Nucleic acid element for enhancing gene expression in liver and applications thereof

Abstract

To provide a liver-targeted gene therapy approach applicable to hemophilia, ornithine transcarbamylase (OTC) deficiency, and other conditions, while ensuring both safety and therapeutic benefit.SOLUTION: The present invention provides an isolated nucleic acid element for enhancing gene expression in liver cells, comprising the full length of intron 1 of the selenoprotein P (SELENOP) gene or a partial sequence thereof that comprises the splicing donor and acceptor sequences; a synthetic promoter in which the nucleic acid element is linked downstream of a functional promoter in liver cells; an expression vector for gene expression in the liver, comprising the synthetic promoter; a gene therapy agent comprising the vector; and a gene therapy that administers the expression vector or gene therapy agent in small doses.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a nucleic acid element that enhances gene expression in the liver, a synthetic promoter that contains the element and is highly expressed in the liver, as well as a liver expression vector (e.g., a viral vector such as adeno-associated virus (AAV)) that contains the promoter, and gene therapy using the vector. [Background technology]

[0002] In recent years, the development of gene therapy drugs using AAV vectors has become active. AAV vectors are currently the most widely used viral vectors because they can efficiently introduce genes into quiescent cells and are safer than other vectors. One of the main organs targeted by AAV vectors is the liver. Genes can be introduced into the liver with high efficiency by intravenous administration of AAV vectors. Gene therapy targeting the liver is intended for many diseases, including hemophilia, a deficiency of coagulation factors, and ornithine transcarbamylase (OTC) deficiency, a urea metabolism disorder.

[0003] However, recently, even AAV vectors, which were believed to be non-pathogenic and highly safe, have been reported to cause serious adverse events, particularly deaths due to severe liver damage, in patients who received high-dose systemic administration via the blood vessels in clinical trials. Therefore, regardless of the type of vector, systemic administration of high-dose vectors can cause serious adverse events, including liver damage, and there is a demand for the development of expression vectors that can highly express therapeutic genes at lower doses.

[0004] Currently, AAV vectors directed to high expression in the liver use the CAG promoter, which is a constitutively high expression promoter, or promoters of liver-specific genes such as transthyretin (TTR) (see, for example, Non-Patent Document 1) and α1-antitrypsin (AAT). In particular, a chimeric promoter HCCRhAAT, in which the liver-type control region (HCR) enhancer of the Apo E / C1 gene is linked upstream of the human AAT promoter, is often used (see, for example, Non-Patent Document 2). [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] N Engl J Med. 2021 Nov 18;385(21):1961-1973. doi: 10.1056 / NEJMoa2104205. [Non-Patent Document 2] N Engl J Med. 2022 Jul 21;387(3):237-247. doi: 10.1056 / NEJMoa2119913. Summary of the Invention [Problem to be solved by the invention]

[0006] Existing liver-specific promoters are not necessarily sufficient when considering both safety and therapeutic effect. Therefore, the object of the present invention is to identify a novel gene expression enhancing nucleic acid element that realizes a sufficient amount of gene expression to achieve a desired therapeutic effect even with a low dose of vector administration, and thus to provide a novel liver-targeting gene therapy means that can achieve both safety and therapeutic effect. [Means for solving the problem]

[0007] In order to achieve the above object, the present inventors first selected genes with high expression in the liver from previously reported proteome analysis data (Cell Proteomics 2016 Oct;15(10):3190-3202. doi: 10.1074 / mcp.M116.060145.) and performed promoter analysis. In the process, they accidentally found that a small insertion of the first intron sequence of the selenoprotein P (SELENOP) gene affects promoter activity. Therefore, they cloned the full length of the first intron (sometimes called "intron 1") and also prepared shortened fragments of various lengths, linked them to previously reported liver-specific promoters, and analyzed their effects of enhancing gene expression in the liver in vitro and in vivo. As a result, it was found that the full length and various fragments of SELENOP intron 1 can significantly enhance the gene expression efficiency of any liver-expressing promoter. Since the effect disappeared when the splicing donor and acceptor sequences were removed, it was revealed that the intron exerts the effect of enhancing gene expression via splicing. Based on these findings, the present inventors conducted further research and have completed the present invention.

[0008] That is, the present invention is as follows. [Section 1] An isolated nucleic acid element for enhancing gene expression in liver cells, comprising the full length of intron 1 of the selenoprotein P (SELENOP) gene or a subsequence thereof including splicing donor and acceptor sequences. [Section 2] Item 2. The nucleic acid element according to Item 1, wherein the entire length of the intron consists of the nucleotide sequence represented by SEQ ID NO: 1 or a variant sequence thereof. [Section 3” 3. The nucleic acid element according to claim 1 or 2, wherein the partial sequence is 50 nucleotides or more in length as a whole. [Section 4] A synthetic promoter comprising the nucleic acid element according to any one of Items 1 to 3 linked downstream of a promoter functional in liver cells. [Section 5] Item 5. The synthetic promoter according to Item 4, wherein the promoter functional in liver cells is selected from the group consisting of a transthyretin (TTR) promoter, an alpha 1-antitrypsin (AAT) promoter, and a chimeric promoter (HCRhAAT) of the human AAT promoter and the liver-type regulatory region (HCR) of the Apo E / C1 gene. [Section 6] Item 6. An expression vector for gene expression in liver cells, comprising the synthetic promoter according to Item 4 or 5. [Section 7] Item 7. The expression vector according to Item 6, comprising a nucleic acid encoding a target protein downstream of a synthetic promoter. [Section 8] Item 8. The expression vector according to Item 7, wherein the target protein is a therapeutic protein. [Section 9] Item 9. The expression vector according to item 8, wherein the therapeutic protein is coagulation factor VIII, coagulation factor IX or ornithine transcarbamylase (OTC). [Section 10] Item 10. The expression vector according to any one of Items 7 to 9, which is a viral vector or a plasmid vector. [Section 11] Item 11. The expression vector according to Item 10, which is a viral vector selected from the group consisting of an adeno-associated viral (AAV) vector, a lentiviral vector, a retroviral vector, an adenoviral vector, a Sindbis viral vector, a rabies viral vector, a Sendai viral vector, and a herpes simplex viral vector. [Section 12] Item 12. A gene therapy agent comprising the expression vector according to any one of Items 8 to 11. [Section 13] A method for expressing a target protein in liver cells, comprising introducing the expression vector according to any one of items 7 to 11 or the gene therapeutic agent according to item 12 into liver cells in vitro. [Section 14] A method for treating a disease caused by a deficiency of a therapeutic protein in a subject in need of expression of the protein in liver cells, comprising administering to the subject a therapeutically effective amount of the expression vector according to any one of items 8 to 11 or the gene therapy agent according to item 12. [Section 15] Item 15. The method according to item 14, wherein the therapeutically effective amount is a lower dose than the therapeutically effective amount in an expression vector comprising a nucleic acid encoding a therapeutic protein downstream of only a promoter functional in liver cells. [Section 16] Item 16. The method according to item 14 or 15, wherein the subject is a human. Effect of the Invention

[0009] According to the present invention, sufficient gene expression in the liver can be achieved even by administration of a low dose of vector, thereby enabling in vivo gene therapy for diseases requiring expression of therapeutic genes in the liver, such as hemophilia and OTC deficiency, while avoiding the risk of adverse events due to high-dose administration and ensuring safety. [Brief description of the drawings]

[0010] [Figure 1]Figure 1 shows that SELENOP intron 1 enhances transgene expression in hepatocytes in vitro. (A) Schematic diagram of the intron sequences used in this study. The indicated intron sequences (SELENOP (1), SELENOP (2), SELENOP (3), SELENOP (full), SERPINA1 (1), SERPINA1 (2), SERPINA1 (3), SERPINA1 (full) or F9 (FIX) intron) were inserted immediately after the HCRhAAT (Apo E / C1 gene liver type control region enhancer and human antitrypsin) promoter in plasmids. (B,C) HEK293 cells (B) or Huh-7 cells (C) were transfected with plasmids driven by the indicated promoters. Luciferase activity in cell lysates was measured using a luminometer and expressed as relative light units. Values ​​are shown as mean ± SD (n=3). Statistical significance was determined by one-way ANOVA with Dunnett's multiple comparison post-hoc test (comparison with HCRhAAT). ns: not significant; ****: P<0.0001. [Diagram 2] Figure 2 shows that SELENOP intron 1 enhances AAV vector-mediated transgene expression in hepatocytes in vitro. (A) Schematic diagram of AAV vectors expressing luciferase driven by HCRhAAT or CAG promoters. The indicated intron sequence (SELENOP (1), SELENOP (2), SELENOP (3), SERPINA1 (1), SERPINA1 (2), SERPINA1 (3) or F9 (FIX) intron) was inserted immediately after the HCRhAAT promoter. (B,C,D) Huh-7 cells (B), HEL293 cells (C) or human umbilical vein endothelial cells (HUVEC) (D) were transfected with the indicated AAV6 vectors at a multiplicity of infection (MOI) of 1 × 104 vector genomes. Luciferase activity in cell lysates was measured using a luminometer and expressed as relative light units. Values ​​are shown as mean ± SD (n = 3). Statistical significance was determined by one-way ANOVA with Dunnett's multiple comparison post-hoc test (comparison with HCRhAAT). ns: not significant; *: P<0.05; ****: P<0.0001. [Diagram 3] FIG. 3 shows that enhanced transgene expression by SELENOP intron 1 requires splicing. The indicated AAV6 vectors carrying luciferase were introduced into Huh-7 cells at a multiplicity of infection (MOI) of 1×104 vector genomes. A schematic diagram of the AAV vectors is shown in the left panel. Luciferase activity in cell lysates was measured with a luminometer and expressed as relative light units. Values ​​are shown as mean ± SD (n=3). (A) Comparison of luciferase expression driven by the HCRhAAT promoter or SELENOP intron 1. (B) SELENOP intron 1 was inserted downstream of HCRhAAT (insertion 5') or SV40 polyA (insertion 3'). (C) Comparison of luciferase expression in the presence and absence of the splice donor and splice acceptor of SELENOP intron 1. Statistical significance was determined by two-tailed Student's test (comparison between two groups) or one-way analysis of variance with Dunnett's multiple comparison post-hoc test (comparison with HCRhAAT). ns: not significant; ***: P<0.001; ****: P<0.0001. [Figure 4]FIG. 4 shows that SELENOP intron 1 enhances AAV vector-mediated transgene expression in liver in vivo. (A) Schematic diagram of AAV vector. (B,C,D) C57BL / 6 mice were treated with AAV8 vector carrying human F9 (clotting factor IX (FIX)) cDNA. (B) Increase in FIX antigen (FIX:Ag) in plasma. (C) F9 mRNA expression in liver assessed by quantitative RT-PCR. (D) AAV genome load in liver assessed by quantitative PCR. (E,F,G) C57BL / 6 mice were treated with AAV8 vector carrying FLAG sequence conjugated with human ornithine transcarbamylase (OTC) cDNA. (E) OTC expression in liver assessed by immunoblotting (left) and quantitative data (right). (F) OTC mRNA expression in liver assessed by quantitative RT-PCR. (G) AAV genome load in liver assessed by quantitative PCR. Values ​​are shown as mean ± SD (n = 3). Statistical significance was tested by two-tailed Student's test (comparison between two groups) or two-way analysis of variance with Sidak's multiple comparison post-hoc test. ns: no significant difference; ***: P<0.001; ****: P<0.0001. [Diagram 5]Figure 5 shows that SELENOP intron 1 enhances transgene expression driven by the mouse transthyretin promoter. (A) Schematic diagram of AAV vectors expressing luciferase driven by mouse transthyretin (mTTRp) with or without the indicated intron sequence (mouse minimal viral intron (MVMi), SELENOP (1), SELENOP (2) or SELENOP (3)). (B) Huh-7 cells were transfected with the indicated AAV6 vectors at a multiplicity of infection (MOI) of 1x104 vg. Luciferase activity in cell lysates was measured in a luminometer and expressed as relative light units. Values ​​are shown as mean ± SD (n=3). Statistical analysis was performed by one-way ANOVA with Dunnett's multiple comparison post-hoc test (compared to mTTR). ***: P<0.001. (C,D) C57BL / 6 mice were treated with AAV8 vectors (1x1010 vector genomes) carrying luciferase driven by mTTRp or SELENOP (3) intron conjugated mTTRp. (C) Representative data from IVIS imaging system 4 weeks after vector administration. (D) Quantitative data expressed in photon units (photons / sec). Values ​​are shown as mean ± SD (n=4). Statistical analysis was performed by two-tailed Student's test. P<0.001; ****: P<0.0001. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] 1. Nucleic acid element containing SELENOP intron 1 sequence The present invention provides an isolated nucleic acid element for enhancing gene expression in liver cells, comprising a full-length or partial sequence of intron 1 of selenoprotein P (SELENOP) gene (hereinafter, sometimes collectively referred to as "SELENOPi sequence"). The present invention is based, at least in part, on the discovery that binding of the SELENOPi sequence downstream of a promoter functional in liver cells significantly enhances gene expression efficiency in the liver compared to the basal promoter activity of the promoter in the liver.

[0012] Selenoprotein P is a protein containing selenocysteine ​​in its molecule and is the major selenium compound found in blood. The protein is encoded by the SELENOP gene (also known as the SEPP1 gene in humans). It is believed that most of the selenoprotein P present in blood is synthesized in the liver, and the SELENOP gene can be said to be a liver-specific gene, but it has not been known until now that the SELENOPi sequence has the effect of enhancing the expression of the gene.

[0013] As shown in the examples described later, the SELENOPi sequence is believed to exert its gene expression enhancing effect through splicing, and therefore, when the sequence is a partial sequence of SELENOP intron 1, the partial sequence is characterized by including a splicing donor sequence (including GT at the 5' end) and a splicing acceptor sequence (including AG at the 3' end).

[0014] Although the SELENOPi sequence alone has weak promoter activity in the liver, its gene expression efficiency is significantly lower than that of existing liver-specific promoters. In addition, since the promoter activity is not significantly enhanced even when the SELENOPi sequence is inserted downstream of the polyA addition signal of the expression vector, the SELENOPi sequence is considered not to have distal enhancer activity.

[0015] The liver is composed of hepatic parenchymal cells (hepatocytes) that are responsible for the main functions of the liver, such as bile production and substance metabolism, as well as non-parenchymal hepatic cells, such as hepatic sinusoidal endothelial cells, Kupffer cells, hepatic stellate cells, pit cells, bile duct epithelial cells, and mesothelial cells. As used herein, "liver cells" refers to one or more of the above cell groups that compose the liver (including cancer cells and cell lines derived from them).

[0016] As used herein, the term "promoter functional in liver cells" refers to a promoter capable of inducing the transcription of a gene (a nucleic acid encoding a protein) linked downstream in liver cells. Considering that the preferred application of the present invention is in vivo gene therapy, it is desirable that the promoter functions specifically in liver cells (induces the transcription of a downstream gene). Here, "specifically in liver cells" and "liver-specific" are used to include cases where expression is limited only to liver cells, as well as cases where expression in liver cells is significantly higher than expression in cells of other organs and tissues. Even when inducing gene expression in cells other than liver cells, for example, by combining with a vector (e.g., various serotypes of viral vectors) having high organ, tissue, and cell tropism, liver-specific gene expression can be substantially achieved (e.g., to the extent that undesirable side effects are not caused).

[0017] As used herein, "enhancing gene expression" means that when the SELENOPi sequence is bound downstream of a promoter functional in liver cells, the expression level of a gene under the control of the resulting synthetic promoter is significantly higher than the expression level of a gene under the control of only the promoter functional in the liver cells.

[0018] As used herein, "isolated" means that the SELENOPi sequence is not present in an intact state as part of the SELENOP gene (genomic DNA) but is provided in a form separate from the rest of the gene.

[0019] The "nucleic acid element" of the present invention may be DNA, RNA, or a DNA / RNA chimera. The nucleic acid element may be single-stranded or double-stranded and may be appropriately selected depending on the application (such as the type of vector used), but is preferably DNA, more preferably double-stranded DNA. The nucleic acid may be a physiologically acceptable salt with an acid or base, for example, a physiologically acceptable acid addition salt is preferred. Examples of such salts include salts with inorganic acids (e.g., hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid) or salts with organic acids (e.g., acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid).

[0020] The SELENOPi sequence contained in the nucleic acid element of the present invention is not particularly limited in origin as long as it has the effect of enhancing gene expression in liver cells of a subject, and may be a sequence derived from intron 1 of the SELENOP gene of a mammal (e.g., human, mouse, rat, dog, cat, cow, horse, pig, sheep, goat, etc.), but is preferably derived from an animal of the same species as the subject. Since the subject of the present invention is preferably a human, the SELENOPi sequence contained in the nucleic acid element of the present invention is preferably the full-length or partial sequence of intron 1 of the human SELENOP gene.

[0021] The full-length nucleotide sequence of human SELENOP intron 1 includes the nucleotide sequence of positions 58 to 3526 of the human SELENOP pre-mRNA sequence (the nucleotide sequence represented by SEQ ID NO: 1), which consists of 12013 nucleotides and is represented by positions 42799880 to 42811892 of the complementary strand of NCBI Reference Sequence: NC_000005.10, or a variant sequence thereof. Here, the term "variant sequence" refers to a corresponding intron 1 sequence in a natural allelic variant or genetic polymorphism (e.g., SNP, etc.) of the RefSeq sequence, or a sequence obtained by artificially modifying such a sequence, which has the same or greater gene expression enhancing effect as the parent sequence. The position of the modified nucleotide is not particularly limited as long as it is other than the splicing donor or splicing acceptor sequence, and examples include modifications (e.g., substitution, deletion, insertion, addition, or a combination thereof) in any one or more nucleotides. For example, the nucleotide sequence may be one in which 1 to 50, preferably 1 to 30, more preferably 1 to 10, and even more preferably 1 to several (2, 3, 4, 5, or 6) nucleotides have been modified from the parent sequence.

[0022] Since the SELENOPi sequence is considered to exert a gene expression enhancing effect via splicing, when the sequence is a partial sequence of SELENOP intron 1, as described above, it is essential that the sequence contains at least the region necessary for splicing (at least the splicing donor and acceptor sequences), but as long as this condition is satisfied, the length of the SELENOPi sequence is not particularly limited. As shown in the Examples described later, the SELENOPi sequence can significantly enhance the basal gene expression efficiency of a functional promoter in liver cells so long as it contains a partial sequence of about 200 bp in total from the 5' and 3' ends of SELENOP intron 1. Thus, the overall length of the SELENOPi sequence in the nucleic acid element of the present invention can be, for example, 50 nucleotides (e.g., 50, 60, 70, 80, 90 nucleotides) or more, preferably 100 nucleotides (e.g., 100, 200, 300, 400 nucleotides) or more, more preferably 500 nucleotides (e.g., 500, 600, 700, 800, 900 nucleotides) or more, and even more preferably 1000 nucleotides (e.g., 1000, 1100, 1200, 1300, 1400, 1500, 2000, 2500, 3000 nucleotides) or more. The length of each partial sequence on the 5'-end (including the splicing donor sequence) and the 3'-end (including the splicing acceptor sequence) is not particularly limited, as long as both partial sequences have a length sufficient to cause splicing of the SELENOPi sequence. For example, each partial sequence on the end may be 20 nucleotides (e.g., 20, 30, 40 nucleotides) or more in length, preferably 50 nucleotides (e.g., 50, 60, 70, 80, 90 nucleotides) or more in length, and more preferably 100 nucleotides (e.g., 100, 110, 120, 130, 140, 150 nucleotides) or more in length. The lengths of both terminal partial sequences may be similar or significantly different; for example, one terminal partial sequence may have a minimum length required for splicing to occur (e.g., 20 to 100 nucleotides) and the other terminal partial sequence may have a length of 200 nucleotides or more (e.g., 200, 300, 400, 500, 1000, 1500, 2000 nucleotides).

[0023] On the other hand, in gene therapy, which is the main purpose of using the nucleic acid element of the present invention, the insert length that can be accommodated by the AAV vector most commonly used at present is small, about 4.7 kb. Therefore, when considering application to AAV vectors, it is unrealistic to use the full length (3469 nucleotides) of SELENOP intron 1, and it is preferable to use a partial sequence thereof. For example, the length of the SELENOPi partial sequence as a whole may be 3000 nucleotides (e.g., 3000, 2500, 2000 nucleotides) or less, preferably 1500 nucleotides (e.g., 1500, 1400, 1300, 1200, 1100, 1000 nucleotides) or less.

[0024] The nucleic acid element of the present invention may contain one or more internal partial sequences of SELENOP intron 1 in addition to the 5'-terminal sequence containing a splicing donor sequence and the 3'-terminal sequence containing a splicing acceptor sequence.

[0025] In addition to the SELENOPi sequence, the nucleic acid element of the present invention may have other nucleotide sequences added to its 5' and / or 3' end, or other nucleotide sequences inserted inside the SELENOPi sequence. For example, a promoter functional in liver cells, as described below, or a linker, adapter, restriction enzyme recognition sequence, etc. for facilitating linkage with a nucleic acid encoding a target protein can be added to each end of the SELENOPi sequence.

[0026] The SELENOP intron 1 sequence can be isolated, for example, by extracting genomic DNA from any cell (e.g., peripheral blood cell, etc.) of a human or other mammal, designing appropriate primers based on the RefSeq sequence information, and performing PCR using the genomic DNA as a template. The SELENOPi partial sequence can be prepared by cloning the full-length intron 1 sequence into an appropriate vector, designing appropriate primers for the 5' and 3' ends of intron 1, performing inverse PCR using the vector as a template, and excising the vector portion from the amplified product with a restriction enzyme or the like, thereby preparing a SELENOPi partial sequence including 5'- and 3'-end sequences.

[0027] Alternatively, the SELENOPi sequence can be prepared by chemically synthesizing a DNA strand of the full-length or partial sequence of intron 1 based on the RefSeq sequence information of the SELENOP gene, or by connecting chemically synthesized partially overlapping oligo DNA short strands using the PCR method or the Gibson Assembly method to construct DNA containing the desired SELENOPi sequence. The advantage of preparing the SELENOPi sequence by chemical synthesis or a combination of the PCR method or the Gibson Assembly method is that when the SELENOPi sequence is synthesized together with the DNA encoding the downstream target protein, the codons used can be designed over the entire length of the CDS in accordance with the cell type (e.g., human liver cells) into which the vector is to be introduced.

[0028] 2. Liver cell-specific synthetic promoter The present invention also provides a synthetic promoter comprising the nucleic acid element of the present invention linked downstream of a promoter functional in liver cells. Here, "promoter functional in liver cells" has the same meaning as above. As long as the promoter contains a minimum nucleotide sequence that exerts basal transcription activity, it may further contain other regulatory sequences (e.g., endogenous proximal or distal enhancer sequences, enhancer sequences derived from other genes, etc.). As used herein, the term "synthetic promoter" refers to an "isolated" promoter that comprises the nucleic acid element of the present invention and is capable of inducing the expression in liver cells of a target gene located downstream, and includes not only promoters that are functional in liver cells derived from a source other than the SELENOP gene (chimeric promoters), but also fragments of the gene that comprise the SELENOP promoter and the SELENOPi sequence for expressing a heterologous gene in liver cells.

[0029] The promoter functional in liver cells is not particularly limited as long as it can induce transcription of downstream genes in liver cells, but is preferably a promoter of a gene highly expressed in the liver, and more preferably a liver-specific promoter. Here, "liver-specific" has the same meaning as above. Examples of promoters functional in liver cells that can be used in the synthetic promoter of the present invention include, but are not limited to, transthyretin (TTR) promoter, α1-antitrypsin (AAT) promoter, albumin promoter, α-fetoprotein promoter, thyroxine-binding globulin promoter, etc. In a preferred embodiment, for example, when the purpose is to use in gene therapy for hemophilia or OTC deficiency using an AAV vector, the TTR promoter or AAT promoter can be used as a promoter functional in liver cells. In another preferred embodiment, a chimeric promoter (HCRhAAT) of the human AAT promoter and the liver-type control region (HCR) of the Apo E / C1 gene can also be used as a promoter functional in liver cells.

[0030] Promoters functional in liver cells can be obtained based on the sequence information of the gene (genomic DNA) from which each promoter is derived, by the same method as described above for SELENOP intron 1. Such sequence information is registered in publicly available databases such as NCBI, EMBL, FASTA, and DDBJ, and is also described in literature. Those skilled in the art can easily access the sequence information.

[0031] As described above, it is believed that the SELENOPi sequence must be spliced ​​in order for the nucleic acid element of the present invention to exert its effect of enhancing gene expression in the liver, and therefore it is desirable to clone a promoter functional in liver cells so that it contains at least a portion of exon 1 from the transcription start point at its 3' end. When a promoter prepared in advance and provided does not contain an endogenous transcription start point and exon 1 or a portion thereof, the SELENOPi sequence can also be cloned so as to contain SELENOP exon 1 at its 5' end, but in terms of ensuring appropriate transcription initiation from the promoter, it is more desirable for the gene from which the promoter is derived to contain an endogenous transcription start point and exon 1 or a portion thereof.

[0032] The promoter functional in liver cells and the nucleic acid element of the present invention can be linked by a method known per se in the art. For example, when each fragment of the promoter and the nucleic acid element has a blunt end, the two fragments can be ligated using DNA ligase. Alternatively, any adapter sequence can be added to the ends of both fragments, treated with an appropriate restriction enzyme to generate sticky ends, and then the two can be ligated. The synthetic promoter thus obtained can be subcloned into an appropriate vector.

[0033] 3. Expression Vector The present invention also provides an expression vector for expressing a gene in a liver cell, comprising the synthetic promoter. The vector into which the synthetic promoter is inserted is not particularly limited as long as it is generally used in gene therapy, and for example, viral vectors such as AAV vectors, lentivirus vectors, retrovirus vectors, adenovirus vectors, Sindbis virus vectors, rabies virus vectors, Sendai virus vectors, and herpes simplex virus vectors, and non-viral vectors such as plasmids for animal cells can be used. From the viewpoints of high gene introduction and expression efficiency, ability to introduce into non-dividing cells, and ability to express the introduced gene for a long time, AAV vectors and lentivirus vectors are preferred, and from the viewpoints of low frequency of chromosomal integration, no risk of insertional mutation, low immunogenicity, and high safety, it is more preferred to use AAV vectors. Although AAV vectors are diluted with cell proliferation in dividing cells, adult hepatocytes hardly divide, so expression of the introduced gene can be maintained for a long period of time. However, since the gene size that can be accommodated is small at 4.7 kb, depending on the size of the target gene to be introduced, it may be preferable to use lentivirus vectors or adenovirus vectors that can accommodate a larger insert.

[0034] For example, when an AAV vector is used as a vector, it is desirable to use a vector derived from a serotype with high liver tropism, such as AAV1, AAV2, AAV3, AAV6, AAV6.2, AAV7, AAV8, AAV9, AAVrh10, AAV-DJ, and AAV-DJ / 8. The procedure for constructing a vector will be described below using an AAV vector as an example. However, those skilled in the art will be able to construct other viral vectors and non-viral vectors (e.g., plasmid vectors) using methods known per se [e.g., Current Protocols in Molecular Biology, F. Ausubel et al. eds. (1994) John Wiley & Sons, Inc.; Molecular Cloning (A Laboratory Manual), 3rd ed. Volumes 1-3, Joseph Sambrook & David W.Russeleds., Cold Spring Harbor Laboratory Press (Cold Spring Harbor, New York) (2001); Culture of Animal Cells; A Manual of Basic Technique, R. Freshney eds., 2nd ed. (1987), Wiley-Liss; Frank L. Graham, Manipulation of adenovirus vector, Chapter 11. p109-p128; EJMurray eds., Methods in Molecular Biology, Vol. 7, Gene Transfer and Expression Protocols (1991); Chen, SH. et al., Combination gene therapy for liver metastases of colon carcinoma in vivo., Proc. Natl. Acad. Sci. USA (1995) 92, 2477-2581, etc.], a desired expression vector can be easily constructed.

[0035] In the preparation of an AAV expression vector, the synthetic promoter of the present invention is first inserted between the 5'- and 3'-ITRs of AAV into a plasmid that can be amplified in a suitable host cell (e.g., Escherichia coli, Bacillus subtilis, yeast, etc.). Examples of such plasmids include plasmids derived from Escherichia coli (e.g., pBR322, pBR325, pUC12, pUC13), plasmids derived from Bacillus subtilis (e.g., pUB110, pTP5, pC194), and plasmids derived from yeast (e.g., pSH19, pSH15). As the AAV ITR, an ITR derived from the desired serotype may be used, but usually an ITR derived from AAV2 can be commonly used. Only the synthetic promoter of the present invention can be inserted between the two ITR sequences, but it is preferable to insert a polyA addition signal (e.g., SV40 polyA addition signal) downstream of it, and it is also preferable to further insert a woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) between the synthetic promoter and the polyA addition signal. A shortened version of the WPRE (0.25 kb; Mol. Brain, 7: 17, 2014) can also be used. Inserting one or more restriction enzyme recognition sites, preferably a multicloning site (MCS), between the synthetic promoter of the present invention and the polyA addition signal (between the synthetic promoter and the WPRE when the WPRE is included) can facilitate the insertion of a nucleic acid encoding a target protein (construction of an expression cassette for a target gene).

[0036] An expression cassette for a target gene is constructed in the plasmid by inserting a nucleic acid encoding a target protein to be expressed in liver cells between the synthetic promoter of the present invention and the polyA addition signal (between the synthetic promoter and WPRE if WPRE is included) of the obtained expression plasmid for AAV vector. That is, the present invention also provides an expression vector comprising a nucleic acid encoding a target protein downstream of the synthetic promoter of the present invention (preferably between the synthetic promoter and the polyA addition signal (between the synthetic promoter and WPRE if WPRE is included)).

[0037] The target protein may be any protein that is desired to be expressed in liver cells, but considering that one of the main uses of the expression vector of the present invention is gene therapy, in a preferred embodiment, the target protein is a therapeutic protein. Here, the term "therapeutic protein" refers to a protein that, when expressed in liver cells of a subject, has the effect of improving symptoms of a disease requiring treatment in the subject, whether directly or indirectly. Examples of therapeutic proteins that can be used in liver-targeting gene therapy include coagulation factor VIII for hemophilia A, coagulation factor IX for hemophilia B, various enzymes for urea cycle disorders (e.g., ornithine transcarbamylase for OTC deficiency, arbamyyl phosphate synthase for CPS1 deficiency, argininosuccinic acid synthase for citrullinemia type I, citrulline for citrullinemia type II, argininosuccinic acid decomposition enzyme for argininosuccinic aciduria, arginase for argininemia, and N-acetylglutamine synthase for NAGS deficiency), and copper transport protein (ATP7B gene) for Wilson's disease. , various enzymes for organic acid metabolism disorders (e.g., propionyl-CoA carboxylase for propionic acidemia, methylmalonyl-CoA mutase for methylmalonic acidemia, branched-chain ketoacid dehydrogenase for maple syrup urine disease, etc.), various proteins for familial intrahepatic cholestasis (PFIC) (e.g., FIC1 (ATP8B1 gene) for PFIC1, BSEP (ABCB11 gene) for PFIC2, MDR3 (ABCB4 gene) for PFIC3, TJP2 for PFIC4, FXR (NR1H4 gene) for PFIC5, etc.), phenylalanine hydroxylase for phenylketonuria, etc., but are not limited to these.

[0038] From the AAV vector expression plasmid containing a nucleic acid encoding a target protein downstream of the synthetic promoter of the present invention, an AAV vector can be produced by a method known per se, for example, a plasmid transfection method, a recombinant baculovirus method, a recombinant herpes virus vector method, a yeast method, etc. For example, in the plasmid transfection method, the AAV vector expression plasmid, a plasmid containing the Rep and Cap genes of AAV, and a pHelper plasmid containing the E2A, E4orf6, and VARNA genes derived from adenovirus can be transfected into HEK293 cells or the like to produce AAV virus particles. The Rep gene does not need to be derived from the target serotype, and the Rep gene derived from AAV2 is generally used. On the other hand, the Cap gene must be derived from the target serotype, and the cell tropism of the AAV serotype is determined by the capsid protein encoded by the Cap gene. That is, in the expression vector of the present invention that targets liver cells, the Cap gene can be derived from AAV1, AAV2, AAV3, AAV6, AAV6.2, AAV7, AAV8, AAV9, AAVrh10, AAV-DJ, AAV-DJ / 8, etc., which have liver tropism.

[0039] On the other hand, when a non-viral vector is used as the expression vector of the present invention, the expression vector can be introduced using a polymer carrier such as a poly-L-lysine-nucleic acid complex or by encapsulation in a liposome. A liposome is a capsule made of phospholipids and has a particle size of several tens to several hundreds of nm, and a plasmid vector can be encapsulated inside it.

[0040] 4. Gene Therapy Agents and Gene Therapy Methods An expression vector comprising a nucleic acid encoding a therapeutic protein downstream of the synthetic promoter of the present invention can be administered to a subject having a disease for which the therapeutic protein exerts a therapeutic effect, thereby treating the disease. Thus, the present invention also provides a gene therapy agent comprising the expression vector of the present invention.

[0041] The gene therapy agent of the present invention may be the expression vector of the present invention used as is, or, if necessary, may be mixed with a pharmacologically acceptable carrier to form various formulations, such as injections, and then used as a medicine.

[0042] Here, as the pharmacologically acceptable carrier, various organic or inorganic carrier substances commonly used as pharmaceutical ingredients are used, and are incorporated in liquid preparations as solvents, solubilizing agents, suspending agents, isotonicity agents, buffering agents, soothing agents, etc. Furthermore, preparation additives such as preservatives, antioxidants, coloring agents, etc. can also be used as necessary.

[0043] Preferable examples of the solvent include water for injection, physiological saline, Ringer's solution, alcohol, propylene glycol, polyethylene glycol, sesame oil, corn oil, olive oil, cottonseed oil, and the like.

[0044] Suitable examples of the solubilizing agent include polyethylene glycol, propylene glycol, D-mannitol, trehalose, benzyl benzoate, ethanol, trisaminomethane, cholesterol, triethanolamine, sodium carbonate, sodium citrate, sodium salicylate, and sodium acetate.

[0045] Suitable examples of suspending agents include surfactants such as stearyl triethanolamine, sodium lauryl sulfate, lauryl aminopropionic acid, lecithin, benzalkonium chloride, benzethonium chloride, and glycerin monostearate; hydrophilic polymers such as polyvinyl alcohol, polyvinylpyrrolidone, sodium carboxymethylcellulose, methylcellulose, hydroxymethylcellulose, hydroxyethylcellulose, and hydroxypropylcellulose; polysorbates; and polyoxyethylene hydrogenated castor oil.

[0046] Preferable examples of the isotonicity agent include sodium chloride, glycerin, D-mannitol, D-sorbitol, glucose, and the like.

[0047] Suitable examples of the buffering agent include buffer solutions such as phosphate, acetate, carbonate, and citrate.

[0048] A suitable example of the soothing agent is benzyl alcohol.

[0049] Suitable examples of the preservative include paraoxybenzoic acid esters, chlorobutanol, benzyl alcohol, phenethyl alcohol, dehydroacetic acid, and sorbic acid.

[0050] Suitable examples of the antioxidant include sulfites and ascorbates.

[0051] Suitable examples of coloring agents include water-soluble edible coal-tar dyes (e.g., food dyes such as Food Red No. 2 and No. 3, Food Yellow No. 4 and No. 5, Food Blue No. 1 and No. 2), water-insoluble lake dyes (e.g., aluminum salts of the above-mentioned water-soluble edible coal-tar dyes), and natural dyes (e.g., β-carotene, chlorophyll, red iron oxide, etc.).

[0052] The dosage form of the pharmaceutical composition includes, for example, parenteral preparations such as injections (e.g., subcutaneous injections, intravenous injections, intramuscular injections, intraperitoneal injections, etc.) and drip infusions.

[0053] The gene therapy agent of the present invention can be produced by a method commonly used in the field of pharmaceutical technology, such as the method described in the Japanese Pharmacopoeia. The content of the viral vector, which is the active ingredient in the formulation, varies depending on the dosage form, the dosage of the active ingredient, etc., but is, for example, about 0.1 to 100% by weight. The viral titer is, for example, 1 x 10 10 ~10 13 It can be appropriately adjusted to about vp / mL, but is not limited to this range.

[0054] Preparations suitable for parenteral administration (e.g., intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal administration, etc.) include aqueous and non-aqueous isotonic sterile injection solutions, which may contain antioxidants, buffers, bacteriostats, isotonicity agents, etc. Also included are aqueous and non-aqueous sterile suspensions, which may contain suspending agents, solubilizers, thickeners, stabilizers, preservatives, etc. The most suitable dosage form in the present invention is an injection solution.

[0055] The dosage of the formulation varies depending on the type of vector, promoter activity (the sum of the basal transcription activity of a promoter functional in liver cells and the gene expression enhancing effect of the nucleic acid element of the present invention), administration route, severity of the disease, the animal species to be administered, the drug tolerance, body weight, age, and the like of the subject to be administered. For example, when an AAV vector (e.g., AAV8) with the HCRhAAT promoter as the basal promoter is used as a coagulation factor IX (FIX) expression vector for hemophilia B, when the AAV vector is administered systemically, particularly via a peripheral vein, a single dose of, for example, about 5×10 12 ~Approx. 5×10 13 At a dose of 100 vp / kg body weight, FIX activity was increased above normal levels. However, by adding the SELENOPi sequence, F9 (FIX) expression increased by about 10-fold at both the mRNA and protein levels, even when the same dose of AAV vector was administered. Therefore, the AAV expression vector of the present invention can increase FIX expression by about 5×10 11 ~Approx. 5×10 12 It is expected that a low dose of vp / kg body weight will have a sufficient therapeutic effect. In recent clinical trials of AAV vectors, a high dose (3 × 10 14 vp / kg body weight) group, deaths due to severe liver damage were reported (Audentes Therapeutics. Letter to the MTM disease community. https: / / myotubulartrust.org / audentes-therapeutics-letter-23-june-2020 / ; Hum Gene Ther 2020; 31: 695-696), but in the same clinical trial, 14vp / kg body weight) group did not experience any adverse events in the liver, despite the presence of liver disease. 14 It is estimated that the gene therapy agent of the present invention can be safely used at a dose of about 1×10 vp / kg body weight or less. 10 ~Approx. 5×10 13 vp / kg body weight, preferably about 1×10 11 ~Approx. 1×10 13 vp / kg body weight, more preferably about 5×10 11 ~Approx. 5×10 12 It can be administered systemically at a dose of vp / kg body weight.

[0056] 5. Induction of gene expression in liver in vitro As shown in the examples below, when liver cells are transfected with an expression vector (plasmid vector and virus vector) containing a nucleic acid encoding a target protein downstream of the synthetic promoter of the present invention, the expression of the target protein is significantly increased compared to the basal transcription activity of a promoter functional in liver cells. Therefore, the present invention also provides a method for expressing a target protein in liver cells, which includes introducing the expression vector of the present invention or the gene therapy agent of the present invention containing a nucleic acid encoding a therapeutic protein as a target gene into liver cells in vitro. In this method, the target protein is not limited to a therapeutic protein, and may be, for example, a reporter protein (e.g., luciferase, green fluorescent protein (GFP), etc.). Such a highly efficient gene introduction / expression system into liver cells in vitro may be useful as a research tool. In addition, in vitro gene introduction using an expression vector expressing a therapeutic protein can be used to elucidate the pathology in liver cells of a target disease. Furthermore, by transplanting liver cells transfected with the gene therapy agent of the present invention into a subject having a target disease of the therapeutic agent, an ex vivo treatment method for the disease can be provided.

[0057] The present invention will be described in more detail below by showing examples. However, these are merely illustrative and do not limit the scope of the present invention in any way. EXAMPLES

[0058] Materials and Methods cell culture HEK293 cells (JRCB Cell Bank), AAVpro 293T cells (Takara Bio) and Huh-7 cells (RIKEN BRC) were maintained in Dulbecco's modified Eagle's medium (DMEM; Sigma Aldcich) supplemented with 10% fetal bovine serum (FBS; Thermo Fischer Scientific) and GlutaMAX (Thermo Fischer Scientific). Human umbilical vein endothelial cells (HUVECs; Cambrex BioScience) were cultured in EGM-2 medium.

[0059] Plasmid constructs and plasmid transfer Genomic DNA fragments of SELENOP intron 1 (3469 bp) or SERPINA1 (human α1-antitrypsin-encoding gene) intron 1 (5318 bp) were cloned into pCRBluntII TOPO (Thermo Fisher Scientific) using PrimeStar HS DNA polymerase (Takara Bio). The shortened introns were generated by inverse PCR (Figure 1). F9 (human clotting factor IX-encoding gene) intron 1 was generated as previously described (J Biol Chem 1995 Mar 10;270(10):5276-81. doi: 10.1074 / jbc.270.10.5276.). The intron fragment was introduced into a pAAV plasmid expressing cDNA [luciferase, coagulation factor IX (FIX) or ornithine transcarbamylase (OTC)] driven by the HCRhAAT promoter (a chimera of the liver-type control region enhancer of the Apo E / C1 gene and the human antitrypsin promoter). The mouse transthyretin (mTTR) promoter and mouse minimal viral intron (MVMi) sequences were synthesized by Eurofins Genomics. Human OTC cDNA was amplified by PCR from human liver RNA reverse transcription cDNA (Takara Bio) using the PrimeScript RT Reagent kit (Takara Bio). A FLAG sequence was introduced into the C-terminus of OTC cDNA using the InFusion cloning kit (Takara Bio). Cells were transfected with the plasmids using Lipofectamine 3000 Reagent (Thermo Fischer Scientific).

[0060] AAV vector production AAV vectors were generated by triple transfection of AAVpro 293T cells (helper-free line) as previously described (Mol Ther. 2013 Feb;21(2):318-23. doi: 10.1038 / mt.2012.258. Epub 2012 Dec 18.). AAV6 and AAV8 vectors were generated for in vitro and in vivo use, respectively. 72 hours after transfection, AAV vectors were purified from transfected cells using the AAVpro Purification Kit (Takara Bio) or ultracentrifugation as previously described (Mol Ther. 2013, supra). The titer of the recombinant AAV vector was measured by quantitative PCR targeting the SV40 polyA sequence (J Gene Med. 2023 Aug;25(8):e3505. doi: 10.1002 / jgm.3505. Epub 2023 Apr 12.). The primer and probe sequences used are shown in Table 1.

[0061] AAV vector transduction and measurement of luciferase activity in vitro The day before transfection, 1 × 10 cells were plated in each well of a 96-well plate coated with type I collagen (Cellmartrix Type IA, Nitta Gelatin). 4 Cells were seeded at a density of 1000 x 1000 cells / well. A vial of vector stock was thawed, diluted in DMEM containing 5% FBS, and added directly to each well. Cells were lysed 72 hours after transfection with 50 μL of 1 × Passive Lysis Buffer (Promega) and stored in a deep freezer. Luciferase activity was measured by a luminometer (Centro LB 960, BERTHOLD Technologies) as previously described (Mol Ther Methods Clin Dev. 2021 Jun 12;22:162-171. doi: 10.1016 / j.omtm.2021.06.004.).

[0062] Animal testing All animal experimental procedures were approved by the Animal Experiment Committee of Jichi Medical University. Animal care was performed in accordance with the committee and the ARRIVE guidelines. Mice were anesthetized with isoflurane (1-3%) and blood samples were collected. Blood was collected from the jugular vein using a 29G microsyringe (Terumo). AAV vectors (100 μL) were administered intravenously via the jugular vein.

[0063] FIX antigen measurement Human coagulation factor IX (hFIX) antigen (FIX:Ag) was measured in mice by ELISA as previously described (Commun Med (Lond). 2023 Apr 19;3(1):56. doi: 10.1038 / s43856-023-00286-w.).

[0064] Quantitative PCR (qPCR) Genomic DNA and RNA from the liver were extracted using the DNeasy Blood & Tissue Kit (QIAGEN) and RNeasy Mini kit (QIAGEN), respectively. RNA samples were reverse transcribed using the PrimeScript RT Reagent kit (Takara Bio). THUNDERBIRD Probe qPCR Mix (Toyobo) or THUNDERBIRD TM Quantitative polymerase chain reaction (qPCR) was performed in a QuantStudio 12K Flex (Thermo Fisher Scientific) using SYBR qPCR Mix (Toyobo). Reactions were analyzed in duplicate, and expression levels were normalized by Hprt1 mRNA levels. The primers and probes used are listed in Table 1.

[0065] [Table 1]

[0066] Immunoblotting Liver specimens were mixed with SDS sample buffer containing 2-mercaptoethanol and boiled for 5 min. Proteins were separated by 12.5% ​​SDS-polyacrylamide gel electrophoresis and transferred to a PVDF membrane. FLAG-conjugated proteins or β-actin were detected with mouse anti-FLAG monoclonal antibody (clone M2, Sigma Aldrich) or mouse anti-β-actin monoclonal antibody (clone C4, Santa Cruz Biotechnology, Santa Cruz). Bound antibodies were detected with peroxidase-labeled anti-mouse IgG antibody (BioRad) and visualized using Immobilon Western Chemiluminescent (Merck Millipore) and ImageQuant LAS4000 digital imaging system (Cytiva). Band intensities of immunoblotting were quantified by ImageQuant TL software (Cytiva) and normalized by the band intensity of the internal control (β-actin).

[0067] Measurement of luciferase activity in mice after AAV vector administration To analyze luciferase expression in the liver in vivo, anesthetized mice were intraperitoneally injected with the substrate luciferin (3 mg). Photons due to luciferase activity were measured using an IVIS® Imaging System and Living Image software (Xenogen Corp.). Quantitative data were expressed in photon units.

[0068] statistical analysis Statistical analysis was performed using Prism 9 software (Graph Pad Software). All data are presented as mean ± standard deviation (SD). Statistical significance was determined by two-tailed Student's test (comparison between two groups), one-way analysis of variance with Dunnett's multiple comparison post-hoc test, or two-way analysis of variance with Sidak's multiple comparison post-hoc test. P < 0.05 was considered statistically significant.

[0069] (result) First, we examined whether the intron sequence had the effect of enhancing gene expression in the liver. We used a plasmid expressing luciferase downstream of the liver-specific promoter HCRhAAT promoter. Intron 1 of SELENOP and SERPINA1, and intron 1 of F9 were inserted downstream of the HCRhAAT promoter (Figure 1A). Although no gene expression enhancing effect was observed in HEK293 cells, gene expression was increased by the insertion of the intron 1 sequence of SELENOP in Huh-7 cells, a liver cell line (Figure 1B). Compared to the full-length intron 1, the fragment also had the effect of enhancing gene expression.

[0070] Next, a similar experiment was performed using an AAV6 vector. Since the gene length that can be inserted into AAV is limited, it is not possible to insert the entire intron sequence, so three types of intron fragments were inserted. As with gene expression by plasmids, insertion of SELENOP intron 1 enhanced gene expression in Huh-7 cells, but not in HEK293 cells or HUVECs (Fig. 2B, 2C, 2D). In particular, the enhancing effect was greater with the SELENOP (2) and SELENOP (3) sequences, which are longer than SELENOP (1). These results demonstrate that the SELENOP intron 1 sequence is effective in increasing the efficiency of gene expression in hepatocytes by AAV vectors.

[0071] To analyze the mechanism of gene expression regulation by the insertion of the SELENOP intron, we examined whether the sequence had promoter activity. Compared to the HCRhAAT promoter, the promoter activity alone was weak (Figure 3A). Furthermore, to examine whether it had enhancer activity, the SELENOP intron was inserted into the 3' side of the polyA sequence. When the SELENOP intron was inserted into the 3' side, the effect of enhancing luciferase expression was weakened (Figure 3B). Next, to examine whether splicing via the intron is important, the splice donor and acceptor sequences at both ends of the intron were eliminated. Under conditions in which splicing does not occur, as shown in Figure 3C, the effect of enhancing luciferase expression was lost. From the above, it is thought that the SELENOP intron exerts its effect on gene expression via splicing.

[0072] Next, we examined whether the in vivo gene therapy effect would actually be improved. We created AAV8 vectors expressing factor IX, which is deficient in hemophilia B, or OTC, which is the most common urea cycle abnormality, downstream of the HCRhAAT promoter (Fig. 4A), and administered them to 7-week-old wild-type mice. The amount of factor IX antigen in the blood and the expression of OTC in the liver were dramatically increased by the insertion of the intron sequence (Fig. 4B, 4E). The intron insertion increased mRNA expression, indicating transcriptional promotion (Fig. 4C, 4F). However, no difference was observed in the genome amount of the AAV vector in the liver (Fig. 4D, 4G).

[0073] Finally, we investigated whether a similar phenomenon would be observed with other liver-specific promoters. Mouse minimal viral intron (MVMi) and SELENOP intron sequences were inserted downstream of the mouse transthyretin promoter sequence (Fig. 5A). Insertion of the SELENOP intron into the transthyretin promoter enhanced luciferase expression in Huh-7 cells by AAV6 vectors (Fig. 5B). No effect of enhancing gene expression was observed with MVMi. An AAV8 vector carrying a similar gene expression cassette was created and administered to wild-type mice, and gene expression was examined by in vivo imaging. As expected, insertion of the SELENOP intron enhanced luciferase activity in vivo (Fig. 5C, 5D). [Industrial Applicability]

[0074] The nucleic acid element of the present invention, the synthetic promoter for expression in liver cells comprising said nucleic acid element, and the expression vector comprising said synthetic promoter exhibit high gene expression efficiency in the liver even at low doses, and are therefore extremely useful in enabling liver-targeted gene therapy that is both safe and effective in treatment.

Claims

1. An isolated nucleic acid element for enhancing gene expression in liver cells, comprising the full length of intron 1 of the selenoprotein P (SELENOP) gene or a subsequence thereof including splicing donor and acceptor sequences.

2. The nucleic acid element according to claim 1 , wherein the entire length of the intron consists of the nucleotide sequence represented by SEQ ID NO: 1 or a variant sequence thereof.

3. The nucleic acid element according to claim 1 or 2, wherein the subsequence is 50 nucleotides or more in length in total.

4. A synthetic promoter comprising the nucleic acid element according to any one of claims 1 to 3 linked downstream of a promoter functional in liver cells.

5. The synthetic promoter of claim 4, wherein the promoter functional in liver cells is selected from the group consisting of a transthyretin (TTR) promoter, an alpha 1-antitrypsin (AAT) promoter, and a chimeric promoter (HCRhAAT) of the human AAT promoter and the liver-type regulatory region (HCR) of the Apo E / C1 gene.

6. 6. An expression vector for gene expression in liver cells, comprising the synthetic promoter according to claim 4 or 5.

7. The expression vector according to claim 6, comprising a nucleic acid encoding a target protein downstream of a synthetic promoter.

8. The expression vector of claim 7 , wherein the target protein is a therapeutic protein.

9. 9. The expression vector of claim 8, wherein the therapeutic protein is coagulation factor VIII, coagulation factor IX or ornithine transcarbamylase (OTC).

10. The expression vector according to any one of claims 7 to 9, which is a viral vector or a plasmid vector.

11. The expression vector according to claim 10, which is a viral vector selected from the group consisting of an adeno-associated viral (AAV) vector, a lentiviral vector, a retroviral vector, an adenoviral vector, a Sindbis viral vector, a rabies viral vector, a Sendai viral vector, and a herpes simplex viral vector.

12. A gene therapy agent comprising the expression vector according to any one of claims 8 to 11.

13. A method for expressing a target protein in liver cells, comprising introducing an expression vector according to any one of claims 7 to 11 or a gene therapy agent according to claim 12 into liver cells in vitro.

14. A method for treating a disease caused by a deficiency of a therapeutic protein in a subject, comprising administering to the subject in need of expression of the therapeutic protein in liver cells a therapeutically effective amount of the expression vector described in any one of claims 8 to 11 or the gene therapy agent described in claim 12.

15. The method of claim 14, wherein the therapeutically effective amount is a lower dose than the therapeutically effective amount in an expression vector comprising a nucleic acid encoding a therapeutic protein downstream of only a promoter functional in liver cells.

16. The method of claim 14 or 15, wherein the subject is a human.

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