Novel RNA-NANO based genetic modulation of fibrosis for heart failure

Abstract

Disclosed are compositions and methods for BMP7 to inhibit endothelial-to-mesenchymal transition. This therapy uses a nanoparticle to deliver BMP7 mRNA to the heart. The BMP7 mRNA can provide a template trigger message to the heart cells to produce higher amounts of BMP7 protein. Increased BMP7 levels can ameliorate heart failure and its progression, thus ultimately improving the quality of life of the patient.

Description

[0001] NOVEL RNA-NANO BASED GENETIC MODULATION OF FIBROSIS FOR HEART

[0002] FAILURE

[0003] SEQUENCE LISTING

[0004] A Sequence Listing conforming to the rules of WIPO Standard ST.26 is hereby incorporated by reference. Said Sequence Listing has been filed as an electronic document via PatentCenter encoded as XML in UTF-8 text. The electronic document, created on December 11, 2024, is entitled “10063-092W01_ST26.xml”, and is 9,953 bytes in size.

[0005] CROSS-REFERENCE TO RELATED APPLICATIONS

[0006] This application claims benefit of U.S. Provisional Application No. 63 / 613,903, filed December 22, 2023, incorporated herein by reference in its entirety.

[0007] BACKGROUND

[0008] Heart failure bears a large burden on the healthcare system with significant morbidity and mortality. While present medical therapies focus on a neurohormonal blockade strategy (Blocking the Renin- Angiotensin-Aldosterone System (RAAS), Sympathetic system, SGLT-2 system) there remains a significant burden of residual risk . Also, the present treatment strategy for heart failure relies on a costly polypharmacy plan limited by patient non-compliance in many situations. Mechanistically, these medications have not been established to have a direct action that impacts fibrosis, an important perpetuator of cardiac inefficiency in the pathophysiology of heart failure.

[0009] At present there are no therapies that directly reduce the amount of fibrosis in organs experiencing endothelial to mesenchymal transition (EndoMT). More specifically, an RNA-based therapy does not exist for heart failure. There are currently no cardiac drugs on the market that have a direct means of reducing fibrosis, such as cardiac fibrosis, and improving organ functionality, such as in the heart.

[0010] What is needed in the art is an mRNA therapeutic with the capability of reducing EndoMT, decreasing fibrosis, and improving function in organs such as the heart.

[0011] SUMMARY

[0012] In accordance with the purposes of the disclosed materials, compounds, compositions, and methods, as embodied and broadly disclosed herein, the disclosed subject matter, in one aspect, relates to compositions and methods of preparing and using them. Specifically, provided herein is a method of reducing endothelial to mesenchymal cell transition (EndoMT) of endothelial cells associated with an organ, the method comprises exposing the endothelial-derived cells to Bone Morphogenic Protein 7 (BMP7) mRNA, wherein BMP7 reduces the transition of endothelial to mesenchymal cells by 5% or more, wherein reduction in the transition of endothelial to mesenchymal cells results in the reduction of fibrosis within the organ.

[0013] Also provided is a method of treating cardiac dysfunction in a subject in need thereof, comprising administering a composition comprising an effective amount of a nanoparticle to the subject, wherein said nanoparticle encapsulates or is associated with BMP7 mRNA, wherein the BMP7 mRNA is administered through a temporary mechanical assist device, wherein the cardiac dysfunction comprises contractile properties and relaxation properties, wherein said contractile properties are ejection fraction and / or fractional shortening, wherein said relaxation properties are diastolic function assessment.

[0014] Further provided is a composition comprising a nanoparticle encapsulating BMP7 mRNA, wherein the BMP7 mRNA is a functional fragment of GenBank Accession No. AK312419.1 or BC008584.1 with SEQ ID NO: 1 or SEQ ID NO: 3, wherein the nanoparticle is a lipid nanoparticle, wherein the nanoparticle is cationic and ionizable, wherein the nanoparticle comprises a PEGylated lipid and cholesterol.

[0015] Additionally, provided is a pharmaceutical composition of a kit comprising a nanoparticle encapsulating BMP7 mRNA, wherein the nanoparticle is designed to target specific cell markers or tissue types indicating the potential for EndoMT of that cell, wherein the nanoparticle is designed to target endothelial cells, wherein the endothelial cells are within the heart, wherein the nanoparticle further comprises one or more additional therapeutics, medicaments, compounds, or one or more adjuvants.

[0016] DESCRIPTION OF DRAWINGS

[0017] FIG. 1A-B shows endoMT induction in HUVECs. (A) Representative images exhibiting the presence of CD31 and Vimentin positive cells indicating the EndoMT process. CD31+ and Vimentin + cells in the MERGE column indicate cells undergoing EndoMT (Vimentin is a mesenchymal marker which is not expressed by endothelial cells and the presence of such a marker along with the endothelial cell marker CD31 suggests that the cells are transitioning into a mesenchymal state i.e Fibroblasts in this situation); Scale bar: 150 pm. (B) Experimental design to show BMP-7 ability to inhibit EndoMT invitro.

[0018] FIG. 2A-C shows endoMT inhibition by rhBMP7. Representative scheme for EndoMT and its inhibition by BMP7 protein. Representative images of immunofluorescent stained cells for CD31, Vimentin, and DAPI in HUVEC with no treatment, with TGF-0, with TGF-f> and BMP7 protein, with L-NAME + Ang II, and with L-NAME + Ang II and BMP7 protein (A) are shown. Fluorescent intensity of CD31 (B) and Vimentin-stained cells (C) exhibit the change in the expression levels with and without BMP7 protein.

[0019] FIG. 3 shows experimental design to demonstrate the potential of BMP7 treatment.

[0020] FIG. 4 shows cardiac function analysis using echocardiography. Echocardiography analysis of the mice exhibiting maintenance / improvement in cardiac function in terms of ejection fraction, fractional shortening and left ventricle volume at diastole.

[0021] FIG. 5A-C shows cardiac morphological changes during heart failure and treatment. (A) Representative image of mouse heart section stained with Masson’s trichrome illustrates reduced fibrosis in BMP7 treated mice. Scale bar denotes 20 pm. (B) Bar graphs represent fibrosis area. (C) Bar graphs represent Cardiomyocyte size.

[0022] FIG. 6 shows a schematic representation of BMP7 loaded lipid nanoparticle.

[0023] FIG. 7 shows an experiment scheme for BMP7 mRNA testing in vitro.

[0024] FIG. 8A-B shows expression of BMP7 protein in cells. (A) Untreated HUVEC cells (top row), EndoMT cells (middle row) and BMP7 mRNA treated cells. Representative images of immunofluorescent stained cells for BMP7 protein, and DAPI in HUVEC with no treatment, with L-NAME + Ang II, and with L-NAME + Ang II and BMP7 mRNA. (B) Dot plot to show fluorescent intensity of BMP7 stained cells change in the expression levels with and without BMP7 mRNA.

[0025] FIG. 9A-B shows expression of Vimentin (Mesenchymal cell marker) in cells. (A) Untreated HUVEC cells (top row), EndoMT cells (middle row) and BMP7 mRNA treated cells. Representative images of immunofluorescent stained cells for CD31, Vimentin, and DAPI in HUVEC with no treatment, with L-NAME + Ang II, and with L-NAME + Ang II and BMP7 mRNA. (B) Dot plot to show fluorescent intensity of Vimentin -stained cells change in the expression levels with and without BMP7 mRNA.

[0026] FIG. 10A-B shows expression of Transgelin (Mesenchymal cell marker) in cells. (A) Untreated HUVEC cells (top row), EndoMT cells (middle row) and BMP7 mRNA treated cells. Representative images of immunofluorescent stained cells for VE Cadherin, Transgelin (TGLN), and DAPI in HUVEC with no treatment, with L-NAME + Ang II, and with L-NAME + Ang II and BMP7 mRNA. (B) Dot plot to show fluorescent intensity of TGLN -stained cells change in the expression levels with and without BMP7 mRNA.

[0027] FIG. 11A-B shows expression of Collagen I (Mesenchymal cell marker) in cells. (A) Untreated HUVEC cells (top row), EndoMT cells (middle row) and BMP7 mRNA treated cells. Representative images of immunofluorescent stained cells for VE Cadherin, Collagen 1 (Coll), and DAPI in HUVEC with no treatment, with L-NAME + Ang II, and with L-NAME + Ang II and BMP7 mRNA. (B) Dot plot to show fluorescent intensity of Coll -stained cells change in the expression levels with and without BMP7 mRNA.

[0028] FIG. 12 shows an experimental scheme to study the functionality of EndoMT cells treated with BMP7 mRNA in vitro.

[0029] FIG. 13 shows morphology of untreated HUVEC cells (top row), EndoMT cells (middle row) and BMP7 mRNA treated cells.

[0030] FIG. 14A-B shows endothelial function. (A) NO Production in HUVEC cells, EndoMT cells and BMP7 mRNA treated cells. (B) Acetylated LDL Uptake (right panel) in HUVEC cells, EndoMT cells and BMP7 mRNA treated cells.

[0031] FIG. 15A-C shows Endothelial function. (A) Matrigel® tube formation assay with HUVEC cells, EndoMT cells and BMP7 mRNA treated cells. (B) Quantification of network segments and (C) segment length.

[0032] FIG. 16A-B shows size and zeta potential characterization of lipid based nanoformulation. (A) Representative plot of BMP7 mRNA nanoconstruct particle size and (B) zeta potential.

[0033] FIG. 17 shows in vivo experimental design to evaluate the efficacy of BMP7 mRNA loaded lipid nanoparticles.

[0034] FIG. 18 shows BMP7 mRNA protects cardiac function when compared to untreated heart failure mice. HF: Heart Failure, HF+rBMP7: HF treated with recombinant BMP7 protein delivery using osmotic pump, HF+mBMP7 (4): HF treated with 4 BMP7 mRNA injections, and HF+mBMP7 (3) treated with 3 BMP7 mRNA injections.

[0035] FIG. 19A-B shows BMP7 mRNA displayed anti-fibrotic effect compared to untreated mice. Representative Mason’s trichrome staining of the different groups and (B) Fibrosis area.

[0036] FIG. 20 shows gene expression data of selected genes that play a role in Endothelial mesenchymal transitioning process from mouse hearts at various stages of the heart failure and recovery protocol. Week 3 and Week 5 are during HF induction phase and Weeks 7 and 9 are during the recovery phase.

[0037] FIG. 21 shows histological sections of kidney (top row) and liver (bottom row) showing minimal changes with no differences noted between control, heart failure and heart failure treated with BMP7 nanogene therapy.

[0038] FIG. 22 shows a list of top differentially expressed genes and their relative expression changes between group comparisons. The scale on the right side of the table reflects the relative up and down regulation of each gene relative to the comparator. BMP-7 seems to be highly expressed in HFpVAD samples compared to HFIABP samples. HFIABP: Heart Failure- Intra- Aortic Balloon Pump; HFp-VAD: Heart failure with Percutaneous Ventricular Assist Device; HFIABP: Heart Failure with Intra-Aortic Balloon Pump. Gene expression comparison shows that BMP7 is one of the top upregulated molecules as evaluated by bulk RNA sequencing.

[0039] DETAILED DESCRIPTION

[0040] General Definitions

[0041] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs.

[0042] Ranges can be expressed herein as from “about” one particular value, and / or to “about” another particular value. By “about” is meant within 10% of the value, e.g., within 9, 8, 7, 6, 5, 4, 3, 2, or 1% of the value. When such a range is expressed, another aspect includes from the one particular value and / or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it can be understood that the particular value forms another aspect. It is further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed.

[0043] The term “comprising” and variations thereof as used herein is used synonymously with the term “including” and variations thereof and are open, non-limiting terms. Although the terms “comprising” and “including” have been used herein to describe various embodiments, the terms “consisting essentially of’ and “consisting of’ can be used in place of “comprising” and “including” to provide for more specific embodiments and are also disclosed. Throughout the description and claims of this specification the word “comprise” and other forms of the word, such as “comprising” and “comprises,” means including but not limited to, and is not intended to exclude, for example, other additives, components, integers, or steps.

[0044] As used in the specification and claims, the singular form “a”, “an”, and “the” include plural references unless the context clearly dictates otherwise. For example, the term “an agent” includes a plurality of agents, including mixtures thereof.

[0045] As used herein, the terms "may," "optionally," and "may optionally" are used interchangeably and are meant to include cases in which the condition occurs as well as cases in which the condition does not occur. Thus, for example, the statement that a formulation "may include an excipient" is meant to include cases in which the formulation includes an excipient as well as cases in which the formulation does not include an excipient.

[0046] "Inhibit," "inhibiting," and "inhibition" mean to decrease an activity, response, condition, disease, or other biological parameter. This can include but is not limited to the complete ablation of the activity, response, condition, or disease. This may also include, for example, a 10% reduction in the activity, response, condition, or disease as compared to the native or control level. Thus, the reduction can be a 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or any amount of reduction in between as compared to native or control levels.

[0047] By “reduce,” or “abrogate,” (used interchangeably) or other forms of the word, such as “reducing” or “reduction,” or “abrogating” or “abrogation” means lowering of an event or characteristic. It is understood that this is typically in relation to some standard or expected value, in other words it is relative, but that it is not always necessary for the standard or relative value to be referred to.

[0048] By “increase” or other forms of the word, such as “increasing,” means raising or elevating. It is understood that this is typically in relation to some standard or expected value, in other words it is relative, but that it is not always necessary for the standard or relative value to be referred to.

[0049] As used herein, by a “subject” means an individual. Thus, the “subject” can include domesticated animals (e.g. cats, dogs, etc.), livestock (e.g., cattle, horses, pigs, chickens, ducks, geese, sheep, goats, etc.), laboratory animals (e.g., mouse, rabbit, rat, guinea pig, etc.), and birds. “Subject” can also include a mammal, such as a primate or a human. Thus, the subject can be a human or veterinary patient. The term “patient” refers to a subject under the treatment of a clinician, e.g., physician.

[0050] “Detecting” is used herein to identify the existence, presence, or fact of something. General methods of detecting are known to the skilled artisan and may be supplemented with the protocols and reagents disclosed herein. Detection can include a physical readout, such as fluorescence output.

[0051] The term “therapeutically effective amount” refers to an amount that is sufficient to affect a therapeutically significant reduction in one or more symptoms of the condition when administered to a typical subject who has the condition. For example, the term “therapeutically effective” is used herein in a broad sense and includes prophylactic effects. A therapeutically significant reduction in a symptom or complication resulting from pathological EndoMT or fibrosis is, e.g. about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, or more (e.g., 1.5 fold, 2 fold, 3 fold, 4 fold, 5 fold, 10 fold, 25 fold, 50 fold, 100 fold, etc.) as compared to a control or non-treated subject. The term “therapeutically effective amount” refers to the amount of an agent determined to produce any therapeutic response in a subject.

[0052] The precise determination of what would be considered a therapeutically effective amount may be based on factors individual to each subject, including their size, age, injury, and / or disease or injury being treated, and amount of time since the injury occurred or the disease began. One skilled in the art will be able to determine the therapeutically effective amount for a given subject based on these considerations which are routine in the art.

[0053] The term “treats” or “treatment” refers to therapeutic treatment wherein the object is to eliminate or lessen symptoms. Beneficial or desired clinical results include, but are not limited to, elimination of symptoms, alleviation of symptoms, diminishment of extent of condition, stabilized (i.e., not worsening) state of condition, delay or slowing of progression of the condition.

[0054] As used herein, the term “amelioration” means the prevention, reduction or palliation of a state, or improvement of the state of a subject. Amelioration includes but does not require complete recovery or complete prevention of a disease condition. In some embodiments, amelioration includes increasing levels of relevant protein or its activity that is deficient in relevant disease tissues.

[0055] As used herein, the phrase “biologically active” refers to a characteristic of any agent that has activity in a biological system, and particularly in an organism. For instance, an agent that, when administered to an organism, has a biological effect on that organism, is considered to be biologically active. In particular embodiments, where a protein or polypeptide is biologically active, a portion of that protein or polypeptide that shares at least one biological activity of the protein or polypeptide is typically referred to as a “biologically active” portion. As used herein, the term “delivery” encompasses both local and systemic delivery. For example, delivery of mRNA encompasses situations in which an mRNA is delivered to a target tissue and the encoded protein is expressed and retained within the target tissue (also referred to as “local distribution” or “local delivery”), and situations in which an mRNA is delivered to a target tissue and the encoded protein is expressed and secreted into patient's circulation system (e.g., serum) and systematically distributed and taken up by other tissues (also referred to as “systemic distribution” or “systemic delivery).

[0056] 1. As used herein, a “functional” biological molecule is a biological molecule in a form in which it exhibits a property and / or activity by which it is characterized. As used herein, the term “half-life” is the time required for a quantity such as nucleic acid or protein concentration or activity to fall to half of its value as measured at the beginning of a time period.

[0057] The terms “improve,” “increase” or “reduce,” or grammatical equivalents, indicate values that are relative to a baseline measurement, such as a measurement in the same individual prior to initiation of the treatment described herein, or a measurement in a control subject (or multiple control subject) in the absence of the treatment described herein. A “control subject” is a subject afflicted with the same form of disease as the subject being treated, who is about the same age as the subject being treated.

[0058] 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, etc., rather than within a multi-cellular organism.

[0059] The term “in vivo” refers to events that occur within a multi-cellular organism, such as a human and a non-human animal. In the context of cell-based systems, the term may be used to refer to events that occur within a living cell (as opposed to, for example, in vitro systems).

[0060] An “isolated” biological component (such as a nucleic acid molecule) has been substantially separated, produced apart from, or purified away from other biological components. Nucleic acid molecules which have been “isolated” include nucleic acids molecules purified by standard purification methods, as well as those chemically synthesized. Isolated does not require absolute purity and can include nucleic acid molecules that are at least 50% isolated, such as at least 75%, 80%, 90%, 95%, 98%, 99% or even 100% isolated.

[0061] A “nucleic acid” is a deoxyribonucleotide or ribonucleotide polymer, which can include analogues of natural nucleotides that hybridize to nucleic acid molecules in a manner similar to naturally occurring nucleotides. In a particular example, a nucleic acid molecule is a single stranded (ss) DNA or RNA molecule, such as a probe or primer. In another particular example, a nucleic acid molecule is a double stranded (ds) nucleic acid, such as a target nucleic acid. Examples of modified nucleic acids are those with altered backbones, such as peptide nucleic acids (PNA).

[0062] As used herein, the term “messenger RNA (mRNA)” refers to a polynucleotide that encodes at least one polypeptide. mRNA as used herein encompasses both modified and unmodified RNA. mRNA may contain one or more coding and non-coding regions. mRNA can be purified from natural sources, produced using recombinant expression systems and optionally purified, chemically synthesized, etc. Where appropriate, e.g., in the case of chemically synthesized molecules, mRNA can comprise nucleoside analogs such as analogs having chemically modified bases or sugars, backbone modifications, etc. An mRNA sequence is presented in the 5' to 3' direction unless otherwise indicated. In some embodiments, an mRNA is or comprises natural nucleosides (e.g., adenosine, guanosine, cytidine, uridine); nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl adenosine, 5 -methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8- oxoguanosine, O(6)-methylguanine, and 2-thiocytidine); chemically modified bases; biologically modified bases (e.g., methylated bases); intercalated bases; modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose); and / or modified phosphate groups (e.g., phosphorothioates and 5'-N-phosphoramidite linkages).

[0063] The identity / similarity between two or more nucleic acid sequences is expressed in terms of the identity between the sequences. Sequence identity can be measured in terms of percentage identity; the higher the percentage, the more identical the sequences are.

[0064] Methods of alignment of sequences for comparison are well known in art. Various programs and alignment algorithms are described in: Smith & Waterman, Adv. Appl. Math. 2:482, 1981; Needleman & Wunsch, J. Mol. Biol. 48:443, 1970; Pearson & Lipman, Proc. Natl. Acad. Sci. USA 85:2444, 1988; Higgins & Sharp, Gene, 73:237-44, 1988; Higgins & Sharp, CABIOS 5:151-3, 1989; Corpet et al., Nuc. Acids Res. 16: 10881-90, 1988; Huang et al. Computer Appls. in Biosciences 8, 155-65, 1992; and Pearson et al., Meth. Mol. Bio. 24:307-31, 1994. Altschul et al., J. Mol. Biol. 215:403-10, 1990, presents a detailed consideration of sequence alignment methods and homology calculations.

[0065] The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. Mol. Biol. 215:403-10, 1990) is available from several sources, including the National Center for Biotechnology (NCBI, National Library of Medicine, building 38A, Room 8N805, Bethesda, Md. 20894) and on the Internet, for use in connection with the sequence analysis programs blastp, blastn, blastx, tblastn, and tblastx. Additional information can be found at the NCBI web site. BLASTN is used to compare nucleic acid sequences. If the two compared sequences share homology, then the designated output file will present those regions of homology as aligned sequences. If the two compared sequences do not share homology, then the designated output file will not present aligned sequences.

[0066] Once aligned, the number of matches is determined by counting the number of positions where an identical nucleotide or amino acid residue is presented in both sequences. The percent sequence identity is determined by dividing the number of matches either by the length of the sequence set forth in the identified sequence, or by an articulated length (such as 100 consecutive nucleotides or amino acid residues from a sequence set forth in an identified sequence), followed by multiplying the resulting value by 100. For example, a nucleic acid sequence that has 1166 matches when aligned with a test sequence having 1554 nucleotides is 75.0 percent identical to the test sequence (1166- 1554* 100=75.0). The percent sequence identity value is rounded to the nearest tenth. For example, 75.11, 75.12, 75.13, and 75.14 are rounded down to 75.1, while 75.15, 75.16, 75.17, 75.18, and 75.19 are rounded up to 75.2. The length value will always be an integer. In another example, a target sequence containing a 20-nucleotide region that aligns with 20 consecutive nucleotides from an identified sequence as follows contains a region that shares 75 percent sequence identity to that identified sequence (that is, 15-^20*100=75). One indication that two nucleic acid molecules are closely related is that the two molecules hybridize to each other under stringent conditions, as described above.

[0067] As used herein, “Congestive heart failure (CHF)” is caused by coronary artery disease, myocardial infarction (heart attack)or non-ischemic heart failure. CHF occurs when the blood vessels that supply the heart muscle with oxygen and nutrients (coronary arteries) become narrowed or blocked, reducing blood flow to the heart muscle. Ischemic heart failure is often associated with reduced blood flow and oxygen delivery to the heart muscle, which can result in a weakened, "stiff," or scarred heart muscle. Non-ischemic heart failure includes genetic causes, viral causes, inflammation, hypertension, diabetes and a combination of various of these factors.

[0068] In one embodiment, “Myocardial infarction” (heart attack) occurs when a coronary artery is completely blocked, resulting in damage or death of part of the heart muscle due to lack of oxygen and nutrients. The damage to the heart muscle caused by CHF or a heart attack can lead to impaired heart function and eventually heart failure.

[0069] General Description

[0070] The accumulation of a large number of myofibroblasts is responsible for exaggerated and uncontrolled production of extracellular matrix during the development and progression of pathological fibrosis (Piera- Velazquez 2011). Endothelial to mesenchymal transition (EndoMT) is a complex biological process in which endothelial cells lose their specific markers and acquire a mesenchymal or myofibroblastic phenotype. EndoMT cell transition can incite fibrosis in various organs such as the heart and act as a contributor to many fibrotic diseases where endothelial cells (under certain stressors) are able to transition into fibroblasts. Fibroblasts are the perpetuators of collagen deposition and hence cause fibrotic scarring. Several targets have been identified that can regulate such mechanisms in the context of heart failure and recovery. One such molecular target is Bone Morphogenic Protein-7 (BMP7) (also known as osteogenic protein- 1 or OP-1).

[0071] Based on these findings, disclosed herein is a method of reducing EndoMT of endothelial cells associated with an organ, wherein the method comprises exposing the endothelial-derived cells to BMP7 mRNA. By “reducing” EndoMT is meant that the amount of endothelial cells which undergo EndoMT is reduced by 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%,

[0072] 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%,

[0073] 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%,

[0074] 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%,

[0075] 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,

[0076] 99%, or 100%, or 2, 3, 4, 5, 6, 7, 8, 9, or 10 fold, or any amount below, above, or in-between these values. In one particular embodiment, by “reduced” means that there are at least 5% less endothelial cells undergoing EndoMT after treatment with BMP7 as compared to a control in which the cells are not exposed to BMP7.

[0077] Furthermore, it is noted that transition of endothelial to mesenchymal cells happens on a gradient as a function of time, and by “reducing” EndoMT is meant that this progression is reduced or halted when treated as compared to untreated cells. In other words, an endothelial cell may have begun undergoing the process of transition to a mesenchymal cell, but this progression was either slowed (reduced) or halted before the full transition occurred upon treatment. This transition can be measured in a variety of ways, as discussed below. Therefore, cells (in vitro or in vivo} undergoing treatment can be compared to control cells (or cells of the subject before treatment) to determine the effectiveness of treatment.

[0078] During EndoMT, the tight cell-cell junctions of endothelial cells (ECs) are disrupted, causing ECs to lose their cobblestone-like and well-structured appearance, reorganize their cytoskeleton and turn into spindle-shaped, fibroblast-like cells. During this transitional process, the expression of cell-cell adhesion proteins, such as vascular endothelial (VE)-cadherin, platelet / EC adhesion molecule- 1 (CD31 / PECAM-1), tyrosine kinase with immunoglobulin- like and epidermal growth factor (EGF)-like domains 1 (TIE1), TIE2, and von Willebrand factor (vWF), are diminished, while mesenchyme-specific factors, including N-cadherin, a-smooth muscle actin (a-SMA), smooth muscle protein 22a (SM22a), vimentin, fibronectin, and fibroblastspecific protein-1 (FSP-1), are upregulated. The ECs transition towards mesenchymal cells, is further accompanied by functional changes of the ECs, where the ECs lose their characteristic functions viz. tube formation, nitric oxide production and Low-density lipoprotein (LDL) uptake and simultaneous acquisition of mesenchymal functionality viz. collagen production etc. Disclosed herein is measuring these markers to determine EndoMT is taking place, and at what rate. Detecting and measuring markers of EndoMT is described in detail in Ma et al. (Ma J, Sanchez-Duffhues G, Goumans M-J and ten Dijke P (2020) TGF-P-Induced Endothelial to Mesenchymal Transition in Disease and Tissue Engineering. Front. Cell Dev. Biol. 8:260), and is hereby incorporated by reference in its entirety for its teaching concerning detecting EndoMT transition. Other methods of determining EndoMT reduction, both in vivo and in vitro, can be found in Krishnamoorthi et al. (2022), herein incorporated by reference in its entirety for this teaching.

[0079] Another way to measure the effectiveness of BMP7 mRNA treatment is to measure a reduction in cardiac dysfunction. This dysfunction can include, but is not limited to, contractile properties such as ejection fraction or fractional shortening. It can also be measured by determining adverse remodeling, such as by determining left ventricle end-diastolic volume. These metrics can be measured, and treatment with BMP7 mRNA can result in a reduction of cardiac dysfunction in a subject of 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%,

[0080] 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%,

[0081] 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%,

[0082] 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%,

[0083] 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or 2, 3, 4, 5, 6, 7, 8, 9, or 10 fold, or any amount below, above, or in-between these values. In one particular embodiment, by “reduced” is meant that the subject has at least a 5% improvement in cardiac function after treatment with BMP7 as compared to a measurement of cardiac dysfunction in the subject prior to exposure to BMP7 mRNA or compared to a control.

[0084] As discussed above, during EndoMT, the endothelial cells convert to a more mesenchymal cell type that can give rise to cells such as fibroblasts. EndoMT is essential during embryonic development and tissue regeneration. It can also play a role in pathological conditions like fibrosis of organs such as the heart and kidney. For example, EndoMT is known to occur in disease settings, and appears to contribute to vascular pathologies, such as in ischemic mitral regurgitation (IMR) after myocardial infarction (MI). IMR, a common complication after MI, induces adaptive cellular responses in the mitral valve (MV) that may be initially beneficial, but eventually lead to leaflet fibrosis and MV dysfunction. Other examples, beyond cardiac valves, are EndoMT in cerebral cavernous malformations, arterial calcification, and intimal thickening. In addition, EndoMT contributes to the generation of cancer associated fibroblasts (CAF) that are known to influence the tumor-microenvironment favorable for the tumor cells. EndoMT is a form of the more widely known and studied Epithelial-to- Mesenchymal Transition (EMT). Like EMT, EndoMT can be induced by transforming growth factor (TGF)-p.

[0085] The predominant cellular mediators of fibrosis are assumed to be (myo)fibroblasts, not only in heart fibrosis but also in fibrosis of organs such as lung, kidney, and the liver. Fibrosis of all these organs share similar pathways. The origin of these (myo)fibroblasts can be, besides resident interstitial fibroblast, cells derived from the bone marrow as well as fibroblastic cells that have transdifferentiated from cells of epithelial origin. These cells can also be derived from endothelial cells that have undergone EndoMT. An example includes human umbilical vascular endothelial cells (HUVECs).

[0086] In cardiac fibrosis, the heart valves abnormally thicken due to inappropriate proliferation of cardiac fibroblasts and of disruption of normal myocardial structure through excessive deposition of extracellular matrix. Several studies have given evidence for the role of EndoMT in cardiac fibrosis. Not only endothelial cells lining the vessel wall have been shown to be able to undergo EndoMT. Circulating endothelial progenitor cells have also been shown to undergo EndoMT. In a specific example, the endothelial cells which are treated can be either inside or outside the blood vessels within the heart.

[0087] As described above, the result of EndoMT in pathological conditions is fibrosis because of the generation of fibroblasts and excessive extracellular matrix. However, EndoMT also leads to the loss of endothelium. The loss of endothelial function can lead to badly perfused tissue and subsequent tissue damage. For example following traumatic spinal cord injury, significant vascular disruption occurs at the site(s) of injury.

[0088] In summary, pathological EndoMT can occur as a result of a disease or injury. Non-limiting examples where pathological EndoMT can occur following a disease, infection or injury include cancer, fibrodysplasia ossificans progressive (FOP), systemic sclerosis, hypertension, cardiac fibrosis (including but not limited to diabetes mellitus-induced cardiac fibrosis), renal fibrosis, hepatic fibrosis, cirrhosis, keloid formation, fibrosis induced transplant antibody mediated rejection, lung infection, kidney infection, liver infection (hepatitis infection), alcohol abuse, or anti-freeze poisoning, etc.

[0089] It is shown herein that EndoMT can be mitigated by providing BMP7 to a subject in need thereof. One way to achieve that is to provide BMP7 mRNA, such as in the form of a loaded lipid nanoparticle. This is discussed in more detail below. As can be seen in Example 1, it is herein demonstrated that BMP7 can inhibit EndoMT and improve cardiac function in vivo in heart failure. Human BMP7 mRNA can yield similar results both in vitro and in vivo. The disclosed invention can effectively enhance recovery from heart failure in such a way that it can used as a convenient therapy for patients in need. Other examples of how BMP7 mRNA can be used to mitigate EndoMT are discussed above. The results seen with this therapy are a significant improvement over currently used therapies, and can further be provided as an intermittent therapy to subjects in need thereof.

[0090] Further disclosed is a method of treatment, which can involve, for example, an intramyocardial injection in a patient undergoing left ventricular assist device (LVAD) surgery, for example, to promote recovery. Recent studies have shown that in a carefully selected patient population, a combination of LVAD with high dose pharmacotherapy can improve the ability of the heart to recover and explant the LVAD. In one example, this injection can be given as a therapy to facilitate recovery of the heart. Other method treatment can involve an intracoronary injection.

[0091] In one specific example, a subject can be diagnosed with having, or being at risk of developing, cardiac dysfunction, and then the subject can be given BMP7 mRNA. Often, patients experiencing cardiac dysfunction undergo surgery. BMP7 mRNA can be given before, after, or during surgery. In some instances, the subject is given a temporary mechanical assist device (MCS device). In some instances, BMP7 mRNA can be administered through the MCS device. Specific examples of such devices can be found in Salter et al. (Salter, B.S., Gross, C.R., Weiner, M.M. et al. Temporary mechanical circulatory support devices: practical considerations for all stakeholders. Nat Rev Cardiol 20, 263-277 (2023)), which is herein incorporated by reference in its entirety for its teaching concerning MCS devices.

[0092] BMP7 mRNA

[0093] Disclosed herein is BMP7 mRNA, which can be used in a variety of ways, including, but not limited to, in a delivery vehicle such as a nanoparticle. The BMP7 mRNA disclosed herein can be obtained from a variety of sources. There are different publicly available mRNA sequences available from NCB1, including, but not limited to, AK094784, AK291186, AK312419, BC004248, and BC008584 (SEQ ID NO: 1). Disclosed herein are methods and compositions including, but not limited to, any of these BMP7 mRNA sources. One of skill in the art will appreciate that any BMP7 mRNA which results in expression of BMP7 or a functional equivalent thereof can be used with the methods and compositions disclosed herein. For example, any number of modifications, additions, or deletions can be made to any of the referenced sources of BMP7 mRNA and the function still be retained of treating the various diseases and disorders outlined herein. When “BMP7 mRNA” is referred to herein, it is understood that this can mean any mRNA which results in the formation of a BMP7 protein, or functional fragment thereof, which is sufficient to abrogate a disease or disorder associated with BMP7, such as cardiac dysfunction.

[0094] Therefore, disclosed herein is mRNA which is 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to mRNA obtained AK094784, AK291186, AK312419, BC004248, or BC008584 (SEQ ID NO: 1). By “functional fragment” is meant that the fragment is capable of being utilized in the methods described herein to treat disease or mitigate the symptoms thereof.

[0095] BMP7 mRNA according to the present invention may be synthesized according to any of a variety of known methods. For example, mRNAs according to the present invention may be synthesized via in vitro transcription (IVT). Briefly, IVT is typically performed with a linear or circular DNA template containing a promoter, a pool of ribonucleotide triphosphates, a buffer system that may include DTT and magnesium ions, and an appropriate RNA polymerase (e.g., T3, T7 or SP6 RNA polymerase), DNAse I, pyrophosphatase, and / or RNAse inhibitor. The exact conditions will vary according to the specific application.

[0096] In some embodiments, for the preparation of mRNA according to the invention, a DNA template is transcribed in vitro. A suitable DNA template typically has a promoter, for example a T3, T7 or SP6 promoter, for in vitro transcription, followed by desired nucleotide sequence for desired mRNA and a termination signal.

[0097] Desired mRNA sequence(s) according to the invention may be determined and incorporated into a DNA template using standard methods. For example, starting from a desired amino acid sequence (e.g., an enzyme sequence), a virtual reverse translation is carried out based on the degenerated genetic code. Optimization algorithms may then be used for selection of suitable codons. Typically, the G / C content can be optimized to achieve the highest possible G / C content on one hand, taking into the best possible account the frequency of the tRNAs according to codon usage on the other hand. The optimized RNA sequence can be established and displayed, for example, with the aid of an appropriate display device and compared with the original (wildtype) sequence. A secondary structure can also be analyzed to calculate stabilizing and destabilizing properties or, respectively, regions of the RNA. Typically, mRNA sequences are codon-optimized for use in accordance with the invention. Codon-optimization is performed to optimize expression in target cells. For example, if the mRNA is for delivery to a human subject, the mRNA will be codon-optimized for expression in human cells. mRNA according to the present invention may be synthesized as unmodified or modified mRNA. Typically, mRNAs are modified to enhance stability. Modifications of mRNA can include, for example, modifications of the nucleotides of the RNA. A modified mRNA according to the invention can thus include, for example, backbone modifications, sugar modifications or base modifications. In some embodiments, mRNAs may be synthesized from naturally occurring nucleotides and / or nucleotide analogues (modified nucleotides) including, but not limited to, purines (adenine (A), guanine (G)) or pyrimidines (thymine (T), cytosine (C), uracil (U)), and as modified nucleotides analogues or derivatives of purines and pyrimidines. The preparation of such analogues is known to a person skilled in the art e.g. from the U.S. Pat. Nos. 4,373,071, 4,401,796, 4,415,732, 4,458,066, 4,500,707, 4,668,777, 4,973,679, 5,047,524, 5,132,418, 5,153,319, 5,262,530 and 5,700,642, the disclosures of which are incorporated by reference in their entirety.

[0098] The mRNAs disclosed herein may contain RNA backbone modifications for example. Typically, a backbone modification is a modification in which the phosphates of the backbone of the nucleotides contained in the RNA are modified chemically. Exemplary backbone modifications typically include, but are not limited to, modifications from the group consisting of methylphosphonates, methylphosphoramidates, phosphoramidates, phosphorothioates (e.g. cytidine 5'-O-(l-thiophosphate)), boranophosphates, positively charged guanidinium groups etc., which means by replacing the phosphodiester linkage by other anionic, cationic or neutral groups. mRNAs can also contain sugar modifications. A typical sugar modification is a chemical modification of the sugar of the nucleotides it contains including, but not limited to, sugar modifications chosen from the group consisting of 2'-deoxy-2'-fluoro-oligoribonucleotide (2'- fluoro-2'-deoxycytidine 5 '-triphosphate, 2'-fluoro-2'-deoxyuridine 5 '-triphosphate), 2'-deoxy-2'- deamine-oligoribonucleotide (2'-amino-2'-deoxycytidine 5 '-triphosphate, 2'-amino-2'- deoxyuridine 5 '-triphosphate), 2'-O-alkyloligoribonucleotide, 2'-deoxy-2'-C- alkyloligoribonucleotide (2'-O-methylcytidine 5 '-triphosphate, 2'-methyluridine 5 '-triphosphate), 2'-C-alkyloligoribonucleotide, and isomers thereof (2'-aracytidine 5 '-triphosphate, 2'-arauridine 5 '-triphosphate), or azidotriphosphates (2'-azido-2'-deoxycytidine 5'-triphosphate, 2'-azido-2'- deoxyuridine 5'-triphosphate). mRNAs can contain modifications of the bases of the nucleotides (base modifications). A modified nucleotide which contains a base modification is also called a base-modified nucleotide. Examples of such base-modified nucleotides include, but are not limited to, 2- amino- 6- chloropurine riboside 5'-triphosphate, 2-aminoadenosine 5 '-triphosphate, 2-thiocytidine 5'- triphosphate, 2-thiouridine 5 '-triphosphate, 4-thiouridine 5 '-triphosphate, 5 -aminoallylcytidine 5'- triphosphate, 5 -aminoallyluridine 5 '-triphosphate, 5-bromocytidine 5 '-triphosphate, 5- bromouridine 5'-triphosphate, 5 -iodocytidine 5'-triphosphate, 5-iodouridine 5'-triphosphate, 5- methylcytidine 5 '-triphosphate, 5 -methyluridine 5 '-triphosphate, 6-azacytidine 5 '-triphosphate, 6- azauridine 5 '-triphosphate, 6-chloropurine riboside 5 '-triphosphate, 7-deazaadenosine 5'- triphosphate, 7-deazaguanosine 5 '-triphosphate, 8-azaadenosine 5 '-triphosphate, 8-azidoadenosine 5 '-triphosphate, benzimidazole riboside 5 '-triphosphate, Nl-methyladenosine 5 '-triphosphate, NI- methylguanosine 5 '-triphosphate, N6-methyladenosine 5 '-triphosphate, 06-methylguanosine 5'- triphosphate, pseudouridine 5 '-triphosphate, puromycin 5 '-triphosphate or xanthosine 5'- triphosphate.

[0099] Typically, mRNA synthesis includes the addition of a “cap” on the N-terminal (5') end, and a “tail” on the C-terminal (3') end. The presence of the cap is important in providing resistance to nucleases found in most eukaryotic cells. The presence of a “tail” serves to protect the mRNA from exonuclease degradation.

[0100] In some embodiments, mRNAs include a 5' cap structure. A 5' cap is typically added as follows: first, an RNA terminal phosphatase removes one of the terminal phosphate groups from the 5' nucleotide, leaving two terminal phosphates; guanosine triphosphate (GTP) is then added to the terminal phosphates via a guanylyl transferase, producing a 5'5'5 triphosphate linkage; and the 7-nitrogen of guanine is then methylated by a methyltransferase. Examples of cap structures include, but are not limited to, m7G(5')ppp (5'(A,G(5')ppp(5')A and G(5')ppp(5')G.

[0101] Naturally occurring cap structures comprise a 7-methyl guanosine that is linked via a triphosphate bridge to the 5 '-end of the first transcribed nucleotide, resulting in a dinucleotide cap of m7G(5')ppp(5')N, where N is any nucleoside. In vivo, the cap is added enzymatically. The cap is added in the nucleus and is catalyzed by the enzyme guanylyl transferase. The addition of the cap to the 5' terminal end of RNA occurs immediately after initiation of transcription. The terminal nucleoside is typically a guanosine, and is in the reverse orientation to all the other nucleotides, i.e., G(5')ppp(5')GpNpNp.

[0102] A common cap for mRNA produced by in vitro transcription is m7G(5')ppp(5')G, which has been used as the dinucleotide cap in transcription with T7 or SP6 RNA polymerase in vitro to obtain RNAs having a cap structure in their 5 '-termini. The prevailing method for the in vitro synthesis of capped mRNA employs a pre- formed dinucleotide of the form m7G(5')ppp(5')G (“m7GpppG”) as an initiator of transcription. To date, a usual form of a synthetic dinucleotide cap used in in vitro translation experiments is the Anti-Reverse Cap Analog (“ARCA”) or modified ARCA, which is generally a modified cap analog in which the 2' or 3' OH group is replaced with — OCH3.

[0103] Additional cap analogs include, but are not limited to, a chemical structures selected from the group consisting of m7GpppG, m7GpppA, m7GpppC; unmethylated cap analogs (e.g., GpppG); dimethylated cap analog (e.g., m2,7GpppG), trimethylated cap analog (e.g., m2,2,7GpppG), dimethylated symmetrical cap analogs (e.g., m7Gpppm7G), or anti reverse cap analogs (e.g., ARCA; m7,2'OmeGpppG, m7,2'dGPPpG, m7,3'OmeGpppG, m7,3'dGpppG and their tetraphosphate derivatives) (see, e.g., Jemielity, J. et al., “Novel ‘anti-reverse’cap analogs with superior translational properties”, RNA, 9: 1108-1122 (2003)).

[0104] In some embodiments, a suitable cap is a 7-methyl guanylate (“m7G”) linked via a triphosphate bridge to the 5 '-end of the first transcribed nucleotide, resulting in m7G(5')ppp(5')N, where N is any nucleoside.

[0105] Typically, the presence of a “tail” serves to protect the mRNA from exonuclease degradation. The poly A tail is thought to stabilize natural messengers and synthetic sense RNA. Therefore, in certain embodiments a long poly A tail can be added to an mRNA molecule thus rendering the RNA more stable. Poly A tails can be added using a variety of art-recognized techniques. For example, long poly A tails can be added to synthetic or in vitro transcribed RNA using poly A polymerase (Yokoe, et al. Nature Biotechnology. 1996; 14: 1252-1256). A transcription vector can also encode long poly A tails. In addition, poly A tails can be added by transcription directly from PCR products. Poly A may also be ligated to the 3' end of a sense RNA with RNA ligase (see, e.g., Molecular Cloning A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press: 1991 edition)).

[0106] In some embodiments, mRNAs include a 3' poly(A) tail structure. Typically, the length of the poly A tail can be at least about 10, 50, 100, 200, 300, 400 at least 500 nucleotides. In some embodiments, a poly- A tail on the 3' terminus of mRNA typically includes about 10 to 300 adenosine nucleotides (e.g., about 10 to 200 adenosine nucleotides, about 10 to 150 adenosine nucleotides, about 10 to 100 adenosine nucleotides, about 20 to 70 adenosine nucleotides, or about 20 to 60 adenosine nucleotides). A poly-A tail of 10 to 100 adenosine nucleotides, for example of about 20 to 70 adenosine nucleotides, or of about 20 to 60 adenosine nucleotides, is suitable for practicing the invention. In some embodiments, a poly(U) tail may be used to instead of a poly(A) tail described herein. In some embodiments, a poly(U) tail may be added to a poly(A) tail described herein. In some embodiments, mRNAs include a 3' poly(C) tail structure. In some embodiments, the length of the poly(A), poly(U) or poly(C) tail is adjusted to control the stability of a modified sense mRNA molecule of the invention and, thus, the transcription of protein. For example, since the length of a tail structure can influence the half-life of a sense mRNA molecule, the length of the tail can be adjusted to modify the level of resistance of the mRNA to nucleases and thereby control the time course of polynucleotide expression and / or polypeptide production in a target cell.

[0107] In some embodiments, mRNAs include a 5' and / or 3' untranslated region. In some embodiments, a 5' untranslated region includes one or more elements that affect an mRNA’s stability or translation, for example, an iron responsive element. In some embodiments, a 5' untranslated region may be between about 50 and 500 nucleotides in length.

[0108] In some embodiments, a 3' untranslated region includes one or more of a polyadenylation signal, a binding site for proteins that affect an mRNA’s stability of location in a cell, or one or more binding sites for miRNAs. In some embodiments, a 3' untranslated region may be between 50 and 500 nucleotides in length or longer.

[0109] Exemplary 3' and / or 5' UTR sequences can be derived from mRNA molecules which are stable (e.g., globin, actin, GAPDH, tubulin, histone, or citric acid cycle enzymes) to increase the stability of the sense mRNA molecule. For example, a 5' UTR sequence may include a partial sequence of a CMV immediate-early 1 (IE1) gene, or a fragment thereof to improve the nuclease resistance and / or improve the half-life of the polynucleotide. Also contemplated is the inclusion of a sequence encoding human growth hormone (hGH), or a fragment thereof to the 3' end or untranslated region of the polynucleotide (e.g., mRNA) to further stabilize the polynucleotide. Generally, these modifications improve the stability and / or pharmacokinetic properties (e.g., halflife) of the polynucleotide relative to their unmodified counterparts, and include, for example modifications made to improve such polynucleotides' resistance to in vivo nuclease digestion.

[0110] Delivery Vehicles for mRNA

[0111] According to the present invention, mRNA described herein may be delivered as naked RNA (unpackaged) or via delivery vehicles. As used herein, the terms “delivery vehicle,” “transfer vehicle,” “nanoparticle” or grammatical equivalent, are used interchangeably.

[0112] In some embodiments, mRNAs may be delivered via a single delivery vehicle. In some embodiments, mRNAs may be delivered via one or more delivery vehicles each of a different composition. According to various embodiments, suitable delivery vehicles include, but are not limited to polymer based carriers, such as polyethyleneimine (PEI), lipid nanoparticles and liposomes, nanoliposomes, ceramide-containing nanoliposomes, proteoliposomes, both natural and synthetically-derived exosomes, natural, synthetic and semi-synthetic lamellar bodies, nanoparticulates, calcium phosphor-silicate nanoparticulates, calcium phosphate nanoparticulates, silicon dioxide nanoparticulates, nanocrystalline particulates, semiconductor nanoparticulates, poly(D-arginine), sol-gels, nanodendrimers, starch-based delivery systems, micelles, emulsions, niosomes, multi-domain-block polymers (vinyl polymers, polypropyl acrylic acid polymers, dynamic polyconjugates).

[0113] In some embodiments, a suitable delivery vehicle is a liposomal delivery vehicle, e.g., a lipid nanoparticle. As used herein, liposomal delivery vehicles, e.g., lipid nanoparticles, are usually characterized as microscopic vesicles having an interior aqua space sequestered from an outer medium by a membrane of one or more bilayers. Bilayer membranes of liposomes are typically formed by amphiphilic molecules, such as lipids of synthetic or natural origin that comprise spatially separated hydrophilic and hydrophobic domains (Lasic, Trends Biotechnol., 16: 307-321, 1998). Bilayer membranes of the liposomes can also be formed by amphophilic polymers and surfactants (e.g., polymerosomes, niosomes, etc.).

[0114] In the context of the present invention, a liposomal delivery vehicle typically serves to transport a desired mRNA to a target cell or tissue. The process of incorporation of a desired mRNA into a liposome is often referred to as “loading”. Exemplary methods are described in Lasic, et al., FEBS Lett., 312: 255-258, 1992, which is incorporated herein by reference. The liposome-incorporated nucleic acids may be completely or partially located in the interior space of the liposome, within the bilayer membrane of the liposome, or associated with the exterior surface of the liposome membrane. The incorporation of a nucleic acid into liposomes is also referred to herein as “encapsulation” wherein the nucleic acid is entirely contained within the interior space of the liposome. The purpose of incorporating a mRNA into a transfer vehicle, such as a nanoparticle, is often to protect the nucleic acid from an environment which may contain enzymes or chemicals that degrade nucleic acids and / or systems or receptors that cause the rapid excretion of the nucleic acids. Accordingly, in some embodiments, a suitable delivery vehicle is capable of enhancing the stability of the mRNA contained therein and / or facilitate the delivery of mRNA to the target cell or tissue.

[0115] In some embodiments, nanoparticles may comprise one or more cationic lipids. As used herein, the phrase “cationic lipid” refers to any of a number of lipid species that have a net positive charge at a selected pH, such as physiological pH. Several cationic lipids have been described in the literature, many of which are commercially available. In some embodiments, provided nanoparticles comprise one or more cholesterol-based lipids. For example, suitable cholesterol-based cationic lipids include, for example, DC-Choi (N,N-dimethyl-N-ethylcarboxamidocholesterol), l,4-bis(3-N-oleylamino-propyl)piperazine (Gao, et al. Biochem. Biophys. Res. Comm. 179, 280 (1991); Wolf et al. BioTechniques 23, 139 (1997); U.S. Pat. No. 5,744,335), or ICE. In some embodiments, the cholesterol-based lipid may comprise a molar ration of about 2% to about 30%, or about 5% to about 20% of the total lipid present in a nanoparticle. In some embodiments, The percentage of cholesterol-based lipid in the lipid nanoparticle may be greater than 5%, 10%, greater than 20%, greater than 30%, or greater than 40%.

[0116] In some embodiments, provided herein are nanoparticles which comprise one or more PEGylated lipids. For example, the use of polyethylene glycol (PEG)-modified phospholipids and derivatized lipids such as derivatized ceramides (PEG-CER), including N-Octanoyl-Sphingosine- l-[Succinyl(Methoxy Polyethylene Glycol)-2000] (C8 PEG-2000 ceramide) is also contemplated by the present invention in combination with one or more of the cationic and, in some embodiments, other lipids together which comprise the nanoparticle. Contemplated PEG-modified lipids include, but are not limited to, a polyethylene glycol chain of up to 5 kDa in length covalently attached to a lipid with alkyl chain(s) of C6-C20 length. In some embodiments, a PEG-modified or PEGylated lipid is PEGylated cholesterol or PEG-2K. The addition of such components may prevent complex aggregation and may also provide a means for increasing circulation lifetime and increasing the delivery of the lipid-nucleic acid composition to the target cell, (Klibanov et al. (1990) FEBS Letters, 268 (1): 235-237), or they may be selected to rapidly exchange out of the formulation in vivo (see U.S. Pat. No. 5,885,613).

[0117] In some embodiments, a suitable delivery vehicle is formulated using a polymer as a carrier, alone or in combination with other carriers including various lipids described herein. Thus, in some embodiments, liposomal delivery vehicles, as used herein, also encompass polymer containing nanoparticles. Suitable polymers may include, for example, polyacrylates, polyalkycyanoacrylates, polylactide, polylactide-polyglycolide copolymers, polycaprolactones, dextran, albumin, gelatin, alginate, collagen, chitosan, cyclodextrins, protamine, PEGylated protamine, PLL, PEGylated PLL, and polyethylenimine (PEI). When PEI is present, it may be branched PEI of a molecular weight ranging from 10 to 40 kDA, e.g., 25 kDa branched PEI (Sigma #408727).

[0118] According to various embodiments, the selection of cationic lipids, non-cationic lipids, PEG-modified lipids and / or polymers which comprise the lipid nanoparticle, as well as the relative molar ratio of such lipids to each other, is based upon the characteristics of the selected lipid(s) / polymers, the nature of the intended target cells, the characteristics of the mRNA to be delivered. Additional considerations include, for example, the saturation of the alkyl chain, as well as the size, charge, pH, pKa, fusogenicity and toxicity of the selected lipid(s). Thus the molar ratios may be adjusted accordingly.

[0119] The liposomal transfer vehicles (such as nanoparticles) for use in the present invention can be prepared by various techniques which are presently known in the art. The nanoparticles for use in provided compositions can be prepared by various techniques which are presently known in the art. For example, multilamellar vesicles (MLV) may be prepared according to conventional techniques, such as by depositing a selected lipid on the inside wall of a suitable container or vessel by dissolving the lipid in an appropriate solvent, and then evaporating the solvent to leave a thin film on the inside of the vessel or by spray drying. An aqueous phase may then added to the vessel with a vortexing motion which results in the formation of MLVs. Uni-lamellar vesicles (ULV) can then be formed by homogenization, sonication or extrusion of the multi-lamellar vesicles. In addition, unilamellar vesicles can be formed by detergent removal techniques.

[0120] In certain embodiments, provided compositions comprise a nanoparticle wherein the mRNA is associated on both the surface of the nanoparticle and encapsulated within the same nanoparticle. For example, during preparation of the compositions of the present invention, cationic nanoparticles may associate with the mRNA through electrostatic interactions.

[0121] In some embodiments, the compositions and methods of the invention comprise mRNA encapsulated in a nanoparticle. In some embodiments, the one or more mRNA species may be encapsulated in the same nanoparticle. In some embodiments, the one or more mRNA species may be encapsulated in different nanoparticles. In some embodiments, the mRNA is encapsulated in one or more nanoparticles, which differ in their lipid composition, molar ratio of lipid components, size, charge (Zeta potential), targeting ligands and / or combinations thereof. In some embodiments, the one or more nanoparticles may have a different composition of cationic lipids, neutral lipid, PEG-modified lipid and / or combinations thereof. In some embodiments the one or more nanoparticles may have a different molar ratio of cationic lipid, neutral lipid, cholesterol and PEG- modified lipid used to create the nanoparticle.

[0122] The process of incorporation of a desired mRNA into a nanoparticle is often referred to as “loading”. Exemplary methods are described in Lasic, et al., FEBS Lett., 312: 255-258, 1992, which is incorporated herein by reference. The nanoparticle-incorporated nucleic acids may be completely or partially located in the interior space of the nanoparticle, within the bilayer membrane of the nanoparticle, or associated with the exterior surface of the nanoparticle membrane. The incorporation of a nucleic acid into nanoparticles is also referred to herein as “encapsulation” wherein the nucleic acid is entirely contained within the interior space of the nanoparticle. The purpose of incorporating a mRNA into a transfer vehicle, such as a nanoparticle, is often to protect the nucleic acid from an environment which may contain enzymes or chemicals that degrade nucleic acids and / or systems or receptors that cause the rapid excretion of the nucleic acids. Accordingly, in some embodiments, a suitable delivery vehicle is capable of enhancing the stability of the mRNA contained therein and / or facilitate the delivery of mRNA to the target cell or tissue.

[0123] Nanoparticle Size

[0124] Suitable liposomes or other nanoparticles in accordance with the present invention may be made in various sizes. In some embodiments, a suitable nanoparticle has a size of or less than about 150 nm, or less than about 100 nm (e.g., of or less than about 90 nm, 80 nm, 70 nm, 60 nm, 50 nm, 40 nm, 30 nm, or 20 nm). In some embodiments, the nanoparticle has a size of or less than about 60 nm (e.g., of or less than about 55 nm, of or less than about 50 nm, of or less than about 45 nm, of or less than about 40 nm, of or less than about 35 nm, of or less than about 30 nm, or of or less than about 25 nm). In some embodiments, a suitable nanoparticle has a size ranging from about 10-100 nm (e.g., ranging from about 10-90 nm, 10-80 nm, 10-70 nm, 10-60 nm, 10-50 nm, 10-40 nm, or 10-30 nm). Nanoparticles with a size of 60-100 nm (Zaverage) and in particular nanoparticles with a size of 70-90 nm (Zaverage) may be used in practicing the invention. The polydispersity index (PDI) of the nanoparticles is typically in the range of 0.1 to 0.5. In a particular embodiment, a PDI is below 0.2. Typically, the PDI is determined by dynamic light scattering.

[0125] A variety of alternative methods known in the art are available for sizing of a population of nanoparticles. One such sizing method is described in U.S. Pat. No. 4,737,323, incorporated herein by reference. Sonicating a nanoparticle suspension either by bath or probe sonication produces a progressive size reduction down to small ULV less than about 0.05 microns in diameter. Homogenization is another method that relies on shearing energy to fragment large liposomes into smaller ones. In a typical homogenization procedure, MLV are recirculated through a standard emulsion homogenizer until selected liposome sizes, typically between about 0.1 and 0.5 microns, are observed. The size of the liposomes may be determined by quasi-electric light scattering (QELS) as described in Bloomfield, Ann. Rev. Biophys. Bioeng., 10:421-150 (1981), incorporated herein by reference. Average liposome diameter may be reduced by sonication of formed liposomes. Intermittent sonication cycles may be alternated with QELS assessment to guide efficient nanoparticle synthesis.

[0126] Pharmaceutical Compositions and Administration

[0127] To facilitate expression of mRNA in vivo, delivery vehicles such as nanoparticles can be formulated in combination with one or more additional nucleic acids, carriers, targeting ligands or stabilizing reagents, or in pharmacological compositions where it is mixed with suitable excipients. Techniques for formulation and administration of drugs may be found in “Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa., latest edition.

[0128] Provided mRNA (naked or nanoparticle-encapsulated or associated), and compositions containing the same, may be administered and dosed in accordance with current medical practice, taking into account the clinical condition of the subject, the site and method of administration, the scheduling of administration, the subject's age, sex, body weight and other factors relevant to clinicians of ordinary skill in the art.

[0129] The “effective amount” for the purposes herein may be determined by such relevant considerations as are known to those of ordinary skill in experimental clinical research, pharmacological, clinical and medical arts. In some embodiments, the amount administered is effective to achieve at least some stabilization, improvement or elimination of symptoms and other indicators as are selected as appropriate measures of disease progress, regression or improvement by those of skill in the art. In some embodiments, a suitable amount and dosing regimen is one that results in protein expression or activity. In some embodiments, the expression and / or activity of the protein is detectable about 6 hours, 12 hours, 18 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months or longer after a single administration.

[0130] Provided methods of the present invention contemplate single as well as multiple administrations of a therapeutically effective amount of mRNA or a composition described herein. mRNA or a composition described herein can be administered at regular intervals, depending on the nature, severity and extent of the subject's condition. In some embodiments, a therapeutically effective amount of mRNA or a composition described herein may be administered periodically at regular intervals (e.g., once every year, once every six months, once every five months, once every four months, once every three months, bimonthly (once every two months), monthly (once every month), once every three weeks, biweekly (once every two weeks), weekly, once every three days, once every two days, daily or continuously). Typical intervals include once every month and every two months. In some embodiments, mRNA or a composition described herein may be administered at variable intervals.

[0131] EXAMPLES

[0132] Example 1: BMP-7 inhibits EndoMT in vitro

[0133] BMP-7 inhibits EndoMT invitro: To assess the in vitro efficacy of BMP7 to inhibit EndoMT, human umbilical vascular endothelial cells (HUVECs) is used as model endothelial cells to induce EndoMT with Angiotensin II and nitro-L-arginine methyl ester (L-NAME) (FIG. 1 A and IB). TGF-P is a well-established inducer of EndoMT and was used as a positive control in this experiment to validate that when compared to the established inducer of EndoMT, L-NAME and Angiotensin II (inducers of heart failure in our mouse model) also did the same to HUVECs (increased the expression of mesenchymal marker Vimentin and reduced expression of CD31, the endothelial marker). This work established that the inducers of heart failure in our non-ischemic mouse model (Ang-II and L-NAME) act as an inducer of EndoMT. Using the above-described platform, recombinant Human Bone Morphogenic Protein-7 (rhBMP7) is added during the EndoMT induction phase (with both L-NAME and TGF-B induction) and a decrease in the Vimentin expression combined with an increase in CD31 expression was observed, showing that the EndoMT transition process was inhibited by BMP-7 (FIG. 2).

[0134] BMP-7 decreases fibrosis in-vivo: As BMP-7 inhibits EndoMT, there can be a reduction in fibrosis content in a mouse model of HF. Utilizing non-ischemic heart failure in vivo model to study the impact of rhBMP7 protein treatment during concomitant induction of heart failure. In this non-ischemic mouse model of HF, the mice develop HF with reduced ejection fraction by Week 5 due to an osmotic pump (that is implanted on the mice’s back as a subcutaneous implant) that infuses angiotensin II and consumption of L-NAME and salt (NaCl) in their drinking water. FIG. 3 details of this experimental design. The animals were imaged by echocardiography to determine the effects of rhBMP7 on contractile properties of the heart with rhBMP7 (HF+BMP7) in comparison to normal mice (Control) and mice which were induced heart failure (HF) without the presence of BMP-7. As shown in FIG. 4, the contractile properties of the heart (ejection fraction, fractional shortening) were preserved in the presence of BMP-7 despite the induction agents attempts to develop heart failure proving that this protein can be protective from the mechanisms that usually perpetuate adverse remodeling of the heart.

[0135] Additionally, LV end diastolic volume, a marker of adverse remodeling in HF, was lesser in the presence of BMP-7 compared to HF mice. These results demonstrate that there was a significant preservation of function in those animals which were given the treatment (BMP7) despite maintaining the injury agents. Histological quantification of cardiac fibrosis using Masson’ s trichrome staining at 5 weeks showed that the area of fibrosis demonstrates an increase in the heart failure group when compared to control and BMP7 treated groups as shown in Fig. 5B. FIG. 5A shows representative pictures of the fibrosis Masson’s trichrome staining of control, HF and HF+BMP7 mice. Further cardiomyocyte size is measured, a marker of myocardial hypertrophy (Fig. 5C) which followed a similar trend as fibrosis. Therefore, promoting BMP-7 is protective to the phenotype of heart failure by a mechanism of inhibiting endothelial to mesenchymal transitioning leading to less maladaptive interstitial fibrosis.

[0136] BMP-7 RNA with a Nanocarrier can be used to promote BMP levels: The synthesized BMP7 mRNA was then encapsulated in a lipid nanoparticle consisting of cationic / ionizable, support, PEGylated lipid and cholesterol as shown in FIG. 6.

[0137] BMP-7mRNA nano construct inhibits EndoMT invitro: To assess the in vitro efficacy of BMP7 mRNA to inhibit EndoMT, human umbilical vascular endothelial cells (HUVECs) is used as model endothelial cells to induce EndoMT with L-NAME and Angiotensin II (agents that induce HF in the mouse model) as shown in FIG. 7. BMP-7mRNA nano-formulation on cultured cells to check if the BMP-7 mRNA in this form can inhibit EndoMT. The results show that the mRNA was successfully transfected into the cells followed by an increased expression of BMP7 protein (FIG. 8A-B). As a result of coincubation of the BMP7 mRNA with the HUVEC cells undergoing EndoMT, it is observed that the functional end result of the BMP7 mRNA was an inhibition of the transition to a mesenchymal state by showing a reduction in the expression of mesenchymal markers vimentin (FIG. 9A-B), Transgelin (FIG. 10A-B) and collagen 1 (FIG. 11A- B).

[0138] Key endothelial cellular functionality assays (NO production, LDL uptake and tube formation) to study the functional capacity of the cells which have gone through various spectrum of transitions (in the control and BMP-7 treated groups) (FIG. 12) to show that the BMP7 mRNA, not only inhibits the endothelial cells from expressing (and becoming) mesenchymal markers (FIG. 13), but also maintains the original function of the Endothelial cells compared to the untreated control cells in terms of nitric oxide production, LDL uptake and tube formation (FIG. 14, and FIG. 15).

[0139] BMP-7mRNA Nano construct reduces fibrosis and inhibits HF Phenotype invivo: To validate the efficacy of the BMP7 mRNA in vivo, an intervention experiment is performed using a mouse model of non-ischemic heart failure (FIG. 17). Using in vzvo-jetRNA®+ transfection reagent (Polyplus, France), which is a lipid based nanoformulation. Both the in vivo transfection agent and the BMP7 mRNA are combined and characterized the nanoparticles for their size and zeta potential, two key characteristics that determine the cell uptake ability and colloidal stability. The average particle size was approximately 150 nm, and the average zeta potential was approximately +50 mV. These physicochemical characteristics (particle size and zeta potential) determine the efficiency of delivery of the mRNA in vivo.

[0140] To examine the in vivo efficiency of this construct (FIG. 7). BMP7 mRNA loaded lipid nanoparticles (nBMP7) were injected subcutaneously to mouse under point the HF induction as described previously (using L-NAME / NaCl and Angiotensin II). Following administration, the mice were monitored and evaluated using echocardiography to examine the cardiac function and after 5 weeks, euthanized and the heart examined for fibrosis, and cardiomyocyte size. BMP7 mRNA loaded lipid nanoparticle treatment would help in alleviating fibrosis and heart failure phenotype when compared to control groups. BMP7 mRNA was subcutaneously injected to the HF mice in two frequency regimens i) Week

[0141] 2,3,4 & 5 and ii) Week 3, 4, & 5 of the HF protocol. In both the dosing regimens, the cardiac function was better than the untreated HF mice. The mice (HF+mBMP7(4)) that received 4 weekly injections from week 2 - 5 during the early period in the HF induction protocol exhibited significant difference compared to the untreated group. On the other hand, mice that received 3 weekly injections from week 3 - 5 (HF+mBMP7(3)) during the study period, showed improved EF, and was not statistically significant when compared to the untreated HF. Further, both the BMP7 mRNA treated mice group performed similar to the BMP7 protein infusion treatment group FIG. 18. histological quantification of cardiac fibrosis using Masson’s trichrome staining. FIG. 19 exhibits fibrosis staining (blue color) in each group and quantification of the fibrosis area demonstrated an increase in fibrosis in heart failure group when compared to control and BMP7 treated groups (both protein and mRNA). In the HF groups treated with BMP7 mRNA started at different times, Week 2 (HF+mBMP7(4)) and Week 3 (HF+mBMP7(3)), the effect on fibrosis reduction was statistically significant in both the groups when compared to the untreated HF group. Hence, BMP-7 mRNA Nanoconstruct is able to reduce fibrosis and avoid onset of Heart failure when given in conjunction to injury inducing agents.

[0142] Clinical applications of this invention:

[0143] Rationale and Background: Heart failure progresses through various stages and biologically the ability to reverse from pathology of heart failure should be better at earlier stages. Yet, when patients receive left ventricular assist device (LVAD) at ACC Stage D or End Stage Heart failure, the ability to rest the heart over prolonged periods has revealed a regression of fibrosis in studies. Yet such findings have not been consistent suggesting the need to add concerted concomitant therapy to promote recovery along with mechanical unloading. While the overall ability to achieve complete recovery leading to a full explant of the LVAD in clinical practice has been low, a recent trial achieved 40% explant rate in a select cohort of patients who did not have coronary artery disease as a cause of their heart failure. This outcome was achieved with a systematic up titration of very high doses of medical therapy making it an unlikely widely translatable option due to individuals’ inability to tolerate these medications post LVAD. Similar mechanical device assisted strategies to promote recovery of the myocardium with minimally invasive percutaneous- LVADs are underway using the Impella 5.5 device (Clinicaltrials.gov NCT05291884). While mechanical unloading provides rest to the heart, there is a need to combine this strategy with other therapies that actively promote recovery with minimal impact on hemodynamics like blood pressure, kidney function, etc. Hence, there is a need for direct genetic modulation.

[0144] Safety and feasibility human clinical trial: 1) LVAD patients: Young patients who are not eligible for transplant receive an LVAD as a destination therapy with an obvious survival benefit compared to the alternative but yet succumb to their adversities as many are unable to reverse their original contraindication for transplant. Hence an ability to reverse remodel and reduce the fibrosis in the heart could increase the chances of a successful LVAD explant. A safety and feasibility study in a specific cohort of patients receiving a LVAD is executable.

[0145] 2) Temporary MCS patients: Temporary percutaneous devices like Intra-aortic balloon pump and Impella are being implanted through the axillary artery providing an opportunity to promote cardiac recovery in certain cohorts of patients who have a short course of illness. Currently most of these patients are needing cardiac replacement as they do not have the ability to tolerate medications that are the only tools available for reverse remodeling. Hence a therapeutic strategy to promote reduction of fibrosis to establish feasibility and safety is executable.

[0146] 3) Ambulatory Heart failure patients: After feasibility and safety studies in the above cohorts of patients, the final and broad potential yield can be in patients in earlier stages (ACC Stage C) heart failure who are ambulatory. This strategy can start with additional therapy with GDMT and potentially in-lieu of pharmacotherapy.

[0147] Methods

[0148] Cell culture: Human umbilical vascular endothelial cells - HUVEC (Lonza, C2519A), of passage 2-5 was maintained in standard culture conditions in complete EGMTM-2 media.

[0149] EndoMT induction and treatment: For EndoMT induction with L-NAME + Ang II, add 3 ml of EBMTM-2 media containing 1 mM L-NAME and 1 pM Ang II to HUVECs. Then, the HUVECs undergoing EndoMT were treated with BMP7 protein or mRNA for the duration of the study (4 days) and fixed with 4% formaldehyde for the immunofluorescence staining.

[0150] EndoMT functionality assay: Nitric oxide (NO) production- After 4 days of EndoMT induction or EndoMT group treated with BMP in an 8- chambered slide, wash the HUVECs with HBSS with calcium and magnesium three times. Incubate cells with 200 pl of 8 pM of DAF-FM™ diacetate solution for 30 min at 37°C in a CO2 incubator. Wash the cells with HBSS with calcium and magnesium three times. Replace with fresh EGMTM-2 media. Incubate cells for 15 min at 37°C in a CO2 incubator. Observe with a fluorescent microscope with a FITC filter cube to detect NO. Acetylated Dil LDL uptake:Afler 4 days of EndoMT induction or EndoMT group treated with BMP in an 8- chambered slide, wash the HUVECs with HBSS with calcium and magnesium three times. Incubate cells with 200 pl of Dil AcLDL working solution for 4 h at 37°C in a CO2 incubator. Wash the cells with HBSS with calcium and magnesium three times. Observe with a fluorescent microscope with a TRITC filter cube to detect Dil AcLDL.

[0151] Angiogenesis assay: After 4 days of EndoMT induction or EndoMT group treated with BMP in an 8- chambered slide, wash the HUVECs with IX DPBS and add 1ml of trypsin EDTA to each well and incubate for 4 min at 37°C. Detach cells using 4 ml of culture media and collect cell suspension in 15 ml tubes Centrifuge the cell suspension at 200-300 x g for 4 min at room temperature. Carefully aspirate the supernatant and suspend the cell pellet in pre-warmed culture media. Count viable cells sing trypan blue solution in the automatic cell counter (or a standard hemocytometer). Thaw Matrigel by removing the Matrigel from -20°C or -80°C freezer and place it in a refrigerator at 4°C. Place a vial of completely thawed Matrigel and labeled a 48-well plate on ice in a laminar flow hood. Mix the vial thoroughly by inverting a few times. Load 140 pl of Matrigel per well of a 48-well plate while avoiding air bubbles. Transfer the 48-well plate to a cell culture incubator and incubate it at 37°C for 30 min to allow the Matrigel to gel. Gently dispense 400 pl (30,000 cells) per well of the single-cell suspensions to corresponding labeled wells of a 48-well plate on top of the gelled Matrigel. Incubate the 48-well plate at 37°C inaCO2 incubator for a period of 4 h or until the desired result is achieved. Examine the plate every hour for tube formation under an inverted microscope with x4 or xlO objective. Once tube formation is observed, photograph the tubular network in the wells using a digital camera attached to the inverted microscope with x4 or xlO objective. Image analysis is performed using ImageJ.

[0152] Animals and treatment: All animal studies were performed in accordance with approved protocols by the Houston Methodist Research Institute’s Institutional Animal Care and Use Committee and carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

[0153] Twelve-week-old C57BL6 mice were randomly divided into four groups: 1. Control, 2. Heart Failure, 3. Heart Failure with BMP7 recombinant protein and 4. Heart Failure with BMP7 mRNA treatment. L-NAME (0.3 mg / mL with 1 % NaCl) was administered in administered in drinking water to Group 2, 3 & 4, whereas Group 1 , control mice received regular drinking water. For Group 2, 3 and 4, 1 week after L-NAME treatment, the mice were anesthetized with inhaled isoflurane and subcutaneous osmotic pump (Alzet, no 1004) were implanted to deliver Angiotensin II (0.7 mg / kg per day) for 4 weeks, while the L-NAME water was continued. All mice were housed in isolation cages under 12h:12h light / dark cycle and were fed a standard chow diet (Harlan Teklad, 2920). At the end of the 5-week heart failure induction, mice are assessed for cardiac function, and euthanized to collect heart tissue for further processing.

[0154] Transthoracic echocardiography was performed using a Vevo 2100 ultrasound system equipped with a MS-400 30 MHz transducer. Mice were anesthetized with 1-1.5 % isoflurane for the duration of the recording and were placed on a heating platform to maintain body temperature at 37°C. Heart was located in B-mode from parasternal long axis and short axis view. Two- dimensional M-mode echocardiographic images were obtained at the level of the papillary muscles from the parasternal short axis view for measurement of LV dimension and systolic function. Echocardiographic acquisition and analysis were performed by different individuals. Echo analysis was performed using Vevo lab version 5.6 to calculate ejection fraction. mRNA synthesis: Open reading frame (ORF) for mRNA synthesis was designed for the target gene (BMP7) based on the amino acid sequence obtained from UniProt Pl 807. The gene reference for BMP7 was obtained from NCBI. There are different publicly available mRNA sequences available in this source (LOCUS: AK094784, AK291186, AK312419, BC004248, BC008584, any of which can be used with the compositions and methods disclosed herein. In one specific example, the LOCUS AK312419 (1398 bp) was used, based on optimal properties for loading in a nanodelivery system. NCBI ORF Finder was used to find the open reading frame of AK312419 consisting of a start codon and ending in a stop codon, that resulted in a nucleic acid sequence of 1296 base pairs and a protein sequence of 431 amino acids.

[0155] The mRNA was manufactured by the RNA core at Houston Methodist. The gene of interest was inserted into their mRNA manufacturing backbone consisting optimal 5’ and 3’ UTR’s and -150 bp PolyA tail. The mRNA was synthesized by in vitro transcription (IVT) and Nl-methyl- pseudouridine added to the nucleotide mix, to increase the stability and translation efficiency of the mRNA. The DNA plasmids used in IVT are encoded for BMP7. The open reading frame is human BMP7 transcript variant NM_001719. The mRNA was purified, and the size and integrity of mRNA constructs were assessed using a TapeStation (Agilent).

[0156] Example 2: BMP7 mRNA loaded lipid nanoparticles toxicity / safety in other organs

[0157] Methods:

[0158] At the end of the 5 -week heart failure induction, mice were euthanized to collect liver and kidney tissue for further processing. The liver and kidney tissue were fixed, mounted and sectioned, followed by staining with Hematoxylin and Eosin (H&E). The stained tissue sections of the tissues from animals (Control n=4; Heart Failure n=6; Heart Failure+BMP7 mRNA n=4) were imaged using a bright field microscope and the slides (1 representative section per animal) were evaluated by a board-certified pathologist to identify any abnormal morphology. These results are shown in Figure 21.

[0159] Results:

[0160] In relevance to the safety of the BMP7 mRNA Nanogene™ modality, the effect of the injected Nanogene™ compound on the major organs involved in drug metabolism viz. liver and kidney were evaluated.

[0161] Evaluation of the H&E slides of the kidney and liver from the BMP7 Nanogene™ therapy when compared to the normal mice by a board-certified pathologist revealed that the BMP7 mRNA loaded lipid nanoparticles did not show any specific pathological findings in the kidney and liver. (Figure 21). A clinical pathologist expert graded (blinded) all control mice as near normal organ morphology with minimal abnormal changes (e.g., liver - lobular inflammation and kidney cytoplasmic vacuolations). In comparison, the BMP7 mRNA treated mice were assessed as having similar morphology findings as the controls. This shows that the therapy has acceptable safety while ameliorating cardiac fibrosis.

[0162] Example 3: BMP7 expression in human heart tissues is supported by two different unloading strategies

[0163] Background:

[0164] Left ventricular unloading has been validated in studies with durable left ventricular assist devices in the past and temporary mechanical devices more recently as a strategy to rest the heart and promote myocardial recovery. In the field of temporary mechanical assist devices there are different devices that unload the heart in different ways. Two prevalent devices are Intra-aortic balloon pump ( IABP) and percutaneous Left ventricular assist device (p-VAD) (Figure 2) The ability of the device to unload varies significantly and studies have shown that in p-VAD unloaded hearts that undergo revascularization in an acute heart attack state have protective genetic programming turned on compared to when they are not unloaded. There are no studies assessing genetic changes in patients who are unloaded with an IABP. It was hypothesized that patients who are unloaded by a direct left ventricular approach i.e., p-VAD, will have a more favorable genetic programming compared to those unloaded in an indirect manner i.e. IABP.

[0165] Methods:

[0166] Myocardial tissue was collected at the time of cardiac surgery from 4 patients bridged with a pVAD (Impella™ 5.5) and 4 bridged with IABP. Tissues from patients who did not receive either of the pumps and were bridged to cardiac replacement were used as HF tissues. Total RNA was extracted, and their quality and quantity were analyzed using a Bioanalyzer 2100 and RNA 6000 Nano LabChip™ Kit (Agilent, CA, USA). Approximately 10 ug of total RNA was subjected to isolate Poly (A) mRNA with poly-T oligo attached magnetic beads (Invitrogen®). Following purification, the poly(A) mRNA fraction was fragmented into small pieces using divalent cations under elevated temperature. The cleaved RNA fragments were then reverse-transcribed to create the final cDNA library in accordance with a strand-specific library preparation by dUTP method. The average insert size for the paired-end libraries was 300+50 bp. Paired-end 2xl50bp sequencing on an Illumina Hiseq 4000™ was performed. StringTie™ was used to perform expression level for mRNAs.

[0167] Results:

[0168] Myocardial tissues of patients’ hearts that were hemodynamically supported and rested with temporary mechanical pumps were investigated. Bulk RNA sequencing was used to evaluate this in order to understand the molecules involved in the unloading mechanism.

[0169] The results of the bulk RNA sequencing of human tissue obtained from patients supported with a transvalvular pump (pVAD) or a Counter pulsation device (IABP) and were bridged to cardiac replacement, exhibited BMP7 protein as one of the top molecules that was differentially expressed and upregulated in hearts supported by either mechanical device compared to heart failure samples and higher in hearts supported by direct unloading strategy with p-VAD compared to IABP (Figure 22). This can show that higher level of BMP7 can serve to support a more favorable myocardial state and hence a m-RNA gene strategy promoting its synthesis as a therapeutic strategy for failing hearts.

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[0188] SEQUENCES

[0189] SEQ ID NO: 1 : BMP7 cDNA sequence (AK312419.1, Homo sapiens bone morphogenetic protein

[0190] 7, mRNA)

[0191] ATGCACGTGCGCTCACTGCGAGCTGCGGCGCCGCACAGCTTCGTGGCGCTCTGGGCA

[0192] CCCCTGTTCCTGCTCGCTCCGCCCTGGCCGACTTCAGCCTGGACAACGAGGTGCACT

[0193] CGAGCTTCATCCACCGGCGCCTCCGCAGCAGGAGCGGCGGGAGATGCAGCGCGAGA

[0194] TCCTCTCCATTTTGGGCTTGCCCCACCGCCCGCGCCCGCACCTCCAGGGCAAGCACA

[0195] ACTCGGCACCCATGTTCATGCTGGACCTGTACAACGCCATGGCGGTGGAGGAGGGC

[0196] GGCGGGCCCGGCGGCCAGGGCTTCTCCTACCCCTACAAGGCCGTCTTCAGTACCCAG

[0197] GGCCCCCCTCTGGCCAGCCTGCAAGATAGCCATTTCCTCACCGACGCCGACATGGTC

[0198] ATGAGCTTCGTCAACCTCGTGGAACATGACAAGGAATTCTTCCACCCACGCTACCAC

[0199] CATCGAGAGTTCCGGTTTGATCTTTCCAAGATCCCAGAAGGGGAAGCTGTCACGGCA

[0200] GCCGAATTCCGGATCTACAAGGACTACATCCGGGAACGCTTCGACAATGAGACGTT

[0201] CCGGATCAGCGTTTATCAGGTGCTCCAGGAGCACTTGGGCAGGGAATCGGATCTCTT

[0202] CCTGCTCGACAGCCGTACCCTCTGGGCCTCGGAGGAGGGCTGGCTGGTGTTTGACAT

[0203] CACAGCCACCAGCAACCACTGGGTGGTCAATCCGCGGCACAACCTGGGCCTGCAGC

[0204] TCTCGGTGGAGACGCTGGATGGGCAGAGCATCAACCCCAAGTTGGCGGGCCTGATT

[0205] GGGCGGCACGGGCCCCAGAACAAGCAGCCCTTCATGGTGGCTTTCTTCAAGGCCAC

[0206] GGAGGTCCACTTCCGCAGCATCCGGTCCACGGGGAGCAAACAGCGCAGCCAGAACC

[0207] GCTCCAAGACGCCCAAGAACCAGGAAGCCCTGCGGATGGCCAACGTGGCAGAGAA

[0208] CAGCAGCAGCGACCAGAGGCAGGCCTGTAAGAAGCACGAGCTGTATGTCAGCTTCC

[0209] GAGACCTGGGCTGGCAGGACTGGATCATCGCGCCTGAAGGCTACGCCGCCTACTAC

[0210] TGTGAGGGGGAGTGTGCCTTCCCTCTGAACTCCTACATGAACGCCACCAACCACGCC

[0211] ATCGTGCAGACGCTGGTCCACTTCATCAACCCGGAAACGGTGCCCAAGCCCTGCTGT

[0212] GCGCCCACGCAGCTCAATGCCATCTCCGTCCTCTACTTCGATGACAGCTCCAACGTC

[0213] ATCCTGAAGAAATACAGAAACATGGTGGTCCGGGCCTGTGGCTGCCACTAG

[0214] SEQ ID NO: 2: BMP7 amino acid sequence (AK312419.1)

[0215] MHVRSLRAAAPHSFVALWAPLFLLRSALADFSLDNEVHSSFIHRRLRSQERREMQREILSI

[0216] LGLPHRPRPHLQGKHNSAPMFMLDLYNAMAVEEGGGPGGQGFSYPYKAVFSTQGPPLA

[0217] SLQDSHFLTDADMVMSFVNLVEHDKEFFHPRYHHREFRFDLSKIPEGEAVTAAEFRIYKD

[0218] YIRERFDNETFRISVYQVLQEHLGRESDLFLLDSRTLWASEEGWLVFDITATSNHWVVNP

[0219] RHNLGLQLSVETLDGQSINPKLAGLIGRHGPQNKQPFMVAFFKATEVHFRSIRSTGSKQR SQNRSKTPKNQEALRMANVAENSSSDQRQACKKHELYVSFRDLGWQDWIIAPEGYAAY

[0220] YCEGECAFPLNSYMNATNHAIVQTLVHFINPETVPKPCCAPTQLNAISVLYFDDSSNVILK KYRNMVVRACGCH

[0221] SEQ ID NO: 3: BMP7 cDNA sequence (BC008584.1 Homo sapiens bone morphogenetic protein

[0222] 7, mRNA)

[0223] GCCCGGAGCCCGGAGCCCGGGTAGCGCGTAGAGCCGGCGCGATGCACGTGCGCTCA

[0224] CTGCGAGCTGCGGCGCCGCACAGCTTCGTGGCGCTCTGGGCACCCCTGTTCCTGCTG

[0225] CGCTCCGCCCTGGCCGACTTCAGCCTGGACAACGAGGTGCACTCGAGCTTCATCCAC

[0226] CGGCGCCTCCGCAGCCAGGAGCGGCGGGAGATGCAGCGCGAGATCCTCTCCATTTTG

[0227] GGCTTGCCCCACCGCCCGCGCCCGCACCTCCAGGGCAAGCACAACTCGGCACCCATG

[0228] TTCATGCTGGACCTGTACAACGCCATGGCGGTGGAGGAGGGCGGCGGGCCCGGCGG

[0229] CCAGGGCTTCTCCTACCCCTACAAGGCCGTCTTCAGTACCCAGGGCCCCCCTCTGGCC

[0230] AGCCTGCAAGATAGCCATTTCCTCACCGACGCCGACATGGTCATGAGCTTCGTCAACC

[0231] TCGTGGAACATGACAAGGAATTCTTCCACCCACGCTACCACCATCGAGAGTTCCGGT

[0232] TTGATCTTTCCAAGATCCCAGAAGGGGAAGCTGTCACGGCAGCCGAATTCCGGATCT

[0233] ACAAGGACTACATCCGGGAACGCTTCGACAATGAGACGTTCCGGATCAGCGTTTATC

[0234] AGGTGCTCCAGGAGCACTTGGGCAGGGAATCGGATCTCTTCCTGCTCGACAGCCGTA

[0235] CCCTCTGGGCCTCGGAGGAGGGCTGGCTGGTGTTTGACATCACAGCCACCAGCAACC

[0236] ACTGGGTGGTCAATCCGCGGCACAACCTGGGCCTGCAGCTCTCGGTGGAGACGCTG

[0237] GATGGGCAGAGCATCAACCCCAAGTTGGCGGGCCTGATTGGGCGGCACGGGCCCCA

[0238] GAACAAGCAGCCCTTCATGGTGGCTTTCTTCAAGGCCACGGAGGTCCACTTCCGCAG

[0239] CATCCGGTCCACGGGGAGCAAACAGCGCAGCCAGAACCGCTCCAAGACGCCCAAGA

[0240] ACCAGGAAGCCCTGCGGATGGCCAACGTGGCAGAGAACAGCAGCAGCGACCAGAG

[0241] GCAGGCCTGTAAGAAGCACGAGCTGTATGTCAGCTTCCGAGACCTGGGCTGGCAGG

[0242] ACTGGATCATCGCGCCTGAAGGCTACGCCGCCTACTACTGTGAGGGGGAGTGTGCCT

[0243] TCCCTCTGAACTCCTACATGAACGCCACCAACCACGCCATCGTGCAGACGCTGGTCC

[0244] ACTTCATCAACCCGGAAACGGTGCCCAAGCCCTGCTGTGCGCCCACGCAGCTCAATG

[0245] CCATCTCCGTCCTCTACTTCGATGACAGCTCCAACGTCATCCTGAAGAAATACAGAAA

[0246] CATGGTGGTCCGGGCCTGTGGCTGCCACTAGCTCCTCCGAGAATTCAGACCCTTTGG

[0247] GGCCAAGTTTTTCTGGATCCTCCATTGCTCGCCTTGGCCAGGAACCAGCAGACCAAC

[0248] TGCCTTTTGTGAGACCTTCCCCTCCCTATCCCCAACTTTAAAGGTGTGAGAGTATTAG

[0249] GAAACATGAGCAGCATATGGCTTTTGATCAGTTTTTCAGTGGCAGCATCCAATGAACA AGATCCTACAAGCTGTGCAGGCAAAACCTAGCAGAAAAAAAAAACAACGCATAAAG AAAAATGGCCGGGCCAGGTCATTGGCTGGGAAGTCTCAGCCATGCACGGACTCGTTT CCAGAGGTAATTATGAGCGCCTACCAGCCAGGCCACCCAGCCGTGGGAGGAAGGGG GCGTGGCAAGGGGTGGGCACATTGGTGTCTGTGCGAAAGGAAAATTGACCCGGAAG TTCCTGTAATAAATGTCACAATAAAACGAATGAATGAAAAAAAAAAAAAAAAA

[0250] SEQ ID NO: 4: BMP7 amino acid sequence (BC008584.1)

[0251] MHVRSLRAAAPHSFVALWAPLFLLRSALADFSLDNEVHSSFIHRRLRSQERREMQREILSIL GLPHRPRPHLQGKHNSAPMFMLDLYNAMAVEEGGGPGGQGFSYPYKAVFSTQGPPLASL QDSHFLTD ADM VMSFVNLVEHDKEFFHPR YHHREFRFDLS KIPEGEA VTAAEFRIYKD YIR ERFDNETFRISVYQVLQEHLGRESDLFLLDSRTLWASEEGWLVFDITATSNHWVVNPRHNL GLQLSVETLDGQSINPKLAGLIGRHGPQNKQPFMVAFFKATEVHFRSIRSTGSKQRSQNRSK TPKNQEALRMANVAENSSSDQRQACKKHELYVSFRDLGWQDWIIAPEGYAAYYCEGECA FPLNSYMNATNHAIVQTLVHFINPETVPKPCCAPTQLNAISVLYFDDSSNVILKKYRNMVV RACGCH

Claims

WHAT IS CLAIMED IS:

1. A method of reducing endothelial to mesenchymal cell transition (EndoMT) of endothelial cells associated with an organ, the method comprises exposing the endothelial-derived cells to Bone Morphogenic Protein 7 (BMP7) mRNA.

2. The method of claim 1, wherein the mRNA is in a nanoparticle.

3. The method of claim 1 , wherein the organ is a heart.

4. The method of claim 1 or 3, wherein the endothelial cells associated with a heart are in vitro.

5. The method of claim 4, wherein the endothelial cells are human umbilical vascular endothelial cells (HUVECs).

6. The method of claim 1 or 3, wherein the endothelial cells associated with a heart are in vivo.

7. The method of claim 6, wherein the endothelial cells are inside and outside the blood vessels within the heart.

8. The method of claim 6 or 7, wherein endothelial cells are within a subject.

9. The method of claim 8, wherein the subject is a mammal.

10. The method of claim 9, wherein the mammal is a human.

11. The method of any one of claims 8-10, wherein the subject has been diagnosed as having cardiac dysfunction before administration of the BMP7 mRNA.

12. The method of any one of claims 8-10, wherein the subject has been diagnosed as being at risk of cardiac dysfunction before administration of the BMP7 mRNA.

13. The method of any one of claims 7-10, wherein the subject has been diagnosed as having, or being at risk for developing, EndoMT within the heart.

14. The method of any one of claims 8-13, wherein the subject does not undergo heart surgery prior to exposure to BMP7 mRNA.

15. The method of any one of claims 8-13, wherein the BMP7 mRNA is administered before, during, or after heart surgery.

16. The method of any one of claims 8-13, wherein the subject has a temporary mechanical assist device.

17. The method of claim 16, wherein the BMP7 mRNA is administered through a temporary mechanical assist device.

18. The method of any one of claims 8-17, wherein BMP7 mRNA improves cardiac function in the subject by 5% or more.

19. The method of any one of claims 8-18, wherein BMP7 mRNA reduces cardiac dysfunction in a subject.

20. The method of claim 19, wherein the cardiac dysfunction comprises contractile properties or relaxation properties.

21. The method of claim 20, wherein said contractile properties are ejection fraction and / or fractional shortening.

22. The method of claim 20, wherein said relaxation properties are diastolic function assessment.

23. The method of any one of claims 1-20, wherein BMP7 reduces the transition of endothelial to mesenchymal cells by 5% or more.

24. The method of claim 23, wherein reduction in the transition of endothelial to mesenchymal cells results in the reduction of fibrosis within the heart.

25. The method of claim 23 or 24, wherein reduction in the transition of endothelial to mesenchymal cells results in a 5% or more decrease in adverse remodeling.

26. The method of claim 25, wherein adverse remodeling is measured by determining left ventricle end-diastolic volume.

27. The method of any one of claims 1-26, wherein the BMP7 mRNA is of human origin.

28. The method of claim 27, wherein the BMP7 mRNA is derived from GenBank Accession No. AK312419.1 or BC008584.1.

29. The method of any one of claims 1-28, wherein the BMP7 mRNA is 90% or more identical to SEQ ID NO: 1 or SEQ ID NO: 3.

30. The method of claim 28 or 29, wherein the BMP7 mRNA is a functional fragment of GenBank Accession No. AK312419.1 or BC008584.1 with SEQ ID NO: 1 or SEQ ID NO: 3.

31. The method of claim 2, wherein the nanoparticle is a lipid nanoparticle.

32. The method of claim 31, wherein the nanoparticle is cationic and ionizable.

33. The method of claim 31 or 32, wherein the nanoparticle comprises a PEGylated lipid.

34. The method of any one of claims 31-33, wherein the nanoparticle comprises cholesterol.

35. The method of claim 2, wherein the nanoparticle further comprises one or more additional therapeutics, medicaments, compounds, or one or more adjuvants.

36. A composition comprising a nanoparticle, wherein said nanoparticle encapsulates or is associated with BMP7 mRNA.

37. The composition of claim 36, wherein the nanoparticle is a lipid nanoparticle.

38. The composition of claim 36 or 37, wherein the nanoparticle is cationic and ionizable.

39. The composition of any one of claims 36-38, wherein the nanoparticle comprises a PEGylated lipid.

40. The composition of any one of claims 36-39, wherein the nanoparticle comprises cholesterol.

41. The composition of any one of claims 36-40, wherein the BMP7 mRNA is of human origin.

42. The composition of claim 41, wherein the BMP7 mRNA is derived from GenBank Accession No. AK312419.1 or BC008584.1 with SEQ ID NO: 1 or SEQ ID NO: 3.

43. The composition of any one of claims 36-42, wherein the BMP7 mRNA is 90% or more identical to SEQ ID NO: 1 or SEQ ID NO: 3.

44. The composition of claims 42 or 43, wherein the BMP7 mRNA is a functional fragment of GenBank Accession No. AK312419.1 or BC008584.1 with SEQ ID NO: 1 or SEQ ID NO: 3.

45. The composition of any one of claims 36-44, wherein the nanoparticle is designed to target specific cell or tissue types.

46. The composition of claim 45, wherein the nanoparticle is designed to target endothelial cells.

47. The composition of claim 46, wherein the endothelial cells are within the heart.

48. The composition of any one of claims 36-47, wherein the nanoparticle is designed to target cellspecific markers.

49. The composition of claim 47, wherein said target cell-specific markers indicate the potential for EndoMT of that cell.

50. The composition of any one of claims 36-49, wherein the nanoparticle further comprises one or more additional therapeutics, medicaments, compounds, or one or more adjuvants.

51. A kit comprising the nanoparticle of any one of claims 36-50.

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