to a method for directing a change in cell state of WAT comprising: contacting a population of cells comprising WAT with mRNA encapsulated inside of a LNP, wherein the mRNA encodes a sequence interpretable by the ribosomes of the adipocytes of the WAT for synthesizing UCP1 or other proteins that may transition adipose tissue from WAT to BAT or BeAT.

[0013] An aspect of the present invention relates to a composition of a treatment for mediating a cell state transition of treated tissue from WAT to BAT or BeAT comprising: a synthetic mRNA of a sequence which provides instructions to the ribosomes of the target cell to synthesize UCP1, said mRNA disposed within a LNP of a composition which is targeted to be introduced into an adipocyte and / or any cellular component of WAT including but not limited to stem cells, fibroblasts, and vascular cells in said WAT.

[0014] In one embodiment, the invention may be a pharmaceutical composition comprising a synthetic mRNA comprising a nucleotide sequence set forth in SEQ ID NO: 1; wherein the synthetic mRNA is packaged in a lipid nanoparticle (LNP).

[0015] In another embodiment, the LNP is of an LNP component molar ratio substantially similar to that of LNP ID I.

[0016] In another embodiment, the LNP is of an LNP component molar ratio substantially similar to that of LNP ID D.

[0017] In another embodiment, the mRNA comprises one or more modified uridine nucleotides.

[0018] In another embodiment, the modified uridine nucleotide is pseudouridine.

[0019] In another embodiment, the modified uridine nucleotide is N1 -Methylpseudouridine.

[0020] In a second embodiment, the invention may be a method of preparing a pharmaceutical composition, the method comprising preparing a synthetic mRNA comprising a nucleotide sequence set forth in SEQ ID NO: 2, SEQ ID NO: 8, or SEQ ID NO: 18; and packaging said synthetic mRNA in a lipid nanoparticle (LNP).

[0021] In another embodiment, the LNP is of an LNP component molar ratio substantially similar to that of LNP ID I.

[0022] In another embodiment, the LNP is of an LNP component molar ratio substantially similar to that of LNP ID D.

[0023] In another embodiment, the mRNA comprises one or more modified uridine nucleotides.

[0024] In another embodiment, the modified uridine nucleotide is pseudouridine.

[0025] In another embodiment, the modified uridine nucleotide is N1 -Methylpseudouridine.

[0026] In a third embodiment, the invention may be a method comprising identifying an area of a subject’s body for targeted fat reduction; and delivering a pharmaceutical composition comprising a synthetic mRNA in the area; wherein the synthetic mRNA comprises a nucleotide sequence set forth in SEQ ID NO: 3, SEQ ID NO: 9, or SEQ ID NO: 17.

[0027] In another embodiment, the area comprises the abdomen, the outer arms, flanks, upper back, lumbar region, calves, submental fat, dorsal cervical fat, axillary fat, lateral chest wall, outer thighs, inner thighs, or combinations thereof.

[0028] In another embodiment, the delivery is carried out in a single bolus injection. In another embodiment, the delivery is carried out in a series of injections in the area.

[0029] In another embodiment, the delivery is carried out transdermally by a penetration enhancer.

[0030] In another embodiment, the delivery is carried out transdermally by a vesicular carrier.

[0031] In another embodiment, the delivery is carried out transdermally by a microneedle injection.

[0032] In another embodiment, the delivery is carried out transdermally by iontophoresis, sonophoresis or jet injection.

[0033] Brief Description of the Drawings

[0034] Figure 1 is a microscope image of human adipose tissue transfected with mRNA via LNP expressing a protein.

[0035] Figure 2 is a first and a last frame of a 6-day time lapse showing a negative control study of human adipose tissue in culture.

[0036] Figure 3 is a first and a last frame of a 6-day time lapse showing a full dose study of human adipose tissue transfected with an mRNA construct expressing UCP1 in human white adipose tissue and causing shrinkage of said white adipose tissue.

[0037] Figure 4 is a first and a last frame of a 6-day time lapse showing a half dose study of human adipose tissue transfected with an mRNA construct expressing UCP1 in human adipose tissue and causing shrinkage of said white adipose tissue.

[0038] Figure 5 is a Western Blot result showing relative protein expression rates for varying doses of an mRNA construct expressing UCP1 in human adipose tissue.

[0039] Figure 6 is a Western Blot result showing relative protein expression rates for varying doses of an mRNA construct of SEQ ID NO: 1 expressing UCP1 encapsulated in LNP I in human adipose tissue.

[0040] Figure 7 is a series of Western Blot results showing protein expression by cells derived from human white adipose tissue transfected with mRNA constructs of SEQ ID NO: 2 through SEQ ID NO: 21.

[0041] Figures 8 through 14 show shrinkage of the transfected human WAT in one day intervals from the start to the end point at t+6 days after treatment with a I g dose of mRNA of SEQ ID NO: 1.

[0042] Figures 15 through 21 show shrinkage of the transfected human WAT in one day intervals from the start to the end point at t+6 days after treatment with a 5 pg dose of mRNA of SEQ ID NO: 1.

[0043] Figure 22 is a Western Blot result showing dose responsiveness of UCP1 expression 24 hours transfection of cells derived from human white adipose tissue with 5 pg and 10 pg doses of SEQ ID NO: 1 encapsulated in LNP D.

[0044] Figure 23 is a Western Blot result showing UCP1 expression 24 hours transfection of cells derived from human white adipose tissue with 1 pg of SEQ ID NO: 1 encapsulated in LNP I.

[0045] Detailed Description

[0046] Detailed descriptions of one or more embodiments are provided herein. It is to be understood, however, that the present disclosure may be embodied in various forms. Therefore, specific details disclosed herein are not to be interpreted as limiting, but rather as a basis for the claims and as a representative basis for teaching one skilled in the art to employ the present invention in any appropriate manner.

[0047] Brown and / or beige adipocytes (BAs) are a type of adipocyte which are able to uncouple the proton motive force from ATP generation in order to produce thermal energy. This mechanism allows mammals, through non-shivering thermogenesis, to regulate body temperature in cold environments. Because of its metabolically active nature, BAs represent a promising pathway for non-surgical intervention for obesity, aesthetics and other indications.

[0048] Provided herein are novel compositions, methods, formulations and devices to: 1) convert white adipocytes (WAs) to BAs thereby enhancing the metabolic activity of the treated tissue and 2) activate the thermogenic activity of the BAs in the treated tissue so as to consume fatty acids through thermogenesis thereby reducing the appearance of adipose tissue in the treated tissue. WAT includes WAs as well as adipose derived stem cells (ASCs) and other resident cell populations, all of which may also be “browned” and thereby exhibit a similar fatty acid consuming effect.

[0049] Referring to the Sequence Listing incorporated by reference herein, SEQ ID NO:1 was created as an optimized version of the human consensus genomic mRNA encoding for UCP1 including modifications to the 5’ and 3’ UTRs such as inserting a ribosome binding site, a Kozak sequence and a double stop codon. SEQ ID NOs: 2-21 were created as further optimizations of SEQ ID NO:1 using GenScript Biotech Corporation GenSmart tool with several modifications including deleting the 5’ UTR, incorporating Translation Initiator of 5’ UTR (TISU), the genomic human UCP1 5’ UTR, the consensus genomic human fatty acid binding protein 4 (FABP4) 5’ UTR, the consensus genomic human UCP1 3’ UTR, the Cominarty 3’ UTR, and the consensus genomic human beta-globin (HBB) 3' UTR in tandem tail-to-head sequence. For example, SEQ ID NO: 2 is 928 nucleotides long which corresponds identically to nucleotides 54 through 981 of SEQ ID NO: 1. As another example, SEQ ID NO: 3 is 932 nucleotides long which corresponds identically to nucleotides 50 through 981 of SEQ ID NO: 1. As another example, SEQ ID NO: 8 is 1397 nucleotides long and nucleotides 1 through 929 correspond exactly to nucleotides 50 through 979 of SEQ ID NO: 1. As another example, SEQ ID NO: 17 is 1142 nucleotides long and nucleotides 1 through 928 correspond exactly to nucleotides 54 through 980 of SEQ ID NO: 1.

[0050] SEQ ID NOs: 22 through 129 are optimizations of SEQ ID NOs: 1 through 21 using known UTR sequences. The optimizations of the UTR regions of SEQ ID NOs: 22 through 129 are designed to prevent the formation of secondary and tertiary RNA structures that can inhibit translation. The 5’ UTR sequences were selected based on proteins with the highest expression level in human WAT.

[0051] SEQ ID NOs: 22 through 24: codon optimized human UCP1 CDS using custom 5' and 3' UTRs. The 5' UTR for these CDS: Modified genomic human FABP4 5' UTR with a Kozak sequence inserted. The 3' UTR for these CDS: double stop codon.

[0052] SEQ ID NOs: 25 through 27: codon optimized human UCP1 CDS using custom 5' and 3' UTRs. The 5' UTR for these CDS: Modified genomic human LIPE 5' UTR with a Kozak sequence inserted. The 3' UTR for these CDS: double stop codon.

[0053] SEQ ID NOs: 28 through 30: codon optimized human UCP1 CDS using custom 5' and 3' UTRs. The 5' UTR for these CDS: Modified genomic human PLIN1 5' UTR with a Kozak sequence inserted. The 3' UTR for these CDS: double stop codon.

[0054] SEQ ID NOs: 31 through 33: codon optimized human UCP1 CDS using custom 5' and 3' UTRs. The 5' UTR for these CDS: Modified genomic human C0L1A 5' UTR with a Kozak sequence inserted. The 3' UTR for these CDS: double stop codon.

[0055] SEQ ID NOs: 34 through 36: codon optimized human UCP1 CDS using custom 5' and 3' UTRs. The 5' UTR for these CDS: Modified genomic human LEP 5' UTR with a Kozak sequence inserted. The 3' UTR for these CDS: double stop codon.

[0056] SEQ ID NOs: 37 through 39: codon optimized human UCP1 CDS using custom 5' and 3' UTRs. The 5' UTR for these CDS: Modified genomic human ADIPOQ 5' UTR with a Kozak sequence inserted. The 3' UTR for these CDS: double stop codon.

[0057] SEQ ID NOs: 40 through 44: codon optimized human UCP1 CDS using custom 5' and 3' UTRs. The 5' UTR for these CDS: Consensus genomic human FABP4 5' UTR. The 3' UTR for these CDS: double stop codon.

[0058] SEQ ID NOs: 45 through 49: codon optimized human UCP1 CDS using custom 5’ and 3’ UTRs. The 5’ UTR for these CDS: Consensus genomic human Lipase E, Hormone Sensitive Type (LIPE) 5' UTR. The 3’ UTR for these CDS: double stop codon.

[0059] SEQ ID NOs: 50 through 54: codon optimized human UCP1 CDS using custom 5’ and 3’ UTRs. The 5’ UTR for these CDS: Consensus genomic human perilipin 1 (PLIN1) 5' UTR. The 3’ UTR for these CDS: double stop codon.

[0060] SEQ ID NOs: 55 through 59: codon optimized human UCP1 CDS using custom 5’ and 3’ UTRs. The 5’ UTR for these CDS: Consensus genomic human collagen 1A (COL1A) 5' UTR. The 3’ UTR for these CDS: double stop codon.

[0061] SEQ ID NOs: 60 through 64: codon optimized human UCP1 CDS using custom 5’ and 3’ UTRs. The 5’ UTR for these CDS: Consensus genomic human leptin (LEP) 5' UTR. The 3’ UTR for these CDS: double stop codon.

[0062] SEQ ID NOs: 65 through 69: codon optimized human UCP1 CDS using custom 5’ and 3’ UTRs. The 5' UTR for his CDS: Consensus genomic human adiponectin (ADIPOQ) 5' UTR. The 3’ UTR for these CDS: double stop codon. SEQ ID NOs: 70 through 74: codon optimized human UCP1 CDS using custom 5’ and 3’ UTRs. The 5’ UTR for these CDS: Modified genomic human FABP4 5' UTR with a Kozak sequence inserted. The 3’ UTR for these CDS: double stop codon.

[0063] SEQ ID NOs: 75 through 79: codon optimized human UCP1 CDS using custom 5’ and 3’ UTRs. The 5’ UTR for these CDS: Modified genomic human LIPE 5' UTR with a Kozak sequence inserted. The 3’ UTR for these CDS: double stop codon.

[0064] SEQ ID NOs: 80 through 84: codon optimized human UCP1 CDS using custom 5’ and 3’ UTRs. The 5’ UTR for these CDS: Modified genomic human PLIN1 5' UTR with a Kozak sequence inserted. The 3’ UTR for these CDS: double stop codon.

[0065] SEQ ID NOs: 85 through 89: codon optimized human UCP1 CDS using custom 5’ and 3’ UTRs. The 5’ UTR for these CDS: Modified genomic human COL1A 5' UTR with a Kozak sequence inserted. The 3’ UTR for these CDS: double stop codon.

[0066] SEQ ID NOs: 90 through 94: codon optimized human UCP1 CDS using custom 5’ and 3’ UTRs. The 5’ UTR for these CDS: Modified genomic human LEP 5' UTR with a Kozak sequence inserted. The 3’ UTR for these CDS: double stop codon.

[0067] SEQ ID NOs: 95 through 99: codon optimized human UCP1 CDS using custom 5’ and 3’ UTRs. The 5’ UTR for these CDS: Modified genomic human ADIPOQ 5' UTR with a Kozak sequence inserted. The 3’ UTR for these CDS: double stop codon.

[0068] SEQ ID NOs: 100 through 104: codon optimized human UCP1 CDS using custom 5’ and 3’ UTRs. The 5’ UTR for these CDS: Consensus genomic human FABP4 5' UTR. The 3’ UTR for these CDS: double stop codon.

[0069] SEQ ID NOs: 105 through 109: codon optimized human UCP1 CDS using custom 5’ and 3’ UTRs. The 5’ UTR for these CDS: Consensus genomic human LIPE 5' UTR. The 3’ UTR for these CDS: double stop codon.

[0070] SEQ ID NOs: 110 through 114: codon optimized human UCP1 CDS using custom 5’ and 3’ UTRs. The 5’ UTR for these CDS: Consensus genomic human PLIN1 5' UTR. The 3’ UTR for these CDS: double stop codon.

[0071] SEQ ID NOs: 115 through 119: codon optimized human UCP1 CDS using custom 5’ and 3’ UTRs. The 5’ UTR for these CDS: Consensus genomic human COL1A 5' UTR. The 3’ UTR for these CDS: double stop codon.

[0072] SEQ ID NOs: 120 through 124: codon optimized human UCP1 CDS using custom 5’ and 3’ UTRs. The 5’ UTR for these CDS: Consensus genomic human LEP 5' UTR. The 3’ UTR for these CDS: double stop codon.

[0073] SEQ ID NOs: 125 through 129: codon optimized human UCP1 CDS using custom 5’ and 3’ UTRs. The 5’ UTR for these CDS: Consensus genomic human ADIPOQ 5' UTR. The 3’ UTR for these CDS: double stop codon.

[0074] The consensus genomic coding sequence of UCP1 for Homo sapiens is known. UCP homologs from non-human animals, such as lower primates, horses, cows, pigs, sheep, goats, chicken can also be used with the lipid nanoparticles. The synthetic mRNA may further comprise siRNA, circular RNA, self-replicating mRNA, or mRNA designed for gene editing including but not limited to CRISPR-Cas9 systems. The LNP vesicle may be tailored specifically to enhance transfection of the target adipocytes and WAT. Tailoring of the LNP vesicle may include adjusting the molar ratios of ionizable lipids, helper phospholipids, polyethylene glycol (PEG)- lipids, and sterols as well as immunogenicity-reducing molecules including but not limited to steroids and steroid pro-drugs.

[0075] The “lipid nanoparticle” comprises one or more lipids (e.g., cationic lipids, non-cationic lipids, and PEG-modified lipids) which are formulated to deliver one or more mRNA to one or more target cells. Suitable lipids include, for example, phosphatidyl (e.g., phosphatidylglycerol, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, sphingolipids, cerebrosides, and gangliosides). Various polymers may also be incorporated into the lipid vehicles, including, for example, polyacrylates, polyalkycyanoacrylates, polylactide, polylactide-polyglycolide copolymers, polycaprolactones, dextran, albumin, gelatin, alginate, collagen, chitosan, cyclodextrins, dendrimers and poly ethylenimine.

[0076] The lipid nanoparticles can incorporate an ionizable cationic lipid (ICL) to encapsulate and / or enhance the delivery of mRNA into the target cell. A “cationic lipid” refers to any of a number of lipid species that carry a net positive charge at a selected pH, such as physiological pH.

[0077] Non-cationic lipids (NCL) may also be used in the lipid nanoparticles. Non-cationic lipids include, but are not limited to, distearoylphosphatidylcholine (DSPC), dioleoylphosphandylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidlylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleol-phosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N- maleimidomethylj-cyclohexane- 1 -carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanoiamine (DMPE), distearoyl-phosphatidyl- ethanolamine (DSPE), 16-0-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, l-stearoyl-2- oleoyl-phosphatidy ethanolamine (SOPE), cholesterol, or a mixture thereof. Such non-cationic lipids may be used alone, but are preferably used in combination with other excipients, for example, cationic lipids. When used in combination with a cationic lipid, the non-cationic lipid may comprise a molar ratio of 5% to about 90%, or preferably about 10% to about 70% of the total lipid present in the transfer vehicle.

[0078] Polyethylene glycol (PEG)-modified phospholipids, PEG / lipid conjugates and derivatized lipids such as DMG- PEG(2000), derivatized ceramides (PEG-CER), including N-Octanoyl- Sphingosine-l-[Succinyl(Methoxy Polyethylene Glycol)-2000] (C8 PEG-2000 ceramide), or ALC-0159 can be incorporated into the lipid nanoparticle. The PEG-modified lipids include, but are not limited to, a polyethylene glycol chain of up to 5 kDa in length covalently attached to a lipid with alkyl chain(s) of C6-C20 length. The PEG-modified phospholipid and derivatized lipids of the present invention can be present at varying molar ratios, e.g., from about 0% to about 20%, about 0.5% to about 20%, about 1% to about 15%, about 4% to about 10%, or about 2% of the total lipid present in the liposomal transfer vehicle.

[0079] LNP Vesicles may include the following:

[0080] LNP Component Molar Ratios

[0081] LNP PEG / Lipid

[0082] ID ICL Molecule ICL NCL Cholesterol Conjugate

[0083] A ALC-0315 50 10 38.5 1.5 46.3 9.4 42.7 1.6 47.2 9.2 42.2 1.4

[0084] B DLin-MC3-DMA 50 10 38.5 1.5 51.2 10 37.4 1.4 49.4 11.2 37.8 1.6

[0085] C LP-01 45 9 44 2 43.5 10 44.3 2.2 46.3 9.4 42.3 2

[0086] D SM-102 50 10 38.5 1.5

[0087] 48.3 9.1 40.5 2.1

[0088] 46.6 10.4 41.2 1.8

[0089] E 4A3-SC8 38.5 30 30 1.5

[0090] 35 16 46.5 2.5

[0091] 45 11 42.5 1.5

[0092] F CKK-E12 35 16 46.5 2.5

[0093] 45 11 42.5 1.5

[0094] 50 10 38.5 1.5

[0095] G 3060110 35 16 46.5 2.5

[0096] 50 10 38.5 1.5

[0097] 40 32.5 25 2.5

[0098] H C3-K2-E14 50 10 38.5 1.5

[0099] 56.5 10 31.8 1.7

[0100] 35 16 46.5 2.5

[0101] I C14-4 35 16 46.5 2.5

[0102] 50 10 38.5 1.5

[0103] 56.5 10 31.8 1.7

[0104] J Lipid A9 50 10 38.5 1.5

[0105] 35 16 46.5 2.5

[0106] 40 32.5 25 2.5

[0107] K OF-02 40.3 14.9 43.7 1.1

[0108] 56.5 10 31.8 1.7

[0109] 52.7 17.5 28.7 1.1

[0110] L TCL053 60 10.6 27.3 2.1

[0111] 50 10 38.5 1.5

[0112] 35 16 46.5 2.5

[0113] The above list is intended to be exemplary and LNPs of other component molar ratios as described above may be appropriate for the specific application contemplated herein.

[0114] For example, the composition of the LNP (e.g. ICL, Cholesterol, DPSC, and DMG-PEG2000) can be combined to yield different molar ratios. In one embodiment, the molar ratio of ICL to Cholesterol to DPSC to PEG / LIPID CONJUGATE are (3 to 80):(3 to 80):(l to 50):(0.2 to 10). In another embodiment, the molar ratio of ICL to Cholesterol to DPSC to PEG / LIPID CONJUGATE are (7 to 80):(7 to 80):(3 to 50):( 1.0 to 10). In another embodiment, the molar ratio of ICL to Cholesterol to DPSC to PEG / LIPID CONJUGATE are (10 to 80):( 10 to 80):(4 to 50):(l .5 to 10). In another embodiment, the molar ratio of ICL to Cholesterol to DPSC to PEG / LIPID CONJUGATE are (13 to 80):(13 to 80):(5 to 50):(2.0 to 10). In another embodiment, the molar ratio of ICL to Cholesterol to DPSC to PEG / LIPID CONJUGATE are (3 to 75):(3 to 75):(1 to 45):(0.2 to 7). In another embodiment, the molar ratio of ICL to Cholesterol to DPSC to PEG / LIPID CONJUGATE are (7 to 75):(7 to 75):(3 to 45):(1.0 to 7). In another embodiment, the molar ratio of ICL to Cholesterol to DPSC to PEG / LIPID CONJUGATE are (10 to 75):(10 to 75):(4 to 45):(1.5 to 7). In another embodiment, the molar ratio of ICL to Cholesterol to DPSC to PEG / LIPID CONJUGATE are (13 to 75):(13 to 75):(5 to 45):(2.0 to 7). In another embodiment, the molar ratio of ICL to Cholesterol to DPSC to PEG / LIPID CONJUGATE are (3 to 70):(3 to 70):( 1 to 40):(0.2 to 5). In another embodiment, the molar ratio of ICL to Cholesterol to DPSC to PEG / LIPID CONJUGATE are (7 to 70):(7 to 70):(3 to 40):(l .0 to 5). In another embodiment, the molar ratio of ICL to Cholesterol to DPSC to PEG / LIPID CONJUGATE are (10 to 70):(10 to 70):(4 to 40):(l .5 to 5). In another embodiment, the molar ratio of ICL to Cholesterol to DPSC to PEG / LIPID CONJUGATE are (13 to 70):( 13 to 70):(5 to 40):(2.0 to 5).

[0115] In a second embodiment, the molar ratio of ICL to Cholesterol to DPSC to PEG / LIPID CONJUGATE are (3 to 8O):(3 to 70):( 1 to 50):(0.2 to 10). In another embodiment, the molar ratio of ICL to Cholesterol to DPSC to PEG / LIPID CONJUGATE are (7 to 80):(7 to 70):(3 to 50):(l .0 to 10). In another embodiment, the molar ratio of ICL to Cholesterol to DPSC to PEG / LIPID CONJUGATE are (10 to 80):(10 to 70):(4 to 50):(1.5 to 10). In another embodiment, the molar ratio of ICL to Cholesterol to DPSC to PEG / LIPID CONJUGATE are (13 to 80):( 13 to 70):(5 to 50):(2.0 to 10). In another embodiment, the molar ratio of ICL to Cholesterol to DPSC to PEG / LIPID CONJUGATE are (3 to 75):(3 to 80):(l to 45):(0.2 to 7). In another embodiment, the molar ratio of ICL to Cholesterol to DPSC to PEG / LIPID CONJUGATE are (7 to 75):(7 to 80):(3 to 45):( 1.0 to 7). In another embodiment, the molar ratio of ICL to Cholesterol to DPSC to PEG / LIPID CONJUGATE are (10 to 75):( 10 to 80):(4 to 45):(1.5 to 7). In another embodiment, the molar ratio of ICL to Cholesterol to DPSC to PEG / LIPID CONJUGATE are (13 to 75):(13 to 80):(5 to 45):(2.0 to 7). In another embodiment, the molar ratio of ICL to Cholesterol to DPSC to PEG / LIPID CONJUGATE are (3 to 70):(3 to 75):( 1 to 40):(0.2 to 5). In another embodiment, the molar ratio of ICL to Cholesterol to DPSC to PEG / LIPID CONJUGATE are (7 to 70):(7 to 75):(3 to 40):( 1.0 to 5). In another embodiment, the molar ratio of ICL to Cholesterol to DPSC to PEG / LIPID CONJUGATE are (10 to 70):(10 to 75):(4 to 40):( 1.5 to 5). In another embodiment, the molar ratio of ICL to Cholesterol to DPSC to PEG / LIPID CONJUGATE are (13 to 70):(13 to 75):(5 to 40):(2.0 to 5).

[0116] In a third embodiment, the molar ratio of ICL to Cholesterol to DPSC to PEG / LIPID CONJUGATE are (3 to 8O):(3 to 80):( 1 to 40):(0.2 to 10). In another embodiment, the molar ratio of ICL to Cholesterol to DPSC to PEG / LIPID CONJUGATE are (7 to 80):(7 to 80):(3 to 40):( 1.0 to 10). In another embodiment, the molar ratio of ICL to Cholesterol to DPSC to PEG / LIPID CONJUGATE are (10 to 80):(10 to 80):(4 to 40):(l .5 to 10). In another embodiment, the molar ratio of ICL to Cholesterol to DPSC to PEG / LIPID CONJUGATE are (13 to 80):( 13 to 8O):(5 to 40):(2.0 to 10). In another embodiment, the molar ratio of ICL to Cholesterol to DPSC to PEG / LIPID CONJUGATE are (3 to 75):(3 to 75):(1 to 50):(0.2 to 7). In another embodiment, the molar ratio of ICL to Cholesterol to DPSC to PEG / LIPID CONJUGATE are (7 to 75):(7 to 75):(3 to 50):( 1.0 to 7). In another embodiment, the molar ratio of ICL to Cholesterol to DPSC to PEG / LIPID CONJUGATE are (10 to 75):( 10 to 75):(4 to 50):(l .5 to 7). In another embodiment, the molar ratio of ICL to Cholesterol to DPSC to PEG / LIPID CONJUGATE are (13 to 75):(13 to 75):(5 to 50):(2.0 to 7). In another embodiment, the molar ratio of ICL to Cholesterol to DPSC to PEG / LIPID CONJUGATE are (3 to 70) : (3 to 70):( 1 to 45):(0.2 to 5). In another embodiment, the molar ratio of ICL to Cholesterol to DPSC to PEG / LIPID CONJUGATE are (7 to 70):(7 to 70):(3 to 45):( 1.0 to 5). In another embodiment, the molar ratio of ICL to Cholesterol to DPSC to PEG / LIPID CONJUGATE are (10 to 70):(10 to 70):(4 to 45):(1.5 to 5). In another embodiment, the molar ratio of ICL to Cholesterol to DPSC to PEG / LIPID CONJUGATE are (13 to 70):(13 to 70):(5 to 45):(2.0 to 5).

[0117] In a fourth embodiment, the molar ratio of ICL to Cholesterol to DPSC to PEG / LIPID CONJUGATE are (3 to 8O):(3 to 80):( 1 to 50):(0.2 to 5). In another embodiment, the molar ratio of ICL to Cholesterol to DPSC to PEG / LIPID CONJUGATE are (7 to 80):(7 to 80):(3 to 50):(l .0 to 5). In another embodiment, the molar ratio of ICL to Cholesterol to DPSC to PEG / LIPID CONJUGATE are (10 to 80):(10 to 80):(4 to 50):(l .5 to 5). In another embodiment, the molar ratio of ICL to Cholesterol to DPSC to PEG / LIPID CONJUGATE are (13 to 80):( 13 to 80):(5 to 50):(2.0 to 5). In another embodiment, the molar ratio of ICL to Cholesterol to DPSC to PEG / LIPID CONJUGATE are (3 to 75):(3 to 75):( 1 to 45):(0.2 to 10). In another embodiment, the molar ratio of ICL to Cholesterol to DPSC to PEG / LIPID CONJUGATE are (7 to 75):(7 to 75):(3 to 45):(1.0 to 10). In another embodiment, the molar ratio of ICL to Cholesterol to DPSC to PEG / LIPID CONJUGATE are (10 to 75):(10 to 75):(4 to 45):(1.5 to 10). In another embodiment, the molar ratio of ICL to Cholesterol to DPSC to PEG / LIPID CONJUGATE are (13 to 75):(13 to 75):(5 to 45):(2.0 to 10). In another embodiment, the molar ratio of ICL to Cholesterol to DPSC to PEG / LIPID CONJUGATE are (3 to 70):(3 to 70):(l to 40):(0.2 to 7). In another embodiment, the molar ratio of ICL to Cholesterol to DPSC to PEG / LIPID CONJUGATE are (7 to 70):(7 to 70):(3 to 40):( 1.0 to 7). In another embodiment, the molar ratio of ICL to Cholesterol to DPSC to PEG / LIPID CONJUGATE are (10 to 70):( 10 to 70):(4 to 40):( 1.5 to 7). In another embodiment, the molar ratio of ICL to Cholesterol to DPSC to PEG / LIPID CONJUGATE are (13 to 70):(13 to 70):(5 to 40):(2.0 to 7).

[0118] LNP ID A:

[0119] ICL: ALC-0315

[0120] Formal Name: 2-hexyl-decanoic acid, l,l'-[[(4-hydroxybutyl)imino]di-6,l- hexanediyl] ester

[0121] Alternative Names: ((4-Hydroxybutyl)azanediyl)bis(hexane-6,l-diyl) bis(2- hexyldecanoate)

[0122] CAS Number: 2036272-55-4

[0123] Molecular Formula: C48H95NO5

[0124] LNP ID B:

[0125] ICL: DLin-MC3-DMA

[0126] Formal Name: 4-(dimethylamino)-butanoic acid, (10Z,13Z)-l-(9Z,12Z)-9,12- octadecadien- l-yl-10, 13-nonadecadien- 1-yl ester

[0127] Alternative Names: (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4- (dimethylamino)butanoate CAS Number: 1224606-06-7 Molecular Formula: C43H79NO2

[0128] LNP ID C:

[0129] ICL: LP-01

[0130] Formal Name: 9Z,12Z-octadecadienoic acid, 3-[4,4-Z?z5'(octyloxy)-l-oxobutoxy]- 2-[[[[3-(diethylamino)propoxy]carbonyl]oxy]methyl]propyl ester Alternative Names: CIN-16645, 9Z,12Z-octadecadienoic acid, 3-[4,4- bis(octyloxy)- l-oxobutoxy]-2-[[[[3- (diethylamino )propoxy] carbonyl]oxy]methyl]propyl ester CAS Number: 1799316-64-5

[0131] Molecular Formula: C50H93NO9

[0132] LNP ID D:

[0133] ICL: SM-102

[0134] Formal Name: 8-[(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino]-octanoic acid, 1 -octylnonyl ester

[0135] Alternative Names: Lipid H, Heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6- (undecyloxy)hexyl)amino)- octanoate

[0136] CAS Number: 2089251-47-6

[0137] Molecular Formula: C44H87NO5

[0138] LNP ID E:

[0139] ICL: 4A3-SC8

[0140] CAS Number: 1857340-78-3

[0141] Molecular Formula: C75H139N3O16S4

[0142] LNP ID F:

[0143] ICL: CKK-E12

[0144] Formal Name: 3,6-bis[4-[bis(2-hydroxydodecyl)amino]butyl]-2,5-piperazinedione Alternative Names: 3,6-bis(4-(bis(2-hydroxydodecyl)amino)butyl)piperazine-2,5- dione

[0145] CAS Number: 1432494-65-9

[0146] Molecular Formula: C60H120N4O6

[0147] LNP ID G:

[0148] ICL: 3060iio

[0149] Formal Name: tetrakis(8-methylnonyl) 3,3',3",3"'-(((methylazanediyl)bA(propane- 3,l-diyl))bz.y(azanetriyl))tetrapropionate

[0150] CAS Number: 2322290-93-5

[0151] Molecular Formula: C59H115N3O8

[0152] LNP ID H: ICL: C3-K2-E14

[0153] Formal Name: 3,3'-(propylazanediyl)bis(N-(2-(bis(2- hydroxytetradecyl)amino)ethyl)propanamide)

[0154] CAS Number: 2933215-86-0

[0155] Molecular Formula: C69H141N5O6

[0156] FNP ID I:

[0157] ICF: C14-4

[0158] Formal Name: l,l'-[[2-[2-[4-[2-[[2-[2-[Z?A(2- hydroxytetradecyl)amino]ethoxy]ethyl](2-hydroxytetradecyl)amino]ethyl]-l- piperazinyl]ethoxy]ethyl]immo]Z>zs-2-tetradecanol

[0159] CAS Number: 2639634-80-1

[0160] Molecular Formula: C84H173N5O7

[0161] FNP ID I:

[0162] ICF: Fipid A9

[0163] Formal Name: &A(2-butyloctyl) 10-(N-(3-

[0164] (dimethylamino)propyl)nonanamido)nonadecanedioate

[0165] Alternative Names: 1, 19-Bis(2-butyloctyl) 10-[[3-(dimethylamino)propyl](l- oxononyl)amino]nonadecanedioate

[0166] CAS Number: 2036272-50-9

[0167] Molecular Formula: C57H112N2O5

[0168] FNP ID K:

[0169] ICF: OF-02

[0170] Formal Name: 3,6-bis[4-[bis[(9Z,12Z)-2-hydroxy-9,12-octadecadien-l- yl]amino]butyl]-2,5-piperazinedione

[0171] Alternative Names: 3,6-bis(4-(bis((9Z,12Z)-2-hydroxyoctadeca-9,12-dien-l- yl)amino)butyl)piperazine-2, 5-dione

[0172] CAS Number: 1883431-67-1

[0173] Molecular Formula: C84H152N4O6

[0174] FNP ID L:

[0175] ICF: TCF053 Formal Name: 2-(((4-(dimethylamino)butanoyl)oxy)methyl)-2-((((Z)-tetradec-9- enoyl)oxy)methyl)propane- 1 , 3 -diyl (9Z,9'Z)- / ? / .s( tctradcc-9-cnoatc) CAS Number: 2361162-70-9

[0176] Molecular Formula: C53H95NO8

[0177] In another embodiment, the 5' UTR region may comprise a ribosome binding site, a Kozak sequence, a modified genomic human LIPE 5' UTR, a modified genomic human LEP 5' UTR, a modified genomic human COL1A 5' UTR, a modified genomic human ADIPOQ 5' UTR, a modified genomic human PLIN1 5' UTR, a translation initiator of short 5' UTR (TISU), a consensus genomic human LIPE 5' UTR, a consensus genomic human UCP1 5' UTR, a consensus genomic human PLIN1 5' UTR, a consensus genomic human COL1A 5' UTR, a consensus genomic human LEP 5' UTR, a consensus genomic human ADIPOQ 5' UTR, a consensus genomic human FABP4 5' UTR, or combinations thereof.

[0178] In another embodiment, the 3' UTR region may comprise a double stop codon, a consensus genomic human UCP1 3' UTR, a Cominarity 3' UTR, or combinations thereof. The Cominarity 3’ UTR is a combination and optimization of segments from the human mitochondrial 12S rRNA (mtRNRl) and the human AES / TLE5 gene. The AES segment of 136 nt with two C— AP mutations was added after two trinucleotides following the second stop codon. The mtRNRl segment of 139 nt was added immediately after.

[0179] In another embodiment, the mRNA of the present disclosure comprises a nucleotide sequence about 80% to about 100%, about 85% to about 100%, about 90% to about 100%, or about 95% to about 100%, identical to the amino acid sequence set forth in SEQ ID NO:1.

[0180] In another embodiment, the mRNA of the present disclosure comprises a nucleotide sequence about 80% to about 100%, about 85% to about 100%, about 90% to about 100%, or about 95% to about 100%, identical to the amino acid sequence set forth in any of SEQ ID NO:2 through SEQ ID NO: 21.

[0181] In an alternative embodiment, the mRNA of the present disclosure comprises a nucleotide sequence about 80% to about 100%, about 85% to about 100%, about 90% to about 100%, or about 95% to about 100%, identical to the amino acid sequence set forth in any of SEQ ID NO: 22 through SEQ ID NO: 129. In another embodiment, a synthetic mRNA, the sequence of may be selected from one of SEQ ID NO: 1 disclosed in the Sequence Listing incorporated by reference herein, encoding for the intracellular synthesis of UCP1 by the ribosomes of a target cell is packaged in a lipid nanoparticle (LNP) vesicle for transfection of WAs or ASCs with the packaged mRNA.

[0182] In another embodiment, cells transfected with a pharmaceutical composition comprising mRNA of SEQ ID NO: 1 will express UCP1 and undergo a process of lipolysis through non-ATP generating respiration, generating heat and reducing the overall FFA content of the transfected and nearby cells.

[0183] In another embodiment, a physician may administer the pharmaceutical composition comprising the mRNA of SEQ ID NO: 1 packaged in an LNP to a patient seeking to accomplish spot fat reduction.

[0184] In another embodiment, a physician administering the mRNA of SEQ ID NO: 1 packaged in an LNP to a patient seeking to accomplish spot fat reduction may administer the pharmaceutical composition in a bolus injection in the targeted area.

[0185] In another embodiment, a physician administering the mRNA of SEQ ID NO: 1 packaged in an LNP to a patient seeking to accomplish spot fat reduction may administer the pharmaceutical composition in a series of injections in the targeted area.

[0186] In another embodiment, a physician administering the mRNA of SEQ ID NO: 1 packaged in an LNP to a patient seeking to accomplish spot fat reduction may administer the pharmaceutical composition transdermally. The transdermal administration may be accomplished through the use of penetration enhancers (e.g. DMSO, oleic acid, lauric acid, sodium lauryl sulfate, sodium ducosate, sodium oleate, cetyltrimethylammonium bromide, benzalkonium chloride, polysorbates, sorbitan esters, polyoxyethylene alkyl ethers, Laurocarpam, propylene glycol monolaurate, lecithin, terpenes), vesicular carriers (e.g. liposomes, ethosomes, niosomes) microneedle injection, iontophoresis, sonophoresis, jet injection or other suitable means.

[0187] Microneedle injectors utilize microscopic needles, typically ranging from 50 to 900 micrometers in length, to bypass the skin's outermost layer, the stratum comeum, and deliver therapeutic agents directly into the underlying epidermal or dermal layers. The microneedles can be disposed in an array on a patch wherein several hundred or several thousand microneedles are disposed. Iontophoresis utilizes a low-level electrical current to facilitate the transport of ions across biological membranes, most notably the skin. The medication is disposed on a patch with an electrode that conducts electricity to drive the ions across the membrane.

[0188] Sonophoresis, also known as phonophoresis, is a non-invasive technique that utilizes ultrasound waves to temporarily increase the permeability of the skin. This enhanced permeability allows for the improved delivery of therapeutic agents, such as medications and cosmetics, across the skin barrier and into deeper tissues.

[0189] Jet injection employs a high-velocity stream of liquid that penetrates the skin to deliver the substance into the underlying tissues. A jet injector creates a very fine, high-pressure jet of liquid that is powerful enough to pierce the skin and reach the desired tissue depth - typically the intradermal, subcutaneous, or intramuscular layer. This is achieved by rapidly forcing a premeasured dose of medication through a tiny orifice in the injector's nozzle.

[0190] In another embodiment, a physician administering the mRNA of SEQ ID NO: 1 packaged in an LNP to a patient seeking to accomplish spot fat reduction may administer the pharmaceutical composition several times over the course of periodic patient encounters.

[0191] In another embodiment, a physician administering the mRNA of SEQ ID NO: 1 packaged in an LNP to a patient seeking to accomplish spot fat reduction may administer the pharmaceutical composition in a manner prescribed by an algorithm, LLM or machine learning implementation to accomplish the desired spot fat reduction.

[0192] In another embodiment, a synthetic mRNA, comprising the nucleotide sequence set forth in one of SEQ ID NO: 2 through SEQ ID NO: 21 disclosed in the Sequence Listing incorporated by reference herein, encoding for the intracellular synthesis of UCP1 by the ribosomes of a target cell is packaged in a lipid nanoparticle (LNP) vesicle. The packaged synthetic mRNA may be for transfection of WAs or ASCs.

[0193] In another embodiment, cells transfected with a pharmaceutical composition comprising mRNA of any of SEQ ID NO: 2 through SEQ ID NO: 21 will express UCP1 and undergo a process of lipolysis through non- ATP generating respiration, generating heat and reducing the overall FFA content of the transfected cells.

[0194] In another embodiment, a physician may administer the pharmaceutical composition comprising the mRNA of any of SEQ ID NOs: 2 through 21 packaged in an LNP to a patient seeking to accomplish spot fat reduction.

[0195] In another embodiment, the mRNA comprising the nucleotide sequence set forth in any of SEQ ID NOs: 2 through 21 packaged in an LNP is administered to a patient seeking to accomplish spot fat reduction. The packaged mRNA may be administered in a bolus injection in the targeted area.

[0196] In another embodiment, the mRNA comprising the nucleotide sequence set forth in any of SEQ ID NOs: 2 through 21 packaged in an LNP is administered to a patient seeking to accomplish spot fat reduction in a series of injections in the targeted area.

[0197] In another embodiment, the mRNA comprising the nucleotide sequence set forth in any of SEQ ID NOs: 2 through 21 packaged in an LNP is administered transdermally to a patient seeking to accomplish spot fat reduction . The transdermal administration may be accomplished through the use of penetration enhancers (e.g. DMSO, oleic acid, lauric acid, sodium lauryl sulfate, sodium ducosate, sodium oleate, cetyltrimethylammonium bromide, benzalkonium chloride, polysorbates, sorbitan esters, polyoxyethylene alkyl ethers, Laurocarpam, propylene glycol monolaurate, lecithin, terpenes), vesicular carriers (e.g. liposomes, ethosomes, niosomes) microneedle injection, iontophoresis, sonophoresis, jet injection or other suitable means.

[0198] In another embodiment, the mRNA comprising the nucleotide sequence set forth in any of SEQ ID NOs: 2 through 21 packaged in an LNP is administered to a patient seeking to accomplish spot fat reduction several times over the course of periodic patient encounters.

[0199] In another embodiment, a physician administering the mRNA comprising the nucleotide sequence set forth in any of SEQ ID NOs: 2 through 21 packaged in an LNP to a patient seeking to accomplish spot fat reduction may administer the pharmaceutical composition in a manner prescribed by an algorithm, LLM or machine learning implementation to accomplish the desired spot fat reduction.

[0200] In alternative embodiment, a synthetic mRNA, comprising the nucleotide sequence set forth in any of SEQ ID NOs: 22 through 129 disclosed in the Sequence Listing incorporated by reference herein, encoding for the intracellular synthesis of UCP1 by the ribosomes of a target cell is packaged in a lipid nanoparticle (LNP) vesicle. In an alternative embodiment, cells transfected with a pharmaceutical composition comprising mRNA comprising the nucleotide sequence set forth in any of SEQ ID NO: 22 through SEQ ID NO: 129 will express UCP1 and undergo a process of lipolysis through non- ATP generating respiration, generating heat and reducing the overall FFA content of the transfected cells,

[0201] In an alternative embodiment, a physician may administer the pharmaceutical composition comprising the mRNA of SEQ ID NOs: 22 through 129 packaged in an LNP to a patient seeking to accomplish spot fat reduction.

[0202] In an alternative embodiment, a physician administering the mRNA comprising the nucleotide sequence set forth in any of SEQ ID NOs: 22 through 129 packaged in an LNP to a patient seeking to accomplish spot fat reduction may administer the pharmaceutical composition in a bolus injection in the targeted area.

[0203] In an alternative embodiment, a physician administering the mRNA comprising the nucleotide sequence set forth in any of SEQ ID NOs: 22 through 129 packaged in an LNP to a patient seeking to accomplish spot fat reduction may administer the pharmaceutical composition in a series of injections in the targeted area.

[0204] In an alternative embodiment, a physician administering the mRNA comprising the nucleotide sequence set forth in any of SEQ ID NOs: 22 through 129 packaged in an LNP to a patient seeking to accomplish spot fat reduction may administer the pharmaceutical composition transdermally. The transdermal administration may be accomplished through the use of penetration enhancers (e.g. DMSO, oleic acid, lauric acid, sodium lauryl sulfate, sodium ducosate, sodium oleate, cetyltrimethylammonium bromide, benzalkonium chloride, polysorbates, sorbitan esters, polyoxyethylene alkyl ethers, Laurocarpam, propylene glycol monolaurate, lecithin, terpenes), vesicular carriers (e.g. liposomes, ethosomes, niosomes) microneedle injection, iontophoresis, sonophoresis, jet injection or other suitable means.

[0205] In an alternative embodiment, a physician administering the mRNA comprising the nucleotide sequence set forth in any of SEQ ID NOs: 22 through 129 packaged in an LNP to a patient seeking to accomplish spot fat reduction may administer the pharmaceutical composition several times over the course of periodic patient encounters.

[0206] In an alternative embodiment, a physician administering the mRNA comprising the nucleotide sequence set forth in any of SEQ ID NOs: 22 through 129 packaged in an LNP to a patient seeking to accomplish spot fat reduction may administer the pharmaceutical composition in a manner prescribed by an algorithm, LLM or machine learning implementation to accomplish the desired spot fat reduction.

[0207] In another embodiment, the synthetic mRNA may in addition to or alternatively encode for the expression of the B3 adrenoreceptor, PR / SET domain 16 (PRDM16), Peroxisome proliferator- activated receptor gamma coactivator- 1 alpha (PGC-1 alpha), CCAAT / enhancer binding protein beta (C / EPB-Beta), Kriippel-like Factor 11 (KLF11), and / or nuclear receptor interacting protein 1 (NRIP1).

[0208] In another embodiment, the synthetic mRNA may be modified with a 5’ cap, a 5’ untranslated region, polymorphisms in the target protein coding sequence, nucleic acid substitutions to reduce immunogenicity and / or enhance mRNA longevity within the cell, and / or a 3’ untranslated region including 1 or more poly- A tails.

[0209] The mRNA can be chemically or biologically modified. Modifications to an mRNA include the depletion of a base (e.g., by deletion or by the substitution of one nucleotide for another) or modification of a base, chemically by covalent addition pendant groups which are not naturally found in such mRNA molecules. For example, the number of C and / or U residues in an mRNA sequence may be reduced. In another embodiment, the number of C and / or U residues is reduced by substitution of one codon encoding a particular amino acid for another codon encoding the same or a related amino acid. The mRNA nucleic acids can also incorporate pseudouridines. Substitutions and modifications to the mRNA of the present invention may be performed by methods readily known to one or ordinary skill in the art.

[0210] Modifications to the mRNA can also be made to the 3' and 5' ends of an mRNA molecule.

[0211] These modifications include a poly A tail or a longer poly A tail, the alteration of the 3' UTR or the 5' UTR or complexing the mRNA with a protein or complementary nucleic acid molecule.

[0212] Preferred mRNA sequences are provided in the Sequence Listing incorporated by reference herein.

[0213] In another embodiment, the synthetic mRNA encoding for the intracellular synthesis of UCP1 by the ribosomes of a target cell packaged in a LNP vesicle is formulated for injectable delivery to a target area. This may include a dilute formulation that may be injected into a large area of subcutaneous fat using a blunt canula or other means fit for such purpose. This injectable delivery may alternatively include direct injection to a target site of adipose tissue. This formulation may include a carrier solution that protects the LNP vesicles during transport and injection and may further comprise a buffering solution fit for that purpose.

[0214] In another embodiment, the synthetic mRNA encoding for the intracellular synthesis of UCP1 by the ribosomes of a target cell packaged in an LNP vesicle is formulated for transdermal delivery to a target area. This formulation may include a transdermal carrier such as a nano structured lipid carrier molecule suspended in a cream or gel to be rubbed on the skin over the target area. Transdermal delivery may be further effectuated by iontophoresis, a microneedle patch or a topical formulation to be applied after disruption of the skin barrier via methods including but not limited to microneedling, laser treatment, and chemical peels.

[0215] In another embodiment, the synthetic mRNA encoding for the intracellular synthesis of UCP1 by the ribosomes of a target cell packaged in an LNP vesicle is formulated for systemic delivery to multiple sites within a patient’s body. This formulation may include a carrier solution that protects the LNP vesicles during transport and injection / intravenous delivery and may further comprise a buffering solution fit for that purpose.

[0216] In a second embodiment, a patient treated with a synthetic mRNA encoding for the intracellular synthesis of UCP1 by the ribosomes of a target cell packaged in an LNP vesicle employs an apparatus to cool the pharmaceutically generated BeAT in order to activate the thermogenic process of BeAT. This apparatus may comprise: ice packs, a cold water tub or a system that circulates chilled water through a pad or patch positioned on the surface of the skin over a target area. This apparatus may comprise an internal cooling mechanism for precisely regulating the temperature of the cooling apparatus or of the pad or patch positioned on the surface of the skin over a target area.

[0217] In another embodiment, the cooling apparatus may be used as part of a protocol prescribed by a treating physician for activating the thermogenic properties of the BeAT for predetermined time periods in order to achieve the specific goals of the patient with regards to reduction of the appearance of adipose tissue in the target area.

[0218] In another embodiment, the BeAT may be activated by B3 agonists (e.g. mirabegron), caffeine and like molecules, ibuprofen and like molecules, 4-heptylbenzoic acid; bromododecanoic acid; dodecanoic acid; nonadecanoic acid; oleic acid; retinoic acid; tetradecylthioacetic acid; TTNPB; TUG-891.

[0219] In another embodiment, the protocol prescribed by the treating physician may include a time frame for repeat treatment of the target area with the synthetic mRNA encoding for the intracellular synthesis of UCP1 by the ribosomes of a target cell packaged in an LNP vesicle. The time frame may reflect the rate of turnover of UCP1 in the target cells.

[0220] Examples

[0221] Example 1: The image of Figure 1 was created as follows: Synthetic mRNA expressing mCherry fluorescent protein was synthesized via in vitro transcription from a linearized DNA plasmid. The synthetic mRNA was encapsulated into lipid nanoparticles, yielding a reporter gene test article. Subcutaneous fat was donated by a patient undergoing elective surgery under an IRB -approved protocol. The fat was stabilized for long-term ex vivo culture. The ex vivo fat was treated with the reporter gene test article. Fluorescence and brightfield microscopy images were captured every 4 to 6 hours using an automated system for a total of 48 hours. New red fluorescence in previously non-fluorescent cells indicated successful transfection of ex vivo human fat.

[0222] Example 2: The image of Figure 2 was created as follows: Subcutaneous fat was donated by a patient undergoing elective surgery under an IRB-approved protocol. The fat was stabilized for long-term ex vivo culture. Brightfield microscopy images were captured every 4 to 6 hours using an automated system for a total of 6 days (144 hours). No significant change in the adipocytes’ diameter was observed. The red line marked the diameter of one representative mature adipocyte within the adipose tissue cluster at the start of the experiment (hour 0). The blue line marked the diameter of the same adipocyte at the end of the experiment (hour 144).

[0223] Example 3: The images of Figure 3 were created as follows: Synthetic mRNA expressing UCP1 was synthesized via in vitro transcription from a linearized DNA plasmid. The synthetic mRNA was encapsulated into lipid nanoparticles of composition LNP D of component molar ratio (SM-102: DPSC: Cholesterol: PEG / Lipid Conjugate) of 50:10:38.5:1.5, yielding a drug candidate test article. Subcutaneous fat was donated by a patient undergoing elective surgery under an IRB-approved protocol. The same donated fat was used in this study as in the negative control study (Figure 2). The fat was stabilized for long-term ex vivo culture. The ex vivo fat was treated with the drug candidate test article at full dose (5ug drug candidate per lOOmg of ex vivo fat). Brightfield microscopy images were captured every 4 to 6 hours using an automated system for a total of 6 days (144 hours). Every treated fat cluster shrank significantly, with a majority of the unilocular adipocytes shrinking to the point where they could no longer be visually identified as mature, unilocular adipocytes.

[0224] Example 4: The images of Figure 4 were created as follows: Synthetic mRNA expressing UCP1 was synthesized via in vitro transcription from a linearized DNA plasmid. The synthetic mRNA was encapsulated into lipid nanoparticles, yielding a drug candidate test article. Subcutaneous fat was donated by a patient undergoing elective surgery under an IRB-approved protocol. The same donated fat was used in this study as in the negative control study (Figure 2) and the full-dose study (Figure 3). The fat was stabilized for long-term ex vivo culture. The ex vivo fat was treated with the drug candidate test article at half dose (2.5 pg drug candidate per lOOmg of ex vivo fat). Brightfield microscopy images were captured every 4 to 6 hours using an automated system for a total of 6 days (144 hours). Every treated fat cluster shrank significantly, but less so than with the full-dose. Figures 3 and 4 demonstrate a dose-response curve. The lines in Figure 4 denote the diameter of one representative mature adipocyte within the adipose tissue cluster at the start of the experiment (hour 0) and at the end of the experiment (hour 144).

[0225] Example 5: The image of figure 5 was created as follows: Synthetic mRNA expressing UCP1 was synthesized via in vitro transcription from a linearized DNA plasmid. The synthetic mRNA was encapsulated using Lipofectamine™ MessengerMAX™ Transfection Reagent. Varying doses of the synthetic mRNA were used to treat human adipose-derived stem cells (ASCs) for 48 hours. After 48 hours, the ASCs were lysed, and total protein was isolated. The protein isolated were assayed via Western Blot to provide a semi-quantitative assessment of the amount of UCP1 protein as a function of synthetic mRNA dose.

[0226] Example 6: The sequence of mRNA of SEQ ID NO: 1 was created using A plasmid Editor by M. Wayne Davis (ApE) available through the University of Utah. The coding DNA sequence (CDS) was optimized using the GenScript Biotech Corporation GenSmart tool. The mRNA was manufactured by GeneFab Inc. using N1 -methylpseudouridine as a substitute for uridine and packaged in LNP I as described above. The packaged mRNA was introduced to cultured, ex vivo human white adipose tissue that was prepared by Keliomics Inc. to induce transfection of the mRNA into the tissue culture. After 24 hours the following protocol was performed:

[0227] Culture media was aspirated from the dish; cells were washed in chilled phosphate buffered saline (PBS) and PBS was aspirated completely after wash. Lysis buffer added to the culture dish and supplemented with protease and phosphatase inhibitors. Cells and cell lysate transferred to a pre-chilled microcentrifuge tube. Lysate was incubated on ice for 15-30 mins and then centrifuged at high speed for 20 minutes to pellet cell debris. Supernatant was loaded into a new microcentrifuge tube and protein quantified. Sodium Dodecyl Sulfate - Polyacrylamide Gel Electrophoresis (SDS-PAGE), blotting, immunodetection and imaging were performed. The resulting images confirmed the presence of UCP1 in the transfected WAT, as well as expected levels of housekeeping genes heat shock protein 90 (HSP90) and beta-actin ( -actin).

[0228] Figure 6 demonstrates the presence of UCP1 24 hours after lug, 5ug and lOug doses for each 100 mg of ex vivo human WAT and the dose response curve at lug, 5ug and lOug doses. Untreated WAT did not express UCP1 protein. Figures 8 through 14 were generated using automated brightfield microscopy. These images show shrinkage of the transfected WAT in one day intervals from the start to the end point at t+6 days after treatment with a lug dose of mRNA of SEQ ID NO: 1. Figures 15 through 21 show shrinkage of the transfected WAT in one day intervals from the start to the end point at t+6 days after treatment with a 5 ug dose of mRNA of SEQ ID NO: 1.

[0229] Example 7: The sequence of mRNA of SEQ ID NO: 2 was created using A plasmid Editor by M. Wayne Davis (ApE) available through the University of Utah. The coding DNA sequence (CDS) was optimized using the GenScript Biotech Corporation GenSmart tool. The mRNA was manufactured by GeneFab Inc. using N1 -methylpseudouridine as a substitute for uridine and packaged in LNP D as described above. The packaged mRNA was introduced to cultured, ex vivo human white adipose tissue that was prepared by Keliomics Inc. to induce transfection of the mRNA into the tissue culture. After 24 hours the following protocol was performed: Culture media was aspirated from the dish; cells were washed in chilled phosphate buffered saline (PBS) and PBS was aspirated completely after wash. Lysis buffer added to the culture dish and supplemented with protease and phosphatase inhibitors. Cells and cell lysate transferred to a pre-chilled microcentrifuge tube. Lysate was incubated on ice for 15- 30 mins and then centrifuged at high speed for 20 minutes to pellet cell debris. Supernatant was loaded into a new microcentrifuge tube and protein quantified. Sodium Dodecyl Sulfate - Polyacrylamide Gel Electrophoresis (SDS-PAGE), blotting, immunodetection and imaging were performed. The resulting images confirmed the presence of UCP1 in the transfected WAT, as well as expected levels of housekeeping gene HSP90.

[0230] Figure 22 demonstrates the presence of UCP1 24 hours after 5 pig and 10 pg doses and the dose response curve at 5 pg and 10 pg doses. Negative control test articles (LNP D encapsulating synthetic mRNA encoding mCherry in lieu of UCP1) and untreated WAT did not express UCP1 protein.

[0231] Example 8: The sequence of mRNA of SEQ ID NO: 1 was created using A plasmid Editor by M. Wayne Davis (ApE) available through the University of Utah. The coding DNA sequence (CDS) was optimized using the GenScript Biotech Corporation GenSmart tool. The mRNA was manufactured by GeneFab Inc. using N1 -methylpseudouridine as a substitute for uridine and packaged in LNP I as described above. One microgram of the packaged mRNA was introduced to a confluent culture of human adipose derived stem cells (ASCs) that was prepared by Keliomics Inc. to induce transfection of the mRNA into the cell culture. After 24 hours the following protocol was performed:

[0232] Culture media was aspirated from the dish; cells were washed in chilled phosphate buffered saline (PBS) and PBS was aspirated completely after wash. Lysis buffer added to the culture dish and supplemented with protease and phosphatase inhibitors. Cells and cell lysate transferred to a pre-chilled microcentrifuge tube. Lysate was incubated on ice for 15- 30 mins and then centrifuged at high speed for 20 minutes to pellet cell debris. Supernatant was loaded into a new microcentrifuge tube and protein quantified. Sodium Dodecyl Sulfate - Polyacrylamide Gel Electrophoresis (SDS-PAGE), blotting, immunodetection and imaging were performed. The resulting images confirmed the presence of UCP1 in the transfected WAT, as well as expected levels of housekeeping genes heat shock protein 90 (HSP90) and beta-actin (0- Figure 23 demonstrates the presence of UCP1 24 hours after a 1 pg dose.

[0233] Sequence Listing

[0234] Sequences are shown 5’ to 3’ Sequence total quantity: 129

[0235] EQUIVALENTS

[0236] Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the present invention described herein. Such equivalents are intended to be encompassed by the following claim.

[0237] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference into the specification to the same extent as if each individual publication, patent or patent application was specifically indicated to be incorporated herein by reference.

Claims

Claims1. A pharmaceutical composition comprising: a synthetic mRNA comprising the nucleotide sequence set forth in SEQ ID NO: 1; wherein the synthetic mRNA is packaged in a lipid nanoparticle (LNP).

2. The pharmaceutical composition of claim 1, wherein the LNP comprises (C14-4: noncationic lipids (NCL): Cholesterol: PEG / Lipid Conjugate) at a molar ratio of (35: 16: 46.5: 2.5), (50: 10: 38.5: 1.5) or (56.5: 10: 31.8: 1.7).

3. The pharmaceutical composition of claim 1, wherein the LNP comprises (SM-102: NCL: Cholesterol: PEG / Lipid Conjugate) at a molar ratio of (50: 10: 38.5: 1.5), (48.3: 9.1: 40.5: 2.1) or (46.6: 10.4: 41.2: 1.8).

4. The pharmaceutical composition of claim 1, wherein all uridine nucleotides of the synthetic mRNA are substituted with pseudouridine.

5. The pharmaceutical composition of claim 1, wherein all uridine nucleotides of the synthetic mRNA are substituted with N1 -Methylpseudouridine6. A method of preparing a pharmaceutical composition, the method comprising: preparing a synthetic mRNA comprising the nucleotide sequence set forth in SEQ ID NO: 2, SEQ ID NO: 8, or SEQ ID NO: 18; and packaging said synthetic mRNA in a lipid nanoparticle (LNP).

7. The method of claim 6, wherein the LNP comprises (C14-4: NCL: Cholesterol: PEG / Lipid Conjugate) at a molar ratio of (35: 16: 46.5: 2.5), (50: 10: 38.5: 1.5) or (56.5: 10: 31.8: 1.7).

8. The method of claim 6, wherein the LNP comprises (SM-102: NCL: Cholesterol: PEG / Lipid Conjugate) at a molar ratio of (50: 10: 38.5: 1.5), (48.3: 9.1: 40.5: 2.1) or (46.6: 10.4: 41.2: 1.8).

9. The method of claim 6, wherein all uridine nucleotides of the synthetic mRNA are substituted with pseudouridine.

10. The method of claim 6, wherein all uridine nucleotides of the synthetic mRNA are substituted with N1 -Methylpseudouridine.

11. A pharmaceutical composition comprising: a synthetic mRNA comprising the nucleotide sequence set forth in SEQ ID NO: 3, SEQ ID NO: 9, or SEQ ID NO: 17.

12. A method for spot fat reduction in a subject, the method comprising delivering the pharmaceutical composition of claim 11 to an area of the subject targeted for spot fat reduction; wherein the area comprises the abdomen, the outer arms, flanks, upper back, lumbar region, calves, submental fat, dorsal cervical fat, axillary fat, lateral chest wall, outer thighs, inner thighs, or combinations thereof.

13. The method of claim 12, wherein the pharmaceutical composition is delivered in a single bolus injection.

14. The method of claim 12, wherein the pharmaceutical composition is delivered in a series of injections in the area.

15. The method of claim 12, wherein the pharmaceutical composition is delivered transdermally by a penetration enhancer.

16. The method of claim 12, wherein the pharmaceutical composition is delivered transdermally by a vesicular carrier.

17. The method of claim 12, wherein the pharmaceutical composition is delivered transdermally by a microneedle injection.

18. The method of claim 12, wherein the pharmaceutical composition is delivered transdermally by iontophoresis, sonophoresis or jet injection.

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