Cut-type ATP7B and its use

Abstract

This disclosure relates to a cleaved ATP7B that contains CRE5 and / or CRE6, and optionally contains CRE1 and / or CRE2. This disclosure further provides an adeno-associated virus vector encoding the cleaved ATP7B for gene therapy to treat Wilson's disease (WD).

Description

[Technical Field]

[0001] Priority This application claims the interests and priority of PCT application number PCT / CN2023 / 079933, filed on 6 March 2023, all of which are incorporated herein by reference.

[0002] This disclosure relates to cleavage-type copper transport ATPase β (ATP7B) and its nucleic acid sequence, recombinant nucleic acid construct, adeno-associated virus vector, and methods of using them in gene therapy for the treatment of Wilson's disease (WD). [Background technology]

[0003] Wilson's disease (WD) is an autosomal recessive copper metabolism disorder that primarily causes copper accumulation in the liver and subsequently in the nervous system and other systems. WD is a rare disease with a global average prevalence of 1:30,000, caused by mutations in the ATP7B gene, which encodes a P-type copper transport ATPase located on chromosome 13.

[0004] ATP7B is primarily expressed in hepatocytes and functions as a copper ion transmembrane transporter. Loss or reduction in ATP7B function leads to decreased copper excretion into bile by hepatocytes and copper accumulation in the liver and other organs. When copper metabolism is impaired, ceruloplasmin is unable to bind to copper, causing additional hemolytic anemia. ATP7B has a basic P-type ATPase structure, consisting of a cytoplasmic region with phosphorylation (P-), ATP-binding (N-), and actuator / dephosphorylation (A-) domains, and a membrane region with eight transmembrane helices (transmembrane domains, TMDs) that form intramembrane copper ion channels. A unique structural feature of this multi-domain protein is its large cytoplasmic N-terminal tail containing six independently folded copper(II) response elements (CREs), each approximately 70 amino acids long. The CREs are named from the N-terminus, with CRE1 being the first domain starting from the N-terminus and CRE6 being the last domain closest to the ATP7B transmembrane region. [Overview of the project]

[0005] This disclosure provides a cleaved ATP7B comprising one or more sequences selected from CRE5, CRE6 and their variants, and one or more sequences selected from CRE1, CRE2 and their variants.

[0006] In some embodiments, the cleaved ATP7B further includes flexible linkers that connect the sequences to each other.

[0007] In some embodiments, the cleaved ATP7B has the structure shown in formula Ia from the N-terminus to the C-terminus. ALBLCLD(Ia) In the formula, A, B, C or D are selected from CRE1, CRE2, CRE5, CRE6 and their variants, A, B, C and D are different from each other, A and B are present, C and / or D are optionally present, and L is either absent or a flexible linker.

[0008] In some embodiments, the present disclosure provides cleavage-type ATP7B in which 1) CRE5 or a variant thereof and / or 2) CRE6 or a variant thereof exists, and 3) CRE1 or a variant thereof and / or 4) CRE2 or a variant thereof exists.

[0009] Wild-type ATP7B consists of several independently folded Cu(II) response elements (CREs) located between the leader peptide (LP) and the C-terminal region (CTR). Six CREs can be identified from the N-terminus to the transmembrane portion of ATP7B. The CREs constituting cleaved ATP7B may be arranged in any order, combined directly without sequence insertion, or have any flexible sequence containing endogenous linkers between them.

[0010] In some embodiments, the disclosure provides cleavage-type ATP7B in which CRE1 or its variants and CRE5 or its variants exist.

[0011] In some embodiments, the disclosure provides cleavage-type ATP7B in which CRE1 or its variants and CRE6 or its variants exist.

[0012] In some embodiments, the disclosure provides cleavage-type ATP7B in which CRE1 or its variants, CRE5 or its variants, and CRE6 or its variants exist.

[0013] In some embodiments, the disclosure provides cleavage-type ATP7B in which CRE2 or its variants and CRE5 or its variants exist.

[0014] In some embodiments, the disclosure provides cleavage-type ATP7B in which CRE2 or its variants and CRE6 or its variants exist.

[0015] In some embodiments, the disclosure provides cleavage-type ATP7B in which CRE2 or its variants, CRE5 or its variants, and CRE6 or its variants exist.

[0016] In some embodiments, the disclosure provides cleavage-type ATP7B in which CRE1 or its variants, CRE2 or its variants, and CRE5 or its variants exist.

[0017] In some embodiments, the disclosure provides cleavage-type ATP7B in which CRE1 or its variants, CRE2 or its variants, and CRE6 or its variants exist.

[0018] In some embodiments, the disclosure provides cleavage-type ATP7B in which CRE1 or its variants, CRE2 or its variants, CRE5 or its variants, and CRE6 or its variants exist.

[0019] In some embodiments, CRE3 is deleted in the cleaved ATP7B. Preferably, the disclosure provides cleaved ATP7B in which CRE1 or its variants and CRE5 or its variants are present, but CRE2, CRE3, CRE4, and CRE6 are deleted. Preferably, the disclosure provides cleaved ATP7B in which CRE1 or its variants and CRE6 or its variants are present, but CRE2, CRE3, CRE4, and CRE5 are deleted. Preferably, the disclosure provides cleaved ATP7B in which CRE1 or its variants, CRE5 or its variants and CRE6 or its variants are present, but CRE2, CRE3, and CRE4 are deleted. Preferably, the disclosure provides cleaved ATP7B in which CRE2 or its variants and CRE5 or its variants are present, but CRE1, CRE3, CRE4, and CRE6 are deleted. Preferably, the disclosure provides cleaved ATP7B in which CRE2 or its variants and CRE6 or its variants are present, but CRE1, CRE3, CRE4, and CRE5 are deleted. Preferably, the disclosure provides cleaved ATP7B in which CRE2 or its variants, CRE5 or its variants, and CRE6 or its variants are present, but CRE1, CRE3, and CRE4 are deleted. Preferably, the disclosure provides cleaved ATP7B in which CRE1 or its variants, CRE2 or its variants, and CRE5 or its variants are present, but CRE3, CRE4, and CRE6 are deleted. Preferably, the disclosure provides cleaved ATP7B in which CRE1 or its variants, CRE2 or its variants, and CRE6 or its variants are present, but CRE3, CRE4, and CRE5 are deleted. Preferably, the disclosure provides cleaved ATP7B in which CRE1 or its variants, CRE2 or its variants, CRE5 or its variants, and CRE6 or its variants are present, but CRE3, CRE4, and CRE4 are deleted.

[0020] In some embodiments, the cleaved ATP7B includes a sequence selected from the group consisting of amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, and at least 99.8% identity with SEQ ID NO: 18, 20, 22, 24, 26, 28, 30, 32, and 34, wherein the cleaved ATP7B retains the function of transmembrane copper transport.

[0021] In some embodiments, CRE3 is omitted.

[0022] In some embodiments, in the truncated ATP7B, CRE3 is not deleted. Preferably, the present disclosure provides a truncated ATP7B in which CRE1 or its variant, CRE3 or its variant, and CRE5 or its variant are present, but CRE2, CRE4, and CRE6 are deleted. Preferably, the present disclosure provides a truncated ATP7B in which CRE1 or its variant, CRE3 or its variant, and CRE6 or its variant are present, but CRE2, CRE4, and CRE5 are deleted. Preferably, the present disclosure provides a truncated ATP7B in which CRE1 or its variant, CRE3 or its variant, CRE5 or its variant, and CRE6 or its variant are present, but CRE2 and CRE4 are deleted. Preferably, the present disclosure provides a truncated ATP7B in which CRE2 or its variant, CRE3 or its variant, and CRE5 or its variant are present, but CRE1, CRE4, and CRE6 are deleted. Preferably, the present disclosure provides a truncated ATP7B in which CRE2 or its variant, CRE3 or its variant, and CRE6 or its variant are present, but CRE1, CRE4, and CRE5 are deleted. Preferably, the present disclosure provides a truncated ATP7B in which CRE2 or its variant, CRE3 or its variant, CRE5 or its variant, and CRE6 or its variant are present, but CRE1 and CRE4 are deleted. Preferably, the present disclosure provides a truncated ATP7B in which CRE1 or its variant, CRE2 or its variant, CRE3 or its variant, and CRE5 or its variant are present, but CRE4 and CRE6 are deleted. Preferably, the present disclosure provides a truncated ATP7B in which CRE1 or its variant, CRE2 or its variant, CRE3 or its variant, and CRE6 or its variant are present, but CRE4 and CRE5 are deleted. Preferably, the present disclosure provides a truncated ATP7B in which CRE1 or its variant, CRE2 or its variant, CRE3 or its variant, CRE5 or its variant, and CRE6 or its variant are present, but CRE4 is deleted.

[0023] In some embodiments, CRE3 is deleted, but CRE5 or its variants and / or CRE6 or its variants are present, and CRE1 or its variants and / or CRE2 or its variants are present, and the cleaved ATP7B further comprises CRE4 or its variants. Preferably, the disclosure provides cleaved ATP7B in which CRE1 or its variants, CRE4 or its variants and CRE5 or its variants are present, but CRE2, CRE3 and CRE6 are deleted. Preferably, the disclosure provides cleaved ATP7B in which CRE1 or its variants, CRE4 or its variants and CRE6 or its variants are present, but CRE2, CRE3 and CRE5 are deleted. Preferably, the disclosure provides cleaved ATP7B in which CRE1 or its variants, CRE4 or its variants, CRE5 or its variants and CRE6 or its variants are present, but CRE2 and CRE3 are deleted. Preferably, the disclosure provides cleaved ATP7B in which CRE2 or its variants, CRE4 or its variants and CRE5 or its variants are present, but CRE1, CRE3 and CRE6 are deleted. Preferably, the disclosure provides cleaved ATP7B in which CRE2 or its variants, CRE4 or its variants, and CRE6 or its variants are present, but CRE1, CRE3, and CRE5 are deleted. Preferably, the disclosure provides cleaved ATP7B in which CRE2 or its variants, CRE4 or its variants, CRE5 or its variants, and CRE6 or its variants are present, but CRE1 and CRE3 are deleted. Preferably, the disclosure provides cleaved ATP7B in which CRE1 or its variants, CRE2 or its variants, CRE4 or its variants, and CRE5 or its variants are present, but CRE3 and CRE6 are deleted. Preferably, the disclosure provides cleaved ATP7B in which CRE1 or its variants, CRE2 or its variants, CRE4 or its variants, and CRE6 or its variants are present, but CRE3 and CRE5 are deleted. Preferably, the disclosure provides cleaved ATP7B in which CRE1 or its variants, CRE2 or its variants, CRE4 or its variants, CRE5 or its variants, and CRE6 or its variants are present, but CRE3 is deleted.

[0024] In another aspect, the present disclosure provides a truncated ATP7B comprising CRE5 or a variant thereof, and further comprising CRE3 or a variant thereof or CRE4 or a variant thereof.

[0025] In another aspect, the present disclosure provides a nucleic acid sequence encoding the truncated ATP7B.

[0026] In some embodiments, the truncated ATP7B nucleotide comprises SEQ ID NO: 17, 19, 21, 23, 25, 27, 29, 31, and 33, and sequences selected from the group consisting of sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, at least 99.8% identity to SEQ ID NO: 17, 19, 21, 23, 25, 27, 29, 31, and 33, and encodes a truncated ATP7B protein that retains the function of transmembrane copper transport.

[0027] In another aspect, the present disclosure provides an RNA polynucleotide encoding the truncated ATP7B in the present disclosure. The RNA includes messenger RNA (mRNA) or circular RNA (circRNA).

[0028] Messenger RNA (mRNA) is any ribonucleic acid that codes for (at least one) protein (a naturally occurring, unnaturally occurring, or modified amino acid polymer) and can produce coded proteins by in vitro, in vivo, or in situ translation. Those skilled in the art will understand that, unless otherwise specified, nucleic acid sequences described herein may include "T" in representative DNA sequences, but when such sequence represents RNA (e.g., mRNA), "T" should be replaced with "U". Accordingly, any DNA disclosed and identified herein by a specific sequence identification number further discloses a corresponding RNA (e.g., mRNA) sequence similar to the DNA, where each "T" in the DNA sequence is replaced with a "U". Similarly, any RNA disclosed and identified herein by a specific sequence identification number also discloses a corresponding DNA sequence similar to the RNA, where each "U" in the RNA sequence is replaced with a "T".

[0029] In some embodiments, the RNA comprises at least one modified nucleotide. The terms “modified” or, where appropriate, “modified” refer to modifications to A, G, U, or C ribonucleotides. Generally, in this specification, these terms are not intended to refer to ribonucleotide modifications in naturally occurring 5'-terminal mRNA cap portions. In polypeptides, the term “modified” refers to modifications compared to the standard 20-amino acid group.

[0030] In another aspect, this disclosure is: (a) 5'-terminal inverted repeat (ITR) sequence, (b) promoter sequence, (c) The nucleic acid sequence encoding the cleaved ATP7B, (d) Poly(A) sequence, and (e) Provides a recombinant nucleic acid construct containing a 3'-ITR sequence.

[0031] In some embodiments, the recombinant nucleic acid construct further comprises one or more enhancer sequences.

[0032] In some embodiments, the recombinant nucleic acid construct further comprises one or more intron sequences.

[0033] In some embodiments, the recombinant nucleic acid construct further comprises one or more signal sequences.

[0034] In another embodiment, the disclosure provides a recombinant vector comprising the recombinant nucleic acid construct.

[0035] In some embodiments, the vector is a viral vector, preferably an AAV vector.

[0036] In another aspect, the Disclosure provides vectors and capsid proteins relating to the Disclosure.

[0037] In some embodiments, the AAV capsid is selected from the group consisting of serotypes 9, 8, 1, 2, 3, 3B, 4, 5, 6, 7, 10, 11, 12, 13, rh10, or hu37, as well as any or variants thereof of AAV serotypes isolated from human and non-human mammals.

[0038] In another embodiment, the disclosure provides a pharmaceutical composition comprising the cleaved ATP7B, the nucleic acid sequence, the recombinant nucleic acid construct, the recombinant vector, or the rAAV.

[0039] In another embodiment, the present disclosure provides a method for treating a disease in a subject, comprising administering an effective amount of the cleaved ATP7B, the nucleic acid sequence, the recombinant nucleic acid construct, the recombinant vector, the rAAV, or the pharmaceutical composition.

[0040] In some embodiments, the disease is an ATP7B-related disease, preferably Wilson's disease.

[0041] In some embodiments, the subject is a mammal, preferably a human.

Brief Description of the Drawings

[0042] The present invention will be more comprehensively understood from the following drawings.

[0043] [Figure 1] Shows the relative copper transport capacity of a truncated ATP7B form with a single CRE evaluated by the dual luciferase assay. A relatively high relative Fluci / Rluci ratio indicates a relatively low copper transport capacity. Among the six CREs, CRE5 shows the highest copper transport capacity. ***: p < 0.001. The plot shows the mean ± SEM, and n = 3 in each group.

[0044] [Figure 2] Combination with CRE5 is shown to significantly improve the copper transport capacity of CRE1, CRE2, CRE3 and CRE4. ***: p < 0.001. The plot shows the mean ± SEM, and n = 3 in each group.

[0045] [Figure 3] Combination with CRE1 is shown to significantly improve the copper transport capacity of CRE5, CRE6, CRE5+CRE6 and CRE4+CRE6. *: p < 0.05; ***: p < 0.001. The plot shows the mean ± SEM, and n = 15 in each group.

[0046] [Figure 4] Combination with CRE2 is shown to significantly improve the copper transport capacity of CRE5, CRE6, CRE5+CRE6 and CRE4+CRE6. **: 0.001 < p < 0.01, ***: p < 0.001. The plot shows the mean ± SEM, and n = 15 in each group.

[0047] [Figure 5]The combination with CRE3 does not further enhance the copper transport ability of CRE6, CRE5 + CRE6, CRE1 + CRE5, and CRE1 + CRE6, and some of these combinations have an adverse effect on the copper transport ability. NS: p > 0.05. The plot shows the mean ± SEM, and n = 15 in each group.

[0048] [Figure 6] The combination with CRE3 or CRE4 has an adverse effect on some CRE combinations, but as a result of adding CRE3 or CRE4 to these CRE combinations, it still exceeds or is equivalent to CRE5 + CRE6 in terms of copper transport ability. NS: p > 0.05. **: 0.001 < p < 0.01, ***: p < 0.001. The plot shows the mean ± SEM, and n = 15 in each group.

[0049] [Figure 7] The combination with CRE1 + CRE2 shows a significant improvement in the copper transport ability of CRE5, CRE6, and CRE5 + CRE6. ***: p < 0.001. The plot shows the mean ± SEM, and n = 15 in each group.

[0050] [Figure 8] It shows that a specific CRE combination has a copper transport ability corresponding to the wild - type form. The plot shows the mean ± SEM, and n = 15 in each group.

[0051] [Figure 9] It shows that the truncated ATP7B with a specific combination of CRE or CRE scheme is efficiently packaged into AAV8, and the yield > 2.0×1011 vg / mL, which is much higher than that of AAV8 encoding the wild - type full - length ATP7B.

[0052] [Figure 10a]This study demonstrates that packaged AAV8 with cleaved ATP7B having a specific CRE combination or scheme can reverse ceruloplasmin (Cp) activity (a) and tissue copper accumulation (b) in a mouse model of Wilson's disease. *: p<0.05, **: p<0.01. Plots show mean ± SEM, with n=3 in each group. [Figure 10b] Same as above [Modes for carrying out the invention]

[0053] Cut-type ATP7B Wild-type ATP7B consists of several independently folded copper(II) response elements (CREs) located between the leader peptide (LP) and the C-terminal region (CTR). Six CREs can be identified from the N-terminus to the transmembrane region of ATP7B. The sequences of the CREs are shown as SEQ ID NO: 1-6. The cleaved ATP7B nucleotides described herein include nucleotide sequences encoding functional fragments or domains of ATP7B. Cleaved ATP7B includes deletions of one or more CREs. Cleaved ATP7B may also be a recombinant polypeptide containing one or more CREs. The CREs constituting cleaved ATP7B may be arranged in any order, combined directly without sequence insertions, or have any flexible sequence including endogenous linkers between the CREs.

[0054] This disclosure provides a cleavage-type ATP7B that contains CRE5 or its variants and / or CRE6 or its variants, and also contains CRE1 or its variants and / or CRE2 or its variants.

[0055] The term “mutant” includes an amino acid sequence that differs from the natural sequence by at least one “amino acid modification” as defined herein. Preferably, the mutant has at least one amino acid substitution or deletion compared to the natural sequence. The mutants described herein preferably have at least about 80% homology to the natural sequence, most preferably at least about 90% homology, more preferably at least about 95% homology, and retain the function of the natural sequence.

[0056] In some embodiments, the Disclosure provides cleaved ATP7B having CRE1 or a variant thereof and CRE5 or a variant thereof, but lacking CRE2, CRE3, CRE4, and CRE6. In some embodiments, the Disclosure provides cleaved ATP7B comprising amino acid sequences of SEQ ID NO:1 and SEQ ID NO:5. The cleaved ATP7B further comprises a leader peptide (LP) and a C-terminal region (CTR). In some embodiments, the Disclosure provides cleaved ATP7B comprising the amino acid sequence of SEQ ID NO:18, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 99.5% identity with SEQ ID NO:18, and retaining the function of transmembrane copper transport.

[0057] In some embodiments, the Disclosure provides cleaved ATP7B having CRE1 or a variant thereof and CRE6 or a variant thereof, but lacking CRE2, CRE3, CRE4, and CRE5. In some embodiments, the Disclosure provides cleaved ATP7B comprising amino acid sequences of SEQ ID NO:1 and SEQ ID NO:6. The cleaved ATP7B further comprises a leader peptide (LP) and a C-terminal region (CTR). In some embodiments, the Disclosure provides cleaved ATP7B comprising the amino acid sequence of SEQ ID NO:20, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 99.5% identity with SEQ ID NO:20, and retaining the function of transmembrane copper transport.

[0058] In some embodiments, the Disclosure provides cleaved ATP7B having CRE1 or its variants, CRE5 or its variants, and CRE6 or its variants, but lacking CRE2, CRE3, and CRE4. In some embodiments, the Disclosure provides cleaved ATP7B comprising the amino acid sequences of SEQ ID NO:1, SEQ ID NO:5, and SEQ ID NO:6. The cleaved ATP7B further comprises a leader peptide (LP) and a C-terminal region (CTR). In some embodiments, the Disclosure provides cleaved ATP7B comprising the amino acid sequence of SEQ ID NO:22, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 99.5% identity with SEQ ID NO:22, and retaining the function of transmembrane copper transport.

[0059] In some embodiments, the Disclosure provides cleaved ATP7B having CRE2 or a variant thereof and CRE5 or a variant thereof, but lacking CRE1, CRE3, CRE4, and CRE6. In some embodiments, the Disclosure provides cleaved ATP7B comprising amino acid sequences of SEQ ID NO:2 and SEQ ID NO:5. The cleaved ATP7B further comprises a leader peptide (LP) and a C-terminal region (CTR). In some embodiments, the Disclosure provides cleaved ATP7B comprising the amino acid sequence of SEQ ID NO:24, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 99.5% identity with SEQ ID NO:24, and retaining the function of transmembrane copper transport.

[0060] In some embodiments, the disclosure provides cleaved ATP7B having CRE2 or a variant thereof and CRE6 or a variant thereof, but lacking CRE1, CRE3, CRE4, and CRE5. In some embodiments, the disclosure provides cleaved ATP7B comprising amino acid sequences of SEQ ID NO:2 and SEQ ID NO:6. The cleaved ATP7B further comprises a leader peptide (LP) and a C-terminal region (CTR). In some embodiments, the disclosure provides cleaved ATP7B comprising the amino acid sequence of SEQ ID NO:26, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 99.5% identity with SEQ ID NO:26, and retaining the function of transmembrane copper transport.

[0061] In some embodiments, the Disclosure provides cleaved ATP7B having CRE2 or its variants, CRE5 or its variants, and CRE6 or its variants, but lacking CRE1, CRE3, and CRE4. In some embodiments, the Disclosure provides cleaved ATP7B comprising the amino acid sequences of SEQ ID NO:2, SEQ ID NO:5, and SEQ ID NO:6. The cleaved ATP7B further comprises a leader peptide (LP) and a C-terminal region (CTR). In some embodiments, the Disclosure provides cleaved ATP7B comprising the amino acid sequence of SEQ ID NO:28, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 99.5% identity with SEQ ID NO:28, and retaining the function of transmembrane copper transport.

[0062] In some embodiments, the Disclosure provides cleaved ATP7B having CRE1 or a variant thereof, CRE2 or a variant thereof, and CRE5 or a variant thereof, but lacking CRE3, CRE4, and CRE6. In some embodiments, the Disclosure provides cleaved ATP7B comprising the amino acid sequences of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:5. The cleaved ATP7B further comprises a leader peptide (LP) and a C-terminal region (CTR). In some embodiments, the Disclosure provides cleaved ATP7B comprising the amino acid sequence of SEQ ID NO:30, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 99.5% identity with SEQ ID NO:30, and retaining the function of transmembrane copper transport.

[0063] In some embodiments, the Disclosure provides cleaved ATP7B having CRE1 or a variant thereof, CRE2 or a variant thereof, and CRE6 or a variant thereof, but lacking CRE3, CRE4, and CRE5. In some embodiments, the Disclosure provides cleaved ATP7B comprising the amino acid sequences of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:6. The cleaved ATP7B further comprises a leader peptide (LP) and a C-terminal region (CTR). In some embodiments, the Disclosure provides cleaved ATP7B comprising the amino acid sequence of SEQ ID NO:32, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 99.5% identity with SEQ ID NO:32, and retaining the function of transmembrane copper transport.

[0064] In some embodiments, the Disclosure provides cleaved ATP7B having CRE1 or a variant thereof, CRE2 or a variant thereof, CRE5 or a variant thereof, and CRE6 or a variant thereof, but lacking CRE3 and CRE4. In some embodiments, the Disclosure provides cleaved ATP7B comprising the amino acid sequences of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:5, and SEQ ID NO:6. The cleaved ATP7B further comprises a leader peptide (LP) and a C-terminal region (CTR). In some embodiments, the Disclosure provides cleaved ATP7B comprising the amino acid sequence of SEQ ID NO:34, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 99.5% identity with SEQ ID NO:34, and retaining the function of transmembrane copper transport.

[0065] [Table 1] JPEG2026509434000002.jpg252168 JPEG2026509434000003.jpg250167 JPEG2026509434000004.jpg252168 JPEG2026509434000005.jpg252168 JPEG2026509434000006.jpg252167 JPEG2026509434000007.jpg252166 JPEG2026509434000008.jpg254168 JPEG2026509434000009.jpg252167 JPEG2026509434000010.jpg253169 JPEG2026509434000011.jpg252168 JPEG2026509434000012.jpg250165 JPEG2026509434000013.jpg250166 JPEG2026509434000014.jpg250166 JPEG2026509434000015.jpg250165 JPEG2026509434000016.jpg250167 JPEG2026509434000017.jpg250165 JPEG2026509434000018.jpg109165

[0066] In some embodiments, CRE3 is deleted, but CRE5 or a variant thereof and / or CRE6 or a variant thereof are present, and the cleaved ATP7B further comprises CRE4 or a variant thereof.

[0067] In some embodiments, CRE3 is deleted, but CRE5 or its variants and / or CRE6 or its variants are present, and CRE1 or its variants and / or CRE2 or its variants are present, and the cleaved ATP7B further comprises CRE4 or its variants.

[0068] In some embodiments, the disclosure provides cleaved ATP7B in which CRE1 or its variants, CRE4 or its variants, and CRE5 or its variants are present, but CRE2, CRE3, and CRE6 are deleted. In some embodiments, the disclosure provides cleaved ATP7B in which CRE1 or its variants, CRE4 or its variants, and CRE6 or its variants are present, but CRE2, CRE3, and CRE5 are deleted. In some embodiments, the disclosure provides cleaved ATP7B in which CRE1 or its variants, CRE4 or its variants, CRE5 or its variants, and CRE6 or its variants are present, but CRE2 and CRE3 are deleted.

[0069] In some embodiments, the disclosure provides cleaved ATP7B in which CRE2 or its variants, CRE4 or its variants, and CRE5 or its variants are present, but CRE1, CRE3, and CRE6 are deleted. In some embodiments, the disclosure provides cleaved ATP7B in which CRE2 or its variants, CRE4 or its variants, and CRE6 or its variants are present, but CRE1, CRE3, and CRE5 are deleted. In some embodiments, the disclosure provides cleaved ATP7B in which CRE2 or its variants, CRE4 or its variants, CRE5 or its variants, and CRE6 or its variants are present, but CRE1 and CRE3 are deleted.

[0070] In some embodiments, the disclosure provides cleaved ATP7B in which CRE1 or its variants, CRE2 or its variants, CRE4 or its variants, and CRE5 or its variants are present, but CRE3 and CRE6 are deleted. In some embodiments, the disclosure provides cleaved ATP7B in which CRE1 or its variants, CRE2 or its variants, CRE4 or its variants, and CRE6 or its variants are present, but CRE3 and CRE5 are deleted. In some embodiments, the disclosure provides cleaved ATP7B in which CRE1 or its variants, CRE2 or its variants, CRE4 or its variants, CRE5 or its variants, and CRE6 or its variants are present, but CRE3 is deleted.

[0071] In some embodiments, CRE3 is not deleted in the cleaved ATP7B. Preferably, the disclosure provides cleaved ATP7B in which CRE1 or its variants, CRE3 or its variants, and CRE5 or its variants are present, but CRE2, CRE4, and CRE6 are deleted. Preferably, the disclosure provides cleaved ATP7B in which CRE1 or its variants, CRE3 or its variants, and CRE6 or its variants are present, but CRE2, CRE4, and CRE5 are deleted. Preferably, the disclosure provides cleaved ATP7B in which CRE1 or its variants, CRE3 or its variants, CRE5 or its variants, and CRE6 or its variants are present, but CRE2 and CRE4 are deleted. Preferably, the disclosure provides cleaved ATP7B in which CRE2 or its variants, CRE3 or its variants, and CRE5 or its variants are present, but CRE1, CRE4, and CRE6 are deleted. Preferably, the disclosure provides cleaved ATP7B in which CRE2 or its variants, CRE3 or its variants, and CRE6 or its variants are present, but CRE1, CRE4, and CRE5 are deleted. Preferably, the disclosure provides cleaved ATP7B in which CRE2 or its variants, CRE3 or its variants, CRE5 or its variants, and CRE6 or its variants are present, but CRE1 and CRE4 are deleted. Preferably, the disclosure provides cleaved ATP7B in which CRE1 or its variants, CRE2 or its variants, CRE3 or its variants, and CRE5 or its variants are present, but CRE4 and CRE6 are deleted. Preferably, the disclosure provides cleaved ATP7B in which CRE1 or its variants, CRE2 or its variants, CRE3 or its variants, and CRE6 or its variants are present, but CRE4 and CRE5 are deleted. Preferably, the disclosure provides cleaved ATP7B in which CRE1 or its variants, CRE2 or its variants, CRE3 or its variants, CRE5 or its variants, and CRE6 or its variants are present, but CRE4 is deleted.

[0072] In some embodiments, CRE3 is deleted, but CRE5 or its variants and / or CRE6 or its variants are present, and CRE1 or its variants and / or CRE2 or its variants are present, and the cleaved ATP7B further comprises CRE4 or its variants. Preferably, the disclosure provides cleaved ATP7B in which CRE1 or its variants, CRE4 or its variants and CRE5 or its variants are present, but CRE2, CRE3 and CRE6 are deleted. Preferably, the disclosure provides cleaved ATP7B in which CRE1 or its variants, CRE4 or its variants and CRE6 or its variants are present, but CRE2, CRE3 and CRE5 are deleted. Preferably, the disclosure provides cleaved ATP7B in which CRE1 or its variants, CRE4 or its variants, CRE5 or its variants and CRE6 or its variants are present, but CRE2 and CRE3 are deleted. Preferably, the disclosure provides cleaved ATP7B in which CRE2 or its variants, CRE4 or its variants and CRE5 or its variants are present, but CRE1, CRE3 and CRE6 are deleted. Preferably, the disclosure provides cleaved ATP7B in which CRE2 or its variants, CRE4 or its variants, and CRE6 or its variants are present, but CRE1, CRE3, and CRE5 are deleted. Preferably, the disclosure provides cleaved ATP7B in which CRE2 or its variants, CRE4 or its variants, CRE5 or its variants, and CRE6 or its variants are present, but CRE1 and CRE3 are deleted. Preferably, the disclosure provides cleaved ATP7B in which CRE1 or its variants, CRE2 or its variants, CRE4 or its variants, and CRE5 or its variants are present, but CRE3 and CRE6 are deleted. Preferably, the disclosure provides cleaved ATP7B in which CRE1 or its variants, CRE2 or its variants, CRE4 or its variants, and CRE6 or its variants are present, but CRE3 and CRE5 are deleted. Preferably, the disclosure provides cleaved ATP7B in which CRE1 or its variants, CRE2 or its variants, CRE4 or its variants, CRE5 or its variants, and CRE6 or its variants are present, but CRE3 is deleted.

[0073] In another embodiment, the disclosure provides a nucleic acid sequence encoding the cleaved ATP7B.

[0074] In some embodiments, the cleaved ATP7B nucleotide comprises a sequence selected from the group consisting of SEQ ID NO: 17, 19, 21, 23, 25, 27, 29, 31 and 33, and sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, and at least 99.8% identity with SEQ ID NO: 17, 19, 21, 23, 25, 27, 29, 31 and 33, thereby encoding a cleaved ATP7B protein that retains the function of transmembrane copper transport.

[0075] In another embodiment, the Disclosure provides RNA encoding cleaved ATP7B as relating to the Disclosure. In some embodiments, the RNA includes messenger RNA (mRNA) or circular RNA (circRNA).

[0076] Recombinant nucleic acid constructs In another aspect, this disclosure is: (a) 5'-terminal inverted repeat (ITR) sequence, (b) promoter sequence, (c) The nucleic acid sequence encoding the cleaved ATP7B, (d) Poly(A) sequence, and (e) Provides a recombinant nucleic acid construct containing a 3'-ITR sequence.

[0077] In some embodiments, the promoter is preferably selected, cleaved, or optimized from the transthyretin (TTR) promoter, the chicken β-actin (CBA) promoter, the cytomegalovirus immediate early gene (CMV) promoter and the thyroxine-binding globulin (TBG) promoter, the α-1 antitrypsin (A1AT) promoter and the CAG promoter.

[0078] In some embodiments, the recombinant nucleic acid construct further comprises one or more enhancer sequences. The enhancers are preferably selected, cleaved, or optimized from transthyretin enhancers (enTTR), cytomegalovirus immediate early gene (CMV) enhancers, chicken β-actin (CBA) enhancers, En34 enhancers, and apolipoprotein (ApoE) enhancers.

[0079] In some embodiments, the recombinant nucleic acid construct further comprises one or more intron sequences. The introns are preferably selected, cleaved, or optimized from endogenous ATP7B introns, SV40 small T introns, rabbit hemoglobin subunit β(rHBB) introns, human β-globulin IV S2 introns, Promega chimeric introns, and hFIX introns.

[0080] In some embodiments, the recombinant nucleic acid construct further comprises one or more signal sequences. The signal sequences are preferably selected from SV40 polyadenylation signal sequences, bovine growth hormone (BGH) polyadenylation signal sequences, and rabbit β-globin polyadenylation signal sequences.

[0081] Recombinant vectors In another embodiment, the disclosure provides a vector comprising the recombinant nucleic acid construct. Preferably, the vector is a viral vector, and more preferably an AAV vector. The term “vector” means a nucleic acid molecule that allows insertion of a foreign nucleic acid but does not impair the retention, replication, and / or integration functions of the vector in a host cell. The vector may include a nucleic acid sequence that allows replication in a host cell, such as an origin of replication. The vector may further include one or more select marker genes and other genetic elements. An expression vector is a vector that includes essential regulatory sequences that allow transcription and translation of the inserted one or more genes. In some embodiments of this specification, the vector is an AAV vector.

[0082] In various embodiments described herein, the recombinant viral vector is an adeno-associated virus (AAV) vector. The AAV vector may be any of the AAV vectors of serotype 1, 2, 3, 3B, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13, such as AAV1, AAV2, AAV3, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, or AAV13, as well as any of more than 100 variants isolated from human and non-human primate tissues (see, for example, Choi et al., 2005, Curr Gene Ther. 5: 299-310, 2005 and Gao et al., 2005, Curr Gene Ther. 5: 285-297). In this invention, any serotype of AAV vector can be used, and the selection of the AAV serotype depends in part on the type of cell targeted in gene therapy. For the treatment of WD, the liver is one of the relevant target organs. In some embodiments, the AAV vector is selected from serotype 9 (AAV9), serotype 8 (AAV8), serotype 5 (AAV5), or a variant thereof. In an exemplary embodiment, the AAV vector is serotype 8 (AAV8) or a variant thereof.

[0083] In another embodiment, the Disclosure provides a recombinant adeno-associated virus (rAAV) comprising an AAV capsid and the AAV vector.

[0084] In some embodiments, the AAV capsid is derived from AAVs of serotypes 9, 8, 1, 2, 3, 3B, 4, 5, 6, 7, 10, 11, 12, 13, rh10, or hu37, as well as any or variants thereof of AAV serotypes isolated from human and non-human mammals.

[0085] In some embodiments, the aforementioned AAVは、AAV1、AAV2、AAV2G9、AAV3、AAV3a、AAV3b、AAV3-3、AAV4、AAV4-4、AAV5、AAV6、AAV6.1、AAV6.2、AAV6.1.2、AAV7、AAV7.2、AAV8 、AAV9、AAV9.11、AAV9.13、AAV9.16、AAV9.24、AAV9.45、AAV9.47、AAV9.61、AAV9.68、AAV9.84、AAV9.9、AAV10、AAV11、AAV12、AAV16.3、AAV24.1、AAV27 .3、AAV42.12、AAV42-1b、AAV42-2、AAV42-3a、AAV42-3b、AAV42-4、AAV42-5a、AAV42-5b、AAV42-6b、AAV42-8、AAV42-10、AAV42-11、AAV42-12、AAV42-1 3、AAV42-15、AAV42-aa、AAV43-1、AAV43-12、AAV43-20、AAV43-21、AAV43-23、AAV43-25、AAV43-5、AAV44.1、AAV44.2、AAV44.5、AAV223.1、AAV223.2 AV223.4、AAV223.5、AAV223.6、AAV223.7、AAV1-7 / rh.48、AAV1-8 / rh.49、AAV2-15 / rh.62、AAV2-3 / rh.61、AAV2-4 / rh.50、AAV2-5 / rh.51、AAV3.1 / hu .6、AAV3.1 / hu.9、AAV3-9 / rh.52、AAV3-11 / rh.53、AAV4-8 / r11.64、AAV4-9 / rh.54、AAV4-19 / rh.55、AAV5-3 / rh.57、AAV5-22 / rh.58、AAV7.3 / rh.57、AAV3. V16.8 / hu.10、AAV16.12 / hu.11、AAV29.3 / bb.1、AAV29.5 / bb.2、AAV106.1 / hu.37、AAV114.3 / hu.40、AAV127.2 / hu.41、AAV127.5 / hu.42、AAV128.3 / hu.44、AAV130.4 / hu.48、AAV145.1 / hu.53、AAV145.5 / hu.54、AAV145.6 / hu.55、AAV161.10 / hu.60、AAV161.6 / hu.61、AAV33.12 / hu.17、AAV33.4 / hu.15、AAV33.8 / hu.16、AAV52 / hu.19、AAV52.1 / hu.20、AAV58.2 / hu.25、AAVA3.3、AAVA3.4、AAVA3.5、AAVA3.7、AAVC1、AAVC2、AAVC5、AAV-DJ、AAV-DJ8、AAV3. AAVF5、AAVH2、AAVrh.72、AAVhu.8、AAVrh.68、AAVrh.70、AAVpi.1、AAVpi.3、AAVpi.2、AAVrh.60、AAVrh.44、AAVrh.65、AAVrh.55、AAVrh.47、AAVrh.69、 AAVrh.45, AAVrh.59, AAVhu.12, AAVH6, AAVLK03, AAVH-1 / hu.1, AAVH-5 / hu.3, AAVLG-10 / rh.40, AAVLG-4 / rh.38, AAVLG-9 / hu.39, AAVN721-8 / rh.43, AAVCh.5, AAVCh.5R1, AAVcy.2, AAVcy.3, AAVcy.4, AAVcy.5, AAVCy.5R1, AAVCy.5R2, AAVCy.5R3, AAVCy.5R4, AAVcy.6, AAVhu.1, AAVhu.2, AAVhu.3, AAVh u.4、AAVhu.5、AAVhu.6、AAVhu.7、AAVhu.9、AAVhu.10、AAVhu.11、AAVhu.13、AAVhu.15、AAVhu.16、AAVhu.17、AAVhu.18、AAVhu.20、AAVhu.21、AAVhu.22 、AAVhu.23.2、AAVhu.24、AAVhu.25、AAVhu.27、AAVhu.28、AAVhu.29、AAVhu.29R、AAVhu.31、AAVhu.32、AAVhu.34、AAVhu.35、AAVhu.37、AAVhu.39、AAVhu. u.40、AAVhu.41、AAVhu.42、AAVhu.43、AAVhu.44、AAVhu.44R1、AAVhu.44R2、AAVhu.44R3、AAVhu.45、AAVhu.46、AAVhu.47、AAVhu.48、AAVhu.48R1、AAVhu. u.48R2、AAVhu.48R3、AAVhu.49、AAVhu.51、AAVhu.52、AAVhu.54、AAVhu.55、AAVhu.56、AAVhu.57、AAVhu.58、AAVhu.60、AAVhu.61、AAVhu.63、AAVhu.64、AAVhu.66、AAVhu.67、AAVhu.14 / 9、AAVhu.t 19、AAVrh.2、AAVrh.2R、AAVrh.8、AAVrh.8R、AAVrh.10、AAVrh.12、AAVrh.13、AAVrh.13R、AAVrh.14、AAVrh.17、AAVrh.18、AAVrh.19、AAVrh.20 、AAVrh.21、AAVrh.22、AAVrh.23、AAVrh.24、AAVrh.25、AAVrh.31、AAVrh.32、AAVrh.33、AAVrh.34、AAVrh.35、AAVrh.36、AAVrh.37、AAVrh.37 2、AAVrh.38、AAVrh.39、AAVrh.40、AAVrh.46、AAVrh.48、AAVrh.48.1、AAVrh.48.1.2、AAVrh.48.2、AAVrh.49、AAVrh.51、AAVrh.52、AAVrh.53、 AAVrh.54、AAVrh.56、AAVrh.57、AAVrh.58、AAVrh.61、AAVrh.64、AAVrh.64R1、AAVrh.64R2、AAVrh.67、AAVrh.73、AAVrh.74、AAVrh.8R、AAVrh.8R. A586R mutation、AAVrh8R R533A mutation、AAAV、BAAV、ヤギAAV、ウシAAV、AAVhE1.1、AAVhEr1.5、AAVhER1.14、AAVhEr1.8、AAVhEr1.16、AAVhEr1.18、AAVhEr1.35、AAVhEr1.7、AAV hEr1.36、AAVhEr2.29、AAVhEr2.4、AAVhEr2.16、AAVhEr2.30、AAVhEr2.31、AAVhEr2.36、AAVhER1.23、AAVhEr3.1、AAV2.5T、AAV-PAEC、AAV-LK0 1、AAV-LK02、AAV-LK03、AAV-LK04、AAV-LK05、AAV-LK06、AAV-LK07、AAV-LK08、AAV-LK09、AAV-LK10、AAV-LK11、AAV-LK12、AAV-LK13、AAV-LK14 、AAV-LK15、AAV-LK16、AAV-LK17、AAV-LK18、AAV-LK19、AAV-PAEC2、AAV-PAEC4、AAV-PAEC6、AAV-PAEC7、AAV-PAEC8、AAV-PAEC11、AAV-PAEC12、AAV-2-pre-miRNA-101, AAV-8h, AAV-8b, AAV-h, AAV-b, AAV SM 10-2, AAV Shuffle 100-1, AAV Shuffle 100-3, AAV Shuffle 100-7, AAV Shuffle 10-2, AAV Shuffle 10-6, AAV Shuffle 10-8, AAV Shuffle 100-2, AAV SM 10-1, AAV SM 10-8, AAV SM 100-3, AAV SM 100-10, BNP61 AAV, BNP62 AAV, BNP63 AAV, AAVrh.50, AAVrh.43, AAVrh.62, AAVrh.48, AAVhu.19, AAVhu.11, AAVhu.53, AAV4-8 / rh.64, AAVLG-9 / hu.39, AAV54.5 / hu.23, AAV54.2 / hu.22, AAV54.7 / hu.24, AAV54.1 / hu.21, AAV54.4R / hu.27, AAV46.2 / hu.28, AAV46.6 / hu.29, AAV128.1 / hu.43, authentic AAV (ttAAV), UPENN AAV 10, Japanese AAV 10 serotype, AAV CBr-7.1, AAV CBr-7.10, AAV CBr-7.2, AAV CBr-7.3, AAV CBr-7.4, AAV CBr-7.5, AAV CBr-7.7, AAV CBr-7.8, AAV CBr-B7.3, AAV CBr-B7.4, AAV CBr-E1, AAV CBr-E2, AAV CBr-E3, AAV CBr-E4, AAV CBr-E5, AAV CBr-e5, AAV CBr-E6, AAV CBr-E7, AAV CBr-E8, AAV CHt-1, AAV CHt-2, AAV CHt-3, AAV CHt-6.1, AAV CHt-6.10, AAV CHt-6.5, AAV CHt-6.6, AAV CHt-6.7, AAV CHt-6.8, AAV CHt-P1, AAV CHt-P2, AAV CHt-P5, AAV CHt-P6, AAV CHt-P8, AAV CHt-P9, AAV CKd-1, AAV CKd-10, AAV CKd-2, AAV CKd-3, AAV CKd-4, AAV CKd-6, AAV CKd-7, AAV CKd-8, AAV CKd-B1, AAV CKd-B2AAV CKd-B3、AAV CKd-B4、AAV CKd-B5、AAV CKd-B6、AAV CKd-B7、AAV CKd-B8、AAV CKd-H1、AAV CKd-H2、AAV CKd-H3、AAV CKd-H4、AAV CKd-H5、AAV CKd-H6、AAV CKd-N3、AAV CKd-N4、AAV CKd-N9、AAV CLg-F1、AAV CLg-F2、AAV CLg-F3、AAV CLg-F4、AAV CLg-F5、AAV CLg-F6、AAV CLg-F7、AAV CLg-F8、AAV CLv-1、AAV CLv1-1、AAV Clv1-10、AAV CLv1-2、AAV CLv-12、AAV CLv1-3、AAV CLv-13、AAV CLv1-4、AAV Clv1-7、AAV Clv1-8、AAV Clv1-9、AAV CLv-2、AAV CLv-3、AAV CLv-4、AAV CLv-6、AAV CLv-8、AAV CLv-D1、AAV CLv-D2、AAV CLv-D3、AAV CLv-D4、AAV CLv-D5、AAV CLv-D6、AAV CLv-D7、AAV CLv-D8、AAV CLv-E1、AAV CLv-K1、AAV CLv-K3、AAV CLv-K6、AAV CLv-L4、AAV CLv-L5、AAV CLv-L6、AAV CLv-M1、AAV CLv-M11、AAV CLv-M2、AAV CLv-M5、AAV CLv-M6、AAV CLv-M7、AAV CLv-M8、AAV CLv-M9、AAV CLv-R1、AAV CLv-R2、AAV CLv-R3、AAV CLv-R4、AAV CLv-R5、AAV CLv-R6、AAV CLv-R7、AAV CLv-R8、AAV CLv-R9、AAV CSp-1、AAV CSp-10、AAV CSp-11、AAV CSp-2、AAV Csp-3、AAV Csp-4、AAV Csp-6、AAV Csp-7、AAV Csp-8、AAV CSp-8、10、AAV CSp-8.2、AAV CSp-8.4、AAV CSp-8.5、AAV CSp-8.6、AAV CSp-8.7、AAV CSp-8.8、AAV CSp-8.9、AAV CSp-9、AAV.hu.48R3、AAV.VR-355、AAV3B, AAV4, AAV5, AAVF1 / HSC1, AAVF11 / HSC11, AAVF12 / HSC12, AAVF13 / HSC13, AAVF14 / HSC14, AAVF15 / HSC15, AAVF16 / HSC16, AAVF17 / HSC17, AAVF2 / HSC2, AAV, F3 / HSC3, AAVF4 / HSC4, AAVF5 / HSC5, AAVF6 / HSC6, AAVF7 / HSC7, AAVF8 / HSC8, AAVF9 / H SC9, AAV-PHP.B (PHP.B), AAV-PHP.A (PHP.A), G2B-26, G2B-13, TH1.1-32, TH1.1-35, A AVPHP.B2, AAVPHP.B3, AAVPHP.N / PHP.B-DGT, AAVPHP.B-EST, AAVPHP.B-GGT, AAVPHP.B-ATP, AAVPHP.B-ATT-T, AAVPHP.B-DGT-T, AAVPHP.B-GGT-T, AAVPHP.B-SGS, AAVPHP The group is selected from .B-AQP, AAVPHP.B-QQP, AAVPHP.B-SNP(3), AAVPHP.B-SNP, AAVPHP.B-QGT, AAVPHP.B-NQT, AAVPHP.B-EGS, AAVPHP.B-SGN, AAVPHP.B-EGT, AAVPHP.B-DST, AAVPHP.B-DST, AAVPHP.B-STP, AAVPHP.B-PQP, AAVPHP.B-SQP, AAVPHP.B-QLP, AAVPHP.B-TMP, AAVPHP.B-TTP, AAVPHP.S / G2A12, AAVG2A15 / G2A3, AAVG2B4, AAVG2B5 and their variants.

[0086] Pharmaceutical composition In some embodiments, the rAAV is formulated into a buffer / carrier suitable for injection into a human subject. The buffer / carrier should contain components that prevent the rAAV from adsorbing to the injection tube but do not interfere with the biological efficacy and transduction activity of the rAAV in the body.

[0087] Treatment methods for diseases In another embodiment, the present disclosure provides a method for treating a disease in a subject, comprising administering an effective amount of the cleaved ATP7B, the nucleic acid sequence, the recombinant nucleic acid construct, the recombinant vector, the rAAV, or the pharmaceutical composition.

[0088] In some embodiments, the disease is an ATP7B-related disease, preferably Wilson's disease.

[0089] In some embodiments, the subject is a mammal, preferably a human. [Examples]

[0090] The embodiments described below follow the general methods described below unless otherwise specified.

[0091] rAAV production AAV8 and AAV9 particles were produced by triple transient transfection of HEK293 T cells or suspended HEK293 cells with plasmids containing recombinant genomes encoding AAV Rep, AAV Cap, adenovirus helper genes, and the ATP7B construct. rAAV particles were purified using density gradient ultracentrifugation with iodine. Subsequently, rAAV was quantified by probe-based droplet digital PCR (ddPCR, Bio-Rad) assay and characterized by silver staining.

[0092] r-AAV Biological Efficacy Assay The rAAV biological efficacy assay was performed by cell transduction using the Huh-7 cell line. The cells were arranged at 8.5 × 10⁶ cells per plate. 6 At a cell density of 15 cm² 2 The cells were seeded on plates. After 24 hours, the cells were transfected with pGL4×MRE-LUC and pCMV-RL plasmid as an internal control. After another 48 hours, the cells were trypsinized and 1.5×10⁶ cells were added 24 hours before transduction. 4Cells were seeded in 96-well plates at a cell density of cells / well. rAAV transduction resulted in a confirmed multiple infection degree (MOI) of 3 × 10⁶. 6 The procedure was performed as follows: 48 hours after transduction, 150 μM CuSO4 was added, and cells were collected 24 hours later. For baseline measurement, cells without copper supplementation were collected, and relative luminospheric units were calculated by normalizing firefly luciferase activity with reniral luciferase activity.

[0093] Dual luciferase assay The functional copper transport capacity of the cleaved ATP7B transgene cloned into the pCMV vector was evaluated using a reporter plasmid (pGL4×MRE-LUC). Such reporter plasmids express the firefly luciferase reporter gene through transcriptional regulation of a metal-responsive element (MRE) that responds to cytoplasmic copper available to the organism. HEK293T cells were co-transfected with a plasmid expressing cleaved ATP7B under the control of pGL4×MRE-LUC and the CMV promoter, or with an empty pcDNA 3.1 plasmid as a control. All cells were co-transfected with the pCMV-RL plasmid and used as an internal control. Cells were incubated in 75 μM CuSO4 for 24 hours, with the absence of copper overload considered the baseline condition. Relative luminospheric units were calculated by normalizing firefly luciferase activity to reniral luciferase activity.

[0094] Research design of mouse models AAV vectors containing cleaved ATP7B transgenes are used in ATP7B baitfish aged 8-12 weeks. - / - The drug was administered to mice by tail vein injection. All mice were housed in separate, ventilated cages in an environment free of specific pathogens, in accordance with the regulations of the IACUC Committee. All cages, rake bedding, and water were sterilized before use. Rake bedding, food, and water were changed twice per week. The AAV dose is 2.0 × 10 12The concentration was vg / kg. To assess the dynamics and persistence of transgene expression, serum ceruloplasmin activity, alanine aminotransferase levels, and metabolized copper content were measured at various time points after injection. Mice were tracked to the end of the study, after which they were killed and used for tissue copper accumulation, biochemical, and pathological analysis.

[0095] Serum metabolites and tissue samples Serum was separated from fresh, anticoagulant-free blood that had been left to stand at 4°C for 4 hours by centrifugation at 12,000 rpm for 15 minutes. The serum was stored at -80°C. Urine and feces were collected over 24 hours using a metabolic cage and used for analysis of metabolic copper content.

[0096] Mice were anesthetized and euthanized, perfused with saline solution, and then tissue was collected from the mice, rapidly frozen, and stored at -80°C. The tissue samples were divided into four parts, and three of these were frozen in separate tubes and stored at -80°C for tissue copper accumulation analysis, vector genome copy number analysis, and mRNA analysis. The remaining part was fixed in 10% neutral buffered formalin solution (NBF, pH 7.4) at 4°C for approximately 24-48 hours and used for further histological analysis.

[0097] Celluloplasmin activity, alanine aminotransferase levels, metabolized copper content, and tissue copper accumulation. In the manner described above, mouse serum, metabolites, and tissues were collected.

[0098] Serum ceruloplasmin activity was detected using a ceruloplasmin activity detection kit (Nanjing Jiancheng Bioengineering Institute, China) with o-dianisidine dihydrochloride as the substrate (Stepien and Guy, 2018), according to the modified manufacturer's instructions. In short, 5 μL of diluted serum was added to a 96-well plate, then 80 μL of reagent I was added, followed by 20 μL of reagent II. The samples were thoroughly mixed and incubated at 37°C for 140 minutes. A 20-minute incubation was performed simultaneously for baseline detection and calculation. Finally, 200 μL of reagent III was added after incubation for termination, and spectrophotometric analysis (Varioskan) was performed. (商標) Absorbance was measured at 540 nm using LUX (ThermoFisher), and ddH2O was used as a blank.

[0099] Serum ALT activity was detected using an alanine aminotransferase detection kit (Nanjing Jiancheng Bioengineering Institute, China) according to the revised manufacturer's instructions. In short, 10 μL of diluted serum was pipettered into a 96-well plate, then 200 μL of reagent R1 was added, followed by incubation at 37°C for 10 minutes. Finally, 50 μL of reagent R2 was added and thoroughly mixed. Spectrophotometric analysis (Varioskan (商標) Absorbance was measured at 340 nm using a LUX (ThermoFisher) instrument, with a total of 10 measurements taken at 2-minute intervals, and ddH2O was used as a blank.

[0100] Solid samples (feces or tissue) were weighed, homogenized, and dried until they reached a constant weight. The samples were then digested overnight in nitric acid solution. Urine samples were first centrifuged to remove insoluble or suspended particles. Before analysis, solid samples were appropriately diluted with 10% nitric acid and 1% hydrogen peroxide, and urine samples were appropriately diluted with 1% nitric acid and 0.5% hydrochloric acid. The copper content of the samples was then measured by inductively coupled plasma mass spectrometry (ICP-MS), and the analysis was completed by direct calibration using aqueous standards.

[0101] Vector genome copy number, relative RNA transcription level To determine the vector genome copy number in tissue samples after rAAV injection, DNA was isolated from frozen liver samples using the DNeasy Blood and Tissue Kit (QIAGEN) according to the manufacturer's instructions. After DNA isolation, qPCR (Roche) with a probe was performed to determine the vector genome copy number per reaction. The cell number per reaction was calculated based on the quantification result of the DNA amount. Then, the vector genome copy number per cell was calculated by normalizing the genome copy per reaction to the cell number per reaction.

[0102] To determine the relative RNA transcription level in tissue samples after rAAV injection, RNA was isolated from frozen liver samples using the RNeasy Kit (QIAGEN) according to the manufacturer's instructions. After RNA isolation, cDNA was synthesized using PrimeScript RT Master Mix (TAKARA). 500 ng of RNA was added to each reverse transcription reaction. Then, the cDNA was diluted and applied to a qPCR (Roche) test with a probe, and mouse Gapdh was used as an endogenous control gene.

[0103] Immunohistochemistry Cells expressing the cleaved ATP7B in mice were detected using a rabbit anti-human ATP7B antibody (1:100, ThermoFisher, PA5-102826). Based on the manufacturer's proposal, formalin-fixed mouse tissues were deparaffinized with xylene and gradient ethanol, and then antigen retrieval was performed using pepsin. The sections were counterstained with hematoxylin. Then, detection was performed using an HRP-labeled secondary antibody. Pierce (商標) The signal was visualized using a Western blot signal enhancer (Thermo Scientific) according to the manufacturer's instructions.

[0104] Statistical analysis Statistical analysis was performed using Prism 8 (GraphPad) software. Column analysis was performed using one-way ANOVA. P-values ​​and sample sizes are as indicated in the figure captions.

[0105] Example 1: Contribution of ATP7B to the copper transport capacity of CRE alone ATP7B is a multi-domain protein consisting of several independently folded copper(II) response elements (CREs) located between the leader peptide (LP) and the C-terminal region (CTR), the sequence of which is shown in Table 2. Based on the protein structure, six CREs and five endogenous linkers connecting them can be identified from the N-terminus to the transmembrane portion of ATP7B, with each CRE containing four β-chains and two helices.

[0106] [Table 2]

[0107] Here, as shown in Table 3, each single CRE was cloned into a pCMV vector as a cleaved ATP7B transgene along with LP and CTR, and the individual contribution of each single CRE to copper transport capacity was determined by a dual luciferase assay in HEK293T cells. Relatively high relative Fluci / Rluci values ​​indicate relatively low copper transport capacity, but a state without copper overload is considered the baseline. The LYM3P016 vector encoding wild-type full-length ATP7B was used as a positive control. The results showed that LYM3P027-CRE5 exhibited the highest copper transport capacity among the six CREs, indicating that CRE5 primarily contributes to the copper transport capacity of ATP7B (Figure 1).

[0108] [Table 3]

[0109] Example 2: Copper transport capacity of the CRE scheme based on CRE5 As described above, CRE5 was confirmed to be the primary contributor to the copper transport capacity of ATP7B. To compensate for the reduced copper transport capacity of cleaved ATP7B possessing CRE1, CRE2, CRE3, or CRE4 alone, CRE5-based CRE combination schemes were constructed as shown in Table 4, cloned into pCMV vectors, and the CREs were linked via endogenous linkers that connect the CREs in ATP7B. The LYM3P016 vector encoding wild-type full-length ATP7B was used as a positive control. In HEK293T cells, the copper transport capacity of the cleaved ATP7B was evaluated by a dual luciferase assay, and relatively high relative Fluci / Rluci values ​​indicated relatively low copper transport capacity. As shown in Figure 2, all constructs with CRE5 combinations significantly improved the copper transport capacity of other CREs that lacked CRE5, further demonstrating that CRE5 significantly contributes to the copper transport capacity of ATP7B.

[0110] [Table 4]

[0111] Example 3: Combining CRE1 or CRE2 with other CRE significantly improves copper transport capacity. To explore the additive effect of CRE1, CRE2, or CRE3 on the copper transport capacity of other CRE or CRE schemes, cleaved ATP7B transgenes with combinations of CRE1, CRE2, or CRE3 and other CRE or CRE schemes were cloned into pCMV vectors, as shown in Tables 5-7, where the CREs are linked via endogenous linkers that connect the CREs in ATP7B. The LYM3P016 vector encoding wild-type full-length ATP7B was used as a positive control. In HEK293T cells, the copper transport capacity of the cleaved ATP7B encoding vectors described in Tables 5-7 was evaluated by dual luciferase assays. The constructs LYM3P059, LYM3P036, LYM3P048, LYM3P062, LYM3P058, LYM3P035, LYM3P047, and LYM3P061, to which CRE1 or CRE2 was added to other CRE or CRE schemes, all significantly improved the copper transport capacity of the constructs LYM3P026, LYM3P024, LYM3P027, and LYM3P055, which encode ATP7B and do not contain CRE1 or CRE2 (Figures 3 and 4). Conversely, the constructs LYM3P034, LYM3P052, LYM3P053, LYM3P057, and LYM3P064 to which CRE3 is added to other CRE or CRE schemes do not further enhance the copper transport capacity of the constructs LYM3P024, LYM3P047, LYM3P048, LYM3P026, and LYM3P059 that encode ATP7B without CRE3, and in some cases even have a negative effect (Figure 5).

[0112] By further combining CRE3 or CRE4 with the constructs LYM3P035, LYM3P036, and LYM3P047, the constructs LYM3P041, LYM3P042, and LYM3P50 were cloned, and the results showed that the addition of CRE3 or CRE4 had a minor adverse effect on the copper transport capacity of LYM3P035, LYM3P036, and LYM3P047. However, the copper transport capacity of the constructs LYM3P041, LYM3P042, and LYM3P050 encoding CRE combinations with CRE3 or CRE4 still exceeded or was equivalent to that of the construct LYM3P024 encoding CRE5+CRE6 (Figure 6).

[0113] [Table 5]

[0114] [Table 6]

[0115] [Table 7]

[0116] Example 4: Combining CRE1+CRE2 with other CRE or CRE schemes significantly improves copper transport capacity. CRE1 and CRE2 can independently enhance copper transport capacity when combined with other CREs or CRE schemes, but the effect of CRE1+CRE2 is not yet established. To evaluate the additive effect of CRE1+CRE2 on copper transport capacity with other CREs or CRE schemes, cleaved ATP7B transgenes with or without CRE1+CRE2 were cloned into pCMV vectors, as shown in Table 8, where the CREs are linked via endogenous linkers that connect the CREs in ATP7B. In HEK293T cells, the copper transport capacity of the cleaved ATP7B transgenes was evaluated by a dual luciferase assay, with the LYM3P016 vector encoding wild-type full-length ATP7B used as a positive control. Compared to the constructs LYM3P027, LYM3P026, and LYM3P024, which encode only CRE5, CRE6, or CRE5+CRE6, the constructs LYM3P054, LYM3P065, and LYM3P043, which have a CRE1+CRE2 scheme, all show improved copper transport capacity, demonstrating that copper transport capacity can be significantly improved by adding CRE1+CRE2 (Figure 7).

[0117] [Table 8]

[0118] Example 5: A CRE combination containing a specific CRE exhibits copper transport capacity equivalent to the wild-type form. The full-length ATP7B transgene is approximately 4.4 kb, which may be too large for packaging in adeno-associated virus (AAV). Therefore, to develop gene therapy vectors for Wilson's disease, a cleaved form with copper transport capacity equivalent to wild-type ATP7B must be obtained. Considering the constraints of AAV packaging, combinations of three or fewer CREs are preferred. Cleaved ATP7B transgenes with combinations of three or fewer CREs were cloned into pCMV vectors. In HEK293T cells, the copper transport capacity of the above constructs was evaluated by dual luciferase assay. As shown in the results, these constructs with the CRE combinations LYM3P051, LYM3P035, LYM3P062, LYM3P036, LYM3P048, LYM3P044, LYM3P065, and LYM3P047 showed copper transport capacity equivalent to LYM3P016, which encodes the wild-type form of ATP7B. At the same time, the constructs LYM3P051, LYM3P035, LYM3P062, LYM3P036, LYM3P048, LYM3P044, LYM3P065, and LYM3P047 demonstrated significantly higher copper transport capacity compared to the LYM3P024 construct encoding CRE5+CRE6. Compared to the LYM3P057 construct containing CRE3+CRE6, the constructs LYM3P051, LYM3P035, LYM3P062, LYM3P036, LYM3P048, LYM3P044, LYM3P065, LYM3P047, LYM3P050, LYM3P061, LYM3P059, and LYM3P058 all demonstrated significantly higher copper transport capacity (Figure 8).

[0119] Example 6: AAVs of cleaved ATP7B transgenes having one or more specific combinations of CRE can be efficiently produced and exhibit therapeutic effects against Wilson's disease in a mouse model. To verify whether the cleaved ATP7B transgenes with the prominent copper transport capacity described above are suitable for mass production of AAV, as shown in Table 9, cleaved ATP7B transgenes with specific combinations of CRE or CRE schemes were cloned into AAV vectors under the control of a liver-specific promoter. Viral particles were produced as described in Example 1 above. The yield of packaged AAV was then evaluated by ddPCR using specific primer and probe sets. As shown in Figure 9, in the suspended HEK293 cell system, constructs of cleaved ATP7B transgenes with three or fewer CREs could all be packaged into AAV8, with an average yield of 2.0 × 10⁶. 11 It exceeds vg / mL and is twice as high as LYM3P283, which has a full-length ATP7B transgene.

[0120] [Table 9]

[0121] Subsequently, Atp7b was constructed as described in Example 1. - / - The in vivo therapeutic effect of the AAV produced above was evaluated in mice. In short, 12-week-old Atp7b mice - / - 2.0 × 10⁶ mice and control mice (naive mice) 12 AAV (copper-containing polymerase) or the solvent alone was administered via tail vein injection at a dose of vg / kg. Serum was collected periodically after AAV administration to evaluate ceruloplasmin (Cp) activity. After 4 weeks, the mice were killed, and tissue copper accumulation was confirmed by histology and ICP-MS.

[0122] AAVs possessing specific CRE schemes with CRE2+CRE5+CRE6 and CRE1+CRE5+CRE6, namely LYM3P313 and LYM3P314, respectively, which have cleaved ATP7B transgenes, can reverse ceruloplasmin activity in a Wilson's disease mouse model, and this activity corresponds to that of LYM3P021, the CRE scheme encoding CRE5+CRE6 (Figure 10a). Furthermore, LYM3P313 and LYM3P314 were shown to reduce copper accumulation in the liver with significantly higher efficiency compared to LYM3P021 (Figure 10b).

Claims

1. A cleaved ATP7B comprising one or more sequences selected from CRE5, CRE6 and their variants, and one or more sequences selected from CRE1, CRE2 and their variants.

2. The cut-type ATP7B according to claim 1, optionally including a flexible linker to connect the arrays to each other.

3. From the N-terminus to the C-terminus, it has the structure shown in formula Ia, A-L-B-L-C-LD (Ia) In the formula, A, B, C, or D are selected from CRE1, CRE2, CRE5, CRE6 and their variants, A, B, C, and D are different from each other, A and B are present, C and / or D are present optionally, and The cut-type ATP7B according to claim 1, wherein L is absent or is a flexible linker.

4. 1) CRE5 or a variant thereof and / or 2) CRE6 or a variant thereof are present, and 3) CRE1 or a variant thereof and / or 4) CRE2 or a variant thereof are present, according to any one of claims 1 to 3.

5. A cleaved ATP7B according to any one of claims 1 to 3, wherein CRE1 or a variant thereof and CRE5 or a variant thereof are present.

6. A cleaved ATP7B according to any one of claims 1 to 3, wherein CRE1 or a variant thereof and CRE6 or a variant thereof are present.

7. A cleaved ATP7B according to any one of claims 1 to 3, wherein CRE1 or a variant thereof, CRE5 or a variant thereof, and CRE6 or a variant thereof are present.

8. A cleaved ATP7B according to any one of claims 1 to 3, wherein CRE2 or a variant thereof and CRE5 or a variant thereof are present.

9. A cleaved ATP7B according to any one of claims 1 to 3, wherein CRE2 or a variant thereof and CRE6 or a variant thereof are present.

10. A cleaved ATP7B according to any one of claims 1 to 3, wherein CRE2 or a variant thereof, CRE5 or a variant thereof, and CRE6 or a variant thereof are present.

11. A cleaved ATP7B according to any one of claims 1 to 3, wherein CRE1 or a variant thereof, CRE2 or a variant thereof, and CRE5 or a variant thereof are present.

12. A cleaved ATP7B according to any one of claims 1 to 3, wherein CRE1 or a variant thereof, CRE2 or a variant thereof, and CRE6 or a variant thereof are present.

13. A cleaved ATP7B according to any one of claims 1 to 3, wherein CRE1 or a variant thereof, CRE2 or a variant thereof, CRE5 or a variant thereof, and CRE6 or a variant thereof are present.

14. The cleaved ATP7B according to any one of claims 1 to 3, wherein the cleaved ATP7B comprises a sequence selected from the group consisting of amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, and at least 99.8% identity with SEQ ID NO: 18, 20, 22, 24, 26, 28, 30, 32, and 34, and the cleaved ATP7B retains the function of transmembrane copper transport.

15. A cleaved ATP7B according to any one of claims 4 to 14, wherein CRE3 is missing.

16. A cleaved ATP7B according to any one of claims 4 to 13 and 15, wherein CRE4 or a variant thereof is present.

17. A cleaved ATP7B according to any one of claims 4 to 13, wherein CRE3 or a variant thereof is present.

18. A nucleic acid sequence encoding cleaved ATP7B according to any one of claims 1 to 17.

19. The nucleic acid sequence according to claim 18, wherein the nucleic acid sequence includes a sequence selected from the group consisting of sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, and at least 99.8% identity with SEQ ID NO: 17, 19, 21, 23, 25, 27, 29, 31, and 33, and the cleaved ATP7B retains the function of transmembrane copper transport.

20. An RNA polynucleotide encoding cleaved ATP7B according to any one of claims 1 to 17.

21. The RNA polynucleotide according to claim 20, wherein the RNA comprises messenger RNA (mRNA) or circular RNA (circRNA).

22. (a) 5'-terminal inverted repeat (ITR) sequence, (b) promoter sequence, (c) The nucleic acid sequence according to any one of claims 18 or 19, (d) Poly(A) sequence, and (e) A recombinant nucleic acid construct containing a 3'-ITR sequence.

23. The recombinant nucleic acid construct according to claim 22, further comprising one or more enhancer sequences.

24. The recombinant nucleic acid construct according to claim 22 or 23, further comprising one or more intron sequences.

25. A vector comprising the nucleic acid sequence according to claim 18 or 19, the RNA polynucleotide according to claim 20 or 21, or the recombinant nucleic acid construct according to any one of claims 22 to 24.

26. The vector according to claim 25, wherein the vector is a viral vector.

27. The vector according to claim 26, wherein the viral vector is an AAV vector.

28. Recombinant adeno-associated virus (rAAV) comprising the vector and capsid protein according to claim 27.

29. The rAAV according to claim 28, wherein the AAV is selected from the group consisting of serotypes 9, 8, 1, 2, 3, 3B, 4, 5, 6, 7, 10, 11, 12, 13, rh10, or hu37, and any or variants thereof of AAV serotypes isolated from human and non-human mammals.

30. A pharmaceutical composition comprising a cleaved ATP7B according to any one of claims 1 to 17, a nucleic acid sequence according to claim 18 or 19, an RNA polynucleotide according to claim 20 or 21, a recombinant nucleic acid construct according to any one of claims 22 to 24, a vector according to any one of claims 25 to 27, or an rAAV according to claim 28 or 29, and a pharmaceutically acceptable carrier.

31. A method for treating a disease in a subject, comprising administering an effective amount of a cleaved ATP7B according to any one of claims 1 to 17, a nucleic acid sequence according to claim 18 or 19, an RNA polynucleotide according to claim 20 or 21, a recombinant nucleic acid construct according to any one of claims 22 to 24, a vector according to any one of claims 25 to 27, an rAAV according to claim 28 or 29, or a pharmaceutical composition according to claim 30.

32. The method according to claim 31, wherein the disease is an ATP7B-related disease.

33. The method according to claim 31, wherein the disease is Wilson's disease.

34. The method according to claim 31, wherein the subject is a mammal, preferably a human.

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