CN118903476A - Composition containing LNP and mRNA and application thereof in treating Gaucher disease - Google Patents

Abstract

The invention belongs to the field of biological medicine, and particularly relates to a composition containing LNP and mRNA and application thereof in treating Gaucher disease. The composition contains LNP and mRNA, the mRNA is encapsulated in or associated with the lipid nanoparticle; wherein the mRNA contains a nucleotide sequence encoding GBA 1. The composition provided by the invention can effectively increase the expression and activity of beta-glucocerebrosidase (beta-GCase) in serum and multiple target organs in a subject, reduce the level of glucose sphingosine (Lyso-GL 1) in the serum and multiple target organs, has a longer half-life period, and has an application prospect in treatment of Gaucher disease.

Description

Composition containing LNP and mRNA and its application in treating Gaucher disease

Technical Field

The invention belongs to the field of biomedicine, and specifically relates to a composition containing LNP and mRNA and application of the composition in treating Gaucher disease.

Background Art

Gaucher Disease (GD) is a lysosomal storage disorder (LSD) caused by mutations in the glucocerebrosidase β (GBA1) gene. This gene is located at the 1q21 locus of human chromosomes and mainly encodes lysosomal β-glucocerebrosidase (β-GCase). More than 400 mutations associated with Gaucher disease have been found. These mutations may lead to β-GCase deficiency, which in turn leads to the accumulation of glucosylceramide (GL1 or GlcCer) and its deacylated form of glucosylceramide (Lyso-GL1 or GlcSph) in the intracellular lysosomes, especially in the cells of the reticuloendothelial system, forming so-called Gaucher cells, which are mainly distributed in the liver, spleen, bone marrow, lymph nodes and other organs of patients with Gaucher disease.

Gaucher disease is the most common lysosomal storage disease, and its disease course is progressive. Because Gaucher disease can affect multiple systems, the symptoms of patients vary greatly from person to person. Gaucher disease includes two main forms: neurological and non-neurological. The non-neurological form, also known as type 1 Gaucher disease, mainly manifests as systemic symptoms, including hepatosplenomegaly, anemia, leukopenia, and bone abnormalities. The neurological form includes types 2 and 3, which are characterized by astrogliosis, neurophagocytosis (i.e., brain inflammation), and neuronal loss. Type 3 has a later and slower onset than type 2.

Once diagnosed with Gaucher disease, patients require regular checkups and usually lifelong treatment. There are two main approaches to the treatment of 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.

The two SRT drugs currently on the market, Miglustat and Eliglustat, are only suitable for adult patients and not for children. Alglucerase, a derivative of glucocerebrosidase from the placenta, is the first ERT drug for the treatment of Gaucher disease. Since then, the drugs developed for the treatment of Gaucher disease include Imiglucerase, Velaglucerase, and Taliglucerase. It should be pointed out that the effective half-life of these drugs after administration is short. For example, the serum half-life of Imiglucerase is 3.6 to 10.4 minutes, that of Velaglucerase is 11 to 12 minutes, and that of Taliglucerase is slightly increased to 18.9 to 28.7 minutes. Therefore, the development of a new ERT drug with a longer half-life will hopefully provide a more ideal option for the treatment of Gaucher disease.

Messenger RNA (mRNA)-based drugs have been extensively studied and are considered to be a therapeutic strategy with great potential in multiple therapeutic areas. At the same time, the achievements in mRNA chemical modification have also significantly improved the safety of non-immunostimulatory mRNA drug therapy. In the case of Gaucher disease, the restoration of β-GCase protein function by delivering mRNA directly into tissues has significant advantages over traditional ERT therapy. Because the encoding mRNA can use intracellular machinery to produce the target protein at the desired cellular location, it brings benefits to treatment. Compared with viral vector-based gene delivery, mRNA therapy can correct protein dysfunction without modifying genomic DNA. In addition, transient protein expression of mRNA can reduce the risk of accidental overdose caused by the continuous function of proteins. At the same time, compared with viral vector-based gene therapy, mRNA therapy presents a linear dose response, which helps to determine the ideal dose for each patient, which is not easy to achieve in gene therapy.

By changing the core principles of molecular biology, preventive vaccines and therapeutic drugs based on mRNA therapeutic strategies have rapidly gained popularity, but the existence of multiple physiological barriers usually limits the uptake of mRNA molecules into cells. Therefore, a delivery system needs to be developed to circumvent these obstacles to achieve precise targeted delivery of drugs. The mRNA delivery system based on lipid nanoparticles (LNPs) has a complex composition, which systematically includes different components and also involves a variety of different molar ratios between them. So far, a relatively mature LNP delivery system usually includes four components, namely ionizable cationic liposomes, phospholipids, PEGylated liposomes and cholesterol. In the LNP formulation, ionizable cationic liposomes play an important role. On the one hand, positively charged ionizable cationic liposomes can form complexes with negatively charged mRNA chains. At the same time, ionizable cationic liposomes should have an appropriate apparent acid dissociation constant (pKa) to ensure the release of mRNA at the right location. On the other hand, relying on the characteristics of ionizable cationic liposomes, lipid nanoparticles loaded with mRNA are more likely to fuse with cell membranes, thereby delivering the payload to the cytoplasm. Recent studies have shown that ionizable liposomes have a key structure-activity relationship between different chemical structures and expression. Optimizing the chemical structure of ionizable liposomes will help achieve higher expression levels in specific cells or tissues. Therefore, it is necessary to screen the chemical structure of lipids to achieve optimal expression.

Recent developments have shown that lipid nanoparticles, as mRNA delivery tools, have successfully solved the problem of long mRNA molecules with negative charges crossing the cell membrane. In animal models, the safety, effectiveness and reproducibility of LNP technology have therapeutic potential in the treatment of liver metabolic diseases, such as methylmalonic acidemia (MMA), acute intermittent porphyria (AIP), Fabry disease, etc. These encouraging preclinical advances show the advantages of mRNA treatment mode in restoring the defective function of intracellular or transmembrane proteins, which is difficult to achieve with current enzyme replacement therapy drugs. In fact, the LNP-mRNA drug mRNA-3927 developed by Moderna, through intravenous injection, can restore the activity of propionyl-CoA carboxylase in the liver and is currently undergoing a phase I/II study in patients with propionic acidemia. Interim results showed that mRNA-3927 was well tolerated and showed potential signs of clinical improvement after multiple doses.

Summary of the invention

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

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

dissolving the lipid contained in the lipid nanoparticles in ethanol to obtain a lipid ethanol solution;

mixing the lipid ethanol solution with an aqueous solution containing the mRNA;

Then the ethanol is removed and the composition is obtained by separation or purification.

The present invention further provides a pharmaceutical composition, which contains the composition as described above and a pharmaceutically acceptable carrier or diluent.

The present invention further provides use of the above-mentioned composition or the above-mentioned pharmaceutical composition in preventing or treating Gaucher disease.

The present invention further provides use of the aforementioned composition in preparing a pharmaceutical composition, wherein the pharmaceutical composition is used to prevent or treat Gaucher disease.

The composition of the present 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, and has a long half-life, and has application prospects in the treatment of Gaucher disease.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

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

FIG2 is a comparison of the protein levels of hGBA-mRNA transfected and expressed in different cell lines (human embryonic kidney cells 293T, African green monkey kidney cells, and hamster kidney cells) by LNP delivery of three different ionizable cationic liposomes including liposome #9, liposome #10, and liposome #13 in an embodiment of the present invention.

FIG3 is a quantitative comparison of the expression levels of hGBA-mRNA delivered by LNPs containing three cationic liposomes, 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 example of the present invention.

FIG. 4 shows the expression level and enzyme activity of β-glucocerebrosidase in the serum of mice after mGBA-mRNA treatment in an example of the present invention.

FIG. 5 shows the expression level and enzyme activity of β-glucocerebrosidase in the liver of mice after mGBA-mRNA treatment in an example of the present invention.

FIG. 6 shows the expression level and enzyme activity of β-glucocerebrosidase in the spleen of mice after mGBA-mRNA treatment in an example of the present invention.

FIG. 7 shows the β-glucocerebrosidase activity in the serum of mice after multiple mGBA-mRNA treatments in an example of the present invention.

8 shows the levels of glucosylceramide in the serum and liver of mice after mGBA-mRNA treatment in an embodiment of the present invention, as well as the relative expression levels of glucosylceramide in the serum and liver of mice in the mGBA-mRNA treatment group relative to the saline treatment group on the third day after the first injection.

FIG. 9 shows the anti-drug antibody response in mice treated with mGBA-mRNA according to an embodiment of the present invention.

FIG. 10 is a graph showing the relative fold difference levels of innate immune-related cytokines in the liver and spleen of mice treated with mGBA-mRNA, with saline-treated control mice as a benchmark in an example of the present invention.

FIG. 11 is a quantitative analysis result of protein level of β-glucocerebrosidase in mouse liver and western blot after single-dose administration of hGBA-mRNA and Cerezyme in an example of the present invention.

FIG. 12 is the protein level of β-glucocerebrosidase in the spleen of mice after single-dose administration of hGBA-mRNA and Cerezyme in an example of the present invention and the results of quantitative analysis by Western blot.

FIG. 13 is a graph showing the comparison of β-glucocerebrosidase activity in mouse serum after intravenous injection of a single dose of hGBA-mRNA and Cerezyme in an example of the present invention.

FIG. 14 is a graph showing the comparison of β-glucocerebrosidase activity in the liver of mice after intravenous injection of a single dose of hGBA-mRNA and Cerezyme in an example of the present invention.

FIG. 15 is a graph showing the comparison of β-glucocerebrosidase activity in the spleen of mice after intravenous injection of a single dose of hGBA-mRNA and Cerezyme in an example of the present invention.

FIG. 16 is a graph showing changes in β-glucocerebrosidase activity in serum of mice after multiple doses of hGBA-mRNA and Cerezyme were administered in accordance with an embodiment of the present invention.

FIG. 17 is a graph showing changes in β-glucocerebrosidase activity in the liver of mice after multiple doses of hGBA-mRNA and Cerezyme were administered in accordance with an embodiment of the present invention.

FIG. 18 is a graph showing changes in β-glucocerebrosidase activity in the spleen of mice after multiple doses of hGBA-mRNA and Cerezyme were administered in an example of the present invention.

FIG. 19 shows the levels of glucosphingosine in serum and liver of mice after the first injection of hGBA-mRNA treatment in an example of the present invention.

FIG. 20 shows the levels of glucosphingosine in serum and liver of mice after the second injection of hGBA-mRNA in an example of the present invention.

FIG. 21 shows the levels of glucose sphingosine in the serum and liver of mice after the third injection of hGBA-mRNA treatment in an example of the present invention.

FIG. 22 is a graph showing the anti-drug antibody response in mice treated with hGBA-mRNA according to an example of the present invention.

FIG. 23 is a graph showing the relative multiples of innate immune-related cytokines in the liver of mice treated with hGBA-mRNA, with the normal saline control group mice as the benchmark in an embodiment of the present invention.

FIG. 24 is a graph showing the relative multiples of innate immune-related cytokines in the spleen of mice treated with hGBA-mRNA, with the normal saline control group mice as the benchmark in an embodiment of the present invention.

FIG. 25 is a graph showing the relative differences in the distribution of mRNA in various tissues of mice that received a single dose of hGBA-mRNA, with the normal saline control group mice as a benchmark in an embodiment of the present invention.

DETAILED DESCRIPTION

The specific embodiments of the present invention are described in detail below. It should be understood that the specific embodiments described herein are only used to illustrate and explain the present invention, and are not used to limit the present invention. Those skilled in the art may make various modifications and changes to the present invention without departing from the scope or spirit of the present invention. For example, a feature described or illustrated as part of one embodiment may be used in another embodiment to produce a further embodiment.

Unless otherwise indicated, the meaning of all terms (including technical and scientific terms) used to disclose the present invention is the same as that commonly understood by those of ordinary skill in the art to which the present invention belongs. By way of further guidance, the following definitions are used to better understand the teachings of the present invention. The terms used herein in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

The terms "and/or", "or/and", and "and/or" used in this article include any one of two or more related listed items, and also include any and all combinations of related listed items, and the 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 that are all connected by "logical and", and undoubtedly includes technical solutions that are all connected by "logical or". For example, "A and/or B" includes three parallel solutions of 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, the technical solution that is all connected by "logical OR"), and also includes any and all combinations of A, B, C, and D, that is, the combination of any two or any three of A, B, C, and D, and also includes the combination of four of A, B, C, and D (that is, the technical solution that is all connected by "logical AND").

As used herein, the terms "comprising", "including" and "comprising" are synonymous and are inclusive or open-ended and do not exclude additional, unrecited members, elements or method steps.

Numerical ranges expressed as endpoints herein include all numbers and fractions subsumed within the range, as well as the recited endpoints.

The present invention relates to concentration values, and the meaning includes fluctuations within a certain range. For example, it can fluctuate within a corresponding precision range. For example, 2%, it is allowed to fluctuate within ±0.1%. For values that are large or do not require too fine control, it is also allowed to have a greater fluctuation. For example, 100mM, it is allowed to fluctuate within the range of ±1%, ±2%, ±5%, etc. Involving molecular weight, it is allowed to have a fluctuation of ±10%.

In the present invention, descriptions such as "plurality" and "multiple" refer to quantities greater than or equal to 2 unless otherwise specified.

In the present invention, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

In the present invention, "preferably", "better", "more preferably", and "suitably" are only used to describe implementation methods or examples with better effects, and it should be understood that they do not constitute a limitation on the scope of protection of the present invention. In the present invention, "optionally", "optional", and "optional" refer to being optional, that is, any one of the two parallel schemes of "yes" or "no". If multiple "options" appear in a technical solution, unless otherwise specified and there is no contradiction or mutual restriction, each "optional" is independent of each other.

The abbreviations and corresponding common names of the terms mentioned in the present invention are as follows in Table 1:

Table 1

Composition

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

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

Formula I Liposome #10;

Formula II Liposome #13;

or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof.

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

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.

In some embodiments, the nucleotide sequence encoding GBA1 is derived from human or mouse.

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

i) the nucleotide sequence as shown in the NCBI GenBank gene ID 2629;

ii) the nucleotide sequence as shown in the NCBI genome database gene number 14466;

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).

The nucleotide sequence mentioned in iii) herein may show one or more nucleotide deletions, insertions, additions and/or substitutions compared to the nucleotide sequence 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).

In some embodiments, part or all of the uridine in the mRNA is replaced by N1-methyl pseudouridine. The "partial uridine" mentioned herein refers to 5% or more uridine, preferably 10% or more uridine, more preferably 30% or more uridine, further preferably 50% or more uridine, further preferably 60% or more uridine, further preferably 70% or more uridine, further preferably 80% or more uridine, further preferably 90% or more uridine.

In some preferred embodiments, all uridines in the mRNA are replaced by N1-methylpseudouridine.

In some embodiments, the 5' end of the mRNA contains a cap structure.

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

In some embodiments, the mRNA contains, from the 5' end to the 3' end, the following: Cap 1 structure, a 5' untranslated region, a nucleotide sequence encoding GBA1, a 3' untranslated region, and a poly(A) tail.

The "cap" mentioned in the present invention refers to a special modified structure attached to the 5' end of the mRNA molecule. It is usually composed of a 7-methylguanylate and connected to the 5' end of the mRNA through a triphosphate chain. Depending on the specific modification type of the cap, it can be divided into different forms, such as Cap 0, Cap 1 and Cap 2.

The "Cap 1 structure" mentioned in the present invention refers to the m7GPPPNm structure formed by adding a methylation modification to the 2'-O position of the first nucleotide based on Cap 0 (m7GPPPN structure).

The "poly(A) tail" referred to in the present 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 a specific embodiment, the poly(A) tail of the present invention contains 125 adenosine nucleotides.

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

The 5'-untranslated region (5'-UTR) mentioned in the present invention is typically understood as a specific segment of messenger RNA (mRNA). It is located at the 5' of the open reading frame of the mRNA. Typically, the 5'-UTR starts at the transcription start site and ends at a nucleotide before the start codon of the open reading frame. The 5'-UTR may include elements for controlling gene expression, also referred to as regulatory elements. Such regulatory elements may be, for example, a ribosome binding site or a 5'-terminal oligopyrimidine tract. The 5'-UTR may be modified post-transcriptionally, for example, by adding a 5'-cap.

In some embodiments, the mRNA also contains a sequence with one or more regulatory functions. For example, in some specific embodiments, the mRNA also contains a functional sequence that can promote the initiation of translation, such as a Kozak sequence, a Shine-Dalgarno sequence, a TISU (Translation Initiation Stimulation Element) sequence, or an optimized 5'UTR sequence (such as UTR7), etc. For another example, in some specific embodiments, the mRNA also contains a sequence that can regulate the stability and translation efficiency of mRNA, such as a Chi-β-Globin sequence, an AU enrichment element (AREs) sequence, an internal ribosome entry site (IRESs) sequence, a miRNA binding site (MicroRNA (miRNA) Binding Sites), etc. In the specific implementation, those skilled in the art can confirm the specific sequence and setting position of the above sequence in combination with common sense.

In some specific embodiments, the mRNA contains, from the 5' end to the 3' end, the following: Cap 1 structure, Chi-β-Globin Δ4 5' untranslated region (improved Chi-β-Globin 5' untranslated region), Kozak sequence, a nucleotide sequence encoding GBA1, Chi-β-Globin 3' untranslated region and a poly(A) tail.

In specific implementation, those skilled in the art can determine the specific sequence of the functional components mentioned in the above mRNA based on common sense, or add, subtract and replace some functional components and regulatory sequences, all of which can achieve the technical effects of the present invention.

In some embodiments, 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) A nucleotide sequence having at least 90% sequence identity with the nucleotide sequence shown in I).

In some specific embodiments, the mRNA contains any of the following nucleotide sequences, and the 5' end contains a Cap 1 structure, and preferably part or all of the uridine in the mRNA is replaced by N1-methyl pseudouridine:

I) a nucleotide sequence as shown in any one of SEQ ID No.3 to 4;

II) is 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).

The nucleotide sequence mentioned in II) herein may show 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).

In the present invention, the term "lipid nanoparticle (LNP)" may include any lipid that can form a particle to which one or more nucleic acid molecules are attached or in which one or more nucleic acid molecules are 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 divided into at least three categories: (1) "simple lipids" including fats and oils and waxes; (2) "complex lipids" including phospholipids and glycolipids; (3) "derivatized lipids" such as steroids.

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

In the present invention, the term "neutral lipid" refers to any of a number of lipid substances that exist in the form of uncharged or neutral zwitterions at physiological pH. In some embodiments, the neutral lipid is selected from distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE) and dioleoylphosphatidylethanolamine 4-(N-maleimidoyl)phosphatidylcholine). One or more of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (trans-DOPE), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE-mal ...

In some preferred embodiments, the neutral lipid is distearoylphosphatidylcholine (DSPC).

PEG lipids within the scope of the present invention are known in the art per se. In some embodiments, the PEG lipid is selected from one or more of PEG-phospholipids and PEG-ceramides, for example, 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, PEG550-ceramide.

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

In some embodiments, the steroid is cholesterol.

In some embodiments, the lipid nanoparticles contain the ionizable cationic lipid, steroid or steroid analog, neutral lipid, and PEG lipid in a molar ratio of (35-45):(43-54):(8-12):(1-2).

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).

In some more 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.

In some embodiments, the N/P ratio of the composition is in the range of from about 0.1 to about 20, preferably in the range of from about 0.5 to about 15, and more preferably in the range of from about 5 to about 15. By way of example, the N/P ratio of the composition can be 0.1, 0.5, 1, 3, 5, 6, 7, 8, 9, 10, 12, 15, 18, or 20.

In the present invention, the N/P ratio is defined as the molar ratio of nitrogen atoms ("N") of the basic nitrogen-containing groups in the lipid nanoparticles to the phosphate groups ("P") in the mRNA. The "N" value in the lipid nanoparticle can be calculated based on its molecular weight and the relative content of permanent cationic groups and cationizable groups, if present.

Preparation method of composition

The present invention further provides a method for preparing the above-mentioned composition, which comprises:

dissolving the lipid contained in the lipid nanoparticles in ethanol to obtain a lipid ethanol solution;

mixing the lipid ethanol solution with an aqueous solution containing the mRNA;

Then the ethanol is removed and the composition is obtained by separation or purification.

During specific implementation, those skilled in the art may confirm other detailed technical features involved in the preparation method of the composition in combination with existing literature.

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

In a specific implementation, ethanol can be removed by any suitable method that does not negatively affect the lipids or the formed composition. In one embodiment of the present invention, ethanol is removed by dialysis. In an alternative embodiment, ethanol is removed by diafiltration.

In a specific implementation, the separation and optional purification of lipid nanoparticles can also be performed by any suitable method. Preferably, the lipid nanoparticles are filtered, more preferably, the lipid nanoparticles are separated or purified by filtering through a sterile filter.

Pharmaceutical composition

The present invention further provides a pharmaceutical composition, which contains the composition as described above and a pharmaceutically acceptable carrier or diluent.

The pharmaceutical compositions of the present invention contain a therapeutically effective amount of active ingredients. Wherein, 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 that will be recognized by those skilled in the art of medicine. More specifically, a therapeutically effective amount refers to an amount of active ingredient that effectively prevents, alleviates, or ameliorates the symptoms of a disease or prolongs the survival of the individual being treated. The determination of a therapeutically effective amount is within the capabilities of those skilled in the art, especially in light of the disclosure of the present invention.

In the present invention, suitable routes of administration of the composition or pharmaceutical composition may include, for example, parenteral delivery, including intramuscular, subcutaneous, intravenous, intramedullary injection, and injection at the location of the corresponding target organ (such as spleen, liver, kidney, lung, brain and bone marrow, etc.). The pharmaceutical composition can also be administered in a sustained release or controlled release dosage form (including depot injections, osmotic pumps, etc.) so as to be administered for a long time and/or regularly, pulsed at a predetermined rate. In addition, the route of administration may be local or systemic.

The pharmaceutical composition can be manufactured in a manner that is itself known, eg, by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or tableting processes.

The pharmaceutical composition can be prepared in any conventional manner using one or more physiologically acceptable pharmaceutical carriers, which include excipients and adjuvants that help process the active substance into a pharmaceutically usable preparation. Suitable formulations depend on the selected route of administration. Any well-known technology, pharmaceutical carriers, excipients and diluents can be used appropriately and as understood in the art.

In some embodiments, the pharmaceutical composition is an injection.

In the present invention, the injection can be prepared in conventional forms: liquid solution or suspension, solid form suitable for preparing solution or suspension in liquid before injection, or emulsion. Suitable excipients are, for example, water, saline, dextrose, mannitol, lactose, lecithin, albumin, sodium glutamate, cysteine hydrochloride, etc. In addition, if necessary, the injectable pharmaceutical preparation can also contain a small amount of non-toxic auxiliary substances, 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. In addition, if necessary, absorption enhancement preparations can also be used.

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

Therapeutic and pharmaceutical uses

The present invention further provides the use of the above-mentioned composition or the above-mentioned pharmaceutical composition in preventing or treating Gaucher disease. That is, the present invention further provides a method for preventing or treating Gaucher disease, which comprises: using the above-mentioned composition or the above-mentioned pharmaceutical composition.

The present invention further provides use of the aforementioned composition in preparing a pharmaceutical composition, wherein the pharmaceutical composition is used to prevent or treat Gaucher disease.

It is known in the art that the use of the composition or the pharmaceutical composition as described above in the present invention is the use of its safe and effective amount, and the "safe and effective amount" in the present invention can be further understood as an amount sufficient to prevent or treat Gaucher disease while avoiding serious side effects. Those skilled in the art can confirm the safe and effective amount of the composition or pharmaceutical composition in combination with known methods such as in vitro cell tests and animal experiments. In clinical applications, doctors can also adjust the dosage of the composition or pharmaceutical composition in combination with weight, diet, concurrent drug treatment and other factors that technicians in the medical field will recognize, so that it reaches a safe and effective amount.

In some embodiments, the Gaucher disease comprises at least one of type 1 Gaucher disease, type 2 Gaucher disease, and type 3 Gaucher disease.

In some embodiments, the pharmaceutical composition is an injection.

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

In some embodiments, 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-GCase in the serum and target organs of the subjects;

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

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

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

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

In some embodiments, the target organs include spleen, liver, kidney, lung, brain, and bone marrow.

In some embodiments, the subject is a vertebrate.

In some preferred embodiments, the subject is a mammal.

In some preferred embodiments, the subject is a chicken, mouse, hamster, rabbit, sheep, cow, pig, dog, cat, donkey, monkey, gorilla, ape, or human.

In some more preferred embodiments, the subject is a human.

Example

The embodiments of the present invention will be described in detail below by taking hGBA-mRNA (containing mRNA encoding human glucosphingolipidase β1 (GBA1)) and mGBA-mRNA (containing mRNA encoding mouse glucosphingolipidase β1 (GBA1)) as examples.

It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. For experimental methods in the following examples where specific conditions are not specified, reference should be made to the instructions given in the present invention, or to experimental manuals or conventional conditions in the art, or to other experimental methods known in the art, or to conditions recommended by manufacturers.

In the following specific embodiments, the measured parameters of raw material components may have slight deviations within the range of weighing accuracy unless otherwise specified. For temperature and time parameters, acceptable deviations caused by instrument test accuracy or operation accuracy are allowed.

1. Methods

1.1 mRNA sequence of hGBA-mRNA

The mRNA sequence of hGBA-mRNA contains a cap structure, Chi-β-Globin Δ4 5' untranslated region (UTR), a Kozak sequence, a nucleotide sequence encoding human glucosphingolipidase β1 (GBA1, gene number 2629 in the NCBI genome database), and Chi-β-Globin 3' untranslated region (UTR) and a polyadenylic acid tail (125A in length).

The mRNA sequence of mGBA-mRNA contains a cap structure, Chi-β-Globin Δ4 5' untranslated region (UTR), a Kozak sequence, a nucleotide sequence encoding mouse glucosphingolipidase β1 (GBA1, gene number 14466 in the NCBI genome database) (in mGBA-mRNA), and Chi-β-Globin 3' untranslated region (UTR) and a polyadenylation tail (length 125A).

1.2 Preparation of hGBA-mRNA and mGBA-mRNA

First, the plasmid was cut by BspQ1 enzyme to produce a linearized DNA template, and the DNA sequences of hGBA and mGBA are shown in SEQ ID No. 1 and SEQ ID No. 2, respectively. Then, mRNA was synthesized by T7 RNA polymerase in vitro, and the sequences of hGBA-mRNA and mGBA-mRNA are shown in SEQ ID No. 3 and SEQ ID No. 4, respectively. After that, the synthesized mRNA was capped by smallpox virus capping enzyme, adding a 7-methylguanylate capping structure (Cap 0) to the 5' end of the transcribed mRNA, and using Cap 2'-O methyltransferase to convert the Cap 0 structure to the Cap 1 structure, and completely replacing the uridine in the mRNA with 1-N-methyl pseudouridine triphosphate (1-N-Me-Pseudo-UTP).

mRNA was encapsulated in LNPs composed of proprietary cationic liposomes, cholesterol, DSPC, and PEG2000-DMG. 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). 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.). mRNA was dissolved in 50 mM citrate buffer, pH 6.0, to achieve N/P = 8. The two solutions were mixed by a nanomedicine system (I-Nano) at a total flow rate of 16 mL per minute and a flow rate ratio of 4: 1 v/v (aqueous phase: organic phase). Subsequently, the resulting suspension was dialyzed twice in RNase-free water for one hour each, and then dialyzed in Tris buffer for at least 16 hours, followed by concentration by Amicon Ultra and filtration using a 0.22 µm Millex-GV filter. The encapsulation efficiency ratio and mRNA concentration were measured by Quant-iT™ RiboGreen™ RNA Assay Kit according to the manufacturer's instructions. The detailed information of hGBA-mRNA is listed in Table 2.

Table 2. hGBA-mRNA information

1.3 Serum and tissue collection

After the whole blood had rested for 1 hour, serum samples were collected from the orbit and centrifuged (16,000 x g, 30 minutes). After the mice were sacrificed, liver and spleen tissues were obtained, immediately frozen and stored at -80°C for further use. The tissues were lysed with a tissue disruptor (60 Hz, 4°C for 180 seconds), followed by centrifugation (16,000 x g, 4°C for 30 minutes) and the supernatant was collected. The protein concentration was determined using the Pierce BCA protein assay kit (Thermo-23225) according to the manufacturer's instructions.

1.4 Western blotting

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

1.5 Analysis of β-GCase activity

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

1.6 Preparation of Lyso_GL1

Lyso_GL1 levels were detected by LC-MS. The extraction of serum Lyso_GL1 was based on a modification of the method of Bligh and Dyer. For serum samples, 50 μL of 80% methanol was added to 5 μL of serum sample, and 500 pg of N,N-dimethyl-D-ergosterol (Matreya-1320) was added as an internal standard (PMID: 21868580). The mixture was vortexed for 3 minutes and then centrifuged at 16,000 x g for 10 minutes to collect the supernatant. The supernatant was transferred to an autosampler vial for LC-MS/MS analysis. The calibration curve was prepared using the same method as the sample preparation above, using Lyso-GL1 standards and internal standards. The injection volume was 10 μL.

For liver samples, approximately 0.1 g of wet weight liver sample was homogenized with 2 mL of chloroform-methanol (volume ratio 2:1), and then centrifuged to obtain the supernatant. The supernatant was dried and dissolved with 40 μL of 80% methanol. The injection volume was 10 μL.

1.7 UPLC-ESI-MS/MS quantitative analysis of Lyso_GL1

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 the components was achieved using an Acquity BEH C18 column (2.1×100 mm, 1.7 μm column) at 50°C. Mobile phase A (0.1% formic acid in water) and mobile phase B (0.1% formic acid in acetonitrile) were gradient eluted as follows: 30% B from 0 to 0.5 min, from 30% B to 70% B from 0.5 to 2 min, 70% B from 2 to 4 min, from 70% B to 30% B from 4 to 4.2 min, and 30% B from 4.2 to 6 min, with a flow rate of 0.4 mL/min. Subsequently, electrospray ionization (ESI) detection was performed on the mass spectrometer in positive mode. The ion source settings were as follows: spray voltage was 3.8 kV; capillary temperature was 320 °C; sheath gas and auxiliary gas were set to 35 units and 8 units, respectively; RF lens was 80%; maximum fill time was 50 ms; and automatic gain control (AGC) target was 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 with the following transitions: 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-erythro-sphingosine with a resolution of 70,000. The system and acquired data were processed by Chromeleon software (Thermo, USA).

1.8 Assessment of cytokine mRNA levels by qPCR

Total RNA was extracted using RNAiso plus (Takara-9108Q) according to the manufacturer's instructions, and cDNA was prepared by reverse transcription using PrimeScript RT Reagent Kit with gDNA Eraser (Takara-RR047A). qPCR analysis was performed on the QuantStudio™ 6 Flex Real-Time PCR System (Applied Biosystems) using ChamQUniversal SYBR qPCR Master Mix (Vazyme, Q711-02).

2. Results

2.1 Screening of ionizable cationic liposomes for LNP encapsulation

The delivery efficiency of LNP systems containing different ionizable cationic liposomes for different target genes is different, so it is necessary to screen to find an LNP system suitable for delivering specific target genes. Three ionizable cationic liposomes, liposome #9, liposome #10 and liposome #13, were analyzed, and their structures are shown in Figure 1 A, Figure 1 B, and Figure 1 C, respectively. In three different cell lines (293T, Vero and BHK), three doses of hGBA-mRNA were transfected by LNP systems containing these three cationic liposomes, and then the protein levels expressed in each group were evaluated and compared at 24 h, 48 h, and 72 h (Figure 2, where the blank control is saline). It can be seen that the LNP systems containing cationic liposomes with different structures have different effects on the protein expression level. The protein expression level of hGBA-mRNA in the three time periods, LNP containing ionizable cationic liposomes of liposome #13 and liposome #10 is better than liposome #9.

Through the quantitative conversion of the above data, it can be intuitively seen that the protein expression levels of liposome #10 and liposome #13 in the three cell lines of 293T, Vero and BHK are better than those of liposome #9 (Figure 3). The results of in vitro experiments show that the LNP system of cationic liposomes containing liposome #10 and liposome #13 is more suitable for delivering hGBA-mRNA and achieving more efficient protein expression in cells. Comparing the results of liposome #10 and liposome #13, except that in the 293T cell line, the hGBA expression when delivered by LNP containing liposome #13 is slightly higher than that when delivered by LNP containing liposome #10, in the Vero and BHK cell lines, the hGBA expression level is higher when delivered by LNP containing liposome #10, which shows that liposome #10 is relatively more suitable as a cationic liposome for the delivery of hGBA-mRNA.

2.2 In vivo studies

The completed nonclinical pharmacology/pharmacokinetic studies have shown (Table 3):

1) In the GBA D427V mouse model of GD, intravenous injection of mouse hGBA-mRNA (or mGBA-mRNA) restored the deficient β-GCase in serum, liver, and spleen, resulting in reduced levels of the metabolic substrate Lyso-GL1 in these target organs.

2) In the GBA D427V mouse model, intravenous injection of hGBA-mRNA can achieve the following effects: 1) Increased β-GCase expression and activity in target organs in a dose-dependent manner; 2) Achieved longer-term β-GCase expression compared to the current Cerezyme standard therapy; 3) Consistently increased β-GCase activity in target organs after repeated administration, while reducing Lyso-GL1 levels; 4) Good tolerance after repeated administration, without significant induction of anti-PEG antibodies and innate immune-related cytokines.

3) hGBA-mRNA reached its peak in the spleen, kidney, lung, brain, liver and bone marrow on the first day after intravenous administration. In all tissues, hGBA-mRNA levels decreased significantly after 7 days.

Table 3. Summary of nonclinical studies of mGBA-mRNA and hGBA-mRNA

2.2.1 Tissue distribution of hGBA-mRNA in mice

Tissue distribution of hGBA-mRNA was evaluated. GBA D427V mice were given a single intravenous injection of saline or 0.5 mg/kg of hGBA-mRNA. RNA was isolated from brain, liver, spleen, kidney, lung, and bone marrow and quantified by qPCR (quantitative polymerase chain reaction) using primers specific for the human β-GCase gene. Relative increase in human β-GCase mRNA levels was observed relative to animals treated with saline.

On the first day after treatment, hGBA-mRNA levels peaked in all tissues tested, including spleen, kidney, lung, brain, liver, and bone marrow. Among them, the spleen had the highest fold increase, followed by kidney, lung, brain, liver, and bone marrow, in descending order. After 7 days of treatment, hGBA-mRNA levels in all tissues decreased significantly. These results are consistent with the results of nonclinical pharmacology studies, which showed that hGBA-mRNA treatment was able to maintain increased β-GCase activity in the target organs for at least 3 days and up to 7 days.

Gaucher disease is known to result in the accumulation of cerebrosides in the liver, spleen, kidney, lung, brain, and bone marrow. The prolonged presence of human β-GCase mRNA suggests that hGBA-mRNA has the potential to compensate for β-GCase activity in these organs, thereby providing patients with a more comprehensive benefit than protein-based ERT.

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

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

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

To evaluate the in vivo effects of mGBA-mRNA treatment, GBA D427V mice were intravenously injected with 0.5 mg/kg mGBA-mRNA once every two weeks (Q2W) for a total of 3 injections on days 0, 14, and 28.

β-GCase protein levels were assessed in serum, liver, and spleen by immunoblotting, using saline-treated mice as a negative control group. In serum, β-GCase levels peaked at 12 hours after mGBA-mRNA injection and remained detectable for the next 3 days, while no β-GCase protein was detected in the saline group (see Figure 4). Similarly, in liver and spleen, β-GCase expression peaked at 12 hours after mGBA-mRNA injection and remained elevated for at least the next 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 defective β-GCase function. mGBA-mRNA therapy resulted in a 14-fold increase in serum β-GCase enzyme activity (1.4 x 10 ⁶ vs. 1 x10 ⁵ ; pmol/min/μg/μL) at 12 hours after injection relative to pretreatment levels, which returned to baseline levels by day 7 (see Figure 4).

The liver and spleen are the main pathogenic organs of GD. After receiving mGBA-mRNA treatment, the β-GCase enzyme activity in the liver reached a peak 1 day after treatment (9-fold increase relative to the pre-dose level) and in the spleen (7-fold increase relative to the pre-dose level), and remained at a high level for at least 3 days, and then returned to the baseline level on the 14th day (see Figures 5 and 6).

Gaucher disease is a lifelong disease that requires repeated drug treatment. To simulate a possible clinical treatment regimen, GBA D427V mutant mice were given multiple doses of mGBA-mRNA (0.5 mg/kg) by intravenous injection. Consistent with observations after a single dose, mGBA-mRNA-treated GBA D427V mice showed a significant increase in β-GCase enzyme activity in serum between 3 hours and 3 days after each treatment compared to baseline levels, reaching a peak 12 hours after each treatment (Figure 7). The levels of enhanced β-GCase enzyme activity did not differ significantly after each injection.

Lyso-GL1, a β-GCase substrate, is a key biomarker for Gaucher disease, and reduced levels of Lyso-GL1 indicate a positive response to treatment. Therefore, measuring the concentration of Lyso-GL1 can verify the ultimate efficacy of mGBA-mRNA treatment. Lyso-GL1 concentrations were elevated in the serum and liver of untreated GBA D427V mice, and within 3 hours after mGBA-mRNA treatment, Lyso-GL1 concentrations began to decline and then reached a nadir within 3 days. On day 3, Lyso-GL1 levels in the serum of mice treated with mGBA-mRNA decreased to 15% and in the liver to less than 10% compared with saline-injected mice (Figure 8). In addition, in animals treated with mGBA-mRNA, after multiple doses, Lyso-GL1 levels in the liver remained reduced to less than 30% of Lyso-GL1 levels in the saline-injected control group.

Polyethylene glycol (PEG) is a component of liposome nanoparticles (LNPs), and previous reports have shown that it can induce antibody responses after repeated administration. The production of anti-drug antibodies (ADA) may affect the pharmacokinetics and pharmacodynamics of drugs, thereby reducing their efficacy. Therefore, the levels of anti-polyethylene glycol (anti-PEG) and anti-β-GCase (anti-β-GCase) IgG antibodies in the serum of GBA D427V mice treated with mGBA-mRNA were measured by ELISA. After multiple intravenous injections of mGBA-mRNA, no anti-PEG antibodies or anti-β-GCase IgG antibodies were detected in the sera of these animals beyond individual differences (Figure 9).

The tolerability of intravenous mGBA-mRNA therapy can be assessed by stimulating the production of innate immune cytokines. Liver and spleen samples were collected at different time points from GBA D427V mice treated every two weeks, 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-inducible gene-I (RIG-I) were measured by quantitative RT-PCR. As shown in Figure 10, the experimental groups were normalized to the data of saline-treated control mice as the baseline. The measured cytokine levels did not show a significant increase in either the liver or spleen at any time point during mGBA-mRNA treatment, indicating that intravenous mGBA-mRNA did not induce activation of innate immunity.

In conclusion, mGBA-mRNA at a dose of 0.5 mg/kg showed significant therapeutic effects in the GBA D427V GD mouse model, such as the expression of β-GCase in the liver and spleen, and the sustained increase in β-GCase activity for 72 hours, while the level of the metabolic substrate Lyso-GL1 in serum and liver continued to decrease. In addition, repeated administration of mGBA-mRNA did not induce anti-PEG or anti-β-GCase IgG antibodies in vivo, and did not stimulate intrinsic cytokines, showing good tolerability characteristics.

2.2.3 Intravenous administration of hGBA-mRNA versus the therapeutic effect of Cerezyme in GBA D427V mutant mice

In view of the results obtained with mGBA-mRNA in GBA D427V mice, this example further investigated LNP-encapsulated mRNA products encoding human GBA, namely hGBA-mRNA, to further evaluate its efficacy and compare it with the current ERT standard of care (SoC) Cerezyme.

In this study, the present example evaluated the therapeutic effect of hGBA-mRNA by single injection of GBA D427V mutant mice at a dose of 0.02 mg/kg, 0.1 mg/kg, or 0.5 mg/kg (body weight). In addition, a group of GBA D427V mice were injected once every two weeks (Q2W) for a total of three intravenous injections of hGBA-mRNA at a dose of 0.5 mg/kg. As the primary comparison object, this example used 60 U/kg of Cerezyme as a control, which was reported in previous mouse studies. The study design is detailed in Table 5.

Table 5. Study design for comparison of the therapeutic effect of hGBA-mRNA intravenous injection with Cerezyme

The expression of β-GCase in the liver (Figure 11) and spleen (Figure 12) was evaluated by Western blotting. The results were consistent with the mGBA-mRNA in the D427V model. The expression 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 value of β-GCase protein in the liver and spleen at a certain time point is a result of a dynamic balance. Under the high, medium and low dose conditions tested in this study, the expression of β-GCase in the liver and spleen reached a peak on the first day (except for the low-dose group in the spleen, which reached a peak on the third day), and there was a significant dose effect. After administration of high, medium and low doses of hGBA-mRNA for 7 days, 3 days and 1 day respectively, the level of β-GCase in the liver was still higher than the baseline (the basal amount of β-GCase in the liver and spleen on day 0 before administration), while 3 days after administration of all 3 dose levels of hGBA-mRNA, the expression level of β-GCase in the spleen showed that the low and medium dose groups were higher than the high dose group. Consistent with the data previously reported in the literature, the β-GCase level peaked within 20-40 minutes after intravenous injection of Cerezyme and then rapidly declined within 12 hours, which is much shorter than the duration of the increase in β-GCase level after administration of hGBA-mRNA. Therefore, in vivo, the β-GCase level after intravenous injection of hGBA-mRNA lasts significantly longer than Cerezyme.

In addition, the present example also measured the β-GCase enzyme activity in serum, liver and spleen to evaluate the effect of hGBA-mRNA treatment in repairing the defective β-GCase function. After a single intravenous injection of hGBA-mRNA or Cerezyme (i.e., one injection on day 0), the β-GCase enzyme activity increased, but the kinetic properties were completely different. The increase in serum β-GCase activity caused by hGBA-mRNA therapy reached a peak within 24 hours after intravenous injection and then returned to baseline levels 3 days after treatment (Figure 13); this effect was dose-dependent. Compared with the pre-treatment levels (2.8x10 ⁵ vs. 5.5x10 ⁴ pmol/min/μg/μL, 1.6 x 10 ⁵ vs 5.5 x 10 ⁴ pmol/min/μg/μL, 1.1 x 10 ⁵ vs 5.5 x 10 ⁴ pmol/min/μg/μL), a 0.5 mg/kg dose of hGBA-mRNA increased β-GCase activity by 5-fold, a 0.1 mg/kg dose group increased β-GCase activity by 2.9-fold, and a 0.02 mg/kg dose group increased β-GCase activity by 2.0-fold. In contrast, after Cerezyme treatment, β-GCase activity in serum increased rapidly, reaching a peak within 5 minutes, approximately 10 times the pre-treatment level (5.7 x10 ⁵ vs. 5.5 x 10 ⁴ pmol/min/μg/μL), and then rapidly decreased to baseline levels within 20 minutes, consistent with previously reported data. Similar to serum, after treatment with 0.02, 0.1, or 0.5 mg/kg of hGBA-mRNA, β-GCase enzyme activity in the liver reached a maximum after 1 day (3.7, 7.3, or 9.0-fold increase relative to pre-treatment levels, respectively), as did that in the spleen (2.7, 5.4, or 6.7-fold increase relative to pre-treatment levels, respectively), remained elevated for at least 3 days, and then returned to baseline levels on the 7th day (Figures 14 and 15). Similar to serum, after Cerezyme treatment, β-GCase enzyme activity in the liver peaked briefly after 20 minutes (5.2-fold increase relative to pre-treatment levels), and β-GCase enzyme activity in the spleen also peaked briefly after 20 minutes (4.9-fold increase relative to pre-treatment levels), and returned to baseline levels in both organs within 12 hours. These data confirm that hGBA-mRNA treatment results in a longer functional duration of β-GCase enzyme activity in the serum, liver, and spleen of treated animals compared to Cerezyme. Compared to Cerezyme's 20-40 minutes, hGBA-mRNA's β-GCase levels lasted for at least 3 days and increased in a dose-dependent manner (Figures 13 to 15).

Multiple doses of 0.5 mg/kg hGBA-mRNA and 60 U/kg Cerezyme were administered intravenously to GBA D427V mutant mice, respectively, once every two weeks for a total of three injections on days 0, 14, and 28. Consistent with single-dose administration (one injection on day 0), hGBA-mRNA-treated GBA D427V mice showed a significant increase in serum β-GCase enzyme activity from 6 hours to 3 days after each drug administration, reaching a peak at 12 hours after each drug administration (see Figure 16). After each hGBA-mRNA injection, the elevated β-GCase activity did not differ significantly between injection intervals. Compared with Cerezyme, mice injected with hGBA-mRNA continued to observe increased β-GCase activity in the spleen and liver for a longer period of time (7 days vs. 12 hours; see Figures 17 and 18).

As mentioned earlier, Lyso-GL1 is the main biomarker for evaluating the therapeutic effect of GD. After intravenous treatment with hGBA-mRNA, a decrease in Lyso-GL1 in serum and liver was observed. A single dose of hGBA-mRNA showed that the reduction in the level of Lyso-GL1 in the liver appeared to be dose-dependent (69%, 60%, and 46% reductions for high, medium, and low doses, respectively), which was similar to or greater than the effect of 60 U/kg of Cerezyme in reducing Lyso-GL1 levels (56% reduction). The levels of glucose sphingosine in the serum and liver of mice after the first, second, and third injections of hGBA-mRNA treatment are shown in Figures 19, 20, and 21, respectively. From the results, it can be seen that after repeated administration of 0.5 mg/kg of hGBA-mRNA, the level of Lyso-GL1 in serum was reduced to about 60% to 70% of the level of the saline-injected control group after the first and second injections, and the relative level of Lyso-GL1 after the third injection did not change as much as the first two. In contrast, with 60 U/kg of Cerezyme, serum lyso-GL1 levels decreased to 60% to 80% of saline control lyso-GL1 levels after each injection. However, hGBA-mRNA treatment at a dose of 0.5 mg/kg showed a trend toward a greater decrease in liver lyso-GL1 levels after the first and second injections compared with Cerezyme. After the third injection, the hGBA-mRNA group had a decrease in lyso-GL1 levels of approximately 50%, while the Cerezyme group had a decrease in lyso-GL1 levels of approximately 70% (see Figures 19 to 21).

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

Although anti-human β-GCase IgG was not detected in mice injected with Cerezyme, the level of anti-human β-GCase IgG in serum increased by about 100-fold after the second hGBA-mRNA injection and remained until day 42 at the end of the study. These results suggest that human β-GCase protein may be immunogenic in mice. This may be the reason for the decrease in Lyso-GL1 levels when mice were injected with the third hGBA-mRNA, but this did not occur when injected with mGBA-mRNA.

It is critical that LNP-mRNA therapeutics avoid detection by the intrinsic innate immune system. The expression of TNF-α, IL6, IFN-α, IFN-β, and IFN-γ in the liver and spleen of mice treated with hGBA-mRNA was measured by quantitative polymerase chain reaction (Q-PCR), and the fold changes were calculated relative to the levels of the relevant cytokines in saline-treated control mice (see Figures 23 and 24). The results showed that there was no significant increase in the expression of these cytokines during hGBA-mRNA treatment, either in the liver or spleen. Similar results were observed in the Cerezyme treatment group.

In summary, intravenous injection of hGBA-mRNA achieved the expression of β-GCase in the serum, liver, and spleen of GBA D427V mice, and the expression was dose-dependent. Pharmacokinetic analysis of GBA D427V mutant mice showed that the duration of β-GCase expression in serum, liver, and spleen by hGBA-mRNA was significantly longer than that of the current standard treatment Cerezyme. At the same time, hGBA-mRNA treatment reduced the β-GCase metabolic substrate Lyso-GL1 in serum and liver, and the therapeutic effect was maintained after repeated administration. In addition, no obvious anti-PEG antibodies or intrinsic cytokines were found after multiple administrations of hGBA-mRNA in vivo.

2.2.4 Pharmacokinetics

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

Table 6. Tissue distribution study design after single intravenous injection of hGBA-mRNA in GBA D427V mice

Note: a. For blood, tumor and other tissue matrices

As shown in Figure 25, in all tissues, the peak of hGBA-mRNA levels occurred on the first day after treatment. The highest increase in hGBA-mRNA distribution was 6923-fold in the spleen. This was followed by kidney, lung, brain, liver, and bone marrow, in descending order (1833 ± 405, 509 ± 129, 92 ± 33, 51 ± 11, and 19 ± 8, respectively). On day 7, the mRNA level of hGBA-mRNA was significantly reduced in all tissues (the fold increase was 177 ± 69 in spleen, 110 ± 31 in kidney, 43 ± 11 in lung, 6.4 ± 2.8 in brain, 0.9 ± 0.3 in liver, and 7.7 ± 6.4 in bone marrow). These results are consistent with the premise that hGBA-mRNA treatment can maintain increased β-GCase activity in the target organ for at least 3 days and up to 7 days. It is well known that Gaucher disease causes accumulation of cerebrosides in the liver, spleen, kidney, lung, 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, thereby providing patients with more comprehensive benefits than protein gene replacement therapy.

The above-mentioned embodiments only express several implementation methods of the present invention, and the description is relatively specific and detailed, but it cannot be understood as limiting the scope of the invention patent. It should be pointed out that for ordinary technicians in this field, several modifications and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention.

Claims (24)

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; Wherein, 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: Formula I Liposome #10; Formula II Liposome #13; or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof. 3 . The composition according to claim 1 , wherein the nucleotide sequence encoding GBA1 is derived from human or mouse. 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 as shown in the NCBI genome database gene number 2629; ii) the nucleotide sequence as shown in the NCBI genome database gene number 14466; iii) a nucleotide sequence having 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 part 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: Cap 1 structure, a 5' untranslated region, a nucleotide sequence encoding GBA1, a 3' untranslated region and a poly(A) tail. 7. The composition according to claim 5, characterized in that the mRNA contains, from the 5' end to the 3' end, the following: Cap1 structure, a 5' untranslated region, a nucleotide sequence encoding GBA1, a 3' untranslated region and a polyadenylic acid 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) A nucleotide sequence having 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 contains 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) A nucleotide sequence having 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 further contain one or more selected from 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 distearoylphosphatidylcholine, dioleoylphosphatidylcholine, dipalmitoylphosphatidylcholine, dioleoylphosphatidylglycerol, dipalmitoylphosphatidylglycerol, dioleoylphosphatidylethanolamine, palmitoyloleoylphosphatidylcholine, palmitoyloleoylphosphatidylethanolamine and dioleoylphosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate, dipalmitoylphosphatidylethanolamine, dimyristoylphosphoethanolamine, distearoylphosphatidylethanolamine, 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoylphosphatidylethanolamine and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine. 12. The composition according to claim 10, characterized in that the PEG lipid is 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 of claim 10, wherein the steroid 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. The method for preparing the composition according to any one of claims 1 to 14, characterized in that it comprises: dissolving the lipid contained in the lipid nanoparticles in ethanol to obtain a lipid ethanol solution; mixing the lipid ethanol solution with an aqueous solution containing the mRNA; Then the ethanol is removed and the composition is obtained by separation or purification. 16. A pharmaceutical composition, characterized in that it contains the composition according to 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. 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 to prevent or treat Gaucher disease. 19. The use according to claim 18, characterized in that the Gaucher disease comprises at least one of type 1 Gaucher disease, type 2 Gaucher disease, and type 3 Gaucher disease. 20. The use according to claim 18, characterized in that the pharmaceutical composition is an intravenous injection. 21. The use according to claim 18, characterized in that the composition or the pharmaceutical composition is used to achieve at least one of the following purposes: 1) Increase the expression and activity of β-glucocerebrosidase in the serum and target organs of the subjects; 2) Reduce the level of glucosphingosine in the serum and target organs of the subjects. 22. The use according to claim 21, characterized in that the target organ comprises at least one of spleen, liver, kidney, lung, brain and bone marrow. 23. The use according to claim 18, characterized in that the subject is a mammal. 24. The use according to claim 18, characterized in that the subject is chicken, mouse, hamster, rabbit, sheep, cow, pig, dog, cat, donkey, monkey, gorilla, ape or human.