Subcutaneous delivery of messenger RNA

Abstract

To provide methods for subcutaneous delivery of a therapeutic mRNA.SOLUTION: A method for subcutaneous delivery of a messenger RNA (mRNA) to a subject in need thereof comprises administering subcutaneously to the subject a composition comprising: an mRNA encoding a protein or polypeptide; and an mRNA encoding a hyaluronidase.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 62 / 671,820, filed May 15, 2018, which is incorporated herein by reference in its entirety.

[0002] Sequence Listing This specification references a Sequence Listing (submitted electronically on May 13, 2019 as a text (.txt) file entitled "MRT-1252WO_ST25"). The text file was created on May 13, 2019 and is 26,169 bytes in size. The entire contents of the Sequence Listing are incorporated herein by reference. [Background technology]

[0003] Messenger RNA therapy (MRT) has become an increasingly important approach for the treatment of various diseases. MRT involves administering messenger RNA (mRNA) to patients in need of treatment, resulting in the production of the protein encoded by the mRNA within the patient's body. Lipid nanoparticles are commonly used to deliver mRNA due to their efficient in vivo delivery, and specific mRNA-loaded lipid nanoparticles can now be delivered systemically via intravenous delivery. However, improvements in subcutaneous delivery of therapeutic mRNA are critically needed to increase patient comfort and compliance. Summary of the Invention [Means for solving the problem]

[0004] The present invention provides, inter alia, improved methods and compositions for effective in vivo delivery of mRNA via subcutaneous administration. Specifically, to efficiently expose the therapeutic mRNA to circulation, mRNA encoding a therapeutic protein is subcutaneously injected together with mRNA encoding an enzyme capable of degrading extracellular matrix, such as hyaluronidase. As described herein, when administered together with a second mRNA encoding hyaluronidase, a first mRNA encoding a therapeutic protein results in unexpectedly efficient delivery of the first therapeutic mRNA, accompanied by efficient protein expression in vivo, particularly in the liver. The mRNA is encapsulated in lipid nanoparticles (LNPs). In some embodiments, the therapeutic mRNA is encapsulated within lipid nanoparticles (LNPs). In some embodiments, both the therapeutic mRNA and hyaluronidase are encapsulated within lipid nanoparticles (LNPs). Hyaluronidase has been used to enhance the subcutaneous delivery of small molecule and protein drugs, but prior to the inventors' recent investigations, it was unclear whether hyaluronidase would be effective in promoting the subcutaneous delivery of mRNA, particularly mRNA encapsulated in lipid nanoparticles (LNPs), given the considerable size variation and complexity of LNP-mRNA formulations. Many mRNA-loaded LNPs have sizes approaching or around 100 nm, which is at least five times larger than typical proteins (proteins containing typical antibodies have an average size of less than 20 nm). It was further unclear whether delivery of mRNA-LNPs in the presence of mRNA encoding hyaluronidase would be effective in enhancing subcutaneous uptake and delivery of mRNA-LNPs. In light of the efficient mRNA delivery and high protein expression in the liver following subcutaneous delivery using hyaluronidase enzymes, first reported recently in applicant's application PCT / US17 / 61176, filed October 11, 2017, which is incorporated herein by reference in its entirety, the present invention provides a method for treating ornithine transcarbamylase (OTC) deficiency and other conditions. These are particularly useful for the treatment of metabolic disorders. Using mRNA encoding hyaluronidase in the same or a separate formulation to deliver therapeutic mRNA allows for robust and sustained delivery and distribution of the therapeutic mRNA with surprising ease and cost-effectiveness. Without wishing to be bound by theory, it is likely that the mRNA encoding hyaluronidase is readily distributed and translated at the administration site, which then aids in the uptake and efficient distribution of the therapeutic mRNA as a result of the function of the in situ translated hyaluronidase. Administration using the hyaluronidase provided in the present application increases the efficiency of subcutaneous delivery of mRNA, which is more patient-friendly than other administration routes, such as intravenous (IV) or intramuscular (IM), and can reduce medical costs and improve patient compliance and throughput in hospitals.

[0005] In one aspect, the invention provides a method for subcutaneous delivery of messenger RNA (mRNA) to a subject in need thereof, the method comprising subcutaneously administering to the subject a composition comprising mRNA encoding a protein or polypeptide and mRNA encoding a hyaluronidase.

[0006] In some embodiments, the mRNA encoding a protein or polypeptide is a therapeutic mRNA. In some embodiments, the protein or polypeptide encoded by the mRNA, i.e., the therapeutic mRNA described herein, encodes a protein or polypeptide selected from the group consisting of erythropoietin (EPO), phenylalanine hydroxylase (PAH), argininosuccinate synthase 1 (ASS1), alpha 1-antitrypsin (A1AT), factor IX (FIX), factor VIII (FVIII), carboxypeptidase N, alpha galactosidase (GLA), ornithine carbamoyltransferase (OTC), human growth hormone (hOtt), SLC3A1-encoded protein, SLC3A9-encoded protein, COL4A5-encoded protein, FXN-encoded protein, GNS-encoded protein, HGSNAT-encoded protein, NAGLU-encoded protein, SGSH-encoded protein, MUT-encoded protein methylmalonyl-CoA mutase and ATP7B-encoded protein ATPase 2.

[0007] In some embodiments, the therapeutic mRNA, an mRNA encoding a protein or polypeptide, has a length of about 0.5 kb, 1 kb, 1.5 kb, 2 kb, 2.5 kb, 3 kb, 3.5 kb, 4 kb, 4.5 kb, 5 kb, 6 kb, 7 kb, 8 kb, 9 kb, 10 kb, 11 kb, 12 kb, 13 kb, 14 kb, or 15 kb or more.

[0008] In some embodiments, the mRNA encoding the hyaluronidase is a helper mRNA encoding a mammalian hyaluronidase selected from bovine hyaluronidase, porcine hyaluronidase, equine hyaluronidase, ovine hyaluronidase, and human hyaluronidase.

[0009] In some embodiments, the mRNA encoding the hyaluronidase comprises a polynucleotide sequence having at least 80% identity to SEQ ID NO: 9, 10, or 12.

[0010] In some embodiments, the mRNA encoding the protein or polypeptide and the mRNA encoding the hyaluronidase enzyme are separately capped and tailed.

[0011] In some embodiments, the mRNA encoding the protein or polypeptide and the mRNA encoding the hyaluronidase enzyme are encapsulated within a lipid nanoparticle (LNP).

[0012] In some embodiments, the lipid nanoparticles comprise cKK-E12(3,6-bis(4-(bis( 2-hydroxydodecyl)amino)butyl)piperazine-2,5-dione), OF-02, Target 23, Target 24, ICE, HGT5000, HGT5001, HGT4003, DOTAP (1,2-dioleyl-3-trimethylammonium propane), DODAP (1,2-dioleyl-3-dimethylammonium propane), DOTMA (1,2-di-O-octadecenyl-3-trimethylammonium propane), DLinDMA, DODAC, The cationic lipid comprises a cationic lipid selected from the group consisting of DDAB, DMRIE, DOSPA, DOGS, DODMA, DMDMA, DODAC, DLenDMA, DMRIE, CLinDMA, CpLinDMA, DMOBA, DOcarbDAP, DLinDAP, DLincarbDAP, DLinCDAP, KK-DMA, DLin-K-XTC2-DMA, DLin-KC2-DMA, dialkylamino-based, imidazole-based, and guanidine-based cationic lipids.

[0013] In some embodiments, the lipid nanoparticles comprise one or more non-cationic lipids selected from the group consisting of DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine), DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine), DOPC (1,2-dioleoyl-sn-glycero-3-phosphotidylcholine), DPPE (1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine), DMPE (1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine), DOPG (1,2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol)), and combinations thereof.

[0014] In some embodiments, the liposome comprises a PEGylated lipid. In some embodiments, the PEGylated lipid comprises at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, or at least 10% of the total lipid in the liposome. In some embodiments, the PEGylated lipid comprises at least 5% of the total lipid in the liposome. In some embodiments, the PEGylated lipid comprises about 5% of the total lipid in the liposome. In some embodiments, the PEGylated lipid comprises no more than 10%, no more than 9%, no more than 8%, no more than 7%, no more than 6%, no more than 5%, no more than 4%, or no more than 3% of the total lipid in the liposome. In some embodiments, the PEGylated lipid comprises no more than 5% of the total lipid in the liposome.

[0015] In some embodiments, the mRNA comprises unmodified nucleotides. In some embodiments, the mRNA comprises one or more modified nucleotides. In some embodiments, the one or more modified nucleotides comprise pseudouridine, N-1-methyl-pseudouridine, 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyladenosine, 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, 4'thiouridine, 4'-thiocytidine, and / or 2-thiocytidine.

[0016] In some embodiments, the mRNA encoding the protein or polypeptide and the mRNA encoding the hyaluronidase enzyme are encapsulated in nanoparticles (LNPs). In some embodiments, the mRNA encoding the protein or polypeptide and the mRNA encoding the hyaluronidase enzyme are encapsulated in separate LNPs. In some embodiments, the mRNA encoding the protein or polypeptide and the mRNA encoding the hyaluronidase enzyme are encapsulated in separate LNPs having non-identical compositions.

[0017] In some embodiments, the therapeutic mRNA and the mRNA encoding hyaluronidase are administered simultaneously. In some embodiments, the therapeutic mRNA and the mRNA encoding hyaluronidase are administered sequentially. In some embodiments, the mRNA encoding hyaluronidase is administered 0.1 hours, 0.2 hours, 0.3 hours, 0.4 hours, 0.5 hours, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, or 6 hours before the administration of the therapeutic mRNA composition. In some embodiments, the mRNA encoding hyaluronidase is administered 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, or 6 hours before the administration of the therapeutic mRNA composition.

[0018] In some embodiments, the protein encoded by the therapeutic mRNA is expressed in the liver. In some embodiments, the protein encoded by the therapeutic mRNA is expressed in the kidney. In some embodiments, the protein encoded by the therapeutic mRNA is expressed in the lung. In some embodiments, the protein encoded by the therapeutic mRNA is detectable in serum. In some embodiments, the expression of the protein encoded by the therapeutic mRNA is detectable at least 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, or 1 week after a single administration.

[0019] In some embodiments, the therapeutic mRNA is administered at a dose of at least 0.5 mg / kg of body weight. In some embodiments, the therapeutic mRNA is administered at about 1 mg / kg, about 2 mg / kg, about 3 mg / kg, about 4 mg / kg, about 5 mg / kg, about 6 mg / kg, about 7 mg / kg, about 8 mg / kg, about 9 mg / kg, about 10 mg / kg, about 11 mg / kg, about 12 mg / kg, about 13 mg / kg, about 14 mg / kg, about 15 mg / kg, about 16 mg / kg, about 17 mg / kg, about 18 mg / kg, about 19 mg / kg, about 20 mg / kg, about 25 mg / kg, about 30 mg / kg, or about 50 mg / kg of body weight.

[0020] In some embodiments, about 0.1 to 100 mg of mRNA encoding a hyaluronidase is administered. In some embodiments, about 0.5 to 90 mg of mRNA encoding a hyaluronidase is administered. In some embodiments, about 1 to 80 mg of mRNA encoding a hyaluronidase is administered. In some embodiments, about 2 to 70 mg of mRNA encoding a hyaluronidase is administered. In some embodiments, about 3 to 60 mg of mRNA encoding a hyaluronidase is administered. In some embodiments, about 4 to 50 mg of mRNA encoding a hyaluronidase is administered. In some embodiments, about 5 to 50 mg of mRNA encoding a hyaluronidase is administered.

[0021] In some embodiments, the mRNA encoding hyaluronidase is administered at an equivalent dosage for translation to produce an expected amount of at least about 1 U of hyaluronidase enzyme per mg of therapeutic RNA delivered. In some embodiments, the hyaluronidase mRNA is administered at a dosage equivalent to at least 2 U / mg of delivered therapeutic RNA, at least 5 U / mg of therapeutic RNA, at least 10 U / mg of therapeutic RNA, at least 20 U / mg of therapeutic RNA, at least 30 U / mg of therapeutic RNA, at least 40 U of therapeutic RNA, at least 50 U of therapeutic RNA, at least 100 U of therapeutic RNA, at least 200 U of therapeutic RNA, at least 300 U of therapeutic RNA, at least 400 U of therapeutic RNA, at least 500 U of therapeutic RNA, at least 1000 U of therapeutic RNA, at least 2000 U of therapeutic RNA, at least 3000 U of therapeutic RNA, at least 4000 U of therapeutic RNA, or at least 5000 U of therapeutic RNA. In one aspect, the present invention provides a method for treating a disease, disorder, or condition in a subject, comprising subcutaneously delivering to the subject a therapeutic mRNA encoding a protein or polypeptide and a helper mRNA encoding a hyaluronidase, wherein the therapeutic mRNA encodes a protein or polypeptide that is deficient in the subject. The disease, disorder, or condition herein is ornithine transcarbamylase (OTC) deficiency. , phenylalanine hydroxylase (PAH) deficiency (phenylketonuria, PKU), argininosuccinate synthase 1 (ASS1) deficiency, erythropoietin (EPO) deficiency, Fabry disease; hemophilic disorders (e.g., hemophilia B (FIX), hemophilia A (FVIII)); SMN1-related spinal muscular atrophy (SMA); amyotrophic lateral sclerosis (ALS); GALT-associated galactosemia; COL4A5-related disorders including Alport syndrome; galactocerebrosidase deficiency; X-linked adrenoleukodystrophy; Friedreich's ataxia; Pelizaeus-Merzbach disease; TSC1- and TSC2-associated tuberous sclerosis; Sanfilippo B syndrome (MPS) FMR1-related disorders, including Fragile X syndrome, Fragile X-associated tremor / ataxia syndrome, and Fragile X premature ovarian failure syndrome; Prader-Willi syndrome; hereditary hemorrhagic telangiectasia (AT); Niemann-Pick disease type C1; neuronal ceroid lipofuscinosis-related disorders, including juvenile neuronal ceroid lipofuscinosis (JNCL), juvenile Batten disease, Santavoli-Hartier disease, Jansky-Bielschowski disease, and PTT-1 and TPP1 deficiency; EIF2B1-, EIF2B2-, EIF2B3-, EIF2B4-, and EIF2B5-related childhood ataxia with central nervous system hypomyelination / vanishing white matter disease; CACNA1A- and CACNB4-related transient ataxia type 2; MECP2-related disorders including classic Rett syndrome, MECP2-related severe neonatal encephalopathy, and PPM-X syndrome; CDKL5-related atypical Rett syndrome; Kennedy disease (SBMA); Notch-3-related autosomal dominant cerebral arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL); SCN1A- and SCN1B-related seizure disorders; polymerase G-related disorders including Alpers-Huttenlocher syndrome, POLG-related sensory ataxic neuropathy, dysarthria, and ophthalmoplegia, and autosomal dominant and recessive progressive external ophthalmoplegia with mitochondrial DNA deletions; X-linked adrenal hypoplasia; X-linked agammaglobulinemia; Wilson disease.

[0022] In one aspect, the present disclosure provides a method for treating ornithine transcarbamylase (OTC) deficiency by mRNA therapy, comprising administering to a subject in need thereof a composition for subcutaneous delivery comprising messenger RNA encoding the OTC protein and mRNA encoding the hyaluronidase enzyme.

[0023] In some embodiments, the OTC mRNA is encapsulated in a nanoparticle. In some embodiments, the nanoparticle is a lipid-based or polymer-based nanoparticle. In some embodiments, the lipid-based nanoparticle is a liposome.

[0024] In some embodiments, the subcutaneous injection results in expression of the OTC protein in the liver of the subject.

[0025] In some embodiments, subcutaneous injection delivers mRNA to hepatocytes. In some embodiments, subcutaneous injection results in OTC expression in hepatocytes.

[0026] In some embodiments, the subcutaneous injection results in expression of the OTC protein in the serum of the subject.

[0027] In some embodiments, expression of the protein encoded by the mRNA is detectable at least 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, or 1 month after administration.

[0028] In some embodiments, OTC expression following mRNA administration can be detected by a functional assay.

[0029] In some embodiments, administration of the composition results in an increase in the expression or activity level of the OTC protein in the serum of the subject compared to a control level. The control level is the baseline serum OTC protein expression or activity level in a subject before treatment. In some embodiments, the control level is a reference value that represents the average serum OTC protein expression or activity level in OTC patients without treatment.

[0030] In some embodiments, administration of the composition results in a decrease in urinary orotic acid levels in the subject compared to a control orotic acid level. In some embodiments, the control orotic acid level is the subject's baseline urinary orotic acid level before treatment. In some embodiments, the control orotic acid level is a reference value representing the average urinary orotic acid level in OTC patients without treatment.

[0031] In some embodiments, administration of the composition causes an increase in the subject's serum citrulline level compared to a control citrulline level.In some embodiments, the control citrulline level is the subject's baseline serum citrulline level before treatment.In some embodiments, the control citrulline level is a reference value representing the average serum citrulline level in OTC patients without treatment.

[0032] In some embodiments, the mRNA encoding the OTC protein and the mRNA encoding the hyaluronidase enzyme are injected simultaneously.

[0033] In some embodiments, the mRNA encoding the OTC protein and the mRNA encoding the hyaluronidase enzyme are injected in a single composition.

[0034] In some embodiments, the mRNA encoding the OTC protein and the mRNA encoding the hyaluronidase enzyme are injected in separate compositions.

[0035] In some embodiments, the mRNA encoding the OTC protein and the mRNA encoding the hyaluronidase enzyme are injected sequentially.

[0036] In some embodiments, the mRNA encoding the OTC protein and the mRNA encoding the hyaluronidase enzyme are injected in a volume of less than 20 ml, less than 15 ml, less than 10 ml, less than 5 ml, less than 4 ml, less than 3 ml, less than 2 ml, or less than 1 ml.

[0037] In some embodiments, subcutaneous injections are administered no more frequently than once a week. In some embodiments, subcutaneous injections are administered no more frequently than twice a month. In some embodiments, subcutaneous injections are administered no more frequently than once a month.

[0038] In another aspect, the present invention provides a composition for treating ornithine transcarbamylase (OTC) deficiency, comprising mRNA encoding an ornithine transcarbamylase (OTC) protein and mRNA encoding a hyaluronidase enzyme.

[0039] In some embodiments, the mRNA encoding the hyaluronidase enzyme is administered at a dose of 20 mg / mL or less. In some embodiments, the mRNA encoding the hyaluronidase enzyme is administered at a dose of 18 mg / mL or less. In some embodiments, the mRNA encoding the hyaluronidase enzyme is administered at a dose of 16 mg / mL or less. In some embodiments, the mRNA encoding the hyaluronidase enzyme is administered at a dose of 14 mg / mL or less. In some embodiments, the mRNA encoding the hyaluronidase enzyme is administered at a dose of 12 mg / mL or less. In some embodiments, the mRNA encoding the hyaluronidase enzyme is administered at a dose of 10 mg / mL or less. In some embodiments, the mRNA encoding the hyaluronidase enzyme is administered at a dose of 9 mg / mL or less. In some embodiments, the mRNA encoding the hyaluronidase enzyme is administered at a dose of 10 mg / mL or less. In some embodiments, the mRNA encoding the hyaluronidase enzyme is administered at a dose of 9 mg / mL or less. In embodiments, the mRNA encoding the hyaluronidase enzyme is administered at a dose of 8 mg / mL or less. In some embodiments, the mRNA encoding the hyaluronidase enzyme is administered at a dose of 7 mg / mL or less. In some embodiments, the mRNA encoding the hyaluronidase enzyme is administered at a dose of 6 mg / mL or less. In some embodiments, the mRNA encoding the hyaluronidase enzyme is administered at a dose of 5 mg / mL or less. In some embodiments, the mRNA encoding the hyaluronidase enzyme is administered at a dose of 4 mg / mL or less. In some embodiments, the mRNA encoding the hyaluronidase enzyme is administered at a dose of 3 mg / mL or less. In some embodiments, the mRNA encoding the hyaluronidase enzyme is administered at a dose of 2 mg / mL or less. In some embodiments, the mRNA encoding the hyaluronidase enzyme is administered at a dose of 1 mg / mL or less. In some embodiments, the mRNA encoding the hyaluronidase enzyme is administered at a dose ranging from 1 to 20 mg / mL.

[0040] In some embodiments, the mRNA is encapsulated within the nanoparticles.

[0041] In some embodiments, the nanoparticles are lipid-based or polymer-based nanoparticles.

[0042] In some embodiments, the composition is in liquid form.

[0043] In another embodiment, the composition is a lyophilized powder.

[0044] In one aspect, the present invention provides a container containing the above-described composition. The container is a vial or a syringe. The syringe may be pre-filled for a single subcutaneous administration. The vial may contain a lyophilized powder or liquid form of the composition.

[0045] In this application, the use of "or" means "and / or" unless stated otherwise. As used in this application, the term "comprise," and variations of terms such as "comprising" and "comprises," are not intended to exclude other additives, components, integers, or steps. As used in this application, the terms "about" and "approximately" are used synonymously. Both terms are meant to cover any normal fluctuations understood by one of ordinary skill in the art.

[0046] Other features, objects, and advantages of the present invention will become apparent in the following detailed description, drawings, and claims. It should be understood, however, that the following detailed description, drawings, and claims, while indicating embodiments of the present invention, are given by way of illustration only, and not by way of limitation. Various changes and modifications within the scope of the present invention will become apparent to those skilled in the art.

[0047] definition In order that the present invention may be more readily understood, certain terms are first defined below. Additional definitions of the following terms, and other terms, are set forth throughout the specification.

[0048] Animal: As used herein, the term "animal" refers to any member of the animal kingdom. In some embodiments, "animal" refers to a human, at any stage of development. In some embodiments, "animal" refers to a non-human animal, at any stage of development. In certain embodiments, the non-human animal is a mammal (e.g., a rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, a sheep, a cow, a primate, and / or a pig). In some embodiments, animals include, but are not limited to, mammals, birds, reptiles, amphibians, fish, insects, and / or worms. In some embodiments, animals are transgenic animals, genetically modified animals, and the like. The offspring may be recombinant animals and / or clones.

[0049] Approximately or About: As used herein, the term "approximately" or "about" as applied to one or more values ​​of interest refers to a value similar to a stated reference value. In certain embodiments, the term "approximately" or "about" refers to a range of values ​​of 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater or less than) the stated reference value, unless otherwise stated or apparent from the context (except when such number exceeds 100% of possible values).

[0050] Delivery: As used herein, the term "delivery" encompasses both local delivery and systemic delivery. For example, the delivery of mRNA encompasses the situation where mRNA is delivered to target tissue, its encoded protein is expressed and retained in the target tissue (also referred to as "local distribution" or "local delivery"); the situation where mRNA is delivered to target tissue, its encoded protein is expressed and secreted into the patient's circulatory system (for example, serum), and is distributed throughout the body and taken up by other tissues (also referred to as "systemic distribution" or "systemic delivery").

[0051] Encapsulation: As used herein, the term "encapsulation" or grammatical equivalents refers to the process of confining individual mRNA molecules within nanoparticles.

[0052] Expression: As used herein, "expression" of a nucleic acid sequence refers to the translation of mRNA into a polypeptide, the assembly of multiple polypeptides into an intact protein (e.g., an enzyme), and / or the post-translational modification of a polypeptide or fully assembled protein (e.g., an enzyme). In this application, the terms "expression" and "production" and grammatical equivalents are used interchangeably.

[0053] Half-life: As used herein, the term "half-life" is the time it takes for a quantity, such as the concentration or activity of a nucleic acid or protein, to fall to half of the value measured at the beginning of a period.

[0054] Hyaluronidase: As used herein, the term "hyaluronidase" refers to a family of enzymes that can degrade hyaluronic acid (hyaluronan).

[0055] Improve, increase, or reduce: As used herein, the terms "improvement," "increase," or "reduction," or grammatical equivalents, refer to a value relative to a baseline measurement, such as a measurement in the same individual prior to the initiation of a treatment described herein, or a measurement in a control subject (or subjects) not receiving a treatment described herein. A "control subject" is a subject suffering from the same form of disease as the subject being treated and of approximately the same age as the subject being treated.

[0056] In vitro: As used herein, the term "in vitro" refers to events that take place not within a multicellular organism but in an artificial environment, such as, for example, in a test tube or reaction vessel, in cell culture, etc.

[0057] In vivo: As used herein, the term "in vivo" refers to events that occur within a multicellular organism, such as humans and non-human animals. In the context of cell-type systems, the term can be used to refer to events that occur within living cells (e.g., as opposed to in vitro systems).

[0058] Local distribution or local delivery: As used herein, "local distribution," "local delivery," or grammatical equivalents thereof, refers to tissue-specific delivery or distribution. Typically, local distribution or local delivery requires an mRNA-encoded protein (e.g., an enzyme) that is translated and expressed intracellularly or that is secreted only to avoid it entering the patient's circulatory system.

[0059] Messenger RNA (mRNA): As used herein, the term "messenger RNA (mRNA)" refers to a polynucleotide that encodes at least one polypeptide. As used herein, mRNA encompasses both modified and unmodified RNA. mRNA can contain one or more coding and non-coding regions. mRNA can be purified from natural sources, produced using recombinant expression systems, or optionally purified, chemically synthesized, etc. Optionally, for example, in the case of chemically synthesized molecules, mRNA can contain nucleoside analogs, such as analogs with chemically modified bases or sugars, backbone modifications, etc. mRNA sequences are presented in the 5' to 3' direction unless otherwise indicated. In some embodiments, the mRNA is selected from natural nucleosides (e.g., adenosine, guanosine, cytidine, uridine); nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyladenosine, 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, 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., phosphorothioate and 5'-N-phosphoramidite linkages).

[0060] Patient: As used herein, the term "patient" or "subject" refers to any organism to which provided compositions can be administered, for example, for experimental, diagnostic, prophylactic, cosmetic, and / or therapeutic purposes. Typical patients include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and / or humans). In some embodiments, the patient is a human. Humans include prenatal and postnatal forms.

[0061] Pharmaceutically acceptable: As used herein, the term "pharmaceutically acceptable" refers to a material that is, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without undue toxicity, inflammatory irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0062] Subcutaneous administration: As used herein, the term "subcutaneous administration" or "subcutaneous injection" refers to a bolus injection into the subcutaneous tissue, which is the layer of tissue between the skin and muscle.

[0063] Subject: As used herein, the term "subject" refers to a human or any non-human animal (e.g., a mouse, rat, rabbit, dog, cat, cow, pig, sheep, horse, or primate). Humans include prenatal and postnatal forms. In many embodiments, a subject is a human. A subject may be a patient, which means a person who visits a medical institution for diagnosis or treatment of a disease. The term "subject" is used interchangeably herein with "individual" or "patient." A subject may be suffering from or susceptible to a disease or disorder, but may or may not exhibit symptoms of the disease or disorder.

[0064] Substantially: As used herein, the term "substantially" refers to the qualitative state of exhibiting all or nearly all extent or degree of a desired characteristic or property. Those skilled in the art of biology will understand that biological and chemical phenomena rarely, if ever, go to completion and / or reach completion, or achieve or avoid absolute results. Thus, the term "substantially" is used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena.

[0065] Systemic distribution or delivery: As used herein, the terms "systemic distribution," "systemic delivery," or grammatical equivalents refer to a delivery or distribution mechanism or approach that affects the entire body or the entire organism. Typically, systemic distribution or delivery is achieved via the body's circulatory system, e.g., the bloodstream. Compare with the definition of "local distribution or delivery."

[0066] Target tissue: As used herein, the term "target tissue" refers to any tissue affected by a disease to be treated. In some embodiments, the target tissue includes tissue that exhibits pathology, symptoms, or characteristics associated with the disease.

[0067] Therapeutic mRNA: As used herein, the term therapeutic mRNA refers to an mRNA for mRNA therapy. Therapeutic mRNA can refer to an mRNA that encodes a protein or polypeptide that is deficient in a subject in need of treatment. It is used interchangeably with the term "first mRNA" throughout this specification, without any presumption regarding the time sequence of delivery, for example, for a second mRNA.

[0068] Therapeutically effective amount: As used herein, the term "therapeutically effective amount" of a therapeutic agent means an amount that, when administered to a subject suffering from or susceptible to a disease, disorder, and / or condition, is sufficient to treat, diagnose, prevent, and / or delay the onset of the disease, disorder, and / or condition. Those skilled in the art will understand that a therapeutically effective amount is typically administered via a dosing regimen comprising at least one unit dose.

[0069] Treatment: As used herein, the terms "treat," "treatment," or "treating" refer to any method used to partially or completely alleviate, ameliorate, relieve, suppress, prevent, delay the onset of, reduce the severity of, and / or reduce the incidence of one or more symptoms or characteristics of a particular disease, disorder, and / or condition. Treatment may be administered to subjects who do not show signs of disease and / or who show only early signs of disease, with the goal of reducing the risk of developing conditions associated with the disease. DETAILED DESCRIPTION OF THE INVENTION

[0070] In particular, the present invention provides improved mRNA delivery methods and compositions for messenger RNA therapy via the subcutaneous route by administering a target mRNA (first mRNA) together with a second mRNA encoding a hyaluronidase enzyme. The second mRNA aids or enhances cellular uptake and distribution of the mRNA. The mRNA payload was efficiently delivered to the liver (and other organs or tissues) of treated animals. This method using hyaluronidase has significant advantages for creating novel delivery profiles for drugs that would otherwise be intolerable.

[0071] The present invention particularly relates to a method for the preparation of a gene encoding an ornithine transcarbamylase (OTC) protein. Methods and compositions are provided for treating ornithine transcarbamylase (OTC) deficiency by administering to a subject in need of treatment via subcutaneous injection an mRNA and a second mRNA encoding a hyaluronidase enzyme. The present invention can also be used to treat a variety of other diseases, disorders, and conditions, particularly metabolic diseases, disorders, and conditions.

[0072] Various aspects of the present invention are described in detail in the following sections. The use of sections is not meant to limit the present invention. Each section may be applicable to any aspect of the present invention. In this application, the use of "or" means "and / or" unless stated otherwise.

[0073] hyaluronidase enzyme Various hyaluronidase enzymes may be used to practice the present invention. For example, there are three groups of hyaluronidases based on their mechanism of action. Two of these groups are endo-β-N-acetyl-hexosaminidases. One group includes vertebrate enzymes that utilize substrate hydrolysis. Vertebrate hyaluronidases (EC 3.2.1.35) are endo-β-N-acetyl-hexosaminidases that utilize substrate hydrolysis as a catalyst. Vertebrate hyaluronans also possess transglycosidase activity, capable of cross-linking HA chains and of cross-linking HA chains to ChS or Ch. Vertebrate hyaluronidases degrade HA through a non-progressive endolytic process, often producing a tetrasaccharide. Mammalian hyaluronidases are members of a group of carbohydrate-active enzymes (CAZy) called glycosidase family 56, defined as endo-β-acetyl-hexosaminidases that catalyze the hydrolysis of HA at the β1,4 glycosidic linkage.

[0074] The second group, primarily bacterial, includes eliminases that function by β-elimination of glycosidic bonds to introduce unsaturated bonds. Bacterial hyaluronidases are also endo-β-acetylhexosaminidases, but they utilize a lyase mechanism. They belong to a distinct CAZy family, polysaccharide lyase family 8. Generally, these polysaccharide lyases (EC 4.2.2.*) cleave by β-elimination, resulting in a double bond at the new non-reducing end. Hyaluronate lyases (EC 4.2.2.1; bacterial Hyal) comprise only one subgroup within family 8, including chondroitin ABC lyases (EC 4.2.2.4), chondroitin AC lyases (EC 4.2.2.5), and xanthan lyases (EC 4.2.2.12). These bacterial enzymes, hyaluronidases, chondroitinases, and xanthanases, all share significant sequence, structural, and mechanistic homology.

[0075] The third group is the endo-β-glucuronidases, which are found in leeches and certain crustaceans.

[0076] Additionally, there are six known genes encoding hyaluronidase-like sequences in the human genome: Hyal-1, Hyal-2, Hyal-3, Hyal-4, and PH-20 / Spam1, as well as the pseudogene Phial1 (not translated), all of which share a high degree of homology. Mice also have six genes encoding hyaluronidases that share a high degree of homology with human genes (Stern et al., Chem. Rev. 2006, 106(3):818-839). In some embodiments, hyaluronidase can also be obtained from bovine or porcine sources as a sterile preparation free of any other animal products.

[0077] Bovine PH-20 is a commonly used hyaluronidase and is commercially available in a reasonably pure form (Sigma catalog number H3631, type VI-S, from bovine testis, 3,0 (Activity ranges from 00 to 15,000 NFU (National Formulary Unit) units / mg).

[0078] Injectable hyaluronidase is commercially available in powder form or as a solution. For example, FDA-approved bovine testicular hyaluronidase enzyme is available as a colorless, odorless solution.

[0079] In some embodiments, an International Unit of Hyaluronidase may be defined as the activity of 0.1 mg of an international standard preparation, which is equal to 1 Turbidity Reducing Unit (TRU) (Humphrey JH et al., "International Standard for Hyaluronidase," Bull World Health Organ. 1957;16(2):291-294) based on the following reaction: hyaluronidase Hyaluronic acid----------------->disaccharides and monosaccharides + small hyaluronic acid fragments Therefore, one unit of hyaluronidase activity is an A of 0.330 per minute at 37°C at pH 5.3 in a 2.0 ml reaction mixture. 600 (45 min assay). % transmittance is calculated at 600 nm, with a light path of 1 cm.

[0080] In some embodiments, artificially synthesized bovine hyaluronidase PH-20 mRNA may be used for this purpose.

[0081] In some embodiments, the bovine hyaluronidase mRNA used herein has greater than 80% sequence identity to SEQ ID NO: 9 (GenBank ID No.: BC110183.1). In some embodiments, the bovine hyaluronidase mRNA used herein has greater than 90% sequence identity to SEQ ID NO: 9. In some embodiments, the mRNA has greater than 91%, greater than 92%, greater than 93%, greater than 94%, greater than 95%, or greater than 98% sequence identity to SEQ ID NO: 9. In some embodiments, the bovine hyaluronidase mRNA used herein has 100% identity to SEQ ID NO: 9. In some embodiments, the bovine hyaluronidase mRNA encodes a PH-20 hyaluronidase that is approximately 90% identical to SEQ ID NO: 10 (GenBank ID No.: BC110183.1, cds sequence). In some embodiments, the mRNA encoding the PH-20 hyaluronidase has greater than 91%, greater than 92%, greater than 93%, greater than 94%, greater than 95%, or greater than 98% sequence identity to the sequence of SEQ ID NO: 10. In some embodiments, the bovine hyaluronidase has 100% identity to SEQ ID NO: 10. In some embodiments, the bovine hyaluronidase mRNA encodes a protein having an amino acid sequence that has at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 11. (GenBank ID No: AAI10184.1). In some embodiments, the mRNA encodes a protein having amino acids that have greater than 91%, greater than 92%, greater than 93%, greater than 94%, greater than 95%, or greater than 98% sequence identity to SEQ ID NO: 11. An exemplary bovine hyaluronidase mRNA sequence is shown below: GGTTTATCTCTGTTCTTGGTGAGGAGACAGACAGAATTGACTGCTGTGCTCATCCGC GAGGGTAATATGTG CTCAGCTCTT TATGGAGTAGTGGAGACGGGCAGAGATGACAAGATGAAGCAACTTGCAAAACATTCCTAAATACGAAGGAAGAAGAATATTTAAATGAAATCATCATTATTCATTTTTATCCATCAAAGTGGCTTCATTCTGTGTTCATATCTTGCATCAAATATTAGGTACACCAAAGCGTGTAGGAGAAAAAAGTGCCTTTCACAGTCATCGCTCTTTGTGATGAGAATGCTGAGGCGCCACCAT TACTTGAACACTACACTGAATCCTTACATAATCAACGTCACCCTAGCCGCCAAAATGTGCAGCCAAGTGCTTTGCCACAATGAAGGAGTGTGTACAAGGAAACACTGGAATTCAAGCGACTATCTTCACCTGAACCCAATGAATTTTGCTATTCAAACTGGGGAAGGTGGAAAATACACAGTACCTGGGACAGTCACACTTGAAGACTTGCAAAAGTTTTCTGATACATTTTATTGCAGTTGTTATGCCAACATCCACTGTAAGAAGAGAGTTGATATAAAAAATGTTCATAGTGTTAACGTGTGTATGGCAGAAGACATTTGTATAGACAGCCCTGTGAAGTTACAACCCAGTGATCATTCCTCCAGCCAGGAGGCATCTACTACCACCTTCAGCAGTATCTCACCCTCCACTACAACTGCCACAGTATCTCCATGTACTCCTGAGAAACACTCCCCTGAGTGCCTCAAAGTCAGGTGTTCGGAAGTCATCCCCAACGTCACCCAAAAGGCGTGTCAAAGTGTTAAATTGAAGAACATTTCCTATCAGTCACCTATTCAAAATATTAAAAATCAAACAACCTATTAAAATTAAATTCAGTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGAAAAAAAAAAAAAAA(SEQ ID NO: 9) Another example of a bovine hyaluronidase mRNA sequence is as follows: ATGAGAATGCTGAGGCGCCACCATATCTCCTTTCGGAGCTTTGCTGGGTCTAGCGGAACACCCCAGGCAGTGTTCACCTT CCTTCTGCTTCCGTGTTGTTTGGCTCTGGACTTCAGAGCACCCCCTCTTATTTCAAACACTTCTTTCCTCTGGGCCTGGAATGCCCCAGTTGAACGTTGTGTTAACAGAAGATTTCAACTACCTCCAGATCTGAGACTCTTCTCTGTAAAAGGAAGCCCCCAGAAAAGTGCTACCGGACAATTTATTACATTATTTTATGCTGATAGACTTGGCTACTATCCTCATATAGATGAAAAAACAGGCAAAACCGTATTCGGAGGAATTCCCCAGTTGGGAAACTTAAAAAGTCATATGGAGAAAGCAAAAAATGACATTGCCTATTACATACCAAATGACAGCGTGGGCTTGGCGGTCATTGACTGGGAAAACTGGAGGCCTACCTGGGCAAGAAACTGGAAACCTAAAGATGTTTACAGGGATGAGTCAGTTGAGTTGGTTCTGCAAAAAAATCCGCAACTCAGTTTCCCAGAGGCTTCCAAGATTGCAAAAGTGGATTTTGAGACAGCAGGAAAGAGTTTCATGCAAGAGACTTTAAAACTGGGAAAATTACTTCGGCCAAATCACTTATGGGGTTATTATCTTTTTCCTGATTGTT ACAATCATAATCATAACCAACCTACTTACAATGGAAATTGCCCTGATGTAGAAAAAAGGAGAAATGATGATCTCGAGTGGTTGTGGAAGGAAAGCACTGCCCTTTTCCCTTCTGTTTATTTGAATATCAGGTTAAAATCTACTCAAAATGCTGCCTTGTATGTTCGTAATCGTGTCCAGGAAGCCATTCGGTTGTCTAAAATAGCGAGTGTCGAAAGTCCACTTCCGGTTTTTGTATATGCCCGTCCAGTTTTTACTGATGGGTCTTCAACATATCTTTCTCAGGGTGACCTTGTGAATTCGGTTGGTGAGATCGTTTCTCTAGGTGCCTCTGGGATTATAATGTGGGGCAGTCTCAATCTAAGCTTATCTATGCAATCTTGCATGAACCTAGGCACTTACTTGAACACTACACTGAATCCTTACATAATCAACGTCACCCTAGCCGCCAAAATGTGCAGCCAAGTGCTTTGCCACAATGAAGGAGTGTGTACAAGGAAACACTGGAATTCAAGCGACTATCTTCACCTGAACCCAATGAATTTTGCTATTCAAACTGGGGAAGGTGGAAAATACACAGTACCTGGGACAGTCACACTTGAAGACTTGCAAAAGTTTTCTGATACATTTTATTGCAGTTGTTATGCCAACATCCACTGTAAGAAGAGAGTTGATATAAAAAATGTTCATAGTGTTAACGTGTGTATGGCAGAAGACATTTGTATAGACAGCCCTGTGAAGTTACAACCCAGTGATCATTCCTCCAGCCAGGAGGCATCTACTACCACCTTCAGCAGTATCTCACCCTCCACTACAACTGCCACAGTATCTCCATGTACTCCTGAGAAACACTCCCCTGAGTGCCTCAAAGTCAGGTGTTCGGAAGTCATCCCCAACGTCACCCAAAAGGCGTGTCAAAGTGTTAAATTGAAGAACATTTCCTATCAGTCACCTATTCAAAATATTAAAAATCAAACAACCTATTA(SEQ ID NO: 10) An exemplary translated protein sequence is as follows: MRMLRRHHISFRSFAGSSGTPQAVFTFLLLPCCLALDFRAPPLISNTSFLWAWNAPVERCVNRRFQLPPDLRLFSVKGSPQKSATGQFITLFYADRLGYYPHIDEKTGKTVFGGIPQLGNLKSHMEKAKNDIAYYIPNDSVGLAVIDWENWRPTWARNWK PKDVYRDESVELVLQKNPQLSFPEASKIAKVDFETAGKSFMQETLKLGKLLRPNHLWGYYLFPDCYNHNHNQPTYNGNCPDVEKRRNDDLEWLWKESTALFPSVYLNIRLKSTQNAALYVRNRVQEAIRLSKIASVESPLPVFVYARPVFTDGSSTYLSQ GDLVNSVGEIVSLGASGIIMWGSLNLSLSMQSCMNLGTYLNTTLNPYIINVTLAAKMCSQVLCHNEGVCTRKHWNSSDYLHLNPMNFAIQTGEGGKYTVPGTVTLEDLQKFSDTFYCSCYANIHCKKRVDIKNVHSVNVCMAEDICIDSPVKLQPSDHSSSQEASTTTFSSISPSTTTATVSPCTPEKHSPECLKVRCSEVIPNVTQKACQSVKLKNISYQSPIQNIKNQTTY (SEQ ID NO: 11).

[0082] In some embodiments, artificially synthesized human hyaluronidase mRNA is administered for subcutaneous delivery of therapeutic mRNA. The human hyaluronidase mRNA administered for subcutaneous delivery of therapeutic mRNA has greater than 80% sequence identity to SEQ ID NO: 12 (GenBank ID No.: AF040710). In some embodiments, the human hyaluronidase mRNA used herein has greater than 90% sequence identity to SEQ ID NO: 12. In some embodiments, the mRNA has greater than 91%, greater than 92%, greater than 93%, greater than 94%, greater than 95%, or greater than 98% sequence identity to SEQ ID NO: 12. In some embodiments, the human hyaluronidase mRNA used herein has 100% identity to SEQ ID NO: 12. In some embodiments, the human hyaluronidase mRNA encodes a protein having an amino acid sequence that has at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 13. (GenBank ID No.: AAC70915.1). In some embodiments, the mRNA encodes a protein having amino acids with greater than 91%, greater than 92%, greater than 93%, greater than 94%, greater than 95%, or greater than 98% sequence identity to SEQ ID NO:13. An exemplary human hyaluronidase mRNA sequence is shown below: ATGACCACGCAACTGGGCCCAGCCCTGGTGCTGGGGGTGGCCCTGTGCCTGGGTTGTGGCCAGCCCCTACCACAGGTCCCTGAACGCCCCTTCTCTGTGCTGTGGAATGTACCCTCAGCACACTGTGAGGCCCGCTTTGGTGTGCACCTGCCACTCAATGCTCTGGGCATCATAGCCAACCGTGGCCAGCATTTTCACGGTCAGAACATGACCATTTTCTACAAGAACCAACTCGGCCTCTATCCCTACTTTGGACCCAGGGGCACAGCTCACAATGGGGGCATCCCCCAGGCTTTGCCCCTTGACCGCCACCTGGCACTGGCTGCCTACCAGATCCACCACAGCCTGAGACCTGGCTTTGCTGGCCCAGCAGTGCTGGATTGGGAGGAGTGGTGTCCACTCTGGGCTGGGAACTGGGGCCGCCGCCGAGCTTATCAGGCAGCCTCTTGGGCTTGGGCACAGCAGGTATTCCCTGACCTGGACCCTCAGGAGCAGCTCTACAAGGCCTATACTGGCTTTGAGCAGGCGGCCCGTGCACTGATGGAGGATACGCTGCGGGTGGCCCAGGCACTACGGCCCCATGGACTCTGGGGCTTCTATCACTACCCAGCCTGTGGCAATGGCTGGCATAGTATGGCTTCCAACTATACCGGCCGCTGCCATGCAGCCACCCTTGCCCGCAACACTCAACTGCATTGGCTCTGGGCCGCCTCCAGTGCCCTCTTCCCCAGCATCTACCTCCCACCCAGGCTGCCACCTGCCCACCACCAGGCCTTTGTCCGACATCGCCTGGAGGAGGCCTTCCGTGTGGCCCTTGTTGGGCACCGACATCCCCTGCCTGTCCTGGCCTATGTCCGCCTCACACACCGGAGATCTGGGAGGTTCCTGTCCCAGGATGACCTTGTGCAGTCCATTGGTGTGAGTGCAGCACTAGGGGCAGCCGGCGTGGTGCTCTGGGGG GACCTGAGCCTCTCCAGCTCTGAGGAGGAGTGCTGGCATCTCCATGACTACCTGGTGGACACCTTGGGCCCCTATGTGATCAATGTGACCAGGGCAGCGATGGCCTGCAGTCACCAGCGGTGCCATGGCCACGGGCGCTGTGCCCGGCGAGATCCAGGACAGATGGAAGCCTTTCTACACCTGTGGCCAGACGGCAGCCTTGGAGATTGGAAGTCCTTCAGCTGCCACTGTTACTGGGGCTGGGCTGGCCCCACCTGCCAGGAGCCCAGCCTGGGCCTAAAGAAGCAGTATAAAGCCAGGGCCCCTGCCACTGCCTCTTCTTTTCCCTGCTGCCACTTTTCCAGTCCTGGAACTACTCTGTCCCACTCTTGCTCTATTCAGTTTACAGTCAACCCTCCCAAGCACACACCCCGCTTCCCTTGGAATCCCTGA(SEQ ID NO: 12) An exemplary human hyaluronidase protein sequence is shown below: MTTQLGPALVLGVALCLGCGQPLPQVPERPFSVLWNVPSAHCEARFGVHLPLNALGIIANRGQHFHGQNMTIFYKNQLGLYPYFGPRGTAHNGGIPQALPLDRHLALAAYQIHHSLRPGFAGPAVLDWEEWCPLWAGNWGRRRAYQAASWAWAQQVFPDLDPQEQLYKAYTGFEQAARALMEDTLRVAQALRPHGLWGFYHYPACGNGWHSMASNYTGRCHAATLARNTQLHWLWAASSALFPSIYLPPRLPPAHHQAFVRHRLEEAFRVALVGHRHPLPVLAYVRLTHRRSGRFLSQDDLVQSIGVSAALGAAGVVLWGDLSLSSSEEECWHLHDYLVDTLGPYVINVTRAAMACSHQRCHGHGRCARRDPGQMEAFLHLWPDGSLGDWKSFSCHCYWGWAGPTCQEPSLGLKKQYKARAPATASSFPCCHFSSPGTTLSHSCSIQFTVNPPKHTPRFPWNP(SEQ ID NO: 13)

[0083] In some embodiments, mRNA encoding full-length or fragments of hyaluronidase is used.

[0084] Exemplary recombinant hyaluronidase dosages of hyaluronidase are between about 1 unit and 50,000 units. Thus, hyaluronidase mRNA is administered in equivalent doses to be translated into less than 40,000 U, less than 30,000 U, less than 20,000 U, less than 10,000 U, less than 9000 U, less than 8000 U, less than 7000 U, less than 6000 U, less than 5000 U, less than 4000 U, less than 3000 U, less than 2000 U, less than 1000 U, less than 900 U, less than 800 U, less than 700 U, less than 600 U, or less than 500 U of protein. In some embodiments, the hyaluronidase mRNA is administered at a dose equivalent to translate into at least 1 U, at least 5 U, at least 10 U, at least 20 U, at least 30 U, at least 40 U, ​​at least 50 U, at least 60 U, at least 70 U, at least 80 U, at least 100 U, or at least 150 U of protein. In other embodiments, the hyaluronidase mRNA is administered at a dose equivalent to translate into at least 160 U, at least 180 U, at least 200 U, at least 220 U, at least 240 U, ​​at least 260 U, at least 280 U, at least 300 U, at least 320 U, at least 340 U, ​​at least 360 U, at least 380 U, or at least 400 U of protein. In one or more embodiments, porcine (pig) hyaluronidase is used in doses ranging from 1 Unit to 50,000 Units. Hyaluronidase mRNA is less than 40,000 U, less than 30,000 U, less than 20,000 U, less than 10,000 U, less than 9000 U, less than 8000 U, less than 7000 U, less than 6000 U, less than 5000 U, less than 4000 U, less than 3000 U, less than 2000 U, less than 1000 U, less than 900 U, less than 800 U, less than 700 U, less than 600 U, or less than 50 The method of any preceding claim, wherein the hyaluronidase mRNA is administered at an equivalent dose to be translated into an amount of protein of at least 1 U, at least 5 U, at least 10 U, at least 20 U, at least 30 U, at least 40 U, ​​at least 50 U, at least 60 U, at least 70 U, at least 80 U, at least 100 U, or at least 150 U. In some other embodiments, the hyaluronidase mRNA is administered at an equivalent dose to be translated into at least 160 U, at least 180 U, at least 200 U, at least 220 U, at least 240 U, ​​at least 260 U, at least 280 U, at least 300 U, at least 320 U, at least 340 U, ​​at least 360 U, at least 380 U, or at least 400 U of protein.

[0085] In one or more embodiments, the hyaluronidase is administered simultaneously with the therapeutic mRNA. In some embodiments, the hyaluronidase may be administered prior to administration of the mRNA. In some embodiments, the mRNA and the hyaluronidase enzyme are part of the same formulation. In some embodiments, the RNA and the hyaluronidase enzyme are injected as separate formulations.

[0086] In some embodiments, the mRNA encoding hyaluronidase can be administered in an aqueous solution.In some embodiments, the mRNA encoding hyaluronidase is in saline.In some embodiments, the hyaluronidase enzyme is part of the mRNA formulation and is contained in the same solution, and this solution contains mRNA-encapsulated lipid nanoparticles.In some embodiments, a lyophilized preparation comprising mRNA-encapsulated lipid and hyaluronidase enzyme is formulated for therapeutic use.

[0087] Messenger RNA (mRNA): The present invention can be used to deliver any mRNA. As used herein, mRNA is a type of RNA that transmits information from DNA to ribosomes for translation of the encoded protein. mRNA can be synthesized by any of a variety of known methods. For example, mRNA according to the present invention can be synthesized via in vitro transcription (IVT). Briefly, IVT is often performed using a linear or circular DNA template containing a promoter, a set of ribonucleotide triphosphates, a buffer system that may contain DTT and magnesium ions, and an appropriate RNA polymerase (e.g., T3, T7, or SP6 RNA polymerase), DNAse I, pyrophosphatase, and / or an RNAse inhibitor. Stringent conditions will vary depending on the specific application.

[0088] In some embodiments, in vitro synthesized mRNA may be purified prior to formulation and encapsulation to remove undesirable impurities, including various enzymes and other reagents used during mRNA synthesis.

[0089] The present invention can be used to deliver mRNAs of various lengths. In some embodiments, the present invention can be used to deliver in vitro synthesized mRNAs of lengths of about 1 kb, 1.5 kb, 2 kb, 2.5 kb, 3 kb, 3.5 kb, 4 kb, 4.5 kb, 5 kb, 6 kb, 7 kb, 8 kb, 9 kb, 10 kb, 11 kb, 12 kb, 13 kb, 14 kb, 15 kb, or 20 kb or more. In some embodiments, the present invention can be used to deliver in vitro synthesized mRNAs of lengths ranging from about 1 to 20 kb, about 1 to 15 kb, about 1 to 10 kb, about 5 to 20 kb, about 5 to 15 kb, about 5 to 12 kb, about 5 to 10 kb, about 8 to 20 kb, or about 8 to 15 kb.

[0090] The present invention can be used to deliver unmodified mRNA or mRNA containing one or more modifications that generally enhance stability. In some embodiments, the modifications include modified nucleosides. The amino acid sequence is selected from a nucleotide sequence, a modified sugar phosphate backbone, and a 5' and / or 3' untranslated region (UTR).

[0091] In some embodiments, modifications of mRNA may include modifications of nucleotides of the RNA. Modified mRNA according to the present invention may include, for example, backbone modifications, sugar modifications, or base modifications. In some embodiments, mRNA may be synthesized from natural nucleotides and / or nucleotide analogs (modified nucleotides), including, but not limited to, purines (adenine (A), guanine (G)) or pyrimidines (thymine (T), cytosine (C), uracil (U)), as well as nucleotide analogs such as 1-methyladenine, 2-methyladenine, 2-methylthio-N-6-isopentenyl-adenine, N6-methyl-adenine, N6-isopentenyl -Adenine, 2-thio-cytosine, 3-methyl-cytosine, 4-acetyl-cytosine, 5-methyl-cytosine, 2,6-diaminopurine, 1-methyl-guanine, 2-methyl-guanine, 2,2-dimethyl-guanine, 7-methyl-guanine, inosine, 1-methyl-inosine, pseudouracil (5-uracil), dihydrouracil, 2-thio-uracil, 4-thio-uracil, 5-carboxymethylaminomethyl-2-thio-uracil Uracil, 5-(carboxyhydroxymethyl)-uracil, 5-fluoro-uracil, 5-bromo-uracil, 5-carboxymethylaminomethyl-uracil, 5-methyl-2-thio-uracil, 5-methyl-uracil, N-uracil-5-oxyacetic acid methyl ester, 5-methylaminomethyl-uracil, 5-methoxyaminomethyl-2-thio-uracil, 5'-methoxycarbonylmethyl-uracil, 5-methoxy-uracil, uracil-5-oxyacetic acid methyl ester They may also be synthesized as modified nucleotide analogs or derivatives of purines and pyrimidines, such as uracil esters, uracil-5-oxyacetic acid (v), 1-methylpseudouracil, queuosine, beta-D-mannosyl-queuosine, wybutoxosine, and phosphoramidates, phosphorothioates, peptide nucleotides, methylphosphonates, 7-deazaguanosine, 5-methylcytosine, and inosine.The preparation of such analogs is known to those skilled in the art from 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 herein by reference in their entirety.

[0092] In some embodiments, mRNA may comprise RNA backbone modification. Typically, backbone modification is a modification that chemically modifies the backbone phosphate of the nucleotide contained in RNA. Exemplary backbone modifications typically include, but are not limited to, modifications from the group consisting of methylphosphonate, methylphosphoramidate, phosphoramidate, phosphorothioate (e.g., cytidine 5'-O-(1-thiophosphate)), boranophosphate, positively charged guanidinium group, etc., which means that phosphodiester bond is replaced with other anionic group, cationic group, or neutral group.

[0093] In some embodiments, the mRNA may contain sugar modifications. Typical sugar modifications are chemical modifications of the sugars of nucleotides contained in the mRNA, including, but not limited to, 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'-arauidine 5'-triphosphate), or azidotriphosphate (2'-azido-2'-deoxycytidine 5'-triphosphate, 2'-azido-2'-deoxyuridine 5'-triphosphate).

[0094] In some embodiments, mRNA may contain a modification of the base of a nucleotide (base modification). Modified nucleotides containing base modifications are also referred to as base-modified nucleotides. 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, 5-aminoallyl ur ...aminoallyl uridine 5'-triphosphate, 5-aminoallyl uridine 5'-triphosphate, 5-aminoallyl uridine 5'-triphosphate, 5-aminoallyl uridine 5'-triphosphate, 5-aminoallyl uridine 5'-triphosphate, 5- thidine 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, N1-methyladenosine 5'-triphosphate, N1-methylguanosine 5'-triphosphate, N6-methyladenosine 5'-triphosphate, O6-methylguanosine 5'-triphosphate, pseudouridine 5'-triphosphate, puromycin 5'-triphosphate, or xanthosine 5'-triphosphate.

[0095] mRNA synthesis typically involves the addition of a "cap" to the 5' end and a "tail" to the 3' end. The presence of the cap is important for conferring resistance to nucleases found in most eukaryotic cells. The presence of the "tail" helps protect the mRNA from exonuclease degradation.

[0096] Thus, in some embodiments, the mRNA comprises a 5' cap structure. The 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; then, guanosine triphosphate (GTP) is added to the terminal phosphate via a guanylyltransferase to generate a 5'5' inverted triphosphate bond; and then, the 7-nitrogen of guanine is methylated by a methyltransferase. 2'-O-methylation can occur at the first and / or second base after the 7-methylguanosine triphosphate residue. Examples of cap structures include, but are not limited to, m7GpppNp-RNA, m7GpppNmp-RNA, and m7GpppNmpNmp-RNA (where m represents a 2'-O-methyl residue).

[0097] In some embodiments, the mRNA comprises a 3' poly(A) tail structure. The poly(A) tail at the 3' end of the mRNA often comprises 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). In some embodiments, the mRNA comprises a 3' poly(C) tail structure. A suitable poly(C) tail at the 3' end of the mRNA often comprises about 10 to 200 cytosine nucleotides (e.g., about 10 to 150 cytosine nucleotides, about 10 to 100 cytosine nucleotides, about 20 to 70 cytosine nucleotides, about 20 to 60 cytosine nucleotides, or about 10 to 40 cytosine nucleotides). The poly-C tail can be added to or replace the poly-A tail.

[0098] In some embodiments, the mRNA comprises a 5' and / or 3' untranslated region. In some embodiments, the 5' untranslated region includes one or more elements that affect mRNA stability or translation, such as an iron-responsive element. In some embodiments, the 5' untranslated region can be about 50-500 nucleotides in length.

[0099] In some embodiments, the 3' untranslated region comprises one or more of a polyadenylation signal, a binding site for a protein that affects the stability of the mRNA's location in the cell, or one or more binding sites for an miRNA. In some embodiments, the 3' untranslated region can be about 50-500 nucleotides in length, or longer.

[0100] Cap Structure In some embodiments, the mRNA comprises a 5' cap structure. The 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; then, guanosine triphosphate (GTP) is added to the terminal phosphate via a guanylyltransferase to generate a 5'-5' inverted triphosphate bond; and then, the 7-nitrogen of guanine is 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] The naturally occurring cap structure contains 7-methylguanosine, which is linked to the 5' end of the first transcribed nucleotide via a triphosphate bridge, resulting in m 7 This results in a dinucleotide cap of G(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 guanylyltransferase. Addition of the cap to the 5' end of RNA occurs immediately after the initiation of transcription. The terminal nucleoside is typically guanosine and is in the opposite orientation to all other nucleotides, i.e., G(5')ppp(5')GpNpNp.

[0102] The universal cap for mRNAs produced by in vitro transcription is m 7G(5')ppp(5')G, which is used as a dinucleotide cap in in vitro transcription using T7 RNA polymerase or SP6 RNA polymerase to obtain RNA with a cap structure at its 5' end. In a common in vitro method for synthesizing capped mRNA, the form m is used as the initiation factor for transcription. 7 G(5')ppp(5')G("m 7 A preformed dinucleotide, GpppG, is used.

[0103] Traditionally, the usual form of synthetic dinucleotide cap used in in vitro translation experiments is the anti-reverse cap analog ("ARCA") or modified ARCA, which is typically a modified cap analog in which the 2' or 3' OH group is replaced with -OCH3.

[0104] Further cap analogues include m 7 GpppG, m 7 GpppA, m 7 GpppC; unmethylated cap analogs (e.g., GpppG); dimethylated cap analogs (e.g., m 2,7 GpppG), trimethylated cap analogs (e.g., m 2,2,7 GpppG), dimethylated symmetric cap analogs (e.g., m 7 Gpppm7G), or anti-reverse cap analogs (e.g., ARCA;m 7、2’Ome GpppG, m 72’d GpppG, m 7,3’Ome GpppG, m 7,3’d GpppG, and their tetraphosphate derivatives) (see, for example, Jemielity, J. et al., "Novel 'anti-reve rse' cap analogs with superior translational properties,” RNA, 9:1108-1122 (2003).

[0105] In some embodiments, a suitable cap is 7-methylguanylic acid ("m 7 G"), which is linked to the 5' end of the first transcribed nucleotide via a triphosphate bridge, resulting in m 7 G(5')ppp(5')N (N is any nucleoside). 7 A preferred embodiment of the G-cap is m 7 G(5')ppp(5')G.

[0106] In some embodiments, the cap is a Cap 0 structure. The Cap 0 structure lacks 2'-O-methyl residues on the riboses attached to base 1 and base 2. In some embodiments, the cap is a Cap 1 structure. The Cap 1 structure has a 2'-O-methyl residue on base 2. In some embodiments, the cap is a Cap 2 structure. The Cap 2 structure has 2'-O-methyl residues attached to both base 2 and base 3.

[0107] Various m 7 G-cap analogs are known in the art, many of which are commercially available. These include the m 7 These include GpppG and ARCA 3'-OCH3 and 2'-OCH3 cap analogs (Jemielity, J. et al., RNA, 9:1108-1122 (2003)). Additional cap analogs for use in embodiments of the present invention include N7-benzylated dinucleoside tetraphosphate analogs (described in Grudzien, E. et al., RNA, 10:1479-1487 (2004)), phosphorothioate cap analogs (described in Grudzien-Nogalska, E. et al., RNA, 13:1745-1755 (2007)), and cap analogs described in U.S. Patent Nos. 8,093,367 and 8,304,529 (incorporated herein by reference), including biotinylated cap analogs.

[0108] Tail Structure The presence of a "tail" often serves to protect mRNA from exonuclease degradation. Poly-A tails are thought to stabilize natural messenger sense RNAs and synthetic sense RNAs. Thus, in certain embodiments, adding a long poly-A tail to an mRNA molecule can further stabilize the RNA. Poly-A tails can be added using a variety of techniques recognized in the art. For example, long poly-A tails can be added to synthetic or in vitro transcribed RNAs using poly-A polymerase (Yokoe, et al. Nature Biotechnology. 1996;14:1252-1256). Transcription vectors can also encode long poly-A tails. In addition, poly-A tails can be added by direct transcription from PCR products. Poly-A can also be ligated to the 3' end of sense RNA using 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).

[0109] In some embodiments, the mRNA comprises a 3' tail structure. The tail structure typically comprises a poly(A) tail and / or a poly(C) tail. The poly(A) tail or poly(C) tail on the 3' end of the mRNA typically comprises at least 50 adenosine or cytosine nucleotides, at least 150 adenosine or cytosine nucleotides, or at least 200 adenosine or cytosine nucleotides. nucleotides, at least 250 adenosine or cytosine nucleotides, at least 300 adenosine or cytosine nucleotides, at least 350 adenosine or cytosine nucleotides, at least 400 adenosine or cytosine nucleotides, at least 450 adenosine or cytosine nucleotides, at least 500 adenosine or cytosine nucleotides, at least 550 adenosine or cytosine nucleotides, at least 600 adenosine or cytosine nucleotides, Each of the fragments contains at least 650 adenosine or cytosine nucleotides, at least 700 adenosine or cytosine nucleotides, at least 750 adenosine or cytosine nucleotides, at least 800 adenosine or cytosine nucleotides, at least 850 adenosine or cytosine nucleotides, at least 900 adenosine or cytosine nucleotides, at least 950 adenosine or cytosine nucleotides, or at least 1 kb of adenosine or cytosine nucleotides.In some embodiments, the poly-A tail or poly-C tail each has between about 10 and 800 adenosine or cytosine nucleotides (e.g., between about 10 and 200 adenosine or cytosine nucleotides, between about 10 and 300 adenosine or cytosine nucleotides, between about 10 and 400 adenosine or cytosine nucleotides, between about 10 and 500 adenosine or cytosine nucleotides, between about 10 and 550 adenosine or cytosine nucleotides, between about 10 and 600 adenosine or cytosine nucleotides, between about 50 and 600 adenosine or cytosine nucleotides, between about 100 and 600 adenosine or cytosine nucleotides, between about 150 and 600 adenosine or cytosine nucleotides, between about 20 ... The poly(A) tail structure may be about 100 adenosine or cytosine nucleotides, about 250-600 adenosine or cytosine nucleotides, about 300-600 adenosine or cytosine nucleotides, about 350-600 adenosine or cytosine nucleotides, about 400-600 adenosine or cytosine nucleotides, about 450-600 adenosine or cytosine nucleotides, about 500-600 adenosine or cytosine nucleotides, about 10-150 adenosine or cytosine nucleotides, about 10-100 adenosine or cytosine nucleotides, about 20-70 adenosine or cytosine nucleotides, or about 20-60 adenosine or cytosine nucleotides. In some embodiments, the tail structure comprises a combination of poly(A) tails and poly(C) tails of various lengths as described herein. In some embodiments, the tail structure comprises at least 50%, 55%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99% adenosine nucleotides.In some embodiments, the tail structure comprises at least 50%, 55%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99% cytosine nucleotides.

[0110] In some embodiments, the length of the poly-A or poly-C tail is adjusted to control the stability of the modified sense mRNA molecules of the invention, and thereby control protein transcription. For example, because the length of the poly-A tail can affect the half-life of the sense mRNA molecule, the length of the poly-A tail may be adjusted to alter the level of resistance of the mRNA to nucleases, thereby controlling the time course of polynucleotide expression and / or polypeptide production in target cells.

[0111] 5' and 3' untranslated regions In some embodiments, the mRNA comprises a 5' and / or 3' untranslated region. In some embodiments, the 5' untranslated region includes one or more elements that affect mRNA stability or translation, such as an iron-responsive element. In some embodiments, the 5' untranslated region can be about 50-500 nucleotides in length.

[0112] In some embodiments, the 3' untranslated region comprises one or more of a polyadenylation signal, a binding site for a protein that affects the stability of the mRNA's location in the cell, or one or more binding sites for an miRNA. In some embodiments, the 3' untranslated region can be about 50-500 nucleotides in length, or longer.

[0113] Exemplary 3' and / or 5' UTR sequences may be derived from stable mRNA molecules (e.g., globin, actin, GAPDH, tubulin, histone, or citric acid cycle enzymes) to enhance the stability of the sense mRNA molecule. For example, the 5' UTR sequence may include a subsequence of the CMV immediate early 1 (IE1) gene or a fragment thereof to improve nuclease resistance and / or improve the half-life of the polynucleotide. It is also contemplated to include a sequence encoding human growth hormone (hGH) or a fragment thereof in 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., half-life) of the polynucleotide compared to their unmodified counterparts, including, for example, modifications made to improve the polynucleotide's resistance to in vivo nuclease digestion.

[0114] While mRNA resulting from an in vitro transcription reaction is desirable in some embodiments, other sources of mRNA are contemplated within the scope of the present invention, including mRNA produced from bacteria, fungi, plants, and / or animals.

[0115] The present invention can be used to deliver mRNA encoding various proteins.Non-limiting examples of mRNA suitable for the present invention include mRNA encoding target proteins such as argininosuccinate synthetase (ASS1), firefly luciferase (FFL), phenylalanine hydroxylase (PAH) and ornithine transcarbamylase (OTC).

[0116] Example of an mRNA sequence In some embodiments, the present invention provides methods and compositions for delivering mRNA encoding a target protein to a subject for the treatment of a target protein deficiency. Exemplary mRNA sequences are shown below. Construct Design: X-mRNA coding sequence-Y 5' and 3' UTR sequences X(5'UTR sequence) = GGACAGAUCGCCUGGAGACGCCAUCCACGCUGUUUUGACCUCCAUAGAAGACACCGGGACCGAUCCAGCCUCCGCGGCCGGGAACGGUGCAUUGGAACGCGGAUUCCCCGUGCCAAGAGUGACUCACCGUCCUUGACACG (SEQ ID NO: 1) Y(3'UTR sequence)= CGGGUGGCAUCCCUGUGACCCCUCCCCAGUGCCUCUCCUGGCCCUGGAAGUUGCCACUCCAGUGCCCACCAGCCUUGUCCUAAUAAAAUUAAGUUGCAUCAAGCU (SEQ ID NO: 2) or GGGUGGCAUCCCUGUGACCCCUCCCCAGUGCCUCUCCUGGCCCUGGAAGUUGCCACUCCAGUGCCCACCAGCCUUGUCCUAAUAAAAUUAAGUUGCAUCAAAGCU (SEQ ID NO: 3) An example of a full-length codon-optimized human ornithine transcarbamylase (OTC) messenger RNA sequence is shown below: An example of a full-length codon-optimized human ornithine transcarbamylase (OTC) messenger RNA sequence is shown below: GGACAGAUCGCCUGGAGACGCCAUCCACGCUGUUUUGACCUCCAUAGAAGACACCGGGACCGAUCCAGCCUCCCGGGCCGGGAACGGUGCAUUGGAACGCGGAUUCCCCGUGCCAAGAGUGACUCACCGUCCUUGACACGAUGCUGUUCAACCUUCGGAUCUUGCUGAAACAACGCUGCGUUCCGGAAUGGUCCACAACUUCAUGGUCCGGAAACUUCAGAUGCGGCAGCCGCUCCAGACCAGAACAAGGUGCAGCUCAAGGGAGGACCUUCCUCUCACCCUGAAAAA Another example of a codon-optimized full-length human ornithine transcarbamylase (OTC) messenger RNA sequence is shown below: GGACAGAUCGCCUGGAGACGCCAUCCACGCUGUUUUGACCUCCAUAGAAGACACCGGGACCGAUCCAGCCUCCGCGGCCGGGAACGGUGCAUUGGAACGCGGAUUCCCCGUGCCAAGAGUGACUCACCGUCCUUGACACGAUGCUGUUUAACCUGAGAAUUCUGCUGAACAACGCCGCGUUCAGGAACGGCCACAAUUUCAUGGUCCGCAACUUUAGAUGCGGACAGCCUCUCCAAAACAAGGUCCAGCUCAAGGGGCGGGACUUGCUGACCCUUAAGAACUUUACCGGCGAAGAGAUCAAGUACAUGCUGUGGUUGUCAGCGGACCUGAAGUUCCGCAUCAAGCAGAAAGGGGAGUAUCUGCCGCUGCUCCAAGGAAAGUCGCUCGGCAUGAUCUUCGAGAAGCGCUCGACCAGAACCCGGCUGUCCACUGAAACUGGUUUCGCCCUUCUGGGUGGACACCCUUGUUUCCUGACAACCCAGGACAUCCAUCUGGGCGUGAACGAAAGCCUCACUGACACCGCCAGGGUGCUGAGCUCCAUGGCCGACGCUGUCCUUGCCCGGGUGUACAAGCAGUCCGAUCUGGACACUCUGGCCAAGGAAGCGUCCAUCCCGAUCAUUAACGGACUGUCCGACCUGUACCACCCGAUCCAGAUUCUGGCCGACUACCUGACCUUGCAAGAGCACUACAGCUCACUGAAGGGCUUGACCCUGAGCUGGAUCGGCGACGGAAACAACAUUCUGCAUUCGAUCAUGAUGUCCGCGGCCAAGUUCGGAAUGCAUCUGCAGGCCGCAACUCCCAAGGGAUACGAACCUGAUGCGUCCGUGACUAAGCUGGCCG AGCAGUACGCAAAGGAAAACGGCACCAAGCUGCUGCUGACCAACGACCCGCCUCGGAGGGAACGUGCCUCUAUAUCCGACACUUGGAUCUCCAUGGGCAGGAAAGAGAAGAAGAGAAAGCGCUCCAGGCAUUCCAGGGUUACCAGCUACCAUGAAAACGGCCAAAGUGGCCGCUUCGGAUUGGACUUUCCUCCCACGCUUCCCCGCAAACCUGAGGGAAGUGGAAUGUUUCUACUCCCACGCUUCGUUCCCGAGGCCGAAUCGGAAGUGGACCAUAUAUGGCCGUGAUGGUGUCACUGACCGACUACAGCCCCCAACUGCAAAAGCCGAAGUUCUCUGGCCAUCGAAGUCCGCACGAGUAGUUGCCCACUGCCACUAGUGCCCACCAGCCUUGUCCUAGCAUGCCUUGCCUUGCAUCCAGCUUGCUUGCUAUAAAAAUUAGUUGCAUCAAGCU (sequence number 6) Example of a codon-optimized human ASS1 (CO-hASS1) coding sequence Example of a codon-optimized human PAH (CO-hPAH) coding sequence AUGAGCACCGCCGUGCUGGAGAACCCCGGCCUGGGCCGCAAGCUGAGCGACUUCGGCCAGGAGACCAGCUACAUCGAGGA

[0117] In some embodiments, suitable mRNA sequences may encode homologs or analogs of a target protein. For example, a target protein homolog or analog may be a modified target protein that contains one or more amino acid substitutions, deletions, and / or insertions compared to a wild-type or naturally occurring target protein while retaining substantial target protein activity. In some embodiments, mRNA suitable for the present invention encodes an amino acid sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homologous to the exemplary sequences listed above. In some embodiments, mRNA suitable for the present invention encodes a protein that is substantially identical to the target protein. In some embodiments, mRNA suitable for the present invention encodes an amino acid sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the exemplary sequences above. In some embodiments, mRNA suitable for the present invention encodes a fragment or portion of a target protein. In some embodiments, mRNA suitable for the present invention encodes a fragment or portion of a target protein, wherein the fragment or portion of the protein is a fragment or portion of the wild-type protein. In some embodiments, mRNA suitable for the present invention has a nucleotide sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the exemplary sequences above.

[0118] In some embodiments, a suitable mRNA encodes a fusion protein comprising a full-length, fragment, or portion of a target protein fused to another protein (e.g., an N-terminal or C-terminal fusion). In some embodiments, the protein fused to the mRNA encoding the full-length, fragment, or portion of the target protein encodes a signal sequence or a cellular targeting sequence.

[0119] lipid nanoparticles According to the present invention, the mRNA can be encapsulated or complexed in a nanoparticle, which in some embodiments is also referred to as a "delivery vehicle," "transfer vehicle," or grammatical equivalents thereof.

[0120] According to various embodiments, suitable nanoparticles include, but are not limited to, polymeric carriers such as polyethyleneimine (PEI), lipid nanoparticles and liposomes, nanoliposomes, ceramide-containing nanoliposomes, proteoliposomes, exosomes of natural and synthetic origin, natural lamellar bodies, synthetic lamellar bodies and semi-synthetic lamellar bodies, nanoparticles, calcium phosphosilicate nanoparticles, calcium phosphate nanoparticles, silicon dioxide nanoparticles, microcrystalline microparticles, semiconductor nanoparticles, poly(D-arginine), sol-gels, nanodendrimers, starch-based delivery systems, micelles, emulsions, niosomes, multi-domain block polymers (vinyl polymers, polypropylacrylic acid polymers, dynamic polyconjugates), dry powder formulations, plasmids, viruses, calcium phosphate nucleotides, aptamers, peptides, and other targeting tags.

[0121] In some embodiments, the mRNA is encapsulated in one or more liposomes. As used herein, the term "liposome" refers to any lamellar, multilamellar, or solid nanoparticle vesicle. Typically, liposomes used herein can be formed by mixing one or more lipids or by mixing one or more lipids with a polymer. Therefore, the term "liposome" used herein encompasses both lipid-based nanoparticles and polymer-based nanoparticles. In some embodiments, liposomes suitable for the present invention include cationic or non-cationic lipids, cholesterol-based lipids, and / or PEG-modified lipids.

[0122] PEGylated lipids In some embodiments, a suitable lipid solution comprises one or more PEGylated lipids. For example, polyethylene glycol (PEG)-modified phospholipids and derivatized lipids, such as derivatized ceramides (PEG-CER), including N-octanoyl-sphingosine-1-[succinyl(methoxypolyethylene glycol)-2000] (C8 PEG-2000 ceramide), are also contemplated by the present invention. Contemplated PEG-modified lipids range in length from C6 to C 20 In some embodiments, the PEG-modified or PEGylated lipid is PEGylated cholesterol or PEG-2K. In some embodiments, particularly useful exchangeable lipids have shorter acyl chains (e.g., C 14 or C 18 ) is a PEG-ceramide.

[0123] PEG-modified phospholipids and derivatized lipids may comprise at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, or at least 10% of the total lipids in the liposomes. do.

[0124] cationic lipids As used herein, the term "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. Some cationic lipids have been described in the literature, many of which are commercially available. Particularly suitable cationic lipids for use in the compositions and methods of the present invention include those described in International Patent Publication No. 2010 / 053572 (specifically, C12-200, as described in paragraph

[0225] ) and International Patent Publication No. 2012 / 170930, both of which are incorporated herein by reference. In certain embodiments, cationic lipids suitable for the compositions and methods of the present invention include, for example, (15Z,18Z)-N,N-dimethyl-6-(9Z,12Z)-octadeca-9,12-dien-1-yl)tetracosa-15,18-dien-1-amine (HGT5000), (15Z,18Z)-N,N-dimethyl-6-((9Z,12Z)-octadeca-9,12-dien-1-yl)tetracosa-4,15,18-tocosa ionizable cationic lipids described in U.S. Provisional Patent Application No. 61 / 617,468, filed March 29, 2012 (hereby incorporated by reference), such as (15Z,18Z)-N,N-dimethyl-6-((9Z,12Z)-octadeca-9,12-dien-1-yl)tetracosa-5,15,18-trien-1-amine (HGT5001), and (15Z,18Z)-N,N-dimethyl-6-((9Z,12Z)-octadeca-9,12-dien-1-yl)tetracosa-5,15,18-trien-1-amine (HGT5002).

[0125] In some embodiments, cationic lipids suitable for the compositions and methods of the present invention include cationic lipids such as 3,6-bis(4-(bis((9Z,12Z)-2-hydroxyoctadeca-9,12-dien-1-yl)amino)butyl)piperazine-2,5-dione (OF-02).

[0126] In some embodiments, cationic lipids suitable for the compositions and methods of the present invention include those cationic lipids described in WO 2015 / 184256 A2 entitled "Biodegradable Lipids for Delivery of Nucleic Acids," which is incorporated herein by reference, such as 3-(4-(bis(2-hydroxydodecyl)amino)butyl)-6-(4-((2-hydroxydodecyl)(2-hydroxyundecyl)amino)butyl)-1,4-dioxane-2,5-dione (Target 23), 3-(5-(bis(2-hydroxydodecyl)amino)pentan-2-yl)-6-(5-((2-hydroxydodecyl)(2-hydroxyundecyl)amino)pentan-2-yl)-1,4-dioxane-2,5-dione (Target 24).

[0127] In some embodiments, cationic lipids suitable for the compositions and methods of the present invention include those cationic lipids described in WO 2013 / 063468 entitled "Lipid Formulations for Delivery of Messenger RNA" and U.S. Provisional Application No. 2013 / 063468, both of which are incorporated herein by reference.

[0128] In some embodiments, one or more cationic lipids suitable for the present invention may be N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride, or "DOTMA" (Feigner et al. (Proc. Nat'l Acad. Sci. 84, 7413 (1987); U.S. Pat. No. 4,897,355). Other suitable cationic lipids include, for example, 5-carboxyspermylglycinedioctadecylamide, or "DOGS," 2,3-dioleyloxy-N-[2(spermine-carboxamido)ethyl]-N,N-dimethyl-1-propanaminium, or "DOSPA" (Behr et al. Proc. Nat'l Acad. Sci. 86, 6982 (1989); U.S. Pat. No. 5,171,678; U.S. Pat. No. 5,333,333). No. 4,761), 1,2-dioleoyl-3-dimethylammonium-propane or "DODAP," and 1,2-dioleoyl-3-trimethylammonium-propane or "DOTAP."

[0129] Further exemplary cationic lipids include 1,2-distearyloxy-N,N-dimethyl-3-aminopropane or "DSDMA," 1,2-dioleyloxy-N,N-dimethyl-3-aminopropane or "DODMA," 1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane or "DLinDMA," 1,2-dilinolenyloxy-N,N-dimethyl-3-aminopropane or "DLenDMA," N-dioleyl-N,N-dimethylammonium chloride, and the like. Distearyl-N,N-dimethylammonium bromide or "DODAC", N,N-distearyl-N,N-dimethylammonium bromide or "DDAB", N-(l,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide or "DMRIE", 3-dimethylamino-2-(cholest-5-ene-3-beta-oxybutan-4-oxy)-l-(cis,cis-9,12-octadecadienoxy)propane or "CLinDMA", 2-[5'-(cholest-5-ene)-4-yl]-1,1-dimethyl-2 ... -3-beta-oxy)-3'-oxapentoxy)-3-dimethyl-l-(cis,cis-9',l-2'-octadecadienoxy)propane or "CpLinDMA", N,N-dimethyl-3,4-dioleyloxybenzylamine or "DMOBA", 1,2-N,N'-dioleylcarbamyl-3-dimethylaminopropane or "DOcarbDAP", 2,3-dilinoleoyloxy-N,N-dimethylpropylamine or "DLinDAP", l,2-N,N'-dilinoleoyloxy Noleylcarbamyl-3-dimethylaminopropane or "DLincarbDAP," l,2-Dilinoleylcarbamyl-3-dimethylaminopropane or "DLinCDAP," 2,2-Dilinoleyl-4-dimethylaminomethyl-[l,3]-dioxolane or "DLin-DMA," 2,2-Dilinoleyl-4-dimethylaminoethyl-[l,3]-dioxolane or "DLin-K-XTC2-DMA," and 2-(2,2-di((9Z,12Z)-octadeca-9,l,2-dien-1-yl)-l,3-dioxolan-4-yl)-N,N-dimethylethanamine (DLin-KC2-DMA)) (WO 2010 / 042877; Sample et al., Nature Biotech. 28:172-176 (2010)), or mixtures thereof. (Heyes, J., et al., J Controlled Release 107:276-287 (2005); Morrissey, DV., et al., Nat. Biotechnol. 23(8):1003-1007 (2005); PCT Publication No. WO2005 / 121348A1). In some embodiments, one or more of the cationic lipids comprises at least one of an imidazole moiety, a dialkylamino moiety, or a guanidinium moiety.

[0130] In some embodiments, the one or more cationic lipids are selected from the group consisting of XTC (2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane), MC3 (((6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate), ALNY-100 ((3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-amine)), NC98-5 (4,7,13-tris(3-oxo-3-(undecylamino)propyl)-N1,N16-diundecyl-4,7,10,13-tetraazahexadecane-1,16-diamide).

[0131] The term "cationic lipid" refers to any of a number of lipids or lipidoids that have a net positive charge at a selected pH, such as physiological pH.

[0132] Suitable cationic lipids for use in the compositions and methods of the present invention include those described herein. The cationic lipids described in International Patent Publication No. 2010 / 14474, which is incorporated by reference, are included. In certain embodiments, the compositions and methods of the present invention include a cationic lipid, (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate, having the following compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0133] Other suitable cationic lipids for use in the compositions and methods of the invention include the ionizable cationic lipids described in International Patent Publication No. 2013 / 149140, which is incorporated herein by reference. In some embodiments, the compositions and methods of the invention comprise a cationic lipid of one of the following formulas: [ka] or a pharmaceutically acceptable salt thereof, wherein R and R are each independently hydrogen, an optionally substituted, variably saturated or unsaturated C-C 20 Alkyl, and optionally substituted, variably saturated or unsaturated C-C 20 acyl, wherein L and L are each independently hydrogen, optionally substituted C-C 30 Alkyl, optionally substituted variably unsaturated C-C 30 Alkenyl, and optionally substituted C-C 30 alkynyl, wherein m and o are each independently zero or any positive integer (e.g., m is 3), and wherein n is zero or any positive integer (e.g., n is 1). In certain embodiments, the compositions and methods of the present invention provide a cationic lipid (15Z,18Z)-N,N-dimethyl-6-(9Z,12Z)-octadeca-9,12-dien-1-yl)tetracosa-15,18-dien-1-amine ("HGT5000"), having the following compound structure: [ka] (HGT-5000) and pharmaceutically acceptable salts thereof. In certain embodiments, the compositions and methods of the present invention include the cationic lipid (15Z,18Z)-N,N-dimethyl-6-((9Z,12Z)-octadeca-9,12-dien-1-yl)tetracosa-4,15,18-trien-1-amine ("HGT5001"), having the following compound structure: [ka] (HGT-5001) and pharmaceutically acceptable salts thereof. In certain embodiments, the compositions and methods of the present invention comprise a cationic lipid and (15Z,18Z)-N,N-dimethyl-6-((9Z,12Z)-octadeca-9,12-dien-1-yl)tetracosa-5,15,18-trien-1-amine ("HGT5002"), having the following compound structure: [ka] (HGT-5002) and pharmaceutically acceptable salts thereof.

[0134] Other suitable cationic lipids for use in the compositions and methods of the present invention include those cationic lipids described as amino alcohol lipidoids in International Patent Publication No. 2010 / 053572, which is incorporated herein by reference. In certain embodiments, the compositions and methods of the present invention comprise cationic lipids having the following compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0135] Other suitable cationic lipids for use in the compositions and methods of the invention include those described in International Patent Publication No. 2016 / 118725, which is incorporated herein by reference. In certain embodiments, the compositions and methods of the invention include cationic lipids having the following compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0136] Other suitable cationic lipids for use in the compositions and methods of the invention include those described in International Patent Publication No. 2016 / 118724, which is incorporated herein by reference. In certain embodiments, the compositions and methods of the invention include cationic lipids having the following compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0137] Other suitable cationic lipids for use in the compositions and methods of the present invention include cationic lipids having the formula 14,25-ditridecyl 15,18,21,24-tetraaza-octatriacontane, and pharmaceutically acceptable salts thereof.

[0138] Other suitable cationic lipids for use in the compositions and methods of the present invention include those described in International Patent Publication Nos. 2013 / 063468 and 2016 / 205691, which are incorporated herein by reference. In some embodiments, the compositions and methods of the present invention comprise cationic lipids of the following formula: [ka] or a pharmaceutically acceptable salt thereof, wherein R L are independently optionally substituted C6-C 40 In certain embodiments, the compositions and methods of the present invention provide cationic lipids having the following compound structure: [ka] and pharmaceutically acceptable salts thereof. In certain embodiments, the compositions and methods of the present invention include cationic lipids having the following compound structure: [ka] and pharmaceutically acceptable salts thereof. In certain embodiments, the compositions and methods of the present invention include cationic lipids having the following compound structure: [ka] and pharmaceutically acceptable salts thereof. In certain embodiments, the compositions and methods of the present invention include cationic lipids having the following compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0139] Other suitable cationic lipids for use in the compositions and methods of the present invention include those described in International Patent Publication No. 2015 / 184256, which is incorporated herein by reference. In some embodiments, the compositions and methods of the present invention comprise cationic lipids of the following formula: [ka] or a pharmaceutically acceptable salt thereof, wherein each X is independently O or S; each Y is independently O or S; each m is independently 0 to 20; each n is independently 1 to 6; and each R A are independently hydrogen, optionally substituted C alkyl, optionally substituted C alkenyl, optionally substituted C alkynyl, optionally substituted C carbocyclyl, optionally substituted 3-14 membered heterocyclyl, optionally substituted C aryl, optionally substituted 5-14 membered heteroaryl or halogen; Bare independently hydrogen, optionally substituted C1-50 alkyl, optionally substituted C2-50 alkenyl, optionally substituted C2-50 alkynyl, optionally substituted C3-10 carbocyclyl, optionally substituted 3-14 membered heterocyclyl, optionally substituted C6-14 aryl, optionally substituted 5-14 membered heteroaryl or halogen. In certain embodiments, the compositions and methods of the invention provide compounds having the following structure: Cationic lipid "Target 23" with: [ka] (Target 23)

[0140] and pharmaceutically acceptable salts thereof.

[0141] Other suitable cationic lipids for use in the compositions and methods of the invention include those described in International Patent Publication No. 2016 / 004202, which is incorporated herein by reference. In some embodiments, the compositions and methods of the invention include cationic lipids having the following compound structure: [ka] or a pharmaceutically acceptable salt thereof. In some embodiments, the compositions and methods of the present invention comprise a cationic lipid having the following compound structure: [ka] or a pharmaceutically acceptable salt thereof. In some embodiments, the compositions and methods of the present invention comprise a cationic lipid having the following compound structure: [ka] or a pharmaceutically acceptable salt thereof.

[0142] Other suitable cationic lipids for use in the compositions and methods of the present invention include those described in J. McClellan, MCKing, Cell 2010, 141, 210-217 and Whitehead et al., Nature Communications (2014) 5:4277, which are incorporated herein by reference.In certain embodiments, the cationic lipid of the compositions and methods of the present invention is a cationic lipid having the following compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0143] Other suitable cationic lipids for use in the compositions and methods of the invention include those described in International Patent Publication No. 2015 / 199952, which is incorporated herein by reference. In some embodiments, the compositions and methods of the invention include cationic lipids having the following compound structure: [ka] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention include a cationic lipid having the following compound structure: [ka] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention include a cationic lipid having the following compound structure: [ka] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention include a cationic lipid having the following compound structure: [ka] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention include a cationic lipid having the following compound structure: [ka] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention include a cationic lipid having the following compound structure: [ka] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention include a cationic lipid having the following compound structure: [ka] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention include a cationic lipid having the following compound structure: [ka] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention include a cationic lipid having the following compound structure: [ka] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention include a cationic lipid having the following compound structure: [ka] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention include a cationic lipid having the following compound structure: [ka] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention include a cationic lipid having the following compound structure: [ka] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention include a cationic lipid having the following compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0144] Other suitable cationic lipids for use in the compositions and methods of the invention include those described in International Patent Publication No. 2017 / 004143, which is incorporated herein by reference. In some embodiments, the compositions and methods of the invention include cationic lipids with the following compound structure: Cationic lipids with the structure: [ka] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention include a cationic lipid having the following compound structure: [ka] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention include a cationic lipid having the following compound structure: [ka] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention include a cationic lipid having the following compound structure: [ka] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention include a cationic lipid having the following compound structure: [ka] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention include a cationic lipid having the following compound structure: [ka] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention include a cationic lipid having the following compound structure: [ka] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention include a cationic lipid having the following compound structure: [ka] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention include a cationic lipid having the following compound structure: [ka] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention include a cationic lipid having the following compound structure: [ka] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention include a cationic lipid having the following compound structure: [ka] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention include a cationic lipid having the following compound structure: [ka] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention include a cationic lipid having the following compound structure: [ka] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention include a cationic lipid having the following compound structure: [ka] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention include a cationic lipid having the following compound structure: [ka] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention include a cationic lipid having the following compound structure: [ka] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention include a cationic lipid having the following compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0145] Other suitable cationic lipids for use in the compositions and methods of the present invention include those described in International Patent Publication No. WO 2017 / 075531, which is incorporated herein by reference. In some embodiments, the compositions and methods of the present invention include cationic lipids of the following formula: [ka] or a pharmaceutically acceptable salt thereof, wherein L 1 or L 2 One of the following is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) x , -SS-, -C(=O)S-, -SC(=O)-, -NR a C(=O)-, -C(=O)NR a -, NR a C(=O)NR a -, -OC(=O)NR a -, or -NR a C(=O)O-; another L 1 or L 2 -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) x , -SS-, -C(=O)S-, SC(=O)-, -NR a C(=O)-, -C(=O)NR a -, NR a C(=O)NR a -, -OC(=O)NR a -or-NR a C(=O)O- or a direct bond; G 1 and G 2 are each independently unsubstituted C-C 12 Alkylene or C1-C 12 Alkenylene; G 3 is C1-C 24 Alkylene, C1-C 24 alkenylene, C3-C8 cycloalkyl, C3-C8 cycloalkenylene; R a is H or C1-C 12 alkyl; R 1 and R 2 are independently C6-C 24 Alkyl or C6-C 24 alkenyl; R 3is H, OR 5 , CN, -C(=O)OR 4 , -OC(=O)R 4 or -NR 5 C(=O)R 4 and R 4 is C1-C 12 alkyl; R 5 is H or C1-C6 alkyl; and x is 0, 1, or 2.

[0146] Other suitable cationic lipids for use in the compositions and methods of the invention include those described in International Patent Publication No. 2017 / 117528, which is incorporated herein by reference. In some embodiments, the compositions and methods of the invention include cationic lipids having the following compound structure: [ka] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention include a cationic lipid having the following compound structure: [ka] and pharmaceutically acceptable salts thereof. In some embodiments, the compositions and methods of the present invention include a cationic lipid having the following compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0147] Other suitable cationic lipids for use in the compositions and methods of the present invention include those described in International Patent Publication No. 2017 / 049245, which is incorporated herein by reference. In some embodiments, the cationic lipid of the compositions and methods of the present invention is a cationic lipid of one of the following formulas: [ka] and pharmaceutically acceptable salts thereof. For any one of these four formulas, R4 is -(CH2) n Q and -(CH2) n CHQR, where Q is -OR, -OH, -O(CH2) n In certain embodiments, the cationic lipid is selected from the group consisting of N(R), -OC(O)R, -CX, -CN, -N(R)C(O)R, -N(H)C(O)R, -N(R)S(O)R, -N(H)S(O)R, -N(R)C(O)N(R), -N(H)C(O)N(R), -N(H)C(O)N(H)(R), -N(R)C(S)N(R), -N(H)C(S)N(R), -N(H)C(S)N(H)(R), and heterocycle, wherein n is 1, 2, or 3. In certain embodiments, the compositions and methods of the present invention provide cationic lipids having the following compound structure: [ka] and pharmaceutically acceptable salts thereof. In certain embodiments, the compositions and methods of the present invention include cationic lipids having the following compound structure: [ka] and pharmaceutically acceptable salts thereof. In certain embodiments, the compositions and methods of the present invention include cationic lipids having the following compound structure: [ka] and pharmaceutically acceptable salts thereof. In certain embodiments, the compositions and methods of the present invention include cationic lipids having the following compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0148] Other suitable cationic lipids for use in the compositions and methods of the invention include those described in International Patent Publication Nos. 2017 / 173054 and 2015 / 095340, which are incorporated herein by reference. In certain embodiments, the compositions and methods of the invention comprise cationic lipids having the following compound structure: [ka] and pharmaceutically acceptable salts thereof. In certain embodiments, the compositions and methods of the present invention is a cationic lipid having the following compound structure: [ka] and pharmaceutically acceptable salts thereof. In certain embodiments, the compositions and methods of the present invention include cationic lipids having the following compound structure: [ka] and pharmaceutically acceptable salts thereof. In certain embodiments, the compositions and methods of the present invention include cationic lipids having the following compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0149] Other suitable cationic lipids for use in the compositions and methods of the present invention include cholesterol-based cationic lipids. In certain embodiments, the compositions and methods of the present invention include imidazole cholesterol esters or "ICEs," which have the following compound structure: [ka] (ICE) and pharmaceutically acceptable salts thereof.

[0150] Other suitable cationic lipids for use in the compositions and methods of the present invention include the cleavable cationic lipids described in International Patent Publication No. 2012 / 170889, which is incorporated herein by reference. In some embodiments, the compositions and methods of the present invention comprise a cationic lipid of the following formula: [ka] , wherein R1 is selected from the group consisting of imidazole, guanidine, amino, imine, enamine, optionally substituted alkylamino (e.g., alkylamino such as dimethylamino), and pyridyl, and R2 is selected from the group consisting of one of the following two formulae: [ka] wherein R and R are each independently an optionally substituted variably saturated or unsaturated C-C 20 Alkyl and optionally substituted variably saturated or unsaturated C6-C 20 acyl, wherein n is 0 or any positive integer (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more). In certain embodiments, the compositions and methods of the present invention provide a cationic lipid "HGT4001" having the following compound structure: [ka] (HGT4001) and pharmaceutically acceptable salts thereof. In certain embodiments, the compositions and methods of the present invention include a cationic lipid "HGT4002" having the following compound structure: [ka] (HGT4002) and pharmaceutically acceptable salts thereof. In certain embodiments, the compositions and methods of the present invention include a cationic lipid "HGT4003" having the following compound structure: [ka] (HGT4003) and pharmaceutically acceptable salts thereof. In certain embodiments, the compositions and methods of the present invention include a cationic lipid "HGT4004" having the following compound structure: [ka] (HGT4004) and pharmaceutically acceptable salts thereof. In certain embodiments, the compositions and methods of the present invention include a cationic lipid "HGT4005" having the following compound structure: [ka] (HGT4005) and pharmaceutically acceptable salts thereof.

[0151] In some embodiments, the compositions and methods of the present invention comprise the cationic lipid, N-[1-(2,3-dioleyloxy)propyl]-N,N,N,N-trimethylammonium chloride ("DOTMA") (Feigner et al. (Proc. Nat'l Acad. Sci. 84, 7413 (1987); U.S. Patent No. 4,897,355, which is incorporated herein by reference. Other suitable cationic lipids for the compositions and methods of the present invention include, for example, 5-carboxyspermylglycinedioctadecylamide (DOGS), 2,3-dioleyloxy-N-[2(spermine-carboxamido)ethyl]-N,N-dimethyl-1-propanaminium (DOSPA) (Behr et al. Proc. Nat. Acad. Sci. 86, 6982 (1989); U.S. Patent No. 5,171,678, U.S. Patent No. 5,334,761), 1,2-dioleoyl-3-dimethylammonium-propane (DODAP), and 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP).

[0152] Additional exemplary cationic lipids suitable for the compositions and methods of the present invention also include 1,2-distearyloxy-N,N-dimethyl-3-aminopropane ("DSDMA"); 1,2-dioleyloxy-N,N-dimethyl-3-aminopropane ("DODMA"), 1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane ("DLinDMA"), 1,2-dilinolenyloxy-N,N-dimethyl-3-aminopropane ("DLenDMA"), N-dioleyl-N,N-dimethylammonium chloride ("DODAC"), N,N-distearyl-N,N-dimethylalnernonium bromide ("DDAB"), N-(1,2-dimyrityloxyprop-3-yl)-N,N-dimethyl -N-hydroxyethylammonium bromide ("DMRIE"), 3-dimethylamino-2-(cholest-5-ene-3-beta-oxybutan-4-oxy)-l-(cis,cis-9,12-octadecadenoxy)propane ("CLinDMA"); 2-[5'-(cholesterol-5-ene-3-beta-oxy)-3'-oxapentoxy)-3-dimethyll-(cis,cis-9',l2'-octadecadenoxy)propane ("CpLinDMA"); N,N-dimethyl-3,4-dioleyloxybenzylamine ("DMOBA"); 1,2-N,N'-dioleylcarbamyl-3-dimethylaminopropane ("DOcarbDAP"); 2,3-Dilinoleoyloxy-N,N-dimethylpropylamine ("DLinDAP"), l,2-N,N'-Dilinoleylcarbamyl-3-dimethylaminopropane ("DLincarbDAP"); l,2-Dilinoleylcarbamyl-3-dimethylaminopropane ("DLinCDAP"); 2,2-Dilinoleyl-4-dimethylaminomethyl-[l,3]-dioxolane ("DLin-K-DMA"); 2-((8-[(3P)-cholest-5-en-3-yloxy]octyl)oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine ("Octyl-CLinDMA(2R)"); (2R)-2-((8-[(3P)- (2S)-2-((8-[(3P)-cholest-5-en-3-yloxy]octyl)oxy)-N,fsl-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine ("Octyl-CLinDMA(2R)"); (2S)-2-((8-[(3P)-cholest-5-en-3-yloxy]octyl)oxy)-N,fsl-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine ("Octyl-CLinDMA(2S)");2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane ("DLin-K-XTC2-DMA"), and 2-(2,2-di((9Z,12Z)-octadeca-9,12-dien-1-yl)-1,3-dioxolan-4-yl)-N,N-dimethylethanamine ("DLin-KC2-DMA") (see WO 2010 / 042877, incorporated herein by reference; Semple et al., Nature Biotech. 28:172-176 (2010)). (Heyes, J., et al., J Controlled Release 107:276-287 (2005); Morrissey, DV., et al., Nat. Biotechnol. 23(8):1003-1007 (2005); International Patent Publication WO 2005 / 121348). In some embodiments, one or more of the cationic lipids comprises at least one of an imidazole moiety, a dialkylamino moiety, or a guanidinium moiety.

[0153] In some embodiments, the one or more cationic lipids suitable for the compositions and methods of the present invention include 2,2-dilinol-4-dimethylaminoethyl-[1,3]-dioxolane ("XTC"); (3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-amine ("ALNY-100") and / or 4,7,13-tris(3-oxo-3-(undecylamino)propyl)-N1,N16-diundecyl-4,7,10,13-tetraazahexadecane-1,16-diamide ("NC98-5").

[0154] In some embodiments, the compositions of the present invention comprise one or more cationic lipids that constitute at least about 5%, 10%, 20%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, or 70% of the total lipid content in the composition, e.g., measured by weight of lipid nanoparticles. In some embodiments, the compositions of the present invention comprise one or more cationic lipids that constitute at least about 5%, 10%, 20%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, or 70% of the total lipid content in the composition, e.g., measured by mole % of lipid nanoparticles. In some embodiments, the compositions of the present invention comprise one or more cationic lipids that constitute at least about 5%, 10%, 20%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, or 70% of the total lipid content in the composition. The lipid content, e.g., measured by weight of the lipid nanoparticles, comprises one or more cationic lipids that constitute about 30-70% (e.g., about 30-65%, about 30-60%, about 30-55%, about 30-50%, about 30-45%, about 30-40%, about 35-50%, about 35-45%, or about 35-40%). In some embodiments, the compositions of the present invention comprise one or more cationic lipids that constitute about 30-70% (e.g., about 30-65%, about 30-60%, about 30-55%, about 30-50%, about 30-45%, about 30-40%, about 35-50%, about 35-45%, or about 35-40%) of the total lipid content in the composition, e.g., measured by molar percentage of the lipid nanoparticles.

[0155] Non-cationic / Helper Lipids As used herein, the term "non-cationic lipid" refers to any neutral lipid, zwitterionic lipid, or anionic lipid. As used herein, the term "cationic lipid" refers to any of a number of lipid species that carry a net negative charge at a selected pH, such as physiological pH. Non-cationic lipids include, but are not limited to, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoylphosphatidylcholine (DPPC), dioleoylphosphatidylethanolamine (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylethanolamine (P ... and mixtures thereof.

[0156] In some embodiments, non-cationic lipids may comprise at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, or 70% by weight or molar of the total lipids in a suitable lipid solution, hi some embodiments, non-cationic lipids comprise about 30-50% (e.g., about 30-45%, about 30-40%, about 35-50%, about 35-45%, or about 35-40%) of the total lipids in a suitable lipid solution, by weight or molar.

[0157] Cholesterol-based lipids In some embodiments, the suitable lipid solution comprises one or more cholesterol-based lipids. For example, suitable cholesterol-based cationic lipids include DC-Choi (N,N-dimethyl-N-ethylcarboxamidocholesterol), 1,4-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. Patent No. 5,744,335), or ICE. In some embodiments, the cholesterol-based lipid comprises at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, or 70% of the total lipids in the suitable lipid solution by weight or molar ratio. In some embodiments, the cholesterol-based lipids constitute about 30-50% (e.g., about 30-45%, about 30-40%, about 35-50%, about 35-45%, or about 35-40%) of the total lipids in a suitable lipid solution, by weight or by mole.

[0158] Exemplary combinations of cationic lipids, non-cationic lipids, cholesterol-based lipids, and PEG-modified lipids are described in the Examples section. For example, a suitable lipid solution may include: The lipid mixture may include cKK-E12, DOPE, cholesterol, and DMG-PEG2K; C12-200, DOPE, cholesterol, and DMG-PEG2K; HGT5000, DOPE, cholesterol, and DMG-PEG2K; HGT5001, DOPE, cholesterol, and DMG-PEG2K; cKK-E12, DPPC, cholesterol, and DMG-PEG2K; C12-200, DPPC, cholesterol, and DMG-PEG2K; HGT5000, DPPC, cholesterol, and DMG-PEG2K; or HGT5001, DPPC, cholesterol, and DMG-PEG2K. The selection of cationic lipids, non-cationic lipids, and / or PEG-modified lipids comprising the lipid mixture, as well as the relative molar ratios of these lipids to each other, is based on the characteristics of the selected lipids and the properties and characteristics of the mRNA to be encapsulated. Additional considerations include, for example, the degree of saturation of the alkyl chain of the selected lipid, as well as the size, charge, pH, pKa, fusogenicity, and toxicity. Thus, the molar ratios may be adjusted accordingly.

[0159] mRNA-loaded nanoparticles Any desired lipids can be mixed in any ratio suitable for encapsulating mRNA. In some embodiments, a suitable lipid solution contains a mixture of desired lipids, including cationic lipids, non-cationic lipids, cholesterol, and / or PEGylated lipids.

[0160] In some embodiments, the process of encapsulating mRNA in lipid nanoparticles comprises mixing an mRNA solution with a lipid solution, wherein the mRNA solution and / or the lipid solution are heated to a predetermined temperature higher than ambient temperature before mixing to form lipid nanoparticles that encapsulate the mRNA (see U.S. Patent Application Publication No. 14 / 790,562, filed July 2, 2015, entitled "Encapsulation of messenger RNA," and U.S. Provisional Patent Application No. 62 / 020,163, filed July 2, 2014, the disclosures of which are incorporated herein by reference in their entireties).

[0161] In some embodiments, the process for encapsulating mRNA in lipid nanoparticles involves mixing preformed lipid nanoparticles with mRNA (see U.S. Provisional Patent Application No. 62 / 420,413, filed November 10, 2016, and U.S. Provisional Patent Application No. 62 / 580,155, filed November 1, 2017, the disclosures of which are incorporated herein by reference). In some embodiments, mixing preformed lipid nanoparticles with mRNA results in lipid nanoparticles that improve the efficiency of mRNA delivery into cells. In some embodiments, mixing preformed lipid nanoparticles with mRNA results in extremely high encapsulation efficiency (i.e., in the range of 90-95%) of mRNA. In some embodiments, the preformed lipid nanoparticles and mRNA are mixed using a pump system that maintains a constant lipid / mRNA (N / P) ratio throughout the process and facilitates large-scale production.

[0162] Suitable liposomes according to the present invention can be made in a variety of sizes. In some embodiments, the provided liposomes can be made smaller than conventionally known mRNA-encapsulating liposomes. In some embodiments, reduced liposome size increases the efficiency of mRNA delivery. The appropriate liposome size can be selected taking into account the location of the target cell or tissue, and in part, the intended use of the liposomes.

[0163] In some embodiments, liposomes of appropriate size are selected to promote the systemic distribution of the antibody encoded by mRNA.In some embodiments, it may be desirable to limit the transfection of mRNA to specific cells or tissues.For example, to target hepatocytes, liposomes can be sized so that their size is smaller than the fenestration of the endothelial layer covering the hepatic sinusoids of the liver, and in this case, liposomes can easily penetrate the endothelial fenestration and reach the target hepatocytes.

[0164] Alternatively or additionally, liposomes may be sized to be of sufficient diameter to limit or intentionally prevent distribution within particular cells or tissues, for example, liposomes may be sized to be larger than the fenestrations in the endothelial layer lining the liver sinusoids, thereby limiting distribution of the liposomes to hepatocytes.

[0165] In some embodiments, the size of a liposome is determined by the maximum diameter of the liposome particle. In some embodiments, suitable liposomes have a size of about 250 nm or less (e.g., about 225 nm, 200 nm, 175 nm, 150 nm, 125 nm, 100 nm, 75 nm, or 50 nm or less). In some embodiments, suitable liposomes have a size in the range of about 10 to 250 nm (e.g., about 10 to 225 nm, 10 to 200 nm, 10 to 175 nm, 10 to 150 nm, 10 to 125 nm, 10 to 100 nm, 10 to 75 nm, or 10 to 50 nm). In some embodiments, suitable liposomes have a size in the range of about 100 to 250 nm (e.g., about 100 to 225 nm, 100 to 200 nm, 100 to 175 nm, or 100 to 150 nm). In some embodiments, suitable liposomes have a size in the range of about 10-100 nm (e.g., in the range of about 10-90 nm, 10-80 nm, 10-70 nm, 10-60 nm, or 10-50 nm). In certain embodiments, suitable liposomes have a size less than about 100 nm.

[0166] Various alternative methods known in the art are available for sizing liposome populations. One such sizing method is described in U.S. Pat. No. 4,737,323, which is incorporated herein by reference. Sonication of liposome suspensions, either by bath or probe sonication, results in a gradual reduction in size to small ULVs with diameters of less than about 0.05 micrometers. Homogenization is another method that utilizes shear energy to fragment large liposomes into smaller ones. In a typical homogenization procedure, MLVs are recirculated using a standard emulsion homogenizer until a selected liposome size, typically about 0.1 to 0.5 micrometers, is observed. Liposome size can be calculated by quasi-electric light scattering (QELS), as described in Bloomfield, Ann. Rev. Biophys. Bioeng., 10:421-150 (1981), incorporated herein by reference. The average liposome diameter can be reduced by sonicating the formed liposomes. Intermittent sonication cycles can be alternated with QELS assessment to guide efficient liposome synthesis.

[0167] Pharmaceutical Composition To promote mRNA expression in vivo, delivery vehicles such as lipid nanoparticles, including liposomes, may be formulated in combination with one or more additional nucleic acids, carriers, targeting ligands, or stabilizing reagents, or may be formulated into pharmacological compositions mixed with suitable excipients. In some embodiments, lipid nanoparticles encapsulating mRNA are administered simultaneously with hyaluronidase. Techniques for drug formulation and administration can be found in "Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, Pa., latest edition.

[0168] The provided liposome-encapsulated or liposome-associated mRNA and compositions containing same may be administered and dosed in accordance with current medical practice, taking into consideration the clinical condition of the subject, the site and method of administration, the administration schedule, the age, sex, and weight of the subject, and other factors relevant to a clinician of the art. For purposes herein, an "effective amount" is defined as an amount determined by experimental clinical trials. Such dosages may be determined by appropriate considerations known to those skilled in the research, pharmacological, clinical, and medical fields. In some embodiments, the amount administered is effective to at least somewhat stabilize, ameliorate, or eliminate symptoms or other indicators selected by those skilled in the art as appropriate measures of disease progression, regression, or improvement. For example, a suitable amount and dosing regimen is one that results in at least transient production of a protein (e.g., an enzyme).

[0169] While the present invention focuses on subcutaneous delivery, such as a bolus injection into the subcutaneous tissue (the tissue layer between the skin and muscle), other suitable routes of administration include, for example, oral, rectal, vaginal, transmucosal, pulmonary, including intratracheal or inhalation, or intestinal administration; intradermal, transdermal (topical), intramuscular, intramedullary, and parenteral delivery, including intrathecal, direct intraventricular, intravenous, intraperitoneal, or intranasal. In certain embodiments, intramuscular administration is into a muscle selected from the group consisting of skeletal muscle, smooth muscle, and cardiac muscle. In some embodiments, administration results in delivery of mRNA to muscle cells. In some embodiments, administration results in delivery of mRNA to hepatocytes (i.e., liver cells). In certain embodiments, intramuscular administration results in delivery of mRNA to muscle cells.

[0170] Alternatively, or additionally, the liposome-encapsulated mRNA and compositions of the present invention may be administered in a local rather than systemic manner.

[0171] The provided methods of the present invention contemplate single administration as well as multiple administrations of a therapeutically effective amount of a therapeutic agent (e.g., an mRNA encoding a therapeutic protein) described herein. The therapeutic agent may be administered at regular intervals depending on the nature, severity, and extent of the subject's condition (e.g., OTC deficiency). In some embodiments, a therapeutically effective amount of a therapeutic agent (e.g., an mRNA encoding a therapeutic protein) of the present invention may be administered subcutaneously periodically at regular intervals (e.g., once a year, once every six months, once every five months, once every three months, every other month (once every two months), monthly (once every month), every other week (once every two weeks), twice a month, once every 30 days, once every 28 days, once every 14 days, once every 10 days, once every 7 days, weekly, twice a week, daily, or continuously).

[0172] In some embodiments, the provided liposomes and / or compositions are formulated for sustained release of the mRNA contained therein. Such sustained-release compositions can be administered to a subject over extended dosing intervals as appropriate. For example, in some embodiments, the compositions of the present invention are administered to a subject twice daily, daily, or every other day. In preferred embodiments, the compositions of the present invention are administered to a subject twice weekly, once weekly, once every 7 days, once every 10 days, once every 14 days, once every 28 days, once every 30 days, once every two weeks, once every three weeks, or more preferably once every four weeks, once a month, twice a month, once every six weeks, once every eight weeks, once every two months, once every three months, once every four months, once every six months, once every eight months, once every nine months, or annually. Compositions and liposomes formulated for depot administration (e.g., intramuscular, subcutaneous, intravitreal) to deliver or release mRNA over an extended period of time are also contemplated. Preferably, the sustained release means employed is combined with modifications made to the mRNA to enhance stability.

[0173] As used herein, the term "therapeutically effective amount" is primarily based on the total amount of the therapeutic agent contained in the pharmaceutical composition of the present invention. Generally, a therapeutically effective amount is an amount sufficient to provide a meaningful benefit to the subject (e.g., treat, regulate, cure, prevent, and / or ameliorate OTC deficiency). For example, a therapeutically effective amount may be an amount sufficient to provide a desired therapeutic and / or prophylactic effect. Typically, the amount of a therapeutic agent (e.g., mRNA encoding a therapeutic protein) administered to a subject in need thereof will vary depending on the characteristics of the subject. Such characteristics include the subject's condition, disease severity, general health, age, sex, and weight. Those skilled in the art will be able to easily determine the appropriate dosage depending on these and other relevant factors. Furthermore, objective and subjective assays can be used to identify optimal dosage ranges. Both can be used optionally.

[0174] A therapeutically effective amount is generally administered in a dosing regimen that may include multiple unit doses. For any particular therapeutic protein, the therapeutic effect (and / or appropriate unit dose within an effective dosing regimen) may vary, for example, depending on the route of administration and in combination with other pharmaceutical agents. Additionally, the therapeutically effective amount (and / or unit dose) specific to any particular patient may depend on a variety of factors, including the disorder being treated and the severity of the disorder; the activity of the particular pharmaceutical agent employed; the particular composition employed; the patient's age, weight, general health, sex, and diet; the time of administration, route of administration, and / or excretion or metabolic rate of the particular protein employed; the duration of treatment; and similar factors well known in the medical field.

[0175] In some embodiments, the therapeutically effective dose is between about 0.005 mg / kg body weight and 500 mg / kg body weight, e.g., between about 0.005 mg / kg body weight and 400 mg / kg body weight, between about 0.005 mg / kg body weight and 300 mg / kg body weight, between about 0.005 mg / kg body weight and 200 mg / kg body weight, between about 0.005 mg / kg body weight and 100 mg / kg body weight, between about 0.005 mg / kg body weight and 90 mg / kg body weight, between about 0.005 mg / kg body weight and 80 mg / kg body weight, between about 0.005 mg / kg body weight and 70 mg / kg body weight, or between about 0.005 mg / kg body weight and 70 mg / kg body weight. The ranges are 0 mg / kg (body weight), approximately 0.005 mg / kg (body weight) to 60 mg / kg (body weight), approximately 0.005 mg / kg (body weight) to 50 mg / kg (body weight), approximately 0.005 mg / kg (body weight) to 40 mg / kg (body weight), approximately 0.005 mg / kg (body weight) to 30 mg / kg (body weight), approximately 0.005 mg / kg (body weight) to 25 mg / kg (body weight), approximately 0.005 mg / kg (body weight) to 20 mg / kg (body weight), approximately 0.005 mg / kg (body weight) to 15 mg / kg (body weight), and approximately 0.005 mg / kg (body weight) to 10 mg / kg (body weight).

[0176] In some embodiments, a therapeutically effective dose is greater than about 0.1 mg / kg body weight, greater than about 0.5 mg / kg body weight, greater than about 1.0 mg / kg body weight, greater than about 3 mg / kg body weight, greater than about 5 mg / kg body weight, greater than about 10 mg / kg body weight, greater than about 15 mg / kg body weight, greater than about 20 mg / kg body weight, greater than about 30 mg / kg body weight, greater than about 40 mg / kg body weight, greater than about 50 mg / kg body weight, greater than about 60 mg / kg body weight, or greater than about 100 mg / kg body weight. In certain embodiments, the therapeutically effective dose is greater than about 1.0 mg / kg body weight. In some embodiments, a therapeutically effective dose of 1.0 mg / kg body weight is administered intramuscularly or intravenously.

[0177] Also contemplated herein are lyophilized pharmaceutical compositions comprising one or more of the liposomes disclosed herein, and related methods for using such compositions, e.g., as disclosed in International Patent Application PCT / US12 / 41663, filed June 8, 2012. The teachings of this application are incorporated herein by reference in their entirety. For example, lyophilized pharmaceutical compositions according to the present invention can be reconstituted prior to administration or reconstituted in vivo. For example, lyophilized pharmaceutical compositions can be formulated into an appropriate dosage form (e.g., an intradermal dosage form such as a disk, rod, or membrane) and administered such that the dosage form is rehydrated in vivo by the individual's bodily fluids over time.

[0178] The provided liposomes and compositions can be administered to any desired tissue. In some embodiments, the provided liposomes and compositions containing mRNA are delivered subcutaneously, and the mRNA is expressed in cells or tissue types other than the subcutaneous tissue. In some embodiments, the provided liposomes The mRNA encoding the target protein delivered by the liposome or composition is expressed in the tissue to which the liposome and / or composition is administered. In some embodiments, the delivered mRNA is expressed in a tissue different from the tissue to which the liposome and / or composition is administered. Examples of tissues to which the delivered mRNA may be delivered and / or expressed include, but are not limited to, the liver, kidney, heart, spleen, serum, brain, skeletal muscle, lymph nodes, skin, and / or cerebrospinal fluid.

[0179] In some embodiments, administration of provided compositions increases the expression of the administered mRNA or increases the activity level of the mRNA-encoded protein in a biological sample from a subject compared to the baseline expression or activity level before treatment or administration. In some embodiments, administration of provided compositions increases the expression or activity level of a therapeutic protein encoded by the mRNA of the provided compositions in a biological sample from a subject compared to the baseline expression or activity level before treatment. Typically, the baseline level is measured immediately before treatment. Biological samples include, for example, whole blood, serum, plasma, urine, and tissue samples (e.g., muscle, liver, skin fibroblasts). In some embodiments, administration of provided compositions increases the expression or activity level of a therapeutic protein (the protein encoded by the administered mRNA) by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% compared to the baseline level immediately before treatment. In some embodiments, administration of provided compositions increases the mRNA expression or activity level in a biological sample from a subject compared to an untreated subject. In some embodiments, administration of a provided composition increases the expression or activity level of a therapeutic protein encoded by the mRNA of a provided composition in a biological sample from a subject compared to an untreated subject.

[0180] According to various embodiments, the timing of expression of the delivered mRNA can be tailored to suit particular medical needs, hi some embodiments, expression of the protein encoded by the delivered mRNA is detectable 1 hour, 2 hours, 3 hours, 6 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 2 weeks, or 1 month after administration of provided liposomes and / or compositions.

[0181] In some embodiments, when a therapeutically effective dose of the provided composition is administered regularly, it causes an increase in citrulline production in a subject compared to the baseline citrulline production before treatment.Typically, the citrulline level before or after treatment can be measured in a biological sample obtained from a subject, such as blood, plasma or serum, urine, or solid tissue extract.In some embodiments, treatment according to the present invention increases the citrulline level in a biological sample (e.g., plasma, serum, or urine) by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 1-fold, 1.5-fold, 2-fold, 2.5-fold, or 3-fold compared to the baseline citrulline level.

[0182] According to the present invention, a therapeutically effective dose of a provided composition, when administered regularly, results in a reduction in the intensity, severity, or frequency of, or a delay in the onset of, at least one symptom or characteristic of a protein deficiency.

[0183] therapeutic use The present invention can be used to treat a variety of diseases, disorders, and conditions. In particular, single gene disorders and disorders in which administration of an mRNA encoding a protein reduces or ameliorates one or more disease-related symptoms are candidates for therapeutic use using the present invention. Exemplary therapeutic messenger RNAs for subcutaneous administration detailed in this disclosure are listed in Tables 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, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99, 100, 101, 102, 103, 104, 105, 106, 107 The gene may be selected from any of the corresponding exemplary genes listed in 4, 5 or 6. [Table 1-1] [Table 1-2] [Table 2-1] [Table 2-2] Table 2-3 Table 2-4 Table 3-1 Table 3-2 Table 3-3 Table 3-4 Table 3-5 Table 3-6 Table 3-7 Table 3-8 Table 3-9 Table 3-10 Table 3-11 Table 4-1 Table 4-2 Table 4-3 Table 4-4 Table 4-5 Table 5-1 Table 5-2 Table 5-3 Table 5-4 Table 5-5 Table 6-1 Table 6-2 Table 6-3 Table 6-4 Table 6-5 Table 6-6 Table 6-7 Table 6-8 Table 6-9 Table 6-10 Table 6-11 Table 6-12 Table 6-13 Table 6-14 Table 6-15 Table 6-16 Table 6-17 Table 6-18 Table 6-19 Table 6-20 Table 6-21 Table 6-22 Table 6-23 Table 6-24 Table 6-25 Table 6-26 Table 6-27 Table 6-28 Table 6-29 Table 6-30 Table 6-31 Table 6-32 Table 6-33 Table 6-34 Table 6-35 Table 6-36 Table 6-37 Table 6-38 Table 6-39 Table 6-40 Table 6-41 Table 6-42 Table 6-43 Table 6-44 Table 6-45 Table 6-46

[0184] In some embodiments, the present invention is useful for treating a disease or disorder listed in Table 1.

[0185] In some embodiments, the present invention is useful for the delivery of vaccines. Subcutaneously delivered vaccines include vaccines against infectious diseases, including, but not limited to, diphtheria, tetanus, pertussis, poliomyelitis, measles, mumps, rubella, Haemophilus influenzae type b, hepatitis B, influenza, pneumococcal disease, cholera, hepatitis A, meningococcal disease, plague, rabies, bat lyssavirus, yellow fever, Japanese encephalitis, Q fever, tuberculosis, typhoid fever, and varicella-zoster. Subcutaneously delivered vaccines may also include vaccines against cell proliferative disorders, such as cancer. In some embodiments, subcutaneously delivered vaccines include cancer vaccines for lymphoproliferative diseases. In some embodiments, cancer vaccines comprise subcutaneously delivered mRNA encoding an immunogenic agent that induces a cellular immune response against cancer cells, using the methods of the present invention. In some embodiments, MHC class II antigens containing one or more cancer antigenic epitopes are used. Vaccines containing mRNA encoding a target peptide are administered subcutaneously along with mRNA encoding hyaluronidase, which can result in superior systemic delivery of the vaccine and a stronger antigen response.

[0186] In some embodiments, the present invention is useful for treating liver diseases, such as OTC deficiency. Co-injection of hyaluronidase enzyme with mRNA encoding OTC protein increases the level of OTC enzyme (protein) in a subject's liver cells (e.g., hepatocytes) compared to baseline levels before treatment. Typically, baseline levels are measured before treatment (e.g., up to 12 months before treatment, and in some cases immediately before treatment). In some embodiments, subcutaneous injection of the present invention increases the level of OTC protein in hepatocytes by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% compared to baseline levels before treatment. In some embodiments, subcutaneous injection of the present invention increases the level of OTC protein in hepatocytes compared to the level of OTC protein in liver cells of a subject not receiving treatment.

[0187] In some embodiments, subcutaneous injection of the present invention increases the level of OTC protein in the subject's plasma or serum compared to the baseline level before treatment. Typically, the baseline level is measured before treatment (e.g., up to 12 months before treatment, and in some cases immediately before treatment). In some embodiments, administration of provided compositions increases the level of OTC protein in the plasma or serum by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% compared to the baseline level immediately before treatment. In some embodiments, administration of provided compositions increases the level of OTC protein in the plasma or serum compared to the level of OTC protein in the plasma or serum of a subject not receiving treatment.

[0188] The compositions and methods of the present invention provide mRNA delivery for treating a variety of disorders. In particular, the compositions and methods of the present invention are suitable for treating diseases or disorders related to the deficiency of proteins and / or enzymes excreted or secreted by the liver. These diseases or disorders include, but are not limited to, phenylalanine hydroxylase (PAH) deficiency (previously known as phenylketonuria (PKU)), argininosuccinate synthetase 1 (ASS1) deficiency, which causes the hepatic urea cycle disorder citrullinemia, and erythropoietin (EPO) deficiency, which causes anemia (erythropoietin is a protein produced both in the kidney and the liver).

[0189] Disorders for which the present invention is useful include, but are not limited to, disorders such as Fabry disease; hemophilic diseases (e.g., hemophilia B (FIX), hemophilia A (FVIII)); SMN1-associated spinal muscular atrophy (SMA); amyotrophic lateral sclerosis (ALS); GALT-associated galactosemia; COL4A5-associated disorders, including Alport syndrome; galactocerebrosidase deficiency; X-linked adrenoleukodystrophy; Friedreich's ataxia; Pelizaeus-Merzbach disease; TSC1- and TSC2-associated tuberous sclerosis; Sanfilippo B syndrome (MPS) FMR1-related disorders, including fragile X syndrome, fragile X-associated tremor / ataxia syndrome, and fragile X premature ovarian failure syndrome; Prader-Willi syndrome; hereditary hemorrhagic telangiectasia (AT); Niemann-Pick disease type C1; neuronal ceroid lipofuscinosis-related disorders, including juvenile neuronal ceroid lipofuscinosis (JNCL), juvenile Batten disease, Santavoli-Hartier disease, Jansky-Bierschowski disease, and PTT-1 and TPP1 deficiency; EIF2B1-, EIF2B2-, EIF2B3-, EIF2B4-, and EIF2B5-related childhood ataxia with central nervous system hypomyelination / vanishing white matter disease; CACNA1A- and CACNB4-related transient ataxia type 2; MECP2-related disorders, including classic Rett syndrome, MECP2-associated severe neonatal encephalopathy, and PPM-X syndrome; CDKL5-related atypical These include Rett syndrome; Kennedy disease (SBMA); Notch-3-related autosomal dominant cerebral arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL); SCN1A- and SCN1B-related seizure disorders; Alpers-Huttenlocher syndrome, polymerase G-related disorders including POLG-related sensory ataxic neuropathy, dysarthria, and ophthalmoplegia, and autosomal dominant and recessive progressive external ophthalmoplegia with mitochondrial DNA deletions; X-linked adrenal hypoplasia; X-linked agammaglobulinemia; and Wilson's disease.

[0190] In some embodiments, the nucleic acids, particularly mRNAs, of the invention may encode functional proteins or enzymes that are secreted into the extracellular space. For example, secreted proteins include clotting factors, complement pathway components, cytokines, chemokines, chemoattractants, protein hormones (e.g., EGF, PDF), serum protein components, antibodies, secreted Toll-like receptors, etc. In some embodiments, the compositions of the invention may contain mRNAs encoding erythropoietin, α1-antitrypsin, carboxypeptidase N, or human growth hormone. [Example]

[0191] While certain compounds, compositions, and methods of the present invention have been specifically described in accordance with certain embodiments, the following examples serve merely to illustrate the compounds of the present invention and are not intended to be limiting thereof.

[0192] lipid material The formulations described in the Examples below, unless otherwise noted, contain multi-component lipid mixtures using one or more cationic lipids, helper lipids (e.g., non-cationic lipids and / or cholesterol-based lipids), and PEGylated lipids in various ratios designed to encapsulate various nucleic acid materials. Cationic lipids for the process include, but are not limited to, cKK-E12 (3,6-bis(4-(bis(2-hydroxydodecyl)amino)butyl)piperazine-2,5-dione), OF-02, Target 23, Target 24, ICE, HGT5000, HGT5001, HGT4003, DOTAP (1,2-dioleyl-3-trimethylammonium propane), DODAP (1,2-dioleyl-3-dimethylammonium propane), DOTMA (1,2-di-O-octadecenyl-3-trimethylammonium propane), DLinDMA (Heyes, J.; Palmer, L.; Bremner, K.; MacLachlan, I. "Cationic lipid saturation influences intracellular delivery of encapsulated nucleic acids" J. Contr. Rel. 2005, 107, 276-287), DLin-KC2-DMA (Semple, SC et al. "Rational Design of Cationic Lipids for siRNA Delivery" Nature Biotech. 2010, 28, 172-176), C12-200 (Love, KT et al. "Lipid-like materials for low-dose in vivo gene silencing" PNAS 2010, 107, 1864-1869), dialkylamino-based, imidazole-based, guanidinium-based, and the like can be mentioned.Helper lipids may include, but are not limited to, DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine), DOPE (1,2-dioleyl-sn-glycero-3-phosphoethanolamine), DPPE (1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine), DMPE (1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine), DOPG (1,2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol)), DOPC (1,2-dioleyl-sn-glycero-3-phosphotidylcholine), cholesterol, etc. PEGylated lipids may include, but are not limited to, C6-C. 20 A lipid having an alkyl chain of length The antibody may comprise a poly(ethylene) glycol chain up to 5 kDa in length covalently attached to the antibody.

[0193] mRNA material In some embodiments, codon-optimized messenger RNA encoding a target protein was synthesized by in vitro transcription from a gene-encoding plasmid DNA template, followed by the addition of a 5' cap structure (Cap1) (F. F. Fechter, P.; Brownlee, GG "Recognition of mRNA cap structures by viral and cellular proteins" J. Gen. Virology 2005, 86, 1239-1249) and a 3' poly(A). The 5' and 3' untranslated regions present in each mRNA product are designated X and Y, respectively, and are defined as above.

[0194] Example 1. In vivo expression of firefly luciferase protein in mice This example shows an exemplary method for administering firefly luciferase (FFL) mRNA-carrying LNPs and for analyzing firefly luciferase in target tissues in vivo.Wild-type mice were treated with 20 mg / kg of FFL mRNA-encoding LNPs formulated with 20 mg / kg of hyaluronidase mRNA via subcutaneous delivery.The luminescence produced by FFL protein was observed 3, 24, and 48 hours after subcutaneous administration. Significant luminescence was observed, indicating successful production of active FFL protein in the livers of these mice. Furthermore, persistent FFL activity was maintained for at least 24 hours with little or no decrease in intensity.

[0195] Example 2. In vivo activity of expressed hOTC in mice This example uses OTC KO spf ashThis example shows a comparison of intravenous administration of human OTC (hOTC) mRNA-loaded lipid nanoparticles without hyaluronidase and subcutaneous administration with or without mRNA encoding hyaluronidase in mice. In this example, hOTC and hyaluronidase mRNA are present in the same formulation and are therefore administered simultaneously. The hOTC protein is shown to be enzymatically active, as determined by measuring citrulline production levels using a custom ex vivo activity assay. Generally, citrulline production can be used to assess the activity of expressed hOTC protein. Citrulline activity of hOTC protein was measured in mouse liver extracts 24 hours after subcutaneous delivery (20 mg / kg) of a single dose of hOTC mRNA-encapsulated lipid nanoparticles with or without hyaluronidase. Citrulline activity was also measured in the livers of saline-treated OTC KO mice. No significant hOTC protein activity was observed following subcutaneous administration of hOTC mRNA without the hyaluronidase mRNA co-formulation. hOTC protein activity in these animals was similar to that seen in animals treated with saline. In contrast, hOTC protein activity (as evidenced by citrulline protein levels) was similar in the livers of mice administered intravenously with the hOTC mRNA LNP composition and in the livers of mice administered subcutaneously with the hOTC mRNA LNP composition co-formulated with mRNA encoding hyaluronidase. The dose-dependence of hyaluronidase mRNA on the robustness of OTC mRNA expression can be tested using various doses of hyaluronidase mRNA in the formulation.

[0196] Example 3. In vivo efficacy of CO-hOTC mRNA delivery in mice This example uses OTC KO spf ashFigure 1 shows a comparison of intravenous administration of CO-hOTC (codon-optimized human OTC) mRNA-loaded lipid nanoparticles without hyaluronidase versus subcutaneous administration with or without hyaluronidase-encoding mRNA in mice. Subcutaneously delivered CO-hOTC mRNA lipid nanoparticles co-formulated with hyaluronidase mRNA were significantly superior to subcutaneously delivered mRNA without hyaluronidase-encoding mRNA. NA is more effective than lipid nanoparticles.

[0197] The efficiency of administration was calculated by comparing the CO-hOTC mRNA copy number in the livers of various treatment groups. The CO-hOTC mRNA copy number in mouse livers was measured 24 hours after a single subcutaneous administration of 20 mg / kg of CO-hOTC mRNA and 20 mg / kg of hyaluronidase mRNA (SEQ ID NO: 12) LNP formulation. A control set contained OTC mRNA without hyaluronidase mRNA. For comparison, the CO-hOTC mRNA copy number was also measured in the livers of mice 24 hours after intravenous injection of 0.50 mg / kg of CO-hOTC mRNA LNP solution. The mOTC mRNA copy number was measured in the livers of saline-treated wild-type (WT) mice, saline-treated OTC KO mice, and OTC KO mice treated intravenously with hOTC LNP solution, subcutaneously with a hyaluronidase-free hOTC LNP formulation, or subcutaneously with hOTC LNPs co-formulated with hyaluronidase.

[0198] Example 4. In vivo expression of human erythropoietin (hEPO) in mice This example shows an exemplary time course of human erythropoietin (hEPO) protein expression following subcutaneous administration of hEPO-encoding mRNA compared to intravenous administration using the disclosed methods.

[0199] Male CD1 mice were administered hEPO mRNA-loaded lipid nanoparticles at a dose of 1 mg / kg intravenously or hEPO mRNA-loaded lipid nanoparticles co-formulated with 5 mg / kg hyaluronidase mRNA at a dose of 5 mg / kg subcutaneously once on day 1. Human EPO protein expression was examined in serum samples by hEPO-specific ELISA for 4 days.

[0200] High levels of EPO protein expression were observed in both the intravenous and subcutaneous administration groups of mice 6 hours after mRNA administration (day 1) and on day 2. This expression level is comparable to that observed with the intravenous administration of the same mRNA LNPs.

[0201] equivalent Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. The scope of the present invention is not intended to be limited to the above description, but rather is as set forth in the following claims.

Claims

1. a) a first messenger RNA (mRNA) encoding a protein or polypeptide; and b) a second mRNA encoding a hyaluronidase 1. A composition for subcutaneous administration comprising: The composition, wherein the first mRNA and the second mRNA are encapsulated in one or more lipid nanoparticles.

2. The composition of claim 1 , wherein the composition is in liquid form.

3. 10. The composition of claim 1, wherein the composition is a lyophilized powder prior to subcutaneous administration.

4. The composition of claim 1 , wherein the first mRNA is a therapeutic mRNA.

5. The composition of claim 1 , wherein the second mRNA encodes a mammalian hyaluronidase.

6. 6. The composition of claim 5, wherein the mammalian hyaluronidase is bovine hyaluronidase.

7. 6. The composition of claim 5, wherein the mammalian hyaluronidase is a human hyaluronidase.

8. The composition of claim 1 , wherein the second mRNA comprises the polynucleotide sequence of SEQ ID NO:9, SEQ ID NO:10, or SEQ ID NO:

12.

9. 2. The composition of claim 1, wherein the first mRNA and / or the second mRNA comprises one or more modified nucleotides.

10. 2. The composition of claim 1, wherein the first mRNA and / or the second mRNA are individually capped and tailed.

11. 10. The composition of claim 1, wherein the one or more lipid nanoparticles comprise a cationic lipid.

12. The cationic lipid may be cKK-E12 (3,6-bis(4-(bis(2-hydroxydodecyl)amino)butyl)piperazine-2,5-dione), OF-02 ((3,6-bis(4-(bis((9Z,12Z)-2-hydroxyoctadeca-9,12-dien-1-yl)amino)butyl)piperazine-2,5-dione)), Target 23 ((3-(4-(bis(2-hydroxydodecyl)amino)butyl)-6-(4-((2-hydroxydodecyl)(2- (hydroxyundecyl)amino)butyl)-1,4-dioxane-2,5-dione)), Target 24 ((3-(5-(bis(2-hydroxydodecyl)amino)pentan-2-yl)-6-(5-((2-hydroxydodecyl)(2-hydroxyundecyl)amino)pentan-2-yl)-1,4-dioxane-2,5-dione)), ICE (imidazole cholesterol ester), HGT5000 ((15Z,18Z)-N,N-dimethyl-6-(9Z, 12Z)-octadeca-9,12-dien-1-yl)tetracosa-15,18-dien-1-amine), HGT5001 ((15Z,18Z)-N,N-dimethyl-6-((9Z,12Z)-octadeca-9,12-dien-1-yl)tetracosa-4,15,18-trien-1-amine), DOTAP (1,2-dioleyl-3-trimethylammonium propane), DODAP (1,2-dioleyl-3-dimethylammonium propane), DOTMA ( 1,2-di-O-octadecenyl-3-trimethylammonium propane), DLinDMA (1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane), DLin-KC2-DMA (2-(2,2-di((9Z,12Z)-octadeca-9,12-dien-1-yl)-1,3-dioxolan-4-yl)-N,N-dimethylethanamine), dialkylamino-based cationic lipids, imidazole-based cationic lipids, guanidinium-based cationic lipids, 【Chemistry 1】 12. The composition of claim 11, comprising HGT4003 having the compound structure:

13. The composition of claim 1 , wherein the one or more lipid nanoparticles comprise a PEG-modified lipid.

14. 14. The composition of claim 13, wherein the PEG-modified lipid comprises at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, or at least 10% of the total lipid of the one or more lipid nanoparticles.

15. 10. The composition of claim 1, wherein the one or more lipid nanoparticles comprise a non-cationic lipid.

16. The non-cationic lipids include distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), and dioleoylphosphatidylethanolamine.

16. The composition of claim 15, comprising 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), distearoylphosphatidylethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), or a combination thereof.

17. 10. The composition of claim 1, wherein the one or more lipid nanoparticles comprise a cholesterol-based lipid.

18. 18. The composition of claim 17, wherein the cholesterol-based lipid comprises DC-Choi (N,N-dimethyl-N-ethylcarboxamido cholesterol), 1,4-bis(3-N-oleylamino-propyl)piperazine, ICE (imidazole cholesterol ester), or a combination thereof.

19. The composition of any one of claims 1 to 18, wherein the first mRNA and the second mRNA are co-encapsulated in the one or more lipid nanoparticles.

20. The composition of any one of claims 1 to 18, wherein the first mRNA and the second mRNA are encapsulated in separate lipid nanoparticles.

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