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 the delivery of proteins, and specifically to lipid nanoparticles for delivering various pharmaceutical proteins, such as therapeutic proteins or enzyme replacement therapy (ERT) enzymes, into cells or the body.

Background Art

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

[0003] On the other hand, lysosomal storage disorders (LSDs) are relatively rare genetic metabolic disorders due to defects in lysosomal function. LSDs are typically caused by a deficiency of a single enzyme involved in the breakdown of metabolites within lysosomes. The accumulation of products due to the lack of enzyme activity affects various organ systems and can lead to severe symptoms and early death. In addition, the majority of LSDs are associated with important neurological structural disorders ranging from progressive neurodegeneration, severe cognitive impairment to interstitial, behavioral disorders and mental disorders.

[0004] Typical diseases such as Fabry disease, Gaucher disease, and mucopolysaccharidosis are representative of such lysosomal storage disorders, and enzyme replacement therapy (ERT) has been established as the standard treatment. Such enzyme replacement therapy plays a role in reducing the harmful levels of substance concentrations accumulated in cells by periodically administering the deficient enzyme protein in the body intravenously (Concolino et al., Italian Journal of Pediatrics, 2018).

[0005] However, in order to improve the therapeutic efficiency of such enzyme replacement therapy, there is a need to develop delivery systems that reduce side effects such as undesirable immune responses and effectively and stably supply enzyme proteins within cells. This is because it would be possible to lower the concentration of the administered enzyme and minimize anti-drug antibody reactions. [Overview of the project] [Problems that the invention aims to solve]

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

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

[0008] A further exemplary object of the present invention is to provide a composition for intracellular or intracellular delivery of proteins, containing lipid nanoparticles encapsulating proteins.

[0009] A further exemplary object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of lysosomal storage disorders containing protein-encapsulated lipid nanoparticles.

[0010] A further exemplary object of the present invention is to provide a method for producing lipid nanoparticles containing proteins.

[0011] A further exemplary object of the present invention is to provide a method for delivering ERT enzymes or therapeutic proteins into cells using lipid nanoparticles.

[0012] A further exemplary object of the present invention is to provide a method for treating a disease using lipid nanoparticles encapsulating an ERT enzyme or therapeutic protein.

[0013] The technical problems that the technical ideas of the inventions disclosed herein seek to solve are not limited to those problems mentioned above, and other problems not mentioned will be clearly understood by an ordinary person of the art from the following description. [Means for solving the problem]

[0014] This can be explained in more detail as follows: On the other hand, each description and embodiment disclosed in this application can be applied to each other description and embodiment. In other words, all combinations of the 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 aspect to achieve the objectives of the present invention, the present invention provides lipid nanoparticles for the intracellular or intracellular delivery of proteins. Specifically, the present invention provides lipid nanoparticles for the intracellular or intracellular delivery of proteins, comprising a lipid mixture including hydrogenated soybean phosphatidylcholine (HSPC); cholesterol; DOTAP; and DSPE-mPEG.

[0016] In this invention, the term "lipid nanoparticle (LNP)" refers to an endoplasmic reticulum having an adjacent lipid bilayer, such as a globular endoplasmic reticulum, which can be used for delivery to a target site. Such lipid nanoparticles may include liposomes, solid lipid nanoparticles (SLNs), nanostructured lipid carriers (NLCs), and monolayer membrane structures (micelles).

[0017] The lipid nanoparticles of the present invention may contain one or more types of lipids. For example, they may contain ionic lipids, PEG-lipids, phospholipids, or cholesterol. Here, ionic lipids may be cationic lipids. 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-dimyristiloxypropane-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide (DMRIE); N1,N3,N5-tris(3- Examples include (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] Furthermore, it can be composed together with other lipid components. For example, phosphatidylcholine (PC) derivatives (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- It may contain other lipid molecules belonging to PEG2000, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-methoxy(polyethylene glycol) (DSPE-mPEG), L-α-phosphatidylcholine (HSPC), dipalmitoylphosphatidylcholine (DPPC), and 1,2-dimiristoyl-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 containing HSPC, cholesterol, DOTAP, and DSPE-mPEG.

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

[0021] Furthermore, the lipid mixture may have a molar ratio of HSPC:cholesterol:DOTAP:DSPE-mPEG of 8-15:30-50:40-60:0.5-5, preferably the lipid mixture has a molar ratio of HSPC:cholesterol:DOTAP:DSPE-mPEG of 8-10:35-45:45-55:0.5-1.5, and more preferably the lipid mixture has a molar ratio of HSPC:cholesterol:DOTAP:DSPE-mPEG of 9:40:50:1, but is not limited thereto.

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

[0023] In the present invention, the particle size of lipid nanoparticles may affect drug release rate, biodistribution, mucosal adhesion, cellular water absorption, buffer exchange into the nanoparticles, and protein diffusion. For example, the LNP diameter may 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 measure of the heterogeneity of molecules or particle sizes in 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 is applicable without particular limitation. As an example, it can include an enzyme or a therapeutic protein. The therapeutic protein means a protein for treating or preventing a disease or disorder, and can be a recombinant protein, an antibody, a biosimilar, a biobetter, or the like. In the present invention, the enzyme is applicable without particular limitation, and may be an enzyme that is deficient or absent in the body. In particular, the enzyme may be an enzyme for enzyme replacement therapy (ERT).

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

[0027] As another aspect for achieving the object of the present invention, the present invention provides lipid nanoparticles encapsulating a protein, which contain a lipid mixture including HSPC; cholesterol; DOTAP; and DSPE-mPEG.

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

[0029] The term "enzyme replacement therapy" in the present invention is a treatment method in which an enzyme lacking in the body is directly administered to the patient's vein. It is known as a typical treatment method for Fabry disease, Gaucher disease, Pompe disease, Hunter syndrome, etc., which are called lysosomal storage disorders (LSD), and is also used for the treatment of rare congenital syndromes such as severe combined immunodeficiency (ADA-SCID) derived from adenosine deaminase deficiency. Furthermore, enzyme replacement therapy can be used when an enzyme or protein is deficient in substrate reduction therapy, gene therapy, bone marrow-derived stem cell transplantation, etc.

[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, pegnigalsidase alpha, imiglucerase, taliglucerase alpha, alglucerase, velaglucerase, alglucosidase alpha, avalglucosidase alpha, and sebelipase alpha. It 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 create an expression plasmid, transducing the expression plasmid into host cells and culturing them, selecting and culturing highly productive enzyme-expressing single-clonal cell lines, and then separating and purifying the protein.

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

[0033] More than 50 types of lysosomal storage disorders are known. These disorders involve genetic defects that inhibit the production of specific enzymes within lysosomes, leading to the accumulation of these products in the body, ultimately causing serious effects on the skeleton, brain, skin, heart, central nervous system, and other organs.

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

[0035] For example, Fabry disease is a progressive, X-related congenital error in sphingoglycolipid metabolism caused by a deficiency of the lysosomal enzyme α-galactosidase A (α-Gal A) due to a mutation in the α-Gal A gene (GLA). Despite being an X-related disease, women may exhibit varying degrees of clinical symptoms. Fabry disease is a rare disease, with an estimated incidence of 1 in 40,000 men and 1 in 117,000 in the overall population. In patients with Fabry disease, if left untreated, the expected lifespan is shortened, and death typically occurs in the 40s or 50s due to vascular disease affecting the kidneys, heart, and / or central nervous system. The enzyme deficiency leads to intracellular accumulation of its substrate, globotriaosylceramide (GL-3), in the vascular endothelium and visceral tissues throughout the body.

[0036] Cardiac disease associated with Fabry disease occurs in most men and a large number of women. Early findings of cardiac disease include left ventricular enlargement, valvular invasion, and conduction abnormalities. Mitral regurgitation is the most common valvular lesion, typically appearing in childhood or adolescence. Cerebrovascular symptoms are mainly due to multifocal small vessel invasion and can include thrombosis, transient ischemic attacks, basal basilar artery ischemia and aneurysms, seizures, hemiplegia, unilateral anesthesia, aphasia, labyrinthine disorder, or cerebral hemorrhage. The mean age of onset for cerebrovascular symptoms is 33.8 years. Changes in character and psychotic behavior may appear with age.

[0037] The current FDA-approved treatment for Fabry disease is enzyme replacement therapy (ERT), which includes 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 compensate for a patient's insufficient α-Gal A activity by intravenously administering recombinant forms of the enzyme. While ERT is effective in high-risk situations, the treatment also has limitations. ERT has not been proven to reduce the risk of stroke, the myocardium responds with a delay, and GL-3 removal in some types of renal cells is limited. Some patients also exhibit an immune response to ERT.

[0038] Another example is Gaucher disease, a representative lysosomal storage disorder. It is caused by a deficiency of beta-glucocerebrosidase (β-GC), a hydrolytic enzyme for lysosomes, which leads to 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 the lipid storage disorders.

[0039] Gaucher disease is a disorder caused by a mutation in the GBA gene, inherited in an autosomal recessive manner, and exhibits various clinical manifestations of varying severity depending on the degree of GBA enzyme deficiency. Based on the classification by Knudsen and Kaplan, Gaucher disease is divided into three clinical types depending on the presence or absence of central nervous system invasion, the rate of disease progression, and the age of onset. However, recently, these are classified into three groups: non-neuronopathic, acute or chronic neuronopathic. In this case, the non-neuronopathic type is called type 1, the acute neuronopathic type is called type 2, and the chronic neuronopathic type is called type 3.

[0040] Enzyme replacement therapy is a typical and proven treatment for Gaucher disease, particularly in non-invasive and chronic neurologically invasive Gaucher disease, where it is known to be highly effective in hepatomegaly / splenomegaly and hematological findings. Gaucher disease was a pioneering disease for recombinant enzyme therapy, and the acidic β-glucosidase enzyme, imiglucerase (Cerezyme®, Genzyme Co.), was commercialized through recombinant DNA technology in 1994 and until recently was used as the standard treatment for non-invasive neurological Gaucher disease type I.

[0041] In Japan, Abcertin® (Isu Abxis, Seongnam, South Korea), a biosimilar of imiglucerase, has been developed, commercialized, and is in use. In addition, VPRIV® (Takeda Pharmaceutical Company, Tokyo, Japan) is also used as a recombinant enzyme preparation. These recombinant enzymes improve symptoms of the reticuloendothelial system and lead to improvement of hepatomegaly / splenomegaly. However, because these enzyme preparations cannot cross the cerebrovascular barrier, they cannot prevent the progression of neurological symptoms in patients with neurologically invasive Gaucher disease. Furthermore, it is known that 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 1 (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 necessary for the liposomal catabolism of the very common complex polysaccharides heparan sulfate and dermatan sulfate. These polysaccharides, called glycosaminoglycans (GAGs), accumulate in the tissues of MPSI patients, leading to characteristic storage lesions and various disease sequelae. Patients may develop short stature, bone and joint malformations, coarse facial features, hepatosplenomegaly, cardia valve disease, obstructive sleep apnea, recurrent upper respiratory tract infections, hearing impairment, 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). Furthermore, many patients experience symptoms related to GAG storage in the central nervous system, which may include hydrocephalus, spinal cord compression, and, in some patients, cognitive impairment.

[0044] Mucopolysaccharidosis type II (Hunter syndrome / MPSII) is a rare X-associated recessive genetic disorder occurring in 0.5 to 1.3 cases per 100,000 male newborns. This progressive and devastating disorder is caused by a mutation in the IDS gene, which leads to a deficiency of iduronate-2-sulfatase, a lysosomal storage enzyme necessary for the lysosomal catabolism of heparan sulfate and dermatan sulfate.

[0045] These ubiquitous polysaccharides, called GAGs (glycosaminoglycans), accumulate in the tissues and organs of MPS II patients, causing characteristic storage lesions and various disease sequelae. This patient population has high morbidity and mortality rates. Death has been reported to occur at a mean age of 11.7 years in patients with the severe phenotype (characterized by impaired neurocognition) and at a mean age of 21.7 years in patients with the mild or diminished phenotype.

[0046] Mucopolysaccharidosis type IVA (MPS IVA; Morquio A syndrome) is an autosomal recessive lysosomal storage disorder caused by a deficiency of N-acetylgalactosamine-6-sulfate sulfatase (GALNS) (Khan et al., Mol Genet Metab., 2017;120(1-2):78-95). The deficiency of this enzyme leads to the gradual accumulation of glycosaminoglycans (GAGs), chondroitin 6-sulfate (C6S), and keratan sulfate (KS), resulting in a distinctive systemic skeletal dysplasia with incomplete ossification and continuous growth imbalances, consequently resulting in a short neck and torso, cervical spinal cord compression, organ closure, protruding chest, joint laxity, kyphosis, genu valgus, and genu valgus. Other clinical findings of the disease may include hearing loss, heart valve complications, and corneal opacity. More than 200 different mutations have been identified in patients, and the prevalence in the United States is approximately 1 in 250,000 people.

[0047] By using the lipid nanoparticles according to the present invention in enzyme replacement therapy, it becomes possible to treat the aforementioned lysosomal storage disease. Therefore, from this viewpoint, the present invention provides a method for treating diseases using ERT enzymes. In addition to the aforementioned lysosomal storage disease, various diseases can be treated and prevented by effectively delivering deficient or low-level proteins into the body. Therefore, the lipid nanoparticles of the present invention, which carry therapeutic proteins, can be used to provide methods for treating various diseases. Such treatment methods can be achieved by delivering ERT enzymes or therapeutic proteins carried within lipid nanoparticles into cells.

[0048] In another aspect to achieve the objectives of the present invention, the present invention provides a composition for intracellular or intracellular delivery of proteins, comprising lipid nanoparticles containing the protein or enzyme.

[0049] The aforementioned "proteins" and "lipid nanoparticles" are as described above.

[0050] In the present invention, the composition can be modified by adding a substance for increasing the efficiency of intracellular or intracellular delivery of lipid nanoparticles containing proteins or enzymes, or by making additional improvements to the composition.

[0051] In another aspect to achieve the objectives of the present invention, the present invention provides a composition for the prevention or treatment of lysosomal storage diseases containing lipid nanoparticles encapsulating the protein.

[0052] The aforementioned "proteins," "lipid nanoparticles," and "lysosomal storage disorders" are as described above.

[0053] In this invention, the term "prevention" means all actions that suppress or delay the onset of lysosomal storage disorders through the administration of the pharmaceutical composition of the present invention, and the term "treatment" means all actions that improve, alleviate, or beneficially alter a disorder by administering the pharmaceutical composition of the present invention.

[0054] In this invention, the term "pharmaceutical composition" means a substance manufactured for the purpose of preventing or treating a disease, and each can be formulated and used in various forms according to conventional methods. For example, depending on the route of administration, it can be formulated into oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, and syrups, and can also be formulated and used in the form of topical preparations and sterile injection solutions. Specifically, the route of administration is any suitable route including local routes, oral routes, intravenous routes, intramuscular routes, and direct absorption through mucosal tissue, and it is also possible to use two or more routes in combination. An example of a combination of two or more routes is when two or more drug formulations are combined depending on the route of administration, for example, when one drug is first administered intravenously and then another drug is administered locally.

[0055] The pharmaceutical compositions of the present invention may be manufactured in the form of pharmaceutical compositions for the treatment or prevention of cancer, further comprising suitable carriers, excipients, or diluents commonly used in the manufacture of pharmaceutical compositions, wherein the carriers may include non-naturally occurring carriers. Specifically, the pharmaceutical compositions can be formulated and used by conventional methods in the form of oral preparations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, and aerosols, as well as topical preparations, suppositories, and sterile injection solutions. Specific formulations of the pharmaceutical compositions are publicly known in the industry and can be found, for example, in the literature "Remington's Pharmaceutical Sciences (19th ed., 1995)," which is considered part of this specification.

[0056] The pharmaceutical compositions of the present invention can be administered to animals such as rats, dogs, cats, cattle, horses, pigs, and humans via various routes, with human administration being preferred. All methods of administration are predictable and are not limited to, for example, oral, intravenous, arterial, intramuscular, or subcutaneous injection.

[0057] In the present invention, the treatment method includes combination with conventionally known therapeutic therapies, or combination administration with other conventionally known therapeutic agents.

[0058] In another aspect to achieve the object of the present invention, the present invention provides a method for producing protein-encapsulated lipid nanoparticles and nanoparticles obtained by this method. Specifically, the method for producing protein-encapsulated lipid nanoparticles includes the steps of (a) producing a lipid mixture comprising 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 protein into another inlet of the microfluidics system; and (d) purifying the protein-encapsulated lipid nanoparticles.

[0059] The aforementioned "proteins" and "lipid nanoparticles" are as described above.

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

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

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

[0063] In the present invention, the step of purifying the lipid nanoparticles in step (d) may include the step of removing the alcoholic solvent and unencapsulated proteins in step (b). The purification of the lipid nanoparticles may be carried out using a tangential flow filtration (TFF) system equipped with a column, specifically by circulating the lipid nanoparticles through the column and purifying them while 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 to achieve the objectives of the present invention, the present invention provides a method for intracellular delivery of ERT enzymes or therapeutic proteins using lipid nanoparticles.

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

[0066] In another aspect to achieve the objectives of the present invention, the present invention provides a method for treating a disease using lipid nanoparticles containing an ERT enzyme or a therapeutic protein.

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

[0068] In the present invention, the "disease" may be a lysosomal storage disorder. Furthermore, in addition to the lysosomal storage disorder, various other diseases can be treated and prevented by effectively delivering deficient or low-level 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 therapeutic methods can be achieved by delivering ERT enzymes or therapeutic proteins encapsulated in lipid nanoparticles into cells. [Effects of the Invention]

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

[0071] [Figure 1] Figure 1 is a schematic diagram of lipid nanoparticle encapsulation using a microfluidic system. [Figure 2] Figure 2 shows the results of measuring the concentration of proteins taken up into cells after treating normal fibroblasts and fibroblasts from Hunter syndrome patients with the IDS and IDS / LNP of the present invention. [Figure 3] Figure 3 shows the results of measuring the amount of heparan sulfate accumulated in cells after treating fibroblasts from Hunter syndrome patients with the IDS and IDS / LNP of the present invention. [Modes for carrying out the invention]

[0072] The present invention will be described in more detail below through 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. Production of lipid nanoparticles with protein encapsulation. 1.1. Enzyme Protein Production 1.1.1. Development of IDS-producing cell lines After synthesizing the human iduronate-2-sulfatase (IDS) gene, an expression plasmid was created by cloning it into PanGen's proprietary expression vector pPGXII (see Figure 2, Registered Patent No. 10-1385223, Republic of Korea). Transduction of the expression plasmid was performed in 24-well CHO DG44 host cell lines using electroporation. After culturing in a 37°C, 5% CO2 incubator, the cells were cultured in selective medium to ensure that only transformed cells proliferated once they had grown sufficiently. Approximately two weeks later, when the cells had grown sufficiently, a portion of the culture medium was taken, and the IDS-expressing cell population was selected by ELISA analysis.

[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. Cells were dispensed into 96-well plates at a concentration of 0.5 cells / well, and after approximately 4 weeks, the formed colonies were analyzed to select and culture cell lines with high expression efficiency. Once a sufficient number of cells were secured, the expression efficiency of single clonal cell lines was compared and analyzed using ELISA, and single clonal cell lines with high productivity were selected as candidate cell lines. The single clonal cell lines were confirmed to stably express proteins during 90 days of long-term subculturing, and the most productive cell line among them was selected as the final cell line. Once a sufficient number of cells were secured, the selected final cell line was dispensed into vials to create a research cell bank, and then stored in a liquid nitrogen storage facility.

[0075] 1.1.2. Cell culture for IDS production After thawing the IDS-producing cell line, it was subcultured at 2-3 day intervals using EX-CELL medium (+8mM L-glutamine + 1% 2X Feed A+). The subculture medium used was EX-CELL medium supplemented with 8mM L-glutamine and 1% 2X Feed A+ additive.

[0076] After securing a sufficient number of cells through a total of 15 days of seed culture, IDS was produced in a 7.5L bioreactor (New Brunswick Scientific, Bioflo320) on a 5L culture medium scale. During production, the cells were 1 × 10⁶ 6 Cells were inoculated at a concentration of cells / mL, and the culture medium used was EX-CELL medium supplemented with 8 mM L-glutamine and 12.5% ​​2X Feed A+ additive. Culture conditions were set to a culture temperature of 37°C, dissolved oxygen (DO) of 30%, pH of 7.0-7.2, and a stirring speed of 150 rpm, and culture was carried out for a total of 11 days. Cell count and viability during the culture period were analyzed using a Vi-CELL® counter, and pH and glucose and lactate content were monitored by measuring them daily, ensuring that the glucose content was maintained at 20 mM or higher. After the end of culture, the culture medium was collected from the bioreactor, cells were removed using a COHC depth filter, and the medium was sterilized using a 0.22 μm PES filter.

[0077] 1.1.3. Purification of IDS protein IDS proteins were isolated and purified using cell culture medium. Chromatography was performed using a column packed with Capto MMC resin. The column was mounted on an AKTA Pure (GE healthcare) and equilibrated with 20 mM sodium acetate / 150 mM sodium chloride (pH 4.3) equilibrium buffer, after which a culture medium adjusted to the same pH was loaded. Subsequently, the column was washed, and 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 mounted on an AKTA Pure (GE healthcare) and equilibrated with 20 mM sodium acetate / 50 mM sodium chloride (pH 4.0) equilibrium buffer, after which a sample diluted 1 / 3 with dilution buffer (20 mM sodium acetate (pH 4.0)) was loaded.

[0078] Subsequently, the column was washed, and the IDS protein was eluted with 20 mM sodium phosphate (pH 6.6) elution buffer. Chromatography was performed using Q Sepharose FF with the eluted IDS protein. The column was set up in AKTA Pure (GE Healthcare), equilibrated with 20 mM sodium phosphate (pH 6.6) equilibrium buffer, and then loaded with the sample. After washing the column with washing buffer (50 mM acetic acid (pH 3.0)), the IDS protein was secured by flushing with 50 mM acetic acid / 150 mM sodium chloride (pH 3.0) elution buffer. Immediately before proceeding to the next purification step, the pH of the Q Sepharose FF eluate was lowered to 3.5 using 10% acetic acid, and the virus inactivation step was performed by reacting at room temperature for 180 minutes. Immediately after virus inactivation was complete, the sample was diluted 1 / 3 with dilution buffer (50 mM sodium acetate / 4,430 mM sodium chloride (pH 4.0)), and then phenyl Sepharose chromatography was performed. A column was set up in AKTA Pure (GE Healthcare), and after equilibrating the column with 50 mM sodium acetate / 3,000 mM sodium chloride (pH 4.0) equilibrium buffer, the sample was loaded. Subsequently, the column was washed, and the IDS protein was eluted with 20 mM sodium phosphate (pH 6.0) elution buffer. The eluted IDS protein was prepared at a concentration of approximately 2 mg / mL in 20 mM sodium phosphate / 137 mM sodium chloride (pH 6.0) buffer via the UF / DF process. After collecting the sample, Polysorbate 20 was added to a concentration of 0.022%.

[0079] 1.2. Manufacturing of Lipid Nanoparticles Lipid nanoparticles (LNPs) were produced using a microfluidic system by dissolving a cationic lipid mixture in ethanol at specific concentrations, mainly in the range of 2.5 to 7.5 mg / mL of total lipids.

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

[0081] 1.3. Production of lipid nanoparticles with protein encapsulation Protein encapsulation via lipid nanoparticles was carried out through a microfluidic system in a herringbone-shaped micromixer chip. A schematic diagram of lipid nanoparticle encapsulation by the microfluidic system flow rate is shown in Figure 1.

[0082] The selected lipid mixture was dissolved in ethanol at specific concentrations, mainly in the range of 2.5–7.5 mg / mL of total lipids, and injected into one of the two inlets of the microfluidic system. The aqueous phase containing dissolved proteins (20 mM sodium phosphate, 137 mM sodium chloride, pH 6±0.2) was then injected into the secondary inlet. Cationic lipid nanoparticles were prepared to have compositions of ≤15% HSPC, ≤50% cholesterol, ≤60% DOTAP, and ≤5% DSPE-mPEG. The size, polydispersity index (PDI), and encapsulation efficiency of the lipid nanoparticles produced for each lipid composition are shown in Table 1 below.

[0083] [Table 1]

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

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

[0086] [Table 2]

[0087] [Table 3]

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

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

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

[0091] Specifically, the cells used in the experiment were cultured at 5% CO2 and 37°C. Fibroblasts from normal humans were cultured in IMDM (Iscove's Modified Dulbecco's Medium) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin antibiotics, while cells from Hunter syndrome patients were cultured in MEM (Minimum Essential Medium with Earle's salts, L-glutamine, sodium bicarbonate) supplemented with 10% fetal bovine serum (FBS). The cells were cultured at 60 mm 24 hours prior to IDS / LNP treatment. 2 Cell culture plate (2.5 × 10 5 Cells inoculated into cells / plates were cultured at 37°C for 24 hours on the day of drug treatment in 5% FBS-IMDM or MEM culture medium with the addition of the IDS control drug (ELAPRASE), IDS, and IDS / LNP. Patient cells used in the experiment had fewer than 10 passages.

[0092] After a 24-hour reaction, the culture medium was removed, washed 1-2 times with PBS, treated with 0.25% Trypsin-EDTA, centrifuged, and washed again with PBS. Only the cells were collected. The collected cells were lysed in a RIPA lysate containing a protease inhibitor and a phosphatase inhibitor (Sigma), centrifuged, and the supernatant was collected and used as the analytical sample. For the analytical sample (total cell lysate), the amount of IDS transferred into the cells was quantified using the Human Iduronate 2-Sulfatase / IDS ELISA kit (R&D systems, Cat. #DY2449-05), and the amount of BCA (Bicinchoninic acid) protein was quantified using the BCA protein analysis kit (Thermo Scientific). The results are expressed as ng of IDS protein transferred into 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 compared to when treated with IDS alone (Figure 2).

[0094] Example 3. Confirmation of cellular activity of IDS / LNP Mucopolysaccharidosis II (Hunter syndrome / MPS II) is a disease in which polysaccharides called glycosaminoglycans (GAGs) accumulate in cells due to an IDS deficiency, and heparan sulfate is a representative 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 decrease in intracellularly accumulated heparan sulfate was quantified by ELISA.

[0096] Specifically, the 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. The cells were 60 mm in size 24 hours before IDS / LNP treatment. 2 Cell culture plate (2.5 × 10 5 Cells inoculated into cells / plates were used, and on the day of drug treatment, the IDS control drug (ELAPRASE), IDS, and IDS / LNP were added to 5% FBS-MEM culture medium and cultured at 37°C for 24 hours. Patient cells used in the experiment had fewer than 10 passages.

[0097] After a 24-hour reaction, the culture medium was removed, washed 1-2 times with PBS, treated with 0.25% Trypsin-EDTA, centrifuged, and washed with PBS again, after which only the cells were collected. The collected cells were lysed in a RIPA lysate containing a protease inhibitor and a phosphatase inhibitor (Sigma), centrifuged, and only the supernatant was collected and used as the analytical sample. The analytical sample (total cell lysate) was used to quantify intracellular heparan sulfate using the Human HS (Heparan Sulfate) ELISA kit (MyBioSource, Cat. #MBS2515971) and protein content using the BCA protein analysis kit (Thermo Scientific). The HS content (ng / mg) was expressed as the corrected HS concentration (ng / ml) divided by the protein concentration (mg / ml).

[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 (Figure 3).

[0099] As described above, specific parts of the present invention have been explained in detail, but it will be clear to those with ordinary skill in the art that these specific techniques are merely preferred embodiments and do not limit the scope of the invention. Therefore, the substantial scope of the invention is defined by the appended claims and their equivalents.

Claims

1. Lipid nanoparticles for intracellular or intracellular delivery of proteins, comprising a lipid mixture containing hydrogenated soybean phosphatidylcholine (HSPC); cholesterol; DOTAP; and DSPE-mPEG, wherein the HSPC is present in an amount of 8 to 15 mol% of the lipid mixture.

2. The lipid nanoparticles according to claim 1, wherein the protein is an enzyme or a therapeutic protein.

3. The lipid nanoparticles according to claim 1, comprising the cholesterol in an amount of 30 to 50 mol% of the lipid mixture.

4. The lipid nanoparticles according to 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.

5. HSPC; cholesterol; Protein-encapsulated lipid nanoparticles comprising a lipid mixture containing DOTAP and DSPE-mPEG, wherein the HSPC is present in an amount of 8 to 15 mol% of the lipid mixture.

6. The lipid nanoparticles according to claim 5, wherein the protein is an enzyme or a therapeutic protein.

7. The lipid nanoparticles according to claim 6, wherein the enzyme is an enzyme replacement therapy (ERT) enzyme.

8. The lipid nanoparticles according to claim 7, wherein the enzyme is a lysosomal enzyme.

9. The enzymes mentioned above are idursulfase, laronidase, elosulfase alpha, galsulfase, vestronidase alpha, agalsidase beta, agalsidase alpha, pegnigalsidase alpha, imiglucerase, taliglucerase alpha, alglucerase, velaglucerase, alglucosidase alpha, avalglucosidase alpha, sebelipase alpha, cerliponase alpha, or adenosine deaminase. Lipid nanoparticles according to claim 8.

10. The lipid nanoparticles according to claim 5, 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.

11. The lipid nanoparticles according to claim 5, wherein the lipid nanoparticles are for treating lysosomal storage disorders.

12. The lipid nanoparticles according to claim 11, wherein the lysosomal storage disorder is MPS I, MPS II, MPS IVA, MPS VI, MPS VII, Fabry disease, Gaucher disease, Gaucher disease type I, Pompe disease, lysosomal lipid deficiency (Wolman disease, CESD), neuronal ceroid lipofuscinosis (CLN2 disease), or severe combined immunodeficiency.

13. A composition for intracellular or intracellular delivery of proteins containing lipid nanoparticles as described in claim 5.

14. A pharmaceutical composition for the prevention or treatment of lysosomal storage diseases, comprising lipid nanoparticles as described in claim 5.

15. The pharmaceutical composition according to claim 14, wherein the lysosomal storage disorder is MPS I, MPS II, MPS IVA, MPS VI, MPS VII, Fabry disease, Gaucher disease, Gaucher disease type I, Pompe disease, lysosomal lipid deficiency (Wolman disease, CESD), neuronal ceroid lipofuscinosis (CLN2 disease), or severe combined immunodeficiency.

16. (a) A step of producing a lipid mixture containing HSPC; cholesterol; DOTAP; and DSPE-mPEG; (b) The step of injecting the first solution, in which the lipid mixture is dissolved in an alcoholic solvent, into the inlet of the microfluidics system; (c) The step of injecting the second solution containing the protein into another inlet of the microfluidic system; and (d) The step of purifying the lipid nanoparticles containing the protein, Herein, the HSPC is characterized by being present in an amount of 8 to 15 mol% of the lipid mixture. A method for producing lipid nanoparticles containing proteins.

17. The method for producing the product according to claim 16, 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.

18. The manufacturing method according to claim 16, wherein the flow rate ratio of the alcohol phase and the aqueous phase of the microfluidic system proceeds at a ratio of 1:3 to 1:

5.

19. The manufacturing method according to claim 16, wherein the flow velocity of the microfluidic system proceeds at a rate of 9 to 18 ml / min.

20. The manufacturing method according to claim 16, wherein the step of purifying the lipid nanoparticles in step (d) includes the step of removing the alcoholic solvent and proteins not encapsulated in the lipid nanoparticles in step (b).

21. A method for delivering an ERT enzyme or therapeutic protein into a cell in vitro using lipid nanoparticles as described in claim 1.

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