CN111088285A

CN111088285A - AAV vector carrying ATP7B gene expression cassette and variant and application

Info

Publication number: CN111088285A
Application number: CN201910753099.5A
Authority: CN
Inventors: 田文洪; 董小岩; 吴小兵; 马思思
Original assignee: Beijing Jinlan Gene Technology Co Ltd
Current assignee: Beijing Jinlan Gene Technology Co Ltd
Priority date: 2019-08-15
Filing date: 2019-08-15
Publication date: 2020-05-01
Anticipated expiration: 2039-08-15
Also published as: CN111088285B

Abstract

The present invention provides recombinant AAV vectors containing an ATP7B gene expression cassette or variants thereof. The AAV vector is injected into a hepatolenticular degeneration model mouse body through a vein, and efficiently expresses and generates ATP7B or variant protein thereof in the liver, so that the copper ion content in the liver and urine of the model mouse is effectively reduced, the glutamic-pyruvic transaminase level in the blood of the model mouse is obviously reduced, adverse symptoms caused by the accumulation of the copper ions of the model mouse are relieved, and the potential for treating hepatolenticular degeneration is shown.

Description

AAV vector carrying ATP7B gene expression cassette and variant and application

Technical Field

The invention relates to the field of biotechnology, in particular to an AAV vector containing an ATP7B gene expression cassette and a variant, and the AAV vector is applied to the development of gene therapy medicines for hepatolenticular degeneration.

Background

Hepatolenticular degeneration, also known as Wilson's disease, is an autosomal monogenic recessive genetic disease characterized by copper metabolism disorder, which mainly affects liver, brain, kidney and cornea, causing progressive cirrhosis, basal ganglia damage, kidney damage and corneal chroman ring (Kodama H, et al, Curr Drug Metab. 2012;13(3): 237-. Hepatolenticular degeneration is caused by mutations in the ATP7B gene, which encodes a copper-transporting P-type ATPase (ATP 7B). ATP7B is a membrane protein expressed in multiple organs, and its main function is to promote the excretion of copper with blood and bile. When the ATP7B gene is mutated, the intracellular localization of ATP7B is changed, the copper transport capacity of the ATP7B is reduced, and the synthesis of serum ceruloplasmin is reduced and the copper excretion of bile duct is blocked. Excessive copper accumulates in the body, causing cell necrosis and organ damage, which later seriously affects the quality of life of the patient and can eventually lead to death. Currently, diagnosis of hepatolenticular degeneration relies mainly on typical clinical manifestations, laboratory tests and genetic testing, and treatments include penicillamine, zinc preparations, and liver transplantation. Early diagnosis and early intervention are critical to delay disease progression and prevent irreversible sequelae. Currently, the global prevalence of hepatolenticular degeneration is about 1/3 ten thousand, and in isolated populations (e.g., the islands of Spanish), the prevalence can reach 1/1 thousand (Dedossis GV, et al, Ann Hum Genet. 2005; 69(3):268- & 274. Gialluisi A, et al Eur J Hum Genet. 2013;21(11): 1308- & 1311.), and the prevalence in Han nationality of hong Kong is as high as 1/5400 (MakCM, et al, J Hum Genet. 2008; 53(1): 55-63.).

Although the specific molecular mechanism by which mutations in the ATP7B gene lead to hepatolenticular degeneration is not fully understood, there is increasing evidence that mutations in the ATP7B gene are the underlying cause of hepatolenticular degeneration.

The ATP7B gene is located in the long arm of chromosome 13 (13 q 14.3-q 21.1), has a total length of about 85kb, comprises 21 exons and 20 introns, and encodes ATP7B consisting of 1465 amino acids. ATP7B belongs to a transmembrane protein, containing 6N-terminal metal-binding domains (MBD), each containing a highly conserved repeat sequence methionine-X-cysteine, 8 transmembrane channels (TM-region), 1 ATP-binding domain (N-region), 1 phosphorylation domain (P-region), 1 phosphatase domain (A-region) (Lutsko S, et al. ArchBiochem Biophys. 2007; 463; 2: 134-. MBD has high affinity for copper, interacts with antioxidant protein 1 (Atox-1), accepts copper ions from the cytoplasm, and promotes the release of copper into the transmembrane pore (Lenartofaciz M, et al. front Mol neurosci. 2016; 9: 68.). MBDs 1-4 mainly regulate their own interactions, MBDs 5-6 are copper binding sites, and can affect the activity and catalytic ability of ATP7B protein (Yu CH, et al IUBMB Life 2017; 69(4): 226-. The cysteine-proline-cysteine sequence is located in the 6 th transmembrane channel and can selectively pass through metallic copper ions (Kumari N, et al. Hum Mutat. 2018; 39(12): 1926-1941.). If the mutated residue of the ATP7B gene is a critical site for binding to ATP or copper, it may cause a complete loss of ATP7B function; ATP7B may retain part of the transport function if the mutation only affects affinity for the substrate, slows conformational transition or affects protein localization (Huster D, et al. gastroenterology 2012; 142(4): 947-.

ATP7B is a transmembrane protein highly expressed in the trans-golgi network of hepatocytes. Copper is mainly absorbed in the duodenum and participates in the liver-intestine circulation through high affinity copper transporter 1. Upon reaching hepatocytes, copper and the metal chaperone Atox-1 bind and form a complex, which then interacts with ATP7B in a protein-protein fashion. Copper is transported to a trans-Golgi network under the action of ATP7B and is combined with related enzymes to form serum ceruloplasmin which is metabolized along with blood; when the level of copper ions in hepatocytes rises to some extent, ATP7B is transported from the trans-Golgi network to lysosomes, transporting excess copper to the vesicles, where it is excreted via exocytosis with the bile (Polishchuk EV, et al. Dev cell. 2014;29(6): 686-700). In this process, ATP7B is phosphorylated by energy released by ATP hydrolysis, and subsequently dephosphorylated to release the energy required for copper to cross-membrane. Therefore, ATP 7B-dependent bile copper excretion is the main way to maintain the balance of copper metabolism.

The hepatolenticular degeneration ATP7B gene mutation types are various, and 780 ATP7B gene mutations are recorded in a human gene mutation database at present, wherein the mutations comprise 491 missense/nonsense mutations, 65 shearing site mutations, 187 small deletion/insertion mutations and 12 large fragment deletions. Mutations may occur anywhere in a gene, including exons, introns, and even promoter regions. The ATP7B gene mutation has genetic heterogeneity in different ethnic groups and regions. The most common type of mutation in the European population is the H1069Q mutation in exon 14, which is common in Italy, Sweden and Romania, with an allele frequency of 30-70% (Zarina A, et al Mol Genomic Med.2017; 5(4): 405-. The most common mutation type of Asian population is missense mutation R778L on exon 8, which is most common in China, Korea and Japan, and has allele frequency of 17.3% -60% (Dong Y, et al Theranostics.2016; 6(5): 638-; the others are P992L, Q1399R and the like.

The ① MBD region (related to No. 1-6 exon) has reported 18 missense mutations in this region, such as G85V in MBD1, which inhibits the interaction of ATP7B with Atox-1, affecting the binding of protein and copper (Yu CH, et al. Sci Rep.2018; 8(1): 581.) the ② cysteine-proline-cysteine transport region (related to 7-10, 12, 13 exons) has reported 103 mutations, such as R778, G93S, which affect the function of protein mainly by changing its localization (Yu CH, et al. Sci Rep.2018; 1: 581; ③ A-region (related to 11 exon) has also found 22 mutations, which cause loss of ATP7, 5, 8, 11, 8, 15, 18, 31A-12, 31A-11 exon related to the phosphorylation of proteins, such as ATP 1-19-11 exon, 18, 11, 18, 11, 8, 11, 8, 24, and 24, three, four.

At present, missense mutation is the main part of ATP7B gene mutation function research, and the mutation mainly causes weakening or deletion of ATP7B protein transport function by influencing processes of protein synthesis, folding, positioning, degradation and the like, and finally causes copper deposition. Mutations in the ATP7B gene may inhibit the farnesoid X receptor/short heterodimer chaperone pathway, leading to increased bile acid production and impaired liver function, resulting in cholestasis. The main pathway of copper metabolism is bile, which, when cholestasis occurs, further affects copper metabolism, thereby causing the vicious circle (Wooton-Kee CR, et al J Clin invest. 2015;125(9): 3449-.

Alternative splicing of precursor mRNA is an important means for eukaryotes to increase protein diversity and is important for gene expression, including 5 'and 3' end splicing, etc. Splicing is regulated by cis-acting elements and trans-acting factors, such as splicing enhancers, silencers, and the like. Approximately 10% of the pathogenic mutations in the ATP7B gene occur at conserved splice sites at the junction of exons and introns. Studies have found that infants with hepatolenticular degeneration carry the missense mutation R919G of ATP7B gene, which is located in the exon splicing regulatory element region, resulting in exon skipping and amino acid changes (Wang C, et al, Liver int 2018;38(8): 1504-. In vitro and in vivo experiments show that the skipping of exon in R919G causes the disruption of splicing enhancer and the appearance of silencer, thereby affecting the splicing of exon 12 and causing the loss of protein function (Wang C, et al, Liver int 2018;38(8): 1504-. Thus, point mutations in the ATP7B gene may cause severe aberrant splicing of mRNA, rather than a direct effect on coding potential. The 1707+5G > A mutation is located at the 5' splice site, affecting the splicing of the precursor mRNA. Researchers use a splicing abnormity in-vitro function verification experiment (minigene technology) to analyze the mutation, and find that the c.1707 +5G & gtA transcription product has exon 4 deletion, the amplification length is 293bp, and the length is obviously shortened compared with 457bp of a wild type; the mRNA expression level is obviously reduced compared with the wild type (Liu M, et al. J Mol Neurosci.2018; 64(1): 20-28.). Exon 4 skipping causes premature appearance of the stop codon, leading to premature termination of ATP7B protein synthesis and inactivation of the copper ion channel. The nonsense-mediated mRNA degradation pathway is an important RNA monitoring mechanism in eukaryotic cells, can identify and degrade mRNA containing a premature stop codon in an open reading frame, eliminates an abnormal splicing form, and avoids toxic hazard to cells due to accumulation of truncated protein products.

The mutation of the ATP7 gene results in a structural change of the ATP7B protein S653Y mutation site is located in a highly conserved region G621-S668 between MBD6 and TM 1. the mutation does not affect the localization of the protein and its folding structure, normally transports copper to trans-Golgi network, does not affect the formation of serum ceruloplasmin, but when intracellular copper levels rise to a certain threshold, ATP7B remains located in Golgi and is unable to carry copper to the cell membrane. the study found that the S653Y mutation causes local deformation in the trans-membrane segment TM1, resulting in an altered interaction between trans-membrane TM1 and TM2, thereby affecting the transport of ATP7B from the trans-Golgi network to lysosomes (Braiterman, LT, Proc Natl Acad Sci USA 2014; 111(14) E1374-E3. the mutation site, the G V, 20124, 20135. Natal Acad Sci 2014; 11, 11 (11) (E4-E3-E) and E24. E11-E35. the mutation site has a reduced stability compared to the wild protein 12, 386, and/95) and/or a reduced stability of the protein found that the protein folding stability of the protein can be significantly reduced by comparison with the wild protein found that the protein folding structure of the protein found that the protein can be more efficiently regulated by reducing the protein folding structure of the protein when the protein found by reducing the protein folding structure of the protein found by reducing the protein found by the protein (MBD-S-9 protein found by reducing the protein found that the protein found by reducing the protein found that the protein found by reducing the protein found by.

The ATP7B gene mutation causes abnormal localization and reduced expression of ATP7B protein. ATP7B is a membrane protein located in trans-Golgi apparatus, and mainly utilizes the energy released by hydrolyzing ATP to discharge copper out of the body and maintain the balance of copper ions in the body; the mutant ATP7B can be retained in endoplasmic reticulum and can not carry copper to transport, thus causing copper to deposit in liver, brain and other parts. The copper transport function of 25 mutant ATP7B proteins (Huster D, et al. gastroenterology, 2012; 142(4): 947-; there are 8 mutations that retain partial transport capacity, such as a874V, I857T located in the a-region; while only the M645R mutation showed copper overload. The researchers transfect wild type and mutant ATP7B gene plasmids into HEK293T and Sf9 cells, detect the positioning of ATP7B protein in the cells by using an immunofluorescence technique, and show that R969Q (mutation site is in MBD region) protein is positioned in trans-Golgi network and is consistent with the wild type; the L1083F (mutation site in N-region) and A874V (mutation site in A-region) proteins localize to the endoplasmic reticulum with altered localization; the expression quantity of the three mutant proteins is obviously reduced compared with that of the wild type.

The protein expression of L168P and S1423N were found to be (34.3. + -. 8)%, respectively, for wild type and (66.0. + -. 8)% (Guttmann S, et al Front peptide 2018;6: 106.). The transport experiment shows that L168P shows copper dependence, and S1423N has no response to copper level increase. Mislocalization and reduced expression of the ATP7B mutant protein can lead to reduced copper transport capacity.

The ATP7B gene mutation accelerates the degradation of ATP7B protein. Protein kinase signaling pathways mediated by p38 and c-Jun amino-terminal kinase (JNK) affect protein stability (e.g., cystic fibrosis transmembrane transduction regulator (Hegde RN, et al. Elife. 2015; 4.pii: e 10365.)) and regulate the endoplasmic reticulum-associated protein degradation pathway. The degradation pathway of endoplasmic reticulum-associated proteins can ensure the output of correctly folded proteins, and identify, classify and degrade misfolded proteins, thereby eliminating non-functional proteins in cells. It was found that p38 and JNK expression was significantly elevated in cells overexpressing the H1069Q mutation (Chesi G, et al hepatology. 2016;63(6): 1842-1859.). The activated mitogen-activated protein kinase signaling pathway induces retention of misfolded H1069Q protein in the endoplasmic reticulum, and accelerates the rate of degradation of the mutein through endoplasmic reticulum-associated protein degradation pathways; meanwhile, the level of active oxygen ions is increased due to the accumulation of copper ions in cells, so that the steady environment of endoplasmic reticulum is further influenced, and a vicious circle is formed. The research results indicate that p38 and JNK inhibitors can correct the mislocalization of H1069Q, D765N, L776V and R778L mutant proteins by regulating transcription factors, and improve the expression and the transport capacity of the ATP7B protein in a trans-Golgi network; reducing the degradation of protein, promoting the metabolism of copper and reducing the level of copper ions in cells; but there was no significant correction for the a874V and L1083F mutations.

Chaperone drugs can correct misfolding of mutant ATP7B proteins α crystallin B chain (α -crystallin bhanin, CRYAB) is a cytoplasmic chaperone, belonging to the class of small heat shock proteins, that can bind to transmembrane proteins, inhibit formation of multimeric complexes, promote proper folding of mutant proteins studies showing that CRYAB can interact with ATP7B-H1069Q at the endoplasmic reticulum, inhibit formation of large oligomers in the mutant proteins (D' Agostino M, et al. JCell sci. 2013;126(Pt18): 4160. 4172.) immunofluorescence results suggest that more than 60% of H1069Q protein returns to its normal localization under cotransfection with CRYAB. when intracellular copper levels increase, crb can promote redistribution of H1069 yab Q to golgi vesicle, but no increase in the capacity of H1069 protein to carry copper in vitro is a significant carrier protein, is a protein that is a stable for intracellular calcium kinase inhibitor, which can be used to correct for intracellular protein deposition, and for the correct for curcumin protein deposition by the intracellular calcium deposition (e protein) via the intracellular calcium kinase binding protein, calcium binding protein, which is reported by the intracellular binding protein, and intracellular binding protein binding (beyg) and intracellular binding protein binding, cd 13).

Hepatolenticular degeneration is an autosomal recessive genetic disease, has various clinical manifestations, is influenced by various factors such as genes, age, diet, lipid metabolism, race and the like, and increases difficulty in diagnosis and treatment of diseases. The ATP7B gene mutation causes encoded protein misfolding, so that ATP7B is retained in endoplasmic reticulum, the functions of the catalysis and transport activities of the ATP7B are weakened or lost, and copper ions are deposited in the liver, the brain and other parts. The mechanism of copper-mediated tissue damage is not yet completely understood. The accumulation of excess copper in tissues may cause a series of oxidative stress reactions that disrupt mitochondrial structure and integrity, leading to cell damage and apoptosis. Both copper-induced increases in reactive oxygen species levels and lipid peroxidation can lead to mitochondrial losses (Sauer SW, et al. Biochim Biophys acta. 2011; 1812(12): 1607-1615.). Meanwhile, the accumulation of copper ions causes the redistribution of zinc within hepatocytes to damage the hepatocytes (Meacham KA, et al. metals, 2018; 10(11): 1595-1606.).

Currently, treatments for hepatolenticular degeneration include drug therapy, surgical therapy, gene and cell therapy, rehabilitation therapy, and the like. In China, the D-penicillamine is economic and effective and is one of the classic medicaments for treating hepatolenticular degeneration; however, D-penicillamine is not suitable for patients with severe neurological symptoms, particularly those with muscle stiffness (Cocos R, et al. PLoS one. 2014; 9(6): e98520. Zhang JW, et al. Biochem Biophys Res Commun.2015; 458(1): 82-85.). Research shows that certain molecular chaperone drugs (such as 4-phenylbutyric acid) and p38 and JNK inhibitors can correct the error localization of mutant proteins and restore the transport function of the proteins; the small molecule substance DPM-1001 can effectively reduce the copper level of the liver and brain in a hepatolenticular degeneration mouse model (Krishnan N, et al. Genes Dev.2018; 32(13-14): 944-952.). Individualized cellular and/or gene therapy is the current focus of research, with the primary objective of restoring ATP 7B-mediated hepatobiliary copper excretion function (Gupta S. Ann NY Acad Sci. 2014; 1315: 70-80), perhaps the most promising treatment in the future.

The invention provides a drug development strategy for treating hepatolenticular degeneration, which is to use a recombinant AAV vector carrying an ATP7B gene expression cassette or a truncated ATP7B gene expression cassette. After intravenous injection, the AAV vector can effectively transduce liver cells, introduce the carried ATP7B gene expression frame or the truncated ATP7B gene expression frame into the liver cells, continuously express and generate ATP7B protein or truncated ATP7B protein with normal physiological functions, recover the copper ion excretion function in a hepatolenticular degeneration model animal body, and thus achieve the purpose of treating hepatolenticular degeneration. The ATP7B gene expression frame selects a liver-specific promoter with short sequence and high expression strength which is artificially designed to regulate the transcription of the ATP7B gene, and the ATP7B is optimized by expression of a humanized codon, so that the ATP7B protein expression efficiency of the ATP7B gene expression frame in hepatocytes is obviously improved. Considering the size limit of the AAV vector carrying genome, a short polyA tailing signal designed artificially is selected to ensure that the designed ATP7B gene expression cassette can be carried by the AAV vector. On this basis, we refer to the literature for the design of a truncated ATP7B gene expression cassette (Guo Y, et al, AmJ Physiol gastroenterest light physiology. 2005; 289: G904-G916.). The length of the coding sequence of the truncated ATP7B gene is obviously shorter than that of the coding sequence of the ATP7B gene, so that under the condition that the two expression cassettes adopt the same promoter, the truncated ATP7B gene expression cassette selects a tailing signal with higher efficiency, and the gene expression efficiency is improved. The high hepatotropic AAV vector is selected to carry the ATP7B gene expression frame or the truncated ATP7B gene expression frame, so that the recombinant AAV is injected intravenously to transduce the hepatocyte efficiently, and the ATP7B protein or the truncated ATP7B protein is expressed and generated, thereby achieving the purpose of treating hepatolenticular degeneration.

Adeno-associated virus (AAV) is known as Atchison RW, et al, as it is found in adenovirus preparations.Science. 1965; 149: 754-756.Hoggan MD, et al.Proc Natl Sci USA1966, 55: 1467-. AAV is a member of the family of parvoviridae (subviruses), and comprises multiple serotypes, the genome of which is single-stranded DNA (Rose JA, et al.Proc Natl Acad Sci USA1969, 64: 863-869), wherein the AAV2 has a genome size of 4682 nucleotides. AAV is a dependent virus, requiring other diseasesViruses such as adenovirus, herpes simplex virus and human papilloma virus (Geoffroy MC, et al.Curr Gene Ther2005, (5), (3) 265 and 271), or an auxiliary factor to provide an auxiliary function. In the absence of helper virus, AAV infects cells and its genome integrates into the cell chromosome to become latent (Chiorini JA, et al.Curr Top Microbiol Immunol1996; 218: 25-33.) without producing progeny virus.

The first AAV virus isolated was AAV serotype 2 (AAV 2) (atcheson RW, et al.Science1965; 149: 754-. AAV2 genome is about 4.7kb long, and has Inverted Terminal Repeat (ITR) with length of 145bp at both ends and has palindromic-hairpin structure (Lusby E, et al.J Virol1980; 34: 402-. There are two large Open Reading Frames (ORFs) in the genome, encoding the rep and cap genes, respectively. The full-length genome of AAV2 has been cloned into an E.coli plasmid (Samulski RJ, et al.Proc Natl Acad Sci USA. 1982; 79: 2077-2081. Laughlin CA, et al.Gene. 1983; 23: 65-73.）。

ITRs are cis-acting elements of the AAV vector genome that play an important role in integration, rescue, replication, and genome packaging of AAV viruses (Xiao X, et al.J Virol1997, (71) (2) 941-948). The ITR sequences include a Rep protein binding site (RBS) and a terminal melting site tr (terminal resolution site) that is recognized by Rep protein binding and nicks at tr (Linden RM, et al).Proc Natl Acad Sci USA1996, 93(15), 7966 and 7972). The ITR sequences may also form unique "T" alphabetical secondary structures that play an important role in the life cycle of AAV viruses (Ashktorab H, et al.J Virol. 1989; 63(7):3034-3039.）。

The remainder of the AAV2 genome can be divided into 2 functional regions, the rep gene region and the cap gene region (Srivastava A, et al.J Virol1983, 45(2) 555-. The Rep gene region encodes four Rep proteins, Rep78, Rep68, Rep52 and Rep 40. Rep proteins play an important role in replication, integration, rescue and packaging of AAV viruses. Wherein Rep78 and Rep68 are in ITRThe terminal melting site of (tr) and the GAGY repeat motif (repeat motif) specifically bind (H ü ser D, et al.PLoS Pathog2010, 6(7) e 1000985), the replication process of AAV genome from single strand to double strand is initiated. The trs and GAGC repeat motifs in the ITRs are central to replication of the AAV genome, and therefore although the ITR sequences are not identical in all serotypes of AAV virus, both hairpin structures are formed and Rep binding sites are present. The AAV2 genome map has p19 promoter at position 19, and expresses Rep52 and Rep40, respectively. Rep52 and Rep40 have no function of binding to DNA, but have ATP-dependent DNA helicase activity. The cap gene encodes the capsid proteins VP1, VP2, and VP3 of AAV virus. Of these, VP3 has the lowest molecular weight but the highest number, and the ratio of VP1, VP2, and VP3 in mature AAV particles is approximately 1:1: 10. VP1 is essential for the formation of infectious AAV; VP2 assists VP3 in entering the nucleus; VP3 is the major protein that makes up AAV particles.

With the understanding of the life cycle of AAV and its related molecular biological mechanism, AAV is transformed into one efficient foreign gene transferring tool, AAV vector. The modified AAV vector genome only contains the ITR sequence of AAV virus and an exogenous gene expression frame carrying transport, Rep and Cap proteins required by virus packaging are provided in trans through exogenous plasmids, and possible harm caused by packaging Rep and Cap genes into AAV vectors is reduced. Moreover, the AAV virus itself is not pathogenic, making the AAV vector one of the most recognized safe viral vectors. Deletion of the D sequence and the trs (tertiary resolution site) sequence in the ITR sequence on one side of the AAV can also enable the packaged recombinant AAV vector to carry genome self-complementation to form double chains, and remarkably improve the in vitro and in vivo transduction efficiency (Wang Z, et al) of the AAV vector.Gene Ther.2003;10(26):2105-2111. McCarty DM, et al.Gene Ther2003, 10(26), 2112 and 2118). The resulting packaged virus becomes a scAAV (self-complementary AAV) virus, a so-called double-stranded AAV virus. Unlike ssAAV (single-stranded AAV), a classical AAV virus, in which neither ITR is mutated at both sides. The packaging capacity of the scAAV virus is smaller, only half of the packaging capacity of the scAAV, about 2.2kb-2.5kb, but transduction effects after infection of cellsThe rate is higher. AAV viruses are numerous serotypes, with different serotypes having different tissue infection tropism, and thus the use of AAV vectors enables the transport of foreign genes to specific organs and tissues (Wu Z, et al.Mol Ther2006, 14(3), 316-. Some serotype AAV vectors can also cross the blood brain barrier, lead exogenous genes into cerebral neurons, and provide possibility for gene transduction targeting the brain (Samaranch L, et al.Hum Gene Ther2012, 23(4) 382 and 389). In addition, AAV vectors are stable in physicochemical properties and exhibit strong tolerance to acids and bases and high temperatures (Gruntman AM, et al.Hum Gene Ther Methods2015, 26(2) and 71-76), biological products with higher stability can be easily developed.

AAV vectors also have relatively mature packaging systems, facilitating large-scale production. At present, the AAV vector packaging system commonly used at home and abroad mainly comprises a three-plasmid cotransfection system, a packaging system taking adenovirus as a helper virus, a packaging system taking herpes simplex virus type 1 (HSV 1) as a helper virus and a packaging system based on baculovirus. Among them, the three plasmid transfection packaging system is the most widely used AAV vector packaging system because of no need of auxiliary virus and high safety, and is also the mainstream production system in the world at present. The lack of efficient large-scale transfection methods has somewhat limited the use of three-plasmid transfection systems for large-scale production of AAV vectors. Yuan et al established an AAV large-scale packaging system with adenovirus as the helper virus (Yuan Z, et al.Hum Gene Ther2011, (22) (613) and 624), the production efficiency of the system is high, but the trace amount of adenovirus in the final AAV finished product in the packaging system influences the safety of the AAV finished product. HSV1 is another type of AAV vector packaging system that has been used more widely as a packaging system for helper viruses. Almost simultaneously, Wushijia and Conway et al internationally proposed the AAV2 vector packaging strategy with HSV1 as helper virus (Wushijia, Wu soldier et al scientific bulletin, 1999; 44(5): 506-.Gene Ther1999, 6: 986-. Subsequently Wusterer et al proposed an AAV5 vector packaging strategy (Wusterer JT, et al) with HSV1 as a helper virus.Mol Ther2002, 6(4) 510-. On the basis of the above, Booth and the likeTwo HSV1 are used for carrying a rep/cap gene of AAV and an Inverted Terminal Repeat (ITR)/exogenous gene expression cassette of AAV respectively, then two recombinant HSV1 viruses are used for co-infecting a production cell, and packaging is carried out to generate AAV (Booth MJ, et al).Gene Ther2004, 11:829- > 837). Thomas et al further established the suspension cell system for AAV production of bis-HSV 1 virus (Thomas DL, et al.Gene Ther2009; 20:861- & 870.) makes possible larger scale production of AAV viruses. In addition, Urabe and the like construct a baculovirus packaging system of AAV vectors by using three baculoviruses to respectively carry AAV structural, non-structural and ITR/exogenous gene expression cassettes. Considering the instability of baculovirus carrying foreign genes, the number of baculoviruses required in the production system is subsequently reduced, gradually going from the first requiring three baculoviruses to the need of two or one baculoviruses (Chen H).Mol Ther. 2008; 16(5): 924-930. Galibert L,et al.J Invertebr Pathol2011; 107 Suppl: S80-93.) and one baculovirus plus one strain of inducible cell line strategy (MietzschM, et al.Hum Gene Ther. 2014; 25: 212-222. Mietzsch M, et al.Hum Gene Ther2015, 26(10) 688-697. Each packaging system has various characteristics, and can be selected as required.

As a result of the above characteristics, AAV vector has gradually become a foreign gene transfer tool widely used in gene therapy, especially gene therapy of genetic diseases, until 12 months 2018, there are 238 approved gene therapy clinical test protocols based on AAV vector in the world (http:// www.abedia.com/willey/vectors. php). more importantly, Glybera, a lipoprotein lipase gene therapy drug based on AAV vector has been approved by European drug administration in 2012 and is the first approved gene therapy drug in the western world (YI ä -Herttuala S).Mol Ther2012, 20(10) 1831 and 1832); the American FDA approved congenital black disease (caused by RPE65 gene mutation) gene therapy medicine Luxturna is marketed in 19.12.2017, and becomes the gene therapy medicine of the first rare disease in the United states (https:// www.fda.gov/news events/news group/presentation uncementes/ucm 589467. htm); us FDA approved spinal muscular atrophy (SM) 24 months 5 in 2019N1 gene mutation) gene therapy medicine Zolgensma is marketed as the gene therapy medicine (https:// www.fda.gov/media/126130/download) administered by the first intravenous injection system in the world, marking that AAV vector gene therapy medicine development enters a new stage. Hemophilia B (Kay MA, et al.Nat GenetThe AAV vector gene therapy medicaments of 2000, 24(3), 257 and 261) all have good clinical test effects, are expected to be sold in the near future and benefit a large number of patients.

In the present invention, the AAV vector is selected to carry the ATP7B gene expression cassette or the truncated ATP7B gene expression cassette, based mainly on the following features of the AAV vector. For one, AAV vectors retain only the two ITR sequences required for viral packaging in wild-type virus, but do not contain the protein-encoding genes in the wild-type virus genome (salenik M, et al.Microbiol Spectr2015, 3(4), low immunogenicity. Secondly, AAV achieves sustained stable expression of the gene-carrying reading frame, usually in the form of non-integrated extrachromosomal genetic material (Chen ZY, et al.Mol Ther2001, 3(3) 403-. Third, AAV vectors have a higher transduction efficiency into the liver by intravenous injection (Sands MS.Methods Mol Biol. 2011; 807: 141-157. Wang L, et al.Mol Ther2015, 23(12) 1877 and 1887. to ensure that the gene expression cassette carrying ATP7B or truncated ATP7B can transfer to liver efficiently and produce ATP7B or truncated ATP7B protein in liver expression.

According to the design thought, a series of recombinant AAV vectors are prepared and used for drug development strategies for treating hepatolenticular degeneration. These recombinant AAV vectors carry an ATP7B gene expression cassette or contain a truncated ATP7B gene expression cassette, respectively. Firstly, a recombinant AAV8 vector carrying an ATP7B gene expression frame is injected into a hepatolenticular degeneration model mouse ATP7b through the tail vein at 3 different doses with high, medium and low levels^-/-A mouse. After virus injection, transaminase level in serum of a model mouse, copper ion content in urine and copper ion content in liver tissues and ATP7B mRNA expression level in liver are detected, and validity of ATP7B gene expression frame is verifiedAnd different virus injection doses for hepatolenticular degeneration model mouse atp7b^-/-Effect of the mice. Next, recombinant AAV8 vector containing ATP7B gene expression cassette or truncated ATP7B gene expression cassette was injected via tail vein into the hepatolenticular degeneration model mouse ATP7b at 3 different doses of high, medium or low level^-/-Mice to verify the function of the truncated ATP7B gene expression cassette. On the basis, AAV vectors of different serotypes are used for carrying a truncated ATP7B gene expression frame, and AAV viruses are injected into a hepatolenticular degeneration model mouse ATP7b through tail veins^-/-Mice to compare the differences in the effects of different serotype AAV vectors. The result shows that after the designed ATP7B gene expression frame or the truncated ATP7B gene expression frame is carried by an AAV vector, the liver can be effectively transduced by intravenous administration, ATP7B protein or the truncated ATP7B protein is expressed and generated, the symptoms in a mouse with a hepatolenticular degeneration model are remarkably relieved, and the potential of developing a drug for treating the hepatolenticular degeneration gene is shown. And different serotype AAV vectors carry truncated ATP7B gene expression frames, and can effectively relieve physiological symptoms of a model mouse after tail vein injection of the mouse with the hepatolenticular degeneration, which indicates that other serotype AAV except AAV8 can also be used for developing drugs for treating the hepatolenticular degeneration genes. In a word, the invention provides a development method of a hepatolenticular degeneration gene therapy drug, and provides a new choice for treating hepatolenticular degeneration.

Disclosure of Invention

In view of this, the present invention provides a series of recombinant AAV vectors carrying ATP7B gene expression cassettes or containing truncated ATP7B gene expression cassettes for drug development strategies for the treatment of hepatolenticular degeneration. Firstly, an ATP7B gene expression frame and a truncated ATP7B gene expression frame are designed, AAV vector plasmids of the two expression frames are constructed, and the two plasmids are packaged to obtain AAV viruses of different serotypes. Then, recombinant AAV8 vector AAV8-LP15-ATP7B carrying ATP7B gene expression cassette is injected into a mouse ATP7b with hepatolenticular degeneration model at 3 different doses with high, medium and low grade through tail vein^-/-Mice were evaluated for the effectiveness of AAV8-LP15-ATP7B and for the effect of different injected doses on its effectiveness.Next, AAV8-LP15-ATP7B was used as a control, and AAV8-LP15- Δ C4ATP7B, which is a recombinant AAV8 vector carrying a truncated ATP7B (Δ C4ATP 7B) gene expression cassette, was injected into a mouse ATP7b, which is a hepatolenticular degeneration model, through the caudal vein at 3 different doses, medium or low, in height^-/-Mice were evaluated for the effectiveness of AAV8-LP15- Δ C4ATP7B and for the effect of different injected doses on its effectiveness. On the basis, AAV vectors of different serotypes are used to carry a delta C4ATP7B gene expression frame, and 3 recombinant viruses such as AAV3B-LP 15-delta C4ATP7B, AAV5-LP 15-delta C4ATP7B and AAV9-LP 15-delta C4ATP7B are obtained. AAV8-LP15- Δ C4ATP7B is used as a control, and 3 AAV viruses such as AAV3B-LP15- Δ C4ATP7B, AAV5-LP15- Δ C4ATP7B, AAV9-LP15- Δ C4ATP7B and the like are injected into a hepatolenticular degeneration model mouse ATP7b through the caudal vein^-/-Mice, evaluation of different AAV serotypes on model animal effect. The experimental results show that after the designed ATP7B gene expression frame or the truncated ATP7B gene expression frame is carried by an AAV vector, the liver can be effectively transduced through intravenous administration, ATP7B protein or the truncated ATP7B protein is generated through expression, the symptoms in a hepatolenticular degeneration model mouse are remarkably relieved, and the potential of developing the drug for treating the hepatolenticular degeneration gene is shown. And different serotype AAV vectors carry truncated ATP7B gene expression frames, and can effectively relieve physiological symptoms of a model mouse after tail vein injection of the mouse with the hepatolenticular degeneration, which indicates that other serotype AAV except AAV8 can also be used for developing drugs for treating the hepatolenticular degeneration genes. In conclusion, the hepatolenticular degeneration gene therapeutic drug candidate provided by the invention shows a great development value and provides a new choice for treating hepatolenticular degeneration.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides an ATP7B gene expression cassette design, which is characterized in that the expression cassette consists of a human designed liver specificity promoter LP15 promoter, a human source expression codon optimized ATP7B coding sequence and a short human designed polyA tailing signal, and the size of the expression cassette is 4.8 kb. The expression cassette plus two ITR sequences, 5.1kb in size, is near the packaging limit of AAV viral vectors. The expression cassette can specifically and highly express and produce ATP7B protein in liver cells. The specific process is detailed in example 1.

The invention provides a truncated ATP7B gene expression cassette design, which is characterized in that the expression cassette consists of a liver-specific promoter LP15 promoter, a truncated ATP7B coding sequence and a bovine growth hormone polyA tailing signal (BGH polyA) sequence which are artificially designed. The truncated ATP7B coding sequence, Δ C4ATP7B reference (Guo Y, et al, Am JPhysiol gastroenterest light physiology. 2005; 289: G904-G916.) was synthesized by deleting 4 copper ion binding domains in the full-length ATP7B protein, shortening the length of the gene coding sequence, but not affecting its biological function. The truncated Δ C4ATP7B coding sequence was 1.1kb shorter than the full-length ATP7B coding sequence, making the sequence size of the Δ C4ATP7B expression cassette "LP 15- Δ C4ATP7B-BGH polyA" 0.9kb shorter than the sequence size of the ATP7B expression cassette "LP 15-ATP 7B-SPA", the expression cassette size was 3.9kb, less than 4.7kb, and was able to be efficiently packaged into various AAV viruses. The expression cassette can specifically and highly express and produce ATP7B protein in liver cells. The specific implementation process is shown in example 1.

The invention provides a recombinant AAV8 virus AAV8-LP15-ATP7B carrying an ATP7B gene expression frame, which is characterized in that the recombinant virus is injected into a hepatolenticular model mouse ATP7b through tail vein by using high, medium and low 3 doses^-/-After mice, the transaminase level in serum of a hepatolenticular model mouse can be effectively reduced, the content of copper ions in blood and urine is reduced, the hepatolenticular degeneration symptom is obviously improved, and the effect shows obvious dose dependence. The expression of ATP7B mRNA was detected in the liver of virus-injected mice by quantitative PCR. The result shows that AAV8-LP15-ATP7B has the potential of being developed into a drug for treating hepatolenticular degeneration genes. See example 3 for a specific implementation.

The invention provides a recombinant AAV8 virus AAV8-LP15- Δ C4ATP7B carrying a Δ C4ATP7B gene expression cassette, which is characterized in that the recombinant virus is injected into a hepatolenticular model mouse ATP7b through tail vein at high, medium and low doses of 3 doses^-/-After mice, the transaminase level in serum of the liver bean-shaped model mice can be effectively reduced, and copper ions in blood and urine can be effectively reducedContent, remarkably improves the hepatolenticular degeneration symptom, and has obvious dosage dependence on the action effect. No obvious difference is found between the action effect of AAV8-LP15- Δ C4ATP7B and the action effect of AAV8-LP15-ATP 7B. Expression of Δ C4ATP7B mRNA was detected in the liver of virus-injected mice by quantitative PCR. The result shows that AAV8-LP15- Δ C4ATP7B also has the potential of being developed into a drug for treating the hepatolenticular degeneration gene. See example 4 for a specific implementation.

The recombinant AAV carrying the gene expression frame of the Δ C4ATP7B provided by the invention comprises AAV3B-LP15- Δ C4ATP7B, AAV5-LP15- Δ C4ATP7B and AAV9-LP15- Δ C4ATP7B, and is characterized in that the recombinant AAV are injected into a hepatolenticular model mouse ATP7b through caudal vein like AAV8-LP15- Δ C4ATP7B viruses^-/-After mice, the transaminase level in serum of the hepatolenticular model mice can be effectively reduced, the copper ion content in blood and urine can be reduced, and the hepatolenticular degeneration symptoms can be obviously improved. Δ C4ATP7B mRNA expression was also detected in the liver of virus-injected mice by quantitative PCR. The result shows that in addition to AAV8, other serotype AAV vectors (such as AAV3B, AAV5 and AAV 9) can be used for the development of medicine for treating hepatolenticular degeneration gene. See example 5 for a specific implementation.

The recombinant AAV carrying the ATP7B gene expression cassette and/or the Δ C4ATP7B gene expression cassette provided by the invention is also characterized in that the recombinant AAV can continuously and effectively express and generate ATP7B mRNA or Δ C4ATP7B mRNA after being injected into a mouse body by veins, is further translated into ATP7B protein or truncated ATP7B protein, recovers the copper metabolism function of a model mouse, reduces the transaminase level in the model mouse body and the copper ion content in urine and blood, and provides a new choice for the development of hepatolenticular degeneration gene drugs. See examples 3, 4 and 5 for specific examples.

The important original experimental materials used in the present invention are as follows:

pHelper plasmid, derived from AAV Helper Free System (Agilent Technologies, USA), was purchased from Agilent Technologies, Inc. and stored. The plasmid contains three plasmids to co-transfect HEK293 cells to prepare adenovirus-derived helper function genes E2A, E4, VA RNA and the like required by recombinant AAV.

The pAAV-R2C3B plasmid was constructed and stored by this company. The plasmid pAAV-RC in AAV Helper Free systems (Agilent technologies, USA) is used as a basic skeleton, and the sequence from 2013 to 4220 in the plasmid pAAV-RC is replaced by the coat protein coding sequence Cap3B (sequence from 2208 to 4418 in the genome) in AAV3B genome (GenBank ID: AF 028705), so that the plasmid pAAV-R2C3B is obtained. The simple construction process is that pAAV-R2C3B plasmid sequence information is obtained according to the above thought, sequences between HindIII and PmeI restriction sites in the pAAV-R2C3B plasmid are artificially synthesized, and the sequences between HindIII and PmeI of the pAAV-RC plasmid are replaced by the synthetic sequences by adopting a standard molecular cloning method to obtain the pAAV-R2C3B plasmid. The pAAV-R2C3B plasmid contains the cap gene of AAV3B and the Rep gene of AAV2 completely, and 4 Rep proteins (Rep 78, Rep68, Rep52 and Rep 40) and AAV3B coat proteins which are necessary for providing packaging in the preparation of recombinant AAV3B virus by three-plasmid co-transfection packaging.

The pAAV-R2C5 plasmid was constructed and stored by this company. The plasmid sequence pAAV-R2C5 is obtained by using pAAV-RC plasmid in AAV Helper Free systems (Agilent technologies, USA) as basic skeleton and replacing sequences 2013 to 4220 in pAAV-RC plasmid with coat protein coding sequence Cap5 (sequences 2207 to 4381 in genome) in AAV genome (GenBank ID: NC-006152.1). The simple construction process is that pAAV-R2C5 plasmid sequence information is obtained according to the above thought, sequences between HindIII and PmeI restriction sites in the pAAV-R2C5 plasmid are artificially synthesized, and the sequences between HindIII and PmeI of the pAAV-RC plasmid are replaced by the synthetic sequences by adopting a standard molecular cloning method to obtain the pAAV-R2C5 plasmid. The pAAV-R2C5 plasmid contains the cap gene of AAV5 and the Rep gene of AAV2 in a complete form, and 4 Rep proteins (Rep 78, Rep68, Rep52 and Rep 40) and AAV5 coat proteins which are necessary for packaging are provided in the preparation of recombinant AAV5 virus by three-plasmid co-transfection packaging.

The pAAV-R2C8 plasmid was constructed and stored by this company. The pAAV-RC plasmid in AAV Helper Free systems (Agilent technologies, USA) is used as a basic skeleton, and the sequence from 2013 to 4220 in the pAAV-RC plasmid is replaced by the coat protein coding sequence Cap8 (sequence from 2121 to 4337 in the genome) in AAV8 genome (GenBank ID: AF 513852), so that the pAAV-R2C8 plasmid is obtained. The simple construction process is that pAAV-R2C8 plasmid sequence information is obtained according to the above thought, sequences between HindIII and PmeI restriction sites in the pAAV-R2C8 plasmid are artificially synthesized, and the sequences between HindIII and PmeI of the pAAV-RC plasmid are replaced by the synthetic sequences by adopting a standard molecular cloning method to obtain the pAAV-R2C8 plasmid. The pAAV-R2C8 plasmid contains the cap gene of AAV8 and the Rep gene of AAV2 in a complete form, and 4 Rep proteins (Rep 78, Rep68, Rep52 and Rep 40) and AAV8 coat proteins which are necessary for packaging are provided in the preparation of recombinant AAV8 virus by three-plasmid co-transfection packaging.

The pAAV-R2C9 plasmid was constructed and stored by this company. The pAAV-RC plasmid in AAV Helper Free System (Agilent technologies, USA) is used as basic skeleton, and the sequences 2013 to 4220 in pAAV-RC plasmid are replaced by AAV9 coat protein coding sequence (GenBank ID: AY 530579), so that pAAV-R2C9 plasmid is obtained. The simple construction process is that pAAV-R2C9 plasmid sequence information is obtained according to the thought, a sequence between HindIII and PmeI restriction sites in the pAAV-R2C9 plasmid is artificially synthesized, and a standard molecular cloning method is adopted to replace the sequence between the HindIII and PmeI restriction sites of the pAAV-RC plasmid by the synthetic sequence to obtain the pAAV-R2C9 plasmid. The pAAV-R2C9 plasmid contains the cap gene of AAV9 and the Rep gene of AAV2 in a complete form, and 4 Rep proteins (Rep 78, Rep68, Rep52 and Rep 40) and AAV9 coat proteins which are necessary for packaging are provided in the preparation of recombinant AAV9 virus by three-plasmid co-transfection packaging.

The hepatolenticular degeneration model breeder mice were prepared: b6 heterozygous for the atp7B gene; 129S1-Atp7b^tm1TcgMale and female mice/LtsnkJ were purchased from Jacksonlaboratory, usa under the trade designation 032624. The mouse ATP7b gene was replaced by neo gene expression cassette using homologous recombination method, and ATP7b gene lacking exon 2 could not be expressed to produce ATP7B protein. Hepatolenticular degeneration model atp7b^-/-Mice were prepared by mating male and female heterozygous mice. The prepared model is identified by a PCR method, and the identification method and the identification process are shown in a Jackson Laboratory website.

C57BL/6 mice: purchased from beijing huafukang biotech inc, used as a wild-type mouse for animal experiments.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.

FIG. 1 is a schematic diagram of the pAAV2neo vector structure. The AAV vector pAAV2neo (Dong X, et al, PLoS ONE. 2010; 5(10): e 13479.) with both ITRs on both sides being 145bp wild-type ITRs was stored by this company. ITR, inverted terminal repeat, length 145 bp. CMV promoter, human cytomegalovirus early promoter. BGH polyA, polynucleotide tailing signal of bovine growth hormone. Amp, reading frame for ampicillin resistance gene. Neo, neomycin resistance gene reading frame. XhoI, KpnI, EcoRI, SalI, BglII, BamHI and ApaI are all restriction sites.

FIG. 2 is a schematic diagram of the structure of pAAV2-LP15 vector. ITR, inverted terminal repeat, length 145 bp. LP15 promoter, a human-designed liver-specific promoter, and the sequence information is detailed in SEQ ID NO. 1. BGH polyA, polynucleotide tailing signal of bovine growth hormone. Amp, reading frame for ampicillin resistance gene. Neo, neomycin resistance gene reading frame. XhoI, KpnI, EcoRI, SalI, BglII, BamHI and ApaI are all restriction sites.

FIG. 3 is a schematic diagram of the structure of pAAV2-LP15-EGFP vector. ITR, inverted terminal repeat, length 145 bp. LP15 promoter, a human-designed liver-specific promoter, the sequence information of which is detailed in SEQ ID NO. 1. BGH polyA, polynucleotide tailing signal of bovine growth hormone. Amp, reading frame for ampicillin resistance gene. Neo, neomycin resistance gene reading frame. EGFP, enhanced green fluorescent protein coding region sequence. XhoI, KpnI, EcoRI, SalI, BglII, BamHI and ApaI are all restriction sites.

FIG. 4 shows a schematic structure of pAAV2-LP15-ATP7B vector. ITR, inverted terminal repeat, length 145 bp. LP15 promoter, a human-designed liver-specific promoter, the sequence information of which is detailed in SEQ ID NO. 1. SPA, artificially designed polynucleotide tailing signal, and the sequence information is shown in SEQ ID NO. 2. Amp, reading frame for ampicillin resistance gene. Neo, neomycin resistance gene reading frame. ATP7B, codon optimized ATP7B coding sequence, and the sequence information is detailed in SEQ ID NO. 3. XhoI, KpnI, EcoRI, SalI, BglII, BamHI and ApaI are all restriction sites.

FIG. 5 shows a schematic diagram of the vector structure of pAAV2-LP15- Δ C4ATP 7B. ITR, inverted terminal repeat, length 145 bp. LP15 promoter, a human-designed liver-specific promoter, the sequence information of which is detailed in SEQ ID NO. 1. BGH polyA, polynucleotide tailing signal of bovine growth hormone, and sequence information is detailed in SEQ ID NO. 2. Amp, reading frame for ampicillin resistance gene. Neo, neomycin resistance gene reading frame. Δ C4ATP7B, truncated and codon optimized ATP7B coding sequence, sequence information is detailed in SEQ ID No. 4. XhoI, KpnI, EcoRI, SalI, BglII, BamHI and ApaI are all restriction sites.

FIG. 6 shows the results of detection of glutamic-pyruvic transaminase level in serum of mice injected with rAAV8-LP15-ATP7B virus. rAAV8-LP15-ATP7B is injected into a hepatolenticular degeneration model mouse (ATP 7 b) through tail vein with high, medium and low (1E +10 vg/mouse, 3E +10 vg/mouse and 9E +10 vg/mouse)^-/-Mouse). Wild-type mice without virus injection are used as positive control, rAAV8-LP15-EGFP virus (9E +10 vg/mouse) is used as negative control, tail vein blood collection is carried out at different time points (4 weeks, 8 weeks, 12 weeks and 24 weeks after virus injection), serum is separated, and the level of glutamic-pyruvic transaminase in the serum is determined by using the kit. Glutamate pyruvate transaminase levels in serum of wild type, non-virus injected wild type mice. Control, levels of glutamate pyruvate transaminase in serum of mice injected with rAAV8-LP15-EGFP virus (9E +10 vg/mouse). 1E +10 vg/mouse, levels of glutamate pyruvate transaminase in serum of mice injected with rAAV8-LP15-ATP7B virus (1E +10 vg/mouse). 3E +10 vg/mouse, levels of glutamate pyruvate transaminase in serum of mice injected with rAAV8-LP15-ATP7B virus (3E +10 vg/mouse). 9E +10 vg/mouse, levels of glutamate pyruvate transaminase in serum of mice injected with rAAV8-LP15-ATP7B virus (9E +10 vg/mouse). IU/L, glutamate pyruvate transaminase activity units.

FIG. 7 shows the result of detecting the level of copper ions in urine of mice injected with rAAV8-LP15-ATP7B virus. rAAV8-LP15-ATP7B is injected into a hepatolenticular degeneration model mouse (ATP 7 b) through tail vein with high, medium and low (1E +10 vg/mouse, 3E +10 vg/mouse and 9E +10 vg/mouse)^-/-Mouse). Wild-type mice without virus injection are used as positive controls, rAAV8-LP15-EGFP virus (9E +10 vg/mouse) is used as negative controls, urine discharged by the mice within 24h is collected at different time points (4 weeks, 8 weeks, 12 weeks and 24 weeks after virus injection), and the total amount of copper ions in the collected urine is determined. Total amount of copper ions in urine of wild type, non-virus injected wild type mice. Control, total amount of copper ions in urine of mice injected with rAAV8-LP15-EGFP virus (9E +10 vg/mouse). 1E +10 vg/mouse, injected with rAAV8-LP15-ATP7B virus (1E +10 vg/mouse) in the urine of total copper ion. 3E +10 vg/mouse, injected with rAAV8-LP15-ATP7B virus (3E +10 vg/mouse) in the urine of total copper ion. 9E +10 vg/mouse, injected with rAAV8-LP15-ATP7B virus (9E +10 vg/mouse) in the urine of total copper ion. ng/24h, the total amount of copper ions in urine excreted by the mice within 24h is expressed by ng.

FIG. 8 shows the results of testing the level of copper ions in the liver of mice injected with rAAV8-LP15-ATP7B virus. rAAV8-LP15-ATP7B is injected into a hepatolenticular degeneration model mouse (ATP 7 b) through tail vein with high, medium and low (1E +10 vg/mouse, 3E +10 vg/mouse and 9E +10 vg/mouse)^-/-Mouse). Wild-type mice without virus injection were positive controls, rAAV8-LP15-EGFP virus (9E +10 vg/mouse) was injected as negative controls, and 24 weeks after virus injection, mice were sacrificed, livers were isolated, and the total amount of copper ions in the desiccated liver tissues was determined. Wild type, wild type mice not injected with virus total amount of copper ions in dry liver tissue. Control, mice injected with rAAV8-LP15-EGFP virus (9E +10 vg/mouse) total copper ions in dry liver tissue. 1E +10 vg/mouse, mice injected with rAAV8-LP15-ATP7B virus (1E +10 vg/mouse) had total copper ion in the dried liver tissue. 3E +10 vg/mouse, rAAV8-LP15-ATP7B virus (3E +10 vg/mouse) was injected to total copper ion in the dried liver tissue. 9E +10 vg/mouse, rAAV8-LP15-ATP7B virus (9E +10 vg/mouse) was injected to total copper ion in the dried liver tissue. Mu g/g dry fractionThe total amount of copper ions per gram of dry liver tissue is expressed in μ g.

FIG. 9 shows the result of detecting the expression level of ATP7B mRNA in the liver of mice injected with rAAV8-LP15-ATP7B virus. rAAV8-LP15-ATP7B is injected into a hepatolenticular degeneration model mouse (ATP 7 b) through tail vein with high, medium and low (1E +10 vg/mouse, 3E +10 vg/mouse and 9E +10 vg/mouse)^-/-Mouse). rAAV8-LP15-EGFP virus (9E +10 vg/mouse) is injected as a negative control, 24 weeks after virus injection, mice are killed, livers are separated, total RNA is extracted, and ATP7B mRNA content in 100ng of total RNA is determined by reverse transcription quantitative PCR (polymerase chain reaction) and used for representing the expression level of the transduction ATP7B gene in the livers of the mice. Control, the expression level of ATP7B mRNA in liver tissue of mice injected with rAAV8-LP15-EGFP virus (9E +10 vg/mouse). 1E +10 vg/mouse, injected with rAAV8-LP15-ATP7B virus (1E +10 vg/mouse) and ATP7B mRNA expression level in liver tissue. 3E +10 vg/mouse, injected with rAAV8-LP15-ATP7B virus (3E +10 vg/mouse) and expressed ATP7B mRNA in liver tissue. 9E +10 vg/mouse, injected with rAAV8-LP15-ATP7B virus (9E +10 vg/mouse) and ATP7B mRNA expression level in liver tissue. copies of ATP7B mRNA in copies/100ng total RNA, 100ng total RNA.

FIG. 10 shows the results of detection of glutamic-pyruvic transaminase level in serum of mice injected with rAAV8-LP15- Δ C4ATP7B virus. The rAAV8-LP15-ATP7B or rAAV8-LP15- Δ C4ATP7B is injected into a hepatolenticular degeneration model mouse (ATP 7 b) through the tail vein with high, medium and low (1E +10 vg/mouse, 3E +10 vg/mouse and 9E +10 vg/mouse)^-/-Mouse). At various time points after injection of the virus (4 weeks, 8 weeks, 12 weeks, and 24 weeks after injection of the virus), tail vein blood was collected, serum was separated, and glutamate pyruvate transaminase levels in serum were measured using the kit. ATP7B-1E +10 vg/mouse, levels of glutamate pyruvate transaminase in sera of mice injected with rAAV8-LP15-ATP7B virus (1E +10 vg/mouse). Delta ATP7B-1E +10 vg/mouse, levels of glutamate pyruvate transaminase in sera of mice injected with rAAV8-LP 15-delta C4ATP7B virus (1E +10 vg/mouse). ATP7B-3E +10 vg/mouse, levels of glutamate pyruvate transaminase in sera of mice injected with rAAV8-LP15-ATP7B virus (3E +10 vg/mouse). Delta ATP7B-3E +10 vg/mouse, levels of glutamate pyruvate transaminase in sera of mice injected with rAAV8-LP 15-delta C4ATP7B virus (3E +10 vg/mouse). ATP7B-9E +10 vg/mouse, injectionGlutamate pyruvate transaminase levels in serum of mice with rAAV8-LP15-ATP7B virus (9E +10 vg/mouse). Delta ATP7B-9E +10 vg/mouse, levels of glutamate pyruvate transaminase in sera of mice injected with rAAV8-LP 15-delta C4ATP7B virus (9E +10 vg/mouse).

FIG. 11 shows the result of detecting the level of copper ions in urine of mice injected with rAAV8-LP15- Δ C4ATP7B virus. The rAAV8-LP15-ATP7B or rAAV8-LP15- Δ C4ATP7B is injected into a hepatolenticular degeneration model mouse (ATP 7 b) through the tail vein with high, medium and low (1E +10 vg/mouse, 3E +10 vg/mouse and 9E +10 vg/mouse)^-/-Mouse). At various time points after injection of the virus (4 weeks, 8 weeks, 12 weeks, and 24 weeks after injection of the virus), urine excreted by the mice was collected for 24 hours, and the total amount of copper ions in the collected urine was determined. ATP7B-1E +10 vg/mouse, rAAV8-LP15-ATP7B virus (1E +10 vg/mouse) injected in urine total copper ion. Delta ATP7B-1E +10 vg/mouse, injected rAAV8-LP 15-delta C4ATP7B virus (1E +10 vg/mouse) in urine total copper ion. ATP7B-3E +10 vg/mouse, rAAV8-LP15-ATP7B virus (3E +10 vg/mouse) injected in urine total copper ion. Delta ATP7B-3E +10 vg/mouse, injected rAAV8-LP 15-delta C4ATP7B virus (3E +10 vg/mouse) in urine total copper ion. ATP7B-9E +10 vg/mouse, rAAV8-LP15-ATP7B virus (9E +10 vg/mouse) injected in urine total copper ion. Delta ATP7B-9E +10 vg/mouse, injected rAAV8-LP 15-delta C4ATP7B virus (9E +10 vg/mouse) urine total copper ion. ng/24h, the total amount of copper ions in urine excreted by the mice within 24h is expressed by ng.

FIG. 12 shows the results of testing the level of copper ions in the liver of mice injected with rAAV8-LP15- Δ C4ATP7B virus. rAAV8-LP15-ATP7B is injected into a hepatolenticular degeneration model mouse (ATP 7 b) through tail vein with high, medium and low (1E +10 vg/mouse, 3E +10 vg/mouse and 9E +10 vg/mouse)^-/-Mouse). 24 weeks after virus injection, mice were sacrificed, livers were isolated, and the total amount of copper ions in the dried liver tissue was determined. ATP7B-1E +10 vg/mouse injected with rAAV8-LP15-ATP7B virus (1E +10 vg/mouse) the total amount of copper ions in the dry liver tissue. Δ ATP7B-1E +10 vg/mouse injected with rAAV8-LP15- Δ C4ATP7B virus (1E +10 vg/mouse) total copper ion in dry liver tissue. ATP7B-3E +10 vg/mouse dried liver injected with rAAV8-LP15-ATP7B virus (3E +10 vg/mouse)Total amount of copper ions in the visceral tissue. Delta ATP7B-3E +10 vg/mouse injected with rAAV8-LP 15-delta C4ATP7B virus (3E +10 vg/mouse) the total amount of copper ions in the dry liver tissue. ATP7B-9E +10 vg/mouse injected with rAAV8-LP15-ATP7B virus (9E +10 vg/mouse) the total amount of copper ions in the dry liver tissue. Delta ATP7B-9E +10 vg/mouse, injected with rAAV8-LP 15-delta C4ATP7B virus (9E +10 vg/mouse) the total amount of copper ions in the dry liver tissue. μ g/g of dried tissue, the total amount of copper ions per gram of dried liver tissue, expressed in μ g.

FIG. 13 shows the results of detecting the expression level of Δ C4ATP7B mRNA in the liver of mice injected with rAAV8-LP15- Δ C4ATP7B virus. The rAAV8-LP15- Δ C4ATP7B or rAAV8-LP15-ATP7B is injected into a hepatolenticular degeneration model mouse (ATP 7 b) through the tail vein with high, medium and low (1E +10 vg/mouse, 3E +10 vg/mouse and 9E +10 vg/mouse)^-/-Mouse). 24 weeks after virus injection, mice were sacrificed, livers were isolated, total RNA was extracted, and the ATP7B mRNA or Δ C4ATP7B mRNA content in 100ng of total RNA was determined by reverse transcription quantitative PCR, which was used to indicate the expression level of the transduced ATP7B gene or Δ C4ATP7B gene in the livers of mice. ATP7B-1E +10 vg/mouse, rAAV8-LP15-ATP7B virus (1E +10 vg/mouse) injected ATP7BmRNA expression level in liver tissue. Delta ATP7B-1E +10 vg/mouse, injected rAAV8-LP 15-delta C4ATP7B virus (1E +10 vg/mouse) in liver tissue of delta C4ATP7B mRNA expression level. ATP7B-3E +10 vg/mouse, rAAV8-LP15-ATP7B virus (3E +10 vg/mouse) injected ATP7B mRNA expression level in liver tissue. Delta ATP7B-3E +10 vg/mouse, injected rAAV8-LP 15-delta C4ATP7B virus (3E +10 vg/mouse) in liver tissue of delta C4ATP7B mRNA expression level. ATP7B-9E +10 vg/mouse, rAAV8-LP15-ATP7B virus (9E +10 vg/mouse) injected ATP7BmRNA expression level in liver tissue. Delta ATP7B-9E +10 vg/mouse, injected rAAV8-LP 15-delta C4ATP7B virus (9E +10 vg/mouse) in liver tissue of delta C4ATP7B mRNA expression level. copies of ATP7BmRNA in copies/100ng total RNA, 100ng total RNA.

FIG. 14 measurement results of glutamic-pyruvic transaminase levels in serum of mice injected with AAV vectors of different serotypes. rAAV8-LP15- Δ C4ATP7B, rAAV3B-LP15- Δ C4ATP7B, rAAV5-LP15- Δ C4ATP7B or rAAV9-LP15- Δ C4ATP7B are dosed at 3E +10 vg/minVein injection hepatolenticular degeneration model mouse (atp 7 b)^-/-Mouse). At various time points after injection of the virus (4 weeks, 8 weeks, 12 weeks, and 24 weeks after injection of the virus), tail vein blood was collected, serum was separated, and glutamate pyruvate transaminase levels in serum were measured using the kit. AAV3B, injected with rAAV3B-LP15- Δ C4ATP7B virus (3E +10 vg/mouse) in serum glutamate pyruvate transaminase level. AAV5, levels of glutamate pyruvate transaminase in serum of mice injected with rAAV5-LP15- Δ C4ATP7B virus (3E +10 vg/mouse). AAV8, levels of glutamate pyruvate transaminase in serum of mice injected with rAAV8-LP15- Δ C4ATP7B virus (3E +10 vg/mouse). AAV9, levels of glutamate pyruvate transaminase in serum of mice injected with rAAV9-LP15- Δ C4ATP7B virus (3E +10 vg/mouse).

FIG. 15 test results of copper ion levels in urine after mice injected with different serotype AAV vectors. rAAV8-LP15- Δ C4ATP7B, rAAV3B-LP15- Δ C4ATP7B, rAAV5-LP15- Δ C4ATP7B or rAAV9-LP15- Δ C4ATP7B is injected into a hepatolenticular degeneration model mouse through tail vein at the dose of 3E +10 vg/mouse (ATP 7 b)^-/-Mouse). At various time points after injection of the virus (4 weeks, 8 weeks, 12 weeks, and 24 weeks after injection of the virus), urine excreted by the mice was collected for 24 hours, and the total amount of copper ions in the collected urine was determined. AAV3B, injected with rAAV3B-LP15- Δ C4ATP7B virus (3E +10 vg/mouse) in urine total copper ion. AAV5, injected with rAAV5-LP15- Δ C4ATP7B virus (3E +10 vg/mouse) in urine total copper ion. AAV8, injected with rAAV8-LP15- Δ C4ATP7B virus (3E +10 vg/mouse) in urine total copper ion. AAV9, injected with rAAV9-LP15- Δ C4ATP7B virus (3E +10 vg/mouse) in urine total copper ion. ng/24h, the total amount of copper ions in urine excreted by the mice within 24h is expressed by ng.

FIG. 16 results of detection of copper ion levels in the liver after mice injected with different serotype AAV vectors. rAAV8-LP15- Δ C4ATP7B, rAAV3B-LP15- Δ C4ATP7B, rAAV5-LP15- Δ C4ATP7B or rAAV9-LP15- Δ C4ATP7B is injected into a hepatolenticular degeneration model mouse through tail vein at the dose of 3E +10 vg/mouse (ATP 7 b)^-/-Mouse). 24 weeks after virus injection, mice were sacrificed, livers were isolated, and the total amount of copper ions in the dried liver tissue was determined. AAV3B, rAAV3B-LP15- Δ C4ATP7B virus (3E +10 vg/mouse) injected mouse dried liver groupTotal amount of copper ions in the fabric. AAV5, rAAV5-LP15- Δ C4ATP7B virus (3E +10 vg/mouse) injected mice total copper ion in dry liver tissue. AAV8, rAAV8-LP15- Δ C4ATP7B virus (3E +10 vg/mouse) injected mice total copper ion in dry liver tissue. AAV9, rAAV9-LP15- Δ C4ATP7B virus (3E +10 vg/mouse) injected mice total copper ion in dry liver tissue. μ g/g of dried tissue, the total amount of copper ions per gram of dried liver tissue, expressed in μ g.

FIG. 17 results of measurement of the expression level of Δ C4ATP7B mRNA in liver after mice injected with AAV vector of different serotypes. rAAV8-LP15- Δ C4ATP7B, rAAV3B-LP15- Δ C4ATP7B, rAAV5-LP15- Δ C4ATP7B or rAAV9-LP15- Δ C4ATP7B is injected into a hepatolenticular degeneration model mouse through tail vein at the dose of 3E +10 vg/mouse (ATP 7 b)^-/-Mouse). 24 weeks after virus injection, mice were sacrificed, livers were isolated, total RNA was extracted, and the content of Δ C4ATP7B mRNA in 100ng of total RNA was determined by reverse transcription quantitative PCR, which was used to indicate the expression level of the transduced Δ C4ATP7B gene in the livers of mice. AAV3B, injected with rAAV3B-LP15- Δ C4ATP7B virus (3E +10 vg/mouse) expression levels of Δ C4ATP7B mRNA in liver tissue of mice. AAV5, injected with rAAV5-LP15- Δ C4ATP7B virus (3E +10 vg/mouse) for Δ C4ATP7BmRNA expression levels in liver tissue of mice. AAV8, injected with rAAV8-LP15- Δ C4ATP7B virus (3E +10 vg/mouse) expression levels of Δ C4ATP7B mRNA in liver tissue of mice. AAV9, injected with rAAV9-LP15- Δ C4ATP7B virus (3E +10 vg/mouse) expression levels of Δ C4ATP7B mRNA in liver tissue of mice. copies of ATP7B mRNA in copies/100ng total RNA, 100ng total RNA.

Detailed Description

The invention discloses a series of recombinant AAV viruses carrying ATP7B gene expression cassette or truncated ATP7B gene expression cassette, comprising gene expression cassette design, recombinant AAV virus minipreparation and function verification. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention. In which, unless otherwise specified, the various reagents mentioned in the examples are commercially available.

The invention is further illustrated by the following examples:

example 1 plasmid vector construction

In order to package the AAV required for the inventive process, a series of AAV plasmid vectors are constructed. The AAV plasmid vectors can be divided into three types, wherein the first type is a plasmid vector pAAV2-LP15-EGFP required by control virus packaging; the second is AAV plasmid vector pAAV2-LP15-ATP7B of ATP7B gene expression frame; the third is the AAV plasmid vector pAAV2-LP15- Δ C4ATP7B with truncated ATP7B gene expression cassette. All AAV plasmid vector construction is based on pAAV2neo vector constructed and stored by the company, and the structure of pAAV2neo is shown in FIG. 1.

(1) Plasmid vector pAAV2-LP15-EGFP construction required for control virus packaging

Firstly, XhoI enzyme cutting site '5' -CTCGAG-3 'and KpnI enzyme cutting site' 5 '-GGTACC-3' are added at the 5 'end and the 3' end of a liver specific promoter LP15 (the sequence information is shown in SEQ ID NO. 1) designed and stored by the company, and an LP15-XK sequence is obtained, wherein the sequence information is shown in SEQ ID NO.5 in detail. The LP15-XK sequence was synthesized by Nanjing Kinsley and cloned into pUC57-1.8K vector (Nanjing Kinsley), resulting in pUC57-1.8K-LP15-XK vector. The pUC57-LP15-XK vector was digested with XhoI and KpnI, and the 0.35kb fragment was recovered for use. The pAAV2neo vector was digested with XhoI and KpnI by double digestion, and a 6.2kb fragment was recovered for use. The two recovered fragments were ligated to transform E.coli JM109 competent cells (Takara Shuzo), and screened and identified to obtain pAAV2-LP15 vector (FIG. 2). The pAAV2-LP15 vector is AAV plasmid vector for replacing CMV promoter in pAAV2neo plasmid vector with LP15 promoter.

Next, using pCMV-C-EGFP (Biyuntian biotechnology limited, China) as a template, designing a primer EGFP-F/EGFP-R, amplifying an EGFP gene coding region sequence by PCR, wherein the EGFP-F and EGFP-R primers respectively contain KpnI and EcoRI enzyme cutting sites. And amplifying to obtain an EGFP coding region sequence fragment, digesting by EcoRI and KpnI double enzyme, and recovering for later use. The vector pAAV2-LP15 was digested with EcoRI and KpnI, respectively, and the linearized vector fragment pAAV2-LP15 (about 6.5 kb) was recovered. The two recovered fragments were ligated to transform E.coli JM109 competent cells (Baozhi, Dalian), and screened and identified to obtain AAV plasmid vector pAAV2-LP15-EGFP containing EGFP gene expression cassette (see FIG. 3).

EGFP-F: 5’-ataggtaccgccaccatggtgagcaag-3’ (SEQ ID NO.6)

EGFP-R: 5’-gcggaattcttacttgtacagctcgtc-3’ (SEQ ID No.7)

(2) Construction of AAV plasmid vector pAAV2-LP15-ATP7B for ATP7B Gene expression cassette

The NCBI Protein database is searched to obtain a human ATP7B sequence (ID: NP-000044.2) which is named ATP7B-P, and the Protein amino acid sequence is detailed in SEQ ID NO. 8. The ATP7B protein sequence is optimized by Nanjing Kingsrei company according to human expression and is named ATP7B, and the sequence information is detailed in SEQ ID NO. 3. According to the principle of 'GGTACC + GCCACC + ATP7B + TGATAA + AGATCT + tcgagaggcctaataaagagctcagatgcatcgatcagagtgtgttggttttttgtgtg + GGATCC', sequences are added to the 5 'end and the 3' end of an ATP7B sequence respectively to obtain an ATP7B-KB sequence, the sequence is cloned into a pUC-1.8K vector to obtain pUC-1.8K-ATP7B-KB, and the sequence information of ATP7B-KB is detailed in SEQ ID NO. 9. The sequence is added, wherein 'GGTACC' is KpnI enzyme cutting site, 'GCCACC' is Kozak sequence, 'TGATAA' is stop codon sequence, 'AGATCT' is BglII enzyme cutting site, 'tcgagaggcctaataaagagctcagatgcatcgatcagagtgtgttggttttttgtgtg' is artificially designed poly A tailing signal, detailed sequence information is shown in SEQ ID NO.2, and 'GGATCC' is BamHI enzyme cutting site. The pUC-1.8K-ATP7B-KB vector was digested with KpnI and BamHI, and a 4.5KB fragment was recovered for use. The pAAV2-LP15 vector was digested with KpnI and BamHI, and the 6.3kb fragment was recovered for use. After the two recovered fragments are connected, E.coli JM109 competent cells (Bao biol, Dalian) are transformed, and AAV plasmid vectors pAAV2-LP15-ATP7B containing an ATP7B gene expression cassette are obtained after screening and identification (see FIG. 4).

(3) Construction of AAV plasmid vector pAAV2-LP15- Δ C4ATP7B for Δ C4ATP7B Gene expression cassette

The NCBI Protein database is searched to obtain a human ATP7B sequence (ID: NP-000044.2) which is named ATP7B-P, and the Protein amino acid sequence is detailed in SEQ ID NO. 8. The amino acid sequence corresponding to the 1 st to 4 th copper ion binding domains of the sequence ATP7B-P was found in the reference (Guo Y, et al, Am J physiol gastroenterest Liver physiology, 2005; 289: G904-G916.), and named ATP7B-C4, for details of the sequence information see SED ID No. 10. The ATP7B-C4 sequence in the ATP7B-P sequence is deleted to obtain a delta C4ATP7B-P sequence, and the amino acid sequence information is shown in SEQ ID NO. 11. The protein sequence of the delta C4ATP7B-P is optimized by Nanjing Kinshiri according to human expression and is named as delta C4ATP7B, and the sequence information is detailed in SEQ ID NO. 4. According to the principle of 'GGTACC + GCCACC + delta C4ATP7B + TGATAA + AGATCT', sequences are added at the 5 'end and the 3' end of a delta C4ATP7B sequence respectively to obtain a delta C4ATP7B-KB sequence, and the sequence is cloned into a pUC-1.8K vector to obtain pUC-1.8K-delta C4ATP7B-KB, and the sequence information of the delta C4ATP7B-KB is shown in SEQ ID NO.12 in detail. The sequence is added, wherein 'GGTACC' is KpnI enzyme cutting site, 'GCCACC' is Kozak sequence, 'TGATAA' is stop codon sequence, and 'AGATCT' is BglII enzyme cutting site. The pUC-1.8K-. DELTA.C 4ATP7B-KB vector was digested with KpnI and BglII, and the 3.3KB fragment was recovered for use. The pAAV2-LP15 vector was digested with KpnI and BglII, and the linearized 6.5kb fragment was recovered for use. The two recovered fragments were ligated to transform E.coli JM109 competent cells (Bao biol., Dalian), and screened and identified to obtain the AAV plasmid vector pAAV2-LP15- Δ C4ATP7B (see FIG. 5) containing the Δ C4ATP7B gene expression cassette.

Example 2 recombinant AAV Virus preparation and assay

As a reference (Xiao X, et al J Virol, 1998;72(3): 2224) -2232), AAV is packaged by a three-plasmid packaging system and packaged by cesium chloride density gradient centrifugation for purification. Briefly, AAV vector plasmids (referred to as plasmids containing the word "pAAV 2" in the present invention) (pAAV 2-LP15-EGFP, pAAV2-LP15-ATP7B or pAAV2-LP15- Δ C4ATP 7B), helper plasmids (pHelper), and Rep and Cap protein expression plasmids (referred to as plasmids containing the word "pAAV-R" in the present invention) of AAV were mixed uniformly at a molar ratio of 1:1:1, HEK293 cells were transfected by a calcium phosphate method, and after 48h of transfection, the cells and culture supernatants were harvested, and recombinant AAV viruses were isolated and purified by cesium chloride density gradient centrifugation. Packaging and purifying to obtain 6 recombinant viruses. In particular to rAAV8-LP15-EGFP, rAAV8-LP15-ATP7B, rAAV8-LP15- Δ C4ATP7B, rAAV3B-LP15- Δ C4ATP7B, rAAV5-LP15- Δ C4ATP7B and rAAV9-LP15- Δ C4ATP 7B.

And determining the genome titer of the prepared AAV by a quantitative PCR method. The quantitative PCR detection process uses TaqMan probe method. Packaging to obtain AAV virus for AAV virus, and determining genome titer with AAV vector universal quantitative PCR detection primer and probe. The sequence of the primer probe for quantitative PCR detection is described in the literature (Aurnhammer C, et al Hum Gene methods 2012;23(1): 18-28.). Specific sequence information is as follows,

ITR-F: 5’-GGAACCCCTAGTGATGGAGTT-3’ (SEQ ID NO.13)

ITR-R: 5’-CGGCCTCAGTGAGCGA-3’ (SEQ ID NO.14)

ITR-P: 5’-CACTCCCTCTCTGCGCGCTCG-3’ (SEQ ID NO.15)

wherein ITR-F and ITR-R are primers, and ITR-P is a probe. The 5 'end of the probe is marked by FAM fluorescent protein, and the 3' end is connected with BlackBerry query. Primers and probes were synthesized by thermolfisher Scientific. The ITR-F and ITR-R are used as primers to specifically amplify a 62bp fragment in the ITR of the packaging virus, a TaqMan probe combination method is adopted, 1 mu g/mu l of pAAV2-LP15-EGFP plasmid and a sample diluted by 10 times of gradient are used as standard substances, Premix Ex Taq (ProbeqPCR) reagent (Takara, Dalian, China) is applied, and a fluorescent quantitative PCR instrument (model: ABI 7500 fast, ABI) is used for detecting the virus genome titer. The procedures are described in Premix Ex Taq (Probe qPCR) reagent Specification. Methods for the treatment of viruses are described in the literature (Aurnhammer C, et al Hum Gene their methods, 2012;23(1): 18-28.).

Example 3 different doses of rAAV8-LP15-ATP7B Virus injection model animal experimental results

By taking rAAV8-LP15-EGFP virus as a control, injecting the recombinant AAV8 virus carrying an ATP7B expression frame into a hepatolenticular degeneration model mouse through tail vein with high, medium and low (1E +10 vg/mouse, 3E +10 vg/mouse and 9E +10 vg/mouse), and detecting transaminase, copper ion content in urine and copper ion content in liver tissues and ATP7B mRNA expression level in liver so as to verify the effectiveness of rAAV8-LP15-ATP7B and the influence of different doses on the effectiveness. Wild type C57BL/6 mice were also set as controls to observe the changes in transaminase levels in normal mice.

Hepatolenticular degeneration mouse atp7b^-/-Prepared by mating of breeding mice prepared by a hepatolenticular degeneration model. Stock mice were purchased from Jackson Laboratory, usa as heterozygous B6; 129S1-Atp7b^tm1TcgFemale and male mice, commercial 032624,/LtsnkJ. The prepared model is identified by a PCR method, and the identification method and the identification process are shown in a Jackson laboratory website.

First, rAAV8-LP15-ATP7B was injected into hepatolenticular degeneration model mice (ATP 7 b) via tail vein at 3 doses of high, medium and low (1E +10 vg/mouse, 3E +10 vg/mouse, and 9E +10 vg/mouse)^-/-Mice) were injected with 5 mice per dose. Wild-type mice without virus injection are used as positive control, rAAV8-LP15-EGFP virus (9E +10 vg/mouse) is used as negative control, tail vein blood collection is carried out at different time points (4 weeks, 8 weeks, 12 weeks and 24 weeks after virus injection), serum is separated, the level of glutamic-pyruvic transaminase in serum is determined by a kit, and the detection process is referred to kit instructions. The results are shown in FIG. 6. From the results shown in fig. 6, the glutamic-pyruvic transaminase activity of the model mice injected with the rAAV8-LP15-ATP7B virus was significantly reduced compared to the group of model mice injected with the rAAV8-LP15-EGFP virus at the same time point, and the glutamic-pyruvic transaminase activity was gradually approached or reached to the glutamic-pyruvic transaminase level of the wild-type mice not injected with the virus as the reduction degree of the glutamic-pyruvic transaminase activity was higher with the increase of the injection dose. Moreover, the level of glutamic-pyruvic transaminase of mice injected with rAAV8-LP15-EGFP virus model gradually increases with time, but the level of glutamic-pyruvic transaminase of mice injected with rAAV8-LP15-ATP7B virus does not change obviously with time.

Next, rAAV8-LP15-ATP7B A hepatolenticular degeneration model mouse (ATP 7 b) was injected via tail vein at 3 doses of high, medium and low (1E +10 vg/mouse, 3E +10 vg/mouse and 9E +10 vg/mouse)^-/-Mice) were injected with 5 mice per dose. Wild-type mice without virus injection are positive controls, rAAV8-LP15-EGFP virus (9E +10 vg/mouse) is negative controls, urine is collected from the mice within 24h at different time points after virus injection (4 weeks, 8 weeks, 12 weeks and 24 weeks after virus injection), the total amount of copper ions in the collected urine is determined, and the determination method and the operation process are described in the literature (Murillo O, et al. Journal of hepatology. 2016; 64(2): 419-426.). The results are shown in FIG. 7. From the results of FIG. 7, it can be seen that the content of copper ions in urine of mice injected with rAAV8-LP15-EGFP virus model gradually increases with time. At the same time point, the urine of the mouse injected with the rAAV8-LP15-ATP7B virus model has obviously lower copper ion content than that of the mouse injected with the rAAV8-LP15-EGFP model, and the content of the copper ions in the urine gradually decreases along with the increase of the injection dosage until the level of the copper ions in the urine of the wild-type mouse is approached or reached. Furthermore, as the time after virus injection is prolonged, the copper ion content in urine is not obviously changed.

Then, rAAV8-LP15-ATP7B was injected into hepatolenticular degeneration model mice (ATP 7 b) via tail vein at 3 doses of high, medium and low (1E +10 vg/mouse, 3E +10 vg/mouse, and 9E +10 vg/mouse)^-/-Mice) were injected with 5 mice per dose. Wild-type mice without virus injection were used as positive control, rAAV8-LP15-EGFP virus (9E +10 vg/mouse) was used as negative control, 24 weeks after virus injection, mice were sacrificed, livers were separated, total copper ion amount in dried liver tissue was determined, and the determination method and procedure were described in the literature (Murillo O, et al, Journal of hepatology. 2016; 64(2): 419-426.). The detection results are shown in fig. 8. From the results shown in FIG. 8, it can be seen that the liver of the model mouse injected with rAAV8-LP15-EGFP virus has significantly higher copper ion content than that of the model mouse injected with rAAV8-LP15-ATP7B virus and the wild-type mouse not injected with the virus. And the content of copper ions in the liver of the rAAV8-LP15-ATP7B virus injection model mouse is gradually reduced along with the increase of the virus injection dosage until the wild mouse is close to or reachedLevel of the adult mouse.

Finally, rAAV8-LP15-ATP7B was injected into hepatolenticular degeneration model mice (ATP 7 b) via tail vein at 3 doses of high, medium and low (1E +10 vg/mouse, 3E +10 vg/mouse, and 9E +10 vg/mouse)^-/-Mouse). rAAV8-LP15-EGFP virus (9E +10 vg/mouse) is injected as a negative control, 24 weeks after virus injection, mice are killed, livers are separated, total RNA is extracted by TRIzol reagent (Invitrogen, 15596018), and ATP7B mRNA content in 100ng of total RNA is determined by reverse transcription quantitative PCR (RT-PCR) and used for representing the expression level of the transduced ATP7B gene in the livers of the mice. The RNA standard substance for artificial synthesis quantitative PCR detection is named as ATP7B-SR, and the sequence information is detailed in SEQ ID NO. 16. RNA standards were synthesized by ThermoFisher Scientific, 100nt in length and diluted to 1X 10 with RNase-free water¹⁰copies/. mu.L for use. The primers for quantitative PCR detection are ATP7B-Q-F (SEQ ID NO. 17) and ATP7B-Q-R (SEQ ID NO. 18), and the probe is ATP7B-Q-P (SEQ ID NO. 19). The primer probe is synthesized by ThermoFisher Scientific, the 5 'end of the probe is marked by FAM fluorescent protein, and the 3' end of the probe is connected with BlackBerrysequencher. The details of the primers and probes are as follows.

ATP7B-Q-F：5’GACACATGAAGCCACTGACC3’ (SEQ ID NO.17)

ATP7B-Q-R：5’CTGTCCCTCCACCTATCGTC3’ (SEQ ID NO.18)

ATP7B-Q-P：5’TGCCGATGTGCACGCTCACCTGA3’ (SEQ ID NO.19)

The copy number of ATP7B mRNA in the total RNA is detected by adopting a one-step method. Using ATP7B-Q-F and ATP7B-Q-R as primers to specifically perform reverse transcription amplification on a fragment with the length of 70bp in an ATP7B mRNA (messenger RNA) sequence by adopting a TaqMan probe binding method and using the TaqMan probe binding method to perform amplification on the fragment with the length of 1 multiplied by 10⁸copies/. mu.l of synthetic ATP7B-SR RNA and 10-fold gradient diluted sample were used as standards, and One Step PrimeScript was applied^TMRT-PCR Kit (Perfect Real Time) reagent (cat # RR 064A) (Takara, Dalian, China) was used to detect the ATP7B mRNA content in total RNA using a fluorescent quantitative PCR instrument (model: ABI 7500 fast, ABI) to indicate ATP7B expression. The specific detection process is shown in One Step PrimeScript^TMRT-PCR Kit (Perfect Real Time) reagent instructions. The results are shown in FIG. 9As shown.

From the results in fig. 9, ATP7b mrna was not detected in the model mice injected with rAAV8-LP15-EGFP virus, indicating that ATP7B gene expression was not present in the model mice, which is consistent with the expectation. In contrast, ATP7B mRNA expression is detected in the liver of a mouse model injected with rAAV8-LP15-ATP7B, and the expression detection value of ATP7B mRNA is gradually increased along with the increase of virus injection dose, which indicates that the mouse model injected with rAAV8-LP15-ATP7B virus can effectively express ATP7B mRNA in the liver.

Example 4 results of animal experiments using different doses of rAAV8-LP15- Δ C4ATP7B virus injection models

rAAV8-LP15-ATP7B virus is taken as a control, recombinant AAV8 virus rAAV8-LP15- Δ C4ATP7B carrying a truncated ATP7B expression frame is injected into a hepatolenticular degeneration model mouse through tail veins at 3 doses of high, medium and low (1E +10 vg/mouse, 3E +10 vg/mouse and 9E +10 vg/mouse), transaminase, copper ion content in urine, copper ion content in liver tissues, ATP7B mRNA or Δ C4ATP7BmRNA expression level in liver are detected, and the effectiveness and the influence of different doses on the effectiveness are verified by rAAV8-LP15- Δ C4ATP 7B.

First, rAAV8-LP15- Δ C4ATP7B was injected into hepatolenticular degeneration model mice (ATP 7 b) via tail vein at 3 doses of high, medium and low (1E +10 vg/mouse, 3E +10 vg/mouse, and 9E +10 vg/mouse)^-/-Mice) were injected with 5 mice per dose. Model mice injected with high, medium and low (1E +10 vg/mouse, 3E +10 vg/mouse and 9E +10 vg/mouse) doses of rAAV8-LP15-ATP7B virus were set as experimental controls. At various time points (4 weeks, 8 weeks, 12 weeks and 24 weeks after injection of the virus), tail vein blood was collected, serum was isolated, glutamic-pyruvic transaminase levels in serum were determined using a kit (Merck), and the assay procedure was described in the kit's instructionsA book. The results are shown in FIG. 10. From the results in fig. 10, it can be seen that the glutamate pyruvate transaminase activity of the model mice injected with the rAAV8-LP15- Δ C4ATP7B virus at the same time point was not significantly different, but was lower, and the glutamate pyruvate transaminase activity was lower as the injection dose was increased, compared to the model mice injected with the rAAV8-LP15-ATP7B virus at the same dose point. Furthermore, the glutamic-pyruvic transaminase activity of mice injected with rAAV8-LP15- Δ C4ATP7B or rAAV8-LP15-ATP7B virus is not obviously changed along with the increase of time.

Next, rAAV8-LP15- Δ C4ATP7B was injected via tail vein into hepatolenticular degeneration model mice (ATP 7 b) at 3 doses of high, medium and low (1E +10 vg/mouse, 3E +10 vg/mouse, and 9E +10 vg/mouse)^-/-Mice) were injected with 5 mice per dose. Model mice injected with high, medium and low (1E +10 vg/mouse, 3E +10 vg/mouse and 9E +10 vg/mouse) doses of rAAV8-LP15-ATP7B virus were set as experimental controls. At various time points after virus injection (4 weeks, 8 weeks, 12 weeks and 24 weeks after virus injection), urine from the mice was collected for 24 hours, and the total amount of copper ions in the collected urine was determined, as well as the procedure described in the literature (Murillo O, et al. Journal of hepatology. 2016; 64(2): 419-426.). The results are shown in FIG. 11. From the results in FIG. 11, it can be seen that the urine copper ion content of the model mice injected with the rAAV8-LP15- Δ ATP7B virus was lower than that of the model mice injected with the rAAV8-LP15-ATP7B virus at the same time point. Furthermore, as the time after virus injection is prolonged, the copper ion content in urine is not obviously changed.

Then, rAAV8-LP15- Δ C4ATP7B was injected via tail vein into hepatolenticular degeneration model mice (ATP 7 b) at 3 doses of high, medium and low (1E +10 vg/mouse, 3E +10 vg/mouse, and 9E +10 vg/mouse)^-/-Mice) were injected with 5 mice per dose. Model mice injected with high, medium and low (1E +10 vg/mouse, 3E +10 vg/mouse and 9E +10 vg/mouse) doses of rAAV8-LP15-ATP7B virus were set as experimental controls. 24 weeks after virus injection, mice were sacrificed, livers were isolated, total copper ion content in dried liver tissue was determined, and the method and procedure were described in the literature (Murillo O, et al, Journal of hepatology, 2016; 64(2): 419-426). The detection results are shown in fig. 12. From the figure12, it is clear that the liver of the model mouse injected with the rAAV8-LP15- Δ C4ATP7B virus has lower copper ion content compared with the model mouse injected with the rAAV8-LP15-ATP7B virus at the same dose. And the content of copper ions is gradually reduced along with the increase of the virus dose of rAAV8-LP15- Δ C4ATP 7B.

Finally, rAAV8-LP15- Δ C4ATP7B was injected caudally into a hepatolenticular degeneration model mouse (ATP 7 b) with high, medium and low levels (1E +10 vg/mouse, 3E +10 vg/mouse, and 9E +10 vg/mouse)^-/-Mice) were injected with 5 mice per dose. Model mice injected with high, medium and low (1E +10 vg/mouse, 3E +10 vg/mouse and 9E +10 vg/mouse) doses of rAAV8-LP15-ATP7B virus were set as experimental controls. 24 weeks after virus injection, mice were sacrificed, livers were separated, total RNA was extracted using TRIzol reagent (Invitrogen, 15596018), and ATP7B mRNA or Δ C4ATP7B mRNA content in 100ng of total RNA was determined by reverse transcription quantitative PCR to indicate the expression level of transduced ATP7B gene or Δ C4ATP7B gene in the livers of mice. ATP7B mRNA or Δ C4ATP7B mRNA detection used the same standards, primer probes, and reagents and methods. The RNA standard substance for artificial synthesis quantitative PCR detection is named as ATP7B-SR, and the sequence information is detailed in SEQ ID NO. 16. RNA standards were synthesized by Thermo Fisher scientific, 100nt in length and diluted to 1X 10 with RNase-free water¹⁰copies/. mu.L for use. The primers for quantitative PCR detection are ATP7B-Q-F (SEQ ID NO. 17) and ATP7B-Q-R (SEQ ID NO. 18), and the probe is ATP7B-Q-P (SEQ ID NO. 19). The primer probe is synthesized by ThermoFisher scientific, the 5 'end of the probe is marked by FAM fluorescent protein, and the 3' end of the probe is connected with BlackBerry query. The details of the primers and probes are as follows.

ATP7B-Q-F：5’GACACATGAAGCCACTGACC3’ (SEQ ID NO.17)

ATP7B-Q-R：5’CTGTCCCTCCACCTATCGTC3’ (SEQ ID NO.18)

ATP7B-Q-P：5’TGCCGATGTGCACGCTCACCTGA3’ (SEQ ID NO.19)

The copy number of ATP7B mRNA or Δ C4ATP7B mRNA in total RNA is detected by a one-step method. ATP7B-Q-F and ATP7B-Q-R are used as primers to specifically perform reverse transcription amplification on the medium and long lengths of ATP7B mRNA or delta C4ATP7B mRNA (messenger RNA)The degree is 70bp fragment, adopting TaqMan probe binding method, and the detection is performed at 1 × 10⁸copies/. mu.l of synthetic ATP7B-SR RNA and 10-fold gradient diluted sample were used as standards, and One Step PrimeScript was applied^TMRT-PCRKit (perfect Real time) reagent (cat # RR 064A) (Takara, Dalian, China) used a fluorescence quantitative PCR instrument (model: ABI 7500 fast, ABI) to detect the ATP7B mRNA or Δ C4ATP7B mRNA content in total RNA, which indicates the expression of ATP7B or Δ C4ATP 7B. The specific detection process is shown in One Step PrimeScript^TMRT-PCR Kit (PerfectReal Time) reagent instructions. The results are shown in FIG. 13.

From the results in fig. 13, it is understood that Δ C4ATP7B mRNA expression was detected in the liver of the rAAV8-LP15- Δ C4ATP 7B-injected model mouse, as with the rAAV8-LP15-ATP7B virus, and the Δ C4ATP7B mRNA expression detection value gradually increased with the increase in the virus injection dose, suggesting that the rAAV8-LP15- Δ C4ATP 7B-virus-injected model mouse can efficiently produce ATP7B Δ C4mRNA in the liver.

Example 5 different serotypes of AAV carry Δ C4ATP7B Gene expression cassette injection model animal test results

By taking rAAV8-LP15- Δ C4ATP7B virus as a control, recombinant AAV3B, AAV5 or AAV9 virus carrying the Δ C4ATP7B expression cassette is injected into a hepatolenticular degeneration model mouse through tail vein at the dose of 3E +10 vg/mouse, and transaminase, copper ion content in urine and copper ion content in liver tissues and the expression level of Δ C4ATP7B mRNA in liver are detected in serum to verify the effectiveness of the different AAV serotypes carrying the Δ C4ATP7B expression cassette.

First, rAAV3B-LP15- Δ C4ATP7B, rAAV5-LP15- Δ C4ATP7B or rAAV9-LP15- Δ C4ATP7B were injected via caudal vein into a hepatolenticular degeneration model mouse (ATP 7 b) at a dose of 3E +10 vg/mouse^-/-Mouse), each time5 mice were injected with one dose. Meanwhile, rAAV8-LP15- Δ C4ATP7B virus (3E +10 vg/mouse) is injected as an experimental control. At various time points after injection of the virus (4 weeks, 8 weeks, 12 weeks and 24 weeks after injection of the virus), tail vein blood was collected, serum was isolated, and glutamate pyruvate transaminase levels in serum were determined using a kit (Merck), the assay procedure being described in the kit instructions. The results are shown in FIG. 14. From the results shown in FIG. 14, it was found that, compared with the group of model mice injected with rAAV8-LP15- Δ C4ATP7B virus, none of the glutamate pyruvate transaminase activities of the model mice injected with rAAV3B-LP15- Δ C4ATP7B, rAAV5-LP15- Δ C4ATP7B, or rAAV9-LP15- Δ C4ATP7B virus were significantly different from each other at the same time point, and the level of glutamate pyruvate transaminase of the model mice injected with virus was not significantly changed with the increase of time.

Next, a hepatolenticular degeneration model mouse (ATP 7 b) was injected into the caudal vein of 3E +10 vg/mouse at the dose of 3E +10 vg/mouse for rAAV3B-LP15- Δ C4ATP7B, rAAV5-LP15- Δ C4ATP7B, or rAAV9-LP15- Δ C4ATP7B^-/-Mice) were injected with 5 mice per dose. Meanwhile, rAAV8-LP15- Δ C4ATP7B virus (3E +10 vg/mouse) is injected as an experimental control. At various time points after virus injection (4 weeks, 8 weeks, 12 weeks and 24 weeks after virus injection), urine from the mice was collected for 24 hours, and the total amount of copper ions in the collected urine was determined, as well as the procedure described in the literature (Murillo O, et al. Journal of hepatology. 2016; 64(2): 419-426.). The results are shown in FIG. 15. From the results in fig. 15, it was found that the urine copper ion content of the model mice injected with rAAV3B-LP15- Δ C4ATP7B, rAAV5-LP15- Δ C4ATP7B, or rAAV9-LP15- Δ C4ATP7B virus was not significantly different from that of the group of model mice injected with rAAV8-LP15- Δ C4ATP7B virus. Furthermore, as the time after virus injection is prolonged, the copper ion content in urine is not obviously changed.

Then, rAAV3B-LP15- Δ C4ATP7B, rAAV5-LP15- Δ C4ATP7B or rAAV9-LP15- Δ C4ATP7B were injected via caudal vein into a hepatolenticular degeneration model mouse (ATP 7 b) at a dose of 3E +10 vg/mouse^-/-Mice) were injected with 5 mice per dose. Meanwhile, rAAV8-LP15- Δ C4ATP7B virus (3E +10 vg/mouse) is injected as an experimental control. 24 weeks after injection of virus, mice were sacrificed, livers were isolated, and dry liver tissue was determinedThe total amount of copper ions in (1), the determination method and the operation process are described in the literature (Murillo O, et al. Journal of biology. 2016; 64(2): 419-426). The detection results are shown in fig. 16. From the results shown in FIG. 16, it was found that there was no significant difference in the content of copper ions in the liver of the model mice injected with the rAAV3B-LP15- Δ C4ATP7B, rAAV5-LP15- Δ C4ATP7B, or rAAV9-LP15- Δ C4ATP7B virus, compared with the group of model mice injected with the rAAV8-LP15- Δ C4ATP7B virus. Moreover, the content of copper ions in the liver did not change significantly with the time after virus injection.

Finally, rAAV3B-LP15- Δ C4ATP7B, rAAV5-LP15- Δ C4ATP7B or rAAV9-LP15- Δ C4ATP7B are injected into a hepatolenticular degeneration model mouse (ATP 7 b) through tail vein at the dose of 3E +10 vg/mouse^-/-Mice) were injected with 5 mice per dose. Meanwhile, rAAV8-LP15- Δ C4ATP7B virus (3E +10 vg/mouse) is injected as an experimental control. 24 weeks after virus injection, mice were sacrificed, livers were isolated, total RNA was extracted using TRIzol reagent (Invitrogen, 15596018), and the Δ C4ATP7B mRNA content in 100ng of total RNA was determined by reverse transcription quantitative PCR to indicate the expression level of transduced Δ C4ATP7B gene in mouse livers. The RNA standard substance for artificial synthesis quantitative PCR detection is named as ATP7B-SR, and the sequence information is detailed in SEQ ID NO. 16. RNA standards were synthesized by ThermoFisher Scientific, 100nt in length and diluted to 1X 10 with RNase-free water¹⁰copies/. mu.L for use. The primers for quantitative PCR detection are ATP7B-Q-F (SEQ ID NO. 17) and ATP7B-Q-R (SEQ ID NO. 18), and the probe is ATP7B-Q-P (SEQ ID NO. 19). The primer probe is synthesized by ThermoFisher scientific, the 5 'end of the probe is marked by FAM fluorescent protein, and the 3' end of the probe is connected with BlackBerry query. The details of the primers and probes are as follows.

ATP7B-Q-F：5’GACACATGAAGCCACTGACC3’ (SEQ ID NO.17)

ATP7B-Q-R：5’CTGTCCCTCCACCTATCGTC3’ (SEQ ID NO.18)

ATP7B-Q-P：5’TGCCGATGTGCACGCTCACCTGA3’ (SEQ ID NO.19)

The copy number of the Δ C4ATP7B mRNA in the total RNA was detected by a "one-step method". Reverse transcription amplification of medium and long delta C4ATP7B mRNA (messenger RNA) sequence by taking ATP7B-Q-F and ATP7B-Q-R as primersThe degree is 70bp fragment, adopting TaqMan probe binding method, and the detection is performed at 1 × 10⁸copies/. mu.l of synthetic ATP7B-SR RNA and 10-fold gradient diluted sample were used as standards, and One Step PrimeScript was applied^TMRT-PCR Kit (Perfect Real Time) reagent (cat # RR 064A) (Takara, Dalian, China) was used to detect the amount of Δ C4ATP7B mRNA in total RNA using a fluorescent quantitative PCR instrument (model: ABI 7500 fast, ABI) to indicate ATP7B expression. The specific detection process is described in One StepPrimeScript^TMRT-PCR Kit (Perfect Real Time) reagent instructions. The results are shown in FIG. 17.

From the results shown in FIG. 17, it was found that Δ C4ATP7B mRNA was detected in total RNA of liver of model mice injected with rAAV3B-LP15- Δ C4ATP7B, rAAV5-LP15- Δ C4ATP7B, or rAAV9-LP15- Δ C4ATP7B virus, as in the model mice injected with rAAV8-LP15- Δ C4ATP7B virus. The AAV viruses prepared by different serotypes of AAV carrying the gene expression frame of the delta C4ATP7B can effectively transduce mouse liver to express and generate the delta C4ATP7B mRNA.

Sequence listing

<110> Beijing brocade basket Gene science and technology Co., Ltd

<120> AAV vector carrying ATP7B gene expression cassette and variant and application

<160>19

<170>SIPOSequenceListing 1.0

<210>1

<211>352

<212>DNA

<213>2 Ambystoma laterale x Ambystoma jeffersonianum

<400>1

gttaattttt aaaaagcaag tggcccttgg cagcatctgt ttgctctggt taataatctc 60

aggagcacaa acattcccag gagaagaaat caacatcctg gacttatcct ctgggcctaa 120

gtatttagtt tggttagtaa ttactaaaca ctgagaacgc caatgaaata caaagatgag 180

tctagttaat aatctacaat tattggttaa agaagtatat tagtgctaat ttccctccgt 240

ttgtcctagc ttttctcttc tgtcaacccc acacgccttt ggcaggtaag ttggcgccgt 300

ttaagggatg gttggttggt ggggtattaa tgtttaatta ccttttttac ag 352

<210>2

<211>59

<212>DNA

<213>2 Ambystoma laterale x Ambystoma jeffersonianum

<400>2

tcgagaggcc taataaagag ctcagatgca tcgatcagag tgtgttggtt ttttgtgtg 59

<210>3

<211>4395

<212>DNA

<213>2 Ambystoma laterale x Ambystoma jeffersonianum

<400>3

atgcccgaac aggaaagaca gatcaccgca agggaaggag caagtcgcaa gattctgagc 60

aaactgagcc tgccaacccg agcctgggag cccgccatga agaagagctt cgcctttgac 120

aacgtgggat acgagggcgg cctggatggc ctgggaccta gctcccaggt ggccacctcc 180

accgtgagaa tcctgggcat gacatgccag agctgcgtga agtccatcga ggacagaatc 240

tctaatctga agggcatcat ctctatgaag gtgagcctgg agcagggctc cgccaccgtg 300

aagtatgtgc cttctgtggt gtgcctgcag caggtgtgcc accagatcgg cgatatgggc 360

ttcgaggcca gcatcgcaga gggcaaggca gcatcctggc cttctcggag cctgccagca 420

caggaggcag tggtgaagct gagagtggaa ggaatgacct gtcagagctg cgtgagcagc 480

atcgagggca aggtgaggaa gctgcagggc gtggtgcgcg tgaaggtgtc cctgtctaac 540

caggaggccg tgatcaccta ccagccctat ctgatccagc ctgaggacct gagggatcac 600

gtgaatgaca tgggcttcga ggccgccatc aagagcaagg tggcaccact gtccctggga 660

ccaatcgaca tcgagcggct gcagtccacc aacccaaagc ggcccctgtc ctctgccaac 720

cagaacttca acaattctga gacactggga caccagggca gccacgtggt gaccctgcag 780

ctgaggatcg acggcatgca ctgcaagagc tgcgtgctga acatcgagga gaatatcggc 840

cagctgctgg gcgtgcagtc catccaggtg tctctggaga acaagacagc ccaggtgaag 900

tacgatcctt cttgcaccag cccagtggcc ctgcagaggg caatcgaggc cctgccccct 960

ggcaatttca aggtgtccct gcctgacgga gcagagggct ctggcaccga tcaccggagc 1020

agcagcagcc actccccagg ctctccacca aggaaccagg tgcagggcac atgttctacc 1080

acactgatcg caatcgcagg aatgacctgc gcaagctgcg tgcactccat cgagggcatg 1140

atcagccagc tggagggcgt gcagcagatc agcgtgtccc tggcagaggg caccgcaaca 1200

gtgctgtaca atcccagcgt gatctcccct gaggagctga gggcagcaat cgaggatatg 1260

ggatttgagg ccagcgtggt gtctgagagc tgctccacaa accccctggg caatcactct 1320

gccggcaaca gcatggtgca gaccacagac ggcaccccta caagcgtgca ggaggtggca 1380

ccacacaccg gccggctgcc agcaaatcac gcaccagata tcctggccaa gtctccccag 1440

agcacaagag ccgtggcccc tcagaagtgt tttctgcaga tcaagggcat gacctgcgcc 1500

tcctgcgtga gcaacatcga gcggaatctg cagaaggagg caggcgtgct gtccgtgctg 1560

gtggccctga tggcaggcaa ggccgagatc aagtacgacc ctgaagtgat ccagccactg 1620

gagatcgccc agttcatcca ggatctgggc tttgaggccg ccgtgatgga ggactatgcc 1680

ggcagcgatg gcaacatcga gctgaccatc acaggcatga cctgcgcctc ttgcgtgcac 1740

aacatcgaga gcaagctgac cagaacaaat ggcatcacat acgcctctgt ggccctggcc 1800

accagcaagg ccctggtgaa gttcgacccc gagatcatcg gccctaggga tatcatcaag 1860

atcatcgagg agatcggctt tcacgcctcc ctggcccagc gcaacccaaa tgcccaccac 1920

ctggaccaca agatggagat caagcagtgg aagaagtcct tcctgtgctc tctggtgttt 1980

ggcatccccg tgatggccct gatgatctac atgctgatcc cttccaacga gccacaccag 2040

tctatggtgc tggatcacaa catcatccct ggcctgagca tcctgaatct gatcttcttt 2100

atcctgtgca cattcgtgca gctgctgggc ggctggtact tttatgtgca ggcttacaag 2160

tccctgcggc accggagcgc caatatggac gtgctgatcg tgctggccac cagcatcgcc 2220

tacgtgtatt ccctggtcat cctggtggtg gcagtggcag agaaggcaga gcggtccccc 2280

gtgaccttct ttgatacacc tccaatgctg ttcgtgttta tcgccctggg cagatggctg 2340

gagcacctgg ccaagagcaa gacctccgag gccctggcca agctgatgag cctgcaggcc 2400

acagaggcca ccgtggtgac actgggcgag gacaacctga tcatcaggga ggagcaggtg 2460

cctatggagc tggtgcagcg cggcgatatc gtgaaggtgg tgccaggcgg caagttccca 2520

gtggacggca aggtgctgga gggcaataca atggccgatg agagcctgat caccggcgag 2580

gccatgcctg tgaccaagaa gccaggctct acagtgatcg caggcagcat caacgcacac 2640

ggctccgtgc tgatcaaggc cacccacgtg ggcaatgaca ccacactggc ccagatcgtg 2700

aagctggtgg aggaggccca gatgtccaag gcccctatcc agcagctggc cgatcggttc 2760

tccggctact tcgtgccctt catcatcatc atgtctaccc tgacactggt ggtgtggatc 2820

gtgatcggct tcatcgactt tggcgtggtg cagaggtatt ttcccaaccc taataagcac 2880

atcagccaga ccgaagtgat catccgcttc gcctttcaga ccagcatcac agtgctgtgc 2940

atcgcatgcc catgttccct gggcctggca accccaacag ccgtgatggt gggcacagga 3000

gtggcagcac agaacggcat cctgatcaag ggcggcaagc ccctggagat ggcccacaag 3060

atcaagaccg tgatgtttga caagaccggc acaatcaccc acggcgtgcc cagagtgatg 3120

agagtgctgc tgctgggcga tgtggccaca ctgcctctga gaaaggtgct ggcagtggtg 3180

ggcaccgcag aggccagcag cgagcaccca ctgggcgtgg ccgtgacaaa gtactgcaag 3240

gaggagctgg gcacagagac actgggctat tgtaccgact tccaggccgt gcccggatgc 3300

ggaatcggct gtaaggtgag caacgtggag ggcatcctgg cacactccga gcggcccctg 3360

agcgcccctg catcccacct gaatgaggca ggctctctgc cagcagagaa ggacgccgtg 3420

cctcagacct tcagcgtgct gatcggcaac agagagtggc tgcggagaaa tggcctgacc 3480

atcagctccg acgtgtccga tgccatgaca gatcacgaga tgaagggcca gaccgcaatc 3540

ctggtggcaa tcgacggcgt gctgtgcggc atgatcgcca tcgccgatgc agtgaagcag 3600

gaggccgccc tggcagtgca caccctgcag agcatgggcg tggacgtggt gctgatcacc 3660

ggcgataaca ggaagacagc aagggcaatc gcaacccaag tgggcatcaa taaggtgttc 3720

gccgaggtgc tgccttccca caaggtggcc aaggtgcagg agctgcagaa caagggcaag 3780

aaggtggcca tggtgggcga cggcgtgaat gattctccag ccctggcaca ggcagacatg 3840

ggagtggcaa tcggcacagg caccgacgtg gcaatcgagg cagcagatgt ggtgctgatc 3900

aggaatgacc tgctggatgt ggtggcctct atccacctga gcaagcggac cgtgaggcgc 3960

atcagaatca acctggtgct ggccctgatc tacaatctgg tgggcatccc aatcgcagca 4020

ggcgtgttta tgccaatcgg catcgtgctg cagccatgga tgggctctgc cgcaatggca 4080

gcctctagcg tgagcgtggt gctgtcctct ctgcagctga agtgctacaa gaagccagac 4140

ctggagcggt acgaggcaca ggcacacgga cacatgaagc cactgaccgc ctctcaggtg 4200

agcgtgcaca tcggcatgga cgataggtgg agggacagcc caagggcaac accatgggat 4260

caggtgtcct acgtgagcca ggtgagcctg agcagcctga cctccgataa gccctcccgc 4320

cactctgccg ccgccgacga cgacggggac aagtggagcc tgctgctgaa cgggagagac 4380

gaggaacagt acatt 4395

<210>4

<211>3309

<212>DNA

<213>2 Ambystoma laterale x Ambystoma jeffersonianum

<400>4

atgcccgaac aggaaagaca gatcaccgca agggaaggag caagtcgcaa gattctgagc 60

aaactgagcc tgccaacccg agcctgggag cccgccatga agaagagctt cgcctttgac 120

aacgtgggat acgagggcgg cctggatggc ctgggaccta gctcccaggt ggccacctcc 180

accgtgagag tggtgtctga gagctgctcc acaaaccccc tgggcaatca ctctgccggc 240

aacagcatgg tgcagaccac agacggcacc cctacaagcg tgcaggaggt ggcaccacac 300

accggccggc tgccagcaaa tcacgcacca gatatcctgg ccaagtctcc ccagagcaca 360

agagccgtgg cccctcagaa gtgttttctg cagatcaagg gcatgacctg cgcctcctgc 420

gtgagcaaca tcgagcggaa tctgcagaag gaggcaggcg tgctgtccgt gctggtggcc 480

ctgatggcag gcaaggccga gatcaagtac gaccctgaag tgatccagcc actggagatc 540

gcccagttca tccaggatct gggctttgag gccgccgtga tggaggacta tgccggcagc 600

gatggcaaca tcgagctgac catcacaggc atgacctgcg cctcttgcgt gcacaacatc 660

gagagcaagc tgaccagaac aaatggcatc acatacgcct ctgtggccct ggccaccagc 720

aaggccctgg tgaagttcga ccccgagatc atcggcccta gggatatcat caagatcatc 780

gaggagatcg gctttcacgc ctccctggcc cagcgcaacc caaatgccca ccacctggac 840

cacaagatgg agatcaagca gtggaagaag tccttcctgt gctctctggt gtttggcatc 900

cccgtgatgg ccctgatgat ctacatgctg atcccttcca acgagccaca ccagtctatg 960

gtgctggatc acaacatcat ccctggcctg agcatcctga atctgatctt ctttatcctg 1020

tgcacattcg tgcagctgct gggcggctgg tacttttatg tgcaggctta caagtccctg 1080

cggcaccgga gcgccaatat ggacgtgctg atcgtgctgg ccaccagcat cgcctacgtg 1140

tattccctgg tcatcctggt ggtggcagtg gcagagaagg cagagcggtc ccccgtgacc 1200

ttctttgata cacctccaat gctgttcgtg tttatcgccc tgggcagatg gctggagcac 1260

ctggccaaga gcaagacctc cgaggccctg gccaagctga tgagcctgca ggccacagag 1320

gccaccgtgg tgacactggg cgaggacaac ctgatcatca gggaggagca ggtgcctatg 1380

gagctggtgc agcgcggcga tatcgtgaag gtggtgccag gcggcaagtt cccagtggac 1440

ggcaaggtgc tggagggcaa tacaatggcc gatgagagcc tgatcaccgg cgaggccatg 1500

cctgtgacca agaagccagg ctctacagtg atcgcaggca gcatcaacgc acacggctcc 1560

gtgctgatca aggccaccca cgtgggcaat gacaccacac tggcccagat cgtgaagctg 1620

gtggaggagg cccagatgtc caaggcccct atccagcagc tggccgatcg gttctccggc 1680

tacttcgtgc ccttcatcat catcatgtct accctgacac tggtggtgtg gatcgtgatc 1740

ggcttcatcg actttggcgt ggtgcagagg tattttccca accctaataa gcacatcagc 1800

cagaccgaag tgatcatccg cttcgccttt cagaccagca tcacagtgct gtgcatcgca 1860

tgcccatgtt ccctgggcct ggcaacccca acagccgtga tggtgggcac aggagtggca 1920

gcacagaacg gcatcctgat caagggcggc aagcccctgg agatggccca caagatcaag 1980

accgtgatgt ttgacaagac cggcacaatc acccacggcg tgcccagagt gatgagagtg 2040

ctgctgctgg gcgatgtggc cacactgcct ctgagaaagg tgctggcagt ggtgggcacc 2100

gcagaggcca gcagcgagca cccactgggc gtggccgtga caaagtactg caaggaggag 2160

ctgggcacag agacactggg ctattgtacc gacttccagg ccgtgcccgg atgcggaatc 2220

ggctgtaagg tgagcaacgt ggagggcatc ctggcacact ccgagcggcc cctgagcgcc 2280

cctgcatccc acctgaatga ggcaggctct ctgccagcag agaaggacgc cgtgcctcag 2340

accttcagcg tgctgatcgg caacagagag tggctgcgga gaaatggcct gaccatcagc 2400

tccgacgtgt ccgatgccat gacagatcac gagatgaagg gccagaccgc aatcctggtg 2460

gcaatcgacg gcgtgctgtg cggcatgatc gccatcgccg atgcagtgaa gcaggaggcc 2520

gccctggcag tgcacaccct gcagagcatg ggcgtggacg tggtgctgat caccggcgat 2580

aacaggaaga cagcaagggc aatcgcaacc caagtgggca tcaataaggt gttcgccgag 2640

gtgctgcctt cccacaaggt ggccaaggtg caggagctgc agaacaaggg caagaaggtg 2700

gccatggtgg gcgacggcgt gaatgattct ccagccctgg cacaggcaga catgggagtg 2760

gcaatcggca caggcaccga cgtggcaatc gaggcagcag atgtggtgct gatcaggaat 2820

gacctgctgg atgtggtggc ctctatccac ctgagcaagc ggaccgtgag gcgcatcaga 2880

atcaacctgg tgctggccct gatctacaat ctggtgggca tcccaatcgc agcaggcgtg 2940

tttatgccaa tcggcatcgt gctgcagcca tggatgggct ctgccgcaat ggcagcctct 3000

agcgtgagcg tggtgctgtc ctctctgcag ctgaagtgct acaagaagcc agacctggag 3060

cggtacgagg cacaggcaca cggacacatg aagccactga ccgcctctca ggtgagcgtg 3120

cacatcggca tggacgatag gtggagggac agcccaaggg caacaccatg ggatcaggtg 3180

tcctacgtga gccaggtgag cctgagcagc ctgacctccg ataagccctc ccgccactct 3240

gccgccgccg acgacgacgg ggacaagtgg agcctgctgc tgaacgggag agacgaggaa 3300

cagtacatt 3309

<210>5

<211>364

<212>DNA

<213>2 Ambystoma laterale x Ambystoma jeffersonianum

<400>5

ctcgaggtta atttttaaaa agcaagtggc ccttggcagc atctgtttgc tctggttaat 60

aatctcagga gcacaaacat tcccaggaga agaaatcaac atcctggact tatcctctgg 120

gcctaagtat ttagtttggt tagtaattac taaacactga gaacgccaat gaaatacaaa 180

gatgagtcta gttaataatc tacaattatt ggttaaagaa gtatattagt gctaatttcc 240

ctccgtttgt cctagctttt ctcttctgtc aaccccacac gcctttggca ggtaagttgg 300

cgccgtttaa gggatggttg gttggtgggg tattaatgtt taattacctt ttttacaggg 360

tacc 364

<210>6

<211>27

<212>DNA

<213>2 Ambystoma laterale x Ambystoma jeffersonianum

<400>6

ataggtaccg ccaccatggt gagcaag 27

<210>7

<211>27

<212>DNA

<213>2 Ambystoma laterale x Ambystoma jeffersonianum

<400>7

gcggaattct tacttgtaca gctcgtc 27

<210>8

<211>1465

<212>PRT

<213>2 Ambystoma laterale x Ambystoma jeffersonianum

<400>8

Met Pro Glu Gln Glu Arg Gln Ile Thr Ala Arg Glu Gly Ala Ser Arg

1 5 10 15

Lys Ile Leu Ser Lys Leu Ser Leu Pro Thr Arg Ala Trp Glu Pro Ala

20 25 30

Met Lys Lys Ser Phe Ala Phe Asp Asn Val Gly Tyr Glu Gly Gly Leu

35 40 45

Asp Gly Leu Gly Pro Ser Ser Gln Val Ala Thr Ser Thr Val Arg Ile

50 55 60

Leu Gly Met Thr Cys Gln Ser Cys Val Lys Ser Ile Glu Asp Arg Ile

65 70 75 80

Ser Asn Leu Lys Gly Ile Ile Ser Met Lys Val Ser Leu Glu Gln Gly

85 90 95

Ser Ala Thr Val Lys Tyr Val Pro Ser Val Val Cys Leu Gln Gln Val

100 105 110

Cys His Gln Ile Gly Asp Met Gly Phe Glu Ala Ser Ile Ala Glu Gly

115 120 125

Lys Ala Ala Ser Trp Pro Ser Arg Ser Leu Pro Ala Gln Glu Ala Val

130135 140

Val Lys Leu Arg Val Glu Gly Met Thr Cys Gln Ser Cys Val Ser Ser

145 150 155 160

Ile Glu Gly Lys Val Arg Lys Leu Gln Gly Val Val Arg Val Lys Val

165 170 175

Ser Leu Ser Asn Gln Glu Ala Val Ile Thr Tyr Gln Pro Tyr Leu Ile

180 185 190

Gln Pro Glu Asp Leu Arg Asp His Val Asn Asp Met Gly Phe Glu Ala

195 200 205

Ala Ile Lys Ser Lys Val Ala Pro Leu Ser Leu Gly Pro Ile Asp Ile

210 215 220

Glu Arg Leu Gln Ser Thr Asn Pro Lys Arg Pro Leu Ser Ser Ala Asn

225 230 235 240

Gln Asn Phe Asn Asn Ser Glu Thr Leu Gly His Gln Gly Ser His Val

245 250 255

Val Thr Leu Gln Leu Arg Ile Asp Gly Met His Cys Lys Ser Cys Val

260 265 270

Leu Asn Ile Glu Glu Asn Ile Gly Gln Leu Leu Gly Val Gln Ser Ile

275 280 285

Gln Val Ser Leu Glu Asn Lys Thr Ala Gln Val Lys Tyr Asp Pro Ser

290 295300

Cys Thr Ser Pro Val Ala Leu Gln Arg Ala Ile Glu Ala Leu Pro Pro

305 310 315 320

Gly Asn Phe Lys Val Ser Leu Pro Asp Gly Ala Glu Gly Ser Gly Thr

325 330 335

Asp His Arg Ser Ser Ser Ser His Ser Pro Gly Ser Pro Pro Arg Asn

340 345 350

Gln Val Gln Gly Thr Cys Ser Thr Thr Leu Ile Ala Ile Ala Gly Met

355 360 365

Thr Cys Ala Ser Cys Val His Ser Ile Glu Gly Met Ile Ser Gln Leu

370 375 380

Glu Gly Val Gln Gln Ile Ser Val Ser Leu Ala Glu Gly Thr Ala Thr

385 390 395 400

Val Leu Tyr Asn Pro Ser Val Ile Ser Pro Glu Glu Leu Arg Ala Ala

405 410 415

Ile Glu Asp Met Gly Phe Glu Ala Ser Val Val Ser Glu Ser Cys Ser

420 425 430

Thr Asn Pro Leu Gly Asn His Ser Ala Gly Asn Ser Met Val Gln Thr

435 440 445

Thr Asp Gly Thr Pro Thr Ser Val Gln Glu Val Ala Pro His Thr Gly

450 455460

Arg Leu Pro Ala Asn His Ala Pro Asp Ile Leu Ala Lys Ser Pro Gln

465 470 475 480

Ser Thr Arg Ala Val Ala Pro Gln Lys Cys Phe Leu Gln Ile Lys Gly

485 490 495

Met Thr Cys Ala Ser Cys Val Ser Asn Ile Glu Arg Asn Leu Gln Lys

500 505 510

Glu Ala Gly Val Leu Ser Val Leu Val Ala Leu Met Ala Gly Lys Ala

515 520 525

Glu Ile Lys Tyr Asp Pro Glu Val Ile Gln Pro Leu Glu Ile Ala Gln

530 535 540

Phe Ile Gln Asp Leu Gly Phe Glu Ala Ala Val Met Glu Asp Tyr Ala

545 550 555 560

Gly Ser Asp Gly Asn Ile Glu Leu Thr Ile Thr Gly Met Thr Cys Ala

565 570 575

Ser Cys Val His Asn Ile Glu Ser Lys Leu Thr Arg Thr Asn Gly Ile

580 585 590

Thr Tyr Ala Ser Val Ala Leu Ala Thr Ser Lys Ala Leu Val Lys Phe

595 600 605

Asp Pro Glu Ile Ile Gly Pro Arg Asp Ile Ile Lys Ile Ile Glu Glu

610 615620

Ile Gly Phe His Ala Ser Leu Ala Gln Arg Asn Pro Asn Ala His His

625 630 635 640

Leu Asp His Lys Met Glu Ile Lys Gln Trp Lys Lys Ser Phe Leu Cys

645 650 655

Ser Leu Val Phe Gly Ile Pro Val Met Ala Leu Met Ile Tyr Met Leu

660 665 670

Ile Pro Ser Asn Glu Pro His Gln Ser Met Val Leu Asp His Asn Ile

675 680 685

Ile Pro Gly Leu Ser Ile Leu Asn Leu Ile Phe Phe Ile Leu Cys Thr

690 695 700

Phe Val Gln Leu Leu Gly Gly Trp Tyr Phe Tyr Val Gln Ala Tyr Lys

705 710 715 720

Ser Leu Arg His Arg Ser Ala Asn Met Asp Val Leu Ile Val Leu Ala

725 730 735

Thr Ser Ile Ala Tyr Val Tyr Ser Leu Val Ile Leu Val Val Ala Val

740 745 750

Ala Glu Lys Ala Glu Arg Ser Pro Val Thr Phe Phe Asp Thr Pro Pro

755 760 765

Met Leu Phe Val Phe Ile Ala Leu Gly Arg Trp Leu Glu His Leu Ala

770 775 780

Lys Ser Lys Thr Ser Glu Ala Leu Ala Lys Leu Met Ser Leu Gln Ala

785 790 795 800

Thr Glu Ala Thr Val Val Thr Leu Gly Glu Asp Asn Leu Ile Ile Arg

805 810 815

Glu Glu Gln Val Pro Met Glu Leu Val Gln Arg Gly Asp Ile Val Lys

820 825 830

Val Val Pro Gly Gly Lys Phe Pro Val Asp Gly Lys Val Leu Glu Gly

835 840 845

Asn Thr Met Ala Asp Glu Ser Leu Ile Thr Gly Glu Ala Met Pro Val

850 855 860

Thr Lys Lys Pro Gly Ser Thr Val Ile Ala Gly Ser Ile Asn Ala His

865 870 875 880

Gly Ser Val Leu Ile Lys Ala Thr His Val Gly Asn Asp Thr Thr Leu

885 890 895

Ala Gln Ile Val Lys Leu Val Glu Glu Ala Gln Met Ser Lys Ala Pro

900 905 910

Ile Gln Gln Leu Ala Asp Arg Phe Ser Gly Tyr Phe Val Pro Phe Ile

915 920 925

Ile Ile Met Ser Thr Leu Thr Leu Val Val Trp Ile Val Ile Gly Phe

930 935 940

Ile Asp Phe Gly Val Val Gln Arg Tyr Phe Pro Asn Pro Asn Lys His

945 950 955 960

Ile Ser Gln Thr Glu Val Ile Ile Arg Phe Ala Phe Gln Thr Ser Ile

965 970 975

Thr Val Leu Cys Ile Ala Cys Pro Cys Ser Leu Gly Leu Ala Thr Pro

980 985 990

Thr Ala Val Met Val Gly Thr Gly Val Ala Ala Gln Asn Gly Ile Leu

995 1000 1005

Ile Lys Gly Gly Lys Pro Leu Glu Met Ala His Lys Ile Lys Thr Val

1010 1015 1020

Met Phe Asp Lys Thr Gly Thr Ile Thr His Gly Val Pro Arg Val Met

1025 1030 1035 1040

Arg Val Leu Leu Leu Gly Asp Val Ala Thr Leu Pro Leu Arg Lys Val

1045 1050 1055

Leu Ala Val Val Gly Thr Ala Glu Ala Ser Ser Glu His Pro Leu Gly

1060 1065 1070

Val Ala Val Thr Lys Tyr Cys Lys Glu Glu Leu Gly Thr Glu Thr Leu

1075 1080 1085

Gly Tyr Cys Thr Asp Phe Gln Ala Val Pro Gly Cys Gly Ile Gly Cys

1090 1095 1100

Lys Val Ser Asn Val Glu Gly Ile Leu Ala His Ser Glu Arg Pro Leu

1105 1110 1115 1120

Ser Ala Pro Ala Ser His Leu Asn Glu Ala Gly Ser Leu Pro Ala Glu

1125 1130 1135

Lys Asp Ala Val Pro Gln Thr Phe Ser Val Leu Ile Gly Asn Arg Glu

1140 1145 1150

Trp Leu Arg Arg Asn Gly Leu Thr Ile Ser Ser Asp Val Ser Asp Ala

1155 1160 1165

Met Thr Asp His Glu Met Lys Gly Gln Thr Ala Ile Leu Val Ala Ile

1170 1175 1180

Asp Gly Val Leu Cys Gly Met Ile Ala Ile Ala Asp Ala Val Lys Gln

1185 1190 1195 1200

Glu Ala Ala Leu Ala Val His Thr Leu Gln Ser Met Gly Val Asp Val

1205 1210 1215

Val Leu Ile Thr Gly Asp Asn Arg Lys Thr Ala Arg Ala Ile Ala Thr

1220 1225 1230

Gln Val Gly Ile Asn Lys Val Phe Ala Glu Val Leu Pro Ser His Lys

1235 1240 1245

Val Ala Lys Val Gln Glu Leu Gln Asn Lys Gly Lys Lys Val Ala Met

1250 1255 1260

Val Gly Asp Gly Val Asn Asp Ser Pro Ala Leu Ala Gln Ala Asp Met

1265 1270 1275 1280

Gly Val Ala Ile Gly Thr Gly Thr Asp Val Ala Ile Glu Ala Ala Asp

1285 1290 1295

Val Val Leu Ile Arg Asn Asp Leu Leu Asp Val Val Ala Ser Ile His

1300 1305 1310

Leu Ser Lys Arg Thr Val Arg Arg Ile Arg Ile Asn Leu Val Leu Ala

1315 1320 1325

Leu Ile Tyr Asn Leu Val Gly Ile Pro Ile Ala Ala Gly Val Phe Met

1330 1335 1340

Pro Ile Gly Ile Val Leu Gln Pro Trp Met Gly Ser Ala Ala Met Ala

1345 1350 1355 1360

Ala Ser Ser Val Ser Val Val Leu Ser Ser Leu Gln Leu Lys Cys Tyr

1365 1370 1375

Lys Lys Pro Asp Leu Glu Arg Tyr Glu Ala Gln Ala His Gly His Met

1380 1385 1390

Lys Pro Leu Thr Ala Ser Gln Val Ser Val His Ile Gly Met Asp Asp

1395 1400 1405

Arg Trp Arg Asp Ser Pro Arg Ala Thr Pro Trp Asp Gln Val Ser Tyr

1410 1415 1420

Val Ser Gln Val Ser Leu Ser Ser Leu Thr Ser Asp Lys Pro Ser Arg

1425 1430 1435 1440

His Ser Ala Ala Ala Asp Asp Asp Gly Asp Lys Trp Ser Leu Leu Leu

1445 1450 1455

Asn Gly Arg Asp Glu Glu Gln Tyr Ile

1460 1465

<210>9

<211>4484

<212>DNA

<213>2 Ambystoma laterale x Ambystoma jeffersonianum

<400>9

ggtaccgcca ccatgcccga acaggaaaga cagatcaccg caagggaagg agcaagtcgc 60

aagattctga gcaaactgag cctgccaacc cgagcctggg agcccgccat gaagaagagc 120

ttcgcctttg acaacgtggg atacgagggc ggcctggatg gcctgggacc tagctcccag 180

gtggccacct ccaccgtgag aatcctgggc atgacatgcc agagctgcgt gaagtccatc 240

gaggacagaa tctctaatct gaagggcatc atctctatga aggtgagcct ggagcagggc 300

tccgccaccg tgaagtatgt gccttctgtg gtgtgcctgc agcaggtgtg ccaccagatc 360

ggcgatatgg gcttcgaggc cagcatcgca gagggcaagg cagcatcctg gccttctcgg 420

agcctgccag cacaggaggc agtggtgaag ctgagagtgg aaggaatgac ctgtcagagc 480

tgcgtgagca gcatcgaggg caaggtgagg aagctgcagg gcgtggtgcg cgtgaaggtg 540

tccctgtcta accaggaggc cgtgatcacc taccagccct atctgatcca gcctgaggac 600

ctgagggatc acgtgaatga catgggcttc gaggccgcca tcaagagcaa ggtggcacca 660

ctgtccctgg gaccaatcga catcgagcgg ctgcagtcca ccaacccaaa gcggcccctg 720

tcctctgcca accagaactt caacaattct gagacactgg gacaccaggg cagccacgtg 780

gtgaccctgc agctgaggat cgacggcatg cactgcaaga gctgcgtgct gaacatcgag 840

gagaatatcg gccagctgct gggcgtgcag tccatccagg tgtctctgga gaacaagaca 900

gcccaggtga agtacgatcc ttcttgcacc agcccagtgg ccctgcagag ggcaatcgag 960

gccctgcccc ctggcaattt caaggtgtcc ctgcctgacg gagcagaggg ctctggcacc 1020

gatcaccgga gcagcagcag ccactcccca ggctctccac caaggaacca ggtgcagggc 1080

acatgttcta ccacactgat cgcaatcgca ggaatgacct gcgcaagctg cgtgcactcc 1140

atcgagggca tgatcagcca gctggagggc gtgcagcaga tcagcgtgtc cctggcagag 1200

ggcaccgcaa cagtgctgta caatcccagc gtgatctccc ctgaggagct gagggcagca 1260

atcgaggata tgggatttga ggccagcgtg gtgtctgaga gctgctccac aaaccccctg 1320

ggcaatcact ctgccggcaa cagcatggtg cagaccacag acggcacccc tacaagcgtg 1380

caggaggtgg caccacacac cggccggctg ccagcaaatc acgcaccaga tatcctggcc 1440

aagtctcccc agagcacaag agccgtggcc cctcagaagt gttttctgca gatcaagggc 1500

atgacctgcg cctcctgcgt gagcaacatc gagcggaatc tgcagaagga ggcaggcgtg 1560

ctgtccgtgc tggtggccct gatggcaggc aaggccgaga tcaagtacga ccctgaagtg 1620

atccagccac tggagatcgc ccagttcatc caggatctgg gctttgaggc cgccgtgatg 1680

gaggactatg ccggcagcga tggcaacatc gagctgacca tcacaggcat gacctgcgcc 1740

tcttgcgtgc acaacatcga gagcaagctg accagaacaa atggcatcac atacgcctct 1800

gtggccctgg ccaccagcaa ggccctggtg aagttcgacc ccgagatcat cggccctagg 1860

gatatcatca agatcatcga ggagatcggc tttcacgcct ccctggccca gcgcaaccca 1920

aatgcccacc acctggacca caagatggag atcaagcagt ggaagaagtc cttcctgtgc 1980

tctctggtgt ttggcatccc cgtgatggcc ctgatgatct acatgctgat cccttccaac 2040

gagccacacc agtctatggt gctggatcac aacatcatcc ctggcctgag catcctgaat 2100

ctgatcttct ttatcctgtg cacattcgtg cagctgctgg gcggctggta cttttatgtg 2160

caggcttaca agtccctgcg gcaccggagc gccaatatgg acgtgctgat cgtgctggcc 2220

accagcatcg cctacgtgta ttccctggtc atcctggtgg tggcagtggc agagaaggca 2280

gagcggtccc ccgtgacctt ctttgataca cctccaatgc tgttcgtgtt tatcgccctg 2340

ggcagatggc tggagcacct ggccaagagc aagacctccg aggccctggc caagctgatg 2400

agcctgcagg ccacagaggc caccgtggtg acactgggcg aggacaacct gatcatcagg 2460

gaggagcagg tgcctatgga gctggtgcag cgcggcgata tcgtgaaggt ggtgccaggc 2520

ggcaagttcc cagtggacgg caaggtgctg gagggcaata caatggccga tgagagcctg 2580

atcaccggcg aggccatgcc tgtgaccaag aagccaggct ctacagtgat cgcaggcagc 2640

atcaacgcac acggctccgt gctgatcaag gccacccacg tgggcaatga caccacactg 2700

gcccagatcg tgaagctggt ggaggaggcc cagatgtcca aggcccctat ccagcagctg 2760

gccgatcggt tctccggcta cttcgtgccc ttcatcatca tcatgtctac cctgacactg 2820

gtggtgtgga tcgtgatcgg cttcatcgac tttggcgtgg tgcagaggta ttttcccaac 2880

cctaataagc acatcagcca gaccgaagtg atcatccgct tcgcctttca gaccagcatc 2940

acagtgctgt gcatcgcatg cccatgttcc ctgggcctgg caaccccaac agccgtgatg 3000

gtgggcacag gagtggcagc acagaacggc atcctgatca agggcggcaa gcccctggag 3060

atggcccaca agatcaagac cgtgatgttt gacaagaccg gcacaatcac ccacggcgtg 3120

cccagagtga tgagagtgct gctgctgggc gatgtggcca cactgcctct gagaaaggtg 3180

ctggcagtgg tgggcaccgc agaggccagc agcgagcacc cactgggcgt ggccgtgaca 3240

aagtactgca aggaggagct gggcacagag acactgggct attgtaccga cttccaggcc 3300

gtgcccggat gcggaatcgg ctgtaaggtg agcaacgtgg agggcatcct ggcacactcc 3360

gagcggcccc tgagcgcccc tgcatcccac ctgaatgagg caggctctct gccagcagag 3420

aaggacgccg tgcctcagac cttcagcgtg ctgatcggca acagagagtg gctgcggaga 3480

aatggcctga ccatcagctc cgacgtgtcc gatgccatga cagatcacga gatgaagggc 3540

cagaccgcaa tcctggtggc aatcgacggc gtgctgtgcg gcatgatcgc catcgccgat 3600

gcagtgaagc aggaggccgc cctggcagtg cacaccctgc agagcatggg cgtggacgtg 3660

gtgctgatca ccggcgataa caggaagaca gcaagggcaa tcgcaaccca agtgggcatc 3720

aataaggtgt tcgccgaggt gctgccttcc cacaaggtgg ccaaggtgca ggagctgcag 3780

aacaagggca agaaggtggc catggtgggc gacggcgtga atgattctcc agccctggca 3840

caggcagaca tgggagtggc aatcggcacaggcaccgacg tggcaatcga ggcagcagat 3900

gtggtgctga tcaggaatga cctgctggat gtggtggcct ctatccacct gagcaagcgg 3960

accgtgaggc gcatcagaat caacctggtg ctggccctga tctacaatct ggtgggcatc 4020

ccaatcgcag caggcgtgtt tatgccaatc ggcatcgtgc tgcagccatg gatgggctct 4080

gccgcaatgg cagcctctag cgtgagcgtg gtgctgtcct ctctgcagct gaagtgctac 4140

aagaagccag acctggagcg gtacgaggca caggcacacg gacacatgaa gccactgacc 4200

gcctctcagg tgagcgtgca catcggcatg gacgataggt ggagggacag cccaagggca 4260

acaccatggg atcaggtgtc ctacgtgagc caggtgagcc tgagcagcct gacctccgat 4320

aagccctccc gccactctgc cgccgccgac gacgacgggg acaagtggag cctgctgctg 4380

aacgggagag acgaggaaca gtacatttga taaagatctt cgagaggcct aataaagagc 4440

tcagatgcat cgatcagagt gtgttggttt tttgtgtggg atcc 4484

<210>10

<211>362

<212>PRT

<213>2 Ambystoma laterale x Ambystoma jeffersonianum

<400>10

Ile Leu Gly Met Thr Cys Gln Ser Cys Val Lys Ser Ile Glu Asp Arg

1 5 10 15

Ile Ser Asn Leu Lys Gly Ile Ile Ser Met Lys Val Ser Leu Glu Gln

20 25 30

Gly Ser Ala Thr Val Lys Tyr Val Pro Ser Val Val Cys Leu Gln Gln

35 40 45

Val Cys His Gln Ile Gly Asp Met Gly Phe Glu Ala Ser Ile Ala Glu

50 55 60

Gly Lys Ala Ala Ser Trp Pro Ser Arg Ser Leu Pro Ala Gln Glu Ala

65 70 75 80

Val Val Lys Leu Arg Val Glu Gly Met Thr Cys Gln Ser Cys Val Ser

85 90 95

Ser Ile Glu Gly Lys Val Arg Lys Leu Gln Gly Val Val Arg Val Lys

100 105 110

Val Ser Leu Ser Asn Gln Glu Ala Val Ile Thr Tyr Gln Pro Tyr Leu

115 120 125

Ile Gln Pro Glu Asp Leu Arg Asp His Val Asn Asp Met Gly Phe Glu

130 135 140

Ala Ala Ile Lys Ser Lys Val Ala Pro Leu Ser Leu Gly Pro Ile Asp

145 150 155 160

Ile Glu Arg Leu Gln Ser Thr Asn Pro Lys Arg Pro Leu Ser Ser Ala

165 170 175

Asn Gln Asn Phe Asn Asn Ser Glu Thr Leu Gly His Gln Gly Ser His

180 185 190

Val Val Thr Leu Gln Leu Arg Ile Asp Gly Met His Cys Lys Ser Cys

195200 205

Val Leu Asn Ile Glu Glu Asn Ile Gly Gln Leu Leu Gly Val Gln Ser

210 215 220

Ile Gln Val Ser Leu Glu Asn Lys Thr Ala Gln Val Lys Tyr Asp Pro

225 230 235 240

Ser Cys Thr Ser Pro Val Ala Leu Gln Arg Ala Ile Glu Ala Leu Pro

245 250 255

Pro Gly Asn Phe Lys Val Ser Leu Pro Asp Gly Ala Glu Gly Ser Gly

260 265 270

Thr Asp His Arg Ser Ser Ser Ser His Ser Pro Gly Ser Pro Pro Arg

275 280 285

Asn Gln Val Gln Gly Thr Cys Ser Thr Thr Leu Ile Ala Ile Ala Gly

290 295 300

Met Thr Cys Ala Ser Cys Val His Ser Ile Glu Gly Met Ile Ser Gln

305 310 315 320

Leu Glu Gly Val Gln Gln Ile Ser Val Ser Leu Ala Glu Gly Thr Ala

325 330 335

Thr Val Leu Tyr Asn Pro Ser Val Ile Ser Pro Glu Glu Leu Arg Ala

340 345 350

Ala Ile Glu Asp Met Gly Phe Glu Ala Ser

355 360

<210>11

<211>1103

<212>PRT

<213>2 Ambystoma laterale x Ambystoma jeffersonianum

<400>11

Met Pro Glu Gln Glu Arg Gln Ile Thr Ala Arg Glu Gly Ala Ser Arg

1 5 10 15

Lys Ile Leu Ser Lys Leu Ser Leu Pro Thr Arg Ala Trp Glu Pro Ala

20 25 30

Met Lys Lys Ser Phe Ala Phe Asp Asn Val Gly Tyr Glu Gly Gly Leu

35 40 45

Asp Gly Leu Gly Pro Ser Ser Gln Val Ala Thr Ser Thr Val Arg Val

50 55 60

Val Ser Glu Ser Cys Ser Thr Asn Pro Leu Gly Asn His Ser Ala Gly

65 70 75 80

Asn Ser Met Val Gln Thr Thr Asp Gly Thr Pro Thr Ser Val Gln Glu

85 90 95

Val Ala Pro His Thr Gly Arg Leu Pro Ala Asn His Ala Pro Asp Ile

100 105 110

Leu Ala Lys Ser Pro Gln Ser Thr Arg Ala Val Ala Pro Gln Lys Cys

115 120 125

Phe Leu Gln Ile Lys Gly Met Thr Cys Ala Ser Cys Val Ser Asn Ile

130 135 140

Glu Arg Asn Leu Gln Lys Glu Ala Gly Val Leu Ser Val Leu Val Ala

145 150 155 160

Leu Met Ala Gly Lys Ala Glu Ile Lys Tyr Asp Pro Glu Val Ile Gln

165 170 175

Pro Leu Glu Ile Ala Gln Phe Ile Gln Asp Leu Gly Phe Glu Ala Ala

180 185 190

Val Met Glu Asp Tyr Ala Gly Ser Asp Gly Asn Ile Glu Leu Thr Ile

195 200 205

Thr Gly Met Thr Cys Ala Ser Cys Val His Asn Ile Glu Ser Lys Leu

210 215 220

Thr Arg Thr Asn Gly Ile Thr Tyr Ala Ser Val Ala Leu Ala Thr Ser

225 230 235 240

Lys Ala Leu Val Lys Phe Asp Pro Glu Ile Ile Gly Pro Arg Asp Ile

245 250 255

Ile Lys Ile Ile Glu Glu Ile Gly Phe His Ala Ser Leu Ala Gln Arg

260 265 270

Asn Pro Asn Ala His His Leu Asp His Lys Met Glu Ile Lys Gln Trp

275 280 285

Lys Lys Ser Phe Leu Cys Ser Leu Val Phe Gly Ile Pro Val Met Ala

290 295 300

Leu Met Ile Tyr Met Leu Ile Pro Ser Asn Glu Pro His Gln Ser Met

305 310 315 320

Val Leu Asp His Asn Ile Ile Pro Gly Leu Ser Ile Leu Asn Leu Ile

325 330 335

Phe Phe Ile Leu Cys Thr Phe Val Gln Leu Leu Gly Gly Trp Tyr Phe

340 345 350

Tyr Val Gln Ala Tyr Lys Ser Leu Arg His Arg Ser Ala Asn Met Asp

355 360 365

Val Leu Ile Val Leu Ala Thr Ser Ile Ala Tyr Val Tyr Ser Leu Val

370 375 380

Ile Leu Val Val Ala Val Ala Glu Lys Ala Glu Arg Ser Pro Val Thr

385 390 395 400

Phe Phe Asp Thr Pro Pro Met Leu Phe Val Phe Ile Ala Leu Gly Arg

405 410 415

Trp Leu Glu His Leu Ala Lys Ser Lys Thr Ser Glu Ala Leu Ala Lys

420 425 430

Leu Met Ser Leu Gln Ala Thr Glu Ala Thr Val Val Thr Leu Gly Glu

435 440 445

Asp Asn Leu Ile Ile Arg Glu Glu Gln Val Pro Met Glu Leu Val Gln

450 455 460

Arg Gly Asp Ile Val Lys Val Val Pro Gly Gly Lys Phe Pro Val Asp

465 470 475 480

Gly Lys Val Leu Glu Gly Asn Thr Met Ala Asp Glu Ser Leu Ile Thr

485 490 495

Gly Glu Ala Met Pro Val Thr Lys Lys Pro Gly Ser Thr Val Ile Ala

500 505 510

Gly Ser Ile Asn Ala His Gly Ser Val Leu Ile Lys Ala Thr His Val

515 520 525

Gly Asn Asp Thr Thr Leu Ala Gln Ile Val Lys Leu Val Glu Glu Ala

530 535 540

Gln Met Ser Lys Ala Pro Ile Gln Gln Leu Ala Asp Arg Phe Ser Gly

545 550 555 560

Tyr Phe Val Pro Phe Ile Ile Ile Met Ser Thr Leu Thr Leu Val Val

565 570 575

Trp Ile Val Ile Gly Phe Ile Asp Phe Gly Val Val Gln Arg Tyr Phe

580 585 590

Pro Asn Pro Asn Lys His Ile Ser Gln Thr Glu Val Ile Ile Arg Phe

595 600 605

Ala Phe Gln Thr Ser Ile Thr Val Leu Cys Ile Ala Cys Pro Cys Ser

610 615 620

Leu Gly Leu Ala Thr Pro Thr Ala Val Met Val Gly Thr Gly Val Ala

625 630 635 640

Ala Gln Asn Gly Ile Leu Ile Lys Gly Gly Lys Pro Leu Glu Met Ala

645 650 655

His Lys Ile Lys Thr Val Met Phe Asp Lys Thr Gly Thr Ile Thr His

660 665 670

Gly Val Pro Arg Val Met Arg Val Leu Leu Leu Gly Asp Val Ala Thr

675 680 685

Leu Pro Leu Arg Lys Val Leu Ala Val Val Gly Thr Ala Glu Ala Ser

690 695 700

Ser Glu His Pro Leu Gly Val Ala Val Thr Lys Tyr Cys Lys Glu Glu

705 710 715 720

Leu Gly Thr Glu Thr Leu Gly Tyr Cys Thr Asp Phe Gln Ala Val Pro

725 730 735

Gly Cys Gly Ile Gly Cys Lys Val Ser Asn Val Glu Gly Ile Leu Ala

740 745 750

His Ser Glu Arg Pro Leu Ser Ala Pro Ala Ser His Leu Asn Glu Ala

755 760 765

Gly Ser Leu Pro Ala Glu Lys Asp Ala Val Pro Gln Thr Phe Ser Val

770 775 780

Leu Ile Gly Asn Arg Glu Trp Leu Arg Arg Asn Gly Leu Thr Ile Ser

785 790 795 800

Ser Asp Val Ser Asp Ala Met Thr Asp His Glu Met Lys Gly Gln Thr

805 810 815

Ala Ile Leu Val Ala Ile Asp Gly Val Leu Cys Gly Met Ile Ala Ile

820 825 830

Ala Asp Ala Val Lys Gln Glu Ala Ala Leu Ala Val His Thr Leu Gln

835 840 845

Ser Met Gly Val Asp Val Val Leu Ile Thr Gly Asp Asn Arg Lys Thr

850 855 860

Ala Arg Ala Ile Ala Thr Gln Val Gly Ile Asn Lys Val Phe Ala Glu

865 870 875 880

Val Leu Pro Ser His Lys Val Ala Lys Val Gln Glu Leu Gln Asn Lys

885 890 895

Gly Lys Lys Val Ala Met Val Gly Asp Gly Val Asn Asp Ser Pro Ala

900 905 910

Leu Ala Gln Ala Asp Met Gly Val Ala Ile Gly Thr Gly Thr Asp Val

915 920 925

Ala Ile Glu Ala Ala Asp Val Val Leu Ile Arg Asn Asp Leu Leu Asp

930 935 940

Val Val Ala Ser Ile His Leu Ser Lys Arg Thr Val Arg Arg Ile Arg

945 950 955 960

Ile Asn Leu Val Leu Ala Leu Ile Tyr Asn Leu Val Gly Ile Pro Ile

965 970 975

Ala Ala Gly Val Phe Met Pro Ile Gly Ile Val Leu Gln Pro Trp Met

980 985 990

Gly Ser Ala Ala Met Ala Ala Ser Ser Val Ser Val Val Leu Ser Ser

995 1000 1005

Leu Gln Leu Lys Cys Tyr Lys Lys Pro Asp Leu Glu Arg Tyr Glu Ala

1010 1015 1020

Gln Ala His Gly His Met Lys Pro Leu Thr Ala Ser Gln Val Ser Val

1025 1030 1035 1040

His Ile Gly Met Asp Asp Arg Trp Arg Asp Ser Pro Arg Ala Thr Pro

1045 1050 1055

Trp Asp Gln Val Ser Tyr Val Ser Gln Val Ser Leu Ser Ser Leu Thr

1060 1065 1070

Ser Asp Lys Pro Ser Arg His Ser Ala Ala Ala Asp Asp Asp Gly Asp

1075 1080 1085

Lys Trp Ser Leu Leu Leu Asn Gly Arg Asp Glu Glu Gln Tyr Ile

1090 1095 1100

<210>12

<211>3333

<212>DNA

<213>2 Ambystoma laterale x Ambystoma jeffersonianum

<400>12

ggtaccgcca ccatgcccga acaggaaaga cagatcaccg caagggaagg agcaagtcgc 60

aagattctga gcaaactgag cctgccaacc cgagcctggg agcccgccat gaagaagagc 120

ttcgcctttg acaacgtggg atacgagggc ggcctggatg gcctgggacc tagctcccag 180

gtggccacct ccaccgtgag agtggtgtct gagagctgct ccacaaaccc cctgggcaat 240

cactctgccg gcaacagcat ggtgcagacc acagacggca cccctacaag cgtgcaggag 300

gtggcaccac acaccggccg gctgccagca aatcacgcac cagatatcct ggccaagtct 360

ccccagagca caagagccgt ggcccctcag aagtgttttc tgcagatcaa gggcatgacc 420

tgcgcctcct gcgtgagcaa catcgagcgg aatctgcaga aggaggcagg cgtgctgtcc 480

gtgctggtgg ccctgatggc aggcaaggcc gagatcaagt acgaccctga agtgatccag 540

ccactggaga tcgcccagtt catccaggat ctgggctttg aggccgccgt gatggaggac 600

tatgccggca gcgatggcaa catcgagctg accatcacag gcatgacctg cgcctcttgc 660

gtgcacaaca tcgagagcaa gctgaccaga acaaatggca tcacatacgc ctctgtggcc 720

ctggccacca gcaaggccct ggtgaagttc gaccccgaga tcatcggccc tagggatatc 780

atcaagatca tcgaggagat cggctttcac gcctccctgg cccagcgcaa cccaaatgcc 840

caccacctgg accacaagat ggagatcaag cagtggaaga agtccttcct gtgctctctg 900

gtgtttggca tccccgtgat ggccctgatg atctacatgc tgatcccttc caacgagcca 960

caccagtcta tggtgctgga tcacaacatc atccctggcc tgagcatcct gaatctgatc 1020

ttctttatcc tgtgcacatt cgtgcagctg ctgggcggct ggtactttta tgtgcaggct 1080

tacaagtccc tgcggcaccg gagcgccaat atggacgtgc tgatcgtgct ggccaccagc 1140

atcgcctacg tgtattccct ggtcatcctg gtggtggcag tggcagagaa ggcagagcgg 1200

tcccccgtga ccttctttga tacacctcca atgctgttcg tgtttatcgc cctgggcaga 1260

tggctggagc acctggccaa gagcaagacc tccgaggccc tggccaagct gatgagcctg 1320

caggccacag aggccaccgt ggtgacactg ggcgaggaca acctgatcat cagggaggag 1380

caggtgccta tggagctggt gcagcgcggc gatatcgtga aggtggtgcc aggcggcaag 1440

ttcccagtgg acggcaaggt gctggagggc aatacaatgg ccgatgagag cctgatcacc 1500

ggcgaggcca tgcctgtgac caagaagcca ggctctacag tgatcgcagg cagcatcaac 1560

gcacacggct ccgtgctgat caaggccacc cacgtgggca atgacaccac actggcccag 1620

atcgtgaagc tggtggagga ggcccagatg tccaaggccc ctatccagca gctggccgat 1680

cggttctccg gctacttcgt gcccttcatc atcatcatgt ctaccctgac actggtggtg 1740

tggatcgtga tcggcttcat cgactttggc gtggtgcaga ggtattttcc caaccctaat 1800

aagcacatca gccagaccga agtgatcatc cgcttcgcct ttcagaccag catcacagtg 1860

ctgtgcatcg catgcccatg ttccctgggc ctggcaaccc caacagccgt gatggtgggc 1920

acaggagtgg cagcacagaa cggcatcctg atcaagggcg gcaagcccct ggagatggcc 1980

cacaagatca agaccgtgat gtttgacaag accggcacaa tcacccacgg cgtgcccaga 2040

gtgatgagag tgctgctgct gggcgatgtg gccacactgc ctctgagaaa ggtgctggca 2100

gtggtgggca ccgcagaggc cagcagcgag cacccactgg gcgtggccgt gacaaagtac 2160

tgcaaggagg agctgggcac agagacactg ggctattgta ccgacttcca ggccgtgccc 2220

ggatgcggaa tcggctgtaa ggtgagcaac gtggagggca tcctggcaca ctccgagcgg 2280

cccctgagcg cccctgcatc ccacctgaat gaggcaggct ctctgccagc agagaaggac 2340

gccgtgcctc agaccttcag cgtgctgatc ggcaacagag agtggctgcg gagaaatggc 2400

ctgaccatca gctccgacgt gtccgatgcc atgacagatc acgagatgaa gggccagacc 2460

gcaatcctgg tggcaatcga cggcgtgctg tgcggcatga tcgccatcgc cgatgcagtg 2520

aagcaggagg ccgccctggc agtgcacacc ctgcagagca tgggcgtgga cgtggtgctg 2580

atcaccggcg ataacaggaa gacagcaagg gcaatcgcaa cccaagtggg catcaataag 2640

gtgttcgccg aggtgctgcc ttcccacaag gtggccaagg tgcaggagct gcagaacaag 2700

ggcaagaagg tggccatggt gggcgacggc gtgaatgatt ctccagccct ggcacaggca 2760

gacatgggag tggcaatcgg cacaggcacc gacgtggcaa tcgaggcagc agatgtggtg 2820

ctgatcagga atgacctgct ggatgtggtg gcctctatcc acctgagcaa gcggaccgtg 2880

aggcgcatca gaatcaacct ggtgctggcc ctgatctaca atctggtggg catcccaatc 2940

gcagcaggcg tgtttatgcc aatcggcatc gtgctgcagc catggatggg ctctgccgca 3000

atggcagcct ctagcgtgag cgtggtgctg tcctctctgc agctgaagtg ctacaagaag 3060

ccagacctgg agcggtacga ggcacaggca cacggacaca tgaagccact gaccgcctct 3120

caggtgagcg tgcacatcgg catggacgat aggtggaggg acagcccaag ggcaacacca 3180

tgggatcagg tgtcctacgt gagccaggtg agcctgagca gcctgacctc cgataagccc 3240

tcccgccact ctgccgccgc cgacgacgac ggggacaagt ggagcctgct gctgaacggg 3300

agagacgagg aacagtacat ttgataaaga tct 3333

<210>13

<211>21

<212>DNA

<213>2 Ambystoma laterale x Ambystoma jeffersonianum

<400>13

ggaaccccta gtgatggagt t 21

<210>14

<211>16

<212>DNA

<213>2 Ambystoma laterale x Ambystoma jeffersonianum

<400>14

cggcctcagt gagcga 16

<210>15

<211>21

<212>DNA

<213>2 Ambystoma laterale x Ambystoma jeffersonianum

<400>15

cactccctct ctgcgcgctc g 21

<210>16

<211>100

<212>RNA

<213>2 Ambystoma laterale x Ambystoma jeffersonianum

<400>16

uacgaggcac aggcacacgg acacaugaag ccacugaccg ccucucaggu gagcgugcac 60

aucggcaugg acgauaggug gagggacagc ccaagggcaa 100

<210>17

<211>20

<212>DNA

<213>2 Ambystoma laterale x Ambystoma jeffersonianum

<400>17

gacacatgaa gccactgacc 20

<210>18

<211>20

<212>DNA

<213>2 Ambystoma laterale x Ambystoma jeffersonianum

<400>18

ctgtccctcc acctatcgtc 20

<210>19

<211>23

<212>DNA

<213>2 Ambystoma laterale x Ambystoma jeffersonianum

<400>19

tgccgatgtg cacgctcacc tga 23

Claims

1. An artificially designed gene expression unit comprising:

(1) the sequence information of the liver specific promoter is shown as SEQ ID NO. 1;

(2) a human source expression optimized human ATP7B coding sequence or a truncated human ATP7B coding sequence, and the sequence information is shown as SEQ ID NO.3 or SEQ ID NO. 4; and/or

(3) Polyadenylic acid tailing signal, wherein one kind of selective sequence information is shown in SEQ ID NO. 2.

2. A recombinant adeno-associated viral vector carrying the gene sequence of claim 1.

3. The recombinant adeno-associated viral vector according to claim 2 comprising:

(1) in vivo transduction may express a human ATP 7B-producing protein or a truncated human ATP7B protein; and/or

(2) Recombinant adeno-associated viral vector serotypes include, but are not limited to, AAV1, AAV2, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, aavrh.10, with preferred serotypes being AAV3B, AAV5, AAV8, and AAV 9.

4. A gene therapy drug comprising the gene expression cassette of claim 1 and the recombinant adeno-associated virus vector of claims 2 and 3.

5. The gene therapy drug according to claim 4, wherein the administration is intravenous injection.

6. The gene therapy drug according to claim 4, wherein the human ATP7B protein or truncated ATP7B protein is produced by expression in the liver for a long period of time in one administration, thereby achieving treatment of diseases caused by mutations in the ATP7B gene.

7. The ATP7B gene mutation-induced disease according to claim 6, preferably hepatolenticular degeneration.