KR102536321B1 - Liver-specific gene expression cassette based on human-derived gene expression module - Google Patents

Abstract

The present invention relates to a liver-specific gene expression system based on a human-derived gene expression induction module, and specifically, based on an alpha-1 antitrypsin core promoter, upstream an albumin enhancer (albumin enhancer) is attached, and a splicing donor/acceptor optimized for expression control and ^a poly A signal are attached downstream. In addition, this gene expression module was developed with an emphasis on minimizing the length to be advantageous when considering the application for gene therapy by loading into an AAV-based vector.

Description

Liver-specific gene expression cassette based on human-derived gene expression module}

The present invention relates to a liver-specific gene expression system based on a human-derived gene expression induction module.

In previous studies on liver uptake of viral vectors used in gene therapy, it was confirmed that the liver has a high ability to uptake viral vectors. That is, it has been confirmed through several studies that many viral vectors used in gene therapy, including adeno-associated virus (AAV) vectors, can be efficiently absorbed in the liver.

Moreover, the liver has been a target organ for gene therapy because of its central role in metabolism and serum protein production. Much of the current work on liver-derived AAV gene therapy development has resulted from preclinical and clinical success in the field of hemophilia B. A number of studies in classic mouse and dog models of hemophilia A and B have demonstrated potent effects due to administration of viral vectors.

However, gene therapy targeting the liver is still not efficient, and off-target delivery and expression may lead to reduced efficacy or complications. Therefore, there is a need to develop a robust liver-specific gene expression system capable of appropriately, specifically or preferentially expressing a gene of interest in the liver.

Korean Patent Publication No. 10-2018-0069054 (published on June 22, 2018)

An object of the present invention is a liver-specific gene expression cassette comprising a liver-specific promoter gene, a splicing donor/acceptor (SD/SA) gene, an expression target gene, and a polyA signal gene in order, An object of the present invention is to provide a recombinant expression vector containing the liver-specific gene expression cassette and a recombinant microorganism transfected with the recombinant expression vector.

Another object of the present invention is to provide a composition for liver-specific gene expression comprising the recombinant microorganism as an active ingredient.

In order to achieve the above object, the present invention provides a liver-specific promoter gene represented by SEQ ID NO: 1, a splicing donor/acceptor (SD/SA) gene represented by SEQ ID NO: 2, and a target gene And it provides a liver-specific gene expression cassette comprising the polyA signal gene represented by SEQ ID NO: 3 in order.

In addition, the present invention provides a recombinant expression vector containing the liver-specific gene expression cassette and a recombinant microorganism transfected with the recombinant expression vector.

In addition, the present invention provides a liver-specific gene expression composition comprising the recombinant microorganism as an active ingredient.

The present invention relates to a liver-specific gene expression system based on a human-derived gene expression induction module, and specifically, based on an alpha-1 antitrypsin core promoter, upstream an albumin enhancer (albumin enhancer) is attached, and a splicing donor/acceptor optimized for expression control and ^a poly A signal are attached downstream. In addition, this gene expression module was developed with an emphasis on minimizing the length to be advantageous when considering the application for gene therapy by loading into an AAV-based vector.

Figure 1 shows the results of characterization of human-derived liver-specific synthetic promoters. A) The structure of the liver-specific synthetic promoter module is shown. B) The result of confirming the ability of the liver-specific synthetic promoter to induce hepatocyte-specific gene expression is shown.
2 shows the poly A signal module characteristic analysis results. A) The structure and modified form of the newly designed polyA signal are shown. B) The result of confirming the effect of polyA signal on gene expression regulation is shown.
3 shows the result of analyzing the characteristics of a synthetic splicing donor/acceptor (SD/SA) module. A) Indicates the structure and transformation of the synthetic enhanced module. B) Gene expression activity of synthetic SD/SA variants is shown.
Figure 4 shows the results of liver tissue-specific expression cassette characteristics analysis through the optimized combination of each module. A) Shows the optimized combination structure of each module. B) Shows the results of gene expression activity investigation of each combination structure. C) Results of investigation of primary hepatocyte-specific gene expression activity of pS-AA1 (pLiver ^TM ) are shown.
5 shows the result of confirming pLiver ^TM dependent therapeutic gene expression. A) The structure of the pS-AA1 (pLiver ^TM )-hAGXT expression vector is shown. B) The results of confirming the expression of hAGXT in primary hepatocytes are shown. C) The result of confirming the expression of hAGXT through immunocytochemistry (ICC) analysis is shown.
Figure 6 shows a cleavage map of the pS-AA1 (pLiver ^™ ) -hAGXT recombinant expression vector.

The present invention is a liver-specific promoter gene represented by SEQ ID NO: 1, a splicing donor / acceptor (SD / SA) gene represented by SEQ ID NO: 2, an expression target gene, and SEQ ID NO: 3 A cassette for liver-specific gene expression containing the polyA signal gene in sequence is provided.

Preferably, the gene to be expressed may be human alanine-glyoxylate and serine-pyruvate aminotransferase (hAGXT), but is not limited thereto.

The "hAGXT" gene is NCBI accession no. It may be NM_000030.3.

In the present invention, the cassette for liver-specific gene expression may include a signal sequence or leader sequence for membrane targeting or secretion in addition to expression control elements such as a promoter, initiation codon, stop codon, polyadenylation signal and enhancer, and may be used for the purpose It can be manufactured in a variety of ways.

In addition, the present invention provides a recombinant expression vector containing the liver-specific gene expression cassette.

Preferably, the recombinant expression vector may be an adeno-associated virus (AAV)-based recombinant expression vector, more preferably, the recombinant expression vector may be the cleavage map of FIG. 6, but is limited thereto It is not.

In the present invention, "vector" means a self-replicating DNA molecule used to transfer clonal genes (or other fragments of clonal DNA).

In the present invention, "expression vector" means a recombinant DNA molecule containing a desired coding sequence and an appropriate nucleic acid sequence essential for expressing the operably linked coding sequence in a specific host organism. Expression vectors may preferably include one or more selectable markers. The marker is a nucleic acid sequence having a characteristic that can be selected by a conventional chemical method, and includes all genes capable of distinguishing transformed cells from non-transformed cells. Examples include, but are not limited to, antibiotic resistance genes such as Ampicillin, Kanamycin, Geneticin (G418), Bleomycin, Hygromycin, and Chloramphenicol. It is not, and can be appropriately selected by those skilled in the art.

In addition, the present invention provides a recombinant microorganism transfected with the recombinant expression vector. Preferably, the recombinant microorganism may be Adeno-associated virus (AAV), but is not limited thereto.

In addition, the present invention provides a liver-specific gene expression composition comprising the recombinant microorganism as an active ingredient.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for explaining the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .

< Experimental example >

The following experimental examples are intended to provide experimental examples commonly applied to each embodiment according to the present invention.

1. Cell culture

HEK 293T (ATCC CRL-1573), HeLa (ATCC CCL2.2), Huh7, HepG2 (ATCC HB-8065), and MRC5 (ATCC CCL-171) cells were all cultured in 10% fetal bovine serum (FBS, Invitrogen , Carlsbad, CA), GlutaMAX-1 (2 mM, Invitrogen) and Penicillin (100 IU/ml)/Streptomycin (50 g/ml, Invitrogen) using DMEM medium (Invitrogen) at 37°C, 5% Cultured in a CO ₂ incubator.

Primary hepatocytes were isolated from 11-week-old male C57BL/6N-tac AGXT KO mice. Cells were cultured in Medium 199 medium (Sigma-Aldrich, St. Louis, Mo) containing 10% fetal bovine serum, 10 nM dexamethasone (Sigma-Aldrich, St. Louis, Mo), and penicillin (100 IU/ml)/streptomycin (50 g/ml). Aldrich) at 37° C., 5% CO ₂ and cultured in an incubator.

2. cloning (cloning)

1) Liver-specific promoter (AA1) module design

The liver-specific minimal promoter module contained the nucleotide sequence of the phAAT portion (positions 1025-1229) from the human-derived alpha-1 antitrypsin (AAT) gene expression control region (genebank #D38257.1).

Positive control LP1 (liver-specific promoter consisting of apolipoprotein enhancer (EhApo), phAAT, 5'-UTR (hAAT), and SV40 intron), and another positive control group T-LP1 (EhApo, ahAAT part of LP1 promoter) were LP -1 By analyzing the nucleotide sequence composition, it was composed of only the enhancer module and promoter region similarly to AA1.

Each module was constructed through nucleotide sequence synthesis, and the gene expression characteristics of the promoter were cloned into a pGL3-basic vector (PR-E1751, promega) with the restriction enzymes KpnI and BglII, and firefly-luciferase activity was determined quantitatively.

2) synthesis polyA signal design

The minimal synthetic polyA module was based on homo sapiens growth hormone (hGH, NCBI sequence ID: NG_011676.1) and then combined with bovine growth hormone (bGH) to create a minimal synthetic polyA signal.

First, truncated hGH (T-hGH) with a length predicted to be absolutely necessary to express the function of the polyA signal was prepared with a total length of 476 bp.

The T-hGH nucleotide sequence was functionally classified into a total of five regions by dividing them into x region (in front of poly A signal), y region (middle of poly A signal and cleavage site), and z region (behind cleavage site). x, y. The z part was compared with the seq of bGH, and some bases were switched. In addition, various modules were produced by applying a modified nucleotide sequence to a specific nucleotide sequence of the y region.

Each module was prepared through nucleotide sequence synthesis and PCR, and the gene expression characteristics of the poly A signal were determined by cloning with the restriction enzymes XbaI and BamHI into the pGL3-pCMV vector and quantitatively measuring the activity of firefly-luciferase.

3) synthetic SD/SA design

A minimal synthetic splicing donor/splicing acceptor (SD/SA) module was introduced to increase gene expression. SD/SA (207 bp) of pSP72-XP7-scAAV2-CMV-GFP (a GFP vector currently used in the laboratory) was used as a reference. A truncated SD/SA (T-SD/SA) with a length predicted to be necessary to represent the function of SD/SA was produced, and functionally further analyzed to determine SD site, branch site, Pyrimidine (Py)-rich site, SA classified by site.

Various new SD/SA modules were created through mutation and modification of each part. Each module was prepared through nucleotide sequence synthesis and PCR, and the gene expression characteristics of SD/SA were cloned into pGL3-pCMV vector with restriction enzymes NheI and XhoI, and firefly-luciferase activity was quantitatively measured.

4) pLiver ^TM design

AA1 (cloning with restriction enzymes SnaBI/EcoRI), T-bSD/SA (cloning with restriction enzymes KpnI/BglII), T-xzhGH (restriction enzymes XbaI/ Cloning with SacI) was introduced.

As a negative control, that is, green fluorescent protein (GFP), the pSP72-scAAV2-GFP vector was applied.

3. Transformation ( transfection ) and luciferase active measurement

For transient transfection, various cells were cultured in 12-well or 6-well for one day. Various plasmids (100-300 ng/6-well) produced by cloning in cultured cells and plasmids expressing renilla-luciferase (20-50 ng/6-well) were simultaneously cloned in serum-free medium using lipofectamine-2000. After treatment for 4 hours, the medium was changed to a fresh medium containing 10% fetal bovine serum. After about two days, cells were lysed using M-PER™ Mammalian Protein Extraction Reagent (78501, Thermo Scientific™). Firefly-luciferase and renilla-luciferase activities were analyzed using the Dual-Luciferase® Reporter Assay System (E1910, Promega) and VICTOR ^TM X4 Multilabel Plate Reader (PerkinElmer). Luciferase activity was evaluated through relative analysis of renilla-luciferase values based on firefly luciferase values.

4. Recombination adeno-associated virus (recombinant adeno -Associated virus production and transfection

The production of rAAV is described as follows, namely, PEI complexes were prepared for a total of three plasmids for virus production and treated with 293T cells. After about two days, when the toxicity of the cells was evident, both the cells and the culture medium were recovered, and the virus was recovered through a freeze-thaw process repeated 3-4 times, and then aliquoted and stored at -80 ° C. The total number of viral particles was quantitatively analyzed through viral genome-specific quantitative PCR analysis.

5. western blot analyze

First, proteins were separated by reducing SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis), which was then transferred to polyvinylidene fluoride using transfer buffer (20% methanol, 25 mM Tris-HCl, 192 mM glycine). (polyvinylidene fluoride, PVDF) membrane. The membrane onto which the protein was transferred was blocked with TBS (Tris-buffered saline) containing 0.1% tween 20 and 5% fetal bovine serum, followed by reaction with a primary antibody and HRP-conjugated secondary antibody Jackson ImmunoResearch Laboratories, West Grove, PA). A mixture of SuperSignal™ West Pico PLUS Chemiluminescent Substrate (34580, Thermo Scientific™) color reagents Ⅰ and Ⅱ in a 1:1 ratio was applied to the membrane, and protein expression was performed using the biomolecular imaging system LAS-4000 (GE FUJIFILM) degree was observed. Anti-AGXT (HPA035370, Atlas antibodies), GFP (33-2600, Thermo Scientific™), and Anti-beta Actin (AB6276, abcam) antibodies were used in the experiment.

6. ICC analysis

For immunocytochemistry (ICC) analysis, AAV-pLiver ^TM -hAGXT (MOI=235,200) and AD5 were cultured about 4 hours after seeding 0.5×10 ⁵ primary hepatocytes on a 6-well basis. (MOI=5) were infected simultaneously. Two days later, it was immersed in 4% paraformaldehyde (PFA) and fixed at room temperature for 20 minutes, and the cells were washed with phosphate buffered saline (PBS) and then treated with 1% bovine serum albumin (BSA, 1322). , Amresco) for 1 hour, and then treated with Anti-AGXT (HPA035370, Atlas antibodies) at a 1:200 dilution at 4°C overnight (O/N). After washing the culture plate with water, reacting with anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 555 (A-31570, Thermo Scientific™) at room temperature for 1 hour, washing the culture plate again, and fluorescence Observed under a microscope.

As a negative control, primary hepatocytes were treated with primary and secondary antibodies without virus treatment (MT; 1'+2').

7. Statistical Analysis

All results were expressed as mean±standard deviation (SD), and values of *p<0.05, **p<0.01, and ***p<0.001 were considered significant. Statistical analysis was performed using Prism5 (GraphPad, San Diego, CA, USA).

< Example 1> Characterization of human-derived liver-specific synthetic promoters

1. Structure of liver-specific synthetic promoter module

It was constructed by attaching an albumin enhancer to the alpha-1 antitrypsin core promoter and upstream, and named AA1 promoter. The AA1 promoter is a promoter for inducing liver tissue-specific expression, and is based on the phAAT part (alpha-1 antitrypsin core promoter; NCBI sequence ID: D38257.1) among the LP1 promoters of previous papers, and a new Human Albumin Enhancer (NCBI sequence ID: M92816.1) was placed upstream to maximize expression. All of these are human-derived gene expression inducing modules. To experimentally compare their activities, each module was cloned into pGL3, a luciferase expression vector, and an existing LP1 promoter and a truncated form of T-LP1 were additionally constructed to be used as a comparison group (Fig. 1A).

2. Hepatocyte-specific gene expression of liver-specific synthetic promoters induction check

Huh7, HepG2, MRC5, 293T, and HeLa cells were treated with luciferase expression vectors including AA1, LP1, and T-LP1, and expression was confirmed by luciferase assay (FIG. 1B). the results are as follow.

1) AA1 promoter induced luciferase activity very specifically in hepatocytes (Huh7). The degree of activity in Huh7 was similar to that of conventional LP1.

2) The AA1 promoter showed a selective activity only in hepatocytes, whereas the LP1 promoter showed significant activity in other cells besides hepatocytes (293T: human embryonic kidney cells). This suggests that the AA1 promoter is superior to the existing LP1 in hepatocyte-specific expression activity.

3) AA1 has a total length of 427bp, 120bp shorter than that of LP-1, which is 547bp, which is advantageous in terms of gene insertion capacity of AAV-based vectors.

< Example 2> poly A signal module characteristic analysis

1. Newly designed polyA Structure and transformation of signal

In the present invention, based on the consideration of polyadenylation and poly A cleavage, we tried to improve the characteristics based on the polyA signal derived from the human body, rather than the non-human SV40 or bGH (bovine growth hormone) used in many vectors. . To this end, the polyA signal of f-hGH (full-length human growth hormone) was structurally analyzed, and various modified forms were prepared by modifying and minimizing.

First, truncated hGH (T-hGH) with only a portion predicted to be essential for functioning in the polyA signal of human growth hormone with a total length of 476 bp was prepared. T-hGH has a length of 201 bp, which is 275 bp shorter than f-hGH, which is 476 bp. This may be advantageous in terms of the gene insertion capacity of AAV-based vectors.

Next, the T-hGH nucleotide sequence was functionally divided into a total of 5 regions. The hGH polyA signal was divided into x region (in front of polyA signal), y region (middle of polyA signal and cleavage site), and z region (behind cleavage site). x, y. The z part was compared with the seq of bGH, and some bases were switched. In addition, referring to the literature that a specific nucleotide sequence of the y region contributes to inducing stabilization of the polyA signal region, various modules were fabricated by applying a modified nucleotide sequence to the y region. The modified modules are gray boxed (Fig. 2A).

2. polyA Confirmation of gene expression regulation effect of signal

Various modified forms of the polyA signal designed above were cloned into pGL3, a luciferase expression vector, and gene expression activity was confirmed through luciferase assay.

As a result, T-xzhGH showed the highest level of gene expression activity among several variants in which the length was minimized and various modifications were applied to each section, and the level of gene expression activity was comparable to that of conventional f-hGH. By minimizing the length of the polyA signal by about 250 bp less than before, an advantage was secured when applied to AAV-based vectors (Fig. 2B).

< Example 3> Analysis of synthetic splicing donor/acceptor (SD/SA) module characteristics

1. Structure and variant of synthetic enhanced module

In the present invention, in order to increase gene expression, a splicing donor/acceptor portion was introduced. SD/SA was developed based on SD/SA (207 bp) of pSP72-XP87-scAAV2-CMV-GFP (a GFP gene expression vector for AAV developed by the present invention).

A truncated SD/SA (T-SD/SA) was first prepared by extracting only the part predicted to be absolutely necessary to represent the function of SD/SA from a total length of 207 bp. T-SD/SA has a shorter length by 106 bp compared to conventional SD/SA. This can be advantageous in terms of gene insertion capacity when applied to AAV-based vectors.

The T-SD/SA sequence was further analyzed functionally and classified into SD site, branch site, Pyrimidine-rich site (Py-rich site), and SA site. Various new SD/SA modules were created through mutation and modification of each part classified in this way. The modified modules are gray boxed (Fig. 3A).

2. Investigation of gene expression activity of synthetic SD/SA variants

Minimized and variously modified SD/SA modules were cloned into pGL3 where luciferase is expressed, and the activity control function was confirmed by the degree of luciferase expression.

As a result, it was confirmed that among the various modular variants, T-bSD/SA induced stable gene expression at almost the same level as f-SD/SA, so it was decided to utilize this (Fig. 3B).

< Example 4> Through optimized combination of each module liver tissue Specific expression cassette characterization

1. Optimized combination structure of each module

Based on the results of each module obtained above, the promoter, polyA signal, and SD/SA parts were combined in various orders. The degree of gene expression activity was confirmed by cloning luciferase into the gene expression modules created through this (FIG. 4A).

pAA1 (combination of promoter and polyA signal only), pS-AA1 (SD/SA location in front of gene, promoter, SD/SA, gene, polyA signal in order), pAA1-S (SD/SA location behind gene, promoter , gene, SD/SA, polyA signal in order)

2. Investigation of gene expression activity of each combination structure

Through the luciferase assay, the level of luciferase expression by each module was confirmed in cell lines derived from various tissues, and the ability to induce hepatocyte-specific expression was evaluated.

Through this, it was confirmed that the gene expression by pAA1 and pS-AA1 among the modules developed in Huh7, a hepatocyte, specifically increased significantly.

In particular, it was confirmed that gene expression by the pS-AA1 module was the most robust in hepatocyte-specificity, and this module was finally determined as a module for liver-specific gene expression. This was named pLiver ^TM (FIG. 4B).

3. pS-AA1 ( pLiver ^TM ) of primary hepatocytes (primary hepatocyte ) Investigation of specific gene expression activity

Gene expression can be effectively induced in normal hepatocytes, not tumor cells, by testing the liver tissue-specific expression of the pS-AA1 (pLiver ^TM ) module on primary hepatocytes obtained from the liver of C57BL/6 mice. was confirmed (Fig. 4C).

< Example 5> pLiver ^TM Confirmation of dependent therapeutic gene expression

The module developed in the present invention can be loaded into a viral vector and used for the development of a treatment for hereditary liver disease. To confirm this possibility, the following experiment was performed to confirm the actual gene expression in liver tissue by cloning the target gene AGXT for treatment into an AAV vector containing the pS-AA1 (referred to as pLiver ^TM ) module.

1. pS-AA1 ( pLiver ^TM ) - hAGXT Structure of expression vectors

Based on the above results, an AAV-based vector was loaded with pLiver ^TM and the human AGXT (hAGXT) gene was cloned as a target gene to construct a vector capable of expressing hepatocyte-specific hAGXT (FIG. 6). AAV-pLiver ^TM -hAGXT virus was produced through the following AAV production technique. Primary hepatocytes were obtained in the same manner as in FIG. 4C and treated with AAV-pLiver ^TM -hAGXT virus to confirm effective expression of hAGXT. Unlike previous studies, the mice used at this time were AGXT K/O mice, and AAV-GFP virus expressing GFP was applied as a negative control group (FIG. 5A).

2. Primary hepatocytes (primary hepatocytes )at hAGXT Expression confirmation (Western blotting method)

In order to confirm the expression of pLiver ^TM- specific, that is, hepatocyte-specific hereditary-metabolic liver disease treatment gene hAGXT, AGXT K/O cultured primary hepatocytes cultured in 6-well were treated with the virus under the following conditions After two days, the cells were harvested and WB was performed. In order to confirm the virus infection efficiency, an experimental group treated separately with AAV-GFP was prepared. AGXT protein was not expressed in primary hepatocytes obtained from AGXT KO mice (FIG. 5B).

Experiment conditions: ~0.5×10 ⁵ cells/6-well (MOI: AAV=235,200 + AD5=5)

1st antibody: hAGXT: #HAP035370 (Atlas antibodies) 1: 1,000

3. Immunocytochemistry ( Immunocytochemistry ; ICC) analysis hAGXT expression confirmation

Samples were prepared under the same conditions as in FIG. 5B. That is, primary hepatocytes obtained from AGXT KO mice were treated with AAV-pLiver ^TM -hAGXT together with AD5, the cells were fixed, and ICC was performed. In order to confirm the virus infection efficiency, an experimental group treated separately with AAV-GFP was prepared.

The experimental results are as follows (Fig. 5C).

1) Characteristically, GFP protein expression due to AAV virus infection was confirmed only in cells treated with the AAV-GFP negative control virus.

2) In hepatocytes obtained from AGXT KO mice in which AGXT is not expressed, a significant level of AGXT expression was confirmed in both WB and ICC, depending on AAV-pLiver ^TM -hAGXT treatment.

In view of these results, it was proved that the present invention successfully developed a new gene expression vector having liver tissue specificity and improved function. In particular, optimization of expression inducing ability in human cells was sought by using the human-derived promoter and polyA signal as the basis.

Furthermore, by minimizing the length of each module, the fact that the gene insertion capacity was greatly improved when inducing liver-specific gene expression of AAV-based vectors suggests that the use value can be very high in the development of AAV vectors for hereditary-metabolic liver diseases. .

Having described specific parts of the present invention in detail above, it is clear that these specific techniques are only preferred embodiments for those skilled in the art, and the scope of the present invention is not limited thereto. Accordingly, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

<110> THE ASAN FOUNDATION University of Ulsan Foundation For Industry Cooperation <120> Liver-specific gene expression cassette based on human-derived gene expression induction module <130> ADP-2020-0491 <160> 3 <170> KopatentIn 2.0 <210> 1 <211> 427 <212> DNA <213> artificial sequence <220> <223> liver-specific minimal promoter <400> 1 tggactaata ttattctttt catttgttaa atgaaaaagt atgcacaaag tatatgtgag 60 agtgacaaag gcctgaattt gtcaattagt aacaattgta ttcaacagta aggattttat 120 gtttgggtag gcctttccca gggacttcta caaggaaaaa gctagagttg gttactgact 180 tctaataaat aatgcctaca atttctagga agttaaaagt tgaatgactc ctttcggtaa 240 gtgcagtgga agctgtacac tgcccaggca aagcgtccgg gcagcgtagg cgggcgactc 300 agatcccagc cagtggactt agcccctgtt tgctcctccg ataactgggg tgaccttggt 360 taatattcac cagcagcctc ccccgttgcc cctctggatc cactgcttaa atacggacga 420 ggacagg 427 <210> 2 <211> 101 <212> DNA <213> artificial sequence <220> <223> T-bSD/SA <400> 2 ctggtaagtt tagtcttttt gtcttttatt tcaggtcccc gatccggtgg tggtgcaaac 60 atcgacactg ctcctcagtg gatgttgcct ttacttctag g 101 <210> 3 <211> 201 <212> DNA <213> artificial sequence <220> <223> T-xzhGH polyA signal <400> 3 agttgccagc catctgtccc ctcccccgtg ccttccttga ccctggaagg tgccactcca 60 gtgcccactg tccttgtcct aataaaatta agttgcatca ttttgtctga ctaggtgtca 120 ttctattctg gtatggggtg gggtggggca ggacggagca agggggaggt tgggaagaca 180 acctagcagg catgctgggg a 201

Claims (8)

Liver-specific promoter gene represented by SEQ ID NO: 1, splicing donor / acceptor (SD / SA) gene represented by SEQ ID NO: 2, expression target gene, and polyA signal gene represented by SEQ ID NO: 3 Liver-specific gene expression cassette comprising in order. The liver-specific gene expression cassette according to claim 1, wherein the target gene is human alanine-glyoxylate and serine-pyruvate aminotransferase (hAGXT). . A recombinant expression vector comprising the liver-specific gene expression cassette of claim 1 or 2. The recombinant expression vector according to claim 3, wherein the recombinant expression vector is an adeno-associated virus (AAV)-based recombinant expression vector. The recombinant expression vector according to claim 4, wherein the recombinant expression vector has the cleavage map of FIG. 6. A cell line transfected with the recombinant expression vector according to claim 3. Recombinant Adeno-associated virus (rAAV) particles produced from the cell line according to claim 6. A liver-specific gene expression composition comprising the recombinant expression vector according to claim 3 as an active ingredient.

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