Lipid nanoparticle compositions and methods for mRNA delivery

Abstract

To provide compositions and methods for regulating protein production in target cells. [Solution] The compositions and methods disclosed herein can improve diseases associated with protein or enzyme deficiency. The present invention provides, for example, a composition comprising (a) at least one mRNA molecule that is at least partly encoding a functionally secreted polypeptide, and (b) a transport vehicle comprising lipid nanoparticles. The present invention also provides a method for treating a subject deficient in a functional polypeptide, comprising administering a composition comprising (a) at least one mRNA that is at least partly encoding the functionally secreted polypeptide, and (b) a transport vehicle comprising lipid nanoparticles, wherein after administration of the composition, the mRNA is expressed in a target cell to produce the functionally secreted polypeptide.

Description

Background Art

[0001] New approaches and therapies are still needed for the treatment of protein and enzyme deficiencies. For example, lysosomal storage diseases are a group of approximately 50 rare genetic metabolic diseases usually caused by a defect in 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 alpha-galactosidase (GLA), which accumulates a glycolipid known as globotriaosylceramide in blood vessels and other tissues, leading to symptoms associated with various pains. In certain diseases such as Fabry disease, there is a need to replace a protein or enzyme that is normally secreted by cells and enters the bloodstream. Treatments such as gene therapy that increase the level or production of the affected protein or enzyme 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 to insert desired genetic information into host cells. The DNA introduced into cells is usually partially integrated into the genome of one or more transfected cells, enabling the long-term effects of the introduced genetic material in the host. While there may be many advantages to such sustained effects, the integration of exogenous DNA into the host genome can also have many adverse effects. For example, introduced DNA may be inserted into intact genes, resulting in mutations that interfere with or even completely eliminate the function of endogenous genes. Therefore, DNA-based gene therapy can lead to impairments of essential genetic functions in the treated host, such as the elimination or detrimental reduction of essential enzymes or interference with genes essential for regulating cell proliferation, potentially resulting in uncontrolled or cancerous cell proliferation. In addition, conventional DNA-based gene therapy requires the inclusion of a strong promoter sequence for the effective expression of the desired gene product, which can also lead to undesirable changes in the regulation of normal gene expression within cells. DNA-based genetic material may also induce undesirable anti-DNA antibodies, which could then trigger a potentially fatal immune response. Gene therapy approaches using viral vectors can also result in adverse immune responses. In some cases, viral vectors can even be incorporated into the host genome. In addition, the production of clinical-grade viral vectors is expensive and time-consuming. Targeted delivery of introduced genetic material using viral vectors can also be difficult to control. Therefore, although DNA-based gene therapy has been evaluated for the delivery of secreted proteins using viral vectors (Patent Document 1 (U.S. Patent No. 6,066,626), Patent Document 2 (U.S. Patent Application Publication No. 2004 / 0110709)), these approaches may be limited for these various reasons.

[0003] Another apparent obstacle in these earlier approaches to the delivery of nucleic acids encoding secretory proteins lies in the level of the protein ultimately produced. Obtaining a significant level of the desired protein in the blood is difficult, and the amount is not maintained over the long term. For example, the amount of protein produced by nucleic acid delivery does not reach normal physiological levels. See, for example, Patent Document 2 (U.S. Patent Application Publication No. 2004 / 0110709).

[0004] In contrast to DNA, using RNA as a gene therapy agent has advantages: (1) it does not involve the risk of RNA being stably integrated into the genome of transfected cells, thus eliminating concerns that the introduced genetic material may interfere with the normal function of essential genes or cause mutations that lead to harmful effects or carcinogenicity; (2) exogenous promoter sequences are not required for the effective translation of the encoded protein, thus avoiding potential harmful side effects; and (3) unlike plasmid DNA (pDNA), messenger RN A(mRNA) is substantially safer because it lacks an immunogenic CpG motif to prevent the generation of anti-RNA antibodies, and (4) due to the relatively short half-life of RNA, any adverse effects caused by mRNA based on gene therapy are limited to a limited period. In addition, while DNA must overcome this major barrier, mRNA does not need to enter the nucleus to perform its function.

[0005] One reason mRNA-based gene therapy has not been widely used in the past is that mRNA stability is far lower than that of DNA, especially when it reaches the cytoplasm of a cell and is exposed to degrading enzymes. The presence of a hydroxyl group on the second carbon of the sugar moiety in mRNA causes steric hindrance, preventing mRNA from forming a more stable DNA double helix structure, and thus increasing the mRNA's tendency to hydrolyze. As a result, until recently, it was widely believed that mRNA was too unstable to withstand transfection protocols. Advances in RNA stabilization modifications have fueled interest in using mRNA instead of plasmid DNA in gene therapy. Delivery carriers, such as cationic lipids or polymer delivery carriers, can also help protect transfected mRNA from endogenous RNases. Furthermore, despite the increased stability of modified mRNA, the delivery of mRNA to cells in vivo in a manner that enables therapeutic-level protein production, particularly full-length protein-coding mRNA, remains a challenge. While the delivery of mRNA encoding secreted proteins is being attempted (Patent Document 3 (U.S. Patent Application Publication No. 2009 / 0286852)), the actual level of full-length secreted protein produced via in vivo mRNA delivery is unknown, and there is no reason to expect that this level will exceed the levels observed in DNA-based gene therapy. To date, significant progress has been made using mRNA gene therapy only in applications where low levels of translation, such as immunization with mRNA-coding antigens, were not a limiting factor. Clinical trials, including intradermal injection of naked or protamine complex mRNA to vaccine against tumor antigens, have demonstrated feasibility, lack of toxicity, and promising results. X. Su et al., Mol. Pharmaceuticals 8:774-787 (2011). Unfortunately, low levels of translation greatly limit the use of mRNA-based gene therapy in other applications that require higher levels of sustained expression of mRNA-coding proteins to achieve biological or therapeutic effects. [Prior art documents] [Patent Documents]

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

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

[0008] The present invention provides compositions and methods for intracellular delivery of mRNA in a liposome transport vehicle to one or more target cells for the production of therapeutically-grade functional secreted proteins.

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

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

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

[0012] Methods for treating subjects comprising administering compositions of the present invention are also contemplated. For example, methods are provided for treating or preventing conditions in which the production and / or utilization of specific secretory proteins is inadequate or impaired. In one embodiment, the methods provided herein can be used to treat subjects who are deficient in one or more urea cycle enzymes or who are deficient in one or more enzymes in lysosomal storage disorders.

[0013] In preferred embodiments, the mRNA in the composition of the present invention is formulated within a liposome transport vehicle to facilitate delivery to target cells. The intended transport vehicle may comprise one or more cationic lipids, non-cationic lipids, and / or PEG-modified lipids. For example, the transport vehicle may comprise at least one of the following cationic lipids: C12-200, DLin-KC2-DMA, DODAP, HGT4003, ICE, HGT5000, or HGT5001. In embodiments, the transport vehicle comprises cholesterol (chol) and / or PEG-modified lipids. In some embodiments, the transport vehicle comprises DMG-PEG2K. In one embodiment, the transport vehicle comprises one of the following lipid preparations: 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 promoting the transfection of target cells that can exhibit a "depot effect" and the delivery of one or more mRNA molecules to those cells. For example, the compositions and methods of the present invention intend to use targeted ligands that can increase the affinity of the composition to one or more target cells. In one embodiment, the targeted ligand is apolipoprotein B or apolipoprotein E, and the corresponding target cell Cells express low-density lipoprotein receptors, thus facilitating the recognition of targeted ligands. A vast number of target cells can be preferentially targeted using the methods and compositions of the present invention. For example, intended target cells include, but are not limited to, hepatocytes, epithelial cells, hematopoietic cells, epithelial cells, endothelial cells, lung cells, osteocytes, stem cells, mesenchymal cells, nerve cells, cardiac cells, adipocytes, vascular smooth muscle cells, cardiomyocytes, skeletal muscle cells, β-cells, pituitary cells, synovial-lining cells, ovarian cells, testicular cells, fibroblasts, B cells, T cells, reticulocytes, leukocytes, granulocytes, and tumor cells.

[0015] In some embodiments, secreted proteins are produced by target cells for a continuous period. For example, secreted proteins 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, polypeptides are expressed at peak levels about 6 hours after administration. In some embodiments, polypeptide expression is maintained at least at therapeutic levels. In some embodiments, polypeptides are expressed at least at therapeutic levels 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, polypeptides are detectable at therapeutic levels in the patient's serum or tissue (e.g., liver or lung). In some embodiments, detectable levels of polypeptides are derived from continuous expression from an 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 one embodiment, secreted proteins are produced at levels exceeding normal physiological levels. Levels of secreted proteins may be increased compared to a control.

[0017] In some embodiments, the control is a baseline physiological level of polypeptide in a normal individual or a population of normal individuals. In other embodiments, the control is a baseline physiological level of polypeptide in an individual or a population of individuals deficient in the relevant protein or polypeptide. In some embodiments, the control may be a normal level of the relevant protein or polypeptide in an individual to which the composition is administered. In other embodiments, the control is a polypeptide at one or more equivalent time points during other therapeutic interventions, for example, direct infusion of the corresponding polypeptide.

[0018] In some embodiments, polypeptides are expressed by target cells at levels 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 control. In some embodiments, the doubling of expression compared to control is maintained for more than 1 hour, more than 4 hours, more than 6 hours, more than 12 hours, more than 24 hours, or more than 48 hours, or more than 72 hours after administration. For example, in one embodiment, levels of secreted proteins are 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 control for at least 48 hours or 2 days. In one embodiment, levels of secreted proteins can be detected on day 3, day 4, day 5, or more than one week after administration. Increased levels of secreted proteins may be observed in serum and / or tissues (e.g., liver, lungs).

[0019] In some embodiments, this method sustains the circulating half-life of a desired secreted protein. This results in, for example, a secreted protein being detected for several hours or days longer than the half-life observed via subcutaneous injection of the secreted protein. In embodiments, the half-life of the secreted 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 includes single or repeated dosing. In certain embodiments, the dosage 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] One embodiment relates to compositions and methods that provide at least a portion encoding a functional protein to a cell or target mRNA in an amount substantially less than the amount of the corresponding functional protein produced from the mRNA. Stated another way, in certain embodiments, the mRNA delivered to the cell can produce a protein that is substantially more 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 the cell or subject, the amount of the corresponding protein produced by that mRNA can be at least 1.5, 2, 3, 5, 10, 15, 20, 25, 50, 100, 150, 200, 250, 300, 400, 500-fold, or more, greater 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 intracellular protein, 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 particular tissue or tissues, e.g., liver, kidney, heart, or brain; a particular cell type, e.g., one of the various cell types of the liver or brain; or a protein measured in any combination of serum, tissue, and / or cell type. Further, the baseline amount of endogenous protein is measured in the cell or subject prior to administration of the mRNA and then subtracted from the protein measured after administration of the mRNA to obtain the amount of the corresponding protein produced from the mRNA. In this way, the mRNA can provide the cell or subject with a reservoir or depot source of a large amount of therapeutic substance, for example, as compared to the amount of mRNA delivered to the cell or subject. The depot source can function as a continuous source for polypeptide expression from the mRNA over a duration.

[0023] In certain embodiments, for example, the following are provided: (Item 1) A composition comprising (a) at least one mRNA molecule that at least partly encodes a functionally secreted polypeptide, and (b) a transport vehicle containing lipid nanoparticles. (Item 2) The composition according to item 1, wherein the mRNA encodes an enzyme that is abnormally deficient in individuals with lysosomal storage disorders. (Item 3) The composition according to item 1, wherein the mRNA encodes a functional erythropoietin or a functional α-galactosidase polypeptide. (Item 4) The RNA molecule includes at least one modification that confers stability to the RNA molecule. The composition described in item 1. (Item 5) The composition according to item 1, wherein the RNA molecule comprises a modification of the 5' untranslated region of the RNA molecule. (Item 6) The composition according to item 5, wherein the modification includes the inclusion of a Cap1 structure. (Item 7) The composition according to item 1, wherein the RNA molecule comprises a modification of the 3' untranslated region of the RNA molecule. (Item 8) The composition according to item 7, wherein the modification includes the inclusion of a poly-A tail. (Item 9) The composition according to item 1, further comprising an active substance for promoting the movement of the RNA molecule into an intracellular compartment of a target cell. (Item 10) The lipid nanoparticles are the composition according to item 1, comprising one or more cationic lipids. (Item 11) The lipid nanoparticles comprise one or more noncationic lipids, as described in item 1. (Item 12) The lipid nanoparticles are the composition described in item 1, comprising one or more PEG-modified lipids. (Item 13) The lipid nanoparticles are the composition described in item 1, comprising C12-200. (Item 14) The lipid nanoparticles are the composition described in item 1, comprising DLinKC2DMA, CHOL, DOPE, and DMG-PEG-2000. (Item 15) The lipid nanoparticles are the composition described in item 1, comprising C12-200, DOPE, CHOL, and DMGPEG2K. (Item 16) The lipid nanoparticles are the composition according to item 1, comprising cleavable lipids. (Item 17) The composition described in item 1 is freeze-dried. (Item 18) The composition described in item 1 is a freeze-dried reconstituted composition. (Item 19) The composition according to item 10, wherein the target cells are selected from the group consisting of hepatocytes, epithelial cells, hematopoietic cells, epithelial cells, endothelial cells, lung cells, osteocytes, stem cells, mesenchymal cells, nerve cells, cardiac cells, adipocytes, vascular smooth muscle cells, cardiomyocytes, skeletal muscle cells, β 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 deficient in a functional polypeptide, comprising administering a composition comprising (a) at least one mRNA that at least partially encodes the functional secretory polypeptide, and (b) a transport vehicle comprising lipid nanoparticles, wherein, after administration of the composition, the mRNA is expressed in a target cell to produce the functional secretory polypeptide. (Item 21) The mRNAs mentioned above include functional erythropoietin, α-galactosidase, LDL receptor, factor VIII, factor IX, α-L-iduronidase, iduronate sulfatase, heparin-N-sulfatase, α-N-acetylglucosaminidase, and galactose-6-sulfatase. The method according to item 21, comprising (b) encoding a sulfatase, β-galactosidase, lysosomal acid lipase, or arylsulfatase-A polypeptide, and a transport vehicle, wherein after administration of the composition, the mRNA is expressed in target cells to produce a functionally secreted polypeptide. (Item 22) The method according to item 21, wherein the functionally secreted polypeptide is an enzyme that is abnormally deficient in individuals with lysosomal storage disorders. (Item 23) The method according to item 21, wherein the mRNA molecule includes at least one modification that confers stability to the mRNA molecule. (Item 24) The method according to item 21, wherein the mRNA molecule includes modification of the 5' untranslated region of the mRNA molecule. (Item 25) The modification is as described in item 25, which includes the inclusion of the Cap1 structure. (Item 26) The method according to item 21, wherein the mRNA molecule includes modification of the 3' untranslated region of the mRNA molecule. (Item 27) The modification is as described in item 27, including the inclusion of a poly-A tail. (Item 28) The method according to item 21, further comprising an active agent for promoting the movement of the mRNA molecule into the intracellular compartment of the target cell. (Item 29) The method according to item 21, wherein the lipid nanoparticles comprise one or more cationic lipids. (Item 30) The method according to item 21, wherein the lipid nanoparticles comprise one or more noncationic lipids. (Item 31) The method according to item 21, wherein the lipid nanoparticles comprise one or more PEG-modified lipids. (Item 32) The lipid nanoparticles are the method described in item 21, comprising C12-200. (Item 33) The lipid nanoparticles are the method described in item 21, comprising DLinKC2DMA, CHOL, DOPE, and DMG-PEG-2000. (Item 34) The lipid nanoparticles are the method described in item 21, comprising C12-200, DOPE, CHOL, and DMGPEG2K. (Item 35) The method according to item 21, wherein the lipid nanoparticles contain cleavable lipids. (Item 36) The composition is freeze-dried according to the method described in item 21. (Item 37) The method according to item 21, wherein the composition is a freeze-dried reconstituted composition. (Item 38) The method according to item 21, wherein the target cells are selected from the group consisting of hepatocytes, epithelial cells, hematopoietic cells, epithelial cells, endothelial cells, lung cells, osteocytes, stem cells, mesenchymal cells, nerve cells, cardiac cells, adipocytes, vascular smooth muscle cells, cardiomyocytes, skeletal muscle cells, β cells, pituitary cells, synovial-lining cells, ovarian cells, testicular cells, fibroblasts, B cells, T cells, reticulocytes, leukocytes, granulocytes, and tumor cells. (Item 39) A method for treating a subject deficient in a functionally secreted polypeptide, comprising administering a composition comprising (a) at least one mRNA that at least partially encodes the functionally secreted polypeptide, and (b) a transport vehicle comprising lipid nanoparticles, wherein, after administration of the composition, the mRNA is translated in a target cell to produce the functional polypeptide in the target cell at least at a minimum therapeutic level more than one hour after administration. (Item 40) A method for producing a functionally secreted polypeptide in a target cell, comprising administering a composition comprising (a) at least one mRNA that at least partially encodes the functionally secreted polypeptide, and (b) a transport vehicle comprising lipid nanoparticles, wherein, after administration of the composition, the mRNA is translated in the target cell to produce a functionally secreted polypeptide at least at a minimum therapeutic level more than one hour after administration. The features discussed above, as well as many other features and incidental advantages of the present invention, will be better understood by referring to the following detailed description of the invention in conjunction with the appended examples. The various embodiments described herein are complementary and may be combined or used together in a manner that will be understood by those skilled in the art, taking into account the teachings contained herein. [Brief explanation of the drawing]

[0024] [Figure 1] The nucleotide sequence of the 5'CMV sequence (SEQ ID NO: 1) is shown, where X is GGA if present. [Figure 2] The nucleotide sequence of the 3'hGH sequence (SEQ ID NO: 2) is shown. [Figure 3] The nucleotide sequence of human erythropoiesis (EPO) mRNA (SEQ ID NO: 3) is shown. This sequence 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. This sequence may be adjacent to the 5' end of SEQ ID NO: 1 and the 3' end of SEQ ID NO: 2. [Figure 5] The nucleotide sequence of human α-1 anti-trypsin (A1AT) mRNA (SEQ ID NO: 5) is shown. This sequence 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. This sequence may be adjacent to the 5' end of SEQ ID NO: 1 and the 3' end of SEQ ID NO: 2. [Figure 7] This section shows the quantification of secreted hEPO protein levels measured using ELISA. The detected proteins are the result of their production from hEPO mRNA delivered intravenously by a single dose of various lipid nanoparticle formulations. Formulations C12-200 (30 ug), HGT4003 (150 ug), ICE (100 ug), and DODAP (200 ug) are represented by the cationic / ionic lipid components of their respective test substances (formulations 1-4). Values ​​are based on blood samples taken 4 hours after administration. [Figure 8] The results of hematocrit measurements in mice treated with a single intravenous administration of human EPO mRNA-loaded lipid nanoparticles (formulations 1-4) are shown. Whole blood samples were collected 4 hours (day 1), 24 hours (day 2), 4 days, 7 days, and 10 days after administration. [Figure 9] The hematocrit measurements of mice treated with human EPO-mRNA-loaded lipid nanoparticles via either a single intravenous dose or three infusions (days 1, 3, and 5) are shown. Whole blood samples were collected before infusion (-4 days), on day 7, and on day 15. Administration of formulation 1: (30 ug single dose) or (3 × 10 ug administered on days 1, 3, and 5); Administration of formulation 2: (3 × 50 ug administered on days 1, 3, and 5). [Figure 10] This shows the quantification of secreted human α-galactosidase (hGLA) protein levels measured using ELISA. The detected protein is a result of production from hGLA mRNA delivered via lipid nanoparticles (Formulation 1: single intravenous administration of 30 ug based on encapsulated mRNA). hGLA protein is detected over 48 hours. [Figure 11] This shows the hGLA activity in serum. hGLA activity was measured at 37°C using the substrate 4-methylumbelliferyl-α-D-galactopyranoside (4-MU-α-gal). The data are the average of the measurement results from 6 to 9 individuals. [Figure 12] This describes the quantification of hGLA protein levels in serum measured using ELISA. The protein is produced from hGLA mRNA delivered via C12-200-based lipid nanoparticles (C12-200:DOPE:Chol:DMGPEG2K, 40:30:25:5 (Formulation 1); 30 ug of mRNA based on encapsulated mRNA, single intravenous administration). hGLA protein levels are monitored for 72 hours (based on encapsulated mRNA, per single intravenous administration). [Figure 13]This report describes the quantification of hGLA protein levels in the liver, kidney, and spleen as measured using ELISA. The protein is produced from hGLA mRNA delivered via C12-200-based lipid nanoparticles (Formulation 1: a single intravenous administration of 30 ug of mRNA based on encapsulated mRNA). hGLA protein levels are monitored for 72 hours. [Figure 14A] This study presents a dose-response analysis monitoring the protein production of hGLA, a secreted MRT-derived human GLA protein, in serum (A) and liver (B). Samples were measured 24 hours after administration (Formulation 1: single intravenous administration, N=4 mice / group) and quantified using ELISA. [Figure 14B] This study presents a dose-response analysis monitoring the protein production of hGLA, a secreted MRT-derived human GLA protein, in serum (A) and liver (B). Samples were measured 24 hours after administration (Formulation 1: single intravenous administration, N=4 mice / group) and quantified using ELISA. [Figure 15] This shows the pharmacokinetic profiles of ERT-based α-galactosidase (dose of 40 ug / kg) and hGLA protein produced from MRT (formulation 1: 1.0 mg / kg mRNA dose) in athymic nude mice. [Figure 16] This report quantifies secreted hGLA protein levels in MRT-treated Fabry mice, measured using ELISA. hGLA protein is produced from hGLA mRNA delivered via C12-200-based lipid nanoparticles (Formulation 1: 10 ug of mRNA per single intravenous dose based on encapsulated mRNA). Serum is monitored for 72 hours. [Figure 17]This report quantifies hGLA protein levels in the liver, kidney, spleen, and heart of MRT-treated Fabry knockout mice, measured using ELISA. The protein is produced from hGLA mRNA delivered via C12-200-based lipid nanoparticles (Formulation 1: a single intravenous administration of 30 ug of mRNA based on encapsulated mRNA). hGLA protein levels are monitored for 72 hours. Literature values ​​representing normal physiological levels are plotted as dotted lines. [Figure 18] This shows the quantification of secreted hGLA protein levels in Fabry mice treated with MRT and α-galactosidase, as measured using ELISA. Both therapeutic agents were administered as a single intravenous dose of 1.0 mg / kg. [Figure 19] This report quantifies hGLA protein levels in the liver, kidney, spleen, and heart of Fabry knockout mice treated with MRT and ERT (α-galactosidase) using ELISA. The protein was produced from hGLA mRNA delivered via lipid nanoparticles (Formulation 1: a single intravenous administration of 1.0 mg / kg of mRNA based on encapsulated mRNA). [Figure 20] This report presents the relative quantification of globotriaosylceramide (Gb3) and lyso-Gb3 in the kidneys 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. The dose reflects the Gb3 / Lyso-Gb3 levels one week after administration. [Figure 21] This report presents the 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 a dose of 1.0 mg / kg. The dose reflects the Gb3 / Lyso-Gb3 ratio one week after administration. [Figure 22]This study presents a dose-response analysis monitoring GLA protein production 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 administration, N=4 mice / group), and quantified using ELISA. [Figure 23A] This shows 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 administration, N=4 mice / group) and quantified using ELISA. [Figure 23B] This shows 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 administration, N=4 mice / group) and quantified using ELISA. [Figure 24] This report quantifies secreted human factor IX protein levels as measured by ELISA (mean ng / mL ± standard deviation). FIX protein is produced from FIX mRNA delivered via C12-200-based lipid nanoparticles (C12-200:DOPE:Chol:DMGPEG2K (formulation 1) in a 40:30:25:5 ratio, 30 ug of mRNA per single intravenous dose based on encapsulated mRNA). FIX protein levels are monitored for 72 hours. (n=24 mice) [Figure 25] This report describes the quantification of secreted human α-1-antitrypsin (A1AT) protein levels measured using ELISA. A1AT protein is produced from A1AT mRNA delivered via C12-200-based lipid nanoparticles (C12-200:DOPE:Chol:DMGPEG2K (formulation 1) in a 40:30:25:5 ratio, with 30 ug of mRNA per single intravenous dose based on encapsulated mRNA). A1AT protein levels are monitored over 24 hours. [Figure 26]This shows ELISA-based quantification of hEPO protein detected in the lungs and serum of treated mice after intratracheal administration of hEPO mRNA-loaded nanoparticles (measured in mIU units) (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). [Modes for carrying out the invention]

[0025] (Description of exemplary embodiments) The present invention provides compositions and methods for intracellular delivery of mRNA in a liposome transport vehicle to one or more target cells for the production of therapeutically-grade functional secreted proteins.

[0026] As used herein to limit proteins or enzymes, the term “functional” means that the protein or enzyme is biologically active or capable of performing the same or similar functions as naturally occurring or normally functioning proteins or enzymes. The mRNA compositions of the present invention are useful for treating a variety of metabolic or genetic disorders, specifically genetic or metabolic disorders involving the non-expression, misexpression, or deficiency of proteins or enzymes. The term “therapeutic level” refers to the level of protein detected in the blood or tissue above the control level, where the control may be a normal physiological level or the level in the subject before administration of the mRNA composition. The term “secreted” refers to the protein detected outside the target cell (extracellular space). Proteins are secreted in the blood or tissue. It can be detected. 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 compositions include a transport vehicle. As used herein, the term “transport vehicle” includes any of the standard pharmaceutical carriers, diluents, excipients, etc., which are generally intended for use in connection with the administration of physiologically active agents, including nucleic acids. The compositions described herein, specifically the transport vehicle, can deliver mRNA to target cells. In embodiments, the transport vehicle is lipid nanoparticles. mRNA

[0027] The mRNA in the composition of the present invention may encode, for example, a normally secreted secretory hormone, enzyme, receptor, polypeptide, peptide, or other target protein. 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 promote protein production.

[0028] The method of the present invention provides, for example, the simultaneous delivery of one or more unique mRNAs to target cells by combining two unique mRNAs and placing them in a single transport vehicle. In one embodiment of the present invention, a first therapeutic mRNA and a second therapeutic mRNA may be formulated and administered in a single transport vehicle. The present invention also intends for the simultaneous delivery and / or administration of a first therapeutic mRNA and a second nucleic acid to promote and / or enhance the function or delivery of the first therapeutic mRNA. For example, such a second nucleic acid (e.g., exogenous or synthetic mRNA) may encode a membrane transporter protein that promotes the delivery of the first mRNA or enhances its biological activity at the time of expression (e.g., at the time of translation of the exogenous or synthetic mRNA). Alternatively, the first therapeutic mRNA may be administered together with a second nucleic acid that performs a “chaperone” function to direct the folding of either of the first therapeutic mRNAs.

[0029] The methods of the present invention also provide the delivery of one or more therapeutic nucleic acids to treat a single disorder or deficiency, each such therapeutic nucleic acid functioning by a different mechanism of action. For example, a composition of the present invention may comprise a first therapeutic mRNA administered, for example, to correct an endogenous protein or enzyme deficiency, and accompanied by a second nucleic acid, the second nucleic acid administered to deactivate or "knock down" the non-functioning endogenous nucleic acid and its protein or enzyme product. Such a "second" nucleic acid may encode, for example, mRNA or siRNA.

[0030] During transfection, the native mRNA in the composition of the present invention may decay with a half-life of 30 minutes to several days. The mRNA in the composition of the present invention preferably retains at least some ability to be translated and therefore produces functional secretory proteins or enzymes. Accordingly, the present invention provides compositions comprising stabilized mRNA and methods for administering them. In some embodiments of the present invention, the activity of the mRNA is prolonged over a long period. For example, the activity of the mRNA can be prolonged so that the composition of the present invention is administered to a subject twice a week or every other week, or more preferably once a month, every other month, four times a year, or once a year. The prolonged or extended activity of the mRNA of the present invention is directly related to the amount of functional secretory proteins or enzymes produced from such mRNA. Similarly, the activity of the composition of the present invention can be further prolonged or extended by modifications that improve or enhance the translation of the mRNA. Furthermore, the amount of functional proteins or enzymes produced by target cells is a function of the amount of mRNA delivered to the target cells and the stability of such mRNA. The half-life, the activity of the secreted protein or enzyme produced, and the frequency of administration of the composition can be further extended to an extent that the stability of the mRNA of the present invention can be improved or enhanced.

[0031] Accordingly, in some embodiments of the present invention, the mRNA in the composition includes 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 “modified” and “modified” as used herein in relation to the nucleic acids provided herein preferably include at least one modification that enhances stability and gives the mRNA greater stability (e.g., resistance to nuclease digestion) than the wild-type or naturally occurring version of the mRNA. The terms “stable” and “resilient” as used herein in relation to the nucleic acids of the present invention, and in particular with respect to mRNA, refer to increased or enhanced resistance to degradation by nucleases (i.e., endonucleases or exonucleases) that can successfully degrade such mRNA. Increased stability may include, for example, hydrolysis or other disruption by endogenous enzymes (e.g., endonucleases or exonucleases), or reduced sensitivity to conditions within target cells or tissues, and thus increase or enhance the retention of such mRNA in target cells, tissues, subjects, and / or cytoplasm. The stabilized mRNA molecules provided herein exhibit longer half-lives compared to their naturally occurring unmodified counterparts (e.g., wild-type versions of mRNA). The terms “modified” and “modified” in relation to mRNA in the present invention also refer to modifications that improve or enhance the translation of mRNA nucleic acids, including, for example, the inclusion of sequences that function at 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 mRNAs of the present invention have undergone chemical or biological modifications to give them greater stability. Exemplary modifications to mRNA include base deficiencies (e.g., deletions or substitution of one nucleotide with another) or base modifications, such as chemical modifications of bases. As used herein, the term "chemical modification" includes modifications that introduce chemical properties different from those found in naturally occurring mRNA, such as the introduction of modified nucleotides (e.g., nucleotide analogs), or covalent modifications such as the inclusion of pendant groups not found in nature in such mRNA molecules.

[0033] In addition, preferred modifications include alterations of one or more nucleotides in a codon such that the codon codes for the same amino acid but is more stable than the codon found in the wild-type version of mRNA. For example, an inverse correlation has been demonstrated between RNA stability and more cytidine (C) and / or uridine (U) residues, 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 substituting a codon coding for a particular amino acid with another codon coding for the same or related amino acid. The intended modifications to mRNA nucleic acids of the present invention also include the incorporation of pseudouridine. Incorporation of pseudouridine into mRNA nucleic acids of the present invention may increase stability and translational capacity and decrease immunogenicity in vivo. For example, Kariko, K., et al. See l., Molecular Therapy 16(11):1833-1840(2008). The mRNA substitution and modification of the present invention can be carried out by methods readily known to those skilled in the art.

[0034] The constraints on reducing the number of C and U residues in a sequence may be greater within the coding region of mRNA than in the uncoding region (i.e., it is probably impossible to eliminate all C and U residues present in a message while still maintaining the ability of the message to encode the desired amino acid sequence). However, the degeneracy of gene coding This presents an opportunity to reduce the number of C and / or U residues present in the sequence while maintaining the same coding ability (i.e., several different possibilities for RNA sequence modification are possible depending on which amino acids are encoded by the codon). For example, the codon of Gly could be changed to GGA or GGG instead of GGU or GGC.

[0035] The term modification also includes, for example, the incorporation of non-nucleotide bonded or modified nucleotides into the mRNA sequence of the present invention (e.g., modification to one or both of the 3' and 5' ends of an mRNA molecule encoding a functional secretory 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, compounding of mRNA with an active substance (e.g., a protein or complementary nucleic acid molecule), and inclusion of elements that alter the structure of the mRNA molecule (e.g., form a secondary structure).

[0036] The poly(A) tail is thought to stabilize natural messengers. Therefore, in one embodiment, a long poly(A) tail can be attached to an mRNA molecule, thereby conferring greater stability to the mRNA. The poly(A) tail can be attached using various techniques recognized in the art. For example, a long poly(A) tail can be attached to synthetic mRNA or in vitro transcribed mRNA using poly(A) polymerase (Yokoe, et al. Nature). (Biotechnology. 1996;14:1252-1256). The transcription vector may also encode a long polyA tail. In addition, the polyA tail can be added by direct transcription from the PCR product. In one embodiment, the polyA tail is at least about 90, 200, 300, 400, or at least 500 nucleotides long. In one embodiment, the length of the polyA tail is adjusted to control the stability of the modified mRNA molecule of the present invention and, consequently, the transcription of the protein. For example, since the length of the polyA tail may affect the half-life of the mRNA molecule, the length of the polyA tail may be adjusted to modify the level of mRNA's resistance to nucleases and thus control the time course of protein expression in cells. 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 target cells without a transport vehicle.

[0037] In one embodiment, mRNA may be modified by the incorporation of 3' and / or 5' untranslated (UTR) sequences not found in nature in wild-type mRNA. In one embodiment, 3' and / or 5' flanking sequences that are naturally adjacent to mRNA and encode a second unassociated protein may be incorporated into and modify the nucleotide sequence of an mRNA molecule encoding a therapeutic or functional protein. For example, 3' or 5' sequences derived from a stable mRNA molecule (e.g., globin, actin, GAPDH, tubulin, histone, or citrate cycle enzymes) may be incorporated into the 3' and / or 5' region of a sense mRNA nucleic acid molecule to increase the stability of the sense mRNA molecule. See, for example, U.S. Patent No. 2003 / 0083272.

[0038] In some embodiments, the mRNA in the compositions of the present invention includes modifications to the 5' end of the mRNA to incorporate a subsequence of the CMV pre-initial 1 (IE1) gene or a fragment thereof (e.g., SEQ ID NO: 1), thereby improving nuclease resistance and / or improving the mRNA half-life. In addition to increasing the stability of the mRNA nucleic acid sequence, it has been unexpectedly found that the inclusion of a subsequence of the CMV pre-initial 1 (IE1) gene enhances the translation and expression of functional proteins or enzymes of the mRNA. To further stabilize the mRNA, inclusion of the 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 considered. Generally, 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, modifications that improve the resistance of such mRNA to in vivo nuclease digestion.

[0039] Further variants of the nucleic acid sequences of SEQ ID NO: 1 and / or SEQ ID NO: 2 are intended to be developed, and these variants will maintain the functional properties of the nucleic acid, including mRNA stabilization, and / or pharmacokinetic properties (e.g., half-life). The variants may have sequence identity exceeding 90%, 95%, 98%, or 99% of SEQ ID NO: 1 or SEQ ID NO: 2.

[0040] In some embodiments, the composition may include stabilizing reagents. The composition may include one or more formulation reagents that bind directly or indirectly to stabilize mRNA and thus increase its residence time in target cells. Such reagents preferably lead to an improvement in the half-life of mRNA in target cells. For example, mRNA stability and translation efficiency may be increased by incorporating “stabilizing reagents” that form complexes with naturally occurring mRNA in cells (see, for example, U.S. Patent No. 5,677,124). Incorporation of stabilizing reagents may be achieved, for example, by combining poly(A) and proteins with mRNA and stabilizing the mRNA in vitro before loading or encapsulating it in a transport vehicle. Exemplary stabilizing reagents include one or more proteins, peptides, aptamers, translation accessory proteins, mRNA-binding proteins, and / or translation initiation factors.

[0041] The stabilization of the composition can also be improved by the use of opsonization inhibitory moieties, typically large hydrophilic polymers, that are chemically or physically bound to the transport vehicle (e.g., by intercalation of lipid-soluble anchors into their own membranes, or by direct binding to the active groups of membrane lipids). These opsonization inhibitory hydrophilic polymers form a protective surface layer that significantly reduces the uptake of liposomes by the macrophage-monocyte system and the reticuloendothelial system (e.g., as described in U.S. Patent No. 4,920,016, the full disclosure of which is incorporated herein by reference). Thus, transport carriers modified with opsonization inhibitory moieties remain in circulation for 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 a nuclease (Krieg, et al. Melton. Methods in Enzymology. 1987; 155, 397-415). The stability of the hybridized mRNA is likely due to the intrinsic single-strand specificity of most RNAses. In some embodiments, the stabilizing reagent chosen for compounding the mRNA is a eukaryotic protein (e.g., a mammalian protein). In yet another embodiment, the mRNA may 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 may be reduced. In some embodiments, the 5' untranslated region and the AUG start region of the mRNA molecule may optionally remain unhybridized. After translation initiation, the unwinding activity of the ribosome complex can function even on high-affinity double helixes, allowing translation to begin (Liebhaber.J.Mol.Biol.1992;226:2-13, Moni, et al.J Biol Chem.1993;268:14514-22).

[0043] It is understood that any of the methods described above for enhancing mRNA stability may be used alone or in combination with one or more of the other methods and / or compositions described above, or any of them.

[0044] The mRNA of the present invention may be optionally combined with a reporter gene (e.g., upstream or downstream of the mRNA coding region) that facilitates the determination of mRNA delivery to target cells or tissues. Suitable reporter genes may include, for example, green fluorescent protein mRNA (GFP mRNA), renirul luciferase mRNA (luciferase mRNA), firefly luciferase mRNA, or any combination thereof. For example, GFP mRNA can be fused with mRNA encoding secreted proteins, potentially facilitating the confirmation of mRNA localization in target cells that function as depots for protein production.

[0045] As used herein, the terms “transfect” or “transfection” mean the intracellular introduction of mRNA into a cell, or preferably into a target cell. The introduced mRNA may be maintained stably or transiently within the target cell. The term “transfection efficiency” refers to the relative amount of mRNA absorbed by the target cell being transfected. 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 having high transfection efficacy, specifically compositions that minimize side effects mediated by transfection of non-target cells. Compositions of the present invention exhibiting high transfection efficacy increase the likelihood that an appropriate dose of mRNA will be delivered to the target cell while minimizing potential systemic side effects. In one embodiment of the present invention, the transport vehicle of the present invention can deliver large mRNA sequences (e.g., mRNA 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 larger). The mRNA may be formulated with one or more acceptable reagents, which provide a carrier for delivering such mRNA to target cells. Suitable reagents are generally selected with respect to several factors, including, among others, the biological or chemical properties of the mRNA, the administration route of interest, the biological environment to which such mRNA is expected to be exposed, and the specificity of the target cells being targeted. In some embodiments, the transport vehicle, such as a liposome, encapsulates the mRNA without impairing its biological activity. In some embodiments, the transport vehicle exhibits selective and / or substantial binding to target cells compared to non-target cells. In a preferred embodiment, the transport vehicle delivers its contents to the target cell so that the mRNA is delivered to an appropriate intracellular compartment, such as the cytoplasm. Transport carrier

[0046] In embodiments, the transport vehicle in the composition of the present invention is a liposome transport vehicle, for example, lipid nanoparticles. In one embodiment, the transport vehicle is selected and / or prepared to optimize the delivery of mRNA to target cells. For example, if the target cells are hepatocytes, the properties of the transport vehicle (e.g., dimensions, charge, and / or pH) may be optimized to effectively deliver such a transport vehicle to target cells to reduce immune clearance and / or promote its retention within those target cells. Alternatively, if the target cells are the central nervous system (e.g., mRNA administered for the treatment of neurodegenerative diseases may specifically target brain or spinal cord tissue), the selection and preparation of the transport vehicle must take into account penetration and retention across the blood-brain barrier, and / or the use of alternative means to directly deliver such a transport vehicle to such target cells. In one embodiment of the present invention, the composition may be combined with an active agent that promotes the movement of exogenous mRNA (e.g., an active agent that reduces or improves the permeability of the blood-brain barrier and thus enhances the movement of exogenous mRNA to target cells).

[0047] The use of liposome transport vehicles to facilitate the delivery of nucleic acids to target cells is intended by the present invention. Liposomes (e.g., liposomal lipid nanoparticles) are generally useful in a variety of applications in research, industry, and medicine, particularly for their use as transport vehicles for diagnostic or therapeutic compounds in vivo (Lasic, Trends Biotechnol., 16:307-321, 1998; Drummond et al., Pharmacol. Rev., 51:691-743, 1999), and are typically considered microvesicles having an internal water space isolated from external media by one or more bilayer membranes. The bilayer membrane of a liposome typically contains spatially separated hydrophilic and hydrophobic domains. Liposomes are formed by amphiphilic molecules such as lipids of synthetic or natural origin (Lasic, Trends Biotechnol., 16:307-321, 1998). The liposome bilayer can also be formed by amphiphilic polymers and surfactants (e.g., polymerosomes, niosomes, etc.).

[0048] In the context of the present invention, liposome transport vehicles typically serve to transport mRNA to target cells. For the purposes of the present invention, liposome transport vehicles are prepared to contain a desired nucleic acid. The process of incorporating a desired entity (e.g., nucleic acid) into a liposome is often referred to as "filling" (Lasic, et al., FEBS Lett., 312:255-258, 1992). The nucleic acid incorporated into the liposome may be located entirely or partially within the internal space of the liposome within the liposome's bilayer membrane, or associated with the outer surface of the liposome membrane. The incorporation of nucleic acid into a liposome is also referred to herein as "encapsulation," in which the nucleic acid is completely contained within the internal space of the liposome. The purpose of incorporating mRNA into a transport vehicle such as a liposome is often to protect the nucleic acid from environments that may contain enzymes or chemicals that degrade the nucleic acid and / or the system, or receptors that cause rapid efflux of the nucleic acid. Therefore, in preferred embodiments of the present invention, the selected transport vehicle can enhance the stability of the mRNA contained therein. Liposomes can enable encapsulated mRNA to reach target cells, and / or preferentially enable encapsulated mRNA to reach target cells, or restrict the delivery of such mRNA to other sites or cells where the presence of the administered mRNA may be unhelpful or undesirable. Furthermore, the incorporation of mRNA into a transport vehicle, such as cationic liposomes, also facilitates the delivery of such mRNA to target cells.

[0049] Ideally, the liposome transport vehicle is prepared to encapsulate one or more desired mRNAs so that the composition exhibits highly efficient transfection and enhanced stability. While liposomes can facilitate the delivery of nucleic acids to target cells, the addition of polycations (e.g., poly-L-lysine and protamine) as copolymers can enhance the transfection efficiency of several types of cationic liposomes in several cell lines, both in vitro and in vivo, and in some cases significantly enhance it by 2 to 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 transport vehicle is formulated as lipid nanoparticles. As used herein, the term “lipid nanoparticles” refers to a transport vehicle comprising 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 transport vehicles, whether alone or in combination with other transport vehicles, is also intended. Suitable polymers may include, for example, polyacrylates, polyalkylcyanoacrylates, polylactides, polylactide-polyglycolide copolymers, polycaprolactones, dextrans, albumins, gelatins, alginates, collagens, chitosans, cyclodextrins, dendrimers, and polyethyleneimines. In one embodiment, the transport vehicle is selected based on its ability to facilitate the transfection of mRNA into target cells.

[0051] The present invention envisions the use of lipid nanoparticles as a transfer vehicle containing cationic lipids to encapsulate mRNA and / or facilitate the delivery of mRNA to target cells that function as a depot for protein production. As used herein, the term “cationic lipid” refers to any of several lipid species that carry a net positive charge at a selected pH, such as physiological pH. The envisioned lipid nanoparticles can be prepared by incorporating multi-component lipid mixtures in various ratios using one or more cationic lipids, non-cationic lipids, and PEG-modified lipids. Several cationic lipids are documented in the literature, and many of them are commercially available.

[0052] Cationic lipids particularly suitable for use in the compositions and methods of the present invention include those described in International Patent Publication WO2010 / 053572, incorporated herein by reference, and most specifically, C12-200 as described in paragraph

[0225] of International Patent Publication WO2010 / 053572. In some embodiments, the compositions and methods of the present invention include, for example, (15Z,18Z)-N,N-dimethyl-6-(9Z,12Z)-octadeca-9,12-dien-1-yl)tetracosa-15,18-dien-1amine (HGT5000), (15Z,18Z)-N,N-dimethyl-6-((9Z,12Z)-octadeca-9,12-dien-1-yl)tetracosa-4,15,18-triene-1 Lipid nanoparticles containing ionic cationic lipids described in U.S. Provisional Patent Application No. 61 / 617,468, filed March 29, 2012 (incorporated herein by reference), such as -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. Patent No. 4,897,355)). DOTMA can be formulated alone or in combination with neutral lipids, dioleoylphosphatidylethanolamine, or "DOPE", or other cationic or non-cationic lipids to a liposome transport vehicle or lipid nanoparticles, and such liposomes can be used to facilitate the 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-carboxyamide)ethyl]-N,N-dimethyl-1-propaneaminium or "DOSPA" (Behr et al. al.Proc.Nat.'l Acad.Sci.86,6982 (1989), U.S. Patent No. 5,171,678, U.S. Patent No. 5,334,761), includes 1,2-diol-eoyl-3-dimethylammonium-propane or "DODAP", and 1,2-diol-eoyl-3-trimethylammonium-propane or "DOTAP".The cationic lipids intended are 1,2-distearyloxy-N,N-dimethyl-3-aminopropane or "DSDMA", 1,2-dioleyloxy-N,N-dimethyl-3-aminopropane or "DODMA", 1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane or "DLinDMA", 1,2-dilinolenyloxy-N,N-dimethyl-3-aminopropane or "DLenDMA", N-dioleyl-N,N-dimethylammonium chloride or "DODAC", and N,N-distearyl Lu-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-ene-3-β-oxybutane-4-oxy)-1-(cis,cis-9,12-octadecadieneoxy)propane or "CLinDMA", 2-[5'-(cholest-5-ene-3-β-oxy)-3'-oxapentoxy)-3-dimethyl-1-(cis,cis-. 9',1-2'-octadecadieneoxy)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-dimethylaminopropane or "DLincarbDAP", 1,2-dilinoleoylcarbamyl-3-dimethyl Aminopropane 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-dioxolane-4-yl)-N,N-dimethylethaneamine (DLin-KC2-DMA)) (International Publication No. WO2010 / 042877, Semple This also includes mixtures thereof (see 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 lipids is also intended by the present invention. Such cholesterol-based cationic lipids may be used alone or in combination with other cationic or non-cationic lipids. Suitable cholesterol-based cationic lipids include, for example, DC-Chol (N,N-dimethyl-N-ethylcarboxamide cholesterol), 1,4-bis(3-N-oleylaminopropyl)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 are commercially available to enhance transfection efficacy. Preferred examples include lipofectin (DOTMA:DOPE) (Invitrogen, Carlsbad, Calif.), lipofectamine (DOSPA:DOPE) (Invitrogen), lipofectamine 2000 (Invitrogen), FUGENE, transfectam (DOGS), and effecten.

[0056] Cationic lipids such as dialkylamino-based, imidazole-based, and guanidinium-based lipids are also intended. For example, one embodiment involves a composition comprising one or more imidazole-based cationic lipids represented by the following structure (I), 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]phenanthrene-3-yl3-(1H-imidazole-4-yl)propanoate. In a preferred embodiment, the transport vehicle for mRNA delivery may comprise one or more imidazole-based cationic lipids represented by structure (I), for example, 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-yl3-(1H-imidazole-4-yl)propanoate. [ka] Without being constrained by any particular theory, it is conceivable that the fusionability of imidazole-based cationic lipids ICE is related to endosomal disruption facilitated by imidazole groups having lower pKas compared to conventional cationic lipids. This endosomal disruption then promotes osmotic expansion and disruption of the liposome membrane, followed by transfection or intracellular release of nucleic acid(s)-containing substances loaded into target cells.

[0057] Imidazole-based cationic lipids are also characterized by reduced toxicity compared to other cationic lipids. Imidazole-based cationic lipids (e.g., ICE) can be used as a standalone cationic lipid in lipid nanoparticles or in combination with conventional cationic lipids, non-cationic lipids, and PEG-modified lipids. Cationic lipids may constitute approximately 1% to 90%, 2% to 70%, 5% to 50%, 10% to 40%, or preferably approximately 20% to 70% of the total lipids present in the transport vehicle.

[0058] Similarly, one embodiment relates to a lipid nanoparticle comprising the HGT4003 cationic lipid 2-((2,3-bis((9Z,12Z)-octadeca-9,12-diene-1-yloxy)propyl)disulfanyl)-N,N-dimethylethaneamine, represented by the following structure (II), and the entire teaching thereof is incorporated herein by reference as a whole. [ka]

[0059] In other embodiments, the compositions and methods described herein relate to lipid nanoparticles containing one or more cleavable lipids, such as one or more cationic lipids or compounds containing cleavable disulfide (SS) functional groups (e.g., HGT4001, HGT4002, HGT4003, HGT4004, and HGT4005), as further described in U.S. Provisional Application No. 61 / 494,745, the entire teaching thereof is incorporated herein by reference as a whole.

[0060] The use of polyethylene glycol (PEG)-modified phospholipids and derivatized lipids, such as derivatized ceramides (PEG-CER) containing N-octanoyl-sphingosine-1-[succinyl(methoxypolyethylene glycol)-2000] (C8 PEG-2000 ceramide), either alone or preferably in combination with other lipids including a transport vehicle (e.g., lipid nanoparticles), is also envisioned by the present invention. The envisioned PEG-modified lipids include C6-C 20 This includes, but is not limited to, polyethylene glycol chains up to 5 kDa covalently bonded to lipids having long alkyl chains (including multiple chains). The addition of such components can inhibit complex aggregation, increase circulating survival time, and deliver lipid nucleic acids to target cells. Means for increasing the delivery of the composition may also be provided (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. Patent No. 5,885,613). Particularly useful exchangeable lipids are PEG-ceramides having shorter acyl chains (e.g., C14 or C18). The PEG-modified phospholipids and derivatized lipids of the present invention may include molar ratios 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 lipids present in the liposome transport vehicle.

[0061] The present invention also intends to utilize noncationic lipids. As used herein, the term "noncationic lipid" refers to any neutral, amphoteric, or anionic lipid. As used herein, the term "anionic lipid" refers to any of several lipid species that carry a net negative charge at a selected pH, such as physiological pH. Noncationic lipids include distearoyl phosphatidylcholine (DSPC), diol eoyl phosphatidylcholine (DOPC), dipalmitoyl phosphatidylcholine (DPPC), diol eoyl phosphatidylglycerol (DOPG), dipalmitoyl phosphatidylglycerol (DPPG), diol eoyl phosphatidylethanolamine (DOPE), palmitoyl oleoyl phosphatidylcholine (POPC), palmitoyl oleoyl-phosphatidylethanolamine (POPE), and diol eoyl-phosphatidyl These include, but are not limited to, tanolamine 4-(N-meleiimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoylphosphatidylethanolamine (DSPE), 16-O-monomethylPE, 16-O-dimethylPE, 18-1-transPE, 1-stearoyl-2-oleoylphosphatidylethanolamine (SOPE), cholesterol, or mixtures thereof. Such noncationic 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 noncationic lipids may constitute 5% to about 90%, or preferably about 10% to about 70%, of the total lipids present in the transport vehicle.

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

[0063] In one 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 embodiments, the transport vehicle comprises cholesterol and / or PEG-modified lipids. In some embodiments, the transport vehicle comprises DMG-PEG2K. In one embodiment, the transport 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 liposome transport vehicles used in the compositions of the present invention can be prepared by various techniques currently known in the art. Multilayer vesicles (MLVs) can be prepared by conventional techniques, for example, by dissolving lipids in a suitable solvent to deposit selected lipids on the inner wall of a suitable container or vessel, and then evaporating the solvent to leave a thin film on the inside of the container, or by spray drying. The aqueous phase can then be added to the container by a vortex motion that results in the formation of MLVs. Monolayer vesicles (ULVs) can then be formed by homogenization, sonication, or extrusion of the multilayer vesicles. In addition, monolayer vesicles can be formed by surfactant removal techniques.

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

[0066] In some embodiments, the compositions of the present invention may be loaded with diagnostic radionuclides, fluorescent substances, or other substances detectable both in vitro and in vivo. For example, suitable diagnostic substances for use in the present invention may include Rhod amine-diol ethylphosphatidylethanolamine (Rh-PE), green fluorescent protein mRNA (GFP mRNA), renyl luciferase mRNA, and firefly luciferase mRNA.

[0067] The selection of a liposome transport vehicle of appropriate dimensions should take into account the site of the target cell or tissue, and to some extent, the application in which the liposomes are produced. In some embodiments, it may be desirable to restrict the transfection of mRNA into certain cells or tissues. For example, for target hepatocytes, the liposome transport vehicle may be sized so that its dimensions are smaller than the fenestrations of the endothelial-lined hepatic sinusoids in the liver, so that the liposome transport vehicle can easily penetrate such endothelial fenestrations and reach the target hepatocytes. Alternatively, the liposome transport vehicle may be sized so that the diameter of the liposomes is sufficient to restrict or clearly avoid the distribution of liposomes into certain cells or tissues. For example, the liposome transport vehicle may be sized so that its dimensions are larger than the fenestrations of the endothelial-lined hepatic sinusoids, so that the distribution of liposome transport vehicles into hepatocytes can be restricted. Generally, the dimensions of the transport vehicle are in the range of approximately 25 to 250 nm, preferably less than approximately 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] Various alternative methods known in the art are available for dimensional determination of liposome transport vehicle populations. One such dimensional determination method is described in U.S. Patent No. 4,737,323, which is incorporated herein by reference. Sonication of liposome suspensions in either bath sonication or probe sonication can reduce the dimensions to within approximately 0.05 microns in diameter. The MLV is gradually reduced to the smallest possible size. Homogenization is another method that relies on shear energy to fragment larger liposomes into smaller ones. In a typical homogenization procedure, the MLV is recycled using a standard emulsion homogenizer until a selected liposome size, typically about 0.1–0.5 microns, is observed. The size of the liposomal vesicles can be determined by pseudo-electric light scattering (QELS) as described in Bloomfield, Ann. Rev. Biophys. Bioeng., 10:421–450 (1981), which is incorporated herein by reference. The average liposome diameter can be reduced by sonication of the formed liposomes. Intermittent sonication cycles may occur alternately with QELS evaluation to induce efficient liposome synthesis. target cell

[0069] As used herein, the term “target cells” refers to cells or tissues that are subject to or targeted by the compositions of the present invention. In some embodiments, the target cells are deficient in the protein or enzyme of interest. For example, if it is desired to deliver nucleic acids to hepatocytes, hepatocytes represent the target cells. In some embodiments, the compositions of the present invention identify and transfect target cells (i.e., do not transfect non-target cells). The compositions of the present invention include, but are not limited to, hepatocytes, epithelial cells, hematopoietic cells, epithelial cells, endothelial cells, lung cells, osteocytes, stem cells, mesenchymal cells, nerve cells (e.g., meninges, 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, cardiomyocytes, skeletal muscle cells, β-cells, pituitary cells, synovial-lining cells, ovarian cells, testicular cells, fibroblasts, B cells, T cells, reticulocytes, leukocytes, granulocytes, and tumor cells, and can also be prepared to preferentially target a variety of target cells.

[0070] The compositions of the present invention can be prepared to preferentially distribute to target cells in, for example, the heart, lungs, kidneys, liver, and spleen. In some embodiments, the compositions of the present invention distribute to liver cells to facilitate the delivery and subsequent expression of mRNA contained therein by those 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 liposome transport vehicle targets hepatocytes and / or preferentially distributes to liver cells upon delivery. After transfection of the target hepatocytes, the mRNA loaded in the liposome carrier is translated, producing, excreting, and systemically distributing functional protein products. In other embodiments, cells other than hepatocytes (e.g., lung, spleen, heart, eye, or central nervous system cells) can function as depot sites 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 of a subject, specifically the production of proteins and / or enzymes exhibiting lower immunogenicity compared to their recombinantly prepared counterparts. In a preferred embodiment of the present invention, the transport vehicle contains mRNA encoding the deficient protein or enzyme. Upon distribution of such a composition to a target tissue and subsequent transfection of such target cells, the exogenous mRNA loaded onto the liposome transport vehicle (e.g., lipid nanoparticles) can be translated in vivo to produce a functional protein or enzyme (e.g., the protein or enzyme deficient in the subject) encoded by the extracorporeally administered mRNA. Thus, the composition of the present invention utilizes the subject's ability to translate extracorporeally or recombinantly prepared mRNA to produce a protein or enzyme translated in vivo, and therefore a functional protein or enzyme (and excreted as necessary). The expressed or translated protein or enzyme may also be characterized by the in vivo inclusion of native post-translational modifications, which are often absent in recombinantly prepared proteins or enzymes, and therefore further reduce the immunogenicity of the translated protein or enzyme.

[0072] Administration of mRNA encoding a deficient protein or enzyme avoids the need to deliver the nucleic acid to a specific organelle (e.g., mitochondria) within the target cell. Rather, during transfection of the target cell and delivery of the nucleic acid to the target cell's cytoplasm, the mRNA content of the transport vehicle can be translated, leading to the expression of the functional protein or enzyme.

[0073] The present invention also aims at the discriminative targeting of target cells and tissues using both passive and active targeting means. Passive targeting utilizes the natural distribution patterns of transport vehicles in vivo without relying on the use of further excipients or means to enhance the recognition of transport vehicles by target cells. For example, transport vehicles exposed to phagocytosis by reticuloendothelial cells are likely to accumulate in the liver or spleen, thus providing means for passively directing the delivery of compositions to such target cells.

[0074] Alternatively, the present invention envisions active targeting involving the use of further excipients, referred herein as “targeting ligands,” which are conjugated (either covalently or non-covalently) to a transport vehicle and can promote the localization of such transport vehicle in certain target cells or tissues. For example, targeting may be mediated by the incorporation of one or more endogenous targeting ligands (e.g., apolipoprotein E) in or on a transport vehicle to promote distribution to target cells or tissues. Recognition of the targeting ligand by the target tissue actively promotes the tissue distribution and cellular uptake of the transport vehicle and / or its components in the target cells and tissues (e.g., incorporation of an apolipoprotein E targeting ligand in or on a transport vehicle promotes the recognition of the transport vehicle and its binding to endogenous low-density lipoprotein receptors expressed by hepatocytes). The compositions provided herein may contain ligands that can enhance the affinity of the composition to target cells. Targeted ligands may be bound to the outer two layers of lipid particles during or after formulation. These methods are well known in the art. In addition, some lipid particle formulations may utilize fusion polymers such as PEAA, hemagglutinin, other lipopeptides (see U.S. Patent Applications No. 08 / 835,281 and 60 / 083,294, 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 demonstrate improved transfection efficacy and / or enhanced selectivity for target cells or tissues of interest. Thus, compositions comprising one or more ligands (e.g., peptides, aptamers, oligonucleotides, vitamins, or other molecules) that can increase the affinity of the composition and its nucleic acid-containing components to target cells or tissues are contemplated. Suitable ligands may optionally be bound or ligated to the surface of the transport vehicle. In some embodiments, the targeted ligand may spread on the surface of the transport vehicle or be encapsulated within the transport vehicle. Suitable ligands are selected based on their physical, chemical, or biological properties (e.g., selective affinity and / or recognition of target cell surface markers or features).Cell-specific target sites and their corresponding targeting ligands can vary considerably. Suitable targeting ligands are selected to leverage the unique characteristics of the target cells and thus enable the composition to distinguish between target and non-target cells. For example, the compositions of the present invention may contain surface markers (e.g., apolipoprotein B or apolipoprotein E) that selectively enhance the recognition or affinity to hepatocytes (e.g., by receptor-mediated recognition of such surface markers and binding to such surface markers). Furthermore, the use of galactose as a targeting ligand is predicted to direct the compositions of the present invention towards parenchymal hepatocytes, or the use of mannose-containing sugar residues as targeting ligands is predicted to direct the compositions of the present invention towards liver endothelial cells (e.g., mannose-containing sugar residues that can preferentially bind to asialoglycoprotein receptors present in hepatocytes) (Hillery AM, et al. "Drug Delivery and Targeting: For Pharmacists and Pharmaceutical Sciences"). See "Tists" (2002) Taylor & Francis, Inc.). Therefore, the presentation of such targeted ligands conjugated to a portion present in a transport 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 targeted ligands include one or more peptides, proteins, aptamers, vitamins, and oligonucleotides. Application and Administration

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

[0076] The compositions and methods of the present invention provide mRNA delivery for the treatment of several disorders. Specifically, the compositions and methods of the present invention are suitable for the treatment of diseases or disorders related to defective proteins and / or enzymes (e.g., mRNA encoding hormones and neurotransmitters) that are excreted or secreted into the surrounding extracellular fluid by target cells. In some embodiments, the disease may involve a defect or deficiency of secretory proteins (e.g., Fabry disease, or ALS). In some embodiments, the disease may not be caused by a defect or deficiency of secretory proteins, but may benefit from the provision of secretory proteins. For example, the symptoms of a disease may be improved by providing the compositions of the present invention (e.g., cystic fibrosis).Disorders for which this invention is useful include Huntington's disease; Parkinson's disease; muscular dystrophy (e.g., Duchenne and Becker types); hemophilia (e.g., type B hemophilia (FIX), type A hemophilia (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 adrenal spinal neuropathy; Friedreich's ataxia; Pelizaeus-Merzbacher disease; TSC1 and TSC2-associated tuberous sclerosis; and Sanfilippo B syndrome (MPS). IIIB); CTNS-associated cystine storage disorders; FMR1-associated disorders including Fragile X syndrome, Fragile X-associated tremor / ataxia syndrome, and Fragile X premature menopause syndrome; Prader-Willi syndrome; Hereditary hemorrhagic capillary dilatation (AT); Niemann-Pick disease type C1; Juvenile neuronal ceroid lipofuscinosis (JNCL), juvenile Batten disease, Hartia-Santavuori disease, Jansky-Birszowski disease, and neuronal ceroid lipofuscinosis-associated disorders including PTT1 and TPP1 deficiencies; Childhood ataxia associated with EIF2B1, EIF2B2, EIF2B3, EIF2B4, and EIF2B5 with central nervous system hypomyelination / white matter disappearance; CACNA1A and CACNB4-associated transient ataxia type 2; Disorders include, but are not limited to, those described above: MECP2-related disorders including Lasiclett syndrome, MECP2-related severe neonatal encephalopathy, and PPM-X syndrome; CDKL5-related atypical Rett syndrome; Kennedy disease (SBMA); Notch 3-related autosomal dominant cerebral arteriovenous disease (CADASIL) with subcortical infarction and leukoencephalopathy; SCN1A and SCN1B-related paroxysmal disorders; polymerase G-related disorders including Alpers-Huttenlocher syndrome, POLG-related ataxic neuropathy, dysarthria, and ophthalmoplegia, as well as autosomal dominant and recessive progressive extraocular myopalsy with mitochondrial DNA deletion; X-linked adrenal hypoplasia; X-linked agammaglobulinemia; Wilson's disease; and Fabry disease. In one embodiment, the nucleic acids of the present invention, and specifically mRNA, may encode functional proteins or enzymes secreted into the extracellular space.For example, secreted proteins include coagulation 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 present invention, compositions may include mRNA encoding erythropoietin, α1-antitrypsin, carboxypeptidase N, or human growth hormone.

[0077] In embodiments, the present invention encodes a secreted protein composed of subunits encoded by two or more genes. For example, a secreted protein may be a heterodimer, with each strand or subunit being encoded by an individual gene. Two or more mRNA molecules may be delivered by a transport vehicle, and the mRNA may encode individual subunits of the secreted protein. Alternatively, a single mRNA may be engineered to encode two or more subunits (e.g., a single-stranded Fv antibody). In one embodiment, individual mRNA molecules encoding individual subunits may be administered by individual transport vehicles. In one 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-stranded Fv (scFv)) to immunize a target. While one embodiment of the present invention relates to a method and composition useful for immunizing a target (e.g., via translation of mRNA encoding a functional antibody), the present invention disclosed herein and contemplated herein is broadly applicable. In alternative embodiments, the compositions of the present invention encode antibodies used to temporarily or prolongedly influence a functional response in a target. For example, the mRNA of the present invention may encode a functional monoclonal or polyclonal antibody that may be useful for targeting and / or inactivating a biological target (e.g., a stimulant cytokine such as tumor necrosis factor) during translation and secretion from target cells. Similarly, the mRNA nucleic acid of the present invention may encode, for example, a functional anti-nephritis factor antibody useful for treating membranoproliferative glomerulonephritis type II or acute hemolytic uremic syndrome, or an anti-vascular endothelial growth factor (VEGF) antibody useful for treating VEGF-mediated diseases such as cancer. In other embodiments, the secreted protein is a cytokine or other secreted protein consisting of two or more subunits (e.g., IL-12 or IL-23).

[0078] The compositions of the present invention can be administered to a target. In some embodiments, the compositions are formulated in combination with one or more further nucleic acids, carriers, targeted ligands, or stabilizing reagents, or formulated in a pharmacological composition mixed with suitable excipients. For example, in one embodiment of the present invention, the composition may be prepared to deliver two or more distinctly different mRNAs encoding 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] Various molecules that may have pharmaceutically or therapeutic effects can be delivered to target cells using the compositions and methods of the present invention. The molecules may be organic or inorganic. Organic molecules may be peptides, proteins, carbohydrates, lipids, sterols, nucleic acids (including peptide nucleic acids), or any combination thereof. Formulations for delivery to target cells may comprise one or more types of molecules, for example, two different nucleotide sequences, or proteins, enzymes, or steroids.

[0080] The compositions of the present invention may be administered and prescribed in accordance with current medical practice, taking into consideration the clinical condition of the subject, the site and method of administration, the administration schedule, the age, sex, and weight of the subject, and other factors appropriate to clinicians in the art. The “effective dose” for the purposes of this specification may be determined by such relevant considerations known to those skilled in the art in the fields of experimental clinical research, pharmacology, clinical medicine, and medicine. In some embodiments, the dose is an effective dose to achieve at least some degree of stabilization, improvement, or elimination of symptoms, and other indicators may be selected by those skilled in the art as appropriate criteria for evaluating disease progression, regression, or improvement. For example, a suitable dose and dosage regimen is one that causes at least transient protein production.

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

[0082] Alternatively, the compositions of the present invention may be administered topically rather than systemically, preferably in a sustained-release formulation, for example, by direct injection of the pharmaceutical composition into a targeted tissue. Topical delivery can act in a variety of ways depending on the targeted tissue. For example, an aerosol containing the composition of the present invention may be inhaled for nasal, tracheal, or bronchial delivery; the composition may be injected, for example, into a site of injury, disease, or pain; the composition may be provided as a lozenge for oral, tracheal, or esophageal application; supplied in liquid, tablet, or capsule form for gastric or intestinal administration; supplied in suppository form for rectal or vaginal application; or even delivered to the eye by cream, droplet, or injection. Formulations containing the composition of the present invention, combined with a therapeutic molecule or ligand, may be administered, for example, by a polymer or other structure or substance that can allow the composition to diffuse from the injection site to surrounding cells, or even surgically. Alternatively, they may be applied surgically without the use of polymers or supports.

[0083] In one embodiment of the present invention, compositions are formulated to be suitable for the sustained release of mRNA contained therein. Such sustained-release compositions can be conveniently administered to a subject at extended dosing intervals. For example, in one embodiment of the present invention, the composition is administered to the subject twice daily, once daily, or once every two days. In a preferred embodiment of the present invention, the composition is administered to the subject twice weekly, once weekly, 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. Compositions and liposome carriers formulated for depot administration (e.g., intramuscular, subcutaneous, intravitreous) to deliver or release mRNA over extended periods are also intended. Preferably, the sustained-release means used are combined with modifications to improve mRNA stability.

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

[0085] While certain compounds, compositions, and methods of the present invention are clearly described according to certain embodiments, the following examples are solely for illustrative purposes of the compounds of the present invention and are not intended to limit the compounds of the present invention. Publications, reference materials, accession numbers, etc., referenced herein to provide background information on the present invention and to offer further details regarding its practice are, by reference, incorporated herein in their entirety.

[0086] The articles "a" and "an" used in the specification and claims should be understood to refer to multiple subjects unless explicitly stated otherwise. Claims or statements that include "or" between one or more members of a group should be understood to refer to multiple subjects. Unless otherwise explicitly stated or evident from the context, one, two or more, or all of the members of the group are deemed satisfied if they are present in, used in, or otherwise related to a given product or process. The present invention includes embodiments in which only one member of the group is present in, used in, or otherwise related to a given product or process. The present invention also includes embodiments in which two or more members of the group, or all members of the group, are present in, used in, or otherwise related to a given product or process. Furthermore, it should be understood that the present invention encompasses all variations, combinations, and rearrangements in which one or more limitations, elements, annotations, descriptive terms, etc., derived from one or more of the listed claims are introduced into another claim (or any other related claim) that depends on the same underlying claim, unless otherwise indicated that such a contradiction or inconsistency would arise, or unless otherwise evident to a person skilled in the art. Where elements are presented in a list (e.g., by a Markush group or similar format), each of those elements is also disclosed as a subgroup, and it should be understood that any element(s) may be removed from that group. In general, where the present invention or an aspect of the present invention is considered to include certain elements, features, etc., it should be understood that a particular embodiment or aspect of the present invention consists of, or is partially composed of, such elements, features, etc. For the sake of simplification, these embodiments are not described in detail in any way in this specification using a great many words. It should also be understood that any embodiment or aspect of the present invention may be expressly excluded from the claims, regardless of whether specific exclusions are enumerated herein. Publications and other reference materials referenced herein to provide context for the present invention and to offer further details on its practice are incorporated herein by reference. Examples Example 1: Protein production depot via intravenous delivery of polynucleotide composition

[0087] Messenger RNA Human erythropoietin (EPO) (SEQ ID NO: 3, Figure 3), human α-galactosidase (GLA) (SEQ ID NO: 4, Figure 4), human α-1 anti-trypsin (A1AT) (SEQ ID NO: 5, Figure 5), and human factor IX (FIX) (SEQ ID NO: 6, Figure 6) were synthesized by in vitro transcription from gene-encoding plasmid DNA templates, and then a 5' cap structure (Cap1) (Fechter & Brownlee, J. Gen. Virology 86:1239-1249 (2005)) and a 3' poly(A) tail of approximately 200 nucleotides, determined by gel electrophoresis, were added. The 5' and 3' untranslated regions are present in the respective mRNA products in the following examples and are defined by SEQ ID NOs: 1 and 2 (Figures 1 and 2), respectively. Lipid nanoparticle formulations

[0088] Formulation 1: A fixed volume of 50 mg / mL ethanol solutions of C12-200, DOPE, Chol, and DMG-PEG2K (40:30:25:5) was 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 mRNA aqueous solution and shaken to obtain a final suspension in 20% ethanol. The resulting nanoparticle suspension was filtered, difiltration through PBS (pH 7.4), concentrated, and stored at 2–8°C.

[0089] Preparation 2: A fixed volume of 50 mg / mL ethanol solutions of DODAP, DOPE, cholesterol, and DMG-PEG2K (18:56:20:6) was mixed and diluted with ethanol to a final volume of 3 mL. Separately, an aqueous buffer 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 mRNA aqueous solution and shaken to obtain a final suspension in 20% ethanol. The resulting nanoparticle suspension was filtered, difiltration with PBS (pH 7.4), concentrated, and stored at 2-8°C. Final concentration = 1.35 mg / mL EPO mRNA (encapsulated).ave =75.9nm(Dv (50) =57.3nm;Dv (90) (=92.1nm).

[0090] Formulation 3: A fixed volume of 50 mg / mL ethanol solutions of HGT4003, DOPE, cholesterol, and DMG-PEG2K (50:25:20:5) was 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 mRNA aqueous solution and shaken to obtain a final suspension in 20% ethanol. The resulting nanoparticle suspension was filtered, difiltration through PBS (pH 7.4), concentrated, and stored at 2–8°C.

[0091] Formulation 4: A fixed volume of 50 mg / mL ethanol solutions of ICE, DOPE, and DMG-PEG2K (70:25:5) was 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 mRNA aqueous solution and shaken to obtain a final suspension in 20% ethanol. The resulting nanoparticle suspension was filtered, difiltration through PBS (pH 7.4), concentrated, and stored at 2–8°C.

[0092] Formulation 5: A fixed volume of 50 mg / mL ethanol solutions of HGT5000, DOPE, cholesterol, and DMG-PEG2K (40:20:35:5) was mixed and diluted with ethanol to a final volume of 3 mL. Separately, an aqueous buffer 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 mRNA aqueous solution and shaken to obtain a final suspension in 20% ethanol. The resulting nanoparticle suspension was filtered, difiltration through 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) (=157nm).

[0093] Formulation 6: A fixed volume of 50 mg / mL ethanol solutions of HGT5001, DOPE, cholesterol, and DMG-PEG2K (40:20:35:5) was mixed and diluted with ethanol to a final volume of 3 mL. Separately, an aqueous buffer 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 mRNA aqueous solution and shaken to obtain a final suspension in 20% ethanol. The resulting nanoparticle suspension was filtered, difiltration through 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 specified, each experiment began with male CD-1 mice approximately 6–8 weeks old. Samples were introduced by a single bolus tail vein injection of encapsulated mRNA with a total equivalent dose of 30–200 micrograms. Mice were sacrificed and perfused with saline at the specified time. Isolation of organ tissue for analysis

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

[0096] All animals were euthanized by CO2 asphyxiation 48 hours after dose administration (±5%), followed by thoracotomy to collect cardiac blood. Whole blood (maximum available volume) was collected via cardiac puncture of euthanized animals, placed in a serum separator, allowed to coagulate at room temperature for at least 30 minutes, and 9300 g was centrifuged at 22°C ± 5°C for 10 minutes to extract serum. For preliminary blood collection, approximately 40–50 μL of whole blood was collected via facial vein puncture or tail transection. Samples collected from untreated animals were used as a 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, catalog number Dep-00). The positive controls used consisted of ultra-high purity and tissue culture grade recombinant human erythropoietin protein (R&D Systems, catalog numbers 286-EP and 287-TC, respectively). Detection was monitored via absorption (450 nm) on a Molecular Device Flex Station instrument.

[0098] GLA ELISA: 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), the standard ELISA procedure was followed. The 3,3',5,5'-tetramethylbenzidine (TMB) substrate solution was activated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG. The reaction was stopped after 20 minutes using 2N H2SO4. Detection was performed using Molecular Absorption (450 nm) was monitored on the Device Flex Station instrument. Untreated mouse serum and human α-galactosidase protein were used as negative and positive controls, respectively.

[0099] FIX ELISA: FIX protein quantification was performed according to the procedure reported for the Human FIX ELISA Kit (AssayMax, Assay Pro, catalog number EF1009-1).

[0100] A1AT ELISA: The A1AT protein was quantified according to the procedure reported for the Human A1AT ELISA Kit (Innovative Research, catalog number IRAPKT015). Western blot analysis

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

[0102] The study described in this embodiment demonstrates the use of mRNA-encapsulated lipid nanoparticles as a depot source for protein production. Such depot effects can be achieved in multiple sites within the body (i.e., liver, kidney, spleen, and muscle). Measurement results of desired exogenous proteins derived from messenger RNA delivered via liposome nanoparticles were obtained and quantified, demonstrating protein secretion from depots using human erythropoietin (hEPO), human α-galactosidase (hGLA), human α-1 anti-trypsin (hA1AT), and human factor IX (hFIX) mRNA. 1A. Results of in vivo human EPO protein production

[0103] The production of hEPO protein was demonstrated 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) yielded 18.3 ug / mL of hEPO protein, which was secreted into the bloodstream. Normal hEPO protein levels in human serum are 3.3–16.6 mIU / mL (NCCLS document C28-P, Vol. 12, No. 2). Based on the specific activity of EPO protein at 120,000 IU / mg, this formulation produces 27.5–138 pg / mL of hEPO protein in a normal human individual. Therefore, a single 30 ug dose of a C12-200-based cationic lipid formulation encapsulating hEPO mRNA resulted in a more than 100,000-fold increase in the respective protein levels compared to physiological levels.

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

[0105] In addition, the obtained proteins were tested to determine if they were active and functioned properly. In the case of mRNA replacement therapy (MRT) using hEPO mRNA, changes in hematocrit levels of five different lipid nanoparticle formulations (Figure 8, Table 1) were monitored over 10 days to evaluate protein activity. During this period, two of the five formulations showed an increase in hematocrit levels (≥15%), indicating the activity of the hEPO protein produced from such systems.

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

[0107] A second exogenous protein system was studied to demonstrate the "depot effect" when mRNA-loaded lipid nanoparticles were used. Animals were intravenously administered a single dose of 30 micrograms of encapsulated human α-galactosidase (hGLA) mRNA using a C12-200-based lipid nanoparticle system and sacrificed after 6 hours (Formulation 1). Secreted hGLA protein was quantified using ELISA. Untreated mouse serum and human α-galactosidase protein were used as controls. The detection of human α-galactosidase protein was monitored for 48 hours.

[0108] Measurable levels of hGLA protein were observed throughout the experimental time course, with the highest level being 2.0 ug / mL at 6 hours (Figure 10). Table 3 lists the specific amounts of hGLA found in serum. Normal activity in healthy human males has been reported to be approximately 3.05 nanomoles / hour / mL. In terms of α-galactosidase activity, recombinant human α-galactosidase protein showed an activity of 3.56 × 10⁶. 6The values ​​are nanomoles / hour / mg. Analysis of these values ​​yields an amount of hGLA protein of approximately 856 pg / mL in normal, healthy male individuals. The amount of hGLA protein observed 6 hours after administration of hGLA mRNA-loaded lipid nanoparticles, 2.0 ug / mL, is more than 2300 times higher than the normal physiological level. Furthermore, a considerable level of hGLA protein (86.2 ng / mL) can still be detected after 48 hours. This level represents nearly 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 after administration of 0.2 mg / kg is approximately 108 minutes. The production of GLA protein via the "depot effect" after administration of GLA mRNA-loaded lipid nanoparticles shows a significant increase in blood retention time compared to direct injection of naked recombinant protein. As mentioned above, a considerable amount of protein remains 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 proteins produced from these nanoparticle systems are quite active and reflect the level of available protein (Figure 12, Table 3). AUC comparisons of mRNA therapy-based hGLA production compared to enzyme substitution therapy (ERT) in mice and humans show increases of 182-fold and 30-fold, respectively (Table 4). [Table 4]

[0111] The ability of mRNA-encapsulated lipid nanoparticles to target organs that can function as depots for desired protein production has been demonstrated. Observed secretion levels were several orders of magnitude higher than normal physiological levels. This “depot effect” is repeatable. Figure 12 shows robust protein production achieved by administering a single 30ug dose of hGLA mRNA-loaded C12-200-based lipid nanoparticles (Formulation 1) to wild-type (CD-1) mice. This reiterates what is observed when administered. In this experiment, hGLA levels were evaluated over 72 hours. Serum with a maximum mean of 4.0 ug of human hGLA protein / mL was detected 6 hours after administration. Based on a normal physiological level of approximately 1 ng / mL of hGLA protein, the hGLA MRT provides approximately 4000 times higher protein levels. As previously mentioned, hGLA protein could be detected up to 48 hours after administration (Figure 12).

[0112] Analysis of tissues isolated from this same experiment provided insights into the distribution of hGLA protein in mice treated with hGLA MRT (Figure 13). Hyperphysiological levels of hGLA protein were detected in the liver, spleen, and kidneys of all treated mice, with peak 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, it was shown that the production of hGLA upon administration of hGLA mRNA-loaded C12-200 nanoparticles exhibited a dose-response in both serum (Figure 14A) and liver (Figure 14B).

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

[0115] In individual experiments, hGLA MRT was applied to hGLA knockout mice (Fabry mice), a mouse disease model. Female knockout mice were administered a single intravenous injection of 0.33 mg / kg of hGLA mRNA-loaded C12-200-based lipid nanoparticles (Formulation 1). Significant amounts of MRT-derived hGLA protein were produced, peaking at 6 hours (approximately 560 ng / mL serum), which is about 600 times higher than normal physiological levels. Furthermore, hGLA protein remained 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 levels observed in major organs with reported normal physiological levels is plotted (normal levels are plotted as dotted lines). While protein levels at 24 hours were higher than at 72 hours post-administration, levels of hGLA protein detected in the liver, kidney, spleen, and heart of treated Fabry mice were comparable to wild-type levels. For example, 3.1 ng of hGLA protein per 1 mg of tissue was observed in the kidneys of treated mice 3 days after a single MRT treatment.

[0117] In subsequent experiments, ERT-based α-galactosidase treatment and hGLA MRT-based treatment were compared in male Fabry knockout mice. A single intravenous dose of 1.0 mg / kg was administered in each therapy, and the mice were sacrificed one week after administration. Serum levels of hGLA protein were monitored at 6 hours and 1 week post-infusion. One week after administration, hGLA protein accumulation was analyzed in the liver, kidneys, spleen, and heart. In addition to in vivo distribution analysis, efficacy criteria included globulin in the kidneys and heart. The results were determined using measurements of riaosylceramide (Gb3) and lyso-Gb3 depletion. Figure 18 shows the 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 at 6 hours and 1 week post-administration. A robust signal was detected at 6 hours in mice treated with MRT, with a serum hGLA protein level of approximately 4.0 ug / mL. In contrast, no detectable α-galactosidase remained in the bloodstream at this time.

[0118] Fabry mice in this experiment were sacrificed one week after the initial injection, and their organs were collected and analyzed (liver, kidney, spleen, heart). Figure 19 shows a comparison of human GLA protein observed in each organ after either hGLA MRT treatment or α-galactosidase ERT treatment. The 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 an hGLA protein accumulation of 2.42 ng / mg of hGLA protein in the kidneys, while mice treated with α-galactosidase had only a residual level (0.37 ng / mg of hGLA protein). This corresponds to a level of hGLA protein approximately 6.5 times higher than that of mice treated with hGLA MRT. In cardiac analysis, 11.5 ng of hGLA protein / mg of hGLA protein was observed in the MRT-treated cohort compared to only 1.0 ng of α-galactosidase / mg of hGLA protein. This corresponds to approximately 11 times higher accumulation in the hearts of mice treated with hGLA MRT compared to ERT-based therapy.

[0119] In addition to the in vivo distribution analysis performed, efficacy was assessed using measurements of globotriaosylceramide (Gb3) and Lyso-Gb3 levels in major organs. A direct comparison of Gb3 reduction after a single intravenous dose of 1.0 mg / kg GLA MRT treatment with that after α-galactosidase ERT-based therapy at an equivalent dose revealed significant differences in Gb3 levels in the kidneys and heart. For example, Gb3 levels for GLA MRT and α-galactosidase decreased by 60.2% and 26.8%, respectively (Figure 20). Furthermore, Gb3 levels in the heart decreased 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 efficiently reduced Lyso-Gb3 more effectively than α-galactosidase in both the kidneys and hearts (Figures 20 and 21, respectively). Specifically, Fabry mice treated with MRT showed 86.1% and 87.9% reductions in Lyso-Gb3 in the kidneys and hearts, respectively, compared to 47.8% and 61.3% reductions in mice treated with α-galactosidase.

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

[0122] Overall, mRNA replacement therapy applied as a depot for protein production produces large quantities of functionally therapeutically active proteins at a hyperphysiological level. This method has been demonstrated to result in a sustained circulating half-life of the desired protein, and this MRT-derived protein The protein is highly effective in the therapy demonstrated with the α-galactosidase enzyme in Fabry mice. 1C. Results of in vivo human FIX protein production

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

[0124] Pharmacokinetic analysis over 72 hours showed that MRT-derived FIX protein could be detected at all time points tested (Figure 24). The peak serum concentration was observed 24 hours post-infusion, with a value of approximately 3 ug (2995 ± 738 ng / mL) of FIX protein / mL serum. This demonstrates another successful example of the depot effect. 1D. Results of in vivo human A1AT protein production

[0125] We conducted a study to determine the amount of A1AT protein secreted into the bloodstream by administering α-1-anti-trypsin (A1AT) mRNA-loaded lipid nanoparticles to wild-type mice (CD-1). Robust protein production was observed after a single intravenous administration of 30ug of C12-200-based A1AT mRNA-loaded lipid nanoparticles (dosage based on encapsulated mRNA) (Formulation 1) (Figure 25).

[0126] As shown in Figure 25, detectable levels of human A1AT protein derived from A1AT MRT were observed over 24 hours after administration. A maximum serum level of approximately 48 ug of A1AT protein / mL serum was detected 12 hours after infusion. Example 2: Protein production depot via pulmonary delivery of polynucleotide composition Injection protocol

[0127] At the beginning of each experiment, all studies were conducted using female CD-1 or BALB / C mice approximately 7–10 weeks old. The test substance was introduced via single intratracheal aerosol administration. The mice were sacrificed and perfused with saline at the specified time. The lungs of each mouse were collected, divided into two, and either stored 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] The depot effect can be obtained via pulmonary delivery (e.g., intranasal or intratracheal spray). Measurement results for desired exogenous proteins derived from messenger RNA delivered via nanoparticle systems were obtained and quantified.

[0129] The production of human EPO protein via hEPO mRNA-loaded lipid nanoparticles in CD-1 mice was investigated via single intratracheal administration (MicroSprayer®). Several formulations were tested using various cationic lipids (Formulations 1, 5, 6). All formulations yielded highly encapsulated human EPO mRNA. At the time of administration, animals were sacrificed 6 hours after administration, and lung and serum samples were collected.

[0130] Human EPO protein was detected at the administration site (lungs) during processing via aerosol delivery. Serum analysis 6 hours after administration showed detectable levels of hEPO protein in the blood. These data (shown in Figure 26) indicate the production of hEPO protein (and This demonstrates the lungs' ability to function as a "depot" for secretion.

Claims

[Claim 1] A composition, etc., comprising the mRNA molecule described herein and lipid nanoparticles.

Images