Liver-specific inducible promoters and methods of use thereof

文档序号：1803708 发布日期：2021-11-05 浏览：25次中文

阅读说明：本技术 肝脏特异性诱导型启动子及其使用方法 (Liver-specific inducible promoters and methods of use thereof ) 是由迈克尔·罗伯茨格雷厄姆·怀特斯德安妮·布雷厄于 2020-01-20 设计创作，主要内容包括：本发明涉及诱导型调控元件、启动子和载体,尤其是基因治疗载体,及其使用方法。(The present invention relates to inducible regulatory elements, promoters and vectors, particularly gene therapy vectors, and methods of use thereof.)

1. A gene therapy vector comprising an expression cassette comprising a synthetic liver-specific inducible promoter operably linked to a gene, said synthetic liver-specific inducible promoter comprising a cis-regulatory element (CRE) capable of being bound and activated by the heterodimer of CAR and RXR.

2. A gene therapy vector according to claim 1, wherein CRE capable of being bound and activated by CAR-RXR heterodimer comprises or consists of SEQ ID NO 1 or SEQ ID NO 2, or a functional variant of SEQ ID NO 1 or SEQ ID NO 2.

3. The gene therapy vector according to claim 1 or 2, wherein SEQ ID NO 1 or a functional variant of SEQ ID NO 2 comprises the NR1 motif from PBREM, preferably the sequence TGTACTTTCCTGACCN (SEQ ID NO:20) or a sequence differing from the sequence TGTACTTTCCTGACCN (SEQ ID NO:20) at NO more than 2, preferably NO more than 1, nucleotide positions.

4. Gene therapy vector according to any of the preceding claims, wherein the functional variant of SEQ ID No. 1 or SEQ ID No. 2 comprises one of the following sequences:

-[TGTACTTTCCTGACCN-S-]_n(SEQ ID NO:29)；

-[CTGTACTTTCCTGACCN-S-]_n(SEQ ID NO: 30); and

-[NCTGTACTTTCCTGACCNTG-S-]_n(SEQ ID NO:31),

wherein S is an optional spacer and n is 1 to 5, optionally 2 to 4, preferably 3.

5. Gene therapy vector according to any of the preceding claims, wherein the functional variant of SEQ ID No. 1 or SEQ ID No. 2 comprises one of the following sequences:

-TGTACTTTCCTGACCN-S-TGTACTTTCCTGACCN(SEQ ID NO:32)；

-TGTACTTTCCTGACCN-S-TGTACTTTCCTGACCN-S-TGTACTTTCCTGACCN(SEQ ID NO:33)；

-CTGTACTTTCCTGACCN-S-CTGTACTTTCCTGACCN(SEQ ID NO:34)；

-CTGTACTTTCCTGACCN-S-CTGTACTTTCCTGACCN-S-CTGTACTTTCCTGACCN (SEQ ID NO: 35); and

-NCTGTACTTTCCTGACCNTG-S-NCTGTACTTTCCTGACCNTG (SEQ ID NO: 36); and

-NCTGTACTTTCCTGACCNTG-S-NCTGTACTTTCCTGACCNTG-S-NCTGTACTTTCCTGACCNTG(SEQ ID NO:37),

wherein S is an optional spacer.

6. Gene therapy vector according to any of the preceding claims, wherein the functional variant of SEQ ID No. 1 or SEQ ID No. 2 comprises one of the following sequences:

-TCTGTACTTTCCTGACCTTG-S-TCTGTACTTTCCTGACCTTG-S-TCTGTACTTTCCTGACCTTG (SEQ ID NO: 38); or

-ACTGTACTTTCCTGACCCTG-S-ACTGTACTTTCCTGACCCTG-S-ACTGTACTTTCCTGACCCTG(SEQ ID NO:39)，

Wherein S is an optional spacer.

7. Gene therapy vector according to any one of the preceding claims, wherein the functional variant of SEQ ID No. 1 or SEQ ID No. 2 comprises a sequence which is at least 60% identical to SEQ ID No. 1 or SEQ ID No. 2, preferably a sequence which is at least 65%, 75%, 80%, 85%, 90%, 95% or 99% identical to SEQ ID No. 1 or SEQ ID No. 2.

8. Gene therapy vector according to any one of the preceding claims, wherein the functional variant of SEQ ID No. 1 or SEQ ID No. 2 comprises a sequence which is at least 90% identical, preferably at least 95% identical, more preferably identical, to SEQ ID No. 1 or SEQ ID No. 2 in the region spanning nucleotides 3 to 18 (preferably the region spanning nucleotides 1 to 20) and at least 70%, 80%, 85%, 90%, 95% or 99% identical to the rest of SEQ ID No. 1 or SEQ ID No. 2.

9. The gene therapy vector according to any one of the preceding claims, wherein the functional variant of SEQ ID NO 1 or SEQ ID NO 2 comprises the sequence:

NCTGTACTTTCCTGACCNTGNNNNNGTGNCANCATNNACTTNCCTGANNCN (SEQ ID NO:41), or a sequence at least 90%, preferably 95%, more preferably 99% identical thereto.

10. The gene therapy vector according to any of the preceding claims, comprising a plurality of CREs, each CRE capable of being bound and activated by a heterodimer of CAR and RXR.

11. The gene therapy vector according to any of the preceding claims, comprising 2 or 3 CREs, each CRE being capable of being bound and activated by a heterodimer of CAR and RXR.

12. Gene therapy vector according to any of the preceding claims, comprising a synthetic liver-specific inducible promoter comprising or consisting of a cis-regulatory module (CRM) comprising or consisting of one of the following sequences:

-NCTGTACTTTCCTGACCNTGNNNNNGTGNCANCATNNACTTNCCTGANNCN-S-NCTGTACTTTCCTGACCNTGNNNNNGTGNCANCATNNACTTNCCTGANNCN (SEQ ID NO: 42); and

-NCTGTACTTTCCTGACCNTGNNNNNGTGNCANCATNNACTTNCCTGANNCN-S-NCTGTACTTTCCTGACCNTGNNNNNGTGNCANCATNNACTTNCCTGANNCN-S-NCTGTACTTTCCTGACCNTGNNNNNGTGNCANCATNNACTTNCCTGANNCN(SEQ ID NO:43),

wherein S is an optional spacer.

13. Gene therapy vector according to any one of the preceding claims, comprising a synthetic liver-specific inducible promoter comprising CRM comprising or consisting of one of the following sequences:

-TCTGTACTTTCCTGACCTTGGCACAGTGCCACCATCAACTTGCCTGACACC-S-TCTGTACTTTCCTGACCTTGGCACAGTGCCACCATCAACTTGCCTGACACC(SEQ ID NO:44)；

-TCTGTACTTTCCTGACCTTGGCACAGTGCCACCATCAACTTGCCTGACACC-S-TCTGTACTTTCCTGACCTTGGCACAGTGCCACCATCAACTTGCCTGACACC-S-TCTGTACTTTCCTGACCTTGGCACAGTGCCACCATCAACTTGCCTGACACC(SEQ ID NO:45)；

-ACTGTACTTTCCTGACCCTGAAGAGGTGGCAGCATGGACTTTCCTGAACCA-S-ACTGTACTTTCCTGACCCTGAAGAGGTGGCAGCATGGACTTTCCTGAACCA-S-ACTGTACTTTCCTGACCCTGAAGAGGTGGCAGCATGGACTTTCCTGAACCA (SEQ ID NO: 46); and

-ACTGTACTTTCCTGACCCTGAAGAGGTGGCAGCATGGACTTTCCTGAACCA-S-ACTGTACTTTCCTGACCCTGAAGAGGTGGCAGCATGGACTTTCCTGAACCA(SEQ ID NO:47)，

wherein S is an optional spacer.

14. The gene therapy vector of any one of the preceding claims, which is substantially free of nucleic acid sequences that result in constitutive expression or expression in non-liver cells.

15. The gene therapy vector according to any of the preceding claims, comprising CRE capable of being bound and activated by heterodimers of CAR and RXR, said heterodimers operably linked to a minimal or proximal promoter.

16. A gene therapy vector according to claim 15, wherein the minimal promoter is HSV thymidine kinase minimal promoter (MinTK), CMV minimal promoter (CMVmp) or SV40 minimal promoter (SV40 mp).

17. Gene therapy vector according to any one of the preceding claims, comprising a sequence according to any one of SEQ ID NOs 7 to 18 and 59 to 71, or a functional variant of any one thereof.

18. Gene therapy vector according to any one of the preceding claims, comprising a sequence according to SEQ ID No. 7, SEQ ID No. 10, SEQ ID No. 11, SEQ ID No. 12, SEQ ID No. 68, SEQ ID No. 69, SEQ ID No. 70 or SEQ ID No. 71, or a functional variant of any of them.

19. The gene therapy vector according to any one of the preceding claims, wherein the synthetic liver-specific expression cassette comprises one or more, preferably all, sequences providing or encoding a ribosome binding site, an initiation codon, a stop codon, a transcription termination sequence, a nucleic acid encoding a post-transcriptional regulatory element and/or a polyA element.

20. The gene therapy vector of any one of the preceding claims, wherein said gene encodes a protein or RNA.

21. Gene therapy vector according to any one of the preceding claims, wherein said gene encodes a therapeutic expression product, preferably a therapeutic protein suitable for the treatment of a disease or condition associated with abnormal gene expression in the liver.

22. The gene therapy vector according to any one of the preceding claims, wherein the gene encodes a site-specific nuclease, such as a meganuclease, a Zinc Finger Nuclease (ZFN), a transcription activator-like effector-based nuclease (TALEN), or an aggregated regularly-spaced short palindromic repeat system (CRISPR-Cas).

23. The gene therapy vector according to any one of the preceding claims, which is a viral vector, such as a retroviral, lentiviral, adenoviral or adeno-associated viral (AAV) vector.

24. The gene therapy vector of any one of the preceding claims, which is a plasmid.

25. An expression cassette comprising a synthetic liver-specific inducible promoter operably linked to a gene, the synthetic liver-specific inducible promoter comprising CRE capable of being bound and activated by a heterodimer of CAR and RXR.

26. The expression cassette of claim 25, wherein the gene is not a reporter gene.

27. The expression cassette of claim 25 or 26, wherein the gene encodes a therapeutic expression product.

28. A synthetic liver-specific inducible promoter comprising a sequence according to any one of SEQ ID NOs:7 to 18 and 59 to 71, or a functional variant of any one thereof.

29. A recombinant virion comprising a gene therapy vector according to any one of claims 1 to 24, an expression cassette according to any one of claims 25 to 27, or a promoter according to claim 28.

30. A pharmaceutical composition comprising a gene therapy vector according to any one of claims 1 to 24, an expression cassette according to any one of claims 25 to 27, a promoter according to claim 28, or a virion according to claim 29.

31. A cell comprising a gene therapy vector according to any one of claims 1 to 24, an expression cassette according to any one of claims 25 to 27, a promoter according to claim 28, or a virion according to claim 29.

32. The cell of claim 31, which is a hepatocyte, optionally a hepatocyte in cell culture or a hepatocyte present in the liver of a subject.

33. Use of a gene therapy vector according to any one of claims 1 to 24, an expression cassette according to any one of claims 25 to 27, a promoter according to claim 28, a virosome according to claim 29 or a pharmaceutical composition according to claim 30 for the treatment of a disease, preferably a disease associated with abnormal gene expression in the liver.

34. A method for producing an expression product, preferably a therapeutic expression product, in a cell, preferably a hepatocyte, the method comprising:

-providing a cell comprising an expression cassette comprising a synthetic liver-specific inducible promoter operably linked to a gene, the synthetic liver-specific inducible promoter comprising a cis-regulatory element (CRE) capable of being bound and activated by the heterodimer of CAR and RXR; and

-administering to said cell an inducing agent capable of inducing expression of an expression product of a gene operably linked to an inducible promoter in said expression cassette.

35. The method of claim 34, comprising maintaining the hepatocyte under suitable conditions to express an expression product from a gene.

36. The method of claim 34 or 35, wherein the inducing agent comprises one or more agents selected from the group consisting of:

phenobarbital (PB); flavonoids, such as flavones, chrysin, baicalein or galangin; 1, 4-bis [2- (3, 5-dichloropyridinyloxy) ] benzene (TCPOBOP); 6- (4-chlorophenyl) imidazo [2,1-b ] [1,3] thiazole-5-carbaldehyde-O- (3, 4-dichlorobenzyl) oxime (CITCO); acetaminophen; buprenorphine; phenytoin; carbamazepine; valproic acid; artemisinin and its derivatives; chlorpromazine; efavirenz; nevirapine; ropinirostrin; etravirine; diazepam; cyclophosphamide; ifosfamide; cerivastatin; simvastatin; lovastatin; substituted sulfonamides; thiazolidin-4-one; estradiol; estrone and analogs thereof; 17 α -ethynyl-3; 17 β -estradiol (EE 2); dehydroepiandrosterone (DHEA); 5 β -pregnane-3, 20-dione; diethylstilbestrol; a ginkgo extract; galangin; chrysin; baicalein; diallyl sulfide; ellagic acid; resveratrol; squalane-1; bilobalide; triclocarban; triclosan; dichlorodiphenyltrichloroethane (DDT); a dieldrin agent; methoxy chloride; metoflutine; permethrin; pyrethrin; sulfoxaflor; diethylhexyl phthalate (DEHP); cyproconazole; fluconazole; propiconazole; FL 81; tri-p-methylphenyl phosphate (TMPP); UM104 and UM 145.

37. The method of any one of claims 34 to 36, wherein the inducing agent comprises one or more agents selected from the group consisting of:

phenobarbital (PB); flavonoids such as flavone, chrysin, baicalein or galangin; acetaminophen; buprenorphine; phenobarbital; phenytoin; carbamazepine; valproic acid; artemisinin and its derivatives; chlorpromazine; efavirenz; nevirapine; ropinirostrin; etravirine; diazepam; cyclophosphamide; ifosfamide; cerivastatin; simvastatin; lovastatin; substituted sulfonamides; and thiazolidin-4-one.

38. The method of any one of claims 34 to 37, comprising discontinuing administration of the inducing agent.

39. The method of any one of claims 34 to 38, comprising varying the concentration of the inducing agent administered to the cells over time.

40. The method of any one of claims 34 to 39, comprising the step of introducing the synthetic liver-specific expression cassette into the cell.

41. A method of expressing a therapeutic transgene in a hepatocyte of a subject, the method comprising introducing the gene therapy vector of any one of claims 1 to 24 into the hepatocyte, and then administering an inducer to the cell.

42. A method of gene therapy of a subject, preferably a human, in need thereof, the method comprising:

-introducing a gene therapy vector according to any one of claims 1 to 24, an expression cassette according to any one of claims 25 to 27, a virosome according to claim 29 or a pharmaceutical composition according to claim 30 comprising a gene encoding a therapeutic product into the liver of a subject; and

-administering an inducing agent to the subject such that a therapeutically effective amount of the therapeutic product is expressed in the subject.

43. The method of claim 42, comprising varying the amount of the inducing agent administered to the subject over time to modulate the dose of the therapeutic gene product provided in the subject.

44. The method of claim 42 or 43, comprising the steps of:

-determining the amount of the therapeutic product expressed in the subject or assessing the response of the subject to the therapeutic product;

a) increasing the amount of inducer administered to the subject when a higher amount of therapeutic product is required in the subject, or

b) When a lower amount of the therapeutic product is needed in the subject, the amount of the inducing agent administered to the subject is reduced.

45. A method according to any one of claims 42 to 44, comprising altering the inducing agent to alter the amount of the therapeutic product in the subject.

46. Use of a gene therapy vector according to any one of claims 1 to 24, an expression cassette according to any one of claims 25 to 27, a promoter according to claim 28, a virosome according to claim 29 for the preparation of a pharmaceutical composition for the treatment of any condition or disease described herein.

47. In another aspect, the present invention provides a method of producing an expression product, the method comprising the steps of:

a) providing a population of eukaryotic cells, preferably animal cells, more preferably mammalian cells, more preferably hepatocytes, comprising an expression cassette comprising a synthetic liver-specific inducible promoter operably linked to a gene, the synthetic liver-specific inducible promoter comprising a cis-regulatory element (CRE) capable of being bound and activated by a heterodimer of CAR and RXR;

b) culturing said population of cells; and

c) administering to the cell an inducing agent capable of inducing expression of an expression product of a gene operably linked to an inducible promoter in the expression cassette; and

d) recovering the expression product.

Technical Field

The present invention relates to liver-specific inducible promoters and vectors, particularly gene therapy vectors, comprising them, and methods of their use.

Background

The following discussion is provided to aid the reader in understanding the present disclosure and does not constitute any admission as to the content or relevance of the prior art.

Metabolism of liver microsomal cytochrome P450 enzymes (CYPs) plays an important role in detoxification of exogenous chemicals such as drugs and environmental pollutants. Gene transcription induced by exposure to xenogenic chemicals is a characteristic of CYPs, which increases the defense of organisms against toxicity and carcinogenicity. Based on the discovery of PB induction of the CYP2B gene, PB has been the prototype for a large group of structurally and functionally diverse xenogeneic chemicals that induce the CYP2B gene. The PB response enhancer module (PBREM) was identified as a 51bp element in mouse Cyp2B10 and rat Cyp2B1, and almost identical DNA elements were identified in rat Cyp2B 2. The PBREM sequence, which is related to CYP2B6, is also found in humans. The nuclear receptor heterodimer CAR-RXR has been identified as a transactivator of PBREM. (see Negishi et al, "The reproduced Nuclear Receptor CAR responses to phenolic in Activating The Human CYP2B6 Gene"; J.biol.chem 1999,274: 6043-.

In many fields, including gene therapy, it is desirable to provide regulatory nucleic acid sequences capable of driving gene expression to produce a protein or nucleic acid expression product in a desired cell, tissue or organ.

Expression in the liver is of particular interest because it is involved in a wide range of basic functions in the body, including synthesis of many proteins involved in metabolism, hemostasis, and prevention of infection. Given that many diseases are associated with disruption of gene expression in the liver, there is great interest in developing gene therapy strategies that allow for expression of transgenes in the liver to produce therapeutic expression products. Examples of liver diseases associated with aberrant gene expression include hemophilia (including hemophilia a or B), familial hypercholesterolemia, ornithine transcarbamylase deficiency, alpha-antitrypsin deficiency, hepatitis virus infection, non-viral hepatitis, liver cancer, and various other liver diseases such as non-alcoholic fatty liver disease (NAFLD) and alcohol-related liver disease (ARLD).

One significant challenge in treating liver disease using gene therapy is the ability to provide liver-specific (also known as liver-specific) therapeutic gene expression. It is known to target mammalian hepatocytes by injection of DNA or viral vectors into the liver parenchyma, hepatic artery or portal vein. It has been reported that adenovirus vectors are directed mainly against the liver of mice. However, they can also infect other tissues, particularly the lung and skeletal muscle, leading to "off-target" effects. Certain forms of adeno-associated viral vectors (AAV) or lentiviral vectors preferentially transduce hepatocytes, but off-target effects can also occur.

Therefore, there is a need to provide systems for modulating gene expression in a liver-specific manner. Ideally, such systems are highly specific to the liver (thereby avoiding or minimizing off-target expression in non-target tissues) and are also powerful, i.e. they drive high expression levels in the liver. The use of cis-acting regulatory elements has been proposed to provide specificity and activity. Typically, this involves cis-regulatory enhancer sequences, i.e. nucleic acid sequences that increase promoter activity in cis-action. Enhancers are generally active, regardless of their orientation, and in some cases they may function at a distance of several kilobases from a promoter, although they generally function closer to a promoter.

It is also desirable to provide an induction system for gene expression so that gene expression can be induced as desired. Inducibility means that expression of the expression product of a therapeutic gene can be induced when desired. Furthermore, if induction is dose-dependent, the expression level of the therapeutic gene expression product can be modulated by modulating the amount of inducer administered.

Therefore, regulatory sequences are required in many cases to control gene expression, especially in therapeutic gene expression in gene therapy and the like. In particular, there is a need to provide regulatory sequences for inducible gene expression. Regulatory sequences that provide inducible gene expression in the liver are of particular interest.

Disclosure of Invention

In a first aspect, the invention provides a gene therapy vector comprising an expression cassette comprising a synthetic liver-specific inducible promoter operably linked to a gene, the synthetic liver-specific inducible promoter comprising a cis-regulatory element (CRE) capable of being bound and activated by the heterodimer of CAR and RXR.

Constitutive Androstane Receptors (CARs) are members of the nuclear receptor superfamily and are key regulators of xenobiotic and endophytic metabolism. Unlike most nuclear receptors, CARs are constitutively active in the absence of ligand and are regulated by agonists and inverse agonists. Activation results in the transfer of the CAR from the cytoplasm into the nucleus where the protein can bind to specific DNA sites (called response elements). Binding occurs both as a monomer and with Retinoid X Receptor (RXR), resulting in activation or inhibition of transcription of target genes.

Retinoid X Receptors (RXRs) are members of the nuclear receptor superfamily and are activated by 9-cis retinoic acid and 9-cis-13, 14-dihydro-retinoic acid.

When the CAR is activated (either directly or indirectly), it translocates to the nucleus where the CAR and RXR form a heterodimer (referred to herein as a "CAR-RXR heterodimer" or "CAR-RXR"), which can bind to an activator gene comprising an appropriate target sequence.

As described above, CAR-RXR binds and induces target genes through the so-called PB response enhancer module (PBREM). Thus, in some preferred embodiments of the invention, CRE capable of being bound and activated by CAR-RXR heterodimers comprises PBREM elements or functional variants thereof.

The mouse PBREM sequence is shown in SEQ ID NO 1. The human PBREM sequence is shown in SEQ ID NO. 2. In some embodiments of the invention, the CRE capable of being bound and activated by CAR-RXR heterodimers comprises or consists of SEQ ID NO 1 or SEQ ID NO 2, or a functional variant of SEQ ID NO 1 or SEQ ID NO 2. Thus, in some embodiments, CRE capable of being bound and activated by CAR-RXR heterodimers comprises or consists of a PBREM element or functional variant thereof.

The sequences and alignments of mouse and human PBREM elements are shown below, with the so-called NR1 motif underlined, the NH1 motif in italics, and the NR2 motif in bold (SEQ ID NO shown in parentheses):

PBREM is a 51-bp DNA inducible enhancer consisting of two nuclear receptor DR4 motifs (NR1 and NR2) flanking the NF1 binding site. From this alignment, it can be seen that the NR1 motif has very high sequence conservation between mouse and human PBREM. The 16-bp NR1 motif of human PBREM differs from the mouse NR1 motif by only 1 base, making NR1 the most conserved sequence between human and mouse PBREM elements. The NR1 sequence is proposed in the literature as a binding site for the CAR-RXR heterodimer, with the remaining sequences being partially or fully redundant. Triple repeats of NR1 were demonstrated to be maintained and induced in Sueyoshi, et al (j.bio.chem.vol.274, 10, pp.6043-6046,1999). However, it is believed that moieties other than NR1 may play a role in making PBREM elements more specific and inducible (e.g., reducing background, constitutive expression and allowing a greater degree of induction).

Thus, CRE capable of being bound and activated by CAR-RXR heterodimers suitably comprises at least one NR1 motif. Suitably, the NR1 motif comprises or consists of the sequence TGTACT-X-TGACC [ C/T ] (SEQ ID NO: #), wherein X represents any sequence of 3-6 nucleotides in length, preferably 4-5 nucleotides in length, suitably 4 nucleotides in length. In some preferred embodiments, X comprises or consists of the sequence TTCC, and suitably comprises or consists of the sequence TTCC or TTTCC. When a nucleotide is shown in square brackets, it indicates that one of the nucleotides indicated in square brackets is present at that position.

In some embodiments, the NR1 motif comprises or consists of sequence TGTACTTTCCTGACCN (SEQ ID NO:20) (e.g., TGTACTTTCCTGACCT (SEQ ID NO:3) or TGTACTTTCCTGACCC (SEQ ID NO:4)) or a sequence that differs from sequence TGTACTTTCCTGACCN (SEQ ID NO:20) at NO more than 2, preferably NO more than 1, nucleotide positions. Preferably, the functional variant of the PBREM element comprises sequence CTGTACTTTCCTGACCN (SEQ ID NO:21) (e.g., CTGTACTTTCCTGACC [ T/C ] (SEQ ID NO:22), i.e., CTGTACTTTCCTGACCT (SEQ ID NO:23) or CTGTACTTTCCTGACCC (SEQ ID NO:24)) or a sequence that differs from sequence SEQ ID NO:21 at NO more than 2, preferably NO more than 1, nucleotide positions; this sequence includes the conserved C in human and mouse PBREM located 5' to the NR1 sequence, as shown above. Suitably, a functional variant of the PBREM sequence comprises sequence NCTGTACTTTCCTGACCNTG (SEQ ID NO:25) (e.g., [ T/A ] CTGTACTTTCCTGACC [ C/T ] TG (SEQ ID NO:26), TCTGTACTTTCCTGACCTTG (SEQ ID NO:27) or ACTGTACTTTCCTGACCCTG (SEQ ID NO:28)), or a sequence that differs from sequence SEQ ID NO:25 at NO more than 2, preferably NO more than 1, nucleotide positions; as mentioned above, this sequence includes two nucleotides located 5 'and 3' to the human and mouse PBREM NR1 sequences. When N is present in a nucleic acid sequence herein, it represents any nucleotide. When a nucleotide is shown in square brackets, it indicates that one of the nucleotides indicated in square brackets is present at that position.

In some embodiments, the functional variant of a PBREM element suitably comprises two or more, suitably three or more, operably linked sequences containing the NR1 motif.

Thus, in some embodiments, CREs capable of being bound and activated by CAR-RXR heterodimers suitably comprise two or more, suitably three or more, operably linked NR1 motifs. In some embodiments, NR1 motifs may be provided adjacent to each other (e.g., in tandem), and they may be immediately adjacent to each other or separated by a spacer. Thus, CRE capable of being bound and activated by CAR-RXR heterodimers suitably comprises the general structure NR1-S-NR1, wherein NR1 represents any of the NR1 motifs discussed herein and S represents an optional spacer. When a spacer is present, it may have any sequence and have any suitable length, for example 2 to 50, 3 to 40, 4 to 30, 5 to 20, 6 to 10, 7 to 9 or 8 nucleotides in length.

In some embodiments of the invention, CRE capable of being bound and activated by CAR-RXR heterodimers comprises or consists of a functional variant of PBREM sequence comprising one of the following sequences:

-[TGTACTTTCCTGACCN-S-]_n(SEQ ID NO:29)；

-[CTGTACTTTCCTGACCN-S-]_n(SEQ ID NO:30)；

-[NCTGTACTTTCCTGACCNTG-S-]_n(SEQ ID NO:31),

wherein S is an optional spacer and n is 1 to 5, optionally 2 to 4, suitably 3. Thus, a functional variant of a PBREM sequence may comprise a multimer comprising the NR1 motif sequence. Various preferred NR1 motifs are discussed above, which of course may be used in this embodiment. In some embodiments, n is 1 to 10, 1 to 6, or 2 to 4. In some embodiments of the invention, n is 3.

In some preferred embodiments, a spacer is present between adjacent NR1 motif-containing sequences. When present, the spacer may be any suitable length, for example, 2 to 50, 3 to 40, 4 to 30, 5 to 20, 6 to 10, 7 to 9, or 8 nucleotides in length.

-TGTACTTTCCTGACCN-S-TGTACTTTCCTGACCN(SEQ ID NO:32)；

-TGTACTTTCCTGACCN-S-TGTACTTTCCTGACCN-S-TGTACTTTCCTGACCN(SEQ ID NO:33)；

-CTGTACTTTCCTGACCN-S-CTGTACTTTCCTGACCN(SEQ ID NO:34)；

-CTGTACTTTCCTGACCN-S-CTGTACTTTCCTGACCN-S-CTGTACTTTCCTGACCN(SEQ ID NO:35)；

-NCTGTACTTTCCTGACCNTG-S-NCTGTACTTTCCTGACCNTG (SEQ ID NO: 36); and

-NCTGTACTTTCCTGACCNTG-S-NCTGTACTTTCCTGACCNTG-S-NCTGTACTTTCCTGACCNTG(SEQ ID NO:37),

wherein S is an optional spacer. When present, the spacer may be any suitable length, for example 2 to 50, 3 to 40, 4 to 30, 5 to 20, 6 to 10, 7 to 9 or 8 nucleotides in length. Various preferred NR1 motifs are discussed above, which of course may be used in this embodiment.

In some embodiments of the invention, CRE capable of being bound and activated by CAR-RXR heterodimers comprises or consists of a functional variant of PBREM sequence suitably comprising one of the following sequences:

-TCTGTACTTTCCTGACCTTG-S-TCTGTACTTTCCTGACCTTG-S-TCTGTACTTTCCTGACCTTG (SEQ ID NO: 38); or

-ACTGTACTTTCCTGACCCTG-S-ACTGTACTTTCCTGACCCTG-S-ACTGTACTTTCCTGACCCTG(SEQ ID NO:39),

Wherein S is an optional spacer, as described above.

In some cases, the spacer may have the sequence GATCGATC (SEQ ID NO:40), but any other suitable spacer sequence may be used.

In other embodiments of the invention, CRE capable of being bound and activated by CAR-RXR heterodimers comprises each of NR1, NF1, and NR2 elements. Suitably they are present in the order NR1-NF1-NR 2. The NR1 element suitably comprises or consists of a sequence as described above. The NF1 element suitably comprises or consists of a sequence TGGCACAGTGCCA (SEQ ID NO:55) or TGAAGAGGTGGCA (SEQ ID NO:56), or a sequence differing from SEQ ID NO:55 or 56 by 7 or less nucleotides (e.g. 6, 5, 4, 3,2 or 1 nucleotide) or a sequence TGGCACAGTGCCA (SEQ ID NO:55) or TGAAGAGGTGGCA (SEQ ID NO:56), or a sequence differing from SEQ ID NO:55 or 56 by 7 or less nucleotides (e.g. 6, 5, 4, 3,2 or 1 nucleotide). The NR2 element suitably comprises or consists of a sequence TCAACTTGCCTGACAC (SEQ ID NO:57) or TGGACTTTCCTGAACC (SEQ ID NO:58), or a sequence which differs from SEQ ID NO:57 or 58 by 5 or less nucleotides (e.g. 4, 3,2 or 1 nucleotide), or a sequence TCAACTTGCCTGACAC (SEQ ID NO:57) or TGGACTTTCCTGAACC (SEQ ID NO:58), or a sequence which differs from SEQ ID NO:57 or 58 by 5 or less nucleotides (e.g. 4, 3,2 or 1 nucleotide).

In some embodiments of the invention, the CRE capable of being bound and activated by CAR-RXR heterodimers comprises or consists of a functional variant of PBREM sequence comprising a sequence at least 60% identical to SEQ ID No. 1 or SEQ ID No. 2, preferably at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% identical to SEQ ID No. 1 or SEQ ID No. 2. Mouse PBREM (SEQ ID NO:1) and human PBREM (SEQ ID NO:2) are 71% identical over the full length (i.e., 51 nucleotides). Mouse PBREM remains functional in human cells, while human PBREM remains functional in mouse cells. Thus, at least this level of overall sequence variation across PBREM elements can be tolerated.

However, as mentioned above, there is a very high level of conservation in the NR1 motif, and it is therefore generally preferred that a functional variant of the PBREM sequence comprises a sequence that is at least 90% identical, preferably at least 95% identical, more preferably identical, to sequence TCTGTACTTTCCTGACCTTG (SEQ ID NO:27) or ACTGTACTTTCCTGACCC TG (SEQ ID NO:28), and at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% identical to the remainder of SEQ ID NO:1 or SEQ ID NO: 2. Preferably, the total sequence is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% identical to SEQ ID NO 1 or SEQ ID NO 2.

Thus, in a preferred embodiment, the CRE capable of being bound and activated by CAR-RXR heterodimers comprises or consists of a functional variant of PBREM sequence comprising a sequence at least 90%, preferably at least 95%, more preferably identical to SEQ ID NO:1 or SEQ ID NO:2 in the region spanning nucleotides 3 to 18 (preferably the region spanning nucleotides 1 to 20), and a sequence at least 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or 99% identical to the remainder of SEQ ID NO:1 or SEQ ID NO:2 (i.e. nucleotides 1, 2 and 19 to 51, or nucleotides 21 to 51). The nucleotides are of course numbered with reference to SEQ ID NO 1 or SEQ ID NO 2. Preferably, the total sequence is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% identical to SEQ ID NO 1 or SEQ ID NO 2.

In some preferred embodiments of the invention, CRE capable of being bound and activated by CAR-RXR heterodimer comprises or consists of the following sequence:

NCTGTACTTTCCTGACCNTGNNNNNGTGNCANCATNNACTTNCCTGANNCN (SEQ ID NO:41), or a sequence at least 90%, preferably 9.5%, more preferably 99% identical thereto. In this case, identity is calculated with respect to a specifically defined nucleotide rather than an undefined "Ns". Functional variants of PBREM sequences, in which one or more nucleotides identified as N are deleted, are specifically contemplated as part of such embodiments of the invention, e.g., wherein up to 7,6, 5, 4, 3,2, or 1 of the nucleotides labeled as N are deleted. In addition, functional variants of PBREM sequences with one or more nucleotide insertions are specifically contemplated as part of these embodiments of the invention, e.g., insertions of up to 7,6, 5, 4, 3,2, or 1 nucleotide. As a specific example, the rat PBREM element from the CYP2B2 gene includes a T inserted between the T and C at positions 10-11, i.e., within NR1 of the PBREM element. Substitutions, deletions or insertions of nucleotides in the region outside the NR1 motif may be well tolerated.

In some cases, functional variants of the PBREM sequence that are more similar to wild-type mouse or human PBREM elements (SEQ ID NO:1 or SEQ ID NO:2) are preferred, as it is believed that they may exhibit particularly desirable characteristics of low background expression and high induction levels. This property is generally desirable when background expression of the genes provided in the vectors of the invention is kept to a minimum, i.e. when induction of expression does not occur.

In some embodiments of the invention, CRE capable of being bound and activated by CAR-RXR heterodimers comprises or consists of a portion of PBREM elements from each of two or more different species, suitably from two or more different genera of species (e.g., two different mammals). Such elements may be referred to as "hybrid PBREM elements". Hybrid PBREM elements typically comprise all of the motifs of wild-type PBREM elements (i.e., NR1, NF1, and NR2 motifs), but these motifs are from two or more different species. In some embodiments, a hybrid PBREM element includes a portion from a first species (e.g., NR1, NF1, and NR2 motifs), and a corresponding portion from a second species (e.g., corresponding NR1, NF1, and NR2 motifs). As non-limiting examples, hybrid PBREM elements may include portions of primate (e.g., human) and rodent (e.g., mouse) PBREM elements (e.g., NR1, NF1, and NR2 motifs). For example, a hybrid PBREM element can comprise a NR1 motif from a primate (e.g., human) and a NR2 motif from a rodent (e.g., mouse), or a NR1 motif from a rodent (e.g., mouse) and a NR2 motif from a primate (e.g., human). The primate or rodent NR1 and NR2 motifs may also bind to the NF1 motif of the corresponding species.

In some embodiments, the hybrid PBREM element comprises one of a combination of PBREM motifs of hNR1-mNF1-hNR2, hNR1-mNF1-mNR2, mNR1-hNF1-mNR2, or mNR1-hNF1-hNR2 (wherein "h" denotes a human motif and m denotes a mouse motif). Exemplary hybrid PBREM element sequences are underlined in SEQ ID nos 61 to 64 of table 3; functional variants of these sequences may of course be used, for example sequences having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% identity to the hybrid PBREM element sequences underlined in SEQ ID Nos 61 to 64, optionally with a higher level of identity in the NR1 motif, as described above.

As described above, and without wishing to be bound by theory, induction of gene expression by PBREM elements or variants thereof is thought to be dependent on binding of CAR-RXR heterodimers thereto. The ability of any given CRE capable of being bound and activated by CAR-RXR heterodimers (e.g., variants of PBREM elements) to function as expected, i.e., to be induced in the same manner as wild-type mouse or human PBREM elements, although not necessarily to the same extent, can be readily determined experimentally. For example, variant CRE can be inserted into the construct described in the examples below in place of SEQ ID NO:1, and the ability of the construct to be induced can be compared when it contains SEQ ID NO: 1. For example, as described in example 2, a variant CRE can be provided in place of SEQ ID NO:1 in the PB1-MinTK construct and tested in hepatocytes, preferably primary hepatocytes, e.g., AXOL assay-ready expanded (ARE) hepatocytes (Axol, ax3701) induced by 1. mu.M CITCO. Alternatively, the variant may be inserted into the pAAV-PB1-MinTk construct, as described in example 4. Alternatively, the relevant putative VARIANT sequences of PBREM can be cloned in front of the tk promoter (BglII site) in pGL 3-based vectors containing the firefly luciferase reporter gene (Promega) to give PBREM VARIANT-tk-luciferase and reporter plasmids using the experimental methods used in Sueyoshi, et al (J.BIOL.CHEM.Vol.274,10, pp.6043-6046,1999). Typically, a functional variant of CRE should provide an inducibility level of at least 50%, preferably 75%, more preferably 80%, 85%, 90% or 95% when compared to an equivalent construct (e.g., SEQ ID NO:1) comprising a wild-type PBREM element (as measured by the fold increase in expression caused by induction, i.e., a 2-fold increase in gene expression reported upon induction is considered to be 50% of a 4-fold increase in inducibility). In general, it is expected that the functional variant should provide at least 2-fold induction, more preferably 3-fold, 4-fold, 5-fold, 6-fold or 7-fold induction, when provided in the above described PB1-MinTK construct in place of mouse wild-type PBREM and tested in hepatocytes, preferably primary hepatocytes, e.g. AXOL assoy-ready expanded (ARE) hepatocytes (AXOL, ax3701) induced by 1 μ M CITCO, as described in example 2 below. The functional variant preferably results in no more than three times, preferably no more than two times, more preferably no more than 1.5 times background expression level compared to an equivalent construct comprising wild type mouse PBREM elements (e.g. again tested in the PB1-MinTK construct and in ARE hepatocytes).

It should be noted that PBREM elements or functional variants thereof may exist in either orientation. Thus, the reverse complement of the PBREM elements described above forms part of the present invention. Notably, human PBREM elements naturally exist in the opposite direction compared to mouse PBREM elements, and in studies of Sueyoshi, et al (j.bio. chem.vol.274,10, pp.6043-6046,1999), human PBREM elements were demonstrated to remain functional in the "opposite" direction (i.e., the same direction as mouse PBREM).

In some embodiments of the invention, the synthetic liver-specific inducible promoter suitably comprises a plurality of CREs, each CRE capable of being bound and activated by a heterodimer of CAR and RXR. Thus, in some embodiments, the invention provides synthetic liver-specific inducible promoters comprising multimers of CRE, each capable of being bound and activated by a heterodimer of CAR and RXR. From another perspective, a synthetic liver-specific inducible promoter suitably comprises a Cis Regulatory Module (CRM) comprising a plurality of CREs, each CRE capable of being bound and activated by a heterodimer of CAR and RXR. CREs that are capable of being bound and activated by heterodimers of CAR and RXR may be the same or different from each other.

In such embodiments, the synthetic liver-specific inducible promoter suitably comprises 2-10 CREs, each CRE capable of being bound and activated by a heterodimer of CAR and RXR. The inducible promoter suitably comprises 2-7 CREs, 2-5 CREs, 2-4 CREs, optionally 2 or 3 CREs, and in some embodiments preferably 3 CREs are each capable of being bound and activated by the heterodimer of CAR and RXR.

As mentioned above, CRE capable of being bound and activated by the heterodimer of CAR and RXR is preferably a PBREM element or a functional variant thereof. Thus, in a preferred embodiment, the synthetic liver-specific inducible promoter suitably comprises 2 to 5, even more preferably 2 to 4, even more preferably 3 PBREM elements comprising SEQ ID NO 1 or SEQ ID NO 2 or functional variants thereof. Various PBREM elements and functional variants thereof are discussed in detail above.

In some preferred embodiments of the invention, the synthetic liver-specific inducible promoter comprises 2 to 4, optionally 2 or 3, operably linked copies of CRE comprising SEQ ID NO 1 or SEQ ID NO 2, or a functional variant of SEQ ID NO 1 or SEQ ID NO 2.

CRE that is capable of being bound and activated by the heterodimer of CAR and RXR may be separated by a spacer sequence. The spacer can be of any suitable length, for example, 2 to 100 nucleotides, 3 to 50 nucleotides, 5 to 30 nucleotides, and 10 to 25 nucleotides. In some embodiments, the length of the spacer is a multiple of 5. Spacers of about 20 nucleotides in length have been found suitable (e.g., 18-22 nucleotides in length).

In some preferred embodiments of the invention, the synthetic liver-specific inducible promoter comprises CRM comprising or consisting of one of the following sequences:

-NCTGTACTTTCCTGACCNTGNNNNNGTGNCANCATNNACTTNCCTGANNCN-S-NCTGTACTTTCCTGACCNTGNNNNNGTGNCANCATNNACTTNCCTGANNCN (SEQ ID NO: 42); or

-NCTGTACTTTCCTGACCNTGNNNNNGTGNCANCATNNACTTNCCTGANNCN-S-NCTGTACTTTCCTGACCNTGNNNNNGTGNCANCATNNACTTNCCTGANNCN-S-NCTGTACTTTCCTGACCNTGNNNNNGTGNCANCATNNACTTNCCTGANNCN(SEQ ID NO:43),

Wherein S is an optional spacer. The spacer separates adjacent PBREM elements or functional variants thereof. The spacers are selected as described above. In some embodiments, the spacer is about 20 nucleotides in length. Variants comprising a sequence at least 90%, preferably 95%, more preferably 99% identical to PBREM element NCTGTACTTTCCTGACCNTGNNNNNGTGNCANCATNNACTTNCCTGANNCN (SEQ ID NO:41) also form part of the invention. In this case, identity is calculated with respect to a specifically defined nucleotide rather than an undefined "Ns". Functional variants of PBREM sequences, in which one or more nucleotides identified as N are deleted, are specifically contemplated as part of such embodiments of the invention, e.g., wherein up to 7,6, 5, 4, 3,2, or 1 of the nucleotides labeled as N are deleted. In addition, functional variants of PBREM sequences with one or more nucleotide insertions are specifically contemplated as part of these embodiments of the invention, e.g., insertions of up to 7,6, 5, 4, 3,2, or 1 nucleotide. As a specific example, the rat PBREM element from the CYP2B2 gene includes a T inserted between the T and C at positions 10-11, i.e., within NR1 of the PBREM element. Substitutions, deletions or insertions of nucleotides in the region outside the NR1 motif may be well tolerated.

In some preferred embodiments of the invention, the synthetic liver-specific inducible promoter comprises CRM comprising or consisting of one of the following sequences:

-TCTGTACTTTCCTGACCTTGGCACAGTGCCACCATCAACTTGCCTGACACC-S-TCTGTACTTTCCTGACCTTGGCACAGTGCCACCATCAACTTGCCTGACACC(SEQ ID NO:44)；

-TCTGTACTTTCCTGACCTTGGCACAGTGCCACCATCAACTTGCCTGACACC-S-TCTGTACTTTCCTGACCTTGGCACAGTGCCACCATCAACTTGCCTGACACC-S-TCTGTACTTTCCTGACCTTGGCACAGTGCCACCATCAACTTGCCTGACACC(SEQ ID NO:45)；

-ACTGTACTTTCCTGACCCTGAAGAGGTGGCAGCATGGACTTTCCTGAACCA-S-ACTGTACTTTCCTGACCCTGAAGAGGTGGCAGCATGGACTTTCCTGAACCA-S-ACTGTACTTTCCTGACCCTGAAGAGGTGGCAGCATGGACTTTCCTGAACCA (SEQ ID NO: 46); or

-ACTGTACTTTCCTGACCCTGAAGAGGTGGCAGCATGGACTTTCCTGAACCA-S-ACTGTACTTTCCTGACCCTGAAGAGGTGGCAGCATGGACTTTCCTGAACCA(SEQ ID NO:47),

Wherein S is an optional spacer. The spacers are selected as described above. In some embodiments, the spacer is about 20 nucleotides in length.

The synthetic liver-specific inducible promoter of the invention suitably does not comprise or comprises only minimal nucleic acid sequences that would result in constitutive expression or expression in non-liver cells (or in cells that do not express CAR and RXR as the case may be). Thus, background expression and expression in non-hepatocytes (or cells that do not express CAR and RXR) is minimized or avoided altogether.

Synthetic liver-specific inducible promoters typically comprise CRE, which is capable of being bound and activated by a heterodimer of CAR and RXR, operably linked to a minimal or proximal promoter, preferably a minimal promoter. When a proximal promoter is used, it is preferably liver-specific. In general, a minimal promoter is preferred because proximal promoters tend to drive at least some degree of background expression. However, in some cases a certain amount of background expression may be required.

The minimal promoter may be any suitable minimal promoter. A wide range of minimal promoters are known in the art. Suitable minimal promoters include, but are not limited to, the HSV thymidine kinase minimal promoter (MinTK), the CMV minimal promoter (CMVmp), and the SV40 minimal promoter (SV40 mp). The minimal promoter may be a synthetic minimal promoter.

In some preferred embodiments of the invention, the synthetic liver-specific inducible promoter comprises CRE that is capable of being bound and activated by the heterodimer of CAR and RXR operably linked to the MinTK minimal promoter. This combination has been shown to provide the ideal combination of low background expression and inducibility. Some particularly preferred embodiments comprise two or three CREs that are capable of being bound and activated by heterodimers of CAR and RXR operably linked to MinTK, such promoters having particularly strong inducibility and low background.

In some preferred embodiments of the invention, the synthetic liver-specific inducible promoter comprises CRE, which is capable of being bound and activated by a heterodimer of CAR and RXR, operably linked to an SV40 minimal promoter. This combination has been shown to provide high levels of inducibility despite increased background expression levels. Some particularly preferred embodiments comprise two or three CREs that are capable of being bound and activated by heterodimers of CAR and RXR operably linked to a minimal promoter, such promoters being particularly strongly inducible.

In some preferred embodiments, CRE that is capable of being bound and activated by the heterodimer of CAR and RXR is separated from the minimal or proximal promoter by a spacer sequence. The spacer sequence may be of any suitable length. For example, the spacer between a CRE (or, when multiple CREs are present, the closest CRE) and the minimal or closest promoter that is capable of being bound and activated by the heterodimer of CAR and RXR can have a length of 10 to 200 nucleotides. Spacers of various lengths (e.g., including 20, 46, 80 and 100 nucleotides) are successfully used in the examples set forth below.

In some embodiments of the invention, the synthetic liver-specific inducible promoter comprises a sequence according to any one of SEQ ID NOs 7 to 18, or a functional variant of any one thereof. The functional variant is suitably at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% identical to any one of SEQ ID NOs 7 to 18. As mentioned above, in the region corresponding to the NR1 motif, a sequence identity of 90%, 95% or 99% or more is preferred.

In some preferred embodiments of the invention, the synthetic liver-specific inducible promoter comprises a sequence according to SEQ ID NO 7, SEQ ID NO 10, SEQ ID NO 11 or SEQ ID NO 12, or a functional variant of any one thereof. The functional variant is suitably at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% identical to any of SEQ ID NO 7, 10, 11 or 12. As mentioned above, in the region corresponding to the NR1 motif, a sequence identity of 90%, 95% or 99% or more is preferred. These promoters contained 1, 2, 3 and 4 copies of mouse PBREM linked to the MinTK minimal promoter, respectively. In view of their particularly desirable properties in terms of high inducibility and low background expression levels, SEQ ID NO 10 and SEQ ID NO 11 or functional variants thereof are particularly preferred embodiments of the present invention.

In some embodiments of the invention, the synthetic liver-specific inducible promoter comprises or consists of a sequence according to any one of SEQ ID NOs 59 to 71 or a functional variant thereof. The functional variant is suitably at least 60%, 70%, 80%, 90%, 95% or 99% identical to any one of SEQ ID NOs 59 to 71. As mentioned above, in the region corresponding to the NR1 motif, a sequence identity of 90%, 95% or 99% or more is preferred.

In some preferred embodiments of the invention, the synthetic liver-specific inducible promoter comprises a sequence according to any one of SEQ ID NOs 68, 69, 70 or 71, or a functional variant of any one thereof. The functional variant is suitably at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% identical to any of SEQ ID NO 60 or 61. As mentioned above, in the region corresponding to the NR1 motif, a sequence identity of 90%, 95% or 99% or more is preferred. These promoters contain 2x MHM or 3x human PBREM elements.

Suitably, the expression cassette comprises a sequence providing or encoding one or more, preferably all, of a ribosome binding site, an initiation codon, a stop codon and a transcription termination sequence. Suitably, the expression cassette comprises a nucleic acid encoding a post-transcriptional regulatory element. Suitably, the expression cassette comprises a nucleic acid encoding a polyA element.

The genes used in the present invention typically encode a desired gene expression product, such as a polypeptide (protein) or RNA. The gene may be a full-length cDNA or genomic DNA sequence, or any fragment, subunit, or mutant thereof, which has at least some desired biological activity.

When a gene encodes a protein, it can be essentially any type of protein. By way of non-limiting example, the protein can be an enzyme, an antibody or antibody fragment (e.g., a monoclonal antibody), a viral protein (e.g., REP, CAP, REV, VSV-G, or RD114), a therapeutic protein, or a toxic protein (e.g., caspase 3, 8, or 9).

In some preferred embodiments of the invention, the gene encodes a therapeutic expression product, preferably a therapeutic protein suitable for use in the treatment of a liver-related disease or disorder. Such genes may be referred to as "therapeutic genes". The therapeutic expression product may be a protein, e.g., a secretable protein, such as a blood coagulation factor (e.g., factor IX or factor VIII), a cytokine, a growth factor, an antibody or nanobody, a chemokine, a plasma factor, insulin, erythropoietin, a lipoprotein lipase, or a toxic protein. Alternatively, the therapeutic expression product may be an RNA, such as an siRNA or miRNA. A non-exhaustive list of therapeutic expression products (and sequences encoding them) contemplated for use in the present invention includes: factor VIII, factor IX, factor VII, factor X, von Willebrand factor (von Willebrand factor), Erythropoietin (EPO), interferon-a, interferon-B, interferon-y, 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), chemokine (C-X-C 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 chemotactic protein-1 (MCP-1), Tumor Necrosis Factor (TNF), Afamin (AFM), a 1-antitrypsin, a-galactosidase A, alpha-L-iduronidase, ATP7b, ornithine transcarbamylase, phenylalanine hydroxylase, lipoprotein lipase, aromatic Amino Acid Decarboxylase (AADC), ATPase sarcoplasm/endoplasmic reticulum Ca2+ transport 2(ATP2A2), cystic fibrosis transmembrane conductance regulator (CTFR), glutamate decarboxylase 65kDa protein (GAD65), glutamate decarboxylase 67kDa protein (GAD67), lipoprotein lipase (LPL) kDa, Nerve Growth Factor (NGF), neurotrophic factor (NTN), protoporphyrinogen deaminase (PBGD), myoglycan alpha (SGCA), soluble fms-like tyrosine kinase-1 (sFLT-1), apolipoprotein, low density lipoprotein receptor (LDL-R), albumin, glucose-6-phosphatase, antibodies, nanobodies, aptamers, antiviral dominant-negative proteins and functional fragments, subunits or mutants thereof. Preferably, the protein is a primate protein, more preferably a human protein.

The protein or polypeptide of interest can be, for example, antibodies, enzymes or fragments thereof, viral proteins, cytokines, lymphokines, adhesion molecules, receptors and derivatives or fragments thereof, protein antibiotics, toxin fusion proteins, carbohydrate-protein conjugates, structural proteins, regulatory proteins, vaccines and vaccine-like proteins or particles, processing enzymes, growth factors, hormones, and any other polypeptide that can be used as an agonist or antagonist and/or has therapeutic or diagnostic use. According to a preferred embodiment, the protein is an immunoglobulin, preferably an antibody or antibody fragment, most preferably a Fab or scFv antibody. According to another preferred embodiment, the protein is a viral protein.

In some embodiments of the invention, the synthetic liver-specific expression cassette comprises a gene for gene editing, for example a gene encoding a site-specific nuclease, such as a meganuclease, a Zinc Finger Nuclease (ZFN), a transcription activator-like effector-based nuclease (TALEN), or an aggregated regularly-spaced short palindromic repeat system (CRISPR-Cas). Suitably, the site-specific nuclease is adapted to edit the desired genomic site of interest by cleavage (typically a site-specific double-strand break) and then repair the cleavage by non-homologous end joining (NHEJ) or homology-dependent repair (HDR), thereby generating the desired edit. Editing may be partial or complete repair of a dysfunctional gene, or knock-down or knock-out of a functional gene.

The product of interest can also be a nucleic acid, e.g., an RNA, such as an antisense RNA, microRNA, siRNA, tRNA, rRNA, guide RNA, or any other regulatory, therapeutic, or other useful RNA.

In some preferred embodiments of the invention, the gene therapy vector is a viral vector, such as a retroviral, lentiviral, adenoviral, or adeno-associated viral (AAV) vector, although other forms of gene therapy vectors are also contemplated. In some preferred embodiments, the vector is an AAV vector. In some preferred embodiments, the AAV has a serotype suitable for liver transduction. In some embodiments, the AAV is selected from the group consisting of AAV2, AAV5, AAV6, AAV7, AAV8, AAV9, or a derivative thereof. To overcome one of the limiting steps in AAV transduction (i.e., single-stranded to double-stranded AAV transformation), AAV vectors are suitable for use as self-complementary double-stranded AAV vectors (scAAV), although the use of single-stranded AAV vectors (ssav) is also encompassed herein. In some embodiments of the invention, the AAV vector is chimeric, meaning that it comprises components from at least two AAV serotypes, such as the ITRs of AAV2 and the capsid protein of AAV 5.

In some embodiments of the invention, the vector is a plasmid. Such plasmids may include a variety of other functional nucleic acid sequences, such as one or more selectable markers, one or more origins of replication, multiple cloning sites, and the like.

In another aspect, the invention provides an expression cassette comprising a synthetic liver-specific inducible promoter operably linked to a gene, the synthetic liver-specific inducible promoter comprising a CRE capable of being bound and activated by a heterodimer of CAR and RXR. Various synthetically inducible promoters are discussed above in the context of gene therapy vectors, and may be used in this aspect of the invention. Thus, the invention also includes the synthetic liver-specific promoter as described in the first aspect in gene therapy vectors (detailed above) and in other cases expression cassettes.

In some embodiments of the invention, the expression cassette is present in an expression vector expressed in a eukaryotic cell. Examples of eukaryotic expression vectors include, but are not limited to, pW-LNEO, pSV2CAT, pOG44, pXTl, and pSG, available from Stratagene; pSVK3, pBPV, pMSG, and pSVL available from Amersham Pharmacia Biotech; and pCMVDsRed2-express, pIRES2-DsRed2, pDsRed2-Mito, pCMV-EGFP, available from Clontech. Many other vectors are well known and commercially available. For mammalian cell adenoviral vectors, the pSV and pCMV series of vectors are particularly well known non-limiting examples. There are many well known yeast expression vectors, including but not limited to yeast integrating plasmids (YIp) and yeast replicating plasmids (YRp). For plants, the Ti plasmid of Agrobacterium is a typical expression vector, and plant viruses also provide suitable expression vectors, such as Tobacco Mosaic Virus (TMV), potexvirus and cowpea mosaic virus.

In an embodiment of this aspect of the invention, the gene is preferably not a reporter gene. Suitably, the gene encodes a therapeutic expression product (e.g. as described above) or another expression product useful in industry or research. Thus, in some preferred embodiments of the invention, the expression cassette is used to express a product that is not a reporter protein (e.g., a fluorescent protein, a luminescent protein, or a chromogenic protein), and preferably is used to express a therapeutic expression product. Various suitable expression products are discussed above. Other useful expression products will be apparent to the skilled person.

In another aspect, the invention also provides a synthetic liver-specific inducible promoter comprising CRE capable of being bound and activated by the heterodimer of CAR and RXR. Various synthetically inducible promoters discussed in the context of gene therapy vectors are contemplated as embodiments of this aspect of the invention. In other words, the various promoters described above are considered embodiments of this aspect of the invention, regardless of whether they are in the context of a gene therapy vector. In particular, various synthetic liver-specific inducible promoters are disclosed, which have beneficial properties and are not disclosed in the art. These promoters are useful in gene therapy, but have broad utility in other settings, such as cell culture and bioprocessing, as discussed further below.

In particular, but not exclusively, embodiments of this aspect of the invention include a synthetic liver-specific inducible promoter comprising a sequence according to any one of SEQ ID NOs 7 to 18, or a functional variant of any one thereof. The functional variant is suitably at least 60%, 70%, 80%, 90%, 95% or 99% identical to any one of SEQ ID NOs 7 to 18. As mentioned above, in the region corresponding to the NR1 motif, a sequence identity of 90%, 95% or 99% or more is preferred.

In some preferred embodiments, the synthetic liver-specific inducible promoter comprises a sequence according to SEQ ID NO 7, SEQ ID NO 10, SEQ ID NO 11 or SEQ ID NO 12, or a functional variant of any one thereof. The functional variant is suitably at least 60%, 70%, 80%, 90%, 95% or 99% identical to any of SEQ ID NO 7, 10, 11 or 12. These promoters contained 1, 2, 3 and 4 copies of mouse PBREM, respectively, linked to the MinTK minimal promoter.

In some preferred embodiments of the invention, the synthetic liver-specific inducible promoter comprises a sequence according to any one of SEQ ID NOs 68, 69, 70 or 71, or a functional variant of any one thereof. The functional variant is suitably at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% identical to any of SEQ ID NOs 68, 69, 70 or 71. As mentioned above, sequence identity of 90%, 95% or 99% or more in the region corresponding to the NR1 motif is preferred. These promoters contain 2x MHM or 3x human PBREM elements.

In another aspect of the invention, there is provided a CRE that is capable of being bound and activated by CAR-RXR heterodimers comprising or consisting of a functional variant of a PBREM element. Suitably, the variant PBREM is a non-naturally occurring variant of PBREM, i.e. it comprises a sequence that does not occur in nature. For example, the variant does not comprise the sequence of a human, mouse or rat PBREM element (e.g., SEQ ID NO:1 or SEQ ID NO: 2). Various non-native CREs capable of being bound and activated by CAR-RXR heterodimers are described above in relation to the first aspect of the invention, and these provide exemplary embodiments of this aspect.

In some embodiments, as described above, CRE of the invention comprise or consist of hybrid PBREM elements. As non-limiting examples, hybrid PBREM elements can include portions of primate (e.g., human) and rodent (e.g., mouse) PBREM elements. For example, a hybrid PBREM element may comprise a NR1 motif from human and a NR2 motif from mouse, or a NR1 motif from mouse and a NR2 motif from human. The mouse or human NR1 and NR2 motifs may also bind to the NR1 motif of the corresponding species. In some embodiments, the hybrid PBREM element comprises one of a combination of PBREM motifs of hNR1-mNF1-hNR2, hNR1-mNF1-mNR2, mNR1-hNF1-mNR2, or mNR1-hNF1-hNR2 (wherein "h" denotes a human motif and m denotes a mouse motif). Exemplary hybrid PBREM element sequences are underlined in SEQ ID NOs 61 to 64 of table 3; of course, functional variants of these sequences may be used, such as sequences having 60%, 70%, 75%, 80%, 85%, 90%, 95% or 99% identity to the hybrid PBREM element sequences underlined in SEQ ID NOS: 61 to 64.

In some embodiments, CRE capable of being bound and activated by CAR-RXR heterodimer comprises or consists of one of the following sequences:

ACTGTACTTTCCTGACCCTGAAGAGACTGTACTTTCCTGACCCTGAAGAGACTGTACTTTCCTGACCCTGAAGAG (SEQ ID NO: #; human NR1x 3);

ACTGTACTTTCCTGACCCTGGCACAGTGCCACCATGGACTTTCCTGAACCA (SEQ ID NO: 72; HMH hybrid);

ACTGTACTTTCCTGACCCTGGCACAGTGCCACCATCAACTTGCCTGACACC (SEQ ID NO: 73; HMM hybrid);

TCTGTACTTTCCTGACCTTGAAGAGGTGGCACCATCAACTTGCCTGACACC (SEQ ID NO: 74; MHM hybrid); or

TCTGTACTTTCCTGACCTTGAAGAGGTGGCAGCATGGACTTTCCTGAACCA (SEQ ID NO: 75; MHH hybrid),

or a functional variant thereof. A functional variant suitably comprises a sequence that is 80%, 85%, 90%, 95% or 99% identical to any of the sequences.

In another aspect of the invention, CRMs are provided comprising two or more CREs capable of being bound and activated by CAR-RXR heterodimers comprising or consisting of a functional variant of PBREM elements. Various CRMs comprising two or more CREs capable of being bound and activated by CAR-RXR heterodimers are described above and in relation to the first aspect of the invention, and these provide exemplary embodiments of this aspect. In some embodiments, the CRM comprises at least one non-natural CRE as described above, e.g., at least one hybrid CRE.

In some embodiments, CRM comprises one of the following sequences:

TCTGTACTTTCCTGACCTTGGCACAGTGCCACCATCAACTTGCCTGACACC-S-CTGTACTTTCCTGACCTTGGCACAGTGCCACCATCAACTTGCCTGACACC (SEQ ID NO: 76; 2 × murine PBREM CREs);

TCTGTACTTTCCTGACCTTGGCACAGTGCCACCATCAACTTGCCTGACACC-S-TCTGTACTTTCCTGACCTTGGCACAGTGCCACCATCAACTTGCCTGACACC-S-TCTGTACTTTCCTGACCTTGGCACAGTGCCACCATCAACTTGCCTGACACC-S-TCTGTACTTTCCTGACCTTGGCACAGTGCCACCATCAACTTGCCTGACACC (SEQ ID NO: 77; 3 × murine PBREM CREs);

CTGTACTTTCCTGACCTTGAAGAGGTGGCACCATCAACTTGCCTGACACC-S-TCTGTACTTTCCTGACCTTGAAGAGGTGGCACCATCAACTTGCCTGACACC (SEQ ID NO: 78; 2X MHM heterozygous CREs);

ACTGTACTTTCCTGACCCTGAAGAGGTGGCAGCATGGACTTTCCTGAACCA-S-ACTGTACTTTCCTGACCCTGAAGAGGTGGCAGCATGGACTTTCCTGAACCA (SEQ ID NO: 79; 2X human PBREM CREs);

ACTGTACTTTCCTGACCCTGAAGAGGTGGCAGCATGGACTTTCCTGAACCA-S-ACTGTACTTTCCTGACCCTGAAGAGGTGGCAGCATGGACTTTCCTGAACCA-S-ACTGTACTTTCCTGACCCTGAAGAGGTGGCAGCATGGACTTTCCTGAACCA (SEQ ID NO: 80; 3X human PBREM CREs);

or any functional variant thereof. A functional variant suitably comprises a sequence that is 80%, 90%, 95% or 99% identical to any of the sequences described, and wherein S is an optional spacer. When present, the spacer can be of any suitable length, for example, 2 to 100 nucleotides, 3 to 50 nucleotides, 5 to 30 nucleotides, and 10 to 25 nucleotides. In some embodiments, the length of the spacer mentioned is a multiple of 5. Spacers of about 20 nucleotides in length have been found suitable (e.g., 18-22 nucleotides in length).

In some embodiments, CRM comprises one of the following sequences:

TCTGTACTTTCCTGACCTTGGCACAGTGCCACCATCAACTTGCCTGACACCCATTACTCGCATCCATTCTCTCTGTACTTTCCTGACCTTGGCACAGTGCCACCATCAACTTGCCTGACACC (SEQ ID NO: 81; 2 × mouse PBREM CREs);

TCTGTACTTTCCTGACCTTGGCACAGTGCCACCATCAACTTGCCTGACACCCATTACTCGCATCCATTCTCTCTGTACTTTCCTGACCTTGGCACAGTGCCACCATCAACTTGCCTGACACCGCACTGAAGGTCCTCAATCGTCTGTACTTTCCTGACCTTGGCACAGTGCCACCATCAACTTGCCTGACACCCTGACCTCCTGCCAGCAATATCTGTACTTTCCTGACCTTGGCACAGTGCCACCATCAACTTGCCTGACACC (SEQ ID NO: 82; 3 × mouse PBREM) CTGTACTTTCCTGACCTTGAAGAGGTGGCACCATCAACTTGCCTGACACCCATTACTCGCATCCATTCTCTCTGTACTTTCCTGACCTTGAAGAGGTGGCACCATCAACTTGCCTGACACC (SEQ ID NO: 83; 2XMHM hybrid);

ACTGTACTTTCCTGACCCTGAAGAGGTGGCAGCATGGACTTTCCTGAACCACATTACTCGCATCCATTCTCACTGTACTTTCCTGACCCTGAAGAGGTGGCAGCATGGACTTTCCTGAACCA (SEQ ID NO: 84; 2X human PBREM); or

ACTGTACTTTCCTGACCCTGAAGAGGTGGCAGCATGGACTTTCCTGAACCACATTACTCGCATCCATTCTCACTGTACTTTCCTGACCCTGAAGAGGTGGCAGCATGGACTTTCCTGAACCAGCACTGAAGGTCCTCAATCGACTGTACTTTCCTGACCCTGAAGAGGTGGCAGCATGGACTTTCCTGAACCA (SEQ ID NO: 85; 3X human PBREM),

or any functional variant thereof. A functional variant suitably comprises a sequence that is 80%, 90%, 95% or 99% identical to any of the sequences.

In one embodiment, a cis regulatory element, cis regulatory module, or promoter, or variant thereof, provided herein can enhance or drive expression of a gene in a tissue of non-liver or non-liver origin (e.g., spleen, muscle, heart, lung, and brain). Gene expression in non-liver tissues may be exclusive or in addition to expression in liver cells. When the expression of the gene is driven in both the non-liver tissue and the liver tissue, the expression level in the non-liver tissue may be equal to or greater than the expression level in the liver tissue. For example, a promoter provided herein or a variant thereof may drive equivalent expression of a gene in the liver and heart.

In another aspect, the present invention provides recombinant viral particles (virions) comprising the gene therapy vector according to the invention. The viral particle may be, for example, an AAV particle, a retroviral particle, a lentiviral particle, or some other form of gene therapy viral particle.

The gene therapy vectors or virosomes of the invention may be formulated into pharmaceutical compositions together with pharmaceutically acceptable excipients (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.

Thus, in another aspect, the present invention provides a pharmaceutical composition comprising a gene therapy vector or viral particle as described above. The gene therapy vectors or virosomes of the invention may be formulated into pharmaceutical compositions together with pharmaceutically acceptable excipients (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.

According to another aspect of the invention there is provided a cell comprising a gene therapy vector, expression cassette, promoter, CRE or CRM according to the various aspects of the invention.

Suitable cells are eukaryotic cells. The eukaryotic cell may suitably be a fungal cell (e.g. a yeast cell), an animal (metazoan) cell (e.g. a mammalian cell) or a plant cell. Alternatively, the cell may be a prokaryotic cell.

In some embodiments of the invention, the cell is ex vivo, e.g., in cell culture. In other embodiments of the invention, the cells may be part of a tissue or a multicellular organism.

In a preferred embodiment, the cell is a hepatocyte (hepatocyte), which may be ex vivo or in vivo. The hepatocytes may be primary hepatocytes or cells of a liver-derived cell line, such as an immortalized cell line. The cell may be present in the environment of the liver tissue (e.g., in the liver), or may be isolated from the liver tissue, e.g., it may be in a cell culture. Suitable cells are human cells. Usually hepatocytes express CAR and RXR simultaneously. However, some hepatogenic cell lines do not express CAR (e.g. Huh7 cell line), in which case it is necessary to provide exogenous CAR. This can be achieved by providing the cell with a suitable expression construct comprising a nucleic acid encoding the CAR operably linked to a promoter, e.g., a constitutively active promoter. Suitable methods are known in the art and are described in the examples.

In some embodiments, the cell has been modified to express a CAR and/or RXR. In such embodiments, the suitable cell is a cell that does not normally express CAR, RXR, or both CAR and RXR. Suitable cells are not hepatocytes. Thus, in some embodiments of the invention, the promoters, expression cassettes, and vectors of the invention are used in non-liver cells, which do not normally (i.e., in their natural state) express CAR and/or RXR; the promoters of the invention are therefore generally not active in such cells. However, the cell has been modified such that it is capable of expressing CAR and RXR, so the promoter can be induced. As a non-limiting example, the cells may be any type of animal primary cell or animal cell line.

The gene therapy vector, expression cassette, promoter, CRE or CRM according to various aspects of the invention may be inserted into the genome of a cell, or it may be present in an episomal vector.

Cells suitable for use in the present invention include, but are not limited to, eukaryotic cells (e.g., yeast), plants, insects, or mammalsA cell. For example, the cell may be any type of differentiated cell, or may be an oocyte, an embryonic stem cell, a hematopoietic stem cell, or other forms. In some embodiments, the cell is an animal (metazoan) cell (e.g., a mammalian cell). In some preferred embodiments, the cells are hepatocytes (e.g., Huh7 cells, HepaRG cells, HEPG2 cells, etc.; various hepatocytes are available from ATCC, DSMZ, and other sources). In some embodiments, the cell is a mammalian cell. In some embodiments, the mammalian cell is a human, simian, murine, rat, rabbit, hamster, goat, bovine, ovine, or porcine cell. Some cells or "host cells" producing the product of interest are human, mouse, rat, monkey or rodent cell lines. In some embodiments, hamster cells are preferred, e.g., BHK21, BHK TK^-CHO, CHO-K1, CHO-DUKX B1, CHO-S and CHO-DG44 cells, or derivatives/progeny of any such cell line. In an alternative embodiment, the cell may be a human cell. In some preferred embodiments, the human cell can be a Human Embryonic Kidney (HEK) cell, preferably a HEK 293 cell. In another preferred embodiment of the invention, the cell may be a retinal cell, such as a retinal pigment epithelial cell, for example ARPE-19(ATCC CRL-2302). Furthermore, murine myeloma cells, preferably NS0 and Sp2/0 cells or derivatives/progeny of any such cell line are also well known biopharmaceutical protein producing cell lines. Non-limiting examples of cell lines and sources thereof that may be used in the present invention are summarized in table 1. Suitable host cells are commercially available, for example from culture collections such as DSMZ (Deutsche Sammlung von Mikroorganismen and Zeilkuiturren GmbH, Braunschweig, Germany) or the American Type Culture Collection (ATCC).

For bioprocessing applications, it may be preferred that the cells are established, adapted and cultured completely under serum-free conditions, and optionally in a medium that is free of any animal-derived proteins/peptides. Commercially available media, such as Ham ' S F12(Sigma, Deisenhofen, Germany), RPMI-1640(Sigma), Dulbecco ' S Modified Eagle ' S Medium (DMEM; Sigma), minimum Essential Medium (MEM; Sigma), Iscove ' S Modified Dulbecco ' S Medium (IMDM; Sigma), CD-CHO (Invitrogen, Carlsbad, CA), CHO-S-SFMII (Invitrogen), CHO Medium without serum (Sigma), protein-free CHO Medium (Sigma), EX-CELL Medium (SAFC), CDM4CHO and SFM4CHO (HyClone) are exemplary suitable nutrients. Any of the media can be supplemented as needed with various compounds, such as hormones and/or other growth factors (e.g., insulin, transferrin, epidermal growth factor, insulin-like growth factor), salts (e.g., sodium chloride, calcium, magnesium, phosphate), buffers (e.g., HEPES), nucleosides (e.g., adenosine, thymidine), glutamine, glucose or other equivalent energy sources, antibiotics, trace elements. In the present invention, serum-free medium is preferably used, but medium supplemented with an appropriate amount of serum may also be used for culturing the host cells. For growth and selection of genetically modified cells expressing selectable genes, a suitable selection agent is added to the culture medium.

The cell may be a prokaryotic cell, such as a bacterial cell. In some embodiments of the invention, the cell may be a prokaryotic cell; although prokaryotic cells do not possess the CAR/RXR system associated with the present invention, prokaryotic cells can still be used to produce vectors or other steps of vector processing, transport or storage.

In some embodiments, the cell is a packaging or producer cell for production of a viral vector. For example, the cell can be a packaging or producer cell for production of an AAV vector. Various packaging or production cell lines are known in the art. In a packaging or production cell line, it is preferred that the promoter of the invention is operably linked to a viral protein, such as Rep or Cap or other viral structural or non-structural gene.

In another aspect of the invention, the promoter of the invention may be operably associated with a gene or RNA encoding a viral protein. The viral protein may be a structural or non-structural protein. By way of non-limiting example, the viral protein may be an AAV protein, such as a Rep or Cap protein, or it may be a viral accessory protein, such as E1A, E1B, E2A, or E4. In other embodiments, the viral protein may be REV, VSV-G or RD 114.

In another aspect of the invention there is provided a gene therapy vector, expression cassette, promoter, virosome or pharmaceutical composition according to the various aspects of the invention for use in the treatment of a disease, preferably a disease associated with aberrant gene expression, optionally in the liver (e.g. inherited liver disease).

Expression in the liver is of particular interest because it is involved in a wide range of basic functions in the body, including synthesis of many proteins involved in metabolism, hemostasis, and prevention of infection. Given that many diseases are associated with disruption of gene expression in the liver, there is great interest in developing gene therapy strategies that allow for expression of transgenes in the liver to produce therapeutic expression products. Diseases associated with aberrant gene expression include, but are not limited to, hemophilia (including hemophilia a or B), familial hypercholesterolemia, ornithine transcarbamylase deficiency, phenylketonuria, ornithine transcarbamylase deficiency, glycogen storage disease, alpha 1-antitrypsin deficiency, hereditary hemochromatosis, tyrosinemia type 1, argininosuccinuria, hepatitis virus infection, non-viral hepatitis, liver cancer, hereditary cholestasis, wilson's disease, and various other liver diseases such as non-alcoholic fatty liver disease (NAFLD) and alcohol-related liver disease (ARLD). For the treatment of hemophilia a or B represent preferred embodiments of the various aspects of the invention.

In another aspect, the invention provides a method of producing an expression product, suitably a therapeutic expression product, in a cell, suitably a hepatocyte, the method comprising:

-administering to said cell an inducing agent capable of inducing expression of an expression product of a gene operably linked to an inducible promoter in said expression cassette.

The method suitably comprises maintaining the cell under suitable conditions to express an expression product from the gene. In culture, this may comprise incubating the cells or tissue comprising the cells under suitable culture conditions. The cell may of course be in vivo, for example in one or more cells of the liver of a subject. Thus, this aspect of the invention provides, inter alia, methods for producing a product of interest or expressing a therapeutic product (e.g., in a subject or ex vivo) in cell culture (e.g., in bioprocessing applications). Various cells suitable for use in this regard are discussed above.

Suitably, the method comprises the step of introducing the expression cassette into the liver. Various methods of transfecting hepatocytes are known in the art. A preferred method of transfecting hepatocytes is to transduce the cells with a viral vector (e.g., an AAV vector) comprising a synthetic liver-specific expression cassette. Details of various vectors used in the present invention are described below.

The method may comprise collecting or isolating the expression product. Suitable methods for collecting or isolating various expression products (e.g., proteins or nucleic acids) are well known to the skilled artisan.

In some embodiments, the cell is a hepatocyte. Usually hepatocytes express CAR and RXR simultaneously. However, some hepatogenic cell lines do not express CAR (e.g. Huh7 cell line), in which case it is necessary to provide exogenous CAR. This can be achieved by providing a suitable expression construct to express the CAR in the cell, for example comprising a nucleic acid encoding the CAR operably linked to a suitable promoter. Suitable expression vectors and other methods for expressing a CAR in any given cell are well known in the art.

Natural expression of CARs is generally restricted to hepatocytes. Thus, in cells that do not express the CAR (which is typical for non-hepatocyte cells), it is often necessary to provide exogenous CAR to the cell. As described above, this can be achieved by providing a suitable expression construct to express the CAR in the cell, e.g., comprising a nucleic acid encoding the CAR operably linked to a suitable promoter. Suitable expression vectors and other methods for expressing a CAR in any given cell are well known in the art.

The inducing agent for use in the present invention may be any agent suitable for inducing CAR activation and CAR-RXR heterodimer formation. Such an inducer can induce expression from the PBREM elements in cells in which the expression cassette is present. A variety of expression inducers from mouse and human PBREM elements are known in the art (see, for example, HONKAKOSKI, et al, Molecular Pharmacology,53: 597-. Typically, these inducers are activating ligands of the CAR. CARs are characterized by a wide range of specificities for a variety of endogenous and exogenous ligands with different chemical structures, making them xenobiotic sensors. Table 1 of Cherian et al lists various activators of CAR activators in various species, which can be used as inducers in any aspect of the invention.

For example, but not limited to, the inducing agent suitably comprises one or more agents selected from the following list:

In some preferred embodiments, the inducing agent is a drug (medicament), such as a drug that has been approved by regulatory approval in at least one country (preferably the us or members of the european union) for use in humans or animals (preferably humans) to treat at least one medical condition. Alternatively, it may be preferred that the inducer has a generally recognized as harmless (GRAS) status in at least one country, preferably the us or members of the european union. In some preferred embodiments, the inducing agent comprises one or more agents selected from the group consisting of:

phenobarbital; flavonoids such as flavone, chrysin, baicalein or galangin; acetaminophen; buprenorphine; phenobarbital; phenytoin; carbamazepine; valproic acid; artemisinin and its derivatives; chlorpromazine; efavirenz; nevirapine; ropinirostrin; etravirine; diazepam; cyclophosphamide; ifosfamide; cerivastatin; simvastatin; lovastatin; substituted sulfonamides; and thiazolidin-4-one.

These compounds are known drugs or are considered GRAS for human use and therefore can be used to induce expression in humans in general with reasonable safety.

In some embodiments of the invention, the inducer is phenobarbital. In some other embodiments, the inducer is CITCO or TCPOBOP. In some other embodiments, the inducing agent is a flavonoid, such as a flavone.

The inducing agent may be administered to the cells in any suitable manner. For example, in cell culture, an inducer may be added to the culture medium. When the cells are in vivo, e.g., in the liver of an animal, the inducing agent can be administered to the cells by systemic administration to the animal or by local administration to a target tissue (e.g., liver). One skilled in the art can readily determine the appropriate dosage rate for any given inducer. Thus, one skilled in the art can readily determine the appropriate manner and appropriate concentration for use to deliver an inducing agent to a cell for any inducing agent. In the case of CITCO, it has been demonstrated in the examples below that administration of a concentration of 0.5. mu.M to 3. mu.M (e.g.about 1. mu.M) to cells is suitable for inducing expression. In the case of TCPOBOP, it has been demonstrated in the examples below that the administration of a concentration of 50nM to 150nM to the cells is suitable for inducing expression. In the case of flavones, it has been demonstrated in the examples below that administration of a concentration of 30. mu.M to cells is suitable for inducing expression. However, other suitable concentrations may be used and the skilled person may determine the necessary dose to administer to the patient. Thus, in some embodiments, the invention contemplates exposing cells comprising the expression cassette to 0.1 μ M to 15 μ M of CITCO to induce expression, e.g., 0.25 μ M to 6 μ M, 0.5 μ M to 3 μ M. In some embodiments, the invention contemplates exposing cells comprising the expression cassette to TCPOBOP at 10nM or 750nM to induce expression, e.g., 25nM to 300nM, or 50nM to 150 nM. In some embodiments, the invention contemplates exposing cells comprising the expression cassette to 6 μ M to 150 μ M of flavone to induce expression, e.g., 15 to 150 μ M, or 25 to 35 μ M.

The method may suitably comprise discontinuing administration of the inducer. Discontinuing administration of the inducing agent will result in at least a reduction in expression of the expression product. Typically, expression of the expression product will return to baseline levels over time.

The method may suitably comprise varying the concentration of the inducing agent administered to the cells over time. This can be used to modulate the expression level of the expression product.

In some embodiments of the invention, the concentration of the inducer administered to the cells is varied over time in order to adjust the dose of the therapeutic gene product provided in the subject or to alter the production of the expression product in cell culture. In the case of treatment of a subject (discussed in more detail below), the concentration of the inducing agent may vary in response to a change in the condition of the subject, the level of a biomarker in the subject, or any other reason.

In some embodiments, the method comprises administering an inhibitor to the cell. The inhibitor can be any agent suitable to inhibit or reduce CAR activation and CAR-RXR heterodimer formation. The addition of an inhibitor may be used to reduce or eliminate expression of the expression product (i.e., to down-regulate or turn off expression). As a non-limiting example, metformin is a known CAR antagonist that can be used as an inhibitor. Furthermore, androstanes and several isomers of androstanes, are known to be endogenous antagonists of CARs, and these can be administered as inhibitors. Various inhibitors of CAR are described in Cherian et al, "Small-molecular models of the constitutive alkane receptor", Expert Opin Drug Metab toxin.2015 July; 11(7): 1099-. Those skilled in the art can, of course, determine other CAR (or RXR) inhibitors suitable for inhibiting or reducing the formation of CAR-RXR heterodimers useful in the present invention. One skilled in the art can readily determine the appropriate dosage rate for any given inhibitor.

In another aspect, the present invention provides a method for expressing a therapeutic transgene in a hepatocyte, the method comprising introducing a gene therapy vector according to the present invention into a hepatocyte, and then administering an inducer to the cell. Suitable inducers are described above. Hepatocytes may be in vivo or ex vivo. As described above, the concentration of the inducing agent administered to the cells can vary over time. Exemplary therapeutic genes for use in this regard have been discussed above.

It will be apparent to those skilled in the art that gene therapy vectors, expression cassettes, virosomes or pharmaceutical compositions according to various aspects of the invention may be used in gene therapy. Thus, the use of such gene therapy vectors, expression cassettes, virosomes or pharmaceutical compositions in gene therapy forms part of the present invention. Accordingly, one aspect of the present invention provides a gene therapy vector, expression cassette, virosome or pharmaceutical composition as described herein for use in gene therapy, preferably gene therapy by liver-specific expression of a therapeutic gene, suitable for the treatment of diseases involving abnormal gene expression in the liver.

In another aspect, the present invention provides a method of gene therapy of a subject, preferably a human, in need thereof, the method comprising:

-introducing into the liver of a subject a gene therapy vector, expression cassette or virosome of the invention comprising a gene encoding a therapeutic product; and

-administering an inducing agent to the subject such that a therapeutically effective amount of the therapeutic product is expressed in the subject.

Suitable therapeutic genes are also discussed above. Conditions that may be treated are also discussed above, including but not limited to hemophilia (including hemophilia a or B), familial hypercholesterolemia, ornithine carbamoylase deficiency, phenylketonuria, ornithine carbamoylase deficiency, glycogen storage disease, alpha 1-antitrypsin deficiency, hereditary hemochromatosis, tyrosinemia type 1, argininuria, hepatitis virus infection, non-viral hepatitis, liver cancer, hereditary cholestasis, wilson's disease, and various other liver diseases (such as non-alcoholic fatty liver disease (NAFLD), alcohol-related liver disease (ARLD), and lysosomal storage disorders).

The method suitably comprises expressing a therapeutically effective amount of a therapeutic product from the gene in the liver of the subject. The therapeutic product may have a therapeutic effect on the liver or other part of the subject. For example, the therapeutic product may be released into the bloodstream.

Suitable inducers, and methods of administration of the inducers, are listed above. As mentioned above, the application of the inducer may be stopped after a period of time, for example after a suitable therapeutic effect has been achieved. Alternatively, the amount of inducing agent administered to the subject can vary over time. The amount of inducing agent administered to the subject can be adjusted to obtain the desired amount (dose) of expression of the therapeutic product. Thus, when an increase in the amount of a therapeutic product is clinically desirable (e.g., due to an inadequate response by the subject), the amount of an induction agent administered to the subject can be increased, and vice versa (e.g., due to an excessive response or undesirable side effects).

In some embodiments, the method may comprise the steps of:

-determining the amount of the therapeutic product expressed in the subject or assessing the response of the subject to the therapeutic product;

a) increasing the amount of induction agent administered to the subject when a higher amount of therapeutic product is desired in the subject, or

b) When a lower amount of the therapeutic product is desired in the subject, the amount of the inducing agent administered to the subject is reduced.

Standard laboratory techniques can be used to determine the amount of the therapeutic product in the subject.

When it is contemplated that the amount of inducing agent administered to a subject may vary over time, it will of course be understood that the inducing agent will not normally be administered to the patient continuously, but will normally be administered at a given dosage level at given time intervals. Thus, the present invention contemplates varying the amount of inducing agent administered to a subject over time by adjusting the dosage, adjusting the time interval between dosages, or both. Thus, for example, to increase the amount of an inducing agent administered to a subject, the dose can be increased while the time period between doses remains constant, the dose can remain constant while the time period between doses decreases, or the dose can be increased and the time period between doses decreases. To reduce the amount of induction agent administered to a subject, the dose can be reduced while the time period between doses is kept constant, the dose can be kept constant while the time period between doses is reduced, or the dose can be reduced and the time period between doses increased.

Alternatively or additionally, the method may comprise altering the inducing agent to alter the amount of the therapeutic product in the subject. For example, a weak inducer may be replaced by a strong inducer and vice versa.

The method can also include, for example, altering the inducer if the subject has an adverse reaction to the inducer or the inducer is found to be ineffective in the subject.

The method may further comprise administering an inhibitor to the subject. Suitable inhibitors for use in the present invention have been discussed above. Inhibitors may be added to reduce or stop the production of the therapeutic product in the subject. The amount of inhibitor administered to the subject can be adjusted to obtain the desired amount (dose) of expression of the therapeutic product.

Genes encoding suitable therapeutic gene products have been discussed above. However, therapeutic proteins, such as factor VIII and IX for the treatment of hemophilia, may be specifically mentioned.

The method suitably comprises administering to the subject a vector or virosome according to the invention. Suitable vectors are viral gene therapy vectors, preferably AAV vectors.

In some embodiments, the method comprises systemically administering the viral gene therapy vector. Systemic administration can be enteral (such as oral, sublingual and rectal) or parenteral (such as injection). Preferred routes of injection include intravenous, intramuscular, subcutaneous, intraarterial, intraarticular, intrathecal and intradermal injection.

In some embodiments, the viral gene therapy vector may be administered simultaneously or sequentially with one or more additional therapeutic agents or one or more saturants designed to prevent clearance of the vector by the reticuloendothelial system.

When the vector is an AAV vector, the dose of the vector may be 1x10¹⁰gc/kg to 1x10¹⁵gc/kg or more, suitably 1X10¹²gc/kg to 1x10¹⁴gc/kg, suitably 5X10¹²gc/kg to 5x10¹³gc/kg。

Generally, the subject in need will be a mammal, preferably a primate, more preferably a human. Typically, a subject in need thereof will exhibit symptoms characteristic of the disease. The methods generally involve ameliorating a symptom exhibited by a subject in need thereof by expressing a therapeutic amount of a therapeutic product.

Gene therapy protocols for therapeutic gene expression in target cells in vitro and in vivo are well known in the art and will not be discussed in detail herein. Briefly, they include intramuscular injection, interstitial injection, airway instillation, application to endothelium, intrahepatic parenchyma, and intravenous or intraarterial administration of plasmid DNA vectors (naked or in liposomes) or viral vectors (e.g., intrahepatic artery, intrahepatic vein). Various devices have been developed to enhance the utilization of DNA by target cells. While a simple method is to physically contact the target cells with a catheter or implantable material containing the relevant carrier, more complex methods may use jet injection devices and the like. Ex vivo and in vivo procedures have been used to transfer genes into mammalian hepatocytes. Ex vivo methods typically require harvesting of hepatocytes, in vitro transduction with a suitable expression vector, and then reintroduction of the transduced hepatocytes into the liver. In vivo gene transfer is achieved by injecting DNA or viral vectors into the liver parenchyma, hepatic artery or portal vein.

In another aspect of the invention there is provided the use of a synthetic inducible promoter, a synthetic expression cassette, a vector or a virosome according to various aspects of the invention in the manufacture of a pharmaceutical composition for the treatment of any condition or disease mentioned herein.

In another aspect, the present invention provides a method of producing an expression product, the method comprising the steps of:

(a) providing a population of eukaryotic cells, preferably animal cells, more preferably mammalian cells, more preferably hepatocytes, comprising an expression cassette comprising a synthetic liver-specific inducible promoter operably linked to a gene, the synthetic liver-specific inducible promoter comprising a cis-regulatory element (CRE) capable of being bound and activated by a heterodimer of CAR and RXR;

(b) culturing said population of cells; and

(c) administering to the cell an inducing agent capable of inducing expression of an expression product of a gene operably linked to an inducible promoter in the expression cassette; and

(d) recovering the expression product.

The method is preferably a bioprocessing method, i.e., a process in which living cells are used to obtain the desired expression product. Preferred transgenes and their encoded products of interest are discussed above. The expression products can be used in therapeutic, cosmetic, research or other industrial processes. Inducers and cells suitable for use in this regard have been discussed above.

Step (b) typically comprises maintaining the population of cells under conditions suitable for cell proliferation. Upon induction in step (c), these conditions generally prime the cell for expression of the expression product from the transgene. Those skilled in the art will know the appropriate conditions for each type of cell. Thus, the method suitably comprises incubating the population of cells under conditions suitable for cell growth prior to treating the population of cells to induce expression in step (c).

The step of recovering the expression product typically comprises isolating the expression product from the cell population, and in some cases from other components of the cell culture medium. The method preferably comprises the step of purifying the expression product. Suitable methods for recovering and/or purifying the expression product are conventional in the art and depend on the specific nature of the expression product.

It is apparent that the present invention allows for the production of the expression product to be delayed to a desired point in time during the cell culture process. This may allow, for example, the cell population to expand until a desired number or concentration of cells is reached, or a desired growth stage is reached. This may be desirable for a number of reasons, for example, to allow cells to grow under optimal conditions before expressing the transgene, which may inhibit growth. For example, in the case of toxic proteins, the production of toxic expression products can be avoided until the cell culture system is at the desired stage. Once the toxic protein is expressed, the cells are of course adversely affected or killed. However, even for non-toxic expression products, there may be considerable efficiency advantages in delaying expression of the transgene to the desired point in time.

These processes may be carried out in any suitable reactor, including but not limited to stirred tanks, airlift reactors, fibers, microfibers, hollow fibers, ceramic matrices, fluidized beds, fixed and/or spouted bed bioreactors. As used herein, "reactor" may include a fermentor or a fermentation unit, or any other reaction vessel, and the term "reactor" may be used interchangeably with "fermentor". For example, in some aspects, an exemplary bioreactor unit may perform one or more or all of the following, supply of nutrients and/or carbon sources, injection of suitable gases (e.g., oxygen), fermentation or in-and-out flow of cell culture media, separation of gas and liquid phases, maintenance of temperature, oxygen and CO₂Maintenance of levels, maintenance of pH levels, agitation (e.g., stirring), and/or cleaning/disinfecting. An exemplary reactor unit, such as a fermentation unit, may contain multiple reactors within the unitThe reactors, for example the unit, may have 1 to 10 or more bioreactors in each unit. In various embodiments, the bioreactor may be adapted for batch, semi-batch fed, fed-batch, perfusion, and/or continuous fermentation processes. In some embodiments, the bioreactor may have a volume of about 100ml to about 50,000 liters, preferably 10 liters or more. In addition, suitable reactors may be multi-purpose, single-purpose, disposable, or non-disposable, and may be formed of any suitable material. U.S. patent publication nos. 2013/0280797, 2012/0077429, 2011/0280797, 2009/0305626 and U.S. patent nos. 8,298,054, 7,629,167 and 5,656,491 (incorporated herein by reference in their entirety) describe exemplary systems that may be used in the present invention.

In some preferred embodiments of the invention, the method is used to produce gene therapy viral vectors, such as rAAV viral particles. In this embodiment, the cell is suitably a packaging cell or a producer cell, and one of the helper functions is under the control of an inducible promoter. By way of non-limiting example, the Rep, Cap, E1A, E1B, E2A, E4, and VA RNA genes can be placed under the control of an inducible promoter. Due to its toxicity, control of Rep expression is of particular interest. Thus, in another aspect of the invention there is provided the use of a promoter as described herein to control the expression of a viral protein in a method of producing a viral gene therapy product, preferably a viral accessory protein, more preferably a Rep gene.

In another aspect, the invention provides a reactor vessel comprising a cell culture comprising cells of the invention and a medium sufficient to support cell growth. Various reactors suitable for use in the present invention are described above.

In another aspect of the invention, there is provided the use of a bioprocessing vector or cell of the invention in a bioprocessing method for the manufacture of a product of interest (e.g., a therapeutic product).

Drawings

FIG. 1A shows measurement of luciferase expression from the PB1-MinTK construct after transfection into Huh7 cells and treatment with DMSO, 50nM, 150nM, and 250nM TCPOBOP.

Figure 1B shows measurement of luciferase expression from PB1-MinTK construct after transfection into Huh7 cells, with or without Huh7 cells transfected with CARs.

FIG. 1C shows the data from FIG. 1A, expressed as the ratio of the strong viral promoter CMV-IE. Figure 1C also shows that in the absence of CAR, there is no luciferase expression. One PBREM element was combined with the MinTK promoter to drive expression up to a maximum of 40% of CMV-IE gene expression.

FIG. 1D shows measurement of EPO expression from the PB1-MinTK construct after transfection into Huh7 cells and treatment with DMSO, 0.5. mu.M, 1. mu.M, 2. mu.M, and 3. mu.M CITCO. The figure also shows that addition of CITCO does not alter EPO expression of the promoter CMV-MP.

FIG. 2A shows the results of measurements of luciferase expression from PB1-MinTK, PB1-CMV-MP and PB1-SV40-MP constructs after transfection into AXOL ARE-hepatocytes and treatment with DMSO (left) or 1 μ M CITCO (right).

FIG. 2B shows the data from FIG. 2A, expressed as the ratio of the strong viral promoter CMV-IE.

FIG. 3A shows luciferase expression from PB1-SV40, PB1-1-SV40, PB1-2-SV40, and PB1-3-SV40 constructs containing 1, 2, 3, and 4 PBREM elements, respectively. The combination of PBREM multimers with the SV40 promoter was inducible and increased expression levels, but only 3 PBREM elements at the maximum.

FIG. 3B shows luciferase expression from PB1-CMV, PB1-1-CMV, PB1-2-CMV and PB1-3-CMV constructs containing 1, 2, 3 and 4 PBREM elements, respectively. The combination of PBREM multimers with CMV promoter is inducible and increases expression levels, but only 3 copies of PBREM elements at the maximum.

FIG. 3C shows luciferase expression from PB1-MinTK, PB1-1-MinTK, PB1-2-MinTK, and PB1-3-MinTK constructs containing 1, 2, 3, and 4 PBREM elements, respectively. The combination of PBREM multimers with the MinTK promoter was inducible and increased expression levels, but only 3 copies of PBREM elements were possible at the maximum.

FIG. 4A shows luciferase expression of PB1-MinTK construct in pGL4.10 vector in AXOL ARE-hepatocytes, PB1-MinTK construct in pAAV vector in Huh7 cells, and PB1-MinTK construct in pAAV vector in AXOL ARE-hepatocytes. The induction of luciferase expression from the PB1-MinTK construct was comparable in different vectors and cell types.

FIG. 4B shows luciferase expression of PB1-2-MinTK construct in pGL4.10 vector in AXOL ARE-hepatocytes, PB1-2-MinTK construct in pAAV vector in Huh7 cells, and PB1-2-MinTK construct in pAAV vector in AXOL ARE-hepatocytes. The induction of luciferase expression from the PB1-2-MinTK construct was comparable in different vectors and cell types.

FIG. 5 shows luciferase expression of pAAV vector in AXOL ARE-hepatocytes without CITCO, induction of CITCO (1 μ M) and withdrawal of CITCO for the PB1-MinTK and PB1-2-MinTK constructs.

FIG. 6 shows murine PBREM elements.

FIG. 7 shows a plasmid map of the pGL4.10 vector.

FIG. 8 shows the in vivo results for PB1 and PB1-2 constructs. a) Bioluminescence imaging of representative mice 0 to 48 hours post-induction, B) bioluminescence mapping to show different induction kinetics, C) fold induction observed for each construct (n ═ 5).

Figure 9 shows the effect of CITCO and flavones on the induction of PBREM hybrids in HUH7 cell line stably expressing human CARs.

Figure 10 shows the effect of CITCO and flavones on the induction of PBREM hybrids in primary hepatocytes.

Figure 11 shows the effect of CITCO and flavones on the induction of multimers of PBREM hybrids in Huh7 cell line stably expressing human CARs.

Figure 12 shows the effect of CITCO and flavones on the induction of multimers of PBREM hybrids in primary hepatocytes.

Detailed Description

While the making and using of various embodiments of the present invention are discussed in detail below, it should be appreciated that the present invention provides many applicable inventive concepts which can be embodied in a wide variety of specific contexts. The specific embodiments discussed herein are merely illustrative of specific ways to make and use the invention, and do not delimit the scope of the present invention.

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. These techniques are explained fully in the literature. For example, see Current Protocols in Molecular Biology (Ausubel,2000, Wiley and son Inc, Library of Congress, USA); molecular Cloning A Laboratory Manual, Third Edition, (Sambrook et al,2001, Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press); oligonucleotide Synthesis (m.j. gate ed., 1984); U.S. Pat. No.4,683,195; nucleic Acid Hybridization (Harries and Higgins eds. 1984); transformation and transformation (Hames and Higgins eds. 1984); culture of Animal Cells (Freshney, Alan R.Liss, Inc., 1987); immobilized Cells and Enzymes (IRL Press, 1986); perbal, A Practical Guide to Molecular Cloning (1984); the series, Methods in Enzymology (Abelson and Simon, eds. -in-coef, Academic Press, Inc., New York), specialty, Vols.154 and 155(Wu et al. eds.) and Vol.185, "Gene Expression Technology" (Goeddel, ed.); gene Transfer Vectors For Mammarian Cells (Miller and Calos eds.,1987, Cold Spring Harbor Laboratory); immunochemical Methods in Cell and Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); handbook of Experimental Immunology, Vols.I-IV (Weir and Blackwell, eds., 1986); and Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986).

The discussion of the background to the invention herein is included to explain the context of the invention. This is not to be taken as an admission that any of the material referred to was published, known or part of the common general knowledge in any country before the priority date of any claim.

Throughout this disclosure, various publications, patents, and published patent specifications are cited by identifying citations. All documents cited in this specification are herein incorporated by reference in their entirety. In particular, the teachings or sections of such documents specifically mentioned herein are incorporated by reference.

To facilitate an understanding of the present invention, a number of terms are defined below. Terms defined herein have meanings as commonly understood by one of ordinary skill in the art to which this invention pertains. Terms such as "a," "an," and "the" are not intended to refer to only a single entity, but include the general class of which a particular example may be used for illustration. The terms used herein are used to describe specific embodiments of the invention, but their use does not limit the invention unless outlined in the claims.

The term "cis-regulatory element" or "CRE" is a term well known to those skilled in the art and refers to a nucleic acid sequence, such as an enhancer, promoter, insulator or silencer, that is capable of regulating or modulating transcription of an adjacent gene (i.e., cis). CREs are found in the vicinity of the genes they regulate. CREs typically regulate gene transcription by binding to TF, i.e., they include TFBS. A single TF can bind many CREs, thereby controlling the expression of many genes (pleiotropic). CREs are usually, but not always, located upstream of the Transcription Start Site (TSS) of the genes they regulate. An "enhancer" is a CRE that enhances (i.e., upregulates) transcription of genes to which it is operably linked, and can be found upstream, downstream, or even in introns of the genes that they regulate. Multiple enhancers may act synergistically to regulate the transcription of a gene. A "silencer" as used herein is associated with CRE that binds to a TF called a repressor, which acts to prevent or down-regulate transcription of a gene. The term "silencer" can also refer to a region in the 3' untranslated region of messenger RNA that binds a protein that inhibits translation of the mRNA molecule, but this usage is different from its usage in describing CRE. Typically, the CRE of the invention is a liver-specific induction enhancer. In this context, CRE is preferably located 1500 nucleotides or less from the transcription start site, more preferably 1000 nucleotides or less from the transcription start site, more preferably 500 nucleotides or less from the transcription start site, suitably 250, 200, 150 or 100 nucleotides or less from the transcription start site. CRE of the invention is preferably relatively short in length, preferably 100 nucleotides or less in length, for example 90, 80, 70, 60 nucleotides or less in length.

The term "cis-regulatory module" or "CRM" refers to a functional module consisting of two or more CREs; in the present invention, CRE is typically a liver-specific inducible enhancer. Thus, in the present application, CRM typically includes multiple liver-specific inducible CREs. Typically, multiple CREs in CRM act together (e.g., additionally or synergistically) to enhance transcription of genes to which CRM is operably linked. In CRM, the spacing in CRE can be adjusted (i.e., reordered), reversed (i.e., reversed direction), and changed. Thus, functional variants of CRMs of the present invention include variants of reference CRMs in which the CREs within them have been adjusted and/or reversed, and/or the spacing between CREs has been altered.

As used herein, the term "promoter" refers to a region of DNA generally upstream of a nucleic acid sequence to be transcribed, which is required for transcription to occur, i.e., a region that initiates transcription. A promoter allows the transcription of a coding sequence to be appropriately activated or repressed under its control. Promoters generally comprise specific sequences that are recognized and bound by multiple transcription factors. TF binds to the promoter sequence, resulting in the recruitment of RNA polymerase, an enzyme that synthesizes RNA from the coding region of a gene. Many promoters are known in the art. Inducible promoters of the invention typically drive low levels of expression prior to being induced, and when induced, they drive significantly higher levels of expression (e.g., increased expression by 2, 3,4, 5,6, 7, 8, 9, or even 10-fold post-induction).

The promoter of the present invention is a synthetic promoter. The term "synthetic promoter" as used herein relates to a promoter that does not occur in nature. In this context, it typically comprises synthetic CRE and/or CRM of the invention operably linked to a minimal (or core) promoter or liver-specific proximal promoter. The CRE and/or CRM of the invention are useful for providing inducible liver-specific transcription of a gene operably linked to a promoter. Portions of a synthetic promoter may be naturally occurring (e.g., the minimal promoter or one or more CREs in the promoter), but the synthetic promoter is not naturally occurring as an entire entity.

As used herein, "minimal promoter" (also referred to as "core promoter") refers to a short DNA fragment that is inactive by itself or is largely inactive, but can mediate transcription when bound to other transcriptional regulatory elements. The minimal promoter sequence may be from a variety of different sources, including prokaryotic and eukaryotic genes. Examples of minimal promoters are described above, including the dopamine β -hydroxylase gene minimal promoter, Cytomegalovirus (CMV), early gene minimal promoter (CMV-MP), SV40 minimal promoter (SV40-MP), and herpes thymidine kinase minimal promoter (MinTK). However, the proximal promoter may be synthetic. A minimal promoter typically comprises a transcription initiation site (TSS) and elements immediately upstream, a binding site for RNA polymerase II, and a general transcription factor binding site (typically a TATA box).

As used herein, "proximal promoter" refers to the minimal promoter plus proximal sequences upstream of the gene that tend to contain primary regulatory elements. It typically extends about 250 base pairs upstream of the TSS and includes the particular TFBS. In the present case, the proximal promoter is suitably a naturally occurring liver-specific proximal promoter, which may be combined with one or more CRE or CRM of the present invention. However, the proximal promoter may be synthetic.

In the context of the present invention, a "functional variant" of a cis-regulatory element, cis-regulatory module, promoter or other nucleic acid sequence is a variant of a reference sequence that retains the ability to function in the same manner as the reference sequence, e.g., as an inducible liver-specific cis-regulatory enhancer element, an inducible liver-specific cis-regulatory module, or an inducible liver-specific promoter. Alternative terms for such functional variants include "biological equivalents" or "equivalents".

It will be appreciated that, as described above, the ability of a given cis-regulatory element to act as an inducible liver-specific enhancer is largely dependent on the ability of the sequence to bind to the CAR-RXR heterodimer and thereby induce expression. Thus, in most cases, functional variants of cis regulatory elements will comprise the appropriate binding site for the CAR-RXR heterodimer. CAR-RXR heterodimers are thought to bind to the NR1 motif in wild-type PBREM elements and therefore a sequence that can function as the NR1 motif is needed. Within the NR1 motif, there is a high degree of sequence conservation between mouse and human PBREM, and it is therefore generally desirable to maintain a high degree of identity to the NR1 motif in any functional variant. Additional sequences in wild-type PBREM elements may help to minimize background expression and provide a high level of inducibility, and therefore it is generally preferred that functional variants comprise at least some degree of sequence identity in these other regions. Thus, the level of sequence identity between a functional variant and a reference sequence can be an indicator or retained function. The high level of sequence identity in the NR1 motif of cis regulatory elements is generally more important than in other regions (e.g., NF1 and NR2, which have much less, if any, requirement for sequence conservation).

The ability of CAR-RXR heterodimers to bind to a given CRE can be determined by any relevant method known in the art, including, but not limited to, electrokinetic mobilization assay (EMSA), binding assay, chromatin immunoprecipitation (ChIP), and ChIP sequencing (ChIP-seq). In a preferred embodiment, the ability of the CAR-RXR heterodimer to bind to a given functional variant is determined by EMSA. Methods of performing EMSA are well known in the art. Suitable methods are described in the above-cited article by Sambrook et al. There are many related articles describing this process, such as Hellman and Fried, Nat protoc.2007; 2(8):1849-1861.

"liver-specific" or "liver-specific expression" refers to the ability of a cis regulatory element, module or promoter to enhance or drive expression of a gene in the liver (or liver-derived cells) in a preferential or predominant manner as compared to other tissues such as the spleen, muscle, heart, lung and brain. In the context of the present invention, expression should be inducible, i.e. expression of the gene will only occur or increase significantly when a suitable inducer (the inducer used in all aspects of the invention has been discussed above) is administered. The expression of the gene may be in the form of mRNA or protein. In a preferred embodiment, liver-specific expression is such that expression in other (i.e. non-liver) tissues or cells is negligible, i.e. expression is highly liver-specific.

The skilled person can easily assess the ability of the promoter to function as a liver-specific inducible promoter. Thus, the skilled person can readily determine whether any variant of a particular promoter exemplified herein is still functional (i.e. it is a functional variant as defined above). For example, any given CRE to be assessed may be operably linked to a minimal promoter (e.g., located upstream of MinTK) and the ability of cis regulatory elements to provide inducible liver-specific expression of genes (typically reporter genes) measured. Alternatively, variants of CRE can be replaced with inducible liver-specific promoters instead of reference CRE, and the effect on recognizable liver-specific expression driven by the modified promoters can be determined and compared to unmodified forms. Similarly, the skilled artisan can readily assess the ability of the promoter to induce liver-specific expression (e.g., as described in the examples below). The expression level and inducibility of a gene driven by a variant of a reference promoter can be compared to the expression level and inducibility of a reference sequence, and suitable methods are discussed above.

Liver specificity can be identified where a gene (e.g., a therapeutic gene or reporter gene) is preferentially or predominantly expressed in cells of hepatic origin when induced. Preferential or predominant expression may be defined, for example, when induced, expression levels in hepatogenic cells are significantly higher than in other types of cells (i.e., non-hepatogenic cells). For example, when induced, expression in a hepatogenic cell is suitably at least 5-fold higher than a non-hepatocyte, preferably at least 10-fold higher than a non-hepatocyte, and in some cases may be 50-fold or more higher. For convenience, liver-specific expression may be suitably demonstrated by comparing the expression levels in a liver cell line (e.g., a liver-derived cell line such as Huh7 and/or HepG2 cell) or a liver primary cell, with the expression levels in a kidney-derived cell line (e.g., HEK-293), a cervical tissue-derived cell line (e.g., HeLa), and/or a lung-derived cell line (e.g., a 549).

The liver-specific inducible promoter of the invention preferably has an expression level which is at least 4-fold lower than the CMV-IE promoter in cells of non-liver origin, suitably HEK-293, HeLa and/or A549 cells.

The liver-specific inducible promoter of the invention is preferably suitable for promoting expression in the liver of a subject, for example driving liver-specific expression of a transgene, preferably a therapeutic transgene.

It is also noted that in some cases, the liver-specific promoters of the present invention may be used in non-hepatocyte cells. Typically, non-liver cells do not express a CAR, and thus the promoter of the invention does not function in these cells. However, when non-hepatocytes are engineered to express a CAR (if they do not naturally express RXR, then RXR is expressed), such non-hepatocytes can induce expression of a gene linked to the promoter of the invention. In other words, in terms of CAR and RXR expression, the liver-specific promoter of the present invention may also function in non-hepatocytes that have been modified to have a liver-like phenotype. The term "liver-specific" should be construed accordingly. When hepatocytes do not express CAR (e.g., Huh-7 cell line) or RXR, they may likewise be modified to express the relevant protein.

The term "nucleic acid" as used herein generally refers to an oligomer or polymer (preferably a linear polymer) of any length consisting essentially of nucleotides. The nucleotide unit typically comprises a heterocyclic base, a sugar group and at least one, for example one, two or three, phosphate groups, including modified or substituted phosphate groups. Heterocyclic bases may include, inter alia, purine and pyrimidine bases, such as adenine (a), guanine (G), cytosine (C), thymine (T) and uracil (U), which are widely found 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 derivatized bases. The sugar groups may in particular comprise pentose (pentofuranose) groups, such as the preferred ribose and/or 2-deoxyribose, or arabinose, 2-deoxyarabinose, threose or hexose sugar groups, as well as modified or substituted sugar groups, which are common in naturally occurring nucleic acids. Nucleic acids contemplated 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 of 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, including specifically hnRNA, pre-mRNA, cDNA, genomic DNA, amplification products, oligonucleotides and synthetic (e.g., chemically synthesized) DNA, RNA or DNA RNA hybrids. Nucleic acids can be naturally occurring, e.g., occurring in or isolated from nature; or may be non-naturally occurring, e.g., recombinant, i.e., produced by recombinant DNA techniques, and/or partially or wholly chemically or biochemically synthesized. A "nucleic acid" may be double-stranded, partially double-stranded, or single-stranded. In the case of single strands, the nucleic acid may be the sense strand or the antisense strand. Furthermore, the nucleic acid may be circular or linear.

The terms "identity" and "identical" and the like refer to sequence similarity between two polymeric molecules, e.g., between two nucleic acid molecules, e.g., between two DNA molecules. Sequence alignments and determination of sequence identity can be determined, for example, using Basic Local Alignment Search Tool (BLAST), as originally described by Altschul et al 1990(J Mol Biol 215:403-10), such as the "BLAST 2 sequences" algorithm described by Tatusova and Madden 1999(FEMS Microbiol Lett 174: 247-250).

Methods for aligning sequences are well known in the art. Various programs and alignment algorithms are described, for example, in Smith and Waterman (1981) adv.Appl.Math.2: 482; needleman and Wunsch (1970) J.mol.biol.48: 443; pearson and Lipman (1988) Proc.Natl.Acad.Sci.U.S.A.85: 2444; higgins and Sharp (1988) Gene 73: 237-44; higgins and Sharp (1989) CABIOS 5: 151-3; corpet et al (1988) Nucleic Acids Res.16: 10881-90; huang et al (1992) Comp.appl.biosci.8: 155-65; pearson et al (1994) Methods mol. biol.24: 307-31; tatiana et al (1999) FEMS Microbiol. Lett.174: 247-50A derived registration of sequence alignment methods and homology calculations can be found as found in, e.g., Altschul et al (1990) J.mol.biol.215: 403-10.

National Center for Biotechnology Information (NCBI) basedThe Local Alignment Search Tool (BLAST)^TM(ii) a Altschul et al (1990)) are available from several sources, including the national center for biotechnology information (Bethesda, MD) and the internet, for use in conjunction with multiple sequence analysis programs. Instructions on how to use this program to determine sequence identity can be found in BLAST on the Internet^TMThe "help" section of (1). For comparison of nucleic acid sequences, BLAST may be utilized using default parameters^TMThe "Blast 2 sequences" function of the (Blastn) program. Nucleic acid sequences having greater similarity to a reference sequence will exhibit an increased percentage of identity when evaluated in this manner. Typically, percent sequence identity is calculated over the entire sequence length.

For example, a globally optimal alignment is suitably found by the Needleman-Wunsch algorithm with the following scoring parameters: matching score: +2, mismatch score: -3; gap penalties: the vacancy is open 5 and the vacancy is extended 2. The percent identity of the resulting optimal global alignment is suitably calculated by the ratio of the number of alignment bases to the total length of the alignment, where the length of the alignment includes matches and mismatches, multiplied by 100.

"synthetic" in this application refers to a nucleic acid molecule that does not occur in nature. The synthetic nucleic acid expression constructs of the invention are typically produced artificially by recombinant techniques. Such synthetic nucleic acids may comprise naturally occurring sequences (e.g., promoters, enhancers, introns, and other such regulatory sequences), but which are present in a non-naturally occurring environment. For example, a synthetic gene (or portion of a gene) typically comprises one or more nucleic acid sequences that are not substantially contiguous (chimeric sequences), and/or may include substitutions, insertions, and deletions, as well as combinations thereof.

"complementary" or "complementarity" as used herein refers to Watson-Crick base pairing of two nucleic acid sequences. For example, for the sequence 5 '-AGT-3', the complementary sequence 3 '-TCA-5' binds. Complementarity between two nucleic acid sequences may be "partial," in which only some bases bind to their complementary sequences, or it may be complete when each base in the sequence binds to its complementary base. The degree of complementarity between nucleic acid strands has a significant effect on the efficiency and strength of hybridization between nucleic acid strands.

"transfection" in this application refers broadly to any process by which nucleic acid is intentionally introduced into a cell and encompasses the introduction of viral and non-viral vectors, and also includes terms and processes of transformation, transduction, and the like. Examples include, but are not limited to: transfection with viral vectors; transforming with a plasmid vector; electroporation (Fromm et al (1986) Nature 319: 791-3); lipofection (Feigner et al (1987) Proc. Natl. Acad. Sci. USA 84: 7413-7); microinjection (Mueller et al (1978) Cell 15: 579-85); agrobacterium-mediated transfer (Fraley et al (1983) Proc. Natl. Acad. Sci. USA 80: 4803-7); direct DNA uptake; whisker-mediated transformation; and microjet bombardment (Klein et al (1987) Nature 327: 70).

As used herein, the phrase "transgene" refers to an exogenous nucleic acid sequence. In one example, a transgene is a gene encoding an industrially or pharmaceutically useful compound, or a gene encoding a desired trait. In yet another example, the transgene encodes an antisense nucleic acid sequence, wherein expression of the antisense nucleic acid sequence inhibits expression of the target nucleic acid sequence.

The term "vector" is well known in the art and as used herein refers to a nucleic acid molecule, e.g. a double stranded DNA, which may have inserted a nucleic acid sequence according to the invention. The vector is suitable for use in transporting the inserted nucleic acid molecule into a suitable host cell. The vector typically contains all the necessary elements to allow transcriptional insertion of the nucleic acid molecule, and preferably translation of the transcript into a polypeptide. The vector typically contains all the necessary elements such that, once the vector enters a host cell, the vector can replicate independently of, or in synchrony with, the host chromosomal DNA; several copies of the vector and its inserted nucleic acid molecule can be generated. The vector of the present invention may be an episomal vector (i.e., one that does not integrate into the genome of the host cell), or may be a vector that integrates into the genome of the host cell. This definition includes non-viral and viral vectors. Non-viral vectors include, but are not limited to, plasmid vectors (e.g., pMA-RQ, pUC vectors, bluescript vectors (pBS) and pBR322 or their derivatives without bacterial sequences (minicircles)), transposon-based vectors (e.g., PigyBac (PB) vectors or Sleeping Beauty (SB) vectors), and the like. Larger vectors such as artificial chromosomes (bacteria (BAC), Yeast (YAC) or Human (HAC)) can be used to accommodate the larger insert. Viral vectors are derived from viruses, including but not limited to retroviruses, lentiviruses, adeno-associated viruses, adenoviruses, herpes viruses, hepatitis viral vectors, and the like. Typically, but not necessarily, viral vectors are replication-defective in that they have lost the ability to propagate in a given cell because the viral genes necessary for replication have been eliminated from the viral vector. However, some viral vectors may also be adapted to replicate specifically in a given cell (e.g. a cancer cell) and are typically used to trigger cell-specific (oncolysis) lysis. Virosomes are non-limiting examples of vectors comprising both viral and non-viral elements, in particular they bind liposomes to inactivated HIV or influenza viruses (Yamada et al, 2003). Another example includes a viral vector mixed with a cationic lipid.

The terms "operably linked," "operably linked," or the equivalent as used herein, refer to the arrangement of various nucleic acid elements relative to one another such that the elements are functionally linked and capable of interacting in the desired manner. These elements may include, but are not limited to, promoters, enhancers and/or regulatory elements, polyadenylation sequences, one or more introns and/or exons, and coding sequences of the gene of interest to be expressed. When properly oriented or operably linked, the nucleic acid sequence elements act together to modulate the activity of each other and, ultimately, may affect the expression level of the expression product. Modulation refers to increasing, decreasing, or maintaining the level of activity 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 element can be represented by the number of nucleotides or base pairs inserted between the elements. As understood by those skilled in the art, operably linked means functionally active and is not necessarily associated with a naturally-located linkage. Indeed, when used in a nucleic acid expression cassette, the cis-regulatory element is typically located immediately upstream of the promoter (although this is usually the case, it should never be construed as limiting or excluding the location within the nucleic acid expression cassette), but this is not necessarily the case in vivo, e.g., regulatory element sequences naturally occurring downstream of a gene whose transcription is affected can function in the same manner when it is located upstream of the promoter. Thus, according to particular embodiments, the modulating or enhancing effect of the regulatory element is location-independent.

A "spacer" or "spacer" as used herein is a nucleic acid sequence that separates two functional nucleic acid sequences (e.g., TFBS, CRE, CRM, minimal promoter, etc.). It can have essentially any sequence so long as it does not prevent a functional nucleic acid sequence (e.g., a cis regulatory element) from functioning as desired (e.g., as may occur if it includes a silencing sequence, prevents binding of a desired transcription factor, etc.). Typically, it is non-functional in that it is present only to space adjacent functional nucleic acid sequences from each other.

The term "pharmaceutically acceptable" as used herein is consistent with the art and refers to being compatible with the other ingredients of the pharmaceutical composition and not deleterious to the recipient thereof.

"therapeutically effective amount" and similar phrases refer to a dose or plasma concentration that provides a particular pharmacological effect desired in a subject, for example, expression of a therapeutic gene in the liver. It is emphasized that a therapeutically effective amount is not always effective in treating the conditions described herein, even though such a dose is considered by those skilled in the art to be a therapeutically effective amount. The therapeutically effective amount may vary depending on the route and dosage form of administration, the age and weight of the subject, and/or the disease or condition being treated.

The term "treating" refers to reducing, ameliorating, or eliminating one or more signs, symptoms, or effects of a disease or condition.

The terms "individual", "subject" and "patient" are used interchangeably to refer to any individual subject suffering from a disease or condition in need of treatment. For the purposes of this disclosure, a subject may be a primate, preferably a human, or another mammal, such as a dog, cat, horse, pig, goat, or cow, among others.

Description of the technology

Nuclear receptors

Nuclear receptors play a crucial role in converting chemical changes in the cellular environment into transcriptional and biological changes. This function is crucial not only for maintaining cellular homeostasis, but also for maintaining homeostasis in the whole organism. Nuclear receptors are found only in metazoans, and vary widely in number between species, e.g., 48 in humans and 270 in c.

The importance and number of nuclear receptors has increased dramatically since their discovery, and these proteins are now considered to be a superfamily of proteins. This family includes receptors that bind to and respond to steroids, thyroid hormones, nutrients, and xenobiotics. Once bound by the ligand, the receptor undergoes a conformational change and binds to the DNA, thereby initiating or inhibiting gene expression. This ability to bind genomic DNA is critical for the function of the receptor and its importance in cell fate, ontogeny and metabolism. The number of ligands that have been shown to bind to nuclear receptors has increased and masked the function of cognate receptors. From endogenous hormones to vitamins and xenobiotics, their importance for cellular metabolism and homeostasis is emphasized. Due to the profound effects of these receptors on the transcriptome, they are excellent targets for drug therapy, with an estimated FDA-approved drug targeting nuclear receptors in about 13%.

Structure of the product

The mass of nuclear receptors is between 50-100kDa, and the mature polypeptide is divided into 5 domains:

A/B is highly variable between receptors. Contains an activation function 1(AF-1) which acts as a weak transcriptional activator in the absence of ligand but as a strong activator upon ligand binding. This is due to the interaction with AF-2 in the E domain.

C: a highly conserved domain containing 2 zinc fingers that bind to a DNA response element.

Flexible domain connecting LBD and DBD and allowing their interaction. Is important in cell trafficking and subcellular distribution

E, structure is highly conserved, but the sequence is only moderately conserved. Contains a ligand binding cavity and confers ligand specificity to the receptor. Forms a dimerization interface with DBD, and also binds coactivators and repressors. Contains activating factor 2(AF2), the action of which is dependent on ligand binding.

F, a highly variable C-terminal domain.

Mechanism of action

Nuclear receptors are classified into 4 classes according to their mechanism of action. The following is a summary of each type:

type I these receptors are found in the cytoplasm of cells in an inactive state. Binding of the ligand results in dissociation, homodimerization, translocation to the nucleus and binding to the receptor's DNA reactive motif of Heat Shock Proteins (HSPs). These receptors bind to a DNA motif consisting of 2¹/₂Sites consisting of variable length DNA (direct repeats 1-5(DR1-5)), where the second 1/2 site is the first inverted repeat. Some of these receptors bind to direct repeat sequences and can bind as monomers/dimers or, in the case of constitutive androstane receptors, as heterodimers to RXRs.

Type II these receptors, either inactive or active, are present in the nucleus. They usually bind to the RXR to DNA in the form of heterodimers. In the absence of a ligand, these receptors are often complexed with a co-inhibitory protein.

Type III-similar to type I receptors but only binds to direct repeats of the DNA sequence.

Type IV they can bind as dimeric monomers, but only a single DNA binding domain binds to a single half-site on DNA.

Constitutive Androstane Receptor (CAR)

Constitutive Androstane Receptor (CAR) -or nuclear receptor subfamily 1, group I, member 3-is a member of the nuclear receptor superfamily, expressed almost exclusively in hepatocytes. It is here that CARs act synergistically with another nuclear receptor, the Pregnane X Receptor (PXR), as sensors for endogenous and exogenous chemicals. Once bound by the activating substance, these receptors regulate the activity of a number of genes, including cytochrome p450s, and are therefore responsible for the metabolism and excretion of these compounds. It is through this binding and gene activation activity that CARs and PXRs play an important role in the detoxification of foreign chemicals in vivo.

The functions are as follows:

as mentioned above, CARs function as key regulators of xenobiotic and endogenous metabolism. It has 24 transcripts in the liver. It has been shown that some of these transcripts are responsible for low levels of constitutive activity of the receptor, while others are inducible. Constitutive activity is thought to be regulated by interaction with a transcriptional co-activator, such as steroid receptor co-activator 1(SRC 1). This activity can be inhibited by the binding of inverse agonists (e.g. androstanes).

Ligand activation:

inactive CARs are phosphorylated and are present in the cytoplasm of the cell. Here, it forms a complex with heat shock protein 90(hsp90) and Cytoplasmic CAR Retention Protein (CCRP), an association that allows the CAR to be retained in the cytoplasm and thus be inactive. This inactive CAR can be activated in 2 ways 1) directly by ligand binding to a mouse CAR ligand, e.g. by TCPOBOP, or 2) indirectly by phenobarbital. Both pathways result in dissociation of the CAR from the polyprotein complex and allow its transfer into the nucleus. In the nucleus, the CAR may act as a monomer, and may also form heterodimers with Retinoid X Receptors (RXRs). Nuclear CARs bind DNA at the phenobarbital response element (PBREM), through which CAR regulatory genes, such as the CYP2B, CYP2C, and CYP3A subfamilies, are activated.

Direct activation:

TCPOBOP is thought to bind directly to mouse CAR and induce its translocation to the nucleus. However, this chemical does not bind to human CAR, so in human studies, CITCO is an equivalent compound.

Indirect activation:

indirect activation of CARs by the anticonvulsant Phenobarbital (PB) is a well-established pattern of indirect CAR activation. Activation of the PB by phosphatase PP2A causes dephosphorylation of the CAR. The exact mechanism of PP2A activation is not clear, however, PB activates AMP-activated protein kinase, which may activate PP2A, which activated PP2A is then recruited by the polyprotein complex.

Another theory is that PB competes with Epidermal Growth Factor (EGF) for its receptor, the Epidermal Growth Factor Receptor (EGFR). Dissociation of epidermal growth factor and EGFR inactivates SRC1, which leads to dephosphorylation of the activated C kinase 1(RACK 1) receptor, stimulating PP 2A.

PBREM element:

the DNA binding site of the mouse CAR homolog was determined by Honkakoski et al (MOLECULAR PHARMACOLOGY,53: 597-. In this study, they found that RXR and CAR heterodimers bind to sites in the phenobarbital response-enhancing submodule (PBREM) of the cytochrome P-450 Cyp2b10 gene in response to phenobarbital induction. Expression of RXR and CAR in mammalian cell lines activates PBREM, indicating that CAR-RXR heterodimer is a trans-acting factor of the Cyp2b10 gene. It has also been shown that this heterodimer binds to 2 incomplete direct repeat-4 motifs, and that such motifs are conserved in humans. PBREM elements as shown in figure 6, NR1 and NR2 are nuclear receptor binding sites, incomplete direct repeats are shown in bold. NFI binding sites are shown. The CAR-RXR heterodimer appears to bind to the NR1 site.

Human PBREM elements have also been identified, Sueyoshi et al (j. bio. chem. vol.274,10, pp.6043-6046,1999).

The sequences and alignments of mouse and human PBREM elements are shown below, with the so-called NR1 motif underlined in each element (SEQ ID NO shown in parentheses):

it can be seen that there is a very high level of conservation in the NR1 motif, while there is a much lower level of identity in other regions.

The present invention is based on the surprising discovery that PBREM elements and functional variants thereof can be used to provide inducible expression, for example, when provided in a gene therapy vector. The present invention allows useful levels of inducibility and low levels of background (constitutive expression).

Example 1

The PBREM enhancer is used with the MinTK promoter to drive the expression of luciferase and EPO.

Material

The Huh7 cell is a human hepatocyte line

-DPBS: does not contain CaCl₂Free of MgCl₂(Gibco,14190-094)

-DMEM(Sigma,D6546)

-FBS(Sigma,F9665)

-Pen-Strep(Sigma,P4333)

-Promega Fugene-HD(E2311)

-TCPOBOP(Sigma,T2320)

-CITCO(Cayman Chemicals Company,16027)

pcDNA6 plasmid containing the beta-galactosidase gene, used as an internal control for transfection efficiency (Thermofisiher, V22020)

Mouse CAR expression plasmid from Joua et al, 2016(BioCat GmbH, EX-Z4288-M51-10-GC). This is because Huh7 lacks a CAR.

-beta-galactosidase substrate solution (Thermofoisher, 75707/75710)

-Pierce BCA kit (23225)

LARII (Dual-luciferase reporter 1000 assay System, Promega, E1980)

EPO ELISA kit (Abcam, ab119522)

The method comprises the following steps:

day 1

Cells seeded at a density of 25,000 cells/300. mu.l in 48-well plates

Day 2

On the day of transfection, the DNA to be transfected (CAR plasmid/PB 1-MinTK operably linked to luciferase or EPO/pcDNA6 plasmid for internal control) was diluted to 100 ng/. mu.l stock solution.

Transfection per 48 wells:

45ng of DNA (15 ng each of plasmid, pcDNA6, CAR and test plasmid) were mixed with 4.1. mu.l of Optimem medium.

Mix 0.5. mu.l of Fusion HD with 4. mu.l of Optimem medium.

The two solutions were mixed and incubated for 15 minutes at room temperature.

-then dropping the final solution into the wells.

3 hours after transfection, the inducer TCPOBOP was added to the appropriate wells at the indicated concentration.

Day 3

24 hours after induction, the medium is removed from the cells.

Cells washed once in 300. mu.l of DPBS

Lysis of cells with 100. mu.l of passive lysis buffer and incubation with shaking for 15 minutes

Sedimentation of cell debris by centrifugation of the plate for 1 minute at maximum speed in a bench top centrifuge

For luciferase, 10 μ Ι of sample are transferred into white 96-well plates and luminescence is measured by injection of 50 μ Ι of LARII substrate.

Beta-galactosidase activity was measured using 25. mu.l of lysate according to the manufacturer's instructions (mammalian. beta. -galactosidase assay kit, 75707/75710, Thermo Scientific). Transfer 25. mu.l of lysate to the well of a microwell plate and mix with 25. mu.l of beta-galactosidase assay reagent and equilibrate to room temperature. The mixture was incubated at 37 ℃ for 30 minutes and the absorbance was measured at 405 nm.

According to the manufacturer's instructions (Pierce)^TMBCA protein assay kit, 23225/23227, Thermo Scientific) protein concentration was measured using 25 μ Ι lysate. 25 μ l of lysate was transferred to the wells of the microplate and mixed with 200 μ l of working solution. The mixture was incubated at 37 ℃ for 30 minutes, cooled to room temperature, and then the absorbance was measured at around 562 nm. Protein concentration was calculated relative to a protein standard curve prepared from assay standards with known protein concentrations.

Luciferase readings were normalized to β -galactosidase and protein concentrations in lysates to produce normalized Relative Light Units (RLUs).

For comparison between experiments, the strength of the promoter was compared to the CMV-IE promoter, which drives the same gene as the construct comprising the PBREM, which was included in each experiment.

Transfection with PB1-MinTk-EPO was performed as described above, except that EPO was secreted into the medium. Therefore, the medium was collected and EPO concentration was measured using ELISA according to the manufacturer's instructions (ab119522 Erythropoetin (EPO) Human ELISA Kit, Abcam). 50 μ l of medium was transferred to prewashed microwell plates and mixed with 50 μ l of 1 Xbiotin-binding antibody. The mixture was incubated at room temperature for 1 hour. The wells were washed, 100. mu.l streptavidin-HRP was added and the plates were incubated for 15 minutes at room temperature. The wells were washed and then washed with 100. mu.l of TMB substrate solution. The mixture was incubated at room temperature for 10 minutes. The enzyme reaction was stopped by adding 100. mu.l of stop solution and the absorbance was read at 450 nm. EPO concentrations were calculated from EPO standard curves prepared from measurement standards of known EPO concentrations.

The PB1-MinTk construct was cloned into the vector pGL4.10 using KpnI and NcoI restriction sites. This places the promoter directly upstream of the firefly luciferase reporter gene. The PB1-MinTK construct contained a 51bp enhancer and a MinTK minimal promoter from the herpes thymidine kinase gene.

As a result:

luciferase expression of the PB1-MinTk construct in fig. 1A shows that the promoter has little measurable luciferase activity when the cells are treated with vector (DMSO). This activity corresponds to leaky expression of the promoter. Addition of 50nM or 150nM TCPOBOP resulted in strong induction of the promoter, measured as high as 6-fold. The addition of 250nM TCPOBOP did not elicit an abnormal response from the promoter, indicating that homeostatic control mechanisms may have been activated to prevent cell overload. Thus, the addition of TCPOBOP to Huh7 cells induced the PB1-MinTK construct. The induction is tunable, depending on the concentration of TCPOBOP, but decreases at higher concentrations.

In fig. 1B, luciferase expression from the PB1-MinTK construct was high in the presence of CAR and 150nM TCPOBOP as previously seen in fig. 1A. However, when cells were transfected with PB1-MinTK but without CAR, there was no measurable activity when 150nm TCPOBOP was added. This can be explained by TCPOBOP having no receptor bound to it when CAR is not present. Therefore, induction was CAR dependent and indicated to be a highly liver specific process.

Taken together, these results indicate that the PB1-MinTk construct can be induced by addition of TCPOBOP in Huh7 cells, that induction is regulatable, that induction is CAR dependent, and that expression from a single element is sufficient to drive 40% of CMV-IE gene expression.

The experimental data shown in FIG. 1A are presented in FIG. 1C as the ratio of luciferase expression from the strong viral promoter CMV-IE. This indicates that luciferase expression from one PBREM element linked to the MinTK promoter is sufficient to drive 40% of the luciferase expression of the CMV-IE promoter. Figure 1C also shows that in the absence of CAR, there is no luciferase expression from the PB1-MInTK construct.

The PB1-MinkTK promoter was then used to drive expression of the protein EPO, which was of therapeutic interest. Expression was driven in Huh7 cells transfected with the PB1-MinTK promoter operably linked to the EPO protein and the CAR-containing plasmid. Transfection was performed as described previously, but EPO expression was induced by the human CAR inducer CITCO.

Human CAR inducer CITCO was used instead of mouse inducer tcpopbo because tcpopbo does not activate human CAR. This means that TCPOBOP cannot be used for human cells, which are the ultimate goal of using the present invention. This was also done to ensure that the mouse PBREM elements could be activated by human CARs induced with the human CAR inducer CITCO.

Expression of EPO from the PB1-MinTK construct after transfection into Huh7 cells and treatment with DMSO, 0.5. mu.M, 1. mu.M, 2. mu.M, and 3. mu.M CITCO is shown in FIG. 1D. The figure shows that EPO expression is very low without drug addition, but EPO production is significantly increased with addition of maximum 2 μ M of CITCO. This again returns to the baseline activity at the highest concentration. The figure also shows that addition of CITCO does not alter EPO expression from the CMV-MP promoter. In this example, the total EPO expression level of PB1-MinTk was approximately 22% of the EPO expression of CMV-IE (data not shown). This is different from 40% of the CMV-IE previously observed in fig. 1C, since CITCO is a weaker inducer of CAR activity than TCPOBOP.

PB1-MinTk was used to successfully drive regulated expression of luciferase and EPO.

Example 2

The PBREM elements were then used in conjunction with two other minimal promoters to test for inducibility and expression. PBREM was cloned before CMV minimal promoter, MinTK promoter and SV40 minimal promoter and introduced into AXOL ARE-hepatocytes.

Axol Assay-Ready Expanded (ARE) hepatocytes ARE primary human hepatocytes that have been Expanded in vitro. Large batches (up to 2000 vials) are available, ensuring a reliable, ready-to-use and consistent source of primary hepatocytes. The ARE liver cells express CYP enzyme, have a metabolic function, ARE in a polarized state and can be infected by hepatitis C virus. AXOL ARE-hepatocytes express CAR without the need for transfection with a plasmid expressing CAR.

Materials:

AXOL assay-ready expanded (ARE) hepatocytes (Axol, ax3701)

ARE hepatocyte thawing medium (Axol, ax3705)

ARE maintenance medium (Axol, ax3710)

Virimer red transfection reagent (Lipocalys, VR04-02-15)

CITCO, luciferase, beta-galactosidase and BCA kit as described above

The method comprises the following steps:

ARE hepatocytes were cultured and transfected as described in the manufacturer's manual.

200,000 cells were seeded in 2ml of culture medium in collagen-coated 6-well plates. Cells were incubated at 37 ℃ and 5% CO₂The following incubations were carried out for 4 hours to obtain sufficient adhesion. 200 μ l of the transfection mixture containing the DNA to be transfected (PB1-MinTK/PB1-CMV/PB1-SV40 operably linked to luciferase and beta-galactosidase containing promoter) was added and the cells were incubated at 37 ℃ and 5% CO₂The following was incubated for 3 hours at 100rpm on an orbital shaker. 3 hours after transfection, CITCO was added as appropriateIn the hole of (a). Cells were incubated at 37 ℃ and 5% CO₂Overnight and in the morning the medium was replaced with fresh ARE hepatocyte maintenance medium. Readings were taken 24 hours after induction.

As a result:

luciferase expression from the PB1-MinTK, PB1-CMV-MP and PB1-SV40-MP constructs in FIG. 2A show that each minimal promoter supports expression from the PBREM element after addition of 1. mu.M CITCO. SV40 and MinTK induced approximately 7-fold, and CMV-IE promoter induced 2-fold. The expression levels of each construct of PB1-MinTK, PB1-CMV and PB1-SV40 were 20%, 10% and 55%, respectively, compared to CMV-IE. SV40 may drive the highest expression, but at the expense of a higher background level. The CMV minimal promoter showed little or no expression. From this data, the original MinTk construct can provide the best compromise between expression level, inducibility, and control stringency (i.e., minimizing background expression).

PBREM can be used in combination with various minimal promoters to drive inducible expression.

Example 3

This experiment was performed to examine whether the poly-NR binding site would subsequently increase the activity of the promoter. Thus, we cloned 2, 3 and 4 repeats of the PBREM element in front of the MinTk, CMV and SV40 minimal promoters. An important consideration here is the spacing between elements, where we follow the general rule of a spacing of 5, i.e., elements that are 5bp apart do not sterically hinder each other. Using internal knowledge, we cloned elements 20bp apart from previous inducible promoter designs. These multimers were cloned into the pGL4.10 plasmid described previously. These constructs were then tested in AXOL ARE hepatocytes as previously described.

MinTK promoter:

PB1-MinTK, PB1-1-MinTK, PB1-2-MinTK and PB1-3-MinTK contain 1, 2, 3 and 4 PBREM elements, respectively, in combination with the MinTK minimal promoter. Luciferase expression from the PB1-1-MinTK, PB1-2-MinTK, and PB1-3-MinTK constructs when induced with 1. mu.l of CITCO showed that the multimers were induced and increased expression levels in FIG. 3C. However, this increase in expression level was only observed in 3 copies of the PBREM element (PB1-2), as the addition of another element appears to have a detrimental effect on induction and expression levels. Each multimer was induced to CMV-IE at 1.5, 4.1 and 2.66, respectively. However, the induction level was similar to the original PB1-MinTK structure described here as PB 1. This is due to the increased background activity of the promoter. The results are expressed as a ratio to CMV-IE. The results are the average of 3 biological replicates.

SV40 minimal promoter:

PB1-SV40, PB1-1-SV40, PB1-2-SV40 and PB1-3-SV40 contain 1, 2, 3 and 4 elements of PBREM in combination with the SV40 minimal promoter, respectively. Luciferase expression from the PB1-1-SV40, PB1-2-SV40, and PB1-3-SV40 constructs showed that the multimers were induced and indeed increased expression levels when induced with 1 μ l of CITCO in FIG. 3A. However, as previously described, only an increase in the expression level of 3 copies of the PBREM element was observed, as the addition of another element appears to have a detrimental effect on induction and expression levels. Each multimer was induced to CMV-IE at 2.6, 3.6 and 2.57, respectively. The induction level was 6-fold higher than that seen with the MinTk minimal promoter, with induction levels up to 9-fold. Also, the background expression level was increased, but this was much lower than the CMV-MP promoter described below. The results are expressed as a ratio to CMV-IE. The results are the average of 3 biological replicates.

The CMV promoter:

PB1-CMV, PB1-1-CMV, PB1-2-CMV and PB1-3-CMV contain 1, 2, 3 and 4 PBREM elements, respectively, which bind to the CMV minimal promoter. Luciferase expression from PB1-1-CMV, PB1-2-CMV, and PB1-3-CMV constructs when induced with 1. mu.l of CITCO in FIG. 3B showed that multimers were induced and indeed increased expression levels. However, as previously described, this increase in expression level was only observed in 3 copies of the PBREM element, as the addition of another element did not appear to have an effect on induction and expression levels. Each multimer was induced to CMV-IE at 1.9, 2.67 and 2.67, respectively. The level of induction was 5-fold lower than that observed with the MinTk or SV40 minimal promoters, maximum. The use of the CMV minimal promoter appears to increase background expression levels to very high levels and thus it is likely to be the worst candidate to be evaluated.

Increasing the number of PBREM elements can increase the expression level to 3 PBREM elements. Further increasing PBREM number to 4 resulted in decreased luciferase expression.

Example 4

PB1-MinTK and PB1-2-MinTk were decided for in vivo studies according to example 3. To facilitate this, the above two constructs were cloned into pAAV vector (Takara, Clontech) to prepare AAV virus. The insert was cloned using restriction digestion of the pAAV plasmid and PCR amplification of the original pgl4.10 construct.

We investigated the effect of Inverted Terminal Repeats (ITRs) on promoter activity. This is because we observed interference of AAV ITRs among other projects. To this end, pAAV-PB1-MinTk and pAAV-PB1-2-MinTk were transfected into Huh7 and ARE primary cells and their activity was evaluated as described previously.

The results of these experiments can be seen in fig. 4A and 4B. These figures represent the average of 3 biological replications and indicate that ITRs do not affect promoter performance. Luciferase expression from the PB1-MinTK and PB1-2-MinTK constructs induced comparability between vector and cell type. These constructs were very robust and the plasmid backbone appeared to have no effect on activity.

Example 5

Luciferase expression of the PB1-MinTK and PB1-2-MinTK constructs in the pAAV vector in AXOL ARE hepatocytes was induced by CITCO (1. mu.M), but decreased after CITCO removal, as shown in FIG. 5. This indicates that removal of the drug reduced the activity of the promoter to almost baseline levels.

Example 6 in vivo experiments with AAV induced including PB1 and PB1-2

From the previous application, constructs PB1 (single mouse PBREM element and Min-TK promoter) and PB1-2(3 xPBREFEM and Min-TK promoter) were selected for in vivo testing in mice. This is done as follows.

The PB1 and PB1-2 AAV constructs from the previous applications, having the following sequences (SEQ ID NOs 49 and 50, table 3), were used to make AAV viruses.

Production of AAV

Day 1:

seeding HEK 293-AAV cells in 15cm plates. The day of transfection was 70-80% confluency-final volume on each plate: 15ml

Day 2: preparation of transfection mixture:

PDG9 (packaging plasmid of AAV 9) 10.5. mu.g/pHGTI (Ad. helper plasmid) 31.5. mu.g/vector plasmid 10.5. mu.g/prepared in serum without DMEM/Optimem

Transfer mix/plate PEI 125. mu.l/prepared in serum without DMEM/Optimem

-adding the DNA mixture to the transfection mixture. Mix and stand at room temperature for 15-20 minutes.

-mixing 3mlThe transfection mixture was added dropwise to each plate and gently dispersed. Incubate for 24 hours.

Day 3:

the medium was changed to 15ml DMEM and P/S and 2% FCS were added. The mixture was left to stand for 48 hours.

Day 5:

the supernatants were collected and stored in 50ml tubes, 25ml per tube. The storage was at-20 ℃.

Cells were harvested by adding 5ml of PBS to each plate → scraping and harvesting in 50ml tubes.

To wash the plate, add 1ml of PBS and collect.

Spin at 1500rpm for 5 minutes.

-removing the supernatant and resuspending the pelleted cells in 1 ml/plate TD lysis buffer → cell

-storage at-80 ℃.

Endonuclease treatment

Cell:

-freezing and thawing the pellet 5 times → about 20 minutes at 37 ℃ and then about 20 minutes in dry ice (or-80 ℃).

Addition of 25. mu.l/ml of 20% deoxycholate of cells (or 50. mu.l/ml of 10% deoxycholate).

Addition of 8. mu.l/ml cell of the benzolase.

Incubation at 37 ℃ for 30 minutes.

-rotation at 4Krpm for 30 minutes.

The supernatant was filtered using a 0.45 μ M filter.

Storage at 4 ℃ for up to 24 hours.

Supernatant fluid:

the benzoase was added at 2.5. mu.l/25 ml of supernatant.

Add 50. mu.l/25 ml of supernatant MgSO 4.

Incubation at 37 ℃ for 30 minutes.

-rotation at 4Krpm for 30 minutes.

The supernatant was filtered using a 0.45 μ M filter.

Storage at 4 ℃ for up to 24 hours.

HPLC purification

Put two lines in 20% ETOH → template → system clean

Place line A in PBS and line B in Glycine → template → System Wash

Placing the column in the machine → manual run → flow rate 5ml/min → run 25ml or until the UV line flattens.

FACS tubes (10 cells, more supernatant) are prepared by adding 30. mu.l/tube Tris → placing in a machine to collect the vector.

-placing the waste line in a separate test tube to enable it to pass through the column again.

Run the sample (low flow rate, depending on the speed of the system and the concentration of the sample; slower for cells and higher for supernatant).

After passing through the column, the waste was washed with PBS → flow rate 5ml/min until the UV line flattened.

Set → fraction size 1 ml; flow rate: 1 ml/min; concentration% B: 100% → run.

Start collection looking for peaks. Peaks indicate carrier purification. Test tubes containing these vectors were labeled.

-saving the program before exiting.

Wash with PBS → 75ml,5ml/min

Washing with Na3PO4 (storing the column therein) → 75ml,5ml/min

-removing the column and storing at 4 ℃

Washing the machine with PBS → template → system wash

Cleaning machine with 20% ETOH → template → system clean

Put both lines in 20% ETOH and then shut down

2L of PBS was added to the vat and placed in a dialysis cassette (Side-A-Lyzer; Thermo scientific) for perfusion.

Using a syringe and needle to collect the vector from the labeled FACS test tube and add to the dialysis cassette → remove excess air on the membrane, carefully place the rubber on top of the cassette and let it float in a bucket containing PBS → overnight at room temperature on a slow rotating rotor.

The next day:

preparation of the membranes by addition of 5ml PBS on the centrifuge filter (Amico Ultra 15; MERCK) → spinning at 4Krpm for 5 minutes

-removing excess PBS inside the membrane.

-removing the carrier from the cassette and loading onto the membrane → spinning at 4Krpm for 5 minutes

Washing the membrane several times with the carrier inside, and then collecting in a 2ml centrifuge tube filter 0.22. mu.M (Spin-X; COSTAR)

-rotation at 13Krpm for 3 minutes.

The filter was removed and aliquoted 1X100 l (for injection) and the remaining 10-2. mu.l.

-storage at-80 ℃.

Viral quantification using primers and probes for qRT-PCR and luciferase genes.

Mouse experiment

The AAV serotype chosen was AAV9, because it is chemotactic for most tissues and organs, so the specificity of our promoter can be understood (avoiding AAV chemotaxis problems). The output of the experiment is the luciferase activity measured visually on day 5 post injection with the first reading. Mice were then monitored weekly, and after 35 days, a baseline of induction profiles was established once the control vector AAV9 with CMVIE showed consistent stable results. An inducer is added at this point and measurements are taken before and after induction. See below for further details.

Mice:

adult (8 weeks old) CD1 male mice were injected with AAV9 vector via tail vein.

Total administration of 5 × 10 per mouse¹¹Number of copies per ml of vector genome.

Mice were imaged 5 days after injection. They were first anesthetized and received an intraperitoneal injection of fluorescein (300. mu.l stock solution of fluorescein 15 mg/ml). After 5 minutes, the mice were placed in the IVIS machine and images were taken.

Imaging:

the exposure time for the image is 1 second and 10 seconds.

Images taken once a week

Furthermore, mice were imaged daily for four days prior to administration of the inducing or repressing agent.

Induction:

the concentration of inducer (phenobarbital) was 5 mg/ml. Mice were intraperitoneally injected with 10. mu.l, i.e. 50 micrograms per mouse (approximately 30g per mouse weight).

Results

The results of the experiment are shown in FIG. 8. Figure 8A shows representative mice from each test construct. Here it can be seen that the expression of PB1 and PB1-2 is restricted to the liver, whereas the CMV-IE promoter is expressed in almost all tissues of the mouse. In addition, at 0 hours, PB1 and PB1-2 mice did not show luciferase gene expression, indicating that expression is tightly controlled. However, we can see that the expression of both PB1 and PB1-2 was increased when the inducer phenobarbital was added. There was some variation in the magnitude and duration of induction. For example, PB1 induced about 10-fold, reached maximal activity at 9 hours, induction was completed at 24 hours, whereas PB1-2 increased maximal activity about 50-fold at 24 hours, induction was not completed until 48 hours post-injection (fig. 8B and 8C). These data confirm the findings observed in model cell lines and further demonstrate the potential of this induction system for in vivo use. The system has low background and good inducibility, even though the inducer is applied at a dose 10 times less than the recommended human dose.

Example 7 variation of PBREM elements

As described above, the nuclear receptor CAR binds to human and mouse DNA sequences. As can be seen from the sequence alignment, there are some sequence differences between the species. The 51bp modular PBREM element itself can be divided into 3 different parts (see table below). 1) The NR1 region (containing NR1 elements) which is believed to be responsible for most of the induction activity, 2) the NF1 region (containing NF1 elements) which binds to other nuclear receptors and is likely to be responsible for reducing background levels in the absence of activated CARs, and 3) the NR2 region (containing NR2 elements) which is again involved in CAR induction activity.

The alignment and delineation of the components that make up the PBREM elements is shown below:

mouse TCTGTACTTTCCTGACCTTGGCACAGTGCCACCATCAACTTGCCTGACACC 51

Human ACTGTACTTTCCTGACCCTGAAGAGGTGGCAGCATGGACTTTCCTGAACCA 51

**************** ** *** ** *** **** ***** *

(mouse ═ SEQ ID NO:1, human ═ SEQ ID NO:2)

Table 4-mouse and human PBREM sequences:

in the previous examples, variants of the promoters used contained mouse PBREM elements. To confirm our expectation that human PBREM elements can also be used, we also evaluated human PBREM inducibility; this may be relevant as one goal of this project is to provide inducible promoters for human gene therapy, and human sequences may have some advantages. Furthermore, we also evaluated human and mouse hybrids of PBREM elements to determine if we could modulate or improve the inducibility of the promoter and the background level. This new combination is not found in any environment in nature and may have new characteristics, such as background and induction levels. The constructs were tested in the PGL4.10 backbone as described previously and are listed in table 2 below. They are human NR1x3-Min TK (3x human NR1 region with MinTK minimal promoter); human PBREM-minTK (human PBREM with minTK minimal promoter); hNR1-mNFI-hNR2-Min TK (human-mouse-human hybrid with MinTK minimal promoter); hNR1-mNFI mNR2-Min TK (human murine hybrid with MinTK minimal promoter); mNR1-hNFI-mNR2-Min TK (murine human murine hybrid with MinTK minimal promoter); mNR1-hNFI-hNR2-Min TK (murine human hybrid with MinTK minimal promoter); hPB-SV40 (human PBREM with SV40 minimal promoter); and MHM-SV40 (murine-murine hybrid with SV40 minimal promoter) -SEQ ID NOs: 59 to 66.

These constructs were tested in Huh7 stable cell line and primary hepatocytes as previously described. Furthermore, to test the constructs with the traditional human CAR activator CITCO, we also tested the natural compound flavone. This is a GRAS (generally regarded as safe) product that has been previously reported to activate human CARs and may be a useful drug for gene therapy applications due to its non-toxic nature and few side effects. It is predicted that CITCO is relatively unstable in vivo and has no data on its safety for use in humans, and is therefore preferred for tissue culture only. In addition, the in vivo results described above used phenobarbital as an inducer. In some cases, the use of phenobarbital may be undesirable, even if 10 times less than the recommended dose (which seems to be possible in view of the above data). Thus, flavones may be a more desirable inducer to alleviate any safety or regulatory issues.

The results of these experiments can be seen in fig. 9 and 10. These numbers show a comparison with the in vivo tested PB 1. Human PBREM (hpb) performed almost identically to mouse PBREM (PB1) in the stable CAR-expressing Huh7 cell line (fig. 9) and primary cells (fig. 10), indicating that they can be interchanged with Min-TK and SV40 minimal promoters. In the tested hybrids, all but MHM hybrids induced similar levels and similar backgrounds as PB 1. It has lower background and lower overall activity but good inducibility, suggesting that it may have more stringent expression control than the PB1 construct; this is independent of the minimal promoter environment, as its hybridization with Min-Tk and SV40 showed similar results. Levels of all constructs induced by flavones were similar to CITCO, indicating that this compound is indeed a useful inducer of PBREM constructs. One slight abnormality in these experiments was the construct consisting of 3x NR1 from human PBREM. It has a relatively high background and is induced only in stable cell lines, and less in primary cells.

From these experiments, a method similar to the above example was applied. The best monomers, human PBREM and MHM hybrid, with Min-Tk minimal promoter and SV40 minimal promoter for multimerization and testing. The sequences of these multimers are shown in Table 3. The promoter is 2xhPB SV40 (2 x human PBREM element with SV40 minimal promoter); 2 xmmhm-MinTK (2 x murine human murine heterozygote with MinTK minimal promoter); 2xMHM-SV40 (2 x murine-murine hybrid with SV40 minimal promoter); 3xhPB minTK (3x human PBREM element with minTK minimal promoter); and xhPB-SV40 (3x human PBREM element with SV40 minimal promoter) -SEQ ID NOs: 67 to 71, respectively.

As previously described, in Huh7 CAR and primary hepatocytes expressing stable cell lines, expression and induction of the promoter was assessed using CITCO and flavones.

The results are shown in FIGS. 11 and 12. The comparison here is PB1-2 from the earlier example. It can be observed that both compounds are highly induced by multimeric promoters and that they have a very low background regardless of the minimal promoter used. Furthermore, 2xMHM and 3xhPB performed best, with overall expression levels higher than our observed PB1-2 and comparable background levels. This suggests that some of the new promoters may perform better in vivo and also provide more options for controlling gene expression as desired.

Sequence of

TABLE 1 PBREM elements, variants and parts thereof

The NR1 motif is underlined, the NF1 motif is bolded, and the NF2 motif is double underlined.

TABLE 2 inducible promoter containing mouse PBREM elements

PBREM element is underlined; the minimal promoter is in bold; the gene sequence is double underlined.

Table 3-constructs and other sequences.

Sequence listing

<110> Sepoproca, Inc

<120> liver-specific inducible promoter and method of use thereof

<130> P265002WO

<150> GB1900741.8

<151> 2019-01-18

<160> 91

<170> PatentIn version 3.5

<210> 1

<211> 51

<212> DNA

<213> muscle of mouse

<400> 1

tctgtacttt cctgaccttg gcacagtgcc accatcaact tgcctgacac c 51

<210> 2

<211> 51

<212> DNA

<213> Intelligent people

<400> 2

actgtacttt cctgaccctg aagaggtggc agcatggact ttcctgaacc a 51

<210> 3

<211> 16

<212> DNA

<213> muscle of mouse

<400> 3

tgtactttcc tgacct 16

<210> 4

<211> 16

<212> DNA

<213> Intelligent people

<400> 4

tgtactttcc tgaccc 16

<210> 5

<211> 84

<212> DNA

<213> Artificial sequence

<220>

<223> 3x NR1 sequence (mouse)

<400> 5

gatctctgta ctttcctgac cttggatcga tctctgtact ttcctgacct tggatcgatc 60

tctgtacttt cctgaccttg gatc 84

<210> 6

<211> 84

<212> DNA

<213> Artificial sequence

<220>

<223> 3x NR1 sequence (human)

<400> 6

gatcactgta ctttcctgac cctggatcga tcactgtact ttcctgaccc tggatcgatc 60

actgtacttt cctgaccctg gatc 84

<210> 7

<211> 262

<212> DNA

<213> Artificial sequence

<220>

<223> PB1-MinTK

<400> 7

tctgtacttt cctgaccttg gcacagtgcc accatcaact tgcctgacac cgatccggcc 60

ccgcccagcg tcttgtcatt ggcgaattcg aacacgcaga tgcagtcggg gcggcgcggt 120

ccgaggtcca cttcgcatat taaggtgacg cgtgtggcct cgaacaccga gcgaccctgc 180

agcgacccgc ttaacagcgt caacagcgtg ccgcagatct cgaggagctt ggcgagattt 240

tcaggagcta aggaagctaa ac 262

<210> 8

<211> 180

<212> DNA

<213> Artificial sequence

<220>

<223> PB1-CMV-MP

<400> 8

tctgtacttt cctgaccttg gcacagtgcc accatcaact tgcctgacac cgctgggagt 60

tcgtagacgg agcgattaat ccatatgcag gtctatataa gcagagctcg tttagtgaac 120

cgtcagatcg cctagatacg ccatccacgc tgttttgacc tccatagaag atcgccaccc 180

<210> 9

<211> 330

<212> DNA

<213> Artificial sequence

<220>

<223> PB1-SV40-MP

<400> 9

tctgtacttt cctgaccttg gcacagtgcc accatcaact tgcctgacac cgctgggagt 60

tcgtagacgg actagcccgg gctcgagatc tgcgatctgc atctcaatta gtcagcaacc 120

atagtcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc cgcccattct 180

ccgccccatc gctgactaat tttttttatt tatgcagagg ccgaggccgc ctcggcctct 240

gagctattcc agaagtagtg aggaggcttt tttggaggcc taggcttttg caaaaagctt 300

ggcattccgg tactgttggt aaagccaccc 330

<210> 10

<211> 353

<212> DNA

<213> Artificial sequence

<220>

<223> PB1-1-MinTk (2xPBREM)

<400> 10

tctgtacttt cctgaccttg gcacagtgcc accatcaact tgcctgacac ccattactcg 60

catccattct ctctgtactt tcctgacctt ggcacagtgc caccatcaac ttgcctgaca 120

ccgctgggag ttcgtagacg gagatccggc cccgcccagc gtcttgtcat tggcgaattc 180

gaacacgcag atgcagtcgg ggcggcgcgg tccgaggtcc acttcgcata ttaaggtgac 240

gcgtgtggcc tcgaacaccg agcgaccctg cagcgacccg cttaacagcg tcaacagcgt 300

gccgcagatc tcgaggagct tggcgagatt ttcaggagct aaggaagcta aac 353

<210> 11

<211> 424

<212> DNA

<213> Artificial sequence

<220>

<223> PB1-2-MinTk (3xPBREM)

<400> 11

tctgtacttt cctgaccttg gcacagtgcc accatcaact tgcctgacac ccattactcg 60

catccattct ctctgtactt tcctgacctt ggcacagtgc caccatcaac ttgcctgaca 120

ccgcactgaa ggtcctcaat cgtctgtact ttcctgacct tggcacagtg ccaccatcaa 180

cttgcctgac accgctggga gttcgtagac ggagatccgg ccccgcccag cgtcttgtca 240

ttggcgaatt cgaacacgca gatgcagtcg gggcggcgcg gtccgaggtc cacttcgcat 300

attaaggtga cgcgtgtggc ctcgaacacc gagcgaccct gcagcgaccc gcttaacagc 360

gtcaacagcg tgccgcagat ctcgaggagc ttggcgagat tttcaggagc taaggaagct 420

aaac 424

<210> 12

<211> 495

<212> DNA

<213> Artificial sequence

<220>

<223> PB1-3-MinTk

<400> 12

tctgtacttt cctgaccttg gcacagtgcc accatcaact tgcctgacac ccattactcg 60

catccattct ctctgtactt tcctgacctt ggcacagtgc caccatcaac ttgcctgaca 120

ccgcactgaa ggtcctcaat cgtctgtact ttcctgacct tggcacagtg ccaccatcaa 180

cttgcctgac accctgacct cctgccagca atatctgtac tttcctgacc ttggcacagt 240

gccaccatca acttgcctga caccgctggg agttcgtaga cggagatccg gccccgccca 300

gcgtcttgtc attggcgaat tcgaacacgc agatgcagtc ggggcggcgc ggtccgaggt 360

ccacttcgca tattaaggtg acgcgtgtgg cctcgaacac cgagcgaccc tgcagcgacc 420

cgcttaacag cgtcaacagc gtgccgcaga tctcgaggag cttggcgaga ttttcaggag 480

ctaaggaagc taaac 495

<210> 13

<211> 401

<212> DNA

<213> Artificial sequence

<220>

<223> PB1-1-SV40

<400> 13

tctgtacttt cctgaccttg gcacagtgcc accatcaact tgcctgacac ccattactcg 60

catccattct ctctgtactt tcctgacctt ggcacagtgc caccatcaac ttgcctgaca 120

ccgctgggag ttcgtagacg gactagcccg ggctcgagat ctgcgatctg catctcaatt 180

agtcagcaac catagtcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt 240

ccgcccattc tccgccccat cgctgactaa ttttttttat ttatgcagag gccgaggccg 300

cctcggcctc tgagctattc cagaagtagt gaggaggctt ttttggaggc ctaggctttt 360

gcaaaaagct tggcattccg gtactgttgg taaagccacc c 401

<210> 14

<211> 472

<212> DNA

<213> Artificial sequence

<220>

<223> PB1-2-SV40

<400> 14

tctgtacttt cctgaccttg gcacagtgcc accatcaact tgcctgacac ccattactcg 60

catccattct ctctgtactt tcctgacctt ggcacagtgc caccatcaac ttgcctgaca 120

ccgcactgaa ggtcctcaat cgtctgtact ttcctgacct tggcacagtg ccaccatcaa 180

cttgcctgac accgctggga gttcgtagac ggactagccc gggctcgaga tctgcgatct 240

gcatctcaat tagtcagcaa ccatagtccc gcccctaact ccgcccatcc cgcccctaac 300

tccgcccagt tccgcccatt ctccgcccca tcgctgacta atttttttta tttatgcaga 360

ggccgaggcc gcctcggcct ctgagctatt ccagaagtag tgaggaggct tttttggagg 420

cctaggcttt tgcaaaaagc ttggcattcc ggtactgttg gtaaagccac cc 472

<210> 15

<211> 543

<212> DNA

<213> Artificial sequence

<220>

<223> PB1-3-SV40

<400> 15

tctgtacttt cctgaccttg gcacagtgcc accatcaact tgcctgacac ccattactcg 60

catccattct ctctgtactt tcctgacctt ggcacagtgc caccatcaac ttgcctgaca 120

ccgcactgaa ggtcctcaat cgtctgtact ttcctgacct tggcacagtg ccaccatcaa 180

cttgcctgac accctgacct cctgccagca atatctgtac tttcctgacc ttggcacagt 240

gccaccatca acttgcctga caccgctggg agttcgtaga cggactagcc cgggctcgag 300

atctgcgatc tgcatctcaa ttagtcagca accatagtcc cgcccctaac tccgcccatc 360

ccgcccctaa ctccgcccag ttccgcccat tctccgcccc atcgctgact aatttttttt 420

atttatgcag aggccgaggc cgcctcggcc tctgagctat tccagaagta gtgaggaggc 480

ttttttggag gcctaggctt ttgcaaaaag cttggcattc cggtactgtt ggtaaagcca 540

ccc 543

<210> 16

<211> 251

<212> DNA

<213> Artificial sequence

<220>

<223> PB1-1-CMV

<400> 16

tctgtacttt cctgaccttg gcacagtgcc accatcaact tgcctgacac ccattactcg 60

catccattct ctctgtactt tcctgacctt ggcacagtgc caccatcaac ttgcctgaca 120

ccgctgggag ttcgtagacg gagcgattaa tccatatgca ggtctatata agcagagctc 180

gtttagtgaa ccgtcagatc gcctagatac gccatccacg ctgttttgac ctccatagaa 240

gatcgccacc c 251

<210> 17

<211> 322

<212> DNA

<213> Artificial sequence

<220>

<223> PB1-2-CMV

<400> 17

tctgtacttt cctgaccttg gcacagtgcc accatcaact tgcctgacac ccattactcg 60

catccattct ctctgtactt tcctgacctt ggcacagtgc caccatcaac ttgcctgaca 120

ccgcactgaa ggtcctcaat cgtctgtact ttcctgacct tggcacagtg ccaccatcaa 180

cttgcctgac accgctggga gttcgtagac ggagcgatta atccatatgc aggtctatat 240

aagcagagct cgtttagtga accgtcagat cgcctagata cgccatccac gctgttttga 300

cctccataga agatcgccac cc 322

<210> 18

<211> 393

<212> DNA

<213> Artificial sequence

<220>

<223> PB1-3-CMV

<400> 18

tctgtacttt cctgaccttg gcacagtgcc accatcaact tgcctgacac ccattactcg 60

catccattct ctctgtactt tcctgacctt ggcacagtgc caccatcaac ttgcctgaca 120

ccgcactgaa ggtcctcaat cgtctgtact ttcctgacct tggcacagtg ccaccatcaa 180

cttgcctgac accctgacct cctgccagca atatctgtac tttcctgacc ttggcacagt 240

gccaccatca acttgcctga caccgctggg agttcgtaga cggagcgatt aatccatatg 300

caggtctata taagcagagc tcgtttagtg aaccgtcaga tcgcctagat acgccatcca 360

cgctgttttg acctccatag aagatcgcca ccc 393

<210> 19

<211> 1023

<212> DNA

<213> Artificial sequence

<220>

<223> PB1-MinTK and EPO

<400> 19

ggcctaactg gccggtactc tgtactttcc tgaccttggc acagtgccac catcaacttg 60

cctgacaccg atccggcccc gcccagcgtc ttgtcattgg cgaattcgaa cacgcagatg 120

cagtcggggc ggcgcggtcc gaggtccact tcgcatatta aggtgacgcg tgtggcctcg 180

aacaccgagc gaccctgcag cgacccgctt aacagcgtca acagcgtgcc gcagatctcg 240

aggagcttgg cgagattttc aggagctaag gaagctaaac atgggggtgc acgaatgtcc 300

tgcctggctg tggcttctcc tgtccctgct gtcgctccct ctgggcctcc cagtcctggg 360

cgccccacca cgcctcatct gtgacagccg agtcctggag aggtacctct tggaggccaa 420

ggaggccgag aatatcacga cgggctgtgc tgaacactgc agcttgaatg agaatatcac 480

tgtcccagac accaaagtta atttctatgc ctggaagagg atggaggtcg ggcagcaggc 540

cgtagaagtc tggcagggcc tggccctgct gtcggaagct gtcctgcggg gccaggccct 600

gttggtcaac tcttcccagc cgtgggagcc cctgcagctg catgtggata aagccgtcag 660

tggccttcgc agcctcacca ctctgcttcg ggctctggga gcccagaagg aagccatctc 720

ccctccagat gcggcctcag ctgctccact ccgaacaatc actgctgaca ctttccgcaa 780

actcttccga gtctactcca atttcctccg gggaaagctg aagctgtaca caggggaggc 840

ctgcaggaca ggggacagat gatctagagt cggggcggcc ggccgcttcg agcagacatg 900

ataagataca ttgatgagtt tggacaaacc acaactagaa tgcagtgaaa aaaatgcttt 960

atttgtgaaa tttgtgatgc tattgcttta tttgtaacca ttataagctg caataaacaa 1020

gtt 1023

<210> 20

<211> 16

<212> DNA

<213> Artificial sequence

<220>

<223> NR1 motif from PBREM

<220>

<221> misc_feature

<222> (16)..(16)

<223> n is a, c, g, or t

<400> 20

tgtactttcc tgaccn 16

<210> 21

<211> 17

<212> DNA

<213> Artificial sequence

<220>

<223> sequences preferably comprised in functional variants of PBREM elements

<220>

<221> misc_feature

<222> (17)..(17)

<223> n is a, c, g, or t

<400> 21

ctgtactttc ctgaccn 17

<210> 22

<211> 17

<212> DNA

<213> Artificial sequence

<220>

<223> sequences preferably comprised in functional variants of PBREM elements

<400> 22

ctgtactttc ctgaccy 17

<210> 23

<211> 17

<212> DNA

<213> Artificial sequence

<220>

<223> sequences preferably comprised in functional variants of PBREM elements

<400> 23

ctgtactttc ctgacct 17

<210> 24

<211> 17

<212> DNA

<213> Artificial sequence

<220>

<223> sequences preferably comprised in functional variants of PBREM elements

<400> 24

ctgtactttc ctgaccc 17

<210> 25

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> sequences suitably comprised in functional variants of the PBREM sequences

<220>

<221> misc_feature

<222> (1)..(1)

<223> n is a, c, g, or t

<220>

<221> misc_feature

<222> (18)..(18)

<223> n is a, c, g, or t

<400> 25

nctgtacttt cctgaccntg 20

<210> 26

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> sequences suitably comprised in functional variants of the PBREM sequences

<400> 26

wctgtacttt cctgaccytg 20

<210> 27

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> sequences suitably comprised in functional variants of the PBREM sequences

<400> 27

tctgtacttt cctgaccttg 20

<210> 28

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> sequences suitably comprised in functional variants of the PBREM sequences

<400> 28

actgtacttt cctgaccctg 20

<210> 29

<211> 16

<212> DNA

<213> Artificial sequence

<220>

<223> sequences suitably comprised in functional variants of the PBREM sequences

<220>

<221> misc_feature

<222> (16)..(16)

<223> optional spacer

<220>

<221> misc_feature

<222> (16)..(16)

<223> n is a, c, g, or t

<400> 29

tgtactttcc tgaccn 16

<210> 30

<211> 17

<212> DNA

<213> Artificial sequence

<220>

<223> sequences suitably comprised in functional variants of the PBREM sequences

<220>

<221> misc_feature

<222> (17)..(17)

<223> optional spacer

<220>

<221> misc_feature

<222> (17)..(17)

<223> n is a, c, g, or t

<400> 30

ctgtactttc ctgaccn 17

<210> 31

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> sequences suitably comprised in functional variants of the PBREM sequences

<220>

<221> misc_feature

<222> (1)..(1)

<223> n is a, c, g, or t

<220>

<221> misc_feature

<222> (18)..(18)

<223> n is a, c, g, or t

<220>

<221> misc_feature

<222> (20)..(20)

<223> optional spacer

<400> 31

nctgtacttt cctgaccntg 20

<210> 32

<211> 32

<212> DNA

<213> Artificial sequence

<220>

<223> sequences suitably comprised in functional variants of the PBREM sequences

<220>

<221> misc_feature

<222> (16)..(17)

<223> optional spacer

<220>

<221> misc_feature

<222> (16)..(16)

<223> n is a, c, g, or t

<220>

<221> misc_feature

<222> (32)..(32)

<223> n is a, c, g, or t

<400> 32

tgtactttcc tgaccntgta ctttcctgac cn 32

<210> 33

<211> 48

<212> DNA

<213> Artificial sequence

<220>

<223> sequences suitably comprised in functional variants of the PBREM sequences

<220>

<221> misc_feature

<222> (16)..(17)

<223> optional spacer

<220>

<221> misc_feature

<222> (16)..(16)

<223> n is a, c, g, or t

<220>

<221> misc_feature

<222> (32)..(32)

<223> n is a, c, g, or t

<220>

<221> misc_feature

<222> (32)..(33)

<223> optional spacer

<220>

<221> misc_feature

<222> (48)..(48)

<223> n is a, c, g, or t

<400> 33

tgtactttcc tgaccntgta ctttcctgac cntgtacttt cctgaccn 48

<210> 34

<211> 34

<212> DNA

<213> Artificial sequence

<220>

<223> sequences suitably comprised in functional variants of the PBREM sequences

<220>

<221> misc_feature

<222> (17)..(18)

<223> optional spacer

<220>

<221> misc_feature

<222> (17)..(17)

<223> n is a, c, g, or t

<220>

<221> misc_feature

<222> (34)..(34)

<223> n is a, c, g, or t

<400> 34

ctgtactttc ctgaccnctg tactttcctg accn 34

<210> 35

<211> 51

<212> DNA

<213> Artificial sequence

<220>

<223> sequences suitably comprised in functional variants of the PBREM sequences

<220>

<221> misc_feature

<222> (17)..(18)

<223> optional spacer

<220>

<221> misc_feature

<222> (17)..(17)

<223> n is a, c, g, or t

<220>

<221> misc_feature

<222> (34)..(34)

<223> n is a, c, g, or t

<220>

<221> misc_feature

<222> (34)..(35)

<223> optional spacer

<220>

<221> misc_feature

<222> (51)..(51)

<223> n is a, c, g, or t

<400> 35

ctgtactttc ctgaccnctg tactttcctg accnctgtac tttcctgacc n 51

<210> 36

<211> 40

<212> DNA

<213> Artificial sequence

<220>

<223> sequences suitably comprised in functional variants of the PBREM sequences

<220>

<221> misc_feature

<222> (1)..(1)

<223> n is a, c, g, or t

<220>

<221> misc_feature

<222> (18)..(18)

<223> n is a, c, g, or t

<220>

<221> misc_feature

<222> (20)..(21)

<223> optional spacer

<220>

<221> misc_feature

<222> (21)..(21)

<223> n is a, c, g, or t

<220>

<221> misc_feature

<222> (38)..(38)

<223> n is a, c, g, or t

<400> 36

nctgtacttt cctgaccntg nctgtacttt cctgaccntg 40

<210> 37

<211> 60

<212> DNA

<213> Artificial sequence

<220>

<223> sequences suitably comprised in functional variants of the PBREM sequences

<220>

<221> misc_feature

<222> (1)..(1)

<223> n is a, c, g, or t

<220>

<221> misc_feature

<222> (18)..(18)

<223> n is a, c, g, or t

<220>

<221> misc_feature

<222> (20)..(21)

<223> optional spacer

<220>

<221> misc_feature

<222> (21)..(21)

<223> n is a, c, g, or t

<220>

<221> misc_feature

<222> (38)..(38)

<223> n is a, c, g, or t

<220>

<221> misc_feature

<222> (40)..(41)

<223> optional spacer

<220>

<221> misc_feature

<222> (41)..(41)

<223> n is a, c, g, or t

<220>

<221> misc_feature

<222> (58)..(58)

<223> n is a, c, g, or t

<400> 37

nctgtacttt cctgaccntg nctgtacttt cctgaccntg nctgtacttt cctgaccntg 60

<210> 38

<211> 60

<212> DNA

<213> Artificial sequence

<220>

<223> sequences suitably comprised in functional variants of the PBREM sequences

<220>

<221> misc_feature

<222> (20)..(21)

<223> optional spacer

<220>

<221> misc_feature

<222> (40)..(41)

<223> optional spacer

<400> 38

tctgtacttt cctgaccttg tctgtacttt cctgaccttg tctgtacttt cctgaccttg 60

<210> 39

<211> 60

<212> DNA

<213> Artificial sequence

<220>

<223> sequences suitably comprised in functional variants of the PBREM sequences

<220>

<221> misc_feature

<222> (20)..(21)

<223> optional spacer

<220>

<221> misc_feature

<222> (40)..(41)

<223> optional spacer

<400> 39

actgtacttt cctgaccctg actgtacttt cctgaccctg actgtacttt cctgaccctg 60

<210> 40

<211> 8

<212> DNA

<213> Artificial sequence

<220>

<223> spacer

<400> 40

gatcgatc 8

<210> 41

<211> 51

<212> DNA

<213> Artificial sequence

<220>

The <223> sequence is preferably comprised in a functional variant of the PBREM element

<220>

<221> misc_feature

<222> (1)..(1)

<223> n is a, c, g, or t

<220>

<221> misc_feature

<222> (18)..(18)

<223> n is a, c, g, or t

<220>

<221> misc_feature

<222> (21)..(25)

<223> n is a, c, g, or t

<220>

<221> misc_feature

<222> (29)..(29)

<223> n is a, c, g, or t

<220>

<221> misc_feature

<222> (32)..(32)

<223> n is a, c, g, or t

<220>

<221> misc_feature

<222> (36)..(37)

<223> n is a, c, g, or t

<220>

<221> misc_feature

<222> (42)..(42)

<223> n is a, c, g, or t

<220>

<221> misc_feature

<222> (48)..(49)

<223> n is a, c, g, or t

<220>

<221> misc_feature

<222> (51)..(51)

<223> n is a, c, g, or t

<400> 41

nctgtacttt cctgaccntg nnnnngtgnc ancatnnact tncctgannc n 51

<210> 42

<211> 102

<212> DNA

<213> Artificial sequence

<220>

<223> CRM

<220>

<221> misc_feature

<222> (1)..(1)

<223> n is a, c, g, or t

<220>

<221> misc_feature

<222> (18)..(18)

<223> n is a, c, g, or t

<220>

<221> misc_feature

<222> (21)..(25)

<223> n is a, c, g, or t

<220>

<221> misc_feature

<222> (29)..(29)

<223> n is a, c, g, or t

<220>

<221> misc_feature

<222> (32)..(32)

<223> n is a, c, g, or t

<220>

<221> misc_feature

<222> (36)..(37)

<223> n is a, c, g, or t

<220>

<221> misc_feature

<222> (42)..(42)

<223> n is a, c, g, or t

<220>

<221> misc_feature

<222> (48)..(49)

<223> n is a, c, g, or t

<220>

<221> misc_feature

<222> (51)..(52)

<223> optional spacer

<220>

<221> misc_feature

<222> (51)..(51)

<223> n is a, c, g, or t

<220>

<221> misc_feature

<222> (52)..(52)

<223> n is a, c, g, or t

<220>

<221> misc_feature

<222> (69)..(69)

<223> n is a, c, g, or t

<220>

<221> misc_feature

<222> (72)..(76)

<223> n is a, c, g, or t

<220>

<221> misc_feature

<222> (80)..(80)

<223> n is a, c, g, or t

<220>

<221> misc_feature

<222> (83)..(83)

<223> n is a, c, g, or t

<220>

<221> misc_feature

<222> (87)..(88)

<223> n is a, c, g, or t

<220>

<221> misc_feature

<222> (93)..(93)

<223> n is a, c, g, or t

<220>

<221> misc_feature

<222> (99)..(100)

<223> n is a, c, g, or t

<220>

<221> misc_feature

<222> (102)..(102)

<223> n is a, c, g, or t

<400> 42

nctgtacttt cctgaccntg nnnnngtgnc ancatnnact tncctgannc nnctgtactt 60

tcctgaccnt gnnnnngtgn cancatnnac ttncctgann cn 102

<210> 43

<211> 153

<212> DNA

<213> Artificial sequence

<220>

<223> CRM

<220>

<221> misc_feature

<222> (1)..(1)

<223> n is a, c, g, or t

<220>

<221> misc_feature

<222> (18)..(18)

<223> n is a, c, g, or t

<220>

<221> misc_feature

<222> (21)..(25)

<223> n is a, c, g, or t

<220>

<221> misc_feature

<222> (29)..(29)

<223> n is a, c, g, or t

<220>

<221> misc_feature

<222> (32)..(32)

<223> n is a, c, g, or t

<220>

<221> misc_feature

<222> (36)..(37)

<223> n is a, c, g, or t

<220>

<221> misc_feature

<222> (42)..(42)

<223> n is a, c, g, or t

<220>

<221> misc_feature

<222> (48)..(49)

<223> n is a, c, g, or t

<220>

<221> misc_feature

<222> (51)..(52)

<223> optional spacer

<220>

<221> misc_feature

<222> (51)..(51)

<223> n is a, c, g, or t

<220>

<221> misc_feature

<222> (52)..(52)

<223> n is a, c, g, or t

<220>

<221> misc_feature

<222> (69)..(69)

<223> n is a, c, g, or t

<220>

<221> misc_feature

<222> (72)..(76)

<223> n is a, c, g, or t

<220>

<221> misc_feature

<222> (80)..(80)

<223> n is a, c, g, or t

<220>

<221> misc_feature

<222> (83)..(83)

<223> n is a, c, g, or t

<220>

<221> misc_feature

<222> (87)..(88)

<223> n is a, c, g, or t

<220>

<221> misc_feature

<222> (93)..(93)

<223> n is a, c, g, or t

<220>

<221> misc_feature

<222> (99)..(100)

<223> n is a, c, g, or t

<220>

<221> misc_feature

<222> (102)..(102)

<223> n is a, c, g, or t

<220>

<221> misc_feature

<222> (102)..(103)

<223> optional spacer

<220>

<221> misc_feature

<222> (103)..(103)

<223> n is a, c, g, or t

<220>

<221> misc_feature

<222> (120)..(120)

<223> n is a, c, g, or t

<220>

<221> misc_feature

<222> (123)..(127)

<223> n is a, c, g, or t

<220>

<221> misc_feature

<222> (131)..(131)

<223> n is a, c, g, or t

<220>

<221> misc_feature

<222> (134)..(134)

<223> n is a, c, g, or t

<220>

<221> misc_feature

<222> (138)..(139)

<223> n is a, c, g, or t

<220>

<221> misc_feature

<222> (144)..(144)

<223> n is a, c, g, or t

<220>

<221> misc_feature

<222> (150)..(151)

<223> n is a, c, g, or t

<220>

<221> misc_feature

<222> (153)..(153)

<223> n is a, c, g, or t

<400> 43

nctgtacttt cctgaccntg nnnnngtgnc ancatnnact tncctgannc nnctgtactt 60

tcctgaccnt gnnnnngtgn cancatnnac ttncctgann cnnctgtact ttcctgaccn 120

tgnnnnngtg ncancatnna cttncctgan ncn 153

<210> 44

<211> 102

<212> DNA

<213> Artificial sequence

<220>

<223> CRM

<220>

<221> misc_feature

<222> (51)..(52)

<223> optional spacer

<400> 44

tctgtacttt cctgaccttg gcacagtgcc accatcaact tgcctgacac ctctgtactt 60

tcctgacctt ggcacagtgc caccatcaac ttgcctgaca cc 102

<210> 45

<211> 153

<212> DNA

<213> Artificial sequence

<220>

<223> CRM

<220>

<221> misc_feature

<222> (51)..(52)

<223> optional spacer

<220>

<221> misc_feature

<222> (102)..(103)

<223> optional spacer

<400> 45

tctgtacttt cctgaccttg gcacagtgcc accatcaact tgcctgacac ctctgtactt 60

tcctgacctt ggcacagtgc caccatcaac ttgcctgaca cctctgtact ttcctgacct 120

tggcacagtg ccaccatcaa cttgcctgac acc 153

<210> 46

<211> 153

<212> DNA

<213> Artificial sequence

<220>

<223> CRM

<220>

<221> misc_feature

<222> (51)..(52)

<223> optional spacer

<220>

<221> misc_feature

<222> (102)..(103)

<223> optional spacer

<400> 46

actgtacttt cctgaccctg aagaggtggc agcatggact ttcctgaacc aactgtactt 60

tcctgaccct gaagaggtgg cagcatggac tttcctgaac caactgtact ttcctgaccc 120

tgaagaggtg gcagcatgga ctttcctgaa cca 153

<210> 47

<211> 102

<212> DNA

<213> Artificial sequence

<220>

<223> CRM

<220>

<221> misc_feature

<222> (51)..(52)

<223> optional spacer

<400> 47

actgtacttt cctgaccctg aagaggtggc agcatggact ttcctgaacc aactgtactt 60

tcctgaccct gaagaggtgg cagcatggac tttcctgaac ca 102

<210> 48

<211> 5716

<212> DNA

<213> Artificial sequence

<220>

<223> pAAV-ZsGreen original plasmid

<400> 48

agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc 60

acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc 120

tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa 180

ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac gaattgcctg 240

caggcagctg cgcgctcgct cgctcactga ggccgcccgg gcaaagcccg ggcgtcgggc 300

gacctttggt cgcccggcct cagtgagcga gcgagcgcgc agagagggag tggccaactc 360

catcactagg ggttcctatc gatatcaagc tttaatagta atcaattacg gggtcattag 420

ttcatagccc atatatggag ttccgcgtta cataacttac ggtaaatggc ccgcctggct 480

gaccgcccaa cgacccccgc ccattgacgt caataatgac gtatgttccc atagtaacgc 540

caatagggac tttccattga cgtcaatggg tggagtattt acggtaaact gcccacttgg 600

cagtacatca agtgtatcat atgccaagta cgccccctat tgacgtcaat gacggtaaat 660

ggcccgcctg gcattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca 720

tctacgtatt agtcatcgct attaccatgg tgatgcggtt ttggcagtac atcaatgggc 780

gtggatagcg gtttgactca cggggatttc caagtctcca ccccattgac gtcaatggga 840

gtttgttttg gcaccaaaat caacgggact ttccaaaatg tcgtaacaac tccgccccat 900

tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta tataagcaga gctggtttag 960

tggatatcct taagggccca gccggcccga atcccggccg ggaacggtgc attggaacgc 1020

ggattccccg tgccaagagt gacgtaagta ccgcctatag agtctatagg cccacaaaaa 1080

atgctttctt cttttaatat acttttttgt ttatcttatt tctaatactt tccctaatct 1140

ctttctttca gggcaataat gatacaatgt atcatgcctc tttgcaccat tctaaagaat 1200

aacagtgata atttctgggt taaggcaata gcaatatttc tgcatataaa tatttctgca 1260

tataaattgt aactgatgta agaggtttca tattgctaat agcagctaca atccagctac 1320

cattctgctt ttattttatg gttgggataa ggctggatta ttctgagtcc aagctaggcc 1380

cttttgctaa tcatgttcat acctcttatc ttcctcccac agctcctggg caacgtgctg 1440

gtctgtgtgc tggcccatca ctttggcaaa gaattgggat tcgcgagaat tcgccaccat 1500

ggcccagtcc aagcacggcc tgaccaagga gatgaccatg aagtaccgca tggagggctg 1560

cgtggacggc cacaagttcg tgatcaccgg cgagggcatc ggctacccct tcaagggcaa 1620

gcaggccatc aacctgtgcg tggtggaggg cggccccttg cccttcgccg aggacatctt 1680

gtccgccgcc ttcatgtacg gcaaccgcgt gttcaccgag tacccccagg acatcgtcga 1740

ctacttcaag aactcctgcc ccgccggcta cacctgggac cgctccttcc tgttcgagga 1800

cggcgccgtg tgcatctgca acgccgacat caccgtgagc gtggaggaga actgcatgta 1860

ccacgagtcc aagttctacg gcgtgaactt ccccgccgac ggccccgtga tgaagaagat 1920

gaccgacaac tgggagccct cctgcgagaa gatcatcccc gtgcccaagc agggcatctt 1980

gaagggcgac gtgagcatgt acctgctgct gaaggacggt ggccgcttgc gctgccagtt 2040

cgacaccgtg tacaaggcca agtccgtgcc ccgcaagatg cccgactggc acttcatcca 2100

gcacaagctg acccgcgagg accgcagcga cgccaagaac cagaagtggc acctgaccga 2160

gcacgccatc gcctccggct ccgccttgcc ctgataagga tccacgggtg gcatccctgt 2220

gacccctccc cagtgcctct cctggccctg gaagttgcca ctccagtgcc caccagcctt 2280

gtcctaataa aattaagttg catcattttg tctgactagg tgtccttcta taatattatg 2340

gggtggaggg gggtggtatg gagcaagggg caagttggga agacaacctg tagggcctgc 2400

ggggtctatt gggaaccaag ctggagtgca gtggcacaat cttggctcac tgcaatctcc 2460

gcctcctggg ttcaagcgat tctcctgcct cagcctcccg agttgttggg attccaggca 2520

tgcatgacca ggctcagcta atttttgttt ttttggtaga gacggggttt caccatattg 2580

gccaggctgg tctccaactc ctaatctcag gtgatctacc caccttggcc tcccaaattg 2640

ctgggattac aggcgtgaac cactgctccc ttccctgtcc ttatcgatag atctaggaac 2700

ccctagtgat ggagttggcc actccctctc tgcgcgctcg ctcgctcact gaggccgggc 2760

gaccaaaggt cgcccgacgc ccgggctttg cccgggcggc ctcagtgagc gagcgagcgc 2820

gcagctgcct gcaggcagct tggcactggc cgtcgtttta caacgtcgtg actgggaaaa 2880

ccctggcgtt acccaactta atcgccttgc agcacatccc cctttcgcca gctggcgtaa 2940

tagcgaagag gcccgcaccg atcgcccttc ccaacagttg cgcagcctga atggcgaatg 3000

gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc gcatacgtca 3060

aagcaaccat agtacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg 3120

cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct 3180

tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta 3240

gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgattt gggtgatggt 3300

tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg 3360

ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat ctcgggctat 3420

tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt 3480

taacaaaaat ttaacgcgaa ttttaacaaa atattaacgt ttacaatttt atggtgcact 3540

ctcagtacaa tctgctctga tgccgcatag ttaagccagc cccgacaccc gccaacaccc 3600

gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc 3660

gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgagacga 3720

aagggcctcg tgatacgcct atttttatag gttaatgtca tgataataat ggtttcttag 3780

acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa 3840

atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat 3900

tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg 3960

gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa 4020

gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt 4080

gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt 4140

ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat 4200

tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg 4260

acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta 4320

cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat 4380

catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag 4440

cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa 4500

ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca 4560

ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc 4620

ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt 4680

atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc 4740

gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat 4800

atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt 4860

tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac 4920

cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc 4980

ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca 5040

actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta 5100

gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct 5160

ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg 5220

gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc 5280

acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta 5340

tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg 5400

gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt 5460

cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg 5520

cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg 5580

ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc 5640

gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg 5700

agcgaggaag cggaag 5716

<210> 49

<211> 5857

<212> DNA

<213> Artificial sequence

<220>

<223> pAAV-PB1-MinTk

<400> 49

tgcctgcagg cagctgcgcg ctcgctcgct cactgaggcc gcccgggcaa agcccgggcg 60

tcgggcgacc tttggtcgcc cggcctcagt gagcgagcga gcgcgcagag agggagtggc 120

caactccatc actaggggtt cctatcgata tcaagcttct gtactttcct gaccttggca 180

cagtgccacc atcaacttgc ctgacaccga tccggccccg cccagcgtct tgtcattggc 240

gaattcgaac acgcagatgc agtcggggcg gcgcggtccg aggtccactt cgcatattaa 300

ggtgacgcgt gtggcctcga acaccgagcg accctgcagc gacccgctta acagcgtcaa 360

cagcgtgccg cagatctcga ggagcttggc gagattttca ggagctaagg aagctaaaca 420

tggaagatgc caaaaacatt aagaagggcc cagcgccatt ctacccactc gaagacggga 480

ccgccggcga gcagctgcac aaagccatga agcgctacgc cctggtgccc ggcaccatcg 540

cctttaccga cgcacatatc gaggtggaca ttacctacgc cgagtacttc gagatgagcg 600

ttcggctggc agaagctatg aagcgctatg ggctgaatac aaaccatcgg atcgtggtgt 660

gcagcgagaa tagcttgcag ttcttcatgc ccgtgttggg tgccctgttc atcggtgtgg 720

ctgtggcccc agctaacgac atctacaacg agcgcgagct gctgaacagc atgggcatca 780

gccagcccac cgtcgtattc gtgagcaaga aagggctgca aaagatcctc aacgtgcaaa 840

agaagctacc gatcatacaa aagatcatca tcatggatag caagaccgac taccagggct 900

tccaaagcat gtacaccttc gtgacttccc atttgccacc cggcttcaac gagtacgact 960

tcgtgcccga gagcttcgac cgggacaaaa ccatcgccct gatcatgaac agtagtggca 1020

gtaccggatt gcccaagggc gtagccctac cgcaccgcac cgcttgtgtc cgattcagtc 1080

atgcccgcga ccccatcttc ggcaaccaga tcatccccga caccgctatc ctcagcgtgg 1140

tgccatttca ccacggcttc ggcatgttca ccacgctggg ctacttgatc tgcggctttc 1200

gggtcgtgct catgtaccgc ttcgaggagg agctattctt gcgcagcttg caagactata 1260

agattcaatc tgccctgctg gtgcccacac tatttagctt cttcgctaag agcactctca 1320

tcgacaagta cgacctaagc aacttgcacg agatcgccag cggcggggcg ccgctcagca 1380

aggaggtagg tgaggccgtg gccaaacgct tccacctacc aggcatccgc cagggctacg 1440

gcctgacaga aacaaccagc gccattctga tcacccccga aggggacgac aagcctggcg 1500

cagtaggcaa ggtggtgccc ttcttcgagg ctaaggtggt ggacttggac accggtaaga 1560

cactgggtgt gaaccagcgc ggcgagctgt gcgtccgtgg ccccatgatc atgagcggct 1620

acgttaacaa ccccgaggct acaaacgctc tcatcgacaa ggacggctgg ctgcacagcg 1680

gcgacatcgc ctactgggac gaggacgagc acttcttcat cgtggaccgg ctgaagagcc 1740

tgatcaaata caagggctac caggtagccc cagccgaact ggagagcatc ctgctgcaac 1800

accccaacat cttcgacgcc ggggtcgccg gcctgcccga cgacgatgcc ggcgagctgc 1860

ccgccgcagt cgtcgtgctg gaacacggta aaaccatgac cgagaaggag atcgtggact 1920

atgtggccag ccaggttaca accgccaaga agctgcgcgg tggtgttgtg ttcgtggacg 1980

aggtgcctaa aggactgacc ggcaagttgg acgcccgcaa gatccgcgag attctcatta 2040

aggccaagaa gggcggcaag atcgccgtgt aatcgcgaga attctctaga gtcgacacta 2100

gtgcggatcc acgggtggca tccctgtgac ccctccccag tgcctctcct ggccctggaa 2160

gttgccactc cagtgcccac cagccttgtc ctaataaaat taagttgcat cattttgtct 2220

gactaggtgt ccttctataa tattatgggg tggagggggg tggtatggag caaggggcaa 2280

gttgggaaga caacctgtag ggcctgcggg gtctattggg aaccaagctg gagtgcagtg 2340

gcacaatctt ggctcactgc aatctccgcc tcctgggttc aagcgattct cctgcctcag 2400

cctcccgagt tgttgggatt ccaggcatgc atgaccaggc tcagctaatt tttgtttttt 2460

tggtagagac ggggtttcac catattggcc aggctggtct ccaactccta atctcaggtg 2520

atctacccac cttggcctcc caaattgctg ggattacagg cgtgaaccac tgctcccttc 2580

cctgtcctta tcgatagatc taggaacccc tagtgatgga gttggccact ccctctctgc 2640

gcgctcgctc gctcactgag gccgggcgac caaaggtcgc ccgacgcccg ggctttgccc 2700

gggcggcctc agtgagcgag cgagcgcgca gctgcctgca ggcagcttgg cactggccgt 2760

cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc caacttaatc gccttgcagc 2820

acatccccct ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca 2880

acagttgcgc agcctgaatg gcgaatggcg cctgatgcgg tattttctcc ttacgcatct 2940

gtgcggtatt tcacaccgca tacgtcaaag caaccatagt acgcgccctg tagcggcgca 3000

ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta 3060

gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt 3120

caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac 3180

cccaaaaaac ttgatttggg tgatggttca cgtagtgggc catcgccctg atagacggtt 3240

tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga 3300

acaacactca accctatctc gggctattct tttgatttat aagggatttt gccgatttcg 3360

gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata 3420

ttaacgttta caattttatg gtgcactctc agtacaatct gctctgatgc cgcatagtta 3480

agccagcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg 3540

gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca 3600

ccgtcatcac cgaaacgcgc gagacgaaag ggcctcgtga tacgcctatt tttataggtt 3660

aatgtcatga taataatggt ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc 3720

ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa 3780

taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc 3840

cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa 3900

acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa 3960

ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg 4020

atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa 4080

gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc 4140

acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc 4200

atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta 4260

accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag 4320

ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca 4380

acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca acaattaata 4440

gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc 4500

tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca 4560

ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca 4620

actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg 4680

taactgtcag accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa 4740

tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt 4800

gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat 4860

cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg 4920

gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga 4980

gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac 5040

tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt 5100

ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag 5160

cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc 5220

gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag 5280

gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca 5340

gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt 5400

cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc 5460

tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc 5520

cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc 5580

cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgccc aatacgcaaa 5640

ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac 5700

tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt agctcactca ttaggcaccc 5760

caggctttac actttatgct tccggctcgt atgttgtgtg gaattgtgag cggataacaa 5820

tttcacacag gaaacagcta tgaccatgat tacgaat 5857

<210> 50

<211> 6044

<212> DNA

<213> Artificial sequence

<220>

<223> pAAV-PB1-2-MinTk

<400> 50

gcctgcaggc agctgcgcgc tcgctcgctc actgaggccg cccgggcaaa gcccgggcgt 60

cgggcgacct ttggtcgccc ggcctcagtg agcgagcgag cgcgcagaga gggagtggcc 120

aactccatca ctaggggttc ctatcgatat caagcttttc tctggcctaa ctggccggta 180

ctctgtactt tcctgacctt ggcacagtgc caccatcaac ttgcctgaca cccattactc 240

gcatccattc tctctgtact ttcctgacct tggcacagtg ccaccatcaa cttgcctgac 300

accgcactga aggtcctcaa tcgtctgtac tttcctgacc ttggcacagt gccaccatca 360

acttgcctga caccgctggg agttcgtaga cggagatccg gccccgccca gcgtcttgtc 420

attggcgaat tcgaacacgc agatgcagtc ggggcggcgc ggtccgaggt ccacttcgca 480

tattaaggtg acgcgtgtgg cctcgaacac cgagcgaccc tgcagcgacc cgcttaacag 540

cgtcaacagc gtgccgcaga tctcgaggag cttggcgaga ttttcaggag ctaaggaagc 600

taaacatgga agatgccaaa aacattaaga agggcccagc gccattctac ccactcgaag 660

acgggaccgc cggcgagcag ctgcacaaag ccatgaagcg ctacgccctg gtgcccggca 720

ccatcgcctt taccgacgca catatcgagg tggacattac ctacgccgag tacttcgaga 780

tgagcgttcg gctggcagaa gctatgaagc gctatgggct gaatacaaac catcggatcg 840

tggtgtgcag cgagaatagc ttgcagttct tcatgcccgt gttgggtgcc ctgttcatcg 900

gtgtggctgt ggccccagct aacgacatct acaacgagcg cgagctgctg aacagcatgg 960

gcatcagcca gcccaccgtc gtattcgtga gcaagaaagg gctgcaaaag atcctcaacg 1020

tgcaaaagaa gctaccgatc atacaaaaga tcatcatcat ggatagcaag accgactacc 1080

agggcttcca aagcatgtac accttcgtga cttcccattt gccacccggc ttcaacgagt 1140

acgacttcgt gcccgagagc ttcgaccggg acaaaaccat cgccctgatc atgaacagta 1200

gtggcagtac cggattgccc aagggcgtag ccctaccgca ccgcaccgct tgtgtccgat 1260

tcagtcatgc ccgcgacccc atcttcggca accagatcat ccccgacacc gctatcctca 1320

gcgtggtgcc atttcaccac ggcttcggca tgttcaccac gctgggctac ttgatctgcg 1380

gctttcgggt cgtgctcatg taccgcttcg aggaggagct attcttgcgc agcttgcaag 1440

actataagat tcaatctgcc ctgctggtgc ccacactatt tagcttcttc gctaagagca 1500

ctctcatcga caagtacgac ctaagcaact tgcacgagat cgccagcggc ggggcgccgc 1560

tcagcaagga ggtaggtgag gccgtggcca aacgcttcca cctaccaggc atccgccagg 1620

gctacggcct gacagaaaca accagcgcca ttctgatcac ccccgaaggg gacgacaagc 1680

ctggcgcagt aggcaaggtg gtgcccttct tcgaggctaa ggtggtggac ttggacaccg 1740

gtaagacact gggtgtgaac cagcgcggcg agctgtgcgt ccgtggcccc atgatcatga 1800

gcggctacgt taacaacccc gaggctacaa acgctctcat cgacaaggac ggctggctgc 1860

acagcggcga catcgcctac tgggacgagg acgagcactt cttcatcgtg gaccggctga 1920

agagcctgat caaatacaag ggctaccagg tagccccagc cgaactggag agcatcctgc 1980

tgcaacaccc caacatcttc gacgccgggg tcgccggcct gcccgacgac gatgccggcg 2040

agctgcccgc cgcagtcgtc gtgctggaac acggtaaaac catgaccgag aaggagatcg 2100

tggactatgt ggccagccag gttacaaccg ccaagaagct gcgcggtggt gttgtgttcg 2160

tggacgaggt gcctaaagga ctgaccggca agttggacgc ccgcaagatc cgcgagattc 2220

tcattaaggc caagaagggc ggcaagatcg ccgtgtaatc gcgagaattc tctagagtcg 2280

acactagtgc ggatccacgg gtggcatccc tgtgacccct ccccagtgcc tctcctggcc 2340

ctggaagttg ccactccagt gcccaccagc cttgtcctaa taaaattaag ttgcatcatt 2400

ttgtctgact aggtgtcctt ctataatatt atggggtgga ggggggtggt atggagcaag 2460

gggcaagttg ggaagacaac ctgtagggcc tgcggggtct attgggaacc aagctggagt 2520

gcagtggcac aatcttggct cactgcaatc tccgcctcct gggttcaagc gattctcctg 2580

cctcagcctc ccgagttgtt gggattccag gcatgcatga ccaggctcag ctaatttttg 2640

tttttttggt agagacgggg tttcaccata ttggccaggc tggtctccaa ctcctaatct 2700

caggtgatct acccaccttg gcctcccaaa ttgctgggat tacaggcgtg aaccactgct 2760

cccttccctg tccttatcga tagatctagg aacccctagt gatggagttg gccactccct 2820

ctctgcgcgc tcgctcgctc actgaggccg ggcgaccaaa ggtcgcccga cgcccgggct 2880

ttgcccgggc ggcctcagtg agcgagcgag cgcgcagctg cctgcaggca gcttggcact 2940

ggccgtcgtt ttacaacgtc gtgactggga aaaccctggc gttacccaac ttaatcgcct 3000

tgcagcacat ccccctttcg ccagctggcg taatagcgaa gaggcccgca ccgatcgccc 3060

ttcccaacag ttgcgcagcc tgaatggcga atggcgcctg atgcggtatt ttctccttac 3120

gcatctgtgc ggtatttcac accgcatacg tcaaagcaac catagtacgc gccctgtagc 3180

ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc 3240

gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt 3300

ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac 3360

ctcgacccca aaaaacttga tttgggtgat ggttcacgta gtgggccatc gccctgatag 3420

acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa 3480

actggaacaa cactcaaccc tatctcgggc tattcttttg atttataagg gattttgccg 3540

atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac 3600

aaaatattaa cgtttacaat tttatggtgc actctcagta caatctgctc tgatgccgca 3660

tagttaagcc agccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg 3720

ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg 3780

ttttcaccgt catcaccgaa acgcgcgaga cgaaagggcc tcgtgatacg cctattttta 3840

taggttaatg tcatgataat aatggtttct tagacgtcag gtggcacttt tcggggaaat 3900

gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg 3960

agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa 4020

catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac 4080

ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac 4140

atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt 4200

ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc 4260

gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca 4320

ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc 4380

ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag 4440

gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa 4500

ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg 4560

gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa 4620

ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg 4680

gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt 4740

gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt 4800

caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag 4860

cattggtaac tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat 4920

ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct 4980

taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct 5040

tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca 5100

gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc 5160

agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc 5220

aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct 5280

gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag 5340

gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc 5400

tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg 5460

agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag 5520

cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt 5580

gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac 5640

gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg 5700

ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc 5760

cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcccaata 5820

cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca cgacaggttt 5880

cccgactgga aagcgggcag tgagcgcaac gcaattaatg tgagttagct cactcattag 5940

gcaccccagg ctttacactt tatgcttccg gctcgtatgt tgtgtggaat tgtgagcgga 6000

taacaatttc acacaggaaa cagctatgac catgattacg aatt 6044

<210> 51

<211> 4242

<212> DNA

<213> Artificial sequence

<220>

<223> pGL4.10 plasmid backbone

<400> 51

ggcctaactg gccggtacct gagctcgcta gcctcgagga tatcaagatc tggcctcggc 60

ggccaagctt ggcaatccgg tactgttggt aaagccacca tggaagatgc caaaaacatt 120

aagaagggcc cagcgccatt ctacccactc gaagacggga ccgccggcga gcagctgcac 180

aaagccatga agcgctacgc cctggtgccc ggcaccatcg cctttaccga cgcacatatc 240

gaggtggaca ttacctacgc cgagtacttc gagatgagcg ttcggctggc agaagctatg 300

aagcgctatg ggctgaatac aaaccatcgg atcgtggtgt gcagcgagaa tagcttgcag 360

ttcttcatgc ccgtgttggg tgccctgttc atcggtgtgg ctgtggcccc agctaacgac 420

atctacaacg agcgcgagct gctgaacagc atgggcatca gccagcccac cgtcgtattc 480

gtgagcaaga aagggctgca aaagatcctc aacgtgcaaa agaagctacc gatcatacaa 540

aagatcatca tcatggatag caagaccgac taccagggct tccaaagcat gtacaccttc 600

gtgacttccc atttgccacc cggcttcaac gagtacgact tcgtgcccga gagcttcgac 660

cgggacaaaa ccatcgccct gatcatgaac agtagtggca gtaccggatt gcccaagggc 720

gtagccctac cgcaccgcac cgcttgtgtc cgattcagtc atgcccgcga ccccatcttc 780

ggcaaccaga tcatccccga caccgctatc ctcagcgtgg tgccatttca ccacggcttc 840

ggcatgttca ccacgctggg ctacttgatc tgcggctttc gggtcgtgct catgtaccgc 900

ttcgaggagg agctattctt gcgcagcttg caagactata agattcaatc tgccctgctg 960

gtgcccacac tatttagctt cttcgctaag agcactctca tcgacaagta cgacctaagc 1020

aacttgcacg agatcgccag cggcggggcg ccgctcagca aggaggtagg tgaggccgtg 1080

gccaaacgct tccacctacc aggcatccgc cagggctacg gcctgacaga aacaaccagc 1140

gccattctga tcacccccga aggggacgac aagcctggcg cagtaggcaa ggtggtgccc 1200

ttcttcgagg ctaaggtggt ggacttggac accggtaaga cactgggtgt gaaccagcgc 1260

ggcgagctgt gcgtccgtgg ccccatgatc atgagcggct acgttaacaa ccccgaggct 1320

acaaacgctc tcatcgacaa ggacggctgg ctgcacagcg gcgacatcgc ctactgggac 1380

gaggacgagc acttcttcat cgtggaccgg ctgaagagcc tgatcaaata caagggctac 1440

caggtagccc cagccgaact ggagagcatc ctgctgcaac accccaacat cttcgacgcc 1500

ggggtcgccg gcctgcccga cgacgatgcc ggcgagctgc ccgccgcagt cgtcgtgctg 1560

gaacacggta aaaccatgac cgagaaggag atcgtggact atgtggccag ccaggttaca 1620

accgccaaga agctgcgcgg tggtgttgtg ttcgtggacg aggtgcctaa aggactgacc 1680

ggcaagttgg acgcccgcaa gatccgcgag attctcatta aggccaagaa gggcggcaag 1740

atcgccgtgt aataattcta gagtcggggc ggccggccgc ttcgagcaga catgataaga 1800

tacattgatg agtttggaca aaccacaact agaatgcagt gaaaaaaatg ctttatttgt 1860

gaaatttgtg atgctattgc tttatttgta accattataa gctgcaataa acaagttaac 1920

aacaacaatt gcattcattt tatgtttcag gttcaggggg aggtgtggga ggttttttaa 1980

agcaagtaaa acctctacaa atgtggtaaa atcgataagg atccgtcgac cgatgccctt 2040

gagagccttc aacccagtca gctccttccg gtgggcgcgg ggcatgacta tcgtcgccgc 2100

acttatgact gtcttcttta tcatgcaact cgtaggacag gtgccggcag cgctcttccg 2160

cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc 2220

actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt 2280

gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc 2340

ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa 2400

acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc 2460

ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg 2520

cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc 2580

tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc 2640

gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca 2700

ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact 2760

acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg 2820

gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt 2880

ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct 2940

tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga 3000

gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa 3060

tctaaagtat atatgagtaa acttggtctg acagcggccg caaatgctaa accactgcag 3120

tggttaccag tgcttgatca gtgaggcacc gatctcagcg atctgcctat ttcgttcgtc 3180

catagtggcc tgactccccg tcgtgtagat cactacgatt cgtgagggct taccatcagg 3240

ccccagcgca gcaatgatgc cgcgagagcc gcgttcaccg gcccccgatt tgtcagcaat 3300

gaaccagcca gcagggaggg ccgagcgaag aagtggtcct gctactttgt ccgcctccat 3360

ccagtctatg agctgctgtc gtgatgctag agtaagaagt tcgccagtga gtagtttccg 3420

aagagttgtg gccattgcta ctggcatcgt ggtatcacgc tcgtcgttcg gtatggcttc 3480

gttcaactct ggttcccagc ggtcaagccg ggtcacatga tcacccatat tatgaagaaa 3540

tgcagtcagc tccttagggc ctccgatcgt tgtcagaagt aagttggccg cggtgttgtc 3600

gctcatggta atggcagcac tacacaattc tcttaccgtc atgccatccg taagatgctt 3660

ttccgtgacc ggcgagtact caaccaagtc gttttgtgag tagtgtatac ggcgaccaag 3720

ctgctcttgc ccggcgtcta tacgggacaa caccgcgcca catagcagta ctttgaaagt 3780

gctcatcatc gggaatcgtt cttcggggcg gaaagactca aggatcttgc cgctattgag 3840

atccagttcg atatagccca ctcttgcacc cagttgatct tcagcatctt ttactttcac 3900

cagcgtttcg gggtgtgcaa aaacaggcaa gcaaaatgcc gcaaagaagg gaatgagtgc 3960

gacacgaaaa tgttggatgc tcatactcgt cctttttcaa tattattgaa gcatttatca 4020

gggttactag tacgtctctc aaggataagt aagtaatatt aaggtacggg aggtattgga 4080

caggccgcaa taaaatatct ttattttcat tacatctgtg tgttggtttt ttgtgtgaat 4140

cgatagtact aacatacgct ctccatcaaa acaaaacgaa acaaaacaaa ctagcaaaat 4200

aggctgtccc cagtgcaagt gcaggtgcca gaacatttct ct 4242

<210> 52

<211> 584

<212> DNA

<213> Artificial sequence

<220>

<223> CMV-IE comparison promoter

<400> 52

gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc 60

catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca 120

acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga 180

ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc 240

aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct 300

ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat 360

tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc 420

ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt 480

ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa 540

tgggcggtag gcgtgtacgg tgggaggtct atataagcag agct 584

<210> 53

<211> 8191

<212> DNA

<213> Artificial sequence

<220>

<223> pcDNA6.0 beta-galactosidase transfection control

<400> 53

gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg 60

ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 120

cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc 180

ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt 240

gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata 300

tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc 360

cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc 420

attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt 480

atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 540

atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca 600

tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg 660

actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 720

aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg 780

gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca 840

ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc 900

gtttaaactt aagcttggta ccgagctcgg atccactagt ccagtgtggt ggaattctgc 960

agatcgaaac gatgatagat cccgtcgttt tacaacgtcg tgactgggaa aaccctggcg 1020

ttacccaact taatcgcctt gcagcacatc cccctttcgc cagctggcgt aatagcgaag 1080

aggcccgcac cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa tggcgctttg 1140

cctggtttcc ggcaccagaa gcggtgccgg aaagctggct ggagtgcgat cttcctgagg 1200

ccgatactgt cgtcgtcccc tcaaactggc agatgcacgg ttacgatgcg cccatctaca 1260

ccaacgtgac ctatcccatt acggtcaatc cgccgtttgt tcccacggag aatccgacgg 1320

gttgttactc gctcacattt aatgttgatg aaagctggct acaggaaggc cagacgcgaa 1380

ttatttttga tggcgttaac tcggcgtttc atctgtggtg caacgggcgc tgggtcggtt 1440

acggccagga cagtcgtttg ccgtctgaat ttgacctgag cgcattttta cgcgccggag 1500

aaaaccgcct cgcggtgatg gtgctgcgct ggagtgacgg cagttatctg gaagatcagg 1560

atatgtggcg gatgagcggc attttccgtg acgtctcgtt gctgcataaa ccgactacac 1620

aaatcagcga tttccatgtt gccactcgct ttaatgatga tttcagccgc gctgtactgg 1680

aggctgaagt tcagatgtgc ggcgagttgc gtgactacct acgggtaaca gtttctttat 1740

ggcagggtga aacgcaggtc gccagcggca ccgcgccttt cggcggtgaa attatcgatg 1800

agcgtggtgg ttatgccgat cgcgtcacac tacgtctgaa cgtcgaaaac ccgaaactgt 1860

ggagcgccga aatcccgaat ctctatcgtg cggtggttga actgcacacc gccgacggca 1920

cgctgattga agcagaagcc tgcgatgtcg gtttccgcga ggtgcggatt gaaaatggtc 1980

tgctgctgct gaacggcaag ccgttgctga ttcgaggcgt taaccgtcac gagcatcatc 2040

ctctgcatgg tcaggtcatg gatgagcaga cgatggtgca ggatatcctg ctgatgaagc 2100

agaacaactt taacgccgtg cgctgttcgc attatccgaa ccatccgctg tggtacacgc 2160

tgtgcgaccg ctacggcctg tatgtggtgg atgaagccaa tattgaaacc cacggcatgg 2220

tgccaatgaa tcgtctgacc gatgatccgc gctggctacc ggcgatgagc gaacgcgtaa 2280

cgcgaatggt gcagcgcgat cgtaatcacc cgagtgtgat catctggtcg ctggggaatg 2340

aatcaggcca cggcgctaat cacgacgcgc tgtatcgctg gatcaaatct gtcgatcctt 2400

cccgcccggt gcagtatgaa ggcggcggag ccgacaccac ggccaccgat attatttgcc 2460

cgatgtacgc gcgcgtggat gaagaccagc ccttcccggc tgtgccgaaa tggtccatca 2520

aaaaatggct ttcgctacct ggagagacgc gcccgctgat cctttgcgaa tacgcccacg 2580

cgatgggtaa cagtcttggc ggtttcgcta aatactggca ggcgtttcgt cagtatcccc 2640

gtttacaggg cggcttcgtc tgggactggg tggatcagtc gctgattaaa tatgatgaaa 2700

acggcaaccc gtggtcggct tacggcggtg attttggcga tacgccgaac gatcgccagt 2760

tctgtatgaa cggtctggtc tttgccgacc gcacgccgca tccagcgctg acggaagcaa 2820

aacaccagca gcagtttttc cagttccgtt tatccgggca aaccatcgaa gtgaccagcg 2880

aatacctgtt ccgtcatagc gataacgagc tcctgcactg gatggtggcg ctggatggta 2940

agccgctggc aagcggtgaa gtgcctctgg atgtcgctcc acaaggtaaa cagttgattg 3000

aactgcctga actaccgcag ccggagagcg ccgggcaact ctggctcaca gtacgcgtag 3060

tgcaaccgaa cgcgaccgca tggtcagaag ccgggcacat cagcgcctgg cagcagtggc 3120

gtctggcgga aaacctcagt gtgacgctcc ccgccgcgtc ccacgccatc ccgcatctga 3180

ccaccagcga aatggatttt tgcatcgagc tgggtaataa gcgttggcaa tttaaccgcc 3240

agtcaggctc tctttcacag atgtggattg gcgataaaaa ccaactgctg acgccgctgc 3300

gcgatcagtt cacccgtgca ccgctggata acgacattgg cgtaagtgaa gcgacccgca 3360

ttgaccctaa cgcctgggtc gaacgctgga aggcggcggg ccattaccag gccgaagcag 3420

cgttgttgca gtgcacggca gatacacttg ctgatgcggt gctgattacg accgctcacg 3480

cgtggcagca tcaggggaaa accttattta tcagccggaa aacctaccgg attgatggta 3540

gtggtcaaat ggcgattacc gttgatgttg aagtggcgag cgatacaccg catccggcgc 3600

ggattggcct gaactgccag ctggcgcagg tagcagagcg ggtaaactgg ctcggattag 3660

ggccgcaaga aaactatccc gaccgcctta ctgccgcctg ttttgaccgc tgggatctgc 3720

cattgtcaga catgtatacc ccgtacgtct tcccgagcga aaacggtctg cgctgcggga 3780

cgcgcgaatt gaattatggc ccacaccagt ggcgcggcga cttccagttc aacatcagcc 3840

gctacagtca acagcaactg atggaaacca gccatcgcca tctgctgcac gcggaagaag 3900

gcacatggct gaatatcgac ggtttccata tggggattgg tggcgacgac tcctggagcc 3960

cgtcagtatc ggcggaattc cagctgagcg ccggtcgcta ccattaccag ttggtctggt 4020

gtcaaaaagc ggccgctcga ggtcacccat tcgaaggtaa gcctatccct aaccctctcc 4080

tcggtctcga ttctacgcgt accggtcatc atcaccatca ccattgagtt taaacccgct 4140

gatcagcctc gactgtgcct tctagttgcc agccatctgt tgtttgcccc tcccccgtgc 4200

cttccttgac cctggaaggt gccactccca ctgtcctttc ctaataaaat gaggaaattg 4260

catcgcattg tctgagtagg tgtcattcta ttctgggggg tggggtgggg caggacagca 4320

agggggagga ttgggaagac aatagcaggc atgctgggga tgcggtgggc tctatggctt 4380

ctgaggcgga aagaaccagc tggggctcta gggggtatcc ccacgcgccc tgtagcggcg 4440

cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc 4500

tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc 4560

gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg 4620

accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg 4680

tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg 4740

gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt 4800

cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat taattctgtg 4860

gaatgtgtgt cagttagggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca 4920

aagcatgcat ctcaattagt cagcaaccag gtgtggaaag tccccaggct ccccagcagg 4980

cagaagtatg caaagcatgc atctcaatta gtcagcaacc atagtcccgc ccctaactcc 5040

gcccatcccg cccctaactc cgcccagttc cgcccattct ccgccccatg gctgactaat 5100

tttttttatt tatgcagagg ccgaggccgc ctctgcctct gagctattcc agaagtagtg 5160

aggaggcttt tttggaggcc taggcttttg caaaaagctc ccgggagctt gtatatccat 5220

tttcggatct gatcagcacg tgttgacaat taatcatcgg catagtatat cggcatagta 5280

taatacgaca aggtgaggaa ctaaaccatg gccaagcctt tgtctcaaga agaatccacc 5340

ctcattgaaa gagcaacggc tacaatcaac agcatcccca tctctgaaga ctacagcgtc 5400

gccagcgcag ctctctctag cgacggccgc atcttcactg gtgtcaatgt atatcatttt 5460

actgggggac cttgtgcaga actcgtggtg ctgggcactg ctgctgctgc ggcagctggc 5520

aacctgactt gtatcgtcgc gatcggaaat gagaacaggg gcatcttgag cccctgcgga 5580

cggtgccgac aggtgcttct cgatctgcat cctgggatca aagccatagt gaaggacagt 5640

gatggacagc cgacggcagt tgggattcgt gaattgctgc cctctggtta tgtgtgggag 5700

ggctaagcac ttcgtggccg aggagcagga ctgacacgtg ctacgagatt tcgattccac 5760

cgccgccttc tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg gctggatgat 5820

cctccagcgc ggggatctca tgctggagtt cttcgcccac cccaacttgt ttattgcagc 5880

ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc 5940

actgcattct agttgtggtt tgtccaaact catcaatgta tcttatcatg tctgtatacc 6000

gtcgacctct agctagagct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg 6060

ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg 6120

tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc 6180

gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt 6240

gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct 6300

gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga 6360

taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc 6420

cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg 6480

ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg 6540

aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt 6600

tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt 6660

gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg 6720

cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact 6780

ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt 6840

cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct 6900

gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac 6960

cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc 7020

tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg 7080

ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta 7140

aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca 7200

atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc 7260

ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc 7320

tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc 7380

agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat 7440

taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt 7500

tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc 7560

cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag 7620

ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt 7680

tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac 7740

tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg 7800

cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat 7860

tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc 7920

gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc 7980

tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa 8040

atgttgaata ctcatactct tcctttttca atattattga agcatttatc agggttattg 8100

tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg 8160

cacatttccc cgaaaagtgc cacctgacgt c 8191

<210> 54

<211> 3196

<212> DNA

<213> Artificial sequence

<220>

<223> pGL4.10-CMV-MP-EPO

<400> 54

ggcctaactg gccggtaccg tcgacgatat cggatccagg tctatataag cagagctcgt 60

ttagtgaacc gtcagatcgc ctagatacgc catccacgct gttttgacct ccatagaaga 120

tcgccaccat gggggtgcac gaatgtcctg cctggctgtg gcttctcctg tccctgctgt 180

cgctccctct gggcctccca gtcctgggcg ccccaccacg cctcatctgt gacagccgag 240

tcctggagag gtacctcttg gaggccaagg aggccgagaa tatcacgacg ggctgtgctg 300

aacactgcag cttgaatgag aatatcactg tcccagacac caaagttaat ttctatgcct 360

ggaagaggat ggaggtcggg cagcaggccg tagaagtctg gcagggcctg gccctgctgt 420

cggaagctgt cctgcggggc caggccctgt tggtcaactc ttcccagccg tgggagcccc 480

tgcagctgca tgtggataaa gccgtcagtg gccttcgcag cctcaccact ctgcttcggg 540

ctctgggagc ccagaaggaa gccatctccc ctccagatgc ggcctcagct gctccactcc 600

gaacaatcac tgctgacact ttccgcaaac tcttccgagt ctactccaat ttcctccggg 660

gaaagctgaa gctgtacaca ggggaggcct gcaggacagg ggacagatga tctagagtcg 720

gggcggccgg ccgcttcgag cagacatgat aagatacatt gatgagtttg gacaaaccac 780

aactagaatg cagtgaaaaa aatgctttat ttgtgaaatt tgtgatgcta ttgctttatt 840

tgtaaccatt ataagctgca ataaacaagt taacaacaac aattgcattc attttatgtt 900

tcaggttcag ggggaggtgt gggaggtttt ttaaagcaag taaaacctct acaaatgtgg 960

taaaatcgat aaggatccgt cgaccgatgc ccttgagagc cttcaaccca gtcagctcct 1020

tccggtgggc gcggggcatg actatcgtcg ccgcacttat gactgtcttc tttatcatgc 1080

aactcgtagg acaggtgccg gcagcgctct tccgcttcct cgctcactga ctcgctgcgc 1140

tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc 1200

acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg 1260

aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat 1320

cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag 1380

gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga 1440

tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg 1500

tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt 1560

cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac 1620

gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc 1680

ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt 1740

ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc 1800

ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc 1860

agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg 1920

aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag 1980

atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg 2040

tctgacagcg gccgcaaatg ctaaaccact gcagtggtta ccagtgcttg atcagtgagg 2100

caccgatctc agcgatctgc ctatttcgtt cgtccatagt ggcctgactc cccgtcgtgt 2160

agatcactac gattcgtgag ggcttaccat caggccccag cgcagcaatg atgccgcgag 2220

agccgcgttc accggccccc gatttgtcag caatgaacca gccagcaggg agggccgagc 2280

gaagaagtgg tcctgctact ttgtccgcct ccatccagtc tatgagctgc tgtcgtgatg 2340

ctagagtaag aagttcgcca gtgagtagtt tccgaagagt tgtggccatt gctactggca 2400

tcgtggtatc acgctcgtcg ttcggtatgg cttcgttcaa ctctggttcc cagcggtcaa 2460

gccgggtcac atgatcaccc atattatgaa gaaatgcagt cagctcctta gggcctccga 2520

tcgttgtcag aagtaagttg gccgcggtgt tgtcgctcat ggtaatggca gcactacaca 2580

attctcttac cgtcatgcca tccgtaagat gcttttccgt gaccggcgag tactcaacca 2640

agtcgttttg tgagtagtgt atacggcgac caagctgctc ttgcccggcg tctatacggg 2700

acaacaccgc gccacatagc agtactttga aagtgctcat catcgggaat cgttcttcgg 2760

ggcggaaaga ctcaaggatc ttgccgctat tgagatccag ttcgatatag cccactcttg 2820

cacccagttg atcttcagca tcttttactt tcaccagcgt ttcggggtgt gcaaaaacag 2880

gcaagcaaaa tgccgcaaag aagggaatga gtgcgacacg aaaatgttgg atgctcatac 2940

tcgtcctttt tcaatattat tgaagcattt atcagggtta ctagtacgtc tctcaaggat 3000

aagtaagtaa tattaaggta cgggaggtat tggacaggcc gcaataaaat atctttattt 3060

tcattacatc tgtgtgttgg ttttttgtgt gaatcgatag tactaacata cgctctccat 3120

caaaacaaaa cgaaacaaaa caaactagca aaataggctg tccccagtgc aagtgcaggt 3180

gccagaacat ttctct 3196

<210> 55

<211> 13

<212> DNA

<213> muscle of mouse

<400> 55

tggcacagtg cca 13

<210> 56

<211> 13

<212> DNA

<213> Intelligent people

<400> 56

tgaagaggtg gca 13

<210> 57

<211> 16

<212> DNA

<213> muscle of mouse

<400> 57

tcaacttgcc tgacac 16

<210> 58

<211> 16

<212> DNA

<213> Intelligent people

<400> 58

tggactttcc tgaacc 16

<210> 59

<211> 273

<212> DNA

<213> Artificial sequence

<220>

<223> human NR1x3-Min TK

<400> 59

actgtacttt cctgaccctg aagagactgt actttcctga ccctgaagag actgtacttt 60

cctgaccctg aagaggatcc ggccccgccc agcgtcttgt cattggcgaa ttcgaacacg 120

cagatgcagt cggggcggcg cggtccgagg tccacttcgc atattaaggt gacgcgtgtg 180

gcctcgaaca ccgagcgacc ctgcagcgac ccgcttaaca gcgtcaacag cgtgccgcag 240

atctcgagga gcttggcgag attttcagga gct 273

<210> 60

<211> 286

<212> DNA

<213> Artificial sequence

<220>

<223> human PBREM-MinTK

<400> 60

actgtacttt cctgaccctg aagaggtggc agcatggact ttcctgaacc agatccggcc 60

ccgcccagcg tcttgtcatt ggcgaattcg aacacgcaga tgcagtcggg gcggcgcggt 120

ccgaggtcca cttcgcatat taaggtgacg cgtgtggcct cgaacaccga gcgaccctgc 180

agcgacccgc ttaacagcgt caacagcgtg ccgcagatct cgaggagctt ggcgagattt 240

tcaggagcta aggaagctaa acatggaaga tgccaaaaac attaag 286

<210> 61

<211> 286

<212> DNA

<213> Artificial sequence

<220>

<223> hNR1-mNFI-hNR2-Min TK (HMH MinTK)

<400> 61

actgtacttt cctgaccctg gcacagtgcc accatggact ttcctgaacc agatccggcc 60

ccgcccagcg tcttgtcatt ggcgaattcg aacacgcaga tgcagtcggg gcggcgcggt 120

ccgaggtcca cttcgcatat taaggtgacg cgtgtggcct cgaacaccga gcgaccctgc 180

agcgacccgc ttaacagcgt caacagcgtg ccgcagatct cgaggagctt ggcgagattt 240

tcaggagcta aggaagctaa acatggaaga tgccaaaaac attaag 286

<210> 62

<211> 286

<212> DNA

<213> Artificial sequence

<220>

<223> hNR1-mNFI mNR2-Min TK (HMM MinTK)

<400> 62

actgtacttt cctgaccctg gcacagtgcc accatcaact tgcctgacac cgatccggcc 60

ccgcccagcg tcttgtcatt ggcgaattcg aacacgcaga tgcagtcggg gcggcgcggt 120

ccgaggtcca cttcgcatat taaggtgacg cgtgtggcct cgaacaccga gcgaccctgc 180

agcgacccgc ttaacagcgt caacagcgtg ccgcagatct cgaggagctt ggcgagattt 240

tcaggagcta aggaagctaa acatggaaga tgccaaaaac attaag 286

<210> 63

<211> 286

<212> DNA

<213> Artificial sequence

<220>

<223> mNR1-hNFI-mNR2-Min TK (MHM MinTK)

<400> 63

tctgtacttt cctgaccttg aagaggtggc accatcaact tgcctgacac cgatccggcc 60

ccgcccagcg tcttgtcatt ggcgaattcg aacacgcaga tgcagtcggg gcggcgcggt 120

ccgaggtcca cttcgcatat taaggtgacg cgtgtggcct cgaacaccga gcgaccctgc 180

agcgacccgc ttaacagcgt caacagcgtg ccgcagatct cgaggagctt ggcgagattt 240

tcaggagcta aggaagctaa acatggaaga tgccaaaaac attaag 286

<210> 64

<211> 286

<212> DNA

<213> Artificial sequence

<220>

<223> mNR1-hNFI hNR2-Min TK (MHH MinTK)

<400> 64

tctgtacttt cctgaccttg aagaggtggc agcatggact ttcctgaacc agatccggcc 60

ccgcccagcg tcttgtcatt ggcgaattcg aacacgcaga tgcagtcggg gcggcgcggt 120

ccgaggtcca cttcgcatat taaggtgacg cgtgtggcct cgaacaccga gcgaccctgc 180

agcgacccgc ttaacagcgt caacagcgtg ccgcagatct cgaggagctt ggcgagattt 240

tcaggagcta aggaagctaa acatggaaga tgccaaaaac attaag 286

<210> 65

<211> 330

<212> DNA

<213> Artificial sequence

<220>

<223> hPB-SV40

<400> 65

actgtacttt cctgaccctg aagaggtggc agcatggact ttcctgaacc agctgggagt 60

tcgtagacgg actagcccgg gctcgagatc tgcgatctgc atctcaatta gtcagcaacc 120

atagtcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc cgcccattct 180

ccgccccatc gctgactaat tttttttatt tatgcagagg ccgaggccgc ctcggcctct 240

gagctattcc agaagtagtg aggaggcttt tttggaggcc taggcttttg caaaaagctt 300

ggcattccgg tactgttggt aaagccaccc 330

<210> 66

<211> 330

<212> DNA

<213> Artificial sequence

<220>

<223> MHM-SV40

<400> 66

tctgtacttt cctgaccttg aagaggtggc accatcaact tgcctgacac cgctgggagt 60

tcgtagacgg actagcccgg gctcgagatc tgcgatctgc atctcaatta gtcagcaacc 120

atagtcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc cgcccattct 180

ccgccccatc gctgactaat tttttttatt tatgcagagg ccgaggccgc ctcggcctct 240

gagctattcc agaagtagtg aggaggcttt tttggaggcc taggcttttg caaaaagctt 300

ggcattccgg tactgttggt aaagccaccc 330

<210> 67

<211> 401

<212> DNA

<213> Artificial sequence

<220>

<223> 2xhPB SV40

<400> 67

actgtacttt cctgaccctg aagaggtggc agcatggact ttcctgaacc acattactcg 60

catccattct cactgtactt tcctgaccct gaagaggtgg cagcatggac tttcctgaac 120

cagctgggag ttcgtagacg gactagcccg ggctcgagat ctgcgatctg catctcaatt 180

agtcagcaac catagtcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt 240

ccgcccattc tccgccccat cgctgactaa ttttttttat ttatgcagag gccgaggccg 300

cctcggcctc tgagctattc cagaagtagt gaggaggctt ttttggaggc ctaggctttt 360

gcaaaaagct tggcattccg gtactgttgg taaagccacc c 401

<210> 68

<211> 353

<212> DNA

<213> Artificial sequence

<220>

<223> 2xMHM-MinTK

<400> 68

tctgtacttt cctgaccttg aagaggtggc accatcaact tgcctgacac ccattactcg 60

catccattct ctctgtactt tcctgacctt gaagaggtgg caccatcaac ttgcctgaca 120

ccgctgggag ttcgtagacg gagatccggc cccgcccagc gtcttgtcat tggcgaattc 180

gaacacgcag atgcagtcgg ggcggcgcgg tccgaggtcc acttcgcata ttaaggtgac 240

gcgtgtggcc tcgaacaccg agcgaccctg cagcgacccg cttaacagcg tcaacagcgt 300

gccgcagatc tcgaggagct tggcgagatt ttcaggagct aaggaagcta aac 353

<210> 69

<211> 401

<212> DNA

<213> Artificial sequence

<220>

<223> 2xMHM-SV40

<400> 69

tctgtacttt cctgaccttg aagaggtggc accatcaact tgcctgacac ccattactcg 60

catccattct ctctgtactt tcctgacctt gaagaggtgg caccatcaac ttgcctgaca 120

ccgctgggag ttcgtagacg gactagcccg ggctcgagat ctgcgatctg catctcaatt 180

agtcagcaac catagtcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt 240

ccgcccattc tccgccccat cgctgactaa ttttttttat ttatgcagag gccgaggccg 300

cctcggcctc tgagctattc cagaagtagt gaggaggctt ttttggaggc ctaggctttt 360

gcaaaaagct tggcattccg gtactgttgg taaagccacc c 401

<210> 70

<211> 424

<212> DNA

<213> Artificial sequence

<220>

<223> 3xhPB-minTK

<400> 70

actgtacttt cctgaccctg aagaggtggc agcatggact ttcctgaacc acattactcg 60

catccattct cactgtactt tcctgaccct gaagaggtgg cagcatggac tttcctgaac 120

cagcactgaa ggtcctcaat cgactgtact ttcctgaccc tgaagaggtg gcagcatgga 180

ctttcctgaa ccagctggga gttcgtagac ggagatccgg ccccgcccag cgtcttgtca 240

ttggcgaatt cgaacacgca gatgcagtcg gggcggcgcg gtccgaggtc cacttcgcat 300

attaaggtga cgcgtgtggc ctcgaacacc gagcgaccct gcagcgaccc gcttaacagc 360

gtcaacagcg tgccgcagat ctcgaggagc ttggcgagat tttcaggagc taaggaagct 420

aaac 424

<210> 71

<211> 472

<212> DNA

<213> Artificial sequence

<220>

<223> 3xhPB-SV40

<400> 71

actgtacttt cctgaccctg aagaggtggc agcatggact ttcctgaacc acattactcg 60

catccattct cactgtactt tcctgaccct gaagaggtgg cagcatggac tttcctgaac 120

cagcactgaa ggtcctcaat cgactgtact ttcctgaccc tgaagaggtg gcagcatgga 180

ctttcctgaa ccagctggga gttcgtagac ggactagccc gggctcgaga tctgcgatct 240

gcatctcaat tagtcagcaa ccatagtccc gcccctaact ccgcccatcc cgcccctaac 300

tccgcccagt tccgcccatt ctccgcccca tcgctgacta atttttttta tttatgcaga 360

ggccgaggcc gcctcggcct ctgagctatt ccagaagtag tgaggaggct tttttggagg 420

cctaggcttt tgcaaaaagc ttggcattcc ggtactgttg gtaaagccac cc 472

<210> 72

<211> 51

<212> DNA

<213> Artificial sequence

<220>

<223> HMH shuffling

<400> 72

actgtacttt cctgaccctg gcacagtgcc accatggact ttcctgaacc a 51

<210> 73

<211> 51

<212> DNA

<213> Artificial sequence

<220>

<223> HMM shuffling

<400> 73

actgtacttt cctgaccctg gcacagtgcc accatcaact tgcctgacac c 51

<210> 74

<211> 51

<212> DNA

<213> Artificial sequence

<220>

<223> MHM shuffling

<400> 74

tctgtacttt cctgaccttg aagaggtggc accatcaact tgcctgacac c 51

<210> 75

<211> 51

<212> DNA

<213> Artificial sequence

<220>

<223> MHH shuffling

<400> 75

tctgtacttt cctgaccttg aagaggtggc agcatggact ttcctgaacc a 51

<210> 76

<211> 101

<212> DNA

<213> Artificial sequence

<220>

<223> 2x mouse PBREM CREs

<220>

<221> misc_feature

<222> (51)..(52)

<223> optional spacer

<400> 76

tctgtacttt cctgaccttg gcacagtgcc accatcaact tgcctgacac cctgtacttt 60

cctgaccttg gcacagtgcc accatcaact tgcctgacac c 101

<210> 77

<211> 204

<212> DNA

<213> Artificial sequence

<220>

<223> 3x mouse PBREM CREs

<220>

<221> misc_feature

<222> (51)..(52)

<223> optional spacer

<220>

<221> misc_feature

<222> (102)..(103)

<223> optional spacer

<220>

<221> misc_feature

<222> (153)..(154)

<223> optional spacer

<400> 77

tctgtacttt cctgaccttg gcacagtgcc accatcaact tgcctgacac ctctgtactt 60

tcctgacctt ggcacagtgc caccatcaac ttgcctgaca cctctgtact ttcctgacct 120

tggcacagtg ccaccatcaa cttgcctgac acctctgtac tttcctgacc ttggcacagt 180

gccaccatca acttgcctga cacc 204

<210> 78

<211> 101

<212> DNA

<213> Artificial sequence

<220>

<223> 2x MHM heterozygous CREs

<220>

<221> misc_feature

<222> (50)..(51)

<223> optional spacer

<400> 78

ctgtactttc ctgaccttga agaggtggca ccatcaactt gcctgacacc tctgtacttt 60

cctgaccttg aagaggtggc accatcaact tgcctgacac c 101

<210> 79

<211> 102

<212> DNA

<213> Artificial sequence

<220>

<223> 2x human PBREM CREs

<220>

<221> misc_feature

<222> (51)..(52)

<223> optional spacer

<400> 79

actgtacttt cctgaccctg aagaggtggc agcatggact ttcctgaacc aactgtactt 60

tcctgaccct gaagaggtgg cagcatggac tttcctgaac ca 102

<210> 80

<211> 153

<212> DNA

<213> Artificial sequence

<220>

<223> 3x human PBREM CREs

<220>

<221> misc_feature

<222> (51)..(52)

<223> optional spacer

<220>

<221> misc_feature

<222> (102)..(103)

<223> optional spacer

<400> 80

actgtacttt cctgaccctg aagaggtggc agcatggact ttcctgaacc aactgtactt 60

tcctgaccct gaagaggtgg cagcatggac tttcctgaac caactgtact ttcctgaccc 120

tgaagaggtg gcagcatgga ctttcctgaa cca 153

<210> 81

<211> 122

<212> DNA

<213> Artificial sequence

<220>

<223> 2x mouse PBREM CREs

<400> 81

tctgtacttt cctgaccttg gcacagtgcc accatcaact tgcctgacac ccattactcg 60

catccattct ctctgtactt tcctgacctt ggcacagtgc caccatcaac ttgcctgaca 120

cc 122

<210> 82

<211> 264

<212> DNA

<213> Artificial sequence

<220>

<223> 3x mouse PBREM

<400> 82

tctgtacttt cctgaccttg gcacagtgcc accatcaact tgcctgacac ccattactcg 60

catccattct ctctgtactt tcctgacctt ggcacagtgc caccatcaac ttgcctgaca 120

ccgcactgaa ggtcctcaat cgtctgtact ttcctgacct tggcacagtg ccaccatcaa 180

cttgcctgac accctgacct cctgccagca atatctgtac tttcctgacc ttggcacagt 240

gccaccatca acttgcctga cacc 264

<210> 83

<211> 121

<212> DNA

<213> Artificial sequence

<220>

<223> 2x MHM shuffling

<400> 83

ctgtactttc ctgaccttga agaggtggca ccatcaactt gcctgacacc cattactcgc 60

atccattctc tctgtacttt cctgaccttg aagaggtggc accatcaact tgcctgacac 120

c 121

<210> 84

<211> 122

<212> DNA

<213> Artificial sequence

<220>

<223> 2x human PBREM

<400> 84

actgtacttt cctgaccctg aagaggtggc agcatggact ttcctgaacc acattactcg 60

catccattct cactgtactt tcctgaccct gaagaggtgg cagcatggac tttcctgaac 120

ca 122

<210> 85

<211> 193

<212> DNA

<213> Artificial sequence

<220>

<223> 3x human PBREM

<400> 85

actgtacttt cctgaccctg aagaggtggc agcatggact ttcctgaacc acattactcg 60

catccattct cactgtactt tcctgaccct gaagaggtgg cagcatggac tttcctgaac 120

cagcactgaa ggtcctcaat cgactgtact ttcctgaccc tgaagaggtg gcagcatgga 180

ctttcctgaa cca 193

<210> 86

<211> 18

<212> DNA

<213> muscle of mouse

<400> 86

tctgtacttt cctgacct 18

<210> 87

<211> 18

<212> DNA

<213> Intelligent people

<400> 87

actgtacttt cctgaccc 18

<210> 88

<211> 16

<212> DNA

<213> muscle of mouse

<400> 88

tggcacagtg ccacca 16

<210> 89

<211> 16

<212> DNA

<213> Intelligent people

<400> 89

tgaagaggtg gcagca 16

<210> 90

<211> 17

<212> DNA

<213> muscle of mouse

<400> 90

tcaacttgcc tgacacc 17

<210> 91

<211> 17

<212> DNA

<213> Intelligent people

<400> 91

tggactttcc tgaacca 17

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