mRNA comprising polynucleotide encoding ATP7b protein, and use thereof

Abstract

Provided are an mRNA comprising a polynucleotide encoding an ATP7B protein, and a use thereof. An mRNA. The mRNA is a non-natural mRNA, and comprises a polynucleotide encoding an ATP7B protein.

Description

mRNAs containing polynucleotides encoding ATP7B protein and their applications Technical Field

[0001] This invention belongs to the field of biotechnology and relates to an mRNA containing a polynucleotide encoding the ATP7B protein and its applications. Background Technology

[0002] Wilson's disease (WD), also known as Wilson's disease, is an autosomal recessive inherited disorder of copper metabolism that commonly affects adolescents. It is caused by a mutation in the gene encoding copper-transporting ATPase 2 (ATP7B) on chromosome 13, leading to the loss or impairment of ATP7B protein function. This results in impaired synthesis of ceruloplasmin in hepatocytes and impaired copper excretion in bile. Excess copper accumulates in tissues and organs such as the liver, brain, and cornea, causing the disease. Clinically, it is characterized by cirrhosis, neurological / psychiatric symptoms, and KF rings in the cornea. Summary of the Invention

[0003] This disclosure provides an mRNA that is a non-natural mRNA containing a polynucleotide encoding the ATP7B protein.

[0004] In some embodiments, the ATP7B protein is the full-length human ATP7B protein, and the mRNA further comprises a 5'-UTR, a 3'-UTR, and a poly(A) tail.

[0005] In some embodiments, the polynucleotide encoding the ATP7B protein is one of the following:

[0006] (1) A polynucleotide with a nucleotide sequence as shown in any one of SEQ ID NO: 8-10; and

[0007] (2) A polynucleotide whose nucleotide sequence has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and less than 100% identity with any of the nucleotide sequences shown in SEQ ID NO: 8–10 and encodes the full-length human ATP7B protein;

[0008] In some embodiments, the ATP7B protein is a truncated ATP7B protein, and the truncated ATP7B protein is one of the following:

[0009] (1) The truncated ATP7B protein of MBD1 to MBD4 is missing;

[0010] (2) A truncated ATP7B protein lacking MBD3;

[0011] (3) The truncated ATP7B protein lacking MBD1 and MBD3;

[0012] (4) The truncated ATP7B protein of MBD1, MBD3 and MBD4 is missing;

[0013] (5) truncated ATP7B protein lacking MBD1 and MBD4;

[0014] (6) The truncated ATP7B protein of MBD1-MBD2 is missing;

[0015] (7) truncated ATP7B proteins lacking MBD1, MBD2 and MBD4;

[0016] (8) The truncated ATP7B protein of MBD1-MBD3 is missing;

[0017] (9) truncated ATP7B protein lacking MBD2 and MBD3;

[0018] (10) truncated ATP7B protein lacking MBD2 and MBD4;

[0019] (11) truncated ATP7B protein lacking MBD3 and MBD4;

[0020] (12) The truncated ATP7B protein lacking MBD2–MBD4; and

[0021] (13) The truncated ATP7B protein of MBD1 to MBD5 is missing.

[0022] In some embodiments, the truncated ATP7B protein is one of the following:

[0023] (1) The truncated ATP7B protein from position 57 to position 485 is missing;

[0024] (2) The truncated ATP7B protein from position 57 to position 486 is missing;

[0025] (3) The truncated ATP7B protein at positions 257 to 355 is missing;

[0026] (4) The truncated ATP7B protein at positions 57-140 and 237-337 is missing;

[0027] (5) The truncated ATP7B protein at positions 57-140 and 237-485 is missing;

[0028] (6) The truncated ATP7B protein at positions 57-140 and 359-485 is missing;

[0029] (7) The truncated ATP7B protein from position 57 to position 236 is missing;

[0030] (8) The truncated ATP7B protein at positions 57-236 and 359-485 is missing;

[0031] (9) The truncated ATP7B protein from position 57 to position 337 is missing;

[0032] (10) The truncated ATP7B protein from position 141 to position 337 is missing;

[0033] (11) The truncated ATP7B protein is missing from positions 141 to 236 and 359 to 485;

[0034] (12) The truncated ATP7B protein from position 237 to position 485 is missing;

[0035] (13) The truncated ATP7B protein from position 141 to position 485 is missing;

[0036] (14) The truncated ATP7B protein from position 57 to position 490 is missing; and

[0037] (15) The truncated ATP7B protein at positions 57 to 495 is missing.

[0038] In some embodiments, the truncated ATP7B protein is one of the following:

[0039] (1) A truncated ATP7B protein with an amino acid sequence as shown in any one of SEQ ID NO: 11–25; and

[0040] (2) A protein having an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% and less than 100% identical to any of the amino acid sequences shown in SEQ ID NO: 11 to 25 and having the function of ATP7B protein.

[0041] In some embodiments, the polynucleotide encoding the truncated ATP7B protein is one of the following:

[0042] (1) A polynucleotide with a nucleotide sequence as shown in SEQ ID NO: 26;

[0043] (2) A polynucleotide sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and less than 100% identity with the nucleotide sequence shown in SEQ ID NO: 26 and encoding an amino acid sequence as shown in SEQ ID NO: 11 of the truncated ATP7B protein;

[0044] (3) A polynucleotide with a nucleotide sequence as shown in any one of SEQ ID NO: 27-30;

[0045] (4) The nucleotide sequence has at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and less than 100% identity with the nucleotide sequence shown in any one of SEQ ID NO: 27–30 and encodes a polynucleotide encoding an amino acid sequence of the truncated ATP7B protein as shown in SEQ ID NO: 12;

[0046] (5) Polynucleotides with nucleotide sequences as shown in SEQ ID NO: 31;

[0047] (6) A polynucleotide sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and less than 100% identity with the nucleotide sequence shown in SEQ ID NO: 31 and encoding an amino acid sequence of a truncated ATP7B protein as shown in SEQ ID NO: 13;

[0048] (7) Polynucleotides with nucleotide sequences as shown in SEQ ID NO: 32;

[0049] (8) A polynucleotide sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and less than 100% identity with the nucleotide sequence shown in SEQ ID NO: 32 and encoding an amino acid sequence as shown in SEQ ID NO: 14;

[0050] (9) Polynucleotides with nucleotide sequences as shown in SEQ ID NO: 33 or 34;

[0051] (10) A polynucleotide sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and less than 100% identity with the nucleotide sequence shown in SEQ ID NO: 33 or 34 and encoding an amino acid sequence as shown in SEQ ID NO: 15, truncated ATP7B protein;

[0052] (11) Polynucleotides with nucleotide sequences as shown in SEQ ID NO: 35;

[0053] (12) A polynucleotide sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and less than 100% identity with the nucleotide sequence shown in SEQ ID NO: 35 and encoding an amino acid sequence of a truncated ATP7B protein as shown in SEQ ID NO: 16;

[0054] (13) Polynucleotides with nucleotide sequences as shown in SEQ ID NO: 36;

[0055] (14) A polynucleotide sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and less than 100% identity with the nucleotide sequence shown in SEQ ID NO: 36 and encoding an amino acid sequence of a truncated ATP7B protein as shown in SEQ ID NO: 17;

[0056] (15) Polynucleotides with nucleotide sequences as shown in SEQ ID NO: 37;

[0057] (16) A polynucleotide sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and less than 100% identity with the nucleotide sequence shown in SEQ ID NO: 37 and encoding an amino acid sequence of a truncated ATP7B protein as shown in SEQ ID NO: 18;

[0058] (17) Polynucleotides with nucleotide sequences as shown in SEQ ID NO: 38;

[0059] (18) A polynucleotide sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and less than 100% identity with the nucleotide sequence shown in SEQ ID NO: 38 and encoding an amino acid sequence of a truncated ATP7B protein as shown in SEQ ID NO: 19;

[0060] (19) Polynucleotides with nucleotide sequences as shown in SEQ ID NO: 39;

[0061] (20) A polynucleotide sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and less than 100% identity with the nucleotide sequence shown in SEQ ID NO: 39 and encoding an amino acid sequence of a truncated ATP7B protein as shown in SEQ ID NO: 20;

[0062] (21) Polynucleotides with nucleotide sequences as shown in SEQ ID NO: 40;

[0063] (22) A polynucleotide sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and less than 100% identity with the nucleotide sequence shown in SEQ ID NO: 40 and encoding an amino acid sequence of a truncated ATP7B protein as shown in SEQ ID NO: 21;

[0064] (23) Polynucleotides with nucleotide sequences as shown in SEQ ID NO: 41;

[0065] (24) A polynucleotide sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and less than 100% identity with the nucleotide sequence shown in SEQ ID NO: 41 and encoding an amino acid sequence of a truncated ATP7B protein as shown in SEQ ID NO: 22;

[0066] (25) Polynucleotides with nucleotide sequences as shown in SEQ ID NO: 42;

[0067] (26) A polynucleotide sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and less than 100% identity with the nucleotide sequence shown in SEQ ID NO: 42 and encoding an amino acid sequence of a truncated ATP7B protein as shown in SEQ ID NO: 23;

[0068] (27) Polynucleotides with nucleotide sequences as shown in SEQ ID NO: 43;

[0069] (28) A polynucleotide sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and less than 100% identity with the nucleotide sequence shown in SEQ ID NO: 43 and encoding an amino acid sequence of a truncated ATP7B protein as shown in SEQ ID NO: 24;

[0070] (29) Polynucleotides with nucleotide sequences as shown in SEQ ID NO: 44; and

[0071] (30) A polynucleotide sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and less than 100% identity with the nucleotide sequence shown in SEQ ID NO: 44 and encoding an amino acid sequence as shown in SEQ ID NO: 25, truncated ATP7B protein.

[0072] In some embodiments, the truncated ATP7B protein is a truncated human ATP7B protein.

[0073] In some embodiments, the mRNA further comprises at least one of a 5'-UTR, a 3'-UTR, and a poly(A) tail.

[0074] In some embodiments, the nucleotide sequence of the 5'-UTR is as shown in SEQ ID NO: 2 or 3.

[0075] In some embodiments, the nucleotide sequence of the 3'-UTR is as shown in SEQ ID NO: 4 or 5.

[0076] In some embodiments, the nucleotide sequence of the poly(A) tail is as shown in SEQ ID NO: 6 or 7.

[0077] In some implementations, the mRNA also includes a microRNA binding site.

[0078] In some embodiments, the microRNA binding site is located in the 5'-UTR;

[0079] In some embodiments, the microRNA binding site is located in the 3'-UTR;

[0080] In some implementations, the microRNA binding site is located after the 3'-UTR and before the poly(A) tail.

[0081] In some implementations, the mRNA contains a modified nucleoside.

[0082] In some embodiments, the modified nucleoside includes at least one of modified uridine, modified cytidine, modified adenosine, and modified guanosine.

[0083] In some embodiments, the mRNA can increase the expression level of ATP7B protein in the test subject by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, or 500% compared to before administration.

[0084] In some embodiments, the mRNA can increase the activity of the ATP7B protein in the test subject by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, or 500% compared to before administration.

[0085] In some implementations, the mRNA reduces copper levels in the liver of test subjects by at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% compared to before administration.

[0086] In some embodiments, the mRNA can increase the level of ceruloplasmin in the serum of the test subject by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, or 500% compared with before administration.

[0087] This disclosure also provides a DNA that can be transcribed into the above-described mRNA.

[0088] This disclosure also provides a genetic engineering vector comprising the aforementioned DNA.

[0089] This disclosure also provides a host cell comprising the DNA described above or the genetic engineering vector described above.

[0090] This disclosure also provides a truncated ATP7B protein encoded by the aforementioned mRNA.

[0091] This disclosure also provides a pharmaceutical composition comprising the above-described mRNA, the above-described DNA, the above-described genetic engineering vector, or the above-described truncated ATP7B protein.

[0092] In some implementations, the mRNA or DNA is formulated in a delivery vector.

[0093] In some embodiments, the delivery carrier is a lipid nanoparticle.

[0094] This disclosure also provides the use of the above-described mRNA, DNA, genetic engineering vector, host cell, truncated ATP7B protein, or pharmaceutical composition in the preparation of a medicament for treating and / or preventing Wilson's disease.

[0095] In some implementations, the drug is a nucleic acid drug. Attached Figure Description

[0096] Figures 1A to 1C show the protein expression of different ATP7B expression plasmids in HuH-7, HepG2, and HEK293T cells. The numbers in Figures 1A to 1C refer to the expression plasmids corresponding to the same mRNA numbers in Table 1. For example, "1695" in Figure 1A refers to the expression plasmid corresponding to mRNA number 1695 in Table 1. The same applies to Figures 2A to 2C.

[0097] Figures 2A-2C show the copper efflux function of ATP7B protein expressed by different ATP7B expression plasmids in HuH-7, HepG2, and HEK293T cells.

[0098] Figures 3A and 3B show the protein expression of ATP7B mRNA-LNP in wild-type mice. The injection dose in Figure 3A is 2 mg / kg, and the injection dose in Figure 3B is 1 mg / kg. "1917" in Figures 3A and 3B refers to 1917 mRNA-LNP, and so on.

[0099] Figures 4A to 4C show the confirmation of biomarkers in Atp7b KO mice. Figure 4A shows the expression of ATP7B, Figure 4B shows the activity of ceruloplasmin, and Figure 4C shows the results of liver copper content. In Figures 4A to 4C, "Atp7b- / -" refers to Atp7b KO mice, and "WT" refers to WT mice.

[0100] Figures 5A to 5D show the therapeutic effects of a single dose in Atp7b KO mice. Figure 5A shows the serum ceruloplasmin activity of Atp7b KO mice after administration, Figure 5B shows the serum total copper content of Atp7b KO mice after administration, Figure 5C shows the liver copper content of Atp7b KO mice after administration, and Figure 5D shows the protein expression of 778mRNA-LNP in Atp7b KO mice. In Figures 5A to 5D, "PBS WT" refers to WT mice treated with PBS, "PBS Atp7b- / -" refers to Atp7b KO mice treated with PBS, "778Atp7b- / -" refers to Atp7b KO mice treated with 778mRNA-LNP, and "1912Atp7b- / -" refers to Atp7b KO mice treated with 1912mRNA-LNP, and so on.

[0101] Figures 6A to 6D show the therapeutic effects of multiple administrations (1918 mRNA-LNP) to Atp7b KO mice. Figure 6A shows the serum ceruloplasmin activity of Atp7b KO mice after multiple administrations, Figure 6B shows the liver copper content of Atp7b KO mice after multiple administrations, Figure 6C shows the urinary copper content of Atp7b KO mice after multiple administrations, and Figure 6D shows the fecal copper content of Atp7b KO mice after multiple administrations.

[0102] Invention Details

[0103] I. Definition

[0104] All patents, patent applications, scientific publications, manufacturers' specifications and guidelines, etc., cited herein, are incorporated herein in their entirety, whether mentioned above or below. Nothing herein should be construed as an admission that this disclosure is not entitled to precede such disclosure.

[0105] Unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the terms related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, and microbiology used herein are all widely used terms in their respective fields (see, for example, *Molecular Cloning: A Laboratory Manual, 2nd Edition*, J. Sambrook et al. eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989). To better understand this invention, definitions and explanations of related terms are provided below.

[0106] As used herein, the expressions “comprising,” “including,” “containing,” and “having” are open-ended, meaning they include the listed elements, steps, or components but do not exclude other unlisted elements, steps, or components. The expression “composed of” excludes any unspecified elements, steps, or components. The expression “substantially composed of” means that the scope is limited to the specified elements, steps, or components, plus optional elements, steps, or components that do not significantly affect the essential and novel nature of the claimed subject matter. It should be understood that the expressions “substantially composed of” and “composed of” are encompassed within the meaning of the expression “including.”

[0107] As used herein, unless the context otherwise indicates, the singular forms of “a,” “an,” and “the,” and similar references used in the context of describing the invention (particularly in the context of the claims) should be interpreted to cover both the singular and plural. The terms “one or more” or “at least one” cover 1, 2, 3, 4, 5, 6, 7, 8, 9, or more.

[0108] The numerical ranges described herein should be understood to encompass any and all subranges contained therein. For example, the range “1 to 10” should be understood to include not only the explicitly stated values ​​of 1 and 10, but also any single value within the range of 1 to 10 (e.g., 2, 3, 4, 5, 6, 7, 8, and 9) and subranges (e.g., 1 to 2, 1.5 to 2.5, 1 to 3, 1.5 to 3.5, 2.5 to 4, 3 to 4.5, etc.). This principle also applies to ranges that use only one numerical value as their minimum or maximum value.

[0109] As used herein, the terms “and / or,” “any combination thereof,” and their grammatical equivalents are used interchangeably. These terms can express any combination specifically. For example, the phrases “A, B, and / or C” or “A, B, C, or any combination thereof” can refer to “A alone; B alone; C alone; A and B; B and C; A and C; and A, B, and C.”

[0110] Unless otherwise stated, all methods described herein may be performed in any suitable order.

[0111] As used herein, the term “naturally occurring” or “naturally existing” refers to the fact that a substance is visible in nature. For example, peptides, amino acids, proteins, or nucleic acids that are present in organisms (including viruses) and can be isolated from natural sources and have not been artificially modified in experiments are naturally occurring.

[0112] As used herein, the term "non-natural" is intended to mean that the mRNA has not been found in nature. For example, a non-naturally occurring mRNA has at least one genetic alteration or chemical modification that is not normally detected compared to the wild type. Such genetic alterations include, for example, nucleotide addition, nucleotide deletion, or nucleotide substitution. Chemical modifications include, for example, one or more functional nucleotide analogs as described herein.

[0113] As used herein, the term "wild-type" means that the sequence is naturally occurring and unmodified, including naturally occurring mutants. The term "variant" in the context of nucleic acids (polynucleotides) refers to a nucleic acid variant in which at least one nucleotide differs from a reference nucleic acid (or "parent"). Variant nucleic acids, compared to the reference nucleic acid, include single or multiple nucleotide deletions, additions, mutations, and / or insertions, wherein: deletions include the removal of one or more nucleotides from the reference nucleic acid; additions include the fusion of one or more nucleotides (e.g., 1, 2, 3, 5, 10, 20, 30, 50, or more nucleotides) to the 5' and / or 3' ends of the reference nucleic acid; mutations may include, but are not limited to, substitutions (e.g., the removal of at least one nucleotide and the insertion of another nucleotide at its position (e.g., transversion and conversion)); insertions include the addition of at least one nucleotide. The term "nucleic acid variant" as used herein includes both naturally occurring variants and engineered variants. Therefore, a "nucleic acid variant" as defined herein may be derived from, isolated from, related to, based on, or homologous to a reference nucleic acid sequence. "Nucleic acid variant" may optionally have at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the corresponding naturally occurring (wild-type) nucleic acid or its homologues, fragments, or derivatives; preferably at least 70%, more preferably at least 80%, even more preferably at least 85%, even more preferably at least 90%, most preferably at least 95%, or even 97%. It is understood that for nucleic acid molecules (e.g., mRNA), the term "variant" includes degenerate nucleic acid sequences, wherein the degenerate nucleic acid sequences according to the invention differ from the reference nucleic acid in the codon sequence due to the degeneracy of the genetic code.

[0114] As used herein, the term "% identity" or "% similarity" refers to the percentage of identical nucleotides or amino acids in the best alignment between sequences to be compared. Differences between the two sequences can be distributed across local regions (segments) or the entire length of the sequences being compared. Identity between two sequences is typically determined after the best alignment of a segment or "comparison window." The best alignment can be performed manually or using algorithms known in the art. Algorithms known in the art include, but are not limited to, the local homology algorithms described in Smith and Waterman, 1981, Ads App. Math. 2, 482 and Neddleman and Wunsch, 1970, J. Mol. Biol. 48, 443; the similarity search methods described in Pearson and Lipman, 1988, Proc. Natl Acad. Sci. USA 88, 2444; or the use of computer programs such as GAP, BESTFIT, FASTA, BLAST P, BLAST N, and TFASTA from the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis. For example, the percentage similarity between two sequences can be determined using the BLASTN or BLASTP algorithms publicly available on the National Center for Biotechnology Information (NCBI) website.

[0115] "% identity" or "% similarity" can be obtained by determining the number of identical positions corresponding to the sequences to be compared, dividing this number by the number of positions being compared (e.g., the number of positions in the reference sequence), and multiplying the result by 100. In some embodiments, a degree of similarity is given for at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% of the region. In some embodiments, a degree of similarity is given for the entire length of the reference sequence. Alignment for determining sequence similarity can be performed using tools known in the art, preferably using optimal sequence alignment, such as Align, using standard settings, preferably EMBOSS::needle, Matrix:Blosum62, Gap Open 10.0, or Gap Extend 0.5.

[0116] Preferably, the fragment or variant of the specific nucleic acid or the nucleic acid having a specific degree of identity with the specific nucleic acid preferably has at least one functional property of the specific nucleic acid, and preferably is functionally equivalent to the specific nucleic acid, for example, a nucleic acid exhibiting the same or similar properties as the specific nucleic acid.

[0117] In this article, "nucleotide" includes deoxyribonucleotides, deoxyribonucleotides, deoxyribonucleotide derivatives, and ribonucleotide derivatives. As used herein, "ribonucleotide" is the building block of ribonucleic acid (RNA), consisting of one base, one pentose sugar, and one phosphate molecule; it refers to a nucleotide with a hydroxyl group at the 2' position of the β-D-ribofuranosyl group. "Deoxyribonucleotide" is the building block of deoxyribonucleic acid (DNA), also consisting of one base, one pentose sugar, and one phosphate molecule; it refers to a nucleotide where the hydroxyl group at the 2' position of the β-D-ribofuranosyl group is replaced by hydrogen, and is a major chemical component of chromosomes.

[0118] Nucleotides are usually identified by a single letter representing the bases in them. "A" or "A nucleotide" refers to adenine deoxyribonucleotide or adenine ribonucleotide containing adenine; "C" or "C nucleotide" refers to cytosine deoxyribonucleotide or cytosine ribonucleotide containing cytosine; "G" or "G nucleotide" refers to guanine deoxyribonucleotide or guanine ribonucleotide containing guanine; "U" or "U nucleotide" refers to uracil ribonucleotide containing uracil; and "T" or "T nucleotide" refers to thymine deoxyribonucleotide containing thymine.

[0119] As used herein, the term "nucleic acid" generally refers to any compound comprising a polymer of deoxyribonucleotides (deoxyribonucleic acid, or DNA) or a polymer of ribonucleotides (ribonucleic acid, or RNA), or a combination thereof. Additionally, "nucleic acid" as used herein also includes derivatives of nucleic acids. The term "derivatives of nucleic acids" includes chemical derivatization of nucleic acids at the bases, sugars, or phosphates of nucleotides, as well as nucleic acids containing non-natural nucleotides and nucleotide analogs. Furthermore, in this document, nucleic acids can be in the form of single-stranded or double-stranded linear or covalently closed circular molecules.

[0120] The terms "polynucleotide sequence," "nucleic acid sequence," and "nucleotide sequence" are used interchangeably to refer to the sequence of nucleotides in a polynucleotide. Those skilled in the art should understand that the DNA coding strand (sense strand) and its encoded RNA can be considered to have the same nucleotide sequence, and the deoxythymidine nucleotide in the DNA coding strand sequence corresponds to the uridine nucleotide in its encoded RNA sequence. The RNA-corresponding DNA refers to a polynucleotide in RNA where all U nucleotides are replaced with T nucleotides.

[0121] A polynucleotide may comprise one or more segments (nucleic acid fragments) (e.g., segments 1, 2, 3, 4, 5, 6, 7, and 8). For example, a polynucleotide may comprise a segment encoding a polypeptide of interest. In a particular embodiment, a polynucleotide may comprise a segment encoding a polypeptide of interest as well as a regulatory segment (including, but not limited to, segments for transcriptional and translational regulation). In one embodiment, the regulatory segment comprises a polynucleotide corresponding to one or more of the following regulatory elements: a promoter, a 5' untranslated region (5'-UTR), a 3' untranslated region (3'-UTR), and a poly(A) tail.

[0122] The term "promoter" refers to a polynucleotide located upstream of the 5' end of the coding region of a gene. It contains a conserved sequence required for the specific binding of RNA polymerase and transcription initiation. It activates RNA polymerase, enabling it to bind accurately to the template DNA and possessing transcription initiation specificity. Promoters can originate from viruses, bacteria, fungi, plants, insects, and animals. Representative examples of promoters include the bacteriophage T7 promoter, bacteriophage T3 promoter, SP6 promoter, lac operon-promoter, tac promoter, SV40 late promoter, SV40 early promoter, RSV-LTR promoter, CMV IE promoter, SV40 early promoter, or SV40 late promoter and CMV IE promoter.

[0123] As used herein, the term "5' untranslated region" or "5'-UTR" can refer to an RNA sequence in mRNA that is upstream of the coding sequence and is not translated into protein. A 5'-UTR in a gene typically begins at the transcription start site and ends with a nucleotide upstream of the translation start codon in the coding sequence. The 5'-UTR can contain elements that control gene expression, such as ribosome binding sites, 5'-terminal oligopyrimidine bundles, and translation initiation signals such as the Kozak sequence. mRNA can undergo post-transcriptional modification by adding a 5' cap. Therefore, the 5'-UTR in mature mRNA can also refer to the RNA sequence between the 5' cap and the start codon.

[0124] As used herein, the term "3' untranslated region" or "3'-UTR" can refer to a region of mRNA located downstream of the coding sequence that is not translated into a protein. The 3'-UTR in mRNA is located between a stop codon and a poly(A) sequence of the coding sequence, for example, starting from a nucleotide downstream of the stop codon and ending at a nucleotide upstream of the poly(A) sequence.

[0125] As used herein, the terms “poly(A) nucleotide,” “poly(A) sequence,” and “poly(A) tail” are used interchangeably. Naturally occurring poly(A) sequences typically consist of adenine ribonucleotides. Poly(A) sequences are usually located at the 3' end of mRNA, such as the 3' end (downstream) of the 3'-UTR.

[0126] As used herein, the term "5' cap structure" refers to a 5' cap structure that is typically located at the 5' end of mature mRNA. In some embodiments, the 5' cap structure is linked to the 5' end of the mRNA via a 5'-5'-triphosphate bond. The 5' cap structure is typically formed from modified (e.g., methylated) ribonucleotides, particularly guanine nucleotide derivatives. For example, m7GpppN (cap0, or "cap0") is a cap structure formed by the reaction of the 5' phosphate group of hnRNA with the 5' phosphate group of m7GTP to form a 5',5'-phosphodiester bond under the action of guanylate transferase, where N is the terminal 5' nucleotide of the nucleic acid carrying the 5' cap structure. In some embodiments, the 5' cap structure includes, but is not limited to, cap 0, cap 1 (a cap structure formed by further methylation of the 2'-OH of the first nucleotide glycosyl group of hnRNA on the basis of cap 0, or "cap1"), cap 2 (a cap structure formed by further methylation of the 2'-OH of the second nucleotide glycosyl group of hnRNA on the basis of cap 1, or "cap2"), cap 4, cap 0 analogue, cap 1 analogue, cap 2 analogue, or cap 4 analogue.

[0127] As used herein, the term “expression” includes the transcription and / or translation of a nucleotide sequence. Therefore, expression can involve the production of transcripts and / or peptides. The term “transcription” refers to the process of transcribing the genetic code in a DNA sequence into RNA (transcription). The term “in vitro transcription” refers to the in vitro synthesis of RNA, particularly mRNA, in a cell-free system (e.g., in a suitable cell extract) (see, e.g., Pardi N., Muramatsu H., Weissman D., Karikó K. (2013). In: Rabinovich P. (eds) Synthetic Messenger RNA and Cell Metabolism Modulation. Methods in Molecular Biology (Methods and Protocols), vol 969. Humana Press, Totowa, NJ.). Vectors that can be used to produce transcripts are also called “transcription vectors,” which contain the regulatory sequences required for transcription. The term “transcription” encompasses “in vitro transcription.”

[0128] As used herein, the term "host cell" refers to a cell used to receive, maintain, replicate, and express polynucleotides or vectors. The term "host cell" includes prokaryotic cells (e.g., *Escherichia coli*) or eukaryotic cells (e.g., yeast cells and insect cells). Examples include cells derived from humans, mice, hamsters, pigs, goats, and primates. Cells can be derived from a variety of tissue types and include primary cells and cell lines. Some specific examples include keratinocytes, peripheral blood leukocytes, bone marrow stem cells, and embryonic stem cells. In other embodiments, the host cell is an antigen-presenting cell, particularly dendritic cells, monocytes, or macrophages. Nucleic acids may be present in the host cell in single or multiple copies. In some embodiments, the host cell may be a cell in which the proteins of the present invention are expressed.

[0129] In the context of this invention, the term "plasmid" generally refers to a circular DNA molecule, but the term can also encompass linearized DNA molecules. Specifically, the term "plasmid" also encompasses molecules obtained by linearizing a circular plasmid, for example, by digesting the circular plasmid with a restriction enzyme, thereby converting the circular plasmid molecule into a linear molecule. Plasmids can replicate, i.e., amplify genetic information in cells independently of chromosomal DNA, and can be used for cloning, i.e., for amplifying genetic information in bacterial cells. Preferably, the DNA plasmid is a medium-copy or high-copy plasmid, more preferably a high-copy plasmid. Examples of such high-copy plasmids include, for example, pUC and pTZ plasmids or any other plasmid (e.g., pMB1, pCoIE1) containing a replication origin that supports high copy number of the plasmid.

[0130] The term "treatment" and similar terms are used herein to generally mean achieving a desired pharmacological and / or physiological effect. Therefore, the treatment of this application may involve the treatment of a disease state, but may also involve preventative treatment with regard to the complete or partial prevention of the disease or its symptoms. Preferably, in some embodiments, the term "treatment" should be understood as therapeutic in terms of partially or completely curing the disease and / or the adverse effects and / or symptoms attributable to the disease. Treatment can also be prophylactic or preventive treatment, i.e., measures taken to prevent disease, such as to prevent infection and / or the onset of disease.

[0131] As used herein, the terms "subject" and "patient" are used interchangeably. In some embodiments, a subject is a mammal (e.g., a human) suffering from an infectious disease or a neoplastic disease. In other embodiments, a subject is a mammal (e.g., a human) at risk of developing an infectious disease or a neoplastic disease.

[0132] As used herein, the term "administration" means the provision or administration of a drug to a subject by any effective route. Exemplary routes of administration include, but are not limited to, one or more of the following: injection (e.g., subcutaneous, intramuscular, intradermal, intraperitoneal, intrathecal, intravenous, intraventricular, or intravenous), oral, intraluminal bile duct, sublingual, rectal, transdermal, intranasal, vaginal, and inhalation. When used to treat a disease, condition, symptom, or symptom, the substance is usually administered after the onset of the disease, condition, symptom, or symptom. When used to prevent a disease, condition, symptom, or symptom, the substance is usually administered before the onset of the disease, condition, symptom, or symptom.

[0133] This document describes some elements of the invention. These elements are listed together with specific embodiments; however, it should be understood that they can be combined in any manner and in any number to produce other embodiments. The examples and preferred embodiments described differently should not be construed as limiting the invention to the explicitly described embodiments. This specification should be understood to support and include embodiments that combine the explicitly described embodiments with any number of the disclosed and / or preferred elements. Furthermore, unless the context otherwise indicates, any arrangement and combination of all descriptive elements in this invention should be considered as disclosed in this specification. For example, in one embodiment, the truncated ATP7B protein is a truncated human ATP7B protein, and in another embodiment, the truncated ATP7B protein lacks MBD3; the following is also an embodiment claimed by this invention: the truncated ATP7B protein is a truncated human ATP7B protein, and the truncated ATP7B protein lacks MBD3.

[0134] 2. mRNA

[0135] Copper-transporting ATPase 2 (ATP7B) is a highly conserved transmembrane protein of the P-type ATPase family. The main function of ATP7B protein is to promote the synthesis of ceruloplasmin and the biliary excretion of copper.

[0136] This disclosure provides an mRNA that is a non-natural mRNA containing a polynucleotide encoding the ATP7B protein.

[0137] In some embodiments, the above-mentioned mRNA also includes at least one of a 5'-cap structure, a 5'-UTR, a 3'-UTR, and a poly(A) tail.

[0138] In some implementations, the ATP7B protein is the full-length ATP7B protein.

[0139] In some implementations, the polynucleotide encoding the full-length ATP7B protein is codon-optimized.

[0140] Codon optimization methods are known in the art and can be used as provided herein. In some embodiments, codon optimization can be used to: match codon frequencies between the target and the host organism to ensure proper folding; bias GC content to increase mRNA stability or reduce secondary structures; minimize tandem repeat codons or base elongations that could impair gene structure or expression; customize transcription and translation control regions; insert or remove protein transport sequences; remove / add post-translational modification sites in encoded proteins (e.g., glycosylation sites); add, remove, or reorganize protein domains; insert or delete restriction sites; modify ribosome binding sites and mRNA degradation sites; modulate translation rates so that various protein domains fold properly; or reduce or eliminate problematic secondary structures within polynucleotides. Codon optimization tools, algorithms, and services are known in the art, and non-limiting examples include services from GeneArt (Life Technologies), DNA2.0 (Menlo Park CA), and / or proprietary methods. In some embodiments, optimization algorithms are used to optimize open reading frame (ORF) sequences.

[0141] In some implementations, the codon-optimized polynucleotide encoding the full-length ATP7B protein has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity with the unoptimized polynucleotide encoding the full-length ATP7B protein.

[0142] In some embodiments, the polynucleotide encoding the full-length ATP7B protein is a polynucleotide whose nucleotide sequence has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the nucleotide sequence shown in any one of SEQ ID NO: 8-10.

[0143] In some implementations, the full-length ATP7B protein is the human full-length ATP7B protein.

[0144] The human ATP7B gene is located on chromosome 13 (chromosome position 13q14.3). Information about the human ATP7B gene is available from the NCBI accession number Gene ID: 540 (http: / / www.ncbi.nlm.nih.gov / gene / 540, updated January 8, 2023). The full-length human ATP7B protein has six metal-binding domains (MBDs): MBD1, MBD2, MBD3, MBD4, MBD5, and MBD6.

[0145] Alternative splicing and ribosomal frameshifting of the human ATP7B protein produce multiple isoforms. For example, Uniprot identifies human ATP7B proteins with identification numbers P35670-1, P35670-2, P35670-3, P35670-4, and P35670-5. In some embodiments, the amino acid sequence of the full-length human ATP7B protein is shown in SEQ ID NO: 1. The full-length human ATP7B protein (hereinafter referred to as "human ATP7B protein subtype 1") with an amino acid sequence as shown in SEQ ID NO: 1 has the identification number P35670-1 in Uniprot, consisting of 1465 amino acids. Its MBD1 is located at positions 58 to 124 of SEQ ID NO: 1, MBD2 is located at positions 143 to 209 of SEQ ID NO: 1, MBD3 is located at positions 257 to 323 of SEQ ID NO: 1, MBD4 is located at positions 359 to 425 of SEQ ID NO: 1, MBD5 is located at positions 488 to 554 of SEQ ID NO: 1, and MBD6 is located at positions 564 to 630 of SEQ ID NO: 1.

[0146] In some implementations, the mRNA contains a 5'-UTR, a 3'-UTR, a poly(A) tail, and a polynucleotide encoding the full-length human ATP7B protein.

[0147] In some implementations, the mRNA contains a 5'-cap structure, a 5'-UTR, a 3'-UTR, a poly(A) tail, and a polynucleotide encoding the full-length human ATP7B protein.

[0148] In some implementations, the mRNA, from the 5' end to the 3' end, includes a 5'-cap structure, a 5'-UTR, a polynucleotide encoding the full-length human ATP7B protein, a 3'-UTR, and a poly(A) tail.

[0149] In some embodiments, the polynucleotide encoding the full-length human ATP7B protein has a nucleotide sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence shown in any one of SEQ ID NO: 8-10.

[0150] In some embodiments, the polynucleotide encoding the full-length human ATP7B protein is a polynucleotide whose nucleotide sequence has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the nucleotide sequence shown in any one of SEQ ID NO: 8-10 and encodes the amino acid sequence shown in SEQ ID NO: 1 for the full-length human ATP7B protein.

[0151] In some embodiments, the nucleotide sequence of the polynucleotide encoding the full-length human ATP7B protein is shown in one of SEQ ID NO: 8 to 10.

[0152] In some implementations, the ATP7B protein is a truncated ATP7B protein that retains the functions of the ATP7B protein.

[0153] In some embodiments, the mRNA comprises at least one of a 5'-cap structure, a 5'-UTR, a 3'-UTR, and a poly(A) tail, and a polynucleotide encoding a truncated ATP7B protein.

[0154] In some embodiments, the mRNA comprises a 5'-UTR, a 3'-UTR, a poly(A) tail, and a polynucleotide encoding a truncated ATP7B protein. In some embodiments, the mRNA comprises a 5'-cap structure, a 5'-UTR, a 3'-UTR, a poly(A) tail, and a polynucleotide encoding a truncated ATP7B protein. In some embodiments, from the 5' end to the 3' end, the mRNA comprises a 5'-cap structure, a 5'-UTR, a polynucleotide encoding a truncated ATP7B protein, a 3'-UTR, and a poly(A) tail.

[0155] In some implementations, the truncated ATP7B protein is a truncated ATP7B protein lacking one or more MBDs.

[0156] In some implementations, the truncated ATP7B protein is one of the following:

[0157] (1) The truncated ATP7B protein of MBD1 to MBD4 is missing;

[0158] (2) A truncated ATP7B protein lacking MBD3;

[0159] (3) The truncated ATP7B protein lacking MBD1 and MBD3;

[0160] (4) The truncated ATP7B protein of MBD1, MBD3 and MBD4 is missing;

[0161] (5) truncated ATP7B protein lacking MBD1 and MBD4;

[0162] (6) The truncated ATP7B protein of MBD1-MBD2 is missing;

[0163] (7) truncated ATP7B proteins lacking MBD1, MBD2 and MBD4;

[0164] (8) The truncated ATP7B protein of MBD1-MBD3 is missing;

[0165] (9) truncated ATP7B protein lacking MBD2 and MBD3;

[0166] (10) truncated ATP7B protein lacking MBD2 and MBD4;

[0167] (11) truncated ATP7B protein lacking MBD3 and MBD4;

[0168] (12) The truncated ATP7B protein lacking MBD2–MBD4; and

[0169] (13) The truncated ATP7B protein of MBD1 to MBD5 is missing.

[0170] In some implementations, the truncated ATP7B protein is one of the following:

[0171] (1) The truncated ATP7B protein, which is missing MBD1-MBD4 and retains MBD5-MBD6;

[0172] (2) A truncated ATP7B protein that lacks MBD3 but retains MBD1–MBD2 and MBD4–MBD6;

[0173] (3) A truncated ATP7B protein that lacks MBD1 and MBD3 but retains MBD2, MBD4, MBD5 and MBD6;

[0174] (4) A truncated ATP7B protein that lacks MBD1, MBD3 and MBD4 but retains MBD2, MBD5 and MBD6;

[0175] (5) A truncated ATP7B protein that lacks MBD1 and MBD4 but retains MBD2, MBD3, MBD5 and MBD6;

[0176] (6) The truncated ATP7B protein, which lacks MBD1-MBD2 but retains MBD3-MBD6, is missing.

[0177] (7) A truncated ATP7B protein that lacks MBD1, MBD2 and MBD4 but retains MBD3, MBD5 and MBD6;

[0178] (8) A truncated ATP7B protein that lacks MBD1 to MBD3 but retains MBD4 to MBD6;

[0179] (9) A truncated ATP7B protein that lacks MBD2 and MBD3 but retains MBD1 and MBD4-MBD6;

[0180] (10) A truncated ATP7B protein that lacks MBD2 and MBD4 but retains MBD1, MBD3, MBD5 and MBD6;

[0181] (11) A truncated ATP7B protein that lacks MBD3 and MBD4 but retains MBD1, MBD2, MBD5 and MBD6;

[0182] (12) A truncated ATP7B protein lacking MBD2–MBD4 but retaining MBD1, MBD5, and MBD6; and

[0183] (13) A truncated ATP7B protein that lacks MBD1 to MBD5 but retains MBD6.

[0184] In this disclosure, the truncated ATP7B protein lacking a certain MBD can be any of the following: (1) a partial amino acid deletion of the MBD; (2) a complete amino acid deletion of the MBD; (3) a partial amino acid deletion of the MBD and a portion or all amino acids of the segment between the MBD and its adjacent MBD; and (4) a complete amino acid deletion of the MBD and a portion or all amino acids of the segment between the MBD and its adjacent MBD. For example, the truncated ATP7B protein is a truncated ATP7B protein lacking MBD1. The loss of MBD1 can be a partial or complete amino acid deletion of MBD1, or a partial or complete amino acid deletion of MBD1 and a partial or complete amino acid deletion of at least one of the following regions: the region between the MBD start position and MBD1, and the segment between MBD1 and MBD2. In this document, "at least a partial amino acid deletion" is equivalent to "partial or complete amino acid deletion," both referring to the partial or complete deletion of amino acids on the peptide chain. When multiple amino acids are deleted, these deleted amino acids can be continuous or discontinuous. For example, 66 amino acids are missing from position 257 to position 323 in human ATP7B protein isotype 1. In some embodiments, these 66 amino acids are from position 257 to position 322, and in other embodiments, they are from position 257 to position 317 and from position 319 to position 323.

[0185] Given the substantial conservation of the amino acid sequence of the ATP7B protein, those skilled in the art can readily compare the amino acid positions between different native ATP7B protein sequences to determine the corresponding amino acid positions of the ATP7B protein across different species and subtypes. Therefore, the conservation of the amino acid sequence of the native ATP7B protein across species and subtypes allows the use of a reference amino acid sequence to compare amino acids at specific positions within the ATP7B protein. For the purposes of this disclosure (unless the context otherwise indicates), the amino acid positions of the ATP7B protein herein refer to that shown in SEQ ID NO: 1. However, those skilled in the art will understand that different ATP7B proteins may have different numbering systems (e.g., with additions or deletions of additional amino acid residues compared to SEQ ID NO: 1). Therefore, it should be understood that when a specific amino acid residue is designated by a number, it is not limited to the amino acid residue that is precisely located at the numbered position when counting from the beginning of a given amino acid sequence, but also includes any and all equivalent / corresponding amino acid residues in the amino acid sequence of the ATP7B protein, even if the amino acid residue is not at the same precise numbered position (e.g., if the amino acid sequence of the ATP7B protein is shorter or longer than SEQ ID NO: 1, or has an insertion or deletion compared to SEQ ID NO: 1).

[0186] In some implementations, the truncated ATP7B protein is a truncated ATP7B protein with positions 57 to 485 missing.

[0187] In some implementations, the truncated ATP7B protein is a truncated ATP7B protein with positions 57 to 486 missing.

[0188] In some implementations, the truncated ATP7B protein is a truncated ATP7B protein with positions 257 to 355 missing.

[0189] In some implementations, the truncated ATP7B protein is a truncated ATP7B protein with positions 57–140 and 237–337 missing.

[0190] In some implementations, the truncated ATP7B protein is a truncated ATP7B protein with positions 57–140 and 237–485 missing.

[0191] In some implementations, the truncated ATP7B protein is a truncated ATP7B protein with positions 57–140 and 359–485 missing.

[0192] In some implementations, the truncated ATP7B protein is a truncated ATP7B protein with positions 57 to 236 missing.

[0193] In some implementations, the truncated ATP7B protein is a truncated ATP7B protein with positions 57–236 and 359–485 missing.

[0194] In some implementations, the truncated ATP7B protein is a truncated ATP7B protein with positions 57 to 337 missing.

[0195] In some implementations, the truncated ATP7B protein is a truncated ATP7B protein with positions 141 to 337 missing.

[0196] In some implementations, the truncated ATP7B protein is a truncated ATP7B protein with positions 141–236 and 359–485 missing.

[0197] In some implementations, the truncated ATP7B protein is a truncated ATP7B protein with positions 237 to 485 missing.

[0198] In some implementations, the truncated ATP7B protein is a truncated ATP7B protein with positions 141 to 485 missing.

[0199] In some implementations, the truncated ATP7B protein is a truncated ATP7B protein with positions 57 to 490 missing.

[0200] In some implementations, the truncated ATP7B protein is a truncated ATP7B protein with positions 57 to 495 missing.

[0201] In some embodiments, the truncated ATP7B protein is one of the following: (1) a protein missing amino acids 57 to 485 of SEQ ID NO: 1; (2) a protein missing amino acids 57 to 486 of SEQ ID NO: 1; (3) a protein missing amino acids 257 to 355 of SEQ ID NO: 1; (4) a protein missing amino acids 57 to 140 and 237 to 337 of SEQ ID NO: 1; (5) a protein missing amino acids 57 to 140 and 237 to 485 of SEQ ID NO: 1; (6) a protein missing amino acids 57 to 140 and 359 to 485 of SEQ ID NO: 1; (7) a protein missing amino acids 57 to 236 of SEQ ID NO: 1; (8) a protein missing amino acids 57 to 236 of SEQ ID NO: 1. (9) Proteins after amino acids 57 to 236 and 359 to 485 of SEQ ID NO: 1; (10) Proteins after amino acids 57 to 337 of SEQ ID NO: 1; (11) Proteins after amino acids 141 to 337 of SEQ ID NO: 1; (12) Proteins after amino acids 237 to 485 of SEQ ID NO: 1; (13) Proteins after amino acids 141 to 236 and 359 to 485 of SEQ ID NO: 1; The protein following amino acids 141 to 485 of NO:1; and (14) the protein whose amino acid sequence has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% and less than 100% similarity to the amino acid sequence of one of the proteins in (1) to (13) and has the function of ATP7B protein.

[0202] In some embodiments, the protein in (14) above has the same length as the corresponding proteins in (1) to (13). For example, the length of the truncated ATP7B protein is the same as the length of the protein whose amino acid sequence is the deletion of amino acids 57 to 486 of SEQ ID NO: 1 (hereinafter referred to as the first protein), and the amino acid sequence of the truncated ATP7B protein has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and less than 100% identity with the amino acid sequence of the first protein and has the function of ATP7B protein.

[0203] In some embodiments, the truncated ATP7B protein is one of the following: (1) a truncated ATP7B protein with an amino acid sequence as shown in any one of SEQ ID NO: 11-25; and (2) a protein with an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% and less than 100% identity with the amino acid sequence shown in any one of SEQ ID NO: 11-25 and having ATP7B protein function.

[0204] In some implementations, the polynucleotide encoding the truncated ATP7B protein is codon-optimized.

[0205] In some implementations, the codon-optimized polynucleotide encoding the truncated ATP7B protein has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity with the original codon-optimized polynucleotide encoding the truncated ATP7B protein.

[0206] In some implementations, the polynucleotide encoding the truncated ATP7B protein is one of the following:

[0207] (1) A polynucleotide with a nucleotide sequence as shown in SEQ ID NO: 26;

[0208] (2) A polynucleotide sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and less than 100% identity with the nucleotide sequence shown in SEQ ID NO: 26 and encoding an amino acid sequence as shown in SEQ ID NO: 11 of the truncated ATP7B protein;

[0209] (3) A polynucleotide with a nucleotide sequence as shown in any one of SEQ ID NO: 27-30;

[0210] (4) The nucleotide sequence has at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and less than 100% identity with the nucleotide sequence shown in any one of SEQ ID NO: 27–30 and encodes a polynucleotide encoding an amino acid sequence of the truncated ATP7B protein as shown in SEQ ID NO: 12;

[0211] (5) Polynucleotides with nucleotide sequences as shown in SEQ ID NO: 31;

[0212] (6) A polynucleotide sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and less than 100% identity with the nucleotide sequence shown in SEQ ID NO: 31 and encoding an amino acid sequence as shown in SEQ ID NO: 13 of the truncated ATP7B protein.

[0213] (7) Polynucleotides with nucleotide sequences as shown in SEQ ID NO: 32;

[0214] (8) A polynucleotide sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and less than 100% identity with the nucleotide sequence shown in SEQ ID NO: 32 and encoding an amino acid sequence as shown in SEQ ID NO: 14;

[0215] (9) Polynucleotides with nucleotide sequences as shown in SEQ ID NO: 33 or 34;

[0216] (10) A polynucleotide sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and less than 100% identity with the nucleotide sequence shown in SEQ ID NO: 33 or 34 and encoding an amino acid sequence as shown in SEQ ID NO: 15, truncated ATP7B protein;

[0217] (11) Polynucleotides with nucleotide sequences as shown in SEQ ID NO: 35;

[0218] (12) A polynucleotide sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and less than 100% identity with the nucleotide sequence shown in SEQ ID NO: 35 and encoding an amino acid sequence of a truncated ATP7B protein as shown in SEQ ID NO: 16;

[0219] (13) Polynucleotides with nucleotide sequences as shown in SEQ ID NO: 36;

[0220] (14) A polynucleotide sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and less than 100% identity with the nucleotide sequence shown in SEQ ID NO: 36 and encoding an amino acid sequence of a truncated ATP7B protein as shown in SEQ ID NO: 17;

[0221] (15) Polynucleotides with nucleotide sequences as shown in SEQ ID NO: 37;

[0222] (16) A polynucleotide sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and less than 100% identity with the nucleotide sequence shown in SEQ ID NO: 37 and encoding an amino acid sequence of a truncated ATP7B protein as shown in SEQ ID NO: 18;

[0223] (17) Polynucleotides with nucleotide sequences as shown in SEQ ID NO: 38;

[0224] (18) A polynucleotide sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and less than 100% identity with the nucleotide sequence shown in SEQ ID NO: 38 and encoding an amino acid sequence of a truncated ATP7B protein as shown in SEQ ID NO: 19;

[0225] (19) Polynucleotides with nucleotide sequences as shown in SEQ ID NO: 39;

[0226] (20) A polynucleotide sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and less than 100% identity with the nucleotide sequence shown in SEQ ID NO: 39 and encoding an amino acid sequence of a truncated ATP7B protein as shown in SEQ ID NO: 20;

[0227] (21) Polynucleotides with nucleotide sequences as shown in SEQ ID NO: 40;

[0228] (22) A polynucleotide sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and less than 100% identity with the nucleotide sequence shown in SEQ ID NO: 40 and encoding an amino acid sequence of a truncated ATP7B protein as shown in SEQ ID NO: 21;

[0229] (23) Polynucleotides with nucleotide sequences as shown in SEQ ID NO: 41;

[0230] (24) A polynucleotide sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and less than 100% identity with the nucleotide sequence shown in SEQ ID NO: 41 and encoding an amino acid sequence of a truncated ATP7B protein as shown in SEQ ID NO: 22;

[0231] (25) Polynucleotides with nucleotide sequences as shown in SEQ ID NO: 42;

[0232] (26) A polynucleotide sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and less than 100% identity with the nucleotide sequence shown in SEQ ID NO: 42 and encoding an amino acid sequence of a truncated ATP7B protein as shown in SEQ ID NO: 23;

[0233] (27) Polynucleotides with nucleotide sequences as shown in SEQ ID NO: 43;

[0234] (28) A polynucleotide sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and less than 100% identity with the nucleotide sequence shown in SEQ ID NO: 43 and encoding an amino acid sequence of a truncated ATP7B protein as shown in SEQ ID NO: 24;

[0235] (29) Polynucleotides with nucleotide sequences as shown in SEQ ID NO: 44; and

[0236] (30) A polynucleotide sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and less than 100% identity with the nucleotide sequence shown in SEQ ID NO: 44 and encoding an amino acid sequence of a truncated ATP7B protein as shown in SEQ ID NO: 25.

[0237] In some embodiments, the polynucleotide encoding the truncated ATP7B protein comprises or is one of the following: (1) a polynucleotide with a nucleotide sequence as shown in one of SEQ ID NO: 27-30; and (2) a polynucleotide with a nucleotide sequence having at least 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the nucleotide sequence shown in any of SEQ ID NO: 27-30 and encoding an amino acid sequence as shown in SEQ ID NO: 12.

[0238] In some implementations, the truncated ATP7B protein is a truncated human ATP7B protein.

[0239] In some embodiments, the truncated human ATP7B protein is a protein or polypeptide that has been truncated from one of the various isoforms of the full-length human ATP7B protein. In some embodiments, the truncated human ATP7B protein is a protein whose amino acid sequence is truncated from the full-length human ATP7B protein as shown in SEQ ID NO: 1.

[0240] In some implementations, the truncated ATP7B protein has no deletions other than the metal-binding domain.

[0241] In other implementations, the truncated ATP7B protein is missing not only at the metal-binding domain but also at other locations.

[0242] In some implementations, the truncated ATP7B protein is 835 to 1455 amino acids in length.

[0243] In some implementations, the truncated ATP7B protein is 850–1390 amino acids, 880–1380 amino acids, 900–1390 amino acids, 920–1380 amino acids, 925–1370 amino acids, 925–1370 amino acids, or 1026–1366 amino acids in length.

[0244] In some implementations, the truncated ATP7B protein is not limited to the human truncated ATP7B protein, but can also be a truncated ATP7B protein from other species. Examples include the western lowland gorilla (Gorilla gorilla gorilla), chimpanzee (Pan troglodytes), bonobo (Pan paniscus), Sumatran orangutan (Pongo abelii), pig-tailed macaque (Macaca nemestrina), crab-eating macaque (Macaca fascicularis), East African baboon (Papio anubis), and Chinese stump-tailed macaque (Macaca thibetana thibetana).

[0245] In some implementations, the 5'-hat structure includes, but is not limited to, m 7 GpppG, m2 7,3′-O GpppG, m 7 Gppp(5')N1 and m 7 Gppp(m 2′-O At least one of N1. 7 "G" represents 7-methylguanosine cap nucleotide, "ppp" represents the triphosphate bond between the 5' carbon of the cap nucleotide and the first nucleotide of the primary RNA transcript, N1 is the most 5' nucleotide, "G" represents guanine nucleoside, and "m" represents the guanine nucleoside. 7 " represents the methyl group at the 7-position of guanine, "m 2′-O " " represents the methyl group at the 2′-O position of the nucleotide.

[0246] In some implementations, the 5'-UTR sequence is as shown in SEQ ID NO: 2 or 3.

[0247] In some implementations, the 5'-UTR sequence is as shown in SEQ ID NO: 3.

[0248] In some implementations, the 3'-UTR sequence is as shown in SEQ ID NO: 4 or 5.

[0249] In some implementations, the 3'-UTR sequence is as shown in SEQ ID NO: 4.

[0250] In some implementations, the 5'-UTR sequence is shown in SEQ ID NO: 3, and the 3'-UTR sequence is shown in SEQ ID NO: 4.

[0251] It is understood that in other embodiments, the 5'-UTR and 3'-UTR of this disclosure are not limited to the above, but may be others, such as the 5'-UTR and 3'-UTR described in patents CN108291230A, CN104321432A, CN107849574A, etc.

[0252] In some embodiments, the nucleotides constituting the poly(A) tail comprise at least 20, at least 40, at least 80, at least 100, or at least 120 A nucleotides. Preferably, the nucleotides constituting the poly(A) tail comprise at least 20, at least 40, at least 80, at least 100, or at least 120 consecutive A nucleotides. In some embodiments, the nucleotides constituting the poly(A) tail include one or more nucleotides other than A nucleotides. Optionally, the nucleotides constituting the poly(A) tail comprise two or more consecutive nucleotides other than A nucleotides. In one optional specific example, the sequence of the poly(A) tail is as shown in SEQ ID NO: 6 or 7. It is understood that in other embodiments, the poly(A) tail is not limited to the above and may be other poly(A) tails, such as those described in patents such as US20170166905A1 and WO2020074642A1.

[0253] In some implementations, the mRNA of any of the above implementations does not contain modified nucleotides.

[0254] In some implementations, the mRNA of any of the above implementations contains modified nucleotides.

[0255] In some embodiments, the mRNA of any of the above embodiments contains a modified nucleoside. In some embodiments, the modified nucleoside includes at least one of modified uridine, modified cytidine, modified adenosine, and modified guanosine.

[0256] In some embodiments, the modified nucleoside is modified uridine. In some embodiments, 0.1% to 100% of the uridine in the above-mentioned mRNA is modified. For example, at least 0.1%, at least 0.5%, at least 1%, at least 2%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 82%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99%, or 100% of the uridine is modified. In some embodiments, 80% to 100% of the uridine is modified. In some embodiments, 100% of the uridine is modified. Exemplary modified uridines include, but are not limited to, one or more of the following: pseudouridine (ψ), N1-methylpseudouridine, pyridine-4-ketoribonucleoside, 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine (s2U), 4-thio-uridine (s4U), 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uridine (ho5U), 5-aminoallyl-uridine, 5-halo-uridine (e.g., 5... 5-Iodine-uridine or 5-bromo-uridine), 3-methyl-uridine (m3U), 5-methoxy-uridine (mo5U), uridine-5-oxyacetic acid (cmo5U), methyl uridine-5-oxyacetic acid (mcmo5U), 5-carboxymethyl-uridine (cm5U), 1-carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl-uridine (chm5U), 5-carboxyhydroxymethyl-uridine methyl ester (mchm5U), 5-methoxycarbonylmethyl-uridine (mc m5U), 5-methoxycarbonylmethyl-2-thio-uridine (mcm5s2U), 5-aminomethyl-2-thio-uridine (nm5s2U), 5-methylaminomethyl-uridine (mnm5U), 5-methylaminomethyl-2-thio-uridine (mnm5s2U), 5-methylaminomethyl-2-seleno-uridine (mnm5se2U), 5-carbamoylmethyl-uridine (ncm5U), 5-carboxymethylaminomethyl-uridine (c mnm5U), 5-carboxymethylaminomethyl-2-thio-uridine (cmnm5s2U), 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyl-uridine (5-taurinomethyl-uridine) (τm5U), 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine (τm5s2U), 1-taurinomethyl-4-thio-pseudouridine, 5-methyl-uridine (m5U,That is, possessing nucleobase deoxythymidine), 1-methyl-pseudouridine (m1ψ), 5-methyl-2-thio-uridine (m5s2U), 1-methyl-4-thio-pseudouridine (m1s4ψ), 4-thio-1-methyl-pseudouridine, 3-methyl-pseudouridine (m3ψ), 2-thio-1-methyl-pseudouridine, 1-methyl-1-deazo-pseudouridine, 2-thio-1-methyl-1-deazo-pseudouridine, dihydrouridine (D), dihydropseudouridine, 5,6-dihydrouridine, 5-methyl-dihydrouridine (m5D) ), 2-thio-dihydrouridine, 2-thio-dihydropseuuridine, 2-methoxy-uridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseuuridine, 4-methoxy-2-thio-pseuuridine, N1-methyl-pseuuridine, 3-(3-amino-3-carboxypropyl)uridine (acp3U), 1-methyl-3-(3-amino-3-carboxypropyl)pseuuridine (acp3ψ), 5-(isopentenylaminomethyl)uridine (inm5U), 5-(isopentenylaminomethyl)-2-thio-uridine ( inm5s2U), α-thiouridine, 2'-O-methyluridine (Um), 5,2'-O-dimethyluridine (m5Um), 2'-O-methyl-pseudouridine (ψm), 2-thio-2'-O-methyluridine (s2Um), 5-methoxycarbonylmethyl-2'-O-methyluridine (mcm5Um), 5-carbamoylmethyl-2'-O-methyluridine (ncm5Um), 5-carboxymethylaminomethyl-2'-O-methyluridine (cmnm5Um), 3 2'-O-dimethyluridine (m3Um), 5-(isopentenylaminomethyl)-2'-O-methyluridine (inm5Um), 1-thiouridine, deoxythymidine, 2'-F-arauridine, 2'-F-uridine, 2'-OH-arauridine, 5-(2-methoxyformylvinyl)uridine, and 5-[3-(1-E-propenylamino)uridine. In some embodiments, the uridine in the above mRNAs is all N1-methylpseudouridine.

[0257] In some embodiments, the modified nucleoside is modified cytidine. In some embodiments, 0.1% to 100% of the cytidine in the above-mentioned mRNA is modified. For example, at least 0.1%, at least 0.5%, at least 1%, at least 2%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 82%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99%, or 100% of the cytidine is modified. In some embodiments, 80% to 100% of the cytidine is modified. In some embodiments, 100% of the cytidine is modified. Exemplary modified cytidines include one or more of the following: 5-aza-cytidine, 6-aza-cytidine, pseudoisocytidine, 3-methylcytidine (m3C), N4-acetylcytidine (ac4C), 5-formylcytidine (f5C), N4-methylcytidine (m4C), 5-methylcytidine (m5C), 5-halo-cytidine (e.g., 5-iodocytidine), 5-hydroxymethylcytidine (hm5C), 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine (s2C), 2-thio-5-methylcytidine, 4-thio-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, 4-thio-1-methyl-1-deazo-pseudoisocytidine, 1-methyl-1-deazo-pseudoisocytidine, zebularin e) 5-aza-zabraline, 5-methylzabraline, 5-aza-2-thio-zabraline, 2-thio-zabraline, 2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-pseudo-cytidine, 4-methoxy-1-methyl-pseudo-cytidine, lysidine (k2C), α-thio-cytidine, 2'-O-methyl-cytidine (Cm), 5,2'-O -Dimethyl-cytidine (m5Cm), N4-acetyl-2'-O-methyl-cytidine (ac4Cm), N4,2'-O-dimethyl-cytidine (m4Cm), 5-formyl-2'-O-methyl-cytidine (f5Cm), N4,N4,2'-O-trimethyl-cytidine (m42Cm), 1-thio-cytidine, 2'-F-arabinocytidine, 2'-F-cytidine and 2'-OH-arabinocytidine.

[0258] In some embodiments, the modified nucleoside is modified adenosine. In some embodiments, 0.1% to 100% of the adenosine in the above-mentioned mRNA is modified. For example, at least 0.1%, at least 0.5%, at least 1%, at least 2%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 82%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99%, or 100% of the adenosine is modified. In some embodiments, 80% to 100% of the adenosine is modified. In some embodiments, 100% of the adenosine is modified.Exemplary modified adenosines include, but are not limited to, one or more of the following: 2-amino-purine, 2,6-diaminopurine, 2-amino-6-halo-purine (e.g., 2-amino-6-chloro-purine), 6-halo-purine (e.g., 6-chloro-purine), 2-amino-6-methyl-purine, 8-azido-adenosine, 7-deadenine, 7-deadenine-8-aza-adenosine, 7-deadenine-2-amino-purine, 7-deadenine-8-aza-2-amino-purine, 7-deadenine-2,6-diaminopurine, 7-deadenine-8-aza-2,6- Diaminopurine, 1-methyl-adenosine (m1A), 2-methyl-adenosine (m2A), N6-methyl-adenosine (m6A), 2-methylthio-N6-methyl-adenosine (ms2m6A), N6-isopentenyl-adenosine (i6A), 2-methylthio-N6-isopentenyl-adenosine (ms2i6A), N6-(cis-hydroxyisopentenyl)adenosine (io6A), 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine (ms2io6A), N6-glycylcarbamoyl-adenosine (g6A), N6-threonylamino N6-methyl-N6-threonylcarbamoyl-adenosine (t6A), N6-methyl-N6-threonylcarbamoyl-adenosine (m6t6A), 2-methylthio-N6-threonylcarbamoyl-adenosine (ms2g6A), N6,N6-dimethyl-adenosine (m62A), N6-hydroxyn-valinecarbamoyl-adenosine (hn6A), 2-methylthio-N6-hydroxyn-valinecarbamoyl-adenosine (ms2hn6A), N6-acetyl-adenosine (ac6A), 7-methyl-adenosine, 2-methylthio-adenosine, 2-methoxy-adenosine α-Thio-adenosine, 2'-O-methyl-adenosine (Am), N6,2'-O-dimethyl-adenosine (m6Am), N6,N6,2'-O-trimethyl-adenosine (m62Am), 1,2'-O-dimethyl-adenosine (m1Am), 2'-O-ribosyl-adenosine (phosphate) (Ar(p)), 2-amino-N6-methyl-purine, 1-thio-adenosine, 8-azido-adenosine, 2'-F-arabinose-adenosine, 2'-F-adenosine, 2'-OH-arabinose-adenosine, and N6-(19-amino-pentaenodecyl)-adenosine.

[0259] In some embodiments, the modified nucleoside is modified guanosine. In some embodiments, 0.1% to 100% of the guanosine in the above-mentioned mRNA is modified. For example, at least 0.1%, at least 0.5%, at least 1%, at least 2%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 82%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99%, or 100% of the guanosine is modified. In some embodiments, 80% to 100% of the guanosine is modified. In some embodiments, 100% of the guanosine is modified.Exemplary modified guanosines include, but are not limited to, one or more of the following: inosine (I), 1-methyl-inosine (m1I), wyosine (imG), methyl-γ-glycoside (mimG), 4-demethyl-γ-glycoside (imG-14), iso-γ-glycoside (imG2), eugenol (yW), peroxyeugenol (o2yW), hydroxyeugenol (OHyW), undermodified hydroxyeugenol (OHyW*), 7-deazo-guanosine, queuosine (... Q), epoxyguanosine (oQ), galactosylguanosine (galQ), mannosylguanosine (manQ), 7-cyano-7-deazo-guanosine (preQ0), 7-aminomethyl-7-deazo-guanosine (preQ1), archapurin (G+), 7-deazo-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deazo-guanosine, 6-thio-7-deazo-8-aza-guanosine, 7-methyl-guanosine (m7G), 6-thio-7-methyl-guanosine, 7-methyl-inosine, 6-methoxy-guanosine, 1 α-Methyl-guanosine (m1G), N2-methyl-guanosine (m2G), N2,N2-dimethyl-guanosine (m22G), N2,7-dimethyl-guanosine (m2,7G), N2,N2,7-dimethyl-guanosine (m2,2,7G), 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, N2,N2-dimethyl-6-thio-guanosine, α-thio-guanosine, 2'-O-methyl-guanosine (Gm), N2-methyl-2'- O-methyl-guanosine (m2Gm), N2,N2-dimethyl-2'-O-methyl-guanosine (m22Gm), 1-methyl-2'-O-methyl-guanosine (m1Gm), N2,7-dimethyl-2'-O-methyl-guanosine (M2,7Gm), 2'-O-methyl-inosine (Im), 1,2'-O-dimethyl-inosine (m1Im), 2'-O-ribosylguanosine (phosphate ester) (Gr(p)), 1-thio-guanosine, O6-methyl-guanosine, 2'-F-arose-guanosine and 2'-F-guanosine.

[0260] In some implementations, the modified nucleotides in the mRNA of any of the above implementations include isotopic nucleotides.

[0261] In some embodiments, the mRNA of any of the above embodiments comprises a nucleotide containing a hydrogen isotope. The hydrogen isotope is not limited to deuterium or tritium. Additionally, in some embodiments, the mRNA of any of the above embodiments also comprises or contains nucleotides containing isotopes of elements other than hydrogen, wherein these other elements include, but are not limited to, carbon, oxygen, nitrogen, and phosphorus.

[0262] In some embodiments, the mRNA further includes a microRNA binding site. The microRNA binding site is used to regulate the expression of the mRNA, for example, to increase or decrease the expression of the mRNA it contains, preferably to decrease the expression of the mRNA in undesirable cells and / or tissues.

[0263] microRNAs (or miRNAs) are small non-coding RNAs that can induce post-transcriptional silencing of specific genes in cells by inhibiting translation or by degrading target mRNAs. MicroRNAs possess a seed sequence, typically the nucleotide sequence from position 2 to 8 of the 5' end of the microRNA. Exemplary microRNAs and their sequences can be found in US2005261218 and US2005059005. A microRNA binding site is a polynucleotide capable of binding to microRNA, enabling the microRNA to bind to the mRNA containing the microRNA binding site.

[0264] In some implementations, the microRNA binding site is sufficiently complementary to the microRNA.

[0265] Sufficient complementarity between the microRNA binding site and the microRNA means that the complementarity between the microRNA and the microRNA binding site is sufficient to achieve microRNA-mediated mRNA regulation. For example, microRNA-mediated translational repression or degradation of mRNA. In some embodiments, sufficient complementarity between the microRNA binding site and the microRNA means that the complementarity between the microRNA binding site and the microRNA binding site is sufficient to achieve microRNA-mediated mRNA degradation. For example, microRNA-guided RISC-mediated mRNA cleavage. For example, the microRNA binding site is complementary to a microRNA that is 19–25 or 19–23 (e.g., 22) nucleotides long. In some embodiments, the microRNA binding site is complementary to only a portion of the microRNA. For example, complementary to a portion that is 1, 2, 3, or 4 nucleotides shorter than naturally occurring microRNA. When the desired regulation is mRNA degradation, complete complementarity (e.g., complete complementarity to all or a significant portion of the natural microRNA) is preferred.

[0266] In some embodiments, the microRNA binding site comprises a polynucleotide that is sufficiently complementary to the microRNA (e.g., partially or completely complementary). In some embodiments, the microRNA binding site comprises a sequence that is completely complementary to the microRNA sequence.

[0267] In some embodiments, the microRNA binding site comprises a polynucleotide that is sufficiently complementary (e.g., partially or completely complementary) to the seed sequence of the microRNA. In some embodiments, the microRNA binding site comprises a polynucleotide that is completely complementary to the seed sequence of the microRNA.

[0268] In some implementations, the microRNA binding site is completely complementary to the microRNA sequence, except for 1, 2, or 3 nucleotide substitutions, end additions, and / or truncations.

[0269] A microRNA binding site is a polynucleotide or a variant thereof that is complementary to a full-length microRNA or a portion of a microRNA. The variant retains the ability of the microRNA binding site to bind to microRNA, enabling the binding of microRNA to the mRNA containing the microRNA binding site. The microRNA binding site can be completely complementary to a full-length microRNA or a portion of a microRNA, or it can be incompletely complementary.

[0270] In some embodiments, the microRNA binding site contains a polynucleotide complementary to the seed sequence of the microRNA or does not contain a polynucleotide complementary to (all or part of) the seed sequence of the microRNA. In some embodiments, the microRNA binding site is a polynucleotide that is completely complementary to the full-length microRNA.

[0271] In some implementations, the mRNA may also contain one or more microRNA binding sites.

[0272] In some implementations, the mRNA described above contains a microRNA binding site.

[0273] In some embodiments, the mRNA contains multiple microRNA binding sites. For example, the mRNA may contain two, three, four, five, six, seven, eight, nine, or ten microRNA binding sites. In some embodiments, the mRNA may contain three microRNA binding sites.

[0274] In some embodiments, the mRNA includes a 3'-UTR and a microRNA binding site, the microRNA binding site being located within the 3'-UTR. In some embodiments, the mRNA includes a 3'-UTR and a microRNA binding site, the location of which is selected from one or more of the following: located within the 3'-UTR and near the 5' end of the 3'-UTR, located in the region between the 5' and 3' ends of the 3'-UTR, and located within the 3'-UTR and near the 3' end of the 3'-UTR.

[0275] In some embodiments, the mRNA includes a 5'-UTR and a microRNA binding site, the microRNA binding site being located within the 5'-UTR. In some embodiments, the mRNA includes a 5'-UTR and a microRNA binding site, the location of which is selected from one or more of the following: located within the 5'-UTR and near the 5' end of the 5'-UTR, located in the region between the 5' end and the 3' end of the 5'-UTR, and located within the 5'-UTR and near the 3' end of the 5'-UTR.

[0276] In some embodiments, the mRNA includes a poly(A) tail and a microRNA binding site located within the poly(A) tail. In some embodiments, the mRNA includes a poly(A) tail and multiple microRNA binding sites located within the poly(A) tail. In some embodiments, the mRNA includes a poly(A) tail and three microRNA binding sites located within the poly(A) tail.

[0277] In some embodiments, the mRNA includes a 5'-UTR, a 3'-UTR, a poly(A) tail, and a microRNA binding site, wherein the microRNA binding site is located in the 5'-UTR, in the 3'-UTR, or after the 3'-UTR and before the poly(A) tail.

[0278] In some implementations, the mRNA includes a 5'-UTR, a 3'-UTR, a poly(A) tail, and a microRNA binding site located in the poly(A) tail.

[0279] In some embodiments, the mRNA comprises a 5'-UTR, a 3'-UTR, and a microRNA binding site, the location of which is selected from one of the following: located in the 3'-UTR and near the 5' end of the 3'-UTR, located in the region between the 5' end and the 3' end of the 3'-UTR, located in the 3'-UTR and near the 3' end of the 3'-UTR, located in the 5'-UTR and near the 5' end of the 5'-UTR, located in the region between the 5' end and the 3' end of the 5'-UTR, and located in the 5'-UTR and near the 3' end of the 5'-UTR.

[0280] In some embodiments, the mRNA comprises a 5'-UTR, a 3'-UTR, and multiple microRNA binding sites, the locations of which are selected from one or more of the following: located in the 3'-UTR and near the 5' end of the 3'-UTR, located in the region between the 5' and 3' ends of the 3'-UTR, located in the 3'-UTR and near the 3' end of the 3'-UTR, located in the 5'-UTR and near the 5' end of the 5'-UTR, located in the region between the 5' and 3' ends of the 5'-UTR, and located in the 5'-UTR and near the 3' end of the 5'-UTR. For example, the multiple microRNA binding sites are all located in the 3'-UTR and near the 3' end of the 3'-UTR, all located in the region between the 5' and 3' ends of the 3'-UTR, all located in the region between the 5' and 3' ends of the 5'-UTR, or all located in the 5'-UTR and near the 3' end of the 5'-UTR. For example, some of the multiple microRNA binding sites may be located in the 5'-UTR and near its 5' end, while the remaining portion may be located in the 3'-UTR and near its 3' end. Alternatively, some of the multiple microRNA binding sites may be located in the 5'-UTR and near its 5' end, some may be located in the region between the 5' and 3' ends of the 5'-UTR, and the remaining portion may be located in the 3'-UTR and near its 3' end. It is understood that the location of multiple microRNA binding sites is not limited to the examples above and can also be a combination of other locations.

[0281] In some implementations, the mRNA contains multiple microRNA binding sites (e.g., 3 microRNA binding sites), which may be the same or different.

[0282] In some embodiments, the mRNA contains multiple microRNA binding sites, and these multiple microRNA binding sites are identical. For example, in some embodiments, the mRNA contains three microRNA binding sites that can specifically bind to miR142, and these three microRNA binding sites that can specifically bind to miR142 are completely identical.

[0283] In some implementations, the mRNA contains multiple microRNA binding sites, which are different from each other, and the multiple microRNA binding sites bind to the same microRNA or different microRNAs.

[0284] In some implementations, the mRNA contains multiple microRNA binding sites, which are not identical and bind to the same microRNA. For example, the mRNA contains three microRNA binding sites that specifically bind to miR142. The first of these three sites specifically binds to the 5' end of miR142, the second binds specifically to the 3' end of miR142, and the third is identical, partially identical, or completely different from one of the aforementioned two sites.

[0285] In some implementations, the mRNA contains multiple microRNA binding sites, which are not identical. These multiple microRNA binding sites bind to different microRNAs originating from the same cell and / or tissue, or from different cells and / or tissues. These multiple microRNA binding sites are used to increase or decrease the expression of their respective mRNAs in a specific cell or tissue, or to increase or decrease the expression of their respective mRNAs in several specific cells and / or tissues.

[0286] In some embodiments, the multiple microRNA binding sites include different microRNA binding sites capable of specifically binding to different microRNAs expressed in the same tissue or the same type of cells. For example, the mRNA described above contains three microRNA binding sites, all of which are capable of specifically binding to microRNAs specifically expressed in immune cells. For example, the first of the three microRNA binding sites is specifically capable of binding to miR-142 specifically expressed in immune cells, the second is specifically capable of binding to miR-125 specifically expressed in immune cells, and the third is specifically capable of binding to miR-142 specifically expressed in immune cells, miR-125 specifically expressed in the liver, or other microRNAs specifically expressed in immune cells.

[0287] In some implementations, multiple microRNA binding sites include different microRNAs that can specifically bind to at least two different tissues or different cell types.

[0288] In some implementations, the microRNA binding site can reduce the expression of the aforementioned mRNA in undesirable cells and / or tissues.

[0289] In some implementations, the microRNA binding site can reduce the expression of the aforementioned mRNA in immune cells.

[0290] In some implementations, the microRNA binding site can reduce the immune response generated by the aforementioned mRNA or lipid nanoparticles containing the aforementioned mRNA.

[0291] In some implementations, the microRNA binding site can reduce or inhibit the production of anti-drug antibodies (ADAs) by proteins expressing the aforementioned mRNA.

[0292] In some implementations, the microRNA binding site can reduce or inhibit the accelerated blood clearance phenomenon of lipid nanoparticles delivering the aforementioned mRNA.

[0293] In some implementations, the microRNA binding site is sufficiently complementary to the microRNA expressed in immune cells to reduce the expression of the aforementioned mRNA in immune cells.

[0294] In some implementations, the microRNAs expressed in immune cells include, but are not limited to, one or more of the following: hsa-let-7a-2-3p, hsa-let-7a-3p, hsa-7a-5p, hsa-let-7c, hsa-let-7e-3p, hsa-let-7e-5p, hsa-let-7g-3p, hsa-let-7g-5p, hsa-let-7i-3p, hsa-let-7i-5p, miR-10a-3p, miR-10a-5p, miR-1184, hsa-let-7f-l-3p, hsa-let-7f-2~5p, hsa-let -7f-5p,miR-125b-l-3p,miR-125b-2-3p,miR-125b-5p,miR-1279,miR-130a-3p,miR-130a-5p,miR-132-3p,miR-132-5p,miR-142-3p,miR-142-5p, miR-143-3p,miR-143-5p,miR-146a-3p,miR-146a-5p,miR-146b-3p,miR-146b-5p,miR-147a,miR-147b,miR-148a-5p,miR-148a-3p,miR-150-3p,m iR-150-5p,miR-151b,miR-155-3p,miR-155-5p,miR-15a-3p,miR-15a-5p,miR-15b-5p,miR-15b-3p,miR-16-l-3p,miR-16-2-3p,miR-16-5p,miR-1 7-5p,miR-181a-3p,miR-181a-5p,miR-181a-2-3p,miR-182-3p,miR-182-5p,miR-197-3p,miR-197-5p,miR-21-5p,miR-21-3p,miR-214-3p,miR-21 4-5p,miR-223-3p,miR-223-5p,miR-221-3p,miR-221-5p,miR-23b-3p,miR-23b-5p,miR-24-l-5p,miR-24-2-5p,miR-24-3p,miR-26a-l-3p,miR-26 a-2-3p,miR-26a-5p,miR-26b-3p,miR-26b-5p,miR-27a-3p,miR-27a-5p,miR-27b-3p,miR-27b-5p,miR-28-3p,miR-28-5p,miR-2909,miR-29a-3p,miR-29a-5p,miR-29b-l-5p,miR-29b-2-5p,miR-29c-3p,miR-29c-5p,miR-30e-3p,miR-30e-5p,miR-331-5p,mi R-339-3p,miR-339-5p,miR-345-3p,miR-345-5p,miR-346,miR-34a-3p,miR-34a-5p,miR-363-3p,miR-363-5p, miR-372, miR-377-3p, miR-377-5p, miR-493-3p, miR-493-5p, miR-542, miR-548b-5p, miR-548c-5p, miR-548i, miR-548j, miR-548n, miR-574-3p, miR-598, miR-718, miR-935, miR-99a-3p, miR-99a-5p, miR-99b-3p, and miR-99b-5p. In addition, microRNAs expressed in immune cells include those identified by Jima DD et al., Blood, 2010, 116: el18-el27 and Vaz C et al., BMC Genomics, 2010, 11, 288.

[0295] In some embodiments, the mRNA contains multiple microRNA binding sites, which are linked by a linker element (e.g., "GCTG"). It is understood that in other embodiments, the multiple microRNA binding sites may be directly linked.

[0296] In some embodiments, the microRNA binding site is the miR142 binding site. In some embodiments, the nucleotide sequence of the miR142 binding site is shown in SEQ ID NO: 49.

[0297] In some implementations, the mRNA contains three miR142 binding sites located in the 5'-UTR, the 3'-UTR, or after the 3'-UTR and before the poly(A) tail.

[0298] In some embodiments, the mRNA contains three miR142 binding sites located in a poly(A) tail. In some embodiments, the nucleotide sequence of the poly(A) tail containing the three miR142 binding sites is shown in SEQ ID NO: 46, 47 or 48.

[0299] In some embodiments, the nucleotide sequence of the polynucleotide encoding the ATP7B protein of the above-mentioned mRNA is shown in SEQ ID NO: 10, and the nucleotide sequence of the poly(A) tail containing the microRNA binding site is shown in SEQ ID NO: 47.

[0300] In some implementations, the above-mentioned mRNA contains an open reading frame (ORF).

[0301] In some embodiments, the mRNA further includes a stop codon. It is understood that in other embodiments, the mRNA does not contain a stop codon. When using the mRNA without a stop codon, those skilled in the art will know that a stop codon (e.g., UGA, UAA, etc.) should be added at an appropriate position. It is understood that the mRNA may contain one or more stop codons.

[0302] In some implementations, the mRNA may further contain polynucleotides encoding proteins or polypeptides other than ATP7B. It is understood that proteins or polypeptides other than ATP7B refer to proteins or polypeptides with biological significance (e.g., immunogenicity, prophylaxis, therapeutic effect, etc.). It is also understood that in the mRNA, both the polynucleotide encoding ATP7B and the polynucleotide encoding other proteins or polypeptides may have multiple (e.g., two or three) repeating units on the same strand. For example, for the polynucleotide encoding ATP7B, if the polynucleotide encoding ATP7B is simply referred to as "fragment A," then the mRNA may contain multiple fragments A. As another example, for polynucleotides encoding both ATP7B and other proteins or polypeptides, if the polynucleotide encoding ATP7B is simply referred to as "fragment A" and the polynucleotide encoding other proteins or polypeptides is simply referred to as "fragment B," then the mRNA may contain multiple fragments A and multiple fragments B, one fragment A and multiple fragments B, or multiple fragments A and one fragment B.

[0303] Furthermore, the polynucleotide encoding the ATP7B protein in the aforementioned mRNA can be a polynucleotide encoding one ATP7B protein or a polynucleotide encoding multiple ATP7B proteins. The polynucleotide encoding the ATP7B protein in the aforementioned mRNA can be one or more polynucleotides encoding the full-length ATP7B protein, one or more polynucleotides encoding a truncated ATP7B protein, or a combination thereof. For example, in some embodiments, the polynucleotide in the aforementioned mRNA encoding the truncated ATP7B protein is a polynucleotide encoding the protein following the deletion of amino acids 257 to 355 of SEQ ID NO: 1. In other embodiments, the aforementioned mRNA contains a polynucleotide encoding the truncated ATP7B protein that encodes the protein following the deletion of amino acids 257 to 355 of SEQ ID NO: 1 (referred to as fragment A1) and a polynucleotide encoding the protein following the deletion of amino acids 57 to 140 and 237 to 485 of SEQ ID NO: 1 (referred to as fragment A2). Of course, in some implementations, the above mRNA may contain multiple A1 fragments and multiple A2 fragments, one A1 fragment and multiple A2 fragments, or multiple A1 fragments and one A2 fragment.

[0304] In some implementations, the mRNA described above is isolated mRNA.

[0305] In some embodiments, the mRNA described above is a non-replicating mRNA. In other embodiments, the mRNA described above is a self-replicating mRNA.

[0306] In some embodiments, the mRNAs mentioned above are the mRNAs listed in Table 1. The mRNAs in Table 1 are mRNAs encoding the ATP7B protein, which contain a Cap1-type cap structure, a 5'-UTR, a polynucleotide encoding the ATP7B protein, a 3'-UTR, and a poly(A) tail. All uridines in all mRNAs in Table 1 are N1-methylpseudouridines. For example, the amino acid sequence of the ATP7B protein encoded by the mRNA numbered 1932 is shown in SEQ ID NO: 1. The mRNA numbered 1932 contains a Cap1-type cap structure, a 5'-UTR as shown in SEQ ID NO: 3, a polynucleotide encoding the ATP7B protein as shown in SEQ ID NO: 8, a 3'-UTR as shown in SEQ ID NO: 4, and a poly(A) tail as shown in SEQ ID NO: 7. All uridines in the mRNA numbered 1932 are N1-methylpseudouridines. In addition, the mRNAs numbered 1691, 1695, 1813, 1644, 1466, 1814, 1816, 1812 and 1888 in Table 1 also contain microRNA binding sites with nucleotide sequences as shown in SEQ ID NO: 45 between their 3'-UTR and poly(A) tail.

[0307] In Table 1, “ / ” indicates that the ATP7B protein encoded by the mRNA has no amino acid deletions or no MBD deletions, and its amino acid sequence is shown in SEQ ID NO:1.

[0308] In some implementations, the aforementioned mRNA can alleviate, improve, or reduce the severity of one or more symptoms of WD. For example, it can be used to increase and / or restore whole blood ceruloplasmin synthesis and / or copper excretion in bile.

[0309] In some implementations, the mRNA can increase the expression level of ATP7B protein in test subjects (e.g., WD patients) by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, or 500% compared to before administration.

[0310] In some implementations, the mRNA can increase the activity of the ATP7B protein in test subjects (e.g., WD patients) by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, or 500% compared to before administration.

[0311] In some implementations, compared with the mRNA administered above, the mRNA can increase the ability of the ATP7B protein to transport copper in test subjects (e.g., WD patients) by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, or 500%.

[0312] In some implementations, compared with before administration of the above-mentioned mRNA, the above-mentioned mRNA can reduce the copper content in the liver of test subjects (e.g., WD patients) by at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%.

[0313] In some implementations, the mRNA can increase the level of ceruloplasmin in the serum of test subjects (e.g., WD patients) by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, or 500% compared to before administration.

[0314] In some implementations, compared with before administration of the above-mentioned mRNA, the above-mentioned mRNA can reduce the level of alanine transaminase (ALT) in the serum of test subjects (e.g., WD patients) by at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%.

[0315] In some implementations, compared with before administration of the above-mentioned mRNA, the above-mentioned mRNA can reduce the level of aspartate aminotransferase (AST) in the serum of test subjects (e.g., WD patients) by at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%.

[0316] III. DNA, gene engineering vectors, host cells, DNA preparation methods, and ATP7B protein preparation methods.

[0317] This disclosure provides a DNA for preparing mRNA according to any of the above embodiments.

[0318] This disclosure also provides a DNA that can be transcribed into mRNA according to any of the above embodiments.

[0319] This disclosure also provides a DNA that can be transcribed and post-transcribed (e.g., capped) into mRNA according to any of the above embodiments.

[0320] In some implementations, the DNA contains an ORF.

[0321] In some implementations, the DNA described above can be transcribed in vitro and processed post-transcriptionally (e.g., capping) to form one of the mRNAs listed in Table 1.

[0322] In some embodiments, the DNA comprises at least one of a 5'-UTR, a 3'-UTR, and a polynucleotide encoding a poly(A) tail, and a DNA sequence corresponding to the polynucleotide encoding the ATP7B protein in any of the above embodiments.

[0323] In some embodiments, the DNA comprises a 5'-UTR, a 3'-UTR, and a polynucleotide encoding a poly(A) tail, as well as a DNA sequence corresponding to the polynucleotide encoding the full-length human ATP7B protein of any of the above embodiments. Preferably, the DNA sequence corresponding to the polynucleotide encoding the full-length human ATP7B protein is one of the following: (1) a DNA sequence corresponding to the polynucleotide as shown in any one of SEQ ID NO: 8 to 10; and (2) a DNA sequence corresponding to the polynucleotide that has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and less than 100% identity with the nucleotide sequence as shown in any one of SEQ ID NO: 8 to 10 and encodes an amino acid sequence as shown in SEQ ID NO: 1 for the ATP7B protein.

[0324] In some embodiments, the DNA comprises at least one of a 5'-UTR, a 3'-UTR, and a polynucleotide encoding a poly(A) tail, and a DNA sequence corresponding to a polynucleotide encoding the truncated human ATP7B protein of any of the above embodiments. Preferably, the DNA sequence corresponding to the polynucleotide encoding the truncated human ATP7B protein is a DNA sequence corresponding to a polynucleotide as shown in any of SEQ ID NO: 26-44.

[0325] In addition, this disclosure also provides a gene engineering vector, which contains DNA of any of the above embodiments, or the gene engineering vector contains a polynucleotide capable of being transcribed into mRNA of any of the above embodiments, or the gene engineering vector can be transcribed or processed by transcription and post-transcriptional processing into mRNA of any of the above embodiments.

[0326] In some embodiments, the genetic engineering vector is an expression vector. In some embodiments, the genetic engineering vector is a plasmid, a granule, a virus (e.g., adenovirus, adeno-associated virus), a bacteriophage, or another vector conventionally used in genetic engineering. In one optional specific example, the genetic engineering vector is a plasmid. In one optional specific example, the genetic engineering vector is adeno-associated virus (AAV). In some embodiments, the genetic engineering vector further comprises at least one or more of the following: origin of replication (ORI), a marker gene or a fragment thereof, a reporter gene or a fragment thereof, and a restriction site allowing the insertion of a DNA element. In one optional specific example, the restriction site allowing the insertion of a DNA element is a multiple cloning site (MCS).

[0327] In some embodiments, the above-mentioned genetic engineering vector comprises a promoter, a 5'-UTR, a DNA sequence corresponding to the polynucleotide encoding the ATP7B protein in any of the above embodiments, a 3'-UTR, and a polynucleotide encoding a poly(A) tail, wherein the polynucleotide encoding the poly(A) tail, the promoter, the 5'-UTR, the DNA sequence corresponding to the polynucleotide encoding the ATP7B protein, and the 3'-UTR are operatively linked to each other.

[0328] In some embodiments, the above-mentioned genetic engineering vector comprises a promoter, a 5'-UTR, a DNA sequence corresponding to the polynucleotide encoding the ATP7B protein in any of the above embodiments, a 3'-UTR, a microRNA binding site, and a polynucleotide encoding a poly(A) tail, wherein the polynucleotide encoding the poly(A) tail, the promoter, the 5'-UTR, the DNA sequence corresponding to the polynucleotide encoding the ATP7B protein, the microRNA binding site, and the 3'-UTR are operatively linked to each other.

[0329] In other implementations, the aforementioned genetic engineering vector is a cloning vector.

[0330] This disclosure also provides a method for preparing DNA according to any of the above embodiments, the method comprising the steps of introducing (e.g., in plasmid form) a genetic engineering vector according to any of the above embodiments into a host cell (e.g., Escherichia coli) and then culturing the host cell containing the genetic engineering vector.

[0331] In addition, this disclosure also provides another method for preparing the above-described DNA, which includes the step of preparing the DNA using a chemical synthesis method based on the nucleotide sequence of the DNA according to any of the above embodiments. It is understood that the specific method of chemical synthesis can be a method known in the art, such as the solid-phase phosphorus amide method.

[0332] It is understood that the method for preparing DNA in any of the above embodiments is not limited to the above, and may also be other methods.

[0333] In addition, this disclosure also provides a host cell comprising RNA of any of the above embodiments, DNA of any of the above embodiments, or a genetic engineering vector of any of the above embodiments.

[0334] In some implementations, the host cell is a separated cell.

[0335] In some implementations, the host cell is used to store and / or amplify the DNA of any of the above implementations.

[0336] In some implementations, the host cell is a bacterial cell. Bacterial host cells include *Escherichia coli* (E. coli) cells, which are well-known to those skilled in the art.

[0337] The host cells of this disclosure can be prepared by transforming competent host cells with a genetically engineered vector according to any of the above embodiments. Competent host cells are cells capable of taking up free extracellular genetic material (e.g., DNA plasmids) in a sequence-independent manner. Many bacterial cells known to those skilled in the art are naturally capable of taking up exogenous DNA from the environment and can therefore serve as bacterial host cells according to this disclosure. Furthermore, those skilled in the art know that competent bacterial host cells can be obtained from naturally non-competent bacterial cells using methods such as electroporation or chemicals (e.g., treatment with calcium ions accompanied by high-temperature exposure). After take-up, the exogenous DNA preferably neither degrades nor integrates into the genome of the bacterial host cell.

[0338] In addition, this disclosure also provides a truncated ATP7B protein encoded by mRNA of any of the above embodiments.

[0339] In addition, this disclosure also provides a method for preparing a truncated ATP7B protein, the method comprising: transcribing a polynucleotide (e.g., DNA or a genetic engineering vector) encoding the truncated ATP7B protein into RNA; and translating the transcribed RNA into the truncated ATP7B protein.

[0340] In addition, this disclosure also provides another method for preparing a truncated ATP7B protein, the method comprising: translating the mRNA encoding the truncated ATP7B protein of any of the above embodiments into a truncated ATP7B protein.

[0341] In some embodiments, the preparation of the truncated ATP7B protein in any of the above embodiments is carried out entirely or partially in vitro.

[0342] IV. mRNA Preparation Methods

[0343] This disclosure also provides a method for preparing mRNA, which includes the step of transcribing the DNA of any of the above embodiments or the gene engineering vector of any of the above embodiments.

[0344] In some implementations, the above-mentioned mRNA is prepared by an in vitro method.

[0345] In some embodiments, the method for preparing the above-described mRNA includes contacting a linearized genetic engineering vector (e.g., a plasmid) of any of the above embodiments with an RNA polymerase.

[0346] In some implementations, the above-mentioned method for preparing mRNA further includes a step of linearizing the genetic engineering vector (e.g., plasmid).

[0347] In some implementations, the supercoilability of the genetic engineering vector (e.g., plasmid) is at least 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, etc.) before linearization.

[0348] In some implementations, the method for preparing mRNA further includes the step of purifying the linearized genetic engineering vector.

[0349] In some implementations, the above-mentioned method for preparing mRNA also includes a step of purifying RNA.

[0350] This disclosure also provides another method for preparing mRNA, which includes the step of preparing mRNA using a chemical synthesis method based on the mRNA sequence according to any of the above embodiments. It is understood that the specific method of chemical synthesis can be a method known in the art, such as the solid-phase phosphorusamide method.

[0351] In some embodiments, the method for preparing any mRNA disclosed herein further includes the steps of capping and optionally purifying the capped product.

[0352] In some implementations, the cap is a Cap1 type cap. The Cap1 type cap has the following structure: cap G 1 G 2 =m 7 G-5'-ppp-5'-Gm2'-3'-p-[m7=7-CH3; m2'=2'-O-CH3; -ppp-=-PO2H-O-PO2H-O-PO2H)-; -p-=-PO2H-].

[0353] The capping reaction is shown below: pppN1(p)Nx-OH(3')→ppN1(pN)x-OH(3')+Pi ppN1(pN)x-OH(3')+GTP→G(5')ppp(5')N1(pN)x-OH(3')+PPi G(5')ppp(5')N1(pN)x-OH(3')+AdoMet→m7G(5')ppp(5')N1(pN)x-OH(3')+AdoHyc m7GpppN1(pN)x-OH(3')+AdoMet→m7Gppp[m2'-O]N1(pN)x-OH(3')+AdoHyc.

[0354] In other embodiments, the method for preparing the above-described mRNA is partially in vitro. The method for preparing the mRNA in this case includes the following steps: preparing a gene-engineered vector according to any of the above embodiments in vitro; and introducing the gene-engineered vector into the body (e.g., in plasmid form). In some embodiments, the gene-engineered vector is encapsulated in a delivery vector. In this case, the gene-engineered vector can be delivered into the body via the delivery vector.

[0355] In some embodiments, the mRNA prepared in the mRNA preparation method of any of the above embodiments contains a modified nucleoside or a modified nucleotide. Correspondingly, the raw materials for preparing this RNA include one or more modified nucleosides or nucleotides. It is understood that the amount and type of modified nucleosides or nucleotides correspond to the RNA to be prepared.

[0356] In addition, this disclosure also provides an mRNA prepared by the method for preparing mRNA according to any of the above embodiments.

[0357] V. Pharmaceutical Compositions

[0358] This disclosure also provides a pharmaceutical composition comprising mRNA of any of the above embodiments, DNA of any of the above embodiments, a genetically engineered vector of any of the above embodiments, a truncated ATP7B protein of any of the above embodiments, or a host cell of any of the above embodiments.

[0359] In some embodiments, the pharmaceutical compositions described above further comprise a pharmaceutically acceptable carrier. As used herein, the term "pharmaceuticalally acceptable" means approved for use in animals and / or humans by a regulatory authority (e.g., the China Food and Drug Administration, the U.S. Food and Drug Administration) or a recognized pharmacopoeia (e.g., the Chinese Pharmacopoeia, the European Pharmacopoeia). The term "pharmaceuticalally acceptable carrier" refers to a substance that can be administered with the mRNA, DNA, genetically engineered vector, host cell, or truncated ATP7B protein of this disclosure, including but not limited to delivery carriers, diluents, sweeteners, flavoring agents, wetting agents, adjuvants, gliding agents, preservatives, dyes / coloring agents, surfactants, dispersants, suspending agents, stabilizers, isotonic agents, solvents, or emulsifiers.

[0360] In some embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable vector and one or more of the above-described embodiments of mRNA, one or more of the above-described embodiments of DNA, one or more of the above-described embodiments of genetically engineered vectors, or one or more of the above-described embodiments of truncated ATP7B protein.

[0361] In some embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable carrier and an mRNA of any of the above embodiments, a DNA of any of the above embodiments, a genetically engineered vector of any of the above embodiments, or a truncated ATP7B protein of any of the above embodiments.

[0362] In some embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable carrier and mRNA of any of the above embodiments, DNA of any of the above embodiments, genetically engineered vector of any of the above embodiments, or truncated ATP7B protein of any of the above embodiments.

[0363] In some implementations, pharmaceutically acceptable carriers include delivery carriers.

[0364] In some embodiments, pharmaceutically acceptable vectors include delivery vectors, wherein the mRNA of any of the above embodiments, the DNA of any of the above embodiments, the genetically engineered vector of any of the above embodiments, or the truncated ATP7B protein of any of the above embodiments is formulated in the delivery vector.

[0365] In some embodiments, the delivery carrier is selected from a combination or one of the following: lipid nanoparticles (LNPs), liposomes, cationic proteins, vesicles, microparticles, polymers, and micelles. In some embodiments, the delivery carrier is selected from one of the following: lipid nanoparticles, liposomes, cationic proteins, vesicles, microparticles, polymers, and micelles.

[0366] In some implementations, the delivery carrier is LNPs.

[0367] In some implementations, LNPs refer to particles having a nanoscale (e.g., 1 nm to 1000 nm) size, which include one or more lipids.

[0368] In some implementations, the average diameter of LNPs is 20nm–800nm, 20nm–500nm, 20nm–400nm, 20nm–300nm, 20nm–200nm, 20nm–100nm, 30nm–700nm, 30nm–500nm, 30nm–300nm, 30nm–200nm, 30nm–100nm, 40nm–800nm, 40nm–600nm, 40nm–500nm, 40nm–300nm, etc. 0nm, 40nm~200nm, 40nm~100nm, 50nm~800nm, 50nm~600nm, 50nm~500nm, 50nm~400nm, 50nm~300nm, 50nm~200nm, 50nm~100nm, 60nm~800nm, 60nm~600nm, 60nm~500nm, 60nm~400nm, 60nm~300nm, 60nm~200nm or 60nm~100nm. In some optional specific examples, the average diameter of LNPs is 26nm, 31nm, 36nm, 41nm, 46nm, 51nm, 56nm, 61nm, 66nm, 71nm, 76nm, 81nm, 86nm, 91nm, 96nm, 101nm, 106nm, 111nm, 116nm, 121nm, 126nm, 131nm, 136nm, 141nm, 146nm, 151nm, 156nm, 161nm, 166nm, 171nm, 176nm, 181nm, 186nm, 191nm, 196nm, 201nm, 206nm, 211nm, 216nm, 221nm, 226nm, 231nm, 236nm, 241nm, 246nm, or 249nm. In this paper, the average diameter of LNPs can be represented by the z-average value determined by dynamic light scattering.

[0369] In some embodiments, LNPs include one of the following: cationic lipid nanoparticles, solid lipid nanoparticles (SLN), nanostructured lipid carriers (NLC), and nonlamellar lipid nanoparticles. In one optional specific example, LNPs are cationic lipid nanoparticles.

[0370] In some embodiments, LNPs contain one or more of the following substances: ionizable lipids (i.e., cationic lipids), accessory lipids, structural lipids, and polymer-lipids. The term "ionizable lipid" refers to a lipid that becomes positively charged when the pH drops below the pKa of its ionizable group, but gradually becomes neutral at higher pH values. Below the pKa, the positively charged lipid can bind to negatively charged nucleic acids. In some embodiments, ionizable lipids include zwitterionic lipids.

[0371] In some embodiments, ionizable lipids include the following compounds (IV), their N-oxides, their salts, or isomers thereof:

[0372] Wherein, A1 is H, C1-C5 hydrocarbon group or C1-C5 heterohydrocarbon group, A2 is H, C1-C5 hydrocarbon group or C1-C5 heterohydrocarbon group, and A3 is C1-C5 alkylene group or bond;

[0373] A4 is a C1-C5 hydrocarbon group or bond;

[0374] A5, A6, A7, and A8 are independently C1-C 18 Hydroxyl group, C1-C 18 heteroalkyl groups or bonds;

[0375] Q1 and Q2 are independently -C(=O)O- and -OC(=O)-;

[0376] Each Z9 is independently C1-C 24 Hydrocarbon group or C1-C containing O or S 24 heterohydrocarbon group.

[0377] Each Z 11 Independently for C1-C 24 Hydrocarbon group or C1-C containing O or S 24 heterohydrocarbon group.

[0378] In some embodiments, each hydrocarbon group in formula (IV) is independently an alkyl, alkenyl, or alkynyl group.

[0379] In some embodiments, each of the alkylene groups in formula (IV) is independently an alkylene group, an alkenylene group, or an alkynylene group.

[0380] In some embodiments, each heteroalkyl group in formula (IV) is independently a heteroalkyl, heteroalkenyl, or heteroynyl group.

[0381] In some embodiments, each heteroalkyl group in formula (IV) is independently a heteroalkyl, heteroalkenyl, or heteroyne group.

[0382] In some embodiments, in the compound represented by formula (IV), Q1 and Q2 are -C(=O)O-.

[0383] In some embodiments, in the compound represented by formula (IV), Q1 and Q2 are -C(=O)O-, A4 is a bond, and A5 and A6 are independently C3-C 10 Alkylenes, A7 and A8 are independently C2-C4 alkylenes, each Z9 is independently C3-C9 alkyl, each Z 11 A1 and A2 are independently C3-C9 alkyl groups, A3 is a C2-C4 alkylene group.

[0384] Ionizable lipids include compounds (IV), or their N-oxides, salts, or isomers thereof, and, where applicable, include one or more of the following characteristics.

[0385] In some implementations, A4 is the bond, and Q1 and Q2 are C(=O)O-.

[0386] In some implementations, A5 and A6 are independently C4-C9 alkylene groups.

[0387] In some implementations, A7 and A8 are independently C2-C4 alkylene groups.

[0388] In some embodiments, each Z9 is independently a C5-C8 alkyl group, such as C6, C8, C9 ... 7、 C8 alkyl.

[0389] In some implementation schemes, each Z 11 Independently C5-C8 alkyl, such as C6, C 7、 C8 alkyl.

[0390] In some embodiments, A1 and A2 are independently C1-C3 alkyl groups.

[0391] In some embodiments, A3 is a C2-C4 alkylene, such as a C3 alkylene.

[0392] In some embodiments, the ionizable lipid is compound 2-1, its salt, or an isomer thereof:

[0393] In some embodiments, the ionizable lipid is compound 2-2, its salt, or an isomer thereof:

[0394] In some implementations, the ionizable lipid is compound 2-3, its salt, or an isomer thereof:

[0395] In some embodiments, the ionizable lipid is compound 2-4, its salt, or an isomer thereof:

[0396] In some implementations, the ionizable lipids are compounds 2-5, their salts, or isomers thereof:

[0397] In some embodiments, the cofactor lipid comprises a phospholipid (phospholipid). Phospholipids are typically semi-synthetic, but may also be of natural origin or chemically modified. In one optional specific example, the cofactor lipid is a phospholipid. In some embodiments, the phospholipid comprises one or more of the following: DSPC (distearylphosphatidylcholine), DOPE (dioleoylphosphatidylethanolamine), DOPC (dioleoyllecithin), DOPS (dioleoylphosphatidylserine), DSPG (1,2-octacosanoyl-sn-glycerol-3-phosphate-(1'-rac-glycerol)), DPPG (dispalmitoylphosphatidylglycerol), DPPC (dispalmitoylphosphatidylcholine), DGTS (1,2-dispalmitoyl-sn-glycerol-3-O-4'-(N,N,N-trimethyl)homoserine), and lysophospholipids. In some embodiments, the cofactor lipid is selected from one or more of the following: DSPC, DOPE, DOPC, and DOPS. In some implementations, the assisting lipids are DSPC and / or DOPE.

[0398] In some embodiments, the structural lipid comprises sterols. In one optional specific example, the structural lipid is a sterol. In some embodiments, the sterol comprises one or more of the following: 20α-hydroxycholesterol, cholesterol, cholesterol esters, sterol hormones, sterol vitamins, bile acids, ergosterol, β-sitosterol, and oxidized cholesterol derivatives. In some embodiments, the structural lipid comprises at least one of cholesterol, cholesterol esters, sterol hormones, sterol vitamins, and bile acids. In some embodiments, the structural lipid is cholesterol. In one optional specific example, the structural lipid is high-purity cholesterol, particularly injectable high-purity cholesterol, such as CHO-HP (produced by AVT). In other embodiments, the structural lipid is 20α-hydroxycholesterol.

[0399] Polymer-lipid conjugates are complexes comprising a polymer and a lipid coupled to that polymer. Polymer-lipid conjugates (e.g., polyethylene glycol-lipid conjugates) in LNPs can improve the stability of LNPs in vivo.

[0400] In some embodiments, the lipids used to form the polymer-lipids of LNPs include one or more of the following: 1,2-dimyristoyl-sn-glycerol (DMG), distearoyl-phosphatidyl-ethanolamine (DSPE), diacylglycerol (DAG), dialkyloxypropyl (DAA), phospholipids, ceramide (Cer), 1,2-distearoyl-rac-glycerol (DSG), and 1,2-dipalmitoyl-rac-glycero (DPG).

[0401] In some embodiments, the polymer-lipid polymers used to form LNPs include one or two of the following: hydrophilic polymers and amphoteric polymers.

[0402] In some embodiments, the polymer-lipid polymer used to form LNPs is a hydrophilic polymer. In other embodiments, the polymer-lipid polymer used to form LNPs is an amphoteric polymer.

[0403] In some embodiments, the hydrophilic polymer includes one or more of the following: polyethylene glycol (PEG), poly(oxazolines) (POX), poly(glycerols) (PGs), poly(hydroxypropyl methacrylate) (PHPMA), poly(2-hydroxyethyl methacrylate) (PHEMA), poly(N-(2-hydroxypropyl)methacrylamide) (HPMA), polyvinylpyrrolidone (PVP), poly(N,N-dimethyl acrylamide) (PDMA), poly(N-acryloyl morpholine) (PAcM), polyamino acids, glycosaminoglycans (GAGs), heparin, and hyaluronic acid. The following are listed: HA (acid), polysialic acid (PSA), elastin-like polypeptides (ELPs), serum albumin, and CD47.

[0404] Correspondingly, polymer-lipids include one or more of the following: polyethylene glycol-lipids (PEG-lipids), polyoxazoline-lipids, polyglycerol-lipids, polyhydroxypropyl methacrylate-lipids, poly-2-hydroxyethyl methacrylate-lipids, poly-N-(2-hydroxypropyl)methacrylamide-lipids, polyvinylpyrrolidone-lipids, poly-N,N-dimethylacrylamide-lipids, poly-N-acryloylmorpholine-lipids, glycosaminoglycan-lipids, heparin-lipids, hyaluronic acid-lipids, polysialic acid-lipids, elastin-like lipids, serum albumin-lipids, and CD47-lipids. It should be noted that "PEG-lipids" are conjugates of polyethylene glycol and lipids, "polyoxazoline-lipids" refer to conjugates formed by coupling polyoxazoline and lipids, and "polyglycerol-lipids" refer to conjugates formed by coupling polyglycerol and lipids; the same applies to other polymer-lipids. In an optional specific example, the hydrophilic polymer includes polyethylene glycol.

[0405] In some embodiments, the polymer-lipid comprises a PEG-lipid. In one optional specific example, the polymer-lipid is a PEG-lipid. In some embodiments, the PEG-lipid comprises one or more of the following: myristoyl glycerol-PEG (DMG-PEG), distearate phosphatidylethanolamine-PEG (DSPE-PEG), diacylglycerol-PEG (DAG-PEG), dialkyloxypropyl-PEG (DAA-PEG), phospholipid-PEG, ceramide-PEG (Cer-PEG), 1,2-distearate-rac-glycerol-PEG (DSG-PEG), and 1,2-dispalmitoyl-rac-glycerol-PEG (DPG-PEG). The PEG-lipid is preferably DMG-PEG, DSG-PEG, or DPG-PEG. DMG-PEG is a polyethylene glycol derivative of 1,2-dimyristoyl glycerol. In some embodiments, the average molecular weight of the PEG in the PEG-lipid is about 2000 to 5000. In one optional specific example, the average molecular weight of PEG in the PEG-lipid is about 2000.

[0406] In some implementations, PEG-lipids can alleviate or prevent accelerated blood clearance (ABC).

[0407] In some embodiments, the PEG-lipid is a compound of formula (V), or a salt thereof, or a stereoisomer thereof:

[0408] in:

[0409] R 3 OR 0 ;

[0410] R 0 It is hydrogen, an optionally substituted alkyl group, or an oxygen protecting group;

[0411] g is an integer between 1 and 100;

[0412] L1 is an optional substituted C1-C 10 Alkylenes, wherein optional substituted C1-C 10 At least one methylene group of the alkylene group is independently substituted with a carbocyclic group, a heterocyclic group, an aryl group, a heteroaryl group, -O-, or -N(R) group. M -, -S-, -C(O)-, -C(O)N(R) M )-、-NR MC(O)-, -C(O)O-, -OC(O)-, -OC(O)O-, -OC(O)N(R M )-、-NR M C(O)O- or -NR M C(O)N(R M )-replace;

[0413] D represents a group obtained through click chemistry or a group that can be cleaved under physiological conditions;

[0414] h is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0415] A is

[0416] Each L2 is independently a C1-C6 alkylene group with a bond or optional substitution; optionally, one methylene group of the optionally substituted C1-C6 alkylene group is surrounded by -O-, -N(R) M -, -S-, -C(O)-, -C(O)N(R) M )-、-NR M C(O)-, -C(O)O-, -OC(O)-, -OC(O)O-, -OC(O)N(R M )-、-NR M C(O)O- or -NR M C(O)N(R M - Optional replacement;

[0417] Each R 2 C1-C independently of optional substitution 30 Alkyl, optionally substituted C1-C 30 alkenyl or optionally substituted C1-C 30 Alkyne group; optionally, R 2 One or more methylene groups are independently substituted with a carbocyclic group, a optionally substituted heterocyclic group, an optionally substituted arylene group, an optionally substituted heteroarylene group, or -N(R M )-, -O-, -S-, -C(O)-, -C(O)N(R M )-、-NR M C(O)-、-NR M C(O)N(R M )-, -C(O)O-, -OC(O)-, -OC(O)O-, -OC(O)N(R M )-、-NR M C(O)O-, -C(O)S-, -SC(O)-, -C(=NR M )-、-C(=NR M )N(R M )-、-NRM C(=NR M )-、-NR M C(=NR M )N(R M -, -C(S)-, -C(S)N(R) M )-、-NR M C(S)-、-NR M C(S)-、-NR M C(S)N(R M )-, -S(O)-, -OS(O)-, -S(O)O-, -OS(O)O-, -OS(O)2-, -S(O)2O-, -OS(O)2O-, -N(R M S(O)-、S(O)N(R) M )-、-N(R M )S(O)N(R M )-、-OS(O)N(R M )-、-N(R M S(O)O-, -S(O)2-, N(R) M S(O)2-、-S(O)2N(R) M )-、-N(R M )S(O)2N(R M )-、-OS(O)2N(R M - or -N(R) M S(O)₂O- substitution;

[0418] Each R M Independently hydrogen, optionally substituted alkyl or nitrogen protecting group;

[0419] Ring B is an optionally substituted carbocyclic ring, an optionally substituted heterocyclic ring, an optionally substituted aryl group, or an optionally substituted heteroaryl group; and

[0420] k equals 1 or 2.

[0421] In some embodiments, the compound of formula (V) is a PEG-OH lipid (i.e., R...). 0 (H).

[0422] In some embodiments, the PEG-lipid is a compound of formula (V-1), or a salt thereof, or a stereoisomer thereof:

[0423] in:

[0424] R 3 OR 0 ;

[0425] R 0It is hydrogen, an optionally substituted alkyl group, or an oxygen protecting group;

[0426] g is an integer between 1 and 100;

[0427] R 5 C is an optional replacement 10 -C 40 Alkyl, optionally substituted C 10 -C 40 alkenyl or optionally substituted C 10 -C 40 Alkyne group; optionally, R 5 One or more methylene groups are independently substituted with a carbocyclic group, a optionally substituted heterocyclic group, an optionally substituted arylene group, an optionally substituted heteroarylene group, or -N(R M )-, -O-, -S-, -C(O)-, -C(O)N(R M )-、-NR M C(O)-、-NR M C(O)N(R M )-, -C(O)O-, -OC(O)-, -OC(O)O-, -OC(O)N(R M )-、-NR M C(O)O-, -C(O)S-, -SC(O)-, -C(=NR M )-、-C(=NR M )N(R M )-、-NR M C(=NR M )-、-NR M C(=NR M )N(R M -, -C(S)-, -C(S)N(R) M )-、-NR M C(S)-、-NR M C(S)-、-NR M C(S)N(R M )-, -S(O)-, -OS(O)-, -S(O)O-, -OS(O)O-, -OS(O)2-, -S(O)2O-, -OS(O)2O-, -N(R M S(O)-、S(O)N(R) M )-、-N(R M )S(O)N(R M )-、-OS(O)N(R M )-、-N(R M S(O)O-, -S(O)2-, N(R) M S(O)2-、-S(O)2N(R)M )-、-N(R M )S(O)2N(R M )-、-OS(O)2N(R M - or -N(R) M S(O)₂O- substitution;

[0428] Each R M It is independently protected by hydrogen, alkyl, or nitrogen.

[0429] In some embodiments, the compound of formula (V-1) is a PEG-OH lipid (i.e., R...). 0 (H).

[0430] In addition, in some embodiments, the polymer lipid may also be any one or more PEG-lipids in Hoang Thi, Thai Thanh et al., Polymers, 12(2), 298(2020), the PEG-lipids in the above literature are incorporated herein by reference.

[0431] In some embodiments, the amphoteric polymer includes one or more of the following: poly(carboxybetaine) (pCB), poly(sulfobetaine) (pSB), phosphobetaine-based polymers, and phosphorylcholine polymers. In some embodiments, the amphoteric polymer includes one or more of the following: poly(carboxybetaine acrylamide, pCBAA), poly(carboxybetaine methacrylate), poly(sulfobetaine methacrylate), poly(methacryloyloxyethyl phosphorylcholine), poly(vinyl-pyridinio propanesulfonate), poly(carboxybetaine) based on vinylimidazole, poly(sulfobetaine) based on vinylimidazole, and poly(sulfobetaine) based on vinylpyridine.

[0432] Correspondingly, the polymer-lipid includes one or more of the following: polyhydroxybetaine-lipid, polysulfobetaine-lipid, phosphate betaine-based polymer-lipid, and phosphate choline polymer-lipid. In some embodiments, the polymer-lipid includes one or more of the following: poly(carboxybetaine acrylamide)-lipid, poly(carboxybetaine methacrylate)-lipid, poly(sulfobetaine methacrylate)-lipid, poly(methacryloyloxyethyl phosphorylcholine)-lipid, poly(vinylpyridinylpropanesulfonate)-lipid, polyvinylimidazolyl betaine-lipid, polyvinylimidazolyl sulfobetaine-lipid, and polyvinylpyridinyl sulfobetaine-lipid.

[0433] In some implementations, LNPs contain ionizable lipids.

[0434] In some implementations, LNPs also contain accessory lipids, structural lipids, and polymer-lipids.

[0435] In some implementations, LNPs contain ionizable lipids, auxiliary lipids, structural lipids, and polymer-lipids.

[0436] In some implementations, LNPs contain ionizable lipids, phospholipids, structured lipids, and PEG-lipids.

[0437] In some implementations, LNPs contain ionizable lipids, phospholipids, cholesterol, and PEG-lipids.

[0438] In some implementations, LNPs contain compounds 2-3, phospholipids, structural lipids, and PEG-lipids.

[0439] In some implementations, LNPs contain compounds 2-3, phospholipids, cholesterol, and PEG-lipids.

[0440] In some embodiments, LNPs comprise ionizable lipids, accessory lipids, structural lipids, and polymer-lipids, wherein ionizable lipids comprise 25 mol% to 75 mol% of the total lipids present in the LNPs, accessory lipids comprise 0 mol% to 45 mol% of the total lipids present in the LNPs, structural lipids comprise 0 mol% to 60 mol% of the total lipids present in the LNPs, and polymer-lipids comprise 0.5 mol% to 5 mol% of the total lipids present in the LNPs.

[0441] In some embodiments, the ionizable lipids in the lipid nanoparticles account for 30 mol% to 65 mol%, 30 mol% to 60 mol%, 35 mol% to 60 mol%, 40 mol% to 60 mol%, 45 mol% to 55 mol% or 50 mol% to 55 mol% of the total lipids present in the lipid nanoparticles.

[0442] In some embodiments, the accessory lipids (e.g., DSPC) in the lipid nanoparticles account for 1 mol% to 40 mol%, 5 mol% to 40 mol%, 5 mol% to 35 mol%, 5 mol% to 30 mol%, 5 mol% to 25 mol%, or 5 mol% to 15 mol% of the total lipids present in the lipid nanoparticles.

[0443] In some embodiments, the structural lipids (e.g., cholesterol) in the lipid nanoparticles account for 1 mol% to 60 mol%, 1 mol% to 55 mol%, 5 mol% to 55 mol%, 10 mol% to 50 mol%, 15 mol% to 50 mol%, 15 mol% to 45 mol%, 20 mol% to 45 mol%, or 25 mol% to 40 mol% of the total lipids present in the lipid nanoparticles.

[0444] In some embodiments, the polymer-lipid (e.g., PEG lipid) in the lipid nanoparticles accounts for 0.5 mol% to 4.5 mol%, 1 mol% to 4.5 mol%, 1 mol% to 4 mol%, 1.5 mol% to 4 mol%, 1.5 mol% to 3.5 mol%, or 1.5 mol% to 3 mol% of the total lipids present in the lipid nanoparticles.

[0445] In some embodiments, ionizable lipids account for 45 mol% to 55 mol% of the total lipids present in the lipid nanoparticles, phospholipids account for 5 mol% to 25 mol% of the total lipids present in the lipid nanoparticles, structural lipids account for 25 mol% to 45 mol% of the total lipids present in the lipid nanoparticles, and PEG-lipids account for 1 mol% to 4.5 mol% of the total lipids present in the lipid nanoparticles.

[0446] In some embodiments, the non-layered lipid nanoparticles are selected from one of the following: ethosomes and echogenic liposomes.

[0447] In some embodiments, the delivery vector is a liposome. The liposome utilizes vesicles formed by a phospholipid bilayer membrane to encapsulate the mRNA, DNA, gene-engineered vector, or truncated ATP7B protein of any of the above embodiments. In some embodiments, the components of the liposome include phospholipids and cholesterol.

[0448] In some embodiments, a delivery carrier cationic protein is used. In some embodiments, the cationic protein includes, but is not limited to, protamine.

[0449] In some embodiments, the delivery carrier is a polymer. In some embodiments, the polymer serving as the delivery carrier is a lipopolyplex (LPP) and / or a hyaluronic acid polymer (e.g., hyaluronic acid gel). In one optional specific example, the polymer serving as the delivery carrier is either a lipopolyplex or a hyaluronic acid gel. A lipopolyplex is a bilayer structure with a polymer-encapsulated nucleic acid (e.g., mRNA) core and a lipid (e.g., phospholipid) outer shell.

[0450] It is understood that the delivery vectors applicable to this disclosure are not limited to those described above, and may also be other substances capable of delivering mRNA of any of the above embodiments, DNA of any of the above embodiments, genetically engineered vectors of any of the above embodiments, or truncated ATP7B protein of any of the above embodiments into the body. For example, vesicles (e.g., exosomes).

[0451] In some embodiments, the pharmaceutical composition comprises a delivery vector (e.g., LNPs) and an mRNA of any of the above embodiments, a DNA of any of the above embodiments, a genetically engineered vector of any of the above embodiments, or a truncated ATP7B protein of any of the above embodiments, formulated (e.g., encapsulated) in the delivery vector.

[0452] In some embodiments, the pharmaceutical composition comprises a delivery vector (e.g., LNPs) and a variety of mRNAs, DNAs, genetically engineered vectors, or truncated ATP7B proteins of any of the above embodiments formulated (e.g., encapsulated) in the delivery vector.

[0453] In some embodiments, the pharmaceutical composition comprises multiple mRNAs of any of the above embodiments co-formulated within a delivery vector (e.g., LNPs). That is, multiple mRNAs are formulated (e.g., encapsulated) within a single delivery vector of the pharmaceutical composition.

[0454] In some embodiments, the pharmaceutical composition comprises multiple mRNAs of any of the above embodiments individually formulated within a delivery carrier (e.g., LNPs). That is, one mRNA is formulated (e.g., encapsulated) in a single delivery carrier of the pharmaceutical composition.

[0455] In some implementations, the above-mentioned pharmaceutical composition is a nucleic acid drug.

[0456] The above-mentioned pharmaceutical composition contains the above-mentioned mRNA and has its corresponding advantages.

[0457] VI. Prevention or Treatment Methods

[0458] This disclosure also provides the use of mRNA of any of the above embodiments, DNA of any of the above embodiments, genetic engineering vector of any of the above embodiments, truncated ATP7B protein of any of the above embodiments, host cell of any of the above embodiments, or pharmaceutical composition of any of the above embodiments in the preparation of a medicament for treating or preventing diseases caused by deficiency or dysfunction of ATP7B protein.

[0459] This disclosure also provides the use of mRNA of any of the above embodiments, DNA of any of the above embodiments, genetic engineering vector of any of the above embodiments, truncated ATP7B protein of any of the above embodiments, host cell of any of the above embodiments, or pharmaceutical composition of any of the above embodiments in the preparation of a medicament for the treatment or prevention of Wilson's disease (WD).

[0460] In some implementations, the drug is used to treat or prevent upregulation of ATP7B protein expression or its activity, which may produce therapeutic benefits or improve any other symptoms and diseases.

[0461] In some implementations, the drug is used to produce proteins associated with the treatment of Wilson's disease.

[0462] In some implementations, the drug is used to generate the ATP7B protein.

[0463] Furthermore, this disclosure also provides a method for expressing functional ATP7B protein in the body of a subject in need, the method comprising administering to the subject in need (e.g., a patient with Wilson's disease) an effective amount of mRNA, DNA, genetically engineered vector, or pharmaceutical composition (containing mRNA, DNA, or genetically engineered vector) of any of the above embodiments. This method, by administering mRNA, DNA, genetically engineered vector, or pharmaceutical composition of any of the above embodiments, enables the expression of ATP7B protein with copper ion transport function in the subject's body, thereby treating the corresponding condition or delaying its further deterioration.

[0464] This disclosure also provides a method for enhancing the activity of ATP7B protein, comprising administering to a subject in need (e.g., a patient with Wilson's disease) an effective amount of mRNA, DNA, genetically engineered vector, truncated ATP7B protein, or pharmaceutical composition of any of the above embodiments. This method, by administering mRNA, DNA, genetically engineered vector, truncated ATP7B protein, or pharmaceutical composition of any of the above embodiments, enables the subject to have ATP7B protein with copper ion transport function, thereby enhancing the activity of ATP7B protein in the subject and thereby improving its copper ion transport function, thus treating the corresponding disease or delaying its further deterioration.

[0465] Furthermore, this disclosure also provides a method for preventing or treating conditions caused by ATP7B protein deficiency or dysfunction, the method comprising administering to a subject an effective dose of mRNA, DNA, genetically engineered vector, truncated ATP7B protein, or pharmaceutical composition of any of the above embodiments. This method, by administering mRNA, DNA, genetically engineered vector, truncated ATP7B protein, or pharmaceutical composition of any of the above embodiments, ensures sufficient ATP7B protein in the patient's body to maintain normal copper metabolism and / or normal ceruloplasmin synthesis, thereby treating the corresponding condition or delaying its further deterioration.

[0466] Furthermore, this disclosure also provides a method for preventing or treating Wilson's disease, the method comprising administering to a subject an effective dose of mRNA, DNA, genetically engineered vector, truncated ATP7B protein, or pharmaceutical composition of any of the above embodiments. This method, by administering mRNA, DNA, genetically engineered vector, truncated ATP7B protein, or pharmaceutical composition of any of the above embodiments, ensures sufficient ATP7B protein in the patient's body to maintain normal copper metabolism and / or normal ceruloplasmin synthesis, thereby preventing or treating Wilson's disease.

[0467] Furthermore, this disclosure also provides a method for preventing or treating copper metabolism disorders, comprising administering to a subject in need (e.g., a patient with Wilson's disease) an effective amount of the mRNA, DNA, genetically engineered vector, truncated ATP7B protein, or pharmaceutical composition of any of the above embodiments. This method, by administering the mRNA, DNA, genetically engineered vector, truncated ATP7B protein, or pharmaceutical composition of any of the above embodiments, enables excess copper ions in the liver to be transported out of the liver by the ATP7B protein (e.g., the truncated ATP7B protein), thereby alleviating or treating copper metabolism disorders.

[0468] Furthermore, this disclosure also provides a method for alleviating or avoiding copper ion poisoning by increasing the efficiency of copper ion transport out of hepatocytes. This method includes administering an effective amount of the mRNA, DNA, genetically engineered vector, truncated ATP7B protein, or pharmaceutical composition of any of the above embodiments to a subject in need (e.g., a patient with Wilson's disease). This method, by administering the mRNA, DNA, genetically engineered vector, truncated ATP7B protein, or pharmaceutical composition of any of the above embodiments, increases the efficiency by which excess copper ions in hepatocytes can be transported out of hepatocytes by ATP7B protein (e.g., truncated ATP7B protein), thereby alleviating or avoiding toxicity caused by excess copper ions in hepatocytes.

[0469] In addition, this disclosure also provides a method for reducing copper ion accumulation in hepatocytes to alleviate or avoid copper ion poisoning. The method includes administering an effective amount of the mRNA, DNA, genetically engineered vector, truncated ATP7B protein, or pharmaceutical composition of any of the above embodiments to a subject in need (e.g., a patient with Wilson's disease). This method, by administering the mRNA, DNA, genetically engineered vector, truncated ATP7B protein, or pharmaceutical composition of any of the above embodiments, increases the efficiency of transporting excess copper ions from hepatocytes out of the liver by ATP7B protein (e.g., truncated ATP7B protein), reducing copper ion accumulation in the liver, thereby alleviating or avoiding toxicity caused by excessive copper ions in the liver.

[0470] In addition, this disclosure also provides a method for increasing serum ceruloplasmin levels, the method comprising administering to a subject in need (e.g., a patient with Wilson's disease) an effective amount of mRNA, DNA, genetically engineered vector, truncated ATP7B protein, or pharmaceutical composition of any of the above embodiments. This method increases serum ceruloplasmin levels by administering mRNA, DNA, genetically engineered vector, truncated ATP7B protein, or pharmaceutical composition of any of the above embodiments to enhance ceruloplasmin synthesis in the liver.

[0471] In some implementations, the subjects of the methods in any of the above implementations are mammals. These include, for example, humans, non-human primates (e.g., apes, chimpanzees, monkeys, and orangutans), domesticated animals (e.g., dogs, cats, and livestock (e.g., horses, cattle, pigs, sheep, and goats)), or other mammals. Other mammals include, but are not limited to, mice, rats, guinea pigs, rabbits, hamsters, etc.

[0472] In some implementations, the subjects are humans.

[0473] In some implementation schemes, the subjects are children, adolescents, or adults.

[0474] In some implementation schemes, the subjects are children, adolescents, or adults with Wilson's disease.

[0475] In some implementation schemes, the method of any of the above implementation schemes is applied once, twice, three times, four times or more.

[0476] In some implementations, based on the subject's weight, the method of any of the above implementations includes administering the mRNA, DNA, genetically engineered vector, truncated ATP7B protein, or pharmaceutical composition (e.g., nucleic acid drug) of any of the above implementations at dosages of 0.0001 mg / kg to 100 mg / kg, 0.001 mg / kg to 0.05 mg / kg, 0.005 mg / kg to 0.05 mg / kg, 0.001 mg / kg to 0.005 mg / kg, 0.05 mg / kg to 0.5 mg / kg, 0.01 mg / kg to 50 mg / kg, or 0.1 mg / kg. The drug is administered to subjects at daily doses of 0.01 mg / kg to 40 mg / kg, 0.5 mg / kg to 30 mg / kg, 0.01 mg / kg to 10 mg / kg, 0.1 mg / kg to 10 mg / kg, 0.1 mg / kg to 5 mg / kg, 0.1 mg / kg to 2.5 mg / kg, 0.1 mg / kg to 2 mg / kg, 0.1 mg / kg to 1.5 mg / kg, 0.1 mg / kg to 1 mg / kg, 0.1 mg / kg to 0.5 mg / kg, or 1 mg / kg to 25 mg / kg, once or multiple times a day, to achieve the desired therapeutic or preventative effect. In some embodiments, the required dose is delivered every other day, every three days, once a week, once every two weeks, once every three weeks, or once every four weeks. Example

[0477] To make the objectives and technical solutions of this disclosure clearer, the following detailed description is provided in conjunction with specific embodiments. Obviously, the described embodiments are merely some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other implementation methods obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise specified, the reagents and instruments used in the embodiments are conventionally selected in the art. Experimental methods not specifying specific conditions in the embodiments are implemented according to conventional conditions, such as those described in literature, books, or methods recommended by the manufacturer.

[0478] Example 1 Plasmid Construction

[0479] The 5'-UTR, coding region of ATP7B protein, 3'-UTR, and poly(A) tail of the mRNA were introduced into the pVAX1 plasmid using subcloning techniques (e.g., subcloning techniques based on PCR and restriction endonuclease digestion or in-fusion techniques) to construct the expression plasmids corresponding to each mRNA in Table 1.

[0480] Example 2: Synthesis of mRNA encoding ATP7B protein (ATP7B mRNA)

[0481] The methods for synthesizing ATP7B mRNA include, but are not limited to, the following steps:

[0482] 1. Extract the expression plasmids corresponding to each mRNA prepared in Example 1, ensuring that the supercoilability of the plasmids reaches more than 90%.

[0483] 2. Linearize the plasmid by enzyme digestion, and then purify it using a DNA fragment purification and recovery kit (Takara, 9761) and DNA magnetic beads (Rebecca, SP703) (to remove RNase, proteins, etc.).

[0484] 3. The linearized plasmid was transcribed in vitro using an IVT reaction.

[0485] 4. Use Hieff The RNACleaner kit (YEASEN, 12602ES56) is used to purify products transcribed in vitro.

[0486] 5. The purified in vitro transcripts were subjected to a Cap1-type capping reaction using a capping kit (Cellscript, C-ASF3507), followed by a Hieff reaction. The RNA Cleaner kit (YEASEN, 12602ES56) was used to purify the product of the above capping reaction using magnetic beads to obtain a variety of ATP7B mRNAs containing a Cap1 cap structure, 5'-UTR, 3'-UTR, poly(A) tail, and uridine completely replaced by N1-methylpseudouridine.

[0487] Example 3: Preparation of ATP7B mRNA-LNP

[0488] (1) Encapsulation: Using a microfluidic device and a microfluidic chip (SN.000038), mRNA was encapsulated at a rate of 9 mL / min for the aqueous phase and 3 mL / min for the alcohol phase to prepare a crude product of mRNA-LNP encapsulated with ATP7B mRNA (hereinafter referred to as "ATP7B mRNA-LNP"). In this example, the mRNA used was prepared according to the method in Example 2. The aqueous phase was an acetate-sodium acetate buffer (pH 5.0) containing ATP7B mRNA, and the alcohol phase contained ionizable lipid compound 2-5, DSPC, cholesterol, and DMG-PEG2000. The molar ratio of compound 2-5, DSPC, cholesterol, and DMG-PEG2000 was 50:10:38.5:1.5.

[0489] (2) Dialysis solution change: The crude product obtained by encapsulation was dialyzed with dialysis solution (PBS solution containing 8% (m / V) sucrose) at 100 rpm for 1 h, and then the dialysis solution was changed and dialyzed for another 1 h.

[0490] (3) Sterilization: After dialysis, a 0.22μm PES filter membrane was used for sterilization filtration to obtain each ATP7B mRNA-LNP. The mRNA concentration of the mRNA-LNP was about 0.2μg / μL, the particle size was 80nm~100nm, and the encapsulation rate was more than 80%.

[0491] Example 4 Cell Expression Experiment

[0492] Protein expression of different ATP7B expression plasmids was detected in HuH-7, HepG2, and HEK293T cells. The expression plasmids corresponding to the mRNAs prepared in Example 1 were transfected into HuH-7, HepG2, or HEK293T cells using Lipofectamine 2000 transfection reagent (Invitrogen, 11668-019). The pVAX1 plasmid expressing eGFP was used as a negative control. Cells were lysed 24 h after transfection, and ATP7B protein expression was detected by ELISA (capture antibody: ATP7B antibody LSBio LS-C196725-100; primary antibody: Anti-ATP7b antibody [EPR6794] (abcam, ab124973); secondary antibody: horseradish peroxidase-labeled goat anti-rabbit IgG (Lianke Biotechnology, GAR007)). The results are shown in Figures 1A-1C.

[0493] As shown in Figures 1A-1C, compared with the negative control, the expression plasmids corresponding to the mRNAs in Table 1 showed a significant increase in ATP7B expression in HuH-7 cells, HepG2 cells, and HEK293T cells.

[0494] Example 5: Detection of Copper Ion Escape Function

[0495] ATP7B protein is involved in the transport and distribution of copper ions within cells, playing a crucial role in maintaining copper homeostasis and preventing copper accumulation to toxic levels. The upstream region of the promoter of metallothionein 1 (MT1) contains multiple metal regulatory elements (MREs) that respond to metal-induced responses. The sequence from -728 to +18 of the mouse MT1 promoter was introduced into the pGL3-Basic vector (purchased from Promega, plasmid number #E1751) to obtain the reporter plasmid pGL3-MRE-LUC (hereinafter referred to as the "reporter plasmid"). This plasmid was used to detect the copper ion transport function of different expressed ATP7B proteins. The copper efflux function of ATP7B protein inhibited the activation of the reporter plasmid by intracellular copper ions, thereby reducing its luciferase expression.

[0496] The expression plasmids corresponding to each mRNA in Table 1 prepared in Example 1, along with the reporter plasmids, were co-transfected into copper-incubated HEK293T, HuH-7, or HepG2 cells using Lipofectamine 2000 transfection reagent (Invitrogen, 11668-019). Ammonium tetrathiomolybdate (Sigma, 323446, working concentration 50 μmol / L) served as a positive control (simultaneously transfected with the pVAX1 plasmid expressing eGFP), while the pVAX1 plasmid expressing eGFP served as a negative control. After 24 h of co-incubation, cells were washed and lysed using a luciferase detection system kit (Promega, catalog number E4550), and the fluorescence intensity of eGFP was detected using a multi-mode microplate reader (Molecular Device, SpectraMax i3x). Fluorescence intensities were normalized, and all values ​​are multiples relative to the negative control. The results are shown in Figures 2A-2C.

[0497] The results showed that, compared with the negative control co-transfected with the pVAX1 plasmid expressing eGFP and the reporter plasmid, cells co-transfected with the ATP7B expression plasmid and the reporter plasmid exhibited significantly reduced luciferase-driven signals in response to copper, indicating increased copper efflux after transfection with the ATP7B expression plasmid. These results suggest that the ATP7B expression plasmid produces a functional copper transporter capable of expelling excess copper from the cell.

[0498] Example 6: Expression of ATP7B mRNA-LNP in wild-type mice

[0499] To evaluate the ability of lipid nanoparticles (LNPs) to deliver ATP7B mRNA in vivo, ATP7B mRNA was encapsulated in lipid nanoparticles to form ATP7B mRNA-LNPs encapsulated with ATP7B mRNA (e.g., "1912mRNA-LNP" refers to an mRNA-LNP encapsulated with mRNA numbered 1912 in Table 1, and others are similar). These LNPs were intravenously injected into 6-8 week old wild-type C57BL / 6 mice (WT mice) at doses of 1 mg / kg or 2 mg / kg (based on mRNA content), with PBS-treated WT mice serving as controls. The ATP7B mRNA-LNP used in the 2 mg / kg dose of this example was prepared in Example 3; the ATP7B mRNA-LNP used in the 1 mg / kg dose of this example was prepared according to Example 3, with the ionizable lipid in its alcohol phase being compound 2-3, and the molar ratio of compound 2-3, DSPC, cholesterol, and DMG-PEG2000 being 49.0:5.2:43.3:2.5.

[0500] Liver samples were collected at 8h, 24h, 48h, and 72h post-injection, and the level of ATP7B protein in the liver was detected by ELISA (refer to Example 4). The results are shown in Figures 3A-3B.

[0501] The results showed that, compared with the PBS-injected control, the expression of ATP7B protein in the liver of mice administered ATP7B mRNA-LNP was significantly increased. Notably, significant levels of ATP7B protein were still detected 72 hours after administration, indicating sustained expression of ATP7B protein. These findings also demonstrate that LNPs can effectively deliver ATP7B mRNA to the liver and achieve protein expression.

[0502] Example 7: Construction of the Atp7b gene knockout (KO) mouse model

[0503] To better evaluate the efficacy of ATP7B mRNA-LNP in patients with Wilson's disease, Atp7b KO C57BL / 6J mice (GemPharmatech, T013709, hereinafter referred to as "Atp7b KO mice") generated by CRISPR / Cas9 gene knockout technology were used to assess the efficacy of ATP7B mRNA-LNP. These Atp7b KO mice targeted exon 2 of the Atp7b-201 (ENSMUST00000006742.10) transcript (this region contains a 1207 bp coding sequence), the deletion of which leads to the disruption of ATP7B protein expression and function. These Atp7b KO mice exhibited elevated liver copper levels and decreased ceruloplasmin activity, which is similar to the phenotype of patients with Wilson's disease (whose impaired ATP7B copper transporter function leads to disordered copper metabolism, elevated liver copper accumulation, and decreased serum ceruloplasmin activity).

[0504] Atp7b KO mice and WT mice that were normally fed were selected to verify the knockout effect of Atp7b KO mice. Serum was collected from mouse blood samples and the activity of serum ceruloplasmin was detected using a ceruloplasmin activity assay kit (Nanjing Jiancheng Bioengineering Institute, A029-1-1). After euthanasia, partial liver tissue was harvested for real-time quantitative PCR to detect the Atp7b mRNA level. Total RNA was extracted using a cell / tissue total RNA isolation kit (Vazyme, RC112), and then reverse transcribed into cDNA using HiScript IIQ RT SuperMix (Vazyme, R223). Real-time quantitative PCR was then performed using ChamQ Universal SYBR qPCR Master Mix (Vazyme, Q711) to detect the Atp7b mRNA level. The real-time quantitative PCR results were normalized using the mouse internal reference gene ACT2. The primers used were: mACT2 5'-GGCTGTATTCCCCTCCATCG-3' and 5'-CCAGTTGGTAACAATGCCATGT-3'; mATP7B The liver copper content was detected by Beijing Zhongke Baice Information Technology Co., Ltd. using ICP-MS (Perkin Elmer NexION 300D ICP-MS, USA). The results are shown in Figures 4A to 4C.

[0505] The results showed that in KO mice, liver Atp7b mRNA expression was completely lost (Fig. 4A), serum ceruloplasmin activity was reduced (Fig. 4B), and liver copper content was significantly increased (Fig. 4C). This confirmed the success of gene deletion, and the model is suitable for studying the efficacy of ATP7B mRNA-LNP therapy.

[0506] Example 8: Evaluation of the efficacy of ATP7B mRNA-LNP in treating Wilson's disease in an Atp7b KO mouse model

[0507] 1. Single dose

[0508] A single-dose pharmacological study was conducted using the Atp7b KO mouse model (preparation method as described in Example 7) to evaluate the efficacy of ATP7B mRNA-LNP. 24-week-old Atp7b KO mice were administered a single dose of ATP7B mRNA-LNP (preparation method as described in Example 3, the ionizable lipid in its alcohol phase being compound 2-3, and the molar ratio of compound 2-3, DSPC, cholesterol, and DMG-PEG2000 being 49.0:5.2:43.3:2.5) via tail vein injection at a dose of 1 mg / kg. Atp7b KO mice and WT mice of the same age treated with PBS served as controls. Blood samples were collected before administration, 24 h after administration, and 72 h after administration. Serum ceruloplasmin activity was measured using a ceruloplasmin activity assay kit (Nanjing Jiancheng Bioengineering Institute, A029-1-1). 72 hours after drug administration, mice were euthanized and their livers were collected. The copper content in the liver and serum was detected by ICP-MS (Perkin Elmer NexION 300D ICP-MS, USA) using Beijing Zhongke Baice Information Technology Co., Ltd. The results are shown in Figures 5A to 5C.

[0509] The results showed that baseline serum ceruloplasmin activity in Atp7b KO mice, whether untreated or treated with PBS, was significantly lower than that in WT mice. 24 h after administration of ATP7B mRNA-LNP, serum ceruloplasmin activity in Atp7b KO mice significantly increased. 72 h after administration of ATP7B mRNA-LNP, the activity increased to a level comparable to that in WT mice (Figure 5A). Furthermore, at 72 h post-administration, serum total copper levels in Atp7b KO mice treated with PBS were significantly lower than those in WT mice, while serum total copper levels in Atp7b KO mice treated with ATP7B mRNA-LNP were significantly higher than those treated with PBS (Figure 5B). In addition, compared to the PBS-treated control group, liver copper levels in KO mice treated with ATP7B-LNP mRNA were significantly lower (Figure 5C).

[0510] In addition, the expression level of ATP7B protein in the liver of mice after a single intravenous administration (50 μg / mouse) of 778 mRNA-LNP (preparation method as described in Example 3, wherein the ionizable lipid in the alcohol phase is compound 2-4, and the molar ratio of compound 2-4, DSPC, cholesterol and DMG-PEG2000 is 50:10:38.5:1.5) was detected by Western blot (human ATP7B antibody: Abcam, ab124973, internal control Actin antibody: CST, 5125S). The results are shown in Figure 5D. The results showed that no ATP7B protein expression was detected in the liver of untreated Atp7b KO mice, while significant ATP7B protein expression was detected in the liver of treated Atp7b KO mice on day 3 after administration.

[0511] The above results indicate that a single dose of ATP7B mRNA-LNP can effectively reduce liver copper levels, with the therapeutic effect lasting for more than 3 days.

[0512] 2. Multiple administrations

[0513] Mice were injected once with PBS or 1918 mRNA-LNP (preparation method as described in Example 3, wherein the ionizable lipid in the alcohol phase is compound 2-3, and the molar ratio of compound 2-3, DSPC, cholesterol, and DMG-PEG2000 is 49.0:5.2:43.3:2.5) at D0, D8, D16, and D24, at a dose of 1 mg / kg. Blood samples were collected from mice before administration, 48 h after the first injection, 96 h after the first injection, 168 h after the first injection, 48 h after the second injection, 96 h after the second injection, 168 h after the second injection, 48 h after the third injection, 96 h after the third injection, 168 h after the third injection, 48 h after the fourth injection, 96 h after the fourth injection, and 168 h after the fourth injection. Serum ceruloplasmin activity was measured using the same method as in the single-dose administration example of this example. Feces and urine were collected from mice before administration, and 24–48 h after the first injection, 48–72 h after the first injection, 24–48 h after the second injection, 48–72 h after the second injection, 24–48 h after the third injection, 48–72 h after the third injection, 24–48 h after the fourth injection, and 48–72 h after the fourth injection. Liver samples were collected on day 31. The copper content in the mouse liver, feces, and urine was detected by ICP-MS (Perkin Elmer NexION 300D ICP-MS, USA) using Beijing Zhongke Baice Information Technology Co., Ltd. The results are shown in Figures 6A–6D.

[0514] The results showed that serum ceruloplasmin activity was significantly improved in Atp7b KO mice after multiple administrations of 1918mRNA-LNP compared to baseline, consistent with the results of a single administration. Serum ceruloplasmin activity peaked at 48 h post-administration and then gradually decreased over the next 5 days. Notably, the improvement in ceruloplasmin activity was sustained and did not diminish with repeated administration (Fig. 6A). Seven days after the last administration (D31), liver copper content was significantly reduced in Atp7b KO mice treated with 1918mRNA-LNP compared to PBS-treated Atp7b KO mice. In contrast, 1918mRNA-LNP treatment had no effect on liver copper content in WT mice (Fig. 6B). Urinary copper content was significantly reduced in Atp7b KO mice treated with 1918mRNA-LNP (Fig. 6C), while fecal copper content was significantly increased (Fig. 6D). These results remained unchanged with repeated administration (Figs. 6C and 6D).

[0515] sequence

[0516] SEQ ID NO:1

[0517] SEQ ID NO:2

[0518] SEQ ID NO:3

[0519] SEQ ID NO:4

[0520] SEQ ID NO:5

[0521] SEQ ID NO:6

[0522] SEQ ID NO:7

[0523] SEQ ID NO:8

[0524] SEQ ID NO:9

[0525] SEQ ID NO:10

[0526] SEQ ID NO:11

[0527] SEQ ID NO:12

[0528] SEQ ID NO:13

[0529] SEQ ID NO:14

[0530] SEQ ID NO:15

[0531] SEQ ID NO:16

[0532] SEQ ID NO:17

[0533] SEQ ID NO:18

[0534] SEQ ID NO:19

[0535] SEQ ID NO:20

[0536] SEQ ID NO:21

[0537] SEQ ID NO:22

[0538] SEQ ID NO:23

[0539] SEQ ID NO:24

[0540] SEQ ID NO:25

[0541] SEQ ID NO:26

[0542] SEQ ID NO:27

[0543] SEQ ID NO:28

[0544] SEQ ID NO:29

[0545] SEQ ID NO:30

[0546] SEQ ID NO:31

[0547] SEQ ID NO:32

[0548] SEQ ID NO:33

[0549] SEQ ID NO:34

[0550] SEQ ID NO:35

[0551] SEQ ID NO:36

[0552] SEQ ID NO:37

[0553] SEQ ID NO:38

[0554] SEQ ID NO:39

[0555] SEQ ID NO:40

[0556] SEQ ID NO:41

[0557] SEQ ID NO:42

[0558] SEQ ID NO:43

[0559] SEQ ID NO:44

[0560] SEQ ID NO:45

[0561] SEQ ID NO:46

[0562] SEQ ID NO:47

[0563] SEQ ID NO:48

[0564] SEQ ID NO:49

Claims

1. An mRNA, said mRNA being a non-natural mRNA, comprising a polynucleotide encoding the ATP7B protein.

2. The mRNA according to claim 1, wherein the ATP7B protein is the full-length human ATP7B protein, and the mRNA further comprises a 5'-UTR, a 3'-UTR, and a poly(A) tail.

3. The mRNA according to claim 1 or 2, wherein the polynucleotide encoding the ATP7B protein is one of the following: (1) A polynucleotide with a nucleotide sequence as shown in any one of SEQ ID NO: 8-10; and (2) A polynucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and less than 100% identity with any of the nucleotide sequences shown in SEQ ID NO: 8–10 and encoding the full-length human ATP7B protein.

4. The mRNA according to claim 1, wherein the ATP7B protein is a truncated ATP7B protein, and the truncated ATP7B protein is one of the following: (1) The truncated ATP7B protein of MBD1 to MBD4 is missing; (2) A truncated ATP7B protein lacking MBD3; (3) The truncated ATP7B protein lacking MBD1 and MBD3; (4) The truncated ATP7B protein of MBD1, MBD3 and MBD4 is missing; (5) truncated ATP7B protein lacking MBD1 and MBD4; (6) The truncated ATP7B protein of MBD1-MBD2 is missing; (7) truncated ATP7B proteins lacking MBD1, MBD2 and MBD4; (8) The truncated ATP7B protein of MBD1-MBD3 is missing; (9) truncated ATP7B protein lacking MBD2 and MBD3; (10) truncated ATP7B protein lacking MBD2 and MBD4; (11) truncated ATP7B protein lacking MBD3 and MBD4; (12) The truncated ATP7B protein lacking MBD2–MBD4; and (13) The truncated ATP7B protein of MBD1 to MBD5 is missing.

5. The mRNA according to claim 4, wherein the truncated ATP7B protein is one of the following: (1) The truncated ATP7B protein from position 57 to position 485 is missing; (2) The truncated ATP7B protein from position 57 to position 486 is missing; (3) The truncated ATP7B protein at positions 257 to 355 is missing; (4) The truncated ATP7B protein at positions 57-140 and 237-337 is missing; (5) The truncated ATP7B protein at positions 57-140 and 237-485 is missing; (6) The truncated ATP7B protein at positions 57-140 and 359-485 is missing; (7) The truncated ATP7B protein from position 57 to position 236 is missing; (8) The truncated ATP7B protein at positions 57-236 and 359-485 is missing; (9) The truncated ATP7B protein from position 57 to position 337 is missing; (10) The truncated ATP7B protein from position 141 to position 337 is missing; (11) The truncated ATP7B protein is missing from positions 141 to 236 and 359 to 485; (12) The truncated ATP7B protein from position 237 to position 485 is missing; (13) The truncated ATP7B protein from position 141 to position 485 is missing; (14) The truncated ATP7B protein from position 57 to position 490 is missing; and (15) The truncated ATP7B protein at positions 57 to 495 is missing.

6. The mRNA according to claim 5, wherein the truncated ATP7B protein is one of the following: (1) A truncated ATP7B protein with an amino acid sequence as shown in any one of SEQ ID NO: 11–25; and (2) A protein having an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% and less than 100% identical to any of the amino acid sequences shown in SEQ ID NO: 11 to 25 and having the function of ATP7B protein.

7. The mRNA according to claim 6, wherein the polynucleotide encoding the truncated ATP7B protein is one of the following: (1) A polynucleotide with a nucleotide sequence as shown in SEQ ID NO: 26; (2) A polynucleotide sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and less than 100% identity with the nucleotide sequence shown in SEQ ID NO: 26 and encoding an amino acid sequence as shown in SEQ ID NO: 11 of the truncated ATP7B protein; (3) A polynucleotide with a nucleotide sequence as shown in any one of SEQ ID NO: 27-30; (4) The nucleotide sequence has at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and less than 100% identity with the nucleotide sequence shown in any one of SEQ ID NO: 27–30 and encodes a polynucleotide encoding an amino acid sequence of the truncated ATP7B protein as shown in SEQ ID NO: 12; (5) Polynucleotides with nucleotide sequences as shown in SEQ ID NO: 31; (6) A polynucleotide sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and less than 100% identity with the nucleotide sequence shown in SEQ ID NO: 31 and encoding an amino acid sequence of a truncated ATP7B protein as shown in SEQ ID NO: 13; (7) Polynucleotides with nucleotide sequences as shown in SEQ ID NO: 32; (8) A polynucleotide sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and less than 100% identity with the nucleotide sequence shown in SEQ ID NO: 32 and encoding an amino acid sequence as shown in SEQ ID NO: 14; (9) Polynucleotides with nucleotide sequences as shown in SEQ ID NO: 33 or 34; (10) A polynucleotide sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and less than 100% identity with the nucleotide sequence shown in SEQ ID NO: 33 or 34 and encoding an amino acid sequence as shown in SEQ ID NO: 15, truncated ATP7B protein; (11) Polynucleotides with nucleotide sequences as shown in SEQ ID NO: 35; (12) A polynucleotide sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and less than 100% identity with the nucleotide sequence shown in SEQ ID NO: 35 and encoding an amino acid sequence of a truncated ATP7B protein as shown in SEQ ID NO: 16; (13) Polynucleotides with nucleotide sequences as shown in SEQ ID NO: 36; (14) A polynucleotide sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and less than 100% identity with the nucleotide sequence shown in SEQ ID NO: 36 and encoding an amino acid sequence of a truncated ATP7B protein as shown in SEQ ID NO: 17; (15) Polynucleotides with nucleotide sequences as shown in SEQ ID NO: 37; (16) A polynucleotide sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and less than 100% identity with the nucleotide sequence shown in SEQ ID NO: 37 and encoding an amino acid sequence of a truncated ATP7B protein as shown in SEQ ID NO: 18; (17) Polynucleotides with nucleotide sequences as shown in SEQ ID NO: 38; (18) A polynucleotide sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and less than 100% identity with the nucleotide sequence shown in SEQ ID NO: 38 and encoding an amino acid sequence of a truncated ATP7B protein as shown in SEQ ID NO: 19; (19) Polynucleotides with nucleotide sequences as shown in SEQ ID NO: 39; (20) A polynucleotide sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and less than 100% identity with the nucleotide sequence shown in SEQ ID NO: 39 and encoding an amino acid sequence of a truncated ATP7B protein as shown in SEQ ID NO: 20; (21) Polynucleotides with nucleotide sequences as shown in SEQ ID NO: 40; (22) A polynucleotide sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and less than 100% identity with the nucleotide sequence shown in SEQ ID NO: 40 and encoding an amino acid sequence of a truncated ATP7B protein as shown in SEQ ID NO: 21; (23) Polynucleotides with nucleotide sequences as shown in SEQ ID NO: 41; (24) A polynucleotide sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and less than 100% identity with the nucleotide sequence shown in SEQ ID NO: 41 and encoding an amino acid sequence of a truncated ATP7B protein as shown in SEQ ID NO: 22; (25) Polynucleotides with nucleotide sequences as shown in SEQ ID NO: 42; (26) A polynucleotide sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and less than 100% identity with the nucleotide sequence shown in SEQ ID NO: 42 and encoding an amino acid sequence of a truncated ATP7B protein as shown in SEQ ID NO: 23; (27) Polynucleotides with nucleotide sequences as shown in SEQ ID NO: 43; (28) A polynucleotide sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and less than 100% identity with the nucleotide sequence shown in SEQ ID NO: 43 and encoding an amino acid sequence of a truncated ATP7B protein as shown in SEQ ID NO: 24; (29) Polynucleotides with nucleotide sequences as shown in SEQ ID NO: 44; and (30) A polynucleotide sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and less than 100% identity with the nucleotide sequence shown in SEQ ID NO: 44 and encoding an amino acid sequence of a truncated ATP7B protein as shown in SEQ ID NO:

25.

8. The mRNA according to any one of claims 4 to 7, wherein the truncated ATP7B protein is a truncated human ATP7B protein.

9. The mRNA according to any one of claims 4 to 8, wherein the mRNA further comprises at least one of a 5'-UTR, a 3'-UTR, and a poly(A) tail.

10. The mRNA according to claim 2, 3 or 9, wherein the nucleotide sequence of the 5'-UTR is as shown in SEQ ID NO: 2 or 3; And / or, the nucleotide sequence of the 3'-UTR is as shown in SEQ ID NO: 4 or 5; And / or, the nucleotide sequence of the poly(A) tail is as shown in SEQ ID NO: 6 or 7.

11. The mRNA according to any one of claims 1 to 10, wherein the mRNA further comprises a microRNA binding site; Preferably, the microRNA binding site is the miR142 binding site; Preferably, the nucleotide sequence of the miR142 binding site is shown in SEQ ID NO:

49.

12. The mRNA according to claim 11, wherein the microRNA binding site is located in the 5'-UTR; And / or, the microRNA binding site is located in the 3'-UTR; And / or, the microRNA binding site is located after the 3'-UTR and before the poly(A) tail.

13. The mRNA according to claim 11 or 12, wherein the microRNA binding site is located in the poly(A) tail; Preferably, the nucleotide sequence containing the poly(A) tail of the microRNA binding site is as shown in SEQ ID NO: 46, 47 or 48; Preferably, the nucleotide sequence of the polynucleotide encoding the ATP7B protein is shown in SEQ ID NO: 10, and the nucleotide sequence of the poly(A) tail containing the microRNA binding site is shown in SEQ ID NO:

47.

14. The mRNA according to any one of claims 1 to 13, wherein the mRNA contains a modified nucleoside; Preferably, the modified nucleoside includes at least one of modified uridine, modified cytidine, modified adenosine, and modified guanosine.

15. The mRNA according to any one of claims 1 to 14, wherein, compared with before administration of the mRNA, the mRNA can increase the expression level of ATP7B protein in the test subject by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, or 500%; And / or, compared with before administration of the mRNA, the mRNA can increase the activity of the ATP7B protein in the test subject by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, or 500%; And / or, compared with before administration of the mRNA, the mRNA can reduce the copper content in the liver of the test subjects by at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%; And / or, compared with before administration of the mRNA, the mRNA can increase the content of ceruloplasmin in the serum of the test subject by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, or 500%.

16. A DNA that can be transcribed into the mRNA according to any one of claims 1 to 15.

17. A gene engineering vector comprising the DNA of claim 16.

18. A host cell comprising the DNA of claim 16 or the genetic engineering vector of claim 17.

19. A truncated ATP7B protein encoded by the mRNA according to any one of claims 4 to 15.

20. A pharmaceutical composition comprising the mRNA of any one of claims 1 to 15, the DNA of claim 16, the genetic engineering vector of claim 17, or the truncated ATP7B protein of claim 19.

21. The pharmaceutical composition of claim 20, wherein the mRNA or the DNA is formulated in a delivery vector.

22. The pharmaceutical composition according to claim 21, wherein the delivery carrier is a lipid nanoparticle.

23. The pharmaceutical composition of claim 22, wherein the lipid nanoparticles comprise ionizable lipids, and the ionizable lipids are:

24. The pharmaceutical composition according to any one of claims 22 to 23, wherein the lipid nanoparticles further comprise phospholipids, structural lipids, and PEG-lipids.

25. The pharmaceutical composition according to claim 24, wherein the ionizable lipid comprises 45 mol% to 55 mol% of the total lipids present in the lipid nanoparticles, the phospholipid comprises 5 mol% to 25 mol% of the total lipids present in the lipid nanoparticles, the structural lipid comprises 25 mol% to 45 mol% of the total lipids present in the lipid nanoparticles, and the PEG-lipid comprises 1 mol% to 4.5 mol% of the total lipids present in the lipid nanoparticles.

26. The use of the mRNA of any one of claims 1 to 15, the DNA of claim 16, the genetic engineering vector of claim 17, the host cell of claim 18, the truncated ATP7B protein of claim 19, or the pharmaceutical composition of any one of claims 20 to 25 in the preparation of a medicament for the treatment and / or prevention of Wilson's disease; Preferably, the drug is a nucleic acid drug.

Images