Lipid formulations for the release of messenger RNA

Description

BACKGROUND

[0001] Delivery of nucleic acids has been explored extensively as a potential therapeutic option for certain disease states. In particular, RNA interference (RNAi) has been the subject of significant research and clinical development. While RNAi, such as short interfering RNA (siRNA), may have therapeutic potential, it is of little use in treating diseases involving deficiency of one or more proteins. Messenger RNA (mRNA) therapy has become an increasingly important option for treatment of various diseases, in particular, for those associated with deficiency of one or more proteins.

[0002] WO 99 / 14346 discloses modified, eukaryotic mRNA molecules encoding a therapeutically relevant protein. These mRNA molecules have a nucleotide sequence which comprises at least one chemical modification such as least one end blocking modification which renders the modified mRNA molecule stable.

[0003] US 5,976,569 discloses diketopiperazine-based delivery systems.

[0004] WO 2007 / 024708 discloses methods of reducing the immunogenicity of RNA molecules including mRNA. It also discloses mRNAs comprising pseudouridine or other modified ribonucleotides as well as methods for in vivo gene replacement therapy employing these mRNAs.

[0005] WO 2013 / 063468 discloses single amino acid-based lipid derivatives. These were tested at a dose of 1 mg / kg in mice in an siRNA screen, which indicated that lysine was a favorable amino acid. Lysine-based peptide and polypeptide-lipid derivatives were then investigated at the same dose. The top hits and their analogs were tested at a lower dose of 0.1 mg / kg, which led to selection of cKK-E12 as the lead compound.SUMMARY OF THE INVENTION

[0006] The present invention provides a composition comprising an mRNA encoding a therapeutic protein encapsulated within a liposome for use in a method of treating a disease or disorder comprising administering to a subject in need of treatment the composition, wherein the composition is injected intradermally, intramuscularly, or subcutaneously or administered intrathecally, intraventricularly, or intravenously and expression of the protein encoded by the mRNA is detectable in the liver, kidney, heart, spleen, serum, brain, skeletal muscle, lymph nodes, skin and / or cerebrospinal fluid, and the liposome comprises a cationic lipid of formula I-g: or a pharmaceutically acceptable salt thereof, wherein each of R L< is optionally substituted C 8-10 alkyl and the liposome further comprises a non-cationic lipid, a cholesterol-based lipid and a PEG-modified lipid, wherein the molar ratio of cationic lipid to non-cationic lipid to cholesterol-based lipid to PEG-modified lipid is 30-50:25-35:20-30:1-15.

[0007] This composition is highly efficient in the delivery and expression of mRNA and encoded protein in vivo. The invention is based, in part, on the surprising discovery that liposomes based on this cationic lipid are unexpectedly effective in delivering mRNA and producing encoded protein in vivo, more effective even as compared to those cationic lipids that were considered to be among the best in delivering mRNA in the prior art. Indeed, prior to the present invention, cationic lipids have been extensively explored as an important component of liposomes typically used to encapsulate nucleic acids including mRNA for in vivo delivery. Due to the uniquely fragile and long structure of mRNA and the complicated in vivo translation process, cationic lipids used in the liposomes typically play two roles. First, cationic lipids promote interaction with negatively charged mRNA during encapsulation, circulation and endocytosis, thereby capturing and protecting the mRNA. Then, once inside cytosol, cationic lipids need to be able to release the mRNA so that the mRNA can be translated to produce encoded protein. Some cationic lipids, in particular, those known as titratable cationic lipids are particularly effective in delivering mRNA. One example of such cationic lipids known to be capable of efficient delivery of mRNA is C12-200. Surprisingly, the present inventors found that cationic lipids described herein can be even more effective in delivering various mRNA in vivo, than those best known in the prior art including C12-200. For example, as shown in the Examples below, liposome particles incorporating a cationic lipid described herein (e.g., cKK-E12) resulted in close to 50% higher protein expression of human Factor IX protein detected in the plasma of administered mice, as compared to C12-200-based liposome particles. Furthermore, the plasma residence time of different proteins expressed from mRNA delivered by cKK-E12 based liposomes is sustained up to 7 days or longer post a single administration. Thus, the present inventors have demonstrated that this class of cationic lipids having a structure of formula I-g as described herein (e.g., cKK-E12) can be uniquely useful in delivering mRNA for highly efficient and sustained production of protein (e.g., therapeutic protein) in vivo. The present invention therefore permits an improved mRNA therapy that can significantly reduce required amount of mRNA and associated lipids, administration frequency, and possible side effects, providing more potent, safer, and patient friendly mRNA therapy for various diseases.

[0008] In some embodiments, each of R L< is C 8 alkyl. In some embodiments, each of R L< is C 9 alkyl. In some embodiments, each of R L< is C 10 alkyl.

[0009] In some embodiments, a suitable cationic lipid is cKK-E12:

[0010] In some embodiments, the non-cationic lipid is selected from distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, and 1-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE).

[0011] In some embodiments, the cholesterol-based lipid is selected from cholesterol or PEGylated cholesterol.

[0012] In some embodiments, the PEG-modified lipid comprises a poly(ethylene) glycol chain of up to 5 kDa in length covalently attached to a lipid with alkyl chain(s) of C 6 -C 20 length. In some embodiments, a PEG-modified lipid is a derivatized ceramide such as N-Octanoyl-Sphingosine-1-[Succinyl(Methoxy Polyethylene Glycol)-2000]. In some embodiments, a PEG-modified or PEGylated lipid is PEGylated cholesterol or Dimyristoylglycerol (DMG) -PEG-2K.

[0013] In some embodiments, a suitable liposome comprises cKK-E12, DOPE, cholesterol and DMG-PEG2K.

[0014] In some embodiments, the cationic lipid (e.g., cKK-E12) constitutes about 30-45%, about 30-40%, about 35-50%, about 35-45%, or about 35-40% of the liposome by molar ratio. In some embodiments, the cationic lipid (e.g., cKK-E12) constitutes about 30%, about 35%, about 40 %, about 45%, or about 50% of the liposome by molar ratio.

[0015] In particular embodiments, the ratio of cKK-E12:DOPE:cholesterol:DMG-PEG2K is approximately 40:30:20:10 by molar ratio. In particular embodiments, the ratio of cKK-E12:DOPE:cholesterol:DMG-PEG2K is approximately 40:30:25:5 by molar ratio. In particular embodiments, the ratio of cKK-E12:DOPE:cholesterol:DMG-PEG2K is approximately 40:32:25:3 by molar ratio.

[0016] In some embodiments, a suitable liposome has a size of or less than about 500nm, 450 nm, 400nm, 350 nm, 300nm, 250 nm, 200 nm, 150 nm, 125 nm, 110 nm, 100 nm, 95 nm, 90 nm, 85 nm, 80 nm, 75 nm, 70 nm, 65 nm, 60 nm, 55 nm, or 50 nm.

[0017] In some embodiments, a composition according to the invention is administered intravenously. In some embodiments, a composition according to the invention is administered intrathecally.

[0018] In some embodiments, the expression of the protein encoded by the mRNA is detectable 6, 12, 18, 24, 30, 36, 42, 48, 54, 60, 66, and / or 72 hours after the administration. In some embodiments, the expression of the protein encoded by the mRNA is detectable 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, and / or 7 days after the administration. In some embodiments, the expression of the protein encoded by the mRNA is detectable 1 week, 2 weeks, 3 weeks, and / or 4 weeks after the administration. In some embodiments, the expression of the protein encoded by the mRNA is detectable after a month after the administration.

[0019] In some embodiments, the protein encoded by the mRNA is a cytosolic protein. In some embodiments, the protein encoded by the mRNA is a secreted protein. In some embodiments, the protein encoded by the mRNA is an enzyme. In some embodiments, the mRNA has a length of or greater than about 0.5kb, 1 kb, 1.5 kb, 2 kb, 2.5 kb, 3 kb, 3.5 kb, 4 kb, 4.5 kb, or 5 kb. In some embodiments, the protein encoded by the mRNA is Argininosuccinate Synthetase (ASS1), Factor IX, survival of motor neuron 1, or phenylalanine hydroxylase.

[0020] In some embodiments, the mRNA is administered at a dose ranging from about 0.1 - 5.0 mg / kg body weight, for example about 0.1 - 4.5, 0.1 - 4.0, 0.1 - 3.5, 0.1 - 3.0, 0.1 - 2.5, 0.1 - 2.0, 0.1 - 1.5, 0.1 - 1.0, 0.1 - 0.5, 0.1 - 0.3, 0.3 - 5.0, 0.3 - 4.5, 0.3 - 4.0, 0.3 - 3.5, 0.3 - 3.0, 0.3 - 2.5, 0.3 - 2.0, 0.3 - 1.5, 0.3 - 1.0, 0.3 - 0.5, 0.5 - 5.0, 0.5-4.5, 0.5 - 4.0, 0.5 - 3.5, 0.5 - 3.0, 0.5 - 2.5, 0.5 - 2.0, 0.5 - 1.5, or 0.5 - 1.0 mg / kg body weight. In some embodiments, the mRNA is administered at a dose of or less than about 5.0, 4.5, 4.0, 3.5, 3.0, 2.5, 2.0, 1.5, 1.0, 0.8, 0.6, 0.5, 0.4, 0.3, 0.2, or 0.1 mg / kg body weight.

[0021] 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-methyl adenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, and / or 2-thiocytidine. In some embodiments, the mRNA is unmodified.

[0022] Other features, objects, and advantages of the present invention are apparent in the detailed description and drawings that follow. It should be understood, however, that the detailed description and the drawings, while indicating embodiments of the present invention, are given by way of illustration only, not limitation.BRIEF DESCRIPTION OF THE DRAWING

[0023] The drawings are for illustration purposes only not for limitation. Figure 1 shows an exemplary graph of the levels of human factor IX (FIX) detected in the serum of treated mice 24 hours after administration of C12-200 or cKK-E12 liposomes containing FIX mRNA. Figure 2 shows an exemplary graph of FIX detected in the plasma of mice treated with 0.1, 0.3, 0.6, 1.0, or 3.0 mg / kg of one of two ratios of FIX mRNA containing cKK-E12 liposomes either 6 or 24 hours after administration. Figure 3 shows an exemplary graph of the level of ASS1 protein detected in the livers of mice treated with 0.1, 0.3, 0.6, 1.0, or 3.0 mg / kg of ASS1 mRNA-containing cKK-E12 liposomes 24 hours after administration. Figure 4 shows exemplary western blot analyses of ASS1 protein levels in the liver 24 hours post administration of 0.1, 0.3, 0.6, 1.0, or 3.0 mg / kg of cKK-E12 liposomes containing ASS1 mRNA. Figure 5 shows an exemplary graph of ASS1 protein levels in the liver of mice 0.5, 3, 6, 12, 24, 48, 72 hours after a single IV injection of ASS1 mRNA containing cKK-E12 liposomes (1mg / kg). Also shown is the level of ASS1 protein 7 days after administration. Figure 6 shows exemplary western blot analyses of ASS1 protein levels in the liver 0.5, 3, 6, 12, 24, 48, 72 hours after a single IV injection of 1mg / kg ASS1 mRNA containing cKK-E12 liposomes. Also shown is the level of ASS1 protein 7 days after administration. Figure 7 - shows detection of human ASS1 messenger RNA via in situ hybridization in the livers of treated mice. Exogenous mRNA is observable for at least 72 hr post-administration after a single dose (1.0 mg / kg) of ASS1 mRNA-loaded MD1-based lipid nanoparticles. Human ASS1 mRNA is detectable in sinusoidal cells as well as hepatocytes. Figure 8 - shows exemplary immunohistochemical staining of ASS1 protein levels in mouse liver 24 hours after administration of 1 mg / kg ASS1 mRNA containing cKK-E12 lipid nanoparticles. Human ASS1 protein is detectable in sinusoidal cells as well as hepatocytes. Figure 9 shows low magnification (4x) immunohistochemical staining of ASS1 protein levels in mouse liver 24 hours after administration of 1 mg / kg ASS1 mRNA containing cKK-E12 liposomes. A comparison to untreated mouse liver (left) demonstrates the widespread distribution of human ASS1 protein throughout the liver. Figure 10 shows exemplary results illustrating that cKK-E12 lipid nanoparticles efficiently delivered FL mRNA via nebulization. Mice were exposed to milligram of encapsulated FL mRNA and analysis was performed 24 hours post-exposure. Figure 11 illustrates detection via western blot of human SMN-1 protein derived from exogenous hSMN-1 mRNA that was transfected into BHK-21 cells. Various antibodies specific to human SMN were employed: (A) anti-SMN 4F11 antibody at 1:1,000 dilution; (B) Pierce PA5-27309 a-SMN antibody at 1:10,000 dilution; and (C) LSBio C138149 a-SMN antibody at 1:10,000 dilution. Figure 12A-Cillustrates multiplex nucleic acid in situ detection of human Survival of Motor Neuron (hSMN-1) mRNA in (A) Cervical, (B) Thoracic and (C) Lumbar spinal tissue, 24 hours post intrathecal delivery. Figure 13 illustrates positive detection of human SMN-1 protein produced in the spinal cord of a rat 24 hours post-intrathecal administration of human SMN-1 mRNA-loaded lipid nanoparticles. Anti-human SMN 4F11 antibody was employed at 1:2500 dilution. Panel A represents treated rat spinal cord tissue and panel B represents untreated rat spinal cord tissue. Figure 14 In vivo transfection of CFTR knockout mice with C-terminal His 10 tagged (SEQ ID NO: 11) codon-optimized human CFTR mRNA encapsulated within either a lipid (cKK-E12) or polymeric (PEI) nanoparticle formulation. Following nebulized delivery of each respective mRNA formulation, Right and Left lung tissue lysate was collected and analyzed for CFTR expression by Western blot using anti-His antibody 1187. Control CFTR knockout lung tissue and CFTR-His 10 HEK293 lysate ("His 10 " disclosed as SEQ ID NO: 11) was used as a negative and positive controls respectively. Figure 15 illustrates positive detection of active firefly luciferase (FFL) protein in a treated pig lung via luminescence upon exposure to FFL / CO-CFTR-C-His 10 mRNA ("His 10 " disclosed as SEQ ID NO: 11) encapsulated cKK-E12 lipid nanoparticles. Pigs were treated with 1 mg FFL + 9 mg CO-CFTR-C-His 10 mRNA ("His 10 " disclosed as SEQ ID NO: 11) encapsulated lipid nanoparticles via nebulization using a Pari jet nebulizer and sacrificed 24 hours post-treatment. FFL luminescence was visualized using an IVIS bioluminometer. DEFINITIONS

[0024] In order for the present invention to be more readily understood, certain terms are first defined below. Additional definitions for the following terms and other terms are set forth throughout the specification. The publications and other reference materials referenced herein to describe the background of the invention and to provide additional detail regarding its practice.

[0025] Amino acid: As used herein, term "amino acid," in its broadest sense, refers to any compound and / or substance that can be incorporated into a polypeptide chain. In some embodiments, an amino acid has the general structure H E N-C(H)(R)-COHO. In some embodiments, an amino acid is a naturally occurring amino acid. In some embodiments, an amino acid is a synthetic amino acid; in some embodiments, an amino acid is a d-amino acid; in some embodiments, an amino acid is an l-amino acid. "Standard amino acid" refers to any of the twenty standard l-amino acids commonly found in naturally occurring peptides. "Nonstandard amino acid" refers to any amino acid, other than the standard amino acids, regardless of whether it is prepared synthetically or obtained from a natural source. As used herein, "synthetic amino acid" encompasses chemically modified amino acids, including but not limited to salts, amino acid derivatives (such as amides), and / or substitutions. Amino acids, including carboxyl- and / or amino-terminal amino acids in peptides, can be modified by methylation, amidation, acetylation, protecting groups, and / or substitution with other chemical groups that can change the peptide's circulating half-life without adversely affecting their activity. Amino acids may participate in a disulfide bond. Amino acids may comprise one or posttranslational modifications, such as association with one or more chemical entities (e.g., methyl groups, acetate groups, acetyl groups, phosphate groups, formyl moieties, isoprenoid groups, sulfate groups, polyethylene glycol moieties, lipid moieties, carbohydrate moieties, biotin moieties, etc.). The term "amino acid" is used interchangeably with "amino acid residue," and may refer to a free amino acid and / or to an amino acid residue of a peptide. It will be apparent from the context in which the term is used whether it refers to a free amino acid or a residue of a peptide.

[0026] Animal: As used herein, the term "animal" refers to any member of the animal kingdom. In some embodiments, "animal" refers to humans, at any stage of development. In some embodiments, "animal" refers to non-human animals, 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, cattle, 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, an animal may be a transgenic animal, genetically-engineered animal, and / or a clone.

[0027] Approximately or about: As used herein, the term "approximately" or "about," as applied to one or more values of interest, refers to a value that is similar to a stated reference value. In certain embodiments, the term "approximately" or "about" refers to a range of values that fall within 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 than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).

[0028] Delivery: As used herein, the term "delivery" encompasses both local and systemic delivery. For example, delivery of mRNA encompasses situations in which an mRNA is delivered to a target tissue and the encoded protein is expressed and retained within the target tissue (aslo referred to as "local distribution" or "local delivery"), and situations in which an mRNA is delivered to a target tissue and the encoded protein is expressed and secreted into patient's circulation system (e.g., serum) and systematically distributed and taken up by other tissues (also referred to as "systemic distribution" or "systemic delivery).

[0029] Expression: As used herein, "expression" of a nucleic acid sequence refers to translation of an mRNA into a polypeptide, assemble multiple polypeptides (e.g., heavy chain or light chain of antibody) into an intact protein (e.g., antibody) and / or post-translational modification of a polypeptide or fully assembled protein (e.g., antibody). In this application, the terms "expression" and "production," and grammatical equivalent, are used inter-changeably.

[0030] Functional: As used herein, a "functional" biological molecule is a biological molecule in a form in which it exhibits a property and / or activity by which it is characterized.

[0031] Half-life: As used herein, the term "half-life" is the time required for a quantity such as nucleic acid or protein concentration or activity to fall to half of its value as measured at the beginning of a time period.

[0032] Improve, increase, or reduce: As used herein, the terms "improve," "increase" or "reduce," or grammatical equivalents, indicate values that are relative to a baseline measurement, such as a measurement in the same individual prior to initiation of the treatment described herein, or a measurement in a control subject (or multiple control subject) in the absence of the treatment described herein. A "control subject" is a subject afflicted with the same form of disease as the subject being treated, who is about the same age as the subject being treated.

[0033] In Vitro: As used herein, the term "in vitro" refers to events that occur in an artificial environment, e.g., in a test tube or reaction vessel, in cell culture, etc., rather than within a multi-cellular organism.

[0034] In Vivo: As used herein, the term "in vivo" refers to events that occur within a multi-cellular organism, such as a human and a non-human animal. In the context of cell-based systems, the term may be used to refer to events that occur within a living cell (as opposed to, for example, in vitro systems).

[0035] Isolated: As used herein, the term "isolated" refers to a substance and / or entity that has been (1) separated from at least some of the components with which it was associated when initially produced (whether in nature and / or in an experimental setting), and / or (2) produced, prepared, and / or manufactured by the hand of man. Isolated substances and / or entities may be separated from about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% of the other components with which they were initially associated. In some embodiments, isolated agents are about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% pure. As used herein, a substance is "pure" if it is substantially free of other components. As used herein, calculation of percent purity of isolated substances and / or entities should not include excipients (e.g., buffer, solvent, water, etc.).

[0036] Local distribution or delivery: As used herein, the terms "local distribution," "local delivery," or grammatical equivalent, refer to tissue specific delivery or distribution. Typically, local distribution or delivery requires a protein (e.g., enzyme) encoded by mRNAs be translated and expressed intracellularly or with limited secretion that avoids entering the patient's circulation system.

[0037] messenger RNA (mRNA): As used herein, the term "messenger RNA (mRNA)" refers to a polynucleotide that encodes at least one polypeptide. mRNA as used herein encompasses both modified and unmodified RNA. mRNA may contain one or more coding and non-coding regions. mRNA can be purified from natural sources, produced using recombinant expression systems and optionally purified, chemically synthesized, etc. Where appropriate, e.g., in the case of chemically synthesized molecules, mRNA can comprise nucleoside analogs such as analogs having chemically modified bases or sugars, backbone modifications, etc. An mRNA sequence is presented in the 5' to 3' direction unless otherwise indicated. In some embodiments, an mRNA is or comprises natural nucleosides (e.g., adenosine, guanosine, cytidine, uridine); nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl adenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, and 2-thiocytidine); chemically modified bases; biologically modified bases (e.g., methylated bases); intercalated bases; modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose); and / or modified phosphate groups (e.g., phosphorothioates and 5'-N-phosphoramidite linkages).

[0038] In some embodiments, the mRNA comprises one or more nonstandard nucleotide residues. The nonstandard nucleotide residues may include, e.g., 5-methyl-cytidine ("5mC"), pseudouridine ("ψU"), and / or 2-thio-uridine ("2sU"). See, e.g., U.S. Patent No. 8,278,036 or WO2011012316 for a discussion of such residues and their incorporation into mRNA. The mRNA may be RNA, which is defined as RNA in which 25% of U residues are 2-thio-uridine and 25% of C residues are 5-methylcytidine. Teachings for the use of RNA are disclosed US Patent Publication US20120195936 and internation publication WO2011012316. The presence of nonstandard nucleotide residues may render an mRNA more stable and / or less immunogenic than a control mRNA with the same sequence but containing only standard residues. In further embodiments, the mRNA may comprise one or more nonstandard nucleotide residues chosen from isocytosine, pseudoisocytosine, 5-bromouracil, 5-propynyluracil, 6-aminopurine, 2-aminopurine, inosine, diaminopurine and 2-chloro-6-aminopurine cytosine, as well as combinations of these modifications and other nucleobase modifications. Certain embodiments may further include additional modifications to the furanose ring or nucleobase. Additional modifications may include, for example, sugar modifications or substitutions (e.g., one or more of a 2'-O-alkyl modification, a locked nucleic acid (LNA)). In some embodiments, the RNAs may be complexed or hybridized with additional polynucleotides and / or peptide polynucleotides (PNA). In embodiments where the sugar modification is a 2'-O-alkyl modification, such modification may include, but are not limited to a 2'-deoxy-2'-fluoro modification, a 2'-O-methyl modification, a 2'-O-methoxyethyl modification and a 2'-deoxy modification. In certain embodiments, any of these modifications may be present in 0-100% of the nucleotides-for example, more than 0%, 1%, 10%, 25%, 50%, 75%, 85%, 90%, 95%, or 100% of the constituent nucleotides individually or in combination.

[0039] Nucleic acid: As used herein, the term "nucleic acid," in its broadest sense, refers to any compound and / or substance that is or can be incorporated into a polynucleotide chain. In some embodiments, a nucleic acid is a compound and / or substance that is or can be incorporated into a polynucleotide chain via a phosphodiester linkage. In some embodiments, "nucleic acid" refers to individual nucleic acid residues (e.g., nucleotides and / or nucleosides). In some embodiments, "nucleic acid" refers to a polynucleotide chain comprising individual nucleic acid residues. In some embodiments, "nucleic acid" encompasses RNA as well as single and / or double-stranded DNA and / or cDNA.

[0040] Patient: As used herein, the term "patient" or "subject" refers to any organism to which a provided composition may be administered, e.g., 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, a patient is a human. A human includes pre and post natal forms.

[0041] Pharmaceutically acceptable: The term "pharmaceutically acceptable" as used herein, refers to substances that, within the scope of sound medical judgment, are suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0042] Systemic distribution or delivery: As used herein, the terms "systemic distribution," "systemic delivery," or grammatical equivalent, refer to a delivery or distribution mechanism or approach that affect the entire body or an entire organism. Typically, systemic distribution or delivery is accomplished via body's circulation system, e.g., blood stream. Compared to the definition of "local distribution or delivery."

[0043] Subject: As used herein, the term "subject" refers to a human or any non-human animal (e.g., mouse, rat, rabbit, dog, cat, cattle, swine, sheep, horse or primate). A human includes pre- and post-natal forms. In many embodiments, a subject is a human being. A subject can be a patient, which refers to a human presenting to a medical provider for diagnosis or treatment of a disease. The term "subject" is used herein interchangeably with "individual" or "patient." A subject can be afflicted with or is susceptible to a disease or disorder but may or may not display symptoms of the disease or disorder.

[0044] Substantially: As used herein, the term "substantially" refers to the qualitative condition of exhibiting total or near-total extent or degree of a characteristic or property of interest. One of ordinary skill in the biological arts will understand that biological and chemical phenomena rarely, if ever, go to completion and / or proceed to completeness or achieve or avoid an absolute result. The term "substantially" is therefore used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena.

[0045] Target tissues: As used herein , the term "target tissues" refers to any tissue that is affected by a disease to be treated. In some embodiments, target tissues include those tissues that display disease-associated pathology, symptom, or feature.

[0046] Therapeutically effective amount: As used herein, the term "therapeutically effective amount" of a therapeutic agent means an amount that is sufficient, when administered to a subject suffering from or susceptible to a disease, disorder, and / or condition, to treat, diagnose, prevent, and / or delay the onset of the symptom(s) of the disease, disorder, and / or condition. It will be appreciated by those of ordinary skill in the art that a therapeutically effective amount is typically administered via a dosing regimen comprising at least one unit dose.

[0047] Treating: As used herein, the term "treat," "treatment," or "treating" refers to any method used to partially or completely alleviate, ameliorate, relieve, inhibit, prevent, delay onset of, reduce severity of and / or reduce incidence of one or more symptoms or features of a particular disease, disorder, and / or condition. Treatment may be administered to a subject who does not exhibit signs of a disease and / or exhibits only early signs of the disease for the purpose of decreasing the risk of developing pathology associated with the disease.DETAILED DESCRIPTION

[0048] The present invention provides compositions for delivering mRNA in vivo using improved liposomes incorporating the cationic lipids described herein.Liposomes for mRNA Delivery

[0049] As used herein, the term "liposome" refers to any lamellar, multilamellar, or solid lipid nanoparticle vesicle. Typically, a liposome as used herein can be formed by mixing one or more lipids or by mixing one or more lipids and polymer(s). Thus, the term "liposome" as used herein encompasses both lipid and polymer based nanoparticles. In particular, a liposome according to the present invention incorporates a cationic lipid described herein. As a non-limiting example, a cationic lipid suitable for the present invention is cKK-E12, or (3,6-bis(4-(bis(2-hydroxydodecyl)amino)butyl)piperazine-2,5-dione), as described in more detail below. A suitable liposome also contains a helper lipids (e.g., non-cationic lipids and / or cholesterol-based lipids) and a PEG-modified lipids.

[0050] The cationic lipid (e.g., cKK-E12) constitutes about 30-50 % (e.g., about 30-45%, about 30-40%, about 35-50%, about 35-45%, or about 35-40%) of the liposome by molar ratio. In some embodiments, the cationic lipid (e.g., cKK-E12) constitutes about 30%, about 35%, about 40 %, about 45%, or about 50% of the liposome by molar ratio.Cationic Lipids

[0051] The cationic lipid for use in compositions of the present invention is a compound of formula I-g: wherein each of R L< is optionally substituted C 8-10 alkyl. In some embodiments, each of R L< is C 8 alkyl. In some embodiments, each of R L< is C 9 alkyl. In some embodiments, each of R L< is C 10 alkyl.Chemical definitions

[0052] Definitions of specific functional groups and chemical terms are described in more detail below. The chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75th Ed., inside cover, and specific functional groups are generally defined as described therein. Additionally, general principles of organic chemistry, as well as specific functional moieties and reactivity, are described in Organic Chemistry, Thomas Sorrell, University Science Books, Sausalito, 1999; Smith and March March's Advanced Organic Chemistry, 5th Edition, John Wiley & Sons, Inc., New York, 2001; Larock, Comprehensive Organic Transformations, VCH Publishers, Inc., New York, 1989; and Carruthers, Some Modern Methods of Organic Synthesis, 3rd Edition, Cambridge University Press, Cambridge, 1987.

[0053] Compounds described herein can comprise one or more asymmetric centers, and thus can exist in various isomeric forms, e.g., enantiomers and / or diastereomers. For example, the compounds described herein can be in the form of an individual enantiomer, diastereomer or geometric isomer, or can be in the form of a mixture of stereoisomers, including racemic mixtures and mixtures enriched in one or more stereoisomer. Isomers can be isolated from mixtures by methods known to those skilled in the art, including chiral high performance liquid chromatography (HPLC) and the formation and crystallization of chiral salts; or preferred isomers can be prepared by asymmetric syntheses. See, for example, Jacques et al., Enantiomers, Racemates and Resolutions (Wiley Interscience, New York, 1981); Wilen et al., Tetrahedron 33:2725 (1977); Eliel, E.L. Stereochemistry of Carbon Compounds (McGraw-Hill, NY, 1962); and Wilen, S.H. Tables of Resolving Agents and Optical Resolutions p. 268 (E.L. Eliel, Ed., Univ. of Notre Dame Press, Notre Dame, IN 1972). The invention additionally contemplates compounds as individual isomers substantially free of other isomers, and alternatively, as mixtures of various isomers.

[0054] When a range of values is listed, it is intended to encompass each value and sub-range within the range. For example "C 1-6 alkyl" is intended to encompass, C 1 , C 2 , C 3 , C 4 , C 5 , C 6 , C 1-6 , C 1-5 , C 1-4 , C 1-3 , C 1-2 , C 2-6 , C 2-5 , C 2-4 , C 2-3 , C 3-6 , C 3-5 , C 3-4 , C 4-6 , C 4-5 , and C 5-6 alkyl.

[0055] As used herein, "alkyl" refers to a radical of a straight-chain or branched saturated hydrocarbon group having from 1 to 50 carbon atoms ("C 1-50 alkyl"). An alkyl group can have 1 to 40 carbon atoms ("C1-40 alkyl"). An alkyl group can have 1 to 30 carbon atoms ("C1-30 alkyl"). An alkyl group can have 1 to 20 carbon atoms ("C1-20 alkyl"). An alkyl group can have 1 to 10 carbon atoms ("C1-10 alkyl"). An alkyl group can have 1 to 9 carbon atoms ("C1-9 alkyl"). An alkyl group can have 1 to 8 carbon atoms ("C1-8 alkyl"). An alkyl group can have 1 to 7 carbon atoms ("C1-7 alkyl"). An alkyl group can have 1 to 6 carbon atoms ("C1-6 alkyl"). An alkyl group can have 1 to 5 carbon atoms ("C1-5 alkyl"). An alkyl group can have 1 to 4 carbon atoms ("C1-4 alkyl"). An alkyl group can have 1 to 3 carbon atoms ("C1-3 alkyl"). An alkyl group can have 1 to 2 carbon atoms ("C1-2 alkyl"). An alkyl group can have 1 carbon atom ("C1 alkyl"). An alkyl group can have 2 to 6 carbon atoms ("C2-6 alkyl"). Examples of C1-6 alkyl groups include, without limitation, methyl (C1), ethyl (C2), n-propyl (C3), isopropyl (C3), n-butyl (C4), tert-butyl (C4), sec-butyl (C4), iso-butyl (C4), n-pentyl (C5), 3-pentanyl (C5), amyl (C5), neopentyl (C5), 3-methyl-2-butanyl (C5), tertiary amyl (C5), and n-hexyl (C6). Additional examples of alkyl groups include n-heptyl (C7), n-octyl (C8) and the like. Unless otherwise specified, each instance of an alkyl group is independently unsubstituted (an "unsubstituted alkyl") or substituted (a "substituted alkyl") with one or more substituents.

[0056] As understood from the above, alkyl groups, as defined herein, are optionally substituted. Optionally substituted refers to a group which may be substituted or unsubstituted (e.g., "substituted" or "unsubstituted" alkyl). In general, the term "substituted" means that at least one hydrogen present on a group is replaced with a permissible substituent, e.g., a substituent which upon substitution results in a stable compound, e.g., a compound which does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, or other reaction. Unless otherwise indicated, a "substituted" group has a substituent at one or more substitutable positions of the group, and when more than one position in any given structure is substituted, the substituent is either the same or different at each position. The term "substituted" is contemplated to include substitution with all permissible substituents of organic compounds, any of the substituents described herein that results in the formation of a stable compound. Any and all such combinations are contemplated in order to arrive at a stable compound.Other definitions

[0057] As used herein, use of the phrase "at least one instance" refers to one instance, but also encompasses more than one instance, e.g., for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 instances, and up to 100 instances.

[0058] As used herein, a "polymer" refers to a compound comprised of at least 3 (e.g., at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, etc.) repeating covalently bound structural units.

[0059] "Attached" refers to the covalent attachment of a group.

[0060] As used herein, "lipophilic" refers to the ability of a group to dissolve in fats, oils, lipids, and lipophilic non-polar solvents such as hexane or toluene. In general, a lipophilic group refers to an unsubstituted n-alkyl or unsubstituted n-alkenyl group having 6 to 50 carbon atoms, e.g., 6 to 40, 6 to 30, 6 to 20, 8 to 20, 8 to 19, 8 to 18, 8 to 17, 8 to 16, or 8 to 15 carbon atoms.

[0061] As used herein, the term "salt" or "pharmaceutically acceptable salt" refers to those salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, S. M. Berge et al., describes pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences (1977) 66:1-19. Pharmaceutically acceptable salts of the compounds of this invention include those derived from suitable inorganic and organic acids and bases. Examples of pharmaceutically acceptable, nontoxic acid addition salts are salts of an amino group formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid or with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid or rnalonic acid or by using other methods used in the art such as ion exchange. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate. digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2- naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate salts, and the like. Salts derived from appropriate bases include alkali metal, alkaline earth metal, ammonium and N+(C1-4alkyl)4 salts. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. Further pharmaceutically acceptable salts include, when appropriate, nontoxic ammonium. quaternary ammonium, and amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, sulfonate and aryl sulfonate. Further pharmaceutically acceptable salts include salts formed from the quarternization of an amine using an appropriate electrophile, e.g., an alkyl halide, to form a quarternized alkylated amino salt.Non-cationic / Helper Lipids

[0062] The present invention provides liposomes containing a non-cationic ("helper") lipid. As used herein, the phrase "non-cationic lipid" refers to any neutral, zwitterionic or anionic lipid. As used herein, the phrase "anionic lipid" refers to any of a number of lipid species that carry a net negative charge at a selected H, 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), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, and l-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE).

[0063] The non-cationic lipid comprises a molar ratio of about 25% to about 35%, of the total lipid present in a liposome. In some embodiments, a non-cationic lipid is a neutral lipid, i.e., a lipid that does not carry a net charge in the conditions under which the composition is formulated and / or administered. In some embodiments, the percentage of non-cationic lipid in a liposome may be greater than 30%.Cholesterol-based Lipids

[0064] The present invention provides liposomes comprising a cholesterol-based lipid. The cholesterol-based lipid comprises a molar ratio of about 20% to about 30% of the total lipid present in the liposome. In some embodiments, the percentage of cholesterol-based lipid in the lipid nanoparticle may be greater than 20%.PEGylated Lipids

[0065] The present invention provides liposomes comprising a PEGylated lipid. For example, the use of polyethylene glycol (PEG)-modified phospholipids and derivatized lipids such as derivatized ceramides (PEG-CER), including N-Octanoyl-Sphingosine-l-[Succinyl(Methoxy Polyethylene Glycol)-2000] (C8 PEG-2000 ceramide) is contemplated by the present invention. Contemplated PEG-modified lipids include, but are not limited to, a polyethylene glycol chain of up to 5 kDa in length covalently attached to a lipid with alkyl chain(s) of C 6 -C 20 length. In some embodiments, a PEG-modified or PEGylated lipid is PEGylated cholesterol or PEG-2K. The addition of such components may prevent complex aggregation and may also provide a means for increasing circulation lifetime and increasing the delivery of the lipid-nucleic acid composition to the target cell, (Klibanov et al. (1990) FEBS Letters, 268 (1): 235-237), or they may be selected to rapidly exchange out of the formulation in vivo (see U.S. Pat. No. 5,885,613).

[0066] In some embodiments, particularly useful exchangeable lipids are PEG-ceramides having shorter acyl chains (e.g., C 14 or C 18 ). The PEG-modified phospholipid and derivitized lipids of the present invention may comprise a molar ratio from about 1% to about 15%, about 4% to about 10%, or about 2% of the total lipid present in the liposome.

[0067] As a non-limiting example, a suitable liposome comprises cKK-E12, DOPE, cholesterol and DMG-PEG2K. According to the present invention, the ratio of cationic lipid to non-cationic lipid to cholesterol-based lipid to PEGylated lipid is between about 30-50:25-35:20-30:1-15, respectively. In some embodiments, the ratio of cationic lipid to non-cationic lipid to cholesterol-based lipid to PEGylated lipid is approximately 40:30:20:10, respectively. In some embodiments, the ratio of cationic lipid to non-cationic lipid to cholesterol-based lipid to PEGylated lipid is approximately 40:30:25:5, respectively. In some embodiments, the ratio of cationic lipid to non-cationic lipid to cholesterol-based lipid to PEGylated lipid is approximately 40:32:25:3, respectively.mRNA

[0068] The present invention can be used to deliver any mRNA. mRNA is typically thought of as the type of RNA that carries information from DNA to the ribosome. The existence of mRNA is usually very brief and includes processing and translation, followed by degradation. Typically, in eukaryotic organisms, mRNA processing comprises the addition of a "cap" on the N-terminal (5') end, and a "tail" on the C-terminal (3') end. A typical cap is a 7-methylguanosine cap, which is a guanosine that is linked through a 5'-5'-triphosphate bond to the first transcribed nucleotide. The presence of the cap is important in providing resistance to nucleases found in most eukaryotic cells. The tail is typically a polyadenylation event whereby a polyadenylyl moiety is added to the 3' end of the mRNA molecule. The presence of this "tail" serves to protect the mRNA from exonuclease degradation. Messenger RNA typically is translated by the ribosomes into a series of amino acids that make up a protein.

[0069] Any mRNA capable of being translated into one or more peptides (e.g., proteins) or peptide fragments is contemplated. In some embodiments, an mRNA encodes one or more naturally occurring peptides. In some embodiments, an mRNA encodes one or more modified or non-natural peptides.

[0070] In some embodiments an mRNA encodes an intracellular protein. In some embodiments, an mRNA encodes a cytosolic protein. In some embodiments, an mRNA encodes a protein associated with the actin cytoskeleton. In some embodiments, an mRNA encodes a protein associated with the plasma membrane. In some specific embodiments, an mRNA encodes a transmembrane protein. In some specific embodiments an mRNA encodes an ion channel protein. In some embodiments, an mRNA encodes a perinuclear protein. In some embodiments, an mRNA encodes a nuclear protein. In some specific embodiments, an mRNA encodes a transcription factor. In some embodiments, an mRNA encodes a chaperone protein. In some embodiments, an mRNA encodes an intracellular enzyme (e.g., mRNA encoding an enzyme associated with urea cycle or lysosomal storage metabolic disorders). In some embodiments, an mRNA encodes a protein involved in cellular metabolism, DNA repair, transcription and / or translation. In some embodiments, an mRNA encodes an extracellular protein. In some embodiments, an mRNA encodes a protein associated with the extracellular matrix. In some embodiments an mRNA encodes a secreted protein. In specific embodiments, an mRNA used in the composition of the invention may be used to express functional proteins or enzymes that are excreted or secreted by one or more target cells into the surrounding extracellular fluid (e.g., mRNA encoding hormones and / or neurotransmitters).

[0071] In some embodiments, the compositions of the invention provide for delivery of mRNA encoding a secreted protein. In some embodiments, the compositions of the invention provide for delivery of mRNA encoding one or more secreted proteins listed in Table 1; thus, compositions of the invention may comprise an mRNA encoding a protein listed in Table 1 (or a homolog thereof) along with other components set out herein. Table 1. Secreted Proteins Uniprot ID Protein Name Gene Name A1E959Odontogenic ameloblast-associated proteinODAMA1KZ92Peroxidasin-like proteinPXDNLA1L453Serine protease 38PRSS38A1L4H1Soluble scavenger receptor cysteine-rich domain-containing protein SSC5DSSC5DA2RUU4Colipase-like protein 1CLPSL1A2VDF0Fucose mutarotaseFUOMA2VEC9SCO-spondinSSPOA3KMH1von Willebrand factor A domain-containing protein 8VWA8A4D0S4Laminin subunit beta-4LAMB4A4D1T9Probable inactive serine protease 37PRSS37A5D8T8C-type lectin domain family 18 member ACLEC18AA6NC86phospholipase A2 inhibitor and Ly6 / PLAUR domain-containing proteinPINLYPA6NCI4von Willebrand factor A domain-containing protein 3AVWA3AA6ND01Probable folate receptor deltaFOLR4A6NDD2Beta-defensin 108B-likeA6NE02BTB / POZ domain-containing protein 17BTBD17A6NEF6Growth hormone 1GH1A6NF02NPIP-like protein LOC730153A6NFB4HCG1749481, isoform CRA_kCSH1A6NFZ4Protein FAM24AFAM24AA6NG13Glycosyltransferase 54 domain-containing proteinA6NGN9IgLON family member 5IGLON5A6NHN0Otolin-1OTOL1A6NHN6Nuclear pore complex-interacting protein-like 2NPIPL2A6NI73Leukocyte immunoglobulin-like receptor subfamily A member 5LILRA5A6NIT4Chorionic somatomammotropin hormone 2 isoform 2CSH2A6NJ69IgA-inducing protein homologIGIPA6NKQ9Choriogonadotropin subunit beta variant 1CGB1A6NMZ7Collagen alpha-6(VI) chainCOL6A6A6NNS2Dehydrogenase / reductase SDR family member 7CDHRS7CA6XGL2Insulin A chainINSA8K0G1Protein WntWNT7BA8K2U0Alpha-2-macroglobulin-like protein 1A2ML 1A8K7I4Calcium-activated chloride channel regulator 1CLCA1A8MTL9Serpin-like protein HMSDHMSDA8MV23Serpin E3SERPINE3A8MZH6Oocyte-secreted protein 1 homologOOSP1A8TX70Collagen alpha-5(VI) chainCOL6A5B0ZBE8Natriuretic peptideNPPAB1A4G9SomatotropinGH1B1A4H2HCG1749481, isoform CRA_dCSH1B1A4H9Chorionic somatomammotropin hormoneCSH2B1AJZ6Protein WntWNT4B1AKI9Isthmin-1ISM1B2RNN3Complement C1q and tumor necrosis factor-related protein 9BC1QTNF9BB2RUY7von Willebrand factor C domain-containing protein 2-likeVWC2LB3GLJ2Prostate and testis expressed protein 3PATE3B4DI03SEC11-like 3 (S. cerevisiae), isoform CRA_aSEC11L3B4DJF9Protein WntWNT4B4DUL4SEC11-like 1 (S. cerevisiae), isoform CRA_dSEC11L1B5MCC8Protein WntWNT10BB8A595Protein WntWNT7BB8A597Protein WntWNT7BB8A598Protein WntWNT7BB9A064Immunoglobulin lambda-like polypeptide 5IGLL5C9J3H3Protein WntWNT10BC9J8I8Protein WntWNT5AC9JAF2Insulin-like growth factor II Ala-25 DelIGF2C9JCI2Protein WntWNT10BC9JL84HERV-H LTR-associating protein 1HHLA1C9JNR5Insulin A chainINSC9JUI2Protein WntWNT2D6RF47Protein WntWNT8AD6RF94Protein WntWNT8AE2RYF7Protein PBMUCL2HCG22E5RFR1PENK(114-133)PENKE7EML9Serine protease 44PRSS44E7EPC3Protein WntWNT9BE7EVP0NociceptinPNOCE9PD02Insulin-like growth factor IIGF1E9PH60Protein WntWNT16E9PJL6Protein WntWNT11F5GYM2Protein WntWNT5BF5H034Protein WntWNT5BF5H364Protein WntWNT5BF5H7Q6Protein WntWNT5BF8WCM5Protein INS-IGF2INS-IGF2F8WDR1Protein WntWNT2H0Y663Protein WntWNT4H0YK72Signal peptidase complex catalytic subunit SEC11ASEC11AH0YK83Signal peptidase complex catalytic subunit SEC11ASEC11AH0YM39Chorionic somatomammotropin hormoneCSH2H0YMT7Chorionic somatomammotropin hormoneCSH1H0YN17Chorionic somatomammotropin hormoneCSH2H0YNA5Signal peptidase complex catalytic subunit SEC11ASEC11AH0YNG3Signal peptidase complex catalytic subunit SEC11ASEC11AH0YNX5Signal peptidase complex catalytic subunit SEC11ASEC11AH7BZB8Protein WntWNT10AH9KV56Choriogonadotropin subunit beta variant 2CGB2I3L0L8Protein WntWNT9BJ3KNZ1Choriogonadotropin subunit beta variant 1CGB1J3KP00Choriogonadotropin subunit betaCGB7J3QT02Choriogonadotropin subunit beta variant 1CGB1O00175C-C motif chemokine 24CCL24O00182Galectin-9LGALS9O00187Mannan-binding lectin serine protease 2MASP2O00230CortistatinCORTO00253Agouti-related proteinAGRPO0027012-(S)-hydroxy-5,8,10,14-eicosatetraenoic acid receptorGPR31O00292Left-right determination factor 2LEFTY2O00294Tubby-related protein 1TULP1O00295Tubby-related protein 2TULP2O00300Tumor necrosis factor receptor superfamily member 11BTNFRSF11BO00339Matrilin-2MATN2O00391Sulfhydryl oxidase 1QSOX1O00468AgrinAGRNO00515Ladinin-1LAD1O00533Processed neural cell adhesion molecule L1-like proteinCHL1O00584Ribonuclease T2RNASET2O0585C-C motif chemokine 21CCL21O0602Ficolin-1FCN1O0622Protein CYR61CYR61O0626MDC(5-69)CCL22O00634Netrin-3NTN3O00744Protein Wnt-10bWNT10BO00755Protein Wnt-7aWNT7AO14498Immunoglobulin superfamily containing leucine-rich repeat proteinISLRO14511Pro-neuregulin-2, membrane-bound isoformNRG2O14594Neurocan core proteinNCANO14625C-X-C motif chemokine 11CXCL11O14638Ectonucleotide pyrophosphatase / phosphodiesterase family member 3ENPP3O14656Torsin-1ATOR1AO14657Torsin-1BTOR1BO14786Neuropilin-1NRP1O14788Tumor necrosis factor ligand superfamily member 11, membrane formTNFSF11O14791Apolipoprotein L1APOL1O14793Growth / differentiation factor 8MSTNO14904Protein Wnt-9aWNT9AO14905Protein Wnt-9bWNT9BO14944ProepiregulinEREGO14960Leukocyte cell-derived chemotaxin-2LECT2O15018Processed PDZ domain-containing protein 2PDZD2O15041Semaphorin-3ESEMA3EO15072A disintegrin and metalloproteinase with thrombospondin motifs 3ADAMTS3O15123Angiopoietin-2ANGPT2O15130Neuropeptide FFNPFFO15197Ephrin type-B receptor 6EPHB6O15204ADAM DEC1ADAMDEC1O15230Laminin subunit alpha-5LAMA5O15232Matrilin-3MATN3O15240Neuroendocrine regulatory peptide-1VGFO15263Beta-defensin 4ADEFB4AO15335ChondroadherinCHADO15393Transmembrane protease serine 2 catalytic chainTMPRSS2O15444C-C motif chemokine 25CCL25O15467C-C motif chemokine 16CCL16O15496Group 10 secretory phospholipase A2PLA2G10O15520Fibroblast growth factor 10FGF10O15537RetinoschisinRS1O43157Plexin-B1PLXNB1O43184Disintegrin and metalloproteinase domain-containing protein 12ADAM12O43240Kallikrein-10KLK10O43278Kunitz-type protease inhibitor 1SPINT1O43320Fibroblast growth factor 16FGF16O43323Desert hedgehog protein C-productDHHO43405CochlinCOCHO43508Tumor necrosis factor ligand superfamily member 12, membrane formTNFSF12O43555Progonadoliberin-2GNRH2O43557Tumor necrosis factor ligand superfamily member 14, soluble formTNFSF14O43692Peptidase inhibitor 15PI15O43699Sialic acid-binding Ig-like lectin 6SIGLEC6O43820Hyaluronidase-3HYAL3O43827Angiopoietin-related protein 7ANGPTL7O43852CalumeninCALUO43854EGF-like repeat and discoidin I-like domain-containing protein 3EDIL3O43866CD5 antigen-likeCD5LO43897Tolloid-like protein 1TLL1O43915Vascular endothelial growth factor DFIGFO43927C-X-C motif chemokine 13CXCL13O60218Aldo-keto reductase family 1 member B10AKR1B10O60235Transmembrane protease serine 11DTMPRSS11DO60258Fibroblast growth factor 17FGF17O60259Kallikrein-8KLK8O60383Growth / differentiation factor 9GDF9O60469Down syndrome cell adhesion moleculeDSCAMO60542PersephinPSPNO60565Gremlin-1GREM1O60575Serine protease inhibitor Kazal-type 4SPINK 4O60676Cystatin-8CST8O60687Sushi repeat-containing protein SRPX2SRPX2O60844Zymogen granule membrane protein 16ZG16O60882Matrix metalloproteinase-20MMP20O60938KeratocanKERAO75015Low affinity immunoglobulin gamma Fc region receptor III-BFCGR3BO75077Disintegrin and metalloproteinase domain-containing protein 23ADAM23O75093Slit homolog 1 proteinSLIT1O75094Slit homolog 3 proteinSLIT3O75095Multiple epidermal growth factor-like domains protein 6MEGF6O75173A disintegrin and metalloproteinase with thrombospondin motifs 4ADAMTS4O75200Nuclear pore complex-interacting protein-like 1NPIPL1O75339Cartilage intermediate layer protein 1 C1CILPO75354Ectonucleoside triphosphate diphosphohydrolase 6ENTPD6O75386Tubby-related protein 3TULP3O75398Deformed epidermal autoregulatory factor 1 homologDEAF1O75443Alpha-tectorinTECTAO75445UsherinUSH2AO75462Cytokine receptor-like factor 1CRLF1O75487Glypican-4GPC4O75493Carbonic anhydrase-related protein 11CA11O75594Peptidoglycan recognition protein 1PGLYRP1O75596C-type lectin domain family 3 member ACLEC3AO75610Left-right determination factor 1LEFTY1O75629Protein CREG1CREG1O75636Ficolin-3FCN3O75711Scrapie-responsive protein 1SCRG1O75715Epididymal secretory glutathione peroxidaseGPX5O75718Cartilage-associated proteinCRTAPO75829Chondrosurfactant proteinLECT1O75830Serpin I2SERPINI2O75882AttractinATRNO75888Tumor necrosis factor ligand superfamily member 13TNFSF13O75900Matrix metalloproteinase-23MMP23AO75951Lysozyme-like protein 6LYZL6O75973C1q-related factorC1QL1O76038SecretagoginSCGNO76061Stanniocalcin-2STC2O76076WNT1-inducible-signaling pathway protein 2WISP2O76093Fibroblast growth factor 18FGF18O76096Cystatin-FCST7O94769Extracellular matrix protein 2ECM2O94813Slit homolog 2 protein C-productSLIT2O94907Dickkopf-related protein 1DKK1O94919Endonuclease domain-containing 1 proteinENDOD1O94964N-terminal formSOGA1O95025Semaphorin-3DSEMA3DO95084Serine protease 23PRSS23O95150Tumor necrosis factor ligand superfamily member 15TNFSF15O95156Neurexophilin-2NXPH2O95157Neurexophilin-3NXPH3O95158Neurexophilin-4NXPH4O95388WNT1-inducible-signaling pathway protein 1WISP1O95389WNT1-inducible-signaling pathway protein 3WISP3O95390Growth / differentiation factor 11GDF11O95393Bone morphogenetic protein 10BMP10O95399Urotensin-2UTS2O95407Tumor necrosis factor receptor superfamily member 6BTNFRSF6BO95428PapilinPAPLNO95445Apolipoprotein MAPOMO95450A disintegrin and metalloproteinase with thrombospondin motifs 2ADAMTS2O95460Matrilin-4MATN4O95467LHAL tetrapeptideGNASO95631Netrin-1NTN1O95633Follistatin-related protein 3FSTL3O95711Lymphocyte antigen 86LY86O95715C-X-C motif chemokine 14CXCL14O95750Fibroblast growth factor 19FGF19O95760Interleukin-3 3IL33O95813CerberusCER1O95841Angiopoietin-related protein 1ANGPTL1O95897Noelin-2OLFM2O95925EppinEPPINO95965Integrin beta-like protein 1ITGBL1O95967EGF-containing fibulin-like extracellular matrix protein 2EFEMP2O95968Secretoglobin family 1D member 1SCGB1D1O95969Secretoglobin family 1D member 2SCGB1D2O95970Leucine-rich glioma-inactivated protein 1LGI1O95972Bone morphogenetic protein 15BMP15O95994Anterior gradient protein 2 homologAGR2O95998Interleukin-18-binding proteinIL18BPO96009Napsin-ANAPSAO96014Protein Wnt-11WNT11P00450CeruloplasminCPP00451Factor VIIIa light chainF8P00488Coagulation factor XIII A chainF13A1P00533Epidermal growth factor receptorEGFRP00709Alpha-lactalbuminLALBAP00734ProthrombinF2P00738Haptoglobin beta chainHPP00739Haptoglobin-related proteinHPRP00740Coagulation factor IXa heavy chainF9P00742Factor X heavy chainF10P00746Complement factor DCFDP00747Plasmin light chain BPLGP00748Coagulation factor XIIa light chainF12P00749Urokinase-type plasminogen activator long chain APLAUP00750Tissue-type plasminogen activatorPLATP00751Complement factor B Ba fragmentCFBP00797ReninRENP009732'-5'-oligoadenylate synthase 1OAS1P00995Pancreatic secretory trypsin inhibitorSPINK1P01008Antithrombin-IIISERPINC1P01009Alpha-1-antitrypsinSERPINA1P01011Alpha-1-antichymotrypsin His-Pro-lessSERPINA3P01019Angiotensin-1AGTP01023Alpha-2-macroglobulinA2MP01024Acylation stimulating proteinC3P01031Complement C5 beta chainC5P01033Metalloproteinase inhibitor 1TIMP1P01034Cystatin-CCST3P01036Cystatin-SCST4P01037Cystatin-SNCST1P01042Kininogen-1 light chainKNG1P01127Platelet-derived growth factor subunit BPDGFBP01135Transforming growth factor alphaTGFAP01137Transforming growth factor beta-1TGFB1P01138Beta-nerve growth factorNGFP01148Gonadoliberin-1GNRH1P01160Atrial natriuretic factorNPPAP01178OxytocinOXTP01185Vasopressin-neurophysin 2-copeptinAVPP01189CorticotropinPOMCP01210PENK(237-258)PENKP01213Alpha-neoendorphinPDYNP01215Glycoprotein hormones alpha chainCGAP01222Thyrotropin subunit betaTSHBP01225Follitropin subunit betaFSHBP01229Lutropin subunit betaLHBP01233Choriogonadotropin subunit betaCGB8P01236ProlactinPRLP01241SomatotropinGH1P01242Growth hormone variantGH2P01243Chorionic somatomammotropin hormoneCSH2P01258KatacalcinCALCAP01266ThyroglobulinTGP01270Parathyroid hormonePTHP01275GlucagonGCGP01282Intestinal peptide PHM-27VIPP01286SomatoliberinGHRHP01298Pancreatic prohormonePPYP01303C-flanking peptide of NPYNPYP01308InsulinINSP01344Insulin-like growth factor IIIGF2P01350Big gastrinGASTP01374Lymphotoxin-alphaLTAP01375C-domain 1TNFP01562Interferon alpha-1 / 13IFNA1P01563Interferon alpha-2IFNA2P01566Interferon alpha-10IFNA10P01567Interferon alpha-7IFNA7P01568Interferon alpha-21IFNA21P01569Interferon alpha-5IFNA5P01570Interferon alpha-14IFNA14P01571Interferon alpha-17IFNA17P01574Interferon betaIFNB 1P01579Interferon gammaIFNGP01583Interleukin-1 alphaIL1AP01584Interleukin-1 betaIL1BP01588ErythropoietinEPOP01591Immunoglobulin J chainIGJP01732T-cell surface glycoprotein CD8 alpha chainCD8AP01833Polymeric immunoglobulin receptorPIGRP01857Ig gamma-1 chain C regionIGHG1P01859Ig gamma-2 chain C regionIGHG2P01860Ig gamma-3 chain C regionIGHG3P01861Ig gamma-4 chain C regionIGHG4P01871Ig mu chain C regionIGHMP01880Ig delta chain C regionIGHDP02452Collagen alpha-1(I) chainCOL1A1P02458ChondrocalcinCOL2A1P02461Collagen alpha-1(III) chainCOL3A1P02462Collagen alpha-1(IV) chainCOL4A1P02647Apolipoprotein A-IAPOA1P02649Apolipoprotein EAPOEP02652Apolipoprotein A-IIAPOA2P02654Apolipoprotein C-IAPOC1P02655Apolipoprotein C-IIAPOC2P02656Apolipoprotein C-IIIAPOC3P02671Fibrinogen alpha chainFGAP02675Fibrinopeptide BFGBP02679Fibrinogen gamma chainFGGP02741C-reactive proteinCRPP02743Serum amyloid P-component(1-203)APCSP02745Complement C1q subcomponent subunit AC1QAP02746Complement C1q subcomponent subunit BC1QBP02747Complement C1q subcomponent subunit CC1QCP02748Complement component C9bC9P02749Beta-2-glycoprotein 1APOHP02750Leucine-rich alpha-2-glycoproteinLRG1P02751Ugl-Y2FN1P02753Retinol-binding protein 4RBP4P02760TrypstatinAMBPP02763Alpha-1-acid glycoprotein 1ORM1P02765Alpha-2-HS-glycoprotein chain AAHSGP02766TransthyretinTTRP02768Serum albuminALBP02771Alpha-fetoproteinAFPP02774Vitamin D-binding proteinGCP02775Connective tissue-activating peptide IIIPPBPP02776Platelet factor 4PF4P02778CXCL10(1-73)CXCL10P02786Transferrin receptor protein 1TFRCP02787SerotransferrinTFP02788Lactoferroxin-CLTFP02790HemopexinHPXP02808StatherinSTATHP02810Salivary acidic proline-rich phosphoprotein 1 / 2PRH2P02812Basic salivary proline-rich protein 2PRB2P02814Peptide D1ASMR3BP02818OsteocalcinBGLAPP03950AngiogeninANGP03951Coagulation factor XIa heavy chainF11P03952Plasma kallikreinKLKB1P0395627 kDa interstitial collagenaseMMP1P03971Muellerian-inhibiting factorAMHP03973AntileukoproteinaseSLPIP04003C4b-binding protein alpha chainC4BPAP04004Somatomedin-BVTNP04054Phospholipase A2PLA2G1BP04085Platelet-derived growth factor subunit APDGFAP04090Relaxin A chainRLN2P04114Apolipoprotein B-100APOBP04118ColipaseCLPSP04141Granulocyte-macrophage colony-stimulating factorCSF2P04155Trefoil factor 1TFF1P04180Phosphatidylcholine-sterol acyltransferaseLCATP04196Histidine-rich glycoproteinHRGP04217Alpha-1B-glycoproteinA1BGP04275von Willebrand antigen 2VWFP04278Sex hormone-binding globulinSHBGP04279Alpha-inhibin-31SEMG1P04280Basic salivary proline-rich protein 1PRB1P04628Proto-oncogene Wnt-1WNT1P04745Alpha-amylase 1AMY1AP04746Pancreatic alpha-amylaseAMY2AP04808Prorelaxin H1RLN1P05000Interferon omega-1IFNW1P05013Interferon alpha-6IFNA6P05014Interferon alpha-4IFNA4P05015Interferon alpha-16IFNA16P05019Insulin-like growth factor IIGF1P05060GAWK peptideCHGBP05090Apolipoprotein DAPODP05109Protein S100-A8S100A8P05111Inhibin alpha chainINHAP05112Interleukin-4IL4P05113Interleukin-5IL5P05120Plasminogen activator inhibitor 2SERPINB2P05121Plasminogen activator inhibitor 1SERPINE1P05154Plasma serine protease inhibitorSERPINA5P05155Plasma protease C1 inhibitorSERPING1P05156Complement factor I heavy chainCFIP05160Coagulation factor XIII B chainF13BP05161Ubiquitin-like protein ISG15ISG15P05230Fibroblast growth factor 1FGF1P05231Interleukin-6IL6P05305Big endothelin-1EDN1P05408C-terminal peptideSCG5P05451Lithostathine-1-alphaREG1AP05452TetranectinCLEC3BP05543Thyroxine-binding globulinSERPINA7P05814Beta-caseinCSN2P05997Collagen alpha-2(V) chainCOL5A2P06276CholinesteraseBCHEP06307Cholecystokinin-12CCKP06396GelsolinGSNP06681Complement C2C2P06702Protein S100-A9S100A9P06727Apolipoprotein A-IVAPOA4P06734Low affinity immunoglobulin epsilon Fc receptor soluble formFCER2P06744Glucose-6-phosphate isomeraseGPIP06850CorticoliberinCRHP06858Lipoprotein lipaseLPLP06881Calcitonin gene-related peptide 1CALCAP07093Glia-derived nexinSERPINE2P07098Gastric triacylglycerol lipaseLIPFP07225Vitamin K-dependent protein SPROS1P07237Protein disulfide-isomeraseP4HBP07288Prostate-specific antigenKLK3P07306Asialoglycoprotein receptor 1ASGR1P07355Annexin A2ANXA2P07357Complement component C8 alpha chainC8AP07358Complement component C8 beta chainC8BP07360Complement component C8 gamma chainC8GP07477Alpha-trypsin chain 2PRSS1P07478Trypsin-2PRSS2P07492Neuromedin-CGRPP07498Kappa-caseinCSN3P07585DecorinDCNP07911UromodulinUMODP07942Laminin subunit beta-1LAMB1P07988Pulmonary surfactant-associated protein BSFTPBP07998Ribonuclease pancreaticRNASE1P08118Beta-microseminoproteinMSMBP08123Collagen alpha-2(I) chainCOL1A2P08185Corticosteroid-binding globulinSERPINA6P08217Chymotrypsin-like elastase family member 2ACELA2AP08218Chymotrypsin-like elastase family member 2BCELA2BP0825372 kDa type IV collagenaseMMP2P08254Stromelysin-1MMP3P08294Extracellular superoxide dismutase [Cu-Zn]SOD3P08476Inhibin beta A chainINHBAP08493Matrix Gla proteinMGPP08572Collagen alpha-2(IV) chainCOL4A2P08581Hepatocyte growth factor receptorMETP08603Complement factor HCFHP08620Fibroblast growth factor 4FGF4P08637Low affinity immunoglobulin gamma Fc region receptor III-AFCGR3AP08697Alpha-2-antiplasminSERPINF2P08700Interleukin-3IL3P08709Coagulation factor VIIF7P08833Insulin-like growth factor-binding protein 1IGFBP1P08887Interleukin-6 receptor subunit alphaIL6RP08949Neuromedin-B-32NMBP08F94FibrocystinPKHD1P09038Fibroblast growth factor 2FGF2P09228Cystatin-SACST2P09237MatrilysinMMP7P09238Stromelysin-2MMP10P09341Growth-regulated alpha proteinCXCL1P09382Galectin-1LGALS1P09466GlycodelinPAEPP09486SPARCSPARCP09529Inhibin beta B chainINHBBP09544Protein Wnt-2WNT2P09603Processed macrophage colony-stimulating factor 1CSF1P09681Gastric inhibitory polypeptideGIPP09683SecretinSCTP09919Granulocyte colony-stimulating factorCSF3P0C091FRAS1-related extracellular matrix protein 3FREM3P0C0L4C4d-AC4AP0C0L5Complement C4-B alpha chainC4BP0C0P6Neuropeptide SNPSP0C7L1Serine protease inhibitor Kazal-type 8SPINK8P0C862Complement C1q and tumor necrosis factor-related protein 9AC1QTNF9P0C8F1Prostate and testis expressed protein 4PATE4P0CG01Gastrokine-3GKN3PP0CG36Cryptic family protein 1BCFC1BP0CG37Cryptic proteinCFC1P0CJ68Humanin-like protein 1MTRNR2L1P0CJ69Humanin-like protein 2MTRNR2L2P0CJ70Humanin-like protein 3MTRNR2L3P0CJ71Humanin-like protein 4MTRNR2L4P0CJ72Humanin-like protein 5MTRNR2L5P0CJ73Humanin-like protein 6MTRNR2L6P0CJ74Humanin-like protein 7MTRNR2L7P0CJ75Humanin-like protein 8MTRNR2L8P0CJ76Humanin-like protein 9MTRNR2L9P0CJ77Humanin-like protein 10MTRNR2L10P0DJD7Pepsin A-4PGA4P0DJD8Pepsin A-3PGA3P0DJD9Pepsin A-5PGA5P0DJI8Amyloid protein ASAA1P0DJI9Serum amyloid A-2 proteinSAA2P10082Peptide YY(3-36)PYYP10092Calcitonin gene-related peptide 2CALCBP10124SerglycinSRGNP10145MDNCF-aIL8P10147MIP-1-alpha(4-69)CCL3P10163Peptide P-DPRB4P10451OsteopontinSPP1P10599ThioredoxinTXNP10600Transforming growth factor beta-3TGFB3P10643Complement component C7C7P10645Vasostatin-2CHGAP10646Tissue factor pathway inhibitorTFPIP10720Platelet factor 4 variant(4-74)PF4V1P10745Retinol-binding protein 3RBP3P10767Fibroblast growth factor 6FGF6P10909Clusterin alpha chainCLUP10912Growth hormone receptorGHRP10915Hyaluronan and proteoglycan link protein 1HAPLN1P10966T-cell surface glycoprotein CD8 beta chainCD8BP10997Islet amyloid polypeptideIAPPP11047Laminin subunit gamma-1LAMC1P11150Hepatic triacylglycerol lipaseLIPCP11226Mannose-binding protein CMBL2P11464Pregnancy-specific beta-1-glycoprotein 1PSG1P11465Pregnancy-specific beta-1-glycoprotein 2PSG2P11487Fibroblast growth factor 3FGF3P11597Cholesteryl ester transfer proteinCETPP11684UteroglobinSCGB1A1P11686Pulmonary surfactant-associated protein CSFTPCP12034Fibroblast growth factor 5FGF5P12107Collagen alpha-1(XI) chainCOL11A1P12109Collagen alpha-1(VI) chainCOL6A1P12110Collagen alpha-2(VI) chainCOL6A2P12111Collagen alpha-3(VI) chainCOL6A3P12259Coagulation factor VF5P12272PTHrP[1-36]PTHLHP12273Prolactin-inducible proteinPIPP12544Granzyme AGZMAP12643Bone morphogenetic protein 2BMP2P12644Bone morphogenetic protein 4BMP4P12645Bone morphogenetic protein 3BMP3P12724Eosinophil cationic proteinRNASE3P12821Angiotensin-converting enzyme, soluble formACEP12838Neutrophil defensin 4DEFA4P12872MotilinMLNP13232Interleukin-7IL7P13236C-C motif chemokine 4CCL4P13284Gamma-interferon-inducible lysosomal thiol reductaseIFI30P13500C-C motif chemokine 2CCL2P13501C-C motif chemokine 5CCL5P13521Secretogranin-2SCG2P13591Neural cell adhesion molecule 1NCAM1P13611Versican core proteinVCANP13671Complement component C6C6P13688Carcinoembryonic antigen-related cell adhesion molecule 1CEACAM1P13725Oncostatin-MOSMP13726Tissue factorF3P13727Eosinophil granule major basic proteinPRG2P13942Collagen alpha-2(XI) chainCOL11A2P13987CD59 glycoproteinCD59P14138Endothelin-3EDN3P14174Macrophage migration inhibitory factorMIFP14207Folate receptor betaFOLR2P14222Perforin-1PRF1P14543Nidogen-1NID1P14555Phospholipase A2, membrane associatedPLA2G2AP14625EndoplasminHSP90B1P14735Insulin-degrading enzymeIDEP14778Interleukin-1 receptor type 1, soluble formIL1R1P1478082 kDa matrix metalloproteinase-9MMP9P15018Leukemia inhibitory factorLIFP15085Carboxypeptidase A1CPA1P15086Carboxypeptidase BCPB1P15151Poliovirus receptorPVRP15169Carboxypeptidase N catalytic chainCPN1P15248Interleukin-9IL9P15291N-acetyllactosamine synthaseB4GALT1P15309PAPf3 9ACPPP15328Folate receptor alphaFOLR1P15374Ubiquitin carboxyl-terminal hydrolase isozyme L3UCHL3P15502ElastinELNP15509Granulocyte-macrophage colony-stimulating factor receptor subunit alphaCSF2RAP15515Histatin-1HTN1P15516His3-(31-51)-peptideHTN3P15692Vascular endothelial growth factor AVEGFAP15814Immunoglobulin lambda-like polypeptide 1IGLL1P15907Beta-galactoside alpha-2,6-sialyltransferase 1ST6GAL1P15941Mucin-1 subunit betaMUC1P16035Metalloproteinase inhibitor 2TIMP2P16112Aggrecan core protein 2ACANP16233Pancreatic triacylglycerol lipasePNLIPP16442Histo-blood group ABO system transferaseABOP16471Prolactin receptorPRLRP16562Cysteine-rich secretory protein 2CRISP2P16619C-C motif chemokine 3-like 1CCL3L1P16860BNP(3-29)NPPBP16870Carboxypeptidase ECPEP16871Interleukin-7 receptor subunit alphaIL7RP17213Bactericidal permeability-increasing proteinBPIP17538Chymotrypsinogen BCTRB1P17931Galectin-3LGALS3P17936Insulin-like growth factor-binding protein 3IGFBP3P17948Vascular endothelial growth factor receptor 1FLT1P18065Insulin-like growth factor-binding protein 2IGFBP2P18075Bone morphogenetic protein 7BMP7P18428Lipopolysaccharide-binding proteinLBPP18509PACAP-related peptideADCYAP1P18510Interleukin-1 receptor antagonist proteinIL1RNP18827Syndecan-1SDC1P19021Peptidylglycine alpha-hydroxylating monooxygenasePAMP19235Erythropoietin receptorEPORP19438Tumor necrosis factor-binding protein 1TNFRSF1AP19652Alpha-1-acid glycoprotein 2ORM2P19801Amiloride-sensitive amine oxidase [copper-containing]ABP1P19823Inter-alpha-trypsin inhibitor heavy chain H2ITIH2P19827Inter-alpha-trypsin inhibitor heavy chain H1ITIH1P19835Bile salt-activated lipaseCELP19875C-X-C motif chemokine 2CXCL2P19876C-X-C motif chemokine 3CXCL3P19883FollistatinFSTP19957ElafinPI3P19961Alpha-amylase 2BAMY2BP20061Transcobalamin-1TCN1P20062Transcobalamin-2TCN2P20142GastricsinPGCP20155Serine protease inhibitor Kazal-type 2SPINK2P20231Tryptase beta-2TPSB2P20333Tumor necrosis factor receptor superfamily member 1BTNFRSF1BP20366Substance PTAC1P20382Melanin-concentrating hormonePMCHP20396ThyroliberinTRHP20742Pregnancy zone proteinPZPP20774MimecanOGNP20783Neurotrophin-3NTF3P20800Endothelin-2EDN2P20809Interleukin-11IL11P20827Ephrin-A1EFNA1P20849Collagen alpha-1(IX) chainCOL9A1P20851C4b-binding protein beta chainC4BPBP20908Collagen alpha-1(V) chainCOL5A1P21128Poly(U)-specific endoribonucleaseENDOUP21246PleiotrophinPTNP21583Kit ligandKITLGP21741MidkineMDKP21754Zona pellucida sperm-binding protein 3ZP3P21781Fibroblast growth factor 7FGF7P21802Fibroblast growth factor receptor 2FGFR2P21810BiglycanBGNP21815Bone sialoprotein 2IBSPP21860Receptor tyrosine-protein kinase erbB-3ERBB3P21941Cartilage matrix proteinMATN1P22003Bone morphogenetic protein 5BMP5P22004Bone morphogenetic protein 6BMP6P22079LactoperoxidaseLPOP22105Tenascin-XTNXBP22301Interleukin-10IL10P22303AcetylcholinesteraseACHEP22352Glutathione peroxidase 3GPX3P22362C-C motif chemokine 1CCL1P22455Fibroblast growth factor receptor 4FGFR4P22466Galanin message-associated peptideGALP22692Insulin-like growth factor-binding protein 4IGFBP4P22749Granuly sinGNLYP22792Carboxypeptidase N subunit 2CPN2P22891Vitamin K-dependent protein ZPROZP22894Neutrophil collagenaseMMP8P23142Fibulin-1FBLN1P23280Carbonic anhydrase 6CA6P23352Anosmin-1KAL1P23435Cerebellin-1CBLN1P23560Brain-derived neurotrophic factorBDNFP23582C-type natriuretic peptideNPPCP23946ChymaseCMA1P24043Laminin subunit alpha-2LAMA2P24071Immunoglobulin alpha Fc receptorFCARP24347Stromelysin-3MMP11P24387Corticotropin-releasing factor-binding proteinCRHBPP24592Insulin-like growth factor-binding protein 6IGFBP6P24593Insulin-like growth factor-binding protein 5IGFBP5P24821TenascinTNCP24855Deoxyribonuclease-1DNASE1P25067Collagen alpha-2(VIII) chainCOL8A2P25311Zinc-alpha-2-glycoproteinAZGP1P25391Laminin subunit alpha-1LAMA1P25445Tumor necrosis factor receptor superfamily member 6FASP25940Collagen alpha-3(V) chainCOL5A3P25942Tumor necrosis factor receptor superfamily member 5CD40P26022Pentraxin-related protein PTX3PTX3P26927Hepatocyte growth factor-like protein beta chainMST1P27169Serum paraoxonase / arylesterase 1PON1P27352Gastric intrinsic factorGIFP27487Dipeptidyl peptidase 4 membrane formDPP4P27539Embryonic growth / differentiation factor 1GDF1P27658VastatinCOL8A1P27797CalreticulinCALRP27918ProperdinCFPP28039Acyloxyacyl hydrolaseAOAHP28300Protein-lysine 6-oxidaseLOXP28325Cystatin-DCST5P28799Granulin-1GRNP29122Proprotein convertase subtilisin / kexin type 6PCSK6P29279Connective tissue growth factorCTGFP29320Ephrin type-A receptor 3EPHA3P29400Collagen alpha-5(IV) chainCOL4A5P29459Interleukin-12 subunit alphaIL12AP29460Interleukin-12 subunit betaIL12BP29508Serpin B3SERPINB3P29622KallistatinSERPINA4P29965CD40 ligand, soluble formCD40LGP30990Neurotensin / neuromedin NNTSP31025Lipocalin-1LCN1P31151Protein S100-A7S100A7P31371Fibroblast growth factor 9FGF9P31431Syndecan-4SDC4P3194714-3-3 protein sigmaSFNP32455Interferon-induced guanylate-binding protein 1GBP1P32881Interferon alpha-8IFNA8P34096Ribonuclease 4RNASE4P34130Neurotrophin-4NTF4P34820Bone morphogenetic protein 8BBMP8BP35030Trypsin-3PRSS3P35052Secreted glypican-1GPC1P35070BetacellulinBTCP35225Interleukin-13IL13P35247Pulmonary surfactant-associated protein DSFTPDP35318ADMADMP35542Serum amyloid A-4 proteinSAA4P35555Fibrillin-1FBN1P35556Fibrillin-2FBN2P35625Metalloproteinase inhibitor 3TIMP3P35858Insulin-like growth factor-binding protein complex acid labile subunitIGFALSP35916Vascular endothelial growth factor receptor 3FLT4P35968Vascular endothelial growth factor receptor 2KDRP36222Chitinase-3-like protein 1CHI3L1P36952Serpin B5SERPINBSP36955Pigment epithelium-derived factorSERPINF 1P36980Complement factor H-related protein 2CFHR2P39059Collagen alpha-1(XV) chainCOL15A1P39060Collagen alpha-1(XVIII) chainCOL18A1P39877Calcium-dependent phospholipase A2PLA2G5P39900Macrophage metalloelastaseMMP12P39905Glial cell line-derived neurotrophic factorGDNFP40225ThrombopoietinTHPOP40967M-alphaPMELP41159LeptinLEPP41221Protein Wnt-5aWNT5AP41222Prostaglandin-H2 D-isomerasePTGDSP41271Neuroblastoma suppressor of tumorigenicity 1NBL1P41439Folate receptor gammaFOLR3P42127Agouti-signaling proteinASIPP42702Leukemia inhibitory factor receptorLIFRP42830ENA-78(9-78)CXCL5P43026Growth / differentiation factor 5GDF5P43251BiotinidaseBTDP43652AfaminAFMP45452Collagenase 3MMP13P47710Casoxin-DCSN1S1P47929Galectin-7LGALS7BP47972Neuronal pentraxin-2NPTX2P47989Xanthine oxidaseXDHP47992LymphotactinXCL1P48023Tumor necrosis factor ligand superfamily member 6, membrane formFASLGP48052Carboxypeptidase A2CPA2P48061Stromal cell-derived factor 1CXCL 12P48304Lithostathine-1-betaREG1BP48307Tissue factor pathway inhibitor 2TFPI2P48357Leptin receptorLEPRP48594Serpin B4SERPINB4P48645Neuromedin-U-25NMUP48740Mannan-binding lectin serine protease 1MASP1P48745Protein NOV homologNOVP48960CD97 antigen subunit betaCD97P49223Kunitz-type protease inhibitor 3SPINT3P49747Cartilage oligomeric matrix proteinCOMPP49763Placenta growth factorPGFP49765Vascular endothelial growth factor BVEGFBP49767Vascular endothelial growth factor CVEGFCP49771Fms-related tyrosine kinase 3 ligandFLT3LGP49862Kallikrein-7KLK7P49863Granzyme KGZMKP49908Selenoprotein PSEPP1P49913Antibacterial protein FALL-39CAMPP50607Tubby protein homologTUBP51124Granzyme MGZMMP51512Matrix metalloproteinase-16MMP16P51654Glypican-3GPC3P51671EotaxinCCL11P51884LumicanLUMP51888ProlarginPRELPP52798Ephrin-A4EFNA4P52823Stanniocalcin-1STC1P53420Collagen alpha-4(IV) chainCOL4A4P53621Coatomer subunit alphaCOPAP54108Cysteine-rich secretory protein 3CRISP3P54315Pancreatic lipase-related protein 1PNLIPRP1P54317Pancreatic lipase-related protein 2PNLIPRP2P54793Arylsulfatase FARSFP55000Secreted Ly-6 / uPAR-related protein 1SLURP1P55001Microfibrillar-associated protein 2MFAP2P55056Apolipoprotein C-IVAPOC4P55058Phospholipid transfer proteinPLTPP55075Fibroblast growth factor 8FGF8P55081Microfibrillar-associated protein 1MFAP1P55083Microfibril-associated glycoprotein 4MFAP4P55107Bone morphogenetic protein 3BGDF10P55145Mesencephalic astrocyte-derived neurotrophic factorMANFP55259Pancreatic secretory granule membrane major glycoprotein GP2GP2P55268Laminin subunit beta-2LAMB2P55773CCL23(30-99)CCL23P55774C-C motif chemokine 18CCL18P55789FAD-linked sulfhydryl oxidase ALRGFERP56703Proto-oncogene Wnt-3WNT3P56704Protein Wnt-3aWNT3AP56705Protein Wnt-4WNT4P56706Protein Wnt-7bWNT7BP56730NeurotrypsinPRSS12P56851Epididymal secretory protein E3-betaEDDM3BP56975Neuregulin-3NRG3P58062Serine protease inhibitor Kazal-type 7SPINK7P58215Lysyl oxidase homolog 3LOXL3P58294Prokineticin-1PROK1P58335Anthrax toxin receptor 2ANTXR2P58397A disintegrin and metalloproteinase with thrombospondin motifs 12ADAMTS12P58417Neurexophilin-1NXPH1P58499Protein FAM3BFAM3BP59510A disintegrin and metalloproteinase with thrombospondin motifs 20ADAMTS20P59665Neutrophil defensin 1DEFA1BP59666Neutrophil defensin 3DEFA3P59796Glutathione peroxidase 6GPX6P59826BPI fold-containing family B member 3BPIFB3P59827BPI fold-containing family B member 4BPIFB4P59861Beta-defensin 131DEFB131P60022Beta-defensin 1DEFB1P60153Inactive ribonuclease-like protein 9RNASE9P60827Complement C1q tumor necrosis factor-related protein 8C1QTNF8P60852Zona pellucida sperm-binding protein 1ZP1P60985Keratinocyte differentiation-associated proteinKRTDAPP61109Kidney androgen-regulated proteinKAPP61278Somatostatin-14SSTP61366OsteocrinOSTNP61626Lysozyme CLYZP61769Beta-2-microglobulinB2MP61812Transforming growth factor beta-2TGFB2P61916Epididymal secretory protein E1NPC2P62502Epididymal-specific lipocalin-6LCN6P62937Peptidyl-prolyl cis-trans isomerase APPIAP67809Nuclease-sensitive element-binding protein 1YBX1P67812Signal peptidase complex catalytic subunit SEC11ASEC11AP78310Coxsackievirus and adenovirus receptorCXADRP78333Secreted glypican-5GPC5P78380Oxidized low-density lipoprotein receptor 1OLR1P78423Processed fractalkineCX3CL1P78509ReelinRELNP78556CCL20(2-70)CCL20P80075MCP-2(6-76)CCL8P80098C-C motif chemokine 7CCL7P80108Phosphatidylinositol-glycan-specific phospholipase DGPLD1P80162C-X-C motif chemokine 6CXCL6P80188Neutrophil gelatinase-associated lipocalinLCN2P80303Nucleobindin-2NUCB2P80511CalciterminS100A12P81172Hepcidin-25HAMPP81277Prolactin-releasing peptidePRLHP81534Beta-defensin 103DEFB 103AP81605DermcidinDCDP82279Protein crumbs homolog 1CRB1P82987ADAMTS-like protein 3ADAMTSL3P83105Serine protease HTRA4HTRA4P83110Serine protease HTRA3HTRA3P83859Orexigenic neuropeptide QRFPQRFPP98088Mucin-5ACMUCSACP98095Fibulin-2FBLN2P98160Basement membrane-specific heparan sulfate proteoglycan core proteinHSPG2P98173Protein FAM3AFAM3AQ00604NorrinNDPQ00796Sorbitol dehydrogenaseSORDQ00887Pregnancy-specific beta-1-glycoprotein 9PSG9Q00888Pregnancy-specific beta-1-glycoprotein 4PSG4Q00889Pregnancy-specific beta-1-glycoprotein 6PSG6Q01523HD5(56-94)DEFA5Q01524Defensin-6DEFA6Q01955Collagen alpha-3(IV) chainCOL4A3Q02297Pro-neuregulin-1, membrane-bound isoformNRG1Q02325Plasminogen-like protein BPLGLB1Q02383Semenogelin-2SEMG2Q02388Collagen alpha-1(VII) chainCOL7A1Q02505Mucin-3AMUC3AQ02509Otoconin-90OC90Q02747GuanylinGUCA2AQ02763Angiopoietin-1 receptorTEKQ02817Mucin-2MUC2Q02985Complement factor H-related protein 3CFHR3Q03167Transforming growth factor beta receptor type 3TGFBR3Q03403Trefoil factor 2TFF2Q03405Urokinase plasminogen activator surface receptorPLAURQ03591Complement factor H-related protein 1CFHR1Q03692Collagen alpha-1(X) chainCOL10A1Q04118Basic salivary proline-rich protein 3PRB3Q04756Hepatocyte growth factor activator short chainHGFACQ04900Sialomucin core protein 24CD164Q05315Eosinophil lysophospholipaseCLCQ05707Collagen alpha-1(XIV) chainCOL14A1Q05996Processed zona pellucida sperm-binding protein 2ZP2Q06033Inter-alpha-trypsin inhibitor heavy chain H3ITIH3Q06141Regenerating islet-derived protein 3-alphaREG3AQ06828FibromodulinFMODQ07092Collagen alpha-1(XVI) chainCOL16A1Q07325C-X-C motif chemokine 9CXCL9Q07507DermatopontinDPTQ075Z2Binder of sperm protein homolog 1BSPH1Q07654Trefoil factor 3TFF3Q07699Sodium channel subunit beta-1SCN1BQ08345Epithelial discoidin domain-containing receptor 1DDR1Q08380Galectin-3-binding proteinLGALS3BPQ08397Lysyl oxidase homolog 1LOXL1Q08431LactadherinMFGE8Q08629Testican-1SPOCK1Q08648Sperm-associated antigen 11BSPAG11BQ08830Fibrinogen-like protein 1FGL1Q10471Polypeptide N-acetylgalactosaminyltransferase 2GALNT2Q10472Polypeptide N-acetylgalactosaminyltransferase 1GALNT1Q11201CMP-N-acetylneuraminate-beta-galactosamide-alpha-2,3-sialyltransferase 1ST3GAL1Q11203CMP-N-acetylneuraminate-beta-1,4-galactoside alpha-2,3-sialyltransferaseST3GAL3Q11206CMP-N-acetylneuraminate-beta-galactosamide-alpha-2,3-sialyltransferase 4ST3GAL4Q12794Hyaluronidase-1HYAL1Q12805EGF-containing fibulin-like extracellular matrix protein 1EFEMP1Q12836Zona pellucida sperm-binding protein 4ZP4Q12841Follistatin-related protein 1FSTL1Q12904Aminoacyl tRNA synthase complex-interacting multifunctional protein 1AIMP1Q13018Soluble secretory phospholipase A2 receptorPLA2R1Q13072B melanoma antigen 1BAGEQ13093Platelet-activating factor acetylhydrolasePLA2G7Q13103Secreted phosphoprotein 24SPP2Q13162Peroxiredoxin-4PRDX4Q13201Platelet glycoprotein Ia*MMRN1Q13214Semaphorin-3BSEMA3BQ13219Pappalysin-1PAPPAQ13231Chitotriosidase-1CHIT1Q13253NogginNOGQ13261Interleukin-15 receptor subunit alphaIL15RAQ13275Semaphorin-3FSEMA3FQ13291Signaling lymphocytic activation moleculeSLAMF1Q13316Dentin matrix acidic phosphoprotein 1DMP1Q13361Microfibrillar-associated protein 5MFAP5Q13410Butyrophilin subfamily 1 member A1BTN1A1Q13421Mesothelin, cleaved formMSLNQ13429Insulin-like growth factor IIGF-IQ13443Disintegrin and metalloproteinase domain-containing protein 9ADAM9Q13519Neuropeptide 1PNOCQ13751Laminin subunit beta-3LAMB3Q13753Laminin subunit gamma-2LAMC2Q13790Apolipoprotein FAPOFQ13822Ectonucleotide pyrophosphatase / phosphodiesterase family member 2ENPP2Q14031Collagen alpha-6(IV) chainCOL4A6Q14050Collagen alpha-3(IX) chainCOL9A3Q14055Collagen alpha-2(IX) chainCOL9A2Q14112Nidogen-2NID2Q14114Low-density lipoprotein receptor-related protein 8LRP8Q14118DystroglycanDAG1Q14314FibroleukinFGL2Q14393Growth arrest-specific protein 6GAS6Q14406Chorionic somatomammotropin hormone-like 1CSHL1Q14507Epididymal secretory protein E3-alphaEDDM3AQ14508WAP four-disulfide core domain protein 2WFDC2Q14512Fibroblast growth factor-binding protein 1FGFBP1Q14515SPARC-like protein 1SPARCL1Q14520Hyaluronan-binding protein 2 27 kDa light chainHABP2Q14563Semaphorin-3 ASEMA3AQ14623Indian hedgehog proteinIHHQ14624Inter-alpha-trypsin inhibitor heavy chain H4ITIH4Q14667UPF0378 protein KIAA0100KIAA0100Q14703Membrane-bound transcription factor site-1 proteaseMBTPS1Q14766Latent-transforming growth factor beta-binding protein 1LTBP1Q14767Latent-transforming growth factor beta-binding protein 2LTBP2Q14773Intercellular adhesion molecule 4ICAM4Q14993Collagen alpha-1(XIX) chainCOL19A1Q14CN2Calcium-activated chloride channel regulator 4, 110 kDa formCLCA4Q15046Lysine--tRNA ligaseKARSQ15063PeriostinPOSTNQ15109Advanced glycosylation end product-specific receptorAGERQ15113Procollagen C-endopeptidase enhancer 1PCOLCEQ15166Serum paraoxonase / lactonase 3PON3Q15195Plasminogen-like protein APLGLAQ15198Platelet-derived growth factor receptor-like proteinPDGFRLQ15223Poliovirus receptor-related protein 1PVRL1Q15238Pregnancy-specific beta-1-glycoprotein 5PSG5Q15363Transmembrane emp24 domain-containing protein 2TMED2Q15375Ephrin type-A receptor 7EPHA7Q15389Angiopoietin-1ANGPT1Q15465Sonic hedgehog proteinSHHQ15485Ficolin-2FCN2Q15517CorneodesmosinCDSNQ15582Transforming growth factor-beta-induced protein ig-h3TGFBIQ15661Tryptase alpha / beta-1TPSAB1Q15726MetastinKISS1Q15782Chitinase-3-like protein 2CHI3L2Q15828Cystatin-MCST6Q15846Clusterin-like protein 1CLUL1Q15848AdiponectinADIPOQQ16206Protein disulfide-thiol oxidoreductaseENOX2Q16270Insulin-like growth factor-binding protein 7IGFBP7Q16363Laminin subunit alpha-4LAMA4Q16378Proline-rich protein 4PRR4Q16557Pregnancy-specific beta-1-glycoprotein 3PSG3Q16568CART(42-89)CARTPTQ16610Extracellular matrix protein 1ECM1Q16619Cardiotrophin-1CTF1Q16623Syntaxin-1ASTX1AQ16627HCC-1(9-74)CCL14Q16651Prostasin light chainPRSS8Q16661Guanylate cyclase C-activating peptide 2GUCA2BQ16663CCL15(29-92)CCL15Q16674Melanoma-derived growth regulatory proteinMIAQ16769Glutaminyl-peptide cyclotransferaseQPCTQ16787Laminin subunit alpha-3LAMA3Q16842CMP-N-acetylneuraminate-beta-galactosamide-alpha-2,3-sialyltransferase 2ST3GAL2Q17RR3Pancreatic lipase-related protein 3PNLIPRP3Q17RW2Collagen alpha-1(XXIV) chainCOL24A1Q17RY6Lymphocyte antigen 6KLY6KQ1L6U9Prostate-associated microseminoproteinMSMPQ1W4C9Serine protease inhibitor Kazal-type 13SPINK13Q1ZYL8Izumo sperm-egg fusion protein 4IZUMO4Q29960HLA class I histocompatibility antigen, Cw-16 alpha chainHLA-CQ2I0M5R-spondin-4RSPO4Q2L4Q9Serine protease 53PRSS53Q2MKA7R-spondin-1RSPO1Q2MV58Tectonic-1TCTN1Q2TAL6BrorinVWC2Q2UY09Collagen alpha-1(XXVIII) chainCOL28A1Q2VPA4Complement component receptor 1-like proteinCR1LQ2WEN9Carcinoembryonic antigen-related cell adhesion molecule 16CEACAM16Q30KP8Beta-defensin 136DEFB136Q30KP9Beta-defensin 135DEFB135Q30KQ1Beta-defensin 133DEFB133Q30KQ2Beta-defensin 130DEFB130Q30KQ4Beta-defensin 116DEFB116Q30KQ5Beta-defensin 115DEFB115Q30KQ6Beta-defensin 114DEFB114Q30KQ7Beta-defensin 113DEFB113Q30KQ8Beta-defensin 112DEFB112Q30KQ9Beta-defensin 110DEFB110Q30KR1Beta-defensin 109DEFB109P1Q32P28Prolyl 3-hydroxylase 1LEPRE1Q3B7J2Glucose-fructose oxidoreductase domain-containing protein 2GFOD2Q3SY79Protein WntWNT3AQ3T906N-acetylgluco samine-1-pho sphotransferase subunits alpha / betaGNPTABQ495T6Membrane metallo-endopeptidase-like 1MMEL1Q49AH0Cerebral dopamine neurotrophic factorCDNFQ4GOG5Secretoglobin family 2B member 2SCGB2B2Q4G0M1Protein FAM132BFAM132BQ4LDE5Sushi, von Willebrand factor type A, EGF and pentraxin domain-containing protein 1SVEP1Q4QY38Beta-defensin 134DEFB134Q4VAJ4Protein WntWNT10BQ4W5P6Protein TMEM155TMEM155Q4ZHG4Fibronectin type III domain-containing protein 1FNDC1Q53H76Phospholipase A1 member APLA1AQ53RD9Fibulin-7FBLN7Q53S33BolA-like protein 3BOLA3Q5BLP8Neuropeptide-like protein C4orf48C4orf48Q5DT21Serine protease inhibitor Kazal-type 9SPINK9Q5EBL8PDZ domain-containing protein 11PDZD11Q5FYB0Arylsulfatase JARSJQ5FYB1Arylsulfatase IARSIQ5GAN3Ribonuclease-like protein 13RNASE13Q5GAN4Ribonuclease-like protein 12RNASE12Q5GAN6Ribonuclease-like protein 10RNASE10Q5GFL6von Willebrand factor A domain-containing protein 2VWA2Q5H8A3Neuromedin-SNMSQ5H8C1FRAS1-related extracellular matrix protein 1FREM1Q5IJ48Protein crumbs homolog 2CRB2Q5J5C9Beta-defensin 121DEFB121Q5JS37NHL repeat-containing protein 3NHLRC3Q5JTB6Placenta-specific protein 9PLAC9Q5JU69Torsin-2ATOR2AQ5JXM2Methyltransferase-like protein 24METTL24Q5JZY3Ephrin type-A receptor 10EPHA10Q5K4E3Polyserase-2PRSS36Q5SRR4Lymphocyte antigen 6 complex locus protein G5cLY6G5CQ5T1H1Protein eyes shut homologEYSQ5T4F7Secreted frizzled-related protein 5SFRP5Q5T4W7ArteminARTNQ5T7M4Protein FAM132AFAM132AQ5TEH8Protein WntWNT2BQ5TIE3von Willebrand factor A domain-containing protein 5B1VWA5B1Q5UCC4ER membrane protein complex subunit 10EMC10Q5VST6Abhydrolase domain-containing protein FAM108B1FAM108B1Q5VTL7Fibronectin type III domain-containing protein 7FNDC7Q5VUM1UPF0369 protein C6orf57C6orf57Q5VV43Dyslexia-associated protein KIAA0319KIAA0319Q5VWW1Complement C1q-like protein 3C1QL3Q5VXI9Lipase member NLIPNQ5VXJ0Lipase member KLIPKQ5VXM1CUB domain-containing protein 2CDCP2Q5VYX0RenalaseRNLSQ5VYY2Lipase member MLIPMQ5W186Cystatin-9CST9Q5W5W9Regulated endocrine-specific protein 18RESP18Q5XG92Carboxylesterase 4ACES4AQ63HQ2PikachurinEGFLAMQ641Q3Meteorin-like proteinMETRNLQ66K79Carboxypeptidase ZCPZQ685J3Mucin-17MUC17Q68BL7Olfactomedin-like protein 2AOLFML2AQ68BL8Olfactomedin-like protein 2BOLFML2BQ68DV7E3 ubiquitin-protein ligase RNF43RNF43Q6B9Z1Insulin growth factor-like family member 4IGFL4Q6BAA4Fc receptor-like BFCRLBQ6E0U4DermokineDMKNQ6EMK4VasorinVASNQ6FHJ7Secreted frizzled-related protein 4SFRP4Q6GPI1Chymotrypsin B2 chain BCTRB2Q6GTS8Probable carboxypeptidase PM20D1PM20D1Q6H9L7Isthmin-2ISM2Q6IE36Ovostatin homolog 2OVOS2Q6IE37Ovostatin homolog 1OVOS1Q6IE38Serine protease inhibitor Kazal-type 14SPINK 14Q6ISS4Leukocyte-associated immunoglobulin-like receptor 2LAIR2Q6JVE5Epididymal-specific lipocalin-12LCN12Q6JVE6Epididymal-specific lipocalin-10LCN10Q6JVE9Epididymal-specific lipocalin-8LCN8Q6KF10Growth / differentiation factor 6GDF6Q6MZW2Follistatin-related protein 4FSTL4Q6NSX1Coiled-coil domain-containing protein 70CCDC70Q6NT32Carboxylesterase 5ACES5AQ6NT52Choriogonadotropin subunit beta variant 2CGB2Q6NUI6Chondroadherin-like proteinCHADLQ6NUJ1Saposin A-likePSAPL1Q6P093Arylacetamide deacetylase-like 2AADACL2Q6P4A8Phospholipase B-like 1PLBD1Q6P5S2UPF0762 protein C6orf58C6orf58Q6P988Protein notum homologNOTUMQ6PCB0von Willebrand factor A domain-containing protein 1VWA1Q6PDA7Sperm-associated antigen 11ASPAG11AQ6PEW0Inactive serine protease 54PRSS54Q6PEZ8Podocan-like protein 1PODNL1Q6PKH6Dehydrogenase / reductase SDR family member 4-like 2DHRS4L2Q6Q788Apolipoprotein A-VAPOA5Q6SPF0AtherinSAMD1Q6UDR6Kunitz-type protease inhibitor 4SPINT4Q6URK8Testis, prostate and placenta-expressed proteinTEPPQ6UW01Cerebellin-3CBLN3Q6UW10Surfactant-associated protein 2SFTA2Q6UW15Regenerating islet-derived protein 3-gammaREG3 GQ6UW32Insulin growth factor-like family member 1IGFL1Q6UW78UPF0723 protein C11orf83C11orf83Q6UW88EpigenEPGNQ6UWE3Colipase-like protein 2CLPSL2Q6UWF7NXPE family member 4NXPE4Q6UWF9Protein FAM180AFAM180AQ6UWM5GLIPR1-like protein 1GLIPR1L1Q6UWN8Serine protease inhibitor Kazal-type 6SPINK6Q6UWP2Dehydrogenase / reductase SDR family member 11DHRS11Q6UWP8SuprabasinSBSNQ6UWQ5Lysozyme-like protein 1LYZL1Q6UWQ7Insulin growth factor-like family member 2IGFL2Q6UWR7Ectonucleotide pyrophosphatase / phosphodiesterase family member 6 soluble formENPP6Q6UWT2AdropinENHOQ6UWU2Beta-galactosidase-1-like proteinGLB1LQ6UWW0Lipocalin-15LCN15Q6UWX4HHIP-like protein 2HHIPL2Q6UWY0Arylsulfatase KARSKQ6UWY2Serine protease 57PRSS57Q6UWY5Olfactomedin-like protein 1OLFML1Q6UX06Olfactomedin-4OLFM4Q6UX07Dehydrogenase / reductase SDR family member 13DHRS13Q6UX39AmelotinAMTNQ6UX46Protein FAM150BFAM150BQ6UX73UPF0764 protein C16orf89C16orf89Q6UXB0Protein FAM131AFAM131AQ6UXB1Insulin growth factor-like family member 3IGFL3Q6UXB2VEGF co-regulated chemokine 1CXCL17Q6UXF7C-type lectin domain family 18 member BCLEC18BQ6UXH0Hepatocellular carcinoma-associated protein TD26C19orf80Q6UXH1Cysteine-rich with EGF-like domain protein 2CRELD2Q6UXH8Collagen and calcium-binding EGF domain-containing protein 1CCBE1Q6UXH9Inactive serine protease PAMR1PAMR1Q6UXI7VitrinVITQ6UXI9NephronectinNPNTQ6UXN2Trem-like transcript 4 proteinTREML4Q6UXS0C-type lectin domain family 19 member ACLEC19AQ6UXT8Protein FAM150AFAM150AQ6UXT9Abhydrolase domain-containing protein 15ABHD15Q6UXV4Apolipoprotein O-likeAPOOLQ6UXX5Inter-alpha-trypsin inhibitor heavy chain H6ITIH6Q6UXX9R-spondin-2RSPO2Q6UY14ADAMTS-like protein 4ADAMTSL4Q6UY27Prostate and testis expressed protein 2PATE2Q6W4X9Mucin-6MUC6Q6WN34Chordin-like protein 2CHRDL2Q6WRI0Immunoglobulin superfamily member 10IGSF10Q6X4U4Sclerostin domain-containing protein 1SOSTDC1Q6X784Zona pellucida-binding protein 2ZPBP2Q6XE38Secretoglobin family 1D member 4SCGB1D4Q6XPR3RepetinRPTNQ6XZB0Lipase member ILIPIQ6ZMM2ADAMTS-like protein 5ADAMTSL5Q6ZMP0Thrombospondin type-1 domain-containing protein 4THSD4Q6ZNF0Iron / zinc purple acid phosphatase-like proteinPAPLQ6ZRI0OtogelinOTOGQ6ZRP7Sulfhydryl oxidase 2QSOX2Q6ZWJ8Kielin / chordin-like proteinKCPQ75N90Fibrillin-3FBN3Q76510Urotensin-2BUTS2DQ76B58Protein FAM5CFAM5CQ76LX8A disintegrin and metalloproteinase with thrombospondin motifs 13ADAMTS13Q76M96Coiled-coil domain-containing protein 80CCDC80Q7L1S5Carbohydrate sulfotransferase 9CHST9Q7L513Fc receptor-like AFCRLAQ7L8A9Vasohibin-1VASH1Q7RTM1Otopetrin-1OTOP1Q7RTW8OtoancorinOTOAQ7RTY5Serine protease 48PRSS48Q7RTY7Ovochymase-1OVCH1Q7RTZ1Ovochymase-2OVCH2Q7Z304MAM domain-containing protein 2MAMDC2Q7Z3S9Notch homolog 2 N-terminal-like proteinNOTCH2NLQ7Z4H4Intermedin-shortADM2Q7Z4P5Growth / differentiation factor 7GDF7Q7Z4R8UPF0669 protein C6orf120C6orf120Q7Z4W2Lysozyme-like protein 2LYZL2Q7Z5A4Serine protease 42PRSS42Q7Z5A7Protein FAM19A5FAM19A5Q7Z5A8Protein FAM19A3FAM19A3Q7Z5A9Protein FAM19A1FAM19A1Q7Z5J1Hydroxysteroid 11-beta-dehydrogenase 1-like proteinHSD11B1LQ7Z5L0Vitelline membrane outer layer protein 1 homologVMO1Q7Z5L3Complement C1q-like protein 2C1QL2Q7Z5L7PodocanPODNQ7Z5P417-beta-hydroxysteroid dehydrogenase 13HSD17B13Q7Z5P9Mucin-19MUC19Q7Z5Y6Bone morphogenetic protein 8ABMP8AQ7Z7B7Beta-defensin 132DEFB132Q7Z7B8Beta-defensin 128DEFB128Q7Z7C8Transcription initiation factor TFIID subunit 8TAF8Q7Z7H5Transmembrane emp24 domain-containing protein 4TMED4Q86SG7Lysozyme g-like protein 2LYG2Q86SI9Protein CEIC5orf38Q86TE4Leucine zipper protein 2LUZP2Q86TH1ADAMTS-like protein 2ADAMTSL2Q86U17Serpin A11SERPINA11Q86UU9Endokinin-ATAC4Q86UW8Hyaluronan and proteoglycan link protein 4HAPLN4Q86UX2Inter-alpha-trypsin inhibitor heavy chain H5ITIH5Q86V24Adiponectin receptor protein 2ADIPOR2Q86VB7Soluble CD163CD163Q86VR8Four-jointed box protein 1FJX1Q86WD7Serpin A9SERPINA9Q86WN2Interferon epsilonIFNEQ86WS3Placenta-specific 1-like proteinPLAC1LQ86X52Chondroitin sulfate synthase 1CHSY1Q86XP6Gastrokine-2GKN2Q86XS5Angiopoietin-related protein 5ANGPTL5Q86Y27B melanoma antigen 5BAGE5Q86Y28B melanoma antigen 4BAGE4Q86Y29B melanoma antigen 3BAGE3Q86Y30B melanoma antigen 2BAGE2Q86Y38Xylosyltransferase 1XYLT1Q86Y78Ly6 / PLAUR domain-containing protein 6LYPD6Q86YD3Transmembrane protein 25TMEM25Q86YJ6Threonine synthase-like 2THNSL2Q86YW7Glycoprotein hormone beta-5GPHB5Q86Z23Complement C1q-like protein 4C1QL4Q8IU57Interleukin-28 receptor subunit alphaIL28RAQ8IUA0WAP four-disulfide core domain protein 8WFDC8Q8IUB2WAP four-disulfide core domain protein 3WFDC3Q8IUB3Protein WFDC10BWFDC10BQ8IUB5WAP four-disulfide core domain protein 13WFDC13Q81UH2Protein CREG2CREG2Q8IUK5Plexin domain-containing protein 1PLXDC1Q8IUL8Cartilage intermediate layer protein 2 C2CILP2Q8IUX7Adipocyte enhancer-binding protein 1AEBP1Q8IUX8Epidermal growth factor-like protein 6EGFL6Q81VL8Carboxypeptidase OCPOQ81VN8Somatomedin-B and thrombospondin type-1 domain-containing proteinSBSPONQ8IVW8Protein spinster homolog 2SPNS2Q8IW75Serpin A12SERPINA12Q8IW92Beta-galactosidase-1-like protein 2GLB1L2Q8IWL1Pulmonary surfactant-associated protein A2SFTPA2Q8IWL2Pulmonary surfactant-associated protein A1SFTPA1Q8IWV2Contactin-4CNTN4Q8IWY4Signal peptide, CUB and EGF-like domain-containing protein 1SCUBE1Q8IX30Signal peptide, CUB and EGF-like domain-containing protein 3SCUBE3Q8IXA5Sperm acrosome membrane-associated protein 3, membrane formSPACA3Q8IXB1DnaJ homolog subfamily C member 10DNAJC10Q8IXL6Extracellular serine / threonine protein kinase Fam20CFAM20CQ8IYD9Lung adenoma susceptibility protein 2LAS2Q8IYP2Serine protease 58PRSS58Q8IYS5Osteoclast-associated immunoglobulin-like receptorOSCARQ8IZC6Collagen alpha-1(XXVII) chainCOL27A1Q8IZJ3C3 and PZP-like alpha-2-macroglobulin domain-containing protein 8CPAMD8Q8IZN7Beta-defensin 107DEFB107BQ8N0V4Leucine-rich repeat LGI family member 2LGI2Q8N104Beta-defensin 106DEFB106BQ8N119Matrix metalloproteinase-21MMP21Q8N129Protein canopy homolog 4CNPY4Q8N135Leucine-rich repeat LGI family member 4LGI4Q8N145Leucine-rich repeat LGI family member 3LGI3Q8N158Glypican-2GPC2Q8N1E2Lysozyme g-like protein 1LYG1Q8N2E2von Willebrand factor D and EGF domain-containing proteinVWDEQ8N2E6ProsalusinTOR2AQ8N2S1Latent-transforming growth factor beta-binding protein 4LTBP4Q8N302Angiogenic factor with G patch and FHA domains 1AGGF1Q8N307Mucin-20MUC20Q8N323NXPE family member 1NXPE1Q8N387Mucin-15MUC15Q8N3Z0Inactive serine protease 35PRSS35Q8N436Inactive carboxypeptidase-like protein X2CPXM2Q8N474Secreted frizzled-related protein 1SFRP1Q8N475Follistatin-related protein 5FSTL5Q8N4F0BPI fold-containing family B member 2BPIFB2Q8N4T0Carboxypeptidase A6CPA6Q8N5W8Protein FAM24BFAM24BQ8N687Beta-defensin 125DEFB125Q8N688Beta-defensin 123DEFB123Q8N690Beta-defensin 119DEFB119Q8N6C5Immunoglobulin superfamily member 1IGSF1Q8N6C8Leukocyte immunoglobulin-like receptor subfamily A member 3LILRA3Q8N6G6ADAMTS-like protein 1ADAMTSL1Q8N6Y2Leucine-rich repeat-containing protein 17LRRC17Q8N729Neuropeptide W-23NPWQ8N8U9BMP-binding endothelial regulator proteinBMPERQ8N907DAN domain family member 5DAND5Q8NAT1Glycosyltransferase-like domain-containing protein 2GTDC2Q8NAU1Fibronectin type III domain-containing protein 5FNDC5Q8NB37Parkinson disease 7 domain-containing protein 1PDDC1Q8NBI3DraxinDRAXINQ8NBM8Prenylcysteine oxidase-likePCYOX1LQ8NBP7Proprotein convertase subtilisin / kexin type 9PCSK9Q8NBQ5Estradiol 17-beta-dehydrogenase 11HSD17B11Q8NBV8Synaptotagmin-8SYT8Q8NCC3Group XV phospholipase A2PLA2G15Q8NCF0C-type lectin domain family 18 member CCLEC18CQ8NCW5NAD(P)H-hydrate epimeraseAPOA1BPQ8NDA2Hemicentin-2HMCN2Q8NDX9Lymphocyte antigen 6 complex locus protein G5bLY6G5BQ8NDZ4Deleted in autism protein 1C3orf58Q8NEB7Acrosin-binding proteinACRBPQ8NES8Beta-defensin 124DEFB124Q8NET1Beta-defensin 108BDEFB108BQ8NEX5Protein WFDC9WFDC9Q8NEX6Protein WFDC11WFDC11Q8NF86Serine protease 33PRSS33Q8NFM7Interleukin-17 receptor DIL17RDQ8NFQ5BPI fold-containing family B member 6BPIFB6Q8NFQ6BPI fold-containing family C proteinBPIFCQ8NFU4Follicular dendritic cell secreted peptideFDCSPQ8NFW1Collagen alpha-1(XXII) chainCOL22A1Q8NG35Beta-defensin 105DEFB105BQ8NG41Neuropeptide B-23NPBQ8NHW6OtospiralinOTOSQ8NI99Angiopoietin-related protein 6ANGPTL6Q8TAA1Probable ribonuclease 11RNASE11Q8TAG5V-set and transmembrane domain-containing protein 2AVSTM2AQ8TAL6Fin bud initiation factor homologFIBINQ8TAT2Fibroblast growth factor-binding protein 3FGFBP3Q8TAX7Mucin-7MUC7Q8TB22Spermatogenesis-associated protein 20SPATA20Q8TB73Protein NDNFNDNFQ8TB96T-cell immunomodulatory proteinITFG1Q8TC92Protein disulfide-thiol oxidoreductaseENOX1Q8TCV5WAP four-disulfide core domain protein 5WFDC5Q8TD06Anterior gradient protein 3 homologAGR3Q8TD33Secretoglobin family 1C member 1SCGB1C1Q8TD46Cell surface glycoprotein CD200 receptor 1CD200R1Q8TDE3Ribonuclease 8RNASE8Q8TDF5Neuropilin and tolloid-like protein 1NETO1Q8TDL5BPI fold-containing family B member 1BPIFB1Q8TE56A disintegrin and metalloproteinase with thrombospondin motifs 17ADAMTS17Q8TE57A disintegrin and metalloproteinase with thrombospondin motifs 16ADAMTS16Q8TE58A disintegrin and metalloproteinase with thrombospondin motifs 15ADAMTS15Q8TE59A disintegrin and metalloproteinase with thrombospondin motifs 19ADAMTS19Q8TE60A disintegrin and metalloproteinase with thrombospondin motifs 18ADAMTS18Q8TE99Acid phosphatase-like protein 2ACPL2Q8TER0Sushi, nidogen and EGF-like domain-containing protein 1SNED1Q8TEU8WAP, kazal, immunoglobulin, kunitz and NTR domain-containing protein 2WFIKKN2Q8WTQ1Beta-defensin 104DEFB104BQ8WTR8Netrin-5NTN5Q8WTU2Scavenger receptor cysteine-rich domain-containing group B proteinSRCRB4DQ8WU66Protein TSPEARTSPEARQ8WUA8TsukushinTSKUQ8WUF8Protein FAM172AFAM172AQ8WUJ1NeuferricinCYBSD2Q8WUY1UPF0670 protein THEM6THEM6Q8WVN6Secreted and transmembrane protein 1SECTM1Q8WVQ1Soluble calcium-activated nucleotidase 1CANT1Q8WWA0Intelectin-1ITLN1Q8WWG1Neuregulin-4NRG4Q8WWQ2Inactive heparanase-2HPSE2Q8WWU7Intelectin-2ITLN2Q8WWY7WAP four-disulfide core domain protein 12WFDC12Q8WWY8Lipase member HLIPHQ8WWZ8Oncoprotein-induced transcript 3 proteinOIT3Q8WX39Epididymal-specific lipocalin-9LCN9Q8WXA2Prostate and testis expressed protein 1PATE1Q8WXD2Secretogranin-3SCG3Q8WXF3Relaxin-3 A chainRLN3Q8WXI7Mucin-16MUC16Q8WXQ8Carboxypeptidase A5CPA5Q8WXS8A disintegrin and metalloproteinase with thrombospondin motifs 14ADAMTS14Q92484Acid sphingomyelinase-like phosphodiesterase 3aSMPDL3AQ92485Acid sphingomyelinase-like phosphodiesterase 3bSMPDL3BQ92496Complement factor H-related protein 4CFHR4Q92520Protein FAM3CFAM3CQ92563Testican-2SPOCK2Q92583C-C motif chemokine 17CCL17Q92626Peroxidasin homologPXDNQ92743Serine protease HTRA1HTRA1Q92752Tenascin-RTNRQ92765Secreted frizzled-related protein 3FRZBQ92819Hyaluronan synthase 2HAS2Q92820Gamma-glutamyl hydrolaseGGHQ92824Proprotein convertase subtilisin / kexin type 5PCSK5Q92832Protein kinase C-binding protein NELL1NELL1Q92838Ectodysplasin-A, membrane formEDAQ92874Deoxyribonuclease-1-like 2DNASE1L2Q92876Kallikrein-6KLK6Q92913Fibroblast growth factor 13FGF13Q92954Proteoglycan 4 C-terminal partPRG4Q93038Tumor necrosis factor receptor superfamily member 25TNFRSF25Q93091Ribonuclease K6RNASE6Q93097Protein Wnt-2bWNT2BQ93098Protein Wnt-8bWNT8BQ95460Major histocompatibility complex class I-related gene proteinMR1Q969D9Thymic stromal lymphopoietinTSLPQ969E1Liver-expressed antimicrobial peptide 2LEAP2Q969H8UPF0556 protein C19orf10C19orf10Q969Y0NXPE family member 3NXPE3Q96A54Adiponectin receptor protein 1ADIPOR1Q96A83Collagen alpha-1(XXVI) chainEMID2Q96A84EMI domain-containing protein 1EMID1Q96A98Tuberoinfundibular peptide of 39 residuesPTH2Q96A99Pentraxin-4PTX4Q96BH3Epididymal sperm-binding protein 1ELSPBP1Q96BQ1Protein FAM3DFAM3DQ96CG8Collagen triple helix repeat-containing protein 1CTHRC1Q96DA0Zymogen granule protein 16 homolog BZG16BQ96DN2von Willebrand factor C and EGF domain-containing proteinVWCEQ96DR5BPI fold-containing family A member 2BPIFA2Q96DR8Mucin-like protein 1MUCL1Q96DX4RING finger and SPRY domain-containing protein 1RSPRY1Q96EE4Coiled-coil domain-containing protein 126CCDC126Q96GS6Abhydrolase domain-containing protein FAM108A1FAM108A1Q96GW7Brevican core proteinBCANQ96HF1Secreted frizzled-related protein 2SFRP2Q96I82Kazal-type serine protease inhibitor domain-containing protein 1KAZALD1Q96ID5Immunoglobulin superfamily member 21IGSF21Q96II8Leucine-rich repeat and calponin homology domain-containing protein 3LRCH3Q96IY4Carboxypeptidase B2CPB2Q96JB6Lysyl oxidase homolog 4LOXL4Q96JK4HHIP-like protein 1HHIPL1Q96KN2Beta-Ala-His dipeptidaseCNDP1Q96KW9Protein SPACA7SPACA7Q96KX0Lysozyme-like protein 4LYZL4Q96L15Ecto-ADP-ribosyltransferase 5ARTSQ96LB8Peptidoglycan recognition protein 4PGLYRP4Q96LB9Peptidoglycan recognition protein 3PGLYRP3Q96LC7Sialic acid-binding Ig-like lectin 10SIGLEC10Q96LR4Protein FAM19A4FAM19A4Q96MK3Protein FAM20AFAM20AQ96MS3Glycosyltransferase 1 domain-containing protein 1GLT1D1Q96NY8Processed poliovirus receptor-related protein 4PVRL4Q96NZ8WAP, kazal, immunoglobulin, kunitz and NTR domain-containing protein 1WFIKKN1Q96NZ9Proline-rich acidic protein 1PRAP1Q96P44Collagen alpha-1(XXI) chainCOL21A1Q96PB7Noelin-3OLFM3Q96PC5Melanoma inhibitory activity protein 2MIA2Q96PD5N-acetylmuramoyl-L-alanine amidasePGLYRP2Q96PH6Beta-defensin 118DEFB118Q96PL1Secretoglobin family 3A member 2SCGB3A2Q96PL2Beta-tectorinTECTBQ96QH8Sperm acrosome-associated protein 5SPACA5Q96QR1Secretoglobin family 3A member 1SCGB3A1Q96QU1Protocadherin-15PCDH15Q96QV1Hedgehog-interacting proteinHHIPQ96RW7Hemicentin-1HMCN1Q96S42Nodal homologNODALQ96S86Hyaluronan and proteoglycan link protein 3HAPLN3Q96SL4Glutathione peroxidase 7GPX7Q96SM3Probable carboxypeptidase X1CPXM1Q96T91Glycoprotein hormone alpha-2GPHA2Q99062Granulocyte colony-stimulating factor receptorCSF3RQ99102Mucin-4 alpha chainMUC4Q99217Amelogenin, X isoformAMELXQ99218Amelogenin, Y isoformAMELYQ99435Protein kinase C-binding protein NELL2NELL2Q99470Stromal cell-derived factor 2SDF2Q99542Matrix metalloproteinase-19MMP19Q99574NeuroserpinSERPINI1Q99584Protein S100-A13S100A13Q99616C-C motif chemokine 13CCL13Q99645EpiphycanEPYCQ99674Cell growth regulator with EF hand domain protein 1CGREF1Q99715Collagen alpha-1(XII) chainCOL12A1Q99727Metalloproteinase inhibitor 4TIMP4Q99731C-C motif chemokine 19CCL19Q99748NeurturinNRTNQ99935Proline-rich protein 1PROL1Q99942E3 ubiquitin-protein ligase RNF5RNF5Q99944Epidermal growth factor-like protein 8EGFL8Q99954Submaxillary gland androgen-regulated protein 3ASMR3AQ99969Retinoic acid receptor responder protein 2RARRES2Q99972MyocilinMYOCQ99983OsteomodulinOMDQ99985Semaphorin-3CSEMA3CQ99988Growth / differentiation factor 15GDF15Q9BPW4Apolipoprotein L4APOL4Q9BQ08Resistin-like betaRETNLBQ9BQ16Testican-3SPOCK3Q9BQ51Programmed cell death 1 ligand 2PDCD1LG2Q9BQB4SclerostinSOSTQ9BQI4Coiled-coil domain-containing protein 3CCDC3Q9BQP9BPI fold-containing family A member 3BPIFA3Q9BQR3Serine protease 27PRSS27Q9BQY6WAP four-disulfide core domain protein 6WFDC6Q9BRR6ADP-dependent glucokinaseADPGKQ9BS86Zona pellucida-binding protein 1ZPBPQ9BSG0Protease-associated domain-containing protein 1PRADC1Q9BSG5RetbindinRTBDNQ9BT30Probable alpha-ketoglutarate-dependent dioxygenase ABH7ALKBH7Q9BT56SpexinC12orf39Q9BT67NEDD4 family-interacting protein 1NDFIP1Q9BTY2Plasma alpha-L-fucosidaseFUCA2Q9BU40Chordin-like protein 1CHRDL1Q9BUD6Spondin-2SPON2Q9BUN1Protein MENTMENTQ9BUR5Apolipoprotein OAPOOQ9BV94ER degradation-enhancing alpha-mannosidase-like 2EDEM2Q9BWP8Collectin-11COLEC11Q9BWS9Chitinase domain-containing protein 1CHID1Q9BX67Junctional adhesion molecule CJAM3Q9BX93Group XIIB secretory phospholipase A2-like proteinPLA2G12BQ9BXI9Complement C1q tumor necrosis factor-related protein 6C1QTNF6Q9BXJ0Complement C1q tumor necrosis factor-related protein 5C1QTNF5Q9BXJ1Complement C1q tumor necrosis factor-related protein 1C1QTNF1Q9BXJ2Complement C1q tumor necrosis factor-related protein 7C1QTNF7Q9BXJ3Complement C1q tumor necrosis factor-related protein 4C1QTNF4Q9BXJ4Complement C1q tumor necrosis factor-related protein 3C1QTNF3Q9BXJ5Complement C1q tumor necrosis factor-related protein 2C1QTNF2Q9BXN1AsporinASPNQ9BXP8Pappalysin-2PAPPA2Q9BXR6Complement factor H-related protein 5CFHR5Q9BXS0Collagen alpha-1(XXV) chainCOL25A1Q9BXX0EMILIN-2EMILIN2Q9BXY4R-spondin-3RSPO3Q9BY15EGF-like module-containing mucin-like hormone receptor-like 3 subunit betaEMR3Q9BY50Signal peptidase complex catalytic subunit SEC11CSEC11CQ9BY76Angiopoietin-related protein 4ANGPTL4Q9BYF1Processed angiotensin-converting enzyme 2ACE2Q9BYJ0Fibroblast growth factor-binding protein 2FGFBP2Q9BYW3Beta-defensin 126DEFB126Q9BYX4Interferon-induced helicase C domain-containing protein 1IFIH1Q9BYZ8Regenerating islet-derived protein 4REG4Q9BZ76Contactin-associated protein-like 3CNTNAP3Q9BZG9Ly-6 / neurotoxin-like protein 1LYNX1Q9BZJ3Tryptase deltaTPSD1Q9BZM1Group XIIA secretory phospholipase A2PLA2G12AQ9BZM2Group IIF secretory phospholipase A2PLA2G2FQ9BZM5NKG2D ligand 2ULBP2Q9BZP6Acidic mammalian chitinaseCHIAQ9BZZ2SialoadhesinSIGLEC1Q9COB6Protein FAM5BFAM5BQ9GZM7Tubulointerstitial nephritis antigen-likeTINAGL1Q9GZN4Brain-specific serine protease 4PRSS22Q9GZP0Platelet-derived growth factor D, receptor-binding formPDGFDQ9GZT5Protein Wnt-10aWNT10AQ9GZU5NyctalopinNYXQ9GZV7Hyaluronan and proteoglycan link protein 2HAPLN2Q9GZV9Fibroblast growth factor 23FGF23Q9GZX9Twisted gastrulation protein homolog 1TWSG1Q9GZZ7GDNF family receptor alpha-4GFRA4Q9GZZ8Extracellular glycoprotein lacritinLACRTQ9H0B8Cysteine-rich secretory protein LCCL domain-containing 2CRISPLD2Q9H106Signal-regulatory protein deltaSIRPDQ9H114Cystatin-like 1CSTL1Q9H173Nucleotide exchange factor SIL1SIL1Q9H1E1Ribonuclease 7RNASE7Q9H1F0WAP four-disulfide core domain protein 10AWFDC10AQ9H1J5Protein Wnt-8aWNT8AQ9H1J7Protein Wnt-5bWNT5BQ9H1M3Beta-defensin 129DEFB129Q9H1M4Beta-defensin 127DEFB127Q9H1Z8AugurinC2orf40Q9H239Matrix metalloproteinase-28MMP28Q9H2A7C-X-C motif chemokine 16CXCL16Q9H2A9Carbohydrate sulfotransferase 8CHST8Q9H2R5Kallikrein-15KLK15Q9H2X0ChordinCHRDQ9H2X3C-type lectin domain family 4 member MCLEC4MQ9H306Matrix metalloproteinase-27MMP27Q9H324A disintegrin and metalloproteinase with thrombospondin motifs 10ADAMTS10Q9H336Cysteine-rich secretory protein LCCL domain-containing 1CRISPLD1Q9H3E2Sorting nexin-25SNX25Q9H3R2Mucin-13MUC13Q9H3U7SPARC-related modular calcium-binding protein 2SMOC2Q9H3Y0Peptidase inhibitor R3HDMLR3HDMLQ9H4A4Aminopeptidase BRNPEPQ9H4F8SPARC-related modular calcium-binding protein 1SMOC1Q9H4G1Cystatin-9-likeCST9LQ9H5V8CUB domain-containing protein 1CDCP1Q9H6B9Epoxide hydrolase 3EPHX3Q9H6E4Coiled-coil domain-containing protein 134CCDC134Q9H741UPF0454 protein C12orf49C12orf49Q9H772Gremlin-2GREM2Q9H7Y0Deleted in autism-related protein 1CXorf36Q9H8L6Multimerin-2MMRN2Q9H9S5Fukutin-related proteinFKRPQ9HAT2Sialate O-acetylesteraseSIAEQ9HB40Retinoid-inducible serine carboxypeptidaseSCPEP1Q9HB63Netrin-4NTN4Q9HBJ0Placenta-specific protein 1PLAC1Q9HC23Prokineticin-2PROK2Q9HC57WAP four-disulfide core domain protein 1WFDC1Q9HC73Cytokine receptor-like factor 2CRLF2Q9HC84Mucin-5BMUCSBQ9HCB6Spondin-1SPON1Q9HCQ7Neuropeptide NPSFNPVFQ9HCT0Fibroblast growth factor 22FGF22Q9HD89ResistinRETNQ9NNX1TuftelinTUFT1Q9NNX6CD209 antigenCD209Q9NP55BPI fold-containing family A member 1BPIFA1Q9NP70AmeloblastinAMBNQ9NP95Fibroblast growth factor 20FGF20Q9NP99Triggering receptor expressed on myeloid cells 1TREM1Q9NPA2Matrix metalloproteinase-25MMP25Q9NPE2NeugrinNGRNQ9NPH0Lysophosphatidic acid phosphatase type 6ACP6Q9NPH6Odorant-binding protein 2bOBP2BQ9NQ30Endothelial cell-specific molecule 1ESM1Q9NQ36Signal peptide, CUB and EGF-like domain-containing protein 2SCUBE2Q9NQ38Serine protease inhibitor Kazal-type 5SPINK5Q9NQ76Matrix extracellular phosphoglycoproteinMEPEQ9NQ79Cartilage acidic protein 1CRTAC1Q9NR16Scavenger receptor cysteine-rich type 1 protein M160CD163L1Q9NR23Growth / differentiation factor 3GDF3Q9NR71Neutral ceramidaseASAH2Q9NR99Matrix-remodeling-associated protein 5MXRA5Q9NRA1Platelet-derived growth factor CPDGFCQ9NRC9OtoraplinOTORQ9NRE1Matrix metalloproteinase-26MMP26Q9NRJ3C-C motif chemokine 28CCL28Q9NRM1EnamelinENAMQ9NRN5Olfactomedin-like protein 3OLFML3Q9NRR1Cytokine-like protein 1CYTL1Q9NS15Latent-transforming growth factor beta-binding protein 3LTBP3Q9NS62Thrombospondin type-1 domain-containing protein 1THSD1Q9NS71Gastrokine-1GKN1Q9NS98Semaphorin-3GSEMA3GQ9NSA1Fibroblast growth factor 21FGF21Q9NT22EMILIN-3EMILIN3Q9NTU7Cerebellin-4CBLN4Q9NVR0Kelch-like protein 11KLHL11Q9NWH7Spermatogenesis-associated protein 6SPATA6Q9NXC2Glucose-fructose oxidoreductase domain-containing protein 1GFOD1Q9NY56Odorant-binding protein 2aOBP2AQ9NY84Vascular non-inflammatory molecule 3VNN3Q9NZ20Group 3 secretory phospholipase A2PLA2G3Q9NZC2Triggering receptor expressed on myeloid cells 2TREM2Q9NZK5Adenosine deaminase CECR1CECR1Q9NZK7Group IIE secretory phospholipase A2PLA2G2EQ9NZP8Complement C1r subcomponent-like proteinC1RLQ9NZV1Cysteine-rich motor neuron 1 proteinCRIM1Q9NZW4Dentin sialoproteinDSPPQ9P0G3Kallikrein-14KLK14Q9P0W0Interferon kappaIFNKQ9P218Collagen alpha-1(XX) chainCOL20A1Q9P2C4Transmembrane protein 181TMEM181Q9P2K2Thioredoxin domain-containing protein 16TXNDC16Q9P2N4A disintegrin and metalloproteinase with thrombospondin motifs 9ADAMTS9Q9UBC7Galanin-like peptideGALPQ9UBD3Cytokine SCM-1 betaXCL2Q9UBD9Cardiotrophin-like cytokine factor 1CLCF1Q9UBM4OpticinOPTCQ9UBP4Dickkopf-related protein 3DKK3Q9UBQ6Exostosin-like 2EXTL2Q9UBR5Chemokine-like factorCKLFQ9UBS5Gamma-aminobutyric acid type B receptor subunit 1GABBR1Q9UBT3Dickkopf-related protein 4 short formDKK4Q9UBU2Dickkopf-related protein 2DKK2Q9UBU3Ghrelin-28GHRLQ9UBV4Protein Wnt-16WNT16Q9UBX5Fibulin-5FBLN5Q9UBX7Kallikrein-11KLK11Q9UEF7KlothoKLQ9UFP1Protein FAM198AFAM198AQ9UGM3Deleted in malignant brain tumors 1 proteinDMBT1Q9UGM5Fetuin-BFETUBQ9UGP8Translocation protein SEC63 homologSEC63Q9UHF0Neurokinin-BTAC3Q9UHF1Epidermal growth factor-like protein 7EGFL7Q9UHG2ProSAASPCSK1NQ9UHI8A disintegrin and metalloproteinase with thrombospondin motifs 1ADAMTS1Q9UHL4Dipeptidyl peptidase 2DPP7Q9UI42Carboxypeptidase A4CPA4Q9UIG4Psoriasis susceptibility 1 candidate gene 2 proteinPSORS 1C2Q9UIK5Tomoregulin-2TMEFF2Q9UIQ6Leucyl-cystinyl aminopeptidase, pregnancy serum formLNPEPQ9UJA9Ectonucleotide pyrophosphatase / phosphodiesterase family member 5ENPP5Q9UJH8MeteorinMETRNQ9UJJ9N-acetylgluco samine-1-pho sphotransferase subunit gammaGNPTGQ9UJW2Tubulointerstitial nephritis antigenTINAGQ9UK05Growth / differentiation factor 2GDF2Q9UK55Protein Z-dependent protease inhibitorSERPINA10Q9UK85Dickkopf-like protein 1DKKL1Q9UKJ1Paired immunoglobulin-like type 2 receptor alphaPILRAQ9UKP4A disintegrin and metalloproteinase with thrombospondin motifs 7ADAMTS7Q9UKP5A disintegrin and metalloproteinase with thrombospondin motifs 6ADAMTS6Q9UKQ2Disintegrin and metalloproteinase domain-containing protein 28ADAM28Q9UKQ9Kallikrein-9KLK9Q9UKR0Kallikrein-12KLK12Q9UKR3Kallikrein-13KLK13Q9UKU9Angiopoietin-related protein 2ANGPTL2Q9UKZ9Procollagen C-endopeptidase enhancer 2PCOLCE2Q9UL52Transmembrane protease serine 11E non-catalytic chainTMPRSS11EQ9ULC0EndomucinEMCNQ9ULI3Protein HEG homolog 1HEG1Q9ULZ1Apelin-13APLNQ9ULZ9Matrix metalloproteinase-17MMP17Q9UM21Alpha-1,3-mannosyl-glycoprotein 4-beta-N-acetylglucosaminyltransferase A soluble formMGAT4AQ9UM22Mammalian ependymin-related protein 1EPDR1Q9UM73ALK tyrosine kinase receptorALKQ9UMD997 kDa linear IgA disease antigenCOL17A1Q9UMX5NeudesinNENFQ9UN73Protocadherin alpha-6PCDHA6Q9UNA0A disintegrin and metalloproteinase with thrombospondin motifs 5ADAMTS5Q9UNI1Chymotrypsin-like elastase family member 1CELA1Q9UNK4Group IID secretory phospholipase A2PLA2G2DQ9UP79A disintegrin and metalloproteinase with thrombospondin motifs 8ADAMTS8Q9UPZ6Thrombospondin type-1 domain-containing protein 7ATHSD7AQ9UQ72Pregnancy-specific beta-1-glycoprotein 11PSG11Q9UQ74Pregnancy-specific beta-1-glycoprotein 8PSG8Q9UQC9Calcium-activated chloride channel regulator 2CLCA2Q9UQE7Structural maintenance of chromosomes protein 3SMC3Q9UQP3Tenascin-NTNNQ9Y223UDP-N-acetylglucosamine 2-epimeraseGNEQ9Y240C-type lectin domain family 11 member ACLEC11AQ9Y251Heparanase 8 kDa subunitHPSEQ9Y258C-C motif chemokine 26CCL26Q9Y264Angiopoietin-4ANGPT4Q9Y275Tumor necrosis factor ligand superfamily member 13b, membrane formTNFSF13BQ9Y287BRI2 intracellular domainITM2BQ9Y2E5Epididymis-specific alpha-mannosidaseMAN2B2Q9Y334von Willebrand factor A domain-containing protein 7VWA7Q9Y337Kallikrein-5KLK5Q9Y3B3Transmembrane emp24 domain-containing protein 7TMED7Q9Y3E2BolA-like protein 1BOLA1Q9Y426C2 domain-containing protein 2C2CD2Q9Y4K0Lysyl oxidase homolog 2LOXL2Q9Y4X3C-C motif chemokine 27CCL27Q9Y5C1Angiopoietin-related protein 3ANGPTL3Q9YSI2Protocadherin alpha-10PCDHA10Q9Y5I3Protocadherin alpha-1PCDHA1Q9Y5K2Kallikrein-4KLK4Q9Y5L2Hypoxia-inducible lipid droplet-associated proteinHILPDAQ9Y5Q5Atrial natriuretic peptide-converting enzymeCORINQ9Y5R2Matrix metalloproteinase-24MMP24Q9Y5U5Tumor necrosis factor receptor superfamily member 18TNFRSF18Q9Y5W5Wnt inhibitory factor 1WIF1Q9Y5X9Endothelial lipaseLIPGQ9Y625Secreted glypican-6GPC6Q9Y646Carboxypeptidase QCPQQ9Y6C2EMILIN-1EMILIN1Q9Y6F9Protein Wnt-6WNT6Q9Y6I9Testis-expressed sequence 264 proteinTEX264Q9Y6L7Tolloid-like protein 2TLL2Q9Y6N3Calcium-activated chloride channel regulator family member 3CLCA3PQ9Y6N6Laminin subunit gamma-3LAMC3Q9Y6R7IgGFc-binding proteinFCGBPQ9Y6Y9Lymphocyte antigen 96LY96Q9Y6Z7Collectin-10COLEC10

[0072] In some embodiments, the compositions of the invention provide for the delivery of one or more mRNAs encoding one or more additional exemplary proteins listed in Table 2; thus, compositions of the invention may comprise an mRNA encoding a protein listed in Table 2 (or a homolog thereof) along with other components set out herein. Table 2. Additional Exemplary Proteins Uniprot ID Protein Name Gene Name A6NGW2Putative stereocilin-like proteinSTRCP1A6NIE9Putative serine protease 29PRSS29PA6NJ16Putative V-set and immunoglobulin domain-containing-like protein IGHV4OR15-8IGHV4OR15-8A6NJS3Putative V-set and immunoglobulin domain-containing-like protein IGHV1OR21-1IGHV1OR21-1A6NMY6Putative annexin A2-like proteinANXA2P2A8MT79Putative zinc-alpha-2-glycoprotein-like 1A8MWS1Putative killer cell immunoglobulin-like receptor like protein KIR3DP1KIR3DP1A8MXU0Putative beta-defensin 108ADEFB108P1C9JUS6Putative adrenomedullin-5-like proteinADMSPOC7V7Putative signal peptidase complex catalytic subunit SEC11BSEC11BP0C854Putative cat eye syndrome critical region protein 9CECR9Q13046Putative pregnancy-specific beta-1-glycoprotein 7PSG7Q16609Putative apolipoprotein(a)-like protein 2LPAL2Q2TV78Putative macrophage-stimulating protein MSTP9MST1P9Q5JQD4Putative peptide YY-3PYY3Q5R387Putative inactive group IIC secretory phospholipase A2PLA2G2CQ5VSP4Putative lipocalin 1-like protein 1LCN1P1Q5W188Putative cystatin-9-like protein CST9LP1CST9LP1Q6UXR4Putative serpin A13SERPINA13PQ86SH4Putative testis-specific prion proteinPRNTQ86YQ2Putative latherinLATHQ8IVG9Putative humanin peptideMT-RNR2Q8NHM4Putative trypsin-6TRY6Q8NHW4C-C motif chemokine 4-likeCCL4L2Q9H7L2Putative killer cell immunoglobulin-like receptor-like protein KIR3DX1KIR3DX1Q9NRI6Putative peptide YY-2PYY2Q9UF72Putative TP73 antisense gene protein 1TP73-AS1Q9UKY3Putative inactive carboxylesterase 4CES1P1

[0073] The Uniprot IDs set forth in Table 1 and Table 2 refer to the human versions the listed proteins and the sequences of each are available from the Uniprot database. Sequences of the listed proteins are also generally available for various animals, including various mammals and animals of veterinary or industrial interest. Accordingly, in some embodiments, compositions of the invention provide for the delivery of one or more mRNAs encoding one or more proteins chosen from mammalian homologs or homologs from an animal of veterinary or industrial interest of the secreted proteins listed in Table 1 or Table 2; thus, compositions of the invention may comprise an mRNA encoding a protein chosen from mammalian homologs or homologs from an animal of veterinary or industrial interest of a protein listed in Table 1 or Table 2 along with other components set out herein. In some embodiments, mammalian homologs are chosen from mouse, rat, hamster, gerbil, horse, pig, cow, llama, alpaca, mink, dog, cat, ferret, sheep, goat, or camel homologs. In some embodiments, the animal of veterinary or industrial interest is chosen from the mammals listed above and / or chicken, duck, turkey, salmon, catfish, or tilapia.

[0074] In embodiments, the compositions of the invention provide for the delivery of mRNA encoding a lysosomal protein chosen from Table 3. In some embodiments, the compositions of the invention provide for the delivery of one or more mRNAs encoding one or more lysosomal and / or related proteins listed in Table 3; thus, compositions of the invention may comprise an mRNA encoding a protein listed in Table 3 (or a homolog thereof) along with other components set out herein. Table 3. Lysosomal and Related Proteinsα-fucosidaseα-galactosidaseα-glucosidaseα-Iduronidaseα-mannosidaseα-N-acetylgalactosaminidase (a-galactosidase B)β-galactosidaseβ-glucuronidaseβ-hexosaminidaseβ-mannosidase3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) lyase3-methylcrotonyl-CoA carboxylase3-O-sulfogalactosyl cerebroside sulfatase (arylsulfatase A)acetyl-CoA transferaseacid alpha-glucosidaseacid ceramidaseacid lipaseacid phosphataseacid sphingomyelinasealpha-galactosidase Aarylsulfatase Abeta-galactosidasebeta-glucocerebrosidasebeta-hexosaminidasebiotinidasecathepsin Acathepsin KCLN3CLN5CLN6CLN8CLN9cystine transporter (cystinosin)cytosolic protein beta3A subunit of the adaptor protein-3 complex, AP3formyl-Glycine generating enzyme (FGE)galactocerebrosidasegalactose-1-phosphate uridyltransferase (GALT)galactose 6-sulfate sulfatase (also known as N-acetylgalactosamine-6-sulfatase)glucocerebrosidaseglucuronate sulfataseglucuronidaseglycoprotein cleaving enzymesglycosaminoglycan cleaving enzymesglycosylasparaginase (aspartylglucosaminidase)GM2-APHeparan-alpha-glucosaminide N-acetyltransferase (HGSNAT, TMEM76)Heparan sulfatasehexosaminidase A lysosomal proteases methylmalonyl-CoA mutasehyaluronidaseIduronate sulfataseLAMP-2lysosomal α-mannosidaseLysosomal p40 (C2orf18)Major facilitator superfamily domain containing 8 protein (MFSD8 or CLN7)N-acetylgalactosamine 4-sulfataseN-acetyl glucosamine 6-sulfataseN-acetyl glucosaminidaseN-acetylglucosamine-1-phosphate transferaseNPC1NPC2palmitoyl-protein thioesterasepalmitoyl-protein thioesterase (CLN1)Saposin A (Sphingolipid activator protein A)Saposin B (Sphingolipid activator protein B)Saposin C (Sphingolipid activator protein C)Saposin D (Sphingolipid activator protein D)sialic acid transporter (sialin)sialidaseSialinsulfataseTransmembrane protein 74 (TMEM74)tripeptidyl-peptidasetripeptidyl-peptidase I (CLN2)UDP-N-acetylglucosamine- phosphotransferase

[0075] Information regarding lysosomal proteins is available from Lubke et al., "Proteomics of the Lysosome," Biochim Biophys Acta. (2009) 1793: 625-635. In some embodiments, the protein listed in Table 3 and encoded by mRNA in the compositions of the invention is a human protein. Sequences of the listed proteins are also available for various animals, including various mammals and animals of veterinary or industrial interest as described above.

[0076] In some embodiments, the compositions of the invention provide for the delivery of mRNA encoding a therapeutic protein (e.g., cytosolic, transmembrane or secreted) such as those listed in Table 4. In some embodiments, the compositions of the invention provide for the delivery of an mRNA encoding a therapeutic protein useful in treating a disease or disorder (i.e., indication) listed in Table 4; thus, compositions of the invention may comprise an mRNA encoding a therapeutic protein listed in Table 4 (or a homolog thereof, as discussed below) along with other components set out herein for treating a disease or disorder (i.e., indication) listed in Table 4. Table 4. Exemplary Indications and Related Proteins Indication Therapeutic Protein 3-Methylcrotonyl-CoA carboxylase deficiencyMethylcrotonoyl-CoA carboxylase3-Methylglutaconic aciduriaMethylglutaconyl-CoA hydrataseActinic keratosisAcute intermittent porphyriaPorphobilinogen deaminaseAcute lymphocytic leukemiaAcute myeloid leukemiaAddison's diseaseAdenosine deaminase deficiencyAdenosine deaminaseAdrenoleukodystrophyABCD1AdrenomyeloneuropathyAIDS / HIVAlcohol use disordersAlkaptonuriaHomogentisate 1,2-dioxygenaseAllergic asthmaAnti-IgE mAbAllergies (dermatitis, rhinitis)Alopecia areataAlpers' diseasePOLGAlpers-Huttenlocher syndromeAlpha 1-antitrypsin deficiencyAlpha 1 protease inhibitorAlpha-mannosidosisAlpha-D-mannosidaseAlport syndromeAlzheimer's diseaseAmyloid light-chain amyloidosisAmyotrophic lateral sclerosis (ALS)AnemiaErythropoietinAortic valve stenosisArgininemiaArginaseArgininosuccinic acidemiaArgininosuccinate lyaseArrhythmogenic right ventricular dysplasiaAutismAutosomal dominant and recessive progressive external ophthalmoplegia with mitochondrial DNA deletionsAutosomal recessive polycystic kidney diseaseARPKDBacterial infectionsBasal cell carcinomaBatten diseaseBattenin + othersB-cell chronic lymphocytic leukemiaBecker muscular dystrophyDystrophinBeta-thalassemiaBeta globinBinge eating disorderBipolar disorderBladder cancerBlepharospasm, Cervical dystonia, Chronic migraine, moreBotulinum toxinBronchiolitis obliteransBrugada syndromeBuerger's diseaseCACNA1ACACNB4-related Episodic Ataxia Type 2Cancer and depressionCancer and sexual dysfunctionCancer in pregnancyCarbamylphosphate synthetase deficiencyCarbamylphosphate synthetaseCarcinoma of the gallbladderCardiomyopathy (diabetic)Cardiomyopathy (hypertrophic)Carnitine uptake defectSLC22A5Catecholaminergic polymorphic ventricular tachycardiaCDKL5-related Atypical Rett SyndromeCeliac diseaseCellulitisCerebrovascular diseaseCervix uteri cancerChronic fatigue syndromeChronic graft versus host diseaseChronic idiopathic urticariaChronic immune thrombocytopeniaThrombopoietinChronic kidney kiseaseChronic liver diseaseChronic lymphocytic leukemiaChronic myeloid leukemiaChronic pancreatitisCirrhosis of the liverCitrullinemia, type IArgininosuccinate synthaseClassic Rett SyndromeClassical galactosemiaGalactose-1-phosphate uridylyltransferaseClostridium difficile associated diarrheaClotting disordersCOAD / COPDCocaine addictionCOL4A5-related disordersCold contact urticariaContraception, femaleCoronary artery diseasesCorpus uteri cancerCorticobasal degenerationCrigler-Najjar syndromeUDP-glucuronosyltransferaseCritical limb ischemiaCTNS-related cystinosisCutaneous lupus erythematosusCutaneous neuroendocrine carcinoma (Merkel Cell)Cystic fibrosisCFTRCystic fibrosisDeoxyribonuclease ICystinosisCystinosinCystinuriaSLC7A9Dementia (Lewy body)DepressionDiabetic foot infectionsDiabetic foot ulcerDiabetic peripheral neuropathyDiabetic ulcersDiarrhoeal diseasesDiffuse large B-cell lymphomaDiGeorge syndromeDiverticulitisDrug use disordersDuchenne muscular dystrophyDystrophinDysarthriaDyskinesia (levodopa-induced)Early-onset autosomal dominant Alzheimer's diseaseEczemaEhlers-Danlos syndrome, type 1EIF2B1EIF2B2EIF2B3EIF2B4EIF2B5-related childhood ataxia with central nervous system hypomyelination / vanishing white matterEosinophilic esophagitisEpilepsyErectile dysfunctionErythropoietic protoporphyriaFerrochelataseEsophageal carcinomaEssential tremorFabry diseaseAlpha galactosidaseFamilial adenomatous polyposisAPCFamilial chylomicronemiaLipoprotein lipaseFamilial dysbetalipoproteinemiaApolipoprotein EFamilial isolated dilated cardiomyopathyFamilial mediterranean feverPyrin (MEFV)Familial melanomaFemale infertilityFollicle stimulating hormoneFemale sexual dysfunctionFibromyalgiaFMR1-related disordersFracture healingFragile X Premature Ovarian Failure SyndromeFragile X syndromeFMRPFragile X-Associated Tremor / Ataxia SyndromeFriedreich's ataxiaFrontotemporal dementiaFryns syndromeGalactocerebrosidase deficienciesGALE deficiencyGalactose epimeraseGALK deficiencyGalactokinaseGALT-related galactosemiaGastric cancerGastroesophageal reflux diseaseGaucher diseaseGlucocerebrosidaseGilbert syndromeUDP-glucuronosyltransferaseGlioblastoma multiformeGlomerulonephritisGlutaric acidemia, type IGlutaryl-CoA dehydrogenaseGM2 gangliosidosisHEXA, HEXBGoutUrate oxidaseGraft versus host diseaseGrowth hormone deficiencyGrowth hormone 1 / Growth hormone 2Head and neck cancer, Metastatic colorectal cancerAnti-EGFr mAbHearing loss, adult onsetHeart failureHemachromatosisHFE proteinHemifacial spasmHemolytic uremic syndromeAnti-complement factor C5 mAbHemophilia AFactor VIIIHemophilia A, Hemophilia BFactor VIIHemophilia BFactor IXHepatitis B, Hepatitis CInterferon alphaHER2+ breast cancer, gastric cancerAnti-HER2 mAbHereditary angioedemaC1 esterase inhibitorHereditary hemorrhagic telangiectasiaHereditary hemorrhagic telangiectasia (AT)Hereditary spherocytosisHidradenitis suppurativaHomocystinuriaCystathionine beta-synthaseHomozygous familial hypercholesterolemiaLDL receptorHunter syndrome (MPS II)Iduronate-2-sulfataseHuntington diseaseHuntingtinHurler syndrome (MPS I)Alpha-L iduronidaseHydrolethalusHyperalgesiaHyperbilirubinemiaHyperhidrosisHyperlipidemiaHypermethioninemiaMethionine adenosyltransferaseHyperoxaluria, type ISerine-pyruvate aminotransferaseHypertensionHyperuricemiaHyponatremiaHypoparathyroidismParathyroid hormoneHypophosphatasiaTNSALPIdiopathic pulmonary fibrosisIminoglycinuriaImmunoglobulin deficiencyImmunoglobulinInfection (adenovirus)Infection (anthrax prophylaxis)Infection (BK virus)Infection (Clostridium difficile prophylaxis)Infection (Dengue fever prophylaxis)Infection (Epstein-Barr virus)Infection (Hepatitis-D)Infection (Lyme disease prophylaxis)Infection (Smallpox virus)Infectious diseases vaccinesInfectious antigenInflammatory heart diseasesInsomniaInterstitial cystitisIron-deficiency anaemiaIrritable bowel diseaseIschaemic heart diseaseIsovaleric aciduriaIsovaleric acid CoA dehydrogenase deficiencyJanskv-Bielschowskv diseaseJuvenile Batten diseaseJuvenile Neuronal Ceroid Lipofuscinosis (JNCL)Juvenile rheumatoid arthritisTNF-alpha inhibitorsKennedv's disease (SBMA)KeratoconusKrabbe diseaseGalactocerebrosidaseLeber's hereditary optic neuropathyNADH dehydrogenaseLeiomyosarcomaLennox-Gastaut syndromeLesch-Nyhan syndromeHypoxanthine phosphoribosyltransferase 1LeukaemiaLi-Fraumeni syndromeTP53LipomaLiposarcomaLiver cancerLong-chain 3-OH acyl-CoA dehydrogenase deficiencyLong-chain-3-hydroxyacyl-CoA dehydrogenaseLower respiratory infectionsLysosomal acid lipase deficiencyLysosomal acid lipaseMacular degenerationMajor depressive disorderMalignant fibrous histiocytomaMantle cell lymphomaMaple syrup urine disease3-methyl-2-oxobutanoate dehydrogenaseMarfan syndromeFBN1Maroteaux-Lamy syndrome (MPS VI)N-acetylgalactosamine 4-sulfataseMastocytosisMcArdle diseaseMuscle glycogen phosphorylaseMECP2-related disordersMECP2-related Severe Neonatal EncephalopathyMedium-chain acyl-CoA dehydrogenase deficiencyAcyl-CoA dehydrogenaseMelanomaAnti-CTLA4 mAbMetachromatic leukodystrophyArylsulfatase AMetastatic colorectal cancer, NSCLC, othersAnti-VEGF mAbMethylmalonyl-CoA mutase deficiencyMethylmalonyl-CoA mutaseMigraineMitochondrial oxidative phosphorylation disordersMorquio syndrome, type A (MPS IVA)Galactose 6-sulfate sulfataseMorquio syndrome, type B (MPS IVB)Beta-galactosidaseMouth and oropharynx cancersMultiple carboxylase deficiencyBiotin-methylcrotonoyl-CoA-carboxylase ligaseMultiple myelomaMultiple sclerosisAnti-VLA-4 mAbMultiple sclerosisInterferon betaMultiple system atrophyMyasthenia gravisMyelofibrosisNarcolepsyNeonatal bronchopulmonary dysplasiaNeonatal infectionsNephritis and nephrosisNeurofibromatosis, type 1NF-1Neuronal ceroid lipofuscinoses-related diseasesNeutropeniaG-CSFNiemann Pick disease, type A / BSMPD1Niemann Pick disease, type CNPC1Niemann-Pick disease Type C1NocturiaNon-alcoholic fatty liver diseaseNon-Hodgkin lymphomaAnti-CD20 mAbNon-small cell lung cancerNotch-3 related cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL)ObesityOphthalmoparesisOpioid induced constipationOrnithine transcarbamylase deficiencyOrnithine transcarbamylaseOsteoarthritisOsteopetrosisOsteoporosisAnti-RANKL mAbOvarian cancerPaget disease of boneSequestosome 1PainPancreatic carcinomaPanic disorderParkinson diseaseParoxysmal nocturnal hemoglobinuriaAnti-complement factor C5 MabPediculosis capitis (head lice)Pelizaeus-Merzbacher diseasePemphigus vulgarisPeptic ulcer diseasePeripheral neuropathyPeyronie's diseasePhenylketonuriaPhenylalanine hydroxylasePneumococcal infection prophylaxisPOLG-related sensory ataxic neuropathyPolycystic kidney diseasePolycystic ovary syndromePolycythaemia veraPolymerase G-related disordersPolymorphous light eruptionPompe diseaseAlpha glucosidasePorphyria cutanea tardaUroporphyrinogen decarboxylasePost herpetic neuralgiaPost-organ transplantPouchitisPPM-X SyndromePrader-Willi syndromePreeclampsiaPremature ejaculationPrematurity and low birth weightPrimary ciliary dyskinesiaPrimary glomerular diseasesPrimary humoral immune deficiencies (e.g., CVID)ImmunoglobulinProctitisProgressive multifocal leukoencephalopathyProgressive supranuclear palsyPropionic acidemiaPropionyl-CoA carboxylaseProstate cancerPsoriasisAnti-IL-12 & IL-23 mAbPsoriatic arthritisTNF-alpha inhibitorsPTT-1Pulmonary arterial hypertensionPulmonary arterial hypertensionRaynaud's phenomenonRefractive errorsRenal cell carcinomaRestless leg syndromeRetinitis pigmentosaRheumatic heart diseaseRheumatoid arthritisAnti-interleukin-6 (IL-6) mAbRheumatoid arthritisT-cell costimulation blockerRheumatoid arthritisTNF-alpha inhibitorRomano-Ward syndromeRosaceaSanfilippo syndrome, type A (MPS IIIA)Heparan N-sulfataseSanfilippo syndrome, type B (MPS IIIB)N-acetyl-alpha-D-glucosaminidaseSantavuori-Haltia diseaseSchizophreniaSchnitzler syndromeSclerodermaSCN1ASCN1B-related seizure disordersShort-chain acyl-CoA dehydrogenase deficiencyButyryl-CoA dehydrogenaseSickle cell diseaseHemoglobinSLC3A1-related disordersSmall cell lung cancerSMN-1-related spinal muscular atrophy (SMA)Spinal muscular atrophySurvival motor neuron proteinSquamous cell carcinoma of head and neckStickler syndromeStomach cancerStroke prophylaxisSynovial sarcomaSystemic lupus erythematosusAnti-BAFFSystemic sclerosisTetrahydrobiopterin-deficient hyperphenylalaninemiaTetrahydrobiopterinThromboangiitis obliteransThrombotic disordersThyroid cancerTPP1 deficienciesTrachea, bronchus, lung cancersTricuspid atresiaTSC1TSC2-related tuberous sclerosisType 2 diabetes mellitusGlucagon-like peptide 1 (GLP-1) agonistType 2 diabetes mellitusInsulinTyrosinemia, type IFumarylacetoacetaseUlcerative colitisUterine fibroidsVaricose veinsVenous thromboembolismVery long-chain acyl-CoA dehydrogenase deficiencyLong-chain-acyl-CoA dehydrogenasevon Gierke's diseaseGlucose-6-phosphataseVon Hippel-Lindau diseasepVHLWegener granulomatosisWilson diseaseWilson disease proteinX-Linked adrenal hypoplasiaX-linked adrenoleukodystrophyX-linked agammaglobulinemiaBruton's tyrosine kinase

[0077] In some embodiments, a composition of the present invention is used to prevent, treat and / or cure a subject affected with a disease or disorder listed or associated with the proteins listed in Tables 1, 2, 3 or 4. In some embodiments, an mRNA encodes one or more of argininosuccinate synthetase (ASS1), Factor IX, survival motor neuron 1 (SMN1), or phenylalanine hydroxylaseSynthesis of mRNA

[0078] mRNAs according to the present invention may be synthesized according to any of a variety of known methods. For example, mRNAs according to the present invention may be synthesized via in vitro transcription (IVT). Briefly, IVT is typically performed with a linear or circular DNA template containing a promoter, a pool of ribonucleotide triphosphates, a buffer system that may include DTT and magnesium ions, and an appropriate RNA polymerase (e.g., T3, T7 or SP6 RNA polymerase), DNAse I, pyrophosphatase, and / or RNAse inhibitor. The exact conditions will vary according to the specific application.

[0079] In some embodiments, for the preparation of mRNA according to the invention, a DNA template is transcribed in vitro. A suitable DNA template typically has a promoter, for example a T3, T7 or SP6 promoter, for in vitro transcription, followed by desired nucleotide sequence for desired mRNA and a termination signal.

[0080] Desired mRNA sequence(s) according to the invention may be determined and incorporated into a DNA template using standard methods. For example, starting from a desired amino acid sequence (e.g., an enzyme sequence), a virtual reverse translation is carried out based on the degenerated genetic code. Optimization algorithms may then be used for selection of suitable codons. Typically, the G / C content can be optimized to achieve the highest possible G / C content on one hand, taking into the best possible account the frequency of the tRNAs according to codon usage on the other hand. The optimized RNA sequence can be established and displayed, for example, with the aid of an appropriate display device and compared with the original (wild-type) sequence. A secondary structure can also be analyzed to calculate stabilizing and destabilizing properties or, respectively, regions of the RNA.Modified mRNA

[0081] In some embodiments, mRNA according to the present invention may be synthesized as unmodified or modified mRNA. Typically, mRNAs are modified to enhance stability. Modifications of mRNA can include, for example, modifications of the nucleotides of the RNA. A modified mRNA according to the invention can thus include, for example, backbone modifications, sugar modifications or base modifications. In some embodiments, mRNAs may be synthesized from naturally occurring nucleotides and / or nucleotide analogues (modified nucleotides) including, but not limited to, purines (adenine (A), guanine (G)) or pyrimidines (thymine (T), cytosine (C), uracil (U)), and as modified nucleotides analogues or derivatives of purines and pyrimidines, such as e.g. 1-methyl-adenine, 2-methyl-adenine, 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), dihydro-uracil, 2-thio-uracil, 4-thio-uracil, 5-carboxymethylaminomethyl-2-thio-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, uracil-5-oxyacetic acid (v), 1-methyl-pseudouracil, queosine, .beta.-D-mannosyl-queosine, wybutoxosine, and phosphoramidates, phosphorothioates, peptide nucleotides, methylphosphonates, 7-deazaguanosine, 5-methylcytosine and inosine. The preparation of such analogues is known to a person skilled in the art e.g. from the U.S. Pat. No. 4,373,071, U.S. Pat. No. 4,401,796, U.S. Pat. No. 4,415,732, U.S. Pat. No. 4,458,066, U.S. Pat. No. 4,500,707, U.S. Pat. No. 4,668,777, U.S. Pat. No. 4,973,679, U.S. Pat. No. 5,047,524, U.S. Pat. No. 5,132,418, U.S. Pat. No. 5,153,319, U.S. Pat. Nos. 5,262,530 and 5,700,642.

[0082] In some embodiments, mRNAs (e.g., enzyme encoding mRNAs) may contain RNA backbone modifications. Typically, a backbone modification is a modification in which the phosphates of the backbone of the nucleotides contained in the RNA are modified chemically. Exemplary backbone modifications typically include, but are not limited to, modifications from the group consisting of methylphosphonates, methylphosphoramidates, phosphoramidates, phosphorothioates (e.g. cytidine 5'-O-(1-thiophosphate)), boranophosphates, positively charged guanidinium groups etc., which means by replacing the phosphodiester linkage by other anionic, cationic or neutral groups.

[0083] In some embodiments, mRNAs (e.g., enzyme encoding mRNAs) may contain sugar modifications. A typical sugar modification is a chemical modification of the sugar of the nucleotides it contains including, but not limited to, sugar modifications chosen from the group consisting of 2'-deoxy-2'-fluoro-oligoribonucleotide (2'-fluoro-2'-deoxycytidine 5'-triphosphate, 2'-fluoro-2'-deoxyuridine 5'-triphosphate), 2'-deoxy-2'-deamine-oligoribonucleotide (2'-amino-2'-deoxycytidine 5'-triphosphate, 2'-amino-2'-deoxyuridine 5'-triphosphate), 2'-O-alkyloligoribonucleotide, 2'-deoxy-2'-C-alkyloligoribonucleotide (2'-O-methylcytidine 5'-triphosphate, 2'-methyluridine 5'-triphosphate), 2'-C-alkyloligoribonucleotide, and isomers thereof (2'-aracytidine 5'-triphosphate, 2'-arauridine 5'-triphosphate), or azidotriphosphates (2'-azido-2'-deoxycytidine 5'-triphosphate, 2'-azido-2'-deoxyuridine 5'-triphosphate).

[0084] In some embodiments, mRNAs (e.g., enzyme encoding mRNAs) may contain modifications of the bases of the nucleotides (base modifications). A modified nucleotide which contains a base modification is also called a base-modified nucleotide. Exemples of such base-modified nucleotides include, but are not limited to, 2-amino-6-chloropurine riboside 5'-triphosphate, 2-aminoadenosine 5'-triphosphate, 2-thiocytidine 5'-triphosphate, 2-thiouridine 5'-triphosphate, 4-thiouridine 5'-triphosphate, 5-aminoallylcytidine 5'-triphosphate, 5-aminoallyluridine 5'-triphosphate, 5-bromocytidine 5'-triphosphate, 5-bromouridine 5'-triphosphate, 5-iodocytidine 5'-triphosphate, 5-iodouridine 5'-triphosphate, 5-methylcytidine 5'-triphosphate, 5-methyluridine 5'-triphosphate, 6-azacytidine 5'-triphosphate, 6-azauridine 5'-triphosphate, 6-chloropurine riboside 5'-triphosphate, 7-deazaadenosine 5'-triphosphate, 7-deazaguanosine 5'-triphosphate, 8-azaadenosine 5'-triphosphate, 8-azidoadenosine 5'-triphosphate, benzimidazole riboside 5'-triphosphate, 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.Cap Structure

[0085] Typically, mRNA synthesis includes the addition of a "cap" on the N-terminal (5') end, and a "tail" on the C-terminal (3') end. The presence of the cap is important in providing resistance to nucleases found in most eukaryotic cells. The presence of a "tail" serves to protect the mRNA from exonuclease degradation.

[0086] Thus, in some embodiments, mRNAs (e.g., enzyme encoding mRNAs) include a 5' cap structure. A 5' cap is typically added as follows: first, an RNA terminal phosphatase removes one of the terminal phosphate groups from the 5' nucleotide, leaving two terminal phosphates; guanosine triphosphate (GTP) is then added to the terminal phosphates via a guanylyl transferase, producing a 5'5'5 triphosphate linkage; and the 7-nitrogen of guanine is then methylated by a methyltransferase. Examples of cap structures include, but are not limited to, m7G(5')ppp (5'(A,G(5')ppp(5')A and G(5')ppp(5')G.

[0087] In some embodiments, naturally occurring cap structures comprise a 7-methyl guanosine that is linked via a triphosphate bridge to the 5'-end of the first transcribed nucleotide, resulting in a dinucleotide cap of m 7< 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 guanylyl transferase. The addition of the cap to the 5' terminal end of RNA occurs immediately after initiation of transcription. The terminal nucleoside is typically a guanosine, and is in the reverse orientation to all the other nucleotides, i.e., G(5')ppp(5')GpNpNp.

[0088] A common cap for mRNA produced by in vitro transcription is m 7< G(5')ppp(5')G, which has been used as the dinucleotide cap in transcription with T7 or SP6 RNA polymerase in vitro to obtain RNAs having a cap structure in their 5'-termini. The prevailing method for the in vitro synthesis of capped mRNA employs a pre-formed dinucleotide of the form m 7< G(5')ppp(5')G ("m 7< GpppG") as an initiator of transcription.

[0089] To date, a usual form of a synthetic dinucleotide cap used in in vitro translation experiments is the Anti-Reverse Cap Analog ("ARCA") or modified ARCA, which is generally a modified cap analog in which the 2' or 3' OH group is replaced with -OCH 3 .

[0090] Additional cap analogs include, but are not limited to, chemical structures selected from the group consisting of m 7< GpppG, m 7< GpppA, m 7< GpppC; unmethylated cap analogs (e.g., GpppG); dimethylated cap analog (e.g., m 2,7< GpppG), trimethylated cap analog (e.g., m 2,2,7< GpppG), dimethylated symmetrical cap analogs (e.g., m 7< Gpppm 7< G), or anti reverse cap analogs (e.g., ARCA; m 7< , 2'Ome< GpppG, m 72'd< GpppG, m 73'Ome< GpppG, m 7,3'd< GpppG and their tetraphosphate derivatives) (see, e.g., Jemielity, J. et al., "Novel 'anti-reverse' cap analogs with superior translational properties", RNA, 9: 1108-1122 (2003)).

[0091] In some embodiments, a suitable cap is a 7-methyl guanylate ("m 7< G") linked via a triphosphate bridge to the 5'-end of the first transcribed nucleotide, resulting in m 7< G(5')ppp(5')N, where N is any nucleoside. A preferred embodiment of a m 7< G cap utilized in embodiments of the invention is m 7< G(5')ppp(5')G.

[0092] In some embodiments, the cap is a Cap0 structure. Cap0 structures lack a 2'-O-methyl residue of the ribose attached to bases 1 and 2. In some embodiments, the cap is a Cap1 structure. Cap1 structures have a 2'-O-methyl residue at base 2. In some embodiments, the cap is a Cap2 structure. Cap2 structures have a 2'-O-methyl residue attached to both bases 2 and 3.

[0093] A variety of m 7< G cap analogs are known in the art, many of which are commercially available. These include the m 7< GpppG described above, as well as the ARCA 3'-OCH 3 and 2'-OCH 3 cap analogs (Jemielity, J. et al., RNA, 9: 1108-1122 (2003)). Additional cap analogs for use in embodiments of the 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 (including biotinylated cap analogs) described in U.S. Patent Nos. 8,093,367 and 8,304,529.Tail Structure

[0094] Typically, the presence of a "tail" serves to protect the mRNA from exonuclease degradation. The poly A tail is thought to stabilize natural messengers and synthetic sense RNA. Therefore, in certain embodiments a long poly A tail can be added to an mRNA molecule thus rendering the RNA more stable. Poly A tails can be added using a variety of art-recognized techniques. For example, long poly A tails can be added to synthetic or in vitro transcribed RNA using poly A polymerase (Yokoe, et al. Nature Biotechnology. 1996; 14: 1252-1256). A transcription vector can also encode long poly A tails. In addition, poly A tails can be added by transcription directly from PCR products. Poly A may also be ligated to the 3' end of a sense RNA with RNA ligase (see, e.g., Molecular Cloning A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press: 1991 edition)).

[0095] In some embodiments, mRNAs (e.g., enzyme encoding mRNAs) include a 3' poly(A) tail structure. Typically, the length of the poly A tail can be at least about 10, 50, 100, 200, 300, 400 at least 500 nucleotides (SEQ ID NO: 12). In some embodiments, a poly-A tail on the 3' terminus of mRNA typically includes about 10 to 300 adenosine nucleotides (SEQ ID NO: 13) (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, mRNAs include a 3' poly(C) tail structure. A suitable poly-C tail on the 3' terminus of mRNA typically include about 10 to 200 cytosine nucleotides (SEQ ID NO: 14) (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 may be added to the poly-A tail or may substitute the poly-A tail.

[0096] In some embodiments, the length of the poly A or poly C tail is adjusted to control the stability of a modified sense mRNA molecule of the invention and, thus, the transcription of protein. For example, since the length of the poly A tail can influence the half-life of a sense mRNA molecule, the length of the poly A tail can be adjusted to modify the level of resistance of the mRNA to nucleases and thereby control the time course of polynucleotide expression and / or polypeptide production in a target cell.5' and 3' Untranslated Region

[0097] In some embodiments, mRNAs include a 5' and / or 3' untranslated region. In some embodiments, a 5' untranslated region includes one or more elements that affect an mRNA's stability or translation, for example, an iron responsive element. In some embodiments, a 5' untranslated region may be between about 50 and 500 nucleotides in length.

[0098] In some embodiments, a 3' untranslated region includes one or more of a polyadenylation signal, a binding site for proteins that affect an mRNA's stability of location in a cell, or one or more binding sites for miRNAs. In some embodiments, a 3' untranslated region may be between 50 and 500 nucleotides in length or longer.

[0099] Exemplary 3' and / or 5' UTR sequences can be derived from mRNA molecules which are stable (e.g., globin, actin, GAPDH, tubulin, histone, or citric acid cycle enzymes) to increase the stability of the sense mRNA molecule. For example, a 5' UTR sequence may include a partial sequence of a CMV immediate-early 1 (IE1) gene, or a fragment thereof to improve the nuclease resistance and / or improve the half-life of the polynucleotide. Also contemplated is the inclusion of a sequence encoding human growth hormone (hGH), or a fragment thereof to the 3' end or untranslated region of the polynucleotide (e.g., mRNA) to further stabilize the polynucleotide. Generally, these modifications improve the stability and / or pharmacokinetic properties (e.g., half-life) of the polynucleotide relative to their unmodified counterparts, and include, for example modifications made to improve such polynucleotides' resistance to in vivo nuclease digestion.

[0100] According to various embodiments, any size mRNA may be encapsulated by provided liposomes. In some embodiments, the provided liposomes may encapsulate mRNA of greater than about 0.5 kb, 1 kb, 1.5 kb, 2 kb, 2.5 kb, 3 kb, 3.5 kb, 4 kb, 4.5 kb, or 5 kb in length.Formation of Liposomes

[0101] The liposomes for use in provided compositions can be prepared by various techniques which are presently known in the art. For example, multilamellar vesicles (MLV) may be prepared according to conventional techniques, such as by depositing a selected lipid on the inside wall of a suitable container or vessel by dissolving the lipid in an appropriate solvent, and then evaporating the solvent to leave a thin film on the inside of the vessel or by spray drying. An aqueous phase may then added to the vessel with a vortexing motion which results in the formation of MLVs. Uni-lamellar vesicles (ULV) can then be formed by homogenization, sonication or extrusion of the multi-lamellar vesicles. In addition, unilamellar vesicles can be formed by detergent removal techniques.

[0102] In certain embodiments, provided compositions comprise a liposome wherein the mRNA is associated on both the surface of the liposome and encapsulated within the same liposome. For example, during preparation of the compositions of the present invention, cationic liposomes may associate with the mRNA through electrostatic interactions. For example, during preparation of the compositions of the present invention, cationic liposomes may associate with the mRNA through electrostatic interactions.

[0103] The compositions of the invention comprise mRNA encapsulated in a liposome. In some embodiments, the mRNA is encapsulated in one or more liposomes, which differ in their lipid composition, molar ratio of lipid components, size, charge (Zeta potential), targeting ligands and / or combinations thereof. In some embodiments, the one or more liposome may have a different composition of cationic lipids, neutral lipid, PEG-modified lipid and / or combinations thereof. In some embodiments the one or more liposomes may have a different molar ratio of cationic lipid, neutral lipid, cholesterol and PEG-modified lipid used to create the liposome.

[0104] The process of incorporation of a desired mRNA into a liposome is often referred to as "loading". Exemplary methods are described in Lasic, et al., FEBS Lett., 312: 255-258, 1992. The incorporation of a nucleic acid into liposomes is also referred to herein as "encapsulation" wherein the nucleic acid is entirely contained within the interior space of the liposome. The purpose of incorporating a mRNA into a liposome is often to protect the nucleic acid from an environment which may contain enzymes or chemicals that degrade nucleic acids and / or systems or receptors that cause the rapid excretion of the nucleic acids. Accordingly, a suitable delivery vehicle is capable of enhancing the stability of the mRNA contained therein and / or facilitate the delivery of mRNA to the target cell or tissue.Liposome Size

[0105] Suitable liposomes in accordance with the present invention may be made in various sizes. In some embodiments, provided liposomes may be made smaller than previously known mRNA encapsulating liposomes. In some embodiments, decreased size of liposomes is associated with more efficient delivery of mRNA. Selection of an appropriate liposome size may take into consideration the site of the target cell or tissue and to some extent the application for which the liposome is being made.

[0106] In some embodiments, an appropriate size of liposome is selected to facilitate systemic distribution of antibody encoded by the mRNA. In some embodiments, it may be desirable to limit transfection of the mRNA to certain cells or tissues. For example, to target hepatocytes a liposome may be sized such that its dimensions are smaller than the fenestrations of the endothelial layer lining hepatic sinusoids in the liver; in such cases the liposome could readily penetrate such endothelial fenestrations to reach the target hepatocytes.

[0107] Alternatively or additionally, a liposome may be sized such that the dimensions of the liposome are of a sufficient diameter to limit or expressly avoid distribution into certain cells or tissues. For example, a liposome may be sized such that its dimensions are larger than the fenestrations of the endothelial layer lining hepatic sinusoids to thereby limit distribution of the liposomes to hepatocytes.

[0108] In some embodiments, the size of a liposome is determined by the length of the largest diameter of the lipososme particle. In some embodiments, a suitable liposome has a size of or less than about 500nm, 450 nm, 400nm, 350 nm, 300nm, 250 nm, 200 nm, 150 nm, 125 nm, 110 nm, 100 nm, 95 nm, 90 nm, 85 nm, 80 nm, 75 nm, 70 nm, 65 nm, 60 nm, 55 nm, or 50 nm. In some embodiments, a suitable liposome has a size no greater than about 250 nm (e.g., no greater than about 225 nm, 200 nm, 175 nm, 150 nm, 125 nm, 100 nm, 75 nm, or 50 nm). In some embodiments, a suitable liposome has a size ranging from about 10 - 250 nm (e.g., ranging from about 10 - 225 nm, 10 - 200 nm, 10 - 175 nm, 10 - 150 nm, 10- 125 nm, 10 - 100 nm, 10 - 75 nm, or 10 - 50 nm). In some embodiments, a suitable liposome has a size ranging from about 100 - 250 nm (e.g., ranging from about 100 - 225 nm, 100 - 200 nm, 100 - 175 nm, 100 - 150 nm). In some embodiments, a suitable liposome has a size ranging from about 10 - 100 nm (e.g., ranging from about 10 - 90 nm, 10 - 80 nm, 10 - 70 nm, 10 - 60 nm, or 10 - 50 nm).

[0109] A variety of alternative methods known in the art are available for sizing of a population of liposomes. One such sizing method is described in U.S. Pat. No. 4,737,323. Sonicating a liposome suspension either by bath or probe sonication produces a progressive size reduction down to small ULV less than about 0.05 microns in diameter. Homogenization is another method that relies on shearing energy to fragment large liposomes into smaller ones. In a typical homogenization procedure, MLV are recirculated through a standard emulsion homogenizer until selected liposome sizes, typically between about 0.1 and 0.5 microns, are observed. The size of the liposomes may be determined by quasi-electric light scattering (QELS) as described in Bloomfield, Ann. Rev. Biophys. Bioeng., 10:421-150 (1981). Average liposome diameter may be reduced by sonication of formed liposomes. Intermittent sonication cycles may be alternated with QELS assessment to guide efficient liposome synthesis.Pharmaceutical Compositions

[0110] To facilitate expression of mRNA in vivo, delivery vehicles such as liposomes can be formulated in combination with one or more additional nucleic acids, carriers, targeting ligands or stabilizing reagents, or in pharmacological compositions where it is mixed with suitable excipients. Techniques for formulation and administration of drugs may be found in "Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, Pa., latest edition.

[0111] Provided liposomally-encapsulated or associated mRNAs, and compositions containing the same, may be administered and dosed in accordance with current medical practice, taking into account the clinical condition of the subject, the site and method of administration, the scheduling of administration, the subject's age, sex, body weight and other factors relevant to clinicians of ordinary skill in the art. The "effective amount" for the purposes herein may be determined by such relevant considerations as are known to those of ordinary skill in experimental clinical research, pharmacological, clinical and medical arts. In some embodiments, the amount administered is effective to achieve at least some stabilization, improvement or elimination of symptoms and other indicators as are selected as appropriate measures of disease progress, regression or improvement by those of skill in the art. For example, a suitable amount and dosing regimen is one that causes at least transient protein (e.g., enzyme) production.

[0112] Routes of administration include intradermal, intramuscular, subcutaneous, intrathecal, intraventricular, or intravenous administration.

[0113] In some embodiments, provided liposomes and / or compositions are formulated such that they are suitable for extended-release of the mRNA contained therein. Such extended-release compositions may be conveniently administered to a subject at extended dosing intervals. For example, in one embodiment, the compositions of the present invention are administered to a subject twice day, daily or every other day. In a preferred embodiment, the compositions of the present invention are administered to a subject twice a week, once a week, every ten days, every two weeks, every three weeks, or more preferably every four weeks, once a month, every six weeks, every eight weeks, every other month, every three months, every four months, every six months, every eight months, every nine months or annually. Also contemplated are compositions and liposomes which are formulated for depot administration (e.g., intramuscularly or subcutaneously) to either deliver or release a mRNA over extended periods of time. Preferably, the extended-release means employed are combined with modifications made to the mRNA to enhance stability.

[0114] Also contemplated herein are lyophilized pharmaceutical compositions comprising one or more of the liposomes disclosed herein and related methods for the use of such compositions as disclosed for example, in United States Provisional Application No. 61 / 494,882, filed June 8, 2011. For example, lyophilized pharmaceutical compositions according to the invention may be reconstituted prior to administration or can be reconstituted in vivo. For example, a lyophilized pharmaceutical composition can be formulated in 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 over time in vivo by the individual's bodily fluids.

[0115] Provided liposomes and compositions may be administered to any desired tissue. In some embodiments, the mRNA delivered by provided liposomes or compositions is expressed in the tissue in which the liposomes and / or compositions were administered. In some embodiments, the mRNA delivered is expressed in a tissue different from the tissue in which the liposomes and / or compositions were administered Tissues in which delivered mRNA is expressed are the liver, kidney, heart, spleen, serum, brain, skeletal muscle, lymph nodes, skin, and / or cerebrospinal fluid.

[0116] According to various embodiments, the timing of expression of delivered mRNAs can be tuned to suit a particular medical need. In some embodiments, the expression of the protein encoded by delivered mRNA is detectable 1, 2, 3, 6, 12, 18, 24, 30, 36, 42, 48, 54, 60, 66, and / or 72 hours in serum or target tissues after a single administration of provided liposomes or compositions. In some embodiments, the expression of the protein encoded by the mRNA is detectable 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, and / or 7 days in serum or target tissues after a single administration of provided liposomes or compositions. In some embodiments, the expression of the protein encoded by the mRNA is detectable 1 week, 2 weeks, 3 weeks, and / or 4 weeks in serum or target tissues after a single administration of provided liposomes or compositions. In some embodiments, the expression of the protein encoded by the mRNA is detectable after a month or longer after a single administration of provided liposomes or compositions.

[0117] The present invention can be used to deliver mRNA at various doses. In some embodiments, an mRNA is administered at a dose ranging from about 0.1 - 5.0 mg / kg body weight, for example about 0.1 - 4.5, 0.1 - 4.0, 0.1 - 3.5, 0.1 - 3.0, 0.1 - 2.5, 0.1 - 2.0, 0.1 - 1.5, 0.1 - 1.0, 0.1 - 0.5, 0.1 - 0.3, 0.3 - 5.0, 0.3 - 4.5, 0.3 - 4.0, 0.3 - 3.5, 0.3 - 3.0, 0.3 - 2.5, 0.3 - 2.0, 0.3 - 1.5, 0.3 - 1.0, 0.3 - 0.5, 0.5 - 5.0, 0.5-4.5, 0.5 - 4.0, 0.5 - 3.5, 0.5 - 3.0, 0.5 - 2.5, 0.5 - 2.0, 0.5 - 1.5, or 0.5 - 1.0 mg / kg body weight. In some embodiments, an mRNA is administered at a dose of or less than about 5.0, 4.5, 4.0, 3.5, 3.0, 2.5, 2.0, 1.5, 1.0, 0.8, 0.6, 0.5, 0.4, 0.3, 0.2, or 0.1 mg / kg body weight.EXAMPLES

[0118] The compositions of the present invention have been described with specificity in accordance with certain embodiments. The following examples serve only to illustrate the compositions of the invention and are not intended to limit the same.Example 1. Exemplary Liposome Formulations for mRNA Delivery and Expression

[0119] This example provides exemplary liposome formulations incorporating the cationic lipids described in this application, for example, cKK-E12, for effective delivery and expression of mRNA encoding therapeutic proteins in vivo.Lipid Materials

[0120] In general, the formulations described herein are based on a multi-component lipid mixture of varying ratios employing one or more cationic lipids, one or more helper lipids (e.g., non-cationic lipids and / or cholesterol-based lipids), and one or more PEGylated lipids designed to encapsulate various nucleic acid-based materials. As a non-limiting example, cKK-E12 (3,6-bis(4-(bis(2-hydroxydodecyl)amino)butyl)piperazine-2,5-dione) is used in various formulations described herein. Exemplary helper lipids include one or more of DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine), DOPE (1,2-dioleyl-sn-glycero-3-phosphoethanolamine), DOPC (1,2-dioleyl-sn-glycero-3-phosphotidylcholine) DPPE (1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine), DMPE (1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine), DOPG (,2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol)), cholesterol, etc. Exemplary PEGylated lipids include a poly(ethylene) glycol chain of up to 5 kDa in length covalently attached to a lipid with alkyl chain(s) of C 6 -C 20 length, for example, PEG-2K. As non-limiting examples, liposome formulations used in various examples described herein include cKK-E12, DOPE, cholesterol and DMG-PEG2K at various ratios. For example, in some cases, the ratio of cKK-E12:DOPE:cholesterol:DMG-PEG2K is approximately 40:30:20:10 by weight. In other cases, the ratio of cKK-E12:DOPE:cholesterol:DMG-PEG2K is approximately 40:32:25:3 by weight. Unless otherwise specified, the below Examples include a mixture in the ratio of cKK-E12:DOPE:cholesterol:DMG-PEG2K of approximately 40:30:25:5 by weight.Messenger RNA Material

[0121] The formulations described herein may be used to deliver any mRNA, in particular, therapeutic mRNA. As used herein, a therapeutic mRNA refers to an mRNA that encodes a therapeutic protein. The formulations described herein can also be used to deliver any modified or unmodified mRNA, or mRNA with naturally occurring sequences or codon-optimized.

[0122] As non-limiting examples, human Factor IX (FIX), codon-optimized Firefly Luciferase (FFL), codon-optimized human argininosuccinate synthetase (ASS1) messenger RNA, codon-optimized human Survival of Motor Neuron 1(SMN) mRNA were synthesized by in vitro transcription from a plasmid DNA template encoding the gene, which was followed by the addition of a 5' cap structure (Cap 1) (Fechter, P.; Brownlee, G.G. "Recognition of mRNA cap structures by viral and cellular proteins" J. Gen. Virology 2005, 86, 1239-1249) and a 3' poly(A) tail of, e.g., approximately 250 nucleotides in length (SEQ ID NO: 15) as determined by gel electrophoresis. Typically, 5' and 3' untranslated regions (UTR) are present in each mRNA product and are represented as X and Y, respectively. Example 5' and 3' UTR sequences are described below. The exemplary sequences of FIX, ASS1, and FFL mRNA used in the examples herein are listed below. Also shown are the 5' and 3' UTR sequences. Human Factor IX (FIX) mRNA: Codon-Optimized Human Argininosuccinate Synthetase (ASS1) mRNA: Codon-Optimized Firefly Luciferase (FFL) mRNA: Codon-Optimized Human Survival of Motor Neuron 1(SMN) mRNA: 5' and 3' UTR Sequences Y (3' UTR Sequence) = or C-terminal His 10 Codon-Optimized Human CFTR mRNA ("His 10 " disclosed as SEQ ID NO: 11): Codon-Optimized Human CFTR mRNA: Codon Optimized Human CFTR mRNA coding sequence with a Growth Hormone Leader Sequence (italisized and underlined):

[0123] Aliquots of 50 mg / mL ethanolic solutions of cKK-E12, DOPE, Chol and DMG-PEG2K were mixed in a molar ratio of 40:30:25:5 and diluted with ethanol to 3 mL final volume. Separately, an aqueous buffered solution (10 mM citrate / 150 mM NaCl, pH 4.5) of FIX, ASS1, or FFL mRNA was prepared from a 1 mg / mL stock. The lipid solution was injected rapidly into the aqueous mRNA solution and shaken to yield a final suspension in 20% ethanol. The resulting nanoparticle suspension was filtered, diafiltrated with 1x PBS (pH 7.4), concentrated and stored at 2-8°C. The final concentration of FIX mRNA was approximately 0.77 mg / mL FIX mRNA (encapsulated), Z ave = 76 nm, PDI = 0.08. The final concentration of ASS1 mRNA was approximately 0.64 mg / mL ASS1 mRNA (encapsulated), Z ave = 78 nm (Dv(50) = 46 nm; Dv(90) = 96 nm). The final concentration of FFL mRNA was approximately 1.31 mg / mL FFL mRNA (encapsulated), Z ave = 75 nm, PDI - 0.11. The final concentration of SMN mRNA was approximately 1.85 mg / mL SMN mRNA (encapsulated). Average particle size (Z ave ) = 71 nm, (particle size for 50% of particles was 44nm or less (Dv(50)) = 44 nm; and the particle size for 90% of the particles was 93n or less (Dv(90) = 93 nm)).Example 2. Administration of mRNA-loaded Liposome Nanoparticles

[0124] This example illustrates exemplary methods of administering mRNA-loaded liposome nanoparticles and methods for analyzing delivered mRNA and subsequently expressed protein in various target tissues in vivo.

[0125] All studies were performed using male CD-1 mice of approximately 6-8 weeks of age at the beginning of each experiment. Samples were introduced by a single bolus tail-vein injection of an equivalent total dose of 1.0 mg / kg (or otherwise specified) of encapsulated FIX, FFL or ASS1 mRNA. Mice were sacrificed and perfused with saline at the designated time points.

[0126] Various organ tissues such as the liver, spleen, kidney and heart of each mouse was harvested, apportioned into separate parts, and stored in either 10% neutral buffered formalin or snap-frozen and stored at -80°C for analysis.

[0127] All animals were euthanized by CO 2 asphyxiation at designated time points post dose administration (± 5%) followed by thoracotomy and terminal cardiac blood collection. Whole blood (maximal obtainable volume) was collected via cardiac puncture on euthanized animals into serum separator tubes, allowed to clot at room temperature for at least 30 minutes, centrifuged at 22°C ± 5°C at 9300 g for 10 minutes, and the serum extracted. For interim blood collections, approximately 40-50µL of whole blood was collected via facial vein puncture or tail snip. Samples collected from non-treatment animals were used as a baseline ASS1 levels for comparison to study animals.Enzyme-Linked Immunosorbent Assay (ELISA) AnalysisA. Human FIX ELISA

[0128] Quantification of FIX protein was performed following procedures reported for human FIX ELISA kit (AssayMax, Assay Pro, Catalog # EF1009-1).B. Human ASS1 ELISA

[0129] Standard ELISA procedures were followed employing mouse anti-ASS1 2D1-2E12 IgG as the capture antibody with rabbit anti-ASS1 #3285 IgG as the secondary (detection) antibody (Shire Human Genetic Therapies). Horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG was used for activation of the 3,3',5,5'-tetramethylbenzidine (TMB) substrate solution. The reaction was quenched using 2N H2SO4 after 20 minutes. Detection was monitored via absorption (450 nm) on a Molecular Device SpectraMax instrument. Untreated mouse serum and organs and human ASS1 protein were used as negative and positive controls, respectively.IVIS Bioluminometer Measurements

[0130] To visual luminescence in treated mice, several steps were followed. Anesthesia using isoflurane vaporizer at 1 - 3 % (usually @2.5%) was initially employed. Using a microsprayer, 50 µL / animal of luciferin in PBS was administered at 60 mg / mL via intratracheal / intranasal. Luciferin was allowed to distribute for 5-10 minutes. Animals were placed in an isoflurane chamber until anesthetized. Anesthetized animals were placed into the IVIS imaging chamber at dorsal recumbency and positioned into the manifold. Pictures of mice were taken. In these Examples, the acquisition settings providing highest sensitivity were: camera height at D level, F / Stop at f1, binning at high resolution, and exposure time at 5 minutes. Exposures were repeated up to 3 times (5, 10 and 15 minutes post Luciferin Injection).In Situ Hybridization (ISH) Analysis

[0131] In situ hybridization was performed using "ZZ" probe technology. Probes were generated based on codon-optimized sequence of human messenger RNA. Tissues were fixed for 24-48 hours in 10% neutral buffered formalin and embedded in paraffin. Positive detection of desired mRNA was achieved through 6 consecutive amplification steps followed by chromagenic visualization using 3,3'-diaminobenzidine (DAB). Positive signal was compared to that of untreated mouse.Example 3. Highly Effective in vivo Production of Therapeutic Proteins

[0132] This example demonstrates highly efficient and sustained production of proteins encoded by mRNA delivered by liposomes incorporating the cationic lipids described herein (e.g., cKK-E12) in serum and various organ tissues.In Vivo Human FIX Protein Production Results

[0133] The production of human FIX protein via hFIX mRNA-loaded cKK-E12-based lipid nanoparticles was tested in CD-1 mice as a single, bolus intravenous injection. Figure 1 represents the amount of human FIX protein detected via ELISA when treating mice with human FIX mRNA-loaded cKK-E12-based lipid nanoparticles as compared to a C12-200-based lipid nanoparticle encapsulating hFIX mRNA. The mice were sacrificed twenty-four hours post-injection and organs were harvested (as described above).

[0134] C12-200-based lipid nanoparticles have been shown to be an effective vehicle to deliver and express mRNA in vivo (see, PCT Application Publication NO. WO2012170930). Surprisingly, as represented in Figure 1, cKK-E12 based lipid nanoparticles are even more effective in delivering human FIX mRNA in vivo, resulting in close to 50% higher protein expression detected in the plasma of the treated mice, as compared to C12-200-based lipid nanoparticles.

[0135] Figure 2 shows the results of a dose-response experiment as represented by the amount of human FIX protein detected via ELISA when treating mice with human FIX mRNA-loaded cKK-E12-based lipid nanoparticles at various doses. The mice were bled at 6 hours and sacrificed twenty-four hours post-injection and organs were harvested (as described above).

[0136] A clear dose response was achieved when measuring liver levels of human FIX protein. The dosing range was from 0.10 - 3.0 mg / kg of encapsulated human FIX mRNA. These data demonstrate the ability of the lipid nanoparticles to efficiently deliver messenger RNA, release the payload and process this exogenous mRNA via translation to produce human FIX protein, which is then subsequently secreted into the bloodstream. Levels of human FIX protein are well above therapeutic levels (>100 ng / mL plasma) and surpass normal physiological levels (~5 ug / mL plasma) when dosing at 1.0 mg / kg or greater. Further, the plasma residence time of this human protein is sustained through at least 24 hours post administration.In Vivo Human ASS1 Protein Production Results

[0137] The production of human ASS1 protein via codon-optimized hASS1 mRNA-loaded cKK-E12-based lipid nanoparticles was tested in CD-1 mice as a single, bolus intravenous injection. Figure 3 represents the amount of human ASS1 protein detected via ELISA when treating mice with human ASS1 mRNA-loaded cKK-E12-based lipid nanoparticles at various doses. The mice were sacrificed twenty-four hours post-injection and organs were harvested (as described above).

[0138] A clear dose response was achieved when measuring liver levels of human ASS1 protein. As shown in Table 5, the dosing range was from 0.10 - 2.0 mg / kg of encapsulated human ASS1 mRNA in cKK-E12 lipid nanoparticles. These data demonstrate the ability of the lipid nanoparticles to accumulate in the liver and release the mRNA payload and the liver to process this exogenous mRNA via translation to produce human ASS1 protein. Table 5. Raw values of human ASS1 protein as measured via ELISA analysis (as depicted in Figure 1). Codon-optimized human ASS1 mRNA was delivered via cKK-E12-based lipid nanoparticles. Doses are based on encapsulated ASS1 mRNA. Values are depicted as nanogram of human ASS1 protein per milligram total protein in liver. BLD = Below Limit of Detection for ELISA.Dose Encapsulated ASS1 mRNA (mg / kg) Human ASS1 Protein (ng / mg total protein) 0.10BLD0.30BLD0.605461.013882.03371

[0139] While the sensitivity of the ELISA has limitations at lower values, western blot analysis allows for clear visualization of the human ASS1 protein at lower doses (0.3 - 3.0 mg / kg) (see Figure 4). Figure 4 depicts a comparison of human ASS1 protein levels in liver as a function of dose via western blot analysis upon a single intravenous dose of human ASS1 mRNA-encapsulated cKK-E12 lipid nanoparticles. CD1 mice were sacrificed at 24 hours post-administration and livers were harvested and analyzed as described above.

[0140] To further understand the ability of ASS1 mRNA-encapsulated lipid nanoparticles to facilitate the delivery of mRNA to selected organs (liver), a pharmacokinetic analysis was performed, monitoring human ASS1 protein levels in the liver over a one week time period. Figure 5 depicts the quantity of human ASS1 protein detected in the liver at various time points up to 7 days after administration of human ASS1-loaded lipid nanoparticles (cKK-E12). This was accomplished as a single dose (1.0 mg / kg encapsulated mRNA) given intravenously.

[0141] In this case, we observed a maximum serum level of human ASS1 protein at approximately 24-48 hours post-administration. Measurable levels of protein were still observed 1 week post-administration as determined by both ELISA and western blot (Figures 5 and 6, respectively). Figure 6 depicts a comparison of human ASS1 protein levels in liver over time via western blot analysis upon a single intravenous dose of human ASS1 mRNA-encapsulated lipid nanoparticles (1.0 mg / kg dose).

[0142] Direct detection of the active pharmaceutical ingredient (ASS1 mRNA) in the livers of the treated mice was achieved using in situ hybridization (ISH) based methods. As demonstrated in Figures 7 & 8, the exogenous human ASS1 messenger RNA could be detected in high levels at the earliest time point tested (30 minutes) and the signal remained strong for 48 hours after dosing. Further, human ASS1 mRNA was still detectable 72 hours post-administration.

[0143] In addition to ISH, detection of the resulting human ASS1 protein was achieved using immunohistochemical (IHC) means. Using a mouse monoclonal antibody (02D2-2E12) for specific binding, the presence of target human ASS1 protein in the cytoplasm of hepatocytes of treated livers can be readily observed. Figure 9 shows the immunohistochemical staining of human ASS1 protein in treated mouse livers 24 hours after administration.In vivo delivery of FFL mRNA via nebulization

[0144] To assess whether additional routes of delivery were feasible, FFL mRNA was encapsulated in cKK-E12 liposomes and those liposomes were nebulized. As shown in Figure 10, it is possible to efficiently nebulize cKK-E12 based lipid nanoparticles encapsulating mRNA. Figure 10 represents mice treated with luciferin 24 hours after exposure to nebulized FFL mRNA loaded cKK-E12 lipid nanoparticles.Example 4. CNS Delivery of hSMN-1 mRNA

[0145] This example provides an exemplary cKK-E12 liposome formulations for effective delivery and expression of mRNA in the CNS. Specifically, the example demonstrates that delivery of human survival of motor neuron-1 (hSMN-1) mRNA into various tissues of the brain and spinal cord.Messenger RNA Material

[0146] Codon-optimized human Survival of Motor Neuron-1(hSMN-1) messenger RNA (see SEQ ID NO: 4) was synthesized by in vitro transcription from a plasmid DNA template encoding the gene, which was followed by the addition of a 5' cap structure (Cap 1) (Fechter, P.; Brownlee, G.G. "Recognition of mRNA cap structures by viral and cellular proteins" J. Gen. Virology 2005, 86, 1239-1249) and a 3' poly(A) tail of approximately 250 nucleotides in length (SEQ ID NO: 15) as determined by gel electrophoresis. The 5' and 3' untranslated regions present in each mRNA product are represented as X and Y, respectively and defined as stated in Example 1.Formulation Protocol

[0147] Lipid nanoparticles (LNP) were formed via standard ethanol injection methods (Ponsa, M.; Foradada, M.; Estelrich, J. "Liposomes obtained by the ethanol injection method" Int. J. Pharm. 1993, 95, 51-56). For the various lipid components, a 50mg / ml ethanolic stock solutions was prepared and stored at -20°C. In preparation of the cKK-E12 lipid nanoparticle formulation listed in Table 6, each indicated lipid component was added to an ethanol solution to achieve a predetermined final concentration and molar ratio, and scaled to a 3 ml final volume of ethanol. Separately, an aqueous buffered solution (10mM citrate / 150 mM NaCl, pH 4.5) of hSMN-1 mRNA was prepared from a 1 mg / ml stock. The lipid solution was injected rapidly into the aqueous mRNA solution and shaken to yield a final suspension in 20% ethanol. The resulting nanoparticle suspension was filtered and dialysed against 1xPBS (pH 7.4), concentrated and stored between 2-8°C. SMN-1 mRNA concentration was determined via the Ribogreen assay (Invitrogen). Encapsulation of mRNA was calculated by performing the Ribogreen assay with and without the presence of 0.1% Triton-X 100. Particle sizes (dynamic light scattering (DLS)) and zeta potentials were determined using a Malvern Zetasizer instrument in 1x PBS and 1mM KCl solutions, respectively. Table 6. Exemplary cKK-E12 Lipid Nanoparticle formulation FormulationsComponentsMolar Ratio of lipidsFinal mRNA ConcentrationZeta Parameters1cKK-E1240:30:25:51.8 mg / mlZ ave = 72 nm;DOPECholesterolDv (50) = 49 nm;DMG-PEG-2KDv (90) = 90 nmhSMN-1 mRNA Intrathecal administration of mRNA loaded liposome nanoparticles

[0148] All in vivo studies were performed using either rats or mice of approximately 6-8 weeks of age at the beginning of each experiment. At the start of the experiment, each animal was anesthetized with isoflurane (1-3%, to effect) by inhalation. Once anesthetized, each animal was shaved at the exact injection site (L4-L5 or L5-L6). Following insertion of the needle, reflexive flick of the tail was used to indicate puncture of the dura and confirm intrathecal placement. Each animal received a single bolus intrathecal injection of the test formulation listed in Table 6. All animals were sacrificed 24 hours post injection and perfused with saline.Isolation of organ tissues for analysis

[0149] All animals had the whole brain and spinal cord harvested. The brain was cut longitudinally and placed in one histology cassette per animal. The whole spinal cord was stored ambient in a 15 ml tube containing 10% neutral buffered formalin (NBF) for at least 24 hours and no more than 72 hours before transfer into 70% histology grade alcohol solution. Each spinal cord sample was cut into cervical, thoracic and lumbar sections. Each spinal cord section cut in half and both halves were placed in individual cassettes per section (cervical, thoracic and lumbar) for processing. All three cassettes were embedded into one paraffin block per animal. When applicable, portions of brain and spinal cord were snap frozen and stored at -80°C.hSMN-1 Western Blot Analysis

[0150] Standard western blot procedures were followed employing various antibodies that recognizes hSMN protein, such as: (A) anti-SMN 4F11 antibody at 1:1,000 dilution; (B) Pierce PA5-27309 a-SMN antibody at 1:1,000 dilution; and (C) LSBio C138149 a-SMN antibody at 1:1,000 dilution. For each experiment one microgram of hSMN mRNA was transfected into ~1x10 6< BHK-21 cells using Lipofectamine 2000. Cells were treated with OptiMem and harvested 16-18 hours post-transfection. Cell lysates were harvested, processed and loaded on to an 8-16% Tris Glycine gel. The gel was transferred using a PVDF membrane and treated with the respective primary antibody. Goat anti-mouse HRP antibody was used as the secondary antibody at 1:10,000 dilution for 45 minutes at room temperature followed by washing and development. The data demonstrates that each antibody tested showed a strong signal for hSMN-1 and was specific for human SMN, as indicated by an absence in a cross-reactive signal for untreated BHK cells (Figure 11).In Situ Hybridzation (ISH) Analysis

[0151] Tissue from each representative sample, was assayed for hSMN-1 mRNA using a manual in situ hybridization analysis, performed using RNAscope ®< (Advanced Cell Diagnostic) "ZZ" probe technology. Probes were generated based on the codon-optimized sequence of human SMN messenger RNA (SEQ ID NO: 4). Briefly, the RNAscope ®< assay is an in situ hybridication assay designed to visualize single RNA molecules per cell in formalin-fixed, paraffin-embedded (FFPE) tissue mounted on slides. Each embedded tissue sample was pretreated according to the manufacturers protocol and incubated with a target specific hSMN-1 RNA probe. The hSMN-1 probe was shown to be specific for human SMN-1 and had little to no cross reactivity with mouse or rat SMN-1. Once bound, the hSMN-1 probe is hybridized to a cascade of signal amplification molecules, through a series of 6 consecutive rounds of amplification. The sample was then treated with an HRP-labeled probe specific to the signal amplification cassette and assayed by chromatic visualization using 3,3'-diaminobenzidine (DAB). A probe specific for Ubiquitin C was used as the positive control. Positive SMN signal was compared to that of untreated and vehicle control treated rat or mouse tissue. Stained samples were visualized under a standard bright field microscope.Immunohistochemical Analysis

[0152] Human SMN-1 mRNA-loaded lipid nanoparticles were administered to rats via intrathecal injection, and tissue samples collected and processed 24 hours post administration in accordance with the methods described above. Rat spinal tissue samples were then assayed for hSMN-1 protein expression. Briefly, fixed tissue embedded in paraffin was processed and placed on slides. The slides were dewaxed, rehydrated and antigen retrieval was performed using a pressure cooker with citrate buffer. Several blocking buffers were employed followed by primary antibody incubation overnight at 4°C, using the 4F11 antibody at a 1:2500 dilution. The resulting slides were washed and incubated at ambient temperature with the secondary antibody polymer followed by washing and subsequent chromagen development. The data demonstrates that in as little as 24 hours post intrathecal adminiatration of hSMN-1 mRNA, staining is observed for human SMN-1 protein when compared to no-treatment control (Figure 13). This supports the previous findings which demonstrate delivery of hSMN-1 mRNA to the spinal tissue. Furthermore, the data demonstrates that once delivered to the cell hSMN-1 mRNA is effectively expressed to generate hSMN-1 protein.Results

[0153] The data presented in this example demonstrates that intrathecal administration of hSMN-1 mRNA loaded liposomes (e.g., lipid or polymer-based nanoparticles) results in successful intracellular delivery of mRNA in neurons in the brain and spinal cord, including those difficult to treat cells, such as anterior horn cells and dorsal root ganglia.

[0154] The results have shown that mRNA encapsulated within a lipid nanoparticle (e.g., lipid nanoparticle comprising cKK-E12) can be effectively delivered to various tissues of the CNS following intrathecal administrations. Using the exemplary formulation disclosed in Table 6, mRNA was effectively delivered and internalized within various neurons of the spinal cord (Figures 12A-12C), as verified by in situ hybridization assay. Surprisingly, intracellular mRNA delivery was demonstrated in the difficult to reach neuronal cells of the anterior horn, located deep within the tissues of the spinal column (Figures 12A-12C). Little to no background was observed with mouse or rat SMN-1, indicating specificity for the human SMN-1 probe. Positive SMN signal was compared to that of untreated and vehicle control treated rat or mouse tissue. Stained samples were visualized under a standard bright field microscope.

[0155] These data demonstrates that the lipid or polymer nanoparticle based mRNA delivery approach described herein was able to successfully permeate the complex and dense cell membrane of the spinal cord neurons and deliver the mRNA payload for the production of encoded proteins inside neurons. It was particularly surprising that the mRNA delivery approach described herein was equally successful in permeating difficult to treat neurons such as anterior horn cell and dorsal root ganglia. Thus, the data presented herein demonstrates that lipid or polymer nanoparticles, such as those comprising cKK-E12, may serve as a promising option for delivering mRNA to neuronal cells in the treatment of a CNS disease. In particular, the present example demonstrates that hSMN mRNA loaded nanoparticles can be effectively delivered to neurons, including those difficult to treat motor neurons in the spinal cord, and can be used for the production of SMN protein and treatment of spinal muscular atrophy.Example 5. In Vivo CO-CFTR-C-His 10 mRNA Delivery to CFTR Knockout Mice

[0156] Messenger RNA Synthesis. For the experiment, C-terminal His 10 tagged codon-optimized human cystic fibrosis transmembrane conductance regulator (CO-CFTR-C-His 10 ) (SEQ ID NO:8) ("His 10 " disclosed as SEQ ID NO: 11) and non-tagged codon-optimized human CFTR (CO-CFTR) (SEQ ID NO:9) mRNA were synthesized by in vitro transcription from a plasmid DNA template using standard method. mRNAs used in this example and Example 6 were produced by IVT in which 25% of U residues were 2-thio-uridine and 25% of C residues were 5-methylcytidine.

[0157] Analysis of human CFTR protein produced via intratracheal administered mRNA-loaded nanoparticles. For the study, CFTR knockout mice were used. CFTR mRNA formulation or vehicle control was introduced using a PARI Boy jet nebulizer. Mice were sacrificed and perfused with saline, after a predetermined period of time, to allow for protein expression from the mRNA.

[0158] PEI Formulation. PEI formulation has been used to deliver CFTR mRNA to the lung and was used as a control in this experiment. Polymeric nanoparticle formulations with 25 kDa branched PEI were prepared as follows. The required amount of mRNA was diluted just before application in water for injection (Braun, Melsungen) to a total volume of 4 ml and added quickly to 4 ml of an aqueous solution of branched PEI 25 kDa using a pipette at an N / P ratio of 10. The solution was mixed by pipetting up and down ten times and nebulized as two separate 4.0 ml fractions one after another to the mouse lungs using the indicated nebulizer.

[0159] cKK-E12 Formulation. For the lipid-based nanoparticle experiment, a lipid formulation was created using CO-CFTR-C-His 10 RNA in a formulation of cKK-E12:DOPE:Chol:PEGDMG2K (relative amounts 50:25:20:5 (mg:mg:mg:mg)). The solution was nebulized to the mouse lungs using the indicated nebulizer.

[0160] Nebulization (Aerosol) Administration of Human CO-CFTR-C-His 10 mRNA. CFTR test materials were administered by a single aerosol inhalation via PARI Boy jet nebulizer (nominal dose volume of up to 8 mL / group). The test material was delivered to a box containing the whole group of animals (n=4) and connected to oxygen flow and scavenger system.

[0161] Administration of Human CO-CFTR-C-His 10 mRNA. CFTR mRNA was prepared in the manner described above. Four CFTR knockout mice were placed in an aerosol chamber box and exposed to 2 mg total codon optimized unmodified human CFTR mRNA (comprising the coding sequence of SEQ ID NO: 8) via nebulization (Pari Boy jet nebulizer) over the course of approximately one hour. Mice were sacrificed 24 hours post-exposure.

[0162] Euthanasia. Animals were euthanized by CO 2 asphyxiation at representative times post-dose administration (± 5%) followed by thoracotomy and exsanguinations. Whole blood (maximal obtainable volume) was collected via cardiac puncture and discarded.

[0163] Perfusion. Following exsanguination, all animals underwent cardiac perfusion with saline. In brief, whole body intracardiac perfusion was performed by inserting 23 / 21 gauge needle attached to 10 mL syringe containing saline set into the lumen of the left ventricle for perfusion. The right atrium was incised to provide a drainage outlet for perfusate. Gentle and steady pressure was applied to the plunger to perfuse the animal after the needle had been positioned in the heart. Adequate flow of the flushing solution was ensured when the exiting perfusate flows clear (free of visible blood) indicating that the flushing solution has saturated the body and the procedure was complete.

[0164] Tissue Collection. Following perfusion, all animals had their lungs (right and left) harvested. Both (right and left) lungs were snap frozen in liquid nitrogen and stored separately at nominally -70°C.

[0165] Expression of human CFTR in CO-CFTR-C-His 10 in CFTR knockout mice. CFTR expression was detected by Western blot analysis of tissue lysate collected from CFTR mRNA-treated mouse lungs. Mature "C" band was detected in left and right lungs of all treated mice, for both the cKK-E12-based and PEI-based formulations (Figure 14). Expression of the mature "C" band was verified by comparison with lysate collected from HEK 293T human CO-CFTR-C-His 10 positive cells. In contrast, no detectable signal was observed in lysate collected from wild type untreated control mice (Figure 14). Taken together, these data suggest that cKK-E12 may be used to deliver mRNA (e.g., CFTR mRNA) to the lung via, e.g., inhalation, as effectively as or even better than PEI based formulations.Example 6: In Vivo Expression in the Lung

[0166] This example further desmonstrates successful in vivo expression in the lung following aerosol delivery of mRNA-loaded ckk-E12 based nanoparticles. All studies were performed using pigs of the German Landrace, obtained from Technical University Munich, Weihenstephan, Germany. The pigs had a body weight ranging from 35-90 kg. FFL / CO-CFTR-C-His10 mRNA formulation or vehicle control was introduced using a Pari jet nebulizer. Pigs were sacrificed and perfused with saline, after a predetermined period of time, to allow for protein expression from the mRNA.

[0167] Messenger RNA Synthesis. In the example, codon optimized fire fly luciferase (CO-FFL) mRNA was synthesized by in vitro transcription from plasmid DNA templates.

[0168] cKK-E12 Formulation. For the lipid-based nanoparticle experiment, a lipid formulation was created using 1 mg FFL + 9 mg of CO-CFTR-C-His 10 mRNA encapsulated in a formulation of cKK-E12:DOPE:Chol:PEGDMG2K (relative amounts 40:30:25:5 (mol ratio). The solution was nebulized to the Pig lungs using the indicated nebulizer.

[0169] Aerosol Application. The aerosol (Saline or CO-FFL cKK-E12 formulation) was nebulized and inhaled into the anaesthetized pig. Sedation in pigs was initiated by premedication with azaperone 2 mg / kg body weight, ketamine 15 mg / kg body weight, atropine 0.1 mg / kg body weight and followed by insertion of an intravenous line to the lateral auricular vein. Pigs were anesthetized by intravenous injection of propofol 3-5 mg / kg body weight as required. Anesthesia was maintained by isoflurane (2-3%) with 1% propofol bolus injection at 4 to 8 mg / kg body weight to enhance anesthesia as required. Duration of the anesthesia was approximately 1-3 hrs. Pigs were killed with bolus injection of pentobarbital (100 mg / kg body weight) and potassium chloride via the lateral ear vein. Lungs were excised and tissue specimens were collected from various lung regions followed by incubation in cell culture medium overnight. The stored samples were subjected to bioluminescence detection.

[0170] Bioluminescence Analysis. For measurement of luciferase activity, tissue specimens were either homogenized and analyzed in a tube luminometer or incubated in a medium bath comprising D-Luciferin substrate and subjected to ex vivo luciferase BLI. The data illustrate that a strong bioluminescence signal was observed for each of the (A) CO-FFL / CO-CFTR-C-His 10 mRNA treated pigs, when compared to (B) control lung tissue samples from control pigs (Saline vehicle control) (Figure 15 A&B).

[0171] These data illustrate that FFL / CFTR mRNA were successfully delivered to and expressed in the lung by aerosol administration of a cKK-E12 based lipid formulation.

Claims

1. A composition comprising an mRNA encoding a therapeutic protein encapsulated within a liposome for use in a method of treating a disease or disorder comprising administering to a subject in need of treatment the composition; wherein the composition is injected intradermally, intramuscularly, or subcutaneously or administered intrathecally, intraventricularly, or intravenously and expression of the protein encoded by the mRNA is detectable in the liver, kidney, heart, spleen, serum, brain, skeletal muscle, lymph nodes, skin, and / or cerebrospinal fluid, and the liposome comprises a cationic lipid of formula I-g: or a pharmaceutically acceptable salt thereof, wherein each of RL is optionally substituted C8-10 alkyl and the liposome further comprises a non-cationic lipid, a cholesterol-based lipid and a PEG-modified lipid, wherein the molar ratio of cationic lipid to non-cationic lipid to cholesterol-based lipid to PEG-modified lipid is 30-50:25-35:20-30:1-15.

2. The composition for use according to claim 1, wherein each of RL is C8 alkyl.

3. The composition for use according to claim 1, wherein each of RL is C9 alkyl.

4. The composition for use according to claim 1, wherein each of RL is C10 alkyl.

5. The composition for use according to any one of claims 1 to 4, wherein RL is a substituted straight-chain alkyl group or RL is an unsubstituted straight-chain alkyl group.

6. The composition for use according to any one of claims 1 to 4, RL is a substituted branched alkyl group or RL is an unsubstituted branched alkyl group.

7. The composition for use according to claim 1, wherein the cationic lipid is cKK-E12:

8. The composition for use according to any one of the preceding claims, wherein: (i) the non-cationic lipid is selected from DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine), DOPE (1,2-dioleyl-sn-glycero-3-phosphoethanolamine), DOPC (1,2-dioleyl-sn-glycero-3-phosphotidylcholine) DPPE (1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine), DMPE (1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine), DOPG (,2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol)); and / or (ii) the cholesterol-based lipid is cholesterol or PEGylated cholesterol; and / or (iii) the PEG-modified lipid comprises a poly(ethylene) glycol chain of up to 5 kDa in length covalently attached to a lipid with alkyl chain(s) of C6-C20 length; and / or (iv) the liposome comprises cKK-E12, DOPE, cholesterol and DMG-PEG2K.

9. The composition for use according to any one of the preceding claims, wherein the cationic lipid constitutes 40 % of the liposome by molar ratio.

10. The composition for use according to any one of the preceding claims, wherein the liposome has a size ranging from 10-100 nm.

11. The composition for use according to any one of the preceding claims, wherein the expression of the protein encoded by the mRNA is detectable: (i) 3 hours after the administration; and / or (ii) 6 hours after the administration; and / or (iii) 12 hours after the administration; and / or (iv) 24 hours after the administration; and / or (v) 1 week after the administration.

12. The composition for use according to any one of the preceding claims, wherein the mRNA has a length of or greater than 0.5kb, 1 kb, 1.5 kb, 2 kb, 2.5 kb, 3 kb, 3.5 kb, 4 kb, 4.5 kb, or 5 kb.

13. The composition for use according to any one of the preceding claims, wherein the protein encoded by the mRNA is: (i) a cytosolic protein or a secreted protein; and / or (ii) an enzyme; and / or (iii) Argininosuccinate Synthetase (ASS1), Factor IX, spinal motor neuron 1, or phenylalanine hydroxylase.

14. The composition for use according to any one of the preceding claims, wherein the mRNA is administered at a dose: (i) ranging from 0.1 - 2.0 mg / kg body weight; or (ii) of or less than 1.0 mg / kg body weight; e.g. of or less than 0.5 mg / kg body weight, or of or less than 0.3 mg / kg body weight.

15. The composition for use according to any one of the preceding claims, wherein the mRNA: (i) comprises one or more modified nucleotides, optionally wherein the one or more modified nucleotides comprise pseudouridine, N-1-methyl-pseudouridine, 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl adenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, and / or 2-thiocytidine; or (ii) is unmodified.