Lipid nanoparticles for protein delivery

Abstract

The present invention relates to lipid nanoparticles for protein delivery, specifically to lipid nanoparticles for delivering various pharmaceutical proteins, such as therapeutic proteins or enzyme replacement therapy (ERT) enzymes, into cells or the body. The present invention can be useful for treating and preventing various diseases, such as lysosomal storage diseases, by stably and effectively delivering various proteins, such as therapeutic proteins or ERT enzymes, into cells through lipid nanoparticles.

Description

[Technical Field]

[0001] The present invention relates to lipid nanoparticles for protein delivery, and more particularly to lipid nanoparticles for intracellular or intrabody delivery of various pharmaceutical proteins, such as therapeutic proteins or enzyme replacement therapy (ERT) enzymes. [Background technology]

[0002] Lipid nanoparticles (LNPs) have recently attracted much attention as a system for effectively delivering biomolecules such as mRNA into the body. These lipid nanoparticles are composed of phospholipids, cholesterol, various ionic lipids, PEG-containing phospholipids, etc., to stably deliver substances into the body.

[0003] Lysosomal storage disorders (LSDs) are relatively rare genetic metabolic disorders caused by defects in lysosomal function. LSDs are typically caused by a deficiency of a single enzyme involved in the breakdown of metabolic products within the lysosome. The accumulation of products due to the lack of enzyme activity can affect various organ systems, causing severe symptoms and premature death. The majority of LSDs are also associated with significant neurological impairments, ranging from progressive neurodegeneration and severe cognitive impairment to interstitial, behavioral, and psychiatric disorders.

[0004] Representative examples of lysosomal storage diseases include Fabry disease, Gaucher disease, and mucopolysaccharidoses, and enzyme replacement therapy (ERT) has been established as the standard treatment. ERT reduces harmful levels of substances accumulated within cells by periodically administering missing enzyme proteins intravenously (Concolino et al., Italian Journal of Pediatrics, 2018).

[0005] However, to improve the therapeutic efficiency of enzyme replacement therapy, it is necessary to develop a delivery system that can effectively and stably supply enzyme proteins intracellularly while reducing side effects such as undesirable immune responses, which would allow for lowering the concentration of the administered enzyme and minimizing anti-drug antibody responses. Summary of the Invention [Problem to be solved by the invention]

[0006] An exemplary object of the present invention is to provide lipid nanoparticles for intracellular or internal delivery of proteins.

[0007] Another exemplary object of the present invention is to provide lipid nanoparticles encapsulating proteins.

[0008] Yet another exemplary object of the present invention is to provide a composition for intracellular or internal delivery of proteins comprising lipid nanoparticles encapsulating the proteins.

[0009] Yet another exemplary object of the present invention is to provide a pharmaceutical composition for preventing or treating lysosomal storage diseases, comprising lipid nanoparticles encapsulating proteins.

[0010] It is yet another exemplary object of the present invention to provide a method for producing protein-encapsulated lipid nanoparticles.

[0011] Yet another exemplary object of the present invention is to provide a method for intracellular delivery of ERT enzymes or therapeutic proteins using lipid nanoparticles.

[0012] Yet another exemplary object of the present invention is to provide a method for treating diseases using lipid nanoparticles encapsulating ERT enzymes or therapeutic proteins.

[0013] The technical problems to be solved based on the technical ideas of the invention disclosed in this specification are not limited to the problems to be solved above, and other problems not mentioned will be clearly understood by those skilled in the art from the following description. [Means for solving the problem]

[0014] This will be explained in more detail as follows. Meanwhile, each description and embodiment disclosed in this application can also be applied to each of the other descriptions and embodiments. In other words, all combinations of various elements disclosed in this application belong to the category of this application. Furthermore, the category of this application is not limited by the specific descriptions described below.

[0015] In one embodiment of the present invention, the present invention provides lipid nanoparticles for intracellular or intracorporeal delivery of proteins. Specifically, the present invention provides lipid nanoparticles for intracellular or intracorporeal delivery of proteins, the lipid nanoparticles comprising a lipid mixture including hydrogenated soybean phosphatidylcholine (HSPC), cholesterol, DOTAP, and DSPE-mPEG.

[0016] The term "lipid nanoparticle (LNP)" as used herein refers to a vesicle having adjacent lipid bilayers, such as a spherical vesicle, which can be used for delivery to a target location. Such lipid nanoparticles include liposomes, solid lipid nanoparticles (SLN), nanostructured lipid carriers (NLC), and unilamellar membrane structures (micelles).

[0017] The lipid nanoparticles of the present invention may contain one or more types of lipids. Examples include ionic lipids, PEG-lipids, phospholipids, and cholesterol. Here, the ionic lipid may be a cationic lipid. Examples of cationic lipids include N-(2,3-dioleoyloxy)propyl-N,N,N-trimethylammonium chloride (DOTAP); N-(2,3-dioleyloxy)propyl-N,N,N-trimethylammonium chloride (DOTMA); N,N-distearyl-N,N-dimethylammonium bromide (DDAB); N,N-dioleyl-N,N-dimethylammonium chloride (DODAC); N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide (DMRIE); N1,N3,N5-tris(3- (Didodecylamino)propyl)benzene-1,3,5-tricarboxamide (TT3), Lipofectamine; 1,2-Dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), Dioctadecyldimethylammonium (DODMA), Distearyldimethylammonium (DSDMA), 3-(N-(N',N'-dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol), and 1,2-Dioleoyl-3-dimethylammonium-propane (DODAP).

[0018] It can also be composed of other lipid components. For example, phosphatidylcholines (PCs) (e.g., 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE)), sterols (e.g., cholesterol) and polyethylene glycol (PEG)-lipid conjugates (e.g., 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[folic acid (polyethylene glycol)-2000] (DSPE- The lipid molecules may include other lipid molecules belonging to the following families: 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (C14-PEG2000), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-methoxy(polyethylene glycol) (DSPE-mPEG), L-α-phosphatidylcholine (HSPC), dipalmitoylphosphatidylcholine (DPPC), and 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (C14-PEG2000).

[0019] Specifically, the lipid nanoparticles of the present invention may comprise a lipid mixture comprising HSPC; cholesterol; DOTAP; and DSPE-mPEG.

[0020] The proportion of each lipid in the lipid nanoparticle is not particularly limited, but may be, for example, 8-15 mol% of the lipid mixture in the lipid nanoparticle, 30-50 mol% of cholesterol, 40-60 mol% of DOTAP, and 0.5-5 mol% of DSPE-mPEG. More preferably, 9 mol% of the lipid mixture in the lipid nanoparticle may be HSPC, 40 mol% of cholesterol, 50 mol% of DOTAP, and 1 mol% of DSPE-mPEG.

[0021] The lipid mixture may also be one in which the molar ratio of HSPC:cholesterol:DOTAP:DSPE-mPEG is 8-15:30-50:40-60:0.5-5, preferably the lipid mixture may be one in which the molar ratio of HSPC:cholesterol:DOTAP:DSPE-mPEG is 8-10:35-45:45-55:0.5-1.5, and more preferably the lipid mixture is one in which the molar ratio of HSPC:cholesterol:DOTAP:DSPE-mPEG is 9:40:50:1, but is not limited to these.

[0022] Within the above range, the optimal molar ratio or molar percentage can be appropriately selected depending on the type of protein to be encapsulated in the lipid nanoparticles. For example, when human iduronate-2-sulfatase (IDS) is encapsulated in lipid nanoparticles, the lipid mixture may preferably be composed of HSPC:cholesterol:DOTAP:DSPE-mPEG in a molar ratio of 9:40:50:1.

[0023] In the present invention, the particle size of lipid nanoparticles can affect drug release rate, biodistribution, mucoadhesion, cellular water absorption, buffer exchange into the nanoparticle interior, and protein diffusion. In one example, the LNP diameter can be in the range of 10 to 500 nm, preferably in the range of 70 to 120 nm, and more preferably in the range of 75 to 95 nm.

[0024] In the present invention, the polydispersity index (PDI) of lipid nanoparticles is a measurement of the molecular or particle size heterogeneity of a lipid mixture. For example, the PDI of lipid nanoparticles may be in the range of 0.1 to 0.4, preferably in the range of 0.1 to 0.2, and more preferably in the range of 0.15 to 0.18.

[0025] In the present invention, the protein can be applied without any particular limitation, and can include, for example, an enzyme or a therapeutic protein. The therapeutic protein refers to a protein for treating or preventing a disease or disorder, and can be a recombinant protein, an antibody, a biosimilar, a biobetter, etc. In the present invention, the enzyme can be applied without any particular limitation, and can be an enzyme that is deficient or absent in the body, and in particular, the enzyme can be an enzyme replacement therapy (ERT) enzyme.

[0026] In the present invention, the enzyme can be applied without any particular limitation, but may be an enzyme that is deficient or absent in the body, and particularly, the enzyme may be an enzyme for enzyme replacement therapy (ERT).

[0027] In another aspect of the present invention, the present invention provides protein encapsulated lipid nanoparticles comprising a lipid mixture comprising HSPC; cholesterol; DOTAP; and DSPE-mPEG.

[0028] The "lipid nanoparticles" and "proteins" are as described above.

[0029] The term "enzyme replacement therapy" as used herein refers to a treatment in which a patient's body is deficient in an enzyme, administered directly into the veins. This therapy is known as a typical treatment for lysosomal storage disorders (LSDs), such as Fabry disease, Gaucher disease, Pompe disease, and Hunter syndrome. It is also used to treat rare congenital syndromes, such as severe combined immunodeficiency syndrome (ADA-SCID), caused by adenosine deaminase deficiency. Furthermore, enzyme replacement therapy can also be used in substrate reduction therapy, gene therapy, bone marrow-derived stem cell transplantation, and other treatments where enzymes or proteins are deficient.

[0030] In the present invention, as an example, the enzyme is a lysosomal enzyme that can be used to treat lysosomal storage disorders, such as idursulfase, laronidase, elosulfase alpha, galsulfase, vestronidase alpha, agalsidase beta, agalsidase alpha, pegunigalsidase alpha, imiglucerase, taliglucerase alpha, alglucerase, velaglucerase, alglucosidase alpha, avalglucosidase alpha, and sebelipase alpha. The enzyme may be, but is not limited to, cerliponase alpha, cerliponase alpha, or adenosine deaminase.

[0031] Specifically, the enzyme can be obtained by cloning the enzyme gene into a vector to prepare an expression plasmid, then transfecting the expression plasmid into host cells and culturing them, selecting and culturing a highly productive enzyme-expressing single clone cell line, and then isolating and purifying the protein.

[0032] In the present invention, the enzyme may be used to treat or prevent lysosomal storage diseases. Lysosomes are intracellular organelles that distribute necessary components and excrete unnecessary components from substances produced in the body. However, if there is a deficiency of endogenous enzymes, lysosomes cannot properly perform this function, resulting in the accumulation of impurities in the body and causing damage to tissues and organs.

[0033] There are more than 50 known types of lysosomal storage diseases, which occur when genetic defects inhibit the production of specific enzymes in the lysosomes, leading to the accumulation of endogenous products, ultimately severely affecting the skeleton, brain, skin, heart, and central nervous system.

[0034] Specifically, the lysosomal storage disease may be, but is not limited to, MPS I, MPS II, MPS IVA, MPS VI, MPS VII, Fabry disease, Gaucher disease, Gaucher disease type I, Pompe disease, lysosomal lipase deficiency (Wolman disease, CESD), neuronal ceroid lipofuscinosis (CLN2 disease), or severe combined immunodeficiency.

[0035] As an example, Fabry disease is a progressive, X-linked inborn error of glycosphingolipid metabolism caused by a deficiency of the lysosomal enzyme α-galactosidase A (α-Gal A) due to mutations in the α-Gal A gene (GLA). Despite being an X-linked disorder, females may exhibit varying degrees of clinical symptoms. Fabry disease is a rare disorder with an estimated incidence of 1 in 40,000 males and 1 in 117,000 in the general population. Untreated, patients with Fabry disease have a shortened life expectancy and typically die in their 40s or 50s from vascular disease affecting the kidneys, heart, and / or central nervous system. The enzyme deficiency leads to the intracellular accumulation of the substrate, globotriaosylceramide (GL-3), in vascular endothelium and visceral tissues throughout the body.

[0036] Cardiac disease in Fabry disease affects most men and many women. Early cardiac findings include left ventricular enlargement, valvular involvement, and conduction abnormalities. Mitral regurgitation is the most common valvular lesion, typically presenting in childhood or adolescence. Cerebrovascular symptoms are primarily due to multifocal small vessel involvement and can include thrombosis, transient ischemic attacks, basilar artery ischemia and aneurysms, stroke, hemiplegia, hemisensory loss, aphasia, labyrinthine disturbances, or cerebral hemorrhage. The mean age at onset of cerebrovascular symptoms is 33.8 years. Changes in temperament and psychotic behavior may occur with age.

[0037] Current FDA-approved treatments for Fabry disease are enzyme replacement therapies (ERT), including agalsidase alfa (Replagal®; Shire Human Genetic Therapies), agalsidase beta (Fabrazyme®; Genzyme Corporation), and pegnigalsidase alfa (Elfabrio®; Protalix BioTherapeutics, Inc.). These types of ERT aim to replace a patient's insufficient α-Gal A activity by administering a recombinant form of the enzyme intravenously. Although effective in many settings, ERT also has limitations. ERT has not been proven to reduce stroke risk, the heart muscle responds slowly, and clearance of GL-3 in some kidney cell types is limited. Some patients also develop an immune response to ERT.

[0038] Another example is Gaucher disease, a typical lysosomal storage disease. It occurs when a deficiency of beta-glucocerebrosidase (β-GC), a hydrolytic enzyme, causes the accumulation of glucocerebroside, a type of sphingolipid, mainly in the reticuloendothelial system, including the liver, spleen, central nervous system, skeleton, and lungs. It is the most common of all lipid storage diseases.

[0039] Gaucher disease is an autosomal recessive disorder caused by mutations in the GBA gene. Clinical manifestations vary in severity depending on the degree of GBA enzyme deficiency. Based on the Knudsen and Kaplan classification, Gaucher disease is divided into three clinical types depending on the presence or absence of central nervous system involvement, the rate of disease progression, and the age of onset. Recently, these have been further subdivided into three groups: non-neuronopathic, acute or chronic neuropathic. Non-neuronopathic type is currently referred to as type 1, acute neuropathic type as type 2, and chronic neuropathic type as type 3.

[0040] Enzyme replacement therapy is a typical treatment proven to be effective in treating Gaucher disease, and is known to be particularly useful for treating liver / spleen enlargement and hematological findings in non-neuronal and chronic neuroinvasive Gaucher disease. Gaucher disease was the pioneering disease for recombinant enzyme therapy, and the acid β-glucosidase enzyme, imiglucerase (Cerezyme®, Genzyme Co.), was commercialized in 1994 through recombinant DNA technology and has been used as the standard treatment for non-neuronal Gaucher disease type I until recently.

[0041] In Japan, Abcertin® (Isu Abxis, Seongnam, Korea), a biosimilar of imiglucerase, has been developed and commercialized. Another recombinant enzyme, VPRIV® (Takeda Pharmaceutical Company, Tokyo, Japan), is also used. These recombinant enzymes improve reticuloendothelial symptoms and liver and spleen enlargement. However, because these enzymes cannot cross the blood-brain barrier, they cannot prevent the progression of neurological symptoms in patients with neuroinvasive Gaucher disease. Furthermore, the therapeutic effect is limited in patients with severe lymph node enlargement in areas such as the abdomen and mediastinum.

[0042] Further examples include mucopolysaccharidosis (MPS) I, II, IVA, VI, and VII.

[0043] Mucopolysaccharidosis type I (MPS I) is a rare recessive genetic disorder with an estimated incidence of 1 in 100,000 births (Moore D et al., 2008, Orphanet Journal of Rare Diseases 3). MPS I is caused by a deficiency of α-l-iduronidase (IDUA), an enzyme required for the liposomal catabolism of the highly prevalent complex polysaccharides heparan sulfate and dermatan sulfate. These polysaccharides, called glycosaminoglycans (GAGs), accumulate in the tissues of MPS I patients, causing distinctive storage lesions and various disease sequelae. Patients may experience short stature, bone and joint deformities, coarse facial features, hepatosplenomegaly, cardiac valve disease, obstructive sleep apnea, recurrent upper respiratory tract infections, hearing loss, carpal tunnel syndrome, and visual impairment due to corneal opacity (Beck M, et al., 2014, The natural history of MPS I: global perspectives from the MPS I Registry. Genetics in medicine: official journal of the American College of Medical Genetics 16(10):759-765). Additionally, many patients develop symptoms related to GAG storage in the central nervous system, which can include hydrocephalus, spinal cord compression, and, in some patients, cognitive impairment.

[0044] Mucopolysaccharidosis type II (Hunter syndrome / MPSII) is a rare X-linked recessive disorder with an incidence of 0.5-1.3 per 100,000 male newborns. This progressive and devastating disease is caused by genetic mutations in the IDS gene, which lead to a deficiency of the lysosomal storage enzyme iduronate-2-sulfatase, an enzyme required for the lysosomal catabolism of heparan sulfate and dermatan sulfate.

[0045] These ubiquitous polysaccharides, called GAGs (glycosaminoglycans), accumulate in the tissues and organs of patients with MPS II, causing distinctive storage lesions and various disease sequelae. Morbidity and mortality are high in this patient population. Death has been reported to occur at a mean age of 11.7 years in patients with the severe phenotype (characterized by neurocognitive deterioration) and at a mean age of 21.7 years in patients with the mild or attenuated phenotype.

[0046] Mucopolysaccharidosis type IVA (MPS IVA; Morquio A syndrome) is an autosomal recessive lysosomal storage disorder caused by deficiency of N-acetylgalactosamine-6-sulfate sulfatase (GALNS) (Khan et al., Mol Genet Metab., 2017;120(1-2):78-95). The enzyme deficiency results in the progressive accumulation of glycosaminoglycans (GAGs), chondroitin 6-sulfate (C6S), and keratan sulfate (KS), resulting in a unique skeletal dysplasia with incomplete ossification and sequential growth imbalance, resulting in a short neck and trunk, cervical spinal cord compression, organ atresia, protruding thorax, joint laxity, kyphoscoliosis, coxa valga, and genu valgum. Other clinical manifestations of the disease include hearing loss, heart valve complications, and corneal opacity. More than 200 different mutations have been identified in patients, with a prevalence in the United States of approximately 1 in 250,000.

[0047] The lipid nanoparticles of the present invention can be used in enzyme replacement therapy to treat the above-mentioned lysosomal storage diseases. Therefore, from this perspective, the present invention provides a method for treating diseases using ERT enzymes. Furthermore, in addition to the above-mentioned lysosomal storage diseases, various diseases can be treated and prevented by effectively delivering deficient or low levels of proteins into the body. Therefore, a method for treating various diseases can be provided using the lipid nanoparticles of the present invention loaded with therapeutic proteins. Such a treatment method can be achieved by delivering ERT enzymes or therapeutic proteins loaded into lipid nanoparticles into cells.

[0048] In another aspect of the present invention, the present invention provides a composition for intracellular or in vivo delivery of a protein, which comprises lipid nanoparticles encapsulating the protein or enzyme.

[0049] The "protein" and "lipid nanoparticle" are as described above.

[0050] In the present invention, the composition may have a modified form by further including a substance for increasing the efficiency of intracellular or in vivo delivery of the lipid nanoparticles encapsulating the protein or enzyme, or by additional improvements.

[0051] In another aspect for achieving the object of the present invention, the present invention provides a composition for preventing or treating a lysosomal storage disease, comprising lipid nanoparticles encapsulating the protein.

[0052] The "protein", "lipid nanoparticle", and "lysosomal storage disease" are as described above.

[0053] The term "prevention" as used herein means any action that suppresses or delays the onset of a lysosomal storage disease through administration of the pharmaceutical composition of the present invention, and the term "treatment" as used herein means any action that improves, reverses, or beneficially alters a disease through administration of the pharmaceutical composition of the present invention.

[0054] The term "pharmaceutical composition" as used herein refers to a substance prepared for the purpose of preventing or treating a disease, and can be formulated into various forms according to conventional methods. For example, depending on the route of administration, the composition can be formulated into oral preparations such as powders, granules, tablets, capsules, suspensions, emulsions, and syrups, and can also be formulated into topical preparations and sterile injectable solutions. Specifically, the administration route can be any appropriate route, including topical, oral, intravenous, intramuscular, and direct absorption through mucosal tissues. Two or more routes can also be used in combination. An example of a combination of two or more routes is when two or more drugs in formulations corresponding to different administration routes are combined, for example, when one drug is first administered intravenously and then the other drug is administered topically.

[0055] The pharmaceutical composition of the present invention can be prepared in the form of a pharmaceutical composition for treating or preventing cancer, further comprising a suitable carrier, excipient, or diluent commonly used in the preparation of pharmaceutical compositions, although the carrier may include a non-naturally occurring carrier. Specifically, the pharmaceutical composition can be formulated into oral preparations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, and aerosols, topical preparations, suppositories, and sterile injectable solutions by conventional methods. Specific formulations of pharmaceutical compositions are known in the art, and reference can be made, for example, to "Remington's Pharmaceutical Sciences (19th ed., 1995)," which is incorporated herein by reference.

[0056] The pharmaceutical compositions of the present invention can be administered to animals such as rats, dogs, cats, cows, horses, pigs, and humans by various routes, with humans being preferred. All methods of administration are foreseeable, including but not limited to oral, intravenous, intraarterial, intramuscular, or subcutaneous injection.

[0057] In the present invention, the above-mentioned therapeutic method also includes a combination with a conventionally known therapeutic method or a combined administration with other conventionally known therapeutic agents.

[0058] In another aspect of the present invention, the present invention provides a method for producing lipid nanoparticles encapsulating proteins and nanoparticles obtained by the method. Specifically, the method for producing lipid nanoparticles encapsulating proteins includes the steps of: (a) preparing a lipid mixture containing HSPC, cholesterol, DOTAP, and DSPE-mPEG; (b) injecting a first solution obtained by dissolving the lipid mixture in an alcoholic solvent into an inlet of a microfluidics system; (c) injecting a second solution containing the protein into another inlet of the microfluidics system; and (d) purifying the lipid nanoparticles encapsulating the protein.

[0059] The "protein" and "lipid nanoparticle" are as described above.

[0060] In the present invention, the alcohol solvent may include, but is not limited to, methanol, ethanol, propyl alcohol, or denatured alcohol, and preferably may be ethanol.

[0061] In the present invention, the flow rate ratio of the alcohol phase (first solution) and the aqueous phase (second solution) in the microfluidic system may be, but is not limited to, 1:3 to 1:5, and may preferably be 1:3.

[0062] In the present invention, the flow rate of the microfluidic system may be, but is not limited to, 9 to 18 ml / min, and preferably 15 ml / min.

[0063] In the present invention, the step of purifying the lipid nanoparticles in step (d) can include removing the alcohol solvent and unencapsulated proteins in step (b). The lipid nanoparticles can be purified using a tangential flow filtration (TFF) system equipped with a column. Specifically, the lipid nanoparticles can be purified by circulating them through the column and adding fresh buffer (20 mM sodium phosphate, 137 mM sodium chloride, pH 6±0.2) at the same rate as the permeate coming out of the column.

[0064] In another aspect of achieving the object of the present invention, the present invention provides a method for intracellular delivery of an ERT enzyme or a therapeutic protein using lipid nanoparticles.

[0065] The "ERT", "enzyme", "therapeutic protein", and "lipid nanoparticle" are as described above.

[0066] In another embodiment for achieving the object of the present invention, the present invention provides a method for treating a disease using lipid nanoparticles encapsulating an ERT enzyme or a therapeutic protein.

[0067] The "ERT", "enzyme", "therapeutic protein", and "lipid nanoparticle" are as described above.

[0068] In the present invention, the "disease" may be a lysosomal storage disease. In addition to the lysosomal storage disease, various other diseases can be treated and prevented by effectively delivering deficient or low levels of proteins into the body. Therefore, the lipid nanoparticles of the present invention encapsulating therapeutic proteins or ERT enzymes can be used to provide methods for treating various diseases.

[0069] Such a therapeutic approach can be achieved by intracellular delivery of ERT enzymes or therapeutic proteins encapsulated in lipid nanoparticles. [Effects of the Invention]

[0070] The present invention can be useful for the treatment and prevention of various diseases such as lysosomal storage diseases by stably and effectively delivering various proteins, such as therapeutic proteins or ERT enzymes, into cells via lipid nanoparticles. [Brief explanation of the drawings]

[0071] [Figure 1] FIG. 1 is a schematic diagram of an overall process for encapsulating lipid nanoparticles using a microfluidics system. [Figure 2] FIG. 2 shows the results of measuring the concentration of protein taken up into cells after treating normal fibroblasts and fibroblasts from a Hunter syndrome patient with the IDS and IDS / LNP of the present invention. [Figure 3] FIG. 3 shows the results of measuring the amount of heparan sulfate accumulated in fibroblasts from a Hunter syndrome patient after treating them with the IDS and IDS / LNP of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0072] The present invention will be described in more detail with reference to the following examples. However, these examples are for illustrative purposes only and the scope of the present invention is not limited to these examples.

[0073] Example 1. Preparation of lipid nanoparticles with protein encapsulation 1.1. Production of enzyme proteins 1.1.1. Development of IDS-producing cell lines The human iduronate-2-sulfatase (IDS) gene was synthesized and cloned into PanGen's proprietary expression vector pPGXII (Korean Patent Registration No. 10-1385223, see Figure 2) to create an expression plasmid. The expression plasmid was transfected into the CHO DG44 host cell line at a 24-well scale using electroporation. After culturing in a 5% CO2 incubator at 37°C, once the cells had grown sufficiently, they were cultured in a selective medium to allow only transformed cells to proliferate. After approximately two weeks, when the cells had grown sufficiently, a portion of the culture medium was taken and the IDS-expressing cells were selected by ELISA.

[0074] Based on the ELISA analysis results, cell populations were selected, and single clonal selection was performed to secure stable cell lines with high expression efficiency. The cells were seeded into 96-well plates at 0.5 cells / well in culture medium. After approximately four weeks, colonies formed were analyzed to select cell lines with high expression efficiency. Once sufficient cells were obtained, the expression efficiency of the single clonal cell lines was compared using ELISA, and highly productive single clonal cell lines were selected as candidate cell lines. Stable protein expression was confirmed during 90 days of long-term subculture, and the most productive cell line was selected as the final cell line. Once a sufficient number of cells were obtained, the selected final cell line was dispensed into vials to prepare a cell bank for research use and stored in a liquid nitrogen storage facility.

[0075] 1.1.2. Cell culture for IDS production After thawing the IDS-producing cell line, subculture was performed every 2–3 days using subculture medium (EX-CELL (+8 mM L-glutamine) + 1% 2X Feed A+). The subculture medium used was EX-CELL medium supplemented with 8 mM L-glutamine and 1% 2X Feed A+ supplement.

[0076] After securing a sufficient number of cells through seed culture for a total of 15 days, IDS was produced at a 5 L culture scale in a 7.5 L bioreactor (New Brunswick Scientific, Bioflo320). 6 The cells were inoculated at a concentration of 1000 cells / mL, and the production medium was EX-CELL medium supplemented with 8 mM L-glutamine and 12.5% ​​2X Feed A+ supplements. The culture conditions were a culture temperature of 37°C, DO (dissolved oxygen) of 30%, pH 7.0-7.2, and agitation speed of 150 rpm, and the culture was continued for a total of 11 days. Cell count and viability were analyzed using a Vi-CELL™ counter during the culture period, and pH, glucose, and lactate contents were monitored daily, with the glucose content maintained above 20 mM. After the culture was completed, the culture medium was collected from the bioreactor, the cells were removed using a COHC depth filter, and the cells were sterilized using a 0.22 μm PES filter.

[0077] 1.1.3. Purification of IDS proteins IDS proteins were isolated and purified using cell culture fluid. Chromatography was performed using a column packed with Capto MMC resin. The column was installed in an AKTA Pure (GE Healthcare) and equilibrated with 20 mM sodium acetate / 150 mM sodium chloride (pH 4.3) equilibration buffer. Culture fluid adjusted to the same pH was then loaded. The column was then washed, and the IDS proteins were eluted with 20 mM sodium acetate / 150 mM sodium chloride (pH 5.1) elution buffer. Chromatography was performed using Blue Sepharose 6FF with the eluate. The column was installed in an AKTA Pure (GE Healthcare) and equilibrated with 20 mM sodium acetate / 50 mM sodium chloride (pH 4.0) equilibration buffer. The sample, diluted 1 / 3 with dilution buffer (20 mM sodium acetate, pH 4.0), was then loaded.

[0078] The column was then washed, and the IDS protein was eluted with 20 mM sodium phosphate (pH 6.6) elution buffer. The eluted IDS protein was then subjected to chromatography using Q Sepharose FF. The column was installed in an AKTA Pure (GE Healthcare) and equilibrated with 20 mM sodium phosphate (pH 6.6) equilibration buffer before loading the sample. After washing the column with wash buffer (50 mM acetic acid (pH 3.0)), the IDS protein was isolated by running 50 mM acetic acid / 150 mM sodium chloride (pH 3.0) elution buffer. Just before the next purification step, the pH of the Q Sepharose FF eluate was lowered to 3.5 with 10% acetic acid, and the mixture was incubated at room temperature for 180 minutes for viral inactivation. Immediately after viral inactivation, the sample was diluted 1 / 3 with dilution buffer (50 mM sodium acetate / 4,430 mM sodium chloride (pH 4.0)) and subjected to Phenyl Sepharose chromatography. The column was installed in an AKTA Pure (GE Healthcare) and equilibrated with 50 mM sodium acetate / 3,000 mM sodium chloride (pH 4.0) equilibration buffer before loading the sample. The column was then washed, and the IDS protein was eluted with 20 mM sodium phosphate (pH 6.0) elution buffer. The eluted IDS protein was purified through the UF / DF process to a concentration of approximately 2 mg / mL in 20 mM sodium phosphate / 137 mM sodium chloride (pH 6.0) buffer. After collecting the sample, Polysorbate 20 was added to a concentration of 0.022%.

[0079] 1.2. Preparation of lipid nanoparticles A cationic lipid mixture was dissolved in ethanol at a specific concentration, mainly in the range of 2.5 to 7.5 mg / mL of total lipids, and lipid nanoparticles (LNPs) were prepared using a microfluidic system.

[0080] Specifically, the selected lipid mixture was injected into one of two inlets of a microfluidic system (a micromixer system with a herringbone-shaped channel) manufactured in-house by Moogene Medi, and cationic lipid nanoparticles were prepared with a composition of 15% or less HSPC (hydrogenated soybean phosphatidycholine), 50% or less cholesterol, 60% or less DOTAP (dioleyl-3-trimethylammonium propane), and 5% or less DSPE-mPEG.

[0081] 1.3. Preparation of lipid nanoparticles with protein encapsulation Protein encapsulation in lipid nanoparticles was carried out through a microfluidics system with a herringbone-shaped micromixer chip. Figure 1 shows an overall schematic diagram of lipid nanoparticle encapsulation using a microfluidics system flow rate.

[0082] Selected lipid mixtures were dissolved in ethanol at specific concentrations, primarily total lipids ranging from 2.5 to 7.5 mg / mL, and injected into one of two inlets of the microfluidic system. The aqueous phase containing the dissolved protein (20 mM sodium phosphate, 137 mM sodium chloride, pH 6 ± 0.2) was injected into the second inlet. Cationic lipid nanoparticles were fabricated with a composition of 15% HSPC, 50% cholesterol, 60% DOTAP, and 5% DSPE-mPEG. The size, polydispersity index (PDI), and encapsulation efficiency of the lipid nanoparticles produced with each lipid composition are shown in Table 1.

[0083] [Table 1]

[0084] *"-" in the inclusion rate indicates NM (Not Measured).

[0085] Various production parameters, including the flow rate ratio (ratio of alcohol phase to aqueous phase) and total flow rate (speed at which the two inlets inject through the chip), were controlled through the microfluidic system software. Specifically, flow rate ratios of alcohol phase to aqueous phase of 1:3 to 1:5 and flow rates of 9 to 18 ml / min were tested to select the optimal production parameters (Tables 2 and 3).

[0086] [Table 2]

[0087] [Table 3]

[0088] 1.4. Purification of lipid nanoparticles The produced lipid nanoparticles were purified using a tangential flow filtration system (Repligen KrosFlo® KR2i TFF System) equipped with a 750 kD modified polyethersulfone (mPES) hollow fiber column. To remove ethanol and proteins not encapsulated in the lipid nanoparticles, the lipid nanoparticle sample was circulated through the column, and fresh buffer (20 mM sodium phosphate, 137 mM sodium chloride, pH 6 ± 0.2) was added at the same rate as the permeate flowing out of the column.

[0089] Example 2. Intracellular uptake of IDS-LNP IDS, an enzyme protein deficient in patients with mucopolysaccharidosis II (Hunter syndrome / MPS II), was encapsulated in cationic lipid nanoparticles [iduronate-2-sulfatase protein-cationic lipid nanoparticles] (hereinafter referred to as "IDS / LNPs"), and was prepared using the method of Example 1.

[0090] Normal human fibroblasts (CCD-986Sk, Korean Cell Line Bank, KCLB21947) and Hunter syndrome patient fibroblasts (GM00615, Coriell Institute, hereafter referred to as "patient cells") were treated with IDS and IDS / LNP at various concentrations (1.25-80 nM) for 6 hours, and the amount of protein uptake in the cells was quantified by ELISA.

[0091] Specifically, the cells used in the experiment were cultured at 5% CO2 and 37°C in IMDM (Iscove's Modified Dulbecco's Medium) medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin antibiotics for normal human fibroblasts, and in MEM (Minimum Essential Medium with Earle's salts, L-glutamine, sodium bicarbonate) medium supplemented with 10% fetal bovine serum (FBS) for Hunter syndrome patient cells. 24 hours before IDS / LNP treatment, cells were cultured at 60 mm 2 Cell culture plate (2.5 x 10 5 On the day of drug treatment, cells seeded on a 500-well plate (100 cells / plate) were cultured at 37°C for 24 hours in 5% FBS-IMDM or MEM culture medium supplemented with IDS control drug (ELAPRASE), IDS, or IDS / LNP. Patient cells used in the experiment were passaged less than 10 times.

[0092] After 24 hours of incubation, the culture medium was removed and the cells were washed once or twice with PBS. After treatment with 0.25% Trypsin-EDTA, centrifugation, and PBS washing, the cells were harvested. The harvested cells were lysed in RIPA buffer containing protease and phosphatase inhibitors (Sigma), centrifuged, and the supernatant was collected for analysis. The total cell lysate was analyzed using a Human Iduronate 2-Sulfatase / IDS ELISA kit (R&D Systems, Cat. #DY2449-05) to determine the amount of IDS delivered to the cells, and a BCA (Bicinchoninic acid) protein assay kit (Thermo Scientific) to quantify the amount of protein. The results are expressed as ng of IDS delivered to the cells per mg of protein.

[0093] As a result, it was confirmed that the amount of IDS protein transferred into normal and patient cells was significantly higher when treated with IDS / LNP than when treated with IDS alone (Figure 2).

[0094] Example 3. Confirmation of cellular activity of IDS / LNP Patients with mucopolysaccharidosis II (Hunter syndrome / MPS II) develop this disease due to the accumulation of intracellular polysaccharides called glycosaminoglycans (GAGs) caused by a deficiency of IDS. Heparan sulfate is a typical type of GAG.

[0095] To measure the cellular activity of IDS / LNP, patient cells were treated with IDS protein and IDS / LNP at concentrations of 5–40 nM for 24 hours, and the amount of intracellularly accumulated heparan sulfate reduced was quantified using ELISA.

[0096] Specifically, patient cells used in the experiment were cultured in MEM medium (Minimum Essential Medium with Ear's salts, L-glutamine, sodium bicarbonate) supplemented with 10% fetal bovine serum (FBS) at 37°C under 5% CO2. Cells were cultured at 60 mm 2 Cell culture plate (2.5 x 10 5 On the day of drug treatment, cells seeded on a 500-well plate (200 cells / plate) were added to 5% FBS-MEM culture medium, and the IDS control drug (ELAPRASE), IDS, and IDS / LNP were cultured at 37°C for 24 hours. Patient cells used in the experiment were passaged less than 10 times.

[0097] After 24 hours of incubation, the culture medium was removed and the cells were washed once or twice with PBS. After treatment with 0.25% Trypsin-EDTA, centrifugation, and PBS washing, the cells were harvested. The harvested cells were lysed in RIPA buffer containing protease and phosphatase inhibitors (Sigma), centrifuged, and the supernatant was collected for analysis. The whole cell lysate was quantified for intracellular heparan sulfate using a Human HS (Heparan Sulfate) ELISA kit (MyBioSource, Cat. #MBS2515971) and protein content using a BCA protein assay kit (Thermo Scientific). The HS concentration (ng / ml) was divided by the protein concentration (mg / ml) to determine the corrected HS content (ng / mg).

[0098] As a result, it was confirmed that the activity of the IDS / LNP-treated group was significantly higher than that of the IDS protein-treated group (FIG. 3).

[0099] Although specific portions of the present invention have been described in detail above, it will be apparent to those skilled in the art that these specific techniques are merely preferred embodiments and do not limit the scope of the present invention. Therefore, the true scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. Lipid nanoparticles for intracellular or in vivo delivery of proteins, comprising a lipid mixture comprising hydrogenated soy phosphatidylcholine (HSPC); cholesterol; DOTAP; and DSPE-mPEG.

2. The lipid nanoparticle of claim 1 , wherein the protein is an enzyme or a therapeutic protein.

3. The lipid nanoparticle of claim 1, wherein the HSPC is present in an amount of 8 to 15 mol% of the lipid mixture.

4. The lipid nanoparticles of claim 1, wherein the cholesterol is present in an amount of 30 to 50 mol% of the lipid mixture.

5. The lipid nanoparticles of claim 1, wherein the lipid mixture is composed of HSPC: cholesterol: DOTAP: DSPE-mPEG in a molar ratio of 8-15: 30-50: 40-60: 0.5-5.

6. HSPC; cholesterol; Protein encapsulated lipid nanoparticles comprising a lipid mixture comprising DOTAP; and DSPE-mPEG.

7. The lipid nanoparticle of claim 6, wherein the protein is an enzyme or a therapeutic protein.

8. The lipid nanoparticle of claim 7, wherein the enzyme is an enzyme replacement therapy (ERT) enzyme.

9. The lipid nanoparticle of claim 8, wherein the enzyme is a lysosomal enzyme.

10. The enzyme is idursulfase, laronidase, elosulfase alpha, galsulfase, vestronidase alpha, agalsidase beta, agalsidase alpha, pegunigalsidase alpha, imiglucerase, taliglucerase alpha, alglucerase, velaglucerase, alglucosidase alpha, avalglucosidase alpha, sebelipase alpha, cerliponase alpha, or adenosine deaminase. The lipid nanoparticle of claim 9.

11. The lipid nanoparticles of claim 6, wherein the lipid mixture is composed of HSPC: cholesterol: DOTAP: DSPE-mPEG in a molar ratio of 8-15: 30-50: 40-60: 0.5-5.

12. The lipid nanoparticle of claim 6, wherein the lipid nanoparticle is for treating a lysosomal storage disease.

13. The lipid nanoparticles of claim 12, wherein the lysosomal storage disease is MPS I, MPS II, MPS IVA, MPS VI, MPS VII, Fabry disease, Gaucher disease, Gaucher disease type I, Pompe disease, lysosomal lipase deficiency (Wolman disease, CESD), neuronal ceroid lipofuscinosis (CLN2 disease), or severe combined immunodeficiency.

14. A composition for intracellular or in vivo delivery of a protein, comprising the lipid nanoparticles described in claim 6.

15. A pharmaceutical composition for preventing or treating a lysosomal storage disease, comprising the lipid nanoparticles of claim 6.

16. 16. The pharmaceutical composition of claim 15, wherein the lysosomal storage disease is MPS I, MPS II, MPS IVA, MPS VI, MPS VII, Fabry disease, Gaucher disease, Gaucher disease type I, Pompe disease, lysosomal lipase deficiency (Wolman disease, CESD), neuronal ceroid lipofuscinosis (CLN2 disease), or severe combined immunodeficiency.

17. (a) preparing a lipid mixture comprising HSPC; cholesterol; DOTAP; and DSPE-mPEG; (b) injecting a first solution obtained by dissolving the lipid mixture in an alcohol solvent into an inlet of a microfluidics system; (c) injecting a second solution containing the protein into another inlet of the microfluidic system; and (d) A method for producing protein-encapsulated lipid nanoparticles, comprising purifying the protein-encapsulated lipid nanoparticles.

18. 18. The method of claim 17, wherein the lipid mixture in step (a) contains HSPC:cholesterol:DOTAP:DSPE-mPEG in a molar ratio of 8-15:30-50:40-60:0.5-5.

19. The method according to claim 17, wherein the flow rate ratio of the alcohol phase and the aqueous phase in the microfluidic system is 1:3 to 1:

5.

20. The method of claim 17, wherein the flow rate of the microfluidic system is 9 to 18 ml / min.

21. The method of claim 17, wherein the step of purifying the lipid nanoparticles in step (d) includes removing the alcoholic solvent in step (b) and proteins not encapsulated in the lipid nanoparticles.

22. A method for delivering ERT enzymes or therapeutic proteins into cells using lipid nanoparticles.

23. A method for treating diseases using lipid nanoparticles encapsulating ERT enzymes or therapeutic proteins.

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