Liver-specific nucleic acid regulatory elements and methods and uses thereof

Abstract

The present invention relates to nucleic acid regulatory elements capable of enhancing liver-specific expression of genes, methods using these regulatory elements, and uses of these elements. Expression cassettes and vectors comprising these nucleic acid regulatory elements are also disclosed. The present invention is particularly useful for applications using gene therapy, more particularly liver-directed gene therapy, and for vaccination purposes.

Description

Technical Field

[0001] This invention relates to nucleic acid regulatory elements capable of enhancing liver-specific expression of genes, methods of employing these regulatory elements, and their uses. The invention also includes expression cassettes, vectors, and pharmaceutical compositions comprising these regulatory elements. This invention is particularly applicable to applications of gene therapy, especially liver-directed gene therapy, and for inoculation purposes. Background Art

[0002] Compelling evidence continues to emerge from clinical trials demonstrating the efficacy of gene therapy in patients with a wide variety of diseases. In particular, liver-directed gene therapy is a promising approach to achieving sustained hepatocyte-specific expression of factors secreted into circulation. Indeed, many acquired, complex genetic diseases (liver disease in a narrow sense, as well as some inherited abnormalities that do not directly cause liver disease but manifest primarily elsewhere in the body, such as hemophilia A or B, familial hypercholesterolemia, ornithine carbamoyltransferase deficiency, or antitrypsin deficiency) are associated with altered gene expression in the liver. Furthermore, the liver is frequently susceptible to infection by pathogens such as hepatitis viruses. Finally, the liver can undergo malignant transformation and lead to liver cancer (hepatocellular carcinoma) or functional degeneration due to drug treatments and chemotherapy, drug or alcohol abuse. Therefore, there is significant and growing interest in using gene therapy to express functional genes in the liver to replace desired proteins or, for example, to block the expression of altered or unwanted gene products through RNA interference or dominant-negative repressor proteins, or to restore hepatocyte function in degenerated livers. Liver-directed gene therapy can also be used to express antiviral compounds, such as the VP22 protein fused to the C-terminus of the hepatitis B virus core protein (i.e., a dominant-negative mutant based on the VP22 fusion protein) which can inhibit hepatitis B virus replication, ISG56 and IFITM1 which can inhibit hepatitis C virus replication, and the interferon-induced MyD88 protein which can inhibit hepatitis B virus replication. Transduction of hepatocytes with immunomodulatory cytokines can be used to induce immune responses against, for example, viral hepatitis or liver tumors, or to suppress immune responses to gene delivery vectors or transgenes. Another application of transgene delivery to the liver is for DNA inoculation. Optimization of antigen expression is an important consideration in DNA vaccine vector design. Clearly, transgene expression can be enhanced by using optimized promoters and polyadenylation (polyA) sequences. However, in some cases, such as to express antigens that induce cell death upon overexpression, it may be necessary to optimize the DNA vaccine to produce reduced transgene expression. Tissue specificity is also considered important. Other aspects of vector design can also affect vaccine efficacy. A rational approach to improving the efficacy of DNA vaccination would optimize the following aspects: (i) the vector backbone DNA sequence; (ii) the transgenic sequence; (iii) the co-expression of the stimulating sequence; (iv) the delivery system for the vector; and (v) the targeting of the vector to a suitable immune stimulus (as reviewed in Garmory et al. 2003). The backbone of DNA vaccine vectors can be further modified to enhance immunogenicity by manipulating the DNA to include certain sequences, thereby giving the DNA itself an adjuvant effect.DNA vaccine vectors contain numerous CpG motifs (composed of unmethylated CpG dinucleotides flanked by two 5' purines and two 3' pyrimidines). These CpG motifs generally induce a Th1-like pattern of cytokine production and are thought to lead to the strong CTL responses often seen after DNA vaccination. Responses to DNA vaccine vectors can be enhanced by incorporating CpG motifs into the DNA backbone of plasmids. Alternatively, immune responses can be modulated or enhanced by stimulating the co-expression of molecules or cytokines or by using localization or secretion signals or ligands to direct antigens to sites suitable for immunomodulation. Finally, various routes of administration of DNA vaccines have been investigated, including intramuscular, intradermal, subcutaneous, intravenous, intraperitoneal, oral, vaginal, intranasal, and more recently, non-invasive delivery to the skin.

[0003] Efforts to deliver transgenes to the liver have focused on vectors and plasmids derived from adenoviruses, retroviruses, lentiviruses, and adeno-associated virus (AAV). AAV vectors are currently the most promising gene delivery vectors for liver-directed gene therapy. AAV vectors possess favorable safety profiles and enable sustained transgene expression. Furthermore, several immunologically distinct AAV serotypes have been isolated from humans and non-human primates, and they induce minimal immune responses, making AAV vectors well-suited for liver-directed gene therapy. The main limitation of AAV vectors is the limited packaging capacity of the vector particles (i.e., approximately 4.7 kb), which restricts the size of the transgene expression cassette used to obtain functional vectors.

[0004] However, concerns remain regarding the efficacy and safety of some gene delivery methods. Key limiting factors include insufficient and / or transient transgene expression levels, and inappropriate expression of the transgene in unwanted cell types. Higher vector doses are typically used in gene therapy clinical trials to enhance efficacy. However, in MHC Class I settings, this often triggers T-cell-mediated immune responses against the vector capsid antigens displayed by transduced cells, particularly hepatocytes. This helps eliminate the genetically modified cells and causes hepatotoxicity, resulting in short-lived gene expression. Furthermore, unintended transgene expression in antigen-presenting cells (APCs) increases the risk of unintended immune responses against genetically modified hepatocytes and / or therapeutic transgene products. Therefore, there is a need for improved gene therapy vectors that allow for lower and safer vector doses, enabling sustained hepatocyte-specific expression of therapeutic genes. The availability of more efficient vectors would also alleviate production demands.

[0005] Traditional vector design methods rely on arbitrary trial and error, combining transcriptional enhancers with promoters to improve expression levels. While this approach may sometimes be effective, it often leads to unproductive combinations, resulting in suboptimal or no expression of the gene of interest and / or loss of tissue specificity. Furthermore, these traditional methods fail to consider the importance of incorporating evolutionarily conserved regulatory motifs into the expression module, which is crucial for clinical translation.

[0006] Computational methods relying on a modified distance difference matrix (DDM)-multidimensional permutation (MDS) strategy (DeBleser et al. 2007, Genome Biol 8, R83) have been demonstrated to be useful for the computational (in silico) identification of clusters of evolutionarily conserved transcription factor binding site (TFBS) motifs associated with robust tissue-specific expression in the liver (WO 2009 / 130208) and heart (WO2011 / 051450). It has been shown that combining one of these liver-specific regulatory elements (particularly the serine protease inhibitor (Serpin) enhancer) with a synthetic codon-optimized functionally enhanced FIX transgene (i.e., PaduaR338L) can significantly increase FIX expression and activity (WO 2014 / 064277).

[0007] The purpose of this invention is to further improve the efficacy and safety of liver-guided gene therapy. Summary of the Invention

[0008] The inventors have used computational methods to identify evolutionarily conserved transcription factor binding site (TFBS) motifs associated with highly expressed liver-specific genes as defined herein as nucleic acid regulatory elements. This requires several consecutive computational steps: (1) identifying highly expressed liver-specific genes based on RNA-seq (RNA sequencing) expression data obtained from normal human tissues; (2) extracting corresponding promoter sequences using publicly available databases; (3) using computational methods to identify clusters of transcription factor binding site (TFBS) motifs; and (4) screening the genomic environment of highly expressed genes against evolutionarily conserved clusters of TFBS (i.e., CREs). These regulatory elements for efficient gene expression were subsequently confirmed in vivo. The nucleic acid regulatory elements identified herein allow for the use of lower and therefore safer vector doses in gene therapy while maximizing efficacy.

[0009] Therefore, the present invention provides the following aspects:

[0010] Aspect 1. A nucleic acid regulatory element for enhancing liver-specific gene expression, comprising a sequence selected from the group consisting of SEQ ID NO:6, SEQ ID NO:4, SEQ ID NO:12, SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:16, and SEQ ID NO:20, a sequence having at least 95% identity with any of said sequences, or a functional fragment thereof; wherein the nucleic acid regulatory element comprises at least SP1 and EP300 transcription factor binding sites (TFBS).

[0011] Aspect 2, the nucleic acid regulatory element according to aspect 1, includes a sequence selected from the group consisting of SEQ ID NO:6, SEQ ID NO:4 and SEQ ID NO:12, a sequence having at least 95% identity with any of the sequences, or a functional fragment thereof, wherein the nucleic acid regulatory element further includes TFBS of HNF 4G, TFBS of CEBPB, TFBS of P300, TFBS of HDAC2, TFBS of JUND, TFBS of FOSL2, TFBS of ZBTB7A, TFBS of CEBPD and TFBS of RXRA.

[0012] Aspect 3, the nucleic acid regulatory element according to aspect 1, includes a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:5 and SEQ ID NO:13, a sequence having at least 95% identity with any of the sequences, or a functional fragment thereof; wherein the nucleic acid regulatory element further includes TFBS of POLR2A, TFBS of MYBL2, TFBS of FOXA1, TFBS of FOXA2, TFBS of ARID3A, TFBS of POLR2A and TFBS of HEY1.

[0013] Aspect 4. The nucleic acid regulatory element according to aspect 1 includes a sequence selected from the group consisting of SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:16 and SEQ ID NO:20, a sequence having at least 95% identity with any of the sequences, or a functional fragment thereof; wherein the nucleic acid regulatory element further includes TFBS of HNF 4G, TFBS of CEBPB, TFBS of P300 and TFBS of HNF 4A.

[0014] Aspect 5. A nucleic acid regulatory element according to any one of Aspects 1 to 4, wherein the functional fragment comprises at least 20, preferably at least 25, consecutive nucleotides from a sequence derived therefrom, and wherein the functional fragment comprises at least 1, preferably at least 5, TFBSs present in a sequence derived therefrom.

[0015] Aspect 6: A nucleic acid regulatory element for enhancing liver-specific gene expression, which hybridizes under stringent conditions with a nucleic acid regulatory element or its complement according to any one of Aspects 1 to 5.

[0016] Aspect 7. The nucleic acid regulatory element according to any one of Aspects 1 to 6 has a maximum length of 600 nucleotides, preferably 500 nucleotides, more preferably 400 nucleotides, and even more preferably 300 nucleotides.

[0017] Aspect 8. A nucleic acid expression cassette comprising at least one nucleic acid regulatory element according to any one of Aspects 1 to 7, said at least one nucleic acid regulatory element being operatively connected to a promoter and a transgene.

[0018] Aspect 9, the nucleic acid expression cassette according to aspect 8, further comprising at least one, preferably three, liver-specific regulatory elements different from the nucleic acid regulatory elements according to any one of aspects 1 to 4, wherein preferably the at least one, preferably three, liver-specific regulatory elements comprise SEQ ID NO:22 or a sequence having at least 95% identity with the sequence.

[0019] Aspect 10. The nucleic acid expression cassette according to aspect 9, comprising: a nucleic acid regulatory element including SEQ ID NO:6, SEQ ID NO:4, SEQ ID NO:12 or a sequence having at least 95% identity with any of the sequences; and at least one, preferably three, nucleic acid regulatory elements including SEQ ID NO:22 or a sequence having at least 95% identity with the sequences.

[0020] Aspect 11. The nucleic acid expression cassette according to any one of Aspects 8 to 10, wherein the transgene encodes a therapeutic protein or an immunogenic protein.

[0021] Aspect 12. The nucleic acid expression cassette according to aspect 11, wherein the transgene encodes coagulation factor IX (FIX), preferably wherein the transgene is codon-optimized coagulation factor FIX, or wherein the coagulation factor FIX contains a hyperactivation mutation, preferably wherein the hyperactivation mutation corresponds to an R338L amino acid substitution, more preferably wherein the transgene encoding coagulation factor FIX has a nucleic acid sequence as defined in SEQ ID NO:25.

[0022] Aspect 13. The nucleic acid expression cassette according to aspect 11, wherein the transgene encodes coagulation factor VIII (FVIII), preferably wherein the transgene is codon-optimized coagulation factor FVIII, or wherein the coagulation factor VIII has a deletion of the B domain, preferably wherein the B domain of the FVIII is replaced by a linker as defined in SEQ ID NO:54, more preferably wherein the transgene encoding coagulation factor VIII has a nucleic acid sequence as defined in SEQ ID NO:26.

[0023] Aspect 14. The nucleic acid expression cassette according to any one of Aspects 8 to 13, wherein the promoter is a liver-specific promoter, preferably a promoter derived from the thyroxine transporter (TTR) promoter, and more preferably the minimum promoter (TTRmin) of the thyroxine transporter gene as defined in SEQ ID NO:27.

[0024] Aspect 15, the nucleic acid expression cassette according to aspect 14, further includes a liver-specific regulatory element, which includes SEQ ID NO:24, thereby including a combination of TTRe and TTRm nucleic acids as defined in SEQ ID NO:28.

[0025] Aspect 16. The nucleic acid expression cassette according to any one of Aspects 8 to 15 further includes a mouse parvovirus (MVM) intron, preferably the MVM intron as defined in SEQ ID NO:29.

[0026] Aspect 17. The nucleic acid expression cassette according to any one of Aspects 8 to 16 further includes a transcription termination signal derived from bovine growth hormone polyadenylation signal (BGHpA), preferably BGHpA as defined in SEQ ID NO:30.

[0027] Aspect 18. A vector comprising a nucleic acid regulatory element according to any one of aspects 1 to 7 or a nucleic acid expression cassette according to any one of aspects 8 to 17, wherein the vector is preferably a viral vector, more preferably a vector derived from adeno-associated virus (AAV), and even more preferably a self-complementary AAV vector.

[0028] Aspect 19. The carrier according to aspect 18 has SEQ ID NO:38, SEQ ID NO:36 or SEQ ID NO:44, preferably SEQ ID NO:38.

[0029] Aspect 20: A pharmaceutical composition comprising a nucleic acid expression cassette according to any one of aspects 8 to 17 or a carrier according to aspect 18 or 19, and a pharmaceutically acceptable carrier.

[0030] Aspect 21: A nucleic acid regulatory element according to any one of Aspects 1 to 7, a nucleic acid expression cassette according to any one of Aspects 8 to 17, a vector according to Aspect 18 or 19, or a pharmaceutical composition according to Aspect 20, for use in gene therapy, preferably liver-directed gene therapy, or for use in vaccination therapy, preferably preventive vaccination.

[0031] Aspect 22. The nucleic acid regulatory element, nucleic acid expression cassette, vector, or pharmaceutical composition used according to aspect 21, wherein the gene therapy is used to treat hemophilia A or hemophilia B.

[0032] Aspect 23. An in vitro or ex vivo method for expressing a transgenic product in hepatocytes, comprising:

[0033] —Introduce the nucleic acid expression cassette according to any one of aspects 8 to 17 or the vector according to aspect 18 or 19 into the hepatocytes;

[0034] —The transgenic product is expressed in the liver cells.

[0035] Aspect 24, a nucleic acid regulatory element according to any one of aspects 1 to 7, a nucleic acid expression cassette according to any one of aspects 8 to 17, or a vector according to any one of aspects 18 to 19, for enhancing liver-specific gene expression of transgenic organisms, preferably wherein the use is in vitro.

[0036] Aspect 25. A method for enhancing liver-specific gene expression in a transgenic organism, comprising using a nucleic acid regulatory element according to any one of aspects 1 to 7, a nucleic acid expression cassette according to any one of aspects 8 to 17, or a vector according to any one of aspects 18 or 19. Attached Figure Description

[0037] Figure 1Schematic diagram of the AAVsc constructs. Designs of AAVsc-HS-CRE-TTRenh-TTRm-MVM-hFIXcoPadua-bghpolyA (SEQ ID NO:33-53) (A), AAVsc-TTRenh-TTRm-MVM-hFIXcoPadua-bghpolyA (SEQ ID NO:31) (B), and AAVsc-3xSERP-TTRenh-TTRm-MVM-hFIXcoPadua-bghpolyA (SEQ ID NO:32) (C). SERP: serine protease inhibitor; TTRm: minimal thyroxine carrier protein; TTRenh: TTR enhancer; MVM: mouse parvovirus intron; hFIXcoPadua: codon-optimized human FIX Padua R338L; bghpolyA: polyadenylation signal derived from bovine growth hormone. All constructs used have AAV inverted terminal repeat sequences flanking them.

[0038] Figure 2A comprehensive in vivo comparison of transgene expression levels obtained using constructs containing HS-CRE and controls without HS-CRE. Adult male C57BL / 6 mice were hydrodynamically transfected with AAVsc-HS-CRE1-TTRenh-TTRm-MVM-hFIXcoPadua-bghpolyA (SEQ ID NO:33), AAVsc-HS-CRE2-TTRenh-TTRm-MVM-hFIXcoPadua-bghpolyA (SEQ ID NO:34), AAVsc-HS-CRE3-TTRenh-TTRm-MVM-hFIXcoPadua-bghpolyA (SEQ ID NO:35), AAVsc-HS-CRE4-TTRenh-TTRm-MVM-hFIXcoPadua-bghpolyA (SEQ ID NO:36), and AAVsc-HS-CRE5-TTRenh-TTRm-MVM-hFIXcoPadua-bghpolyA (SEQ ID NO:36). NO:37), AAVsc-HS-CRE6-TTRenh-TTRm-MVM-hFIXcoPadua-bghpolyA (SEQ ID NO:38), AAVsc-HS-CRE7-TTRenh-TTRm-MVM-hFIXcoPadua-bghpolyA (SEQ ID NO: 39), AAVsc-HS-CRE8-TTRenh-TTRm-MVM-hFIXcoPadua-bghpolyA (SEQ ID NO: 40), AAVsc-HS-CRE9-TTRenh-TTRm-MVM-hFIXcoPadua-bghpolyA (SEQ ID NO: 41), AAVsc-HS-CRE10-TTRenh-TTRm-MVM-hFIXcoPadua-bghpolyA (SEQ ID NO: 42), AAVsc-HS-CRE11-TTRenh-TTRm-MVM-hFIXcoPadua-bghpolyA (SEQ ID NO: 43), AAVsc-HS-CRE12-TTRenh-TTRm-MVM-hFIXcoPadua-bghpolyA (SEQ ID NO: 44), AAVsc-HS-CRE13-TTRenh-TTRm-MVM-hFIXcoPadua-bghpolyA (SEQ ID NO: 45), AAVsc-HS-CRE14-TTRenh-TTRm-MVM-hFIXcoPadua-bghpolyA (SEQ IDNO: 46), AAVsc-HS-CRE15-TTRenh-TTRm-MVM-hFIXcoPadua-bghpolyA (SEQ ID NO: 47), AAVsc-HS-CRE16-TTRenh-TTRm-MVM-hFIXcoPadua-bghpolyA (SEQ ID NO: 48), AAVsc-HS-CRE17-TTRenh-TTRm-MVM-hFIXcoPadua-bghpolyA (SEQ ID NO: 49), AAVsc-HS-CRE18-TTRenh-TTRm-MVM-hFIXcoPadua-bghpolyA (SEQ ID NO:50), AAVsc-HS-CRE19-TTRenh-TTRm-MVM-hFIXcoPadua-bghpolyA (SEQ ID NO:51), AAVsc-HS-CRE20-TTRenh-TTRm-MVM-hFIXcoPadua-bghpolyA (SEQ ID NO:52), AAVsc-HS-CRE21-TTRenh-TTRm-MVM-hFIXcoPadua-bghpolyA (SEQ ID NO:53), which are respectively abbreviated as HS-CRE1, HS-CRE2, HS-CRE3, HS-CRE4, HS-CRE5, HS-CRE6, HS-CRE7, HS-CRE8, HS-CRE9, HS-CRE10, HS-CRE11, HS-CRE12, HS-CRE13, HS-CRE14, HS-CRE15, HS-CRE16, HS-CRE17, HS-CRE18, HS-CRE19, HS-CRE20 and HS-CRE21. As controls, AAVsc-3XSERP-TTRenh-TTRm-MVM-hFIXcoPadua-bghpolyA (SEQ ID NO:32) and AAVsc-TTRenh-TTRm-MVM-hFIXcoPadua-bghpolyA (SEQ ID NO:31) were used, denoted as 3XSERP-TTRenh-TTRmin and TTRenh-TTRmin, respectively. On day 1 (Group A) and day 2 (Group B) post-transfection, human FIX-specific ELISA was used to quantify the production of the transgenic product (i.e., human FIX-Padua) in plasma.

[0039] Figure 3 Comparison of FIX levels (measured as ng / ml mouse plasma) in adult C57BL / 6 mice injected via tail vein with 1 x 10 9Vector genome (vg)(A) or 5x10 9 The vectors vg(B)AAV9sc-HS-CRE4-TTRenh-TTRm-MVM-hFIXcoPadua-bghpolyA, AAV9sc-HS-CRE6-TTRenh-TTRm-MVM-hFIXcoPadua-bghpolyA, AAV9sc-TTRenh-TTRm-MVM-hFIXcoPadua-bghpolyA, or AAV9sc-SERP3X-TTRenh-TTRm-MVM-hFIXcoPadua-bghpolyA are represented in abbreviated form as HS-CRE4, HS-CRE6, HS-CRE-free, and 3XSERP, respectively. Blood was collected in citrate buffer for several consecutive days after injection, and the concentration of hFIX antigen in citrate plasma was measured by ELISA. Detailed Description of the Invention

[0041] As used herein, the singular forms “a,” “an,” and “the” include both singular and plural indicators, unless the context clearly indicates otherwise.

[0042] As used herein, the term "comprising" is synonymous with "including" or "containing" and is inclusive or open-ended, not excluding additional, unlisted components, elements, or method steps. These terms also cover "consisting of" and "substantially consisting of," which have explicit meanings in patent terminology.

[0043] The enumeration of numerical ranges by endpoints includes all numerical values ​​and fractions included in the corresponding range, as well as the listed endpoints.

[0044] As used herein when referring to measurable values ​​such as parameters, quantities, time intervals, etc., the term "about" or "approximately" is intended to include the specified value and variations from the specified value, such as the specified value and variations from the specified value by + / -10% or less, preferably + / -5% or less, more preferably + / -1% or less, and still more preferably + / -0.1% or less, within which such variations are suitable to function in the disclosed invention. It should be understood that the values ​​referred to by the modifier "about" are themselves specifically and preferably disclosed.

[0045] Although the terms “one or more” or “at least one” (such as one or more components or at least one component in a set of components) are inherently explicit, by further illustration, the term specifically includes reference to any one of the components or to any two or more of the components (such as any ≥3, ≥4, ≥5, ≥6, or ≥7 of the components, and up to all of the components). In another example, “one or more” or “at least one” may refer to 1, 2, 3, 4, 5, 6, 7, or more.

[0046] To explain the context of this invention, a discussion of the background of the invention is included herein. This should not be construed as an admission that any material involved has been published, is known, or is part of general common knowledge in any country from the priority date of any claim.

[0047] Throughout this disclosure, references are made to various publications, patents, and published patent specifications by way of identification. All references cited in this specification are incorporated herein by reference in their entirety. Specifically, the teachings or sections of such references specifically mentioned herein are incorporated by reference.

[0048] Unless otherwise defined, all terms used in disclosing this invention, including technical and scientific terms, shall have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains. Further guidance includes terminology definitions to better understand the teachings of this invention. When specific terms are defined with respect to a particular aspect or embodiment of the invention, such meaning is intended to apply throughout that specification, and also in the context of other aspects or embodiments of the invention, unless otherwise defined.

[0049] In the following paragraphs, different aspects or embodiments of the invention are defined in more detail. Each aspect or embodiment thus defined may be combined with any other aspect or embodiment unless expressly indicated to the contrary. Specifically, any feature indicated as preferred or advantageous may be combined with any other feature indicated as preferred or advantageous.

[0050] Throughout this specification, the reference to "an embodiment" or "an embodiment" means that a particular feature, structure, or characteristic described with respect to that embodiment is included in at least one embodiment of the invention. Therefore, the appearance of the phrase "in one embodiment" or "in an embodiment" in various places throughout this specification does not necessarily refer to the same embodiment, but may refer to the same embodiment. Furthermore, particular features, structures, or characteristics can be combined in any suitable manner in one or more embodiments, as will be apparent to those skilled in the art upon reading this disclosure. Moreover, while some embodiments described herein include certain features but not others included in other embodiments, as will be understood by those skilled in the art, combinations of features from different embodiments are intended to be within the scope of the invention and form different embodiments. For example, in the appended claims, any of the claimed embodiments can be used in any combination.

[0051] The terms and definitions provided herein are for the purpose of understanding the invention. Unless specifically defined herein, all terms used herein have the same meaning as defined by one of ordinary skill in the art to which this invention pertains. For definitions and terms in this art, practitioners will pay particular attention to: Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Press, Plainsview, New York (1989); and Ausubel et al., Current Protocols in Molecular Biology (Supplement 47), John Wiley & Sons, New York (1999).

[0052] In one aspect, the present invention relates to a nucleic acid regulatory element for enhancing liver-specific gene expression, comprising, substantially consisting of (i.e., the regulatory element may, for example, additionally include sequences for cloning purposes, but the indicated sequences constitute a fundamental part of the regulatory element, for example, they do not form part of a larger regulatory region such as a promoter) or consisting of: selected from SEQ ID NO:6, SEQ ID NO:4, SEQ ID NO:12, SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID The sequence consisting of the group NO:21; or the sequence having at least 80%, preferably at least 85%, more preferably at least 90%, or even more preferably at least 95% (such as 95%, 96%, 97%, 98%, or 99%) identity with any of these sequences; or a functional segment thereof (i.e., a functional segment of a sequence selected from the group consisting of SEQ ID NO:6, SEQ ID NO:4, SEQ ID NO:12, SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, or a functional segment of a sequence having a high percentage sequence identity with any of the sequences and still performing the function of the regulatory element (i.e., enhancing expression)).

[0053] As used herein, “nucleic acid regulatory elements” or “regulatory elements,” also referred to as “CREs” (cis-regulatory elements), “CRMs” (cis-regulatory modules), or “HS-CREs” (hepatocyte-specific cis-regulatory elements), are transcriptional control elements capable of regulating and / or controlling gene transcription, particularly tissue-specific transcription of genes, and especially non-coding cis-acting transcriptional control elements. Regulatory elements include at least one transcription factor binding site (TFBS), more particularly at least one binding site for tissue-specific transcription factors, and most particularly at least one binding site for liver-specific transcription factors. Typically, regulatory elements, as used herein, enhance or amplify promoter-driven gene expression compared to transcription from a single promoter without a regulatory element. Therefore, regulatory elements specifically include enhancer sequences; however, it should be understood that regulatory elements that enhance transcription are not limited to typical distal upstream enhancer sequences but can appear at any distance from the gene they regulate. In fact, it is known in the art that sequences regulating transcription can be located upstream (e.g., in the promoter region) or downstream (e.g., in the 3'UTR) of the gene they regulate in vivo, and can be located in immediate or distant regions of the gene. It is noteworthy that although regulatory elements as disclosed herein typically comprise naturally occurring sequences, combinations (partial) or multiple copies of such regulatory elements (i.e., regulatory elements including non-naturally occurring sequences) are also contemplated as regulatory elements on their own. Regulatory elements as used herein may include a portion of a larger sequence involved in transcriptional control, such as a portion of a promoter sequence. However, a regulatory element alone is generally insufficient to initiate transcription, and a promoter is required for this purpose. The regulatory elements disclosed herein are provided as nucleic acid molecules, i.e., isolated nucleic acids or isolated nucleic acid molecules. Thus, the nucleic acid regulatory elements have only a small fraction of the naturally occurring genomic sequence, and are therefore not naturally occurring, but rather sequences isolated from the naturally occurring genomic sequence.

[0054] As used herein, the term "nucleic acid" generally refers to oligomers or polymers (preferably linear polymers) of any length that are essentially composed of nucleotides. A nucleotide unit typically comprises a heterocyclic base, a glycosyl group, and at least one (e.g., one, two, or three) phosphate group, including modified or substituted phosphate groups. Heterocyclic bases may in particular include purine and pyrimidine bases of adenine (A), guanine (G), cytosine (C), thymine (T), and uracil (U), which are widely distributed in naturally occurring nucleic acids; other naturally occurring bases (e.g., xanthine, inosine, hypoxanthine); and chemically or biochemically modified (e.g., methylated) non-natural or derived bases. Glycosyl groups may in particular include pentose (furanopentose) groups, such as ribose and / or 2-deoxyribose, or arabinose, 2-deoxyarabinose, threonose, or hexose glycosyl groups, which are preferably common in naturally occurring nucleic acids; and modified or substituted glycosyl groups. Nucleic acids as used herein may include naturally occurring nucleotides, modified nucleotides, or mixtures thereof. Modified nucleotides may include modified heterocyclic bases, modified sugar moieties, modified phosphate groups, or combinations thereof. Modifications to phosphate groups or sugars may be introduced to improve stability, resistance to enzymatic degradation, or some other useful property. The term "nucleic acid" further preferably includes DNA, RNA, and DNA / RNA hybrid molecules, specifically including hnRNA, pre-mRNA, mRNA, cDNA, genomic DNA, amplification products, oligonucleotides, and synthetic (e.g., chemically synthesized) DNA, RNA, or DNA / RNA hybrids. Nucleic acids may be naturally occurring, such as those found in or isolated from nature; or they may be non-natural, such as recombinants, i.e., produced by recombinant DNA technology, and and / or partially or wholly chemically or biochemically synthesized. "Nucleic acid" may be double-stranded, partially double-stranded, or single-stranded. In the case of single-stranded nucleic acids, the nucleic acid may be a sense strand or an antisense strand. Additionally, nucleic acids may be circular or linear.

[0055] As used in this article, “transcription factor binding site,” “transcription factor binding sequence,” or “TFBS” refers to the sequence of the nucleic acid region to which a transcription factor binds. Non-limiting examples of TFBS include the following protein and factor binding sites: E-box binding proteins E47 and E12; CCAAT / enhancer binding proteins (also known as CEB / P, C / EPB, or CEPB); hepatocyte nuclear factor 1 homeobox A (also known as HNF-1A, HNF1, IDDM20, LFB1, MODY3, TCF-1, or TCF); interferon regulatory factor 1 (also known as IRF1, IRF-1, or MAR); lymphocyte enhancer binding factor 1 (also known as LEF1, LEF-1, TCF10, TCF1ALPHA, or TCF7L3); forkhead box protein O4 (also known as FOXO4, AFX, AFX1, or MLLT7); forkhead box protein O1 (also known as forkhead in rhabdomyosarcoma, FOXO1, FKH1, FKHR, or FOXO1A). These can be found in, for example... Transcription factor binding sites were found in databases such as [database name missing].

[0056] The sequences disclosed herein may be part of sequences of regulatory elements that control the transcription of liver-specific genes in vivo, particularly those controlling the transcription of the following genes: albumin (also known as ALB), apolipoprotein A2 (also known as ApoA2), apolipoprotein C2 (also known as ApoC2), apolipoprotein C1 (also known as ApoC1), apolipoprotein A1 (also known as ApoA1), apolipoprotein C3 (also known as ApoC3, APOCIII), transthyretin (also known as TTR), retinaldehyde-binding protein 4 (also known as RBP4), cytochrome P450 2E1 (also known as CYP2E1), and α-1-acid glycoprotein 1 (also known as serum mucin 1 or ORM1).

[0057] Therefore, in embodiments, the nucleic acid regulatory elements disclosed herein include sequences from ALB regulatory elements, which are regulatory elements controlling the expression of ALB genes in vivo, such as regulatory elements comprising SEQ ID NO:1 and SEQ ID NO:2 or functional fragments thereof as described elsewhere herein. In embodiments, the nucleic acid regulatory elements disclosed herein include sequences from APOA2 regulatory elements, which are regulatory elements controlling the expression of APOA2 genes in vivo, such as regulatory elements comprising SEQ ID NO:3 and SEQ ID NO:4 or functional fragments thereof as described elsewhere herein. In embodiments, the nucleic acid regulatory elements disclosed herein include sequences from TTR regulatory elements, which are regulatory elements controlling the expression of TTR genes in vivo, such as regulatory elements comprising SEQ ID NO:5 or functional fragments thereof as described elsewhere herein. In the embodiments, the nucleic acid regulatory elements disclosed herein include sequences from APOC1 regulatory elements, which are regulatory elements controlling the expression of the APOC1 gene in vivo, such as regulatory elements of SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11 or functional fragments thereof as described elsewhere herein. In the embodiments, the nucleic acid regulatory elements disclosed herein include sequences from RBP4 regulatory elements, which are regulatory elements controlling the expression of the RBP4 gene in vivo, such as regulatory elements of SEQ ID NO:12, SEQ ID NO:13 or functional fragments thereof as described elsewhere herein. In the embodiments, the nucleic acid regulatory elements disclosed herein include sequences from APOC3 regulatory elements, which are regulatory elements controlling the expression of the APOC3 gene in vivo, such as regulatory elements of SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18 or functional fragments thereof as described elsewhere herein. In the implementation scheme, the nucleic acid regulatory elements disclosed herein include sequences from CYP2E1 regulatory elements, which are regulatory elements that control the expression of the CYP2E1 gene in vivo, such as regulatory elements including SEQ ID NO:19 or its functional fragments as described elsewhere in this document.In the implementation scheme, the nucleic acid regulatory elements disclosed herein include sequences from ORM1 regulatory elements, which are regulatory elements that control the expression of the ORM1 gene in vivo, such as regulatory elements including SEQ ID NO:20, SEQ ID NO:21 or their functional fragments as described elsewhere in this document.

[0058] Nucleic acid regulatory elements including SEQ ID NO:6, SEQ ID NO:4, SEQ ID NO:12, SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:16 or SEQ ID NO:20 include the TFBS of SP1 and the TFBS of EP300.

[0059] Nucleic acid regulatory elements including SEQ ID NO:6, SEQ ID NO:4, and SEQ ID NO:12 include TFBS of SP1, TFBS of EP300, TFBS of HNF 4G, TFBS of CEBPB, TFBS of P300, TFBS of HDAC2, TFBS of JUND, TFBS of FOSL2, TFBS of ZBTB7A, TFBS of CEBPD, and TFBS of RXRA.

[0060] Nucleic acid regulatory elements including SEQ ID NO:1, SEQ ID NO:5 and SEQ ID NO:13 include TFBS of SP1, TFBS of EP300, TFBS of POLR2A, TFBS of MYBL2, TFBS of FOXA1, TFBS of FOXA2, TFBS of ARID3A, TFBS of POLR2A and TFBS of HEY1.

[0061] Nucleic acid regulatory elements including SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:16 and SEQ ID NO:20 include TFBS of SP1, TFBS of EP300, TFBS of HNF 4G, TFBS of CEBPB, TFBS of P300 and TFBS of HNF 4A.

[0062] As used herein, the terms “sequence identity” and “identical sequence,” etc., refer to the degree of sequence identity or similarity between two polymer molecules (e.g., between two nucleic acid molecules, such as two DNA molecules). Sequence alignment and determination of sequence identity can be accomplished, for example, using a basic local alignment search tool (BLAST) originally described by Altschul et al. 1990 (J Mol Biol 215:403-10) (such as the “Blast 2 sequence” algorithm described by Tatusova and Madden 1999 (FEMS Microbiol Lett 174:247-250). Typically, percentage sequence identity is calculated over the entire length of the sequence. As used herein, the term “substantially identical” means at least 90%, preferably at least 95% (such as 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0063] As used herein, the term "functional fragment" refers to a segment of the regulatory element sequence disclosed herein that retains the ability to regulate liver-specific expression; that is, it still confers tissue specificity and it is able to regulate (trans)gene expression in the same manner (although possibly not to the same extent) as the sequences from which it is derived. Functional fragments may preferably comprise at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 120, at least 150, at least 200, at least 250, at least 300, at least 350, or at least 400 consecutive nucleotides from the sequences from which they are derived. Also preferably, functional fragments may comprise at least one, more preferably at least two, at least three, or at least four, or even more preferably at least five or all of the transcription factor binding sites (TFBS) present in the sequences from which they are derived.

[0064] As used herein, the term "liver-specific expression" refers to the preferential or significant expression of a (transgenic) gene (such as RNA and / or polypeptide) in the liver, liver tissue, or hepatocytes compared to other (i.e., non-liver) tissues or cells. According to a particular embodiment, at least 50%, more particularly at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% of the (transgenic) gene expression occurs within liver tissue or hepatocytes. According to a particular embodiment, liver-specific expression requires that the expressed gene product not "leaks" into other organs or tissues besides the liver, such as the lungs, muscles, brain, kidneys, and / or spleen.

[0065] With appropriate modifications, the above also applies to hepatocyte-specific expression and hepatoblast-specific expression, which can be considered specific forms of liver-specific expression. Throughout this application, whenever liver-specificity is mentioned in the context of expression, hepatocyte-specific expression and hepatoblast-specific expression are explicitly envisioned.

[0066] As used herein, the term "hepatocyte" includes the cells that predominate in the liver and primarily includes hepatocytes, oval cells, hepatic sinusoidal endothelial cells (LSEC), and bile duct cells (epithelial cells that form the bile ducts).

[0067] As used herein, the term "hepatocyte" refers to a cell that differentiates from a progenitor hepatocyte and is able to express a liver-specific phenotype under appropriate conditions. The term "hepatocyte" also refers to dedifferentiated hepatocytes. This term includes cells in vivo and cells cultured in vitro, whether primary or passaged cells.

[0068] As used herein, the term "hepatocyte" refers to embryonic cells in the mesoderm that differentiate into hepatocytes, oval cells, or bile duct cells. This term includes cells in vivo as well as cells cultured in vitro, whether primary or passaged.

[0069] In embodiments, the present invention relates to a nucleic acid regulatory element for enhancing liver-specific gene expression, comprising, substantially consisting of, or consisting of functional fragments of the following sequences: sequences selected from the group consisting of SEQ ID NO:6, SEQ ID NO:4, SEQ ID NO:12, SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, and SEQ ID NO:21; and sequences having at least 80%, preferably at least 85%, more preferably at least 90%, and even more preferably at least 95% (such as 95%, 96%, 97%, 98%, or 99%) identity with any of these sequences. The “functional fragment” of the regulatory element identified herein is defined as comprising at least 20, preferably at least 25, more preferably at least 50, at least 100, at least 200 or at least 250 consecutive nucleotides from a sequence derived therefrom, and / or is defined as comprising at least 1, preferably at least 5, more preferably all of the transcription factor binding sites (TFBSs) present in a sequence derived therefrom.

[0070] In a further embodiment, the present invention provides a nucleic acid regulatory element for enhancing liver-specific gene expression, comprising, substantially comprising, or comprising the following: a sequence selected from the group consisting of SEQ ID NO:6, SEQ ID NO:4, SEQ ID NO:12, SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:16, and SEQ ID NO:20; a sequence having at least 80%, preferably at least 85%, more preferably at least 90%, and even more preferably at least 95% (such as 95%, 96%, 97%, 98%, or 99%) identity with any of these sequences; or a functional fragment thereof. The “functional fragment” of the regulatory element identified herein is defined as including the TFBS of SP1 and the TFBS of EP300, and / or including at least 20, preferably at least 25, more preferably at least 50, at least 100, at least 200 or at least 250 consecutive nucleotides from the sequence derived therefrom of the functional fragment, and / or is defined as including at least 1, preferably at least 5, more preferably all of the TFBS present in the sequence derived therefrom of the functional fragment.

[0071] In a further embodiment, the present invention provides a nucleic acid regulatory element for enhancing liver-specific gene expression, comprising, substantially comprising, or comprising: a sequence selected from the group consisting of SEQ ID NO:6, SEQ ID NO:4, SEQ ID NO:12, SEQ ID NO:1, SEQ ID NO:5, and SEQ ID NO:13; a sequence having at least 80%, preferably at least 85%, more preferably at least 90%, and even more preferably at least 95% (such as 95%, 96%, 97%, 98%, or 99%) identity with any of these sequences; or a functional fragment thereof. The “functional fragment” of the regulatory element identified herein is defined as comprising at least SP1 TFBS and EP300 TFBS, and / or is defined as comprising at least 20, preferably at least 25, more preferably at least 50, at least 100, at least 200, or at least 250 consecutive nucleotides from a sequence derived therefrom of the functional fragment, and / or is defined as comprising at least one, preferably all, of the TFBS present in the sequence derived therefrom of the functional fragment.

[0072] In a further embodiment, the present invention provides a nucleic acid regulatory element for enhancing liver-specific gene expression, comprising, substantially comprising, or comprising: a sequence selected from the group consisting of SEQ ID NO:6, SEQ ID NO:4, and SEQ ID NO:12; or a sequence having at least 80%, preferably at least 85%, more preferably at least 90%, or even more preferably at least 95% (such as 95%, 96%, 97%, 98%, or 99%) identity with any of these sequences; or a functional fragment thereof, wherein the nucleic acid regulatory element comprises TFBS of SP1, TFBS of EP300, or HNF. The TFBS of 4G, TFBS of CEBPB, TFBS of P300, TFBS of HDAC2, TFBS of JUND, TFBS of FOSL2, TFBS of ZBTB7A, TFBS of CEBPD, and TFBS of RXRA; and wherein the functional fragment comprises at least 20, preferably at least 25, more preferably at least 50, at least 100, at least 200, or at least 250 consecutive nucleotides derived therefrom from the functional fragment, and wherein the functional fragment comprises at least 1, preferably at least 5, more preferably at least 10, or at least 15 TFBSs present in the sequence derived therefrom from the functional fragment.

[0073] In a further embodiment, the present invention provides a nucleic acid regulatory element for enhancing liver-specific gene expression, comprising, substantially comprising, or comprising of: selected from SEQ ID NO:1, SEQ ID NO:5, and SEQ ID NO:6. The sequence of group NO:13; or a sequence having at least 80%, preferably at least 85%, more preferably at least 90%, or even more preferably at least 95% (such as 95%, 96%, 97%, 98%, or 99%) identity with any of these sequences; or a functional fragment thereof, wherein the nucleic acid regulatory element comprises TFBS of SP1, TFBS of EP300, TFBS of POLR2A, TFBS of MYBL2, TFBS of FOXA1, TFBS of FOXA2, TFBS of ARID3A, TFBS of POLR2A, and TFBS of HEY1; and wherein the functional fragment comprises at least 20, preferably at least 25, more preferably at least 50, at least 100, at least 200, or at least 250 consecutive nucleotides from a sequence derived therefrom, and wherein the functional fragment comprises at least 1, preferably at least 5, more preferably at least 10, or at least 15 TFBSs present in a sequence derived therefrom.

[0074] In a further embodiment, the present invention provides a nucleic acid regulatory element for enhancing liver-specific gene expression, comprising, substantially comprising, or comprising the following: a sequence selected from the group consisting of SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:16, and SEQ ID NO:20; or a sequence having at least 80%, preferably at least 85%, more preferably at least 90%, or even more preferably at least 95% (such as 95%, 96%, 97%, 98%, or 99%) identity with any of these sequences; or a functional fragment thereof, wherein the nucleic acid regulatory element comprises TFBS of SP1, TFBS of EP300, TFBS of HNF 4G, TFBS of CEBPB, TFBS of P300, and HNF 4A TFBS; and wherein the functional fragment comprises at least 20, preferably at least 25, more preferably at least 50, at least 100, at least 200 or at least 250 consecutive nucleotides from a sequence derived therefrom, and wherein the functional fragment comprises at least 1, preferably at least 5, more preferably at least 10 or at least 15 TFBSs present in a sequence derived therefrom.

[0075] In a further embodiment, the present invention provides a nucleic acid regulatory element for enhancing liver-specific gene expression, comprising, substantially comprising, or comprising the following: a sequence selected from the group consisting of SEQ ID NO:6; or a sequence having at least 80%, preferably at least 85%, more preferably at least 90%, or even more preferably at least 95% (such as 95%, 96%, 97%, 98%, or 99%) identity with SEQ ID NO:6; or a functional fragment thereof as described herein.

[0076] In a further embodiment, the present invention provides a nucleic acid regulatory element for enhancing liver-specific gene expression, comprising, substantially comprising, or comprising the following: a sequence selected from the group consisting of SEQ ID NO:4; or a sequence having at least 80%, preferably at least 85%, more preferably at least 90%, or even more preferably at least 95% (such as 95%, 96%, 97%, 98%, or 99%) identity with SEQ ID NO:4; or a functional fragment thereof as described herein.

[0077] In a further embodiment, the present invention provides a nucleic acid regulatory element for enhancing liver-specific gene expression, comprising, substantially comprising, or comprising the following: a sequence selected from the group consisting of SEQ ID NO:12; or a sequence having at least 80%, preferably at least 85%, more preferably at least 90%, or even more preferably at least 95% (such as 95%, 96%, 97%, 98%, or 99%) identity with SEQ ID NO:12; or a functional fragment thereof as described herein.

[0078] Nucleic acid regulatory elements comprising artificial sequences can also be created by combining two or more (e.g., two, three, four, five or more) identical or different sequences disclosed herein or their functional fragments as described herein.

[0079] Therefore, in some embodiments, a nucleic acid regulatory element for enhancing liver-specific gene expression is provided, comprising at least two sequences selected from SEQ ID NO:6, SEQ ID NO:4, SEQ ID NO:12, SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, or sequences having at least 90%, preferably at least 95% (such as 95%, 96%, 97%, 98%, or 99%) identity with any of these sequences, or functional fragments thereof, preferably selected from SEQ ID NO:6, SEQ ID NO:4, SEQ ID NO:12, SEQ ID NO:1, SEQ ID NO:2 ... SEQ ID NO:5 and SEQ ID NO:13, or sequences having at least 90%, preferably at least 95% (such as 95%, 96%, 97%, 98% or 99%) identity with any of these sequences, or the group consisting of their functional segments as described herein, more preferably selected from SEQ ID NO:6, SEQ ID NO:4, SEQ ID NO:12, or sequences having at least 90%, preferably at least 95% (such as 95%, 96%, 97%, 98% or 99%) identity with any of these sequences, or the group consisting of their functional segments as described elsewhere herein.

[0080] For example, this document discloses nucleic acid regulatory elements comprising SEQ ID NO:6, SEQ ID NO:4, and SEQ ID NO:12, substantially composed of or composed of SEQ ID NO:6, SEQ ID NO:4, and SEQ ID NO:12; nucleic acid regulatory elements comprising 2, 3, 4, or 5 repeat sequences of SEQ ID NO:6 (e.g., tandem repeat sequences), substantially composed of 2, 3, 4, or 5 repeat sequences of SEQ ID NO:6 (e.g., tandem repeat sequences), substantially composed of 2, 3, 4, or 5 repeat sequences of SEQ ID NO:6 (e.g., tandem repeat sequences); nucleic acid regulatory elements comprising 2, 3, 4, or 5 repeat sequences of SEQ ID NO:4 (e.g., tandem repeat sequences), substantially composed of 2, 3, 4, or 5 repeat sequences of SEQ ID NO:4 (e.g., tandem repeat sequences), substantially composed of 2, 3, 4, or 5 repeat sequences of SEQ ID NO:4 (e.g., tandem repeat sequences); or nucleic acid regulatory elements comprising SEQ ID NO:6, SEQ ID NO:4, and SEQ ID NO:12; Nucleic acid regulatory elements consisting of 2, 3, 4, or 5 repeat sequences (e.g., tandem repeat sequences) of SEQ ID NO:12, substantially consisting of 2, 3, 4, or 5 repeat sequences (e.g., tandem repeat sequences) of SEQ ID NO:12, or consisting of 2, 3, 4, or 5 repeat sequences (e.g., tandem repeat sequences) of SEQ ID NO:12. Nucleic acid regulatory elements comprising artificial sequences can also be fabricated by combining one or more (e.g., one, two, three, four, five, or more) sequences disclosed herein or their functional fragments as described herein with one or more liver-specific regulatory elements known in the art. Non-limiting examples of known liver-specific regulatory elements are disclosed in WO2009 / 130208, WO 2016 / 146757, or WO01 / 98482, which are incorporated herein by reference in their entirety.

[0081] Particularly preferred examples of liver-specific regulatory elements used in combination with the nucleic acid regulatory elements disclosed herein are nucleic acid regulatory elements derived from the SERPINA1 gene promoter, and more particularly, regulatory elements comprising a sequence as defined in SEQ ID NO:22, a sequence having at least 85%, preferably at least 90%, more preferably at least 95% (such as 96%, 97%, 98%, or 99%) identity with said sequence, or a functional fragment thereof as described herein. Liver-specific regulatory elements comprising sequences as defined in SEQ ID NO:22 are referred herein to as “serine protease inhibitor enhancer,” “SerpEnh,” or “Serp.”

[0082] Therefore, in some embodiments, a nucleic acid regulatory element for enhancing liver-specific gene expression is provided, comprising the sequence SEQ ID NO:22, a sequence having at least 85%, preferably at least 90%, more preferably at least 95% (such as 96%, 97%, 98%, or 99%) identity with said sequence or a functional fragment thereof as described herein, and at least one or two or more (e.g., two, three, four, five, or more) identical or different sequences, wherein said at least one or two or more identical or different sequences are selected from SEQ ID NO:6, SEQ ID NO:4, SEQ ID NO:12, SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:19, SEQ ID NO:19, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:19 ... NO:21 or a sequence having at least 90%, preferably at least 95% (such as 95%, 96%, 97%, 98%, or 99%) identity with any of these sequences, or a group consisting of their functional segments as described herein; preferably selected from SEQ ID NO:6, SEQ ID NO:4, SEQ ID NO:12, SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:20 or a sequence having at least 90%, preferably at least 95% (such as 95%, 96%, 97%, 98%, or 99%) identity with any of these sequences, or a group consisting of their functional segments as described herein; more preferably selected from SEQ ID NO:6, SEQ ID NO:4, SEQ ID NO:12, SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:20 ... NO:13 or a sequence having at least 90%, preferably at least 95% (such as 95%, 96%, 97%, 98% or 99%) identity with any of these sequences or a group consisting of their functional segments as described herein; more preferably a sequence selected from SEQ ID NO:6 or a sequence having at least 90%, preferably at least 95% (such as 95%, 96%, 97%, 98% or 99%) identity with any of these sequences or a group consisting of their functional segments as described herein;Even more preferably, the sequence is selected from SEQ ID NO:6, SEQ ID NO:4, and SEQ ID NO:12, or sequences having at least 90%, preferably at least 95% (such as 95%, 96%, 97%, 98%, or 99%) identity with any of these sequences, or the group consisting of their functional segments as described herein; more preferably, the sequence is selected from SEQ ID NO:6, or sequences having at least 90%, preferably at least 95% (such as 95%, 96%, 97%, 98%, or 99%) identity with any of these sequences, or their functional segments as described herein.

[0083] In a particular embodiment, a nucleic acid regulatory element for enhancing liver-specific gene expression is provided, comprising, substantially comprising, or comprising: SEQ ID NO:22, more particularly three repeat sequences (e.g., tandem repeat sequences) of SEQ ID NO:22 (Serp), and SEQ ID NO:6. In a more particular embodiment, a nucleic acid regulatory element for enhancing liver-specific gene expression is provided, comprising SEQ ID NO:23 (3xSerp) and SEQ ID NO:6, substantially comprising, or comprising SEQ ID NO:23 (3xSerp) and SEQ ID NO:6.

[0084] In a particular embodiment, a nucleic acid regulatory element for enhancing liver-specific gene expression is provided, comprising, substantially comprising, or comprising: SEQ ID NO:22, more particularly three repeat sequences (e.g., tandem repeat sequences) of SEQ ID NO:22 (Serp), and SEQ ID NO:4. In a more particular embodiment, a nucleic acid regulatory element for enhancing liver-specific gene expression is provided, comprising SEQ ID NO:23 (3xSerp) and SEQ ID NO:4, substantially comprising, or comprising SEQ ID NO:23 (3xSerp) and SEQ ID NO:4.

[0085] In a particular embodiment, a nucleic acid regulatory element for enhancing liver-specific gene expression is provided, comprising, substantially comprising, or comprising: SEQ ID NO:22, more particularly three repeat sequences (e.g., tandem repeat sequences) of SEQ ID NO:22 (Serp), and SEQ ID NO:12. In a more particular embodiment, a nucleic acid regulatory element for enhancing liver-specific gene expression is provided, comprising SEQ ID NO:23 (3xSerp) and SEQ ID NO:12, substantially comprising, or comprising SEQ ID NO:23 (3xSerp) and SEQ ID NO:12.

[0086] Another particularly preferred example of a liver-specific regulatory element for use in combination with the nucleic acid regulatory elements disclosed herein is a “TTR enhancer,” “TTRe,” or “TTREnh,” more particularly a regulatory element comprising a sequence as defined in SEQ ID NO:24, a sequence having at least 85%, preferably at least 90%, more preferably at least 95% (such as 96%, 97%, 98%, or 99%) identity with said sequence, or a functional fragment thereof as described herein. Therefore, in some embodiments, a nucleic acid regulatory element for enhancing liver-specific gene expression is provided, comprising the sequence SEQ ID NO:24, a sequence having at least 85%, preferably at least 90%, more preferably at least 95% (such as 96%, 97%, 98%, or 99%) identity with said sequence or a functional fragment thereof as described herein, and at least one or two or more (e.g., two, three, four, five, or more) identical or different sequences, wherein said at least one or two or more identical or different sequences are selected from SEQ ID NO:6, SEQ ID NO:4, SEQ ID NO:12, SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20 ... NO:21 or a sequence having at least 90%, preferably at least 95% (such as 95%, 96%, 97%, 98%, or 99%) identity with any of these sequences, or a group consisting of their functional segments as described herein; preferably selected from SEQ ID NO:6, SEQ ID NO:4, SEQ ID NO:12, SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:20 or a sequence having at least 90%, preferably at least 95% (such as 95%, 96%, 97%, 98%, or 99%) identity with any of these sequences, or a group consisting of their functional segments as described herein;More preferably, the sequence is selected from SEQ ID NO:6, SEQ ID NO:4, SEQ ID NO:12, SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:13, or sequences having at least 90%, preferably at least 95% (such as 95%, 96%, 97%, 98%, or 99%) identity with any of these sequences, or the group consisting of their functional segments as described herein; more preferably, the sequence is selected from SEQ ID NO:6 or sequences having at least 90%, preferably at least 95% (such as 95%, 96%, 97%, 98%, or 99%) identity with any of these sequences, or the group consisting of their functional segments as described herein; even more preferably, the sequence is selected from SEQ ID NO:6, SEQ ID NO:4, and SEQ ID NO:12, or sequences having at least 90%, preferably at least 95% (such as 95%, 96%, 97%, 98%, or 99%) identity with any of these sequences, or the group consisting of their functional segments as described herein; more preferably, the sequence is selected from SEQ ID NO:13. NO:6 or a sequence having at least 90%, preferably at least 95% (such as 95%, 96%, 97%, 98%, or 99%) identity with any of these sequences, or a functional fragment thereof as described herein.

[0087] Another particularly preferred example of a liver-specific regulatory element for use in combination with the nucleic acid regulatory elements disclosed herein is a regulatory element comprising a “liver locus control element” as described in WO 01 / 98482 (which is incorporated herein by reference), more particularly a regulatory element comprising a sequence as defined in SEQ ID NO:57 or SEQ ID NO:58, a sequence having at least 85%, preferably at least 90%, more preferably at least 95% (such as 96%, 97%, 98% or 99%) identity with said sequence, or a functional fragment thereof as described herein.

[0088] Another particularly preferred example of a liver-specific regulatory element for use in combination with the nucleic acid regulatory elements disclosed herein is a regulatory element comprising at least one, preferably three tandem repeat sequences of a serine protease inhibitor enhancer (e.g., SEQ ID NO:22), such as a regulatory element comprising SEQ ID NO:23 (3xSerp) and a thyroxine carrier protein enhancer (TTRe) as defined in SEQ ID NO:24. Examples of liver-specific regulatory elements comprising at least one, preferably three tandem repeat sequences of a serine protease inhibitor enhancer and TTRe are described in WO 2016 / 146757, which is incorporated herein by reference in its entirety.

[0089] One or more liver-specific regulatory elements known in the art may be located upstream or downstream of one or more (e.g., one, two, three, four, five or more) nucleic acid regulatory elements disclosed herein. Preferably, one or more (e.g., one, two, three, four, five or more) nucleic acid regulatory elements disclosed herein, or their functional fragments as described herein, are located upstream of one or more liver-specific regulatory elements known in the art.

[0090] Specific examples of nucleic acid regulatory elements including artificial sequences include regulatory elements obtained by rearranging transcription factor binding sites (TFBSs) present in the sequences disclosed herein. Such rearrangements may include altering the order of the TFBSs and / or altering the position of one or more TFBSs relative to other TFBSs and / or altering the copy number of one or more TFBSs. For example, nucleic acid regulatory elements for enhancing liver-specific gene expression are also disclosed herein, including ATF3, AR1D3A, 2BTB7A, BHLHE40, CEBPB, CEBPD, CHD2, CTCF, EP300, ELF1, EZH2, ESRRA, FOXA1, FOXA2, FOSL2, HEY1, GATA2, GABPA, HEY1, HDAC2, HNF4A, HNF Binding sites for 4G, HSF1, JUN, JUND, MAFF, MAFK, MAX, MYBL2, MBD4, MAZ, MXI1, MYC, NF1C, NR2C2, NR2F2, POLR2A, PO12, p300, PPARGC1A, RAD21, RXR5, RXRA, RCOR1, REST, SMC3, SUZ12, SHC3, SP1, SRF, SREBP1, SIN3AK20, TAF1, TCF7L2, TBP, TEAD4, TBP, TCF12, TCF4, USF1, USF2, YY1, ZBTB7A, and / or ZBTB33. In other examples, these nucleic acid regulatory elements include at least two (e.g., 2, 3, 4, or more) copies of one or more of the TFBS.

[0091] If the regulatory element is provided as a single-stranded nucleic acid, for example when using a single-stranded AAV vector, the complementary strand is considered to be equal to the published sequence. Therefore, this document also discloses nucleic acid regulatory elements for enhancing liver-specific gene expression, comprising, substantially comprising, or comprising the following: sequences described herein, especially sequences selected from SEQ ID NO:6, SEQ ID NO:4, SEQ ID NO:12, SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, sequences having at least 80%, preferably at least 85%, more preferably at least 90%, even more preferably at least 95% (such as 95%, 96%, 97%, 98%, or 99%) identity with any of these sequences, or sequences comprising the group of functional fragments as described herein.

[0092] This document also discloses nucleic acid regulatory elements for enhancing liver-specific gene expression, which hybridize under stringent conditions with the nucleic acid regulatory elements described herein, particularly with nucleic acid regulatory elements comprising, substantially comprising, or consisting of, or their complements thereof: sequences selected from SEQ ID NO:6, SEQ ID NO:4, SEQ ID NO:12, SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, sequences having at least 90%, preferably at least 95% (such as 95%, 96%, 97%, 98%, or 99%) identity with any of these sequences, or sequences comprising the group of functional fragments as described herein. The nucleic acid regulatory elements do not need to have the same length as the sequences they hybridize with. In a preferred embodiment, the size of the hybridized nucleic acid regulatory elements does not differ from the hybridized sequence by more than 25% of the length, particularly 20% of the length, more particularly 15% of the length, and most particularly 10% of the length.

[0093] The phrase "hybridization under stringent conditions" refers to the ability of a nucleic acid molecule to hybridize with a target nucleic acid molecule under specific temperature and salt concentration conditions. Typically, stringent hybridization conditions are defined as 25°C to 30°C below the melting temperature (Tm) of the native duplex (e.g., 20°C, 15°C, 10°C, or 5°C). Methods for calculating Tm are well known in the art. As a non-limiting example, typical salt and temperature conditions for achieving stringent hybridization are: 1x SSC, 0.5% SDS at 65°C. The abbreviation SSC refers to the buffer used in the nucleic acid hybridization solution. One liter of 20x (twenty-fold concentrated) stock SSC buffer solution (pH 7.0) contains 175.3 g of sodium chloride and 88.2 g of sodium citrate. The typical time length for achieving hybridization is 12 hours.

[0094] Preferably, the regulatory elements described herein, while having only a limited length, possess full functionality. This allows them to be used in vectors or nucleic acid expression cassettes without unduly limiting their effective loading capacity. Therefore, in embodiments, the regulatory elements disclosed herein are nucleic acids of 600 or fewer nucleotides, 500 or fewer nucleotides, 400 or fewer nucleotides, 300 or fewer nucleotides, 250 or fewer nucleotides, 200 or fewer nucleotides, 190 or fewer nucleotides, or 180 or fewer nucleotides, preferably 500 or fewer nucleotides, more preferably 300 or fewer nucleotides.

[0095] However, it should be understood that the disclosed nucleic acid regulatory elements retain regulatory activity (i.e., with regard to specificity and / or transcriptional activity) and therefore they specifically have a minimum length of 20, 25, 30, 35, 40, 45, 50, 100, 150, 160, 170 or all nucleotides of the corresponding sequence of the regulatory element as defined in SEQ ID NO:1 to 21.

[0096] In some embodiments, the present invention provides nucleic acid regulatory elements of 600 or fewer, 500 or fewer, 400 or fewer, 300 or fewer, 250 or fewer, 200 or fewer, 190 or fewer, or 180 or fewer, preferably 500 or fewer, more preferably 300 or fewer, for enhancing liver-specific gene expression, comprising nucleic acid regulatory elements selected from SEQ ID NO:6, SEQ ID NO:4, SEQ ID NO:12, SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:19, SEQ ID NO:19, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:19 ... Sequences comprising the group consisting of IDNO:21, sequences having at least 80%, preferably at least 85%, more preferably at least 90%, even more preferably at least 95% (such as 95%, 96%, 97%, 98%, or 99%) identity with any of these sequences, or functional fragments thereof as described herein. The nucleic acid regulatory elements disclosed herein can be used in nucleic acid expression cassettes. Therefore, in one aspect, the present invention provides for the use of nucleic acid regulatory elements as described herein in nucleic acid expression cassettes.

[0097] In one aspect, the present invention provides a nucleic acid expression cassette comprising a nucleic acid regulatory element as described herein, the nucleic acid regulatory element being operatively linked to a promoter. In an embodiment, the nucleic acid expression cassette does not contain a transgene. Such a nucleic acid expression cassette can be used to drive the expression of an endogenous gene. In a preferred embodiment, the nucleic acid expression cassette comprises a nucleic acid regulatory element as described herein, the nucleic acid regulatory element being operatively linked to a promoter and a transgene.

[0098] As used herein, the term "nucleic acid expression cassette" refers to a nucleic acid molecule that includes one or more transcriptional control elements (such as, but not limited to, promoters, enhancers and / or regulatory elements, polyadenylated sequences, and introns) that guide (transgenic) gene expression in one or more desired cell types, tissues, or organs. Typically, they also contain transgenes, but it is also envisioned that the nucleic acid expression cassette guides the expression of endogenous genes in cells in which the cassette is inserted.

[0099] As used herein, the term "operably linked" refers to the arrangement of various nucleic acid molecular elements relative to each other such that these elements are functionally linked and able to interact with each other. These elements may include, but are not limited to, promoters, enhancers and / or regulatory elements, polyadenylated sequences, one or more introns and / or exons, and the coding sequence of the gene of interest to be expressed (i.e., transgene). When properly oriented or operably linked, nucleic acid sequence elements work together to modulate each other's activity and ultimately affect the expression level of the transgene. Modulation refers to increasing, decreasing, or maintaining the activity level of a particular element. The position of each element relative to other elements can be represented by the 5' end and 3' end of each element, and the distance between any particular elements can be referred to by the number of nucleotides or base pairs between the elements. As understood by those skilled in the art, operably linked implies functional activity and is not necessarily related to the natural position of the link. In practice, when used in nucleic acid expression cassettes, regulatory elements are typically located immediately upstream of the promoter (although this is usually the case, it should absolutely not be interpreted as a restriction or exclusion of a position within the nucleic acid expression cassette), but this need not be the case in vivo. For example, regulatory element sequences naturally located downstream of a gene (which affect the transcription of that gene) can function in the same way as when located upstream of a promoter. Therefore, depending on the specific implementation, the regulatory or enhancing effect of a regulatory element is location-independent.

[0100] In certain embodiments, the nucleic acid expression cassette includes one nucleic acid regulatory element or its functional fragment as described herein. In alternative embodiments, the nucleic acid expression cassette includes two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) nucleic acid regulatory elements or their functional fragments as described herein, i.e., they are modularly combined to enhance their regulatory (and / or enhancing) effects. In other alternative embodiments, the nucleic acid expression cassette includes: at least one nucleic acid regulatory element or its functional fragment as described herein; and at least one liver-specific nucleic acid regulatory element or its functional fragment as described herein known in the art. In further embodiments, at least two of the two or more nucleic acid regulatory elements or their functional fragments as described herein are identical or substantially identical. In even further embodiments, all of the two or more regulatory elements or their functional fragments as described herein are identical or substantially identical. Copies of identical or substantially identical nucleic acid regulatory elements or their functional fragments as described herein may be provided as tandem repeat sequences in the nucleic acid expression cassette. In further alternative embodiments, two or more nucleic acid regulatory elements, or at least two of their functional fragments as described herein, are different from each other. The nucleic acid expression cassette may also include combinations of identical and substantially identical nucleic acid regulatory elements, or their functional fragments as described herein, and different nucleic acid regulatory elements, or their functional fragments as described herein.

[0101] For example, a nucleic acid expression cassette may include a nucleic acid regulation element comprising SEQ ID NO:6 and a nucleic acid regulation element comprising SEQ ID NO:22 (SERP); a nucleic acid expression cassette may include a nucleic acid regulation element comprising SEQ ID NO:6 and 2, 3, or 4 nucleic acid regulation elements comprising SEQ ID NO:22 (SERP); a nucleic acid expression cassette may include 2, 3, or 4 nucleic acid regulation elements comprising SEQ ID NO:6 and SEQ ID NO:22 (SERP); a nucleic acid expression cassette may include 2, 3, or 4 nucleic acid regulation elements comprising SEQ ID NO:6 and 2, 3, or 4 nucleic acid regulation elements comprising SEQ ID NO:22 (SERP). Similarly, a nucleic acid expression cassette may include 2, 3, or 4 nucleic acid regulation elements comprising SEQ ID NO:6 and a nucleic acid regulation element comprising SEQ ID NO:23 (3xSERP).

[0102] As used herein, the term "promoter" refers to a nucleic acid sequence that directly or indirectly regulates the transcription of a corresponding nucleic acid coding sequence (e.g., a transgene or endogenous gene) to which it is operatively linked. A promoter may function alone to regulate transcription or may function in cooperation with one or more other regulatory sequences (e.g., enhancers or silencers, or regulatory elements). In the context of this application, a promoter is generally operatively linked to a regulatory element as disclosed herein to regulate the transcription of a (trans)gene. When a regulatory element as described herein is operatively linked to both a promoter and a transgene, the regulatory element may (1) confer a significant degree of liver-specific expression of the transgene in vivo (and / or specific expression in hepatoblasts, hepatocytes, oval cells, cholangioblasts, liver stem cells, or in vitro liver-derived cell lines) and / or (2) enhance the expression level of the transgene in the liver and / or in hepatoblasts, hepatocytes, oval cells, liver stem cells, or cholangioblasts (in vivo) or in cell lines derived therefrom (in vitro).

[0103] The promoter can be homologous (i.e., from the same species as the animal, especially a mammal, to be transfected with the nucleic acid expression cassette) or heterologous (i.e., from a source other than the animal, especially a mammal, to be transfected with the expression cassette). Accordingly, the promoter can be from any virus, any single-celled prokaryote or eukaryote, any vertebrate or invertebrate, or any plant, or even a synthetic promoter (i.e., having a non-naturally occurring sequence), as long as the promoter functions in combination with the regulatory elements described herein. In a preferred embodiment, the promoter is a mammalian promoter, especially a murine or human promoter.

[0104] The promoter can be an inductive or constitutive promoter.

[0105] The enrichment of liver-specific TFBS in the nucleic acid regulatory elements disclosed herein allows, in principle, the regulatory elements to guide liver-specific expression, even by promoters that themselves do not possess liver specificity. Therefore, the regulatory elements disclosed herein can be used in nucleic acid expression cassettes in combination with their natural promoters and another promoter. Preferably, the nucleic acid expression cassettes disclosed herein include liver-specific promoters. This will improve liver specificity and / or prevent leakage of expression from other tissues. Non-limiting examples of liver-specific promoters are provided in the Liver-Specific Gene Promoter Database (LSPD, http: / / rulai.cshl.edu / LSPD / ), including, for example, albumin (ALB) promoter, apolipoprotein A2 (APOA2) promoter, thyroxine transporter (TTR) promoter, apolipoprotein C1 (APOC1) promoter, retinal binding protein 4 (RBP4) promoter, apolipoprotein C3 (APOC3) promoter, and cytochrome P450. The promoters include: CYP2E1 (CYP2E1), α-1-acid glycoprotein 1 (ORM1), serine protease inhibitor A1 (SERPINA1), apolipoprotein A1 (APOA1), complementary factor B (CFB), hexokinase (KHK), hemobinin (HPX), nicotinamide N-methyltransferase (NNMT), (liver) carboxylesterase 1 (CES1), protein C (PROC), apolipoprotein C3 (APOC3), mannan-binding lectin serine protease 2 (MASP2), hepcidin antimicrobial peptide (HAMP), serine protease inhibitor peptidase inhibitor class C (antithrombin) member 1 (SERPINC1), and α-1-antitrypsin (AAT).

[0106] In a particularly preferred embodiment, the promoter is a mammalian liver-specific promoter, especially a murine or human liver-specific promoter.

[0107] In a preferred embodiment, the promoter is derived from a thyroxine transporter gene, particularly a murine or human thyroxine transporter gene, such as the minimal thyroxine transporter promoter (TTRm) as defined in SEQ ID NO:27.

[0108] In a particular embodiment, if a nucleic acid regulatory element for enhancing liver-specific gene expression, as taught herein, is combined with a TTRe enhancer (e.g., as defined in SEQ ID NO:24), the promoter is preferably TTRm (e.g., as defined in SEQ ID NO:27), and the combination of the TTRe and TTRm nucleic acid components is preferably as described in SEQ ID NO:28. Therefore, in a particular embodiment, the promoter is a thyroxine transporter (TTR) promoter, thereby comprising a combination of TTRe and TTRm nucleic acids as defined in SEQ ID NO:28.

[0109] In a particular embodiment, the promoter is preferably an AAT promoter if the nucleic acid regulatory element for enhancing liver-specific gene expression as taught herein is combined with a “liver locus control element” as described in WO 01 / 98482 (which is incorporated herein by reference), and more particularly with a regulatory element comprising a sequence as defined in SEQ ID NO:57 or SEQ ID NO:58.

[0110] Furthermore, the promoter does not need to be the promoter of the transgene in the nucleic acid expression cassette, although the transgene can be transcribed from its own promoter.

[0111] To minimize the length of the nucleic acid expression cassette, regulatory elements can be attached to a minimal promoter or a shortened form of the promoter described herein. As used herein, a “minimal promoter” (also referred to as a basal promoter or core promoter) is part of a full-size promoter that still drives expression but lacks at least a portion of a sequence that contributes to the regulation (e.g., tissue-specific) of expression. This definition encompasses: promoters with (tissue-specific) regulatory elements deleted, which are capable of driving gene expression but have lost their ability to express the gene in a tissue-specific manner; and promoters with (tissue-specific) regulatory elements deleted, which are capable of driving (potentially reduced) gene expression but have not necessarily lost their ability to express the gene in a tissue-specific manner. Preferably, the promoters included in the nucleic acid expression cassettes disclosed herein are 1000 nucleotides or less, 900 nucleotides or less, 800 nucleotides or less, 700 nucleotides or less, 600 nucleotides or less, 500 nucleotides or less, 400 nucleotides or less, 300 nucleotides or less, or 250 nucleotides or less in length.

[0112] As used herein, the term “transgenic” refers to a specific nucleic acid sequence that encodes a polypeptide or a portion thereof to be expressed in a cell in which the nucleic acid sequence has been introduced. However, transgenics can also be referred to as RNA, typically used to control (e.g., reduce) the amount of a specific polypeptide in a cell in which the nucleic acid sequence has been inserted. These RNA molecules include, but are not limited to, gRNA, shRNA, circRNA, siRNA, antisense RNA, and the like. How the nucleic acid sequence is introduced into the cell is not critical to the present invention, for example, by integration into the genome or as a free plasmid. It is noteworthy that the expression of the transgenic can be restricted to a subset of the cells in which the nucleic acid sequence has been introduced. The term “transgenic” is intended to include (1) a nucleic acid sequence that is not naturally present in the cell (i.e., a heterologous nucleic acid sequence); (2) a nucleic acid sequence that is a mutant form of a naturally present nucleic acid sequence in the cell in which the nucleic acid sequence has been introduced; (3) a nucleic acid sequence for increasing an additional copy of the same (i.e., homologous) or similar nucleic acid sequence naturally present in the cell in which the nucleic acid sequence has been introduced; or (4) a silenced naturally present or homologous nucleic acid sequence whose expression is induced in the cells in which the nucleic acid sequence has been introduced.

[0113] Transgenic organisms can be homologous or heterologous to promoters (and / or, for example, to the animals, especially mammals, in which the transgenic organisms are introduced, in cases where nucleic acid expression cassettes are used in gene therapy).

[0114] Genetically modified organisms can be codon-optimized.

[0115] A transgene can be a full-length cDNA or genomic DNA sequence, or any segment, subunit, or mutant thereof possessing at least one biological activity. Specifically, a transgene can be a small gene, i.e., a gene sequence lacking a portion, a majority, or all of its intron sequences. Therefore, a transgene may optionally contain intron sequences. Optionally, a transgene can be a heterozygous nucleic acid sequence, i.e., a nucleic acid sequence composed of homologous and / or heterologous cDNA and / or genomic DNA segments. A 'mutant form' refers to a nucleic acid sequence containing one or more nucleotides that differ from the wild-type or naturally occurring sequence; that is, a mutant nucleic acid sequence contains one or more nucleotide substitutions, deletions, and / or insertions. Nucleotide substitutions, deletions, and / or insertions can produce a gene product (i.e., a protein or nucleic acid) that differs from the wild-type amino acid / nucleic acid sequence in its amino acid / nucleic acid sequence. The preparation of such mutants is well known in the art. In some cases, a transgene may also include a sequence encoding a leader peptide or signal sequence, such that the transgene product will be secreted from the cell.

[0116] The transgenes that may be included in the nucleic acid expression cassettes described herein typically encode gene products, such as RNA or polypeptides (proteins).

[0117] In the implementation plan, the transgene encodes a therapeutic protein or an immunogenic protein.

[0118] In the implementation plan, the transgene encodes a therapeutic protein. Non-limiting examples of therapeutic proteins include factor VIII, factor IX, factor VII, factor VIIa, factor X, von Willebrand factor, phenylalanine hydroxylase (PAH), α-glucosidase (GAA), C1 esterase inhibitor (C1-INH), lysosomal enzymes, lysosomal enzyme iduronate 2-sulfatase (I2S), erythropoietin (EPO), interferon-α, interferon-β, interferon-γ, interleukin-1 (IL-1), interleukin-2 (IL-2), interleukin-3 (IL-3), interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-6 (IL-6), interleukin-7 (IL-7), interleukin-8 (IL-8), interleukin-9 (IL-9), interleukin-10 (IL-10), interleukin-11 (IL-11), interleukin-12 (IL-12), and chemokines (CXC). Motif ligand 5 (CXCL5), granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage colony-stimulating factor (M-CSF), stem cell factor (SCF), keratinocyte growth factor (KGF), monocyte chemoattractant protein-1 (MCP-1), tumor necrosis factor (TNF), afamin (AFM), α1-antitrypsin, α-galactosidase A, α-L-iduronidase, ATP7b, ornithine transcarbamate, phenylalanine hydroxylase, lipoprotein lipase, apolipoprotein, low-density lipoprotein receptor (LDL-R), albumin, glucose-6-phosphatase, dominant-negative mutants based on VP22 fusion protein, ISG56, IFITM1, transgenic MyD88 protein encoding antibody-encoded interferon-induced protein, nanobodies, antiviral dominant-negative proteins, and their fragments, subunits, or mutants, etc.

[0119] In the implementation plan, the transgene encodes an immunogenic protein. Non-limiting examples of immunogenic proteins include epitopes derived from pathogens and antigens, such as hepatitis A, B, or C surface antigen proteins.

[0120] As used herein, the term “immunogenicity” refers to a substance or composition that can elicit an immune response.

[0121] In some embodiments, the transgene encodes FIX. The term “coagulation factor IX” has the meaning as known in the art. Synonyms for coagulation factor IX are “FIX” or “Christmas factor” or “F9”, and they may be used interchangeably. Specifically, the term “coagulation factor IX” includes a human protein encoded by an mRNA sequence as defined in Genbank accession number NM_000133. Preferably, the FIX is a mutant FIX that is hyperactive or overfunctional compared to wild-type FIX. Modifying the functional activity of human coagulation factors can be accomplished by bioengineering, for example, by introducing point mutations. By this method, hyperactive R338A variants have been reported, which showed a 3-fold increase in coagulation activity compared to wild-type human FIX in the activated partial thromboplastin time assay (APPT) (Chang et al., 1998) and a 2- to 6-fold higher specific activity in hemophilia B mice transfected with the mutant FIX gene (Schuettrumpf et al., 2005). Other exemplary FIX point mutants or domain-exchange mutants with even higher coagulation activity have been described: FIX whose EGF-1 domain is replaced by an EGF-1 domain from FVII, either alone or in combination with the following mutants: R338A point mutation (Brunetti-Pierri et al., 2009); V86A / E277A / R338A triple mutant (Lin et al., 2010); Y259F, K265T and / or Y345T single, double or triple mutants (Milanov et al., 2012); and G190V point mutant (Kao et al., 2010); all of which are incorporated herein by reference. Other exemplary FIX variants include factor IXdalcinonacog alfa (“CB 2679d / ISU304”) (Catalyst Biosciences).

[0122] In a particularly preferred embodiment, the FIX mutant is described by Simioni et al. in 2009 and named the “Factor IX Padua” mutant, which causes an X-linked thrombotic tendency. The mutant Factor IX is excessively active and carries an R338L amino acid substitution. In embodiments of the invention, the FIX transgene encodes a human FIX protein, preferably a Padua mutant of the human FIX protein. In an embodiment, the FIX transgene encodes a Padua mutant (co-FIX-R338L) of the human FIX protein having the sequence described in SEQ ID NO:25.

[0123] In some embodiments, the transgene encodes FVIII. The term “coagulation factor VIII” has the meaning as known in the art. Synonyms for coagulation factor VIII are “FVIII” or “antihemophilic factor” or “AHF”, and may be used interchangeably herein. The term “coagulation factor VIII” includes, for example, human proteins having an amino acid sequence as defined in Uniprot accession number P00451. Preferably, the FVIII is an FVIII in which the B domain is missing (i.e., a B domain-deficient FVIII, also referred to herein as BDD FVIII or FVIII B). The term “B domain-deficient FVIII” includes, for example, but not limited to, FVIII mutants in which all or part of the B domain is missing, and FVIII mutants in which the B domain is replaced by a linker. Non-limiting examples of B domain-deficient FVIIIs are described in Ward et al. (2011), WO 2011 / 005968, and McIntosh et al. (2013), which are specifically incorporated herein by reference. In some embodiments, the FVIII is a B-domain-deficient FVIII, wherein the B-domain is replaced by a connector having the sequence SFSQNPPVLTRHQR (SEQ ID NO:54) (i.e., SQ FVIII as defined in Ward et al. (2011)). In some embodiments, the FVIII has SEQ ID NO:26 (i.e., codon-optimized B-domain-deficient human FVIII or hFVIIIcopt), also disclosed in WO 2011 / 0059, which is specifically incorporated herein by reference.

[0124] Other sequences may also be included in the nucleic acid expression cassettes disclosed herein, typically for further enhancement or stabilization of transgenic product expression (e.g., introns and / or polyadenylated sequences).

[0125] Any intron can be used in the expression cassette described herein. The term "intron" includes any portion of the entire intron that is large enough to be recognized and spliced ​​by a nuclear splicing apparatus. Generally, short functional intron sequences are preferred to keep the size of the expression cassette as small as possible, which facilitates the construction and manipulation of the expression cassette. In some embodiments, introns are obtained from a gene encoding a protein that is encoded by a coding sequence within the expression cassette. Introns can be located at the 5', 3', or within the coding sequence. The advantage of locating introns at the 5' of the coding sequence is that it minimizes the possibility of the intron interfering with the function of polyadenylation signaling. In embodiments, the nucleic acid expression cassettes disclosed herein also include introns. Non-limiting examples of suitable introns are mouse parvovirus (MVM) introns, β-globulin introns (βIVS-II), factor IX (FIX) intron A, simian virus 40 (SV40) small t introns, and β-actin introns. Preferably, the intron is a mouse parvovirus (MVM) intron, more preferably an MVM intron as defined in SEQ ID NO:29.

[0126] Any polyadenylation signal that guides the synthesis of the polyadenylation tail can be used in the expression cassette described herein, examples of which are well known to those skilled in the art. Exemplary polyadenylation signals include, but are not limited to, polyadenylation sequences derived from the late gene of simian virus 40 (SV40), bovine growth hormone (BGH) polyadenylation signals, the minimal rabbit β-globulin (mRBG) gene, and synthetic polyadenylation (SPA) sites as described in Levitt et al. (1989, Genes Dev 3:1019-1025). Preferably, the polyadenylation signal is the bovine growth hormone (BGH) polyadenylation signal (SEQ ID NO:30).

[0127] In a specific implementation, a nucleic acid expression kit is disclosed, which includes:

[0128] — A liver-specific nucleic acid regulatory element comprising a sequence selected from the group consisting of SEQ ID NO:6, SEQ ID NO:4, SEQ ID NO:12, SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, and SEQ ID NO:21, a sequence having at least 95% identity with any of said sequences, or a functional fragment thereof as described herein;

[0129] —As defined in SEQ ID NO:24, the thyroxine transporter enhancer (TTRe);

[0130] — A liver-specific TTRm promoter (e.g., defined by SEQ ID NO:27); and

[0131] -genetically modified;

[0132] Preferably, the combination of TTRe and TTRm nucleic acids is defined by SEQ ID NO:28.

[0133] In a specific implementation, a nucleic acid expression kit is disclosed, which includes:

[0134] — A liver-specific nucleic acid regulatory element comprising a sequence selected from the group consisting of SEQ ID NO:6, SEQ ID NO:4, SEQ ID NO:12, SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, and SEQ ID NO:21, a sequence having at least 95% identity with any of said sequences, or a functional fragment thereof as described herein;

[0135] —As defined in SEQ ID NO:24, the thyroxine transporter enhancer (TTRe);

[0136] — A liver-specific TTRm promoter (e.g., defined by SEQ ID NO:27); and

[0137] —Introns, preferably MVM introns (e.g., as defined by SEQ ID NO:29); and

[0138] —A transgene, preferably wherein the transgene encodes coagulation factor IX (FIX), preferably wherein the transgene is a codon-optimized coagulation factor FIX, or wherein the coagulation factor FIX contains a hyperactivation mutation, preferably wherein the hyperactivation mutation corresponds to an R338L amino acid substitution, more preferably wherein the transgene encoding coagulation factor FIX has a nucleic acid sequence as defined in SEQ ID NO:25; or wherein the transgene encodes coagulation factor VIII (FVIII), preferably wherein the transgene is a codon-optimized coagulation factor FVIII, or wherein the coagulation factor VIII has a deletion of the B domain, preferably wherein the B domain of the FVIII is replaced by an adapter as defined in SEQ ID NO:54, more preferably wherein the transgene encoding coagulation factor VIII has a nucleic acid sequence as defined in SEQ ID NO:26;

[0139] Preferably, the combination of TTRe and TTRm nucleic acids is defined by SEQ ID NO:28.

[0140] The nucleic acid regulatory elements and nucleic acid expression cassettes disclosed herein can be used accordingly, or typically, they can be part of a nucleic acid vector. Therefore, another aspect relates to the use of nucleic acid regulatory elements or nucleic acid expression cassettes as described herein in vectors, particularly nucleic acid vectors.

[0141] In one aspect, the present invention also provides a vector comprising a nucleic acid regulatory element as disclosed herein. In a further embodiment, the vector comprises a nucleic acid expression cassette as disclosed herein.

[0142] As used herein, the term "vector" refers to a nucleic acid molecule (e.g., double-stranded DNA) into which another nucleic acid molecule (the inserted nucleic acid molecule), such as, but not limited to, cDNA, may be inserted. A vector is used to transport the inserted nucleic acid molecule into a suitable host cell. A vector may contain essential elements that allow transcription of the inserted nucleic acid molecule and optionally translate the transcript into a polypeptide. The inserted nucleic acid molecule may be derived from the host cell or from a different cell or organism. Once in the host cell, the vector may replicate independently of or simultaneously with the host chromosomal DNA, and multiple copies of the vector and its inserted nucleic acid molecule may be produced. The vector may be an episomal vector (i.e., one that is not integrated into the host cell's genome) or may be a vector integrated into the host cell's genome. Therefore, the term "vector" may also be defined as a gene delivery vector that facilitates the delivery of genes to target cells. This definition includes both non-viral and viral vectors. Non-viral vectors include, but are not limited to, cationic lipids, liposomes, nanoparticles, PEG, PEI, plasmid vectors (e.g., pUC vectors, bluescript vectors (pBS), and pBR322 or derivatives thereof lacking bacterial sequences (microcircles), transposon-based vectors (e.g., PiggyBac (PB) vectors or Sleeping Beauty (SB) vectors), etc. Viral vectors are derived from viruses and include, but are not limited to, retroviruses, lentiviruses, adeno-associated viruses, adenoviruses, herpesviruses, hepatitis virus vectors, etc. Typically, but not necessarily, viral vectors are replication-deficient because they have lost the ability to reproduce in a given cell, since the viral genes essential for replication have been removed from the viral vector. However, some viral vectors can also be adapted to replicate specifically in a given cell, such as cancer cells, and are often used to trigger (cancer) cell-specific (tumor cell) lysis. Virosomes are non-limiting examples of vectors that include both viral and non-viral elements; specifically, they combine liposomes with inactivated HIV or influenza virus (Yamada et al., 2003). Another example includes viral vectors mixed with cationic lipids.

[0143] In a preferred embodiment, the vector is a viral vector, such as a retrovirus, lentivirus, adenovirus, or adeno-associated virus (AAV) vector, more preferably an AAV vector.

[0144] AAV vectors are preferably used as self-complementary double-stranded AAV vectors (scAAV) to overcome one of the limiting steps in AAV transduction (i.e., single-stranded to double-stranded AAV conversion) (McCarty, 2001, 2003; Nathwani et al., 2002, 2006, 2011; Wu et al., 2008), but the use of single-stranded AAV vectors (ssAAV) is also included herein.

[0145] Different serotypes of AAV, such as AAV serotype 2, AAV serotype 5, AAV serotype 8, and AAV serotype 9, have been isolated and characterized, and all AAV serotypes are envisioned herein. AAV serotype 9 (AAV9) is ideally suited for efficient transduction in the liver. Therefore, in some embodiments, the vector is an AAV9 vector, more particularly a self-complementary AAV9 vector (scAAV9). Alternatively, engineered AAV capsids that influence hepatic transduction efficacy may be employed, including those obtained through molecular evolution and selection, as described by Grimm et al. (2008) or Lisowski et al. (2014).

[0146] In other embodiments, the vector is a non-viral vector, preferably a plasmid, microcircle, or transposon-based vector. Preferably, the transposon-based vector is derived from Sleeping Beauty (SB) or PiggyBac (PB). Preferred SB transposons have been described in Ivics et al. (1997), and their overactive forms (including SB100X) are as described in Mates et al. (2009). PiggyBac-based transposons are safe vectors because they do not increase the risk of tumorigenesis. Furthermore, liver-guided gene therapy using these vectors has shown the induction of immune tolerance to transgenes included in the vector, particularly hFIX or hFVIII transgenes.

[0147] Transposon-based vectors are preferably administered in combination with vectors encoding transposases for gene therapy. For example, a transposon-based vector derived from PiggyBac can be administered with wild-type PiggyBac transposase (Pbase) or mouse codon-optimized PiggyBac transposase (mPBase). Preferably, the transposase is an overactive transposase, such as the overactive PB (hyPB) transposase containing seven amino acid substitutions (I30V, S103P, G165S, M282V, S509G, N538K, N570S) as described by Yusa et al. (2011), which is specifically incorporated herein by reference.

[0148] Transposon / transposase constructs can be delivered via hydrodynamic injection or by transfecting hepatocytes with non-viral nanoparticles.

[0149] In other embodiments, the vector includes viruses and non-viral components.

[0150] In specific embodiments, the following vectors are provided: AAVsc-HS-CRE1-TTRenh-TTRm-MVM-hFIXcoPadua-bghpolyA (SEQ ID NO:33), AAVsc-HS-CRE2-TTRenh-TTRm-MVM-hFIXcoPadua-bghpolyA (SEQ ID NO:34), AAVsc-HS-CRE3-TTRenh-TTRm-MVM-hFIXcoPadua-bghpolyA (SEQ ID NO:35), AAVsc-HS-CRE4-TTRenh-TTRm-MVM-hFIXcoPadua-bghpolyA (SEQ ID NO:36), AAVsc-HS-CRE5-TTRenh-TTRm-MVM-hFIXcoPadua-bghpolyA (SEQ ID NO:37), AAVsc-HS-CRE6-TTRenh-TTRm-MVM-hFIXcoPadua-bghpolyA (SEQ ID NO:38), AAVsc-HS-CRE7-TTRenh-TTRm-MVM-hFIXcoPadua-bghpolyA (SEQ ID NO:39), AAVsc-HS-CRE8-TTRenh-TTRm-MVM-hFIXcoPadua-bghpolyA (SEQ ID NO:40), AAVsc-HS-CRE9-TTRenh-TTRm-MVM-hFIXcoPadua-bghpolyA (SEQ ID NO:41), AAVsc-HS-CRE10-TTRenh-TTRm-MVM-hFIXcoPadua-bghpolyA (SEQ ID NO:42), AAVsc-HS-CRE11-TTRenh-TTRm-MVM-hFIXcoPadua-bghpolyA (SEQ ID NO:43), AAVsc-HS-CRE12-TTRenh-TTRm-MVM-hFIXcoPadua-bghpolyA (SEQ ID NO:44), AAVsc-HS-CRE13-TTRenh-TTRm-MVM-hFIXcoPadua-bghpolyA (SEQ ID NO:45), AAVsc-HS-CRE14-TTRenh-TTRm-MVM-hFIXcoPadua-bghpolyA (SEQ ID NO:46), AAVsc-HS-CRE15-TTRenh-TTRm-MVM-hFIXcoPadua-bghpolyA (SEQ IDNO: 47), AAVsc-HS-CRE16-TTRenh-TTRm-MVM-hFIXcoPadua-bghpolyA (SEQ ID NO: 48), AAVsc-HS-CRE17-TTRenh-TTRm-MVM-hFIXcoPadua-bghpolyA (SEQ ID NO: 49), AAVsc-HS-CRE18-TTRenh-TTRm-MVM-hFIXcoPadua-bghpolyA (SEQ ID NO: 50), AAVsc-HS-CRE19-TTRenh-TTRm-MVM-hFIXcoPadua-bghpolyA (SEQ ID NO: 51), AAVsc-HS-CRE20-TTRenh-TTRm-MVM-hFIXcoPadua-bghpolyA (SEQ ID NO:52), AAVsc-HS-CRE21-TTRenh-TTRm-MVM-hFIXcoPadua-bghpolyA (SEQ ID NO:53).

[0151] Therefore, in a particular embodiment, the vector has a sequence as described in SEQ ID NO:38, 36, 44, 33, 37, 41, 43, 45, 46, 48 or 52, preferably as described in SEQ ID NO:38, 36, 44, 33, 37 or 45, more preferably as described in SEQ ID NO:38, 36 or 44, and even more preferably as described in SEQ ID NO:38.

[0152] In another respect, the present invention relates to the nucleic acid regulatory elements, nucleic acid expression cassettes, carriers or pharmaceutical compositions described herein, which are used in pharmaceuticals.

[0153] More specifically, the nucleic acid expression cassettes and vectors disclosed herein can be used, for example, to express proteins that are normally expressed and used in the liver (i.e., structural proteins) or proteins that are expressed in the liver and subsequently exported into the bloodstream for transport to other parts of the body (i.e., secretory proteins). For instance, the expression cassettes and vectors disclosed herein can be used to express therapeutic amounts of gene products (such as peptides, especially therapeutic proteins or RNA) for therapeutic purposes, particularly for gene therapy. Typically, the gene product is encoded by a transgene within the expression cassette or vector, although in principle, the expression of endogenous genes can also be enhanced for therapeutic purposes.

[0154] Therefore, in certain implementations, the nucleic acid regulatory elements, nucleic acid expression cassettes, vectors, or pharmaceutical compositions described herein can be used in gene therapy, particularly liver-guided gene therapy.

[0155] This article also discloses the use of the nucleic acid regulatory elements, nucleic acid expression cassettes, vectors or pharmaceutical compositions described herein in the preparation of medicaments for gene therapy, particularly liver-guided gene therapy.

[0156] Gene therapy protocols, which aim to achieve the expression of therapeutic gene products in target cells in vitro and, more particularly, in vivo, have been extensively described in the art. These include, but are not limited to, intramuscular injection, interstitial injection, airway infusion, application to the endothelium, liver parenchyma, and intravenous or arterial administration (e.g., intrahepatic artery, intrahepatic vein) of plasmid DNA (naked or in liposomes). Various devices have been developed to improve the availability of DNA to target cells. A simple approach is to physically contact the target cells with a catheter or implantable material containing DNA. Another approach is to use needle-free jet injection devices, which directly inject a jet of liquid into the target tissue under high pressure. These delivery paradigms can also be used to deliver viral vectors. Another approach to targeted gene delivery is to use molecular conjugates consisting of proteins or synthetic ligands linked to nucleic acid binders or DNA binders to enable nucleic acid-specific targeting of cells (Cristiano et al., 1993a&b).

[0157] According to a specific implementation, it is envisioned that the nucleic acid expression cassette and vector described herein be used for gene therapy of hepatocytes (i.e., liver-guided gene therapy). According to another specific implementation, regulatory elements, expression cassettes, or vectors are used for in vivo gene therapy, particularly liver-guided gene therapy. According to yet another specific implementation, methods for gene therapy, particularly liver-guided gene therapy, are used to treat hemophilia, particularly hemophilia B or hemophilia A.

[0158] Gene delivery into mammalian hepatocytes utilizes both in vitro and in vivo procedures. In vitro methods involve harvesting hepatocytes, transducing them in vitro using long-term expression vectors, and reintroducing the transduced hepatocytes into the portal circulation (Kay et al., 1992; Chowdhury et al., 1991). In vivo targeting is achieved by injecting DNA or viral vectors into the liver parenchyma, hepatic artery, or portal vein, and by transcriptional targeting (Kuriyama et al., 1991; Kistner et al., 1996). Recent methods also include intravenous delivery of naked DNA (Budker et al., 1996) and hydrodynamic tail vein transfection (Liu et al., 1999; Zhang et al., 1999).

[0159] This document also discloses the use of the nucleic acid regulatory elements, nucleic acid expression cassettes, or vectors disclosed herein for protein expression in hepatocytes, either in vivo or in vitro, and more particularly for transfecting or transducing hepatocytes in vitro or in vivo using the nucleic acid expression cassettes or vectors described herein. According to another aspect, a method for expressing proteins in hepatocytes is provided, comprising the steps of: introducing, for example, the nucleic acid regulatory elements, nucleic acid expression cassettes, or vectors described herein into hepatocytes by transfection or transduction; and expressing the transgenic protein product in the hepatocytes. These methods can be performed in vitro, ex vivo, and in vivo.

[0160] On the other hand, nucleic acid regulatory elements, nucleic acid expression cassettes, or vectors as taught herein are provided for enhancing liver-specific gene expression of transgenes, preferably wherein said use is in vitro. On the other hand, nucleic acid regulatory elements, nucleic acid expression cassettes, or vectors as taught herein are provided for enhancing liver-specific expression of proteins or peptides encoded by transgenes, preferably wherein said use is in vitro.

[0161] On the other hand, methods are provided for enhancing liver-specific gene expression of transgenic organisms using nucleic acid regulatory elements, nucleic acid expression cassettes, or vectors as taught herein.

[0162] On the other hand, methods are provided for enhancing liver-specific expression of proteins or peptides encoded by transgenes using nucleic acid regulatory elements, nucleic acid expression cassettes, or vectors as taught herein.

[0163] An in vivo method for gene therapy for subjects in need is also provided, comprising the steps of: introducing a nucleic acid expression cassette containing a transgene encoding a therapeutic protein into the liver of a subject; and expressing a therapeutic amount of the therapeutic protein in the liver. According to a further embodiment, the method comprises the steps of: introducing a vector including a nucleic acid expression cassette containing a transgene encoding a therapeutic protein into the liver of a subject; and expressing a therapeutic amount of the therapeutic protein in the liver.

[0164] Non-viral transfection or viral vector-mediated transduction of hepatocytes can also be performed via in vitro or ex vivo procedures. In vitro methods require in vitro transfection or transduction of hepatocytes (e.g., hepatocytes previously harvested from the subject), hepatocyte lines, or hepatocytes differentiated from, for example, induced pluripotent stem cells or embryonic cells. Ex vivo methods require harvesting hepatocytes from the subject, in vitro transfection or transduction, and optionally reintroducing the transfected hepatocytes into the subject.

[0165] This article also discloses in vitro or ex vivo methods for generating therapeutic proteins, the methods comprising:

[0166] — Transfect or transduce hepatocytes, preferably hepatocyte lines, using the nucleic acid expression cassettes or vectors disclosed herein, wherein the nucleic acid expression cassettes or vectors include the nucleic acid regulatory elements disclosed herein, which are operatively linked to a promoter and a transgene, wherein the transgene encodes a therapeutic protein;

[0167] —Culturing hepatocytes under conditions suitable for expressing transgenes in hepatocytes; and

[0168] — Harvesting therapeutic proteins from liver cells or culture media.

[0169] The in vitro uses and methods disclosed herein for expressing transgenic products in hepatocytes, and more particularly in hepatocyte cell lines, are particularly applicable to the recombinant production of therapeutic proteins, such as therapeutic agents used in protein replacement therapies (e.g., factor VIII, factor IX, factor VIIa). The use of nucleic acid regulatory elements disclosed herein allows for enhanced liver-specific expression of transgenes, which advantageously increases the yield of transgenic products, and more particularly therapeutic proteins.

[0170] Those skilled in the art should understand that the uses of the nucleic acid regulatory elements, nucleic acid expression cassettes, and vectors disclosed herein extend beyond gene therapy, such as inducing stem cells to differentiate into hepatogenic cells and for transgenic models of protein overexpression in the liver.

[0171] Transgenic products can be polypeptides, especially therapeutic proteins, such as factor VIII, factor IX, factor VII or factor VIIa, factor X, α-glucosidase (secretory GAA), von Willebrand factor, phenylalanine hydroxylase (PAH), C1 esterase inhibitor (C1-INH), lysosomal enzymes, lysosomal enzyme iduronate 2-sulfatase (I2S), erythropoietin (EPO), interferon-α, interferon-β, interferon-γ, interleukin-1 (IL-1), interleukin-2 (IL-2), interleukin-3 (IL-3), interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-6 (IL-6), interleukin-7 (IL-7), interleukin-8 (IL-8), interleukin-9 (IL-9), interleukin-10 (IL-10), and interleukin-11. (IL-11), interleukin-12 (IL-12), chemokine (CXC motif) ligand 5 (CXCL5), granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage colony-stimulating factor (M-CSF), stem cell factor (SCF), keratinocyte growth factor (KGF), monocyte chemoattractant protein-1 (MCP-1), tumor necrosis factor (TNF), afamin (AFM), α1-antitrypsin, α-galactosidase A, α-L-iduronidase, ATP7b, ornithine transcarbamate, phenylalanine hydroxylase, lipoprotein lipase, apolipoprotein, low-density lipoprotein receptor (LDL-R), albumin, glucose-6-phosphatase, transgenic antibodies encoding antibodies, nanobodies, antiviral dominant / negative proteins, and their fragments, subunits, or mutants. Alternatively, the transgenic product may be RNA, such as siRNA.

[0172] Examples of diseases and conditions that may benefit from gene therapy using the nucleic acid regulatory elements, nucleic acid expression cassettes, vectors, or pharmaceutical compositions described herein include liver diseases and conditions such as hepatitis infections, hemophilia (including hemophilia A and B), and glycogen storage disorders (GSD) such as Pompeii (e.g., GSD II, GSD III, and GSD). IVall), (non-alcoholic) fatty liver (NAFLD), lysosomal storage disease, Hunt syndrome, phenylketonuria (PKU), hereditary angioedema (HAE), cirrhosis, liver fluke disease, alcoholic liver disease, liver cancer (including primary liver cancer (e.g., hepatocellular carcinoma and / or cholangiocarcinoma, angiosarcoma or hemangioendothelioma) and liver metastases), biliary cirrhosis, sclerosing cholangitis, centrilobular necrosis, Budd-Chiari syndrome, hemochromatosis, Wilson's disease, thyroxine-associated hereditary amyloidosis, Gilbert's syndrome, and pediatric liver diseases and conditions (such as biliary atresia, alpha-1 antitrypsin deficiency, Alagille syndrome, and progressive familial intrahepatic cholestasis).

[0173] "Hepatic fasciolopsiasis" refers to a parasitic infection of the liver caused by liver flukes, primarily those belonging to the genus Fasciolopsis. "Hepatitis" refers to inflammation of the liver, which can be caused by various viruses (viral hepatitis), as well as by some hepatotoxins (e.g., alcoholic hepatitis), autoimmune diseases (autoimmune hepatitis), or genetic conditions. "Alcoholic liver disease" in this context refers to any liver manifestation of excessive alcohol consumption, including fatty liver, alcoholic hepatitis, and cirrhosis. Similar terms such as "drug-induced" or "toxic" liver disease are also used in this context to refer to the range of conditions caused by various drugs and environmental chemicals. "Fatty liver" (or hepatic lipidosis) in this context refers to a reversible condition in which large vacuoles of triglycerides accumulate in hepatocytes. "Non-alcoholic fatty liver" refers to a spectrum of diseases associated with obesity and metabolic syndrome, among other things. Fatty liver can cause inflammatory disease (i.e., steatohepatitis) and eventually lead to cirrhosis. Cirrhosis refers to the formation of fibrous tissue (fibrosis) in the location of liver cells that have died due to various causes, including viral hepatitis, excessive alcohol consumption, and other forms of hepatotoxicity. Cirrhosis can lead to chronic liver failure. Primary liver cancer most commonly manifests as hepatocellular carcinoma and / or cholangiocarcinoma; rarer forms include angiosarcoma of the liver and hemangioendothelioma. Many liver malignancies are secondary damage resulting from metastases of primary cancers in the gastrointestinal tract or other organs such as the kidneys, lungs, breast, or prostate. Primary biliary cirrhosis refers to a severe autoimmune disease of the capillary bile ducts. Primary sclerosing cholangitis refers to a severe chronic inflammatory disease of the bile ducts, which is believed to originate from an autoimmune disease. Centrilobular necrosis can be caused by the leakage of enterotoxins into the bloodstream. For example, Salmonella toxins in the ileum have been shown to cause severe damage to liver cells. Budd-Chiari syndrome refers to a clinical phenomenon caused by obstruction of the hepatic veins, which in some cases can lead to cirrhosis. Hereditary diseases that damage the liver include hemochromatosis, which involves the accumulation of iron in the body, and Wilson's disease, which causes the body to retain copper. Liver damage is also a clinical feature of alpha-1 antitrypsin deficiency and glycogen storage disease type II. In thyroxine-associated hereditary amyloidosis, the liver produces mutated thyroxine, which has severe neurodegenerative and / or cardiac effects. Gilbert's syndrome is a hereditary disorder of bilirubin metabolism that can cause mild jaundice.

[0174] According to a very specific implementation, the therapeutic protein encoded by the transgene in a nucleic acid expression cassette or vector is factor IX, and the method is used to treat hemophilia B. Hemophilia B can be treated by expressing factor IX in the liver via gene therapy (Snyder et al., 1999). According to another very specific implementation, the therapeutic protein encoded by the transgene in a nucleic acid expression cassette or vector is factor VIII, and the method is used to treat hemophilia A.

[0175] Unless otherwise noted, the terms “subject” or “patient” are used interchangeably and refer to an animal, preferably a vertebrate, more preferably a mammal, and specifically include human patients as well as non-human mammals such as mice. The preferred patient or subject is a human subject.

[0176] As used herein, the term “treatment” refers to therapeutic procedures and preventative or protective measures aimed at preventing or mitigating (alleviating) undesirable physiological changes or conditions, such as the development or spread of proliferative diseases (e.g., cancer). Favorable or desired clinical outcomes include, but are not limited to, symptom relief, reduction in the extent of disease, stabilization of the disease (i.e., no worsening), delay or slowing of disease progression, improvement or mitigation of the disease state, and relief (whether partial or complete), whether detectable or undetectable. “Treatment” can also refer to extended survival compared to expected survival without treatment.

[0177] As used herein, phrases such as “subjects requiring treatment” include subjects such as mammalian subjects who will benefit from treatment for a given condition (such as hemophilia B or hemophilia A). These subjects generally include, but are not limited to, those who have been diagnosed with the condition, those who are susceptible to or develop the condition, and / or those for whom prevention of the condition is desired.

[0178] The term "therapeuticly effective amount" refers to the amount of a compound or pharmaceutical composition that is capable of effectively treating a given condition (i.e., achieving the desired local or systemic effects and behavioral manifestations) in a subject. Therefore, this term refers to the amount of a compound or pharmaceutical composition sought by researchers, veterinarians, medical doctors, or other clinicians to induce a biological or pharmaceutical response in tissues, systems, animals, or humans, including the reduction of symptoms of the disease or condition being treated. Specifically, these terms refer to the amount of a compound or pharmaceutical composition according to the invention required, in a single or multiple dose manner, to prevent, cure, improve, or at least reduce clinical damage, symptoms, or complications associated with a given condition (such as hemophilia if the therapeutic protein encoded by the transgene is factor IX or VIII).

[0179] In a particular embodiment, if the therapeutic protein encoded by the transgene is factor IX, the term means that the level of factor IX in plasma is equal to or higher than a therapeutic concentration of at least about 1% physiological activity (i.e., 10 mU / ml plasma), which can be obtained by transducing or transfecting a vector according to any of the embodiments described herein into a subject to at least 5% physiological activity or 50 mU / ml plasma, at least 10% physiological activity or 100 mU / ml plasma, at least 15% physiological activity or 150 mU / ml, at least 20% physiological activity or 200 mU / ml plasma, at least 25% physiological activity or 250 mU / ml plasma, or at least 30% physiological activity or 3% physiological activity. 00 mU / ml plasma, at least 35% physiological activity or 350 mU / ml, at least 40% physiological activity or 400 mU / ml, at least 45% physiological activity or 400 mU / ml, at least 45% physiological activity or 450 mU / ml, at least 50% physiological activity or 500 mU / ml, at least 65% physiological activity or 650 mU / ml, at least 70% physiological activity or 700 mU / ml, at least 75% physiological activity or 750 mU / ml, at least 80% physiological activity or 800 mU / ml, at least 85% physiological activity or 850 mU / ml, at least 95% physiological activity or 950 mU / ml, or at least 100% physiological activity or 1000 mU / ml. Because the nucleic acid expression cassette and vector of the present invention are highly efficient, even relatively low doses of the vector can be used to obtain such high therapeutic levels of Factor IX in subjects.

[0180] In another specific implementation, if the therapeutic protein encoded by the transgene is factor VIII, the term means that a trough level of factor VIII in plasma at therapeutic concentrations equal to or higher than 10 mU / ml, 50 mU / ml, 100 mU / ml, 150 mU / ml, 200 mU / ml, 250 mU / ml, 300 mU / ml, 350 mU / ml, 400 mU / ml, 450 mU / ml, 500 mU / ml, 550 mU / ml, 600 mU / ml, 650 mU / ml, 750 mU / ml, 800 mU / ml, 850 mU / ml, 900 mU / ml, 950 mU / ml, or higher can be obtained by transducing or transfecting any of the vectors disclosed herein into a subject. Because the vector and nucleic acid expression cassette disclosed in this paper are highly efficient, it is even possible to obtain such high therapeutic levels of factor VIII in subjects by administering relatively low doses of the vector.

[0181] In a specific implementation, if the transgene encodes factor IX or factor VIII, it can be less than 6 x 10^6. 13 A dose of vg / kg (viral genome per kilogram) will be transduced into the subject using a vector according to any of the embodiments defined herein to achieve a therapeutic factor IX level of 100 mU / ml plasma or higher in the subject. For example, it can be administered at a dose of less than 5 x 10 11 A dose of vg / kg yields a factor IX level of 300 mU / ml or higher in the subject's plasma. For hemophilia therapy, efficacy can be measured, for example, by assessing hemophilic bleeding in the subject. In in vitro assays (such as, but not limited to, the activated partial thromboplastin time assay (APPT) and the factor IX colorimetric activity assay), clotting time, factor IX, or human factor VIII-specific ELISA can also be obtained. Of course, any other assays known in the art for assessing therapeutic efficacy can be used.

[0182] The nucleic acid expression cassettes, vectors, or pharmaceutical compositions of the present invention can be used alone or in combination with any known hemophilia therapy (such as the administration of recombinant or purified clotting factors). Therefore, the nucleic acid expression cassettes, vectors, or pharmaceutical compositions of the present invention can be administered alone or in combination with one or more active compounds. The latter can be administered before, after, or simultaneously with the administration of the pharmaceutical agent.

[0183] Another object of the present invention is a pharmaceutical formulation comprising a therapeutically effective amount of a nucleic acid expression cassette or expression vector as defined herein, and a pharmaceutically acceptable carrier (i.e., one or more pharmaceutically acceptable carrier substances and / or additives, such as buffers, carriers, excipients, stabilizers, etc.). The pharmaceutical composition may be provided in the form of a kit. The term “pharmaceuticalally acceptable” as used herein is consistent with the art and means compatible with other components of the pharmaceutical composition and will not be harmful to the recipient. The term “pharmaceuticalally acceptable salt” as used herein refers to an inorganic acid addition salt such as hydrochloride, sulfate, and phosphate, or an organic acid addition salt such as acetate, maleate, fumarate, tartrate, and citrate. Examples of pharmaceutically acceptable metal salts are alkali metal salts such as sodium and potassium salts, alkaline earth metal salts such as magnesium and calcium salts, aluminum salts, and zinc salts. Examples of pharmaceutically acceptable ammonium salts are ammonium salts and tetramethylammonium salts. Examples of pharmaceutically acceptable organic amine addition salts are salts containing morpholine and piperidine. Examples of pharmaceutically acceptable amino acid addition salts are salts containing lysine, glycine, and phenylalanine. The pharmaceutical compositions according to the invention can be administered orally, for example, in the form of pills, tablets, varnished tablets, sugar-coated tablets, granules, hard and soft capsules, water, alcohol or oil solutions, syrups, emulsions, or suspensions, or rectally, for example, in the form of suppositories. They can also be administered without the intestines, for example, subcutaneously, intramuscularly, or intravenously in the form of injections or infusion solutions. Other suitable forms of administration include, for example, transdermal or topical administration in the form of ointments, tinctures, sprays, or transdermal therapeutic systems, or inhalation in the form of nasal sprays or aerosol mixtures, or, for example, microcapsules, implants, or rods. The pharmaceutical compositions can be prepared in ways known per se to those skilled in the art. For this purpose, a suitable form of administration or dosage form is made, such as a nucleic acid expression cassette or expression vector as defined herein, one or more solid or liquid pharmaceutically acceptable excipients, and, if desired, in combination with other pharmaceutically active compounds, so that it can be used as a medicine in human or veterinary medicine.

[0184] According to another embodiment, a pharmaceutical composition is provided comprising a nucleic acid expression cassette and a pharmaceutically acceptable carrier, the nucleic acid expression cassette containing a transgene encoding a therapeutic protein. According to another embodiment, the pharmaceutical composition comprises a carrier containing a nucleic acid expression cassette and a pharmaceutically acceptable carrier, the nucleic acid expression cassette containing a transgene encoding a therapeutic protein. According to a further specific embodiment, the transgene encodes factor IX and the pharmaceutical composition is for treating hemophilia B, or the transgene encodes factor VIII and the pharmaceutical composition is for treating hemophilia A.

[0185] The use of the nucleic acid expression cassette, its regulatory element, and carrier components as disclosed herein in the preparation of pharmaceutical compositions for the treatment of hemophilia, preferably hemophilia B or hemophilia A, is also envisioned.

[0186] In alternative examples, the expression cassettes and vectors disclosed herein can be used to express gene products (such as peptides, especially immunogenic proteins or RNA) for inoculation purposes.

[0187] In an embodiment, the pharmaceutical composition may be a vaccine. The vaccine may further include one or more adjuvants to enhance the immune response. Suitable adjuvants include, for example, but not limited to, saponins, mineral gels such as aluminum hydroxide, surfactants such as lysophosphatidylcholine, polyols, polyanionic peptides, oil or hydrocarbon emulsions, BCG, Corynebacterium pumilus, and the synthetic adjuvant QS-21. Optionally, the vaccine may further include one or more immunostimulatory molecules. Non-limiting examples of immunostimulatory molecules include various cytokines, lymphokines, and chemokines having immunostimulatory, immunoenhancing, and pro-inflammatory activities, such as interleukins (e.g., IL-1, IL-2, IL-3, IL-4, IL-12, IL-13); growth factors (e.g., granulocyte-macrophage (GM) colony-stimulating factor (CSF)); and other immunostimulatory molecules such as macrophage inflammatory factors, Flt3 ligands, B7.1, B7.2, etc.

[0188] In the implementation plan, the nucleic acid regulatory elements, nucleic acid expression cassettes, vectors or pharmaceutical compositions described herein can be used as vaccines, and more particularly as preventive vaccines.

[0189] This article also discloses the use of the nucleic acid regulatory elements, nucleic acid expression cassettes, vectors or pharmaceutical compositions described herein in the preparation of vaccines, especially prophylactic vaccines.

[0190] This article also discloses methods for administering vaccinations, especially preventative vaccinations, to subjects who require it, including:

[0191] —Introducing the nucleic acid expression cassette, vector, or pharmaceutical composition described herein into a subject, particularly into the liver of a subject, wherein the nucleic acid expression cassette, vector, or pharmaceutical composition includes the nucleic acid regulatory element described herein, which is operatively linked to a promoter and a transgene; and

[0192] —Expressing immunologically effective amounts of the transgenic product in subjects, particularly in the liver of subjects.

[0193] As used herein, “immunely effective amount” refers to the amount of transgenic product that effectively enhances the immune response of a subject to subsequent exposure to an immunogen encoded by the transgenic gene. The level of induced immunity can be determined, for example, by measuring the amount of neutralizing secretory antibodies and / or serum antibodies (e.g., by plaque neutralization, complement binding, enzyme-linked immunosorbent assay, or microneutralization assay).

[0194] Typically, when using an expression cassette or vector as taught herein (i.e., having at least one liver-specific nucleic acid regulatory element, including those selected from SEQ ID NO:6, SEQ ID NO:4, SEQ ID NO:12, SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:19, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:19, SEQ ID NO:19, SEQ ID NO:10 ... The amount of (transgenic) product expressed when using a sequence comprising group NO:21, a sequence having at least 95% identity with any of the sequences described herein, or a functional fragment thereof as described elsewhere herein, is higher than the amount of (transgenic) product expressed when using the same expression cassette or vector but without at least one liver-specific nucleic acid regulatory element as taught herein (e.g., including TTRe and / or Serp). More specifically, the expression described herein is approximately 1.1 to 1.5 times higher than that obtained by using the same nucleic acid expression cassette or vector that does not have at least one liver-specific nucleic acid regulatory element as taught herein but includes 3xSerp; or approximately 2 to 6 times higher than that obtained by using the same nucleic acid expression cassette or vector that does not have at least one liver-specific nucleic acid regulatory element as taught herein (e.g., including TTRe and / or Serp). Furthermore, the higher expression maintains liver specificity. In addition, the expression cassettes and vectors described herein guide the expression of therapeutic amounts of gene product over extended time periods. Typically, therapeutic expression is envisioned to last for at least 20 days, at least 50 days, at least 100 days, at least 200 days, and in some cases, 300 days or more. The expression of the gene product (e.g., peptide) can be measured, for example, by any method recognized in the art (such as by antibody-based assays, such as Western blotting or ELISA), to evaluate whether therapeutic expression of the gene product has been achieved. The expression of the gene product can also be measured in a bioassay that detects the enzymatic or biological activity of the gene product.

[0195] It should be understood that although specific embodiments, constructions and configurations, and materials have been discussed herein with respect to the methods and applications according to the invention, various changes or modifications in form and detail may be made without departing from the scope and spirit of the invention.

[0196] The following embodiments are provided to better illustrate particular implementations, and these embodiments should not be construed as limiting this application. This application is limited only by the claims. Example

[0197] Example 1: Recognition of liver-specific regulatory elements

[0198] Materials and methods

[0199] Computational methods were used to identify evolutionarily conserved transcription factor binding site (TFBS) motifs associated with highly expressed liver-specific genes as defined in this paper as nucleic acid regulatory elements. This requires several consecutive computational steps: (1) Identifying highly expressed liver-specific genes based on RNAseq (RNA sequencing) expression data obtained from normal human tissues; the mRNA expression levels of top liver-expressing genes are as follows (in any unit): ALB (46017,49); APOA2 (21859,94); TTR (9551,38); APOC1 (9505,84); APOA1 (9277,21); RBP4 (8322,91); APOC3 (8105,76); CYP2E1 (5932,55); ORM1 (5860,23); (2) Extracting the corresponding promoter sequences using publicly available databases; (3) Using computational methods to identify clusters of transcription factor binding site (TFBS) motifs; (4) Screening the genomic environment of highly expressed genes for evolutionarily conserved clusters of TFBS (i.e., CRE).

[0200] result

[0201] RNA sequence (RNAseq) analysis yielded a detailed list of highly expressed hepatocyte-specific genes. Based on these expression data, the aforementioned computational methods were applied to the promoters of these highly expressed genes, resulting in the identification of 21 liver-specific cis-regulatory sequences (SEQ ID NO: 1 to 21), as shown in Table 1.

[0202] Table 1

[0203]

[0204]

[0205] Example 2: In vivo validation of identified hepatocyte-specific cis-regulatory elements (CREs) via hydrodynamic plasmid transfection

[0206] Materials and methods

[0207] Generation of AAV plasmid construct (pAAV-HS-CRE-TTRenh-TTRm-MVM-hFIXcoPadua-bghpolyA)

[0208] The hepatocyte-specific (HS) cis-regulatory elements (CREs) identified in Example 1, HS-CRE1, HS-CRE2, HS-CRE3, HS-CRE4, HS-CRE5, HS-CRE6, HS-CRE7, HS-CRE8, HS-CRE9, HS-CRE10, HS-CRE11, HS-CRE12, HS-CRE13, HS-CRE14, HS-CRE15, HS-CRE16, HS-CRE17, HS-CRE18, HS-CRE19, HS-CRE20, and HS-CRE21, identified in Example 1, were synthesized using conventional oligonucleotide synthesis and cloned into the AAVsc-TTRenh-TTRm-MVM-hFIXcoPadua-bghpolyA construct (SEQ IDs 1-1, 2-3, 3-4, 4-5, 4-6, 4-7, 4-8, 4-9, 4-10, 4-11, 4-12, 4-13, 4-14, 4-15, 4-16, 4-17, 4-18, 4-19, 4-10, and 4-11, respectively). NO:33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, and 53). This construct was designed based on a self-complementary adeno-associated virus vector (scAAV) and contains a minimal thyroxine carrier promoter (TTRm) (SEQ ID NO:27) coupled to a TTR enhancer (TTRe or TTRenh) (SEQ ID NO:24), which drives codon-optimized, overactive human factor IX Padua (R338L) (SEQ ID NO:25) cDNA expression. Different HS-CREs ​​were cloned upstream of the TTRm / TTRe promoter / enhancer. The construct also contains an intron (SEQ ID NO:29) from mouse parvovirus (MVM) and a bovine growth hormone polyadenylation site (bghpolyA) (SEQ ID NO:30).

[0209] An additional plasmid construct (equivalent to HS-CRM8 as described in Nair et al., Blood, 2014 and Chuah et al., Mol Ther, 2014) containing a triplet repeat of a previously identified serine protease inhibitor (SERP) cis-regulatory element (SEQ ID NO: 22) (named pAAVsc-3xSERP-TTRe-TTRm-MVM-FIXcoPadua-bghpA (SEQ ID NO: 32)) is included for comparison (WO 2009 / 130208 and WO 2016 / 146757, which are specifically incorporated herein by reference). Figure 1 Different constructs are illustrated schematically.

[0210] Hydrodynamic gene transfer

[0211] The AAV plasmid construct AAVsc-HS-CRE-TTRenh-TTRm-MVM-hFIXcoPadua-bghpolyA (containing the following HS-CREs ​​identified in Example 1: HS-CRE1, HS-CRE2, HS-CRE3, HS-CRE4, HS-CRE5, HS-CRE6, HS-CRE7, HS-CRE8, HS-CRE9, HS-CRE10, HS-CRE11, HS-CRE12, HS-CRE13, HS-CRE14, HS-CRE15, HS-CRE16, HS-CRE17, HS-CRE18, HS-CRE19, HS-CRE20, or HS-CRE21) was hydrodynamically injected via the tail vein into adult C57BL / 6 mice (18 to 19 g) at a dose of 750 ng per mouse in a total volume of 2 ml phosphate-buffered saline (PBS). The AAVsc-TTRenh-TTRm-MVM-hFIXcoPadua-bghpolyA plasmid (SEQ ID NO:31), which does not have any HS-CRE element upstream of TTRenh-TTRm, was used as a reference construct to evaluate the effect of different cis-regulatory elements on FIX expression levels. Two days after hydrodynamic injection, blood was collected from all mice via exsanguination of the posterior orbital plexus into citrate buffer. The concentration of hFIX antigen in citrate plasma was measured by enzyme-linked immunosorbent assay (ELISA) according to the manufacturer's protocol (Diagnostica Stago, France). Each cohort consisted of 3 mice / vectors.

[0212] result

[0213] The effect of the cis-regulatory elements identified in Example 1 on in vivo expression was assessed by measuring FIX protein levels in mice injected with a plasmid construct expressing human coagulation factor IX (hFIX) via a chimeric promoter consisting of an effective liver-specific minimal thyroxine carrier promoter in conjunction with a thyroxine carrier enhancer (TTRenh / TTRmin), wherein a distinct CRE is cloned upstream of TTRenh / TTRmin. FIX expression was assessed 1 or 2 days post-transfection, demonstrating a significant increase in FIX expression via some of the novel HS-CREs. Specifically, approximately 52% (11 of the 21 HS-CREs) of the identified cis-regulatory elements induced a significant increase in hFIX expression on day 2 post-transfection, compared to the control plasmid construct without any cis-regulatory elements (i.e., AAVsc-TTRenh-TTRm-MVM-hFIXcoPadua-bghpolyA (SEQ ID NO: 31)). Figure 2 Furthermore, the newly identified regulatory element was compared with a previously identified liver-specific regulatory element named the serine protease inhibitor enhancer (WO 2009 / 130208, which is specifically incorporated herein by reference), which is present in the vector as a triple repeat sequence (i.e., pAAVsc-3xSERP-TTRe-TTRm-MVM-FIXcoPadua-bghpA (SEQ ID NO:32)). We have previously demonstrated that repeating the SERP element three times enhances the expression of the gene of interest (e.g., FIX). Surprisingly, we found that three of the new CREs (i.e., HS-CRE4, HS-CRE6, and HS-CRE12) outperformed all other CREs (including SERP3X). Specifically, the FIX levels expressed by HS-CRE4, HS-CRE6, and HS-CRE12 were comparable to or even higher than the FIX levels achievable by incorporating the triple SERP repeat sequence into the AAV vector. Taking into account the known effects of doubling SERP elements on transgene expression levels, this shows that, surprisingly, HS-CRE4 (ApoA2; SEQ ID NO:4), HS-CRE6 (ApoC1; SEQ ID NO:6), and HS-CRE12 (RBP4; SEQ ID NO:12) are therefore significantly superior to single SERP elements in enhancing transgene expression levels. HS-CRE6 is the most efficient HS-CRE compared to all CREs (including SERPs).

[0214] Example 3: In vivo validation of the identified hepatocyte-specific cis-regulatory elements (CREs) via AAV viral vector transduction

[0215] To confirm the in vivo results obtained via hydrodynamic (i.e., non-viral) plasmid transfection and to broaden the scope of the invention to include viral vectors, AAV serotype 9 (designated AAV9) vector particles were generated based on non-viral, semi-high-throughput in vivo screening and validation. These particles contain the most effective CREs identified in Example 2 (i.e., HS-CRE4 and HS-CRE6). The corresponding AAV9 vectors are based on self-complementary (sc) structures and are named AAV9sc-HS-CRE4-TTRenh-TTRm-MVM-hFIXcoPadua-bghpolyA and AAV9sc-HS-CRE6-TTRenh-TTRm-MVM-hFIXcoPadua-bghpolyA. As a control, AAV9 vectors without any CRE (named AAV9sc-TTRenh-TTRm-MVM-hFIXcoPadua-bghpolyA) or AAV9 vectors containing a triple repeat sequence of SERP element as a reference (named AAV9sc-3XSERP-TTRenh-TTRm-MVM-hFIXcoPadua-bghpolyA) were generated.

[0216] Carrier generation, purification, and titration:

[0217] As previously described, the above-mentioned AAV serotype 9 vectors (i.e., AAV9sc-HS-CRE4-TTRenh-TTRm-MVM-hFIXcoPadua-bghpolyA, AAV9sc-HS-CRE6-TTRenh-TTRm-MVM-hFIXcoPadua-bghpolyA, AAV9sc-TTRenh-TTRm-MVM-hFIXcoPadua-bghpolyA, AAV9sc-3XSERP-TTRenh-TTRm-MVM-hFIXcoPadua-bghpolyA) were generated by co-transfecting AAV-293 cells with AAV plasmids, chimeric packaging constructs, and adenovirus helper plasmids using calcium phosphate (Thermo Fisher Scientific, Waltham, MD, USA). (VandenDriessche T, Thorrez L, Acosta-Sanchez A, et al., Efficacy and safety of adeno-associated virus vectors based on serotypes 8 and 9 and lentiviral vectors for gene therapy of hemophilia B, J Thromb Haemost. 2007; 5(1):16-24). Two days after transfection, cells were harvested and lysed by continuous freeze-thaw cycles and sonication, followed by ultracentrifugation using benzonase (Novagen, Madison, Wisconsin, USA), deoxycholic acid (Sigma Aldrich (MERCK, Check, St. Louis, Missouri, USA), and three rounds of cesium chloride (Thermo Fisher Scientific, Waltham, Massachusetts, USA) density gradient. Fractions containing AAV particles were collected and dialyzed in Dulbecco phosphate-buffered saline (D-PBS) (Gibco, IRL) containing 1 mg MgCl2.

[0218] To determine the carrier titration amount, use Quantitative real-time PCR was performed using primers for the FIX gene (ABI Prism 7900HT, Applied Biosystems, Foster City, California, USA) from Green (Thermo Fisher Scientific, Waltham, MD, USA). The forward primer sequence was 5'-CACGAGAACGCCAACAAGAT-3' (SEQ ID NO:55), and the reverse primer sequence was 5'-CACTTCTCCTCCATGCACTC-3' (SEQ ID NO:56). Standard curves were generated using known copy numbers of the corresponding vector plasmids.

[0219] AAV9 vector injection

[0220] With 1x10 9 vg / mouse dose ( Figure 3 A) and 5x10 9 vg / mouse dose ( Figure 3 B) The vectors AAV9sc-HS-CRE4-TTRenh-TTRm-MVM-hFIXcoPadua-bghpolyA, AAV9sc-HS-CRE6-TTRenh-TTRm-MVM-hFIXcoPadua-bghpolyA, AAV9sc-TTRenh-TTRm-MVM-hFIXcoPadua-bghpolyA, and AAV9sc-3XSERP-TTRenh-TTRm-MVM-hFIXcoPadua-bghpoly were injected into the tail vein of C57BL / 6 mice (4-5 weeks old). Each cohort consisted of 3 mice. Blood was collected at multiple time points after injection, and plasma FIX protein levels were analyzed by ELISA.

[0221] Compared with control mice injected with the AAV9sc-TTRenh-TTRm-MVM-hFIXcoPadua-bghpolyA vector without any HS-CRE, mice injected with 5x10 9 Mice injected with either AAV9sc-HS-CRE4-TTRenh-TTRm-MVM-hFIXcoPadua-bghpolyA or AAV9sc-HS-CRE6-TTRenh-TTRm-MVM-hFIXcoPadua-bghpolyA produced significantly higher FIX levels. This indicates that the HS-CRE4 and HS-CRE6 elements resulted in a 5- to 6-fold increase in FIX levels, which remained relatively stable over at least 3 months. Similarly, compared to control mice injected with AAV9sc-TTRenh-TTRm-MVM-hFIXcoPadua-bghpolyA without any HS-CRE, mice injected with lower doses (i.e., 10) showed significantly higher FIX levels. 9 Mice using the vectors AAV9sc-HS-CRE4-TTRenh-TTRm-MVM-hFIXcoPadua-bghpolyA or AAV9sc-HS-CRE6-TTRenh-TTRm-MVM-hFIXcoPadua-bghpolyA produced significantly higher FIX levels. For all vectors tested, compared to 10... 9 Compared to vg, when injected with 5x10 9 Following vg, the dose response was evident, consistent with higher stable FIX levels.

[0222] The newly identified regulatory element was further compared with a previously identified liver-specific regulatory element named the serine protease inhibitor enhancer (WO 2009 / 130208, which is specifically incorporated herein by reference), which is present in the vector as a triple repeat sequence (i.e., AAV9sc-3xSERP-TTRe-TTRm-MVM-FIXcoPadua-bghpA). We have previously demonstrated that repeating the SERP element three times increases the expression of the gene of interest (e.g., FIX). Consistent with the hydrodynamic transfection data shown in Example 2, we found that the FIX levels expressed by HS-CRE4 and HS-CRE6 were comparable to or even higher than the FIX levels achievable by incorporating the triple SERP repeat sequence into the AAV vector. Taking into account the known effects of doubling SERP elements on transgene expression levels, this further confirms that HS-CRE4 (ApoA2; SEQ ID NO:4) and HS-CRE6 (ApoC1; SEQ ID NO:6) are superior to single SERP elements in enhancing transgene expression levels. In the case of AAV9-based liver transduction, HS-CRE6 is also the most effective HS-CRE compared to all CREs (including SERPs).

[0223] The inventors conducted experimental studies to test which transcription factors bind to HS-CRE 1-21.

[0224] Experimental studies revealed that (1) HS-CRE6, HS-CRE4, and HS-CRE12 (which are the HS-CREs ​​that cause the greatest increase in liver-specific protein levels) all have transcription factor binding sites for SP1, EP300, HNF4G, CEBPB, P300, HDAC2, JUND, FOSL2, ZBTB7A, CEBPD, and RXRA; (2) HS-CRE1, HS-CRE5, and HS-CRE13 (which are the HS-CREs ​​that show a moderate increase in liver-specific protein levels) all have transcription factor binding sites for SP1, EP300, POLR2A, MYBL2, FOXA1, FOXA2, ARID3A, POLR2A, and HEY1; and (3) HS-CRE9, HS-CRE11, HS-CRE14, HS-CRE16, and HS-CRE20 (which are the HS-CREs ​​that show the lowest increase in liver-specific protein levels) all have transcription factor binding sites for SP1, EP300, HNF4G, CEBPB, P300, HDAC2, JUND, FOSL2, ZBTB7A, CEBPD, and RXRA. HS-CRE6, HS-CRE4, HS-CRE12, HS-CRE1, HS-CRE5, HS-CRE13, HS-CRE9, HS-CRE11, HS-CRE14, HS-CRE16, and HS-CRE20 all possess transcription factor binding sites for SP1 and EP300.

[0225] References

[0226] Budker V, Zhang G, Knechtle S, Wolff JA. Naked DNA delivered intraportally expresses efficiently in hepatocytes. (1996) Gene Ther. Jul; 3(7):593-8.

[0227] BRUNETTI-PIERRI N,GROVE NC,ZUO Y,EDWARDS R,PALMER D,CERULLO V,TERUYAJ,NG P.Bioengineered factor IX molecules with increased catalytic activityimprove the therapeutic index of gene therapy vectors for hemophilia B.HumGene Ther.2009May;20(5):479-85.

[0228] Chang,J.,Jin,J.,Lollar,P.,Bode,W.,Brandstetter,H.,Hamaguchi,N.,Straight,D.L.&Stafford,D.W.(1998).Changing residue 338in human factor IX fromarginine to alanine causes an increase in catalytic activity.J Biol Chem273(20):12089-12094.

[0229] CRISTIANO RJ,Smith LC,Kay MA,Brinkley BR,Woo SL.(1993a)

[0230] Hepatic gene therapy: efficient gene delivery and expression in primary hepatocytes utilizing a conjugated adenovirus-DNA complex. Proc Natl Acad Sci USA.90(24):11548-11552.

[0231] CRISTIANO RJ,Smith LC,Woo SL.(1993b)Hepatic gene therapy:adenovirusenhancement of receptor-mediated gene delivery and expression in primaryhepatocytes.90(6):2122-2126.

[0232] GRIMM D,LEE JS,WANG L,DESAI T,AKACHE B,STORM TA,KAY MA (2008).JVirol.82(12):5887-5991.

[0233] IVICS Z,HACKETT PB,PLASTERK RH, Z.(1997)Molecularreconstruction of Sleeping Beauty,a Tc1-like transposon from fish,and itstransposition in human cells.Cell.91(4):501-510.

[0234] Kay MA,Baley P,Rothenberg S,Leland F,Fleming L,Ponder KP,Liu T,Finegold M,Darlington G,Pokorny W,Woo SLC.(1992)Expression of human alpha 1-antitrypsin in dogs after autologous transplantation of retroviral transducedhepatocytes.Proc Natl Acad Sci U S A.Jan 1;89(1):89-93.

[0235] Kao,C.Y.,Lin,C.N.,Yu,I.S.,Tao,M.H.,Wu,H.L.,Shi,G.Y.,Yang,Y.L.,Kao,J.T.&Lin,S.W.(2010).FIX-Triple,a gain-of-function factor IX variant,improveshaemostasis in mouse models without increased risk of thrombosis.ThrombHaemost 104(2):355-365.

[0236] Kistner A,Gossen M,Zimmermann F,Jerecic J,Ullmer C,Lybbert H,BujardH.(1996)Doxycycline-mediated quantitative and tissue-specific controlof geneexpression in transgenic mice.Proc Natl Acad Sci U S A.Oct 1;93(20):10933-8.

[0237] Lin,C.N.,Kao,C.Y.,Miao,C.H.,Hamaguchi,N.,Wu,H.L.,Shi,G.Y.,Liu,Y.L.,High,K.A.&Lin,S.W.(2010).Generation of a novel factor IX with augmentedclotting activities in vitro and in vivo.J Thromb Haemost 8(8):1773-1783.

[0238] LISOWSKI L,DANE AP,CHU K,ZHANG Y,CUNNINGHAM SC,WILSON EM,NYGAARD S,GROMPE M,ALEXANDER IE,KAY MA.(2014)Nature.506(7488):382-386.

[0239] Liu F,Song Y,Liu D.(1999)Hydrodynamics-based transfection in animalsby systemic administration of plasmid DNA.Gene Ther.Jul;6(7):1258-66.

[0240] McCARTY DM,MONAHAN PE,and SAMULSKI RJ.(2001).Self-complementaryrecombinant adeno-associated virus(scAAV)vectors promote efficienttransduction independently of DNA synthesis.Gene Ther.8,1248-54.

[0241] McCARTY DM, FU H, MONAHAN PE, TOULSON CE, NAIK P, and SAMULSKI RJ.(2003).Adeno-associated virus terminal repeat(TR) mutant generates self-complementary vectors to overcome the rate-limiting step to transduction invivo.Gene Ther.10,2112-8.

[0242] L,CHUAH MK,BELAY E,JERCHOW B,MANOJ N,ACOSTA-SANCHEZ A,GRZELADP,SCHMITT A,BECKER K,MATRAI J,MA L,SAMARA-KUKO E,GYSEMANS C,PRYPUTNIEWICZ D,MISKEYC,FLETCHER B,VANDENDRIESSCHE T,,IVICS Z,and IZSVAK Z.(2009).Molecular evolution of a novel hyperactive Sleeping Beauty transposase enables robust stable gene transfer in vertebrates.Nat Genet.41,753-61.

[0243] Milanov,et al.,2012 Engineered factor IX variants bypass FVIII andcorrect hemophilia A phenotype in mice Blood 119:602-611.

[0244] Nair N,Rincon MY,Evens H,Sarcar S,Dastidar S,Samara-Cock E,Ghandeharian O,Man Viecelli H, B,De Bleser P,VandenDriessche T,Chuah MK.(2014).Computationally designed liver-specific transcriptional modules andhyperactive factor IX improve hepatic gene therapy.Blood123,3195-9.

[0245] NATHWANI AC,DAVIDOFF AM,HANAWA H,YUNYU HU,HOFFERFA,NIKANOROV A,SLAUGHTER C,NG CYC,ZHOU J,LOZIER J,MANDRELL TD,VANIN EF,and NIENHUIS AW.(2002).Sustained high-level expression of human factor IX(hFIX)after liver-targeted delivery of recombinant adeno-associated virus encoding the hFIXgene in rhesus macaques.Blood 100,1662–1669.

[0246] NATHWANI AC,GRAY JT,NG CY,ZHOU J,SPENCE Y,WADDINGTON SN,TUDDENHAM EG,KEMBALL COOK G,McINTOSH J,BOON-SPIJKER M,MERTENS K,DAVIDOFF AM.(2006).Self-complementary adeno-associated virus vectors containing a novel liver-specific human factor IX expression cassette enable highly efficienttransduction of murine and nonhuman primate liver.Blood 107,2653-61.

[0247] NATHWANI AC,TUDDENHAM EG,RANGARAJAN S,ROSALES C,MCINTOSH J,LINCH DC,CHOWDARY P,RIDDELL A,PIE AJ,HARRINGTON C,O'BEIRNE J,SMITH K,PASI J,GLADER B,RUSTAGI P,NG CY,KAY MA,ZHOU J,SPENCE Y,MORTON CL,ALLAY J, COLEMANJ, SLEEP S, CUNNINGHAM JM, SRIVASTAVA D, BASNER-TSCHAKARJAN E, MINGOZZI F, HIGH KA, GRAY JT, REISS UM, NIENHUIS AW, and DAVIDOFF AM Med.365,2357–2365.

[0248] (2008).Optimization of self-complementary AAV vectors for liver-directedexpression results in sustained correction of hemophilia B at low vectordose.Mol Ther.16,280-9.

[0249] Schuettrumpf,J.,Herzog,RW,Schlachterman,A.,Kaufhold,A.,Stafford,DW&Arruda,VR(2005).Factor IX variants improve gene therapy efficacy forhemophilia B.Blood 105(6):2316-2323.

[0250] Simioni , P , Tormene , D , Tognin , G , Gavasso , S , Bulato , C , Iacobelli , NP , Finn , JD , Spiezia , L , Radu , C & Arruda , VR(2009).X-linked thrombophilia with amutant factor IX(factor IX Padua).N Engl J Med 361(17):1671–1675.

[0251] SNYDER RO, MIAO C, MEUSE L, TUBB J, DONAHUE BA, HUI-FENG LIN, STAFFORD DW, PATEL S, THOMPSON AR, NICHOLS T, READ MS, BELLINGER DA, BRINKHOUS KM, and KAY MA adeno-associated viral vectors.Nat Med.5,64–70.

[0252] WARD,NJET AL.Codon optimization of human factor VIII cDNAs leads to high-level expression.Blood 117,798-807(2011).

[0253] Yamada T,Iwasaki Y,Tada H,Iwabuki H,Chuah MK,VandenDriessche T,FukudaH,Kondo A,Ueda M,Seno M,Tanizawa K,Kuroda S.(2003)Nanoparticles for thedelivery of genes and drugs to human hepatocytes.Nat Biotechnol.Aug21(8):885-90.

[0254] YUSA ET AL.A hyperactive piggyBac transposase for mammalianapplications.Proc Natl Acad Sci U S A.2011;108(4):1531-6.

[0255] Zhang G,Budker V,Wolff JA.(1999)High levels of foreign geneexpression in hepatocytes after tail vein injections of naked plasmid DNA.HumGene Ther.JuI 1;10(10):1735-7.

[0256] DE BLESER P,HOOGHE B,VLIEGHE D,VAN ROY F.(2007)A distance differencematrix approach to identifying transcription factors that regulatedifferential gene expression.Genome Biol.8(5):R83.

[0257] CHUAH,M.K.,PETRUS,I.,DE BLESER,P.,LE GUINER,C.,GERNOUX,G.,ADJALI,O.,NAIR,N.,WILLEMS,J.,EVENS,H., M.Y.,MATRAI,J.,DI MATTEO,M.,SAMARA-KUKO,E.,YAN,B.,ACOSTA-SANCHEZ,A.,MELIANI,A.,CHEREL,G.,BLOUIN,V.,CHRISTOPHE,O.,MOULLIER,P.,MINGOZZI,F.,VANDENDRIESSCHE,T.(2014)Liver-specifictranscriptional modules identified by genome-wide in silico analysis enableefficient gene therapy in mice and non-humans primates.Mol.Ther.22(9):1605-13

[0258] GARMORY HS1, BROWN KA, TITBALL RW. (2003) DNA vaccines: improving expression of antigens. Genet Vaccines Ther.1(1):2Wu C, Macleod I, Su AI. (2013) BioGPS and MyGene.info: organizing online, gene-centric information. NucleicAcids Res.41 (Database issue): D561-5.

[0259] MCINTOSH J,LENTING PJ,ROSALES C,LEE D,RABBANIAN S,RAJD,PATEL N,TUDDENHAM EG,CHRISTOPHE OD,MCVEY JH,WADDINGTON S,NIENHUIS AW,GRAY JT,FAGONEP,MINGOZZI F,ZHOU SZ,HIGH KA,CANCIO M,NG CY,ZHOU J,MORTON CL,DAVIDOFF AM,NATHWANI AC.(2003) Therapeutic levels of FVIII following a single peripheral vein administration of rAAV vector encoding a novel human factor VIII variant. Blood.121(17):3335-44XIAO SJ,ZHANG C,ZOU Q,JI ZL.(2010)TiSGeD: database for tissue-specific genes.Bioinformatics 26(9):1273-5. sequence list <110> VRIJE University of Brussels <120> Liver-specific nucleic acid regulatory elements, their methods and applications <130> VUB-076-PCT <150> 18202986.8 <151> 2018-10-26 <160> 58 <170> PatentIn version 3.5 <210> 1 <211> 336 <212> DNA <213> Homo sapiens <400> 1 tgggaaatta cagttaaata ccatggtaat gaataaaagg tacaaatcgt ttaaactctt 60 atgtaaaatt tgataagatg ttttacacaa ctttaataca ttgacaaggt cttgtggaga 120 aaacagttcc agatggtaaa tatacacaag ggatttagtc aaacaatttt ttggcaagaa 180 tattatgaat tttgtaatcg gttggcagcc aatgaaatac aaagatgagt ctagttaata 240 atctacaatt attggttaaa gaagtatatt agtgctaatt tccctccgtt tgtcctagct 300 tttctcttct gtcaacccca cacgcctttg gcacaa 336 <210> 2 <211> 3(4)7 <212> DNA <213> Homo sapiens <400> 2 ttttttcttt tctaaatgat cagtacactt attctttcta aagaaaatac ttttcttaac 60 tactctctat ttttaaactt ctcccacaaa gatgagaaaa catttaaaaa tcattggggc 120 tatttttctg tttaccgagt aaagagaatc tctaaaccat atttataact cttactctaa 180 atatttgcat ttaccctcat gccagagccc gttgatgact gactaaacag agtttcaaag 240 tttgaagaac aggaaattta gaaatgacta acaattatgt aggtttattt ctctcagtat 300 Note: There seems to be an unclear notation <211> 3(4)7 in the original. I've translated it as best as possible while keeping the original format. If this is incorrect, more context might be needed to accurately translate it. agaatgttca tatagaatta atgccagagg ttttcagaga aaaatgc 347 <210> 3 <211> 386 <212> DNA <213> Homo sapiens <400> 3 gggacactga agccaaatgg gcttcagctc ctccccaggg atctccctac ctgctgccca 60 ttccaacctg gctctctccc tctccacaac tgaagcccag gcctctttgg atgctgctca 120 cacatcttgc ctccttatct tacctagcca gcgtctctgt ccttggtgtc tgtgcctgcc 180 ctggctggag aggaaggggc tatatacctg tctgtttacc caccccctgt caggtgacag 240 ggactatgga tgggcccagt ggggcaggga ttatgtgagg ataaacaagt tggagaaatg 300 ggggaggaga ggacaagcac atggaagact catggtgatg gggtgagagg cagggggtac 360 agctcttagt ggaggctaag ggggta 386 <210> 4 <211> 426 <212> DNA <213> Homo sapiens <400> 4 gagttaatgt gtaatgagac ggggagaaaa caggagagcc cagaatgacc tggatgctga 60 tcactgaaca taccctaccc ccagtaaaac aaattcagaa aacagcttcc gcccgtcccc 120 tcccaatgga gggctctggc aggaaaagag gtgaataaga ggcttctacc agggtaaagg 180 ttgaaggcac ctggtcattt gatcacctta tcagttctag gcagtgatta gccaatattg 240 agtcagcagg ggcaatagcc ctggcccttg tctcactcct gttgggggtg ggggaggggg 300 agaggtacat tcccaggttc aaagcatttg ggtgaaatca gttaaataga tatcagaagc 360 ttttgtatct ttcacccttt tgccccccaa gcatactcgc tgagtatgtg gaacattcct 420 gagggt 426 <210> 5 <211> 260 <212> DNA <213> Homo sapiens <400> 5 tgttttgttt tggtgaccta actggtcaaa tgacctatta agaatatttc atagaacgaa 60 tgttccgatg ctctaatctc tctagacaag gttcatattt gtatgggtta cttattctct 120 ctttgttgac taagtcaata atcagaatca gcaggtttgc agtcagattg gcagggataa 180 gcagcctagc tcaggagaag tgagtataaa agccccaggc tgggagcagc catcacagaa 240 gtccactcat tcttggcagg 260 <210> 6 <211> 267 <212> DNA <213> Homo sapiens <400> 6 aggggggccc ctggggctga cagggactgg aagctctgag ctggccagag ggatgttgca 60 atcctgccag ggtcttgtct atgctgtcct tttcacaacc atccccctac tgccaggctg 120 acacgtggtt gcgggggcac aaggccagcc aacctagagt ctgaggctag gcggaggaca 180 ccctccccac cagctgccag ggtcactggc ggtcaaaggc agctggtggg gaaggcattg 240 gactccagcc ttgggggacg gatgtag 267 <210> 7 <211> 313 <212> DNA <213> Homo sapiens <400> 7 tacaggcctg gggtcccggc ttagaggacc tctgagagct ccggggcccc ttctgggtcg 60 tggttgcctc atcgtggtcg ggtgggtctc caggttctcc caggctcagt cccgcaggcg 120 ccaaatctgc gcaggagagc actagcaacc gatgacgtat tgaggcccac acctctggga 180 ttggctgtcc tgcttcgaca gccttgaaag tgggtaagct gggtgggggg ctctgggaga 240 ggtcagtgct gagtaaggca attcccagca gcttgagccc caccaggtca ctccagtatt 300[[ID=​​​​​​​​​ <400> 8 cccgaacgaa taaacccctt ccttaactca gcgtctgagg aattttgtct gcggctcctc 60 ctgctacatt ctgagtgggg aaagggacta aggtggtctg aggaccccac agagtcagga 120 agattgagag gtgagagtgc tgaacgggga ggggctttgg ggctaaggga agtgcccggg 180 accccacctg accccaacgc tcacgggaca ggggcagagg agaaaaacgt gggtggacag 240 agggaggcag gcggtcaggg gaaggctcag gaggagggag atcaacatca acctgccccg 300 ccccctcccc agcctgataa aggtcctgcg ggcaggacag gacctcccaa ccaagccctc 360 cagcaaggat tcaggttggt gctgagtgcc tgggagggac acccgcctac actctgcaag 420 aaactcaaaa agggagatga ggggatcgtg ggagggaggt agggagggag gagggtgcca 480 ctgatcccct gaacccctgc ctctgcctcc aga 513 <210> 9 <211> 306 <212> DNA <213> Homo sapiens <400> 9 gatgagtgca gggaaccccg accccacccg ggagacctgc aagcctgcag acactcccct 60 cccgccccca ctgaaccctt gacccctgcc ctgcagcccc cgcagcttgc tgtttgccca 120 ctctatttgc ccagccccag ggacagagct gatccttgaa ctcttaagtt ccacattgcc 180 aggaccagtg agcagcaaca gggccggggc tgggcttatc agcctcccag cccagaccct 240 ggctgcagac ataaataggc cctgcaagag ctggctgctt agagactgcg agaaggaggt 300 gcgtcc 306 <210> 10 <211> 406 <212> DNA <213> Homo sapiens <400> 10 ttctatcatt atcttcaatc agaaactgat gtgtggggag gtgatgtatc ataactcatc 60 cagggtcagg cagctggatt tgaacctgcc tgacccttag atttgaggtt ttctggctcc 120 agaatcttct ctgaagtgaa aggcatgagg ccgaccactc cctgatctgg taaacagaga 180 tgtcagcctg gtttctagtg ttagggagtt tcctggagtg atggtacagg gtacatttct 240 gccctgcatc ccaagtccag agactgggtt ctaggtccag cctttcttct aactccctga 300 gagatgacag cctctgggca aagtccctct gtgctcttca gtctcttcat ctgtgagatg 360 gtggcatggg gagaggctgg agtgatgtca cagttcctct cagcct 406 <210> 11 <211> 504 <212> DNA <213> Homo sapiens <400> 11 acaaaagctg cagcaaaatt gccaaaagta ggaactactg ggggtggggg tcctgtctcc 60 cctgctcagt caccacctcc cctccctaac tctcattctg gctcatacca cgtgctccca 120 gaagaaacgt tctgagcagg ggctggtatg agactgcctc ccctcccagg acccactcct 180 ccctatgacc aggctggtga ccctccctct agggtgccag tgccattcac tgagcagatg 240 tttggcaggt gtctgttatg tgccagacct agcaggggac agagtcctcc cgatacagat 300 ggaccagtat gtttggacat aaattgtgaa aggagttgct aatctttctg tgcaaaatcc 360 actcttgcgg tcaccgccaa cactcctaca catgtcaggg catccagggc caggacttgg 420 taaccctgtt caggagcagt cgctctggag actcccaaga cgagggaaag aaatgggtgg 480 aaacaggggc ctacactgtg ggcc 504 <210> 12 <211> 431 <212> DNA <213> Homo sapiens <400> 12 gtgaggccac ggtcttcccg ccaggttgac tcgagcctcc tgccagagcc actggccccg 60 gaggccaccc tagaccgcag ctggcggccg ctggcacgag tgcagggtaa ctgagccagg 120 gccgctggcg catttggcct ggccgaggcc accccgcgcg gccgctccac tgtgcccgag 180 gctgtcctgg aggtgaggcc ggcccacagg gaccctgccc gtgcccgggc tccggtgagt 240 cagggcgcgt tatgcaagtg cccccggcgc ctccccttcg gtctttcacc ccgcgcggtt 300 acgaaagcgc gaccccctcc ccccggcgct ataaagcagc ggggcggccg cggcgcgctc 360 gcctccctcg ctccacgcgc gcccggactc ggcggccagg cttgcgcgcg gttcccctcc 420 cggtggtgag t 431 <210> 13 <211> 448 <212> DNA <213> Homo sapiens <400> 13 aaatacctaa actagtcaca ctgttttgat tcaattggct actgaagtta tagaatgttg 60 tttactcttc tctcctttgt ctactcccca gccaacaaaa caaccgacct tagctgtttt 120 gaaaataaat gaaaattcca acatgggttt gaaataaaat tgcatcataa acaatcggta 180 ggtgtttttc aaagtggttt cagggaagtg ccacggagta agcaggcgac caccgaggct 240 gctaaaatat ttcctgtcct gaccagggtt gcgtttctgg agaatattta acagggaggg 300 ttttaacgct tttaaagatg ttgaaactaa agaacaaata ttgaccagag ggcaccacaa 360 cgctcctgaa agagagtaaa atacatcctt tataaaatga aaaactactt ggatgaatta 420 ttccaaaatt cctgcacaag tggacctc 448 <210> 14 <211> 335 <212> DNA <213> Homo sapiens <400> 14 ctgagctcat ctgggctgca gggctggcgg gacagcagcg tggactcagt ctcctaggga 60 tttcccaact ctcccgcccg cttgctgcat ctggacaccc tgcctcaggc cctcatctcc 120 actggtcagc aggtgacctt tgcccagcgc cctgggtcct cagtgcctgc tgccctggag 180<000,0731>atgatataaa acaggtcaga accctcctgc ctgtctgctc agttcatccc tagaggcagc 240 tgctccaggt aatgccctct ggggagggga aagaggaggg gaggaggatg aagaggggca 300 agaggagctc cctgcccagc ccagccagca agcct 335 <210><15 <211> 431 <212> DNA <213> Homo sapiens <400> 15 agaagggtca gcggcccctc ctggaccacc gactccccgc agaactcctc tgtgccctct 60 cctcaccaga ccttgttcct cccagttgct cccacagcca gggggcagtg agggctgctc 120 ttcccccagc cccactgagg aacccaggaa ggtgaacgag agaatcagtc ctggtggggg 180 ctggggaggg ccccagacat gagaccagct cctcccccag gggatgttat cagtgggtcc 240 agagggcaaa atagggagcc tggtggaggg aggggcaaag gcctcgggct ctgagcggcc 300 ttggcccttc tccaccaacc cctgccctac actaaggggg aggcagcggg gggcacacag 360 ggtgggggcg ggtggggggc tgctgggtga gcagcactcg cctgcctgga ttgaaaccca 420 gagatggagg t 431 <210> 16 <211> 559 <212> DNA <213> Homo sapiens <400> 16 gtagctgatg ggaagagcag actgccttcc agccaggcct ggtcctgtga gtcagggacg 60 tccatcttag tgggcatgaa aggcctgtgt gatctcgagg gagacatcgc ctctccaagc 120 ctctccttat ctgtgcaaca ggcagactta atgattggtg aggcaatgag gctgatagct 180 cagcattagc tacagccacc cctcctggcc aaccacacag ggatcaaacc aggggtcagt 240 ccagaggtca gagtcaggag cagacaactc agatccagcc agggacaggc aggtcacacg 300 gacatgtgcc tcacgtatgc ttcaaggggc cctcccccgg gcagaactga aggacagctc 360 ctgttgccat aggagggagc tgggtgagat actaggagga acttccggca tgatgatgtg 420 tgatgaacaa gggcctctgg ccaacaggtc tgaatcaggg ctgcccagcc cagcctggtg 480 ggaagggcat ggagcatggg ggctcatgta ctaaacctca cctggacaca aggtgaaaca 540 gcccaacccc agaggacca 559 <210> 17 <211> 370 <212> DNA <213> Homo sapiens <400> 17 gcttgcaaca atgtctggca cataggaaaa gtgatcacta aatgttagcc acgtcttact 60 cctgcaaggc tcacctccct ggaacccatc ggtcccaacc ctgctcctga atcaggcaca 120 gtccagcttg cagcgggagc aaaggtcagt actcagtgcc cctgtccctt ccccaggcca 180 gaggggagga ggagactgag tcacgaatga cacctcagcc gcagtttgac ctccaggact 240 tacagtccta gcagccggtg ccactagcat gtgagaggtc cagaggcgct tctgtctcac 300 ccgcccgcct gggtgcaccc atgctgggag cgcctgcacc atttgagcat gtccgagagc 360 atccaccaga 370 <210> 18 <211> 431 <212> DNA <213> Homo sapiens <400> 18 ctgggcagga gcagctggct tgagcagaat cttgggacct gaggctctca ggggacctcc 60 cattggggga tggagggcaa tggtggtggt gcccaggagg tcttctactt agatgtctat 120 tggatctcta aatgaggctg catgcataat cacacacaaa catccactga gaaggtgaca 180 caccacgtca gcatgggtcc ctctgccgga ccacaccact cctagtgact atgaggtgac 240 atccaggcac gttgcactat tggctcctgt cggtgagtgc agtgcctgac aacagtgagc 300 tacatttatt tgtaaaaatg aacgccatca gagtagacca caattgtact aactctaatt 360 tgctttgtgt tcattttttc agtttccaga agtggcttaa tgtttcctag ggtcaaaggc 420 agtcaaatga c 431 <210> 19 <211> 481 <212> DNA <"213"> Homo sapiens <400> 19 gaagggggaa gagacccact gaaatcctat ctcccagcct cacctctgct gtctcctcca 60 gaagggggaa gagacccact gaaatcctat ctcccagcct cacctctgct gtctcctcca 60 cgcttcctgt ctccagagcc ccgagttcag cataagcaga aagcggcctg ttccctctct 120 cgcttcctgt ctccagagcc ccgagttcag cataagcaga aagcggcctg ttccctctct 120 agggagagga gggttgcggt ctggaggtct ggctcgtctt tatctgcgca ttctcccagc 180 agggagagga gggttgcggt ctggaggtct ggctcgtctt tatctgcgca ttctcccagc 180 ctcctggctt cagacctcag cgaggcggcg gctgccggcc ggctctcctc ttcctgcctg 240 ctcctggctt cagacctcag cgaggcggcg gctgccggcc ggctctcctc ttcctgcctg 240 cagacctggc ctgctgcttc tttctccttc ctccctccct gcctgccctg cggtttcaaa 300 cagacctggc ctgctgcttc tttctccttc ctccctccct gcctgccctg cggtttcaaa 300 gtagattaga aataacagtg tcccacatgg aagcctctac ttcttcctgg gtcaactttg 360 gtagattaga aataacagtg tcccacatgg aagcctctac ttcttcctgg gtcaactttg 360 atgacgaggc tccagaaaac ctttgcaatg ctgtgtggaa tttttaaatc ggtgagctcg 420 atgacgaggc tccagaaaac ctttgcaatg ctgtgtggaa tttttaaatc ggtgagctcg 420 tgctcttgcc ctatttattt gtccagcgta catttctgaa cattgtgaac gtcgaatggg 480 tgctcttgcc ctatttattt gtccagcgta catttctgaa cattgtgaac gtcgaatggg 480 c 481 c 481 <210> 20<210> 20 <211> 345<211> 345 <212> DNA<212> DNA <213> 智人<213> Homo sapiens <400> 20<400> 20 ggaaaacagg gagacagttt cctgtttgag atgttgggag atgcttcgag tagtatattt 60 ggaaaacagg gagacagttt cctgtttgag atgttgggag atgcttcgag tagtatattt 60 actggaaata gacattcaac ttggatgtcc ctttttggaa atgtgcctgc gtccagggct 120 actggaaata gacattcaac ttggatgtcc ctttttggaa atgtgcctgc gtccagggct 120 gggttggggc cccaatgaac tttggctctg acacagctgt tgccacactc agtggaactg 180 aatctatgtt tgtcttcccc ggcatccttc accccaactc tccccgccac aacatacatc 240 ccatgccagc ctggggaccc tcaaaggtgg cttcatcatt aggtttgtgg ctgggtccca 300 ctgaagtaag tcttggcact cagagggata ggaattgaat gaaga 345 <210> 21 <211> 386 <212> DNA <213> Homo sapiens <400> 21 ggaggttgca gtaagccaag attgtgccac tgcactccaa cctgggtgac agagccagac 60 tctgtctcaa gaaaataaag agaaaaagaa ctagattcag gctgtatgtg ggcaccatct 120 acagacatca gacacacaca tttaggagta ataatgatgc aatattgcca aatgtttctc 180 agcaaccatg tgcaactgtt gcatgtactg tttatttcta actcaggagg atctctctaa 240 ccacacattg caagaaaata gactttcagt gtttctggca aaacaaagag tttttttgtt 300 ggcatttatg ctgaaaggga agagttaaaa tgttaatttg tcttttttct tacctccaag 360 ttcactcaaa tcctctcttg aagcag 386 <210> 22 <211> 72 <212> DNA <213> Artificial sequence <220> <223> SerpEnh <400> 22 gggggaggct gctggtgaat attaaccaag gtcaccccag ttatcggagg agcaaacagg 60 ggctaagtcc ac 72 <210> 23 <211> 218 <212> DNA <213> Artificial sequence <220> <223> 3xSerpEnh <400> 23 gggggaggct gctggtgaat attaaccaag gtcaccccag ttatcggagg agcaaacagg 60 ggctaagtcc accgggggag gctgctggtg aatattaacc aaggtcaccc cagttatcgg 120 aggagcaaac aggggctaag tccaccgggg gaggctgctg gtgaatatta accaaggtca 180 ccccagttat cggaggagca aacaggggct aagtccac 218 <210> 24 <211> 100<00​​​​​​​​​​​​​​​​​ <212> DNA <213> Artificial Sequence <220> <223> co-FIX-R338L <400> 25 atgcagcgcg tgaacatgat catggccgag agccccggcc tgatcaccat ctgcctgctg 60 ggctacctgc tgagcgccga gtgcaccgtg ttcctggacc acgagaacgc caacaagatc 120 ctgaaccgcc ccaagcgcta caacagcggc aagctggagg agttcgtgca gggcaacctg 180 gagcgcgagt gcatggagga gaagtgcagc ttcgaggagg cccgcgaggt gttcgagaac 240 accgagcgca ccaccgagtt ctggaagcag tacgtggacg gcgaccagtg cgagagcaac 300 ccctgcctga acggcggcag ctgcaaggac gacatcaaca gctacgagtg ctggtgcccc 360 ttcggcttcg agggcaagaa ctgcgagctg gacgtgacct gcaacatcaa gaacggccgc 420 tgcgagcagt tctgcaagaa cagcgccgac aacaaggtgg tgtgcagctg caccgagggc 480 taccgcctgg ccgagaacca gaagagctgc gagcccgccg tgcccttccc ctgcggccgc 540 gtgagcgtga gccagaccag caagctgacc cgcgccgagg ccgtgttccc cgacgtggac 600 tacgtgaaca gcaccgaggc cgagaccatc ctggacaaca tcacccagag caccagagc 660 ttcaacgact tcacccgcgt ggtgggcggc gaggacgcca agcccggcca gttcccctgg 720 caggtggtgc tgaacggcaa ggtggacgcc ttctgcggcg gcagcatcgt gaacgagaag 780 tggatcgtga ccgccgccca ctgcgtggag accggcgtga agatcaccgt ggtggccggc 840 gagcacaaca tcgaggagac cgagcacacc gagcagaagc gcaacgtgat ccgcatcatc 900 ccccaccaca actacaacgc cgccatcaac aagtacaacc acgacatcgc cctgctgggag 960 ctggacgagc ccctggtgct gaacagctac gtgaccccca tctgcatcgc cgacaaggag 1020 tacaccaaca tcttcctgaa gttcggcagc ggctacgtga gcggctgggg ccgcgtgttc 1080 cacaagggcc gcagcgccct ggtgctgcag tacctgcgcg tgcccctggt ggaccgcgcc 1140 acctgcctgc tgagcaccaa gttcaccatc tacaacaaca tgttctgcgc cggcttccac 1200 gagggcggcc gcgacagctg ccagggcgac agcggcggcc cccacgtgac cgaggtggag 1260 ggcaccagct tcctgaccgg catcatcagc tggggcgagg agtgcgccat gaagggcaag 1320 tacggcatct acaccaaggt gagccgctac gtgaactgga tcaaggagaa gaccaagctg 1380 acctaatga 1389 <210> 26 <211> 4377 <212> DNA <213> Artificial Sequence <220> <223> co-FVIIIdeltaB <400> 26 atgcagatcg agctgtccac ctgctttttt ctgtgcctgc tgcggttctg cttcagcgcc 60 acccggcggt actacctggg cgccgtggag ctgtcctggg actacatgca gagcgacctg 120 ggcgagctgc ccgtggacgc ccggttcccc cccagagtgc ccaagagctt ccccttcaac 180 accagcgtgg tgtacaagaa aaccctgttc gtggagttca ccgaccacct gttcaatatc 240 gccaagccca ggcccccctg gatgggcctg ctgggcccca ccatccaggc cgaggtgtac 300 gacaccgtgg tgatcaccct gaagaacatg gccagccacc ccgtgagcct gcacgccgtg 360 ggcgtgagct actggaaggc cagcgagggc gccgagtacg acgaccagac cagccagcgg 420 gagaaagaag atgacaaggt gttccctggc ggcagccaca cctacgtgtg gcaggtgctg 480 aaagaaaacg gccccatggc ctccgacccc ctgtgcctga cctacagcta cctgagccac 540 gtggacctgg tgaaggacct gaacagcggc ctgatcggcg ctctgctcgt ctgccgggag 600 ggcagcctgg ccaaagagaa aacccagacc ctgcacaagt tcatcctgct gttcgccgtg 660 ttcgacgagg gcaagagctg gcacagcgag acaaagaaca gcctgatgca ggaccgggac 720 gccgcctctg ccagagcctg gcccaagatg cacaccgtga acggctacgt gaacagaagc 780 ctgcccggcc tgattggctg ccaccggaag agcgtgtact ggcacgtgat cggcatgggc 840 accacacccg aggtgcacag catctttctg gaagggcaca cctttctggt ccggaaccac 900 cggcaggcca gcctggaaat cagccctatc accttcctga ccgcccagac actgctgatg 960 gacctgggcc agttcctgct gttttgccac atcagctctc accagcacga cggcatggaa 1020 gcctacgtga aggtggactc ttgccccgag gaaccccagc tgcggatgaa gaacaacgag 1080 gaagccgagg actacgacga cgacctgacc gacagcgaga tggacgtggt gcggttcgac 1140 gacgacaaca gccccagctt catccagatc agaagcgtgg ccaagaagca ccccaagacc 1200 tgggtgcact atatcgccgc cgaggaagag gactgggact acgcccccct ggtgctggcc 1260 cccgacgaca gaagctacaa gagccagtac ctgaacaatg gcccccagcg gatcggccgg 1320 aagtacaaga aagtgcggtt catggcctac accgacgaga cattcaagac ccgggaggcc 1380 atccagcacg agagcggcat cctgggcccc ctgctgtacg gcgaagtggg cgacacactg 1440 ctgatcatct tcaagaacca ggctagccgg ccctacaaca tctaccccca cggcatcacc 1500 gacgtgcggc ccctgtacag caggcggctg cccaagggcg tgaagcacct gaaggacttc 1560 cccatcctgc ccggcgagat cttcaagtac aagtggaccg tgaccgtgga ggacggcccc 1620 accaagagcg accccagatg cctgacccgg tactacagca gcttcgtgaa catggaacgg 1680 gacctggcct ccgggctgat cggacctctg ctgatctgct acaaagaaag cgtggaccag 1740 cggggcaacc agatcatgag cgacaagcgg aacgtgatcc tgttcagcgt gttcgatgag 1800 aaccggtcct ggtatctgac cgagaacatc cagcggtttc tgcccaaccc tgccggcgtg 1860 cagctggaag atcccgagtt ccaggccagc aacatcatgc actccatcaa tggctacgtg 1920 ttcgactctc tgcagctctc cgtgtgtctg cacgaggtgg cctactggta catcctgagc 1980 atcggcgccc agaccgactt cctgagcgtg ttcttcagcg gctacacctt caagcaag 2040 atggtgtacg aggacaccct gaccctgttc cctttcagcg gcgagacagt gttcatgagc 2100 atggaaaacc ccggcctgtg gattctgggc tgccaacaca gcgacttccg gaaccggggc 2160 atgaccgccc tgctgaaggt gtccagctgc gaagaaca ccggcgacta ctacgaggac 2220 agctacgagg atatcagcgc ctacctgctg tccaagaaca acgccatcga accccggagc 2280 2340 cagtccgacc aggagagat cgattacgac gacaccatca gcgtggagat gaaaagag 2400 gatttcgata tctacgacga ggacgagaac cagagcccca gaagcttcca gaaaaaacc 2460 cggcactact tcattgccgc cgtggagagg ctgtgggact acggcatgag ttctagcccc 2520 cacgtgctgc ggaaccgggc ccagagcggc agcgtgcccc agttcaagaa agtggtgttc 2580 2640 ctggggctgc tggggcccta catcagggcc gaagtggagg acaacatcat ggtgaccttc 2700 cggaatcagg ccagcagacc ctactccttc tacagcagcc tgatcagcta cgaagaggac 2760 cagcggcagg gcgccgaacc ccggaagaac ttcgtgaagc ccaacgaaac caagacctac 2820 ttctggaaag tgcagcacca catggccccc accaaggacg agttcgactg caaggcctgg 2880 gcctacttca gcgacgtgga tctggaaaag gacgtgcact ctggactgat tggcccactc 2940 ctggtctgcc acactaacac cctcaacccc gcccacggcc gccaggtgac cgtgcaggaa 3000 ttcgccctgt tcttcaccat cttcgacgag acaaagtcct ggtacttcac cgagaatatg 3060 gaacggaact gcagagcccc ctgcaacatc cagatggaag atcctacctt caaagagaac 3120 taccggttcc acgccatcaa cggctacatc atggacaccc tgcctggcct ggtgatggcc 3180 caggaccaga gaatccggtg gtatctgctg tccatgggca gcaacgagaa tatccacagc 3240 atccacttca gcggccacgt gttcaccgtg cggaagaaag aagagtacaa gatggccctg 3300 tacaacctgt accccggcgt gttcgagaca gtggagatgc tgcccagcaa ggccggcatc 3360 tggcgggtgg agtgtctgat cggcgagcac ctgcacgctg gcatgagcac cctgtttctg 3420 gtgtacagca acaagtgcca gaccccactg ggcatggcct ctggccacat ccgggacttc 3480 cagatcaccg cctccggcca gtacggccag tgggccccca agctggccag actgcactac 3540 agcggcagca tcaacgcctg gtccaccaaa gagcccttca gctggatcaa ggtggacctg 3600 ctggccccta tgatcatcca cggcattaag acccagggcg ccaggcagaa gttcagcagc 3660 ctgtacatca gccagttcat catcatgtac agcctggacg gcaagaagtg gcagacctac 3720 cggggcaaca gcaccggcac cctgatggtg ttcttcggca atgtggacag cagcggcatc 3780 aagcacaaca tcttcaaccc ccccatcatt gcccggtaca tccggctgca ccccacccac 3840 tacagcatta gatccacact gagaatggaa ctgatgggct gcgacctgaa ctcctgcagc 3900 atgcctctgg gcatggaaag caaggccatc agcgacgccc agatcacagc cagcagctac 3960 ttcaccaaca tgttcgccac ctggtccccc tccaaggcca ggctgcacct gcagggccgg 4020 tccaacgcct ggcggcctca ggtcaacaac cccaaagaat ggctgcaggt ggactttcag 4080 aaaaccatga aggtgaccgg cgtgaccacc cagggcgtga aaagcctgct gaccagcatg 4140 tacgtgaaag agtttctgat cagcagctct caggatggcc accagtggac cctgttcttt 4200 cagaacggca aggtgaaagt gttccagggc aaccaggact ccttcacccc cgtggtgaac 4260 tccctggacc cccccctgct gacccgctac ctgagaatcc acccccagtc ttgggtgcac 4320 cagatcgccc tcaggatgga agtcctggga tgtgaggccc aggatctgta ctgatga 4377 <210> 27 <211> 202 <212> DNA <213> Artificial sequence <220> <223> TTRm <400> 27 gtctgtctgc acatttcgta gagcgagtgt tccgatactc taatctccct aggcaaggtt 60 catatttgtg taggttactt attctccttt tgttgactaa gtcaataatc agaatcagca 120 ggtttggagt cagcttggca gggatcagca gcctgggttg gaaggagggg gtataaaagc 180 cccttcacca ggagaagccg tc 202 <210> 28 <211> 317 <212> DNA <213> Artificial sequence <220> <223> TTRe-TTRm <400> 28 cactgggagg atgttgagta agatggaaaa ctactgatga cccttgcaga gacagagtat 60 taggacatgt ttgaacaggg gccgggcgat cagcaggtag ctctagagga tccccgtctg 120 tctgcacatt tcgtagagcg agtgttccga tactctaatc tccctaggca aggttcatat 180 ttgtgtaggt tacttattct ccttttgttg actaagtcaa taatcagaat cagcaggttt 240 ggagtcagct tggcagggat cagcagcctg ggttggaagg agggggtata aaagcccctt 300 caccaggaga agccgtc 317 <210> 29 <211> 92 <212> DNA <213> Artificial sequence <220> <223> MVM intron <400> 29 aagaggtaag ggtttaaggg atggttggtt ggtggggtat taatgtttaa ttacctggag 60 cacctgcctg aaatcacttt ttttcaggtt gg 92 <210> 30 <211> 295 <212> DNA <213> Artificial sequence <220> <223> BGH polyA <400> 30 gatctgagcc gaattcctgc agcccggggg atcagcctcg actgtgcctt ctagttgcca 60 gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg ccactcccac 120 tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt gtcattctat 180 tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagaca atagcaggca 240 tgctggggat gcggtgggct ctatggcttc tgaggcggaa agaaccagct gggga 295 <210> 31 <211> 6228 <212> DNA <213> Artificial sequence <220> <223> pAAVsc-TTRe-TTRm-MVM-co-FIX-R338L-BGHpA <400> 31 agctggcgta atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg 60 aatggcgaat ggcgattccg ttgcaatggc tggcggtaat attgttctgg atattaccag 120 caaggccgat agtttgagtt cttctactca ggcaagtgat gttattacta atcaaagaag 180 tattgcgaca acggttaatt tgcgtgatgg acagactctt ttactcggtg gcctcactga 240 ttataaaaac acttctcagg attctggcgt accgttcctg tctaaaatcc ctttaatcgg 300 cctcctgttt agctcccgct ctgattctaa cgaggaaagc acgttatacg tgctcgtcaa 360 agcaaccata gtacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc 420 gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt 480 cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag 540 ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt 600 cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt 660 tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt 720 cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt 780 aacaaaaatt taacgcgaac tttaacaaaa tattaacgtt tacaatttaa atatttgctt 840 atacaatctt cctgtttttg gggcttttct gattatcaac cggggtacat atgattgaca 900 tgctagtttt acgattaccg ttcatcgccc tgcgcgctcg ctcgctcact gaggccgccc 960 gggcaaagcc cgggcgtcgg gcgacctttg gtcgcccggc ctcagtgagc gagcgagcgc 1020 gcagagaggg agtggaattc acgcgtggat ctgaattcaa ttcacgcgtg gtacggccgc 1080 ggtacccact gggaggatgt tgagtaagat ggaaaactac tgatgaccct tgcagagaca 1140 gagtattagg acatgtttga acaggggccg ggcgatcagc aggtagctct agaggatccc 1200 cgtctgtctg cacattcgt agagcgagtg ttccgatact ctaatctccc taggcaggt 1260 tcatatttgt gtaggttact tattctcctt tgttgacta agtcaataat cagaatcagc 1320 agttttggag tcagcttggc aggatcagc agcctggtt ggaaggaggg gtataaaag 1380 ccccttcacc aggagaagcc gtcacacaga tccacaagct cctggctaga agaggtaag 1440 ggtttaaggg atggttggtt ggtggggtat taatgtttta ttacctggag cacctgcctg 1500 aaatcacttt tttcaggtt gggctagcccc accatgcagc gcgtgaacat gatcatggcc 1560 gagagccccg gcctgatcac catctgcctg ctgggctacc tgctgagcgc cgagtgcacc 1620 gtgttcctgg accacgagaa cgccacaag atcctgacc gcccaagcg ctacacacagc 1680 ggcaagctgg aggagttcgt gcaggggcaac ctggagcgcg agtgcatgga ggagaagtgc 1740 agctcgagg agccccgcga gttgttcgag aacaccgagc gcaccaccga gttctggaag 1800 cagtacgtgg acggcgacca gtgcgagagc aacccctgcc tgaacggcgg cagctgcaag 1860 gacgacatca acagctacga gtgctggtgc cccttcggct tcgagggcaa gaactgcgag 1920 ctggacgtga cctgcaacat caagaacggc cgctgcgagc agttctgcaa gaacagcgcc 1980 gacaacaagg tggtgtgcag ctgcaccgag ggctaccgcc tggccgagaa ccagaagagc 2040 tgcgagcccg ccgtgccctt cccctgcggc cgcgtgagcg tgagccagac cagcaagctg 2100 acccgcgccg aggccgtgtt ccccgacgtg gactacgtga acagcaccga ggccgagacc 2160 atcctggaca acatcaccca gagcacccag agcttcaacg acttcacccg cgtggtgggc 2220 ggcgaggacg ccaagcccgg ccagttcccc tggcaggtgg tgctgaacgg caaggtggac 2280 gccttctgcg gcggcagcat cgtgaacgag aagtggatcg tgaccgccgc ccactgcgtg 2340 gagaccggcg tgaagatcac cgtggtggcc ggcgagcaca acatcgagga gaccgagcac 2400 accgagcaga agcgcaacgt gatccgcatc atcccccacc acaactacaa cgccgccatc 2460 aacaagtaca accacgacat cgccctgctg gagctggacg agcccctggt gctgaacagc 2520 tacgtgaccc ccatctgcat cgccgacaag gagtacacca acatcttcct gaagttcggc 2580 agcggctacg tgagcggctg gggccgcgtg ttccacaagg gccgcagcgc cctggtgctg 2640 cagtacctgc gcgtgcccct ggtggaccgc gccacctgcc tgctgagcac caagttcacc 2700 atctacaaca acatgttctg cgccggcttc cacgagggcg gccgcgacag ctgccagggc 2760 gacagcggcg gcccccacgt gaccgaggtg gagggcacca gcttcctgac cggcatcatc 2820 agctggggcg aggagtgcgc catgaagggc aagtacggca tctacaccaa ggtgagccgc 2880 tacgtgaact ggatcaagga gaagaccaag ctgacctaat gaaagatgga tttccaaggt 2940 taattcattg gaattgaaaa ttaacagccc cccccccccc ccccctgcag atctgagccg 3000 aattcctgca gcccggggga tcagcctcga ctgtgccttc tagttgccag ccatctgttg 3060 tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc cactcccact gtcctttcct 3120 aataaaatga ggaaattgca tcgcattgtc tgagtaggtg tcattctatt ctggggggtg 3180 gggtggggca ggacagcaag ggggaggatt gggaagacaa tagcaggcat gctggggatg 3240 cggtgggctc tatggcttct gaggcggaaa gaaccagctg gggaccggtg gatctcgata 3300 gcaggcatgc tggggagaga tcgatctgag gaaccctag tgatggagtt ggccactccc 3360 tctctgcgcg ctcgctcgct cactgaggcc gcccgggcaa agcccgggcg tcgggcgacc 3420 tttggtcgcc cggcctcagt gagcgagcga gcgcgcagag agggatggc caaccccccc 3480 cccccccccc cccggcgatt ctcttgtttg ctccagactc tcaggcaatg acctgatagc 3540 ctttgtagag acctctcaaa aatagctacc ctctccggca tgaatttatc agctagaacg 3600 gttgaatatc atattgatgg tgatttgact gtctccggcc tttctcaccc gtttgaatct 3660 ttacctacac attactcagg cattgcattt aaaatatatg agggttctaa aaatttttat 3720 ccttgcgttg aaataaaggc ttctcccgca aaagtattac agggtcataa tgtttttggt 3780 acaaccgatt tagctttatg ctctgaggct ttattgctta attttgctaa ttctttgcct 3840 tgcctgtatg atttattgga tgttggaatc gcctgatgcg gtattttctc cttacgcatc 3900 tgtgcggtat ttcacaccgc atatggtgca ctctcagtac aatctgctct gatgccgcat 3960 agttatatgg tgcactctca gtacaatctg ctctgatgcc ccatagttaa gccagccccg 4020 acaccgcca acacagccag ccccgacacc cgccaacacc cgctgacgcg ccctgacggg 4080 cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt 4140 gtcagaggtt ttcaccgtca tcaccgaaac gcgcgagacg aaagggcctc gtgatacgcc 4200 tatttttata ggttaatgtc atgataataa tggtttctta gacgtcaggt ggcacttttc 4260 ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc 4320 cgctcatgag acaataaccc tgataaatgc tcaataatat tgaaaaagga agagtatgag 4380 tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt 4440 tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt 4500 gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga 4560 acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat 4620 tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga 4680 gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag 4740 tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg 4800 accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg 4860 ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt 4920 agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg 4980 gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc 5040 ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg 5100 tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac 5160 ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact 5220 gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa 5280 acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa 5340 aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg 5400 atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc 5460 gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac 5520 tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca 5580 ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt 5640 ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc 5700 ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg 5760 aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc 5820 cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac 5880 gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct 5940 ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc 6000 cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt 6060 tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac 6120 cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg 6180 cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt cattaatg 6228 <210> 32 <211> 6448 <212> DNA <213> Artificial Sequence <220> <223> pAAVsc-3xSerpEnh-TTRe-TTRm-MVM-co-FIX-R338L-BGHpA <400> 32 agctggcgta atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg 60 aatggcgaat ggcgattccg ttgcaatggc tggcggtaat attgttctgg atattaccag 120 caaggccgat agtttgagtt cttctactca ggcaagtgat gttattacta atcaaagaag 180 tattgcgaca acggttaatt tgcgtgatgg acagactctt ttactcggtg gcctcactga 240 ttataaaaac acttctcagg attctggcgt accgttcctg tctaaaatcc ctttaatcgg 300 cctcctgttt agctcccgct ctgattctaa cgaggaaagc acgttatacg tgctcgtcaa 360 agcaaccata gtacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc 420 gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt 480 cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag 540 ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt 600 cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt 660 tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt 720 cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt 780 aacaaaaatt taacgcgaac tttaacaaaa tattaacgtt tacaatttaa atatttgctt 840 atacaatctt cctgtttttg gggcttttct gattatcaac cggggtacat atgattgaca 900 tgctagtttt acgattaccg ttcatcgccc tgcgcgctcg ctcgctcact gaggccgccc 960 gggcaaagcc cgggcgtcgg gcgacctttg gtcgcccggc ctcagtgagc gagcgagcgc 1020 gcagagaggg agtggaattc acgcgtggat ctgaattcaa ttcacgcgtg gtacggccgc 1080 gggggggagg ctgctggtga atattaacca aggtcacccc agttatcgga ggagcaaaca 1140 ggggctaagt ccaccggggg aggctgctgg tgaatattaa ccaaggtcac cccagttatc 1200 ggaggagcaa acaggggcta agtccaccgg gggaggctgc tggtgaatat taaccaaggt 1260 caccccagtt atcggaggag caacagggg ctaagtccac ggtaccact gggaggatgt 1320 tgagtagat ggaaaactac tgatgaccct tgcagagaca gagtattagg acatgttttga 1380 acaggggccg ggcgatcagc aggtagctct agaggatccc cgtctgctg cacatttcgt 1440 agagcgagtg ttccgatact ctaatctccc taggcaggt tcatatttgt gtaggttact 1500 tattctcctt ttgttgacta agtcaataat cagaatcagc agttttggag tcagcttggc 1560 agggatcagc agcctgggtt ggaggaggg gtataaaag ccccttcacc aggagaagcc 1620 gtcacacaga tccacaagct cctggctaga aagaggtaag gttttaaggg atggttggtt 1680 ggtggggtat taatgtttaa ttacctggag cacctgcctg aaatcacttt tttcaggtt 1740 gggctagccc accatgcagc gcgtgaacat gatcatggcc gagagccccg gcctgatcac 1800 catctgcctg ctgggctacc tgctgagcgc cgagtgcacc gtgttcctgg accacgagaa 1860 cgccacaag atcctgacc gcccaagcg ctacacagc ggcaagctgg aggattcgt 1920 gcagggcaac ctggagcgcg agtgcatgga ggagaagtgc agctcgagg agggcccgcga 1980 ggtgttcgag aacaccgagc gcaccaccga gttctggaag cagtacgtgg acggcgacca 2040 gtgcgagagc aacccctgcc tgaacggcgg cagctgcaag gacgacatca acagctacga 2100 gtgctggtgc cccttcggct tcgagggcaa gaactgcgag ctggacgtga cctgcaacat 2160 caagaacggc cgctgcgagc agttctgcaa gaacagcgcc gacaacaagg tggtgtgcag 2220 ctgcaccgag ggctaccgcc tggccgagaa ccagaagagc tgcgagcccg ccgtgccctt 2280 cccctgcggc cgcgtgagcg tgagccagac cagcaagctg acccgcgccg aggccgtgtt 2340 ccccgacgtg gactacgtga acagcaccga ggccgagacc atcctggaca acatcaccca 2400 gagcacccag agcttcaacg acttcacccg cgtggtgggc ggcgaggacg ccaagcccgg 2460 ccagttcccc tggcaggtgg tgctgaacgg caaggtggac gccttctgcg gcggcagcat 2520 cgtgaacgag aagtggatcg tgaccgccgc ccactgcgtg gagaccggcg tgaagatcac 2580 cgtggtggcc ggcgagcaca acatcgagga gaccgagcac accgagcaga agcgcaacgt 2640 gatccgcatc atcccccacc acaactacaa cgccgccatc aacaagtaca accacgacat 2700 cgccctgctg gagctggacg agcccctggt gctgaacagc tacgtgaccc ccatctgcat 2760 cgccgacaag gagtacacca acatcttcct gaagttcggc agcggctacg tgagcggctg 2820 gggccgcgtg ttccacaagg gccgcagcgc cctggtgctg cagtacctgc gcgtgcccct 2880 ggtggaccgc gccacctgcc tgctgagcac caagttcacc atctacaaca acatgttctg 2940 cgccggcttc cacgagggcg gccgcgacag ctgccagggc gacagcggcg gcccccacgt 3000 gaccgaggtg gagggcacca gcttcctgac cggcatcatc agctggggcg aggagtgcgc 3060 catgaagggc aagtacggca tctacaccaa ggtgagccgc tacgtgaact ggatcaagga 3120 gaagaccaag ctgacctaat gaaagatgga tttccaaggt taattcattg gaattgaaaa 3180 ttaacagccc cccccccccc ccccctgcag atctgagccg aattcctgca gcccggggga 3240 tcagcctcga ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct 3300 tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca 3360 tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag 3420 ggggaggatt gggaagacaa tagcaggcat gctggggatg cggtgggctc tatggcttct 3480 gaggcggaaa gaaccagctg gggaccggtg gatctcgata gcaggcatgc tggggagaga 3540 tcgatctgag gaacccctag tgatggagtt ggccactccc tctctgcgcg ctcgctcgct 3600 cactgaggcc gcccgggcaa agcccgggcg tcgggcgacc tttggtcgcc cggcctcagt 3660 gagcgagcga gcgcgcagag agggagtggc caaccccccc cccccccccc cccggcgatt 3720 ctcttgtttg ctccagactc tcaggcaatg acctgatagc ctttgtagag acctctcaaa 3780 aatagctacc ctctccggca tgaatttatc agctagaacg gttgaatatc atattgatgg 3840 tgatttgact gtctccggcc tttctcaccc gtttgaatct ttacctacac attactcagg 3900 cattgcattt aaaatatatg agggttctaa aaatttttat ccttgcgttg aaataaaggc 3960 ttctcccgca aaagtattac agggtcataa tgtttttggt acaaccgatt tagctttatg 4020 ctctgaggct ttattgctta attttgctaa ttctttgcct tgcctgtatg atttattgga 4080 tgttggaatc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc 4140 atatggtgca ctctcagtac aatctgctct gatgccgcat agttatatgg tgcactctca 4200 gtacaatctg ctctgatgcc gcatagttaa gccagccccg acacccgcca acacagccag 4260 ccccgacacc cgccaacacc cgctgacgcg cctgacggg cttgtctgct cccggcatcc 4320 gcttacagac aagctgtgac cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca 4380 tcaccgaaac gcgcgagacg aaagggcctc gtgatacgcc tatttttata ggttaatgtc 4440 atgataataa tggtttctta gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc 4500 cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc 4560 tgataaatgc tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg 4620 cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg 4680 tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc 4740 tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca 4800 cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac 4860 tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa 4920 agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg 4980 ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt 5040 ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg 5100 aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc 5160 gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga 5220 tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta 5280 ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc 5340 cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg 5400 atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt 5460 cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa 5520 ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt 5580 cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt 5640 ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt 5700 tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga 5760 taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag 5820 caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata 5880 agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg 5940 gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga 6000 gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca 6060 ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa 6120 acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt 6180 tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac 6240 ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt 6300 ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga 6360 ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cccaatacgc aaaccgcctc 6420 tccccgcgcg ttggccgatt cattaatg 6448 <210> 33 <211> 6566 <212> DNA <213> Artificial sequence <220> <223> AAVsc-HS-CRE1-TTRenh-TTRm-MVM-hFIXcoPadua-bghpolyA <400> 33 agctggcgta atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg 60 aatggcgaat ggcgattccg ttgcaatggc tggcggtaat attgttctgg atattaccag 120 caaggccgat agtttgagtt cttctactca ggcaagtgat gttattacta atcaaagaag 180 tattgcgaca acggttaatt tgcgtgatgg acagactctt ttactcggtg gcctcactga 240 ttataaaaac acttctcagg attctggcgt accgttcctg tctaaaatcc ctttaatcgg 300 cctcctgttt agctcccgct ctgattctaa cgaggaaagc acgttatacg tgctcgtcaa 360 agcaaccata gtacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc 420 gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt 480 cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag 540 ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt 600 cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt 660 tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt 720 cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt 780 aacaaaaatt taacgcgaac tttaacaaaa tattaacgtt tacaatttaa atatttgctt 840 atacaatctt cctgtttttg gggcttttct gattatcaac cggggtacat atgattgaca 900 tgctagtttt acgattaccg ttcatcgccc tgcgcgctcg ctcgctcact gaggccgccc 960 gggcaaagcc cgggcgtcgg gcgacctttg gtcgcccggc ctcagtgagc gagcgagcgc 1020 gcagagaggg agtggaattc acgcgtggat ctgaattctg tacaggcgtg gtacggccgc 1080 ggtgggaaat tacagttaaa taccatggta atgaataaaa ggtacaaatc gtttaaactc 1140 tttgtaaaa tttgataaga tgttttacac aactttaata cattgacaag gtcttgtgga 1200 gaaaacagtt ccagatggta aatatacaca agggatttag tcaaacaatt ttttggcaag 1260 aatattatga atttgtaat cggttggcag ccaatgaaat acaaagatga gtctagttaa 1320 taatctacaa ttattggtta aagaagtata ttagtgctaa tttccctccg tttgtcctag 1380 cttttctctt ctgtcaaccc cacacgcctt tggcacaagg tacccactgg gaggatgttg 1440 agtaagatgg aaaactactg atgacccttg cagagacaga gtattaggac atgtttgaac 1500 aggggccggg cgatcagcag gtagctctag agatccccg tctgtctgca catttcgtag 1560 agcgagtgtt ccgatactct aatctcccta ggcaaggttc atatttgtgt aggttactta 1620 ttctcctttt gttgactaag tcaataatca gaatcagcag gtttggagtc agcttggcag 1680 ggatcagcag cctgggttgg aaggaggggg tataaaagcc ccttcaccag gagaagccgt 1740 cacacagatc cacaagctcc tggctagaaa gaggtaaggg tttaagggat ggttggttgg 1800 tggggtatta atgtttaatt acctggagca cctgcctgaa atcacttttt ttcaggttgg 1860 gctagcccac catgcagcgc gtgaacatga tcatggccga gagccccggc ctgatcacca 1920 tctgcctgct gggctacctg ctgagcgccg agtgcaccgt gttcctggac cacgagaacg 1980 ccaacaagat cctgaaccgc cccaagcgct acaacagcgg caagctggag gagttcgtgc 2040 agggcaacct ggagcgcgag tgcatggagg agaagtgcag cttcgaggag gcccgcgagg 2100 tgttcgagaa caccgagcgc accaccgagt tctggaagca gtacgtggac ggcgaccagt 2160 gcgagagcaa cccctgcctg aacggcggca gctgcaagga cgacatcaac agctacgagt 2220 gctggtgccc cttcggcttc gagggcaaga actgcgagct ggacgtgacc tgcaacatca 2280 agaacggccg ctgcgagcag ttctgcaaga acagcgccga caacaaggtg gtgtgcagct 2340 gcaccgaggg ctaccgcctg gccgagaacc agaagagctg cgagcccgcc gtgcccttcc 2400 cctgcggccg cgtgagcgtg agccagacca gcaagctgac ccgcgccgag gccgtgttcc 2460 ccgacgtgga ctacgtgaac agcaccgagg ccgagaccat cctggacaac atcacccaga 2520 gcacccagag cttcaacgac ttcacccgcg tggtgggcgg cgaggacgcc aagcccggcc 2580 agttcccctg gcaggtggtg ctgaacggca aggtggacgc cttctgcggc ggcagcatcg 2640 tgaacgagaa gtggatcgtg accgccgccc actgcgtgga gaccggcgtg aagatcaccg 2700 tggtggccgg cgagcacaac atcgaggaga ccgagcacac cgagcagaag cgcaacgtga 2760 tccgcatcat ccccaccac aactacaacg ccgccatcaa caagtacaac cacgacatcg 2820 ccctgctgga gctggacgag cccctggtgc tgaacagcta cgtgaccccc atctgcatcg 2880 ccgacaagga gtacaccaac atcttcctga agttcggcag cggctacgtg agcggctggg 2940 gccgcgtgtt ccacaagggc cgcagcgccc tggtgctgca gtacctgcgc gtgcccctgg 3000 tggaccgcgc cacctgcctg ctgagcacca agttcaccat ctacaacaac atgttctgcg 3060 ccggcttcca cgagggcggc cgcgacagct gccagggcga cagcggcggc ccccacgtga 3120 ccgaggtgga gggcaccagc ttcctgaccg gcatcatcag ctggggcgag gagtgcgcca 3180 tgaagggcaa gtacggcatc tacaccaagg tgagccgcta cgtgaactgg atcaaggaga 3240 agaccaagct gacctaatga aagatggatt tccaaggtta attcattgga attgaaaatt 3300 aacagccccc cccccccccc ccctgcagat ctgagccgaa ttcctgcagc ccgggggatc 3360 agcctcgact gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc 3420 cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc 3480 gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg 3540 ggaggattgg gaagacaata gcaggcatgc tggggatgcg gtgggctcta tggcttctga 3600 ggcggaaaga accagctggg gaccggtgga tctcgatagc aggcatgctg gggagagatc 3660 gatctgagga acccctagtg atggagttgg ccactccctc tctgcgcgct cgctcgctca 3720 ctgaggccgc ccgggcaaag cccgggcgtc gggcgacctt tggtcgcccg gcctcagtga 3780 gcgagcgagc gcgcagagag ggagtggcca accccccccc cccccccccc cggcgattct 3840 cttgtttgct ccagactctc aggcaatgac ctgatagcct ttgtagagac ctctcaaaaa 3900 tagctaccct ctccggcatg aatttatcag ctagaacggt tgaatatcat attgatggtg 3960 atttgactgt ctccggcctt tctcacccgt ttgaatcttt acctacacat tactcaggca 4020 ttgcatttaa aatatatgag ggttctaaaa atttttatcc ttgcgttgaa ataaaggctt 4080 ctcccgcaaa agtattacag ggtcataatg tttttggtac aaccgattta gctttatgct 4140 ctgaggcttt attgcttaat tttgctaatt cttgccttg cctgtatgat ttattggatg 4200 ttggaatcgc ctgatgcggt atttctcct tacgcatctg tgcggtattt cacaccgcat 4260 atggtgcact ctcagtacaa tctgctctga tgccgcatag ttatatggtg cactctcagt 4320 acaatctgct ctgatgccgc atagttaagc cagccccgac acccgccaac acagccagcc 4380 ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctc cggcatcgc 4440 ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc 4500 accgaaacgc gcgagacgaa agggcctcgt gatacgccta tttttatagg ttaatgtcat 4560 gataataatg gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc 4620 tatttgttta ttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg 4680 ataaatgctc aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc 4740 cttatccct tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg 4800 aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc 4860 4920 tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc 4980 ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag 5040 catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat 5100 aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt 5160 5220 gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc 5280 5340 gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt 5400 gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca 5460 gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat 5520 gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca 5580 gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg 5640 atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg 5700 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt 5760 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg 5820 ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata 5880 ccaaatactg tccttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca 5940 ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag 6000 tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc 6060 tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga 6120 tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg 6180 tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac 6240 gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg 6300 tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg 6360 ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct 6420 gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc 6480 gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc caatacgcaa accgcctctc 6540 cccgcgcgtt ggccgattca ttaatg 6566 <210> 34 <211> 6577 <212> DNA <213> Artificial Sequence <220> <223> AAVsc-HS-CRE2-TTRenh-TTRm-MVM-hFIXcoPadua-bghpolyA <400> 34 agctggcgta atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg 60 aatggcgaat ggcgattccg ttgcaatggc tggcggtaat attgttctgg atattaccag 120 caaggccgat agtttgagtt cttctactca ggcaagtgat gttattacta atcaaagaag 180 tattgcgaca acggttaatt tgcgtgatgg acagactctt ttatcggtg gcctcactga 240 ttataaaac acttctcagg attctggcgt accgttcctg tctaaaatcc ctttaatcgg 300 cctcctgttt agctcccgct ctgattctaa cgaggaaagc acgttatacg tgctcgtcaa 360 agcaaccata gtacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc 420 gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt 480 cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcgggg ctccctttag 540 ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt 600 cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt 660 tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt 720 cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt 780 aacaaaaatt taacgcgaac tttaacaaaa tattaacgtt tacaatttaa atatttgctt 840 atacaatctt cctgtttttg gggcttttct gattatcaac cggggtacat atgattgaca 900 tgctagtttt acgattaccg ttcatcgccc tgcgcgctcg ctcgctcact gaggccgccc 960 gggcaaagcc cgggcgtcgg gcgaccttg gtcgcccggc ctcagtgagc gagcgagcgc 1020 gcagagagggg agtggaatttc acgcgtggat ctgaatttctg tacaggcgtg gtacggccgc 1080 ggttttttct tttctaaatg atcagtacac ttattctc taaagaaaat acttttctta 1140 actactctct atttttaac ttctcccaca aagatgagaa actactttaa atcattggg 1200 gctattttc tgtttaccga gtaagagaa tctctaacc atatttataa ctcttactct 1260 aaatatttgc atttaccctc atgccagagc ccgttgatga ctgactaac agagtttca 1320 agtttgaga acaggaatt tagaaatgac taacattat gtaggtttt ttctctcagt 1380 atagaatgtt catatagaat taatgccaga ggttttcaga gaaaaatgcg gtacccactg 1440 ggaggatgtt gagtagatg gaaaactact gatgaccctt gcagagacag agtattagga 1500 catgtttgaa caggggccgg gcgatcagca ggtagctcta gaggatcccc gtctgtctgc 1560 acatttcgta gagcgagtgt tccgatactc taatctccct aggcaggtt catatttgtg 1620 taggttactt attctccttt tgttgactaa gtcaataatc agaatcagca ggtttggagt 1680 cagcttggca gggatcagca gcctgggttg gaaggagggg gtataaaagc cccttcacca 1740 ggagaagccg tcacacagat ccacaagctc ctggctagaa agaggtaagg gtttaaggga 1800 tggttggttg gtggggtatt aatgtttaat tacctggagc acctgcctga aatcactttt 1860 tttcaggttg ggctagccca ccatgcagcg cgtgaacatg atcatggccg agagccccgg 1920 cctgatcacc atctgcctgc tgggctacct gctgagcgcc gagtgcaccg tgttcctgga 1980 ccacgagaac gccaacaaga tcctgaaccg ccccaagcgc tacaacagcg gcaagctgga 2040 ggagttcgtg cagggcaacc tggagcgcga gtgcatggag gagaagtgca gcttcgagga 2100 ggcccgcgag gtgttcgaga acaccgagcg caccaccgag ttctggaagc agtacgtgga 2160 cggcgaccag tgcgagagca acccctgcct gaacggcggc agctgcaagg acgacatcaa 2220 cagctacgag tgctggtgcc ccttcggctt cgagggcaag aactgcgagc tggacgtgac 2280 ctgcaacatc aagaacggcc gctgcgagca gttctgcaag aacagcgccg acaacaaggt 2340 ggtgtgcagc tgcaccgagg gctaccgcct ggccgagaac cagaagagct gcgagcccgc 2400 cgtgcccttc ccctgcggcc gcgtgagcgt gagccagacc agcaagctga cccgcgccga 2460 ggccgtgttc cccgacgtgg actacgtgaa cagcaccgag gccgagacca tcctggacaa 2520 catcacccag agcacccaga gcttcaacga cttcacccgc gtggtgggcg gcgaggacgc 2580 caagcccggc cagttcccct ggcaggtggt gctgaacggc aaggtggacg ccttctgcgg 2640 cggcagcatc gtgaacgaga agtggatcgt gaccgccgcc cactgcgtgg agaccggcgt 2700 gaagatcacc gtggtggccg gcgagcacaa catcgaggag accgagcaca ccgagcagaa 2760 gcgcaacgtg atccgcatca tcccccacca caactacaac gccgccatca acaagtacaa 2820 ccacgacatc gccctgctgg agctggacga gcccctggtg ctgaacagct acgtgacccc 2880 catctgcatc gccgacaagg agtacaccaa catcttcctg aagttcggca gcggctacgt 2940 gagcggctgg ggccgcgtgt tccacaaggg ccgcagcgcc ctggtgctgc agtacctgcg 3000 cgtgcccctg gtggaccgcg ccacctgcct gctgagcacc aagttcacca tctacaacaa 3060 catgttctgc gccggcttcc acgagggcgg ccgcgacagc tgccagggcg acagcggcgg 3120 cccccacgtg accgaggtgg agggcaccag cttcctgacc ggcatcatca gctggggcga 3180 ggagtgcgcc atgaagggca agtacggcat ctacaccaag gtgagccgct acgtgaactg 3240 gatcaaggag aagaccaagc tgacctaatg aaagatggat ttccaaggtt aattcattgg 3300 aattgaaaat taacagcccc cccccccccc cccctgcaga tctgagccga attcctgcag 3360 cccgggggat cagcctcgac tgtgccttct agttgccagc catctgttgt ttgcccctcc 3420 cccgtgcctt ccttgaccct ggaaggtgcc actcccactg tcctttccta ataaaatgag 3480 gaaattgcat cgcattgtct gagtaggtgt cattctattc tggggggtgg ggtggggcag 3540 gacagcaagg gggaggattg ggaagacaat agcaggcatg ctggggatgc ggtgggctct 3600 atggcttctg aggcggaaag aaccagctgg ggaccggtgg atctcgatag caggcatgct 3660 ggggagagat cgatctgagg aacccctagt gatggagttg gccactccct ctctgcgcgc 3720 tcgctcgctc actgaggccg cccgggcaaa gcccgggcgt cgggcgacct ttggtcgccc 3780 ggcctcagtg agcgagcgag cgcgcagaga gggagtggcc aacccccccc cccccccccc 3840 ccggcgattc tcttgtttgc tccagactct caggcaatga cctgatagcc tttgtagaga 3900 cctctcaaaa atagctaccc tctccggcat gaatttatca gctagaacgg ttgaatatca 3960 tattgatggt gatttgactg tctccggcct ttctcacccg tttgaatctt tacctacaca 4020 ttactcaggc attgcattta aaatatatga gggttctaaa aatttttatc cttgcgttga 4080 aataaaggct tctcccgcaa aagtattaca gggtcataat gtttttggta caaccgattt 4140 agctttatgc tctgaggctt tattgcttaa ttttgctaat tctttgcctt gcctgtatga 4200 tttattggat gttggaatcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt 4260 tcacaccgca tatggtgcac tctcagtaca atctgctctg atgccgcata gttatatggt 4320 gcactctcag tacaatctgc tctgatgccg catagttaag ccagccccga cacccgccaa 4380 cacagccagc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc 4440 ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt 4500 tcaccgtcat caccgaaacg cgcgagacga aagggcctcg tgatacgcct atttttatag 4560 gttaatgtca tgataataat ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg 4620 cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga 4680 caataaccct gataaatgct caataatatt gaaaaaggaa gagtatgagt attcaacatt 4740 tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag 4800 aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg 4860 aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa cgtttccaa 4920 tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc 4980 aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag 5040 tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa 5100 ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc 5160 taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg 5220 agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa 5280 caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg caacaattaa 5340 tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg 5400 gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag 5460 cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg 5520 caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt 5580 ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt 5640 aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac 5700 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag 5760 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg 5820 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca 5880 gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga 5940 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca 6000 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc 6060 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca 6120 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa 6180 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc 6240 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc 6300 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg 6360 cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat 6420 cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca 6480 gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca 6540 aaccgcctct ccccgcgcgt tggccgattc attaatg 6577 <210> 35 <211> 6616 <212> DNA <213> Artificial Sequence <220> <223> AAVsc-HS-CRE3-TTRenh-TTRm-MVM-hFIXcoPadua-bghpolyA <400> 35 agctggcgta atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg 60 aatggcgaat ggcgattccg ttgcaatggc tggcggtaat attgttctgg atattaccag 120 caaggccgat agtttgagtt cttctactca ggcaagtgat gttatacta atcaaagaag 180 tattgcgaca acggttaatt tgcgtgatgg acagactctt ttatcggtg gcctcactga 240 ttataaaac acttctcagg attctggcgt accgttcctg tctaaaatcc ctttaatcgg 300 cctcctgttt agctcccgct ctgattctaa cgaggaaagc acgttatacg tgctcgtcaa 360 agcaaccata gtacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc 420 gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt 480 cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcgggg ctccctttag 540 ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt 600 cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt 660 tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt 720 cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt 780 aacaaaaatt taacgcgaac tttaacaaaa tattaacgtt tacaatttaa atatttgctt 840 atacaatctt cctgtttttg gggcttttct gattatcaac cggggtacat atgattgaca 900 tgctagtttt acgattaccg ttcatcgccc tgcgcgctcg ctcgctcact gaggccgccc 960 gggcaaagcc cgggcgtcgg gcgacctttg gtcgcccggc ctcagtgagc gagcgagcgc 1020 gcagagaggg agtggaattc acgcgtggat ctgaattctg tacaggcgtg gtacggccgc 1080 gggggacact gaagccaaat gggcttcagc tcctccccag ggatctccct acctgctgcc 1140 cattccaacc tggctctctc cctctccaca actgaagccc aggcctcttt ggatgctgct 1200 cacacatctt gcctccttat cttacctagc cagcgtctct gtccttggtg tctgtgcctg 1260 ccctggctgg agaggaaggg gctatatacc tgtctgttta cccaccccct gtcaggtgac 1320 agggactatg gatgggccca gtggggcagg gattatgtga ggataaacaa gttggagaaa 1380 tgggggagagagagaagc acatggaaga ctcatggtgaga tggggtgaga ggcagggggt 1440 acagctctta gtggaggcta agggggtagg taccactgg gaggatgttg agtaagatgg 1500 aaaactactg atgacccttg gtattaggac atgtttgaac aggggccggg 1560 cgatcagcag gtagctctag aggatccccg tctgtctgca catttcgtag agcgagtgtt 1620 ccgatactct aatctcccta ggcaaggttc atatttgtgt aggttactta ttctccttt 1680 1740 cctgggttgg aaggagggg tataaaagcc ccttcaccag gagaagccgt cacacagatc 1800 caaagctcc tggctagaaa gaggtaaggg tttaagggat ggttggttgg tggggtatta 1860 atgtttaatt acctggagca cctgcctgaa atcacttttt ttcaggttgg gctagcccac 1920 catgcagcgc gtgaacatga tcatggccga gagccccggc ctgatcacca tctgcctgct 1980 gggctacctg ctgagcgccg agtgcaccgt gttcctggac cacgagaacg ccaacaagat 2040 cctgaaccgc cccaagcgct aaacagcgg caagctgggag gagttcgtgc agggcaacct 2100 ggagcgcgag tgcatggagg agaagtgcag cttcgaggag gcccgcgagg tgttcgagaa 2160 caccgagcgc accaccgagt tctggaagca gtacgtggac ggcgaccagt gcgagagcaa 2220 cccctgcctg aacggcggca gctgcaagga cgacatcaac agctacgagt gctggtgccc 2280 cttcggcttc gagggcaaga actgcgagct ggacgtgacc tgcaacatca agaacggccg 2340 ctgcgagcag ttctgcaaga acagcgccga caacaaggtg gtgtgcagct gcaccgaggg 2400 ctaccgcctg gccgagaacc agaagagctg cgagcccgcc gtgcccttcc cctgcggccg 2460 cgtgagcgtg agccagacca gcaagctgac ccgcgccgag gccgtgttcc ccgacgtgga 2520 ctacgtgaac agcaccgagg ccgagaccat cctggacaac atcacccaga gcacccagag 2580 cttcaacgac ttcacccgcg tggtgggcgg cgaggacgcc aagcccggcc agttcccctg 2640 gcaggtggtg ctgaacggca aggtggacgc cttctgcggc ggcagcatcg tgaacgagaa 2700 gtggatcgtg accgccgccc actgcgtgga gaccggcgtg aagatcaccg tggtggccgg 2760 cgagcacaac atcgaggaga ccgagcacac cgagcagaag cgcaacgtga tccgcatcat 2820 cccccaccac aactacaacg ccgccatcaa caagtacaac cacgacatcg ccctgctgga 2880 gctggacgag cccctggtgc tgaacagcta cgtgaccccc atctgcatcg ccgacaagga 2940 gtacaccaac atcttcctga agttcggcag cggctacgtg agcggctggg gccgcgtgtt 3000 ccacaagggc cgcagcgccc tggtgctgca gtacctgcgc gtgcccctgg tggaccgcgc 3060 cacctgcctg ctgagcacca agttcaccat ctacaacaac atgttctgcg ccggcttcca 3120 cgagggcggc cgcgacagct gccagggcga cagcggcggc ccccacgtga ccgaggtgga 3180 gggcaccagc ttcctgaccg gcatcatcag ctggggcgag gagtgcgcca tgaagggcaa 3240 gtacggcatc tacaccaagg tgagccgcta cgtgaactgg atcaaggaga agaccaagct 3300 gacctaatga aagatggatt tccaaggtta attcattgga attgaaaatt aacagccccc 3360 cccccccccc ccctgcagat ctgagccgaa ttcctgcagc ccgggggatc agcctcgact 3420 gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc cttgaccctg 3480 gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc gcattgtctg 3540 agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg ggaggattgg 3600 gaagacaata gcaggcatgc tggggatgcg gtgggctcta tggcttctga ggcggaaaga 3660 accagctggg gaccggtgga tctcgatagc aggcatgctg gggagagatc gatctgagga 3720 acccctagtg atggagttgg ccactccctc tctgcgcgct cgctcgctca ctgaggccgc 3780 ccgggcaaag cccgggcgtc gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc 3840 gcgcagagag ggagtggcca accccccccc cccccccccc cggcgattct cttgtttgct 3900 ccagactctc aggcaatgac ctgatagcct ttgtagagac ctctcaaaaa tagctaccct 3960 ctccggcatg aatttatcag ctagaacggt tgaatatcat attgatggtg atttgactgt 4020 ctccggcctt tctcacccgt ttgaatcttt acctacacat tactcaggca ttgcatttaa 4080 aatatatgag ggttctaaaa atttttatcc ttgcgttgaa ataaaggctt ctcccgcaaa 4140 agtattacag ggtcataatg tttttggtac aaccgattta gctttatgct ctgaggcttt 4200 attgcttaat tttgctaatt ctttgccttg cctgtatgat ttattggatg ttggaatcgc 4260 ctgatgcggt atttctcct tacgcatctg tgcggtattt cacaccgcat atggtgcact 4320 ctcagtacaa tctgctctga tgccgcatag ttatatggtg cactctcagt acaatctgct 4380 ctgatgccgc atagttaagc cagccccgac acccgccaac acagccagcc ccgacacccg 4440 ccaacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa 4500 gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc 4560 gcgagacgaa agggcctcgt gatacgccta tttttatagg ttaatgtcat gataataatg 4620 gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta 4680 ttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctc 4740 aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct 4800 ttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag 4860 atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta 4920 agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc 4980 tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca 5040 tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg 5100 atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg 5160 ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca 5220 tggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa 5280 acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa 5340 ctggcgaact acttactcta gcttccccggc aaaattaat agactggatg gaggcggata 5400 aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttat gctgataaat 5460 ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc 5520 5580 gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt 5640 actcatatat actttagatt gatttaaaac ttcattttta atttaaaaagg atctaggtga 5700 agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag 5760 cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa 5820 tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag 5880 agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg 5940 tccttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat 6000 acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta 6060 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg 6120 gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc 6180 gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa 6240 gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc 6300 tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt 6360 caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct 6420 tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc 6480 gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg 6540 agtcagtgag cgaggaagcg gaagagcgcc caatacgcaa accgcctctc cccgcgcgtt 6600 ggccgattca ttaatg 6616 <210> 36 <211> 6656 <212> DNA <213> Artificial sequence <220> <223> AAVsc-HS-CRE4-TTRenh-TTRm-MVM-hFIXcoPadua-bghpolyA <400> 36 agctggcgta atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg 60 aatggcgaat ggcgattccg ttgcaatggc tggcggtaat attgttctgg atattaccag 120 caaggccgat agtttgagtt cttctactca ggcaagtgat gttattacta atcaaagaag 180 tattgcgaca acggttaatt tgcgtgatgg acagactctt ttactcggtg gcctcactga 240 ttataaaaac acttctcagg attctggcgt accgttcctg tctaaaatcc ctttaatcgg 300 cctcctgttt agctcccgct ctgattctaa cgaggaaagc acgttatacg tgctcgtcaa 360 agcaaccata gtacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc 420 gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt 480 cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcgggg ctccctttag 540 ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt 600 cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt 660 tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt 720 cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt 780 aacaaaaatt taacgcgaac tttaacaaaa tattaacgtt tacaatttaa atatttgctt 840 atacaatctt cctgtttttg gggcttttct gattatcaac cggggtacat atgattgaca 900 tgctagtttt acgattaccg ttcatcgccc tgcgcgctcg ctcgctcact gaggccgccc 960 gggcaaagcc cgggcgtcgg gcgacctttg gtcgcccggc ctcagtgagc gagcgagcgc 1020 gcagagagg agtggaattc acgcgtggat ctgaattctg tacaggcgtg gtacggccgc 1080 gggagttaat gtgtaatgag acggggagaa aacaggagag cccagaatga cctggatgct 1140 gatcactgaa cataccctac ccccagtaaa acaaattcag aaaacagctt ccgccccgtcc 1200 cctcccaatg gagggctctg gcaggaaaag aggtgaataa gaggcttcta ccagggtaaa 1260 ggttgaaggc acctggtcat ttgatcacct tatcagttct aggcagtgat tagccaatat 1320 tgagtcagca ggggcaatag ccctggccct tgtctcactc ctgttgggg tggggaggg 1380 ggagaggtac attcccaggt tcaaagcatt tgggtgaaat cagttaaata gatatcagaa 1440 gcttttgtat ctttcaccct tttgcccccc aagcatactc gctgagtatg tggaacattc 1500 ctgagggtgg taccactgg gaggatgttg agtaagatgg aaaactactg atgacccttg 1560 cagagacaga gtattaggac atgtttgaac aggggccggg cgatcagcag gtagctctag 1620 aggatccccg tctgtctgca catttcgtag agcgagtgtt ccgatactct aatctcccta 1680 ggcaaggttc atatttgtgt aggttactta ttctccttt gttgactaag tcaataatca 1740 gaatcagcag gtttggagtc agcttggcag ggatcagcag cctgggttgg aaggggggg 1800 tataaaagcc ccttcaccag gagaagccgt cacacagatc cacaagctcc tggctagaaa 1860 gaggtaaggg tttaagggat ggttggttgg tggggtatta atgtttaatt acctggagca 1920 cctgcctgaa atcacttttt ttcaggttgg gctagcccac catgcagcgc gtgaacatga 1980 tcatggccga gagccccggc ctgatcacca tctgcctgct gggctacctg ctgagcgccg 2040 agtgcaccgt gttcctggac cacgagaacg ccaacaagat cctgaaccgc cccaagcgct 2100 acaacagcgg caagctggag gagttcgtgc agggcaacct ggagcgcgag tgcatggagg 2160 agaagtgcag cttcgaggag gcccgcgagg tgttcgagaa caccgagcgc accaccgagt 2220 tctggaagca gtacgtggac ggcgaccagt gcgagagcaa cccctgcctg aacggcggca 2280 gctgcaagga cgacatcaac agctacgagt gctggtgccc cttcggcttc gagggcaaga 2340 actgcgagct ggacgtgacc tgcaacatca agaacggccg ctgcgagcag ttctgcaaga 2400 acagcgccga caacaaggtg gtgtgcagct gcaccgaggg ctaccgcctg gccgagaacc 2460 agaagagctg cgagcccgcc gtgcccttcc cctgcggccg cgtgagcgtg agccagacca 2520 gcaagctgac ccgcgccgag gccgtgttcc ccgacgtgga ctacgtgaac agcaccgagg 2580 ccgagaccat cctggacaac atcacccaga gcacccagag cttcaacgac ttcacccgcg 2640 tggtgggcgg cgaggacgcc aagcccggcc agttcccctg gcaggtggtg ctgaacggca 2700 aggtggacgc cttctgcggc ggcagcatcg tgaacgagaa gtggatcgtg accgccgccc 2760 actgcgtgga gaccggcgtg aagatcaccg tggtggccgg cgagcacaac atcgaggaga 2820 ccgagcacac cgagcagaag cgcaacgtga tccgcatcat cccccaccac aactacaacg 2880 ccgccatcaa caagtacaac cacgacatcg ccctgctgga gctggacgag cccctggtgc 2940 tgaacagcta cgtgaccccc atctgcatcg ccgacaagga gtacaccaac atcttcctga 3000 agttcggcag cggctacgtg agcggctggg gccgcgtgtt ccacaagggc cgcagcgccc 3060 tggtgctgca gtacctgcgc gtgcccctgg tggaccgcgc cacctgcctg ctgagcacca 3120 agttcaccat ctacaacaac atgttctgcg ccggcttcca cgagggcggc cgcgacagct 3180 gccagggcga cagcggcggc ccccacgtga ccgaggtgga gggcaccagc ttcctgaccg 3240 gcatcatcag ctggggcgag gagtgcgcca tgaagggcaa gtacggcatc tacaccaagg 3300 tgagccgcta cgtgaactgg atcaaggaga agaccaagct gacctaatga aagatggatt 3360 tccaaggtta attcattgga attgaaaatt aacagccccc cccccccccc ccctgcagat 3420 ctgagccgaa ttcctgcagc ccgggggatc agcctcgact gtgccttcta gttgccagcc 3480 atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt 3540 cctttcctaa taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct 3600 ggggggtggg gtggggcagg acagcaaggg ggaggattgg gaagacaata gcaggcatgc 3660 tggggatgcg gtgggctcta tggcttctga ggcggaaaga accagctggg gaccggtgga 3720 tctcgatagc aggcatgctg gggagagatc gatctgagga acccctagtg atggagttgg 3780 ccactccctc tctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccggcgtc 3840 gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca 3900 accccccccc cccccccccc cggcgattct cttgtttgct ccagactctc aggcaatgac 3960 ctgatagcct ttgtagagac ctctcaaaaa tagctaccct ctccggcatg aatttatcag 4020 ctagaacggt tgaatatcat attgatggtg atttgactgt ctccggcctt tctcacccgt 4080 ttgaatcttt acctacacat tactcaggca ttgcatttaa aatatatgag ggttctaaaa 4140 atttttatcc ttgcgttgaa ataaaggctt ctcccgcaaa agtattacag ggtcataatg 4200 tttttggtac aaccgattta gctttatgct ctgaggcttt attgcttaat tttgctaatt 4260 ctttgccttg cctgtatgat ttattggatg ttggaatcgc ctgatgcggt attttctcct 4320 tacgcatctg tgcggtattt cacaccgcat atggtgcact ctcagtacaa tctgctctga 4380 tgccgcatag ttatatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc 4440 cagccccgac acccgccaac acagccagcc ccgacacccg ccaacacccg ctgacgcgcc 4500 ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag 4560 ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgagacgaa agggcctcgt 4620 gatacgccta tttttatagg ttaatgtcat gataataatg gtttcttaga cgtcaggtgg 4680 cactttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa 4740 4800 agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt 4860 cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt 4920 gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga gagttttcgc 4980 cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta 5040 tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac 5100 ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa 5160 ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg 5220 atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc 5280 5340 atgcctgtag caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta 5400 gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg accacttctg 5460 cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg 5520 tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc 5580 tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt 5640 gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt 5700 gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc 5760 atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag 5820 atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa 5880 aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg 5940 aaggtaactg gcttcagcag agcgcagata ccaaatactg tccttctagt gtagccgtag 6000 ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg 6060 ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga 6120 tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc 6180 ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc 6240 acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga 6300 gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt 6360 cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg 6420 aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac 6480 atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga 6540 gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg 6600 gaagagcgcc caatacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatg 6656 <210> 37 <211> 6490 <212> DNA <213> Artificial Sequence <220> <223> AAVsc-HS-CRE5-TTRenh-TTRm-MVM-hFIXcoPadua-bghpolyA <400> 37 agctggcgta atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg 60 aatggcgaat ggcgattccg ttgcaatggc tggcggtaat attgttctgg atattaccag 120 caaggccgat agtttgagtt cttctactca ggcaagtgat gttatacta atcaaagaag 180 tattgcgaca acggttaatt tgcgtgatgg acagactctt ttatcggtg gcctcactga 240 ttataaaac acttctcagg attctggcgt accgttcctg tctaaaatcc ctttaatcgg 300 cctcctgttt agctcccgct ctgattctaa cgaggaaagc acgttatacg tgctcgtcaa 360 agcaaccata gtacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc 420 gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt 480 cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcgggg ctccctttag 540 ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt 600 cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt 660 tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt 720 cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt 780 aacaaaaatt taacgcgaac tttaacaaaa tattaacgtt tacaatttaa atatttgctt 840 atacaatctt cctgtttttg gggcttttct gattatcaac cggggtacat atgattgaca 900 tgctagtttt acgattaccg ttcatcgccc tgcgcgctcg ctcgctcact gaggccgccc 960 gggcaaagcc cgggcgtcgg gcgacctttg gtcgccccggc ctcagtgagc gagcgagcgc 1020 gcagagagg agtggaattc acgcgtggat ctgaattctg tacaggcgtg gtacggccgc 1080 ggtgttttgt tttggtgacc taactggtca aatgacctat taagaatatt tcatagaacg 1140 aatgttccga tgctctaatc tctctagaca aggttcatat ttgtatgggt tacttattct 1200 ctctttgttg actaagtcaa taatcagaat cagcaggttt gcagtcagat tggcagggat 1260 aagcagccta gctcaggaga agtgagtata aaagccccag gctgggagca gccatcacag 1320 aagtccactc attcttggca ggggtaccca ctgggaggat gttgagtaag atggaaaact 1380 actgatgacc cttgcagaga cagagtatta ggacatgttt gaacaggggc cgggcgatca 1440 gcaggtagct ctagaggatc cccgtctgtc tgcacatttc gtagagcgag tgttccgata 1500 ctctaatctc cctaggcaag gttcatattt gtgtaggtta cttattctcc ttttgttgac 1560 taagtcaata atcagaatca gcaggtttgg agtcagcttg gcagggatca gcagcctggg 1620 ttggaaggag ggggtataaa agccccttca ccaggagaag ccgtcacaca gatccacaag 1680 ctcctggcta gaaagaggta agggtttaag ggatggttgg ttggtggggt attaatgttt 1740 aattacctgg agcacctgcc tgaaatcact ttttttcagg ttgggctagc ccaccatgca 1800 gcgcgtgaac atgatcatgg ccgagagccc cggcctgatc accatctgcc tgctgggcta 1860 cctgctgagc gccgagtgca ccgtgttcct ggaccacgag aacgccaaca agatcctgaa 1920 ccgccccaag cgctacaaca gcggcaagct ggaggagttc gtgcagggca acctggagcg 1980 cgagtgcatg gaggagaagt gcagcttcga ggaggcccgc gaggtgttcg agaacaccga 2040 gcgcaccacc gagttctgga agcagtacgt ggacggcgac cagtgcgaga gcaacccctg 2100 cctgaacggc ggcagctgca aggacgacat caacagctac gagtgctggt gccccttcgg 2160 cttcgagggc aagaactgcg agctggacgt gacctgcaac atcaagaacg gccgctgcga 2220 gcagttctgc aagaacagcg ccgacaacaa ggtggtgtgc agctgcaccg agggctaccg 2280 cctggccgag aaccagaaga gctgcgagcc cgccgtgccc ttcccctgcg gccgcgtgag 2340 cgtgagccag accagcaagc tgacccgcgc cgaggccgtg ttccccgacg tggactacgt 2400 gaacagcacc gaggccgaga ccatcctgga caacatcacc cagagcaccc agagcttcaa 2460 cgacttcacc cgcgtggtgg gcggcgagga cgccaagccc ggccagttcc cctggcaggt 2520 ggtgctgaac ggcaaggtgg acgccttctg cggcggcagc atcgtgaacg agaagtggat 2580 cgtgaccgcc gcccactgcg tggagaccgg cgtgaagatc accgtggtgg ccggcgagca 2640 caacatcgag gagaccgagc acaccgagca gaagcgcaac gtgatccgca tcatccccca 2700 ccacaactac aacgccgcca tcaacaagta caaccacgac atcgccctgc tggagctgga 2760 cgagcccctg gtgctgaaca gctacgtgac ccccatctgc atcgccgaca aggagtacac 2820 caacatcttc ctgaagttcg gcagcggcta cgtgagcggc tggggccgcg tgttccacaa 2880 gggccgcagc gccctggtgc tgcagtacct gcgcgtgccc ctggtggacc gcgccacctg 2940 cctgctgagc accaagttca ccatctacaa caacatgttc tgcgccggct tccacgaggg 3000 cggccgcgac agctgccagg gcgacagcgg cggcccccac gtgaccgagg tggagggcac 3060 cagcttcctg accggcatca tcagctgggg cgaggagtgc gccatgaagg gcaagtacgg 3120 catctacacc aaggtgagcc gctacgtgaa ctggatcaag gagaagacca agctgaccta 3180 atgaaagatg gatttccaag gttaattcat tggaattgaa attaacagc cccccccccc 3240 cccccctgc agatctgagc cgaattcctg cagcccgggg gatcagcctc gactgtgcct 3300 tctagttgcc agccatctgt tgtttgcccc tccccgtgc cttccttgac cctggaaggt 3360 gccactcccca ctgtcctttc ctaataaaat gaggaaattg catcgcattg tctgagtagg 3420 tgtcattcta ttctgggggg tggggtgggg caggacagca agggggagga ttgggaagac 3480 aatagcaggc atgctgggga tgcggtgggc tctatggctt ctgaggcgga aagaaccagc 3540 tggggaccgg tggatctcga tagcaggcat gctggggaga gatcgatctg aggaacccct 3600 agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg ccgcccgggc 3660 aaagcccggg cgtcgggcga cctttggtcg cccggcctca gtgagcgagc gagcgcgcag 3720 agagggagtg gccaaccccc cccccccccc cccccggcga ttctcttgtt tgctccagac 3780 tctcaggcaa tgacctgata gcctttgtag agacctctca aaaatagcta ccctctccgg 3840 catgaattta tcagctagaa cggttgaata tcatattgat ggtgatttga ctgtctccgg 3900 cctttctcac ccgtttgaat ctttacctac acattactca ggcattgcat ttaaaatata 3960 tgagggttct aaaaattttt atccttgcgt tgaaataaag gcttctcccg caaaagtatt 4020 acagggtcat aatgtttttg gtacaaccga tttagcttta tgctctgagg ctttattgct 4080 taattttgct aattctttgc cttgcctgta tgatttattg gatgttggaa tcgcctgatg 4140 cggtattttc tccttacgca tctgtgcggt atttcacacc gcatatggtg cactctcagt 4200 acaatctgct ctgatgccgc atagttatat ggtgcactct cagtacaatc tgctctgatg 4260 ccgcatagtt aagccagccc cgacacccgc caacacagcc agccccgaca cccgccaaca 4320 cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg 4380 accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgaga 4440 cgaaagggcc tcgtgatacg cctattttta taggttaatg tcatgataat aatggtttct 4500 tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc 4560 taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat gctcaataat 4620 attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg 4680 cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg 4740 aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc 4800 ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat 4860 gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact 4920 attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca 4980 tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact 5040 tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatgggg 5100 atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg 5160 agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaacta ttaactggcg 5220 aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg 5280 caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag 5340 ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc 5400 gtatcgtagt tatctacacg acggggagagtc aggcaactat ggatgaacga aatagacaga 5460 tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat 5520 atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc 5580 tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag 5640 accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct 5700 gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac 5760 caactcttt tccgaaggta actggcttca gcagagcgca throw actgtccttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga tagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaagggga gaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatcttata 6180. gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg 6240 ggcggagcct atggaaaaac gccagcaacg cggcctttttt acggttcctg gccttttgct 6300. ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta 6360 ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag 6420. tgagcgagga agcggagag cgcccaatac gcaaaccgcc tctccccgcg cgttggccga 6480. ttcattaatg 6490 <210> 38 <211> 6497 <212> DNA <213> Artificial Sequence <220> <223> AAVsc-HS-CRE6-TTRenh-TTRm-MVM-hFIXcoPadua-bghpolyA <400> 38 agctggcgta atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg 60 aatggcgaat ggcgattccg ttgcaatggc tggcggtaat attgttctgg atattaccag 120 caaggccgat agtttgagtt cttctactca ggcaagtgat gttattacta atcaaagaag 180 tattgcgaca acggttaatt tgcgtgatgg acagactctt ttactcggtg gcctcactga 240 ttataaaaac acttctcagg attctggcgt accgttcctg tctaaaatcc ctttaatcgg 300 cctcctgttt agctcccgct ctgattctaa cgaggaaagc acgttatacg tgctcgtcaa 360 agcaaccata gtacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc 420 gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt 480 cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag 540 ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt 600 cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt 660 tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt 720 cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt 780 aacaaaaatt taacgcgaac tttaacaaaa tattaacgtt tacaatttaa atatttgctt 840 atacaatctt cctgtttttg gggcttttct gattatcaac cggggtacat atgattgaca 900 tgctagtttt acgattaccg ttcatcgccc tgcgcgctcg ctcgctcact gaggccgccc 960 gggcaaagcc cgggcgtcgg gcgacctttg gtcgcccggc ctcagtgagc gagcgagcgc 1020 gcagagaggg agtggaattc acgcgtggat ctgaattctg tacaggcgtg gtacggccgc 1080 ggaggggggc ccctggggct gacagggact ggaagctctg agctggccag agggatgttg 1140 caatcctgcc agggtcttgt ctatgctgtc cttttcacaa ccatccccct actgccaggc 1200 tgacacgtgg ttgcgggggc acaaggccag ccaacctaga gtctgaggct aggcggagga 1260 caccctcccc accagctgcc agggtcactg gcggtcaaag gcagctggtg gggaaggcat 1320 tggactccag ccttggggga cggatgtagg gtacccactg ggaggatgtt gagtaagatg 1380 gaaaactact gatgaccctt gcagagacag agtattagga catgtttgaa caggggccgg 1440 gcgatcagca ggtagctcta gaggatcccc gtctgtctgc acatttcgta gagcgagtgt 1500 tccgatactc taatctccct aggcaaggtt catatttgtg taggttactt attctccttt 1560 tgttgactaa gtcaataatc agaatcagca ggtttggagt cagcttggca gggatcagca 1620 gcctgggttg gaaggagggg gtataaaagc cccttcacca ggagaagccg tcacacagat 1680 ccacaagctc ctggctagaa agaggtaagg gtttaaggga tggttggttg gtggggtatt 1740 aatgtttaat tacctggagc acctgcctga aatcactttt tttcaggttg ggctagccca 1800 ccatgcagcg cgtgaacatg atcatggccg agagccccgg cctgatcacc atctgcctgc 1860 tgggctacct gctgagcgcc gagtgcaccg tgttcctgga ccacgagaac gccaacaaga 1920 tcctgaaccg ccccaagcgc tacaacagcg gcaagctgga ggagttcgtg cagggcaacc 1980 tggagcgcga gtgcatggag gagaagtgca gcttcgagga ggcccgcgag gtgttcgaga 2040 acaccgagcg caccaccgag ttctggaagc agtacgtgga cggcgaccag tgcgagagca 2100 acccctgcct gaacggcggc agctgcaagg acgacatcaa cagctacgag tgctggtgcc 2160 ccttcggctt cgagggcaag aactgcgagc tggacgtgac ctgcaacatc aagaacggcc 2220 gctgcgagca gttctgcaag aacagcgccg acaacaaggt ggtgtgcagc tgcaccgagg 2280 gctaccgcct ggccgagaac cagaagagct gcgagcccgc cgtgcccttc ccctgcggcc 2340 gcgtgagcgt gagccagacc agcaagctga cccgcgccga ggccgtgttc cccgacgtgg 2400 actacgtgaa cagcaccgag gccgagacca tcctggacaa catcacccag agcacccaga 2460 gcttcaacga cttcacccgc gtggtgggcg gcgaggacgc caagcccggc cagttcccct 2520 ggcaggtggt gctgaacggc aaggtggacg ccttctgcgg cggcagcatc gtgaacgaga 2580 agtggatcgt gaccgccgcc cactgcgtgg agaccggcgt gaagatcacc gtggtggccg 2640 gcgagcacaa catcgaggag accgagcaca ccgagcagaa gcgcaacgtg atccgcatca 2700 tcccccacca caactacaac gccgccatca acaagtacaa ccacgacatc gccctgctgg 2760 agctggacga gcccctggtg ctgaacagct acgtgacccc catctgcatc gccgacaagg 2820 agtacaccaa catcttcctg aagttcggca gcggctacgt gagcggctgg ggccgcgtgt 2880 tccacaaggg ccgcagcgcc ctggtgctgc agtacctgcg cgtgcccctg gtggaccgcg 2940 ccacctgcct gctgagcacc aagttcacca tctacaacaa catgttctgc gccggcttcc 3000 acgagggcgg ccgcgacagc tgccagggcg acagcggcgg cccccacgtg accgaggtgg 3060 agggcaccag cttcctgacc ggcatcatca gctggggcga ggagtgcgcc atgaagggca 3120 agtacggcat ctacaccaag gtgagccgct acgtgaactg gatcaaggag aagaccaagc 3180 tgacctaatg aaagatggat ttccaaggtt aattcattgg aattgaaaat taacagcccc 3240 cccccccccc cccctgcaga tctgagccga attcctgcag cccgggggat cagcctcgac 3300 tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct 3360 ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct 3420 gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg 3480 ggaagacaat agcaggcatg ctggggatgc ggtgggctct atggcttctg aggcggaaag 3540 aaccagctgg ggaccggtgg atctcgatag caggcatgct ggggagagat cgatctgagg 3600 aacccctagt gatggagttg gccactccct ctctgcgcgc tcgctcgctc actgaggccg 3660 cccgggcaaa gcccgggcgt cgggcgacct ttggtcgccc ggcctcagtg agcgagcgag 3720 cgcgcagaga gggagtggcc aacccccccc cccccccccc ccggcgattc tcttgtttgc 3780 tccagactct caggcaatga cctgatagcc tttgtagaga cctctcaaaa atagctaccc 3840 tctccggcat gaatttatca gctagaacgg ttgaatatca tattgatggt gatttgactg 3900 tctccggcct ttctcacccg tttgaatctt tacctacaca ttactcaggc attgcattta 3960 aaatatatga gggttctaaa aatttttatc cttgcgttga aataaaggct tctcccgcaa 4020 aagtattaca gggtcataat gtttttggta caaccgattt agctttatgc tctgaggctt 4080 tattgcttaa ttttgctaat tctttgcctt gcctgtatga tttattggat gttggaatcg 4140 cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca tatggtgcac 4200 tctcagtaca atctgctctg atgccgcata gttatatggt gcactctcag tacaatctgc 4260 tctgatgccg catagttaag ccagccccga cacccgccaa cacagccagc cccgacaccc 4320 gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca 4380 agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg 4440 cgcgagacga aagggcctcg tgatacgcct atttttatag gttaatgtca tgataataat 4500 ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt 4560 atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct 4620 caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc 4680 ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa 4740 gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt 4800 aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt 4860 ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc 4920 atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg 4980 gatggcatga cattaagaga attatgcagt gctgccataa ccatgagtga taacactgcg 5040 5100 atggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca 5160 aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta 5220 actggcgaac tacttactct agcttccccgg aacaattaa tagactggat ggaggcggat 5280 aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa 5340 tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag 5400 5460 agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt 5520 tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg 5580 aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga 5640 gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta 5700 atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa 5760 gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact 5820 gtccttctag tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca 5880 tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt 5940 accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg 6000 ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag 6060 cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta 6120 agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat 6180 ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg 6240 tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc 6300 ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac 6360 cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc 6420 gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt 6480 tggccgattc attaatg 6497 <210> 39 <211> 6543 <212> DNA <213> Artificial Sequence <220> <223> AAVsc-HS-CRE7-TTRenh-TTRm-MVM-hFIXcoPadua-bghpolyA <400> 39 agctggcgta atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg 60 aatggcgaat ggcgattccg ttgcaatggc tggcggtaat attgttctgg atattaccag 120 caaggccgat agtttgagtt cttctactca ggcaagtgat gttattacta atcaaagaag 180 tattgcgaca acggttaatt tgcgtgatgg acagactctt ttactcggtg gcctcactga 240 ​​​​agcaaccata gtacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc 420 gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt 480 cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcgggg ctccctttag 540 ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt 600 cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt 660 tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt 720 cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt 780 aacaaaaatt taacgcgaac tttaacaaaa tattaacgtt tacaatttaa atatttgctt 840 atacaatctt cctgtttttg gggcttttct gattatcaac cggggtacat atgattgaca 900 tgctagtttt acgattaccg ttcatcgccc tgcgcgctcg ctcgctcact gaggccgccc 960 gggcaaagcc cgggcgtcgg gcgacctttg gtcgcccggc ctcagtgagc gagcgagcgc 1020 gcagagagg agtggaattc acgcgtggat ctgaattctg tacaggcgtg gtacggccgc 1080 ggtacaggcc tggggtcccg gcttagagga cctctgagag ctccggggcc ccttctgggt 1140 cgtggttgcc tcatcgtggt cgggtgggtc tccaggttct cccaggctca gtcccgcagg 1200 cgccaaatct gcgcaggaga gcactagcaa ccgatgacgt attgaggccc acacctctgg 1260 gattggctgt cctgcttcga cagccttgaa agtgggtaag ctgggtgggg ggctctggga 1320 gaggtcagtg ctgagtaagg caattcccag cagcttgagc cccaccaggt cactccagta 1380 ttcctcccca ttcttggtac ccactgggag gatgttgagt aagatggaaa actactgatg 1440 acccttgcag agacagagta ttaggacatg tttgaacagg ggccgggcga tcagcaggta 1500 gctctagagg atccccgtct gtctgcacat ttcgtagagc gagtgttccg atactctaat 1560 ctccctaggc aaggttcata tttgtgtagg ttacttattc tccttttgtt gactaagtca 1620 ataatcagaa tcagcaggtt tggagtcagc ttggcaggga tcagcagcct gggttggaag 1680 gagggggtat aaaagcccct tcaccaggag aagccgtcac acagatccac aagctcctgg 1740 ctagaaagag gtaagggttt aagggatggt tggttggtgg ggtattaatg tttaattacc 1800 tggagcacct gcctgaaatc actttttttc aggttgggct agcccaccat gcagcgcgtg 1860 aacatgatca tggccgagag ccccggcctg atcaccatct gcctgctggg ctacctgctg 1920 agcgccgagt gcaccgtgtt cctggaccac gagaacgcca acaagatcct gaaccgcccc 1980 aagcgctaca acagcggcaa gctggaggag ttcgtgcagg gcaacctgga gcgcgagtgc 2040 atggaggaga agtgcagctt cgaggaggcc cgcgaggtgt tcgagaacac cgagcgcacc 2100 accgagttct ggaagcagta cgtggacggc gaccagtgcg agagcaaccc ctgcctgaac 2160 ggcggcagct gcaaggacga catcaacagc tacgagtgct ggtgcccctt cggcttcgag 2220 ggcaagaact gcgagctgga cgtgacctgc aacatcaaga acggccgctg cgagcagttc 2280 tgcaagaaca gcgccgacaa caaggtggtg tgcagctgca ccgagggcta ccgcctggcc 2340 gagaaccaga agagctgcga gcccgccgtg cccttcccct gcggccgcgt gagcgtgagc 2400 cagaccagca agctgacccg cgccgaggcc gtgttccccg acgtggacta cgtgaacagc 2460 accgaggccg agaccatcct ggacaacatc acccagagca cccagagctt caacgacttc 2520 acccgcgtgg tgggcggcga ggacgccaag cccggccagt tcccctggca ggtggtgctg 2580 aacggcaagg tggacgcctt ctgcggcggc agcatcgtga acgagaagtg gatcgtgacc 2640 gccgcccact gcgtggagac cggcgtgaag atcaccgtgg tggccggcga gcacaacatc gaggagaccg agcacaccg gcagaagcgc aacgtgatcc gcatcatccc ccaccacac 2820. 2820. 2820. 2820. 2820. 2820. 2820. 2820. 2820 ctggtgctga acagctacgt gacccccatc tgcatcgccg acaaggagta caccaacatc ttcctgaagt tcggcagcgg ctacgtgagc ggctggggcc gcgtgttcca caagggccgc 2940 agcgccctgg tgctgcagta cctgcgcgtg cccctggtgg accgcgccac ctgcctgctg 3000 agcaccaagt tcaccatcta caacaacatg ttctgcgccg gcttccacga gggcggccgc 3120. 3120. 3120. 3120. 3120. 3120. 3120. 3120. 3120. ctgaccggca tcatcagctg gggcgaggag tgcgccatga agggcaagta cggcatctac 3180. accaaggtga gccgctacgt gaactggatc aaggagaga ccaagctgac ctaatgaag atggatttcc aaggttaatt cattggaatt gaaaattaac agcccccccc cccccccccc 3300 tgcagatctg agccgaattc ctgcagcccg ggggatcagc ctcgactgtg ccttttagtt 3360 gccagccatc tgttgtttgc ccctcccccg tgccttcct gaccctggaa ggtgccactc 3420 ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt 3480 ctattctggg gggtggggtg gggcaggaca gcaagggga ggattgggaa gacaatagca 3540 ggcatgctgg ggatgcggtg ggctctatgg cttctgaggc ggaaagaacc agctggggac 3600 cggtggatct cgatagcagg catgctgggg agagatcgat ctgaggaacc cctagtgatg 3660 gagttggcca ctccctctct gcgcgctcgc tcgctcactg aggccgcccg ggcaaagccc 3720 gggcgtcggg cgacctttgg tcgcccggcc tcagtgagcg agcgagcgcg cagagaggga 3780 gtggccaacc cccccccccc ccccccccgg cgattctctt gtttgctcca gactctcagg 3840 caatgacctg atagcctttg tagagacctc tcaaaaatag ctaccctctc cggcatgaat 3900 ttatcagcta gaacggttga atatcatatt gatggtgatt tgactgtctc cggcctttct 3960 cacccgtttg aatctttacc tacacattac tcaggcattg catttaaaat atatgagggt 4020 tctaaaaatt tttatccttg cgttgaaata aaggcttctc ccgcaaaagt attacagggt 4080 cataatgttt ttggtacaac cgatttagct ttatgctctg aggctttatt gcttaatttt 4140 gctaattctt tgccttgcct gtatgattta ttggatgttg gaatcgcctg atgcggtatt 4200 ttctccttac gcatctgtgc ggtatttcac accgcatatg gtgcactctc agtacaatct 4260 gctctgatgc cgcatagtta tatggtgcac tctcagtaca atctgctctg atgccgcata 4320 gttaagccag ccccgacacc cgccaacaca gccagccccg acacccgcca acacccgctg 4380 acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct 4440 ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg agacgaaagg 4500 gcctcgtgat acgcctattt ttataggtta atgtcatgat aataatggtt tcttagacgt 4560 caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac 4620 attcaaatat gtatccgctc atgagacaat aaccctgata aatgctcaat aatattgaaa 4680 aaagaagt atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt 4740 ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca 4800 gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag 4860 ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc 4920 ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca 4980 gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt 5040 aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct 5100 ggaacgatc ggaggaccga aggagctaac cgctttttttg caacacatgg gggatcatgt 5160 aactcgcctt gatcgttgg aaccggagct gaatgaagcc ataccaaacg acgagcgtga 5220 caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact 5280 tactctagct tcccggcaac aattaataga ctggatggag gcggataag ttgcaggacc 5340 acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga 5400 gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt 5460 agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga 5520 gataggtgcc tcactgatta agcattggta actgtcagac caagtttact catatatact 5580 ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga 5640 taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt 5700 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca 5760 aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct 5820 ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc ttctagtgta 5880 gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct 5940 aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc 6000 aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca 6060 gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga 6120 aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg 6180 aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt 6240 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag 6300 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt 6360 tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt 6420 tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga 6480 ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc cgattcatta 6540 atg 6543 <210> 40 <211> 6743 <212> DNA <213> Artificial Sequence <220> <223> AAVsc-HS-CRE8-TTRenh-TTRm-MVM-hFIXcoPadua-bghpolyA <400> 40 agctggcgta atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg 60 aatggcgaat ggcgattccg ttgcaatggc tggcggtaat attgttctgg atattaccag 120 caaggccgat agtttgagtt cttctactca ggcaagtgat gttatacta atcaaagaag 180 tattgcgaca acggttaatt tgcgtgatgg acagactctt ttatcggtg gcctcactga 240 ttataaaac acttctcagg attctggcgt accgttcctg tctaaaatcc ctttaatcgg 300 cctcctgttt agctcccgct ctgattctaa cgaggaaagc acgttatacg tgctcgtcaa 360 agcaaccata gtacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc 420 gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt 480 cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcgggg ctccctttag 540 ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt 600 cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt 660 tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt 720 cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt 780 aacaaaaatt taacgcgaac tttaacaaaa tattaacgtt tacaatttaa atatttgctt 840 attackctt cctgtttttg gggcttttct cggggtacat atgattgaca tgctagtttt acgattaccg ttcatcgccc tgcgcgctcg ctcgctcact gaggccgccc 960 gggcaaagcc cggggcgtcgg gcgaccttttg gtcgcccggc ctcagtgagc gagcgagcgc 1020. gcagagaggg agtggaattc acgcgtggat ctgaattctg tacaggcgtg gtacggccgc 1080 ggcccgaacg aataaacccc ttccttaact cagcgtctga ggaattttgt ctgcggctcc tcctgctaca ttctgagtgg ggaaagggac taaggtggtc tgaggacccc acagagtcag gaagattgag aggtgagagt gctgaacggg gaggggcttt ggggctaagg gaagtgcccg ggaccccacc tgaccccaac gctcacggga caggggcaga ggagaaaaac gtgggtggac agggaggc aggcggtcag gggaaggctc aggagggg agatcaacat caacctgccc cgccccctcc ccagcctgat aaaggtcctg cgggcaggac aggacctccc aaccaagccc 1440 tccagcaagg attcaggttg gtgctgagtg cctgggaggg acacccgcct acactctgca agaaactcaa aaagggagat gaggggatcg tgggagggag gtagggaggg aggagggtgc 1560. cactgatccc ctgaacccct gcctctgcct ccagaggtac ccactgggag gatgttgagt 1620 aagatggaaa actactgatg acccttgcag agacagagta ttaggacatg tttgaacagg 1680 ggccgggcga tcagcaggta gctctagagg atccccgtct gtctgcacat ttcgtagagc 1740 gagtgttccg atactctaat ctccctaggc aaggttcata tttgtgtagg ttacttattc 1800 tccttttgtt gactaagtca ataatcagaa tcagcaggtt tggagtcagc ttggcaggga 1860 tcagcagcct gggttggaag gagggggtat aaaagcccct tcaccaggag aagccgtcac 1920 acagatccac aagctcctgg ctagaaagag gtaagggttt aagggatggt tggttggtgg 1980 ggtattaatg tttaattacc tggagcacct gcctgaaatc actttttttc aggttgggct 2040 agcccaccat gcagcgcgtg aacatgatca tggccgagag ccccggcctg atcaccatct 2100 gcctgctggg ctacctgctg agcgccgagt gcaccgtgtt cctggaccac gagaacgcca 2160 acaagatcct gaaccgcccc aagcgctaca acagcggcaa gctggaggag ttcgtgcagg 2220 gcaacctgga gcgcgagtgc atggaggaga agtgcagctt cgaggaggcc cgcgaggtgt 2280 tcgagaacac cgagcgcacc accgagttct ggaagcagta cgtggacggc gaccagtgcg aggcaaccc ctgcctgac ggcggcagct gcaaggacga catcaacagc tacgagtgct ggtgcccctt cggcttcgag ggcaagaact gcgagctgga cgtgacctgc aacatcaaga 2460 acggccgctg cgagcagttc tgcaagaaca gcgccgacaa caaggtggtg tgcagctgca ccgagggcta ccgcctggcc gagaccga agagctgcga gcccgccgtg cccttcccct gcggccgcgt gagcgtgagc cagaccagca agctgacccg cgccgaggcc gtgttccccg 2640 acgtggacta cgtgaacagc accgaggccg agaccatcct ggacaacatc acccagagca cccagagctt caacgacttc acccgcgtgg tgggcggcga ggacgccaag cccggccagt 2760. tcccctggca ggtggtgctg aacggcaagg tggacgcctt ctgcggcggc agcatcgtga 2820 acgagaagtg gatcgtgacc gccgcccact gcgtggagac cggcgtgaag atcaccgtgg 2880. tggccggcga gcacaacatc gaggagaccg agcacaccga gcagaagcgc aacgtgatcc gcatcatccc ccaccacaac tacaacgccg ccatcaac gtacaaccac gacatcgccc tgctggagct ggacgagccc ctggtgctga acagctacgt gacccccatc tgcatcgccg 3060 aaaggaga caccaacatc ttcctgaagt tcggcagcgg ctacgtgagc ggctggggcc 3120 gcgtgttcca caagggccgc agcgccctgg tgctgcagta cctgcgcgtg cccctggtgg 3180 accgcgccac ctgcctgctg agcaccaagt tcaccatcta caacaacatg ttctgcgccg 3240 gcttccacga gggcggccgc gacagctgcc agggcgacag cggcggcccc cacgtgaccg 3300 aggtggaggg caccagcttc ctgaccggca tcatcagctg gggcgaggag tgcgccatga 3360 agggcaagta cggcatctac accaaggtga gccgctacgt gaactggatc your area 3420 ccaagctgac ctaatgaaag atggatttcc aaggttaatt cattggaatt gaaattaac 3480 agcccccccc cccccccccc tgcagatctg agccgaattc ctgcagcccg ggggatcagc 3540 ctcgactgtg ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt 3600 gaccctggaa ggtgccactc ccactgtcct ttcctaataa aatgagaaa ttgcatcgca 3660 ttgtctgagt aggtgtcatt ctattctggg gggtggggtg gggcaggaca gcaagggga 3720 ggattgggaa gacaatagca ggcatgctgg ggatgcggtg ggctctatgg cttctgaggc 3780 ggaaagaacc agctggggac cggtggatct cgatagcagg catgctgggg agagatcgat 3840 ctgaggaacc cctagtgatg gagttggcca ctccctctct gcgcgctcgc tcgctcactg 3900 aggccgcccg ggcaaagccc gggcgtcggg cgacctttgg tcgcccggcc tcagtgagcg 3960 agcgagcgcg cagagaggga gtggccaacc cccccccccc ccccccccgg cgattctctt 4020 gtttgctcca gactctcagg caatgacctg atagcctttg tagagacctc tcaaaaatag 4080 ctaccctctc cggcatgaat ttatcagcta gaacggttga atatcatatt gatggtgatt 4140 tgactgtctc cggcctttct cacccgtttg aatctttacc tacacattac tcaggcattg 4200 catttaaaat atatgagggt tctaaaaatt tttatccttg cgttgaaata aaggcttctc 4260 ccgcaaaagt attacagggt cataatgttt ttggtacaac cgatttagct ttatgctctg 4320 aggctttatt gcttaatttt gctaattctt tgccttgcct gtatgattta ttggatgttg 4380 gaatcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcatatg 4440 gtgcactctc agtacaatct gctctgatgc cgcatagtta tatggtgcac tctcagtaca 4500 atctgctctg atgccgcata gttaagccag ccccgacacc cgccaacaca gccagccccg 4560 acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta 4620 cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc 4680 gaaacgcgcg agacgaaagg gcctcgtgat acgcctattt ttataggtta atgtcatgat 4740 aataatggtt tcttagacgt caggtggcac ttttcggga aatgtgcgcg gaacccctat 4800 ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata 4860 aatgctcaat aatattgaaa aaagaagagt atgagtattc aacatttccg tgtcgccctt 4920 attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa 4980 gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac 5040 agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt 5100 aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt 5160 cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat 5220 cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac 5280 actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac cgctttttg 5340 cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc 5400 ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa 5460 ctattaactg gcgaactact tactctagct tcccggcaac attaataga ctggatggag 5520 gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg gttattgct 5580 gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat 5640 ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa 5700 cgaatagac agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac 5760 caagtttact catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc 5820 taggtgaaga tccttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc 5880 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg 5940 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg 6000 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca 6060 aatactgtcc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg 6120 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg 6180 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga 6240 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac 6300 ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat 6360 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc 6420 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga 6480 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc 6540 ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg 6600 gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag 6660 cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc 6720 gcgcgttggc cgattcatta atg 6743 <210> 41 <211> 6536 <212> DNA <213> Artificial Sequence <220> <223> AAVsc-HS-CRE9-TTRenh-TTRm-MVM-hFIXcoPadua-bghpolyA <400> 41 agctggcgta atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg 60 aatggcgaat ggcgattccg ttgcaatggc tggcggtaat attgttctgg atattaccag 120 caaggccgat agtttgagtt cttctactca ggcaagtgat gttattacta atcaaagaag 180 tattgcgaca acggttaatt tgcgtgatgg acagactctt ttactcggtg gcctcactga 240 ttataaaaac acttctcagg attctggcgt accgttcctg tctaaaatcc ctttaatcgg 300 cctcctgttt agctcccgct ctgattctaa cgaggaaagc acgttatacg tgctcgtcaa 360 agcaaccata gtacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc 420 gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt 480 cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag 540 ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt 600 cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt 660 tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt 720 cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt 780 aacaaaaatt taacgcgaac tttaacaaaa tattaacgtt tacaatttaa atatttgctt 840 atacaatctt cctgtttttg gggcttttct gattatcaac cggggtacat atgattgaca 900 tgctagtttt acgattaccg ttcatcgccc tgcgcgctcg ctcgctcact gaggccgccc 960 gggcaaagcc cgggcgtcgg gcgacctttg gtcgcccggc ctcagtgagc gagcgagcgc 1020 gcagagaggg agtggaattc acgcgtggat ctgaattctg tacaggcgtg gtacggccgc 1080 gggatgagtg cagggaaccc cgaccccacc cgggagacct gcaagcctgc agacactccc 1140 ctcccgcccc cactgaaccc ttgacccctg ccctgcagcc cccgcagctt gctgtttgcc 1200 cactctattt gcccagcccc aggcagag ctgatccttg aactcttaag ttccacattg 1260 ccaggaccag tgagcagcaa cagggccggg gctgggctta tcagcctccc agcccagacc 1320 ctggctgcag acataatag gccctgcaag agctggctgc ttagagactg cgagaaggag 1380 gtgcgtccgg taccactgg gaggatgttg agtaagatgg aaaactactg atgacccttg 1440 cagagacaga gtattaggac atgtttgaac aggggccggg cgatcagcag gtagctctag 1500 aggatccccg tctgtctgca catttcgtag agcgagtgtt ccgatactct aatctcccta 1560 ggcaaggttc atatttgtgt aggttactta ttctccttt gttgactaag tcaataatca 1620 gaatcagcag gtttggagtc agcttggcag ggatcagcag cctgggttgg aaggggggg 1680 tataaaagcc ccttcaccag gagaagccgt cacacagatc caaagctcc tggctagaaa 1740 gaggtaaggg tttaagggat ggttggttgg tggggtatta atgtttaatt acctggagca 1800 cctgcctgaa atcacttttt ttcaggttgg gctagcccac catgcagcgc gtgaacatga 1860 tcatggccga gagccccggc ctgatcacca tctgcctgct gggctacctg ctgagcgccg 1920 agtgcaccgt gttcctggac cacgagaacg ccaacaagat cctgaaccgc cccaagcgct 1980 acaacagcgg caagctggag gagttcgtgc agggcaacct ggagcgcgag tgcatggagg 2040 agaagtgcag cttcgaggag gcccgcgagg tgttcgagaa caccgagcgc accaccgagt 2100 tctggaagca gtacgtggac ggcgaccagt gcgagagcaa cccctgcctg aacggcggca 2160 gctgcaagga cgacatcaac agctacgagt gctggtgccc cttcggcttc gagggcaaga 2220 actgcgagct ggacgtgacc tgcaacatca agaacggccg ctgcgagcag ttctgcaaga 2280 acagcgccga caacaaggtg gtgtgcagct gcaccgaggg ctaccgcctg gccgagaacc 2340 agaagagctg cgagcccgcc gtgcccttcc cctgcggccg cgtgagcgtg agccagacca 2400 gcaagctgac ccgcgccgag gccgtgttcc ccgacgtgga ctacgtgaac agcaccgagg 2460 ccgagaccat cctggacaac atcacccaga gcacccagag cttcaacgac ttcacccgcg 2520 tggtgggcgg cgaggacgcc aagcccggcc agttcccctg gcaggtggtg ctgaacggca 2580 aggtggacgc cttctgcggc ggcagcatcg tgaacgagaa gtggatcgtg accgccgccc 2640 actgcgtgga gaccggcgtg aagatcaccg tggtggccgg cgagcacaac atcgaggaga 2700 ccgagcacac cgagcagaag cgcaacgtga tccgcatcat cccccaccac aactacaacg 2760 ccgccatcaa caagtacaac cacgacatcg ccctgctgga gctggacgag cccctggtgc 2820 tgaacagcta cgtgaccccc atctgcatcg ccgacaagga gtacaccaac atcttcctga 2880 agttcggcag cggctacgtg agcggctggg gccgcgtgtt ccacaagggc cgcagcgccc 2940 tggtgctgca gtacctgcgc gtgcccctgg tggaccgcgc cacctgcctg ctgagcacca 3000 agttcaccat ctacaacaac atgttctgcg ccggcttcca cgagggcggc cgcgacagct 3060 gccagggcga cagcggcggc ccccacgtga ccgaggtgga gggcaccagc ttcctgaccg 3120 gcatcatcag ctggggcgag gagtgcgcca tgaagggcaa gtacggcatc tacaccaagg 3180 tgagccgcta cgtgaactgg atcaaggaga agaccaagct gacctaatga aagatggatt 3240 tccaaggtta attcattgga attgaaaatt aacagccccc cccccccccc ccctgcagat 3300 ctgagccgaa ttcctgcagc ccgggggatc agcctcgact gtgccttcta gttgccagcc 3360 atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt 3420 cctttcctaa taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct 3480 ggggggtggg gtggggcagg acagcaaggg ggaggattgg gaagacaata gcaggcatgc 3540 tggggatgcg gtgggctcta tggcttctga ggcggaaaga accagctggg gaccggtgga 3600 tctcgatagc aggcatgctg gggagagatc gatctgagga acccctagtg atggagttgg 3660 ccactccctc tctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc 3720 gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca 3780 accccccccc cccccccccc cggcgattct cttgtttgct ccagactctc aggcaatgac 3840 ctgatagcct ttgtagagac ctctcaaaaa tagctaccct ctccggcatg aatttatcag 3900 ctagaacggt tgaatatcat attgatggtg atttgactgt ctccggcctt tctcacccgt 3960 ttgaatcttt acctacacat tactcaggca ttgcatttaa aatatatgag ggttctaaaa 4020 attttatcc ttgcgttgaa ataaaggctt ctcccgcaaa agtattacag ggtcataatg 4080 tttttggtac aaccgattta gctttatgct ctgaggcttt attgcttaat tttgctaatt 4140 cttgccttg cctgtatgat ttattggatg ttggaatcgc ctgatgcggt atttctcct 4200 tacgcatctg tgcggtattt cacaccgcat atggtgcact ctcagtacaa tctgctctga 4260 tgccgcatag ttatatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc 4320 cagccccgac acccgccaac acagccagcc ccgacaccg ccaacccg ctgacgcgcc 4380 ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag 4440 ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgagacgaa agggcctcgt 4500 gatacgccta tttttatagg ttaatgtcat gataataatg gtttcttaga cgtcaggtgg 4560 cactttcgg ggaaatgtgc gcggaacccc tatttgttta ttttctaaa tacattcaaa 4620 tatgtatccg ctcatgagac aataaccctg ataaatgctc aataatattg aaaaaggaag 4680 agtatgagta ttcaacattt ccgtgtcgcc cttattccct ttttgcggc attttgcctt 4740 cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt 4800 gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga gagttttcgc 4860 cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta 4920 tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac 4980 ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa 5040 ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg 5100 atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc 5160 cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg 5220 atgcctgtag caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta 5280 gcttccccggc aaaattaat agactggatg gaggcggata aagttgcagg accacttctg 5340 cgctcggccc ttccggctgg ctggtttat gctgataaat ctggagccgg tgagcgtggg 5400 tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc 5460 tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt 5520 gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt 5580 gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc 5640 atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag 5700 atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa 5760 aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg 5820 aaggtaactg gcttcagcag agcgcagata ccaaatactg tccttctagt gtagccgtag 5880 ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg 5940 ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga 6000 tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc 6060 ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc 6120 acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga 6180 gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt 6240 cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg 6300 aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac 6360 atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga 6420 gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg 6480 gaagagcgcc caatacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatg 6536 <210> 42 <211> 6636 <212> DNA <213> Artificial Sequence <220> <223> AAVsc-HS-CRE10-TTRenh-TTRm-MVM-hFIXcoPadua-bghpolyA <400> 42 agctggcgta atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg 60 aatggcgaat ggcgattccg ttgcaatggc tggcggtaat attgttctgg atattaccag 120 caaggccgat agtttgagtt cttctactca ggcaagtgat gttattacta atcaaagaag 180 tattgcgaca acggttaatt tgcgtgatgg acagactctt ttatcggtg gcctcactga 240 ttataaaac acttctcagg attctggcgt accgttcctg tctaaaatcc ctttaatcgg 300 cctcctgttt agctcccgct ctgattctaa cgaggaaagc acgttatacg tgctcgtcaa 360 agcaaccata gtacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc 420 gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt 480 cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcgggg ctccctttag 540 ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt 600 cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt 660 tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt 720 cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt 780 aacaaaaatt taacgcgaac tttaacaaaa tattaacgtt tacaatttaa atatttgctt 840 atacaatctt cctgtttttg gggcttttct gattatcaac cggggtacat atgattgaca 900 tgctagtttt acgattaccg ttcatcgccc tgcgcgctcg ctcgctcact gaggccgccc 960 gggcaaagcc cgggcgtcgg gcgacctttg gtcgccccggc ctcagtgagc gagcgagcgc 1020 gcagagagg agtggaattc acgcgtggat ctgaattctg tacaggcgtg gtacggccgc 1080 ggttctatca ttatcttcaa tcagaaactg atgtgtgggg aggtgatgta tcataactca 1140 tccagggtca ggcagctgga tttgaacctg cctgaccctt agatttgagg tttctggct 1200 ccagaatctt ctctgaagtg aaaggcatga ggccgaccac tccctgatct ggtaaaagaga 1260 gatgtcagcc tggtttctag tgttagggag tttcctggag tgatggtaca gggtacattt 1320 ctgccctgca tcccaagtcc agagactggg ttctaggtcc agcctttctt ctaactccct 1380 gagagatgac agcctctggg caaagtccct ctgtgctctt cagtctcttc atctgtgaga 1440 tggtggcatg gggagaggct ggagtgatgt cacagttcct ctcagcctgg taccactgg 1500 gaggatgttg agtaagatgg aaaactactg atgacccttg cagagacaga gtattaggac 1560 atgtttgaac aggggccggg cgatcagcag gtagctctag aggatccccg tctgtctgca 1620 catttcgtag agcgagtgtt ccgatactct aatctcccta ggcaaggttc atatttgtgt 1680 aggttactta ttctccttt gttgactaag tcaataatca gaatcagcag gtttggagtc 1740 agcttggcag ggatcagcag cctgggttgg aaggagggggg tataaaagcc ccttcaccag 1800 gagaagccgt cacacagatc caaagctcc tggctagaaa gaggtaaggg tttaagggat 1860 ggttggttgg tggggtatta atgtttaatt acctggagca cctgcctgaa atcacttttt 1920 ttcaggttgg gctagcccac catgcagcgc gtgaacatga tcatggccga gagccccggc 1980 ctgatcacca tctgcctgct gggctacctg ctgagcgccg agtgcaccgt gttcctggac 2040 cacgagaacg ccaacaagat cctgaaccgc cccaagcgct aaacagcgg caagctgggag 2100 gagttcgtgc agggcaacct ggagcgcgag tgcatggagg agaagtgcag cttcgaggag 2160 gcccgcgagg tgttcgagaa caccgagcgc accaccgagt tctggaagca gtacgtggac 2220 ggcgaccagt gcgagagcaa cccctgcctg aacggcggca gctgcaagga cgacatcaac 2280 agctacgagt gctggtgccc cttcggcttc gagggcaaga actgcgagct ggacgtgacc 2340 tgcaacatca agaacggccg ctgcgagcag ttctgcaaga acagcgccga caacaaggtg 2400 gtgtgcagct gcaccgaggg ctaccgcctg gccgagaacc agaagagctg cgagcccgcc 2460 gtgcccttcc cctgcggccg cgtgagcgtg agccagacca gcaagctgac ccgcgccgag 2520 gccgtgttcc ccgacgtgga ctacgtgaac agcaccgagg ccgagaccat cctggacaac 2580 atcacccaga gcacccagag cttcaacgac ttcacccgcg tggtgggcgg cgaggacgcc 2640 aagcccggcc agttcccctg gcaggtggtg ctgaacggca aggtggacgc cttctgcggc 2700 ggcagcatcg tgaacgagaa gtggatcgtg accgccgccc actgcgtgga gaccggcgtg 2760 aagatcaccg tggtggccgg cgagcacaac atcgaggaga ccgagcacac cgagcagaag 2820 cgcaacgtga tccgcatcat cccccaccac aactacaacg ccgccatcaa caagtacaac 2880 cacgacatcg ccctgctgga gctggacgag cccctggtgc tgaacagcta cgtgaccccc 2940 atctgcatcg ccgacaagga gtacaccaac atcttcctga agttcggcag cggctacgtg 3000 agcggctggg gccgcgtgtt ccacaagggc cgcagcgccc tggtgctgca gtacctgcgc 3060 gtgcccctgg tggaccgcgc cacctgcctg ctgagcacca agttcaccat ctacaacac 3120 atgttctgcg ccggcttcca cgagggcggc cgcgacagct gccagggcga cagcggcggc 3180 ccccacgtga ccgaggtgga gggcaccagc ttcctgaccg gcatcatcag ctggggcgag 3240 gagtgcgcca tgaagggcaa gtacggcatc tacaccaagg tgagccgcta cgtgaactgg 3300 atcaaggaga agaccaagct gacctaatga aagatggatt tccaaggtta attcattgga 3360 attgaaaatt aacagccccc cccccccccc ccctgcagat ctgagccgaa ttcctgcagc 3420 ccgggggatc agcctcgact gtgccttcta gttgccagcc atctgttgtt tgcccctccc 3480 ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg 3540 aaattgcatc gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg 3600 acagcaaggg ggaggattgg gaagacaata gcaggcatgc tggggatgcg gtgggctcta 3660 tggcttctga ggcggaaaga accagctggg gaccggtgga tctcgatagc aggcatgctg 3720 gggagagatc gatctgagga accctagtg atggagttgg ccactccctc tctgcgcgct 3780 cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc gggcgacctt tggtcgcccg 3840 gcctcagtga gcgagcgagc gcgcagagag ggagtggcca accccccccc cccccccccc 3900 cggcgattct cttgtttgct ccagactctc aggcaatgac ctgatagcct ttgtagagac 3960 ctctcaaaaa tagctaccct ctccggcatg aatttatcag ctagaacggt tgaatatcat 4020 attgatggtg atttgactgt ctccggcctt tctcacccgt ttgaatcttt acctacacat 4080 tactcaggca ttgcatttaa aatatatgag ggttctaaaa atttttatcc ttgcgttgaa 4140 ataaaggctt ctcccgcaaa agtattacag ggtcataatg tttttggtac aaccgattta 4200 gctttatgct ctgaggcttt attgcttaat tttgctaatt ctttgccttg cctgtatgat 4260 ttattggatg ttggaatcgc ctgatgcggt attttctcct tacgcatctg tgcggtattt 4320 cacaccgcat atggtgcact ctcagtacaa tctgctctga tgccgcatag ttatatggtg 4380 cactctcagt acaatctgct ctgatgccgc atagttaagc cagccccgac acccgccaac 4440 acagccagcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc 4500 cggcatccgc ttacagacaa gctgtgaccg tctccggg ctgcatgtgt cagaggtttt 4560 caccgtcatc accgaaacgc gcgagacgaa agggctcgt gatacgccta ttttattagg 4620 ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cactttcgg ggaatgtgc 4680 gcggaacccc tattgttta ttttctaaa tacattcaa tatgtatccg ctcatgagac 4740 aaaccctg aataatgctc aaatattg aaaaggaag agtatgagta ttcacaattt 4800 ccgtgtcgcc cttattccct tttgcggc atttgcctt cctgttttg ctcaccaga 4860 aacgctggtg aaagtaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga 4920 actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat 4980 gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acggccgggca 5040 agagcaactc gtcgccgca tacactattc tcagaatgac tggttgagt actcaccagt 5100 cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac 5160 catgagtgat aacactgcgg cacattact tctgacacg atcggaggac cgaggagct 5220 aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga 5280 gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac 5340 aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat 5400 agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg 5460 ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc 5520 actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc 5580 aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg 5640 gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta 5700 atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg 5760 tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga 5820 tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt 5880 ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag 5940 agcgcagata ccaaatactg tccttctagt gtagccgtag ttaggccacc acttcaagaa 6000 ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag 6060 tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca 6120 gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac 6180 cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa 6240 ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc 6300 agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg 6360 tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc 6420 ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc 6480 ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag 6540 ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc caatacgcaa 6600 accgcctctc cccgcgcgtt ggccgattca ttaatg 6636 <210> 43 <211> 6734 <212> DNA <213> Artificial Sequence <220> <223> AAVsc-HS-CRE11-TTRenh-TTRm-MVM-hFIXcoPadua-bghpolyA <400> 43 agctggcgta atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg 60 aatggcgaat ggcgattccg ttgcaatggc tggcggtaat attgttctgg atattaccag 120 caaggccgat agtttgagtt cttctactca ggcaagtgat gttattacta atcaaagaag 180 tattgcgaca acggttaatt tgcgtgatgg acagactctt ttactcggtg gcctcactga 240 ttataaaaac acttctcagg attctggcgt accgttcctg tctaaaatcc ctttaatcgg 300 cctcctgttt agctcccgct ctgattctaa cgaggaaagc acgttatacg tgctcgtcaa 360 agcaaccata gtacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc 420 gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt 480 cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag 540 ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt 600 cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt 660 tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt 720 cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt 780 aacaaaaatt taacgcgaac tttaacaaaa tattaacgtt tacaatttaa atatttgctt 840 atacaatctt cctgtttttg gggcttttct gattatcaac cggggtacat atgattgaca 900 tgctagtttt acgattaccg ttcatcgccc tgcgcgctcg ctcgctcact gaggccgccc 960 gggcaaagcc cgggcgtcgg gcgacctttg gtcgcccggc ctcagtgagc gagcgagcgc 1020 gcagagaggg agtggaattc acgcgtggat ctgaattctg tacaggcgtg gtacggccgc 1080 ggacaaaagc tgcagcaaaa ttgccaaaag taggaactac tgggggtggg ggtcctgtct 1140 cccctgctca gtcaccacct cccctcccta actctcattc tggctcatac cacgtgctcc 1200 cagaagaaac gttctgagca ggggctggta tgagactgcc tcccctccca ggacccactc 1260 ctccctatga ccaggctggt gaccctccct ctagggtgcc agtgccattc actgagcaga 1320 tgtttggcag gtgtctgtta tgtgccagac ctagcagggg acagagtcct cccgatacag 1380 atggaccagt atgtttggac aaattgtg aaaggagttg ctaatctttc tgtgcaaaat 1440 ccactctgc gtcaccgcc aacactccta cacatgtcag ggcatccagg gccaggactt 1500 ggtaaccctg ttcaggagca gtcgctctgg agactcccca gacgaggga agaatgggt 1560 ggaaacagggg gcctacactg tggccggta cccactggga ggatgttgag tagatgga 1620 aactactgat gacccttgca gagacagagt attaggacat gtttgacag gggccggggcg 1680 atcagcaggt agctctagag gatccccgtc tgtctgcaca ttcgtag cgagtgttcc 1740 gatactctaa tctccctagg caggttcat atttgtgtag gttacttatt ctccttttgt 1800 tgactaagtc aaatcaga atcagcaggt tggagtcag cttggcaggg atcagcagcc 1860 tgggttggaa ggaggggta taaaagcccc ttcaccagga gaagccgtca cacagatcca 1920 caagctcctg gctagaaaga ggtaagggtt taagggatgg ttggttggtg gggtattaat 1980 gtttaattac ctggagcacc tgcctgaat cacttttt caggttgggc tagcccacca 2040 tgcagcgcgt gaacatgatc atggccgaga gccccggcct gatcaccatc tgcctgctgg 2100 gctacctgct gagcgccgag tgcaccgtgt tcctggacca cgagaacgcc aacaagatcc 2160 tgaaccgccc caagcgctac aacagcggca agctggagga gttcgtgcag ggcaacctgg 2220 agcgcgagtg catggaggag aagtgcagct tcgaggaggc ccgcgaggtg ttcgagaaca 2280 ccgagcgcac caccgagttc tggaagcagt acgtggacgg cgaccagtgc gagagcaacc 2340 cctgcctgaa cggcggcagc tgcaaggacg acatcaacag ctacgagtgc tggtgcccct 2400 tcggcttcga gggcaagaac tgcgagctgg acgtgacctg caacatcaag aacggccgct 2460 gcgagcagtt ctgcaagaac agcgccgaca acaaggtggt gtgcagctgc accgagggct 2520 accgcctggc cgagaaccag aagagctgcg agcccgccgt gcccttcccc tgcggccgcg 2580 tgagcgtgag ccagaccagc aagctgaccc gcgccgaggc cgtgttcccc gacgtggact 2640 acgtgaacag caccgaggcc gagaccatcc tggacaacat cacccagagc acccagagct 2700 tcaacgactt cacccgcgtg gtgggcggcg aggacgccaa gcccggccag ttcccctggc 2760 aggtggtgct gaacggcaag gtggacgcct tctgcggcgg cagcatcgtg aacgagaagt ggatcgtgac cgccgcccac tgcgtggaga ccggcgtgaa gatcaccgtg gtggccggcg cgaggagacc gagcacaccg caacgtgatc cgcatcatcc cccaccacaa ctacaacgcc gccatcaaca agtacaacca cgacatcgcc ctgctggagc tggacgagcc cctggtgctg aacagctacg tgaccccat ctgcatcgcc gacaaggagt 3060 acaccaacat cttcctgag ttcggcagcg gctacgtgag cggctggggc cgcgtgttcc acaagggccg cagcgccctg gtgctgcagt acctgcgcgt gcccctggtg gaccgcgcca 3180 cctgcctgct gagcaccag ttcaccatct acaacaacat gttctgcgcc ggcttccacg agggcggccg cgacagctgc cagggcgaca gcggcggccc ccacgtgacc gaggtggagg 3300 gcaccagctt cctgaccggc atcatcagct ggggcgagga gtgcgccatg aagggcaagt 3360 acggcatcta caccaaggtg agccgctacg tgaactggat caaggagaag accaagctga cctaatgaaa gatggatttc caaggttaat tcattggaat tgaaaattaa cagccccccc cccccccccc ctgcagatct gagccgaatt cctgcagccc gggggatcag cctcgactgt 3540 gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct tgaccctgga 3600 aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc attgtctgag 3660 taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg aggattggga 3720 agacaatagc aggcatgctg gggatgcggt gggctctatg gcttctgagg cggaaagaac 3780 cagctgggga ccggtggatc tcgatagcag gcatgctggg gagagatcga tctgaggaac 3840 ccctagtgat ggagttggcc actccctctc tgcgcgctcg ctcgctcact gaggccgccc 3900 gggcaaagcc cgggcgtcgg gcgacctttg gtcgcccggc ctcagtgagc gagcgagcgc 3960 gcagagaggg agtggccaac cccccccccc cccccccccg gcgattctct tgtttgctcc 4020 agactctcag gcaatgacct gatagccttt gtagagacct ctcaaaaata gctaccctct 4080 ccggcatgaa tttatcagct agaacggttg aatatcatat tgatggtgat ttgactgtct 4140 ccggcctttc tcacccgttt gaatctttac ctacacatta ctcaggcatt gcatttaaaa 4200 tatatgaggg ttctaaaaat tttatcctt gcgttgaaat aaaggcttct cccgcaaaag 4260 tattacaggg tcataatgtt tttggtacaa ccgatttagc tttatgctct gaggctttat 4320 tgcttaattt tgctaattct ttgccttgcc tgtatgattt attggatgtt ggaatcgcct 4380 gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcatat ggtgcactct 4440 cagtacaatc tgctctgatg ccgcatagtt atatggtgca ctctcagtac aatctgctct 4500 gatgccgcat agttaagcca gccccgacac ccgccaacac agccagcccc gacacccgcc 4560 aacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc 4620 tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc 4680 gagacgaaag ggcctcgtga tacgcctatt tttataggtt aatgtcatga tataatggt 4740 ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt 4800 tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat aaatgctcaa 4860 tatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt 4920 tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctgggtgaa agtaaaagat 4980 gctgaagaatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag 5040 atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg 5100 ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata 5160 cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat 5220 ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc 5280 aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg 5340 ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac 5400 gacgagcgtg acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact 5460 ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa 5520 gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct 5580 ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc 5640 tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga 5700 cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac 5760 tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag 5820 atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg 5880 tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc 5940 tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag 6000 ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtc 6060 cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac 6120 ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc 6180 gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt 6240 tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt 6300 gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc 6360 ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt 6420 tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca 6480 ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt 6540 tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt 6600 attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag 6660 tcagtgagcg aggaagcgga agagcgccca atacgcaaac cgcctctccc cgcgcgttgg 6720 ccgattcatt aatg 6734 <210> 44 <211> 6661 <212> DNA <213> Artificial Sequence ​​​​​​​​​​​​tattgcgaca acggttaatt tgcgtgatgg acagactctt ttatcggtg gcctcactga 240 ttataaaac acttctcagg attctggcgt accgttcctg tctaaaatcc ctttaatcgg 300 cctcctgttt agctcccgct ctgattctaa cgaggaaagc acgttatacg tgctcgtcaa 360 agcaaccata gtacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc 420 gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt 480 cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcgggg ctccctttag 540 ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt 600 cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt 660 tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt 720 cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt 780 aacaaaaatt taacgcgaac tttaacaaaa tattaacgtt tacaatttaa atatttgctt 840 atacaatctt cctgtttttg gggcttttct gattatcaac cggggtacat atgattgaca 900 tgctagtttt acgattaccg ttcatcgccc tgcgcgctcg ctcgctcact gaggccgccc 960 gggcaaagcc cgggcgtcgg gcgacctttg gtcgcccggc ctcagtgagc gagcgagcgc 1020 gcagagaggg agtggaattc acgcgtggat ctgaattctg tacaggcgtg gtacggccgc 1080 gggtgaggcc acggtcttcc cgccaggttg actcgagcct cctgccagag ccactggccc 1140 cggaggccac cctagaccgc agctggcggc cgctggcacg agtgcagggt aactgagcca 1200 gggccgctgg cgcatttggc ctggccgagg ccaccccgcg cggccgctcc actgtgcccg 1260 aggctgtcct ggaggtgagg ccggcccaca gggaccctgc ccgtgcccgg gctccggtga 1320 gtcagggcgc gttatgcaag tgcccccggc gcctcccctt cggtctttca ccccgcgcgg 1380 ttacgaaagc gcgaccccct ccccccggcg ctataaagca gcggggcggc cgcggcgcgc 1440 tcgcctccct cgctccacgc gcgcccggac tcggcggcca ggcttgcgcg cggttcccct 1500 cccggtggtg agtggtaccc actgggagga tgttgagtaa gatggaaaac tactgatgac 1560 ccttgcagag acagagtatt aggacatgtt tgaacagggg ccgggcgatc agcaggtagc 1620 tctagaggat ccccgtctgt ctgcacattt cgtagagcga gtgttccgat accttaatct 1680 ccctaggcaa gttcatatt tgtgtaggtt acttattctc cttttgttga ctaagtcaat 1740 aatcagaatc agcaggttg gagtcagctt ggcagggatc agcagcctgg gttggaagga 1800 ggggtataa aagccccttc accaggagaa gccgtcacac agatccaca gctcctggct 1860 agaaagaggt aagggtttta gggatggttg gttggtgggg tattaatgtt taattacctg 1920 gagcacctgc ctgaaatcac tttttcag gttgggctag cccaccatgc agcgcgtgaa 1980 catgatcatg gccgagagcc ccggcctgat caccatctgc ctgctggggct acctgctgag 2040 cgccgagtgc accgtgttcc tggaccacga gaacgccaac aagatcctga accgccccaa 2100 gcgctacaac agcggcaagc tggagtt cgtgcaggc aacctggagc gcgagtgcat 2160 ggaggagaag tgcagcttcg aggaggcccg cgaggtgttc gagacaccg agcgcaccac 2220 cgagttctgg aagcagtacg tggacggcga ccagtgcgag agcaacccct gcctgaacgg 2280 cggcagctgc aaggacgaca tcacagcta cgagtgctgg tgcccttcg gcttcgaggg 2340 caagaactgc gagctggacg tgacctgcaa catcaagaac ggccgctgcg agcagttctg 2400 caagaacagc gccgacaaca aggtggtgtg cagctgcacc gagggctacc gcctggccga 2460 gaaccagaag agctgcgagc ccgccgtgcc cttcccctgc ggccgcgtga gcgtgagcca 2520 gaccagcaag ctgacccgcg ccgaggccgt gttccccgac gtggactacg tgaacagcac 2580 cgaggccgag accatcctgg acaacatcac ccagagcacc cagagcttca acgacttcac 2640 ccgcgtggtg ggcggcgagg acgccaagcc cggccagttc ccctggcagg tggtgctgaa 2700 cggcaaggtg gacgccttct gcggcggcag catcgtgaac gagagtgga tcgtgaccgc 2760 cgccactgc gtggagaccg gcgtgaagat caccgtggtg gccggcgagc acaacatcga 2820 ggagaccgag cacaccgagc agaagcgcaa cgtgatccgc atcatcccccc accacaacta 2880 caacgccgcc atcaacaagt acaaccagga catcgccctg ctggagctgg acgagcccct 2940 ggtgctgaac agctacgtga cccccatctg catcgccgac aaggatca ccaacatctt 3000 cctgaagttc ggcagcggct acgtgagcgg ctggggccgc gtgttccaca agggccgcag 3060 cgccctggtg ctgcagtacc tgcgcgtgcc cctggtggac cgcgccacct gcctgctgag 3120 caccaagttc accatctaca acaacatgtt ctgcgccggc ttccacgagg gcggccgcga 3180 cagctgccag ggcgacagcg gcggccccca cgtgaccgag gtggagggca ccagcttcct 3240 gaccggcatc atcagctggg gcgaggagtg cgccatgaag ggcaagtacg gcatctacac 3300 caaggtgagc cgctacgtga actggatcaa ggagaagacc aagctgacct aatgaaagat 3360 ggatttccaa ggttaattca ttggaattga aaattaacag cccccccccc cccccccctg 3420 cagatctgag ccgaattcct gcagcccggg ggatcagcct cgactgtgcc ttctagttgc 3480 cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg tgccactccc 3540 actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct 3600 attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga caatagcagg 3660 catgctgggg atgcggtggg ctctatggct tctgaggcgg aaagaaccag ctggggaccg 3720 gtggatctcg atagcaggca tgctggggag agatcgatct gaggaacccc tagtgatgga 3780 gttggccact ccctctctgc gcgctcgctc gctcactgag gccgccccggg caaagcccgg 3840 gcgtcgggcg acctttggtc gcccggcctc agtgagcgag cgagcgcgca gagagggagt 3900 ggccaacccc cccccccccc ccccccggcg attctcttgt ttgctccaga ctctcaggca 3960 atgacctgat agcctttgta gagacctctc aaaaatagct accctctccg gcatgaattt 4020 atcagctaga acggttgaat atcatattga tggtgatttg actgtctccg gcctttctca 4080 cccgtttgaa tctttaccta cacattactc aggcattgca tttaaaatat atgagggttc 4140 taaaatttt tatccttgcg ttgaaataaa ggcttctccc gcaaaagtat tacagggtca 4200 taatgttttt ggtacaaccg atttagcttt atgctctgag gctttattgc ttaattttgc 4260 taattctttg ccttgcctgt atgatttatt ggatgttgga atcgcctgat gcggtatttt 4320 ctccttacgc atctgtgcgg tatttcacac cgcatatggt gcactctcag tacaatctgc 4380 tctgatgccg catagttata tggtgcactc tcagtacaat ctgctctgat gccgcatagt 4440 taagccagcc ccgacacccg ccaacacagc cagccccgac acccgccaac acccgctgac 4500 gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc 4560 gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag acgaaagggc 4620 ctcgtgatac gcctattttt ataggttaat gtcatgataa taatggtttc ttagacgtca 4680 ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat 4740 tcaaatatgt atccgctcat gagacaataa ccctgataaa tgctcaataa tattgaaaaa 4800 ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt 4860 gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt 4920 tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt 4980 ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg 5040 tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga 5100 atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa 5160 gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga 5220 caacgatcgg aggaccgaag gagctaccg cttttttgca caacatgggg gatcatgtaa 5280 ctcgccttga tcgttgggaa ccggagctga atgaagccat accaacgac gagcgtgaca 5340 ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaact atttactggc gaactactta 5400 ctctagcttc ccggcacaa ttatagact ggatggaggc ggataagtt gcaggaccac 5460 ttctgcgctc gcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc 5520 gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtacgtag 5580 ttactacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga 5640 taggtgcctc actgattaag cattgtaac tgtcagacca agtttactca tatatacttt 5700 agattgattt aaacttcat ttttaattta aaaggatcta ggtgaagagatc cttttgata 5760 atctcatgac caaatccct taacgtgagt ttcgttcca ctgagcgtca gaccccgtag 5820 aaaagatcaa aggatctct tgagatcctt ttttctgcg cgtaatctgc tgcttgcaaa 5880 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcagagcta ccaactctt 5940 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc 6000 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa 6060 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa 6120 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc 6180 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa 6240 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa 6300 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg 6360 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc 6420 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg 6480 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg 6540 agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg 6600 aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat 6660 g 6661 <210> 45 <211> 6678 <212> DNA <213> Artificial sequence <220> <223> AAVsc-HS-CRE13-TTRenh-TTRm-MVM-hFIXcoPadua-bghpolyA <400> 45 agctggcgta atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg 60 aatggcgaat ggcgattccg ttgcaatggc tggcggtaat attgttctgg atattaccag 120 caaggccgat agtttgagtt cttctactca ggcaagtgat gttattacta atcaaagaag 180 tattgcgaca acggttaatt tgcgtgatgg acagactctt ttactcggtg gcctcactga 240 ttataaaaac acttctcagg attctggcgt accgttcctg tctaaaatcc ctttaatcgg 300 cctcctgttt agctcccgct ctgattctaa cgaggaaagc acgttatacg tgctcgtcaa 360 agcaaccata gtacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc 420 gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt 480 cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag 540 ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt 600 cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt 660 tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt 720 cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt 780 aacaaaaatt taacgcgaac tttaacaaaa tattaacgtt tacaatttaa atatttgctt 840 atacaatctt cctgtttttg gggcttttct gattatcaac cggggtacat atgattgaca 900 tgctagtttt acgattaccg ttcatcgccc tgcgcgctcg ctcgctcact gaggccgccc 960 gggcaaagcc cgggcgtcgg gcgacctttg gtcgcccggc ctcagtgagc gagcgagcgc 1020 gcagagaggg agtggaattc acgcgtggat ctgaattctg tacaggcgtg gtacggccgc 1080 ggaaatacct aaactagtca cactgttttg attcaattgg ctactgaagt tatagaatgt 1140 tgtttactct tctctccttt gtctactccc cagccaacaa aacaaccgac cttagctgtt 1200 ttgaaaataa atgaaaattc caacatgggt ttgaaataaa attgcatcat aaacaatcgg 1260 taggtgtttt tcaagtggt tcagggag tgscaggggg tagggaggcg accagggag 1320 ctgctaaaat atttcctgtc ctgaccaggg tgcgttctct ggagaatatt taacagggag 1380 ggttttaacg cttttaaaga tgttgaact aaagacaaa tattgaccag agggcaccac 1440 aacgctcctg aaagagagta aaatacatcc tttataaaat gaaaaactac ttggatgaat 1500 tattccaaaa ttcctgcaca gtggaccctc ggtaccact gggaggatgt tgagtagat 1560 ggaaaactac tgatgaccct tgcagagaca gagtattagg acatgtttga acaggggccg 1620 ggcgatcagc aggtagctct agaggatccc cgtctgtctg cacattcgt agagcgagtg 1680 ttccgatact ctaatctccc taggcaggt tcatatttgt gtaggttact tattctcctt 1740 ttgttgacta agtcaataat cagaatcagc agttttggag tcagcttggc agggatcagc 1800 agcctgggtt ggaaggagggg ggtataaag ccccttcacc aggaagcc gtcacacaga 1860 tccacaagct cctggctaga aagaggtaag ggtttaaggg atggttggtt ggtggggtat 1920 taatgttaa ttacctggag cacctgcctg aaatcacttt tttcaggtt gggctagccc 1980 accatgcagc gcgtgaacat gatcatggcc gagagccccg gcctgatcac catctgcctg 2040 ctgggctacc tgctgagcgc cgagtgcacc gtgttcctgg accacgagaa cgccaacaag 2100 atcctgaacc gccccaagcg ctacaacagc ggcaagctgg aggagttcgt gcagggcaac 2160 ctggagcgcg agtgcatgga ggagaagtgc agcttcgagg aggcccgcga ggtgttcgag 2220 aacaccgagc gcaccaccga gttctggaag cagtacgtgg acggcgacca gtgcgagagc 2280 aacccctgcc tgaacggcgg cagctgcaag gacgacatca acagctacga gtgctggtgc 2340 cccttcggct tcgagggcaa gaactgcgag ctggacgtga cctgcaacat caagaacggc 2400 cgctgcgagc agttctgcaa gaacagcgcc gacaacaagg tggtgtgcag ctgcaccgag 2460 ggctaccgcc tggccgagaa ccagaagagc tgcgagcccg ccgtgccctt cccctgcggc 2520 cgcgtgagcg tgagccagac cagcaagctg acccgcgccg aggccgtgtt ccccgacgtg 2580 gactacgtga acagcaccga ggccgagacc atcctggaca acatcaccca gagcacccag 2640 agcttcaacg acttcacccg cgtggtgggc ggcgaggacg ccaagcccgg ccagttcccc 2700 tggcaggtgg tgctgaacgg caaggtggac gccttctgcg gcggcagcat cgtgaacgag 2760 aagtggatcg tgaccgccgc ccactgcgtg gagaccggcg tgaagatcac cgtggtggcc 2820 ggcgagcaca acatcgagga gaccgagcac accgagcaga agcgcaacgt gatccgcatc 2880 atcccccacc acaactacaa cgccgccatc aacaagtaca accacgacat cgccctgctg 2940 gagctggacg agcccctggt gctgaacagc tacgtgaccc ccatctgcat cgccgacaag 3000 gagtacacca acatcttcct gaagttcggc agcggctacg tgagcggctg gggccgcgtg 3060 ttccacaagg gccgcagcgc cctggtgctg cagtacctgc gcgtgcccct ggtggaccgc 3120 gccacctgcc tgctgagcac caagttcacc atctacaaca acatgttctg cgccggcttc 3180 cacgagggcg gccgcgacag ctgccagggc gacagcggcg gcccccacgt gaccgaggtg 3240 gagggcacca gcttcctgac cggcatcatc agctggggcg aggagtgcgc catgaagggc 3300 aagtacggca tctacaccaa ggtgagccgc tacgtgaact ggatcaagga gaagaccaag 3360 ctgacctaat gaaagatgga tttccaaggt taattcattg gaattgaaaa ttaacagccc 3420 cccccccccc ccccctgcag atctgagccg aattcctgca gcccggggga tcagcctcga 3480 ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc 3540 tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc 3600 tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt 3660 gggaagacaa tagcaggcat gctggggatg cggtgggctc tatggcttct gaggcggaaa 3720 gaaccagctg gggaccggtg gatctcgata gcaggcatgc tggggagaga tcgatctgag 3780 gaacccctag tgatggagtt ggccactccc tctctgcgcg ctcgctcgct cactgaggcc 3840 gcccgggcaa agcccgggcg tcgggcgacc tttggtcgcc cggcctcagt gagcgagcga 3900 gcgcgcagag agggagtggc caaccccccc cccccccccc cccggcgatt ctcttgtttg 3960 ctccagactc tcaggcaatg acctgatagc ctttgtagag acctctcaaa aatagctacc 4020 ctctccggca tgaatttatc agctagaacg gttgaatatc atattgatgg tgatttgact 4080 gtctccggcc tttctcaccc gtttgaatct ttacctacac attactcagg cattgcattt 4140 aaaatatatg agggttctaa aaatttttat ccttgcgttg aaataaaggc ttctcccgca 4200 aaagtattac agggtcataa tgtttttggt acaaccgatt tagctttatg ctctgaggct 4260 ttattgctta attttgctaa ttctttgcct tgcctgtatg atttattgga tgttggaatc 4320 gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc atatggtgca 4380 ctctcagtac aatctgctct gatgccgcat agttatatgg tgcactctca gtacaatctg 4440 ctctgatgcc ccatagttaa gccagccccg acacccgcca acacagccag ccccgacacc 4500 cgccaacacc cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac 4560 aagctgtgac cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac 4620 gcgcgagacg aaagggcctc gtgatacgcc tatttttata ggttaatgtc atgataataa 4680 tggtttctta gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt 4740 tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc 4800 tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc 4860 cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa 4920 agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg 4980 taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt 5040 tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg 5100 catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac 5160 ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc 5220 ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa 5280 catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc 5340 aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt 5400 aactggcgaa ctacttactc tagcttccg gcaacaatta atagactgga tggaggcgga 5460 taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa 5520 atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa 5580 gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa 5640 tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt 5700 ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt 5760 gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg 5820 agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt 5880 aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca 5940 agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac 6000 tgtccttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac 6060 atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct 6120 taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg 6180 gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga gatacctaca 6240 gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt 6300 aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa...

Claims

1. A nucleic acid regulatory element for enhancing liver-specific gene expression, comprising a sequence defined by SEQ ID NO:

6.

2. The nucleic acid regulatory element according to claim 1, having a maximum length of 600 nucleotides.

3. A nucleic acid expression cassette comprising at least one nucleic acid regulatory element according to claim 1 or 2, said nucleic acid regulatory element being operably linked to a promoter and a transgene, wherein said transgene encodes a therapeutic protein or an immunogenic protein.

4. The nucleic acid expression cassette according to claim 3, further comprising at least one liver-specific regulatory element different from the nucleic acid regulatory element according to claim 1 or 2.

5. The nucleic acid expression cassette of claim 4, wherein the at least one liver-specific regulatory element is defined by SEQ ID NO:

22.

6. The nucleic acid expression cassette according to claim 4 or 5, comprising at least one nucleic acid regulatory element defined by SEQ ID NO:

22.

7. The nucleic acid expression cassette of claim 3, wherein the transgene encodes coagulation factor IX (FIX) or coagulation factor VIII (FVIII).

8. The nucleic acid expression cassette of claim 7, wherein the transgene encodes codon-optimized FIX and consists of the nucleic acid sequence defined by SEQ ID NO: 25; or wherein the transgene encodes codon-optimized FVIII and consists of the nucleic acid sequence defined by SEQ ID NO:

26.

9. The nucleic acid expression cassette of claim 3, wherein the promoter is a liver-specific promoter.

10. The nucleic acid expression cassette of claim 9, wherein the promoter is derived from the transthyretin (TTR) promoter.

11. The nucleic acid expression cassette according to claim 10, comprising a combination of TTRe and TTRm nucleic acids as defined in SEQ ID NO:

28.

12. The nucleic acid expression cassette according to claim 5, further comprising - Minute virus of mice (MVM) intron; and / or - A transcription termination signal derived from the bovine growth hormone polyadenylation signal (BGHpA).

13. A vector comprising the nucleic acid regulatory element according to claim 1 or 2 or the nucleic acid expression cassette according to any one of claims 3 to 12.

14. The vector according to claim 13, wherein - the vector is a viral vector; and / or —The vector is defined by SEQ ID NO:

38.

15. A pharmaceutical composition comprising the nucleic acid expression cassette according to any one of claims 3 to 12 or the vector according to claim 13 or 14, and a pharmaceutically acceptable carrier.

16. Use of a vector according to claim 13 or 14 in the preparation of a medicament for treating hemophilia B, wherein the vector comprises a nucleic acid expression cassette according to any one of claims 3 to 12 and wherein the transgene encodes FIX.

17. Use of the vector according to claim 13 or 14 in the preparation of a medicament, wherein the medicament is for treating hemophilia A, wherein the vector comprises a nucleic acid expression cassette according to any one of claims 3 to 12 and wherein the transgene encodes FVIII.

18. An in vitro or ex vivo method for expressing a transgene product in hepatocytes, comprising: - introducing the nucleic acid expression cassette according to any one of claims 3 to 12 or the vector according to claim 13 or 14 into the hepatocytes; - expressing said transgene product in said hepatocytes.

Images