WO2023084013A1

WO2023084013A1 - Compositions comprising modified rna encoding vegf-a and methods of use

Info

Publication number: WO2023084013A1
Application number: PCT/EP2022/081592
Authority: WO
Inventors: Li-Ming Gan; Jaya Birgitte Rosenmeier
Original assignee: Modernatx, Inc.
Priority date: 2021-11-12
Filing date: 2022-11-11
Publication date: 2023-05-19

Abstract

This disclosure relates to compositions comprising modified RNA encoding VEGF-A, and the use of such compositions in methods of treating patients suffering from dilated cardiomyopathy and heart failure associated with dilated cardiomyopathy.

Description

COMPOSITIONS COMPRISING MODIFIED RNA ENCODING VEGF-A AND

METHODS OF USE

1. FIELD

[001] The disclosure relates to compositions comprising modified RNA encoding VEGF-A, and the use of such compositions in methods of treating patients suffering from dilated cardiomyopathy and heart failure associated with dilated cardiomyopathy.

2. BACKGROUND

[002] Dilated cardiomyopathy is a disease of the myocardium in which the myocardium gradually becomes weaker; whereby the left ventricle becomes enlarged and the ventricle wall becomes thinner, and therefore less able to pump blood effectively. Cardiomyocytes becomes increasingly dysfunctional causing the dilatation (Mitrut et al., Curr Health Sci J. 2018, 44, 243-249). Dilated cardiomyopathy is one of the most frequent causes of heart failure with reduced ejection fraction (HFrEF) (Seferovic et al, European Journal of Heart Failure (2019) 21, 553-576).

[003] Dilated cardiomyopathy has different etiology including genetic causes, myocarditis, and exposure to chemotherapy, alcohol and toxins (Weintraub et al., The Lancet, 2017, 390(10092), 400-414).

[004] Options for treatment of dilated cardiomyopathy are still very limited, and severe cases where left ventricular function is severely impaired (HFrEF) may require left ventricular assist devices or heart transplantations. Accordingly, there is a need for new therapies, particularly cardiac regenerative therapies, for patients with dilated cardiomyopathy. 3. SUMMARY

[005] Disclosed herein are compositions comprising a modified RNA that encodes a VEGF-A polypeptide for use in the methods disclosed herein.

[006] Also disclosed is the use of a composition disclosed herein in the manufacture of a medicament for use in the methods disclosed herein.

[007] In one embodiment, there is provided a method of treating dilated cardiomyopathy or heart failure associated with dilated cardiomyopathy in a patient in need thereof comprising administering to the patient an effective amount of a composition comprising a modified RNA in accordance with the present disclosure.

[008] Certain embodiments of the present disclosure are summarized in the following paragraphs. This list is only exemplary and not exhaustive of all of the embodiments provided by this disclosure.

4. DESCRIPTION OF DRAWINGS

[009] Those of skill in the art will understand that the drawings, described below, are for illustrative purposes only. The drawings are not intended to limit the scope of the present teachings in any way.

[010] Fig. 1 shows the LVEF (Echocardiography (rest/hyperaemia), Biplane Simpson) of individual patients in the EPICCURE study as measured at Visit 1 (baseline) and at Visits 5, 6 and 7.

[011] Fig. 2 shows the ¹⁵O-water PET global stress myocardial blood flow of individual patients in the EPICCURE study as measured at Visit 2 (baseline) and at Visits

5 and 6. [012] Fig. 3 shows the ¹⁵O-water PET regional stress myocardial blood flow in the treated area of individual patients in the EPICCURE study as measured at Visit 2 (baseline) and at Visits 5 and 6.

[013] Fig. 4 shows the ¹⁵O-water PET global rest myocardial blood flow of individual patients in the EPICCURE study as measured at Visit 2 (baseline) and at Visits 5 and 6.

[014] Fig. 5 shows the ¹⁵O-water PET regional rest myocardial blood flow in the treated area of individual patients in the EPICCURE study as measured at Visit 2 (baseline) and at Visits 5 and 6.

[015] Fig. 6 shows the ¹⁵O-water PET treated area coronary flow reserve of individual patients in the EPICCURE study as measured at Visit 2 (baseline) and at Visits 5 and 6.

[016] Figs. 7A, 7B, 7C and 7D show the mean SAQ Physical limitation score (PLS), SAQ Treatment satisfaction score (TSS), SAQ Quality of life score (QoLS) and SAQ Angina stability score (ASS) over time, respectively, of patients in the EPICCURE study.

[017] Fig. 8. Shows the mean KCCQ Overall summary score over time of patients in the EPICCURE study.

[018] Fig. 9A is the ¹⁵O-water PET image from Patient 1 recorded at Visit 2 showing the pre-operative suggested area for treatment. Fig. 9B is the ¹⁵O-water PET image showing the injection map for Patient 1.

[019] Fig. 10A shows the ¹⁵O-water PET myocardial blood flow (divided into segments of the left ventricle) of Patient 1 at Baseline (Visit 2) at rest (left image) and under stress (right image). Fig. 10B shows the ¹⁵O-water PET myocardial blood flow (divided into segments of the left ventricle) of Patient 1 at 3 months after surgery (Visit 6) at rest (left image) and under stress (right image).

[020] Fig. 11 A shows the change in LVEF of Patient 1 over time. Fig. 1 IB shows the KCCQ and SAQ scores of Patient 1 over time.

5. DETAILED DESCRIPTION

[021] All references referred to are incorporated herein by reference in their entireties.

[022] Many modifications and other embodiments of the disclosures set forth herein will come to mind to one skilled in the art to which these disclosures pertain having the benefit of the teachings presented in the foregoing descriptions and the associated drawings. Therefore, it is to be understood that the disclosures are not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims. Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.

[023] Units, prefixes and symbols may be denoted in their SI accepted form. Unless otherwise indicated, nucleic acids are written left to right in 5' to 3' orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively. Numeric ranges are inclusive of the numbers defining the range. Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes. The terms defined below are more fully defined by reference to the specification as a whole.

5.1. Definitions

[024] Unless specifically defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Unless mentioned otherwise, the techniques employed or contemplated herein are standard methodologies well known to one of ordinary skill in the art. The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of microbiology, tissue culture, molecular biology, chemistry, biochemistry and recombinant DNA technology, which are within the skill of the art. The materials, methods and examples are illustrative only and not limiting. The following is presented by way of illustration and is not intended to limit the scope of the disclosure.

[025] In some embodiments, the numerical parameters set forth in the specification (into which the claims are incorporated in their entirety) are approximations that can vary depending upon the desired properties sought to be obtained by a particular embodiment. In some embodiments, the numerical parameters should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some embodiments of the present disclosure are approximations, the numerical values set forth in the specific examples are reported as precisely as practicable. The numerical values presented in some embodiments of the present disclosure may contain certain errors necessarily resulting from the standard deviation found in their respective testing measurements. The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein.

[026] For convenience, certain terms employed in the entire application (including the specification, examples, and appended claims) are collected here. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[027] As used herein, the term “administering” refers to the placement of a composition comprising a modified RNA, into a patient by a method or route that results in at least partial localization of the composition, at a desired site or tissue location. In some embodiments, the composition comprising modified RNA can be administered by any appropriate route that results in effective treatment in the patient, i.e. administration results in delivery to a desired location or tissue in the patient where at least a portion of the protein expressed by the modified RNA is located at a desired target tissue or target cell location.

[028] Administration can be through a portal vein catheter, through a coronary sinus catheter, and/or direct administration into the area to be treated.

[029] The term “composition” used herein is generally understood to mean a combination of at least two parts or elements that make up something. For example, a composition as used herein usually comprises at least a modified RNA according to the disclosure and a suitable carrier or excipient such as citrate saline buffer.

[030] The terms “comprise,” “have” and “include” are open-ended linking verbs. Any forms or tenses of one or more of these verbs, such as “comprises,” “comprising,” “has,” “having,” “includes” and “including,” are also open-ended. For example, any method that “comprises,” “has” or “includes” one or more steps is not limited to possessing only those one or more steps and can also cover other unlisted steps. Similarly, any composition that “comprises,” “has” or “includes” one or more features is not limited to possessing only those one or more features and can cover other unlisted features. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g. “such as”) provided with respect to certain embodiments herein is intended merely to better illuminate the present disclosure and does not pose a limitation on the scope of the present disclosure otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the present disclosure.

[031] The term “consisting essentially of’ limits the scope of a claim to the specified materials or steps “and those that do not materially affect the basic and novel characteristic(s)” of the claimed invention. In re Herz, 537 F.2d 549, 551-52, 190 USPQ 461, 463 (CCPA 1976) (emphasis in original) (Prior art hydraulic fluid required a dispersant which appellants argued was excluded from claims limited to a functional fluid “consisting essentially of’ certain components). [032] The term “consisting of’ refers to compositions, methods, and respective components thereof as described herein, which are exclusive of any element not recited in that description of the embodiment.

[033] Wherever aspects are described herein with the language "comprising," otherwise analogous aspects described in terms of "consisting of and/or "consisting essentially of are also provided.

[034] The terms “disease” or “disorder” are used interchangeably herein, and refers to any alternation in state of the body or of some of the organs, interrupting or disturbing the performance of the functions and/or causing symptoms such as discomfort, dysfunction, distress, or even death to the person afflicted or those in contact with a person. A disease or disorder can also related to a distemper, ailing, ailment, malady, sickness, illness, complaint, indisposition, or affection.

[035] The term “effective amount” as used herein refers to the amount of therapeutic agent (for example, a modified RNA), or composition, sufficient to reduce at least one or more symptom(s) of the disease or disorder, or to provide the desired effect. For example, it can be the amount that effects a therapeutically or prophylactically significant reduction in a symptom or clinical marker associated with a cardiac dysfunction or other disorder when administered to a typical patient who has a cardiovascular condition, or other disease or disorder.

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

[037] The term “pharmaceutical composition” as used herein refers to a type of composition that comprises a pharmaceutical mixture or solution containing an active pharmaceutical ingredient (for example, a modified RNA), together with pharmaceutically acceptable excipients suitable to be administered to a mammal (e.g., a human in need thereof) via a particular route of administration. For example, a “pharmaceutical composition” as used herein can be specifically formulated to include suitable delivery agents and/or other pharmaceutically acceptable carriers for administration via one or more of a number of routes, such as via intramuscular, intradermal, subcutaneous, or intracardiac route, through a portal vein catheter, through a coronary sinus catheter, and/or by direct administration into the area to be treated.

[038] As used herein, the term “modified RNA” refers to RNA molecules containing one, two, or more than two nucleoside modifications comparing to adenosine (A) ((2A,3A,45',5A)-2-(6-amino-9J/-purin-9-yl)-5-(hydroxymethyl)oxolane-3,4-diol), guanosine (G) (2-Amino-9-[3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-3J/-purin-6- one), cytidine (C) (4-amino-l-[3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2- yl]pyrimidin-2-one), and uridine (U) (l-[(3A,45,5A)-3,4-dihydroxy-5- (hydroxymethyl)oxolan-2-yl]pyrimidine-2, 4-dione), or compared to AMP, GMP, CMP, and UMP, in RNA molecules, or a portion thereof. Non-limiting examples of nucleoside modifications are provided elsewhere in this specification. Where the nucleotide sequence of a particular claimed RNA is otherwise identical to the sequence of a naturally-existing RNA molecule, the modified RNA is understood to be an RNA molecule with at least one modification different from those existing in the natural counterpart. The difference can be either in the chemical change to the nucleoside/nucleotide or in the position of that change within the sequence. In one embodiment, the modified RNA is modified messenger RNA (or “modified mRNA”).

[039] As used herein, the term “nucleic acid,” in its broadest sense, includes any compound and/or substance that comprises a polymer of nucleotides linked via a phosphodiester bond. These polymers are often referred to as oligonucleotides or polynucleotides, depending on the size. The terms “polynucleotide sequence” and “nucleotide sequence” are also used interchangeably herein.

[040] The phrase “pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. Drugapproval agencies (e.g., EMA, US-FDA) provide guidance and approve pharmaceutically acceptable compounds, materials, compositions, and/or dosage forms. Examples can be listed in Pharmacopeias.

[041] The phrase “pharmaceutically acceptable excipient” is employed herein to refer to a pharmaceutically acceptable material chosen from a solvent, dispersion media, diluent, dispersion, suspension aid, surface active agent, isotonic agent, thickening or emulsifying agent, preservative, core-shell nanoparticles, polymer, peptide, protein, cell, hyaluronidase, and mixtures thereof. In some embodiments, the solvent is an aqueous solvent. [042] As used herein, “polypeptide” means a polymer of amino acid residues

(natural or unnatural) linked together most often by peptide bonds. The term, as used herein, refers to proteins, polypeptides, and peptides of any size, structure, or function. A polypeptide may be a single molecule or may be a multi-molecular complex such as a dimer, trimer or tetramer. They may also comprise single chain or multichain polypeptides such as antibodies or insulin and may be associated or linked. Most commonly disulfide linkages are found in multichain polypeptides. The term polypeptide may also apply to amino acid polymers in which one or more amino acid residues are an artificial chemical analogue of a corresponding naturally occurring amino acid.

[043] As used herein, “protein” is a polymer consisting essentially of any of the 20 amino acids. Although “polypeptide” is often used in reference to relatively large polypeptides, and “peptide” is often used in reference to small polypeptides, usage of these terms in the art overlaps and is varied. The terms “peptide(s)”, “protein(s)” and “polypeptide(s)” are sometime used interchangeably herein.

[044] The term “recombinant,” as used herein, means that a protein is derived from a prokaryotic or eukaryotic expression system through the use of a nucleic acid that has been genetically manipulated by the introduction of a “heterologous nucleic acid” or the alteration of a native nucleic acid.

[045] The term “statistically significant” or “significantly” refers to statistical significance. The term refers to statistical evidence that there is a difference. It can be defined as the probability of making a decision to reject the null hypothesis when the null hypothesis is actually true. The decision is often made using the p-value. Any other measure of significant significance that is well-known in the art can be used. [046] The term “patient” refers to an animal, for example a human, to whom treatment, including prophylactic treatment, with methods and compositions described herein, is or are provided. For treatment of those conditions or disease states which are specific for a specific animal such as a human patient, the term “patient” refers to that specific animal.

[047] As used herein, a patient who is “suffering from” a disease, disorder, and/or condition has been diagnosed with or displays one or more symptoms of a disease, disorder, and/or condition. In some embodiments, a patient may be at risk of suffering from a disease, disorder and/or condition.

[048] The term “tissue” refers to a group or layer of similarly specialized cells which together perform certain special functions. The term “tissue-specific” refers to a source or defining characteristic of cells from a specific tissue.

[049] As used herein, the terms “treat” or “treatment” or “treating” refers to therapeutic treatment, wherein the object is to prevent or slow the development of the disease, such as slow down the development of a cardiac disorder, or reducing at least one adverse effect or symptom of a vascular condition, disease or disorder, such as, any disorder characterized by insufficient or undesired cardiac function.

[050] It should be understood that this disclosure is not limited to the particular methodology, protocols, and reagents, etc., described herein and as such can vary. The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present disclosure, which is defined solely by the claims. 5.2. Compositions

[051] In one embodiment there is provided a composition for use in the methods disclosed herein, wherein the composition comprises a modified RNA that encodes a VEGF-A polypeptide.

[052] It will be appreciated by those of skill in the art that for any particular VEGF gene there may exist one or more variants or isoforms. Non-limiting examples of VEGF-A polypeptides in accordance with the present disclosure are listed in Table 1. It will be appreciated by those of skill in the art that the sequences disclosed in the Table 1 contain potential flanking regions. These are encoded in each nucleotide sequence either to the 5’ (upstream) or 3’ (downstream) of the open reading frame. The open reading frame is definitively and specifically disclosed by teaching the nucleotide reference sequence. It is also possible to further characterize the 5' and 3' flanking regions by utilizing one or more available databases or algorithms. Databases have annotated the features contained in the flanking regions of the NCBI sequences and these are available in the art.

Table 1: Homo sapiens VEGF-A mRNA isoforms.

[053] It will be appreciated by those of skill in the art that modified RNA encoding a VEGF-A polypeptide, e.g., a human VEGF-A polypeptide, can be designed according to the VEGF-A mRNA isoforms listed in the Table 1. One of ordinary of skill in the art is generally familiar with the multiple isoforms of the remaining VEGF family members.

[054] In one embodiment, the VEGF-A polypeptide comprises an amino acid sequence at least 95%, or at least 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 3, wherein the amino acid sequence has VEGF-A activity. In one embodiment, the VEGF-A polypeptide is VEGF-Aies. In one embodiment, the VEGF-A polypeptide consists of an amino acid sequence identical to SEQ ID NO: 3.

[055] In one embodiment, the modified RNA has an open reading frame (ORF) comprising or consisting of the nucleotide sequence of SEQ ID NO: 2. In one embodiment, the modified RNA comprises a nucleotide sequence at least 95%, or at least 98%, or 100% identical to a nucleotide sequence selected from SEQ ID NOs: 1 and 4-7. In one embodiment, the modified RNA consists of a nucleotide sequence selected from SEQ ID NOs: 1 and 4-7. In one embodiment, the modified RNA consists of the nucleotide sequence of SEQ ID NO: 1. In one embodiment, the modified RNA consists of the nucleotide sequence of SEQ ID NO: 4.

[056] Naturally occurring RNAs are synthesized from four basic ribonucleotides: ATP, CTP, UTP and GTP, but may contain post-transcriptionally modified nucleotides. Further, approximately one hundred different nucleoside modifications have been identified in RNA (Rozenski, J, Crain, P, and McCloskey, J., The RNA Modification Database: 1999 update, Nucl Acids Res, (1999) 27: 196-197).

[057] According to the present disclosure, these RNAs are preferably modified as to avoid the deficiencies of other RNA molecules of the art (e.g., activating the innate immune response and rapid degradation upon administration). Hence, these polynucleotides are referred to as modified RNA. In some embodiments, the modified RNA contains one or more chemical modifications to one or more of the natural nucleotides AMP, CMP, UMP and GMP. In some embodiments, the modified RNA avoids the innate immune response upon administration to a patient. In some embodiments, the half-life of the modified RNA is extended compared to an unmodified RNA.

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

[059] Traditionally, the basic components of an mRNA molecule include at least a coding region (including an open reading frame (ORF)), a 5’ untranslated region (UTR), a 3’ untranslated region (UTR), a 5’ cap and a poly-(A) tail. Building on this wild type modular structure, the present disclosure expands the scope of functionality of traditional mRNA molecules by providing polynucleotides or primary RNA constructs which maintain a modular organization, but which comprise one or more structural and/or chemical modifications or alterations that impart useful properties to the polynucleotide including, in some embodiments, the lack of a substantial induction of the innate immune response of a cell into which the polynucleotide is introduced.

[060] The modified RNAs can include any useful modification relative to the standard RNA nucleotide chain, such as to the sugar, the nucleobase (e.g., one or more modifications of a nucleobase, such as by replacing or substituting an atom of a pyrimidine nucleobase with optionally substituted amino, optionally substituted thiol, optionally substituted alkyl (e.g., methyl or ethyl), or halo (e.g., chloro or fluoro), or the internucleoside linkage (e.g., one or more modification to the phosphodiester backbone). The modified RNAs can optionally include other agents (e.g., RNAi-inducing agents, RNAi agents, siRNA, shRNA, miRNA, antisense RNA, ribozymes, catalytic DNA, tRNA, RNA that induce triple helix formation, aptamers, vectors, etc.).

[061] U.S. Patent Application Publication No. 2014/0073687 discloses exemplary modified RNAs with several useful modifications, for example, at least one or more modified nucleosides chosen from 5-methylcytidine (5mC), N6-methyladenosine (m6A), 3,2’-O-dimethyluridine (m4U), 2-thiouridine (s2U), 2’ fluorouridine, pseudouridine, 2 ’-O-m ethyluridine (Um), 2’ deoxy uridine (2’ dU), 4-thiouridine (s4U), 5 -methyluridine (m5U), 2’-O-methyladenosine (m6A), N6,2’-O-dimethyladenosine (m6Am), N6,N6,2’-O-trimethyladenosine (m62Am), 2’-O-methylcytidine (Cm), 7- methylguanosine (m7G), 2’ -O-m ethyl guanosine (Gm), N2,7-dimethylguanosine (m- 2,7G), N2,N2,7-trimethylguanosine (m-2,2,7G). Additional modifications are described in U.S. Patent Application Publication No. 2015/0051268, filed on October 7, 2014 and U.S. Patent No. 9,061,059, filed on February 3, 2014. Accordingly, all of these modifications are incorporated herein in their entirety by reference. Additional modifications are described herein.

[062] In one embodiment, the present disclosure provides for a modified RNA encoding a VEGF-A polypeptide (e.g., SEQ ID NO: 3). In some embodiments, a modified RNA encodes a VEGF-A polypeptide, wherein the modified RNA comprises a nucleotide sequence selected from SEQ ID NOs: 1 and 4-7. In some embodiments, the modified RNA further comprises a 5’ cap, a 5’ UTR, a 3’ UTR, a poly(A) tail, or any combination thereof. In some embodiments, the 5’ cap, the 5’ UTR, the 3’ UTR, the poly(A) tail, or any combination thereof may include one or more modified nucleotides.

[063] In some embodiments, a modified RNA encoding a VEGF-A polypeptide can include, for example, at least one of the UMP is modified to form Nl- methyl-pseudo-UMP. In some embodiments, the Nl-methyl-pseudo-UMP is present instead of UMP in a percentage of the UMPs in the sequence chosen from 0.1%, 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99.9%, and 100%. In some embodiments, all UMP have been replaced by Nl-methyl-pseudo-UMP.

[064] In some embodiments, the modified RNA encoding a VEGF-A polypeptide can include, for example, at least one of the CMP is modified to form methyl-CMP. In some embodiments, the methyl-CMP is present instead of CMP in a percentage of the CMPs in the sequence chosen from 0.1%, 2%, 3%, 4%, 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99.9%, and 100%. In some embodiments, all CMP have been replaced by 5- methyl-CMP.

[065] In some embodiments, the modified RNA encoding a VEGF-A polypeptide can include, for example, at least one of the AMP is modified to form N6- methyl-AMP. In some embodiments, the N6-methyl-AMP is present instead of AMP in a percentage of the AMPs in the sequence chosen from 0.1%, 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99.9%, and 100%. In some embodiments, all AMP have been replaced by N6- methyl-AMP.

[066] In some embodiments, the modified RNA can include, for example, at least one of the GMP is modified to form 7-methyl-GMP. In some embodiments, the 7- methyl-GMP is present instead of GMP in a percentage of the GMPs in the sequence chosen from 0.1%, 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99.9%, and 100%. In some embodiments, all GMP have been replaced by 7-methyl-GMP.

[067] In some embodiments, the modified RNA encoding a VEGF-A polypeptide can include, for example, at least one or more modified nucleosides chosen from 5-methylcytidine (5mC), N6-methyladenosine (m6A), 3,2’-O-dimethyluridine (m4U), 2-thiouridine (s2U), 2’ fluorouridine, pseudouridine, 2’ -O-m ethyluridine (Um), 2’ deoxy uridine (2’ dU), 4-thiouridine (s4U), 5-methyluridine (m5U), 2’-O- methyladenosine (m6A), N6,2’-O-dimethyladenosine (m6Am), N6,N6,2’-O- trimethyladenosine (m62Am), 2’ -O-m ethylcytidine (Cm), 7-methylguanosine (m7G), 2’- O-methylguanosine (Gm), N2,7-dimethylguanosine (m-2,7G), N2,N2,7- trimethylguanosine (m-2,2,7G), and Nl-methyl-pseudouridine, or any combination thereof. Each possibility and combination represents a separate embodiment of the present disclosure.

[068] In some embodiments, modified RNAs comprise a modification to a 5' cap, such as a 5’ diguanosine cap or 5’ 7-methylguanosine cap. In some embodiments, modified RNAs have a Capl 5’ terminal cap. In some embodiments, modified RNAs comprise a modification to a coding region. In some embodiments, modified RNAs comprise a modification to a 5’ UTR. In some embodiments, modified RNAs comprise a modification to a 3’ UTR. In some embodiments, modified RNAs comprise a modification to a poly-(A) tail. In some embodiments, modified RNAs comprise any combination of modifications to a coding region, 5’ cap, 5’ UTR, 3’ UTR, or poly-(A) tail. In some embodiments, a modified RNA can optionally be treated with an alkaline phosphatase.

[069] In some embodiments, the modified RNA comprises a Capl 5 '-terminal cap, a 5' UTR, the ORF sequence encodes a VEGF-A polypeptide (e.g., consisting of the amino acid sequence of SEQ ID NO: 3), a 3' UTR, and a poly(A) tail (e.g., about 100 nucleotides in length), wherein all uracils in the modified RNA are N1 -methylpseudouracils.

[070] In one embodiment the composition is a pharmaceutical composition further comprising a pharmaceutically acceptable excipient.

[071] In one embodiment, the composition may further comprise a buffer. In one embodiment, the composition may further comprise a citrate saline buffer. In one embodiment, the citrate saline buffer has a pH of pH 6 to pH 7. In one embodiment, the citrate saline buffer has a pH of about pH 6.5. In one embodiment, the citrate saline buffer has a citrate concentration of 5 to 15 mmol/L, optionally about 10 mmol/L. In one embodiment, the citrate saline buffer has a sodium chloride concentration of 100 to 160 mmol/L, optionally about 130 mmol/L. In one embodiment, the citrate saline buffer is a sodium citrate/sodium chloride buffer.

[072] In some embodiments, the composition does not comprise a lipid. In some embodiments, the composition does not comprise a lipid nanoparticle. In some embodiments, the citrate saline buffer is substantially free of divalent cations, including calcium and magnesium. In some embodiments, the citrate saline buffer contains no calcium or magnesium.

5.3. Treating Patients Suffering from Diseases Responsive to VEGF-A Therapy

[073] In one embodiment, there is provided a method of treating dilated cardiomyopathy or heart failure associated with dilated cardiomyopathy in a patient in need thereof comprising administering to the patient an effective amount of a composition comprising a modified RNA in accordance with the present disclosure. In some embodiments, the condition is dilated cardiomyopathy. In some embodiments, the condition is heart failure associated with dilated cardiomyopathy. In some embodiments, the condition is heart failure with reduced ejection fraction (HFrEF) associated with dilated cardiomyopathy.

[074] Heart failure (HF) occurs when the heart is weakened and is not filled with, or cannot pump, enough blood to meet the body's needs for blood and oxygen and the heart itself is perhaps poorly perfused. Patients with dilated cardiomyopathy have heart problems caused by enlargement and weaking of heart muscle resulting in the heart being able to pump blood less effectively. Such conditions are routinely clinically diagnosed based on various physical examinations with confirmation by echocardiography, blood tests, magnetic resonance imaging (MRI) electrocardiography, and other suitable tests. Accordingly, a treatment for any one of these conditions is working if the patient shows less severe symptoms by physical examinations and/or improvements in testing results from echocardiography, blood tests, MRI, electrocardiography, or any other suitable and/or routine tests.

[075] In some embodiments, the patient has a left ventricular ejection fraction (LVEF) of less than or equal to 40% prior to administration of the composition. In some embodiments, the patient has a left ventricular ejection fraction (LVEF) of less than or equal to 35% prior to administration of the composition; optionally less than or equal to 30% prior to administration of the composition; optionally less than or equal to 25% prior to administration of the composition.

[076] In some embodiments, the LVEF of the patient is increased by greater than 10% after administration of the composition. In some embodiments, the LVEF of the patient is increased by 15% or greater after administration of the composition; optionally 20% or greater after administration of the composition. In some embodiments, the increase in LVEF occurs within 6 months after administration of the composition; optionally within 3 months after administration of the composition.

[077] In some embodiments, the LVEF of the patient is increased to 55% or greater after administration of the composition. In some embodiments, the LVEF of the patient is increased to 60% or greater after administration of the composition. In some embodiments, the LVEF of the patient is increased to a normal level after administration of the composition. In some embodiments, the increased LVEF occurs within 6 months after administration of the composition; optionally within 3 months after administration of the composition. [078] In some embodiments, the LVEF of the patient is determined by echocardiography, radionuclide ventriculography, contrast angiography, and/or cardiac MRI. In some embodiments, the LVEF of the patient is determined by echocardiography.

[079] In some embodiments, the method improves the quality of life of the patient as characterised by an increase in one or more patient reported outcome scores relative to before administration of the composition, wherein the patient reported outcome scores are (a) KCCQ Overall Summary Score; (b) SAQ Physical Limitation Score; (c) SAQ Quality of Life Score; and (d) SAQ Treatment Satisfaction Score.

[080] In some embodiments, the method improves the quality of life of the patient as characterised by an increase in the patient’s KCCQ Overall Summary Score relative to before administration of the composition. In some embodiments, the method improves the quality of life of the patient as characterised by an increase in the patient’s SAQ Physical Limitation Score relative to before administration of the composition. In some embodiments, the method improves the quality of life of the patient as characterised by an increase in the patient’s SAQ Quality of Life Score relative to before administration of the composition. In some embodiments, the method improves the quality of life of the patient as characterised by an increase in the patient’s SAQ Treatment Satisfaction Score relative to before administration of the composition.

[081] In some embodiments, the KCCQ Overall Summary Score is increased by at least 5 points relative to before administration of the composition; optionally increased by at least 10 points; optionally increased by at least 12 points; optionally increased by at least 14 points. In some embodiments, the SAQ Physical Limitation Score is increased by at least 10 points relative to before administration of the composition; optionally increased by at least 15 points; optionally increased by at least 20 points; optionally increased by at least 30 points. In some embodiments, the SAQ Quality of Life Score is increased at least 10 points relative to before administration of the composition; optionally at least 20 points; optionally at least 30 points. In some embodiments, the SAQ Treatment Satisfaction Score is increased by at least 5 points relative to before administration of the composition.

[082] In some embodiments, the method improves the quality of life of the patient as characterised by an increase in one or more patient reported outcome scores relative to placebo, wherein the patient reported outcome scores are (a) KCCQ Overall Summary Score; (b) SAQ Physical Limitation Score; (c) SAQ Quality of Life Score; and (d) SAQ Treatment Satisfaction Score.

[083] In some embodiments, the method improves the quality of life of the patient as characterised by an increase in the patient’s KCCQ Overall Summary Score relative to placebo. In some embodiments, the method improves the quality of life of the patient as characterised by an increase in the patient’s SAQ Physical Limitation Score relative to placebo. In some embodiments, the method improves the quality of life of the patient as characterised by an increase in the patient’s SAQ Quality of Life Score relative to placebo. In some embodiments, the method improves the quality of life of the patient as characterised by an increase in the patient’s SAQ Treatment Satisfaction Score relative to placebo.

[084] In some embodiments, the KCCQ Overall Summary Score is increased by at least 5 points relative to placebo; optionally increased by at least 10 points; optionally increased by at least 12 points; optionally increased by at least 14 points. In some embodiments, the SAQ Physical Limitation Score is increased by at least 10 points relative to placebo; optionally increased by at least 15 points; optionally increased by at least 20 points; optionally increased by at least 30 points. In some embodiments, the SAQ Quality of Life Score is increased at least 10 points relative to placebo; optionally at least 20 points; optionally at least 30 points. In some embodiments, the SAQ Treatment Satisfaction Score is increased by at least 5 points relative to placebo.

[085] In some embodiments, the increase in one or more patient reported outcome scores occurs within 6 months after administration of the composition. In some embodiments, the increase in one or more patient reported outcome scores occurs within 3 months after administration of the composition, optionally within 1 month after administration of the composition.

[086] In some embodiments, the patient has a New York Heart Association (NYHA) heart failure classification of I, II, III or IV prior to administration of the composition. In some embodiments, the patient has a NYHA heart failure classification of II, III or IV prior to administration of the composition. In some embodiments, the patient has a NYHA heart failure classification of III or IV prior to administration of the composition. In some embodiments, the patient has a NYHA heart failure classification of II prior to administration of the composition. In some embodiments, the patient has a NYHA heart failure classification of III prior to administration of the composition. In some embodiments, the patient has a NYHA heart failure classification of IV prior to administration of the composition.

[087] In some embodiments, the patient’s NYHA heart failure classification improves within 6 months after administration of the composition, optionally within 3 months after administration of the composition, optionally within 1 month after administration of the composition. In some embodiments, the patient has a NYHA heart failure classification of I or II within 6 months after administration of the composition, optionally within 3 months after administration of the composition, optionally within 1 month after administration of the composition. In some embodiments, the patient has a NYHA heart failure classification of I within 6 months after administration of the composition, optionally within 3 months after administration of the composition, optionally within 1 month after administration of the composition.

[088] In some embodiments, the patient has a Canadian Cardiovascular Society (CCS) angina classification of I, II, III or IV prior to administration of the composition. In some embodiments, the patient has a CCS angina classification of II, III or IV prior to administration of the composition. In some embodiments, the patient has a CCS angina classification of III or IV prior to administration of the composition. In some embodiments, the patient has a CCS angina classification of II prior to administration of the composition. In some embodiments, the patient has a CCS angina classification of III prior to administration of the composition. In some embodiments, the patient has a CCS angina classification of IV prior to administration of the composition.

[089] In some embodiments, the patient’s CCS angina classification improves within 6 months after administration of the composition, optionally within 3 months after administration of the composition, optionally within 1 month after administration of the composition. In some embodiments, the patient has a CCS angina classification of I or II within 6 months after administration of the composition, optionally within 3 months after administration of the composition, optionally within 1 month after administration of the composition. In some embodiments, the patient has a CCS angina classification of I within 6 months after administration of the composition, optionally within 3 months after administration of the composition, optionally within 1 month after administration of the composition.

[090] In some embodiments, the patient has a coronary flow reserve of less than 2.5 prior to administration of the composition. In some embodiments, the patient has a coronary flow reserve of less than 2.0 prior to administration of the composition.

[091] In some embodiments, the composition is administered to the patient by epicardial injection. In some embodiments, the composition is administered to the patient by epicardial injection into the left ventricle. In some embodiments, the composition is administered to the patient by epicardial injection at a fixed-dosage in multiple administrations. In some embodiments, the composition is administered to the patient by epicardial injection at a fixed-dosage in 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 or more administrations. In some embodiments, the composition is administered to the patient by epicardial injection at a fixed-dosage in 30 administrations. The “multiple administrations” can be separated from each other by short (1 second to 1 minute) medium (more than 1 minute to 30 minutes), or long (more than 30 minutes to 1 hour) intervals of time.

[092] The composition may be administered to the patient using any amount of the composition effective for treating a disease, disorder, and/or condition. The exact amount required will vary from patient to patient, depending on the species, age, and general condition of the patient, the severity of the disease, the particular composition, its mode of administration, its mode of activity, and the like. It will be understood, however, that the total daily usage of the compositions may be decided by the attending physician within the scope of sound medical judgment. The specific pharmaceutically effective, dose level for any particular patient will depend upon a variety of factors including the disease being treated and the severity of the disease; the activity of the specific compound employed; the specific formulation employed; the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs (for example, a modified RNA) used in combination or coincidental with the specific compound employed; and like factors well known in the medical arts.

[093] In some embodiments, the concentration of the modified RNA in the composition is between 0.1 and 5 mg/mL. In some embodiments, the concentration of the modified RNA in the composition is between 0.5 and 5 mg/mL. In some embodiments, the concentration of the modified RNA in the composition is about 0.5 mg/mL. In some embodiments, the concentration of the modified RNA in the composition is about 5 mg/mL.

[094] In some embodiments, the composition may be administered at fixed- dosage levels of modified RNA. For example, the composition may be administered at fixed-dosage levels from about 0.1 mg to about 1 mg of modified RNA, per administration. In some embodiments, the composition is administered at a fixed-dosage level of about 0.1 mg of modified RNA per administration. In some embodiments, the composition is administered at a fixed-dosage level of about 1 mg of modified RNA per administration. In some embodiments, the composition is administered at a fixed-dosage level of about 1 mg to about 30 mg total dose of modified RNA. In some embodiments, the composition is administered at a fixed-dosage level of about 3 mg to about 30 mg total dose of modified RNA. In some embodiments, the composition is administered at a fixed-dosage level of about 3 mg total dose of modified RNA. In some embodiments, the composition is administered at a fixed-dosage level of about 30 mg total dose of modified RNA. In some embodiments, the composition is administered as about 30 administrations of about 0.1 mg of modified RNA per administration.

[095] All of the claims in the claim listing are herein incorporated by reference into the specification in their entireties as additional embodiments.

6. EXAMPLES

EXAMPLE 1

EPICCURE Phase 2a Clinical Trial Results

Overview

[096] EPICCURE (ClinicalTrials.gov: NCT03370887) was a randomized, placebo-controlled, double-blind, multicenter, 6-month, phase 2a clinical trial of the safety, tolerability and exploratory efficacy of epicardial injections of a modified mRNA that encodes a VEGF-A polypeptide in patients with stable coronary artery disease and moderately decreased left ventricular ejection fraction (LVEF) who are undergoing coronary artery bypass grafting (CABG) surgery. In this Example, results from the EPICCURE phase 2a clinical study are provided.

VEGF-A modified mRNA

[097] The VEGF-A mRNA referred to in this Example is modified mRNA

(SEQ ID NO: 1) encoding VEGF-Aies (SEQ ID NO: 3), and was manufactured in compliance with current Good Manufacturing Practices (GMP) by in vitro transcription using a linearized plasmid DNA template, nucleotide triphosphates and purified enzymes, followed by purification and filtration. The VEGF-A mRNA was used in this Example as a citrate/saline solution (pH 6.5, 10 mmol/L sodium citrate, 130 mmol/L sodium chloride) at a concentration of 0.5 mg/mL VEGF-A mRNA. The composition of the formulation is as described in Table 2.

Table 2: VEGF-A mRNA composition

Ethics and Conduct

[098] EPICCURE is registered on ClinicalTrials.gov (identifier: NCT03370887), and conformed to the principles of the Declaration of Helsinki, the International Conference on Harmonisation Good Clinical Practice, the AstraZeneca policy on Bioethics and Human Biological Samples and all applicable regulatory requirements. Local ethics committees reviewed and approved the study protocol, and participants give their written informed consent before study enrollment. A 10-minute extension of cardioplegia was considered ethically acceptable to allow epicardial injections after completion of the peripheral anastomoses.

Objectives [099] The primary objective of EPICCURE was to assess the safety and tolerability of VEGF-A mRNA. Primary outcome measures included the following:

• Serious adverse events

• Adverse events

• Vital signs (blood pressure, pulse, pulse oximetry)

• ECG

• Physical examination

• Laboratory assessments (hematology, clinical chemistry and urinalysis)

• Hemopericardium and/or tamponade assessment, and LVEF with echocar di ography

• Coagulation and use of concomitant medication

[0100] Exploratory objectives included the following:

• To assess the effect of VEGF-A mRNA in patients undergoing CABG surgery on regional and global sMBF measured with ¹⁵O-water PET imaging.

• To assess the effect of VEGF-A mRNA on global LVEF by echocar di ography .

• To assess the effect of VEGF-A mRNA on regional myocardial wall motion measured by echocardiography and strain analysis in patients undergoing CABG surgery

• To assess the effect of VEGF-A mRNA on clinical symptoms in terms of New York Heart Association (NYHA) class, Seattle Angina Questionnaire (SAQ) and Kansas City Cardiomyopathy Questionnaire (KCCQ) Participants

• For inclusion in the study patients should have fulfilled the following criteria at Visit 1 and/or 2:

■ Provision of signed and dated informed consent prior to any study specific procedures;

• Males and females:

• a. Males must be surgically sterile or using an acceptable method of contraception

• b. Females must be of non-childbearing potential confirmed at screening by fulfilling one of the following criteria a) postmenopausal defined as amenorrhoea for at least 12 months or more following cessation of all exogenous hormonal treatments and follicle-stimulating hormone (FSH) levels in the postmenopausal range, b) documentation of irreversible surgical sterilisation by hysterectomy, bilateral oophorectomy or bilateral salpingectomy but not tubal ligation

• Age >18 years

• Indication for elective CABG surgery enrolled at least 15 days before the planned surgery

• Moderately reduced global LVEF at rest (30% < LVEF < 50%) from medical records

• If patient is on statin, ACE inhibitor/ ARB, and/or beta-blocker, the dose should be stable at least 2 weeks prior to Visit 1

• Patients who are blood donors should not donate blood during the study and for 3 months following their last dose of VEGF-A mRNA.

Exclusion criteria [0101] Patients should not enter the study if any of the following exclusion criteria are fulfilled:

• Involvement in the planning and/or conduct of the study

• Previous randomisation in the present study

• Participation in another clinical study with an investigational product during the last 3 months

• BMI > 35 kg/m² OR poor image window for echocardiography

• Need for CABG emergency operation. (Emergency operation is defined as significant symptom status worsening in CAD, such as crescendo angina, unstable angina or ACS requiring rescheduling the revascularization. CAD should be stable at least 3 months prior to Visit 3.)

• History of ventricular arrhythmia (> Lown III) without Implantable Cardiac Defibrillator (ICD)

• History of any clinically significant disease or disorder which, in the opinion of the PI, may either put the patient at risk because of participation in the study, or influence the results or the patient’s ability to participate in the study

• Severe co-morbidities that can interfere with the execution of the study, interpretation of study results or affect the safety of the patient, in judgement of the investigator

• eGFR < 30 mL/min (derived from creatinine clearance, calculated by local lab)

• For CFVR (Visit 1) and sMBF (Visit 2) measurement: o Known severe adverse reactions to adenosine o Known elevated intracranial pressure o AV block > second degree and/or sick sinus syndrome in patient without pacemaker o Heart rate < 40 bpm (ECG verified) o Systolic blood pressure < 90 mmHg o Asthma or COPD with strong reactive component in judgement of investigator o Treatment with dipyridamole (e.g. Persantin or Asasantin), theophyllamine or fluvoxamine that cannot be paused

• Inability to comply with the protocol

• History of severe allergy/hypersensitivity or ongoing clinically important allergy /hypersensitivity to drugs with a similar chemical structure or class as study drugs

• Patients unable to give their consent or communicate reliably with the investigator or vulnerable patients e.g., kept in detention, protected adults under guardianship, trusteeship, or committed to an institution by governmental or juridical order

• Positive hepatitis C antibody hepatitis B virus surface antigen or hepatitis B virus core antibody or human immunodeficiency virus, at Visit 1

• Known history of drug or alcohol abuse

• Any concomitant medications that are known to be associated with Torsades de Pointes

• History of QT prolongation associated with other medications that required discontinuation of that medication

Congenital long QT syndrome • History of arrhythmia (multifocal premature ventricular contractions, bigeminy, trigeminy, ventricular tachycardia), which is symptomatic or requires treatment (CTCAE Grade 3).

• Current atrial fibrillation as well as paroxysmal atrial fibrillation.

Randomization and Blinding

[0102] 11 participants were enrolled into the study. Enrolled participants were randomized to receive VEGF-A mRNA (3 mg total dose) or placebo in a ratio of 2: 1 at least 14 days before coronary artery bypass grafting. Within the cohort, two sentinel participants were randomized 1 : 1 to VEGF-A mRNA or placebo, followed by another two sentinel participants randomized 1 : 1 to VEGF-A mRNA or placebo, followed by the remaining seven participants randomized 3 : 1 to VEGF-A mRNA (n = 5) or placebo (n = 2). In total, 7 patients received VEGF-A mRNA (3 mg total dose), and 4 patients received placebo (n = 4). Safety data from up to 1 month after administration were reviewed by a safety review committee before the treatment of participants in the next sentinel subgroup.

[0103] Participants and the study team remained blinded to treatment assignments throughout the study. Investigators remained blinded unless they needed to know a patient’s assigned treatment in a medical emergency. The safety review committee was unblinded. Placebo solution for injection matched the appearance of VEGF-A mRNA, so the identity of the treatment cannot be discerned.

Procedures

[0104] The Schedule of assessments for enrollment, treatment and follow-up periods is described in Table 3: Table 3: Schedule of assessments for enrollment, treatment and follow-up periods

[0105] At baseline (Visit 2), participants had a ¹⁵O-water PET scan to quantify pre-operative myocardial blood flow at rest and during adenosine stress. This was paired with the baseline contrast-enhanced coronary computed tomography (CT) angiogram to generate a perfusion map that combines coronary anatomy and myocardial blood flow. Ischemic regions were identified as those with stress myocardial blood flow below 2.3 mL/g/min (± 0.3 mL/g/min), or below 80% (± 10%) of the segment with highest stress myocardial blood flow on ¹⁵O-water PET (see Kajander, SA et al, Circ Cardiovasc

31 Imaging, 2011, 4, 678-684; and Danad, I et al., J Am Coll Cardiol, 2014, 64, 1464- 1475). Target regions of ischemic but viable myocardium were identified as ischemic regions with resting myocardial blood flow above 0.6 mL/g/min (i.e. >60% of normal myocardium resting blood flow of 1 mL/g/min) on ¹⁵O-water PET. On the individualized injection map, 30 epicardial injection sites with approximately 1 cm spacing were identified within the target regions (Fig. 9B provides an example of such an injection map).

[0106] During surgery, participants received 30 epicardial injections of 200 pL volume each, guided by their individualized injection map to target ischemic but viable myocardial regions. Injections were given under cardioplegia immediately after bypass grafting and before reperfusion. Participants received their randomly assigned treatment of VEGF-A mRNA (3 mg total of VEGF-A mRNA; 0.1 mg per injection site; 0.5 mg/mL solution), or matching placebo.

[0107] Injections were performed using a 30G (approximately 0.3 mm diameter) x 13 mm hypodermic needle coupled to a 1 mL syringe. Comments were recorded on the presence or absence of potential or suspected perforation, potential sustained bleeding at the injection site, and other adverse events.

[0108] Patients remained in hospital for at least 4 days after surgery, and then attended follow-up Visits 5, 6 and 7 to assess safety and efficacy. Adverse events were also assessed by telephone interviews at Visit 4.

Safety Outcomes

[0109] Safety outcomes included monitoring of adverse events and serious adverse events, physical examinations, electrocardiography, monitoring of vital signs (blood pressure, pulse, oxygen saturation), laboratory assessments, and echocardiographic assessment of hemopericardium, tamponade, and left ventricular ejection fraction. Adverse events were recorded from coronary artery bypass grafting until the end of follow-up and serious adverse events were recorded from informed consent until the end of follow-up. All adverse events were followed up by the investigator until resolved or for as long as medically indicated. Investigators assessed whether adverse events were causally related to the investigational medical product and also whether serious adverse events were causally related to other medications, to study procedures and to the drug injection procedure.

Exploratory Outcomes

Cardiac Imaging

[0110] Regional and global stress myocardial blood flow and myocardial flow reserve (stress-to-rest ratio in myocardial blood flow) was assessed using ¹⁵O-water PET at baseline (Visit 2) and at Visits 5 and 6 (see Kajander, SA et al, Circ Cardiovasc Imaging, 2011, 4, 678-684; and Danad, I et al., J Am Coll Cardiol, 2014, 64, 1464- 1475). Participants also had follow-up CT angiograms 3 months after surgery.

[0111] Regional wall motion, global longitudinal strain, and cardiac volumes were assessed using comprehensive echocardiography at baseline (Visit 1) and at Visits 5, 6 and 7. Comprehensive echocardiographic examination was performed with patients in the left recumbent position at rest. Standard clinical cardiac transducers were used for B-mode, colour Doppler, and tissue Doppler imaging. CINE-loops (a CINE-loop is a period of images, stored digitally as a sequence of individual frames) of the parasternal long- and short- axis views, apical 4-, 2-, and 3-chamber views, and subcostal views were obtained and stored for off-line analysis of cardiac structure and function. Doppler and tissue Doppler echocardiography was performed to obtain indices necessary for comprehensive assessment of left ventricular (LV) diastolic function and non-invasive hemodynamic measurements.

Clinical and Functional Outcomes

[0112] Patients completed the Kansas City Cardiomyopathy Questionnaire (Green, CP et al., J Am Coll Cardiol 2000, 35, 1245-1255) and the Seattle Angina Questionnaire (Spertus, JA et al, J Am Coll Cardiol 1995, 25, 333-341), and investigators performed New York Heart Association classifications (The Criteria Committee of the New York Heart Association (1994). Nomenclature and Criteria for Diagnosis of Diseases of the Heart and Great Vessels (9th Ed.). Little, Brown & Co.: Boston) at baseline (Visit 1) and at Visits 5, 6 and 7 after surgery.

RESULTS

[0113] The characteristics of the patients in the clinical study are described in

Table 4:

Table 4 - Patient characteristics:

Study Outcomes

Safety

[0114] No deaths were reported during the study. Two serious adverse events were recorded in the group receiving VEGF-A mRNA, but neither of these serious adverse events was related to the drug. No serious adverse events were recorded in the placebo group. No unexpected adverse events were observed in the group receiving VEGF-A mRNA. Ventricular arrhythmia was observed in one patient in the group receiving VEGF-A mRNA, but this was prior to giving the drug and related to acute thrombosis in a grafted artery. No severe arrhythmia was otherwise observed.

Exploratory outcomes

[0115] Change in LVEF (LSMean) from baseline was greater in the group receiving VEGF-A mRNA (3 mg) by 9.367% relative to placebo (95% CI, -2.251 to 20.986; P, 0.108). LVEF change from baseline for each patient group is shown in Table

5. LVEF was measured by echocardiography using Simpson’s biplane method.

Table 5: Echocardiography assessment: left ventricular ejection fraction

(LVEF) at Visit 7, mixed model repeated measures (MMRM)

[0116] Fig. 1 shows the LVEF (Echocardiography (rest/hyperaemia), Biplane Simpson) of individual patients in the study as measured at Visit 1 (baseline) and at Visits 5, 6 and 7.

[0117] Change in global stress myocardial blood flow (as measured by ¹⁵O- water PET) was less in the group receiving VEGF-A mRNA (3 mg) by 0.3075 mL/g/min relative to placebo (95% CI, -0.8788 to 0.2639; P, 0.254). Change in global stress myocardial blood flow from baseline for each patient group is shown in Table 6.

Table 6: ¹⁵O-water PET assessment: global stress myocardial blood flow

(gMBFs) at Visit 6, mixed model repeated measures (MMRM)

[0118] Fig. 2 shows the ¹⁵O-water PET global stress myocardial blood flow of individual patients in the study as measured at Visit 2 (baseline) and at Visits 5 and 6.

[0119] Change in regional stress myocardial blood flow (as measured by ¹⁵O- water PET) in the treated area was similar for the group treated with VEGF-A mRNA (3 mg) relative to placebo (LSmean of difference = 0.0272 mL/g/min; 95% CI, -0.5912 to 0.6456; P, 0.923). Change in regional stress myocardial blood flow in the treated area from baseline for each patient group is shown in Table 7.

Table 7: ¹⁵O-water PET assessment: regional stress myocardial blood flow

(tMBFs) in the treated area at Visit 6, mixed model repeated measures

(MMRM)

[0120] Fig. 3 shows the ¹⁵O-water PET regional stress myocardial blood flow in the treated area of individual patients in the study as measured at Visit 2 (baseline) and at Visits 5 and 6.

[0121] Fig. 4 shows the global rest myocardial blood flow of individual patients in the study as measured at Visit 2 (baseline) and at Visits 5 and 6.

[0122] Fig. 5 shows the regional rest myocardial blood flow in the treated area of individual patients in the study as measured at Visit 2 (baseline) and at Visits 5 and 6.

[0123] Fig. 6 shows the treated area coronary flow reserve of individual patients in the study as measured at Visit 2 (baseline) and at Visits 5 and 6.

Patient Reported Outcomes

[0124] Patients completed the Kansas City Cardiomyopathy Questionnaire and the Seattle Angina Questionnaire at baseline (Visit 1) and at Visits 5, 6 and 7 after surgery. The Physical Limitation Scores (PLS), Treatment Satisfaction Scores (TSS), Quality of Life Scores (QoLS), Angina Stability Scores (ASS) and Angina Frequency Scores (AFS) from the Seattle Angina Questionnaires are provided in Table 8, Table 9, Table 10, Table 11 and Table 12, respectively. The Overall Summary Scores from the Kansas City Cardiomyopathy Questionnaires are presented in Table 13. Table 8: SAQ Physical limitation score (PLS):

Table 9: SAQ Treatment satisfaction score (TSS):

Table 10: SAQ Quality of life score (QoLS):

Table 11: SAQ Angina stability score (ASS):

Table 12: SAQ Angina frequency score (ATS):

[0125] The mean SAQ Physical limitation score (PLS), SAQ Treatment satisfaction score (TSS), SAQ Quality of life score (QoLS) and SAQ Angina stability score (ASS) over time are presented in Figs. 7A, 7B, 7C and 7D, respectively.

Table 13: KCCQ Overall summary score:

[0126] The mean KCCQ Overall summary score over time is presented in Fig.

8.

Patient 1

[0127] One patient in the study had a LVEF of less than 40% at baseline (Visit 1), and this patient received treatment with VEGF-A mRNA (3 mg). This patient is identified as “Patient 1” herein, and the data corresponding to Patient 1 are marked in bold in Figs. 1-6.

[0128] Patient 1 is a 61-years old male with coronary artery disease, an exsmoker, and had hypertension and positive family history. Patient 1 also had a history of pulmonary emphysema and impaired lung functions.

[0129] A coronary angiogram of Patient 1 indicated significant stenosis in LAD ostium, LCX and chronic total occlusion in RCA. In a preoperative echo study, the LVEF was 30-35%, and the low LVEF was due to akinetic anterior wall and anterior septum. Patient l’s ECG revealed signs of previous anterior infarct.

[0130] Fig. 9A is the ¹⁵O-water PET image from Patient 1 recorded at Visit 2 showing the pre-operative suggested area for treatment. Fig. 9B is the ¹⁵O-water PET image showing the injection map for Patient 1. The injections (30 points between the LAD and the LOM coronary arteries) were spaced about 1 cm from each other in midmyocardium, but not in the ventricular septum (the injected area was within the following left ventricular segments: basal anterior, basal inferolateral, basal anterolateral, mid anterior, mid inferolateral and mid anterolateral). The area with most damage (apex and ventricular septum) were not treated directly by injections as this area is not accessible during CABG surgery.

[0131] Following surgery, a remarkable increase was observed in the LVEF of Patient 1, increasing from 26.92% at Baseline (Visit 1), to 39.62% at 1 month post treatment (Visit 5), 47.63% at 3 months post treatment (Visit 6), and 59.48% at 6 months post treatment (Visit 7). The increase in the LVEF of Patient 1 over time is illustrated in Fig. 11 A. The increase in LVEF of Patient 1 is particularly remarkable since it is known that LVEF is not necessarily increased by CABG surgery (Adachi et al. Int Heart J, 2016, 57(5), 565-72).

[0132] The ¹⁵O-water PET blood flow measurements for Patient 1 at Baseline (Visit 2), Visit 5 and Visit 6 are shown in Table 14. The observed global myocardial blood flow at rest and stress, and the observed myocardial blood flow in the treated area at rest and stress changed from severely reduced to moderately reduced. There was an increase from baseline, but not of the magnitude to explain the significant increase in LVEF of Patient 1. This suggests that cardiomyocyte proliferation is providing a significant contribution to the increase in cardiac function of Patient 1, in addition to angiogenesis. [0133] Accordingly, VEGF-A mRNA may have utility in the treatment of dilated cardiomyopathy (a non-ischemic cardiomyopathy where improved blood flow may be less beneficial), in which an increase in cardiac function by cardiomyocyte proliferation would be desirable, particularly for patients with dilated cardiomyopathy with HFrEF.

Table 14: ¹⁵O-water PET blood flow measurements for Patient 1

[0134] Fig. 10A shows the ¹⁵O-water PET myocardial blood flow (divided into segments of the left ventricle) of Patient 1 at Baseline (Visit 2) at rest (left image) and under stress (right image). Fig. 10B shows the ¹⁵O-water PET myocardial blood flow (divided into segments of the left ventricle) of Patient 1 at 3 months after surgery (Visit 6) at rest (left image) and under stress (right image).

[0135] The left-ventricular regional wall motion of Patient 1 was recorded by echocardiography, and scored according to: 0 = Unable to score; 1 = Normal; 2 = Hypokinetic; 3 = Akinetic; 4 = Dyskinetic; and 5 = Aneurysmal. The regional wall motion scores at Baseline (Visit 1), 3 months after treatment (Visit 6) and 6 months after treatment (Visit 7) are provided in Table 15.

Table 15: Left-ventricular regional wall motion scores for Patient 1

[0136] Patient 1 completed the Kansas City Cardiomyopathy Questionnaire and the Seattle Angina Questionnaire at baseline (Visit 1) and at Visits 5, 6 and 7 after surgery. The scores from the two questionnaires are provided in Table 16.

Table 16: KCCQ and SAQ scores for Patient 1 at Visits 1, 5, 6 and 7

[0137] The KCCQ and SAQ scores of Patient 1 over time are illustrated in Fig. 11B.

[0138] Investigators performed New York Heart Association classifications for Patient 1 at baseline (Visit 1) and at Visits 5, 6 and 7 after surgery. The NYHA score of Patient 1 was III at Visit 1; I at Visit 5; I at Visit 6; and II at Visit 7.

7. SEQUENCE LISTING

[0139] SEQ ID NO: 1: Modified RNA encoding VEGF-A used in Example

1: 5 ’ ^7MeGpp_PG2’OMeGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGA GCCACCAUGAACUUUCUGCUGUCUUGGGUGCAUUGGAGCCUUGCCUUGCU GCUCUACCUCCACCAUGCCAAGUGGUCCCAGGCUGCACCCAUGGCAGAAGG AGGAGGGCAGAAUCAUCACGAAGUGGUGAAGUUCAUGGAUGUCUAUCAGC GCAGCUACUGCCAUCCAAUCGAGACCCUGGUGGACAUCUUCCAGGAGUACC CUGAUGAGAUCGAGUACAUCUUCAAGCCAUCCUGUGUGCCCCUGAUGCGA UGCGGGGGCUGCUGCAAUGACGAGGGCCUGGAGUGUGUGCCCACUGAGGA GUCCAACAUCACCAUGCAGAUUAUGCGGAUCAAACCUCACCAAGGCCAGCA CAUAGGAGAGAUGAGCUUCCUACAGCACAACAAAUGUGAAUGCAGACCAA AGAAAGAUAGAGCAAGACAAGAAAAUCCCUGUGGGCCUUGCUCAGAGCGG AGAAAGCAUUUGUUUGUACAAGAUCCGCAGACGUGUAAAUGUUCCUGCAA AAACACAGACUCGCGUUGCAAGGCGAGGCAGCUUGAGUUAAACGAACGUA CUUGCAGAUGUGACAAGCCGAGGCGGUGAUAAUAGGCUGGAGCCUCGGUG GCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGC ACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGCAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAUCUAGo H3’ (SEQ ID NO: 1)

Wherein:

A, C, G & U= AMP, CMP, GMP & Nl-methyl-pseudoUMP, respectively Me = methyl p = inorganic phosphate

[0140] SEQ ID NO: 2: ORF of modified RNA encoding VEGF-A used in the Examples (excluding the stop codon):

AUGAACUUUCUGCUGUCUUGGGUGCAUUGGAGCCUUGCCUUGCUGCUCUA CCUCCACCAUGCCAAGUGGUCCCAGGCUGCACCCAUGGCAGAAGGAGGAGG GCAGAAUCAUCACGAAGUGGUGAAGUUCAUGGAUGUCUAUCAGCGCAGCU ACUGCCAUCCAAUCGAGACCCUGGUGGACAUCUUCCAGGAGUACCCUGAUG AGAUCGAGUACAUCUUCAAGCCAUCCUGUGUGCCCCUGAUGCGAUGCGGG GGCUGCUGCAAUGACGAGGGCCUGGAGUGUGUGCCCACUGAGGAGUCCAA CAUCACCAUGCAGAUUAUGCGGAUCAAACCUCACCAAGGCCAGCACAUAGG AGAGAUGAGCUUCCUACAGCACAACAAAUGUGAAUGCAGACCAAAGAAAG AUAGAGCAAGACAAGAAAAUCCCUGUGGGCCUUGCUCAGAGCGGAGAAAG CAUUUGUUUGUACAAGAUCCGCAGACGUGUAAAUGUUCCUGCAAAAACAC AGACUCGCGUUGCAAGGCGAGGCAGCUUGAGUUAAACGAACGUACUUGCA GAUGUGACAAGCCGAGGCGG (SEQ ID NO: 2)

Wherein:

A, C, G & U= AMP, CMP, GMP & Nl-methyl-pseudoUMP, respectively [0141] SEQ ID NO: 3: Amino acid sequence of human VEGF-A isoform

VEGF-A165

MNFLLSWVHWSLALLLYLHHAKWSQAAPMAEGGGQNHHEVVKFMDVYQRSY CHPIETLVDIFQEYPDEIEYIFKPSCVPLMRCGGCCNDEGLECVPTEESNITMQIMR IKPHQGQHIGEMSFLQHNKCECRPKKDRARQENPCGPCSERRKHLFVQDPQTCK CSCKNTDSRCKARQLELNERTCRCDKPRR (SEQ ID NO: 3)

[0142] SEQ ID NO: 4: A modified RNA encoding VEGF-A

5 ’ ^7MeGp_PP A2’o_MeGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGA GCCACCAUGAACUUUCUGCUGUCUUGGGUGCAUUGGAGCCUUGCCUUGCU GCUCUACCUCCACCAUGCCAAGUGGUCCCAGGCUGCACCCAUGGCAGAAGG AGGAGGGCAGAAUCAUCACGAAGUGGUGAAGUUCAUGGAUGUCUAUCAGC GCAGCUACUGCCAUCCAAUCGAGACCCUGGUGGACAUCUUCCAGGAGUACC CUGAUGAGAUCGAGUACAUCUUCAAGCCAUCCUGUGUGCCCCUGAUGCGA UGCGGGGGCUGCUGCAAUGACGAGGGCCUGGAGUGUGUGCCCACUGAGGA GUCCAACAUCACCAUGCAGAUUAUGCGGAUCAAACCUCACCAAGGCCAGCA CAUAGGAGAGAUGAGCUUCCUACAGCACAACAAAUGUGAAUGCAGACCAA AGAAAGAUAGAGCAAGACAAGAAAAUCCCUGUGGGCCUUGCUCAGAGCGG AGAAAGCAUUUGUUUGUACAAGAUCCGCAGACGUGUAAAUGUUCCUGCAA AAACACAGACUCGCGUUGCAAGGCGAGGCAGCUUGAGUUAAACGAACGUA CUUGCAGAUGUGACAAGCCGAGGCGGUGAUAAUAGGCUGGAGCCUCGGUG GCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGC ACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGCAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAUCUAGo H3’ (SEQ ID NO: 4)

Wherein:

[0143] SEQ ID NO: 5: A modified RNA encoding VEGF-A

5 ’ ^7MeGpp_PG2’OMe AGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAG

AGCCACCAUGAACUUUCUGCUGUCUUGGGUGCAUUGGAGCCUUGCCUUGC

UGCUCUACCUCCACCAUGCCAAGUGGUCCCAGGCUGCACCCAUGGCAGAAG GAGGAGGGCAGAAUCAUCACGAAGUGGUGAAGUUCAUGGAUGUCUAUCAG CGCAGCUACUGCCAUCCAAUCGAGACCCUGGUGGACAUCUUCCAGGAGUAC

CCUGAUGAGAUCGAGUACAUCUUCAAGCCAUCCUGUGUGCCCCUGAUGCG

AUGCGGGGGCUGCUGCAAUGACGAGGGCCUGGAGUGUGUGCCCACUGAGG

AGUCCAACAUCACCAUGCAGAUUAUGCGGAUCAAACCUCACCAAGGCCAGC

ACAUAGGAGAGAUGAGCUUCCUACAGCACAACAAAUGUGAAUGCAGACCA

AAGAAAGAUAGAGCAAGACAAGAAAAUCCCUGUGGGCCUUGCUCAGAGCG

GAGAAAGCAUUUGUUUGUACAAGAUCCGCAGACGUGUAAAUGUUCCUGCA

AAAACACAGACUCGCGUUGCAAGGCGAGGCAGCUUGAGUUAAACGAACGU

ACUUGCAGAUGUGACAAGCCGAGGCGGUGAUAAUAGGCUGGAGCCUCGGU

GGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCU

GCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGCAAAAA

AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAUCU

AGOH3’ (SEQ ID NO: 5)

Wherein:

A, C, G & U= AMP, CMP, GMP & Nl-methyl-pseudoUMP, respectively

Me = methyl p = inorganic phosphate

[0144] SEQ ID NO: 6: A modified RNA encoding VEGF-A

5 ’ ^7MeGpp_PGGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCC

ACCAUGAACUUUCUCCUUUCUUGGGUGCAUUGGAGCCUUGCCUUGUUACU

CUACCUCCACCACGCCAAGUGGUCCCAGGCCGCACCCAUGGCAGAAGGAGG

AGGGCAGAAUCAUCACGAAGUGGUGAAGUUCAUGGACGUCUAUCAGCGCA

GCUACUGCCAUCCAAUCGAGACACUGGUGGACAUCUUCCAGGAGUACCCUG

AUGAGAUCGAGUACAUCUUCAAGCCAUCCUGUGUGCCCCUGAUGCGAUGC

GGCGGCUGCUGCAAUGACGAGGGCCUGGAGUGUGUGCCUACUGAGGAGUC

CAACAUCACCAUGCAGAUUAUGCGGAUCAAACCUCACCAAGGCCAGCACAU

AGGAGAGAUGAGCUUCCUACAGCACAACAAAUGUGAAUGCAGACCAAAGA

AAGAUAGAGCAAGACAAGAGAAUCCCUGUGGGCCUUGCUCAGAGCGGAGA

AAGCAUUUGUUUGUACAAGAUCCGCAGACGUGUAAAUGUUCCUGCAAGAA

CACAGACUCGCGUUGCAAGGCGAGGCAGCUUGAGUUAAACGAACGUACUU

GCAGAUGUGACAAGCCGAGGCGGUGAUAAUAGGCUGGAGCCUCGGUGGCC

AUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACC

CGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGCAAAAAAAAAA

AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAUCUAG3’

(SEQ ID NO: 6)

Wherein: A, C, G & U= AMP, CMP, GMP & Nl-methyl-pseudoUMP, respectively

Me = methyl p = inorganic phosphate

[0145] SEQ ID NO: 7: A modified RNA encoding VEGF-A

5 ’ ^7MeGp_PP AGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAA AUAUAAGAGCC

ACCAUGAACUUUCUGCUGUCUUGGGUGCAUUGGAGCCUUGCCUUGCUGCU

CUACCUCCACCAUGCCAAGUGGUCCCAGGCUGCACCCAUGGCAGAAGGAGG

AGGGCAGAAUCAUCACGAAGUGGUGAAGUUCAUGGAUGUCUAUCAGCGCA

GCUACUGCCAUCCAAUCGAGACCCUGGUGGACAUCUUCCAGGAGUACCCUG

AUGAGAUCGAGUACAUCUUCAAGCCAUCCUGUGUGCCCCUGAUGCGAUGC

GGGGGCUGCUGCAAUGACGAGGGCCUGGAGUGUGUGCCCACUGAGGAGUC

CAACAUCACCAUGCAGAUUAUGCGGAUCAAACCUCACCAAGGCCAGCACAU

AGGAGAGAUGAGCUUCCUACAGCACAACAAAUGUGAAUGCAGACCAAAGA

AAGAUAGAGCAAGACAAGAAAAUCCCUGUGGGCCUUGCUCAGAGCGGAGA

AAGCAUUUGUUUGUACAAGAUCCGCAGACGUGUAAAUGUUCCUGCAAAAA

CACAGACUCGCGUUGCAAGGCGAGGCAGCUUGAGUUAAACGAACGUACUU

GCAGAUGUGACAAGCCGAGGCGGUGAUAAUAGGCUGGAGCCUCGGUGGCC

AUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACC

CGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGCAAAAAAAAAA

AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAUCUAG3’

(SEQ ID NO: 7)

Wherein:

A, C, G & U= AMP, CMP, GMP & Nl-methyl-pseudoUMP, respectively

Me = methyl p = inorganic phosphate

Claims

1. A method of treating dilated cardiomyopathy or heart failure associated with dilated cardiomyopathy in a patient in need thereof comprising administering to the patient an effective amount of a composition comprising a modified RNA that encodes a VEGF-A polypeptide.

2. The method of claim 1, wherein the patient has a left ventricular ejection fraction (LVEF) of less than or equal to 40% prior to administration of the composition.

3. The method of claim 2, wherein the LVEF is less than or equal to 35%, 30%, or 25% prior to administration of the composition.

4. The method of any one of the preceding claims, wherein the LVEF of the patient is increased by greater than 10%, optionally 15% or greater, optionally 20% or greater after administration of the composition.

5. The method of any one of the preceding claims, wherein the LVEF of the patient is increased to 55% or greater after administration of the composition, optionally 60% or greater after administration of the composition.

6. The method of any one of claims 4-5, wherein the increased LVEF occurs within 6 months after administration of the composition; optionally within 3 months after administration of the composition.

7. The method of any one of claims 2-6, wherein the LVEF is determined by echocardiography, radionuclide ventriculography, contrast angiography, and/or cardiac MRI; optionally by echocardiography.

8. The method of any one of the previous claims, wherein the method improves the quality of life of the patient as characterised by an increase in one or more

58 patient reported outcome scores relative to before administration of the composition, wherein the patient reported outcome scores are (a) KCCQ Overall Summary Score; (b) SAQ Physical Limitation Score; (c) S AQ Quality of Life Score; and (d) S AQ Treatment Satisfaction Score.

9. The method of any one of the previous claims, wherein the method improves the quality of life of the patient as characterised by an increase in one or more patient reported outcome scores relative to placebo, wherein the patient reported outcome scores are (a) KCCQ Overall Summary Score; (b) SAQ Physical Limitation Score; (c) SAQ Quality of Life Score; and (d) SAQ Treatment Satisfaction Score.

10. The method of any one of claims 8-9, wherein the increase in one or more patient reported outcome scores occurs within 6 months after administration of the composition, optionally within 3 months, optionally within 1 month.

11. The method of any one of claims 8-10, wherein the KCCQ Overall Summary Score is increased by at least 5 points relative to before administration of the composition; optionally increased by at least 10 points; optionally increased by at least 12 points; optionally increased by at least 14 points.

12. The method of any one of claims 8-11, wherein the SAQ Physical Limitation Score is increased by at least 10 points relative to before administration of the composition; optionally increased by at least 15 points; optionally increased by at least 20 points; optionally increased by at least 30 points.

13. The method of any one of claims 8-12, wherein the SAQ Quality of Life

Score is increased at least 10 points relative to before administration of the composition; optionally at least 20 points; optionally at least 30 points.

59

14. The method of any one of claims 8-13, wherein the SAQ Treatment Satisfaction Score is increased by at least 5 points relative to before administration of the composition.

15. The method of any one of the preceding claims, wherein the patient has a New York Heart Association (NYHA) heart failure classification of II, III or IV.

16. The method of any one of the preceding claims, wherein the patient has a NYHA heart failure classification of III or IV.

17. The method of any one of the preceding claims, wherein the composition is administered to the patient by epicardial injection.

18. The method of any one of the preceding claims, wherein the composition is administered to the patient by epicardial injection into the left ventricle.

19. The method of any one of the preceding claims, wherein the composition is administered to the patient by epicardial injection at a fixed-dosage in multiple administrations.

20. The method of any one of the preceding claims, wherein the VEGF-A polypeptide comprises an amino acid sequence at least 95%, or at least 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 3, wherein the amino acid sequence has VEGF-A activity.

21. The method of any one of the preceding claims, wherein the VEGF-A polypeptide consists of an amino acid sequence identical to SEQ ID NO: 3.

22. The method of any one of the preceding claims, wherein the modified RNA has an open reading frame (ORF) comprising or consisting of the nucleotide sequence of

SEQ ID NO: 2.

60

23. The method of any one of the preceding claims, wherein the modified RNA comprises a nucleotide sequence at least 95%, or at least 98%, or 100% identical to a nucleotide sequence selected from SEQ ID NOs: 1 and 4-7.

24. The method of any one of the preceding claims, wherein the modified RNA consists of a nucleotide sequence selected from SEQ ID NOs: 1 and 4-7, optionally wherein the modified RNA consists of the nucleotide sequence of SEQ ID NO: 1, optionally wherein the modified RNA consists of the nucleotide sequence of SEQ ID NO: 4.

25. The method of any one of the preceding claims, wherein the modified RNA is a modified mRNA.

26. The method of any one of the preceding claims, wherein the composition comprises a citrate saline buffer.

27. The method of claim 26, wherein the citrate saline buffer is pH 6 to pH 7, optionally about pH 6.5.

61