Composition containing LNP and mrna, and use thereof in treatment of gaucher disease

Abstract

Provided are a composition containing a lipid nanoparticle (LNP) and an mRNA, and use thereof in the treatment of Gaucher disease. The composition contains an LNP and an mRNA, and the mRNA is encapsulated in the LNP or associated with the LNP, wherein the mRNA contains a nucleotide sequence encoding GBA1. The composition can effectively increase the expression and activity of β-glucocerebrosidase (β-GCase) in the serum and multiple target organs of a subject, and reduce the level of glucosylsphingosine (Lyso-GL1) therein. In addition, the composition possesses a relatively long half-life and has application prospects in the treatment of Gaucher disease.

Description

Compositions containing LNP and mRNA and their application in the treatment of Gaucher disease

[0001] Cross-reference information

[0002] This application claims priority to Chinese patent application No. 2024114066667, filed on October 10, 2024, the entire contents of which are incorporated herein by reference. Technical Field

[0003] This invention belongs to the field of biomedicine, specifically relating to a composition containing LNP and mRNA and its application in the treatment of Gaucher disease. Background Technology

[0004] Gaucher disease (GD) is a lysosomal storage disorder (LSD) caused by mutations in the glucocerebrosidase β (GBA1) gene. This gene, located at chromosome 1q21, primarily encodes lysosomal β-glucocerebrosidase (β-GCase). More than 400 mutations have been identified as being associated with Gaucher disease. These mutations can lead to a deficiency of β-GCase, resulting in the accumulation of glucosylsphingosine (GL1 or GlcCer) and its deacylated form, glucosphingosine (Lyso-GL1 or GlcSph), in intracellular lysosomes, particularly in reticuloendothelial cells, forming so-called Gaucher cells. These cells are mainly distributed in organs such as the liver, spleen, bone marrow, and lymph nodes of patients with Gaucher disease.

[0005] Gaucher disease is the most common lysosomal storage disorder, and its course is progressive. Because Gaucher disease can affect multiple systems, symptoms vary greatly among individuals. Gaucher disease mainly includes two forms: neurological and non-neurological. The non-neurological form, also known as type 1 Gaucher disease, is mainly characterized by systemic symptoms, including hepatosplenomegaly, anemia, leukopenia, and skeletal abnormalities. Neurological Gaucher disease includes types 2 and 3, characterized by astrocyte proliferation, neurophagocytosis (i.e., brain inflammation), and neuronal reduction. Type 3 has a later and slower onset than type 2.

[0006] Once diagnosed with Gaucher disease, patients require regular checkups and typically need lifelong treatment. There are two main approaches to treating Gaucher disease: enzyme replacement therapy (ERT) and substrate reduction therapy (SRT). ERT aims to slow disease progression by providing exogenous β-GCase, while SRT reduces GL1 production by inhibiting glucocerebroside synthase. Both approaches have proven effective in treating type 1 Gaucher disease, but have not improved neurological symptoms in patients with type 2 and 3 Gaucher disease and are costly.

[0007] Currently, the two commercially available SRT drugs, Miglustastat and Eligliustastat, are only suitable for adult patients and not for children. Alglucerase, a placental glucocerebrosidase derivative, was the first ERT drug for treating Gaucher disease. Subsequently, drugs developed for treating Gaucher disease include Imiglucerase, Velaglucerase, and Taliglucerase. It should be noted that these drugs have relatively short effective half-lives after administration. For example, the serum half-life of Imiglucerase is 3.6 to 10.4 minutes, Velaglucerase is 11 to 12 minutes, while Taliglucerase is slightly longer, at 18.9 to 28.7 minutes. Therefore, developing a new ERT drug with a longer half-life would hopefully provide a more ideal option for the treatment of Gaucher disease.

[0008] Messenger RNA (mRNA)-based drugs have been extensively studied and are considered a treatment strategy with great potential in multiple therapeutic areas. Meanwhile, advancements in mRNA chemical modification have significantly improved the safety of non-immunostimulatory mRNA drug therapies. In the case of Gaucher disease, directly delivering mRNA into tissues to restore β-GCase protein function offers significant advantages over traditional ERT therapy. This is because the encoding mRNA can utilize intracellular mechanisms to produce the target protein at the desired cellular location, thus providing therapeutic benefits. Compared to viral vector-based gene delivery, mRNA therapy can correct protein functional abnormalities without modifying genomic DNA. Furthermore, transient mRNA protein expression reduces the risk of accidental overdose associated with sustained protein function. Simultaneously, compared to viral vector-based gene therapy, mRNA therapy exhibits a linear dose-response, facilitating the determination of the ideal dose for each patient, which is not easily achieved in gene therapy.

[0009] By altering the core principles of molecular biology, preventative vaccines and therapeutic drugs based on mRNA therapeutic strategies have rapidly gained widespread adoption. However, the presence of various physiological barriers typically limits the intracellular uptake of mRNA molecules. Therefore, a delivery system is needed to circumvent these barriers and achieve precise targeted drug delivery. Lipid nanoparticle (LNP)-based mRNA delivery systems have complex compositions, systematically incorporating different components and involving various molar ratios between them. To date, a relatively mature LNP delivery system typically comprises four components: ionizable cationic liposomes, phospholipids, polyethylene glycol-modified liposomes, and cholesterol. Ionizable cationic liposomes play a crucial role in LNP formulations. On one hand, positively charged ionizable cationic liposomes can form complexes with negatively charged mRNA chains. Simultaneously, ionizable cationic liposomes should possess an appropriate apparent acid dissociation constant (pKa) to ensure the release of mRNA at the correct location. On the other hand, due to the properties of ionizable cationic liposomes, mRNA-loaded lipid nanoparticles can more easily fuse with the cell membrane, thereby delivering the payload into the cytoplasm. Recent studies have shown a crucial structure-activity relationship between different chemical structures and expression of ionizable liposomes. Optimizing the chemical structure of ionizable liposomes can help achieve higher expression levels in specific cells or tissues. Therefore, it is necessary to screen the chemical structures of lipids to optimize expression.

[0010] Recent developments demonstrate that lipid nanoparticles, as mRNA delivery tools, have successfully overcome the challenge of crossing cell membranes for negatively charged long-chain mRNA molecules. In animal models, the safety, efficacy, and reproducibility of LNP technology show therapeutic potential for treating hepatic metabolic diseases such as methylmalonic acidemia (MMA), acute intermittent porphyria (AIP), and Fabry disease. These encouraging preclinical advances highlight the advantages of mRNA therapy in restoring deficient function of intracellular or transmembrane proteins, which is difficult to achieve with current enzyme replacement therapies. In fact, the LNP-mRNA drug mRNA-3927, developed by Moderna, has already restored the activity of propionyl-CoA carboxylase in the liver via intravenous injection and is currently undergoing a Phase I / II study in patients with propionateemia. Interim results show that mRNA-3927 is well-tolerated and exhibits potential signs of clinical improvement after multiple doses. Summary of the Invention

[0011] The present invention first provides a composition comprising lipid nanoparticles (LNPs) and mRNA, wherein the mRNA is encapsulated in or associated with the lipid nanoparticles; wherein the mRNA contains a nucleotide sequence encoding GBA1.

[0012] The present invention further provides a method for preparing the composition as described above, comprising:

[0013] The lipids contained in the lipid nanoparticles are dissolved in ethanol to obtain a lipid ethanol solution;

[0014] The lipid ethanol solution is mixed with an aqueous solution containing the mRNA;

[0015] The ethanol is then removed, and the composition is obtained by separation or purification.

[0016] The present invention further provides a pharmaceutical composition comprising the composition as described above, and a pharmaceutically acceptable carrier or diluent.

[0017] The present invention further provides the use of the compositions or pharmaceutical compositions described above in the prevention or treatment of Gaucher disease.

[0018] The present invention further provides the use of the composition as described above in the preparation of a pharmaceutical composition used for the prevention or treatment of Gaucher disease.

[0019] The composition of this invention can effectively increase the expression and activity of β-GCase in the serum and multiple target organs of the subject, and reduce the level of Lyso-GL1 therein. It also has a long half-life and has promising applications in the treatment of Gaucher disease. Attached Figure Description

[0020] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0021] Figure 1 shows the chemical structural formulas of three ionizable cationic liposomes in the embodiments of the present invention. In Figure 1, A is the chemical structural formula of liposome #9, B is the chemical structural formula of liposome #10, and C is the chemical structural formula of liposome #13.

[0022] Figure 2 shows a comparison of the protein levels of hGBA-mRNA delivered by LNP in three different ionizable cationic liposomes, including liposome #9, liposome #10, and liposome #13, in different cell lines (human embryonic kidney cells 293T, African green monkey kidney cells, and hamster kidney cells) in an embodiment of the present invention.

[0023] Figure 3 shows a quantitative comparison of the expression levels of LNP-delivered hGBA-mRNA in different cell lines (human embryonic kidney cells 293T, African green monkey kidney cells, and hamster kidney cells) in an embodiment of the present invention, analyzed and compared with those of three cationic liposomes: liposome #9, liposome #10, and liposome #13.

[0024] Figure 4 shows the expression level and enzyme activity of β-glucocerebroside lipase in the serum of mice after treatment with mGBA-mRNA in this embodiment of the invention.

[0025] Figure 5 shows the expression level and enzyme activity of β-glucocerebroside lipase in the liver of mice after treatment with mGBA-mRNA in this embodiment of the invention.

[0026] Figure 6 shows the expression level and enzyme activity of β-glucocerebroside lipase in the spleen of mice after treatment with mGBA-mRNA in this embodiment of the invention.

[0027] Figure 7 shows the β-glucocerebroside lipase activity in mouse serum after multiple mGBA-mRNA treatments in an embodiment of the present invention.

[0028] Figure 8 shows the levels of glucosamine in the serum and liver of mice after mGBA-mRNA treatment in this embodiment of the invention, and the relative expression levels of glucosamine in the serum and liver of mice in the mGBA-mRNA treatment group relative to those in the saline treatment group on day 3 after the first injection.

[0029] Figure 9 shows the anti-drug antibody response in mice treated with mGBA-mRNA in an embodiment of the present invention.

[0030] Figure 10 shows the relative fold difference levels of innate immune-related cytokines in the liver and spleen of mice treated with mGBA-mRNA in this embodiment of the invention, using saline-treated control mice as a baseline.

[0031] Figure 11 shows the protein levels and quantitative analysis results of β-glucocerebroside lipase in mouse liver after single-dose administration of hGBA-mRNA and Cerezyme in the embodiments of the present invention.

[0032] Figure 12 shows the protein levels and quantitative analysis results of β-glucocerebroside lipase in the spleen of mice after single-dose administration of hGBA-mRNA and Cerezyme in the embodiments of the present invention.

[0033] Figure 13 shows the comparison of β-glucocerebrosidase activity in mouse serum after single-dose intravenous injection of hGBA-mRNA and Cerezyme in an embodiment of the present invention.

[0034] Figure 14 shows the comparison of β-glucocerebrosidase activity in mouse liver after single-dose intravenous injection of hGBA-mRNA and Cerezyme in an embodiment of the present invention.

[0035] Figure 15 shows the comparison of β-glucocerebroside lipase activity in mouse spleens after single-dose intravenous injection of hGBA-mRNA and Cerezyme in an embodiment of the present invention.

[0036] Figure 16 shows the changes in β-glucocerebroside lipase activity in mouse serum after multiple doses of hGBA-mRNA and Cerezyme in the embodiments of the present invention.

[0037] Figure 17 shows the changes in β-glucocerebroside lipase activity in the liver of mice after multiple doses of hGBA-mRNA and Cerezyme in the embodiments of the present invention.

[0038] Figure 18 shows the changes in β-glucocerebroside lipase activity in the spleen of mice after multiple doses of hGBA-mRNA and Cerezyme in the embodiments of the present invention.

[0039] Figure 19 shows the levels of glucosamine in the serum and liver of mice after the first injection of hGBA-mRNA in this embodiment of the invention.

[0040] Figure 20 shows the levels of glucosamine in the serum and liver of mice after the second injection of hGBA-mRNA in this embodiment of the invention.

[0041] Figure 21 shows the levels of glucosamine in the serum and liver of mice after the third injection of hGBA-mRNA in this embodiment of the invention.

[0042] Figure 22 shows the anti-drug antibody response in mice treated with hGBA-mRNA in an embodiment of the present invention.

[0043] Figure 23 shows the relative fold increase in the expression of natural immune-related cytokines in the liver of mice treated with hGBA-mRNA in this embodiment of the invention, using mice in the saline control group as a benchmark.

[0044] Figure 24 shows the relative fold increase in the expression of natural immune-related cytokines in the spleen of mice treated with hGBA-mRNA in this embodiment of the invention, using mice in the saline control group as a benchmark.

[0045] Figure 25 shows the relative fold difference in the distribution of mRNA in various tissues of mice receiving a single dose of hGBA-mRNA, based on the saline control group mice in this embodiment of the invention. Detailed Implementation

[0046] The following provides a detailed description of specific embodiments of the present invention. It should be understood that the specific embodiments described herein are for illustrative and explanatory purposes only and are not intended to limit the invention. Those skilled in the art can make various modifications and variations to the invention without departing from its scope or spirit. For example, features described or illustrated as part of one embodiment can be used in another embodiment to produce further embodiments.

[0047] Unless otherwise stated, all terms used to disclose this invention (including technical and scientific terms) have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Further guidance is provided below for a better understanding of the teachings of this invention. The terminology used herein in the specification of this invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0048] The terms "and / or," "or / and," and "and / or" as used herein include any one of two or more of the related listed items, as well as any and all combinations of the related listed items. These arbitrary and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or," "or / and," and "and / or," it should be understood that in this application, the technical solution undoubtedly includes technical solutions connected by "logical AND," and also undoubtedly includes technical solutions connected by "logical OR." For example, "A and / or B" includes three parallel solutions: A, B, and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, a technical solution that is connected by "logical OR"), as well as any and all combinations of A, B, C, and D, that is, combinations of any two or three of A, B, C, and D, and also combinations of all four of A, B, C, and D (that is, a technical solution that is connected by "logical AND").

[0049] The terms “containing,” “comprising,” and “including” as used in this invention are synonyms and are inclusive or open-ended, not excluding additional, uncited members, elements, or method steps.

[0050] In this invention, the numerical range represented by endpoints includes all numerical values ​​and fractions contained within that range, as well as the endpoints mentioned.

[0051] This invention relates to concentration values, which include fluctuations within a certain range. For example, fluctuations are allowed within a corresponding precision range. For instance, 2% may fluctuate within ±0.1%. For larger values ​​or values ​​that do not require overly precise control, even greater fluctuations are permitted. For example, 100mM may fluctuate within ranges of ±1%, ±2%, ±5%, etc. Regarding molecular weight, fluctuations of ±10% are allowed.

[0052] In this invention, the terms "multiple" or "various" are used unless otherwise specified, referring to a quantity of 2 or more.

[0053] In this invention, the technical features described in an open-ended manner include both closed-ended technical solutions composed of the listed features and open-ended technical solutions that include the listed features.

[0054] In this invention, terms such as "preferred," "better," "more suitable," and "ideal" merely describe implementation methods or embodiments with better effects and should be understood not to limit the scope of protection of this invention. In this invention, terms such as "optionally," "optionally," and "optional" mean that something is optional, that is, selected from either "with" or "without" a parallel solution. If multiple "optional" statements appear in a technical solution, unless otherwise specified and without contradiction or mutual constraint, each "optional" statement is independent.

[0055] The abbreviations and corresponding common names of the terms mentioned in this invention are shown in Table 1 below:

[0056] Table 1

[0057] Composition

[0058] The present invention first provides a composition comprising lipid nanoparticles (LNPs) and mRNA, wherein the mRNA is encapsulated in or associated with the lipid nanoparticles; wherein the mRNA contains a nucleotide sequence encoding GBA1.

[0059] In some embodiments, the lipid nanoparticles contain ionizable cationic lipids as shown in Formula I and / or Formula II:

[0060] Or a pharmaceutically acceptable salt, tautomer, prodrug, or stereoisomer thereof.

[0061] The present invention has found that the above-mentioned cationic lipids can significantly improve the delivery effect of the mRNA described in the present invention.

[0062] In some preferred embodiments, the lipid nanoparticles contain an ionizable cationic lipid as shown in Formula I, or a pharmaceutically acceptable salt, tautomer, prodrug, or stereoisomer thereof.

[0063] In some embodiments, the nucleotide sequence encoding GBA1 is derived from humans or mice.

[0064] In some embodiments, the nucleotide sequence encoding GBA1 is any of the following sequences:

[0065] i) The nucleotide sequence as shown by GeneID 2629 in the NCBI GenBank database;

[0066] ii) The nucleotide sequence shown as gene number 14466 in the NCBI Genome Database;

[0067] iii) A nucleotide sequence having at least 90%, preferably at least 95%, more preferably at least 97%, further preferably at least 98%, and most preferably at least 99% sequence identity with the nucleotide sequence shown in i) or ii).

[0068] The nucleotide sequence mentioned in iii) here may exhibit one or more nucleotide deletions, insertions, additions, and / or substitutions compared to the nucleotide sequences shown in i) or ii). In a preferred embodiment, the nucleotide sequence in iii) still has the same or similar function as the nucleotide sequence shown in i) or ii).

[0069] In some embodiments, some or all of the uridine in the mRNA is replaced by N1-methylpseudouridine. "Some uridine" as used herein refers to 5% or more uridine, preferably 10% or more uridine, more preferably 30% or more uridine, even more preferably 50% or more uridine, even more preferably 60% or more uridine, even more preferably 70% or more uridine, even more preferably 80% or more uridine, and even more preferably 90% or more uridine.

[0070] In some preferred embodiments, all uridine in the mRNA is replaced by N1-methylpseuuridine.

[0071] In some implementations, the 5' end of the mRNA contains a cap structure.

[0072] In some embodiments, the 5' end of the mRNA contains a Cap 1 structure.

[0073] In some embodiments, the mRNA contains, from the 5' end to the 3' end, the following in sequence: a Cap 1 structure, a 5' untranslated region, a nucleotide sequence encoding GBA1, a 3' untranslated region, and a polyadenylated tail.

[0074] In this invention, the "cap" refers to a special modified structure attached to the 5' end of an mRNA molecule. It typically consists of a 7-methylguanylate and is linked to the 5' end of the mRNA via a triphosphate chain. Depending on the specific modification type, the cap can be classified into different forms, such as Cap 0, Cap 1, and Cap 2.

[0075] The “Cap 1 structure” mentioned in this invention refers to the m7GPPPNm structure formed by adding methylation modification at the 2'-O position of the first nucleotide based on the Cap 0 (m7GPPPN structure).

[0076] The "polyadenylated tail" mentioned in this invention is typically a long sequence of cytosine nucleotides, typically about 25 to about 400 adenosine nucleotides, preferably about 50 to about 200 adenosine nucleotides, more preferably about 100 to about 150 adenosine nucleotides, or even more preferably about 120 to about 130 adenosine nucleotides. In one specific embodiment, the polyadenylated tail of this invention contains 125 adenosine nucleotides.

[0077] The 3'-untranslated region (3'-UTR) mentioned in this invention is typically a portion of mRNA, located between the protein-coding region (i.e., open reading frame) and the poly(A) sequence of the mRNA. The 3'-UTR of mRNA is not translated into an amino acid sequence. The 3'-UTR sequence is usually encoded by a gene transcribed into the corresponding mRNA during gene expression.

[0078] The 5'-untranslated region (5'-UTR) mentioned in this invention is typically understood as a specific segment of messenger RNA (mRNA). It is located at the 5' end of the open reading frame of the mRNA. Typically, the 5'-UTR begins at the transcription start site and terminates one nucleotide before the start codon of the open reading frame. The 5'-UTR may contain elements for controlling gene expression, also known as regulatory elements. Such regulatory elements can be, for example, ribosome binding sites or 5'-terminal oligopyrimidine tracts. The 5'-UTR can be posttranscribed, for example, by adding a 5'-cap.

[0079] In some embodiments, the mRNA further contains sequences with one or more regulatory functions. For example, in some specific embodiments, the mRNA also contains functional sequences that promote translation initiation, such as Kozak sequences, Shine-Dalgarno sequences, TISU (Translation Initiation Stimulation Element) sequences, or optimized 5' UTR sequences (such as UTR7). In other specific embodiments, the mRNA also contains sequences that regulate mRNA stability and translation efficiency, such as Chi-β-Globin sequences, AU enrichment element (ARE) sequences, internal ribosome entry sites (IRESs) sequences, and miRNA binding sites (microRNA (miRNA) binding sites). In specific implementations, those skilled in the art can confirm the specific sequences and locations of the above-mentioned sequences based on common sense.

[0080] In some specific embodiments, the mRNA contains, from the 5' end to the 3' end, the following in sequence: Cap1 structure, Chi-β-GlobinΔ4 5' untranslated region (modified Chi-β-Globin 5' untranslated region), Kozak sequence, nucleotide sequence encoding GBA1, Chi-β-Globin 3' untranslated region and polyadenylated tail.

[0081] In practical implementation, those skilled in the art can determine the specific sequences of the functional components mentioned in the above-mentioned mRNA by combining common sense, or add, subtract, or replace some functional components and regulatory sequences, all of which can achieve the technical effects of the present invention.

[0082] In some embodiments, the mRNA contains any of the following nucleotide sequences:

[0083] I) A nucleotide sequence as shown in any one of SEQ ID No. 3 to 4;

[0084] II) is a nucleotide sequence that has at least 90% sequence identity with the nucleotide sequence shown in I).

[0085] In some specific embodiments, the mRNA contains any of the following nucleotide sequences and has a Cap 1 structure at its 5' end; preferably, some or all of the uridine in the mRNA is replaced by N1-methylpseudouridine:

[0086] I) A nucleotide sequence as shown in any one of SEQ ID No. 3 to 4;

[0087] The nucleotide sequences shown in II) and I) have at least 90%, preferably at least 95%, more preferably at least 97%, further preferably at least 98%, and most preferably at least 99% sequence identity.

[0088] The nucleotide sequence mentioned in II) here may exhibit one or more nucleotide deletions, insertions, additions, and / or substitutions compared to the nucleotide sequence shown in I). In a preferred embodiment, the nucleotide sequence in II) still has the same or similar function as the nucleotide sequence shown in I).

[0089] In this invention, the term "lipid nanoparticles (LNPs)" can refer to any lipid capable of forming particles to which one or more nucleic acid molecules are attached or encapsulated. The term "lipid" refers to a group of organic compounds that are derivatives of fatty acids (e.g., esters) and are generally characterized by being insoluble in water but soluble in many organic solvents. Lipids are generally classified into at least three categories: (1) "simple lipids" including fats and oils as well as waxes; (2) "complex lipids" including phospholipids and glycolipids; and (3) "derived lipids" such as steroids.

[0090] In some embodiments, the lipid nanoparticles further contain one or more selected from a) to c): a) neutral lipids; b) PEG lipids; c) steroids or steroid analogs.

[0091] In this invention, the term "neutral lipid" refers to any of a variety of lipid substances that exist as uncharged or neutral zwitterionic forms at physiological pH. In some embodiments, the neutral lipid is selected from distearylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoylphosphatidylcholine (POPC), palmitoylphosphatidylethanolamine (POPE), and dioleoylphosphatidylethanolamine 4-(N-maleimide methyl) It is one or more of the following: 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), distearate phosphatidylethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoylphosphatidylethanolamine (SOPE), and 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine (trans DOPE).

[0092] In some preferred embodiments, the neutral lipid is distearate phosphatidylcholine (DSPC).

[0093] PEG lipids within the scope of this invention are known in the art. In some embodiments, the PEG lipids are selected from one or more of PEG-phospholipids and PEG-ceramides, for example, from one or more of PEG2000-DMG, PEG2000-DSPE, PEG2000-DPPE, PEG2000-DMPE, PEG2000-DOPE, PEG1000-DSPE, PEG1000-DPPE, PEG1000-DMPE, PEG1000-DOPE, PEG550-DSPE, PEG550-DPPE, PEG-550DMPE, PEG-1000DOPE, PEG-BML, PEG-cholesterol, PEG2000-ceramide, PEG1000-ceramide, PEG750-ceramide, and PEG550-ceramide.

[0094] In some preferred embodiments, the PEG lipid is PEG2000-DMG.

[0095] In some embodiments, the steroid is cholesterol.

[0096] In some embodiments, the lipid nanoparticles contain ionizable cationic lipids, steroids or steroid analogs, neutral lipids, and PEG lipids in a molar ratio of (35–45):(43–54):(8–12):(1–2).

[0097] In some preferred embodiments, the molar ratio of the ionizable cationic lipid, steroid or steroid analog, neutral lipid, and PEG lipid is (37-43):(45-52):(9-11):(1.2-1.7).

[0098] In some preferred embodiments, the molar ratio of the ionizable cationic lipid, steroid or steroid analog, neutral lipid, and PEG lipid is 40:48.5:10:1.5.

[0099] In some embodiments, the N / P ratio of the composition is in the range of about 0.1 to about 20, preferably in the range of about 0.5 to about 15, and more preferably in the range of about 5 to about 15. As an example, the N / P ratio of the composition may be 0.1, 0.5, 1, 3, 5, 6, 7, 8, 9, 10, 12, 15, 18 or 20.

[0100] In this invention, the N / P ratio is defined as the molar ratio of nitrogen atoms (“N”) in the basic nitrogen-containing groups of lipid nanoparticles to phosphate groups (“P”) in mRNA. The “N” value in the lipid nanoparticles can be calculated based on their molecular weight and the relative abundance of permanent cationic groups and, if present, cationizable groups.

[0101] Preparation method of the composition

[0102] The present invention further provides a method for preparing the composition as described above, comprising:

[0103] The lipids contained in the lipid nanoparticles are dissolved in ethanol to obtain a lipid ethanol solution;

[0104] The lipid ethanol solution is mixed with an aqueous solution containing the mRNA;

[0105] The ethanol is then removed, and the composition is obtained by separation or purification.

[0106] In practice, those skilled in the art can identify other detailed technical features involved in the preparation method of the composition by referring to existing literature.

[0107] In some specific embodiments, the aqueous solution of the mRNA is a mixture of mRNA and citrate buffer solution with a concentration of 50±5mM and pH 6.0±1.0.

[0108] In practice, ethanol can be removed by any suitable method that does not negatively affect the lipids or the resulting composition. In one embodiment of the invention, ethanol is removed by dialysis. In an alternative embodiment, ethanol is removed by perfiltration.

[0109] In practice, the separation and optional purification of lipid nanoparticles can be carried out by any suitable method. Preferably, the lipid nanoparticles are filtered; more preferably, they are separated or purified by filtration through a sterile filter.

[0110] Pharmaceutical Composition

[0111] The present invention further provides a pharmaceutical composition comprising the composition as described above, and a pharmaceutically acceptable carrier or diluent.

[0112] The pharmaceutical compositions of the present invention contain a therapeutically effective amount of an active ingredient. The therapeutically effective amount depends on the route of administration, the type of animal (including humans) being treated, and the physical characteristics of the particular animal under consideration. The dosage can be adjusted to achieve the desired effect, but the dosage depends on factors such as body weight, diet, concurrent drug therapy, and other factors recognized by those skilled in the medical field. More specifically, a therapeutically effective amount refers to the amount of active ingredient that effectively prevents, alleviates, or improves the symptoms of a disease or prolongs the survival of the treated individual. The determination of a therapeutically effective amount is within the capabilities of those skilled in the art, especially in light of the disclosure of this invention.

[0113] In this invention, suitable routes of administration for the composition or pharmaceutical composition may include, for example, parenteral delivery, including intramuscular, subcutaneous, intravenous, and intramedullary injection, as well as injection at the corresponding target organ (such as spleen, liver, kidney, lung, brain, and bone marrow). The pharmaceutical composition may also be administered in sustained-release or controlled-release formulations (including depot injections, osmotic pumps, etc.) to allow for long-term and / or timed, pulsatile administration at a predetermined rate. Furthermore, the route of administration may be local or systemic.

[0114] The pharmaceutical composition may be prepared in a manner known per se, for example by conventional mixing, dissolving, granulating, tableting, grinding, emulsifying, encapsulating, embedding, or tableting processes.

[0115] The pharmaceutical composition may be formulated in any conventional manner using one or more physiologically acceptable drug carriers, said drug carriers comprising excipients and adjuvants that facilitate the processing of the active substance into a pharmaceutically usable formulation. A suitable formulation depends on the chosen route of administration. Any well-known techniques, drug carriers, excipients, and diluents may be used appropriately and as understood in the art.

[0116] In some embodiments, the pharmaceutical composition is an injection.

[0117] In this invention, the injectable formulation can be prepared in conventional forms: a liquid solution or suspension, suitable for preparation into a solid form of solution or suspension in a liquid prior to injection, or an emulsion. Suitable excipients are, for example, water, saline, dextran, mannitol, lactose, lecithin, albumin, monosodium glutamate, cysteine ​​hydrochloride, etc. Furthermore, if desired, the injectable pharmaceutical preparation may also contain small amounts of non-toxic excipients, such as wetting agents, pH buffers, etc. Physiologically compatible buffers include, but are not limited to, Hanks' solution, Ringer's solution, or physiological saline buffer. Additionally, if desired, absorption-enhancing agents may be used.

[0118] In some preferred embodiments, the pharmaceutical composition is an intravenous injection.

[0119] Therapeutic and pharmaceutical uses

[0120] The present invention further provides the use of the composition or pharmaceutical composition described above in the prevention or treatment of Gaucher disease. That is, the present invention further provides a method for the prevention or treatment of Gaucher disease, comprising: using the composition or pharmaceutical composition described above.

[0121] The present invention further provides the use of the composition as described above in the preparation of a pharmaceutical composition used for the prevention or treatment of Gaucher disease.

[0122] As is known in the art, the application of the compositions or pharmaceutical compositions described above in this invention refers to their safe and effective dosage. In this invention, "safe and effective dosage" can be further understood as a dosage sufficient to prevent or treat Gaucher disease while avoiding serious side effects. Those skilled in the art can confirm the safe and effective dosage of the compositions or pharmaceutical compositions using known methods such as in vitro cell experiments and animal studies. In clinical applications, physicians can also adjust the dosage of the compositions or pharmaceutical compositions to achieve a safe and effective dosage by considering factors such as weight, diet, concurrent drug therapy, and other factors recognized by medical professionals.

[0123] In some embodiments, the Gaucher disease includes at least one of Gaucher disease type 1, Gaucher disease type 2, and Gaucher disease type 3.

[0124] In some embodiments, the pharmaceutical composition is an injection.

[0125] In some preferred embodiments, the pharmaceutical composition is an intravenous injection.

[0126] In some embodiments, the composition or the pharmaceutical composition is used to achieve at least one of the following uses:

[0127] 1) Increase the expression and activity of β-GCase in the serum and target organs of the subjects;

[0128] 2) Reduce the level of Lyso-GL1 in the serum and target organs of the subjects.

[0129] In some embodiments, the target organ includes at least one of the spleen, liver, kidney, lung, brain, and bone marrow.

[0130] In some preferred embodiments, the target organs include the spleen and liver.

[0131] In some preferred embodiments, the target organ further includes at least one of the kidney, lung, brain, and bone marrow.

[0132] In some embodiments, the target organs include the spleen, liver, kidneys, lungs, brain, and bone marrow.

[0133] In some implementations, the subjects are vertebrates.

[0134] In some preferred embodiments, the subject is a mammal.

[0135] In some preferred embodiments, the subjects are chickens, mice, hamsters, rabbits, sheep, cattle, pigs, dogs, cats, donkeys, monkeys, orangutans, apes, or humans.

[0136] In some preferred embodiments, the subject is a human being.

[0137] Example

[0138] The embodiments of the present invention will be described in detail below using hGBA-mRNA (containing mRNA encoding human glucose sphingolipase β1 (GBA1)) and mGBA-mRNA (containing mRNA encoding murine glucose sphingolipase β1 (GBA1)) as examples.

[0139] It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments that do not specify specific conditions should preferably be referred to the guidelines given in this invention, or may be performed according to experimental manuals or conventional conditions in the art, or other experimental methods known in the art, or according to the conditions recommended by the manufacturer.

[0140] In the specific embodiments described below, the measurement parameters involving raw material components may have slight deviations within the weighing accuracy range unless otherwise specified. Temperature and time parameters are subject to acceptable deviations due to instrument testing accuracy or operational precision.

[0141] 1. Method

[0142] 1.1hGBA-mRNA mRNA sequence

[0143] The mRNA sequence of hGBA-mRNA includes a cap structure, a Chi-β-Globin Δ45' untranslated region (UTR), a Kozak sequence, a nucleotide sequence encoding human glucosylsphingolipid β1 (GBA1, gene number 2629 in the NCBI Genome Database), a Chi-β-Globin 3' untranslated region (UTR), and a polyadenylated tail (125 Å in length).

[0144] The mRNA sequence of mGBA-mRNA includes a cap structure, a Chi-β-Globin Δ45' untranslated region (UTR), a Kozak sequence, a nucleotide sequence encoding murine glucose sphingolipase β1 (GBA1, gene number 14466 in the NCBI Genome Database) (in mGBA-mRNA), a Chi-β-Globin 3' untranslated region (UTR), and a polyadenylated tail (125 Å in length).

[0145] 1.2 Preparation of hGBA-mRNA and mGBA-mRNA

[0146] First, linearized DNA templates were generated by cleaving the plasmid with the BspQ1 enzyme. The DNA sequences for hGBA and mGBA are shown in SEQ ID No. 1 and SEQ ID No. 2, respectively. Then, mRNA was synthesized in vitro using T7 RNA polymerase. The sequences of hGBA-mRNA and mGBA-mRNA are shown in SEQ ID No. 3 and SEQ ID No. 4, respectively. Next, the synthesized mRNA was capped using a smallpox virus capping enzyme, adding a 7-methylguanylate cap structure (Cap 0) to the 5' end of the transcribed mRNA. The Cap 0 structure was then converted to a Cap 1 structure using Cap 2'-O methyltransferase, and the uridine in the mRNA was completely replaced with 1-N-methylpseudo-uridine triphosphate (1-N-Me-Pseudo-UTP).

[0147] The mRNA was encapsulated in an LNP consisting of proprietary cationic liposomes, cholesterol, DSPC, and PEG2000-DMG. The cationic liposomes are ionizable cationic lipids that are highly degradable by lipases. In the formulation, the molar ratio of the four lipid components was 40:48.5:10:1.5 (cationic liposomes:cholesterol:DSPC:PEG2000-DMG). The lipids were dissolved in ethanol, as described by Sabnis, S et al. in *A Novel Amino Lipid Series for mRNA Delivery: Improved Endosomal Escape and Sustained Pharmacology and Safety in Non-human Primates* (Mol Ther, 2018, 26(6): p.1509-1519). The mRNA was dissolved in 50 mM citrate buffer at pH 6.0 to achieve an N / P ratio of 8. The two solutions were mixed using a nanomedicine system (I-Nano) at a total flow rate of 16 mL / min, with a flow ratio of 4:1 v / v (aqueous phase: organic phase). The resulting suspension was then dialyzed twice in RNase-free water for one hour each time, followed by dialyzing in Tris buffer for at least 16 hours. The solution was then concentrated using Amicon Ultra and filtered through a 0.22 μm Millex-GV filter. Encapsulation efficiency and mRNA concentration were measured using Quant-iT... TM RiboGreen TM The RNA Assay Kit was used according to the manufacturer's instructions. Detailed information on hGBA-mRNA is listed in Table 2.

[0148] Table 2. hGBA-mRNA Information

[0149] 1.3 Serum and tissue collection

[0150] After whole blood was allowed to stand for 1 hour, serum samples were collected from the orbital cavity and centrifuged (16,000 x g, 30 min). Liver and spleen tissues were obtained from euthanized mice, immediately frozen, and stored at -80°C for further use. Tissues were lysed using a tissue homogenizer (60 Hz, 4°C for 180 s), then centrifuged (16,000 x g, 4°C for 30 min), and the supernatant was collected. Protein concentrations were determined using the Pierce BCA Protein Assay Kit (Thermo-23225) according to the manufacturer's instructions.

[0151] 1.4 Protein Blotting

[0152] Two days after gene transfection, the culture medium was removed, cells were washed with PBS, and then lysed with RIPA lysis buffer. Cells were then scraped using a cell scraper and centrifuged at 4°C for 30 minutes. The total protein concentration was determined using a BCA protein quantification kit (Beyotime Biotechnology, Shanghai, China). Samples were boiled at 95°C for 15 minutes, and then 40 μg of total protein was loaded for SDS-polyacrylamide gel electrophoresis. The protein was then transferred to a PVDF membrane. The PVDF membrane was blocked with 5% nonfat dry milk powder for one hour, and then incubated overnight at 4°C with primary antibodies against β-GCase (Sigma-G4171), β-actin (Sigma-A2228), and GAPDH (Proteintech-60004), respectively. The membrane was washed three times. Then, anti-rabbit antibody (Beyotime-A0208) and anti-mouse antibody (Invitrogen-35519) were added to bind to the primary antibodies at room temperature for one hour. Proteins were visualized using the Bio-RAD ChemiDoc™ MP imaging system via the Alexa 680 fluorescence channel and enhanced chemiluminescence (ECL, Thermo-32106) substrate.

[0153] 1.5β-GCase enzyme activity analysis

[0154] Tissues were homogenized using a tissue homogenizer at 60 Hz for 180 seconds, followed by centrifugation for 30 minutes to obtain the supernatant. β-GCase enzyme activity was analyzed according to the literature description (PMID: 34106956). Tissue lysates or serum samples were mixed with the substrate 4-methylumbelliferone-glucan (TCI-M3022) (10 mM) in a 150 mM citrate-phosphate buffer (pH 5.4) containing taurocholic acid (0.25%, w / w) and Triton X-100 (0.25%, w / w) and incubated at 37 °C for 60 minutes. The reaction was terminated by adding 1 M glycine at pH 10.5. The formation of the 4-methylumbelliferone-glucan product was measured using a microplate reader (Thermo-VarioSkan lux) at an excitation wavelength of 365 nm and an emission wavelength of 445 nm.

[0155] 1.6 Preparation of Lyso_GL1

[0156] Lyso_GL1 levels were detected by LC-MS. Serum Lyso_GL1 extraction was based on a modified Bligh and Dyer method. For serum samples, 50 μL of 80% methanol was added to 5 μL of serum sample, along with 500 pg of N,N-dimethyl-D-ergosterone (Matreya-1320) as an internal standard (PMID: 21868580). The mixture was vortexed for 3 min and then centrifuged at 16,000 x g for 10 min, and the supernatant was collected. The supernatant was transferred to an autosampler vial for LC-MS / MS analysis. Calibration curves were prepared using the same method as the sample preparation described above, using the Lyso-GL1 standard and internal standard. The injection volume was 10 μL.

[0157] For liver samples, take approximately 0.1 g of wet weight liver sample, homogenize it with 2 mL of chloroform-methanol (volume ratio 2:1), and then centrifuge to obtain the supernatant. After drying the supernatant, dissolve it in 40 μL of 80% methanol. The injection volume is 10 μL.

[0158] 1.7 Quantitative analysis of Lyso_GL1 by UPLC-ESI-MS / MS

[0159] UPLC-MS / MS analysis was performed using a Thermo UPLC system equipped with a Q-exactive orbitrap mass spectrometer (Thermo Fisher Scientific, USA). Separation of components was achieved using an Acquity BEH C18 column (2.1 × 100 mm, 1.7 μm column) at 50 °C. Gradient elution was used between mobile phase A (0.1% formic acid aqueous solution) and mobile phase B (0.1% formic acid acetonitrile solution), as follows: 0–0.5 min 30% B, 0.5–2 min from 30% B to 70% B, 2–4 min 70% B, 4–4.2 min from 70% B to 30% B, 4.2–6 min 30% B, at a flow rate of 0.4 mL / min. Electrospray ionization (ESI) was then performed on the mass spectrometer in positive charge mode for detection. The ion source settings were as follows: spray voltage 3.8 kV; capillary temperature 320 °C; sheath gas and auxiliary gas set to 35 units and 8 units respectively; RF lens 80%; maximum fill time 50 ms; automatic gain control (AGC) target 3E6. The optimal collision energies for Lyso-GL1 and N,N-dimethyl-D-ergosterol were 35% and 10%, respectively. Scan event data acquisition was performed in PRM mode using the following conversions: m / z 462.34253 > 282.27901 / 264.26848 for Lyso-GL1, and m / z 328.32101 > 310.30960 / 280.29920 for N,N-dimethyl-D-erythrosphospirin, at a resolution of 70,000. The system and acquired data were processed using Chromeleon software (Thermo, USA).

[0160] 1.8 Assessing Cytokine mRNA Levels by qPCR

[0161] Total RNA was extracted using RNAiso plus (Takara-9108Q) according to the manufacturer's instructions, and cDNA was prepared by reverse transcription using the PrimeScript RT kit containing gDNA Eraser (Takara-RR047A). CDNA was prepared using ChamQ Universal SYBR qPCR Master Mix (Vazyme, Q711-02) in QuantStudio. TM qPCR analysis was performed on the 6Flex real-time PCR system (Applied Biosystems).

[0162] 2. Results

[0163] 2.1 Screening of LNP-encapsulated ionizable cationic liposomes

[0164] LNP systems containing different ionizable cationic liposomes exhibit varying delivery efficiencies for different target genes, necessitating screening to identify suitable LNP systems for specific gene delivery. Three ionizable cationic liposomes, Liposome #9, Liposome #10, and Liposome #13, were analyzed, their structures shown in Figures 1A, 1B, and 1C, respectively. In three different cell lines (293T, Vero, and BHK), three doses of hGBA-mRNA were transfected using LNP systems containing these three cationic liposomes. Protein expression levels were then assessed and compared at 24h, 48h, and 72h (Figure 2, with saline as the blank control). It can be seen that LNP systems containing different cationic liposome structures produced varying effects on protein expression levels. For hGBA-mRNA protein expression levels at all three time points, LNP systems containing Liposome #13 and Liposome #10 ionizable cationic liposomes showed superior performance compared to Liposome #9.

[0165] Quantitative conversion of the above data clearly shows that liposomes #10 and #13 exhibit superior protein expression levels compared to liposome #9 in the 293T, Vero, and BHK cell lines (Figure 3). In vitro experiments demonstrate that the LNP system containing cationic liposomes #10 and #13 is more suitable for delivering hGBA-mRNA and achieving more efficient protein expression within cells. Comparing the results of liposomes #10 and #13, except for a slightly higher hGBA expression when using LNPs containing liposome #13 in the 293T cell line, the hGBA expression level was higher when using LNPs containing liposome #10 in the Vero and BHK cell lines. This indicates that liposome #10 is relatively more suitable as a cationic liposome delivery carrier for hGBA-mRNA.

[0166] 2.2 In vivo studies

[0167] Completed non-clinical pharmacology / pharmacokinetic studies (Table 3):

[0168] 1) In the GBA D427V mouse model of GD, intravenous injection of mouse hGBA-mRNA (or mGBA-mRNA) restored the β-GCase deficiency in serum, liver and spleen, resulting in a decrease in the level of the metabolic substrate Lyso-GL1 in these target organs.

[0169] 2) In the GBA D427V mouse model, intravenous injection of hGBA-mRNA achieved the following effects: 1) increased β-GCase expression and activity in target organs in a significant dose-dependent manner; 2) achieved longer-lasting β-GCase expression compared to the current Cerezyme standard therapy; 3) consistently increased β-GCase activity in target organs with reduced Lyso-GL1 levels after repeated administration; 4) was well-tolerated after repeated administration, without significantly inducing anti-PEG antibodies or innate immune-related cytokines.

[0170] 3) hGBA-mRNA levels peaked in the spleen, kidney, lung, brain, liver, and bone marrow on day 1 after intravenous administration. In all tissues, hGBA-mRNA levels significantly decreased after 7 days.

[0171] Table 3. Summary of non-clinical studies of mGBA-mRNA and hGBA-mRNA

[0172] 2.2.1 Tissue distribution of hGBA-mRNA in mice

[0173] The tissue distribution of hGBA-mRNA was assessed. GBA D427V mice were administered a single intravenous injection of either saline or 0.5 mg / kg hGBA-mRNA. RNA was isolated from the brain, liver, spleen, kidney, lung, and bone marrow, and quantitative analysis was performed using qPCR (quantitative polymerase chain reaction) with primers specific to the human β-GCase gene. A relative increase in human β-GCase mRNA levels was observed compared to animals treated with saline.

[0174] On day 1 post-treatment, hGBA-mRNA levels peaked in all tested tissues, including the spleen, kidney, lung, brain, liver, and bone marrow. The spleen showed the greatest fold increase, followed by the kidney, lung, brain, liver, and bone marrow, decreasing in that order. Seven days after treatment, hGBA-mRNA levels significantly decreased in all tissues. These results are consistent with non-clinical pharmacology studies demonstrating that hGBA-mRNA treatment maintains increased β-GCase activity in target organs for at least 3 days and up to 7 days.

[0175] It is well known that Gaucher disease leads to the accumulation of cerebrosides in the liver, spleen, kidneys, lungs, brain, and bone marrow. The long-term presence of human β-GCase mRNA suggests that hGBA-mRNA has the potential to compensate for β-GCase activity in these organs, thus providing patients with a more comprehensive benefit than protein-based ERT.

[0176] 2.2.2 Therapeutic effect of intravenous injection of mGBA-mRNA in GBA D427V mutant mice

[0177] The goal of this experiment was to serve as a proof-of-concept, demonstrating the in vivo effects of mGBA-mRNA in GBA D427V mutant mice (carrying the GBA gene D427V mutation on a C57BL / 6 background), as an alternative to hGBA-mRNA in mice. Table 4 shows the study design of this experiment.

[0178] Table 4. Study design of the effect of intravenous injection of mGBA-mRNA in GBA D427V mutant mice.

[0179] To evaluate the in vivo efficacy of mGBA-mRNA treatment, GBA D427V mice were intravenously injected with 0.5 mg / kg of mGBA-mRNA every two weeks (Q2W) for a total of three injections on days 0, 14, and 28.

[0180] β-GCase protein levels in serum, liver, and spleen were assessed by Western blotting, with mice treated with saline serving as a negative control. In serum, β-GCase levels peaked 12 hours after mGBA-mRNA injection and remained detectable for the next 3 days, while β-GCase protein was undetectable in the saline group (see Figure 4). Similarly, in liver and spleen, β-GCase expression peaked 12 hours after mGBA-mRNA injection and remained elevated for at least 3 days (see Figures 5 and 6). In addition to protein expression levels, β-GCase enzyme activity was measured to further evaluate the effect of mGBA-mRNA therapy on improving deficient β-GCase function. mGBA-mRNA therapy resulted in a 14-fold increase (1.4 x 10⁻⁶) in serum β-GCase enzyme activity at peak 12 hours after injection, relative to pretreatment levels. 6 vs. 1x 10 5 (Units are pmol / min / μg / μL), and returned to baseline levels on day 7 (see Figure 4).

[0181] The liver and spleen are the main pathogenic organs of GD. After treatment with mGBA-mRNA, β-GCase enzyme activity in the liver peaked on day 1 post-treatment (9-fold increase relative to pre-dose levels), as did in the spleen (7-fold increase relative to pre-dose levels), and remained at high levels for at least 3 days before returning to baseline levels on day 14 (see Figures 5 and 6).

[0182] Gaucher disease is a lifelong condition requiring repeated drug therapy. To mimic a potential clinical treatment regimen, GBA D427V mutant mice were administered multiple intravenous injections of mGBA-mRNA (0.5 mg / kg). Consistent with observations following a single dose, mGBA-mRNA-treated GBA D427V mice showed a significant increase in serum β-GCase enzyme activity between 3 hours and 3 days after each treatment, peaking at 12 hours after each treatment (Figure 7). The enhanced β-GCase enzyme activity levels did not differ significantly after each injection.

[0183] Lyso-GL1, a β-GCase substrate, is a key biomarker for Gaucher disease, and decreased Lyso-GL1 levels indicate a positive response to treatment. Therefore, measuring Lyso-GL1 concentration can validate the ultimate efficacy of mGBA-mRNA therapy. In untreated GBA D427V mice, serum and liver Lyso-GL1 concentrations were elevated. After mGBA-mRNA treatment, Lyso-GL1 concentrations began to decline within 3 hours and then reached their lowest point over 3 days. On day 3, compared to saline-treated mice, mice treated with mGBA-mRNA showed a 15% reduction in serum Lyso-GL1 levels and a reduction to below 10% in liver levels (Figure 8). Furthermore, in animals treated with mGBA-mRNA, liver Lyso-GL1 levels remained reduced to below 30% of the levels in the saline-treated control group even after multiple administrations.

[0184] Polyethylene glycol (PEG) is a component of liposome nanoparticles (LNPs), and previous reports have shown that it can trigger antibody responses after repeated administration. The generation of anti-drug antibodies (ADAs) can affect the pharmacokinetics and pharmacodynamics of the drug, thereby reducing its efficacy. Therefore, the levels of anti-PEG and anti-β-GCase IgG antibodies in the serum of GBA D427V mice treated with mGBA-mRNA were detected by ELISA. After multiple intravenous injections of mGBA-mRNA, no anti-PEG or anti-β-GCase IgG antibodies exceeding individual variability were detected in the serum of these animals (Figure 9).

[0185] The tolerability of intravenously administered mGBA-mRNA therapy can be assessed by stimulating the production of innate immune cytokines. Liver and spleen samples were collected from GBA D427V mice treated every two weeks at different time points, and the levels of interferon-α (IFN-α), interferon-β (IFN-β), interferon-γ (IFN-γ), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and retinoic acid-induced gene-I (RIG-I) were measured by quantitative RT-PCR. As shown in Figure 10, the experimental group samples were normalized and compared with the data of saline-treated control mice. At any time point during mGBA-mRNA treatment, no significant increase was observed in the measured cytokine levels in either the liver or spleen, indicating that intravenous administration of mGBA-mRNA did not induce activation of the innate immune system.

[0186] In summary, a dose of 0.5 mg / kg of mGBA-mRNA demonstrated significant therapeutic effects in the GBA D427V GD mouse model, including a sustained increase in β-GCase expression and activity in the liver and spleen for 72 hours, while serum and liver levels of the metabolic substrate Lyso-GL1 remained persistently low. Furthermore, repeated administration of mGBA-mRNA did not induce anti-PEG or anti-β-GCase IgG antibodies in vivo, nor did it stimulate innate cytokines, demonstrating good tolerability.

[0187] 2.2.3 Comparison of intravenous administration of hGBA-mRNA with the therapeutic effect of Cerezyme in GBA D427V mutant mice

[0188] Based on the results obtained from mGBA-mRNA in GBA D427V mice, this embodiment further investigated the mRNA product encapsulated by LNP that encodes human GBA, namely hGBA-mRNA, in order to further evaluate its efficacy and compare it with the current ERT standard treatment (SoC) Cerezyme.

[0189] In this study, the therapeutic effect of hGBA-mRNA was evaluated by administering single injections of 0.02 mg / kg, 0.1 mg / kg, or 0.5 mg / kg (body weight) to GBA D427V mutant mice. Additionally, a group of GBA D427V mice received three intravenous injections of hGBA-mRNA at a dose of 0.5 mg / kg, administered every two weeks (Q2W). As the primary comparison, 60 U / kg of Cerezyme was used as a control, a dose previously reported in mouse studies. The study design is detailed in Table 5.

[0190] Table 5. Study Design Comparing the Efficacy of hGBA-mRNA Intravenous Injection with Cerezyme

[0191] The expression of β-GCase in the liver (Figure 11) and spleen (Figure 12) was assessed using Western blotting. The results were consistent with those of mGBA-mRNA in the D427V model. The expression level of β-GCase protein in the liver and spleen was positively correlated with the dose of mGBA-mRNA; and the degradation rate of β-GCase protein was positively correlated with the protein presence level in the liver and spleen. Therefore, the amount of β-GCase protein in the liver and spleen at a given time point is a dynamic equilibrium. Under the high, medium, and low dose conditions tested, the expression of β-GCase in the liver and spleen peaked on day 1 (except for the low-dose spleen group, which peaked on day 3), showing a significant dose-response effect. Following high, medium, and low doses of hGBA-mRNA, liver β-GCase levels remained higher than baseline (basal levels of β-GCase in the liver and spleen on day 0 before administration) at days 7, 3, and 1, respectively. However, at day 3 after administration of all three hGBA-mRNA dose levels, spleen β-GCase expression levels showed a higher concentration in the low and medium dose groups than in the high dose group. Consistent with previously reported data, β-GCase levels peaked within 20–40 minutes after intravenous injection of Cerezyme and then rapidly declined over 12 hours, a much shorter duration of increase than that following hGBA-mRNA administration. Therefore, in vivo, the duration of β-GCase level increase following intravenous hGBA-mRNA administration is significantly longer than that following Cerezyme administration.

[0192] In addition, this embodiment also measured β-GCase enzyme activity in serum, liver, and spleen to evaluate the efficacy of hGBA-mRNA therapy in repairing defective β-GCase function. β-GCase enzyme activity increased after a single intravenous injection of hGBA-mRNA or Cerezyme (i.e., a single injection on day 0), but with completely different kinetic characteristics. The increase in serum β-GCase enzyme activity induced by hGBA-mRNA therapy peaked within 24 hours after intravenous injection and then returned to baseline levels 3 days after treatment (Figure 13); this effect was dose-dependent, relative to pre-treatment levels (2.8 x 10⁻⁶). 5 vs. 5.5x10 4 pmol / min / μg / μL, 1.6 x 10 5 vs 5.5x 10 4 pmol / min / μg / μL, 1.1 x 10 5 vs 5.5x 10 4hGBA-mRNA at a dose of 0.5 mg / kg increased β-GCase activity by 5-fold (pmol / min / μg / μL), 2.9-fold (0.1 mg / kg), and 2.0-fold (0.02 mg / kg). Conversely, after Cerezyme treatment, serum β-GCase activity increased rapidly, peaking within 5 minutes, approximately 10-fold (5.7 x 10⁻⁶) of pre-treatment levels. 5 vs. 5.5x 10 4 The levels of β-GCase in the liver (pmol / min / μg / μL) were elevated and then rapidly decreased to baseline levels within 20 minutes, consistent with previously reported data. Similar to serum, after treatment with hGBA-mRNA at 0.02, 0.1, or 0.5 mg / kg, β-GCase activity in the liver reached its maximum after 1 day (an increase of 3.7, 7.3, or 9.0-fold, respectively, relative to pre-treatment levels), and the same was true in the spleen (an increase of 2.7, 5.4, or 6.7-fold, respectively, relative to pre-treatment levels), remaining elevated for at least 3 days before returning to baseline levels on day 7 (Figures 14 and 15). Similarly to serum, after Cerezyme treatment, β-GCase activity in the liver transiently peaked after 20 minutes (a 5.2-fold increase relative to pre-treatment levels), and β-GCase activity in the spleen also transiently peaked after 20 minutes (a 4.9-fold increase relative to pre-treatment levels), returning to baseline levels in both organs within 12 hours. These data confirm that, compared with Cerezyme, hGBA-mRNA treatment resulted in a longer duration of functional β-GCase activity in the serum, liver, and spleen of treated animals. Compared to Cerezyme's 20–40 minutes, hGBA-mRNA β-GCase levels persisted for at least 3 days and increased in a dose-dependent manner (Figures 13–15).

[0193] In GBA D427V mutant mice, multiple doses of 0.5 mg / kg hGBA-mRNA and 60 U / kg Cerezyme were administered intravenously every two weeks, for a total of three injections on days 0, 14, and 28. Consistent with single-dose administration (once on day 0), hGBA-mRNA-treated GBA D427V mice showed a significant increase in serum β-GCase activity from 6 hours to 3 days after each administration, peaking at 12 hours after each administration (see Figure 16). There was no significant difference in elevated β-GCase activity between different injection intervals after each hGBA-mRNA injection. Compared to Cerezyme, mice injected with hGBA-mRNA showed a longer duration of sustained increase in β-GCase activity in the spleen and liver (7 days vs. 12 hours; see Figures 17 and 18).

[0194] As mentioned earlier, Lyso-GL1 is a key biomarker for assessing the efficacy of GD treatment. Following intravenous hGBA-mRNA treatment, a decrease in Lyso-GL1 levels was observed in both serum and liver. A single dose of hGBA-mRNA showed a seemingly dose-dependent reduction in liver Lyso-GL1 levels (69%, 60%, and 46% reductions at high, medium, and low doses, respectively), which was similar to or greater than the effect of 60 U / kg Cerezyme in reducing Lyso-GL1 levels (56% reduction). Serum and liver glucosamine levels in mice after the first, second, and third injections of hGBA-mRNA are shown in Figures 19, 20, and 21, respectively. These results indicate that after repeated administration of 0.5 mg / kg hGBA-mRNA, serum Lyso-GL1 levels decreased to approximately 60% to 70% of the levels in the saline-injected control group after the first and second injections, while the relative change in Lyso-GL1 levels after the third injection was not as significant as the first two. In contrast, with cerezyme at 60 U / kg, serum lyso-GL1 levels decreased to 60% to 80% of the levels in the saline control group after each injection. However, compared to cerezyme, treatment with hGBA-mRNA at a dose of 0.5 mg / kg showed a greater trend toward a reduction in liver lyso-GL1 levels after the first and second injections. After the third injection, lyso-GL1 levels decreased by approximately 50% in the hGBA-mRNA group, while they decreased by approximately 70% in the cerezyme group (see Figures 19–21).

[0195] To assess anti-drug immune responses, serum samples were collected every 14 days during the study period from mice that received repeated doses of 0.5 mg / kg hGBA-mRNA or 60 U / kg Cerezyme (Figure 22). Anti-PEG and anti-human β-GCase IgG titers were determined in each mouse. Similar to the obtained mGBA-mRNA results, no anti-PEG IgG antibodies exceeding individual variability were detected in these animals after two or three intravenous injections of hGBA-mRNA or Cerezyme.

[0196] Although anti-human β-GCase IgG was not detected in mice injected with Cerezyme, serum levels of anti-human β-GCase IgG increased approximately 100-fold after the second hGBA-mRNA injection and remained elevated until day 42 at the end of the study. These results suggest that human β-GCase proteins may be immunogenic in mice. This may explain the reduced change in Lyso-GL1 levels in mice after the third hGBA-mRNA injection, but not after the mGBA-mRNA injection.

[0197] It is crucial that LNP-mRNA therapeutics avoid detection by the innate immune system. The expression of TNF-α, IL-6, IFN-α, IFN-β, and IFN-γ in the liver and spleen of mice treated with hGBA-mRNA was measured using quantitative polymerase chain reaction (Q-PCR), and the fold changes relative to the levels of these cytokines in saline-treated control mice were calculated (see Figures 23 and 24). The results showed that the expression of these cytokines did not significantly increase in either the liver or spleen during hGBA-mRNA treatment. Similar results were observed in the Cerezyme treatment group.

[0198] In summary, intravenous injection of hGBA-mRNA induced dose-dependent expression of β-GCase in the serum, liver, and spleen of GBA D427V mice. Pharmacokinetic analysis in GBA D427V mutant mice revealed that the duration of β-GCase expression in serum, liver, and spleen induced by hGBA-mRNA was significantly longer than that induced by the current standard treatment, Cerezyme. Furthermore, treatment with hGBA-mRNA reduced the levels of the β-GCase metabolite Lyso-GL1 in serum and liver, and this therapeutic effect was maintained after repeated administration. In addition, no significant anti-PEG antibodies or intrinsic cytokines were observed after multiple in vivo administrations of hGBA-mRNA.

[0199] 2.2.4 Pharmacokinetics

[0200] The tissue distribution of hGBA-mRNA in GBA D427V mice was assessed. On day 0, GBA D427V mice were administered a single intravenous injection of either saline or 0.5 mg / kg hGBA-mRNA. RNA was isolated from the brain, liver, spleen, kidney, lung, and bone marrow before administration and on days 1, 3, and 7 post-administration. Quantitative polymerase chain reaction (qPCR) was performed using primers specific to the human β-GCase gene. The relative increase in human β-GCase mRNA levels was assessed compared to the saline control group. The study design is detailed in Table 6.

[0201] Table 6. Study Design of Tissue Distribution After Single Intravenous Injection of hGBA-mRNA in GBA D427V Mice

[0202] Note: a. For blood, tumors, and other tissue matrix

[0203] As shown in Figure 25, the peak hGBA-mRNA level occurred on day 1 post-treatment in all tissues. The spleen showed the highest increase in hGBA-mRNA distribution, at 6923-fold. This was followed by the kidneys, lungs, brain, liver, and bone marrow, decreasing in that order (1833±405, 509±129, 92±33, 51±11, and 19±8, respectively). On day 7, hGBA-mRNA levels significantly decreased in all tissues (the doubling was 177±69 in the spleen, 110±31 in the kidneys, 43±11 in the lungs, 6.4±2.8 in the brain, 0.9±0.3 in the liver, and 7.7±6.4 in the bone marrow). These results are consistent with the premise that hGBA-mRNA treatment can maintain increased β-GCase activity in target organs for at least 3 days and at most 7 days. Gaucher disease is known to cause the accumulation of cerebrosides in the liver, spleen, kidneys, lungs, brain, and bone marrow. The presence of human β-glucosidase (β-GCase) mRNA suggests that hGBA-mRNA has the potential to compensate for β-GCase activity in these organs, thus providing patients with a more comprehensive benefit than protein gene replacement therapy.

[0204] The embodiments described above are merely examples of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention.

Claims

1. A composition, characterized in that, It contains lipid nanoparticles and mRNA, wherein the mRNA is encapsulated in the lipid nanoparticles or associated with the lipid nanoparticles; The mRNA contains a nucleotide sequence encoding GBA1.

2. The composition according to claim 1, characterized in that, The lipid nanoparticles contain ionizable cationic lipids as shown in Formula I and / or Formula II: Or a pharmaceutically acceptable salt, tautomer, prodrug, or stereoisomer thereof.

3. The composition according to claim 1, characterized in that, The nucleotide sequence encoding GBA1 is derived from humans or mice.

4. The composition according to claim 3, characterized in that, The nucleotide sequence encoding GBA1 is any of the following sequences: i) The nucleotide sequence shown as gene number 2629 in the NCBI Genome Database; ii) The nucleotide sequence shown as gene number 14466 in the NCBI Genome Database; iii) A nucleotide sequence that has at least 90% sequence identity with the nucleotide sequence shown in i) or ii).

5. The composition according to claim 3 or 4, characterized in that, Some or all of the uridine in the mRNA is replaced by N1-methylpseudouridine.

6. The composition according to claim 3 or 4, characterized in that, The mRNA contains, from the 5' end to the 3' end, the following structures in sequence: Cap 1 structure, 5' untranslated region, nucleotide sequence encoding GBA1, 3' untranslated region, and polyadenylated tail.

7. The composition according to claim 5, characterized in that, The mRNA contains, from the 5' end to the 3' end, the following structures in sequence: Cap 1 structure, 5' untranslated region, nucleotide sequence encoding GBA1, 3' untranslated region, and polyadenylated tail.

8. The composition according to claim 1, characterized in that, The mRNA contains any of the following nucleotide sequences: I) A nucleotide sequence as shown in any one of SEQ ID No. 3 to 4; II) is a nucleotide sequence that has at least 90% sequence identity with the nucleotide sequence shown in I).

9. The composition according to claim 5, characterized in that, The mRNA contains any of the following nucleotide sequences and has a Cap 1 structure at the 5' end: I) A nucleotide sequence as shown in any one of SEQ ID No. 3 to 4; II) is a nucleotide sequence that has at least 90% sequence identity with the nucleotide sequence shown in I).

10. The composition according to claim 1, characterized in that, The lipid nanoparticles also contain one or more of the following: a) to c): a) neutral lipids; b) PEG lipids; c) steroids or steroid analogs.

11. The composition according to claim 10, characterized in that, The neutral lipid is selected from one or more of the following: distearylphosphatidylcholine, dioleoylphosphatidylcholine, dipalmitoylphosphatidylcholine, dioleoylphosphatidylglycerol, dipalmitoylphosphatidylglycerol, dioleoylphosphatidylethanolamine, palmitoylphosphatidylcholine, palmitoylphosphatidylethanolamine, and dioleoylphosphatidylethanolamine 4-(N-maleimidemethyl)-cyclohexane-1-carboxylic acid ester, dipalmitoylphosphatidylethanolamine, dimyristoylphosphatidylethanolamine, distearylphosphatidylethanolamine, 16-O-monomethylPE, 16-O-dimethylPE, 18-1-transPE, 1-stearoyl-2-oleoylphosphatidylethanolamine, and 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine.

12. The composition according to claim 10, characterized in that, The PEG lipids are selected from one or more of PEG2000-DMG, PEG2000-DSPE, PEG2000-DPPE, PEG2000-DMPE, PEG2000-DOPE, PEG1000-DSPE, PEG1000-DPPE, PEG1000-DMPE, PEG1000-DOPE, PEG550-DSPE, PEG550-DPPE, PEG-550DMPE, PEG-1000DOPE, PEG-BML, PEG-cholesterol, PEG2000-ceramide, PEG1000-ceramide, PEG750-ceramide, and PEG550-ceramide.

13. The composition according to claim 10, characterized in that, The steroid in question is cholesterol.

14. The composition according to any one of claims 10 to 13, characterized in that, The lipid nanoparticles contain ionizable cationic lipids, steroids or steroid analogs, neutral lipids, and PEG lipids in a molar ratio of (35-45):(43-54):(8-12):(1-2).

15. A method for preparing the composition according to any one of claims 1 to 14, characterized in that, It includes: The lipids contained in the lipid nanoparticles are dissolved in ethanol to obtain a lipid ethanol solution; The lipid ethanol solution is mixed with an aqueous solution containing the mRNA; The ethanol is then removed, and the composition is obtained by separation or purification.

16. A pharmaceutical composition, characterized in that, It contains the composition as described in any one of claims 1 to 14, and a pharmaceutically acceptable carrier or diluent.

17. The pharmaceutical composition according to claim 16, characterized in that, The pharmaceutical composition is an intravenous injection.

18. The use of the composition according to any one of claims 1 to 14 in the preparation of a pharmaceutical composition, characterized in that, The pharmaceutical composition is used for the prevention or treatment of Gaucher disease.

19. The application according to claim 18, characterized in that, The Gaucher disease includes at least one of Gaucher disease type 1, Gaucher disease type 2, and Gaucher disease type 3.

20. The application according to claim 18, characterized in that, The pharmaceutical composition is an intravenous injection.

21. The application according to claim 18, characterized in that, The composition or the pharmaceutical composition is used to achieve at least one of the following uses: 1) Increase the expression and activity of β-glucocerebroside lipase in the serum and target organs of the subjects; 2) Reduce the level of glucosamine in the serum and target organs of the subjects.

22. The application according to claim 21, characterized in that, The target organs include at least one of the spleen, liver, kidney, lung, brain, and bone marrow.

23. The application according to claim 18, characterized in that, The subjects were mammals.

24. The application according to claim 18, characterized in that, The subjects were chickens, mice, hamsters, rabbits, sheep, cattle, pigs, dogs, cats, donkeys, monkeys, orangutans, apes, or humans.

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