Lipid nanoparticle compositions and methods for mRNA delivery

Abstract

To provide a composition and a method for modulating the production of proteins in target cells.SOLUTION: The composition and method disclosed herein can ameliorate diseases associated with protein or enzyme deficiency. The present invention provides, for example, a composition comprising (a) at least one mRNA molecule at least partially encoding a functional secreted polypeptide and (b) a delivery vehicle including lipid nanoparticles. The present invention also relates to a method for treating a subject lacking a functional polypeptide, the method comprising: administering a composition comprising (a) at least one mRNA molecule at least partially encoding the functional secreted polypeptide and (b) a delivery vehicle including lipid nanoparticles; and allowing the mRNA to be expressed in target cells after the administration of the composition to produce the functional secreted polypeptide.SELECTED DRAWING: None

Description

[Background technology]

[0001] Novel approaches and therapies remain needed for the treatment of protein and enzyme deficiencies. For example, lysosomal storage diseases are a group of about 50 rare inherited metabolic diseases that usually result from defective lysosomal function due to a deficiency of an enzyme required for metabolism. Fabry disease is a lysosomal storage disease caused by a deficiency of the enzyme α-galactosidase (GLA), which causes a glycolipid known as globotriaosylceramide to accumulate in blood vessels and other tissues, leading to a variety of painful symptoms. In certain diseases, such as Fabry disease, there is a need for replacement of proteins or enzymes that are normally secreted by cells and enter the bloodstream. Treatments such as gene therapy that increase the levels or production of affected proteins or enzymes may provide treatment or even a cure for such disorders. However, there are several limitations to the use of conventional gene therapy for this purpose.

[0002] Conventional gene therapy uses DNA for the insertion of desired genetic information into host cells. DNA introduced into cells is usually integrated to some extent into the genome of one or more transfected cells, allowing for the long-term action of the introduced genetic material in the host. Although there may be many advantages to such a long-lasting action, the integration of foreign DNA into the host genome may also have many adverse effects. For example, the introduced DNA may be inserted into an intact gene, resulting in a mutation that interferes with or even completely eliminates the function of the endogenous gene. Thus, gene therapy with DNA may result in the impairment of essential genetic functions in the treated host, such as, for example, the elimination or harmful reduction of the production of an essential enzyme, or the interference with a gene essential for the regulation of cell growth, resulting in uncontrolled proliferation or cancerous cell growth. In addition, during conventional DNA-based gene therapy, it is necessary to include a strong promoter sequence for the effective expression of the desired gene product, which again may lead to undesirable changes in the regulation of normal gene expression in the cell. It is also possible that DNA-based genetic material may result in the induction of undesirable anti-DNA antibodies, which may then trigger a possibly fatal immune response. Gene therapy approaches using viral vectors may also result in adverse immune responses. In some circumstances, viral vectors may even integrate into the host genome. In addition, the production of clinical grade viral vectors is expensive and time-consuming. Targeted delivery of transgenetic material using viral vectors may also be difficult to control. Thus, although DNA-based gene therapy has been evaluated for the delivery of secreted proteins using viral vectors (US Pat. No. 6,066,626, US 2004 / 0110709), these approaches may be limited by these various reasons.

[0003] Another obvious obstacle in these previous approaches to the delivery of nucleic acid encoding secretory protein is the level of protein that is ultimately produced. It is difficult to obtain a significant level of desired protein in blood, and the amount is not maintained for a long time. For example, the amount of protein produced by nucleic acid delivery does not reach normal physiological level. For example, see US Patent Application Publication No. 2004 / 0110709.

[0004] In contrast to DNA, the use of RNA as a gene therapy agent is substantially safer because (1) there is no risk of RNA being stably integrated into the genome of the transfected cell, thus eliminating the concern that the introduced genetic material will disrupt the normal function of essential genes or cause mutations that result in harmful or oncogenic effects, (2) no exogenous promoter sequence is required for effective translation of the encoded protein, again avoiding possible harmful side effects, (3) in contrast to plasmid DNA (pDNA), messenger RNA (mRNA) lacks immunogenic CpG motifs so that anti-RNA antibodies are not generated, and (4) due to the relatively short half-life of RNA, any adverse effects resulting from mRNA based gene therapy will only be of limited duration. In addition, mRNA does not need to enter the nucleus to perform its function, whereas DNA must overcome this major barrier.

[0005] One of the reasons why mRNA-based gene therapy has not been used much in the past is that the stability of mRNA is much lower than that of DNA, especially when it reaches the cytoplasm of a cell and is exposed to degradative enzymes. The presence of a hydroxyl group on the second carbon of the sugar moiety in mRNA causes steric hindrance that prevents the mRNA from forming a more stable DNA double helix structure, thus increasing the tendency of the mRNA to hydrolyze. As a result, until recently, it was widely believed that mRNA was too unstable to withstand transfection protocols. Advances in RNA stabilizing modifications have sparked increased interest in using mRNA instead of plasmid DNA in gene therapy. Certain delivery vehicles, such as cationic lipids or polymeric delivery vehicles, can also help protect the transfected mRNA from endogenous RNases. Furthermore, despite the increased stability of modified mRNA, the delivery of mRNA, especially full-length protein-encoding mRNA, to cells in vivo in a manner that allows for therapeutic levels of protein production remains a challenge. Although delivery of mRNA encoding secreted proteins has been contemplated (U.S. Patent Application Publication No. 2009 / 0286852), the levels of full-length secreted protein actually produced via in vivo mRNA delivery are unknown, and there is no reason to expect that they will exceed those observed with DNA-based gene therapy. To date, significant progress has been made using mRNA gene therapy only in applications where low-level translation was not a limiting factor, such as immunization with mRNA-encoded antigens. Clinical trials involving vaccination against tumor antigens by intradermal injection of naked or protamine-complexed mRNA have demonstrated feasibility, lack of toxicity, and promising results. X.Su et al., Mol.Pharmaceutics 8:774-787(2011). Unfortunately, low-level translation has greatly limited the use of mRNA-based gene therapy in other applications that require sustained expression of higher levels of mRNA-encoded proteins to have biological or therapeutic effects. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] U.S. Patent No. 6,066,626 [Patent Document 2] US Patent Application Publication No. 2004 / 0110709 [Patent Document 3] US Patent Application Publication No. 2009 / 0286852 Summary of the Invention [Means for solving the problem]

[0007] The present invention provides a method for the delivery of mRNA gene therapy drugs that leads to the production of therapeutically effective levels of secreted proteins via the "depot effect". In an embodiment of the present invention, the mRNA encoding the secreted protein is loaded into lipid nanoparticles and delivered to target cells in vivo. The target cells then act as a depot source for the production of soluble secreted proteins into the circulation at therapeutic levels. In some embodiments, the level of secreted protein produced is above normal physiological levels.

[0008] The present invention provides compositions and methods for the intracellular delivery of mRNA in a liposomal transfer vehicle to one or more target cells for the production of therapeutic levels of a functional secreted protein.

[0009] The compositions and methods of the present invention are useful in the management and treatment of many diseases, particularly diseases caused by protein and / or enzyme deficiency, where protein or enzyme is normally secreted.Individuals suffering from such diseases may have underlying genetic defects that lead to defective expression of protein or enzyme, including, for example, non-synthesis of secretory protein, reduced synthesis of secretory protein, or synthesis of secretory protein that lacks or has reduced biological activity.Specifically, the methods and compositions of the present invention are useful for treating lysosomal storage disorders and / or urea cycle metabolic disorders that result from one or more defects in the biosynthesis of secretory enzymes involved in the urea cycle.

[0010] The composition of the present invention comprises an mRNA, a transfer vehicle, and optionally an agent that facilitates contact with and subsequent transfection of target cells. The mRNA may code for a clinically useful secretory protein. For example, the mRNA may code for a functional secretory urea cycle enzyme or a secretory enzyme involved in lysosomal storage disorders. The mRNA may code for, for example, erythropoietin (e.g., human EPO) or α-galactosidase (e.g., human α-galactosidase (human GLA)).

[0011] In some embodiments, the mRNA may contain one or more modifications that confer stability to the mRNA (e.g., compared to a wild-type or naturally occurring version of the mRNA) and may also contain one or more modifications relative to the wild-type that correct defects involved in the associated aberrant expression of the protein. For example, the nucleic acid of the invention may contain modifications of one or both of the 5' or 3' untranslated regions. Such modifications may include, but are not limited to, a partial sequence of the cytomegalovirus (CMV) immediate early 1 (IE1) gene, a polyA tail, a Cap1 structure, or the inclusion of a sequence encoding human growth hormone (hGH). In some embodiments, the mRNA is modified to reduce mRNA immunogenicity.

[0012] Also contemplated is the method of treating a subject, comprising administering the composition of the present invention.For example, a method of treating or preventing the condition in which the production of specific secretory protein and / or the utilization of specific secretory protein is inappropriate or impaired is provided.In one embodiment, the method provided herein can be used to treat a subject who is deficient in one or more urea cycle enzymes, or who is deficient in one or more enzymes in lysosomal storage disorder.

[0013] In a preferred embodiment, the mRNA in the composition of the present invention is formulated in a liposome transfer vehicle to facilitate delivery to target cells.The transfer vehicle contemplated may comprise one or more cationic lipids, non-cationic lipids, and / or PEG-modified lipids.For example, the transfer vehicle may comprise at least one of the following cationic lipids: C12-200, DLin-KC2-DMA, DODAP, HGT4003, ICE, HGT5000, or HGT5001.In an embodiment, the transfer vehicle comprises cholesterol (chol) and / or PEG-modified lipid.In some embodiments, the transfer vehicle comprises DMG-PEG2K. In certain embodiments, the transfer vehicle comprises one of the following lipid formulations: C12-200, DOPE, chol, DMG-PEG2K; DODAP, DOPE, cholesterol, DMG-PEG2K; HGT5000, DOPE, chol, DMG-PEG2K, HGT5001, DOPE, chol, DMG-PEG2K.

[0014] The present invention also provides compositions and methods useful for facilitating the transfection of target cells that can exhibit a "depot effect" and the delivery of one or more mRNA molecules to the cells. For example, the compositions and methods of the present invention contemplate the use of targeting ligands that can increase the affinity of the composition to one or more target cells. In one embodiment, the targeting ligand is apolipoprotein B or apolipoprotein E, and the corresponding target cells express low-density lipoprotein receptors, thus facilitating recognition of the targeting ligand. The methods and compositions of the present invention can be used to preferentially target a vast number of target cells. For example, contemplated target cells include, but are not limited to, hepatocytes, epithelial cells, hematopoietic cells, epithelial cells, endothelial cells, lung cells, bone cells, stem cells, mesenchymal cells, neural cells, cardiac cells, adipocytes, vascular smooth muscle cells, cardiac muscle cells, skeletal muscle cells, beta cells, pituitary cells, synovial lining cells, ovarian cells, testicular cells, fibroblasts, B cells, T cells, reticulocytes, leukocytes, granulocytes, and tumor cells.

[0015] In embodiments, the secreted protein is produced by the target cell for a sustained period of time. For example, the secreted protein may be produced more than 1 hour, more than 4 hours, more than 6 hours, more than 12 hours, more than 24 hours, more than 48 hours, or more than 72 hours after administration. In some embodiments, the polypeptide is expressed at a peak level about 6 hours after administration. In some embodiments, the expression of the polypeptide is maintained at at least a therapeutic level. In some embodiments, the polypeptide is expressed at at least a therapeutic level more than 1 hour, more than 4 hours, more than 6 hours, more than 12 hours, more than 24 hours, more than 48 hours, or more than 72 hours after administration. In some embodiments, the polypeptide is detectable at a therapeutic level in the serum or tissues (e.g., liver or lung) of the patient. In some embodiments, the detectable level of the polypeptide is derived from continuous expression from the mRNA composition more than 1 hour, more than 4 hours, more than 6 hours, more than 12 hours, more than 24 hours, more than 48 hours, or more than 72 hours after administration.

[0016] In certain embodiments, the secreted protein is produced at levels that are greater than normal physiological levels. The level of the secreted protein can be increased as compared to a control.

[0017] In some embodiments, the control is the baseline physiological level of the polypeptide in a normal individual or a population of normal individuals. In other embodiments, the control is the baseline physiological level of the polypeptide in an individual lacking the relevant protein or polypeptide or a population of individuals lacking the relevant protein or polypeptide. In some embodiments, the control can be the normal level of the relevant protein or polypeptide in the individual to whom the composition is administered. In other embodiments, the control is the expression level of the polypeptide at one or more equivalent time points during other therapeutic interventions, such as direct injection of the corresponding polypeptide.

[0018] In some embodiments, the polypeptide is expressed by target cells at a level at least 1.5 times, at least 2 times, at least 5 times, at least 10 times, at least 20 times, 30 times, at least 100 times, at least 500 times, at least 5000 times, at least 50,000 times, or at least 100,000 times higher than the control. In some embodiments, the fold increase in expression above the control is maintained for more than 1 hour, more than 4 hours, more than 6 hours, more than 12 hours, more than 24 hours, more than 48 hours, or more than 72 hours after administration. For example, in one embodiment, the level of secreted protein is detected in serum at least 1.5 times, at least 2 times, at least 5 times, at least 10 times, at least 20 times, 30 times, at least 100 times, at least 500 times, at least 5000 times, at least 50,000 times, or at least 100,000 times higher than the control for at least 48 hours or 2 days. In certain embodiments, the level of secreted protein is detectable 3 days, 4 days, 5 days, or 1 week or more after administration. Increased levels of secreted protein may be observed in serum and / or tissues (e.g., liver, lung).

[0019] In some embodiments, the method results in the sustained circulating half-life of desired secretory protein.For example, secretory protein can be detected for hours or days longer than the half-life observed through subcutaneous injection of secretory protein.In embodiments, the half-life of secretory protein is sustained for more than 1 day, more than 2 days, more than 3 days, more than 4 days, more than 5 days, or more than 1 week.

[0020] In some embodiments, administration comprises a single dose or multiple doses, hi certain embodiments, the dose is administered intravenously or by pulmonary delivery.

[0021] The polypeptide can be, for example, one or more of erythropoietin, α-galactosidase, LDL receptor, Factor VIII, Factor IX, α-L-iduronidase (for MPS I), iduronate sulfatase (for MPS II), heparin-N-sulfatase (for MPS IIIA), α-N-acetylglucosaminidase (for MPS IIIB), galactose 6-sulfatase (for MPS IVA), lysosomal acid lipase, arylsulfatase-A.

[0022] An embodiment relates to compositions and methods that provide at least a portion of a functional protein to a cell or subject mRNA in an amount that is substantially less than the amount of the corresponding functional protein produced from the mRNA. In other words, in an embodiment, the mRNA delivered to a cell can produce substantially more protein than the amount of mRNA delivered to the cell. For example, after a given time, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, or 24 hours after administration of the mRNA to a cell or subject, the amount of the corresponding protein produced by the mRNA can be at least 1.5, 2, 3, 5, 10, 15, 20, 25, 50, 100, 150, 200, 250, 300, 400, 500 times, or more, more than the amount of mRNA actually administered to the cell or subject. This can be measured on a mass-to-mass basis, a mole-to-mole basis, and / or a molecule-to-molecule basis. Proteins are measured in a variety of ways. For example, in the case of a cell, the measured protein can be measured as an intracellular protein, an extracellular protein, or a combination of the two. In the case of a subject, the measured protein can be a protein measured in serum; a specific tissue or tissues, such as liver, kidney, heart, or brain; a specific cell type, such as one of the various cell types of the liver or brain; or any combination of serum, tissue, and / or cell type. Furthermore, a baseline amount of endogenous protein can be measured in a cell or subject before administration of mRNA, and then subtracted from the protein measured after administration of mRNA to obtain the corresponding amount of protein produced from the mRNA. In this way, the mRNA can provide a reservoir or depot source of a large amount of therapeutic substance to the cell or subject, for example, compared to the amount of mRNA delivered to the cell or subject. The depot source can act as a continuous source for polypeptide expression from the mRNA over a sustained period of time.

[0023] In certain embodiments, for example, the following are provided: (Item 1) A composition comprising: (a) at least one mRNA molecule, at least a portion of which encodes a functional secreted polypeptide; and (b) a transfer vehicle comprising a lipid nanoparticle. (Item 2) 2. The composition of claim 1, wherein the mRNA encodes an enzyme that is abnormally deficient in an individual having a lysosomal storage disorder. (Item 3) The composition of claim 1, wherein the mRNA encodes a functional erythropoietin or a functional alpha-galactosidase polypeptide. (Item 4) 2. The composition of claim 1, wherein the RNA molecule comprises at least one modification that confers stability to the RNA molecule. (Item 5) The composition of claim 1, wherein the RNA molecule comprises a modification in the 5' untranslated region of the RNA molecule. (Item 6) 6. The composition of claim 5, wherein the modification comprises the inclusion of a Cap1 structure. (Item 7) The composition of claim 1, wherein the RNA molecule comprises a modification in the 3' untranslated region of the RNA molecule. (Item 8) 8. The composition of claim 7, wherein the modification comprises the inclusion of a polyA tail. (Item 9) 2. The composition of claim 1, further comprising an agent for promoting the transfer of the RNA molecule into an intracellular compartment of a target cell. (Item 10) 2. The composition of claim 1, wherein the lipid nanoparticles comprise one or more cationic lipids. (Item 11) 2. The composition of claim 1, wherein the lipid nanoparticles comprise one or more non-cationic lipids. (Item 12) The composition of claim 1, wherein the lipid nanoparticles comprise one or more PEG-modified lipids. (Item 13) 2. The composition of claim 1, wherein the lipid nanoparticles comprise C12-200. (Item 14) 2. The composition of claim 1, wherein the lipid nanoparticles comprise DLinKC2DMA, CHOL, DOPE, and DMG-PEG-2000. (Item 15) The composition of claim 1, wherein the lipid nanoparticles comprise C12-200, DOPE, CHOL, and DMGPEG2K. (Item 16) 2. The composition of claim 1, wherein the lipid nanoparticles comprise a cleavable lipid. (Item 17) 2. The composition of claim 1, wherein the composition is lyophilized. (Item 18) 2. The composition of claim 1, wherein the composition is a lyophilized reconstituted composition. (Item 19) 11. The composition according to item 10, wherein the target cell is selected from the group consisting of hepatocytes, epithelial cells, hematopoietic cells, epithelial cells, endothelial cells, lung cells, bone cells, stem cells, mesenchymal cells, neuronal cells, cardiac cells, adipocytes, vascular smooth muscle cells, cardiac myocytes, skeletal muscle cells, beta cells, pituitary cells, synovial lining cells, ovarian cells, testicular cells, fibroblasts, B cells, T cells, reticulocytes, leukocytes, granulocytes, and tumor cells. (Item 20) A method for treating a subject lacking a functional polypeptide, comprising administering a composition comprising: (a) at least one mRNA, at least a portion of which encodes the functional secreted polypeptide; and (b) a transfer vehicle comprising a lipid nanoparticle, wherein after administration of the composition, the mRNA is expressed in a target cell to produce the functional secreted polypeptide. (Item 21) 22. The method of claim 21, wherein the mRNA encodes a functional erythropoietin, alpha-galactosidase, LDL receptor, factor VIII, factor IX, alpha-L-iduronidase, iduronate sulfatase, heparin-N-sulfatase, alpha-N-acetylglucosaminidase, galactose 6-sulfatase, beta-galactosidase, lysosomal acid lipase or arylsulfatase-A polypeptide, and (b) a transfer vehicle, and wherein after administration of the composition, the mRNA is expressed in the target cell to produce a functional secreted polypeptide. (Item 22) 22. The method of claim 21, wherein the functional secreted polypeptide is an enzyme that is abnormally deficient in an individual having a lysosomal storage disorder. (Item 23) 22. The method of claim 21, wherein the mRNA molecule contains at least one modification that confers stability to the mRNA molecule. (Item 24) 22. The method of claim 21, wherein the mRNA molecule comprises a modification in the 5' untranslated region of the mRNA molecule. (Item 25) 26. The method of claim 25, wherein the modification comprises the inclusion of a Cap1 structure. (Item 26) 22. The method of claim 21, wherein the mRNA molecule comprises a modification in the 3' untranslated region of the mRNA molecule. (Item 27) 28. The method of claim 27, wherein the modification comprises the inclusion of a polyA tail. (Item 28) 22. The method of claim 21, further comprising an agent for facilitating the transfer of the mRNA molecule into an intracellular compartment of the target cell. (Item 29) 22. The method of claim 21, wherein the lipid nanoparticles comprise one or more cationic lipids. (Item 30) 22. The method of claim 21, wherein the lipid nanoparticles comprise one or more non-cationic lipids. (Item 31) 22. The method of claim 21, wherein the lipid nanoparticles comprise one or more PEG-modified lipids. (Item 32) 22. The method of claim 21, wherein the lipid nanoparticles comprise C12-200. (Item 33) 22. The method of claim 21, wherein the lipid nanoparticles comprise DLinKC2DMA, CHOL, DOPE, and DMG-PEG-2000. (Item 34) 22. The method of claim 21, wherein the lipid nanoparticles comprise C12-200, DOPE, CHOL, and DMGPEG2K. (Item 35) 22. The method of claim 21, wherein the lipid nanoparticle comprises a cleavable lipid. (Item 36) 22. The method of claim 21, wherein the composition is lyophilized. (Item 37) 22. The method of claim 21, wherein the composition is a lyophilized, reconstituted composition. (Item 38) 22. The method of claim 21, wherein the target cell is selected from the group consisting of a hepatocyte, an epithelial cell, a hematopoietic cell, an epithelial cell, an endothelial cell, a lung cell, a bone cell, a stem cell, a mesenchymal cell, a neuronal cell, a cardiac cell, an adipocyte, a vascular smooth muscle cell, a cardiac muscle cell, a skeletal muscle cell, a beta cell, a pituitary cell, a synovial lining cell, an ovarian cell, a testicular cell, a fibroblast, a B cell, a T cell, a reticulocyte, a leukocyte, a granulocyte, and a tumor cell. (Item 39) A method for treating a subject lacking a functional secreted polypeptide, comprising administering a composition comprising: (a) at least one mRNA, at least a portion of which encodes said functional secreted polypeptide; and (b) a transfer vehicle comprising a lipid nanoparticle, wherein after administration of the composition, the mRNA is translated in a target cell to produce said functional polypeptide in said target cell at least at a minimum therapeutic level more than one hour after administration. (Item 40) A method for producing a functional secreted polypeptide in a target cell, comprising administering a composition comprising: (a) at least one mRNA, at least a portion of which encodes the functional secreted polypeptide; and (b) a transfer vehicle comprising a lipid nanoparticle, wherein after administration of the composition, the mRNA is translated in the target cell to produce the functional secreted polypeptide at at least a minimum therapeutic level more than one hour after administration. The above-discussed and many other features and attendant advantages of the present invention will become better understood by reference to the following detailed description of the invention in conjunction with the accompanying examples. The various embodiments described herein are complementary and may be combined or used together in a manner understood by those skilled in the art in view of the teachings contained herein. [Brief description of the drawings]

[0024] [Figure 1] The nucleotide sequence of the 5'CMV sequence (SEQ ID NO:1) is shown, where X, if present, is GGA. [Diagram 2] The nucleotide sequence of the 3'hGH sequence (SEQ ID NO:2) is shown. [Diagram 3] The nucleotide sequence of human erythropoietin (EPO) mRNA (SEQ ID NO:3) is shown, which may be adjacent to the 5' end of SEQ ID NO:1 and the 3' end of SEQ ID NO:2. [Figure 4] The nucleotide sequence of human α-galactosidase (GLA) mRNA (SEQ ID NO:4) is shown, which may be adjacent to the 5' end of SEQ ID NO:1 and the 3' end of SEQ ID NO:2. [Diagram 5] The nucleotide sequence of human alpha-1 antitrypsin (A1AT) mRNA (SEQ ID NO:5) is shown, which may be adjacent to the 5' end of SEQ ID NO:1 and the 3' end of SEQ ID NO:2. [Figure 6] The nucleotide sequence of human factor IX (FIX) mRNA (SEQ ID NO:6) is shown, which may be adjacent to the 5' end of SEQ ID NO:1 and the 3' end of SEQ ID NO:2. [Figure 7]Quantification of secreted hEPO protein levels measured using ELISA is shown. The detected protein is the result of its production from hEPO mRNA delivered intravenously by a single dose of the various lipid nanoparticle formulations. Formulations C12-200 (30 ug), HGT4003 (150 ug), ICE (100 ug), DODAP (200 ug) are represented by the cationic / ionic lipid components of the respective test substances (formulations 1-4). Values ​​are based on blood samples 4 hours after administration. [Figure 8] Hematocrit measurements of mice treated with a single intravenous dose of human EPO mRNA-loaded lipid nanoparticles (formulations 1-4) are shown. Whole blood samples were taken 4 h (day 1), 24 h (day 2), 4 days, 7 days, and 10 days after dosing. [Figure 9] Hematocrit measurements are shown for mice treated with human EPO-mRNA loaded lipid nanoparticles either as a single intravenous dose or three injections (days 1, 3, 5). Whole blood samples were taken pre-injection (day -4), day 7, and day 15. Formulation 1 administration: (30ug single dose) or (3x10ug administered on days 1, 3, 5), Formulation 2 administration: (3x50ug administered on days 1, 3, 5). [Figure 10] Quantification of secretory human alpha-galactosidase (hGLA) protein levels measured using ELISA. The protein detected is the result of production from hGLA mRNA delivered via lipid nanoparticles (Formulation 1: single intravenous dose of 30ug based on encapsulated mRNA). hGLA protein is detected over a 48 hour period. [Figure 11] Figure 1 shows hGLA activity in serum. hGLA activity was measured at 37°C using the substrate 4-methylumbelliferyl-α-D-galactopyranoside (4-MU-α-gal). Data are the average of measurements from 6-9 individuals. [Figure 12]Quantification of hGLA protein levels in serum measured using ELISA. Protein is produced from hGLA mRNA delivered via C12-200 based lipid nanoparticles (C12-200:DOPE:Chol:DMGPEG2K, 40:30:25:5 (formulation 1); 30ug of mRNA based on encapsulated mRNA, single intravenous dose). hGLA protein is monitored over 72 hours (based on encapsulated mRNA, per single intravenous dose). hGLA protein is monitored over 72 hours. [Figure 13] Quantification of hGLA protein levels in liver, kidney, and spleen measured using ELISA. Protein is produced from hGLA mRNA delivered via C12-200-based lipid nanoparticles (Formulation 1: single intravenous dose of 30ug of mRNA based on encapsulated mRNA). hGLA protein is monitored over 72 hours. [Figure 14A] Dose-response study monitoring protein production of hGLA as secreted MRT-derived human GLA protein in serum (A) and liver (B). Samples were measured 24 hours after dosing (Formulation 1: single intravenous dose, N=4 mice / group) and quantified using ELISA. [Figure 14B] Dose-response study monitoring protein production of hGLA as secreted MRT-derived human GLA protein in serum (A) and liver (B). Samples were measured 24 hours after dosing (Formulation 1: single intravenous dose, N=4 mice / group) and quantified using ELISA. [Figure 15] FIG. 1 shows the pharmacokinetic profile of ERT-based α-galactosidase (40 ug / kg dose) and MRT-produced hGLA protein (Formulation 1: 1.0 mg / kg mRNA dose) in athymic nude mice. [Figure 16]Quantification of secreted hGLA protein levels in Fabry mice treated with MRT measured using ELISA. hGLA protein is produced from hGLA mRNA delivered via C12-200 based lipid nanoparticles (Formulation 1: 10ug of mRNA per single intravenous dose based on encapsulated mRNA). Serum is monitored over 72 hours. [Figure 17] Quantification of hGLA protein levels in liver, kidney, spleen, and heart of Fabry knockout mice treated with MRT measured using ELISA. Protein is produced from hGLA mRNA delivered via C12-200 based lipid nanoparticles (formulation 1: single intravenous dose of 30ug mRNA based on encapsulated mRNA). hGLA protein is monitored over 72 hours. Literature values ​​representing normal physiological levels are graphed with dotted lines. [Figure 18] Quantification of secreted hGLA protein levels in Fabry mice treated with MRT and α-galactosidase as measured using ELISA, both of which were administered as a single intravenous dose of 1.0 mg / kg. [Figure 19] Quantification of hGLA protein levels in liver, kidney, spleen, and heart of Fabry knockout mice treated with MRT and ERT (α-galactosidase) measured using ELISA. Protein was produced from hGLA mRNA delivered via lipid nanoparticles (Formulation 1: single intravenous dose of 1.0 mg / kg mRNA based on encapsulated mRNA). [Figure 20] Relative quantification of globotriaosylceramide (Gb3) and Lyso-Gb3 in kidneys of treated and untreated mice. Male Fabry knockout mice were treated with a single 1.0 mg / kg dose of either GLA mRNA-loaded lipid nanoparticles or α-galactosidase. The amounts reflected the amount of Gb3 / Lyso-Gb3 one week after administration. [Figure 21]Relative quantification of globotriaosylceramide (Gb3) and Lyso-Gb3 in the hearts of treated and untreated mice. Male Fabry knockout mice were treated with a single dose of either GLA mRNA-loaded lipid nanoparticles or α-galactosidase at 1.0 mg / kg. Amounts reflect Gb3 / Lyso-Gb3 one week after administration. [Figure 22] Dose-response study monitoring protein production of GLA as secreted MRT-derived human GLA protein in serum. Samples were measured 24 hours after administration of either HGT4003 (formulation 3) or HGT5000-based lipid nanoparticles (formulation 5) (single intravenous dose, N=4 mice / group) and quantified using ELISA. [Figure 23A] Shown is hGLA protein production measured in serum (A) or liver, kidney, and spleen (B). Samples were measured 6 and 24 hours after administration of HGT5001-based lipid nanoparticles (Formulation 6) (single intravenous dose, N=4 mice / group) and quantified using ELISA. [Figure 23B] Shown is hGLA protein production measured in serum (A) or liver, kidney, and spleen (B). Samples were measured 6 and 24 hours after administration of HGT5001-based lipid nanoparticles (Formulation 6) (single intravenous dose, N=4 mice / group) and quantified using ELISA. [Figure 24] Quantification of secretory human factor IX protein levels measured using ELISA (mean ng / mL ± standard deviation). FIX protein is produced from FIX mRNA delivered via C12-200 based lipid nanoparticles (40:30:25:5 C12-200:DOPE:Chol:DMGPEG2K (formulation 1), 30ug of mRNA per single intravenous dose based on encapsulated mRNA). FIX protein is monitored over 72 hours. (n=24 mice) [Diagram 25]Figure 1 shows quantification of secreted human alpha-1-antitrypsin (A1AT) protein levels measured using ELISA. A1AT protein is produced from A1AT mRNA delivered via C12-200-based lipid nanoparticles (40:30:25:5 C12-200:DOPE:Chol:DMGPEG2K (formulation 1), 30ug of mRNA per single intravenous dose based on encapsulated mRNA). A1AT protein is monitored over 24 hours. [Figure 26] Figure 1 shows ELISA-based quantification of hEPO protein (measured in mIU) detected in lungs and serum of treated mice following intratracheal administration of hEPO mRNA-loaded nanoparticles (C12-200, HGT5000, or HGT5001-based lipid nanoparticles, formulations 1, 5, and 6, respectively). Animals were sacrificed 6 hours after administration (n=4 mice per group). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0025] Description of exemplary embodiments The present invention provides compositions and methods for the intracellular delivery of mRNA in a liposomal transfer vehicle to one or more target cells for the production of therapeutic levels of a functional secreted protein.

[0026] The term "functional" as used herein to qualify a protein or enzyme means that the protein or enzyme has biological activity or can perform the same or similar function as a natural or normally functioning protein or enzyme. The mRNA compositions of the present invention are useful for treating various metabolic or genetic disorders, particularly genetic or metabolic disorders involving non-expression, misexpression, or deficiency of a protein or enzyme. The term "therapeutic level" refers to a level of protein detected in blood or tissue that is above a control level, where the control can be a normal physiological level, or a level in a subject prior to administration of the mRNA composition. The term "secreted" refers to a protein that is detected outside the target cell (extracellular space). The protein can be detected in blood or tissue. In the context of the present invention, the term "produced" is used broadly to refer to the translation of at least one mRNA into a protein or enzyme. As provided herein, the composition includes a delivery vehicle. The term "delivery vehicle" as used herein includes any of the standard pharmaceutical carriers, diluents, excipients, and the like, which are generally intended for use in connection with the administration of a physiologically active agent, including nucleic acids. The compositions described herein, specifically the transfer vehicles, can deliver mRNA to target cells. In embodiments, the transfer vehicle is a lipid nanoparticle. mRNA

[0027] The mRNA in the composition of the present invention may, for example, code for a normally secreted secretory hormone, an enzyme, a receptor, a polypeptide, a peptide, or other protein of interest. In one embodiment of the present invention, the mRNA may optionally have chemical or biological modifications that, for example, improve the stability and / or half-life of such mRNA, or improve or otherwise facilitate the production of the protein.

[0028] The method of the present invention provides for optional simultaneous delivery of one or more unique mRNAs to a target cell, for example, by combining two unique mRNAs in a single transfer vehicle. In one embodiment of the present invention, a first therapeutic mRNA and a second therapeutic mRNA can be formulated and administered in a single transfer vehicle. The present invention also contemplates simultaneous delivery and / or administration of a first therapeutic mRNA and a second nucleic acid to facilitate and / or enhance the function or delivery of the first therapeutic mRNA. For example, such a second nucleic acid (e.g., an exogenous or synthetic mRNA) may code for a membrane transporter protein that upon expression (e.g., upon translation of the exogenous or synthetic mRNA) facilitates delivery of the first mRNA or enhances its biological activity. Alternatively, the first therapeutic mRNA can be administered with a second nucleic acid that acts as a "chaperone", for example, to direct the folding of any of the first therapeutic mRNA.

[0029] The method of the present invention also provides for the delivery of one or more therapeutic nucleic acids to treat a single disorder or deficiency, and each such therapeutic nucleic acid functions by a different mechanism of action.For example, the composition of the present invention can comprise a first therapeutic mRNA, which is administered, for example, to correct an endogenous protein or enzyme deficiency, and is accompanied by a second nucleic acid, which is administered to inactivate or "knock down" the malfunctioning endogenous nucleic acid and its protein or enzyme product.Such a "second" nucleic acid can, for example, code for an mRNA or siRNA.

[0030] Upon transfection, the native mRNA in the composition of the invention may decay with a half-life of 30 minutes to several days. The mRNA in the composition of the invention preferably retains at least some ability to be translated, thus producing a functional secreted protein or enzyme. Thus, the invention provides compositions comprising stabilized mRNA and methods of administering same. In some embodiments of the invention, the activity of the mRNA is extended for an extended period of time. For example, the activity of the mRNA can be prolonged such that the composition of the invention is administered to a subject twice weekly or every other week, or more preferably once a month, every other month, quarterly, or annually. The extended or prolonged activity of the mRNA of the invention is directly related to the amount of functional secreted protein or enzyme produced from such mRNA. Similarly, the activity of the composition of the invention can be further extended or prolonged by modifications that improve or enhance the translation of the mRNA. Furthermore, the amount of functional protein or enzyme produced by a target cell is a function of the amount of mRNA delivered to the target cell and the stability of such mRNA. To the extent that the stability of the mRNA of the present invention can be improved or enhanced, the half-life, the activity of the secreted protein or enzyme produced, and the administration frequency of the composition can be further extended.

[0031] Thus, in some embodiments of the present invention, the mRNA in the composition comprises at least one modification that confers increased or enhanced stability to the nucleic acid, including, for example, improved resistance to nuclease digestion in vivo. The terms "modification" and "modified" as used herein in relation to the nucleic acids provided herein preferably include at least one alteration that enhances stability and renders the mRNA more stable (e.g., resistant to nuclease digestion) than wild-type or naturally occurring versions of the mRNA. The terms "stable" and "stability" as used herein in relation to the nucleic acids of the present invention, and particularly with respect to mRNA, refer to, for example, increased or enhanced resistance to degradation by nucleases (i.e., endonucleases or exonucleases) that can normally degrade such mRNA. Increased stability can include, for example, reduced susceptibility to hydrolysis or other destruction by endogenous enzymes (e.g., endonucleases or exonucleases) or conditions within a target cell or tissue, thus increasing or enhancing the residence of such mRNA in a target cell, tissue, subject, and / or cytoplasm. The stabilized mRNA molecules provided herein show longer half-life compared to their naturally occurring unmodified counterparts (e.g., wild-type version of mRNA).The terms "modification" and "modified" in relation to the mRNA of the present invention also contemplate alterations that improve or enhance the translation of mRNA nucleic acid, including, for example, the inclusion of sequences that function in the initiation of protein translation (e.g., Kozak consensus sequences) (Kozak, M., Nucleic Acids Res 15(20):8125-48(1987)).

[0032] In some embodiments, the mRNA of the present invention undergoes chemical or biological modification to give them higher stability.Exemplary modifications to mRNA include base deficiency (e.g., by deletion or replacement of one nucleotide with another) or base modification, e.g., chemical modification of base.As used herein, the phrase "chemical modification" includes modification that introduces a different chemical property from that found in naturally occurring mRNA, e.g., introduction of modified nucleotide (e.g., nucleotide analogue), or covalent modification such as inclusion of pendant group that is not found in nature in such mRNA molecule.

[0033] In addition, suitable modifications include changing one or more nucleotides of a codon so that the codon codes for the same amino acid but is more stable than the codon found in the wild-type version of the mRNA. For example, an inverse correlation between RNA stability and more cytidine (C) and / or uridine (U) residues has been demonstrated, and RNA lacking C and U residues has been found to be stable against most RNases (Heidenreich, et al. J Biol Chem 269,2131-8 (1994)). In some embodiments, the number of C and / or U residues in the mRNA sequence is reduced. In another embodiment, the number of C and / or U residues is reduced by replacing one codon that codes for a particular amino acid with another codon that codes for the same or related amino acid. Contemplated modifications to the mRNA nucleic acid of the present invention also include the incorporation of pseudouridines. The incorporation of pseudouridines into the mRNA nucleic acid of the present invention can increase stability and translational capacity, and reduce immunogenicity in vivo. See, for example, Kariko, K., et al. See, I., Molecular Therapy 16(11):1833-1840 (2008). Substitutions and modifications of the mRNA of the present invention can be carried out by methods readily known to those of skill in the art.

[0034] The constraints on reducing the number of C and U residues in a sequence may be greater in the coding regions of an mRNA than in untranslated regions (i.e., it is probably not possible to eliminate all of the C and U residues present in the message while still retaining the capacity of the message to code for the desired amino acid sequence). However, the degeneracy of the genetic code presents an opportunity to reduce the number of C and / or U residues present in a sequence while maintaining the same coding capacity (i.e., several different possibilities for modification of an RNA sequence may be considered, depending on which amino acid is coded for by the codon). For example, the codon for Gly may be changed to GGA or GGG instead of GGU or GGC.

[0035] The term modification also includes, for example, the incorporation of non-nucleotide linkages or modified nucleotides into the mRNA sequences of the invention (e.g., modifications to one or both of the 3' and 5' ends of an mRNA molecule encoding a functional secreted protein or enzyme). Such modifications include the addition of bases to the mRNA sequence (e.g., inclusion of a polyA tail or a longer polyA tail), alteration of the 3'UTR or 5'UTR, complexing of the mRNA with an agent (e.g., a protein or a complementary nucleic acid molecule), and the inclusion of elements that alter the structure of the mRNA molecule (e.g., forming a secondary structure).

[0036] PolyA tails are believed to stabilize natural messengers. Thus, in one embodiment, long polyA tails can be added to mRNA molecules, thus conferring greater stability to the mRNA. PolyA tails can be added using a variety of art-recognized techniques. For example, long polyA tails can be added to synthetic or in vitro transcribed mRNAs using polyA polymerase (Yokoe, et al. Nature Biotechnology. 1996; 14: 1252-1256). Transcription vectors can also encode long polyA tails. In addition, polyA tails can be added by direct transcription from PCR products. In one embodiment, the polyA tail is at least about 90, 200, 300, 400, at least 500 nucleotides in length. In one embodiment, the length of the polyA tail is adjusted to control the stability of the modified mRNA molecules of the present invention, and thus the transcription of the protein. For example, since the length of the poly-A tail can affect the half-life of an mRNA molecule, the length of the poly-A tail can be adjusted to modify the level of resistance of the mRNA to nucleases and thus control the time course of protein expression in a cell. In one embodiment, the stabilized mRNA molecule is sufficiently resistant to in vivo degradation (e.g., by nucleases) so that it can be delivered to a target cell without a transfer vehicle.

[0037] In one embodiment, mRNA can be modified by incorporating 3' and / or 5' untranslated (UTR) sequences that are not naturally found in wild-type mRNA. In one embodiment, 3' and / or 5' flanking sequences that naturally flank the mRNA and code for a second unlinked protein can be incorporated into and modify the nucleotide sequence of the mRNA molecule that codes for a therapeutic or functional protein. For example, 3' or 5' sequences from a stable mRNA molecule (e.g., globin, actin, GAPDH, tubulin, histone, or citric acid cycle enzymes) can be incorporated into the 3' and / or 5' regions of a sense mRNA nucleic acid molecule to increase the stability of the sense mRNA molecule. See, for example, US 2003 / 0083272.

[0038] In some embodiments, the mRNA in the composition of the present invention includes a modification of the 5' end of the mRNA to include a partial sequence of the CMV immediate early 1 (IE1) gene or a fragment thereof (e.g., SEQ ID NO: 1), improving nuclease resistance and / or improving the half-life of the mRNA. In addition to increasing the stability of the mRNA nucleic acid sequence, it has been surprisingly found that the inclusion of a partial sequence of the CMV immediate early 1 (IE1) gene enhances the translation of the mRNA and the expression of a functional protein or enzyme. To further stabilize the mRNA, the inclusion of a human growth hormone (hGH) gene sequence or a fragment thereof (e.g., SEQ ID NO: 2) at the 3' end of the nucleic acid (e.g., mRNA) is also contemplated. In general, preferred modifications include modifications that improve the stability and / or pharmacokinetic properties (e.g., half-life) of the mRNA compared to their unmodified counterparts, for example, improving the resistance of such mRNA to in vivo nuclease digestion.

[0039] Variants of the nucleic acid sequences of SEQ ID NO:1 and / or SEQ ID NO:2 are further contemplated, which variants maintain the functional and / or pharmacokinetic properties (e.g., half-life) of the nucleic acid, including stabilization of the mRNA. Variants may have greater than 90%, greater than 95%, greater than 98%, or greater than 99% sequence identity to SEQ ID NO:1 or SEQ ID NO:2.

[0040] In some embodiments, the composition may include a stabilization reagent. The composition may include one or more formulation reagents that directly or indirectly bind to stabilize the mRNA and thus increase its residence time in the target cell. Such reagents preferably lead to an improvement in the half-life of the mRNA in the target cell. For example, the stability of the mRNA and the efficiency of translation may be increased by the incorporation of a "stabilization reagent" that forms a complex with the naturally occurring mRNA in the cell (see, for example, U.S. Patent No. 5,677,124). The incorporation of a stabilization reagent may be achieved, for example, by combining polyA and a protein with the mRNA to stabilize the mRNA ex vivo before loading or encapsulating it into a transfer vehicle. Exemplary stabilization reagents include one or more proteins, peptides, aptamers, translation accessory proteins, mRNA binding proteins, and / or translation initiation factors.

[0041] Stability of the compositions can also be improved by the use of opsonization-inhibiting moieties, which are typically large hydrophilic polymers that are chemically or physically bound to the transfer vehicle (e.g., by intercalation of a lipid-soluble anchor into its membrane or by direct binding to active groups of membrane lipids). These opsonization-inhibiting hydrophilic polymers form a protective surface layer that significantly reduces uptake of liposomes by the macrophage monocyte and reticuloendothelial systems (e.g., as described in U.S. Pat. No. 4,920,016, the entire disclosure of which is incorporated herein by reference). Thus, transfer carriers modified with opsonization-inhibiting moieties remain in the circulation much longer than their unmodified counterparts.

[0042] When RNA hybridizes to a complementary nucleic acid molecule (e.g., DNA or RNA), it can be protected by nucleases (Krieg, et al. Melton. Methods in Enzymology. 1987; 155, 397-415). The stability of the hybridized mRNA is likely due to the inherent single-stranded specificity of most RNases. In some embodiments, the stabilizing reagent selected to complex the mRNA is a eukaryotic protein (e.g., a mammalian protein). In yet another embodiment, the mRNA can be modified by hybridization to a second nucleic acid molecule. When the entire mRNA molecule is hybridized to a complementary nucleic acid molecule, translation initiation can be reduced. In some embodiments, the 5' untranslated region and the AUG initiation region of the mRNA molecule can optionally remain unhybridized. After translation initiation, the unwinding activity of the ribosome complex can function even on high affinity duplexes so that translation can begin (Liebhaber. J. Mol. Biol. 1992; 226:2-13; Moni, et al. J Biol Chem. 1993; 268:14514-22).

[0043] It will be understood that any of the methods for enhancing mRNA stability described above can be used either alone or in combination with one or more of the other above-mentioned methods and / or compositions or any of them.

[0044] The mRNA of the present invention can be optionally combined with a reporter gene (e.g., upstream or downstream of the coding region of the mRNA), which facilitates the determination of the mRNA delivery to target cells or tissues.Suitable reporter genes can include, for example, green fluorescent protein mRNA (GFP mRNA), Renilla luciferase mRNA (luciferase mRNA), firefly luciferase mRNA, or any combination thereof.For example, GFP mRNA can be fused with the mRNA that codes for secretory protein, which facilitates the confirmation of the localization of the mRNA in target cells that act as a depot for protein production.

[0045] The term "transfect" or "transfection" as used herein refers to the intracellular introduction of mRNA into a cell, or preferably, into a target cell. The introduced mRNA may be stably or transiently maintained in the target cell. The term "transfection efficiency" refers to the relative amount of mRNA taken up by the target cell subject to transfection. In practice, transfection efficiency is estimated by the amount of reporter nucleic acid product expressed by the target cell after transfection. Preferred embodiments include compositions with high transfection efficacy, specifically compositions that minimize side effects mediated by transfection of non-target cells. Compositions of the invention that exhibit high transfection efficacy increase the likelihood that an adequate dose of mRNA is delivered to the target cell while minimizing potential systemic side effects. In one embodiment of the present invention, the transfer vehicle of the present invention can deliver large mRNA sequences (e.g., mRNAs of at least 1 kDa, 1.5 kDa, 2 kDa, 2.5 kDa, 5 kDa, 10 kDa, 12 kDa, 15 kDa, 20 kDa, 25 kDa, 30 kDa, or more). The mRNA can be formulated with one or more acceptable reagents, which provide a carrier for delivering such mRNA to target cells. Appropriate reagents are generally selected with respect to several factors, including, among others, the biological or chemical properties of the mRNA, the intended route of administration, the expected biological environment to which such mRNA will be exposed, and the specificity of the intended target cells. In some embodiments, the transfer vehicle, such as a liposome, encapsulates the mRNA without compromising biological activity. In some embodiments, the transfer vehicle exhibits selective and / or substantial binding to target cells compared to non-target cells. In a preferred embodiment, the transfer vehicle delivers its contents to a target cell such that the mRNA is delivered to the appropriate intracellular compartment, such as the cytoplasm. Transport Carrier

[0046] In an embodiment, the transfer vehicle in the composition of the present invention is a liposomal transfer vehicle, e.g., lipid nanoparticle. In one embodiment, the transfer vehicle is selected and / or prepared to optimize the delivery of mRNA to the target cell. For example, when the target cell is a liver cell, the characteristics of the transfer vehicle (e.g., dimensions, charge, and / or pH) can be optimized to effectively deliver such transfer vehicle to the target cell to reduce immune clearance and / or promote retention in the target cell. Alternatively, when the target cell is the central nervous system (e.g., the mRNA administered for neurodegenerative disease treatment may specifically target brain or spinal cord tissue), the selection and preparation of the transfer vehicle must take into account the penetration of the blood-brain barrier and retention in the blood-brain barrier, and / or the use of alternative means to directly deliver such transfer vehicle to such target cells. In one embodiment of the present invention, the composition can be combined with an agent that promotes the transfer of exogenous mRNA (e.g., an agent that reduces or improves the permeability of the blood-brain barrier, thus enhancing the transfer of exogenous mRNA to the target cell).

[0047] The use of liposomal transfer vehicles to facilitate the delivery of nucleic acid to target cells is contemplated by the present invention.Liposomes (e.g., liposomal lipid nanoparticles) are generally useful in various applications in research, industry, and medicine, particularly in their use as vehicles for the transfer of diagnostic or therapeutic compounds in vivo (Lasic, Trends Biotechnol., 16:307-321, 1998; Drummond et al., Pharmacol.Rev., 51:691-743, 1999), and are generally considered as microscopic vesicles with an internal aqueous space that is separated from the external medium by one or more bilayer membranes.The bilayer membrane of liposomes is typically formed by amphiphilic molecules such as lipids of synthetic or natural origin, which contain spatially separated hydrophilic and hydrophobic domains (Lasic, Trends Biotechnol., 16:307-321, 1998). The liposomal bilayer can also be formed by amphiphilic polymers and surfactants (eg, polymersomes, niosomes, etc.).

[0048] In the context of the present invention, liposomal transfer vehicles typically serve to transfer mRNA to target cells. For the purposes of the present invention, liposomal transfer vehicles are prepared to contain the desired nucleic acid. The process of incorporating a desired entity (e.g., nucleic acid) into liposomes is often referred to as "loading" (Lasic, et al., FEBS Lett., 312:255-258, 1992). The liposomal incorporated nucleic acid may be located entirely or partially in the interior space of the liposome within the liposomal bilayer membrane, or may be associated with the outer surface of the liposomal membrane. The incorporation of nucleic acid into liposomes is also referred to herein as "encapsulation", where the nucleic acid is contained entirely within the interior space of the liposome. The purpose of incorporating mRNA into transfer vehicles such as liposomes is often to protect the nucleic acid from an environment that may contain enzymes or chemicals that degrade the nucleic acid and / or system, or receptors that cause the nucleic acid to be rapidly excreted. Thus, in a preferred embodiment of the present invention, the transfer vehicle selected can enhance the stability of the mRNA contained therein. Liposomes may allow encapsulated mRNA to reach target cells and / or may preferentially allow encapsulated mRNA to reach target cells, or alternatively may limit the delivery of such mRNA to other sites or cells where the presence of the administered mRNA may be useless or undesirable.Furthermore, incorporation of mRNA into a transfer vehicle, such as, for example, cationic liposomes, also facilitates the delivery of such mRNA to target cells.

[0049] Ideally, liposomal transfer vehicles are prepared to encapsulate one or more desired mRNAs, so that the composition exhibits high transfection efficiency and enhanced stability.While liposomes can promote the introduction of nucleic acid into target cells, adding polycations (e.g., poly-L-lysine and protamine) as copolymers can promote the transfection efficiency of some types of cationic liposomes in some cell lines both in vitro and in vivo, and in some cases significantly enhance it by 2-28 times (see NJ Caplen, et al., Gene Ther. 1995; 2: 603, S. Li, et al., Gene Ther. 1997; 4, 891). Lipid Nanoparticles

[0050] In a preferred embodiment of the present invention, the transfer vehicle is formulated as lipid nanoparticles. The phrase "lipid nanoparticles" as used herein refers to a transfer vehicle that comprises one or more lipids (e.g., cationic lipids, non-cationic lipids, and PEG-modified lipids). Preferably, the lipid nanoparticles are formulated to deliver one or more mRNAs to one or more target cells. Examples of suitable lipids include, for example, phosphatidyl compounds (e.g., phosphatidylglycerol, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, sphingolipids, cerebrosides, and gangliosides). The use of polymers as transfer vehicles, whether alone or in combination with other transfer vehicles, is also contemplated. Suitable polymers can include, for example, polyacrylates, polyalkylcyanoacrylates, polylactides, polylactide-polyglycolide copolymers, polycaprolactones, dextran, albumin, gelatin, alginates, collagens, chitosans, cyclodextrins, dendrimers, and polyethyleneimines. In one embodiment, a transfer vehicle is selected based on its ability to facilitate transfection of mRNA into a target cell.

[0051] The present invention contemplates the use of lipid nanoparticles as transfer vehicles that contain cationic lipids to encapsulate mRNA and / or facilitate the delivery of mRNA to target cells that act as depots for protein production.The phrase "cationic lipid" as used herein refers to any of several lipid species that carry a net positive charge at a selected pH, such as physiological pH.The contemplated lipid nanoparticles can be prepared by including a multi-component lipid mixture in various ratios with one or more cationic lipids, non-cationic lipids, and PEG-modified lipids.Several cationic lipids have been described in the literature, and many of them are commercially available.

[0052] Particularly suitable cationic lipids for use in the compositions and methods of the invention include those described in International Patent Publication No. WO2010 / 053572, which is incorporated herein by reference, and most particularly, C12-200, as described in paragraph

[0225] of International Patent Publication No. WO2010 / 053572. In certain embodiments, the compositions and methods of the invention include, for example, (15Z,18Z)-N,N-dimethyl-6-(9Z,12Z)-octadeca-9,12-dien-1-yl)tetracosa-15,18-dien-1-amine (HGT5000), (15Z,18Z)-N,N-dimethyl-6-((9Z,12Z)-octadeca-9,12-dien-1-yl)tetracosa-4,15,18-trien-1 No. 61 / 617,468, filed March 29, 2012 (herein incorporated by reference), such as (15Z,18Z)-N,N-dimethyl-6-((9Z,12Z)-octadeca-9,12-dien-1-yl)tetracosa-5,15,18-trien-1-amine (HGT5001), and (15Z,18Z)-N,N-dimethyl-6-((9Z,12Z)-octadeca-9,12-dien-1-yl)tetracosa-5,15,18-trien-1-amine (HGT5002).

[0053] In some embodiments, the cationic lipid N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride or "DOTMA" is used (Felgner et al. (Proc. Nat'l Acad. Sci. 84,7413 (1987), U.S. Pat. No. 4,897,355). DOTMA can be formulated alone or combined with neutral lipids, dioleoylphosphatidyl-ethanolamine, or "DOPE", or other cationic or non-cationic lipids into liposomal transfer vehicles or lipid nanoparticles, and such liposomes can be used to facilitate delivery of nucleic acids to target cells. Other suitable cationic lipids include, for example, 5-carboxyspermylglycine dioctadecylamide or "DOGS", 2,3-dioleyloxy-N-[2(spermine-carboxyamido)ethyl]-N,N-dimethyl-1-propanaminium or "DOSPA" (Behr et al. al. Proc. Nat.'l Acad. Sci. 86, 6982 (1989), U.S. Pat. No. 5,171,678, U.S. Pat. No. 5,334,761), 1,2-dioleoyl-3-dimethylammonium-propane or "DODAP", 1,2-dioleoyl-3-trimethylammonium-propane or "DOTAP". Contemplated cationic lipids include 1,2-distearoyloxy-N,N-dimethyl-3-aminopropane or "DSDMA", 1,2-dioleoyloxy-N,N-dimethyl-3-aminopropane or "DODMA", 1,2-dilinoleyloxy-N,N-dimethyl -3-aminopropane or "DLinDMA", 1,2-dilinolenyloxy-N,N-dimethyl-3-aminopropane or "DLenDMA", N-dioleyl-N,N-dimethylammonium chloride or "DODAC", N,N-distearyl-N,N-dimethylammonium bromide or "DDAB", N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide or "DMRIE", 3-dimethylamino-2-(cholest-5-en-3-β-oxybutane-4-oxy)-1-(cis,cis-9,12-Octadecadienoyl)propane or "CLinDMA", 2-[5'-(cholest-5-en-3-β-oxy)-3'-oxapentoxy)-3-dimethyl-1-(cis,cis-9',1-2'-octadecadienoyl)propane or "CpLinDMA", N,N-Dimethyl-3,4-dioleyloxybenzylamine or "DMOBA", 1,2-N,N'-Dioleylcarbamyl-3-dimethylaminopropane or "DOcarbDAP", 2,3-Dilinoleoyloxy-N,N-dimethylpropylamine or "DLinDAP", 1,2-N,N'-Dilinoleylcarbamyl-3-dimethylamino propane or "DLincarbDAP", 1,2-dilinoleoylcarbamyl-3-dimethylaminopropane or "DLinCDAP", 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane or "DLin-K-DMA", 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane or "DLin-K-XTC2-DMA", and 2-(2,2-di((9Z,12Z)-octadeca-9,12-dien-1-yl)-1,3-dioxolan-4-yl)-N,N-dimethylethanamine (DLin-KC2-DMA)) (International Publication No. WO 2010 / 042877, Semple et al., Nature Biotech. 28:172-176 (2010)), or mixtures thereof (Heyes, J., et al., J Controlled Release 107:276-287 (2005); Morrissey, DV., et al., Nat. Biotechnol. 23(8):1003-1007 (2005); PCT Publication No. WO2005 / 121348A1).

[0054] The use of cholesterol-based cationic lipid is also contemplated by the present invention.Such cholesterol-based cationic lipid can be used alone or in combination with other cationic or non-cationic lipid.Suitable cholesterol-based cationic lipid includes, for example, DC-Chol (N,N-dimethyl-N-ethylcarboxamidocholesterol), 1,4-bis(3-N-oleylamino-propyl)piperazine (Gao, et al. Biochem. Biophys. Res. Comm. 179, 280 (1991); Wolf et al. BioTechniques 23, 139 (1997); U.S. Patent No. 5,744,335) or ICE.

[0055] In addition, several reagents for enhancing transfection efficiency are commercially available. Suitable examples include Lipofectin (DOTMA:DOPE) (Invitrogen, Carlsbad, Calif.), Lipofectamine (DOSPA:DOPE) (Invitrogen), Lipofectamine 2000 (Invitrogen), FUGENE, Transfectam (DOGS), and Effectene.

[0056] Cationic lipids such as dialkylamino-based, imidazole-based and guanidinium-based lipids are also contemplated.For example, one embodiment is directed to a composition comprising one or more imidazole-based cationic lipids, such as imidazole cholesterol ester or "ICE" lipid (3S, 10R, 13R, 17R)-10, 13-dimethyl-17-((R)-6-methylheptan-2-yl)-2, 3, 4, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl 3-(1H-imidazol-4-yl)propanoate, as represented by the following structure (I): In a preferred embodiment, a transfer vehicle for delivery of mRNA may comprise one or more imidazole-based cationic lipids, such as imidazole cholesterol ester or the "ICE" lipid (3S,10R,13R,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl 3-(1H-imidazol-4-yl)propanoate, represented by structure (I). [ka] Without wishing to be bound by a particular theory, it is believed that the fusogenicity of imidazole-based cationic lipid ICE is related to endosomal disruption promoted by the imidazole group having a lower pKa compared to conventional cationic lipids. Endosome disruption then promotes osmotic swelling and rupture of liposome membrane, followed by transfection or intracellular release of the nucleic acid(s) contained therein loaded into target cells.

[0057] Imidazole-based cationic lipids also feature reduced toxicity compared to other cationic lipids. Imidazole-based cationic lipids (e.g., ICE) can be used as the sole cationic lipid in lipid nanoparticles, or alternatively can be combined with conventional cationic lipids, non-cationic lipids, and PEG-modified lipids. The cationic lipid can comprise a molar ratio of about 1% to about 90%, about 2% to about 70%, about 5% to about 50%, about 10% to about 40%, or preferably about 20% to about 70% of the total lipid present in the transfer vehicle.

[0058] Similarly, certain embodiments are directed to lipid nanoparticles comprising HGT4003 cationic lipid 2-((2,3-bis((9Z,12Z)-octadeca-9,12-dien-1-yloxy)propyl)disulfanyl)-N,N-dimethylethanamine, represented by structure (II) below and further described in U.S. Provisional Application No. 61 / 494,745, filed June 8, 2011, the entire teachings of which are incorporated herein by reference in their entirety. [ka]

[0059] In other embodiments, the compositions and methods described herein are directed to lipid nanoparticles comprising one or more cleavable lipids, such as one or more cationic lipids or compounds comprising a cleavable disulfide (SS) functional group (e.g., HGT4001, HGT4002, HGT4003, HGT4004, and HGT4005), further described in U.S. Provisional Application No. 61 / 494,745, the entire teachings of which are incorporated herein by reference in their entirety.

[0060] The use of polyethylene glycol (PEG)-modified phospholipids and derivatized lipids, such as derivatized ceramides (PEG-CER) including N-octanoyl-sphingosine-1-[succinyl(methoxypolyethylene glycol)-2000] (C8 PEG-2000 ceramide), either alone or, preferably, in combination with other lipids comprising a delivery vehicle (e.g., lipid nanoparticles), is also contemplated by the present invention. Contemplated PEG-modified lipids include those having C6-C 20 These include, but are not limited to, polyethylene glycol chains of up to 5 kDa covalently attached to lipids having long alkyl chain(s). The addition of such components may prevent complex aggregation, increase circulation lifetime, and also provide a means for increasing delivery of lipid nucleic acid compositions to target cells (Klibanov et al. (1990) FEBS Letters, 268(1):235-237), or they may be selected to be rapidly exchanged from the formulation in vivo (see U.S. Pat. No. 5,885,613). Particularly useful exchangeable lipids are PEG-ceramides with shorter acyl chains (e.g., C14 or C18). The PEG-modified phospholipids and derivatized lipids of the present invention may comprise a molar ratio of about 0% to about 20%, about 0.5% to about 20%, about 1% to about 15%, about 4% to about 10%, or about 2% of the total lipid present in the liposome transfer vehicle.

[0061] The present invention also contemplates the use of non-cationic lipids. 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 several lipid species that carry a net negative charge at a selected pH, such as physiological pH. Non-cationic lipids include distearoyl phosphatidylcholine (DSPC), dioleoyl phosphatidylcholine (DOPC), dipalmitoyl phosphatidylcholine (DPPC), dioleoyl phosphatidylglycerol (DOPG), dipalmitoyl phosphatidylglycerol (DPPG), dioleoyl phosphatidylethanolamine (DOPE), palmitoyl oleoyl phosphatidylcholine (POPC), palmitoyl oleoyl-phosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine (D ... Examples of non-cationic lipids include, but are not limited to, ethanolamine 4-(N-merimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), cholesterol, or mixtures thereof. Such non-cationic lipids may be used alone, but are preferably used in combination with other excipients, such as cationic lipids. When used in combination with cationic lipids, the non-cationic lipids may comprise a molar ratio of 5% to about 90% of the total lipids present in the transfer vehicle, or preferably, about 10% to about 70%.

[0062] Preferably, the transfer vehicle (e.g., lipid nanoparticle) is prepared by combining multiple lipid and / or polymer components.For example, the transfer vehicle can be prepared by using C12-200, DOPE, chol, DMG-PEG2K at a molar ratio of 40:30:25:5, or DODAP, DOPE, cholesterol, DMG-PEG2K at a molar ratio of 18:56:20:6, or HGT5000, DOPE, chol, DMG-PEG2K at a molar ratio of 40:20:35:5, or HGT5001, DOPE, chol, DMG-PEG2K at a molar ratio of 40:20:35:5.The selection of PEG-modified lipids, including cationic lipids, non-cationic lipids, and / or lipid nanoparticles, and the relative molar ratios of such lipids to each other, are based on the properties of the lipid(s) selected, the properties of the target cell of interest, and the properties of the mRNA to be delivered. Further considerations include, for example, the saturation of the alkyl chain, as well as the size, charge, pH, pKa, fusogenicity, and toxicity of the lipid(s) selected. Thus, the molar ratio can be adjusted accordingly. For example, in embodiments, the percentage of cationic lipid in lipid nanoparticles can be greater than 10%, greater than 20%, greater than 30%, greater than 40%, greater than 50%, greater than 60%, or greater than 70%. The percentage of non-cationic lipid in lipid nanoparticles can be greater than 5%, greater than 10%, greater than 20%, greater than 30%, or greater than 40%. The percentage of cholesterol in lipid nanoparticles can be greater than 10%, greater than 20%, greater than 30%, or greater than 40%. The percentage of PEG-modified lipid in lipid nanoparticles can be greater than 1%, greater than 2%, greater than 5%, greater than 10%, or greater than 20%.

[0063] In a preferred embodiment, the lipid nanoparticles of the present invention comprise at least one of the following cationic lipids: C12-200, DLin-KC2-DMA, DODAP, HGT4003, ICE, HGT5000, or HGT5001. In an embodiment, the transfer vehicle comprises cholesterol and / or a PEG-modified lipid. In some embodiments, the transfer vehicle comprises DMG-PEG2K. In an embodiment, the transfer vehicle comprises one of the following lipid formulations: C12-200, DOPE, chol, DMG-PEG2K, DODAP, DOPE, cholesterol, DMG-PEG2K, HGT5000, DOPE, chol, DMG-PEG2K, HGT5001, DOPE, chol, DMG-PEG2K.

[0064] The liposomal transfer vehicle used in the composition of the present invention can be prepared by various techniques currently known in the art.Multilamellar vesicles (MLVs) can be prepared by conventional techniques, for example, by dissolving lipids in a suitable solvent and depositing selected lipids on the inner wall of a suitable container or vessel, and then evaporating the solvent to leave a thin film inside the vessel, or by spray drying.Then, aqueous phase can be added to the vessel with a vortexing motion, which leads to the formation of MLVs.Then, unilamellar vesicles (ULVs) can be formed by homogenizing, sonicating, or extruding the multilamellar vesicles.In addition, unilamellar vesicles can be formed by detergent removal techniques.

[0065] In one embodiment of the present invention, the composition of the present invention comprises a transfer vehicle, and the mRNA is associated with both surfaces of the transfer vehicle and encapsulated within the same transfer vehicle. For example, during preparation of the composition of the present invention, a cationic liposome transfer vehicle can be associated with the mRNA via electrostatic interactions.

[0066] In some embodiments, the compositions of the present invention can be loaded with diagnostic radionuclides, fluorescent substances, or other substances that can be detected in both in vitro and in vivo applications.For example, diagnostic substances suitable for use in the present invention can include Rhodamine-dioleoylphospha-tidylethanolamine (Rh-PE), green fluorescent protein mRNA (GFP mRNA), Renilla luciferase mRNA, and firefly luciferase mRNA.

[0067] The selection of the appropriate size of the liposome transfer vehicle should take into account the site of the target cell or tissue, and to some extent the application for which the liposome is made. In some embodiments, it may be desirable to restrict the transfection of mRNA to certain cells or tissues. For example, for targeting hepatocytes, the liposome transfer vehicle may be dimensioned so that its size is smaller than the fenestrations of the endothelial layer lining hepatic sinusoids in the liver, and thus the liposome transfer vehicle may easily penetrate such endothelial fenestrations to reach the target hepatocytes. Alternatively, the liposome transfer vehicle may be dimensioned so that the size of the liposome is sufficient diameter to restrict or even avoid distribution to certain cells or tissues. For example, the liposome transfer vehicle may be dimensioned so that its size is larger than the fenestrations of the endothelial layer lining hepatic sinusoids, and thus restrict the distribution of the liposome transfer vehicle to hepatocytes. Generally, the dimensions of the transport vehicles are in the range of about 25 to 250 nm, preferably less than about 250 nm, less than 175 nm, less than 150 nm, less than 125 nm, less than 100 nm, less than 75 nm, less than 50 nm, less than 25 nm, or less than 10 nm.

[0068] A variety of alternative methods known in the art for sizing liposomal transfer vehicle populations are available. One such sizing method is described in U.S. Pat. No. 4,737,323, incorporated herein by reference. Sonication of liposome suspensions, either bath or probe sonication, gradually reduces the size to small ULVs less than about 0.05 microns in diameter. Homogenization is another method that relies on shear energy to fragment large liposomes into smaller liposomes. In a typical homogenization procedure, MLVs are recirculated using a standard emulsion homogenizer until a selected liposome size is observed, typically about 0.1 to 0.5 microns. The size of liposomal vesicles can be determined by pseudoelectric light scattering (QELS) as described in Bloomfield, Ann. Rev. Biophys. Bioeng., 10:421-450 (1981), incorporated herein by reference. The average liposome diameter can be reduced by sonication of the formed liposomes. Intermittent sonication cycles, alternating with QELS evaluation, can induce efficient liposome synthesis. target cell

[0069] The term "target cell" as used herein refers to a cell or tissue that is the object or target of the composition of the present invention. In some embodiments, the target cell lacks a protein or enzyme of interest. For example, if it is desired to deliver nucleic acid to a hepatocyte, the hepatocyte represents the target cell. In some embodiments, the composition of the present invention distinguishes and transfects the target cell (i.e., does not transfect non-target cells). The compositions of the invention may also be prepared to preferentially target a variety of target cells, including, but not limited to, hepatocytes, epithelial cells, hematopoietic cells, epithelial cells, endothelial cells, lung cells, bone cells, stem cells, mesenchymal cells, neural cells (e.g., meningeal, astrocytes, motor neurons, dorsal root ganglion cells, and anterior horn motor neurons), photoreceptor cells (e.g., rods and cones), retinal pigment epithelial cells, secretory cells, cardiac cells, adipocytes, vascular smooth muscle cells, cardiac myocytes, skeletal muscle cells, beta cells, pituitary cells, synovial lining cells, ovarian cells, testicular cells, fibroblasts, B cells, T cells, reticulocytes, leukocytes, granulocytes, and tumor cells.

[0070] The compositions of the present invention can be prepared to distribute preferentially to target cells in, for example, the heart, lung, kidney, liver, and spleen. In some embodiments, the compositions of the present invention distribute to liver cells to facilitate delivery and subsequent expression of the mRNA contained therein by the liver cells (e.g., hepatocytes). Targeted hepatocytes can function as biological "reservoirs" or "depots" that can produce and systemically excrete functional proteins or enzymes. Thus, in one embodiment of the present invention, the liposomal transfer vehicle targets hepatocytes and / or distributes preferentially to liver cells upon delivery. After transfection of the target hepatocytes, the mRNA loaded in the liposomal carrier is translated, and functional protein products are produced, excreted, and distributed systemically. In other embodiments, cells other than hepatocytes (e.g., lung, spleen, heart, eye, or central nervous system cells) can serve as depot locations for protein production.

[0071] In one embodiment of the present invention, the composition promotes the endogenous production of one or more functional proteins and / or enzymes in a subject, specifically the production of proteins and / or enzymes that exhibit lower immunogenicity compared to their recombinantly prepared counterparts. In a preferred embodiment of the present invention, the transfer vehicle comprises an mRNA that encodes a missing protein or enzyme. Upon distribution of such compositions to a target tissue and subsequent transfection of such target cells, the exogenous mRNA loaded into the liposomal transfer vehicle (e.g., lipid nanoparticles) can be translated in vivo to produce a functional protein or enzyme (e.g., a protein or enzyme that the subject is deficient in) encoded by the exogenously administered mRNA. Thus, the composition of the present invention utilizes the subject's ability to translate exogenously or recombinantly prepared mRNA to produce a translated protein or enzyme in vivo, thus producing (and optionally excreting) a functional protein or enzyme. The expressed or translated protein or enzyme can also feature in vivo inclusion of native post-translational modifications that may often be absent in recombinantly prepared proteins or enzymes, thus further reducing the immunogenicity of the translated protein or enzyme.

[0072] Administration of mRNA encoding the missing protein or enzyme avoids the need to deliver the nucleic acid to a specific organelle (e.g., mitochondria) within the target cell. Rather, upon transfection of the target cell and delivery of the nucleic acid to the cytoplasm of the target cell, the mRNA content of the transfer vehicle can be translated and a functional protein or enzyme can be expressed.

[0073] The present invention also contemplates the differential targeting of target cells and tissues using both passive and active targeting means.Passive targeting phenomenon utilizes the natural distribution pattern of transfer vehicle in vivo, without relying on the use of additional excipients or means to enhance the recognition of transfer vehicle by target cells.For example, transfer vehicle that is subject to phagocytosis by reticuloendothelial cells is likely to accumulate in the liver or spleen, and therefore can provide a means to passively direct the delivery of composition to such target cells.

[0074] Alternatively, the present invention contemplates active targeting with the use of additional excipients, referred to herein as "targeting ligands", that can be bound (either covalently or non-covalently) to the transfer vehicle to promote the localization of such transfer vehicle in a target cell or tissue. For example, targeting can be mediated by the inclusion of one or more endogenous targeting ligands (e.g., apolipoprotein E) in or on the transfer vehicle to promote distribution to the target cell or tissue. The recognition of the targeting ligand by the target tissue actively promotes the tissue distribution and cellular uptake of the transfer vehicle and / or its contents in the target cells and tissues (e.g., the inclusion of an apolipoprotein E targeting ligand in or on the transfer vehicle promotes the recognition of the transfer vehicle and its binding to the endogenous low-density lipoprotein receptor expressed by hepatocytes). The compositions provided herein can include ligands that can increase the affinity of the composition to the target cell. Targeting ligands can be attached to the outer bilayer of lipid particles during or after formulation. These methods are well known in the art. In addition, some lipid particle formulations can use fusion polymers such as PEAA, hemagglutinin, other lipopeptides (see U.S. Patent Application Nos. 08 / 835,281 and 60 / 083,294, which are incorporated herein by reference), and other features useful for in vivo and / or intracellular delivery. In some other embodiments, the compositions of the present invention show improved transfection efficacy and / or enhanced selectivity for the intended target cells or tissues. Thus, compositions are contemplated that include one or more ligands (e.g., peptides, aptamers, oligonucleotides, vitamins, or other molecules) that can increase the affinity of the compositions and their nucleic acid content for the target cells or tissues. Suitable ligands can be optionally bound or linked to the surface of the transfer vehicle. In some embodiments, the targeting ligands can be spread on the surface of the transfer vehicle or encapsulated within the transfer vehicle. Suitable ligands are selected based on their physical, chemical, or biological properties (eg, selective affinity and / or recognition of a target cell surface marker or feature).Cell-specific targeting sites and their corresponding targeting ligands can vary widely. Suitable targeting ligands are selected so that the unique properties of target cells are utilized, thus allowing the composition to distinguish between target cells and non-target cells. For example, the composition of the present invention can include a surface marker (e.g., apolipoprotein B or apolipoprotein E) that selectively enhances the recognition or affinity to hepatocytes (e.g., by receptor-mediated recognition of and binding to such surface markers). Furthermore, the use of galactose as a targeting ligand is expected to direct the composition of the present invention to parenchymal hepatocytes, or alternatively, the use of mannose-containing sugar residues as a targeting ligand is expected to direct the composition of the present invention to liver endothelial cells (e.g., mannose-containing sugar residues that can preferentially bind to asialoglycoprotein receptors present in hepatocytes) (see Hillery AM, et al. "Drug Delivery and Targeting: For Pharmacists and Pharmaceutical Scientists" (2002) Taylor & Francis, Inc.). Thus, the presentation of such targeting ligands conjugated to moieties present in the transfer vehicle (e.g., lipid nanoparticles) facilitates the recognition and uptake of the compositions of the present invention in target cells and tissues. Examples of suitable targeting ligands include one or more peptides, proteins, aptamers, vitamins, and oligonucleotides. Application and Administration

[0075] The term "subject" as used herein refers to any animal (e.g., mammal) to which the compositions and methods of the invention are administered, including, but not limited to, humans, non-human primates, rodents, etc. Typically, the terms "subject" and "patient" are used interchangeably herein in reference to human subjects.

[0076] The compositions and methods of the present invention provide for the delivery of mRNA to treat several disorders. Specifically, the compositions and methods of the present invention are suitable for the treatment of diseases or disorders associated with defective proteins and / or enzymes (e.g., mRNAs encoding hormones and neurotransmitters) that are excreted or secreted by target cells into the surrounding extracellular fluid. In embodiments, the disease may involve a defect or deficiency in secreted proteins (e.g., Fabry disease, or ALS). In some embodiments, the disease may not be caused by a defect or deficiency in secreted proteins, but may benefit from the provision of secreted proteins. For example, the symptoms of the disease may be improved by providing the compositions of the present invention (e.g., cystic fibrosis).Disorders in which the invention is useful include Huntington's disease; Parkinson's disease; muscular dystrophies (e.g., Duchenne and Becker); hemophilia (e.g., hemophilia B (FIX), hemophilia A (FVIII), etc.); SMN1-associated spinal muscular atrophy (SMA); amyotrophic lateral sclerosis (ALS); GALT-associated galactosemia; cystic fibrosis (CF); SLC3A1-associated disorders, including cystinuria; COL4A5-associated disorders, including Alport syndrome; galactocerebrosidase deficiency; X-linked adrenoleukodystrophy and adrenomyeloneuropathy; Friedreich's ataxia; Pelizaeus-Merzbacher disease; TSC1- and TSC2-associated tuberous sclerosis; Sanfilippo B syndrome (MPS IIIB); CTNS-associated cystinosis; FMR1-associated disorders including fragile X syndrome, fragile X-associated tremor / ataxia syndrome, and fragile X early ovarian syndrome; Prader-Willi syndrome; hereditary hemorrhagic telangiectasia (AT); Niemann-Pick disease type C1; neuronal ceroid lipofuscinosis-associated disorders including juvenile neuronal ceroid lipofuscinosis (JNCL), juvenile Batten disease, Hartia-Santavuori disease, Jansky-Bierschowsky disease, and PTT1 and TPP1 deficiency; EIF2B1, EIF2B2, EIF2B3, EIF2B4, and EIF2B5-associated childhood ataxia with central nervous system hypomyelination / white matter loss; CACNA1A- and CACNB4-associated transient ataxia type 2; These disorders include, but are not limited to, disorders such as Lassic-Rett syndrome, MECP2-associated severe neonatal encephalopathy, and PPM-X syndrome; CDKL5-associated A atypical Rett syndrome; Kennedy's disease (SBMA); Notch3-associated cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL); SCN1A and SCN1B-associated seizure disorder; Alpers-Huttenlocher syndrome, polymerase G-associated disorders including POLG-associated sensory ataxic neuropathy, dysarthria, and ophthalmoplegia, and autosomal dominant and recessive progressive external ophthalmoplegia with mitochondrial DNA deletions; X-linked adrenal hypoplasia; X-linked agammaglobulinemia; Wilson's disease; and Fabry disease. In one embodiment, the nucleic acid of the invention, and specifically, the mRNA, may encode a functional protein or enzyme that is secreted into the extracellular space.For example, secreted proteins include clotting factors, complement pathway components, cytokines, chemokines, chemoattractants, protein hormones (e.g., EGF, PDF), serum protein components, antibodies, secreted Toll-like receptors, etc. In some embodiments of the invention, the composition may include mRNA encoding erythropoietin, α1-antitrypsin, carboxypeptidase N, or human growth hormone.

[0077] In an embodiment, the invention encodes a secreted protein composed of subunits encoded by two or more genes. For example, the secreted protein may be a heterodimer, with each chain or subunit encoded by an individual gene. Two or more mRNA molecules may be delivered in a transfer vehicle, and the mRNAs may encode individual subunits of the secreted protein. Alternatively, a single mRNA may be engineered to encode two or more subunits (e.g., in the case of a single chain Fv antibody). In an embodiment, individual mRNA molecules encoding separate subunits may be administered in individual transfer vehicles. In an embodiment, the mRNA may encode a full-length antibody (both heavy and light chains of the variable and constant regions) or a fragment of an antibody (e.g., Fab, Fv, or single chain Fv (scFv)) to immunize a subject. Although one embodiment of the present invention relates to methods and compositions useful for immunizing a subject (e.g., via translation of an mRNA encoding a functional antibody), the invention disclosed herein and contemplated by the present invention is broadly applicable. In alternative embodiments, the compositions of the invention encode antibodies that are used to affect functional responses in a subject, either transiently or long-term. For example, the mRNA of the invention may encode functional monoclonal or polyclonal antibodies that, upon translation and secretion from a target cell, may be useful for targeting and / or inactivating biological targets (e.g., stimulatory cytokines such as tumor necrosis factor). Similarly, the mRNA nucleic acids of the invention may encode functional anti-nephritic factor antibodies, useful, for example, for treating membranoproliferative glomerulonephritis type II or acute hemolytic uremic syndrome, or alternatively, may encode anti-vascular endothelial growth factor (VEGF) antibodies, useful for treating VEGF-mediated diseases, such as cancer. In other embodiments, the secreted protein is a cytokine or other secreted protein that is composed of two or more subunits (e.g., IL-12 or IL-23).

[0078] The composition of the present invention can be administered to a subject. In some embodiments, the composition is formulated in combination with one or more additional nucleic acids, carriers, targeting ligands, or stabilizing reagents, or is formulated into a pharmacological composition mixed with a suitable excipient. For example, in one embodiment of the present invention, the composition can be prepared to deliver mRNA that codes for two or more distinct proteins or enzymes. Techniques for drug formulation and administration can be found in "Remington's Pharmaceutical Sciences" (Mack Publishing Co., Easton, Pa., latest edition).

[0079] A variety of molecules that can have pharmaceutical or therapeutic effects can be delivered to target cells using the compositions and methods of the present invention. Molecules can be organic or inorganic. Organic molecules can be peptides, proteins, carbohydrates, lipids, sterols, nucleic acids (including peptide nucleic acids), or any combination thereof. Formulations for delivery to target cells can include one or more types of molecules, for example, two different nucleotide sequences, or proteins, enzymes, or steroids.

[0080] The composition of the present invention may be administered and dosed according to current medical practice, taking into consideration the subject's clinical condition, administration site and method, administration schedule, subject's age, sex, weight, and other factors appropriate to a clinician in the art. For the purposes of this specification, the "effective amount" may be determined by such relevant considerations known to those skilled in the art in experimental clinical research, pharmacology, clinical, and medical fields. In some embodiments, the dosage is effective to achieve at least some stabilization, improvement, or elimination of symptoms, and other indicators are selected by the skilled artisan as appropriate evaluation criteria for disease progression, regression, or improvement. For example, a suitable amount and dosage regimen is one that causes at least transient protein production.

[0081] Suitable routes of administration include, for example, oral, rectal, vaginal, transmucosal, intratracheal or pulmonary, including inhalation, or intestinal administration; parenteral delivery, including intramuscular, subcutaneous, intramedullary injection, and intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injection.

[0082] Alternatively, the compositions of the present invention may be administered locally, rather than systemically, for example, via direct injection of the pharmaceutical composition into the targeted tissue, preferably in a sustained release formulation. Local delivery may work in various ways depending on the targeted tissue. For example, an aerosol containing the compositions of the present invention may be inhaled for nasal, tracheal, or bronchial delivery, the compositions of the present invention may be injected, for example, into the site of injury, disease symptoms, or pain, the compositions may be provided in a lozenge for oral, tracheal, or esophageal application, may be provided in liquid, tablet, or capsule form for administration to the stomach or intestine, may be provided in suppository form for rectal or vaginal application, or may even be delivered to the eye by using creams, drops, or even by injection. Formulations containing the compositions of the present invention conjugated with therapeutic molecules or ligands may even be administered surgically, for example, by polymers or other structures or materials that may allow the compositions to diffuse from the injection site to surrounding cells. Alternatively, they may be applied surgically without the use of polymers or supports.

[0083] In one embodiment of the present invention, the compositions are formulated so that they are suitable for sustained release of the mRNA contained therein. Such sustained release compositions can be conveniently administered to subjects at extended administration intervals. For example, in one embodiment of the present invention, the compositions are administered to subjects twice a day, once a day, or once every two days. In a preferred embodiment of the present invention, the compositions are administered to subjects twice a week, once a week, every 10 days, every 2 weeks, every 3 weeks, or more preferably, every 4 weeks, once a month, every 6 weeks, every 8 weeks, once every 2 months, every 3 months, every 4 months, every 6 months, every 8 months, every 9 months, or once a year. Also contemplated are compositions and liposomal carriers that are formulated for depot administration (e.g., intramuscular, subcutaneous, intravitreal) and either deliver or release the mRNA over an extended period of time. Preferably, the sustained release means used is combined with a modification that improves the stability of the mRNA.

[0084] Also contemplated herein are lyophilized pharmaceutical compositions comprising one or more of the liposomal nanoparticles disclosed herein, and related methods of using such lyophilized compositions, for example, as disclosed in U.S. Provisional Application No. 61 / 494,882, filed June 8, 2011, the teachings of which are incorporated herein by reference in their entirety.For example, the lyophilized pharmaceutical compositions according to the present invention can be reconstituted before administration or reconstituted in vivo.For example, the lyophilized pharmaceutical compositions can be formulated in a suitable dosage form (e.g., intradermal dosage form, such as intervertebral disc, rod, or membrane), and administered such that the dosage form is rehydrated in vivo by the body fluids of an individual over time.

[0085] Although certain compounds, compositions, and methods of the present invention have been clearly described according to certain embodiments, the following examples are merely intended to illustrate the compounds of the present invention and are not intended to limit the compounds of the present invention. Each of the publications, reference materials, accession numbers, etc. referenced herein to describe the background of the invention and to provide further details regarding its practice are hereby incorporated by reference in their entirety.

[0086] The articles "a" and "an" used in the specification and claims should be understood to include plural referents unless clearly indicated to the contrary. A claim or description including "or" between one or more members of a group is considered to be satisfied if one, more than one, or all of the members of the group are present, employed, or otherwise relevant in a given product or process, unless clearly indicated to the contrary or otherwise evident from the context. The invention includes embodiments in which only one member of a group is present, employed, or otherwise relevant in a given product or process. The invention also includes embodiments in which more than one member of a group, or all members of a group are present, employed, or otherwise relevant in a given product or process. Furthermore, it is to be understood that the present invention encompasses all variations, combinations, and permutations of one or more limitations, elements, clauses, descriptive terms, etc., from one or more of the enumerated claims, which are introduced into another claim (or any other related claims) that relies on the same underlying claim, unless otherwise indicated to result in a contradiction or inconsistency, or unless otherwise apparent to a person skilled in the art. When elements are presented in a list (e.g., in a Markush group or similar format), it is to be understood that each subgroup of those elements is also disclosed, and that any element(s) may be removed from the group. When the invention or aspects of the invention are generally considered to include certain elements, features, etc., it is to be understood that an embodiment or aspect of the invention consists or consists in part of such elements, features, etc. For simplicity, those embodiments have not been specifically described in so many words in every instance herein. It is also to be understood that any embodiment or aspect of the invention may be specifically excluded from the claims, regardless of whether a specific exclusion is recited herein. The publications and other reference materials referred to herein to describe the background of the invention and to provide further details regarding its practice are hereby incorporated by reference. Working Example Example 1: Protein production depot via intravenous delivery of polynucleotide compositions

[0087] Messenger RNA Human erythropoietin (EPO) (SEQ ID NO:3, FIG. 3), human alpha-galactosidase (GLA) (SEQ ID NO:4, FIG. 4), human alpha-1 antitrypsin (A1AT) (SEQ ID NO:5, FIG. 5), and human factor IX (FIX) (SEQ ID NO:6, FIG. 6) were synthesized by in vitro transcription from plasmid DNA templates encoding the genes, followed by the addition of a 5' cap structure (Cap1) (Fechter & Brownlee, J. Gen. Virology 86:1239-1249 (2005)) and a 3' poly(A) tail of approximately 200 nucleotides in length as determined by gel electrophoresis. The 5' and 3' untranslated regions are present in each mRNA product in the following examples and are defined by SEQ ID NOs:1 and 2 (FIGS. 1 and 2), respectively. Lipid nanoparticle formulations

[0088] Formulation 1: Aliquots of 50 mg / mL ethanolic solutions of C12-200, DOPE, Chol, and DMG-PEG2K (40:30:25:5) were mixed and diluted with ethanol to a final volume of 3 mL. Separately, a buffered aqueous solution of mRNA (10 mM citrate / 150 mM NaCl, pH 4.5) was prepared from a 1 mg / mL stock solution. The lipid solution was rapidly injected into the aqueous mRNA solution and shaken to obtain a final suspension in 20% ethanol. The resulting nanoparticle suspension was filtered, diafiltered with one aliquot of PBS (pH 7.4), concentrated, and stored at 2-8 °C.

[0089] Formulation 2: Aliquots of 50 mg / mL ethanolic solutions of DODAP, DOPE, cholesterol, and DMG-PEG2K (18:56:20:6) were mixed and diluted with ethanol to a final volume of 3 mL. Separately, a buffered aqueous solution of EPO mRNA (10 mM citrate / 150 mM NaCl, pH 4.5) was prepared from a 1 mg / mL stock solution. The lipid solution was rapidly injected into the aqueous mRNA solution and shaken to obtain a final suspension in 20% ethanol. The resulting nanoparticle suspension was filtered, diafiltered with one aliquot of PBS (pH 7.4), concentrated, and stored at 2-8 °C. Final concentration = 1.35 mg / mL EPO mRNA (encapsulated). Z ave =75.9nm(Dv (50) = 57.3 nm; Dv (90) =92.1 nm).

[0090] Formulation 3: Aliquots of 50 mg / mL ethanolic solutions of HGT4003, DOPE, cholesterol, and DMG-PEG2K (50:25:20:5) were mixed and diluted with ethanol to a final volume of 3 mL. Separately, a buffered aqueous solution of mRNA (10 mM citrate / 150 mM NaCl, pH 4.5) was prepared from a 1 mg / mL stock solution. The lipid solution was rapidly injected into the aqueous mRNA solution and shaken to obtain a final suspension in 20% ethanol. The resulting nanoparticle suspension was filtered, diafiltered with one aliquot of PBS (pH 7.4), concentrated, and stored at 2-8 °C.

[0091] Formulation 4: Aliquots of 50 mg / mL ethanolic solutions of ICE, DOPE and DMG-PEG2K (70:25:5) were mixed and diluted with ethanol to a final volume of 3 mL. Separately, a buffered aqueous solution of mRNA (10 mM citrate / 150 mM NaCl, pH 4.5) was prepared from a 1 mg / mL stock solution. The lipid solution was rapidly injected into the aqueous mRNA solution and shaken to obtain a final suspension in 20% ethanol. The resulting nanoparticle suspension was filtered, diafiltered with one aliquot of PBS (pH 7.4), concentrated, and stored at 2-8 °C.

[0092] Formulation 5: Aliquots of 50 mg / mL ethanolic solutions of HGT5000, DOPE, cholesterol, and DMG-PEG2K (40:20:35:5) were mixed and diluted with ethanol to a final volume of 3 mL. Separately, a buffered aqueous solution of EPO mRNA (10 mM citrate / 150 mM NaCl, pH 4.5) was prepared from a 1 mg / mL stock solution. The lipid solution was rapidly injected into the aqueous mRNA solution and shaken to obtain a final suspension of 20% ethanol. The resulting nanoparticle suspension was filtered, diafiltered with one aliquot of PBS (pH 7.4), concentrated, and stored at 2-8 °C. Final concentration = 1.82 mg / mL EPO mRNA (encapsulated). Z ave =105.6nm(Dv (50) =53.7nm, Dv (90) =157 nm).

[0093] Formulation 6: Aliquots of 50 mg / mL ethanolic solutions of HGT5001, DOPE, cholesterol, and DMG-PEG2K (40:20:35:5) were mixed and diluted with ethanol to a final volume of 3 mL. Separately, a buffered aqueous solution of EPO mRNA (10 mM citrate / 150 mM NaCl, pH 4.5) was prepared from a 1 mg / mL stock solution. The lipid solution was rapidly injected into the aqueous mRNA solution and shaken to obtain a final suspension in 20% ethanol. The resulting nanoparticle suspension was filtered, diafiltered with one aliquot of PBS (pH 7.4), concentrated, and stored at 2-8 °C. Analysis of proteins produced via intravenously delivered mRNA-loaded nanoparticles Injection protocol

[0094] Unless otherwise indicated, studies were performed with male CD-1 mice approximately 6-8 weeks of age at the beginning of each experiment. Samples were introduced by a single bolus tail vein injection of encapsulated mRNA at a total equivalent dose of 30-200 micrograms. Mice were sacrificed and perfused with saline at the indicated time points. Isolation of organ tissue for analysis

[0095] The liver and spleen of each mouse were harvested, divided into triplicates, and either preserved in 10% neutral buffered formalin or snap frozen and stored at -80°C for analysis. Isolation of serum for analysis

[0096] All animals were euthanized by CO2 asphyxiation 48 h after dose administration (± 5%), followed by thoracotomy and terminal cardiac blood collection. Whole blood (maximum amount available) was collected via cardiac puncture from euthanized animals into serum separator tubes, allowed to clot for at least 30 min at room temperature, and centrifuged at 9300g for 10 min at 22 °C ± 5 °C to extract serum. For interim blood collection, approximately 40-50 µL of whole blood was collected via facial vein puncture or tail cut. Samples collected from untreated animals were used as baseline for comparison in animal studies. Enzyme-linked immunosorbent assay (ELISA) analysis

[0097] EPO ELISA: EPO protein quantification was performed according to the procedure reported for the Human EPO ELISA kit (Quantikine IVD, R&D Systems, Cat. No. Dep-00). The positive controls used consisted of ultrapure and tissue culture grade recombinant human erythropoietin protein (R&D Systems, Cat. Nos. 286-EP and 287-TC, respectively). Detection was monitored via absorbance (450 nm) on a Molecular Device Flex Station instrument.

[0098] GLA ELISA: Standard ELISA procedures were followed using sheep anti-α-galactosidase G-188 IgG as the capture antibody and rabbit anti-α-galactosidase TK-88 IgG as the secondary (detection) antibody (Shire Human Genetic Therapies). Horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG was used to activate 3,3',5,5'-tetramethylbenzidine (TMB) substrate solution. The reaction was stopped after 20 min with 2N H2SO4. Detection was monitored via absorbance (450 nm) on a Molecular Device Flex Station instrument. Untreated mouse serum and human α-galactosidase protein were used as negative and positive controls, respectively.

[0099] FIX ELISA: Quantification of FIX protein was performed according to the procedure reported for the human FIX ELISA kit (AssayMax, Assay Pro, Cat. No. EF1009-1).

[0100] A1AT ELISA: Quantification of A1AT protein was performed according to the procedure reported for the human A1AT ELISA kit (Innovative Research, Cat. No. IRAPKT015). Western blot analysis

[0101] (EPO): Western blot analysis was performed using anti-hEPO antibody (R&D Systems, No. MAB2871) and ultra-pure human EPO protein (R&D Systems, No. 286-EP) as a control. result

[0102] The studies described in this example demonstrate the use of mRNA-encapsulated lipid nanoparticles as a depot source for protein production. Such a depot effect can be achieved at multiple sites within the body (i.e., liver, kidney, spleen, and muscle). Measurements of desired exogenous proteins derived from messenger RNA delivered via liposomal nanoparticles were obtained and quantified, and secretion of proteins from the depot was demonstrated using human erythropoietin (hEPO), human alpha-galactosidase (hGLA), human alpha-1 antitrypsin (hA1AT), and human factor IX (hFIX) mRNA. 1A. In vivo human EPO protein production results

[0103] We demonstrated the production of hEPO protein using various lipid nanoparticle formulations. Of the four different cationic lipid systems, C12-200-based lipid nanoparticles produced the most abundant amount of hEPO protein 4 hours after intravenous administration as measured by ELISA (Figure 7). This formulation (formulation 1) resulted in 18.3ug / mL of hEPO protein secreted into the bloodstream. Normal hEPO protein levels in human serum are 3.3-16.6mIU / mL (NCCLS Document C28-P, Vol. 12, No. 2). Based on a specific activity of 120,000IU / mg of EPO protein, this formulation produces 27.5-138pg / mL of hEPO protein in normal human individuals. Thus, a single dose of 30ug of C12-200-based cationic lipid formulation encapsulating hEPO mRNA resulted in an increase of the respective physiological levels of protein by more than 100,000-fold.

[0104] Of the lipid systems tested, the DODAP-based lipid nanoparticle formulation was the least effective, however, the observed amount of human EPO protein resulting from delivery via DODAP-based lipid nanoparticles encapsulating EPO mRNA was 4.1 ng / mL, which is still 30-fold higher than normal physiological levels of EPO protein (Table 1). [Table 1]

[0105] Additionally, the resulting protein was tested to determine if it was active and functioned properly. For mRNA replacement therapy (MRT) with hEPO mRNA, the change in hematocrit of five different lipid nanoparticle formulations (Figure 8, Table 1) was monitored over a 10 day period to assess protein activity. During this period, two of the five formulations showed an increase in hematocrit (greater than 15%), indicating activity of the hEPO protein produced from such a system.

[0106] In a separate experiment, the change in hematocrit was monitored over a 15 day period (Figure 9, Table 2). The lipid nanoparticle formulation (Formulation 1) was administered either in a single dose of 30 μg or in three smaller doses of 10 μg (injected on days 1, 3, and 5). Similarly, Formulation 2 was administered in three doses of 50 μg (on days 1, 3, and 5). C12-200 resulted in a significant increase in hematocrit. Overall, changes of up to about 25% increase were observed, indicating activity of the human EPO protein produced from such a system. [Table 2] 1B. In vivo human GLA protein production results

[0107] A second exogenous protein system was studied to demonstrate a "depot effect" when using mRNA-loaded lipid nanoparticles. Animals were intravenously injected with a single 30 microgram dose of encapsulated human α-galactosidase (hGLA) mRNA using a C12-200-based lipid nanoparticle system and sacrificed 6 hours later (Formulation 1). Quantification of secreted hGLA protein was performed using ELISA. Untreated mouse serum and human α-galactosidase protein were used as controls. Detection of human α-galactosidase protein was monitored over a 48-hour period.

[0108] Measurable levels of hGLA protein were observed throughout the time course of the experiment, with a maximum level of 2.0 ug / mL of hGLA protein at 6 hours (Figure 10). Table 3 lists the specific amounts of hGLA found in serum. Normal activity in healthy human males is reported to be approximately 3.05 nmoles / hour / mL. In terms of α-galactosidase activity, recombinant human α-galactosidase protein produced 3.56 x 10 6 The analysis of these values ​​results in an amount of hGLA protein of about 856 pg / mL in normal and healthy male individuals. The amount of hGLA protein of 2.0 ug / mL observed 6 hours after administration of hGLA mRNA-loaded lipid nanoparticles is more than 2300 times higher than normal physiological levels. Furthermore, after 48 hours, significant levels of hGLA protein (86.2 ng / mL) can still be detected. This level represents almost 100 times more hGLA protein than the physiological amount still present at 48 hours. [Table 3]

[0109] In addition, the half-life of α-galactosidase at 0.2 mg / kg is approximately 108 minutes. The production of GLA protein via the "depot effect" when GLA mRNA-loaded lipid nanoparticles are administered shows a significant increase in blood residence time compared to direct injection of naked recombinant protein. As mentioned above, significant amounts of protein are present after 48 hours.

[0110] The activity profile of α-galactosidase protein produced from GLA mRNA-loaded lipid nanoparticles was measured as a function of 4-methylumbelliferyl-α-D-galactopyranoside (4-MU-α-gal) metabolism. As shown in Figure 11, the protein produced from these nanoparticle systems is fairly active and reflects the levels of available protein (Figure 12, Table 3). AUC comparison of mRNA therapy-based hGLA production versus enzyme replacement therapy (ERT) in mice and humans shows a 182-fold and 30-fold increase, respectively (Table 4). [Table 4]

[0111] The ability of mRNA-encapsulated lipid nanoparticles to target organs that can act as depots for desired protein production has been demonstrated. The secreted protein levels observed were several orders of magnitude greater than normal physiological levels. This "depot effect" is repeatable. Figure 12 recapitulates that robust protein production is observed when a single 30ug dose of hGLA mRNA-loaded C12-200-based lipid nanoparticles (formulation 1) is administered to wild-type (CD-1) mice. In this experiment, hGLA levels were assessed over a 72-hour period. A maximum average of 4.0ug of human hGLA protein / mL of serum is detected 6 hours after administration. Based on a value of approximately 1ng / mL of hGLA protein at normal physiological levels, the hGLA MRT provides approximately 4000-fold higher protein levels. As before, hGLA protein could be detected up to 48 hours after administration (Figure 12).

[0112] Analysis of tissues isolated from this same experiment provided insight into the distribution of hGLA protein in mice treated with hGLA MRT (Figure 13). Supraphysiological levels of hGLA protein were detected in the liver, spleen, and kidneys of all treated mice, with maximum levels observed 12-24 hours after administration. Detectable levels of MRT-derived protein could be observed 3 days after a single injection of hGLA-loaded lipid nanoparticles.

[0113] In addition, production of hGLA upon administration of hGLA mRNA-loaded C12-200 nanoparticles was shown to exhibit a dose response in serum (FIG. 14A) as well as in the liver (FIG. 14B).

[0114] One of the unique properties of lipid nanoparticle-mediated mRNA replacement therapy is the pharmacokinetic profile of the respective protein produced. For example, mice treated with ERT using α-galactosidase results in a plasma half-life of approximately 100 minutes. In contrast, MRT-derived α-galactosidase has a blood residence time of approximately 72 hours, peaking at 6 hours. This allows for much higher exposure to organs involved in the expected continuous uptake of the desired protein. A comparison of the PK profiles is shown in Figure 15, showing clear differences in clearance rates and, ultimately, large changes in area under the curve (AUC) can be achieved via MRT-based therapy.

[0115] In separate experiments, hGLA MRT was applied to hGLA knockout mice (Fabry mice) in a mouse disease model. A dose of 0.33 mg / kg of hGLA mRNA-loaded C12-200-based lipid nanoparticles (formulation 1) was administered to female knockout mice in a single intravenous injection. Significant amounts of MRT-derived hGLA protein were produced, peaking at 6 hours (approximately 560 ng / mL serum), which is approximately 600-fold higher than normal physiological levels. Moreover, hGLA protein was still detectable 72 hours after administration (Figure 16).

[0116] Quantification of MRT-derived GLA protein in vital organs showed significant accumulation, as shown in Figure 17. Comparison of MRT-derived hGLA protein found in observed major organs with reported normal physiological levels is plotted (normal levels are plotted with dotted lines). While the protein levels at 24 hours are higher than those at 72 hours after administration, the levels of hGLA protein detected in liver, kidney, spleen and heart of treated Fabry mice are comparable to wild-type levels. For example, 3.1ng of hGLA protein per mg of tissue was found in the kidney of treated mice 3 days after a single MRT treatment.

[0117] Subsequent experiments compared ERT-based α-galactosidase treatment with hGLA MRT-based treatment in male Fabry knockout mice. A single intravenous dose of 1.0 mg / kg of each regimen was administered, and mice were sacrificed one week after administration. Serum levels of hGLA protein were monitored 6 hours and one week after injection. One week after administration, liver, kidney, spleen, and heart were analyzed for hGLA protein accumulation. In addition to biodistribution analysis, efficacy measures were determined using measurements of globotriaosylceramide (Gb3) and Lyso-Gb3 reduction in kidney and heart. Figure 18 shows serum levels of hGLA protein in male Fabry mice after treatment with either α-galactosidase or GLA mRNA-loaded lipid nanoparticles (Formulation 1). Serum samples were analyzed 6 hours and one week after administration. A robust signal was detected in MRT-treated mice after 6 hours, with hGLA protein serum levels of approximately 4.0 ug / mL In contrast, no detectable α-galactosidase remained in the bloodstream at this time point.

[0118] Fabry mice in this experiment were sacrificed one week after the first injection and organs were harvested and analyzed (liver, kidney, spleen, heart). Figure 19 shows a comparison of human GLA protein found in each respective organ after either hGLA MRT treatment or α-galactosidase ERT treatment. Levels correspond to the hGLA present one week after administration. hGLA protein was detected in all organs analyzed. For example, mice treated with MRT resulted in hGLA protein accumulation of 2.42 ng hGLA protein / mg protein in the kidney, while mice treated with α-galactosidase only had residual levels (0.37 ng / mg protein). This corresponds to approximately 6.5-fold higher levels of hGLA protein when treated with hGLA MRT. In the analysis of the heart, 11.5 ng hGLA protein / mg protein was found in the MRT-treated cohort compared to only 1.0 ng α-galactosidase / mg protein. This corresponds to approximately 11-fold higher accumulation in the hearts of mice treated with hGLA MRT than with ERT-based therapy.

[0119] In addition to the biodistribution analysis performed, efficacy assessment was determined using measurements of globotriaosylceramide (Gb3) and Lyso-Gb3 levels in major organs. Direct comparison of Gb3 reduction after GLA MRT treatment at a single intravenous dose of 1.0 mg / kg with α-galactosidase ERT-based therapy at comparable doses resulted in significant differences in Gb3 levels in the kidney as well as the heart. For example, Gb3 levels of GLA MRT and α-galactosidase resulted in a 60.2% and 26.8% reduction, respectively (Figure 20). Furthermore, Gb3 levels in the heart were reduced by 92.1% and 66.9% for MRT and α-galactosidase, respectively (Figure 21).

[0120] A second relevant biomarker for efficacy measurement is Lyso-Gb3. GLA MRT reduced Lyso-Gb3 more efficiently than α-galactosidase in the kidney and heart as well (Figures 20 and 21, respectively). Specifically, Fabry mice treated with MRT showed 86.1% and 87.9% Lyso-Gb3 reduction in the kidney and heart, respectively, whereas mice treated with α-galactosidase produced a reduction of 47.8% and 61.3%.

[0121] The results with hGLA in C12-200-based lipid nanoparticles extend to other lipid nanoparticle formulations. For example, hGLA mRNA loaded in HGT4003 (formulation 3) or HGT5000-based (formulation 5) lipid nanoparticles administered in a single intravenous dose results in the production of hGLA at 24 hours after administration (Figure 22). hGLA production showed a dose response. Similarly, hGLA production was observed at 6 and 24 hours after administration of hGLA mRNA loaded in HGT5001-based (formulation 6) lipid nanoparticles administered in a single intravenous dose. hGLA production was observed in serum (Figure 23A) as well as in organs (Figure 23B).

[0122] Overall, mRNA replacement therapy applied as a depot for protein production produces large amounts of functionally therapeutic active proteins at supraphysiological levels. It has been demonstrated that this method results in a sustained circulating half-life of the desired protein, and this MRT-derived protein is highly effective in therapy as demonstrated with the α-galactosidase enzyme in Fabry mice. 1C. In vivo human FIX protein production results

[0123] A study was conducted to administer Factor IX (FIX) mRNA-loaded lipid nanoparticles to wild-type mice (CD-1) to determine FIX protein secreted into the bloodstream.Robust protein production was observed upon intravenous injection of a single dose of 30ug C12-200-based (40:30:25:5 ratio of C12-200:DOPE:Chol:PEG) FIX mRNA-loaded lipid nanoparticles (dosage based on encapsulated mRNA) (Formulation 1) (Figure 24).

[0124] Pharmacokinetic analysis over 72 hours showed that MRT-derived FIX protein could be detected at all time points tested (Figure 24). Peak serum concentrations were observed at 24 hours post-infusion, with values ​​of approximately 3ug (2995±738ng / mL) FIX protein / mL serum. This represents another successful example of the depot effect. 1D. In vivo human A1AT protein production results

[0125] A study was conducted to administer alpha-1-antitrypsin (A1AT) mRNA-loaded lipid nanoparticles to wild-type mice (CD-1) and determine A1AT protein secreted into the bloodstream. Robust protein production was observed upon intravenous injection of a single dose of 30ug C12-200-based A1AT mRNA-loaded lipid nanoparticles (dosage based on encapsulated mRNA) (Formulation 1) (Figure 25).

[0126] Detectable levels of human A1AT protein derived from the A1AT MRT were observed for 24 hours after administration, as shown in Figure 25. Maximum serum levels of approximately 48ug A1AT protein / mL serum were detected 12 hours after injection. Example 2: Protein production depot via pulmonary delivery of polynucleotide compositions Injection protocol

[0127] All studies were performed with female CD-1 or BALB / C mice approximately 7-10 weeks of age at the beginning of each experiment. Test substances were introduced via a single intratracheal aerosolized dose. Mice were sacrificed and perfused with saline at the indicated time points. Lungs from each mouse were harvested, divided into two, and either preserved in 10% neutral buffered formalin or snap frozen and stored at -80°C for analysis. Serum was isolated as described in Example 1. EPO ELISA: As described in Example 1. result

[0128] A depot effect can be achieved via pulmonary delivery (e.g., intranasal, intratracheal spray).Measurements of desired exogenous protein derived from messenger RNA delivered via nanoparticle systems have been obtained and quantified.

[0129] Human EPO protein production via hEPO mRNA-loaded lipid nanoparticles in CD-1 mice was tested via single intratracheal administration (MicroSprayer®). Several formulations were tested using different cationic lipids (formulations 1, 5, 6). All formulations resulted in highly encapsulated human EPO mRNA. Upon administration, animals were sacrificed 6 hours after administration and lungs as well as serum were collected.

[0130] Upon treatment via aerosol delivery, human EPO protein was detected at the administration site (lungs). Analysis of serum 6 hours after administration showed detectable amounts of hEPO protein in the blood. These data (shown in FIG. 26) demonstrate the ability of the lungs to act as a "depot" for the production (and secretion) of hEPO protein.

Claims

1. 1. A composition for delivery of messenger RNA (mRNA) for in vivo production of a protein or peptide, comprising: the composition comprises an mRNA encoding the protein or peptide and a lipid nanoparticle, the mRNA being encapsulated within the lipid nanoparticle; Administration of the composition results in expression of the protein or peptide encoded by the mRNA, and the lipid nanoparticles (i) a PEG-modified lipid at a molar ratio of greater than 1% of the total lipid in the lipid nanoparticle; (ii) a cationic lipid at a molar ratio of 20% to 70% of the total lipid in the lipid nanoparticle; (iii) a molar ratio of non-cationic lipids greater than 5% of the total lipids in the lipid nanoparticles; and (iv) cholesterol is greater than 10% of the total lipid in the lipid nanoparticles; Including, The non-cationic lipid comprises DOPE or DSPC; composition.

2. The composition of claim 1 , wherein the non-cationic lipid comprises DSPC.

3. The composition of claim 2, wherein the lipid nanoparticles comprise a PEG-modified lipid in a molar ratio of 1% to 20% of the total lipid.

4. The composition of claim 3, wherein the lipid nanoparticles comprise a molar ratio of non-cationic lipid of 5% to 90% of the total lipid.

5. The composition of claim 1 , wherein the non-cationic lipid comprises DOPE.

6. The composition of claim 5, wherein the one or more PEG-modified lipids comprise DMG-PEG-2000.

7. The composition of claim 1, which is administered systemically.

8. The composition of claim 1, which is administered intramuscularly.

9. The composition of claim 1, which is administered subcutaneously.

10. The composition of claim 1, wherein administration of the composition results in detectable expression of the protein or peptide encoded by the mRNA in a target tissue for at least 48 hours after administration.

11. The composition of claim 1 , wherein the mRNA is modified.

12. The composition of claim 11 , wherein the mRNA comprises a pseudouridine.

13. The composition of claim 1 , wherein the mRNA comprises a 3′ untranslated region.

14. The composition of claim 1 , wherein the mRNA comprises a cap structure.

15. The composition of claim 1 , wherein the mRNA comprises a polyA tail.

16. The composition of claim 1 , wherein the lipid nanoparticles have dimensions of less than 150 nm.

17. 17. The composition of claim 16, wherein the lipid nanoparticles have dimensions of less than 125 nm.

18. 20. The composition of claim 17, wherein the lipid nanoparticles have dimensions less than 100 nm.

19. The composition of claim 1 , wherein administration of the composition confers immunity to a subject.

20. The composition of claim 1 , wherein the target tissue comprises muscle cells.

21. 21. The composition of claim 20, wherein the muscle cells comprise skeletal muscle cells or vascular smooth muscle cells.

22. 1. A composition comprising an mRNA encoding a protein or peptide encapsulated within a lipid nanoparticle, The lipid nanoparticles have a dimension of less than 150 nm; (i) a PEG-modified lipid at a molar ratio of greater than 1% of the total lipid in the lipid nanoparticle; (ii) a cationic lipid at a molar ratio of 20% to 70% of the total lipid in the lipid nanoparticle; (iii) a molar ratio of non-cationic lipids greater than 5% of the total lipids in the lipid nanoparticles; and (iv) cholesterol is greater than 10% of the total lipid in the lipid nanoparticles; Including, The mRNA is (i) a 5′-UTR, (ii) 3'-UTR, (iii) a 5′ cap structure, and (iv) a poly A tail A composition comprising:

23. The composition of claim 22, wherein the lipid nanoparticles comprise a PEG-modified lipid in a molar ratio of 1% to 15% of the total lipid.

24. 24. The composition of claim 23, wherein the lipid nanoparticles comprise a molar ratio of non-cationic lipid of 5% to 90% of the total lipid.

25. 25. The composition of claim 24, wherein the one or more non-cationic lipids comprises DSPC.

26. 25. The composition of claim 24, wherein the one or more non-cationic lipids comprise DOPE.

27. 25. The composition of claim 24, wherein the one or more PEG-modified lipids comprise DMG-PEG-2000.

28. 23. The composition of claim 22, wherein the polyA tail comprises at least 90 nucleotides.

29. 30. The composition of claim 28, wherein the polyA tail comprises at least 200 nucleotides.

30. 23. The composition of claim 22, wherein the lipid nanoparticles have dimensions of less than 125 nm.

31. 31. The composition of claim 30, wherein the lipid nanoparticles have dimensions less than 100 nm.

32. 23. The composition of claim 22, wherein the mRNA is modified.

33. The composition of claim 32, wherein the mRNA comprises a pseudouridine.

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