WNT7a MRNA LIPID NANOPARTICLES FOR THERAPEUTIC USES

Abstract

The present disclosure relates to a lipid nanoparticle (LNP) composition for the delivery of a polynucleotide encoding WNT7a and methods of treating skeletal muscle fibro-adipogenic progenitor (FAP) adipogenesis.

Description

WNT7a MRNA LIPID NANOPARTICLES FOR THERAPEUTIC USESCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims priority to U.S. provisional patent application 63 / 711,530, filed on October 24, 2024 and U.S. provisional patent application 63 / 719,822, filed on November 13, 2024.INCORPORATION BY REFERENCE OF AN ELECTRONIC SEQUENCE LISTING

[0002] This application contains a sequence listing that has been submitted in a computer readable format and is hereby incorporated by reference in its entirety. The computer readable file, created on October 22, 2025, is named 771005_MTST-599PC.xml and is 11,321 bytes in size.ACKNOWLEDGEMENT OF GOVERNMENT SUPPORT

[0003] This invention was made with government support under Grants No. R01AR080616 awarded by the National Institutes of Arthritis and Musculoskeletal and Skin Diseases. The government has certain rights in this invention.BACKGROUND

[0004] Skeletal muscle fatty infiltration is observed in chronic skeletal muscle injuries and degenerative myopathies, correlating with increased disease severity, diminished function, and poor surgical outcomes. Fibro-adipogenic progenitor (FAP) cells are known to contribute to fatty infiltration through unchecked differentiation into adipocytes. Recombinant human protein WNT7a can prevent muscle degeneration by reducing FAP adipogenesis and fatty infiltration without fibrosis, making it a promising therapeutic candidate for muscle regeneration. However, Recombinant protein therapies face challenges related to production scalability and therapeutic efficacy. A need exists for a lipid nanoparticle (LNP) delivery of mRNA offers as an alternative with scalable production and effective therapeutic delivery.SUMMARY

[0005] One aspect of the disclosure includes A lipid nanoparticle composition for administration of a WNT7a polynucleotide, the lipid nanoparticle composition comprising a polynucleotide encoding WNT7a and, a lipid composition, wherein the lipid composition comprises an ionizable lipid, a PEG-lipid conjugate, cholesterol, and phospholipid; and wherein the polynucleotide encoding WNT7a is encapsulated by the lipid composition.

[0006] In an aspect, the polynucleotide encoding WNT7a comprises a nucleic acid sequence with at least about 80% sequence identity to SEQ ID NO: 1 or SEQ ID NO: 5.

[0007] In an aspect, the polynucleotide encoding WNT7a comprises a nucleic acid sequence with at least about 85%, alternatively at least about 90%, alternatively at least about 95%, alternatively at least about 96%, alternatively at least about 97%, alternatively at least about 98%, alternatively at least about 99%, or 100% nucleic acid sequence identity to SEQ ID NO: 1.

[0008] In an aspect, the polynucleotide encoding WNT7a comprises a nucleic acid sequence with at least about 85%, alternatively at least about 90%, alternatively at least about 95%, alternatively at least about 96%, alternatively at least about 97%, alternatively at least about 98%, alternatively at least about 99%, or 100% nucleic acid sequence identity to SEQ ID NO: 5.

[0009] In an aspect, the WNT7a comprises an amino acid sequence with at least about 85%, alternatively at least about 90%, alternatively at least about 95%, alternatively at least about 96%, alternatively at least about 97%, alternatively at least about 98%, alternatively at least about 99%, or 100% nucleic acid sequence identity to SEQ ID NO:6.

[0010] In an aspect, the ionizable lipid comprises heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate (SM-102), [(4-hydroxybutyl)azanediyl]bis(hexane-6, 1-diyl)bis(2 -hexyldecanoate) (ALC-0315), [(6Z,9Z)-hexadeca-6,9-dien-l-yl]-[2- (dimethylamino)ethyl]carbamate (DLin-MC3-DMA), N,N-dimethyl-2-(2-dodecyloxyethyl)ethanamine (Cl 2-200), or combinations thereof.

[0011] In an aspect, the ionizable lipid is present in the lipid composition at a molar percentage of between about 40% to about 60%.

[0012] In an aspect, the ionizable lipid is present in the lipid composition at molar percentage of about 42%, alternatively about 44%, alternatively about 46%, alternatively about 48%, alternatively about 50%, alternatively about 52%, alternatively about 54%, alternatively about 56%, or alternatively about 58%.

[0013] In an aspect, the PEG-lipid conjugate comprises PEG-modified cholesterol, N-octanoyl-sphingosine-l-{succinyl[methoxy(poly ethylene glycol)]}, N-palmitoyl-sphingosine-l-{succinyl[methoxy(polyethylene glycol)]}, PEG-modified DMPE (DMPE-PEG), PEG-modified DSPE (DSPE-PEG), PEG-modified DPPE (DPPE-PEG), PEG-modified DOPE (DOPE-PEG), dimyristoylglycerol-polyethylene glycol (DMG-PEG), distearoylglycerolpolyethylene glycol (DSG-PEG), dipalmitoylglycerol-polyethylene glycol (DPG-PEG), dioleoylglycerol-polyethylene glycol (DOG-PEG), or a combination thereof.

[0014] In an aspect, the PEG-lipid conjugate comprises l,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2000), Distearoyl glycerol-PEG2000 (DSG-PEG2000), l,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(poly ethylene glycol)-2000] (DSPE-PEG2000), Ceramide-PEG2000, or combinations thereof.

[0015] In an aspect, the PEG-lipid conjugate is present in the lipid composition at a molar percentage of between about 0.75% to about 2.5%.

[0016] In an aspect, the PEG-lipid conjugate is present in the lipid composition at a molar percentage of about 1.0%, alternatively about 1.25%, alternatively about 1.5%, alternatively about 1.75%, alternatively about 2.0%, or alternatively about 2.25%.

[0017] In an aspect, the phospholipid comprises l,2-Distearoyl-sn-glycero-3-phosphocholine (DSPC), l,2-Dioleoyl-sn-glycero-3 -phosphoethanolamine (DOPE), Dipalmitoylphosphatidylcholine (DPPC), l,2-dilinoleoyl-sn-glycero-3 -phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3 -phosphocholine (DOPC), ,2- diundecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3 -phosphocholine (POPC), l,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), l-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), l-hexadecyl-sn-glycero-3 -phosphocholine (C 16LysoPC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, l,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1 ,2-didocosahexaenoyl-sn-glycero-3 -phosphocholine, 1 ,2-dipalmitoyl-sn-glycero-3 -phosphoethanolamine (DPPE), l,2-diphytanoyl-sn-glycero-3 -phosphoethanolamine (ME 16.0 PE), l,2-distearoyl-sn-glycero-3-phosphoethanolamine, l,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1 ,2-dilinolenoyl-sn-glycero-3 -phosphoethanolamine, 1 ,2-diarachidonoyl- sn-glycero-3 -phosphoethanolamine, l,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, l,2-dioleoyl-sn-glycero-3-phospho-rac-(l-glycerol) sodium salt (DOPG), sphingomyelin, and combinations thereof.

[0018] In an aspect, the phospholipid is present in the lipid composition at a molar percentage of between about 5% to about 15%.

[0019] In an aspect, the phospholipid is present in the lipid composition at a molar percentage of about 6%, alternatively about 7%, alternatively about 8%, alternatively about 9%, alternatively about 10%, alternatively about 11%, alternatively about 12%, alternatively about 13%, or alternatively about 14%.

[0020] In an aspect, the cholesterol is present in the lipid composition at a molar percentage of between about 30% to about 50%.

[0021] In an aspect, the cholesterol is present in the lipid composition at a molar percentage of about 31.5%, alternatively about 33.0%, alternatively about 34.5%, alternatively about 36.0%, alternatively about 37.5%, alternatively about 38%, alternatively about 38.5%, alternatively about 39.0%, alternatively about 40.5%, alternatively about 42.0%, alternatively about 43.5%, alternatively about 45.0%, alternatively about 46.5%, alternatively about 48.0%, or alternatively about 49.5%.

[0022] In an aspect, the lipid nanoparticle composition has a mean diameter of between about 68 nm and about 85 nm.

[0023] In an aspect, the lipid nanoparticle composition has a PDI between about 0.0845 and about 0.11 nm.

[0024] In an aspect, lipid nanoparticle composition has an encapsulation efficiency of between about 85% to about 100%.

[0025] One aspect of the disclosure is a therapeutic composition comprising the lipid nanoparticle composition disclosed and at least one therapeutically acceptable carrier, diluent, or excipient.

[0026] One aspect of the disclosure is a method of treating skeletal muscle fibro-adipogenic progenitor (FAP) adipogenesis in a subject in need thereof, the method comprising administering a lipid nanoparticle composition as disclosed or the therapeutic composition as disclosed.

[0027] One aspect of the disclosure is use of a lipid nanoparticle composition as disclosed or the therapeutic composition as disclosed, for the treatment of skeletal muscle fibro-adipogenic progenitor (FAP) adipogenesis in a subject in need thereof.

[0028] In an aspect, the lipid nanoparticle composition or the therapeutic composition is administered intracutaneously, subcutaneously, intramuscularly, intratracheally, intravenously, or via infusion.

[0029] In an aspect, the lipid nanoparticle composition or the therapeutic composition is administered at a dose of about 10 ng / mLto about 100 ng / mL of lipid nanoparticle composition.

[0030] In an aspect, the lipid nanoparticle composition or the therapeutic composition is administered at least once monthly, alternatively at least every two weeks, or alternatively at least once a week.

[0031] In an aspect, the lipid nanoparticle composition or the therapeutic composition is administered one day post-injury, alternatively two days post-injury, alternatively three days post-injury, alternatively four days post-injury, or alternatively five days post-injury.

[0032] These and other advantages, aspects, and novel features of the present disclosure, as well as details of illustrated embodiments thereof, will be more fully understood from the following description and drawings.BRIEF DESCRIPTION OF THE DRAWINGS

[0033] FIGs. 1A and IB show that W7a-LNPs demonstrate excellent size uniformity and complete mRNA encapsulation. FIG. 1A is a schematic illustration of W7a-LNP delivery and function. Upon uptake by endogenous muscle cells, W7a-LNP enables transient production and secretion of native WNT7A protein, affecting local MuSCs, myofibers, and FAPs. FIG. IB is a characterization of mRNALNPs used in this study, including W7a-LNP (three batches) and cre-LNPs (two batches).

[0034] FIGs. 2A-2G show efficient ere mRNALNP delivery enables robust recombination in vitro and in vivo. FIG. 2A is a schematic representation of the loxP-flanked STOP cassette upstream of tdTomato, shown before and after cre-LNP -mediated recombination. c / v-LNP delivery induces excision of the STOP cassette, enabling tdTomato expression. FIG.2B shows a primary FAPs isolated from Ail4-tdTomato reporter mice treated with ere mRNA LNPs (1 or 5 pg / mL) or vehicle for 24 hours. Scale bar: 100 pm. FIG. 2C shows quantification of TdT expressing FAPs. One image per well was taken from the center of each well. One-way ANOVA with Tukey’s post-hoc. n=10 technical replicates / group. **** pO.OOOl. FIG. 2D is a representative TA muscles of Ail4-tdTomato mice intramuscularly injected with ere mRNALNPs (15 pg / mL) or PBS vehicle. Two weeks post-injection, TdT+ myofibers were detected in the treated muscle but not in the vehicle-treated contralateral muscle. Scale bar: 100 pm.FIG. 2E shows quantification of TdT expressing muscle fibers. Five images were analyzed per muscle. Two-tailed paired t-test. n=5 mice / group. * p<0.05. Colors represent biological donors / replicates. FIG. 2F are representative images of WNT7A-labeled C2C12. Cells were treated with vehicle or 2 pg / mL W7a-LNP for 24 hours. Scale bar: 100 pm. FIG. 2G shows quantification of mean fluorescence intensity (MFI) of WNT7A normalized by number of nuclei. One-way ANOVA with Tukey’s post-hoc. n=3-5 technical replicates / group. * p<0.05; *** p<0.001; **** pO.OOOl.

[0035] FIGs. 3A-3E show W7a-LNP suppresses FAP adipogenesis in vitro. FIG. 3A is an experimental timeline of FAPs expansion, differentiation induction in adipogenic differentiation media (ADM), and treatment. FIG. 3B are representative images of perilipin-labeled FAPs. Scale bar: 100 pm. FIG. 3C is perilipin area normalized by cell number. Oneway ANOVA with Tukey’s post-hoc. n=8 / group. * p<0.05; ** p<0.01. Colors represent biological donors / replicates. FIG. 3D are representative images of perilipin-labeled FAPs. Scale bar: 100 pm. FIG. 3E is perilipin area normalized by cell number. One-way ANOVA with Tukey’s post-hoc. n=6 technical replicates / group. **** pO.OOOl.

[0036] FIGs. 4A-4E show W7a-LNP increase fusion and myotube size in vitro. FIG. 4A is an experimental timeline of C2C12 myoblast expansion, differentiation induction in differentiation media (DM), and treatment. FIG. 4B are representative images of myosin heavy chain (MyHC) labeled myotubes. Scale bar: 100 pm. FIG.4C shows mean myotube area. FIG.4D shows fusion index (%). FIG. 4E show total number of nuclei. One-way ANOVA with Tukey’s post-hoc. n=7 technical replicates / group. * p<0.05; ** p<0.01; *** p<0.001; **** pO.OOOl .

[0037] FIGs. 5A-5D show W7a-LNPs promotes myofiber hypertrophy without inducing fibrosis in vivo. FIG. 5A is an experimental timeline of W7a-LNPs administration into uninjured supraspinatus muscle. Fig. 5B are representative supraspinatus muscles of C57B1 / 6J mice intramuscularly injected with W7a-LNPs (10 pg / 30 pL) or PBS vehicle. FIG. 5C shows minimum Feret’s diameter of myofibers. One-way ANOVA with Tukey’s post-hoc. n=3-4 mice / group. * p<0.05; ** p<0.01. FIG. 5D are representative supraspinatus muscles stained with Picrosirius red. No qualitative differences were observed between groups.

[0038] FIGs. 6A-6H show W7a-LNP efficacy in glycerol -injured muscle depends on delivery timing and myofiber viability. FIG. 6A is an experimental timeline of W7a-LNPsadministration into glycerol-injured TA muscle. FIG. 6B show representative glycerol-injured TA muscles treated with rWNT7A (2.5 pg / 30 pL), W7a-LNPs (10 pg / 30 pL), or PBS vehicle labeled for perilipin and Oil Red O (ORO). Day 14 post-injury. Scale bar: 1 mm. FIG. 6C depicts the perilipin area and FIG. 6D depicts the ORO area normalized by TA cross sectional area. One-way ANOVA with Tukey’s post-hoc. n=6 mice / group. * p<0.05. FIG. 6E is an experimental timeline of delayed W7a-LNP administration. FIG. 6F shows representative images of ORO-stained muscle sections. Scale bar: 500 pm. Inset scale bar: 250 pm. FIG. 6G shows ORO area normalized by TA cross sectional area. Two-tailed unpaired t-test. n=8 mice / group. ** p<0.01. FIG.6H shows TA wet weight normalized by body weight. Two-tailed unpaired t-test. n=8 mice / group. * p<0.05.

[0039] FIG. 7 shows representative H&E staining of W7a-LNPs administered, uninjured supraspinatus muscles.

[0040] FIG. 8A-8C show Stability of W7a-LNPs after 6 weeks of storage at -80°C in 10% sucrose. FIG. 8A are size distribution profiles of freshly prepared (dashed lines) and stored (solid lines) LNPs measured by dynamic light scattering. FIG. 8B shows Z-average diameter and FIG. 8C shows poly dispersity index (PDI) before and after storage. n=3 independent measurements. Slight increases in size and PDI were observed after storage.

[0041] FIGs. 9A and 9B show W7a-LNP (10 ng / mL), but not Cre or luciferase mRNALNPs (10 ng / mL), increases myotube size in vitro. FIG. 9A is a representative images of myosin heavy chain (MyHC) labeled myotubes. Scale bar: 100 pm. FIG. 9B shows mean myotube area. One-way ANOVA with Tukey’s post-hoc. n=4-5 technical replicates / group. * p<0.05.

[0042] FIGs. 10A and 10B show the effect of glycerol on LNP size and dispersity. W7a-LNPs were incubated in PBS, 25% glycerol, or 50% glycerol for 10 or 30 minutes and analyzed by dynamic light scattering (DLS). FIG. 10A shows the Z-average diameter increased in a concentration-dependent manner at both time points. 2-way ANOVA with Sidak’s post-hoc tests. Groups not sharing a letter are significantly different (p<0.05). FIG. 10B shows the Polydispersity index (PDI) remained low across all conditions, indicating maintained monodispersity despite changes in apparent particle size. Data represent n=3 independent measurements.

[0043] FIG. 11 is an exemplary mRNA sequence encoding WNT7a.DETAILED DESCRIPTION OF THE INVENTIONI. Introduction

[0044] Myosteatosis, characterized by intramuscular adipose tissue (IMAT), and muscle atrophy are pathologic features observed in chronic skeletal muscle injuries, degenerative myopathies, and aging. Both conditions contribute to reduced muscle function, impaired regeneration, and poor surgical repair outcomes. For instance, in Duchenne muscular dystrophy, progressive fatty infiltration is correlated with disease severity and declining ambulatory performance, while concomitant muscle atrophy exacerbates functional decline. Similarly, in rotator cuff tears, tear size is associated with IMAT severity and muscle atrophy, which persists post-repair and contribute to higher repair failure rates and diminished muscle strength. Beyond these conditions, increased IMAT and atrophy are also observed in prolonged bed rest, knee osteoarthritis, low back pain, sarcopenia, and spaceflight, highlighting its widespread impact and the need for engineered therapies that target both IMAT and muscle atrophy to preserve muscle function.

[0045] Fibro-adipogenic progenitors (FAPs), a mesenchymal stromal cell population residing in the interstitial space between myofibers, are key regulators of IMAT development and muscle atrophy. In healthy muscle, FAPs support muscle homeostasis and regeneration by sensing injury signals and coordinating muscle stem cell (MuSC) activation. Conditional ablation of FAPs in uninjured muscles leads to muscle atrophy and loss of MuSCs. Upon muscle injury or exercise, infiltrating immune cells clear debris and release IL-4, which activates FAPs. Activated FAPs secrete pro-myogenic factors that stimulate MuSC activation and expansion, enabling their differentiation into myofibers. As the inflammation resolves, macrophage-derived tumor necrosis factor a (TNF-a), induces apoptotic clearance of FAPs to prevent excessive remodeling. However, in chronic muscle pathology, dysregulated FAP expansion leads to differentiation into adipocytes, contributing to IMAT, or myofibroblasts, leading to fibrosis. These pathologic changes not only disrupt muscle architecture, but also promote myofiber loss, reducing contractile force. Importantly, fatty infiltration and muscle atrophy often progress irreversibly, and no effective treatments exist to restore lost muscle mass or prevent IMAT accumulation. Given the pivotal role of FAPs in IMAT formation and homeostasis, targeting FAPs while simultaneously supporting myofibers represents a critical therapeutic strategy to mitigate IMAT, preserve muscle structure, and improve functional recovery.

[0046] The inventors previously demonstrated that recombinant wingless-type MMTV integration site family member 7A (WNT7a) counteracts muscle degeneration by inhibiting FAP adipogenesis and reducing skeletal muscle fatty infiltration without inducing fibrosis. Beyond suppressing FAP adipogenesis, WNT7a also promotes muscle regeneration by stimulating MuSC expansion, enhancing myofiber hypertrophy, and increasing muscle strength. These combined effects make recombinant WNT7a a promising therapeutic candidate for muscle repair. However, recombinant protein therapies face significant limitations, including production scalability, therapeutic efficacy, and immunogenicity. These challenges highlight the need for alternative, cost-effective strategies to deliver WNT7a efficiently and effectively.

[0047] Lipid nanoparticle (LNP)-mediated mRNA delivery offers a scalable and efficient alternative for therapeutic applications. Compared to plasmid DNA, viral vectors, or recombinant proteins, in vitro-transcribed (IVT) mRNA offers several advantages: it eliminates the risk of insertional mutagenesis, has transient activity, and is naturally degraded through physiological metabolic pathways. Additionally, IVT mRNA synthesis is a simple, cost-effective, single-container process, making it more scalable than recombinant protein production. However, mRNA’s inherent instability, immunogenicity, and susceptibility to nucleases present significant challenges. These can be mitigated by incorporating a 5’ cap, poly(A) tail, 5’ - and 3 ’-untranslated regions (UTRs), and chemically modified nucleosides that reduce the immunostimulatory effect of IVT mRNA. To overcome delivery barriers, LNPs have emerged as a leading technology, efficiently protecting mRNA from nuclease degradation, facilitating cellular uptake, and promoting endosomal escape for cytosolic translation.

[0048] Disclosed herein is localized skeletal muscle delivery of LNP-encapsulated WNT7a mRNA (W7a-LNP) and its efficacy in mitigating both intramuscular fatty infiltration and muscle atrophy.

[0049] To facilitate an understanding of the present disclosure, a number of terms and phrases are defined below.II. Definitions

[0050] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the methods described herein belong. Any reference to standard methods refers to the most recent available version of the method at the time of filing of this disclosure unless otherwise indicated.

[0051] For any method disclosed herein that includes discrete steps, the steps may be conducted in any feasible order. And, as appropriate, any combination of two or more steps may be conducted simultaneously.

[0052] All headings are for the convenience of the reader and should not be used to limit the meaning of the text that follows the heading, unless so specified.

[0053] The words "preferred" and "preferably" refer to embodiments of the invention that may afford certain benefits, under certain circumstances. However, other embodiments may also be preferred, under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful and is not intended to exclude other embodiments from the scope of the invention.

[0054] The terms “comprising,” “having,” “including,” and “containing” are to be construed as open-ended terms (i.e., meaning “including, but not limited to,”) unless otherwise noted. Such terms will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements.

[0055] The singular form "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. These articles refer to one or to more than one (i.e., to at least one). 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. As used herein, the term "or" is generally employed in its usual sense including "and / or" unless the content clearly dictates otherwise. The term "and / or" means any one or more of the items in the list joined by "and / or". As an example, "x and / or y" means any element of the three-element set {(x), (y), (x, y)}. In other words, "x and / or y" means "one or both of x and y". As another example, "x, y, and / or z" means any element of the sevenelement set {(x), (y), (z), (x, y), (x, z), (y, z), (x, y, z)}. In other words, "x, y and / or z" means "one or more of x, y and z".

[0056] Where ranges are given, endpoints include all numbers subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.). Furthermore, unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or subrange within the stated ranges in different embodiments of the disclosure, to the tenth of the unit of the lower limit ofthe range, unless the context clearly dictates otherwise. Herein, "up to" a number (for example, up to 50) includes the number (for example, 50). The term "in the range" or "within a range" (and similar statements) includes the endpoints of the stated range.

[0057] Reference throughout this specification to “one aspect (or embodiment),” “an aspect (or embodiment),” “certain aspects (or embodiments),” or “some aspects (or embodiments),” etc., means that a particular feature, configuration, composition, or characteristic described in connection with the aspect is included in at least one aspect of the disclosure. Thus, the appearances of such phrases in various places throughout this specification are not necessarily referring to the same embodiment of the disclosure. Furthermore, the particular features, configurations, compositions, or characteristics may be combined in any suitable manner in one or more aspects.

[0058] Unless otherwise indicated, all numbers expressing quantities of components, molecular weights, and so forth used in the specification and claims are to be understood as being modified in all instances by the term "about." As used herein in connection with a measured quantity, the term "about" refers to that variation in the measured quantity as would be expected by the skilled artisan making the measurement and exercising a level of care commensurate with the objective of the measurement and the precision of the measuring equipment used. The term "about" as used in connection with a numerical value throughout the specification and the claims denotes an interval of accuracy, familiar and acceptable to a person skilled in the art. In general, such interval of accuracy is + / -10%. Thus, "about" can be understood to be within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1 %, 0.5%, 0.1 %, 0.05%, 0.01 %, or 0.001 % greater or less than the stated value. Accordingly, unless otherwise indicated to the contrary, the numerical parameters set forth in the specification and claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0059] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. All numerical values, however, inherently contain a range necessarily resulting from the standard deviation found in their respective testing measurements.

[0060] The term "exemplary" means serving as a non-limiting example, instance, or illustration. As utilized herein, the terms "e.g.," and "for example" set off lists of one or more non-limiting aspects, examples, instances, or illustrations.

[0061] As used herein, the term "substantially" refers to the qualitative condition of exhibiting total or near-total extent or degree of a characteristic or property of interest. Biological and chemical phenomena rarely, if ever, go to completion and / or proceed to completeness or achieve or avoid an absolute result. The term "substantially" is therefore used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena. For example, "substantially" may refer to being within at least about 20%, alternatively at least about 10%, alternatively at least about 5% of a characteristic or property of interest.

[0062] The term "nucleic acid" refers to any polymeric chain of nucleotides. A nucleic acid may be DNA, RNA, or a combination thereof. In some embodiments, a nucleic acid comprises one or more natural nucleic acid residues. In some embodiments, a nucleic acid comprises of one or more nucleic acid analogs. In some embodiments, nucleic acids are prepared by one or more of isolation from a natural source, enzymatic synthesis by polymerization based on a complementary template (in vivo or in vitro), reproduction in a recombinant cell or system, and chemical synthesis. In some embodiments, a nucleic acid is at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 20, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000 or more residues long (e.g., 20 to 100, 20 to 500, 20 to 1000, 20 to 2000, or 20 to 5000 or more residues). In some embodiments, a nucleic acid is partly or wholly single stranded; in some embodiments, a nucleic acid is partly or wholly double stranded. In some embodiments a nucleic acid has a nucleotide sequence comprising at least one element that encodes, or is the complement of a sequence that encodes, a polypeptide.

[0063] Expression: As used herein, “expression” of a nucleic acid sequence refers to one or more of the following events: (1) production of an RNA template from a DNA sequence (e.g., by transcription); (2) processing of an RNA transcript (e.g., by splicing, editing, 5' cap formation, and / or 3' end processing); (3) translation of an RNA into a polypeptide or protein; and (4) post-translational modification of a polypeptide or protein.

[0064] Identity: As used herein, the term “identity” refers to the overall monomer conservation between polymeric molecules, e.g., between polynucleotide molecules (e.g., DNA moleculesand / or RNA molecules) and / or between polypeptide molecules. Calculation of the percent identity of two polynucleotide sequences, for example, can be performed by aligning the two sequences for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second nucleic acid sequence for optimal alignment and non-identical sequences can be disregarded for comparison purposes). In certain aspects, the length of a sequence aligned for comparison purposes is at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 100% of the length of the reference sequence. The nucleotides at corresponding nucleotide positions are then compared. When a position in the first sequence is occupied by the same nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which needs to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. When comparing DNA and RNA, thymine (T) and uracil (U) can be considered equivalent.

[0065] Suitable software programs are available from various sources and for alignment of both protein and nucleotide sequences. One suitable program to determine percent sequence identity is bl2seq, part of the BLAST suite of programs available from the U.S. government's National Center for Biotechnology Information BLAST website (blast.ncbi.nlm.nih.gov). B12seq performs a comparison between two sequences using either the BLASTN or BLASTP algorithm. BLASTN is used to compare nucleic acid sequences, while BLASTP is used to compare amino acid sequences. Other suitable programs are, e.g., Needle, Stretcher, Water, or Matcher, part of the EMBOSS suite of bioinformatics programs and also available from the European Bioinformatics Institute (EBI).

[0066] Sequence alignments can be conducted using methods such as, but not limited to, MAFFT, Clustal (ClustalW, Clustal X or Clustal Omega), or MUSCLE.

[0067] Different regions within a single polynucleotide or polypeptide target sequence that aligns with a polynucleotide or polypeptide reference sequence can each have their own percent sequence identity.

[0068] In Vitro: As used herein, the term “in vitro” refers to events that occur in an artificial environment, e.g., in a test tube or reaction vessel, in cell culture, in a Petri dish, etc., rather than within an organism (e.g., animal, plant, or microbe).

[0069] In Vivo: As used herein, the term “in vivo” refers to events that occur within an organism (e.g., animal, plant, or microbe or cell or tissue thereof).

[0070] Isolated: As used herein, the term “isolated” refers to a substance or entity that has been separated from at least some of the components with which it was associated (whether in nature or in an experimental setting). Isolated substances (e.g., nucleotide sequence or protein sequence) can have varying levels of purity in reference to the substances from which they have been associated. Isolated substances and / or entities can be separated from at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or more of the other components with which they were initially associated. In some aspects, isolated agents are more than about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% pure. As used herein, a substance is “pure” if it is substantially free of other components. The term “substantially isolated” means that the compound is substantially separated from the environment in which it was formed or detected. Partial separation can include, for example, a composition enriched in the compound of the present disclosure. Substantial separation can include compositions containing at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 97%, or at least about 99% by weight of the compound of the present disclosure, or salt thereof.

[0071] A polynucleotide, vector, polypeptide, cell, or any composition disclosed herein which is “isolated” is a polynucleotide, vector, polypeptide, cell, or composition which is in a form not found in nature. Isolated polynucleotides, vectors, polypeptides, or compositions include those that have been purified to the degree that they are no longer in a form in which they are found in nature. In some aspects, a polynucleotide, vector, polypeptide, or composition that is isolated is substantially pure.

[0072] Nucleotides are referred to by their commonly accepted single-letter codes. Unless otherwise indicated, nucleic acids are written left to right in 5' to 3' orientation. Nucleotides are referred to herein by their commonly known one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Accordingly, A represents adenine, C represents cytosine, G represents guanine, T represents thymine, and U represents uracil.

[0073] Messenger RNA (mRNA): As used herein, the term “messenger RNA” (mRNA) refers to any polynucleotide that encodes a polypeptide of interest and is capable of being translated to produce the encoded polypeptide in vitro, in vivo, in situ, or ex vivo.

[0074] Native or naturally occurring: As used herein, a “native” or “naturally occurring” polynucleotide sequence means a polynucleotide sequence existing in nature without artificial aid.

[0075] Nucleic acid sequence: The terms “nucleic acid sequence,” “nucleotide sequence,” or “polynucleotide sequence” are used interchangeably and refer to a continuous nucleic acid sequence. The sequence can be either single stranded or double stranded DNA or RNA, e.g., an mRNA.

[0076] The phrase “nucleotide sequence encoding” refers to the nucleic acid (e.g., an mRNA or DNA molecule) coding sequence that encodes a polypeptide. As used herein, the terms “coding region” and “coding sequence”, refer to an Open Reading Frame (ORF) in a polynucleotide that upon expression, yields a polypeptide or protein. The coding sequence can further include initiation and termination signals operably linked to regulatory elements, including a promoter and polyadenylation signal capable of directing expression in the cells of an individual or mammal to which the nucleic acid is administered. The coding sequence can further include sequences that encode signal peptides.

[0077] Open reading frame: As used herein, “open reading frame” or “ORF” refers to a sequence that does not contain a stop codon in a given reading frame.

[0078] Part: As used herein, a “part” or “region” of a polynucleotide is defined as any portion of the polynucleotide that is less than the entire length of the polynucleotide. Likewise, a “part” or “region” of a polypeptide is defined as any portion of the polypeptide that is less than the entire length of the polynucleotide.

[0079] Point mutation: As used herein, “point mutation” refers to a genetic mutation in which a single nucleobase is substituted, inserted, or deleted from a polynucleotide sequence. The term “nucleobase substitution”, “substitution”, or “substitution mutation” as used herein refers to replacing a single nucleobase present in a reference polynucleotide sequence (e.g., a wild type or native sequence) with another nucleobase. Accordingly, a reference to a “substitutionat position X” refers to the substitution of a nucleobase present at position X with an alternative nucleobase.

[0080] As used herein, “nucleobase insertion”, “insertion”, or “insertion mutation” refers to inserting a single nucleobase immediately adjacent to a nucleobase at a particular position of a reference polynucleotide sequence. As used herein, “nucleobase deletion”, “deletion”, or “deletion mutation” refers to deleting a single nucleobase immediately adj acent to a nucleobase at a particular position of a reference polynucleotide sequence.

[0081] Polynucleotide: The term “polynucleotide” as used herein refers to polymers of nucleotides of any length, including ribonucleotides, deoxyribonucleotides, analogs thereof, or mixtures thereof. This term refers to the primary structure of the molecule. Thus, the term includes triple-, double- and single-stranded deoxyribonucleic acid (“DNA”), as well as triple-, double- and single-stranded ribonucleic acid (“RNA”). More particularly, the term “polynucleotide” includes polydeoxyribonucleotides (containing 2-deoxy-D-ribose), polyribonucleotides (containing D-ribose), including transfer RNA (tRNA), ribosomal RNA (rRNA), heterogeneous RNA (hRNA), small interfering RNA (siRNA), small RNA (sRNA), mRNA (messenger RNA), complementary DNA (cDNA), or single-stranded DNA (ssDNA), whether spliced or unspliced, any other type of polynucleotide which is an N- or C-glycoside of a purine or pyrimidine base, and other polymers containing non-nucleotidic backbones, for example, polyamide (e.g., peptide nucleic acids “PNAs”) and polymorpholino polymers, and other synthetic sequence-specific nucleic acid polymers providing that the polymers contain nucleobases in a configuration which allows for base pairing and base stacking, such as is found in DNA and RNA. In particular aspects, the polynucleotide comprises an mRNA.

[0082] The T bases in the codon maps disclosed herein are present in DNA, whereas the T bases may be replaced by U bases in corresponding RNAs. For example, a codon-nucleotide sequence disclosed herein in DNA form, e.g., a vector or an in vitro translation (IVT) template, may have its T bases transcribed as U based in its corresponding transcribed mRNA. In this respect, both codon-optimized DNA sequences (comprising T) and their corresponding RNA sequences (comprising U) are considered codon-optimized nucleotide sequences of the present disclosure. Equivalent codon-maps can be generated by replacing one or more bases with nonnatural bases. Thus, e.g., a TTC codon (DNA map) may correspond to a UUC codon (RNA map).

[0083] Standard A-T and G-C base pairs form under conditions that allow the formation of hydrogen bonds between the N3-H and C4-oxy of thymidine and the N1 and C6-NH2, respectively, of adenosine and between the C2-oxy, N3, and C4-NH2, of cytidine and the C2-NH2, N' — H and C6-oxy, respectively, of guanosine. Thus, for example, guanosine (2-amino-6-oxy-9-P-D-ribofuranosyl-purine) can be modified to form isoguanosine (2-oxy-6-amino-9-P-D-ribofuranosyl-purine). Such modification results in a nucleoside base, which will no longer effectively form a standard base pair with cytosine. However, modification of cytosine (l-P-D-ribofuranosyl-2-oxy-4-amino-pyrimidine) to form isocytosine (l-P-D-ribofuranosyl-2-amino-4-oxy -pyrimidine-) results in a modified nucleotide which will not effectively base pair with guanosine but will form a base pair with isoguanosine.

[0084] Polypeptide: The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to polymers of amino acids of any length. The polymer can comprise modified amino acids. The terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. Also included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids such as homocysteine, ornithine, p-acetylphenylalanine, D-amino acids, and creatine).

[0085] The term “polypeptide,” as used herein, refers to proteins, polypeptides, and peptides of any size, structure, or function. Polypeptides include gene products, naturally occurring polypeptides, synthetic polypeptides, homologs, orthologs, paralogs, fragments and other equivalents, variants, and analogs of the foregoing. A polypeptide can be a single polypeptide or can be a multi-molecular complex such as a dimer, trimer, or tetramer. They can also comprise single chain or multichain polypeptides. Most commonly, disulfide linkages are found in multichain polypeptides. The term polypeptide can also apply to amino acid polymers in which one or more amino acid residues are an artificial chemical analogue of a corresponding naturally occurring amino acid. In some aspects, a “peptide” can be less than or equal to about 50 amino acids long, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids long.

[0086] Reference Nucleic Acid Sequence: The term “reference nucleic acid sequence”, “reference nucleic acid”, or “reference nucleotide sequence” or “reference sequence” refers to a starting nucleic acid sequence (e.g., a RNA, e.g., an mRNA sequence) that can be sequenceoptimized. In some aspects, the reference nucleic acid sequence is a wild type or native nucleic acid sequence, a fragment or a variant thereof.

[0087] Sequence Optimization: As used herein, “sequence optimization” refers to a process or series of processes by which nucleobases in a reference nucleic acid sequence are replaced with alternative nucleobases, resulting in a nucleic acid sequence with improved properties. In the context of the present disclosure, sequence optimization refers to modifications in a nucleotide sequence of a 3’ UTR that result in improved translation of a downstream gene target when 3’ UTR is incorporated into a suitable expression system.

[0088] Similarity: As used herein, the term “similarity” refers to the overall relatedness between polymeric molecules, e.g., between polynucleotide molecules (e.g., DNA molecules and / or RNA molecules) and / or between polypeptide molecules. Calculation of percent similarity of polymeric molecules to one another can be performed in the same manner as a calculation of percent identity, except that calculation of percent similarity takes into account conservative substitutions.

[0089] Substantially: As used herein, the term “substantially” refers to the qualitative condition of exhibiting total or near-total extent or degree of a characteristic or property of interest. Biological and chemical phenomena rarely, if ever, go to completion and / or proceed to completeness or achieve or avoid an absolute result. The term “substantially” is therefore used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena. For example, “substantially” may refer to being within at least about 20%, alternatively at least about 10%, alternatively at least about 5% of a characteristic or property of interest.

[0090] Synthetic: The term “synthetic” means produced, prepared, and / or manufactured by the hand of man. Synthesis of polynucleotides or other molecules of the present disclosure can be chemical or enzymatic.

[0091] Terminus: As used herein, the terms “termini” or “terminus,”, when referring to polypeptides, refers to an extremity of a peptide or polypeptide. Such extremity is not limited only to the first or final site of the peptide or polypeptide but can include additional amino acids in the terminal regions. The polypeptide based molecules of the disclosure can be characterized as having both an N-terminus (terminated by an amino acid with a free amino group (NH2)) and a C-terminus (terminated by an amino acid with a free carboxyl group (COOH)). Proteins of the disclosure are in some cases made up of multiple polypeptide chains brought togetherby disulfide bonds or by non-covalent forces (multimers, oligomers). These sorts of proteins will have multiple N- and C-termini. Alternatively, the termini of the polypeptides can be modified such that they begin or end, as the case can be, with a non-polypeptide-based moiety such as an organic conjugate.

[0092] Transfection: As used herein, “transfection” refers to the introduction of a polynucleotide into a cell wherein a polypeptide encoded by the polynucleotide is expressed (e.g., mRNA) or the polypeptide modulates a cellular function (e.g., siRNA, miRNA). As used herein, “expression” of a nucleic acid sequence refers to the translation of a polynucleotide (e.g., an mRNA) into a polypeptide or protein and / or post-translational modification of a polypeptide or protein.

[0093] Unmodified: As used herein, “unmodified” refers to any substance, compound or molecule prior to being changed in any way. Unmodified can, but does not always, refer to the wild type or native form of a biomolecule. Molecules can undergo a series of modifications whereby each modified molecule can serve as the “unmodified” starting molecule for a subsequent modification.

[0094] Untranslated region: As used herein “untranslated region” or “UTR” refer to regions located at the 5 ’ and 3 ’ ends of a messenger RNA (mRNA) construct that do not form a proteincoding region. In the context of the present disclosure, a 3' UTR corresponds to the sequence of a mature mRNA or sRNA which is located downstream from the coding sequence but before the poly(A) tail. The 5’ UTR is upstream from a coding sequence. In the context of the present disclosure, a 5' UTR corresponds to the sequence of a mature mRNA or sRNA which is located between the 5'-CAP and the start codon. The term "corresponds to" means that the UTR sequence may be an RNA sequence, such as in the mRNA sequence used for defining the UTR sequence, or a DNA sequence which corresponds to such RNA sequence.

[0095] “Poly(A)” or “Poly(A) tail” refers to the polyadenylation site of the mRNA. The poly(A) tail is a stretch of adenine nucleotides added to the 3' end of eukaryotic messenger RNA (mRNA) molecules during post-transcriptional processing. This modification is catalyzed by the enzyme poly(A) polymerase and plays a critical role in mRNA stability, nuclear export, translation efficiency, and protection from exonucleases. The length of the poly(A) tail can influence gene expression, as longer tails typically enhance translation, while shorter tails may signal mRNA decay. Additionally, the poly(A) tail interacts with poly(A)-binding proteins (PABPs), which help mediate its functions.

[0096]

[0097] The term "administering" as used herein refers to the physical introduction of an agent to a subject, such as a lipid nanoparticle disclosed herein, using any of the various methods and delivery systems known to those skilled in the art. Exemplary routes of administration for the formulations disclosed herein include intravenous, intramuscular, subcutaneous, intraperitoneal, spinal, or other parenteral routes of administration, for example by injection or infusion. The phrase "parenteral administration" means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion, as well as in vivo electroporation. In some embodiments, the formulation is administered via a non-parenteral route, e.g., orally. Other non-parenteral routes include a topical, epidermal or mucosal route of administration, for example, intranasally, vaginally, rectally, sublingually or topically. Administering can also be performed, for example, once, a plurality of times, and / or over one or more extended periods.

[0098] The term "pharmaceutically acceptable" refers to a molecule or composition that, when administered to a recipient, is not deleterious to the recipient thereof, or that any deleterious effect is outweighed by a benefit to the recipient thereof. With respect to a carrier, diluent, or excipient used to formulate a composition as disclosed herein, a pharmaceutically acceptable carrier, diluent, or excipient must be compatible with the other ingredients of the composition and not deleterious to the recipient thereof, or any deleterious effect must be outweighed by a benefit to the recipient. The term "pharmaceutically acceptable carrier" means a pharmaceutically- acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, or solvent encapsulating material, involved in carrying or transporting an agent from one portion of the body to another (e.g., from one organ to another). Each carrier present in a pharmaceutical composition must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not deleterious to the patient, or any deleterious effect must be outweighed by a benefit to the recipient. Some examples of materials which may serve as pharmaceutically acceptable carriers comprise: sugars, such as lactose, glucose and sucrose; starches, such as com starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppositorywaxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, com oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogenfree water; isotonic saline; Ringer's solution; ethyl alcohol; pH buffered solutions; polyesters, polycarbonates and / or polyanhydrides; and other non-toxic compatible substances employed in pharmaceutical formulations.

[0099] " Treatment" or "treating" of a subject refers to any type of intervention or process performed on, or the administration of an active agent to, the subject with the objective of reversing, alleviating, ameliorating, inhibiting, slowing down or preventing the onset, progression, development, severity or recurrence of a symptom, complication or condition, or biochemical indicia associated with a disease. In one embodiment, "treatment" or "treating" includes a partial remission. In another embodiment, "treatment" or "treating" includes a complete remission. In some embodiments, treatment may be of a subject who does not exhibit signs of the relevant disease, disorder and / or condition and / or of a subject who exhibits only early signs of the disease, disorder, and / or condition. In some embodiments, such treatment may be of a subject who exhibits one or more established signs of the relevant disease, disorder and / or condition. In some embodiments, treatment may be of a subject who has been diagnosed as suffering from the relevant disease, disorder, and / or condition. In some embodiments, treatment may be of a subject known to have one or more susceptibility factors that are statistically correlated with increased risk of development of the relevant disease, disorder, and / or condition.

[0100] A "disease", as used herein, is a state of health of a subject wherein the subject cannot maintain homeostasis, and wherein if the disease is not ameliorated, the subject's health continues to deteriorate. In contrast, a "disorder" is a state of health in which the subject is able to maintain homeostasis, but in which the subject's state of health is less favorable than it would be in the absence of the disorder. Left untreated, a disorder does not necessarily cause a further decrease in the subject's state of health. A disease or disorder is "alleviated" if the severity of a sign or symptom of the disease or disorder, the frequency with which such a sign or symptom is experienced by a subject, or both, is reduced.

[0101] As used herein, the terms “subject”, “individual”, and “patient” are interchangeable, and relate to vertebrates, preferably mammals. For example, mammals in the context of the disclosure are humans, non-human primates, domesticated animals such as dogs, cats, sheep,cattle, goats, pigs, horses, etc., laboratory animals such as mice, rats, rabbits, guinea pigs, etc., as well as animals in captivity such as animals in zoos. The term "animal" as used herein includes humans. The term "subject" may also include a patient, i.e., an animal, having a disease. In exemplary aspects, a subject, individual, or patient refers to a human (e.g., a man, a woman, or a child).

[0102] As used herein, the term “preventing a disease” in a subject means, for example, to stop the development of one or more clinical symptoms of a disease or disorder in a subject before they occur or are detectable. Preferably, the disease or disorder does not develop at all, i.e., no symptoms of the disease or disorder are detectable. In some aspects, it can also mean delaying or slowing of the development of one or more symptoms of the disease or disorder. Alternatively, or in addition, it can mean decreasing the severity of one or more subsequently developed symptoms.

[0103] The invention is defined in the claims. However, below is a non-exhaustive listing of non-limiting exemplary aspects. Any one or more of the features of these aspects may be combined with any one or more features of another example, embodiment, or aspect described herein.

[0104] The invention is defined in the claims. However, below is a non-exhaustive listing of non-limiting exemplary aspects. Any one or more of the features of these aspects may be combined with any one or more features of another example, embodiment, or aspect described herein.III. WNT7a mRNA-LNPs

[0105] WNT7a promotes myofiber hypertrophy, MuSC expansion, and suppresses FAP-induced fatty infiltration (FIG. 1A), highlighting its therapeutic potential for muscle regeneration and disease treatment.

[0106] However, recombinant WNT7a (rWNT7A) protein therapy faces significant translational challenges, including stability, delivery efficiency, and bioavailability. The production process requires the development of recombinant cell lines, which is both capital-and labor-intensive. These technologies involve multiple complex steps that are susceptible to contamination. Additionally, proteins expressed in non-mammalian systems often lack essential post-translational modifications, leading to variability that can pose serious health risks, including immunogenic reactions such as anaphylaxis. As a result, stringent quality control measures are necessary to ensure patient safety. Furthermore, recombinant proteins aresubject to degradation post-synthesis, complicating storage and distribution, and making large-scale production challenging.

[0107] In contrast, mRNA therapeutics offer several advantages. mRNA can be synthesized in a single, cell-free reaction vessel, reducing the need for cell culture infrastructure and minimizing contamination risks. Lower doses of mRNA are typically sufficient to elicit therapeutic effects, reducing the risk of overdose toxicity. The sequence of mRNA is easily modifiable, enabling rapid development and iteration. Proteins translated from mRNA tend to have longer half-lives than recombinant proteins, potentially extending therapeutic efficacy. Importantly, mRNA platforms are more scalable. However, challenges remain in delivering mRNA to target cells or tissues due to its susceptibility to extracellular nucleases and poor cellular uptake. Additionally, mRNA can activate innate immune responses, a legacy of the body’s defense against RNA viruses. This issue has been mitigated through the use of chemically modified nucleotides that reduce immune sensing and enhance translational efficiency, though delivery remains a key hurdle.

[0108] To overcome these limitations, the inventors designed W7a-LNP to enable endogenous muscle cells to transiently produce native WNT7a, acting as “in vivo protein factories” (Figure 1A).

[0109] Lipid nanoparticles (LNPs) address many of the delivery challenges associated with mRNA therapeutics. LNPs encapsulate mRNA, shielding it from nucleases and facilitating cellular uptake. Structural modifications to LNPs can enhance their stability, promote endosomal escape, and direct biodistribution to specific tissues or cell types. Compared to viral vectors such as adeno-associated viruses (AAVs), LNPs exhibit lower immunogenicity, allowing for repeated administration without triggering neutralizing immune responses.

[0110] LNP compositions typically consist of four lipid components which encapsulate nucleic acid, typically forming particles less than 100 nm in diameter. The key component is an ionizable lipid, which contains a cationic head group (e.g., tertiary amine) and hydrophobic tails. At neutral pH, the head group is uncharged, but becomes cationic in acidic environments such as endosomes. This pH-dependent ionization facilitates two critical functions: (i) efficient encapsulation of negatively charged nucleic acids, and (ii) disruption of the endosomal membrane to release nucleic acid into the cytosol. Electrostatic interactions between ionizable lipids and anionic lipids in the endosomal membrane are believed to mediate this disruption. Optimal ionizable lipids typically exhibit a pKa between 6.2 and 6.5.[oni] In some embodiments, the ionizable lipid comprises a synthetic lipid. In some embodiments, the ionizable lipid comprises a dendritic poly(amido amine) core conjugated with C14 alkyl chains, a biodegradable ionizable lipid with ester linkages, a branched-tail ionizable lipid with tertiary amine head group, a hydroxylated ionizable lipid with multiple hydroxyl groups and tertiary amine head group, or combinations thereof. In some embodiments, the ionizable lipid comprises heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate (SM-102), [(4-hydroxybutyl)azanediyl]bis(hexane-6, 1-diyl)bis(2 -hexyldecanoate) (ALC-0315), [(6Z,9Z)-hexadeca-6,9-dien-l-yl]-[2- (dimethylamino)ethyl]carbamate (DLin-MC3-DMA), N,N-dimethyl-2-(2-dodecyloxyethyl)ethanamine (Cl 2-200), or combinations thereof.

[0112] Additional LNP components include polyethylene glycol (PEG)-lipid conjugates, cholesterol, and phospholipids. PEG-lipid conjugates are present in the smallest molar percentage (-1.5%), these stabilize particles during formulation and storage, prevent aggregation, and influence size distribution. They also affect encapsulation efficiency, circulation half-life, biodistribution, transfection efficiency, and immune response.

[0113] In some aspects, the PEG-lipid conjugate compises a PEG-modified phosphatidylethanolamine, a PEG-modified phosphatidic acid, a PEG-modified ceramide, a PEG-modified dialkylamine, a PEG-modified diacylglycerol, a PEG-modified dialkylglycerol, a PEG-modified sterol, and a PEG-modified phospholipid. In various implementations, the PEG-modified lipid is selected from the group consisting of PEG-modified cholesterol, N-octanoyl-sphingosine- 1 - { succinyl [methoxy (poly ethylene glycol)] } , N-palmitoyl-sphingosine- 1 - {succinyl [methoxy (poly ethylene glycol)]}, PEG-modified DMPE (DMPE-PEG), PEG-modified DSPE (DSPE-PEG), PEG-modified DPPE (DPPE-PEG), PEG-modified DOPE (DOPE-PEG), dimyristoylglycerol-polyethylene glycol (DMG-PEG), di stearoylglycerolpolyethylene glycol (DSG-PEG), dipalmitoylglycerol-polyethylene glycol (DPG-PEG), dioleoylglycerol-polyethylene glycol (DOG-PEG), or a combination thereof.

[0114] In some embodiments, the PEG-lipid conjugate comprises 1,2-dimyristoyl-rac-glycero-3 -methoxypolyethylene glycol-2000 (DMG-PEG2000), Distearoyl glycerol-PEG2000 (DSG-PEG2000), l,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG2000), Ceramide-PEG2000, or combinations thereof. In some aspects, the PEG-lipid conjugate comprises dimyristoylglycerol-polyethylene glycol 2000 (DMG-PEG 2000).

[0115] Cholesterol enhances membrane fluidity and rigidity when combined with phospholipids. In some aspects the phospholipids comprise l,2-Distearoyl-sn-glycero-3-phosphocholine (DSPC), l,2-Dioleoyl-sn-glycero-3 -phosphoethanolamine (DOPE), Dipalmitoylphosphatidylcholine (DPPC), l,2-dilinoleoyl-sn-glycero-3 -phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3 -phosphocholine (DOPC), ,2- diundecanoyl-sn-glycero-phosphocholine (DUPC), l-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), l,2-di-O-octadecenyl-sn-glycero-3 -phosphocholine (18:0 Diether PC), l-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3 -phosphocholine (C 16 Lyso PC), l,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1 ,2-diarachidonoyl-sn-glycero-3 -phosphocholine, 1 ,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, l,2-dipalmitoyl-sn-glycero-3 -phosphoethanolamine (DPPE), 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), l,2-distearoyl-sn-glycero-3-phosphoethanolamine, l,2-dilinoleoyl-sn-glycero-3 -phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3 -phosphoethanolamine, 1,2-diarachidonoyl- sn-glycero-3 -phosphoethanolamine, l,2-didocosahexaenoyl-sn-glycero-3 -phosphoethanolamine, l,2-dioleoyl-sn-glycero-3-phospho-rac-(l -glycerol) sodium salt (DOPG), sphingomyelin, and combinations thereof. In some aspects, the phospholipid is DOPE, DSPC, or a combination thereof. DSPC may be used for its stability and encapsulation properties. DOPE may be used to enhance delivery efficiency of nucleic acids into cells. In various aspects, the phospholipid is DOPE.

[0116] The poly dispersity index (PDI) is used to quantify the size distribution of LNPs, with lower values indicating more uniform particle populations; an important parameter for consistent performance and regulatory compliance.

[0117] In some aspects, the ionizable lipid is present in the LNP lipid composition at a molar percentage of between about 40% to about 60% and the total mol% of components in the lipid composition is 100 mol%. In some embodiments, the ionizable lipid is present in the LNP lipid composition at molar percentage of about 40%, alternatively about 42%, alternatively about 44%, alternatively about 46%, alternatively about 48%, alternatively about 50%, alternatively about 52%, alternatively about 54%, alternatively about 56%, alternatively about 58%, or alternatively about 60%.

[0118] In some aspects, the phospholipid is present in the LNP lipid composition at a molar percentage of between about 5% to about 15% and the total mol% of components in the lipidcomposition is 100 mol%. In some embodiments, the phospholipid is present in the LNP lipid composition at molar percentage of about 5%, alternatively about 6%, alternatively about 7%, alternatively about 8%, alternatively about 9%, alternatively about 10%, alternatively about 11%, alternatively about 12%, alternatively about 13%, alternatively about 14%, or alternatively about 15%.

[0119] In some aspects, the cholesterol is present in the LNP lipid composition at a molar percentage of between about 30% to about 50% and the total mol% of components in the lipid composition is 100 mol%. In some embodiments, the cholesterol is present in the LNP lipid composition at molar percentage of about 30.0%, alternatively about 31.5%, alternatively about 33.0%, alternatively about 34.5%, alternatively about 36.0%, alternatively about 37.5%, alternatively about 38%, alternatively about 38.5%, alternatively about 39.0%, alternatively about 40.5%, alternatively about 42.0%, alternatively about 43.5%, alternatively about 45.0%, alternatively about 46.5%, alternatively about 48.0%, alternatively about 49.5%, alternatively about 50%.

[0120] In some aspects, the PEG-lipid conjugates are present in the LNP lipid composition at a molar percentage of between about 0.75% to about 2.5% and the total mol% of components in the lipid composition is 100 mol%. In some embodiments, the PEG-lipid conjugates are present in the LNP lipid composition at molar percentage of about 0.75%, alternatively about 1.0%, alternatively about 1.25%, alternatively about 1.5%, alternatively about 1.75%, alternatively about 2.0%, alternatively about 2.25%, or alternatively about 2.5%.

[0121] In an aspect, the LNP composition is used to encapsulate a polynucleotide that comprises the following formula:5’UTR— Coding Region— 3’ UTR-PolyA

[0122] where “UTRs” are the untranslated regions located at the 5’ and 3’ ends of an mRNA construct, “Poly A” refers to the polyadenylation site of the mRNA, “coding region”, refer to an Open Reading Frame (ORF) in a polynucleotide that upon expression, yields WNT7a. In one example, the 3’UTR is modified. In another example, the 5’UTR is modified. In a further example, both the 3’UTR and 5’UTR are modified.

[0123] In an aspect, the polynucleotide includes a suitable signal sequence, leader sequence, sorting sequence, in frame with and upstream of the coding region.

[0124] Non-limiting examples of the nucleic acid sequence of an isolated mRNA that encodes WNT7a and amino acid sequences of WNT7a according to an aspect of this disclosure are listed in Table 1. The amino acid sequence of WNT7a is listed in Table 1.

[0125] Table 1SEQ Sequence DescriptorID NO1 AUGAACCGGAAAGCGCGGCGCUGCCUGGGCCACCUCUU Polynucleotide UCUCAGCCUGGGCAUGGUCUACCUCCGGAUCGGUGGCU Sequence:WNT7a UCUCCUCAGUGGUAGCUCUGGGCGCAAGCAUCAUCUGU Coding region AACAAGAUCCCAGGCCUGGCUCCCAGACAGCGGGCGAU CUGCCAGAGCCGGCCCGACGCCAUCAUCCUCAUAGGAG AAGGCUCACAAAUGGGCCUGGACGAGUGUCAGUUUCA GUUCCGCAAUGGCCGCUGGAACUGCUCUGCACUGGrAG AGCGCACCGUCUUCGGGAAGGAGCUCAAAGUGGGGAG CCGGGAGGCUGCGUUCACCUACGCCAUCAUUGCCGCCG GCGUGGCCCACGCCAUCACACCUGCCUGUACCCAGGGC AACCUGAGCGACUGUGGCUGCGACAAAGAGAAGCAAG GCCAGUACCACCGGGACGAGGGCUGGAAGUGGGGUGG CUGCUCUGCCGACAUCCGCUACGGCAUCGGCUUCGCCA AGGUCUUUGUGGAUGCCCGGGAGAUCAAGCAGAAUGC CCGGACUCUCAUGAACUUGCACAACAACGAGGCAGGCC GAAAGAUCCUGGAGGAGAACAUGAAGCUGGAAUGUAA GUGCCACGGCGUGUCAGGCUCGUGCACCACCAAGACGU GCUGGACCACACUGCCACAGUUUCGGGAGCUGGGCUAC GUGCUCAAGGACAAGUACAACGAGGCCGUUCACGUGG AGCCUGUGCGUGCCAGCCGCAACAAGCGGCCCACCUUC CUGAAGAUCAAGAAGCCACUGUCGUACCGCAAGCCCAU GGACACGGACCUGGUGUACAUCGAGAAGUCGCCCAACU ACUGCGAGGAGGACCCGGUGACCGGCAGUGUGGGCACC CAGGGCCGCGCCUGCAACAAGACGGCUCCCCAGGCCAG CGGCUGUGACCUCAUGUGCUGUGGGCGUGGCUACAAC ACCCACCAGUACGCCCGCGUGUGGCAGUGCAACUGUAA GUUCCACUGGUCCUGCUAUGUCAAGUGCAACACGUGCAGCGAGCGCACGGAGAUGUACACGUGCAAGUGAGUGAAGUAA GGCAAAAAUCAAAAUCAAUCAUCAUCACAACAUCAAC Polynucleotide AAUCAAUCAUCAACACAUCAUCAAGACACCACC Sequence: 5’UTR GUGAAGUAAUUGUGUAUGCGUUAAUAAAAGAAGGAAC Polynucleotide UCGUAAAAACUCAAUGUAUUUCUGAGGAAGCGUGGUG Sequence: 3’UTR CAVAAUGCCACGCAGCGUCUCCAUAACUUUUAUUAUU UCUUUUAUUAAUCAACAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA Polynucleotide AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA Sequence: PolyA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAA GGCAAAAAUCAAAAUCAAUCAUCAUCACAACAUCAAC Polynucleotide AAUCAAUCAUCAACACAUCAUCAAGACACCACCAUGAA Sequence: Mature CCGGAAAGCGCGGCGCUGCCUGGGCCACCUCUUUCUCA mRNA sequence GCCUGGGCAUGGUCUACCUCCGGAUCGGUGGCUUCUCC UCAGUGGUAGCUCUGGGCGCAAGCAUCAUCUGUAACA AGAUCCCAGGCCUGGCUCCCAGACAGCGGGCGAUCUGC CAGAGCCGGCCCGACGCCAUCAUCCUCAUAGGAGAAGG CUCACAAAUGGGCCUGGACGAGUGUCAGUUUCAGUUC CGCAAUGGCCGCUGGAACUGCUCUGCACUGGRAGAGCG CACCGUCUUCGGGAAGGAGCUCAAAGUGGGGAGCCGG GAGGCUGCGUUCACCUACGCCAUCAUUGCCGCCGGCGU GGCCCACGCCAUCACACCUGCCUGUACCCAGGGCAACC UGAGCGACUGUGGCUGCGACAAAGAGAAGCAAGGCCA GUACCACCGGGACGAGGGCUGGAAGUGGGGUGGCUGC UCUGCCGACAUCCGCUACGGCAUCGGCUUCGCCAAGGU CUUUGUGGAUGCCCGGGAGAUCAAGCAGAAUGCCCGG ACUCUCAUGAACUUGCACAACAACGAGGCAGGCCGAAA GAUCCUGGAGGAGAACAUGAAGCUGGAAUGUAAGUGC CACGGCGUGUCAGGCUCGUGCACCACCAAGACGUGCUG GACCACACUGCCACAGUUUCGGGAGCUGGGCUACGUGC UCAAGGACAAGUACAACGAGGCCGUUCACGUGGAGCC UGUGCGUGCCAGCCGCAACAAGCGGCCCACCUUCCUGAAGAUCAAGAAGCCACUGUCGUACCGCAAGCCCAUGGAC ACGGACCUGGUGUACAUCGAGAAGUCGCCCAACUACUG CGAGGAGGACCCGGUGACCGGCAGUGUGGGCACCCAGG GCCGCGCCUGCAACAAGACGGCUCCCCAGGCCAGCGGC UGUGACCUCAUGUGCUGUGGGCGUGGCUACAACACCCA CCAGUACGCCCGCGUGUGGCAGUGCAACUGUAAGUUCC ACUGGUCCUGCUAUGUCAAGUGCAACACGUGCAGCGA GCGCACGGAGAUGUACACGUGCAAGUGAGUGAAGUAA GUGAAGUAAUUGUGUAUGCGUUAAUAAAAGAAGGAAC UCGUAAAAACUCAAUGUAUUUCUGAGGAAGCGUGGUG CAVAAUGCCACGCAGCGUCUCCAUAACUUUUAUUAUU UCUUUUAUUAAUCAACAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA6 MNRKARRCLGHLFL SLGMVYLRIGGF S S VVALGASIIC Amnio Acid Sequence:NKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQF WNT7a RNGRWNCSALGERTVFGKELKVGSREAAFTYAIIAAG VAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKW GGCSADIRYGIGFAKVFVDAREIKQNARTLMNLHNNE AGRKILEENMKLECKCHGVSGSCTTKTCWTTLPQFRE LGYVLKDKYNEAVHVEPVRASRNKRPTFLKIKKPLSY RKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKT APQASGCDLMCCGRGYNTHQYARVWQCNCKFHWCC YVKCNTCSERTEMYTCK

[0126] In certain embodiments, the 5’UTR has at least 50%, alternatively at least about 60%, alternatively at least about 65%, alternatively at least about 70%, alternatively at least about 75%, alternatively at least about 80%, alternatively at least about 85%, alternatively at least about 90%, alternatively at least about 95%, alternatively at least about 96%, alternatively at least about 97%, alternatively at least about 98%, alternatively at least about 99%, or 100% nucleic acid sequence identity to SEQ ID NO:2.

[0127] In certain embodiments, the 3’UTR has at least 50%, alternatively at least about 60%, alternatively at least about 65%, alternatively at least about 70%, alternatively at least about 75%, alternatively at least about 80%, alternatively at least about 85%, alternatively at least about 90%, alternatively at least about 95%, alternatively at least about 96%, alternatively at least about 97%, alternatively at least about 98%, alternatively at least about 99%, or 100% nucleic acid sequence identity to SEQ ID NO:3.

[0128] In certain embodiments, the PolyA has at least 50%, alternatively at least about 60%, alternatively at least about 65%, alternatively at least about 70%, alternatively at least about 75%, alternatively at least about 80%, alternatively at least about 85%, alternatively at least about 90%, alternatively at least about 95%, alternatively at least about 96%, alternatively at least about 97%, alternatively at least about 98%, alternatively at least about 99%, or 100% nucleic acid sequence identity to SEQ ID NO:4.

[0129] In an aspect, the polynucleotide comprises a coding region having at least 50%, alternatively at least about 60%, alternatively at least about 65%, alternatively at least about 70%, alternatively at least about 75% nucleic acid sequence identity to SEQ ID NO:1.

[0130] In an aspect, the polynucleotide comprises a nucleic acid sequence having at least 50%, alternatively at least about 60%, alternatively at least about 65%, alternatively at least about 70%, alternatively at least about 75% nucleic acid sequence identity to SEQ ID NO:5.

[0131] In certain embodiments, the disclosed polynucleotide may exhibit at least 80% sequence identity to SEQ ID NO: 1 (coding region) or SEQ ID NO: 5 (mature mRNA sequence) while still encoding the same functional protein. This is possible due to the degeneracy of the genetic code, wherein multiple codons can encode the same amino acid. For example, the amino acid leucine can be encoded by six different codons, allowing for substantial variability at the nucleotide level without altering the resulting polypeptide. Such sequence variation may arise from synonymous substitutions, codon optimization for expression in different host systems, or natural polymorphisms. Accordingly, polynucleotides with reduced sequence identity to the reference sequence can still produce proteins with identical amino acid sequences and biological activity, thereby supporting the scope of the claimed invention.

[0132] In an aspect, the polynucleotide comprises a coding region having at least about 80%, alternatively at least about 85%, alternatively at least about 90%, alternatively at least about 95%, alternatively at least about 96%, alternatively at least about 97%, alternatively at leastabout 98%, alternatively at least about 99%, or 100% nucleic acid sequence identity to SEQ ID NO:1.

[0133] In an aspect, the polynucleotide comprises a nucleic acid sequence having at least about 80%, alternatively at least about 85%, alternatively at least about 90%, alternatively at least about 95%, alternatively at least about 96%, alternatively at least about 97%, alternatively at least about 98%, alternatively at least about 99%, or 100% nucleic acid sequence identity to SEQ ID NO:5.

[0134] In an aspect, the polynucleotide comprises the nucleic acid sequence as shown in FIG.11

[0135] In an aspect, the LNP composition is used to encapsulate a polynucleotide that encodes WNT7a, generating W7a-LNPs. In an aspect, the polynucleotide encodes a WNT7a having at least 50%, alternatively at least about 60%, alternatively at least about 65%, alternatively at least about 70%, alternatively at least about 75%, alternatively at least about 80%, alternatively at least about 85%, alternatively at least about 90%, alternatively at least about 95%, alternatively at least about 96%, alternatively at least about 97%, alternatively at least about 98%, alternatively at least about 99%, or 100% amino acid sequence identity with the sequence of SEQ ID NO: 6.

[0136] One aspect of the disclosure is a W7a-LNP using a lipid composition of SM-102, DOPE, cholesterol, and DMG-PEG2000. In embodiment, the lipids are present at a molar ratio of 49.08 (SM-102) : 9.29 (DOPE): 40.21 (cholesterol) : 1.42(DMG-PEG2000), alternatively 46.66 : 11.00 : 40.66 : 1.68, alternatively 50: 10: 38.5: 1.5, alternatively 50.57 : 9.92 : 37.95 : 1.56, alternatively 51.14 : 9.52 : 37.68 : 1.66, alternatively 54.08 : 9.80 : 34.65 : 1.47; wherein a polynucleotide encoding WNT7a is encapsulated by the lipid composition.

[0137] In an aspect, the lipids are complexed with in v ro-transcribed WNT7a mod-RNA using a microfluidic device for precise and reproducible assembly.

[0138] In an aspect, the resulting W7a-LNPs have a mean diameter of between about 68 nm and about 85 nm. In certain embodiments, the W7a-LNPs have a mean diameter of about 68.25 nm, alternatively about 69.01 nm, alternatively about 69.76 nm, alternatively about 70.52 nm, alternatively about 71.28 nm, alternatively about 72.04 nm, alternatively about 72.80 nm, alternatively about 73.56 nm, alternatively about 74.31 nm, alternatively about 75.07 nm, alternatively about 75.83 nm, alternatively about 76.59 nm, alternatively about 77.35 nm, alternatively about 78.10 nm, alternatively about 78.86 nm, alternatively about 79.62 nm,alternatively about 80.38 nm, alternatively about 81.14 nm, alternatively about 81.90 nm, alternatively about 82.65 nm, or alternatively about 83.41 nm.

[0139] In an aspect, the resulting W7a-LNPs have a poly dispersity index (PDI) between about 0.0845 and about 0.11 nm. In certain embodiments, the W7a-LNPs have a PDI of about 0.0846, alternatively about 0.0855, alternatively about 0.0865, alternatively about 0.0874, alternatively about 0.0884, alternatively about 0.0893, alternatively about 0.0902, alternatively about 0.0912, alternatively about 0.0921, alternatively about 0.0931, alternatively about 0.0940, alternatively about 0.0950, alternatively about 0.0960, alternatively about 0.0970, alternatively about 0.0978, alternatively about 0.0987, alternatively about 0.0996, alternatively about 0.1006, alternatively about 0.1015, alternatively about 0.1025, alternatively about 0.1034.

[0140] In an aspect, the resulting W7a-LNPs have an encapsulation efficiency of between about 85% to about 100%. Encapsulation efficiency refers to the percentage of a therapeutic agent successfully enclosed within the nanoparticles during formulation. In certain embodiments, the W7a-LNPs have an encapsulation efficiency of about 87.78%, alternatively about 88.76%, alternatively about 89.73%, alternatively about 90.71%, alternatively about 91.68%, alternatively about 92.66%, alternatively about 93.63%, alternatively about 94.61%, alternatively about 95.58%, alternatively about 96.56%, alternatively about 97.53%, alternatively about 98.51%, alternatively about 99.48%, or alternatively about 100%.

[0141] The W7a-LNPs can be administered via any parenteral or non-parenteral (enteral) route that is therapeutically effective for mRNA therapeutics. Parenteral application methods include, for example, intracutaneous, subcutaneous, intramuscular, intratracheal, intranasal, intravitreal or intravenous injection and infusion techniques, e.g. in the form of injection solutions, infusion solutions or tinctures, as well as aerosol installation and inhalation, e.g. in the form of aerosol mixtures, sprays or powders. An overview of pulmonary drug delivery, i.e. either via inhalation of aerosols (which can also be used in intranasal administration) or intracheal instillation is given by J.S. Patton et al. The lungs as a portal of entry for systemic drug delivery. Proc. Amer. Thoracic Soc. 2004 Vol. 1 pages 338-344, for example). Non-parenteral delivery modes are, for instance, orally, e.g. in the form of pills, tablets, capsules, solutions or suspensions, or rectally, e.g. in the form of suppositories. The W7a-LNPs of the invention can be administered systemically or topically in formulations containing conventional non-toxic pharmaceutically acceptable excipients or carriers, additives and vehicles as desired.

[0142] The W7a-LNPs may be formulated as a pharmaceutical or therapeutic composition to be delivered to a mammal, in particular to a human. Corresponding administration methods include, but are not limited to, for example, intracutaneous, subcutaneous, intramuscular, intratracheal or intravenous injection and infusion techniques, e.g. in the form of injection solutions, infusion solutions or tinctures as well as aerosol installation and inhalation, e.g. in the form of aerosol mixtures, sprays or powders. A combination of different routes of administration in the immunized subject, for example intramuscular and intranasal administration at the same time, is also contemplated by the disclosure.

[0143] In some aspects, the W7a-LNPs are formulated into a therapeutic composition, such as an mRNA-based therapeutic composition. The therapeutic composition may optionally include one or more therapeutically acceptable carriers, diluents, or excipients such as salts, buffering agents, preservatives, antiadherents, antioxidants, binders, coatings, compression aids, disintegrants, dyes, emollients, emulsifiers, fillers, film formers or coatings, flavors, fragrances, glidants, lubricants, sorbents, suspending or dispersing agents, sweeteners, waters of hydration, and / or other therapeutic agents. As used herein, the term “excipient” means any pharmaceutically acceptable additive, carrier, diluent, adjuvant, or other ingredient, other than the active pharmaceutical ingredient (API) (and typically in addition to components of the delivery vehicle compositions), suitably selected with respect to the intended form of administration, and consistent with conventional pharmaceutical practices. The disclosed compounds can be administered to a subject or patient in a therapeutically effective amount. The complexes can be administered alone or as part of a pharmaceutically acceptable composition or formulation. In addition, the compositions can be administered all at once, as for example, by a bolus injection, multiple times, or delivered substantially uniformly over a period of time. It is also noted that the dose of the compound can be varied over time.

[0144] Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions, may be formulated according to the known art using suitable dispersing agents, wetting agents, and / or suspending agents. Sterile injectable preparations may be sterile injectable solutions, suspensions, and / or emulsions in nontoxic parenterally acceptable diluents and / or solvents, for example, as a solution in 1,3 -butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, U.S.P., and isotonic sodium chloride solution. Sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil can be employed, including synthetic mono- or diglycerides. Fatty acids such as oleic acid can be used in the preparation of injectables.Exemplary buffers include citrate, succinate, acetate, malate, succinate, and histidine. In addition, stabilizers such as sucrose may be included.

[0145] Injectable formulations may be sterilized, for example, by filtration through a bacterial-retaining filter and / or by incorporating sterilizing agents in the form of sterile solid compositions, which can be dissolved or dispersed in sterile water or other sterile injectable media prior to use. Adjuvants such as local anesthetics, preservatives and buffering agents can also be added to the compositions.

[0146] In certain embodiments, the disclosure relates to the treatment of skeletal muscle fibro-adipogenic progenitor (FAP) adipogenesis, which is implicated in pathological fat infiltration and fibrosis following musculoskeletal injury or in degenerative muscle conditions. FAPs are a population of mesenchymal progenitor cells residing in skeletal muscle that possess the capacity to differentiate into adipocytes and fibroblasts. Under pathological conditions, such as acute trauma, chronic muscle degeneration, or disease-associated muscle wasting, FAPs may become dysregulated, contributing to impaired muscle regeneration and the accumulation of fibrofatty tissue.

[0147] The present disclosure provides a therapeutic approach comprising the administration of a lipid nanoparticle formulation or a therapeutic composition containing an active agent capable of modulating FAP differentiation. The administration may be carried out via a suitable route, including but not limited to intravenous, intramuscular, or subcutaneous injection, and may be performed in accordance with a dosing regimen appropriate for the condition being treated. The treatment may be administered at defined time points post-injury, including but not limited to one, two, three, four, five, six, or seven days following the onset of injury, and may be repeated according to a clinically appropriate dosing schedule. By targeting FAP adipogenesis, the disclosed method aims to preserve muscle architecture, enhance regenerative outcomes, and reduce the progression of muscle fibrosis and fatty infiltration.

[0148] In some aspects, the mRNA-based therapeutic may be part of a kit. The kit may include a pharmaceutically acceptable carrier and / or a package insert comprising instructions for intratumoral administration (e.g., injection) of the mRNA-based therapeutic composition.

[0149] The disclosure demonstrates that W7a-LNP effectively recapitulates key functions of recombinant WNT7a in vitro, reducing FAP adipogenesis while promoting myofiber hypertrophy. In vivo, repeated W7a-LNP delivery induced muscle hypertrophy without fibrosis, supporting its safety and efficacy in uninjured skeletal muscle. Delayed delivery at 4days post-injury significantly reduced fatty infiltration, supporting the importance of timing and target cell availability for therapeutic efficacy. Together, these results provide proof-of-concept that W7a-LNP can be leveraged to transiently reprogram the muscle environment in a context-dependent manner.

[0150] The disclosed W7a-LNPs effectively recapitulated the key functions of recombinant WNT7a in vitro, suppressing FAP adipogenesis (FIGs. 3A-3E) and increasing myofiber size (FIGs. 4A-4D) These results indicate that muscle cells can take up W7a-LNP, translate WNT7a mRNA, and secrete functional protein capable of modulating both FAP and myofiber function. Using a cre-LNP system, we showed that mRNA-LNP delivery enables efficient transgene expression in both primary FAPs in vitro and myofibers in vivo, with robust TdT reporter activation in both settings (FIGs. 2A-2G). This highlights the feasibility of localized mRNA delivery for skeletal muscle applications.

[0151] A dose-dependent effect on FAP adipogenesis was also observed, with 100 ng / mL W7a-LNP exerting a stronger anti-adipogenic response than 1 ng / mL (FIGs. 3B, 3C). Notably, 10-100 ng / mL W7a-LNP suppressed adipogenesis more consistently than recombinant WNT7a, as reflected by reduced variability in adipogenic inhibition (FIGs. 3B, 3C).

[0152] In some embodiments, the dose may range from a minimum of about 10 ng / mL to a maximum of about 100 ng / mL. In some aspects, the dose may range from about 10-19 ng / mL, alternatively about 20-29 ng / mL, alternatively about 30-39 ng / mL, alternatively about 40-49 ng / mL, alternatively about 50-59 ng / mL, alternatively about 60-69 ng / mL, alternatively about 70-79 ng / mL, alternatively about 80-89 ng / mL, or alternatively about 90-99 ng / mL.

[0153] In some embodiments, the dose may be about 10 ng / mL, alternatively about 20 ng / mL, alternatively about 30 ng / mL, alternatively about 40 ng / mL, alternatively about 50 ng / mL, alternatively about 60 ng / mL, alternatively about 70 ng / mL, alternatively about 80 ng / mL, alternatively about 90 ng / mL, or alternatively about 100 ng / mL.

[0154] Similarly, the inventors observed a robust fusion and hypertrophic response in myofibers starting at 10 ng / mL W7a-LNP, further supporting the dose-dependent efficacy of W7a-LNP in enhancing myofiber differentiation and growth (FIGs.4A-4D). This suggests that WNT7a levels produced from transfected cells can be modulated by mRNA dose, providing a tunable strategy for regulating both FAP and myofiber differentiation and hypertrophy.

[0155] In certain embodiments, the disclosed W7a-LNPs may be administered according to various dosing regimens tailored to the therapeutic needs of the patient and the pharmacological properties of the active agent. Dosing schedules may include administration once per week, twice per week, every other week, every ten days, every fourteen days, or at other intervalssuch as once every three days, once every five days, three times per week, or once monthly. These flexible dosing options enable sustained therapeutic levels, reduce the risk of adverse effects, and enhance patient adherence. The formulation may be delivered via injection, infusion, or other suitable routes, with each dose containing a therapeutically effective amount of the active compound. Such regimens are particularly advantageous for chronic conditions or therapies requiring repeated administration over extended periods.

[0156] In certain embodiments, the disclosed W7a-LNPs may be administered following an injury according to a time-dependent dosing schedule designed to enhance recovery and therapeutic outcomes. The dosing may begin at approximately one day post-injury and continue at intervals such as two days, three days, four days, five days, six days, or seven days postinjury, depending on the severity of the injury, the pharmacodynamics of the active agent, and the desired therapeutic effect.

[0157] To evaluate the in vivo efficacy of W7a-LNP, the inventors tested both single and multiple intramuscular injections (FIGs. 5A-5D). A single injection had no significant effect on myofiber hypertrophy, whereas multiple injections were required to induce hypertrophy, likely due to the transient nature of mRNA expression necessitating repeated dosing to sustain WNT7a levels. The inventors administered three injections over four weeks (every two weeks) to allow sufficient time for muscle recovery between transfections. Following muscle injury, such as needle puncture, regeneration typically completes within 10-14 days, and the tissue returns to a homeostatic condition by week 4. Evaluating tissues 4 weeks after the final injection ensured the inventors captured stable hypertrophic outcomes, not transient regenerative or inflammatory effects. These findings suggest that dose and delivery frequency are critical parameters to optimize, particularly for chronic degenerative conditions. Repeated W7a-LNP administration did not induce fibrosis or other observable pathological changes, supporting its safety and tolerability in skeletal muscle. Altogether, these results support the safety and feasibility of mRNA-LNP technology for muscle regeneration.

[0158] The inventors cre-LNP study demonstrated that W7a-LNP delivery remained localized within the injected muscle without detectable leakage into the contralateral muscle (FIGs. 2D and 2E). Within the injected muscle, the inventors findings of broad TdT expression are consistent with recent reports showing that mRNA LNPs can disseminate locally following intramuscular injection. This broad distribution may reflect passive diffusion of LNPs through the extracellular matrix, as well as proximal-to-distal trafficking of mRNA within multinucleated syncytium of myofibers. Further optimizing the dose and delivery parametersmay help minimize unintended accumulation in other organs while maintaining effective WNT7a expression in skeletal muscle.

[0159] Intramuscular delivery of recombinant WNT7a significantly reduced IMAT following acute glycerol injury (FIGs. 6A-6H). However, the inventors observed variability in response to injury following W7a-LNP administration, with some animals exhibiting reduced fatty infiltration while others showed no significant change (FIGs. 6A-6H). This suggests that the therapeutic effects of W7a-LNP may be influenced by injury severity, host-specific factors, or delivery timing. To investigate whether residual glycerol interferes with LNP stability, the inventors performed DLS analysis and found that although particle diameter increased with higher glycerol concentrations, poly dispersity remained low, suggesting the LNPs remained stable and monodisperse (FIG. 7). Since glycerol ablates all multi-nucleated myofibers, which are presumed to perform the bulk of protein synthesis, administering W7a-LNP one day postinjury may not have allowed for sufficient uptake, translation, and secretion of WNT7a. To test this, we administered W7a-LNP at 4 days post-injury, when regenerating myofibers begin to re-emerge. In this setting, W7a-LNP significantly reduced fatty infiltration (FIGs. 6F and 6G), supporting the idea that viable myofibers are required for effective mRNA LNP-mediated protein production and function. In clinically relevant degenerative conditions like rotator cuff injuries, where myofibers remain viable despite undergoing atrophy, LNP -based delivery may be more effective due to sustained cellular uptake and protein production. Further optimization of the LNP formulation and mRNA structural elements may be needed to improve LNP uptake and sustained expression of the therapeutic protein in injured muscle. Additionally, the effects of W7a-LNP on immune cells remain unknown, and future studies should investigate how immune modulation may influence its therapeutic potential in skeletal muscle regeneration.

[0160] The disclosure demonstrates W7a-LNPs successfully promote myofiber hypertrophy without inducing fibrosis while reducing the adipogenic potential of FAPs. By transiently reprogramming the muscle microenvironment, this approach holds potential for mitigating muscle atrophy and pathological fatty infiltration in degenerative conditions. These findings support LNP-mediated delivery of WNT7a mRNA as a viable, scalable, and cost-effective alternative to recombinant protein therapy for localized muscle treatment. Further research is needed to optimize dosing, delivery timing, biodistribution, LNP formulation, RNA structural elements, and long-term therapeutic outcomes for clinical translation.

[0161] The presently described technology and its advantages will be better understood by reference to the following examples. These examples are provided to describe specificimplementations of the present technology. By providing these specific examples, it is not intended limit the scope and spirit of the present technology. It will be understood by those skilled in the art that the full scope of the presently described technology encompasses the subject matter defined by the claims appending this specification, and any alterations, modifications, or equivalents of those claims.

[0162] EXAMPLES

[0163] 1. W7a-LNPs Production

[0164] MATERIALS AND METHODS

[0165] Animals

[0166] Animal procedures were conducted under the approval of the Institutional Animal Care and Use Committee at the Icahn School of Medicine at Mount Sinai (#PROTO202100002). Mice were housed and maintained in the Center for Comparative Medicine and Surgery Facility at the Icahn School of Medicine at Mount Sinai. C57B1 / 6J mice (12-14 weeks old, #000664) and Ail4-TdTomato mice (22 weeks old, #007914) were acquired from the Jackson Laboratory. Both male and female mice were included in the study in a randomized manner.

[0167] DNA Templates for RNA In Vitro Transcription

[0168] Plasmid templates for RNA in vitro transcription (IVT) were synthetized by Twist Bioscience incorporating a T7 promoter sequence, a 5' UTR, an open reading frame, and a 3' UTR. Our construct employed NASAR UTRs for enhanced gene expression

[0034] , IVT templates were generated from plasmids by amplifying DNA with Q5 High-Fidelity Master Mix (NEB). A 120-nucleotide poly-Atail (SEQ ID NO: 7) was encoded into the PCR template using a reverse primer containing a 120-nucleotide poly-T(SEQ ID NO: 8). Following PCR, Dpnl (NEB) was used to digest plasmid contaminants at 37°C for 30 minutes. The PCR products were purified using the QIAquick PCR Purification Kit (Qiagen), and concentrations were measured with the NanoDrop™ 2000 spectrophotometer (Thermo Fisher Scientific) before IVT.

[0169] Synthesis and Purification of mRNAs

[0170] Modified mRNAs (modRNA) were synthesized using the HiScribe T7 High Yield RNA Synthesis Kit (NEB). Nl-methylpseudouridine-5’ -triphosphate (Nlm), sourced from TriLink Biotechnologies, was fully substituted for UTP in the reaction to produce modRNAs. A 1:4 premix of CleanCap reagent AG (TriLink) was included for co-transcriptional capping. Reaction mixtures were incubated at 37°C for 5 hours, followed by a 30-minute incubation with TURBO DNase I (Thermo Fisher Scientific) to remove residual DNA template. RNAproducts were then purified using the Monarch RNA Cleanup Kit (NEB). RNA concentration was determined with the NanoDrop™ 2000, and species purity and size were verified using a denaturing RNA gel.

[0171] Lipid Nanoparticle Formation

[0172] mRNA encapsulation in LNP was performed using the NanoAssembler™ Ignite™ microfluidic mixing device (Cytiva). SM-102 particles were formulated with helper lipids DSPC (l,2-disteraroyl-sn-glycero-3-phosphocholine), cholesterol, and DMG-PEG2000 (1,2-dimyristoyl-sn-glycerol methoxypolyethylene glycol) at a molar ratio of 50:10:38.5:1.5, with modRNA dissolved in a citrate buffer. The freshly formed RNA-LNPs were dialyzed overnight against PBS buffer using Slide-A-Lyzer dialysis cassettes (3.5K MWCO, Life Technologies) and concentrated to the desired concentration using Amicon® Ultra Centrifugal Filters (10 kDa MWCO, Sigma). Particle size and zeta potential were measured using a Zetasizer Advance (Malvern Panalytical) at 25°C and a scattering angle of 173°. Encapsulation efficiency was determined using the Quant-it™ RiboGreen RNA Assay Kit following the manufacturer's protocol, with quantification performed on a Cytation 3 Cell Imaging Reader (BioTek).

[0173] Isolation of Primary FAPs

[0174] FAPs were isolated from the hindlimb muscles of mice (5-10 weeks old) using magnetic-activated cell sorting (MACS). Muscles were dissected, minced, and digested for 1.5 hours at 37°C in DMEM containing 0.2% (w / v) collagenase type II and 2.5 U / mL Dispase II. Digestion was stopped by adding Ham’s F-10 supplemented with 20% fetal bovine serum (FBS). The resulting cell suspension was filtered through a 70 pm cell strainer, centrifuged at 300g for 5 minutes at 4°C, and resuspended in staining buffer (0.5% BSA and 2 mM EDTA in PBS). The suspension was then filtered through a 35 pm strainer. Cells were incubated at 4°C for 45 minutes with biotin-conjugated antibodies targeting CD31 (BioLegend, cat. no. 102503; 1:150), CD45 (BioLegend, cat. no. 103103; 1:150), and integrin a7 (Miltenyi, cat. no. 130-101-979; 1:10). After centrifugation, cells were incubated with streptavidin beads (1:30) for 15 minutes at 4°C and passed through an LD column (Miltenyi, cat. no. 130-042-901) for negative selection. The remaining cells were labeled with a biotin-conjugated Ly-6A / E (SCA-1) antibody (BioLegend, cat. no. 122504; 1:75) for 20 minutes at 4°C, followed by streptavidin beads (1:30) for 10 minutes. Positively labeled SCA-1+ cells were enriched using an LS column (Miltenyi, cat. no. 130-042-401). Finally, the purified cells were filtered through a 35 pm strainer and prepared for downstream cell culture assays.

[0175] FAPs and C2C12 Culture and Differentiation

[0176] Cultures were maintained at 37°C with 5% CO2. FAPs were expanded in growth medium (DMEM with 10% FBS and IX penicillin-streptomycin) on laminin (10 pg / mL) and collagen I (5 pg / mL)-coated plates. Adipogenic differentiation was induced by switching to differentiation medium (DMEM supplemented with 10% FBS, IX penicillin-streptomycin, 0.5 mM 3 -isobutyl- 1 -methylxanthine, 0.25 pM dexamethasone, and 1 pg / mL insulin). C2C12 myoblasts were expanded in growth medium (DMEM with 10% FBS and IX penicillin-streptomycin) on laminin (10 pg / mL) and collagen I (5 pg / mL)-coated plates. Differentiation was induced by switching -80% confluent cells to differentiation medium (DMEM with 2% horse serum and IX penicillin-streptomycin).

[0177] mRNA LNP Administration and Dose Selection

[0178] Intramuscular mRNA LNP injections were performed in multiple experimental conditions using either cre-LNP for transgene expression validation or W7a-LNP for therapeutic evaluation. A dose of 10-15 pg per injection was selected based on previous SARS-CoV-2 mRNA LNP vaccine and CRISPR-Cas9 mRNA LNP muscular dystrophy studies [36,37], which demonstrated efficient mRNA delivery in skeletal muscle.

[0179] Intramuscular cre-LNP Administration

[0180] Ail4-TdTomato mice were anesthetized with isoflurane, and a single subcutaneous dose of Ethiqa XR (extended-release buprenorphine) was administered on the day of the procedure for pain management. The TA muscle was injected intramuscularly with 15 pg / 30 pL cre-LNP, while 30 pL of PBS vehicle was injected into the contralateral TA muscle. Mice were euthanized at week 2 for histological analysis.

[0181] W7a-LNP Administration in Uninjured Supraspinatus Muscles

[0182] C57B1 / 6J mice were anesthetized with isoflurane, and a single subcutaneous dose of Ethiqa XR was administered on the day of the procedure for pain management. The left forelimb was positioned on a 2 mm elevated surface, extended, and externally rotated to have the palm facing upward. Mouse fur overlying the supraspinatus muscle was removed with Nair. A 1 cm linear incision was made to the skin and the supraspinatus muscle was injected intramuscularly with 10 pg / 30 pL W7a-LNP, while 30 pL of PBS vehicle was injected into the contralateral supraspinatus muscle, and incision was sutured. For the single-injection cohort, mice received injections at week 0 and were euthanized at week 4 for histological analysis. For the multiple-injection cohort, mice received injections at week 0, 2, and 4, then were euthanized at week 8 for histological analysis.

[0183] Glycerol Injuries and W7a-LNP Administration

[0184] C57B1 / 6J mice were anesthetized with isoflurane, and a single subcutaneous dose of Ethiqa XR was administered on the day of the procedure. 50 pL of 50% (v / v) glycerol in saline was injected intramuscularly into the tibialis anterior (TA) muscle to induce injury

[0023] , After 24 hours, the TA muscles were injected with 2.5 pg / 30 pL recombinant human WNT7a (PeproTech; cat. no. 120-31-15UG), 10 pg / 30 pL W7a-LNP, or 30 pL PBS. Mice were euthanized on day 14 for histological analysis.

[0185] Immunocytochemistry Staining

[0186] Cells were fixed with 4% paraformaldehyde (PF A) for 20 min at room temperature. Samples were washed three times with IX PBS and incubated in blocking / permeabilization buffer (5.0% goat serum, 2.0% bovine serum albumin, 0.5% Triton X-100 in PBS) overnight at 4°C. The following primary and secondary antibodies were used for immunocytochemistry in this study: anti -perilipin- 1 (Cell Signaling Technology; cat. no. 9349S; 1:200); MF20 (DSHB; cat. no. AB 2147781; 1:100); human Wnt-7a / b antibody (R&D Systems; cat. no. AF3008; 10 pg / mL); goat anti-rabbit Alexa Fluor 488 (Thermo Fisher Scientific; cat. no. Al 1008; 1:500); goat anti -mouse Alexa Fluor 488 (Thermo Fisher Scientific; cat. no. A32723; 1:500); rabbit anti -goat Alexa Fluor 488 (Thermo Fisher Scientific; cat. no A11078; 1:500). Hoechst 33342 (Thermo Fisher Scientific; cat. no. 62249, 1:1,000) was used to stain nuclei.

[0187] Immunohistochemistry Staining

[0188] TA and supraspinatus muscles were dissected and frozen in liquid nitrogen-chilled methylbutane. 10 pm sections were obtained from the frozen muscle using a cryostat. Tissue sections were fixed with 4% PFAfor 10 min at room temperature, then washed three times with IX PBS and incubated using blocking / permeabilization buffer (5.0% goat serum, 2.0% bovine serum albumin, 0.5% Triton X-100 in PBS) for 1 hour at room temperature. The following primary and secondary antibodies were used for tissue immunohistochemistry in this study: anti-perilipin- 1 (Cell Signaling Technology; cat. no. 9349S; 1:200), anti-laminin (Sigma Aldrich; cat. no. L9393; 1:100), and goat anti-rabbit Alexa Fluor 488 (Thermo Fisher Scientific; cat. no. Al 1008; 1:500). Hoechst 33342 (Thermo Fisher Scientific; cat. no. 62249, 1:1000) and Alexa Fluor 647 Phalloidin (Thermo Fisher Scientific; cat. no. A22287; 1:1000) were used to stain nuclei and F-actin, respectively. Tissue sections were also processed for routine Oil Red O and Picrosirius red staining.

[0189] Imaging and Image Analysis

[0190] Images were captured using a Leica Microsystems THUNDER DMi8 microscope and processed with LAS-X software. Quantitative image analysis was performed using ImageJ / FIJI.

[0191] Statistical Analysis

[0192] Statistical analyses were performed using GraphPad Prism. Two-tailed t-test and oneway analysis of variance (ANOVA) with Tukey’s post-hoc analysis was performed depending on the number of comparisons. Statistical significance was set at p<0.05.

[0193] 2. W7a-LNPs: size uniformity and complete mRNA encapsulation.

[0194] The resulting W7a-LNPs had a mean diameter of 75.83 nm, a poly dispersity index (PDI) of 0.0940, and an encapsulation efficiency of 97.53% at 570.45 pg / mL total mRNA (FIG. IB). Notably, W7a-LNPs remained stable for over six weeks at -80°C in 10% sucrose (FIG. 8A-8C), following a storage protocol similar to Modema’s SARS-CoV-2 vaccine. The z-average diameter remained below 100 nm, and the PDI stayed well below 0.3, indicating minimal aggregation and a uniform particle population (FIG. 8A-8C).

[0195] 3. Feasibility of the disclosed LNP platform for use in skeletal muscle applications,

[0196] The inventors have shown efficient mRNA LNP delivery drives transgene expression in muscle cells in vitro and in vivo. To assess the feasibility of the disclosed LNP platform for use in skeletal muscle applications, the inventors delivered ere mRNA LNPs (characteristics summarized in FIG. IB) to primary FAPs isolated from Ail4-tdTomato (TdT) reporter mice, a model in which Cre-mediated recombination activates TdT expression (FIG. 2A). After 24 hours in culture, both 1 and 5 pg / mL ere mRNA LNP achieved nearly 100% recombination efficiency, as indicated by strong TdT expression in FAPs (FIGs. 2B and 2C). These results confirm that LNP -mediated mRNA delivery is highly efficient for driving functional gene recombination in primary muscle-resident cells.

[0197] To further evaluate the disclosed LNP platform in vivo, the inventors administered a single intramuscular injection of ere mRNA LNPs (15 pg / 30 pL) into the TA muscle of Ail 4-TdTomato mice. Two weeks post-injection, the treated muscle exhibited a significant increase in TdT+ myofibers, with over 50% of myofibers expressing TdT, while the PBS vehicle-treated contralateral muscle showed no TdT expression (FIGs.2D and E). These findings demonstrate the feasibility of localized mRNA delivery in skeletal muscle and confirm that LNP-encapsulated mRNA can be effectively delivered, translated, and drive gene recombination in vivo.

[0198] To further confirm that LNP delivery results in functional protein expression, we treated C2C12 cells with 2 pg / mL W7a-LNP for 24 hours and observed a marked increase in WNT7a staining intensity compared to vehicle-treated controls (FIGs. 2F and 2G). While vehicle-treated cells exhibited low baseline signal, likely due to endogenous expression, the enhanced signal in the W7a-LNP group supports successful mRNA uptake and translation. Together with the observed functional effects in vitro, these results validate that W7a-LNP induces increased WNT7a production and activity in muscle cells.

[0199] 4. W7a-LNP suppression of FAP adipogenesis in vitro.

[0200] Building on the demonstration of efficient mRNA delivery and translation in muscle cells, the inventors investigated whether W7a-LNP could replicate the anti-adipogenic effects of recombinant WNT7a in vitro. To test this, the inventors isolated primary FAPs from the skeletal muscles of C57B1 / 6J mice, expanded them in growth media for 4 days, and then switched to adipogenic differentiation media with either vehicle (PBS), rWNT7A (200 ng / mL; positive control), or W7a-LNP at 1, 10, or 100 ng / mL (FIG. 3A). Prior work established that 200 ng / mL of recombinant WNT7a effectively suppresses FAP adipogenesis. Perilipin immunostaining on day 11 revealed that W7a-LNP at both 10 and 100 ng / mL significantly reduced adipogenesis, matching or exceeding the inhibition achieved by rWNT7A (FIGs. 3B and 3C).

[0201] To ensure that the effect is due to Wnt7a mRNA and not LNP formulation or nonspecific mRNA translation, the inventors repeated the experiment and compared W7a-LNPs’ ability to suppress FAP adipogenesis against Cre mRNA LNP. Here, the inventors used Cre mRNA LNP as a control because it encodes a nuclear recombinase not expected to influence adipogenesis and serves as a reference mRNA-loaded LNP with no known activity on this pathway. Cre mRNA-LNP-treated FAPs showed no reduction in perilipin+ adipocytes compared to vehicle, whereas W7a-LNP significantly reduced adipogenesis relative to both vehicle and Cre-LNP groups (FIGs. 3D and 3E). This supports that the observed anti-adipogenic effect is specific to WNT7a mRNA and not due to nonspecific effects of LNP delivery or mRNA translation. Collectively, these findings demonstrate that W7a-LNP effectively recapitulates rWNT7A’s anti-adipogenic function in vitro, supporting its potential as a therapeutic strategy to modulate FAP behavior.

[0202] 5. W7a-LNP increase fusion and myotube size in vitro.

[0203] Having established that W7a-LNP suppresses FAP adipogenesis, the inventors next examined its effects on myotube formation and growth. WNT7a promotes myofiber growth by activating the non-canonical AKT / mTOR pathway. To determine whether W7a-LNP replicates this effect, the inventors expanded C2C12 myoblasts for 3 days, and then induceddifferentiation in the presence of either vehicle (PBS), rWNT7A (50 ng / mL; positive control), or W7a-LNP at 1, 10, or 100 ng / mL for 3 additional days (FIG. 4A). As expected, rWNT7A significantly increased myotube size compared to the vehicle control (FIGs. 4B and 4C).Notably, W7a-LNP at both 10 and 100 ng / mL produced comparable increases in myotube size, demonstrating that W7a-LNP effectively enhances myotube growth in vitro. Treatment with Cre and luciferase mRNA LNPs had no effect on myotube size, supporting that the observed increase is specific to WNT7a mRNA (FIGs. 9A and 9B).

[0204] To further determine whether W7a-LNP impacts proliferation and fusion, the inventors quantified total nuclear counts and the fusion index (FIGs. 4D and 4E). W7a-LNP significantly increased fusion index at all doses compared to vehicle, while rWNT7A did not have a significant effect on fusion (FIG. 4D). Total nuclear counts were significantly elevated in the rWNT7A-, 10 ng / mL-, and 100 ng / mL-W7a-LNP conditions (FIG. 4E). These results suggest that W7a-LNP may promote both myoblast expansion and enhanced myoblast fusion, which together contribute to increased myotube size under these conditions.

[0205] 6. W7a-LNP promotes myofiber hypertrophy without inducing fibrosis in vivo.

[0206] Given that W7a-LNP promotes myofiber hypertrophy in vitro, the inventors next investigated whether it elicits similar effects in vivo without negatively impacting muscle quality. To test this, the inventors injected W7a-LNP (10 pg / 30 pL) into the supraspinatus muscle of mice, with the contralateral muscle receiving vehicle as a control. Mice received either a single inj ection, with analysis at 4 weeks, or three inj ections over 4 weeks, with analysis at 8 weeks (FIG. 5A). A single injection had no detectable effect on myofiber size at 4 weeks. However, repeated W7a-LNP administration significantly increased myofiber size at 4 weeks post-final injection, as quantified by minimum Feret’s diameter, suggesting that sustained W7a-LNP delivery is required for inducing hypertrophy (FIG. 5B and 5C). Picrosirius red and hematoxylin / eosin (H&E) staining revealed no signs of fibrosis, abnormal muscle morphology, and inflammation, indicating that repeated W7a-LNP administration does not promote fibrotic remodeling or tissue damage (FIG. 5D, FIG. 7). These findings confirm that repeated W7a-LNP delivery promotes muscle hypertrophy without compromising tissue integrity, supporting its therapeutic potential in muscle degeneration.

[0207] 7. W7a-LNP efficacy in glycerol-injured muscle depends on delivery timing and myofiber viability.

[0208] To assess the efficacy of W7a-LNP in an acute injury setting, the inventors used a glycerol injury model, which induces myofiber necrosis through osmotic stress and primes endogenous FAPs for adipogenic differentiation, leading to transient fatty infiltration. One-day post-injury, the inventors administered 2.5 pg / 30 pL rWNT7A, 10 pg / 30 pL W7a-LNP, or 30 pL PBS vehicle to the injured TA muscles. (FIG. 6A - 6D).

[0209] To test whether residual glycerol impacts LNP stability, we performed DLS analysis after incubating W7a-LNPs in PBS, 25% glycerol, or 50% glycerol for 10 or 30 minutes. The inventors observed an increase in z-average particle diameter with increasing glycerol concentration (FIGs. 10A and 10B), likely due to altered hydration or solvent viscosity effects. However, the PDI remained low across all conditions, indicating that LNPs remained monodisperse and did not aggregate. These data suggest that while glycerol may influence apparent particle size, it does not drastically compromise LNP integrity.

[0210] An alternative explanation is that glycerol injury acutely ablates myofibers, eliminating the “biofactory” required for WNT7a production following LNP delivery. To test this, the inventors performed an in vivo experiment in which W7a-LNPs were administered 4 days postinjury (FIG. 6E), a time point when regenerating myofibers begin to reappear. In this setting, W7a-LNP significantly reduced intramuscular fatty infiltration (FIGs. 6F and 6G) and increased muscle wet weight (FIG. 6H), supporting the notion that effective delivery and function require the presence of viable, transfectable myofibers.

[0211] Other embodiments of the disclosure include:1. A lipid nanoparticle comprising a WNT7A mRNA.2. The lipid nanoparticle of embodiment 1, wherein the WNT7A mRNA is encapsulated by the lipid nanoparticle.3. The lipid nanoparticle of embodiment 1 or 2, wherein the WNT7A mRNA is human WNT7A mRNA.4. The lipid nanoparticle of any one of embodiments 1 to 3, wherein the encapsulation efficiency is greater than 90%, the average diameter is from about 76 nm to about 96 nm, or the poly dispersity index is from about 0.0263 to about 0.0463.5. A pharmaceutical composition comprising the lipid nanoparticle of any one of embodiments 1 to 4, and a pharmaceutically acceptable carrier.6. A method of treating skeletal muscle fibro-adipogenic progenitor (FAP) adipogenesis, the method comprising administering the lipid nanoparticle of any oneof embodiments 1 to 4 or the pharmaceutical composition of embodiment 5 to a subject in need thereof.7. The method of embodiment 6, wherein administering is intramuscularly.

[0212] All features disclosed in the specification, including the claims, abstracts, and drawings, and all the steps in any method or process disclosed, may be combined in any combination, except combinations where at least some of such features and / or steps are mutually exclusive. Each feature disclosed in the specification, including the claims, abstract, and drawings, can be replaced by alternative features serving the same, equivalent or similar purpose, unless expressly stated otherwise. Thus, unless expressly stated otherwise, each feature disclosed is one example only of a generic series of equivalent or similar features.

[0213] It will be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

CLAIMS1. A lipid nanoparticle composition for administration of a WNT7a polynucleotide, the lipid nanoparticle composition comprising:a polynucleotide encoding WNT7a and,a lipid composition, wherein the lipid composition comprises an ionizable lipid, a PEG-lipid conjugate, cholesterol, and phospholipid; andwherein the polynucleotide encoding WNT7a is encapsulated by the lipid composition.

2. The lipid nanoparticle composition of claim 1, wherein the polynucleotide encoding WNT7a comprises a nucleic acid sequence with at least about 80% sequence identity to SEQ ID NO: 1 or SEQ ID NO: 5.

3. The lipid nanoparticle composition of claim 2, wherein the polynucleotide encoding WNT7a comprises a nucleic acid sequence with at least about 85%, alternatively at least about 90%, alternatively at least about 95%, alternatively at least about 96%, alternatively at least about 97%, alternatively at least about 98%, alternatively at least about 99%, or 100% nucleic acid sequence identity to SEQ ID NO:1.

4. The lipid nanoparticle composition of claim 2, wherein the polynucleotide encoding WNT7a comprises a nucleic acid sequence with at least about 85%, alternatively at least about 90%, alternatively at least about 95%, alternatively at least about 96%, alternatively at least about 97%, alternatively at least about 98%, alternatively at least about 99%, or 100% nucleic acid sequence identity to SEQ ID NO: 5.

5. The lipid nanoparticle composition any one of the preceding claims, wherein the WNT7a comprises an amino acid sequence with at least about 85%, alternatively at least about 90%, alternatively at least about 95%, alternatively at least about 96%, alternatively at least about 97%, alternatively at least about 98%, alternatively at least about 99%, or 100% nucleic acid sequence identity to SEQ ID NO:6.

6. The lipid nanoparticle composition any one of the preceding claims, wherein the ionizable lipid comprises heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6- (undecyloxy)hexyl)amino)octanoate (SM-102), [(4- hydroxybutyl)azanediyl]bis(hexane-6,l-diyl)bis(2 -hexyldecanoate) (ALC-0315), [(6Z,9Z)-hexadeca-6,9-dien-l-yl]-[2-(dimethylamino)ethyl]carbamate (DLin-MC3- DMA), N,N-dimethyl-2-(2-dodecyloxyethyl)ethanamine (Cl 2-200), or combinations thereof.

7. The lipid nanoparticle composition of claim 6, wherein the ionizable lipid is present in the lipid composition at a molar percentage of between about 40% to about 60%.

8. The lipid nanoparticle composition of claim 7, wherein the ionizable lipid is present in the lipid composition at molar percentage of about 42%, alternatively about 44%, alternatively about 46%, alternatively about 48%, alternatively about 50%, alternatively about 52%, alternatively about 54%, alternatively about 56%, or alternatively about 58%.

9. The lipid nanoparticle composition any one of the preceding claims, wherein the PEG-lipid conjugate comprises PEG-modified cholesterol, N-octanoyl-sphingosine-1- { succinyl [methoxy (poly ethylene glycol)] } , N-palmitoyl-sphingosine- 1 - {succinyl [methoxy (poly ethylene glycol)]}, PEG-modified DMPE (DMPE-PEG), PEG- modified DSPE (DSPE-PEG), PEG-modified DPPE (DPPE-PEG), PEG-modified DOPE (DOPE-PEG), dimyristoylglycerol-polyethylene glycol (DMG-PEG), di stearoylglycerol -poly ethylene glycol (DSG-PEG), dipalmitoylglycerol-polyethylene glycol (DPG-PEG), dioleoylglycerol-polyethylene glycol (DOG-PEG), or a combination thereof.

10. The lipid nanoparticle composition any one of the preceding claims, wherein the PEG- lipid conjugate comprises l,2-dimyristoyl-rac-glycero-3 -methoxypoly ethylene gly col- 2000 (DMG-PEG2000), Distearoyl glycerol-PEG2000 (DSG-PEG2000), 1,2- distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)- 2000] (DSPE-PEG2000), Ceramide-PEG2000, or combinations thereof.

11. The lipid nanoparticle composition of claim 9 or claim 10, wherein the PEG-lipid conjugate is present in the lipid composition at a molar percentage of between about 0.75% to about 2.5%.

12. The lipid nanoparticle composition of claim 11, wherein the PEG-lipid conjugate is present in the lipid composition at a molar percentage of about 1.0%, alternatively about 1.25%, alternatively about 1.5%, alternatively about 1.75%, alternatively about 2.0%, or alternatively about 2.25%.

13. The lipid nanoparticle composition any one of the preceding claims, wherein the phospholipid comprises l,2-Distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2- Dioleoyl-sn-glycero-3 -phosphoethanolamine (DOPE), Dipalmitoylphosphatidylcholine (DPPC), l,2-dilinoleoyl-sn-glycero-3- phosphocholine (DLPC), 1 ,2-dimyristoyl-sn-gly cero-phosphocholine (DMPC), l,2-dioleoyl-sn-glycero-3 -phosphocholine (DOPC), ,2- diundecanoyl-sn- gly cero-phosphocholine (DUPC), l-palmitoyl-2-oleoyl-sn-glycero-3- phosphocholine (POPC), l,2-di-O-octadecenyl-sn-glycero-3 -phosphocholine (18:0 Diether PC), l-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), l-hexadecyl-sn-glycero-3 -phosphocholine (C 16 Ly so PC), 1,2- dilinolenoyl-sn-glycero-3 -phosphocholine, l,2-diarachidonoyl-sn-glycero-3- phosphocholine, 1 ,2-didocosahexaenoyl-sn-glycero-3 - phosphocholine, l,2-dipalmitoyl-sn-glycero-3 -phosphoethanolamine (DPPE), 1,2- diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1,2-distearoyl-sn- glycero-3 -phosphoethanolamine, l,2-dilinoleoyl-sn-glycero-3 -phosphoethanolamine, 1.2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl- sn-glycero- 3 -phosphoethanolamine, l,2-didocosahexaenoyl-sn-glycero-3 -phosphoethanolamine, 1.2-dioleoyl-sn-glycero-3-phospho-rac-(l-glycerol) sodium salt (DOPG), sphingomyelin, and combinations thereof.

14. The lipid nanoparticle composition of claim 13, wherein the phospholipid is present in the lipid composition at a molar percentage of between about 5% to about 15%.

15. The lipid nanoparticle composition of claim 14, wherein the phospholipid is present in the lipid composition at a molar percentage of about 6%, alternatively about 7%,alternatively about 8%, alternatively about 9%, alternatively about 10%, alternatively about 11%, alternatively about 12%, alternatively about 13%, or alternatively about 14%.

16. The lipid nanoparticle of any one of the preceding claims, wherein the cholesterol is present in the lipid composition at a molar percentage of between about 30% to about 50%.

17. The lipid nanoparticle of claim 16, wherein the cholesterol is present in the lipid composition at a molar percentage of about 31.5%, alternatively about 33.0%, alternatively about 34.5%, alternatively about 36.0%, alternatively about 37.5%, alternatively about 38%, alternatively about 38.5%, alternatively about 39.0%, alternatively about 40.5%, alternatively about 42.0%, alternatively about 43.5%, alternatively about 45.0%, alternatively about 46.5%, alternatively about 48.0%, or alternatively about 49.5%.

18. The lipid nanoparticle composition of any one of the preceding claims, wherein the lipid nanoparticle composition has a mean diameter of between about 68 nm and about 85 nm.

19. The lipid nanoparticle composition of any one of the preceding claims, wherein the lipid nanoparticle composition has a PDI between about 0.0845 and about 0.11 nm.

20. The lipid nanoparticle composition of any one of the preceding claims, wherein lipid nanoparticle composition has an encapsulation efficiency of between about 85% to about 100%.

21. A therapeutic composition comprising the lipid nanoparticle composition of any one of the preceding claims, and at least one therapeutically acceptable carrier, diluent, or excipient.

22. A method of treating skeletal muscle fibro-adipogenic progenitor (FAP) adipogenesis in a subject in need thereof, the method comprising administering the lipid nanoparticlecomposition of any one of claims 1 to 20 or the therapeutic composition of claim 21 to the subject.

23. Use of a lipid nanoparticle composition according to any one of claims 1 to 20, or a therapeutic composition according to claim 21, for the treatment of skeletal muscle fibro-adipogenic progenitor (FAP) adipogenesis in a subject in need thereof.

24. The method of claim 22 or the use of claim 23, wherein the lipid nanoparticle composition or the therapeutic composition is administered intracutaneously, subcutaneously, intramuscularly, intratracheally, intravenously, or via infusion.

25. The method of claim 22 or the use of claim 23, wherein the lipid nanoparticle composition or the therapeutic composition is administered at a dose of about 10 ng / mL to about 100 ng / mL.

26. The method of claim 22 or the use of claim 23, wherein the lipid nanoparticle composition or the therapeutic composition is administered at least once monthly, alternatively at least every two weeks, or alternatively at least once a week.

27. The method of claim 22 or the use of claim 23, wherein the lipid nanoparticle composition or the therapeutic composition is administered one day post-injury, alternatively two days post-injury, alternatively three days post-injury, alternatively four days post-injury, or alternatively five days post-injury.

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