Expression vectors for high level expression of foreign genes

Abstract

This disclosure relates to an expression vector for high-level expression of exogenous genes, the expression vector comprising a regulatory nucleic acid molecule that enhances gene expression, said regulatory nucleic acid molecule comprising, in a 5' to 3' sequence, an adenovirus triple leader sequence (TPL) and an enhancer element (eMLP) of the major late promoter of adenovirus. This expression vector can significantly increase the expression level of exogenous genes, increasing the expression level of intracellular exogenous gene-encoded proteins by tens of times or more, and the expression level of secretory exogenous gene-encoded proteins by several times.

Description

Technical Field

[0001] This disclosure relates to a gene expression vector, and more particularly to a vector that mediates the efficient expression of exogenous genes in cells and its applications. Background Technology

[0002] Using vectors to deliver exogenous genes to specific tissues or cells, and then using the expressed exogenous proteins or non-coding RNA molecules to treat or prevent genetic diseases, tumors, degenerative diseases, and other conditions, is a promising treatment approach.

[0003] Viral vectors are a rapidly developing type of gene delivery vector in recent years. Among them, adeno-associated virus (AAV) vectors have gradually become the best choice for gene therapy vectors due to their low immunogenicity, non-integration, and ability to mediate long-term expression of exogenous genes.

[0004] Researchers have developed promoters with high transcriptional activity, such as CMV and CAG, to improve the efficiency of exogenous gene expression. However, gene expression levels in mammalian cells are regulated at multiple levels, including transcription, post-transcriptional mRNA stability, translation, and post-translational protein stability. Therefore, when delivering exogenous genes to mammalian cells, such as in gene therapy, optimization can be achieved from multiple perspectives, including gene transcription and translation regulatory elements, the copy number of the exogenous gene, the integration site of the exogenous gene in the host cell genome, RNA processing and mRNA stability, and the host cell's own translational modifications of proteins.

[0005] Therefore, there is a need for improved methods and optimized expression vectors for expressing genes in cells, especially in mammalian cells, to ensure long-term high-level expression of exogenous genes. Summary of the Invention

[0006] To address the problems existing in the prior art, this disclosure provides a method that includes multiple sequence elements that promote and maintain the expression of exogenous genes, thereby ensuring the long-term high-level expression of exogenous genes.

[0007] Therefore, in one aspect, this disclosure provides a regulatory nucleic acid molecule for enhancing gene expression, said regulatory nucleic acid molecule comprising, in a 5' to 3' sequence, a triple leader sequence (TPL) of adenovirus and an enhancer element (eMLP) of the major late promoter of adenovirus, wherein:

[0008] The adenovirus triple leader sequence (TPL) has a sequence selected from the TPL sequences shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4 or a sequence having at least 85% identity with them;

[0009] The enhancer element (eMLP) of the major late promoter of the adenovirus has an eMLP sequence selected from SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8 or a sequence having at least 85% identity with it.

[0010] In one aspect, this disclosure provides an expression vector comprising, in a 5' to 3' order:

[0011] (a) Start the sub-region;

[0012] (b) 5'UTR region;

[0013] (c) The coding sequence that encodes the polypeptide gene product;

[0014] (d) Polyadenylation region (polyA);

[0015] The 5'UTR region contains the aforementioned regulatory nucleic acid molecules;

[0016] The encoded sequence is operatively linked to the promoter region.

[0017] In one aspect, this disclosure provides a recombinant virus comprising:

[0018] a) Capsid proteins; and

[0019] b) The aforementioned expression carriers.

[0020] In one aspect, this disclosure provides a pharmaceutical composition comprising the aforementioned expression vector and / or recombinant virus and pharmaceutically acceptable excipients.

[0021] In one aspect, this disclosure provides isolated host cells transfected or transduced using the aforementioned expression vector.

[0022] In one aspect, this disclosure provides an isolated host cell infected with the aforementioned recombinant virus.

[0023] In one aspect, this disclosure provides a method for expressing a transgene in mammalian cells, the method comprising contacting one or more mammalian cells with an amount of the aforementioned expression vector and / or recombinant virus, wherein the secreted polypeptide is expressed at a certain level in the one or more mammalian cells.

[0024] In one aspect, this disclosure provides a method for treating or preventing a disease in a mammal that requires treatment or prevention of a disease, the method comprising administering an effective amount of the aforementioned expression vector, recombinant virus, pharmaceutical composition and / or host cell to the mammal.

[0025] This invention can significantly improve the expression level of aflibercept. Compared with vectors that simply use CMV promoters / enhancers to start the aflibercept expression cassette, it can increase the intracellular aflibercept protein expression level by tens of times or more and the secretory aflibercept protein level by several times. Attached Figure Description

[0026] Figure 1 The structure of the AO expression vector of this disclosure is shown.

[0027] Figure 2 This demonstrates the transfection of 293T cells with the Aflibercept expression vector ( Figure 2 A) and ARPE-19 cells ( Figure 2 B) The level of Aflibercept protein in the cell after the infection.

[0028] Figure 3 This demonstrates the transfection of 293T cells with the Aflibercept expression vector ( Figure 3 A) and ARPE-19 cells ( Figure 3 B) Aflibercept protein levels in the post-supernatant.

[0029] Figure 4 The levels of aflibercept protein in ARPE-19 cells after AAV virus infection are shown.

[0030] Figure 5 A shows the average AAV genome copy number in ARPE-19 cells after AAV virus infection; Figure 5 B shows the level of secreted Aflibercept protein in the supernatant corresponding to each copy of the AAV virus genome in ARPE-19 cells after AAV virus infection.

[0031] Figure 6 The correlation analysis of intracellular and secreted Aflibercept after transfection with Aflibercept expression vector or infection of 293T cells and ARPE-19 cells with AAV virus is shown.

[0032] Figure 7The levels of intracellular and secreted Aflibercept protein in the retina of BN rats after AO virus injection are shown. Among them, BLK is an eye sample taken from a control rat that was not injected with AO virus; B2_20R, B2_21R, and B2_22R are eye samples taken from rats injected with AO-1 virus; B2_26R, B2_26L, and B2_29L are eye samples taken from rats injected with AO-2 virus; B2_30L, B2_31L, and B2_32R are eye samples taken from rats injected with AO-3 virus; and B2_37R, B2_36L, B2_38R, and B2_39L are eye samples taken from rats injected with AO-4 virus. Detailed Implementation

[0033] definition

[0034] In this invention, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the terms and laboratory procedures related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology used herein are all widely used terms and routine procedures in their respective fields. To better understand this invention, definitions and explanations of relevant terms are provided below.

[0035] As used herein, "vector" refers to a polymer or polymeric association that includes or is associated with a polynucleotide and can be used to mediate the delivery of the polynucleotide to a cell. Illustrative vectors include, for example, plasmids, viral vectors (i.e., viruses, such as adeno-associated virus), liposomes, and other gene delivery agents.

[0036] The term "AAV" is an abbreviation for adeno-associated virus and can be used to refer to the virus itself or its derivatives. The term covers all subtypes, as well as naturally occurring and recombinant forms, unless otherwise required. The term "AAV" includes AAV type 1 (AAV-1), AAV type 2 (AAV-2), AAV type 3 (AAV-3), AAV type 4 (AAV-4), AAV type 5 (AAV-5), AAV type 6 (AAV-6), AAV type 7 (AAV-7), AAV type 8 (AAV-8), avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV, and sheep AAV. "Primate AAV" refers to AAV that infects primates, "non-primate AAV" refers to AAV that infects non-primate mammals, and "bovine AAV" refers to AAV that infects bovine mammals, etc.

[0037] A "viral AAV," "viral AAV particle," or "rAAV vector particle" refers to a viral particle composed of at least one AAV capsid protein (typically composed of all capsid proteins of wild-type AAV) and capsidated polynucleotides. If the particle includes heterologous polynucleotides (i.e., polynucleotides other than the wild-type AAV genome, such as transgenes to be delivered to mammalian cells), it is usually referred to as a recombinant AAV vector or rAAV. Typically, the heterologous polynucleotides are flanked by an AAV inverted terminal repeat (ITR).

[0038] As used herein, the term "replication defect" in relation to the AAV viral vector of the present invention means that the AAV vector cannot independently replicate and package its genome. For example, when a subject's cells are infected with rAAV viral particles, the heterologous gene is expressed in the infected cells; however, rAAV cannot replicate further due to the fact that the infected cells lack the AAV rep and cap genes and helper genes.

[0039] As used herein, “AAV variant” or “AAV mutant” refers to a viral particle composed of a variant AAV capsid protein, wherein the variant AAV capsid protein includes at least one amino acid difference (e.g., amino acid substitution, amino acid insertion, amino acid deletion) relative to the corresponding parental AAV capsid protein, and wherein the variant capsid protein confers increased infectivity to retinal cells compared to retinal cells containing AAV viral particles comprising the corresponding parental AAV capsid protein, wherein the AAV capsid protein does not include the amino acid sequence present in naturally occurring AAV capsid proteins. The polynucleotide expression vectors of this disclosure can be packaged in variant AAV particles to facilitate delivery of the expression vector to specific cell types (e.g., retinal cells) in target tissues.

[0040] As used herein, the term “packaging” refers to a series of intracellular events that lead to the assembly and shelling of AAV particles.

[0041] As used herein, the terms AAV “rep” and “cap” refer to the polynucleotide sequences encoding the replication and capsiding proteins of adeno-associated virus. AAV rep and cap are referred to as AAV “packaging genes” in this article.

[0042] As used herein, the term "helper virus" in AAV refers to a virus that allows mammalian cells to replicate and package AAV (e.g., wild-type AAV). Many such helper viruses for AAV are known in the art, including adenoviruses, herpesviruses, and poxviruses (e.g., vaccinia). While subclass C type 5 adenoviruses are most commonly used, adenoviruses encompass many different subclasses. Many adenoviruses are known from human, non-human mammals, and avian sources and are available from repositories such as the ATCC. Viruses of the herpes family include, for example, herpes simplex virus (HSV) and Epstein-Barr viruses (EBV), as well as cytomegalovirus (CMV) and pseudorabies virus (PRV); these are also available from repositories such as the ATCC.

[0043] As used herein, the term "helper viral function" refers to the function encoded in the helper viral genome that allows AAV replication and packaging (along with other requirements for replication and packaging as described herein). As described herein, "helper viral function" can be provided in a variety of ways, including by providing a helper virus or providing the production cell with, for example, a trans polynucleotide sequence encoding the essential function. For example, a plasmid or other expression vector containing a nucleotide sequence encoding one or more adenoviral proteins can be transfected into the production cell along with an rAAV vector.

[0044] As used herein, the term "gene" or "coding sequence" refers to a nucleotide sequence that encodes a gene product, either in vitro or in vivo. The term "transgenic" refers to a coding sequence or gene delivered into cells via a vector. A coding sequence or gene may encode a peptide or polypeptide molecule.

[0045] As used herein, "therapeutic gene" and "therapeutic protein" refer to a gene or protein that, when expressed, confers a beneficial effect on the cells, tissues, or mammals in which it is present, and which are expressed in said mammals. Examples of beneficial effects may include alleviating or improving signs or symptoms of a symptom or disease, preventing or inhibiting a symptom or disease, or conferring a desired characteristic. Therapeutic genes and proteins include genes and proteins that correct genetic defects in cells or mammals.

[0046] The "therapeutic effective amount" or "effective amount" of the expression vector, recombinant virus, or pharmaceutical composition of the present invention is an amount sufficient to cause a reduction in one or more signs or symptoms of a disease or medical condition in a subject, wherein the subject may be a human or a non-human mammal.

[0047] As used herein, the term "gene product" refers to the desired expression product of a polynucleotide sequence, such as a peptide or protein.

[0048] As used herein, the terms “polypeptide” and “protein” refer to amino acid polymers of any length. The term “peptide” refers to an amino acid polymer of about 50 or fewer amino acids. The terms also cover amino acid polymers that have been modified, for example, by disulfide bond formation, glycosylation, esterification, or phosphorylation. In some instances, polypeptides may have a length greater than 50 amino acids.

[0049] As used herein, "secretory protein" or "secretory polypeptide" refers to any protein that is secreted by or exported from living cells. A non-limiting example of a secretory protein used with the expression vector described herein is sFLT-1.

[0050] "Comprising" means that the listed elements are essential to, for example, a composition, method, kit, etc., but may include other elements to form, for example, a composition, method, kit, etc., within the scope of the claims. For example, an expression vector "comprising" an expression vector encoding a gene that is operatively linked to a promoter is an expression vector that may include elements other than the gene and promoter (e.g., polyadenylated sequences, enhancer elements, other genes, linker domains, etc.).

[0051] "Substantially composed of..." refers to a limitation on the scope of a particular material or step described, such as a composition, method, kit, etc., that does not substantially affect one or more essential and novel features, such as those of a composition, method, kit, etc. For example, an expression vector "substantially composed of" a gene encoding a therapeutic polypeptide operatively linked to a promoter and a polyadenylated sequence may contain additional sequences, such as linker sequences, as long as they do not substantially affect the transcription or translation of the gene. As another example, a variant or mutant polypeptide fragment "substantially composed of" the described sequence has an amino acid sequence at the sequence boundary based on the full-length untreated polypeptide derived from, with about 10 amino acid residues added or subtracted, for example, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 residue less than the binding amino acid residues, or 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 residues more than the binding amino acid residues.

[0052] "Comprising of..." means excluding from the composition, method, or kit any element, step, or component not specified in the claims. For example, an expression vector "comprising" a gene encoding a therapeutic polypeptide operatively linked to a promoter and a polyadenylated sequence consists only of a promoter, a polynucleotide sequence encoding the therapeutic polypeptide, and a polyadenylated sequence. As another example, a polypeptide "comprising" a described sequence contains only the described sequence.

[0053] As used herein, “expression vector” encompasses vectors that encode the gene product of interest, such as plasmids, microcircles, viral vectors, liposomes, etc., and are used to deliver polynucleotides to intended target cells.

[0054] As used herein, "promoter" encompasses a DNA sequence that guides RNA polymerase binding and thereby promotes RNA synthesis. Promoters and corresponding protein or polypeptide expression can be ubiquitous (meaning highly active in a wide range of cells, tissues, and species) or cell-type specific, tissue-specific, or species-specific. Promoters can be "constitutive" (meaning persistently active) or "inducible" (meaning that the promoter can be activated or inactivated by the presence or absence of biological or abiotic factors). Also included in the nucleic acid constructs or vectors of the present invention is an enhancer sequence, which may or may not be adjacent to the promoter sequence. The enhancer sequence affects promoter-dependent gene expression and may be located in the 5' or 3' region of the native gene.

[0055] As used in this article, "enhancer" encompasses cis-acting elements that stimulate or repress transcription of adjacent genes. Enhancers that repress transcription are also known as "silencers." Enhancers can act in any orientation at a distance of several thousand base pairs (kb) downstream of the coding sequence and the transcribed region (i.e., they can be associated with the coding sequence).

[0056] As used herein, the “polyadenylation signal sequence” encompasses the recognition region required for endonuclease cleavage of RNA transcripts, followed by the polyadenylation concordance sequence AATAAA. The polyadenylation signal sequence provides the “polyA site,” a site on the RNA transcript where adenine residues are added via posttranscriptional polyadenylation.

[0057] As used herein, the term "operably linked" refers to the juxtaposition of genetic elements (e.g., promoters, enhancers, termination signal sequences, polyadenylation sequences, etc.) in a relationship that allows them to operate in a intended manner. For example, if a promoter helps initiate transcription of a coding sequence, then the promoter is operably linked to the coding region. Intercalation residues may exist between the promoter and the coding region as long as this functional relationship is maintained.

[0058] As used herein, the term "heterologous" refers to an entity that is genotyped differently from the entity being compared. For example, a polynucleotide introduced into a plasmid or vector derived from a different species via genetic engineering is a heterologous polynucleotide. As another example, a promoter that has its natural coding sequence removed and operatively linked to a coding sequence not found to be linked naturally is a heterologous promoter. Thus, for example, an rAAV containing a heterologous nucleic acid encoding a heterologous gene product is an rAAV containing nucleic acids not typically contained in naturally occurring wild-type AAVs, and the encoded heterologous gene product is a gene product not typically encoded by naturally occurring wild-type AAVs.

[0059] As used herein, the term “endogenous” in relation to nucleotide molecules or gene products refers to nucleic acid sequences (e.g., genes or genetic elements) or gene products (e.g., RNA, proteins) that are naturally present in or associated with a host virus or cell.

[0060] As used herein, the term “natural” refers to a nucleotide sequence (e.g., a gene) or a gene product (e.g., RNA, protein) present in wild-type viruses or cells.

[0061] As used herein, the term "variant" refers to a mutant of a reference polynucleotide or polypeptide sequence, such as a native polynucleotide or polypeptide sequence, that is, one that has less than 100% sequence identity with the reference polynucleotide or polypeptide sequence. In other words, a polypeptide variant includes at least one amino acid difference (e.g., amino acid substitution, amino acid insertion, amino acid deletion) relative to a reference polypeptide sequence (e.g., a native polypeptide sequence), and a polynucleotide variant includes at least one nucleotide or nucleoside difference (e.g., nucleotide or nucleoside substitution, insertion, or deletion) relative to a reference polynucleotide sequence (e.g., a native polynucleotide sequence).

[0062] As used herein, the terms “sequence identity” or “percentage identity” refer to the degree of identity between two or more polynucleotides when aligned using a nucleotide sequence alignment program; or the degree of identity between two or more polypeptide sequences when aligned using an amino acid sequence alignment program. Similarly, when used in the context of two or more nucleotide or amino acid sequences, the terms “identical” or “percentage identity” refer to sequences that are identical or have a specific percentage of amino acid residues or nucleotides when compared and aligned for maximum correspondence, for example, as measured by using a sequence comparison algorithm (e.g., the Smith-Waterman algorithm) or by visual inspection. For example, the Needleman and Wunsch (1970, *Journal of Molecular Biology*, 48:444-453) algorithm, using a Blossum 62 or PAM250 matrix with vacancy weights of 16, 14, 12, 10, 8, 6, or 4 and length weights of 1, 2, 3, 4, 5, or 6, can be used to determine the percentage identity between two amino acid sequences. This algorithm is incorporated into the GAP program in the GCG software package. As another example, the GAP program in the GCG software package can be used with an NWSgapdna.CMP matrix with vacancy weights of 40, 50, 60, 70, or 80 and length weights of 1, 2, 3, 4, 5, or 6 to determine the percentage identity between two nucleotide sequences. A particularly preferred set of parameters (which should be used unless otherwise specified) is a Blossum 62 scoring matrix with a vacancy penalty of 12, a vacancy extension penalty of 4, and a frameshift vacancy penalty of 5. The percentage identity between two amino acid or nucleotide sequences can also be determined using the algorithm of E. Meyers and W. Miller (1989, *Computer Applications in Biological Sciences (Cabios)*, 4:11-17), using a PAM120 weighted residue table, a vacancy length penalty of 12, and a vacancy penalty of 4, which has been incorporated into the ALIGN program (version 2.0). The nucleic acid and protein sequences described herein can be used as “query sequences” to search public databases for, for example, identifying other family members or related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul et al. (1990, *Journal of Molecular Biology*, 215:403-10). A BLAST nucleotide search can be performed using the NBLAST program (score = 100, word length = 12) to obtain nucleotide sequences homologous to the nucleic acid molecules of this invention. A BLAST protein search can be performed using the XBLAST program (score = 50, word length = 3) to obtain amino acid sequences homologous to the protein molecules of this invention.To obtain vacancy-containing alignments for comparative purposes, vacancy-containing BLAST can be used, as described by Altschul et al. (1997, Nucleic Acids Res, 25:3389-3402). When using BLAST and vacancy-containing BLAST procedures, the default parameters of the respective procedures (e.g., XBLAST and NBLAST) can be used.

[0063] As used herein, the terms "biological activity" and "biologically active" refer to the activity attributed to a specific biological element in a cell. For example, the "biological activity" of an "immunoglobulin," "antibody," or fragment or variant thereof refers to its ability to bind to antigenic determinants and thereby promote immune function. As another example, the biological activity of a polypeptide or a functional fragment or variant thereof refers to its ability to perform its native functions, such as binding, enzymatic activity, etc. As a third example, the biological activity of a gene regulatory element (e.g., promoter, enhancer, kozak sequence, etc.) refers to the ability of the regulatory element or a functional fragment or variant thereof to regulate the expression of the gene to which it is operatively linked (i.e., promote, enhance, or activate its translation).

[0064] As used herein, the terms “application” or “introduction” refer to the delivery of a vector for recombinant protein expression to cells, a subject’s cells and / or organs, or the subject. Such application or introduction can occur in vivo, in vitro, or ex vivo. A vector for expressing a gene product can be introduced into cells via transfection, which typically means the insertion of heterologous DNA into cells by physical means (e.g., calcium phosphate transfection, electroporation, microinjection, or lipid transfection); or via infection or transduction, which typically refers to the introduction of nucleic acid molecules into cells via an infectious agent (i.e., a virus or viral vector).

[0065] Cells are typically referred to as “transduced,” “infected,” “transfected,” or “converted” depending on the method used to administer, introduce, or insert heterologous DNA (i.e., vectors) into them. When DNA is introduced into cells via a virus or viral vector, the cells are transduced with exogenous or heterologous DNA. When DNA is introduced into cells via non-viral methods, the cells are transfected with exogenous or heterologous DNA. Non-viral methods include chemical methods (e.g., lipid transfection) and non-chemical methods. The terms “transduced” and “infected” are used interchangeably herein to refer to cells that have received heterologous DNA or heterologous polynucleotides from a virus or viral vector.

[0066] As used herein, the term "host cell" refers to a cell that has been transduced, infected, transfected, or transformed with a vector. The vector may be a plasmid, viral particle, bacteriophage, etc. Culture conditions (such as temperature, pH, etc.) are those previously used with the host cells selected for expression and will be apparent to those skilled in the art. It should be understood that the term "host cell" refers to the original transduced, infected, transfected, or transformed cell and its progeny.

[0067] The terms “treatment” and “treating” refer to the relief of one or more signs or symptoms of a disease or condition.

[0068] The terms “disease,” “symptom,” and “medical condition” are synonyms and are used interchangeably in this article.

[0069] "Eye disease" refers to any disease, ailment, or condition that affects or involves the eye or one or more parts or areas of the eye. Therefore, eye diseases include retinal diseases or diseases affecting the light-sensitive layer of the tissues at the back of the eye. The eye comprises the eyeball and the tissues and fluids that make up the eyeball, the periocular muscles (such as the oblique and rectus muscles), and the portion of the optic nerve within or near the eyeball.

[0070] The tissue "explant" is a tissue block that has been transferred from the animal to a nutrient culture medium.

[0071] The terms “individual,” “host,” “subject,” and “patient” are used interchangeably in this document and refer to mammals, including but not limited to: human and non-human primates, including apes and humans; mammalian locomotion animals (e.g., horses); mammalian farm animals (e.g., sheep, goats, etc.); mammalian pets (dogs, cats, etc.); and rodents (e.g., mice, rats, etc.).

[0072] Various compositions and methods of the present invention are described below. Although specific compositions and methods are illustrated herein, it should be understood that any of the numerous alternative compositions and methods are applicable and suitable for practicing the present invention. It should also be understood that expression constructs and methods of the present invention can be evaluated using standard procedures in the art.

[0073] Unless otherwise specified, the practice of this invention will employ conventional techniques of cell biology, molecular biology (including recombinant technologies), microbiology, biochemistry, and immunology, which are within the scope of those skilled in the art. This technique is well described in the literature, such as in *Molecular Cloning: A Laboratory Manual*, 2nd edition (Sambrook et al., 1989); *Oligonucleotide Synthesis* (edited by M.J. Gait, 1984); *Animal Cell Culture* (edited by R.R. Freshney, 1987); *Methods in Enzymology* (Academic Press, Inc.); *Handbook of Experimental Immunology* (edited by D.M. Weir and C.C. Blackwell); *Gene Transfer Vectors for Mammalian Cells* (edited by J.M. Miller and M.C. Calos, 1987); *Current Protocols in Molecular Biology* (edited by F.M. Ausubel et al., 1987); and *PCR: The Polymerase Chain Reaction*. The references cited in the references are: "Reaction" (edited by Mullis et al., 1994); and "Current Protocols in Immunology" (edited by JEColigan et al., 1991), each of which is explicitly incorporated herein by reference.

[0074] The following description, using examples for illustration, describes several aspects of the invention. It should be understood that numerous specific details, relationships, and methods are set forth to provide a full understanding of the invention. However, those skilled in the art will readily recognize that the invention can be practiced without one or more of these specific details or in other ways. The invention is not limited to the order of the described actions or events, as some actions may occur in a different order and / or simultaneously with other actions or events. Furthermore, implementing the method according to the invention does not require all of the described actions or events.

[0075] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the invention. As used herein, unless the context clearly indicates otherwise, the singular forms “a,” “an,” and “the” are intended to also include the plural forms. Furthermore, to the extent that the terms “including,” “include,” “having,” “has,” “with,” or variations thereof are used in the detailed description and / or claims, such terms are intended to be inclusive (in a manner similar to the term “comprising”).

[0076] The terms "about" or "approximately" mean that a particular value, as determined by a person skilled in the art, is within an acceptable range of error, which will depend in part on how the value was measured or determined, i.e., the limitations of the measurement system. For example, according to practice in the art, "about" may mean within a standard deviation of 1 or greater than 1. Alternatively, "about" or "approximately" may mean a range up to 20%, preferably up to 10%, more preferably up to 5%, and still more preferably up to 1% of a given value. Alternatively, particularly for biological systems or methods, the term may mean within an order of magnitude of a value, preferably within 5 times the value, and more preferably within 2 times. When a particular value is described in this application and claims, unless otherwise stated, it should be assumed that the term "about" means within an acceptable range of error for the particular value.

[0077] All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and / or materials relating to the cited publications. It should be understood that, in the event of conflict, this disclosure supersedes any disclosure incorporated herein.

[0078] It should be further noted that the claims may be drafted to exclude any optional elements. Therefore, this statement is intended to serve as a premise for the use of exclusive terms such as “alone,” “only,” etc., or the use of “negative” restrictions in relation to the description of the elements of the claims.

[0079] Only disclosures in publications discussed herein prior to the filing date of this application are provided. Nothing herein should be construed as an admission that the invention is not entitled to any prior art prior to such publications. Furthermore, the disclosure date provided may differ from the actual disclosure date, which may need to be determined independently.

[0080] Unless otherwise specified, all terms used herein have the same meaning as they would be to be understood by one of those skilled in the art, and the practice of this invention will employ conventional microbiological techniques and recombinant DNA techniques, which are within the knowledge of one of those skilled in the art.

[0081] II. Detailed Implementation

[0082] In one aspect, this disclosure provides a regulatory nucleic acid molecule for enhancing gene expression, said regulatory nucleic acid molecule comprising, in a 5' to 3' sequence, a triple leader sequence (TPL) of adenovirus and an enhancer element (eMLP) of the major late promoter of adenovirus, wherein:

[0083] The adenovirus triple leader sequence (TPL) has a sequence selected from the TPL sequences shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4 or a sequence having at least 85% identity with them;

[0084] The enhancer element (eMLP) of the major late promoter of the adenovirus has an eMLP sequence selected from SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8 or a sequence having at least 85% identity with it.

[0085] In some embodiments of this disclosure, the regulatory nucleic acid molecule has a sequence selected from or having at least 85% identity with the sequence shown in SEQ ID NO:19-34.

[0086] In some preferred embodiments of this disclosure, the regulatory nucleic acid molecule has the sequence shown in SEQ ID NO:22, 25, 26 or 33 or a sequence having at least 85% identity with it.

[0087] In some preferred embodiments of this disclosure, the regulatory nucleic acid molecule has the sequence shown in SEQ ID NO:25 or a sequence having at least 85% identity with it.

[0088] In one aspect, this disclosure provides an expression vector comprising, in a 5' to 3' order:

[0089] (a) Start the sub-region;

[0090] (b) 5'UTR region;

[0091] (c) The coding sequence that encodes the polypeptide gene product;

[0092] (d) Polyadenylation region (polyA);

[0093] The 5'UTR region contains the aforementioned regulatory nucleic acid molecules;

[0094] The encoded sequence is operatively linked to the promoter region.

[0095] In some embodiments of this disclosure, the promoter region (a) is selected from the cytomegalovirus (CMV) promoter, actin promoter, elongation factor 1α (EF1α) promoter, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter; the promoter region (a) comprises the cytomegalovirus (CMV) promoter sequence shown in SEQ ID NO:9 or a sequence having at least 85% identity with it.

[0096] In some embodiments of this disclosure, the polypeptide gene product is a therapeutic protein.

[0097] In some preferred embodiments of this disclosure, the therapeutic protein is selected from anti-angiogenic peptides or α-1 antitrypsin.

[0098] In some preferred embodiments of this disclosure, the anti-angiogenic polypeptide comprises soluble fms-like tyrosine kinase-1 (sFLT-1) or a VEGF-binding fragment of sFLT-1.

[0099] In some preferred embodiments of this disclosure, the anti-angiogenic polypeptide is aflibercept; preferably, the amino acid composition of aflibercept is as shown in SEQ ID NO:10; more preferably, the encoding nucleic acid of aflibercept is as shown in SEQ ID NO:11.

[0100] In some embodiments of this disclosure, the polyadenylated region is selected from human growth hormone (HGH or hGH), bovine growth hormone (BGH or bGH), or β-globin (β-globin) polyA sequence.

[0101] In some preferred embodiments of this disclosure, the polyadenylation region comprises the bovine growth hormone (BGH or bGH) polyA sequence shown in SEQ ID NO:12 or a sequence having at least 85% identity with it.

[0102] In some embodiments of this disclosure, the expression vector further includes (i) a first enhancer region, which is located upstream of the (a) promoter region.

[0103] In some preferred embodiments of this disclosure, the (i) first enhancer region contains a sequence selected from CMV enhancers or EF1α enhancers.

[0104] In some preferred embodiments of this disclosure, the (i) first enhancer region contains the CMV enhancer sequence shown in SEQ ID NO:13 or a sequence having at least 85% identity with it.

[0105] In some embodiments of this disclosure, the expression vector further includes (ii) an intron region located downstream of the (b) 5'UTR region and upstream of the (c) coding sequence encoding the polypeptide gene product.

[0106] In some preferred embodiments of this disclosure, the (ii) intron region comprises a sequence selected from the SV40 intron, the elongation factor 1α (EF1α) intron, the actin intron, or the CMVc intron.

[0107] In some preferred embodiments of this disclosure, the (ii) intron region comprises the SV40 intron sequence shown in SEQ ID NO:14 or a sequence having at least 85% identity with it.

[0108] In some embodiments of this disclosure, the expression vector further includes (iii) a second enhancer region, which is located downstream of the coding sequence encoding the polypeptide gene product in (c) and upstream of the polyadenylation region in (d).

[0109] In some preferred embodiments of this disclosure, the (iii) second enhancer region contains an expression enhancer sequence (EES).

[0110] In some preferred embodiments of this disclosure, the (iii) second enhancer region includes the interferon scaffold attachment region (SAR).

[0111] In some preferred embodiments of this disclosure, the stent attachment region sequence (SAR) is the human stent attachment region (IFNB SAR) of human beta-interferon.

[0112] In some preferred embodiments of this disclosure, the human β-interferon scaffold attachment region (IFNB SAR) comprises the SAR sequence shown in SEQ ID NO:15 or a sequence having at least 85% identity with it.

[0113] In some embodiments of this disclosure, the expression vector further comprises an inverted terminal repeat (ITR) at the 5' end upstream of (i) the first enhancer and an inverted terminal repeat (ITR) at the 3' end downstream of (d) the polyadenylation region.

[0114] In some preferred embodiments of this disclosure, the (iv) 5' end inverted terminal repeat (ITR) or the (v) 3' end inverted terminal repeat (ITR) comprises an inverted terminal repeat (ITR) selected from adenovirus (AV) or adeno-associated virus (AAV).

[0115] In some preferred embodiments of this disclosure, the (iv) 5' inverted terminal repeat (ITR) sequence comprises the AAV ITR shown in SEQ ID NO:16 or a sequence having at least 85% identity with it.

[0116] In some preferred embodiments of this disclosure, the inverted terminal repeat (ITR) sequence at the (v)3' end comprises the AAV ITR shown in SEQ ID NO:17 or a sequence having at least 85% identity with it.

[0117] In some embodiments of this disclosure, the expression vector further comprises (vi) a selection marker gene.

[0118] In some preferred embodiments of this disclosure, the (vi) selection marker gene is located downstream of the inverted terminal repeat (ITR) at the 3' end of the (v) gene.

[0119] In some preferred embodiments of this disclosure, the (vi) selector gene is selected from ampicillin resistance gene, hygromycin resistance gene, neomycin resistance gene, diammonium phosphate resistance gene and dihydrofolate reductase gene.

[0120] In some preferred embodiments of this disclosure, the ampicillin resistance gene comprises the sequence shown in SEQ ID NO:18 or a sequence having at least 85% identity with it.

[0121] In some embodiments of this disclosure, the expression vector comprises, from 5' to 3':

[0122] (i) A first enhancer region, wherein the first enhancer region contains the CMV enhancer sequence shown in SEQ ID NO:13 or a sequence having at least 85% identity with it;

[0123] (a) A promoter region comprising the cytomegalovirus (CMV) promoter sequence shown in SEQ ID NO:9 or a sequence having at least 85% identity with it;

[0124] (b) 5'UTR region, wherein the 5'UTR region contains a regulatory nucleic acid molecule or a sequence having at least 85% identity with the TPL sequence shown in SEQ ID NO:2 and the eMLP sequence shown in SEQ ID NO:7;

[0125] (c) The coding sequence that encodes the polypeptide gene product;

[0126] (iii) a second enhancement sub-region, the second enhancement sub-region comprising the SAR sequence shown in SEQ ID NO:15 or a sequence having at least 85% identity with it; and

[0127] (d) A polyadenylated region comprising the bovine growth hormone (BGH or bGH) polyA sequence shown in SEQ ID NO:12 or a sequence having at least 85% identity with it, and

[0128] Optionally, the expression vector described herein does not contain an RNA output signal.

[0129] In some embodiments of this disclosure, the expression vector comprises, from 5' to 3':

[0130] (i) A first enhancer region, wherein the first enhancer region contains the CMV enhancer sequence shown in SEQ ID NO:13 or a sequence having at least 85% identity with it;

[0131] (a) A promoter region comprising the cytomegalovirus (CMV) promoter sequence shown in SEQ ID NO:9 or a sequence having at least 85% identity with it;

[0132] (ii) an intron region comprising the SV40 intron sequence shown in SEQ ID NO:14 or a sequence having at least 85% identity with it;

[0133] (b) 5'UTR region, wherein the 5'UTR region contains a regulatory nucleic acid molecule or a sequence having at least 85% identity with the TPL sequence shown in SEQ ID NO:2 and the eMLP sequence shown in SEQ ID NO:7;

[0134] (c) A coding sequence encoding a polypeptide gene product, wherein the coding sequence is operatively linked to the promoter region;

[0135] (iii) A second enhancement sub-region, the second enhancement sub-region comprising the SAR sequence shown in SEQ ID NO:15 or a sequence having at least 85% identity with it;

[0136] (d) Polyadenylation region, wherein the polyadenylation region comprises the bovine growth hormone (BGH or bGH) polyA sequence shown in SEQ ID NO:12 or a sequence having at least 85% identity with it.

[0137] In some embodiments of this disclosure, the expression vector comprises, from 5' to 3':

[0138] (iv) 5' inverted terminal repeat (ITR) sequence, wherein the 5' inverted terminal repeat (ITR) sequence comprises the AAV ITR shown in SEQ ID NO:16 or a sequence having at least 85% identity with it;

[0139] (i) A first enhancer region, wherein the first enhancer region contains the CMV enhancer sequence shown in SEQ ID NO:13 or a sequence having at least 85% identity with it;

[0140] (a) A promoter region comprising the cytomegalovirus (CMV) promoter sequence shown in SEQ ID NO:9 or a sequence having at least 85% identity with it;

[0141] (ii) an intron region comprising the SV40 intron sequence shown in SEQ ID NO:14 or a sequence having at least 85% identity with it;

[0142] (b) 5'UTR region, wherein the 5'UTR region contains a regulatory nucleic acid molecule or a sequence having at least 85% identity with the TPL sequence shown in SEQ ID NO:2 and the eMLP sequence shown in SEQ ID NO:7;

[0143] (c) The coding sequence that encodes the polypeptide gene product;

[0144] (iii) A second enhancement sub-region, the second enhancement sub-region comprising the SAR sequence shown in SEQ ID NO:15 or a sequence having at least 85% identity with it;

[0145] (d) Polyadenylation region, wherein the polyadenylation region comprises the bovine growth hormone (BGH or bGH) polyA sequence shown in SEQ ID NO:12 or a sequence having at least 85% identity with it.

[0146] In some embodiments of this disclosure, it comprises from 5' to 3':

[0147] (iv) 5' inverted terminal repeat (ITR) sequence, wherein the 5' inverted terminal repeat (ITR) sequence comprises the AAV ITR shown in SEQ ID NO:16 or a sequence having at least 85% identity with it;

[0148] (i) A first enhancer region, wherein the first enhancer region contains the CMV enhancer sequence shown in SEQ ID NO:13 or a sequence having at least 85% identity with it;

[0149] (a) A promoter region comprising the cytomegalovirus (CMV) promoter sequence shown in SEQ ID NO:9 or a sequence having at least 85% identity with it;

[0150] (ii) an intron region comprising the SV40 intron sequence shown in SEQ ID NO:14 or a sequence having at least 85% identity with it;

[0151] (b) 5'UTR region, wherein the 5'UTR region contains a regulatory nucleic acid molecule or a sequence having at least 85% identity with the TPL sequence shown in SEQ ID NO:2 and the eMLP sequence shown in SEQ ID NO:7;

[0152] (c) The coding sequence that encodes the polypeptide gene product;

[0153] (iii) A second enhancement sub-region, the second enhancement sub-region comprising the SAR sequence shown in SEQ ID NO:15 or a sequence having at least 85% identity with it;

[0154] (d) Polyadenylation region, wherein the polyadenylation region comprises the bovine growth hormone (BGH or bGH) polyA sequence shown in SEQ ID NO:12 or a sequence having at least 85% identity with it;

[0155] (v)3' inverted terminal repeat (ITR) sequence, wherein the (v)3' inverted terminal repeat (ITR) sequence comprises the AAV ITR shown in SEQ ID NO:17 or a sequence having at least 85% identity with it.

[0156] In some embodiments of this disclosure, it comprises from 5' to 3':

[0157] (iv) 5' inverted terminal repeat (ITR) sequence, wherein the 5' inverted terminal repeat (ITR) sequence comprises the AAV ITR shown in SEQ ID NO:16 or a sequence having at least 85% identity with it;

[0158] (i) A first enhancer region, wherein the first enhancer region contains the CMV enhancer sequence shown in SEQ ID NO:13 or a sequence having at least 85% identity with it;

[0159] (a) A promoter region comprising the cytomegalovirus (CMV) promoter sequence shown in SEQ ID NO:9 or a sequence having at least 85% identity with it;

[0160] (ii) an intron region comprising the SV40 intron sequence shown in SEQ ID NO:14 or a sequence having at least 85% identity with it;

[0161] (b) 5'UTR region, wherein the 5'UTR region contains a regulatory nucleic acid molecule or a sequence having at least 85% identity with the TPL sequence shown in SEQ ID NO:2 and the eMLP sequence shown in SEQ ID NO:7;

[0162] (c) A coding sequence encoding a polypeptide gene product, wherein the polypeptide gene product is aflibercept; preferably, the amino acid composition of aflibercept is as shown in SEQ ID NO:10; more preferably, the coding nucleic acid of aflibercept is as shown in SEQ ID NO:11;

[0163] (iii) A second enhancement sub-region, the second enhancement sub-region comprising the SAR sequence shown in SEQ ID NO:15 or a sequence having at least 85% identity with it;

[0164] (d) Polyadenylation region, wherein the polyadenylation region comprises the bovine growth hormone (BGH or bGH) polyA sequence shown in SEQ ID NO:12 or a sequence having at least 85% identity with it;

[0165] (v)3' inverted terminal repeat (ITR) sequence, wherein the (v)3' inverted terminal repeat (ITR) sequence comprises the AAV ITR shown in SEQ ID NO:17 or a sequence having at least 85% identity with it.

[0166] In some preferred embodiments of this disclosure, the expression vector comprises the sequence shown in SEQ ID NO:35, 36, 37 or 38 or a sequence having at least 85% identity with it.

[0167] In some preferred embodiments of this disclosure, the expression vector comprises the sequence shown in SEQ ID NO:35 or a sequence having at least 85% identity with it.

[0168] In one aspect, this disclosure provides a recombinant virus comprising:

[0169] a) Capsid proteins; and

[0170] b) The aforementioned expression carriers.

[0171] In some embodiments of this disclosure, the recombinant virus is a recombinant adeno-associated virus; preferably, the adeno-associated virus is selected from AAV 1 (AAV-1), AAV 2 (AAV-2), AAV 3 (AAV-3), AAV 4 (AAV-4), AAV 5 (AAV-5), AAV 6 (AAV-6), AAV 7 (AAV-7), AAV 8 (AAV-8), avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV, and sheep AAV.

[0172] In some preferred embodiments of this disclosure, the adeno-associated virus is selected from AAV 5 (AAV-5) or AAV 6 (AAV-6).

[0173] In some preferred embodiments of this disclosure, the capsid protein is an AAV variant 7m8 capsid protein or is derived from the AAV variant 7m8 capsid protein.

[0174] In one aspect, this disclosure provides a pharmaceutical composition comprising the aforementioned expression vector and / or recombinant virus and pharmaceutically acceptable excipients.

[0175] In one aspect, this disclosure provides isolated host cells transfected or transduced using the aforementioned expression vector.

[0176] In one aspect, this disclosure provides an isolated host cell infected with the aforementioned recombinant virus.

[0177] In one aspect, this disclosure provides a method for expressing a transgene in mammalian cells, the method comprising contacting one or more mammalian cells with an amount of the aforementioned expression vector and / or recombinant virus, wherein the secreted polypeptide is expressed at a certain level in the one or more mammalian cells.

[0178] In one aspect, this disclosure provides a method for treating or preventing a disease in a mammal that requires treatment or prevention of a disease, the method comprising administering an effective amount of the aforementioned expression vector, recombinant virus, pharmaceutical composition and / or host cell to the mammal.

[0179] In some preferred embodiments of this disclosure, the disease is an eye disease, and the pharmaceutical composition is applied to the eye of the mammal.

[0180] In some preferred embodiments of this disclosure, the pharmaceutical composition is administered to the eye of the mammal via intraocular injection or intravitreal injection.

[0181] In some preferred embodiments of this disclosure, the eye disease is selected from age-related macular degeneration (AMD), wet AMD, dry AMD, retinal neovascularization, choroidal neovascularization, and diabetic retinopathy.

[0182] In one aspect, this disclosure provides the use of the aforementioned expression vectors, recombinant viruses, pharmaceutical compositions and / or host cells in the preparation of treatment or prevention of diseases in mammals in need of treatment or prevention of disease.

[0183] In some preferred embodiments of this disclosure, the disease is an eye disease, and the pharmaceutical composition is applied to the eye of the mammal.

[0184] In some preferred embodiments of this disclosure, the pharmaceutical composition is administered to the eye of the mammal via intraocular injection or intravitreal injection.

[0185] In some preferred embodiments of this disclosure, the eye disease is selected from age-related macular degeneration (AMD), wet AMD, dry AMD, retinal neovascularization, choroidal neovascularization, and diabetic retinopathy.

[0186] In some aspects of this disclosure, compositions for expressing transgenes in one or more eukaryotic cells are provided. In some aspects, the eukaryotic cells are mammalian cells. In some aspects, the mammalian cells are retinal cells, such as retinal ganglion cells, amacrine cells, horizontal cells, bipolar cells, photoreceptor cells, cone cells, rod cells, Müller glial cells, or retinal pigment epithelial cells.

[0187] In some embodiments of this disclosure, the composition is an expression vector. An "expression vector" refers to a polynucleotide sequence comprising two or more functional polynucleotide sequences typically operatively linked together, such as regulatory elements, translation initiation sequences, coding sequences, termination sequences, etc. Typically, the polynucleotide sequence is composed of DNA. Similarly, an "expression vector for expressing a transgene in mammalian cells" refers to a combination of two or more functional polynucleotide sequences (e.g., promoters, enhancers, 5'UTRs, translation initiation sequences, coding sequences, termination sequences, etc.) that facilitate the expression of a transgene in cells.

[0188] In some embodiments, the expression vectors of this disclosure provide enhanced transgene expression in mammalian cells. In some embodiments, the arrangement of two or more functional polynucleotide sequences within the expression vectors of this disclosure provides enhanced transgene expression in mammalian cells. “Enhanced” means that the expression of the transgene is increased, strengthened, or stronger in cells carrying the expression vectors of this disclosure relative to cells carrying a transgene operatively linked to a comparable regulatory element. In other words, transgene expression by the expression vectors of this disclosure is increased, strengthened, or stronger relative to expression by expression vectors that do not include one or more of the optimized elements of this disclosure (i.e., reference control vectors, such as the CMV reference control vectors described herein). In some embodiments, the enhancement of expression is specific to or limited to one or more desired cell types. In one embodiment, the transgene encodes a protein that is secreted by the cell into an aqueous environment surrounding the cell.

[0189] For example, transgene expression in cells including expression vectors containing promoters disclosed herein may be enhanced, strengthened, or stronger than transgene expression in cells carrying transgenes operatively linked to different promoters. As another example, transgene expression in cells including expression vectors containing enhancer sequences disclosed herein may be enhanced, increased, strengthened, or stronger than transgene expression in cells carrying transgenes operatively linked to different enhancer sequences. As another example, transgene expression in cells including expression vectors encoding the 5'UTR disclosed herein may be enhanced, increased, strengthened, or stronger than transgene expression in cells carrying transgenes operatively linked to different 5'UTR coding sequences. As another example, transgene expression in cells including expression vectors containing introns disclosed herein may be enhanced, increased, strengthened, or stronger than transgene expression in cells carrying transgenes operatively linked to different intron sequences. In yet another instance, transgene expression in cells including intron-containing expression vectors disclosed herein may be enhanced, increased, strengthened, or more intense than in cells carrying transgenes operatively linked to a reference control vector (such as the CMV reference control vector disclosed herein).

[0190] In a preferred embodiment, the polynucleotide expression vector promotes the expression (or a higher level of expression compared to a reference vector) of the transgene in one or more specific cell or tissue types, both in vitro and in vivo. Examples of cell types include, but are not limited to, HeLa cells, HEK-293 cells, ARPE-19 cells (human retinal pigment epithelial cell line), retinal ganglion cells, amacrine cells, horizontal cells, bipolar cells, photoreceptor cells, cone cells, rod cells, Müller glial cells, and retinal pigment epithelial cells. In another embodiment, enhanced expression is observed in cells of retinal tissue explants.

[0191] In some embodiments, the expression of the secretory polypeptide by the expression vector in mammalian cells is at least about 2, 3, 5, 9, 10, 20, or 50 times that of the secretory polypeptide expressed by a reference vector in mammalian cells in vitro or in vivo. More generally, the expression of the secretory polypeptide is 2 to 10, 5 to 10, 9 to 10, at least 2, at least 5, or at least 10 times that of the polypeptide expressed by the reference vector in mammalian cells. In other words, the expression vector expresses the secretory protein at a level in mammalian cell culture that is at least 2, 5, 10, 50 times, or about 5 to about 10 times the expression level obtained by the reference vector in mammalian cell culture.

[0192] Without being bound by theory, it is assumed that enhanced transgene expression in the intracellular or extracellular environment (e.g., culture supernatant or tissue matrix) is due to a faster accumulation of gene product in the cell or a more stable gene product in the cell. Therefore, enhanced transgene expression via the expression vectors of this disclosure can be observed in a variety of ways. For example, if the transgene is operatively linked to a comparable regulatory element (such as those in the CMV reference control vectors described herein), enhanced expression can be observed by detecting transgene expression followed by detection of contact between the expression vector and the cell earlier than expression (e.g., 7 days, 2 weeks, 3 weeks, 4 weeks, 8 weeks, 12 weeks, or more). Enhanced expression can also be observed with increasing amounts of gene product per cell. For example, the amount of gene product per mammalian cell may increase by 2-fold or more, such as 3-fold or more, 4-fold or more, 5-fold or more, or 10-fold or more. Enhanced expression can also be observed with increasing numbers of mammalian cells expressing detectable levels of the transgene carried by the expression vector. For example, the number of mammalian cells expressing detectable levels of the transgene may increase by 2-fold or more, such as 3-fold or more, 4-fold or more, 5-fold or more, or 10-fold or more. As another example, the polynucleotides of the present invention can promote detectable levels of the transgene in a larger percentage of cells compared to conventional expression vectors; for example, while conventional vectors can promote detectable levels of transgene expression in, for example, less than 5% of cells in a certain region, the polynucleotides of the present invention promote detectable levels of expression in 5% or more cells in said region; for example, 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, and in some cases 50% or more, 55% or more; 60% or more, 65% or more, 70% or more, 75% or more (e.g., 80% or more, 85% or more, 90% or more, 95% or more) of the cells will express detectable levels of the gene product. Enhanced expression with changes in cell viability and / or function can also be observed.

[0193] The expression vectors disclosed herein typically include a promoter region. In some embodiments, the promoter region promotes the expression of coding sequences in mammalian cells. In some cases, the promoter is a ubiquitous promoter, i.e., a promoter that is active in a wide range of cells, tissues, and species. Suitable examples include actin, cytomegalovirus (CMV), elongation factor 1α (EF1α), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoters.

[0194] In some embodiments, the polynucleotide includes one or more enhancers. An enhancer is a nucleic acid element that enhances transcription. In some embodiments, the expression vector includes a first enhancer upstream of the coding sequence and a second enhancer downstream of the coding sequence. Suitable exemplary enhancers include, but are not limited to, EF1α, CMV, complete EES or portions thereof, such as 410-564EES or 511-810EES. The EES (expression enhancer sequence) corresponds to the human scaffold attachment region or SAR of human β-interferon (Agarwal, M et al. (1998) "Scaffold attachment region-mediated enhancement of retroviral vector expression in primary T cells" J. Virol. 72(5):3720-3728). The scaffold associated region (SAR) is an AT-rich DNA sequence, also known as the matrix attachment region (MAR). Studies have found that adding the SAR region sequence of the human interferon gene to the expression cassette of a foreign gene helps prevent methylation silencing of the foreign gene and maintains long-term stable expression of the foreign gene.

[0195] The tripartite leader sequence (TPL) of adenovirus is the UTR sequence at the 5' end of the major late-transcribed adenovirus mRNA. Studies have shown that the TPL sequence can promote the translation of mRNA, and the translation level of mRNA containing the TPL sequence at the 5' end is significantly higher than that of mRNA without the TPL sequence at the 5' end.

[0196] The major late promoter (MLP) of adenovirus is a common promoter that regulates the transcription of most late-stage genes after the adenovirus genome begins replication. The activity of the MLP increases significantly once the adenovirus genome begins replication. Adding an enhancer sequence (eMLP) of the adenovirus majorlate protein promoter near the MLP may significantly increase the expression level of exogenous genes.

[0197] In some embodiments, the expression vector includes a sequence encoding a 5' untranslated region, i.e., a polynucleotide sequence encoding the 5' untranslated region of the coding sequence, also referred to as the 5'UTR. In some embodiments, the 5'UTR does not contain the polynucleotide ATG. Suitable exemplary 5' UTR sequences include, but are not limited to, sequences selected from: i) a triplet leader sequence (TPL) from adenovirus (Logan, J et al. (June 1984), “Adenovirus tripartite leader sequence enhances translation of mRNAslate after infection,” Proceedings of the National Academy of Sciences of the United States of America (Proc. Natl. Acad. Sci. USA) 81:3655-3659); ii) enhancer element sequences from the major late promoter (eMLP) of adenovirus (Durocher, Y et al. (2002), “High-level and high-throughput recombinant protein production by transient transfection of suspension-growing human 293-EBNA1 cells”). 293-EBNA1cells), Nucleic Acids Research (Nucl.Acids.Res.) 30(2):e9); (iii) UTR1; and (iv) UTR2. In a preferred embodiment, the 5' UTR includes the TPL and eMLP sequences in 5' to 3' order.

[0198] In some embodiments, the expression vector further includes introns, which include splice donor / recipient regions. In some embodiments, the introns are located downstream of the promoter region and upstream of the translation initiation sequence of the gene. Introns are DNA polynucleotides that are transcribed into RNA via intron splicing and removed during mRNA processing. Expression vectors containing introns generally exhibit higher expression levels than those without introns. Introns can stimulate expression levels between 2 and 500-fold (Buchman and Berg, 1988, *Molecular Cell Biology*, 8(10):4395). Effectively spliced ​​introns contain a pre-splicing donor, a branching point, and a Py-rich region (Senapathy et al., 1990; *Meth. Enzymol.*, 183, 252-78; Wu and Kraner, 1999; *Molecular Cell Biology*, 19(5):3225-36). 5' introns are generally more efficient than introns located at the 3' end (Huang and Gorman, 1990; Molecular Cell Biology, 10:1805). Although introns are known to generally increase gene expression levels, specific increases for a given cDNA (if any) are empirical and must be tested; for example, chimeric introns in pSI vectors increased CAT expression by 21-fold, but only luciferase expression by 3-fold. Exemplary intron sequences include, but are not limited to, sequences from actin, elongation factor 1α (EF1α), enhancer elements from the adenovirus major late promoter (eMLP), and CMVc.

[0199] The coding sequence to be expressed in the cell can be any polynucleotide sequence, such as a gene or cDNA encoding a gene product, such as a polypeptide. The coding sequence can be heterologous to a promoter sequence and / or 5'UTR sequence operably linked to the coding sequence, i.e., the coding sequence does not naturally operably associate with the promoter or 5'UTR. Alternatively, the coding sequence can be endogenous to a promoter sequence and / or 5'UTR sequence operably linked to the coding sequence, i.e., the coding sequence naturally associates with the promoter or 5'UTR. The gene product can act intrinsically on mammalian cells, or it can act extrinsically, for example, it may be secreted. For example, when the transgene is a therapeutic gene, the coding sequence can be any gene encoding a desired gene product or a functional fragment or variant thereof, which can be used as a therapeutic agent for treating a disease or condition. Thus, the coding sequence in the expression vector can encode, for example, opsins or proteins that inhibit VEGF, or the polynucleotide can encode a protein or enzyme that effectively alleviates one or more signs or symptoms of a disease.

[0200] In various preferred embodiments, the transgene encodes a peptide or protein secreted from the cell. In some embodiments, the secreted protein is a therapeutic protein or a protein that effectively treats a disease in the subject. In some embodiments, the therapeutic protein is an anti-angiogenic peptide or a peptide that inhibits new blood vessel growth (angiogenesis). In some forms, the secreted protein is an anti-VEGF protein or a protein that inhibits vascular endothelial growth factor (VEGF). Examples of anti-VEGF proteins include ranibizumab, bevacizumab, and aflibercept. Another example of an anti-VEGF peptide is soluble fms-like tyrosine kinase-1 (sFLT-1). In other cases, the secreted protein comprises a VEGF-binding protein or a functional fragment thereof (such as any one disclosed in U.S. Patent Nos. 5,712,380, 5,861,484, and 7,071,159) or a VEGF-binding fusion protein, such as, for example, disclosed in U.S. Patent No. 7,635,474, or is composed of the thereof. In some forms, secreted proteins include or consist of single-chain antibodies (such as, for example, single-chain anti-VEGF antibodies). According to one embodiment, the transgene encodes sFLT-1, and in a more specific embodiment, the transgene encodes human sFLT-1. Alternatively, the transgene may include a sequence encoding a functional VEGF-binding fragment of sFLT-1 (Wiesmann et al., 1997; Cell, 91:695-704). According to another embodiment, the transgene encodes A1AT or α-1 antitrypsin (Chiuchiolo et al., 2013, 24(4):161-173; Stoller and Aboussouan (2012) American Journal of Respiratory and Critical Care Medicine, 185(3):246-59), said A1AT or α-1 antitrypsin being used in methods for treating diseases associated with A1AT deficiency.

[0201] sFLT-1 is a soluble truncated form of the VEGF receptor FLT-1 and is also known as soluble vascular endothelial growth factor receptor-1 (sVEGFR-1). Recombinant sFLT-1 binds to and inhibits VEGF (Kendall and Thomas, 1993; Proceedings of the National Academy of Sciences 90(22):10705-10709). In nature, recombinant sFLT-1 is generated via alternative mRNA splicing and lacks the proximal immunoglobulin-like domain, transmembrane spanning region, and intracellular tyrosine kinase domain. As described herein, “soluble” FLT-1 or sFLT-1 refers to FLT-1, which is not limited to the cell membrane. Unbound sFLT-1 can diffuse freely in the extracellular space or in solution.

[0202] In one embodiment of the invention, the transgenic coding sequence is modified or “codon-optimized” to enhance expression by replacing infrequently represented codons with more frequently represented ones. The coding sequence is a portion of an mRNA sequence that encodes the amino acids used for translation. During translation, each of the 61 trinucleotide codons is translated into one of 20 amino acids, resulting in degeneracy or redundancy in the genetic code. However, different cell types and different animal species utilize tRNAs (each carrying anticodons) that encode the same amino acids at different frequencies. When a gene sequence contains codons that are infrequently represented by the corresponding tRNA, the ribosomal translation mechanism may be slowed, thus hindering efficient translation. Expression can be improved through species-specific “codon optimization,” where the coding sequence is altered to encode the same protein sequence while utilizing highly represented and / or codons utilized by highly represented human proteins (Cid-Arregui et al., 2003; Journal of Virology 77:4928). On one hand, the coding sequence is optimized for translation in primates. In one aspect of the invention, the coding sequence of the transgene is modified to replace codons that are not frequently expressed in mammals or primates with codons that are frequently expressed in primates. For example, in some embodiments, the coding sequence encoded by the transgene encodes a polypeptide that has at least 85% sequence identity, such as at least 90%, such as at least 95%, at least 98%, or at least 99%, with a sequence identity of at least 85%, 90%, or 95%, with a sequence identity of at least 98%, or at least 99%, and wherein at least one codon of the coding sequence has a higher tRNA frequency in humans than the corresponding codon in the sequence disclosed above or herein.

[0203] In some embodiments, the expression vector of the present invention further includes an RNA output signal. The RNA output signal is a cis-acting post-transcriptional regulatory element that enhances the output of RNA from the cell nucleus. Exemplary RNA output sequences include, but are not limited to, sequences from hepatitis B virus post-transcriptional regulatory elements (HPRE) and woodchuck hepatitis virus post-transcriptional regulatory elements (WPRE) (Higashimoto, T et al., “The woodchuck hepatitis virus post-transcriptional regulatory element reduces readthrough transcription from retroviral vectors”, Gene Ther., September 2007, 14(17):1298-1304).

[0204] In some embodiments, the expression vector of the present invention further includes a polyadenylated region. As understood in the art, RNA polymerase II transcripts are terminated by cleavage and addition of a polyadenylated region, which may also be referred to as a poly(A) signal, poly(A) region, or poly(A) tail. A poly(A) region contains multiple consecutive adenosine monophosphates, typically with repeats of the motif AAUAAA. Several effective polyadenylated sites have been identified, including those derived from SV40, bovine growth hormone, human growth hormone, and rabbit β-globin (Xu et al., 2001; *Gene* 272(1-2):149-156; Xu et al., 2002; *J Control Rel.* 81(1-2):155-163). The most effective polyA signal for expressing transgenes in mammalian cells can vary depending on cell type, species of interest, and the specific vector used. In some embodiments of the present invention, the expression vector includes a polyA region selected from the group consisting of bovine growth hormone (BGH), human growth hormone (HGH), and β-globin (β-globin).

[0205] As will be understood by those skilled in the art, two or more of the polynucleotide elements mentioned above can be combined to produce the expression vector of this disclosure. Thus, for example, the expression vector may, in 5' to 3' order, include an operatively linked CMV enhancer, a CMV or EF1α promoter, optionally a CMVc or EF1α intron, a coding sequence or secreted polypeptide of UTR1, UTR2 or TPL and eMLP 5'UTR, sFLT1, an intact EES, a 410-564EES or 511-810EES enhancer, optionally an HPRE or WPRE RNA output sequence, and a BGH, HGH or β-globin polyadenylation signal sequence.

[0206] Another expression vector may, in 5' to 3' order, include an operatively linked CMV enhancer, a CMV promoter, a 5' UTR including a TPL sequence and an eMLP sequence, a coding sequence encoding a therapeutic agent (e.g., a therapeutic peptide), a full-length EES enhancer, and an HGH polyA signaling sequence. In a particular embodiment, the coding sequence encodes an anti-angiogenic peptide. In a particular embodiment, the coding sequence is codon-optimized. In some of these embodiments, the expression vector includes one or more sequences selected from SEQ ID NO:35-38.

[0207] As will be appreciated by those skilled in the art, expression vectors may optionally contain other elements, including, but not limited to, restriction sites that promote cloning and regulatory elements for a particular gene expression vector. Examples of regulatory sequences include the ITR of an AAV vector, a bacterial sequence of a plasmid vector, an attP or attB site of a phage integrase vector, and transposon elements of a transposon.

[0208] As disclosed herein, in some aspects of the invention, expression vectors are used to deliver genes into animal cells, for example, to determine the effects of genes on cell viability and / or function, to treat cellular diseases, etc. Therefore, in some aspects of the invention, compositions for expressing transgenes in mammalian cells are provided as gene delivery vectors, wherein the gene delivery vectors include the expression vectors disclosed herein.

[0209] The gene delivery vectors disclosed herein encompass any convenient gene delivery vector for delivering polynucleotide sequences into mammalian cells. For example, the vector may comprise a single-stranded or double-stranded nucleic acid, such as single-stranded or double-stranded DNA. For example, the gene delivery vector may be DNA, such as naked DNA, such as plasmids or microcircles. The vector may comprise single-stranded or double-stranded RNA, containing a modified form of RNA. In another instance, the gene delivery vector may be RNA, such as mRNA or modified mRNA.

[0210] As another example, the gene delivery vector can be a viral vector derived from a virus, such as adenovirus, adeno-associated virus (AAV), lentivirus, herpesvirus, alpha virus, or retrovirus, such as Moloney murine leukemia virus (M-MuLV), Moloney murine sarcoma virus (MoMSV), Harvey murine sarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), gibberish leukemia virus (GaLV), feline leukemia virus (FLV), foam virus, Friend murine leukemia virus, murine stem cell virus (MSCV), and Rous sarcoma virus (RSV), or lentivirus. While embodiments covering the use of adeno-associated viruses are described in more detail below, it is expected that those skilled in the art will recognize that similar knowledge and skills in the art can also be applied to non-AAV gene delivery vectors. See, for example, the discussion of retroviral vectors in U.S. Patent Nos. 7,585,676 and 8,900,858, and the discussion of adenoviral vectors in U.S. Patent No. 7,858,367, the entire disclosure of which is incorporated herein by reference.

[0211] In some embodiments, the gene delivery vector is a recombinant adeno-associated virus (rAAV). In this embodiment, the expression vector has functional AAV inverted terminal repeat (ITR) sequences flanked at the 5' and 3' ends. A “functional AAV ITR sequence” refers to an ITR sequence intended for rescuing, replicating, and packaging AAV viral particles. Therefore, the AAV ITR used in the gene delivery vector of the present invention does not need to have a wild-type nucleotide sequence and can be altered by nucleotide insertion, deletion, or substitution, or the AAV ITR can be derived from any of several AAV serotypes, such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, and AAV10. Preferred AAV vectors have all or part of the wild-type Rep and Cap genes deleted, but retain the functional flanked ITR sequences. In a particular embodiment, the AAV viral vector is the AAV2 variant 7m8.

[0212] In some embodiments, the expression vector is capsidated within an AAV capsid, which can be derived from any adeno-associated virus (AAV) serotype, including but not limited to AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, etc., and any of these AAV serotypes can serve as a gene delivery vector. For example, the AAV capsid can be a wild-type capsid or a natural capsid. Wild-type AAV capsids of particular interest include AAV2, AAV5, and AAV9. However, similar to ITRs, the capsid does not need to have a wild-type nucleotide sequence, but can be altered relative to the wild-type sequence by inserting, deleting, or substituting nucleotides in the VP1, VP2, or VP3 sequences, as long as the capsid is capable of transducing mammalian cells. In other words, the AAV capsid can be a variant AAV capsid comprising one or more amino acid substitutions, deletions, or insertions relative to the derived parental capsid protein or AAV capsid protein. The variant AAV of particular concern includes those variant AAVs disclosed in U.S. Patent 9,193,956, the entire disclosure of which is incorporated herein by reference. In some embodiments, the variant AAV includes the 7m8 variant capsid protein disclosed in U.S. Patent 9,193,956 (which may be referred to herein as AAV2.7m8 or 7m8.AAV2).

[0213] Preferably, rAAV is replication-defective because the AAV vector cannot independently replicate and package its genome further. For example, when cone cells are transduced with rAAV viral particles, the gene is expressed in the transduced cone cells; however, rAAV cannot replicate because the transduced cone cells lack the AAV rep and cap genes, as well as the helper genes.

[0214] Gene delivery vectors (e.g., rAAV viral particles) that capsidate the expression vectors of this disclosure can be generated using standard methods. For example, in the case of rAAV viral particles, the AAV expression vector according to the invention can be introduced into production cells, followed by the introduction of an AAV helper construct, wherein the helper construct contains an AAV coding region capable of expression in the production cells and said AAV coding region supplements AAV helper functions not present in the AAV vector. A helper virus and / or additional vectors are then introduced into the production cells, wherein the helper virus and / or additional vectors provide helper functions capable of supporting efficient rAAV viral production. The production cells are then cultured to produce rAAV. These steps are performed using standard methods. Replication-deficient AAV viral particles that capsidate the recombinant AAV vectors of the invention are prepared using AAV packaging cells and packaging techniques known in the art. Examples of these methods can be found, for example, in U.S. Patent No. 5,436,146; U.S. Patent Nos. 5,753,500, 6,040,183, 6,093,570, and 6,548,286, all of which are expressly incorporated herein by reference in their entirety. Wang et al. (US 2002 / 0168342) describe additional compositions and methods for packaging, which are also incorporated herein by reference in their entirety.

[0215] Viral particles of any concentration suitable for efficient transduction into mammalian cells can be prepared for contact with mammalian cells in vitro or in vivo. For example, at a concentration of 10-1 / mL... 8 The concentration of one or more vector genomes (vg / mL) is used to adjust the viral particles, for example, 5 × 10⁻⁶ per milliliter. 8 One vector genome; 10 per milliliter 9 One vector genome; 5 × 10⁵ per milliliter 9 One vector genome, 10 per milliliter 10 One vector genome, 5 × 10 per milliliter 10 One vector genome; 10 per milliliter 11 One vector genome; 5 × 10⁵ per milliliter 11 One vector genome; 10 per milliliter 12 One vector genome; 5 × 10⁵ per milliliter 12 One vector genome; 10 per milliliter 13 One vector genome; 1.5 × 10⁻⁶ per milliliter 13 One vector genome; 3 × 10 per milliliter 13 One vector genome; 5 × 10⁵ per milliliter 13 One vector genome; 7.5 × 10⁻⁶ per milliliter 13 One vector genome; 9 × 10⁹ per milliliter13 One vector genome; 1×10 per milliliter 14 One vector genome, 5 × 10 per milliliter 14 One or more vector genomes, but typically no more than 1 × 10^6 per milliliter. 15 A vector genome. Similarly, any total number of viral particles suitable for providing appropriate cellular transduction to confer the desired effect or treat a disease can be administered to mammals. In various preferred embodiments, at least 10 8 5×10 8 10 9 5×10 9 10 10 5×10 10 10 11 5×10 11 10 12 5×10 12 10 13 1.5 × 10 13 3×10 13 5×10 13 7.5 × 10 13 9×10 13 1×10 14 One or 5×10 14 One or more viral particles, but the number of viral particles injected into each eye usually does not exceed 1 × 10⁻⁶. 15 Any suitable number of carriers can be applied to the eyes of mammals or primates. In one embodiment, the method includes a single application; in other embodiments, multiple applications may be performed over time as deemed appropriate by the attending clinician.

[0216] The viral vector can be formulated into a pharmaceutical composition comprising any suitable unit dose of the vector, and the pharmaceutical composition can be administered to a subject to produce changes in the subject or treat a disease in the subject. In some embodiments, the unit dose includes, but is not limited to, 1 × 10⁻⁶ of the viral vector. 8 One or more vector genomes, for example at least about 1 × 10⁻⁶ 9 1×10 10 1×10 11 1×10 12 1×10 13 1×10 14 One or at least about 3 × 10 14 One or more vector genomes, in some cases at least about 1 × 10⁻⁶. 14 One vector genome, but usually no more than 4 × 10 15One vector genome. In some cases, the unit dose includes up to approximately 5 × 10⁻⁶. 15 One vector genome, for example 1×10 14 One or 5×10 14 One or fewer vector genomes, for example, 1 × 10 13 1×10 12 1×10 11 1×10 10 One or 1×10 9 One or fewer vector genomes, in some cases, 1 × 10 8 One or fewer vector genomes, and usually no less than 1 × 10⁻⁶. 8 One vector genome. In some cases, the unit dose includes 1×102 10 One to 1×10 11 One vector genome. In some cases, the unit dose includes 1×102 10 3×10 12 One vector genome. In some cases, the unit dose includes 1×102 9 3×10 13 One vector genome. In some cases, the unit dose includes 1×102 8 3×10 14 One vector genome. In some cases, the unit dose comprises approximately 1 × 10⁻⁶. 10 Approximately 5 x 10 14 One vector genome.

[0217] In some cases, the multiple of infection (MOI) can be used to measure the unit dose of a drug composition. MOI refers to the ratio or fold of the vector or viral genome to nucleic acid that can be delivered to cells. In some cases, the MOI can be 1 × 10⁻⁶. 6 In some cases, the MOI can be 1×10. 5 -1×10 7 In some cases, the MOI can be 1×10. 4 -1×10 8 In some cases, the recombinant virus disclosed herein is at least about 1 × 10⁻⁶. 1 1×10 2 1×10 3 1×10 4 1×10 5 1×10 6 1×10 7 1×10 8 1×10 9 1×10 10 1×10 111×10 12 1×10 13 1×10 14 1×10 15 1×10 16 1×10 17 and 1×10 18 One MOI. In some cases, the recombinant virus disclosed herein is 1×10 8 3×10 14 One MOI. In some cases, the recombinant virus disclosed herein has a maximum size of approximately 1 × 10⁻⁶. 1 1×10 2 1×10 3 1×10 4 1×10 5 1×10 6 1×10 7 1×10 8 1×10 9 1×10 10 1×10 11 1×10 12 1×10 13 1×10 14 1×10 15 1×10 16 1×10 17 and 1×10 18 One MOI.

[0218] In some respects, the amount of the pharmaceutical composition includes about 1 × 10⁻⁶. 8 Approximately 1×10 15 One recombinant virus, approximately 1×10 9 Approximately 1×10 14 One recombinant virus, approximately 1×10 10 Approximately 1×10 13 One recombinant virus or approximately 1×10 11 Approximately 3 x 10 12 A recombinant virus.

[0219] In preparing the rAAV composition, any host cell used to produce rAAV viral particles can be used, including, but not limited to, mammalian cells (e.g., 293 cells), insect cells (e.g., SF9 cells), microorganisms, and yeast. The host cell can also be a packaging cell or a production cell, in which the AAV rep and cap genes are stably maintained within the host cell, and the AAV vector genome is stably maintained and packaged in the production cell. Exemplary packaging and production cells are derived from SF-9, 293, A549, or HeLa cells. The AAV vector is purified and formulated using standard techniques known in the art.

[0220] This invention comprises pharmaceutical compositions including expression vectors or gene delivery vectors described herein, and pharmaceutically acceptable carriers, diluents, or excipients. For example, one embodiment is a pharmaceutical composition comprising a recombinant virus, said recombinant virus comprising the polynucleotides of this disclosure and pharmaceutically acceptable excipients. In a specific embodiment, the recombinant virus is a recombinant adeno-associated virus (AAV). Expression vectors or gene delivery vectors can be combined with pharmaceutically acceptable carriers, diluents, and reagents that can be used to prepare formulations that are generally safe, non-toxic, and desirable, and contain acceptable excipients for primate use. Such excipients can be solid, liquid, semi-solid, or gaseous (in the case of aerosol compositions). Examples of such carriers or diluents include, but are not limited to, water, saline, Ringer's solution, dextran solution, and 5% human serum albumin. Complementary active compounds may also be incorporated into these formulations. The solution or suspension used for formulation may contain: a sterile diluent, such as water for injection, saline solution, non-volatile oil, polyethylene glycol, glycerol, propylene glycol, or other synthetic solvents; an antimicrobial compound, such as benzyl alcohol or methylparaben; an antioxidant, such as ascorbic acid or sodium bisulfite; a chelating compound, such as ethylenediaminetetraacetic acid (EDTA); a buffer, such as acetate, citrate, or phosphate; a detergent, such as Tween 20 to prevent aggregation; and a compound for adjusting tension, such as sodium chloride or dextrose. The pH may be adjusted with an acid or base (such as hydrochloric acid or sodium hydroxide). In certain embodiments, the pharmaceutical composition is sterile.

[0221] In cases where the cells come into contact with cone cells in vivo, expression vectors or gene delivery vectors containing expression vectors can be considered suitable for delivery to the eye.

[0222] Suitable pharmaceutical compositions for use in this invention further comprise a sterile aqueous solution or dispersion and a sterile powder for the ad hoc preparation of a sterile injectable solution or dispersion. Thus, the pharmaceutical composition can be in the form of a sterile injectable solution. For intravenous administration, suitable carriers include physiological saline, antibacterial water, or phosphate-buffered saline (PBS). In some cases, the composition is sterile and should have a flowability sufficient for easy injection. In some embodiments, the composition is stable under the conditions of manufacture and storage and is preserved against contamination by microorganisms such as bacteria and fungi. The carrier can be, for example, a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.), and suitable mixtures thereof. Appropriate flowability can be maintained, for example, by using a coating (e.g., lecithin), by maintaining the desired particle size in the case of a dispersion, and by using a surfactant. Antimicrobial activity can be achieved by various antibacterial and antifungal agents (e.g., parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc.). In many cases, it is preferred to include an isotonic agent in the composition, such as sugar, polyol (e.g., mannitol, sorbitol), or sodium chloride. Prolonged absorption of the internal composition can be achieved by including agents that delay absorption (e.g., aluminum monostearate and gelatin) in the composition.

[0223] As needed, a sterile solution can be prepared by incorporating the desired amount of the active compound with one or a combination of the ingredients listed above into a suitable solvent, followed by sterilization by filtration. Typically, a dispersion is prepared by incorporating the active compound into a sterile medium containing a base dispersion medium and other desired ingredients from those listed above. In the case of sterile powders used to prepare sterile injectable solutions, the preparation methods are vacuum drying and freeze-drying, which produce a powder of the active ingredient plus any other desired ingredients from a previously sterile filtered solution.

[0224] In one embodiment, the composition is prepared together with a carrier that prevents the expression vector from rapid elimination from the body, such as a controlled-release formulation comprising an implant and a microencapsulated delivery system. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydride, polyglycolic acid, collagen, polyorthoester, and polylactic acid can be used. Methods for preparing such formulations will be apparent to those skilled in the art. The materials are also commercially available. Liposome suspensions (containing liposomes targeting infected cells with monoclonal antibodies against viral antigens) can also be used as pharmaceutically acceptable carriers. These formulations can be prepared according to methods known to those skilled in the art, such as those described in U.S. Patent No. 4,522,811.

[0225] Particularly advantageous is the formulation of oral, ocular, or parenteral compositions in dose units for ease of administration and dosage uniformity. As used herein, dose units refer to physically discrete units suitable as a single dose for a subject to be treated; each unit contains a predetermined amount of a gene delivery vector or expression vector calculated to produce the desired therapeutic effect associated with the desired drug delivery agent. The specifications of the dose unit form of the present invention are determined by the unique characteristics of the gene delivery vector, expression vector, and the specific therapeutic effect to be achieved.

[0226] The pharmaceutical composition may be included in a container, package, or dispenser (e.g., a syringe, such as a pre-filled syringe) along with the instructions for use.

[0227] "Pharmaceutically acceptable excipients" are materials, substances, diluents, or carriers that are substantially non-toxic to cells or the subject to which they are administered. In other words, pharmaceutically acceptable excipients may be incorporated into a pharmaceutical composition and administered to cells or a patient without substantially causing undesirable biological effects or interacting in a harmful manner with any other component of the composition containing the pharmaceutically acceptable excipient.

[0228] Expression vectors or gene delivery vectors (e.g., recombinant viruses (viral particles)) can be incorporated into pharmaceutical compositions for administration to mammalian patients, specifically primates and more specifically humans. The expression vectors or gene delivery vectors (e.g., viral particles) can be formulated with a non-toxic, inert, pharmaceutically acceptable aqueous carrier, preferably with a pH in the range of 3 to 8, more preferably in the range of 6 to 8, or even more preferably in the range of 7 to 8. Such sterile compositions will comprise vectors or viral particles containing nucleic acids encoding a therapeutic molecule dissolved in an aqueous buffer solution having an acceptable pH during reconstitution.

[0229] In some embodiments, the pharmaceutical compositions provided herein comprise a therapeutically effective amount of a carrier or viral particle mixed with a pharmaceutically acceptable carrier and / or excipient, such as saline, phosphate-buffered saline, phosphate, and optionally one or more other agents, such as amino acids, polymers, polyols, sugars, buffers, preservatives, proteins, and inorganic salts (e.g., sodium chloride). Exemplary amino acids, polymers, and sugars include octylphenoxypolyethoxyethanol compounds, polyethylene glycol monostearate compounds, polyoxyethylene sorbitan fatty acid esters, sucrose, fructose, dextrose, maltose, glucose, mannitol, dextran, sorbitol, inositol, galactitol, xylitol, lactose, trehalose, bovine or human serum albumin, citrate, acetate, Ringer's and Hank's solutions, cysteine, arginine, carnitine, alanine, glycine, lysine, valine, leucine, polyvinylpyrrolidone, polyethylene, and ethylene glycol. Preferably, this formulation is stable at 4°C for at least six months.

[0230] In some embodiments, the pharmaceutical compositions provided herein include buffers such as phosphate-buffered saline (PBS) or sodium phosphate / sodium sulfate, Tris buffer, glycine buffer, sterile water, and other buffers known to those skilled in the art, such as those described in Good et al., (1966) Biochemistry 5(2):467-477. The pH of the buffer may be in the range of 6.5 to 7.75, preferably 7 to 7.5, and most preferably 7.2 to 7.4. The pharmaceutical compositions may include an adenovirus or adeno-associated adenovirus vector delivery system containing the expression vector of this disclosure.

[0231] The ability to deliver the expression vectors of the present invention in vivo to selected target cells and to obtain therapeutically effective amounts of the gene product in the cells and extracellular environment after a transduction event could be beneficial for treating many different diseases, including those dependent on angiogenesis, where the goal of treatment could be to enable target cells to secrete therapeutically effective amounts of anti-angiogenic proteins. While not wishing to be bound by any theory, expression vectors capable of enhancing expression and ultimately enhancing the secretion of therapeutic proteins can help provide significant clinical benefit to patients, even when the gene delivery vector only successfully transduces a subset of target cells. High levels of secretion of therapeutic proteins by transduced cells can help balance the infectivity or transduction efficiency achieved with any given dose or cycle of gene therapy.

[0232] Therefore, the expression vectors and gene delivery vectors collectively referred to herein as "compositions" can be used to express transgenes in animal cells. For example, the compositions can be used in studies, such as determining the effect of genes on cell viability and / or function. As another example, the compositions can be used in medicine to treat conditions, for example. The methods and compositions disclosed herein can be used to treat any condition that can be at least partially resolved by gene therapy via cells. Cells include, but are not limited to, blood, eyes, liver, kidneys, heart, muscles, stomach, intestines, pancreas, and skin.

[0233] Therefore, the present invention provides a method for treating or preventing a disease or condition (e.g., an eye disease or condition) in a subject in need, the method comprising administering to the subject in need a viral vector or viral particle comprising an expression vector of the present invention encoding a therapeutic gene product. In a preferred embodiment, the therapeutic gene product is a secreted polypeptide or a protein secreted or exported from a cell after synthesis in the cell, and the expression vector comprises, in 5' to 3' order: (i) a first enhancer region comprising a CMV sequence (SEQ ID NO: 13); (a) a promoter region comprising a CMV sequence (SEQ ID NO: 9); (b) a 5' UTR region comprising, in 5' to 3' order, a TPL sequence and an eMLP sequence (SEQ ID NO: 2 and SEQ ID NO: 7, respectively); (c) a coding sequence encoding a peptide or polypeptide; (iii) a second enhancer region comprising human IFNB SAR (SEQ ID NO: 15); and (d) an HGH polyadenylation site (SEQ ID NO: 12).

[0234] In related embodiments, some methods provide gene expression in cells in vitro or in vivo, the methods including contacting the cells with the composition of this disclosure. In some embodiments, the contact occurs in vitro. In some embodiments, the contact occurs in vivo, i.e., administering the composition to a subject. The composition can be administered parenterally by intravenous injection or oral infusion. In some embodiments, the composition is administered to the eye by injection, for example, to the retina, lower retina, or vitreous humor. In some embodiments, the composition is administered by retinal injection, lower retinal injection, or intravitreal injection. In some embodiments, the composition is administered locally or directly to the tissue or organ of interest, for example, by injection into the liver.

[0235] Subjects can be mammals, including human subjects who require treatment for specific diseases or conditions.

[0236] For cases where mammalian cells are contacted in vitro with an expression vector or a gene delivery vector containing the expression vector, the cells can originate from any mammalian species, such as rodents (e.g., mice, rats, gerbils, squirrels), rabbits, cats, dogs, goats, sheep, pigs, horses, cattle, primates, and humans. The cells can originate from established cell lines or can be primary cells.

[0237] In this document, the terms "primary cells," "primary cell lines," and "primary cultures" are used interchangeably to refer to cells and cell cultures derived from the subject and permitted for a limited number of passages, i.e., divisions, in vitro. For example, a primary culture is a culture that may have been passaged 0, 1, 2, 4, 5, 10, or 15 times, but the number of passages is insufficient to reach a critical stage. Typically, the primary cell lines of this invention are maintained in vitro for fewer than 10 passages.

[0238] If the cells are primary cells, they can be collected from mammals by any convenient method, such as whole explants, biopsies, etc. The collected cells can be dispersed or suspended in a suitable solution. This solution will typically be a balanced salt solution, such as physiological saline, PBS, Hank's balanced salt solution, etc., conveniently supplemented with fetal bovine serum or other naturally occurring factors bound at low concentrations (typically 5-25 mM) to an acceptable buffer. Convenient buffers include HEPES, phosphate buffer, lactate buffer, etc. The cells can be used immediately or stored, frozen for extended periods, thawed, and reused. In this case, cells are typically frozen in 10% DMSO, 50% serum, 40% buffered medium, or some other such solutions commonly used in the art to preserve the cells at this freezing temperature and thawed in a manner as commonly known in the art for thawing frozen cultured cells.

[0239] To promote the expression of transgenes, the expression vector or gene delivery vector including the expression vector is contacted with cells for approximately 30 minutes to 24 hours or longer, such as 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, 3.5 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 12 hours, 16 hours, 18 hours, 20 hours, 24 hours, etc. The expression vector or gene delivery vector including the expression vector can be provided to the subject cells once or multiple times (e.g., once, twice, three times, or more than three times), and the cells are allowed to be cultured with one or more agents for a certain period of time after each contact event (e.g., 16-24 hours), after which the medium is replaced with fresh medium and the cells are further cultured. Cell contact can occur in any medium and under any culture conditions that promote cell survival. For example, cells can be suspended in any suitable and convenient nutrient medium, such as Iscove's modified DMEM or RPMI 1640, supplemented with fetal bovine serum or heat-inactivated goat serum (approximately 5-10%), L-glutamine, thiols, particularly 2-mercaptoethanol, and antibiotics such as penicillin and streptomycin. The culture may contain cell-responsive growth factors. As defined herein, growth factors are molecules capable of promoting cell survival, growth, and / or differentiation in culture or intact tissues through specific action on transmembrane receptors. Growth factors include peptide factors and non-peptide factors.

[0240] Typically, an effective amount of the expression vector or gene delivery vector comprising the expression vector is provided to produce transgenic expression in cells. As discussed elsewhere herein, the effective amount can be readily determined empirically, for example, by detecting the presence or level of the transgenic product, by detecting its effect on cell viability or function, etc. Typically, expression will be enhanced by 2-fold or more, such as 3-fold, 4-fold, or 5-fold or more, and in some cases, 10-fold, 20-fold, or 50-fold or more, such as 100-fold, relative to expression performed with a reference or control expression vector. An example of a reference expression vector for comparative purposes is the CMV reference expression vector described herein. In a specific embodiment, the transgene encodes a secretory protein, and the expression vector expresses the secretory protein at a level in mammalian cells that is at least 2-fold, 5-fold, 10-fold, 5-fold to 10-fold, 5-fold to 15-fold, or 10-fold to 15-fold higher than the expression level of the secretory protein performed in mammalian cells by the CMV reference control expression vector. According to some embodiments, when the transgene is a transgene encoding a non-secretory protein, the expression vector of the present invention expresses the non-secretory protein at a level in mammalian cells that is approximately the same as, within 10-20% of, the expression level of the non-secretory protein expressed by the CMV reference control expression vector in mammalian cells, and is about 1.5 to 2 times lower. The expression level of the secretory protein of each expression vector can be measured by immunoassay or antigen capture assay, and the expression level can be expressed as the amount or concentration of protein per volume of supernatant (e.g., cell culture medium or supernatant) in the extracellular environment.

[0241] Immunoassays for measuring the presence and quantity (and therefore expression level) of proteins in biological or cellular samples are known in the art (e.g., Hage, DS (1999), “Immunoassays”, Analytical Chemistry 71(12):294-304; The Immunoassay Handbook, 4th edition: Theory and Application of Ligand Binding, ELISA and Related Techniques, Elsevier Science (2013)). Typically, immunoassays are based on the reaction between a target protein and an antibody or antibody fragment that specifically binds to the protein. Immunoassays can be performed in liquid or solid phase systems, but a solid phase may be preferred for ease of detection. Suitable immunoassays include, but are not limited to, sandwich and competitive assays, Western blotting, ELISA (enzyme-linked immunosorbent assay), radioimmunoassay (RIA), fluorescence immunoassay (FIA), etc. Biological samples can be cell culture media or supernatants (samples taken from cultures without cell lysis), cell lysates, whole cells, blood, serum, plasma, or other body fluids or tissues. In some embodiments, such as when the transgene is an optional marker, cell populations can be enriched to include those containing the expression vector by separating the modified cells from the remaining population. Separation can be performed using any convenient separation technique suitable for the optional marker used. For example, if the transgene is a fluorescent marker, cells can be separated by fluorescence-activated cell sorting, while if the transgene is a cell surface marker, cells can be separated from heterogeneous populations by affinity separation techniques, such as magnetic separation, affinity chromatography, “panning” with affinity reagents attached to a solid matrix, or other convenient techniques. Techniques providing precise separation include fluorescence-activated cell sorters, which can have varying degrees of complexity, such as multicolor channels, low-angle and obtuse-angle light scattering detection channels, impedance channels, etc. Cells can be selected for dead cells by employing dyes that associate with dead cells (e.g., propidium iodide). Any technique that does not excessively harm cell viability can be used. This method achieves a cell composition with a high enrichment of polynucleotides. "High enrichment" means that the genetically modified cells will constitute 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, for example, about 95% or more or 98% or more of the cell composition.

[0242] In cases where cells come into contact with the expression vector or a gene delivery vector including the expression vector in vivo, the subject can be any mammal, such as rodents (e.g., mice, rats, gerbils), rabbits, cats, dogs, goats, sheep, pigs, horses, cattle, humans, or non-human primates.

[0243] The methods and compositions of the present invention can be used to treat any condition that can be at least partially resolved by gene therapy of cells. Cells include, but are not limited to, blood, eyes, liver, kidneys, heart, muscles, stomach, intestines, pancreas, and skin. One embodiment is a method for treating a medical condition in a subject with therapeutic need, the method comprising administering to the subject a gene delivery vector containing an expression vector as disclosed herein, wherein the expression vector encodes a polypeptide that effectively alleviates one or more signs or symptoms of the medical condition. In some embodiments, the medical condition is an ocular disease, the gene delivery vector is an adeno-associated virus, and the polypeptide is a polypeptide secreted by cells transduced by the vector. In one embodiment, a secreted protein inhibits VEGF signaling. For example, the secreted protein may be a VEGF-binding protein. In some embodiments, the ocular disease is choroidal neovascularization or macular degeneration. Specific forms of macular degeneration may include acute macular degeneration, non-exudative age-related macular degeneration, and exudative age-related macular degeneration. It can be administered by any suitable means, including, for example, ocular delivery, intravitreal injection, intraocular injection, retinal injection, subretinal injection, parenteral administration, intravenous injection or infusion, and injection into the liver.

[0244] Age-related macular degeneration (AMD) is a common eye disease and a major cause of vision loss in the elderly. Based on different fundus manifestations, progressive AMD can be divided into dry AMD and wet AMD. VEGF-induced pathological choroidal neovascularization is the core mechanism of the pathogenesis and progression of wet AMD, leading to exudation, hemorrhage, and fibrous scarring. Anti-VEGF therapy is the most effective treatment for wet AMD, but the efficacy of anti-VEGF antibody injections is only short-lived, thus requiring repeated ocular injections. Using gene therapy vectors such as adeno-associated virus (AAV) to deliver protein-coding sequences that block VEGF biological activity to the fundus can achieve sustained expression of proteins that block VEGF biological activity, thereby achieving sustained inhibition of pathological choroidal neovascularization with a single dose.

[0245] In some embodiments, the gene delivery vector is administered to the eye of a subject in need of treatment. In some embodiments, the gene delivery vector is administered to the subject via intraocular injection, intravitreal injection, or any other convenient mode of administration or route. In some embodiments, the subject is a human subject with macular degeneration or ocular neovascularization or who may be at risk of developing it.

[0246] In some embodiments, the method produces a therapeutic benefit, such as preventing disease progression, stopping disease progression, reversing disease progression, etc. In some embodiments, the method includes a step of detecting whether the therapeutic benefit has been achieved. Those skilled in the art will understand that such a measurement of therapeutic efficacy will be applicable to the specific disease being modified, and will recognize appropriate detection methods for measuring therapeutic efficacy.

[0247] It is expected that the transgenic expression obtained using the transgene will be robust. Therefore, in some cases, transgenic expression can be observed (e.g., detected by measuring the level of the gene product) two months or less after application (e.g., 4 weeks, 3 weeks, or 2 weeks or less after application, e.g., 1 week after application of the composition) by measuring therapeutic efficacy, etc. It is also expected that transgenic expression will persist over time. Therefore, in some cases, transgenic expression can be observed (e.g., detected by measuring the level of the gene product) two months or more after application of the composition by measuring therapeutic efficacy, etc. (e.g., 4 months, 6 months, 8 months, or 10 months or more, in some cases 1 year or more, e.g. 2 years, 3 years, 4 years, or 5 years, and in some cases more than 5 years).

[0248] In some embodiments, the method includes the step of detecting transgene expression in cells, wherein the expression is enhanced relative to expression using an expression vector that does not include one or more of the modified elements of this disclosure (i.e., a reference control). Typically, as demonstrated by, for example, earlier detection, higher levels of gene product, or stronger functional effects on cells, the expression will be enhanced by 2-fold or more, such as 3-fold, 4-fold, or 5-fold or more, and in some cases, 10-fold, 20-fold, or 50-fold or more (e.g., 100-fold), relative to expression using a reference (i.e., a control expression vector). On one hand, the transgene encodes a secreted polypeptide (such as sFLT1).

[0249] Typically, if the subject composition is a virus (e.g., rAAV including the polynucleotide expression vector of this disclosure), the effective amount for achieving changes or producing a therapeutic effect in a subject will be about 1 × 10⁻⁶. 8 One vector genome or more, in some cases, 1×10 9 1×10 10 1×10 11 1×10 12 Or 1×10 13 One vector genome or more, in some cases, 1×10 14 One or more vector genomes, and usually no more than 1 × 10⁻⁶. 16 One vector genome. In some cases, the amount of vector genome delivered is at most about 1 × 10⁻⁶.16 One vector genome, for example 1×10 15 One vector genome or fewer, such as 1×10 13 1×10 12 1×10 11 1×10 10 Or 1×10 9 One vector genome or fewer, in some cases 1×10 8 One vector genome, and usually no less than 1×10 8 One vector genome. In some cases, the amount of vector genome delivered is 1 × 10⁻⁶. 10 One to 1×10 11 One vector genome. In some cases, the amount of vector genome delivered is 1 × 10⁻⁶. 10 3×10 12 One vector genome. In some cases, the amount of vector genome delivered is 1 × 10⁻⁶. 9 3×10 13 One vector genome. In some cases, the amount of vector genome delivered is 1 × 10⁻⁶. 8 3×10 14 One vector genome.

[0250] In some cases, the multiple of infection (MOI) can be used to measure the amount of a drug composition to be administered. In some cases, MOI can refer to the ratio or fold increase in the number of cells to which the vector particle or viral genome can be delivered to the polynucleotide expression vector. In some cases, MOI can be 1 × 10⁻⁶. 6 In some cases, the MOI can be 1×10. 5 One to 1×10 7 In some cases, the MOI can be 1×10. 4 One to 1×10 8 In some cases, the recombinant virus disclosed herein is approximately 1 × 10⁻⁶. 1 1×10 2 1×10 3 1×10 4 1×10 5 1×10 6 One or 1×10 7 One MOI.

[0251] In some respects, the individual dose is generally not less than the amount required to produce a measurable effect in the subject and can be determined based on the pharmacokinetics and pharmacology of the absorption, distribution, metabolism, and excretion (“ADME”) of the composition or its byproducts and therefore on the disposal of the composition within the subject. This includes consideration of the route of administration and dosage. Effective doses and / or dosage regimens can be readily determined empirically based on preclinical assays, safety and escalation and dose range trials, individual clinician-patient relationships, and in vitro and in vivo assays.

[0252] In many embodiments, the recombinant virus can be administered in combination with one or more additional compounds or therapies, including a second recombinant virus, chemotherapeutic agents, surgery, catheter devices, and radiation. Combination therapy includes administration of a single-dose formulation comprising the recombinant virus and one or more additional agents; it also includes administration of the recombinant virus and one or more additional agents in their own separate drug dosage form. For example, the recombinant virus and cytotoxic agents, chemotherapeutic agents, or growth inhibitors can be administered to a patient together as a single-dose composition, such as a combination formulation, or each agent can be administered in a separate dosage form. When using separate dosage forms, the VEGF-specific fusion peptide of the present invention and one or more additional agents can be administered simultaneously or sequentially at staggered times.

[0253] As used herein, the term "cytotoxic agent" refers to a substance that inhibits or impairs cell function and / or causes cell damage. This term includes radioactive isotopes (e.g., I131, I125, Y90, and Re186), chemotherapeutic agents, and toxins such as enzymatically active toxins of bacterial, fungal, plant, or animal origin, or fragments thereof.

[0254] Chemotherapy agents are chemical compounds used to treat cancer. Examples of chemotherapy agents include alkylating agents such as thiotepa and cyclophosphamide preparations; alkyl sulfonates such as busulfan, indomethacin, and piperazine; aziridines such as benzodopa, carboquinone, meturedopa, and uredopa; ethyleneimine and methylamelamines including hexamethylmelamine, tratamine, trietylenephosphoramide, trimethylolomeleamine; nitrogen mustard gases such as chlorambucil, naphthiamethoxam, cholophosphamide, estradiol, ifosfamide, nitrogen mustard, oxynitrogen mustard hydrochloride, melphalan, neonitrogen mustard, nitrogen mustard cholesterol, prednimustine, trazophosphamide, uramustine; nitrosoureas such as carmustine, chloramphenicol, formustine, lomustine, nimustine, and ramustine; and antibiotics such as aclacinomysins, actinomycin, and authraquinone. Mycin, Diazoserine, Bleomycin, Actinomycin C, Calcihamnine, Carabicin, Erythromycin, Carcinomycin, Chromomycin, Actinomycin D, Daunorubicin, Ditorubicin, 6-Diazo-5-O-L-Leucine, Doxorubicin, Epirubicin, Isorubicin, Idarubicin, Maceroxam, Mitomycin, Mycophenolic acid, Nopramine, Oligomycin, Pelomycin, Potfiromycin, Puromethromycin, Doxorubicin, Romaine Doxorubicin, streptomycin, strepzotocin, tuberculin, ubenmex, fenestrated succinate (new carcinogen), zorubicin, antimetabolites such as methotrexate, 5-fluorouracil, folic acid analogs such as denopterin, methotrexate, pteroxate, trimethoprim, purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine, pyrimidine analogs such as ancitabine, azacitidine, 6-azouridine, carmoflurane, cytarabine, dideoxyuridine, deoxyfluorouridine, enoxabin, fluorouridine;

[0255] Androgens such as capprotestone, drotalbuterone propionate, cyclothiosterol, meandrazan, testrolide; adrenocorticotropic agents such as aminoglutethimide, mitotane, trelostan; folic acid reuptake agents such as frolinic acid; aceglucanolactone; aldophosphamide glycoside; 5-aminolevulinic acid; acridine; bestrabucil; bisacodyl; edatraxate; defofamine; colchicine; diacodyl; elfornithine; elifetrazol; etogluconol; gallium nitrate; hydroxyurea; lentinan; chlordamine; mitoxantrone; mopidazole; nitraceridine; pentostatin; phenamet; pirarubicin; podophyllin; 2-ethylhydrazine Procarbazine; Razosen; Cizonan; Germonespirolamine; Sciglioside; Triaminoquinone; 2,2',2”-Trichlorotriethylamine; Urethan; Vinpocetine; Dacarbazine; Mannomustine; Dibromomannitol; Dibromoeusol; Piperbromopropane; Gacytosine; Adenosine arabinoside; Cyclophosphamide; Thiotepa; Taxanes, such as Bristol-Myers Squibb. SquibbOncology, Princeton, NJ); Docetaxel (Aventis Antony, France); Chlorobutazone; Gemcitabine; 6-Thioguanine; Mercaptopurine; Methotrexate; Platinum-based drugs such as cisplatin and carboplatin; Vincristine; Platinum; Etoposide (VP-16); Ifosfamide; Mitomycin C; Mitoxantrone; Vincristine; Vinorelbine; Noviben; Norfloxacin; Teniposide; Donomycin; Ambuterol; Seroda; Ibandronate; CPT-11; Topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoic acid; esperamicins; capecitabine; and any pharmaceutically acceptable salts, acids, or derivatives of the above-mentioned drugs. Antihormonal agents used to regulate or inhibit the effects of hormones on tumors are also included in this definition, including, for example, antiestrogens; such as tamoxifen, raloxifene, aromatase inhibitor 4(5)-imidazole, hydroxytamoxifen, trivoxifen, keoxifene, LY 117018, onanasone, and toremifene (Fareston); and antiandrogens such as flutamide, nilumethamide, bicalutamide, leuprorelin, and goserelin; and any pharmaceutically acceptable salts, acids, or derivatives of the above-mentioned drugs.

[0256] When used here, "growth inhibitor" refers to a compound or composition that inhibits cell growth, particularly cancer cell growth, in vitro or in vivo. Examples of growth inhibitors include agents that block the cell cycle (at stages other than S phase), such as those that induce G1 phase arrest and M phase arrest. Classic M phase blockers include Vincas (vincristine and vinblastine) and topoisomerase II inhibitors such as doxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin. Those that block G1 phase also extend to S phase arrest, such as DNA alkylating agents like tamoxifen, prednisone, dacarbazine, nitrogen mustard, cisplatin, methotrexate, 5-fluorouracil, and cytarabine.

[0257] All of the above U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications, and non-patent publications mentioned in this specification are incorporated herein by reference in their entirety.

[0258] As should be understood from the foregoing, although specific embodiments of the invention have been described herein for illustrative purposes, various modifications may be made without departing from the spirit and scope of the invention. Therefore, the invention is not limited except for the appended claims.

[0259] Example

[0260] The following embodiments are provided to provide a complete disclosure and description of how to make and use the present invention to those skilled in the art, and the following examples are not intended to limit the scope of the invention as believed by the inventors, nor are they intended to represent all or only the experiments conducted.

[0261] Example 1: Construction of expression vector

[0262] To investigate the effects of adenovirus triplet leader (TPL) sequences and adenovirus major late promoter enhancer element (eMLP) sequences on gene expression, regulatory nucleic acid molecules composed of TPL and eMLP sequences were designed to regulate gene expression. Four TPL sequences were selected: TPL1, TPL2, TPL3, and TPL4, with nucleotide sequences shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4, respectively. Four eMLP sequences were selected: eMLP1, eMLP2, eMLP3, and eMLP4, with nucleotide sequences shown in SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8, respectively. Based on the combinations of these four TPL and four eMLP sequences, the following 16 regulatory nucleic acid molecules were designed:

[0263] The regulatory nucleic acid molecule 11 is composed of TPL1 and eMLP1, and its nucleotide sequence is shown in SEQ ID NO:19;

[0264] Regulatory nucleic acid molecule 12, composed of TPL1 and eMLP2, has its nucleotide sequence as shown in SEQ ID NO:20;

[0265] The regulatory nucleic acid molecule 13, composed of TPL1 and eMLP3, has the nucleotide sequence shown in SEQ ID NO:21;

[0266] Regulatory nucleic acid molecule 14, composed of TPL1 and eMLP4, has its nucleotide sequence shown in SEQ ID NO:22;

[0267] The regulatory nucleic acid molecule 21 is composed of TPL2 and eMLP1, and its nucleotide sequence is shown in SEQ ID NO:23;

[0268] The regulatory nucleic acid molecule 22, composed of TPL2 and eMLP2, has the nucleotide sequence shown in SEQ ID NO:24;

[0269] The regulatory nucleic acid molecule 23 is composed of TPL2 and eMLP3, and its nucleotide sequence is shown in SEQ ID NO:25;

[0270] The regulatory nucleic acid molecule 24 is composed of TPL2 and eMLP4, and its nucleotide sequence is shown in SEQ ID NO:26;

[0271] The regulatory nucleic acid molecule 31 is composed of TPL3 and eMLP1, and its nucleotide sequence is shown in SEQ ID NO:27;

[0272] The regulatory nucleic acid molecule 32 is composed of TPL3 and eMLP2, and its nucleotide sequence is shown in SEQ ID NO:28;

[0273] The regulatory nucleic acid molecule 33 is composed of TPL3 and eMLP3, and its nucleotide sequence is shown in SEQ ID NO:29;

[0274] The regulatory nucleic acid molecule 34 is composed of TPL3 and eMLP4, and its nucleotide sequence is shown in SEQ ID NO:30;

[0275] The regulatory nucleic acid molecule 41 is composed of TPL4 and eMLP1, and its nucleotide sequence is shown in SEQ ID NO:31;

[0276] The regulatory nucleic acid molecule 42 is composed of TPL4 and eMLP2, and its nucleotide sequence is shown in SEQ ID NO:32;

[0277] The regulatory nucleic acid molecule 43 is composed of TPL4 and eMLP3, and its nucleotide sequence is shown in SEQ ID NO:33;

[0278] The regulatory nucleic acid molecule 44 is composed of TPL4 and eMLP4, and its nucleotide sequence is shown in SEQ ID NO:34.

[0279] An expression vector for aflibercept was constructed, the structure of which is as follows: Figure 1 As shown. The expression vector comprises, in 5' to 3' order: CMV enhancer (its nucleotide sequence is shown in SEQ ID NO:13), CMV promoter (its nucleotide sequence is shown in SEQ ID NO:9), TPL sequence, eMLP sequence, intron sequence (its nucleotide sequence is shown in SEQ ID NO:14), Kozak sequence, aflibercept coding sequence (its encoded protein is shown in SEQ ID NO:10; its nucleotide sequence is shown in SEQ ID NO:11), human IFNB SAR sequence (its nucleotide sequence is shown in SEQ ID NO:15), and bGHpolyA sequence (its nucleotide sequence is shown in SEQ ID NO:12).

[0280] Sixteen AAV-aflibercept expression vectors containing different combinations of TPL and eMLP were constructed (see Table 1), numbered 11, 12, 13, 14, 21, 22, 23, 24, 31, 32, 33, 34, 41, 42, 43, and 44, respectively. These 16 AAV-aflibercept expression vectors are identical in sequence except for the regulatory nucleic acid molecule composed of TPL and eMLP.

[0281] A baseline control vector, AAV-Afibercept-basic, containing only the CMV enhancer and promoter, the afibercept coding sequence, and the bGH polyA sequence, was also constructed and designated as basic (hereinafter referred to as the basic vector). The basic vector is identical to the sequences of the aforementioned 16 AAV-aflibercept expression vectors, except for the absence of the TPL, eMLP, and Intron sequences.

[0282] Table 1. Sequence element combination methods of AAV vectors

[0283]

[0284] Example 2 Cell Transfection Experiment

[0285] 1.293T cell transfection model

[0286] Sixteen AAV-aflibercept expression vectors containing regulatory nucleic acid molecules composed of different TPLs and eMLPs, constructed in Example 1, along with the basic vector, were transfected into 293T cells using PEI. Forty-eight hours after transfection, cell culture supernatant and total cell protein were harvested. Western blot analysis was used to analyze the expression level of aflibercept protein in 293T cells, and HRP anti-human IgG (which recognizes the Fc fragment of aflibercept) was used for capture and detection. Figure 2 A) HRP anti-human GAPDH was used as an internal control to detect GAPDH protein. Secretory aflibercept protein in the 293T supernatant was analyzed using ELISA, and anti-human IgG (recognizing the Fc fragment of aflibercept) antibody was used for capture and detection. Figure 3 A). The results showed that, compared with the basic vector, various AAV-Aflibercept vectors containing TPL and eMLP elements had significantly increased intracellular and supernatant aflibercept levels, including vectors numbered 11, 13, 14, 21, 22, 23, 24, 31, 41, 42, 43 and 44.

[0287] 2. ARPE-19 cell transfection model

[0288] Sixteen AAV-aflibercept expression vectors containing regulatory nucleic acid molecules composed of different TPLs and eMLPs, constructed in Example 1, along with the basic vector, were transfected into ARPE-19 cells using PEI. Forty-eight hours after transfection, cell culture supernatant and total cell protein were harvested. Western blot analysis was used to analyze the expression level of aflibercept protein in ARPE-19 cells, and HRP anti-human IgG (which recognizes the Fc fragment of aflibercept) was used for capture and detection. Figure 2 B) HRP anti-human GAPDH was used as an internal control to detect GAPDH protein. Secretory aflibercept protein in the 293T supernatant was analyzed using ELISA, with anti-human IgG (recognizing the Fc fragment of aflibercept) antibody used for capture and detection. Figure 3 B). The results showed that, compared with the basic vector, various AAV-Aflibercept vectors containing TPL and eMLP elements had significantly increased intracellular aflibercept levels, including vectors numbered 11, 13, 14, 21, 22, 23, 24, 31, 41, 42, 43 and 44.

[0289] Example 3 Cell Infection Experiment

[0290] Considering the significant differences in DNA states containing exogenous Aflibercept coding sequences after plasmid transfection and AAV viral particle infection, 293T cells were used to further simulate the application scenario of AAV gene therapy. CRL-3216 TM Transfect AAV-DJ, AAV Helper packaging system (Cell Biolabs, VPK-400-DJ), and any Afibercep expression vector to package and produce AAV virus particles.

[0291] The steps for packaging and purifying AAV virus are as follows:

[0292] 1) Day 0: Cell inoculation

[0293] 293T cells were inoculated into 15cm culture dishes (the inoculation quantity was determined by the expected viral load).

[0294] 2) Day 1: Plasmid transfection

[0295] 15cm culture dish transfection system: 15μg AAV expression plasmid + 15μg AAV Helper + 15μg coating

[0296] 3) Day 3: 48 hours after transfection

[0297] A. Prepare liquid nitrogen

[0298] B. Digest AAV293 and collect it into a 50ml centrifuge tube (Corning tubes are more resistant to freezing), wash twice with PBS, centrifuge at 1200rpm for 5min, and aspirate the PBS.

[0299] C. Resuspend cells in ~2ml of cell lysis buffer in each 15cm culture dish (total lysis buffer volume for each virus should not exceed 6ml). After freezing cells with liquid nitrogen, immediately thaw them in a 37°C water bath. Repeat this process 3 times to ensure complete cell lysis. After freezing cells with liquid nitrogen again, they can be stored at -80°C or the purification process can continue.

[0300] 4) After thawing the cell lysis buffer in a water bath at 37°C, add Benzonase to a final concentration of 50 U / ml (add 8 μl of 25 U / ul Benzonase stock solution to 4 ml), add sodium deoxycholate to a final concentration of 0.5%, and incubate in a water bath at 37°C for 30 min.

[0301] 5) Preparation of Iodixanol density gradient solution (OptPrep is 60% iodixanol, and the capacity of 361625 centrifuge tubes is 32.4).

[0302] Table 2. Preparation of Iodixanol density gradient solutions

[0303]

[0304] 6) Use a 10ml syringe to infuse a density gradient: infuse 9ml of 15% iodixanol, 6ml of 25% iodixanol, 5ml of 40% iodixanol, and 2ml of 60% iodixanol in sequence, from low to high density. Insert the syringe needle to the bottom of the centrifuge tube each time to avoid generating air bubbles during infusion.

[0305] 7) Centrifuge at 4500 rpm and 4℃ for 30 min;

[0306] 8) Spread approximately 6 ml of the cell lysis supernatant containing the virus onto the top layer of the density gradient, seal with cell lysis buffer, and level the surface.

[0307] 9) Using a Beckman Ti70 rotor, centrifuge at 67,000 rpm and 18°C ​​for 1 hour, maximum acceleration and braking;

[0308] 10) Using a 10ml syringe and an 18g needle, puncture 3–5mm below the 60–40% interface (with the needle bevel facing up) and collect 3–4ml of 40% iodixanol containing the virus (avoid protein contamination at the 40–25% interface); approximately 500ul can be collected per tube.

[0309] 11) Take 1-2 μL of the collected solution from each tube, dilute it 20 times, and measure the absorbance at 340 nm. The tubes with the highest absorbance can be retained and mixed.

[0310] 12) Using a 100K protein concentration tube, centrifuge at the highest speed to concentrate the viral supernatant, add 20ml of PBS to rinse once, and centrifuge again to concentrate to the desired volume;

[0311] 13) The virus can be further purified using a HiTrap heparin affinity column, or further concentrated using a small-volume protein concentration tube at 100K.

[0312] After purification and quantification of AAV virus, ARPE-19 cells were infected in vitro using viruses carrying different AAV-Aflibercept vectors. 72 hours after infection, cell culture supernatant and total cellular protein were harvested. Western blot analysis was used to analyze the expression level of aflibercept protein in the cells, and anti-human IgG (which recognizes the Fc fragment of aflibercept) was used for capture and detection. Figure 4 Secretory aflibercept protein in culture supernatant was analyzed using ELISA, with anti-human IgG (which recognizes the Fc fragment of aflibercept) used for capture and detection. Figure 5 B). The results showed that, compared with the basic vector, various AAV-Aflibercept vectors containing TPL and eMLP elements had significantly increased intracellular and supernatant aflibercept levels.

[0313] Simultaneously, DNA was extracted from a portion of ARPE-19 cells 72 hours after infection, and then real-time PCR was performed using AAV genome ITR-specific primers to determine the average AAV genome copy number in cells of each experimental group, and to quantitatively analyze the viral copy number in cells after viral infection. Figure 5 A), and calculated the Aflibercept expression level per viral copy in ARPE-19 cells after infection with the corresponding aflibercept expression plasmid packaged with AAV virus, and the average amount of aflibercept protein in the supernatant corresponding to the viral genome in each cell, in order to further accurately evaluate the expression level of various AAV-Aflibercept vectors. Figure 5 B).

[0314] Example 4: Integrated Analysis of Data from Different Cell Models

[0315] To integrate the expression levels of the vectors in the three cell models described in Examples 2 and 3, and to screen the most promising vectors for in vivo evaluation, a correlation analysis was performed on the expression levels of aflibercept protein in the intracellular and supernatant of different AO vectors after transfection and infection. Figure 6Bubble plots were created using Aflibercept protein levels in three different cell models. The X-axis represents the level of secreted Aflibercept protein in the supernatant of the ARPE-19 cell transfection model, and the Y-axis represents the level of secreted Aflibercept protein in the supernatant of the 293T cell transfection model. The size of the bubbles represents the level of secreted Aflibercept protein in the supernatant of the ARPE-19 cell infection model, and the depth of bubble staining represents the expression level of Aflibercept protein in the cells of the ARPE-19 cell infection model. The font size of the numbers above the bubbles represents the expression level of Aflibercept protein in the cells of the 293T cell transfection model, and the font size of the numbers to the right of the bubbles represents the expression level of Aflibercept protein in the cells of the 293T cell transfection model.

[0316] The results showed that both AAV-Aflibercept-23 and AAV-Aflibercept-24 had high protein expression levels in 293T cells and ARPE-19 cells. AAV-Aflibercept-14 showed the highest protein expression in 293T cells, while AAV-Aflibercept-43 showed the highest protein expression in ARPE-19 cells.

[0317] Example 5: Assessment of AAV-Aflibercept vector expression levels in the rat retina

[0318] Four AAV vectors, AAV-Aflibercept-23, AAV-Aflibercept-24, AAV-Aflibercept-43, and AAV-Aflibercept-14, were selected for further development and named AO-1 (nucleotide sequence shown in SEQ ID NO:35), AO-2 (nucleotide sequence shown in SEQ ID NO:36), AO-3 (nucleotide sequence shown in SEQ ID NO:37), and AO-4 (nucleotide sequence shown in SEQ ID NO:38), respectively. The AAV8 packaging system was used, and 293T transfections were performed with AAV8, AAV Helper, and any Afibercep expression vector to package and produce AAV viral particles.

[0319] Table 3. AAV Vector Name Comparison Table

[0320]

[0321] BN rats were injected subretinically with four viruses: AO-1, ​​AO-2, AO-3, and AO-4. Each rat's eye was also injected with 4E10 GC (Genome Copies) virus. Four weeks after injection, aqueous humor was separated for ELISA analysis of secretory aflibercept protein levels. Figure 7 A); Total protein was extracted from retinal and choroidal tissues respectively, and Western blot analysis was performed to analyze the expression level of aflibercept protein in cells. Figure 7 B, 7C).

[0322] Simultaneously, DNA was extracted from retinal tissue, and the AAV genome copy number in the retinal tissue was detected using real-time PCR to quantitatively analyze the viral copy number in cells after viral infection. Figure 7 D), and calculate the expression level of Aflibercept corresponding to each viral copy in the cell to further accurately assess the level of secretory Aflibercept protein corresponding to each viral genome in the aqueous humor of the rat retina after injection of AO-1, ​​AO-2, AO-3 and AO-4 viruses. Figure 7 E); and the level of intracellular aflibercept protein per viral genome in the rat retina and choroid. The results showed that rats injected with AO-1 virus had the highest average intracellular AAV genome copy number; rats injected with AO-2 virus had the highest levels of secretory aflibercept in the aqueous humor, and the highest average intracellular aflibercept levels in the retina and choroid. Figure 7 F, G). sequence list <110> Beijing Anlong Biomedical Co., Ltd. <120> Expression vectors for high-level expression of exogenous genes <130> MTI20135 <160> 38 <170> SIPOSequenceListing 1.0 <210> 1 <211> 202 <212> DNA <213> TPL1 (Artificial Sequence) <400> 1 actctcttcc gcatcgctgt ctgcgagggc cagctgttgg gctcgcggtt gaggacaaac 60 tcttcgcggt ctttccagta ctcttggatc ggaaacccgt cggcctccga acaggtactc 120 cgccgccgag ggacctgagc gagtccgcat cgaccggatc ggaaaacctc tcgagaaagg 180 cgtctaacca gtcacagtcg ca 202 <210> 2 <211> 373 <212> DNA <213> TPL2(Artificial Sequence) <400> 2 cttccgcatc gctgtctgcg agggccagct gttggggtga gtactccctc tcaaaagcgg 60 gcatgacttc tgcgctaaga ttgtcagttt ccaaaaacga ggaggatttg atattcactg 120 gccgcggtg atgcctttga gggtggccgc gtccatctgg tcagaaaaga caatcttttt 180 gttgtcaagc ttccttgatg atgtcatact tatcctgtcc cttttttttc cacagctcgc 240 ggttgaggac aaactcttcg cggtctttc agtactcttg gatcggaaac ccgtcggcct 300 ccgaacggta ctccgccacc gagggacctg agcgagtccg catcgaccgg atcggaaaac 360 ctctcgaggt acc 373 <210> 3 <211> 200 <212> DNA <213> TPL3(Artificial Sequence) <400> 3 actctcttcc gcatcgctgt ctgcgagggc cagctgttgg gctcgcggtt gaggacaaac 60 tcttcgcggt ctttccagta ctcttggatc ggaaacccgt cggcctccga acgtactccg 120 ccaccgaggg acctgagcga gtccgcatcg accggatcgg aaaacctctc gagaaaggcg 180 tctaaccagt cacagtcgca 200 <210> 4 <211> 200 <212> DNA <213> TPL4(Artificial Sequence) <400> 4 actgtcttcc ggatcgctgt ccaggagcgc cagctgttgg gctcgcggtt gagaaggtat 60 tcttcgcgat ccttccagta ctcttcgagg ggaaacccgt ctttttctgc acggtactcc 120 gcgcaaggac ctgatcgtct caagatccac gggatctgaa aacctttcga cgaaagcgtc 180 taaccagtcg caatcgcaag 200 <210> 5 <211> 400 <212> DNA <213> eMLP1(Artificial Sequence) <400> 5 cccccatgct ttttgatgcg tttcttacct ctggtttcca tgagccggtg tccacgctcg 60 gtgacgaaaa ggctgtccgt gtccccgtat acagacttga gaggcctgtc ctcgagcggt 120 gttccgcggt cctcctcgta tagaaactcg gaccactctg agacgaaggc tcgcgtccag 180 gccagcacga aggaggctaa gtgggagggg tagcggtcgt tgtccactag ggggtccact 240 cgctccaggg tgtgaagaca catgtccccc tcttcggcat caaggaaggt gattggttta 300 taggtgtatg ccacgtgacc gggtgttcct gaaggggggg tataaaaggg ggtgggggcg 360 cgtcgtcct cactctcttc cgcatcgctg tctgcgaggg 400 <210> 6 <211> 400 <212> DNA <213> eMLP2(Artificial Sequence) <400> 6 ccgcggcatg gcccttggcg cgcagcttgc ccttggagga ggcgccgcac gaggggcagt 60 gcagactttt gagggcgtag agcttgggcg cgagaaatac cgattccggg gagtaggcat 120 ccgcgccgca ggccccgcag acggtctcgc attccacgag ccaggtgagc tctggccgtt 180 cggggtcaaa aaccaggttt cccccatgct ttttgatgcg tttcttacct ctggtttcca 240 tgagccggtg tccacgctcg gtgacgaaaa ggctgtccgt gtccccgtat acagacttga 300 gaggcctgtc ctcgagcggt gttccgcggt cctcctcgta tagaaactcg gaccactctg 360 agcgaaggc tcgcgtccag gccagcacga aggaggctaa <210> 7 <211> 400 <212> DNA <213> eMLP3(Artificial Sequence) <400> 7 cagacgggcc agggtcatgt ctttccacgg gcgcagggtc ctcgtcagcg tagtctgggt cacggtgaag gggtgcgctc cgggctgcgc gctggccagg gtgcgcttga ggctggtcct 120 gctggtgctg aagcgctgcc ggtcttcgcc ctgcgcgtcg gccaggtagc atttgaccat 180 ggtgtcatag tccagcccct ccgcggcatg gcccttggcg cgcagcttgc ccttggagga 240 ggcgccgcac gaggggcagt gcagactttt gaggcgtag agcttgggcg cgagaaatac 300. cgattccggg gagtaggcat ccgcgccgca ggccccgcag acggtctcgc attccacgag 360 ccaggtgagc tctggccgtt cggggtcaaa aaccaggttt <210> 8 <211> 400 <212> DNA <213> eMLP4(Artificial Sequence) <400> 8 cgatagcagt tcttgcaagg aagcaaagtt tttcaacggt ttgaggccgt ccgccgtagg catgcttttg agcgtttgac caagcagttc caggcggtcc cacagctcgg tcacgtgctc 120 tacggcatct cgatccagca tatctcctcg tttcgcgggt tggggcggct ttcgctgtac 180 ggcagtagtc ggtgctcgtc cagacgggcc agggtcatgt ctttccacgg gcgcagggtc 240 ctcgtcagcg tagtctgggt cacggtgaag gggtgcgctc cgggctgcgc gctggccagg 300 gtgcgcttga ggctggtcct gctggtgctg aagcgctgcc ggtcttcgcc ctgcgcgtcg 360 gccaggtagc atttgaccat ggtgtcatag tccagcccct 400 <210> 9 <211> 204 <212> DNA <213> CMV promoter (Artificial Sequence) <400> 9 gtgatgcggt tttggcagta catcaatggg cgtggatagc ggtttgactc acggggattt 60 ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac 120 tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg 180 tgggaggtct atataagcag agct 204 <21 "0"> 10 <211> 431 <212> PRT <213> Aflibercept (Artificial Sequence) <400> 10 Ser Asp Thr Gly Arg Pro Phe Val Glu Met Tyr Ser Glu Ile Pro Glu 1 5 10 15 Ile Ile His Met Thr Glu Gly Arg Glu Leu Val Ile Pro Cys Arg Val 20 25 30 Thr Ser Pro Asn Ile Thr Val Thr Leu Lys Lys Phe Pro Leu Asp Thr 35 40 45 Leu Ile Pro Asp Gly Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly Phe 50 55 60 Ile Ile Ser Asn Ala Thr Tyr Lys Glu Ile Gly Leu Leu Thr Cys Glu 65 70 75 80 Ala Thr Val Asn Gly His Leu Tyr Lys Thr Asn Tyr Leu Thr His Arg 85 90 95 Gln Thr Asn Thr Ile Ile Asp Val Val Leu Ser Pro Ser His Gly Ile 100 105 110 Glu Leu Ser Val Gly Glu Lys Leu Val Leu Asn Cys Thr Ala Arg Thr 115 120 125 Glu Leu Asn Val Gly Ile Asp Phe Asn Trp Glu Tyr Pro Ser Ser Lys 130 135 140 His Gln His Lys Lys Leu Val Asn Arg Asp Leu Lys Thr Gln Ser Gly 145 150 155 160 Ser Glu Met Lys Lys Phe Leu Ser Thr Leu Thr Ile Asp Gly Val Thr 165 170 175 Arg Ser Asp Gln Gly Leu Tyr Thr Cys Ala Ala Ser Ser Gly Leu Met 180 185 190 Thr Lys Lys Asn Ser Thr Phe Val Arg Val His Glu Lys Asp Lys Thr 195 200 205 His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser 210 215 220 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 225 230 235 240 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 245 250 255 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 260 265 270 Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 275 280 285 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 290 295 300 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 305 310 315 320 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 325 330 335 Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys 340 345 350 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 355 360 365 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 370 375 380 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 385,390,395,400 Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 405 410 415 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 420 425 430 <210> 11 <211> 1377 <212> DNA <213> Artificial Sequence (Artificial Sequence) <400> 11 atggtctctt attgggacac gggagttctc ctgtgtgcac tgctgagctg cttctcctc 60 accggaagtt caagtggttc cgatacaggg cggccgttcg ttgagatgta ctccgaaatt 120 ccggaaatta ttcatatgac agaaggtcgc gaactcgtta ttccgtgtcg cgtaacgtct 180 ccaaacatca cggtaacact caaaaaattc ccacttgaca cgttgatccc ggacggcaaa 240 cggattatct gggatagcag gaaaggtttt atcatttcta acgcgacgta taaagaaatc 300 gggctcctga catgcgaagc tactgtaaat ggccacttgt ataaaaccaa ttacctgacg 360 catcggcaga cgaacaccat tatagacgta gtcctgagtc cgagccacgg cattgaactt 420 agtgttggcg agaaacttgt attgaactgt acggctcgga ctgagctgaa cgtcggcata 480 gattttaatt gggagtatcc tagttcaaaa catcagcata agaaactcgt caatagggac 540 ctcaaaaccc agagtggttc tgagatgaag aagtttttgt caaccctgac gatcgatggt 600 gttacgcgct cagatcaagg gctctatacg tgtgccgcgt cttcagggct catgaccaaa 660 aagaactcca cgtttgtacg cgtgcacgaa aaagacaaga ctcatacatg cccaccttgc 720 cccgcccctg aactgcttgg cggtccctct gtatttcttt tccctcctaa accgaaagat 780 actttgatga tatcccggac ccccgaagtg acatgtgtag ttgtcgacgt atcacatgaa 840 gatccggagg ttaaatttaa ctggtacgtt gatggcgttg aagttcacaa tgctaagact 900 aaaccgaggg aagagcaata taacagtaca tatcgagtcg tatccgtatt gactgtgctc 960 caccaggact ggctgaacgg aaaggagtac aagtgcaagg tatccaataa ggccctcccg 1020 gctcccatcg aaaagaccat atcaaaggcg aaaggccagc cgagggagcc gcaggtttat 1080 actctccccc cgtccaggga cgaattgaca aagaatcagg tgagcctcac atgccttgtg 1140 aaggggttct accccagtga tattgcagtg gagtgggagt ctaacggtca acccgaaaat 1200 aattacaaga cgacacctcc ggtcttggat agcgatgggt ctttcttcct ctattcaaag 1260 ctcacggtag ataagtccag atggcaacag ggaaacgttt tttcctgctc tgtgatgcat 1320 gaagcacttc ataatcacta cacgcagaag tcactttcac tgtcaccggg aaagtaa 1377 <210> 12 <211> 208 <212> DNA <213> bGH polyA (Artificial Sequence) <400> 12 ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc 60 tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc 120 tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt 180 gggaagagaa tagcaggcat gctgggga 208 <210> 13 <211> 380 <212> DNA <213> CMV enhancer (Artificial Sequence) <400> 13 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 380 <210> 14 <211> 189 <212> DNA <213> SV40 intron (Artificial Sequence) <400> 14 gatccggtac tcgaggaact gaaaaaccag aaagttaact ggtaagttta gtctttttgt 60 cttttatttc aggtcccgga tccggtggtg gtgcaaatca aagaactgct cctcagtgga 120 tgttgccttt acttctaggc ctgtacggaa gtgttacttc tgctctaaaa gctgcggaat 180 tgtacccgc 189 <210> 15 <211> 790 <212> DNA <213> Human β-interferon human scaffold attachment region (Artificial Sequence) <400> 15 actgaagtca tgatggcatg cttctatatt attttctaaa agatttaaag ttttgccttc 60 tccatttaga cttataattc actggaattt ttttgtgtgt atggtatgac atatgggttc 120 ccttttattt tttacatata aatatatttc cctgtttttc taaaaaagaa aaagatcatc 180 attttcccat tgtaaaatgc catatttttt tcataggtca cttacatata tcaatgggtc 240 tgtttctgag ctctactcta ttttatcagc ctcactgtct atccccacac atctcatgct 300 ttgctctaaa tcttgatatt tagtggaaca ttctttccca ttttgttcta caagaatatt 360 tttgttattg tcttttgggc ttctatac attttagaat gaggttggca agttaacaaa 420 cagcttttt ggggtgaaca tattgactac aaatttatgt ggaaagaaag tataccttca 480 caatattaag tcttttagtt catgaatata gtatgtctct ccgtttctgc attaacttag 540 acattcatta atttctctca caatttataa gtttatttag atcttcattc atttaaatct 600 tcactaacct ctcatttaca atttgtaagt tttctgggta acagtcttgc acttctttgc 660 ctagattat ttccaagtag attattttca tacatcgtct atggtgtcat ttttaaaatg 720 taatttttca cctttttatt gctaaagaga gatgactgat tgttaatatt gatcttgtgc 780 gtggcgacct 790 <210> 16 <211> 130 <212> DNA <213> 5'‐ITR(Artificial Sequence) <400> 16 cctgcaggca gctgcgcgct cgctcgctca ctgaggcgc cgggcgtcg ggcgacctt 60 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact 120 aggggttcct 130 <210> 17 <211> 141 <212> DNA <213> 3'-ITR (Artificial Sequence) <400> 17 aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg 60 ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc 120 gagcgcgcag ctgcctgcag g 141 <210> 18 <211> 861 <212> DNA <213> Ampicillin Resistance Gene (Artificial Sequence) <400> 18 atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct 60 gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca 120 cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc 180 gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc 240 cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg 300 gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta 360 tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc 420 ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt 480 gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg 540 cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct 600 tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc 660 tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga gcgtggaagc 720 cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac 780 acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc 840 tcactgatta agcattggta a 861 <210> 19 <211> 602 <212> DNA <213> Regulatory nucleic acid molecule 11 (Artificial Sequence) <400> 19 actctcttcc gcatcgctgt ctgcgagggc cagctgttgg gctcgcggtt gaggacaaac 60 tcttcgcggt ctttccagta ctcttggatc ggaaacccgt cggcctccga acaggtactc 120 cgccgccgag ggacctgagc gagtccgcat cgaccggatc ggaaaacctc tcgagaaagg 180 cgtctaacca gtcacagtcg cacccccatg ctttttgatg cgtttcttac ctctggtttc 240 catgagccgg tgtccacgct cggtgacgaa aaggctgtcc gtgtccccgt atacagactt 300 gagaggcctg tcctcgagcg gtgttccgcg gtcctcctcg tatagaaact cggaccactc 360 tgagacgaag gctcgcgtcc aggccagcac gaaggaggct aagtgggagg ggtagcggtc 420 gttgtccact agggggtcca ctcgctccag ggtgtgaaga cacatgtccc cctcttcggc 480 atcaaggaag gtgattggtt tataggtgta tgccacgtga ccgggtgttc ctgaaggggg 540 ggtataaaag ggggtggggg cgcgttcgtc ctcactctct tccgcatcgc tgtctgcgag 600 gg 602 <210> 20 <211> 602 <212> DNA <213> Regulatory nucleic acid molecule 12 (Artificial Sequence) <400> 20 actctcttcc gcatcgctgt ctgcgagggc cagctgttgg gctcgcggtt gaggacaaac 60 tcttcgcggt ctttccagta ctcttggatc ggaaacccgt cggcctccga acaggtactc 120 cgccgccgag ggacctgagc gagtccgcat cgaccggatc ggaaaacctc tcgagaaagg 180 cgtctaacca gtcacagtcg caccgcggca tggcccttgg cgcgcagctt gcccttggag 240 gaggcgccgc acgaggggca gtgcagactt ttgagggcgt agagcttggg cgcgagaaat accgattccg gggagtaggc atccgcgccg caggccccgc agacggtctc gcattccacg 360 agccaggtga gctctggccg ttcggggtca aaaaccaggt ttcccccatg ctttttgatg 420 cgtttcttac ctctggtttc catgagccgg tgtccacgct cggtgacgaa aaggctgtcc 480 gtgtccccgt atacagactt gagaggcctg tcctcgagcg gtgttccgcg gtcctcctcg 540 tatagaaact cggaccactc tgagacgaag gctcgcgtcc aggccagcac gaaggaggct aa 602 <210> 21 <211> 602 <212> DNA <213> Episode 13(Artificial Sequence) <400> 21 actctcttcc gcatcgctgt ctgcgagggc cagctgttgg gctcgcggtt gaggacaaac tcttcgcggt ctttccagta ctcttggatc ggaaacccgt cggcctccga acaggtactc 120 cgccgccgag ggacctgagc gagtccgcat cgaccggatc ggaaaacctc tcgagaaagg cgtctaacca gtcacagtcg cacagacggg ccagggtcat gtctttccac gggcgcaggg 240 tcctcgtcag cgtagtctgg gtcacggtga aggggtgcgc tccgggctgc gcgctggcca 300 gggtgcgctt gaggctggtc ctgctggtgc tgaagcgctg ccggtcttcg ccctgcgcgt 360 cggccaggta gcatttgacc atggtgtcat agtccagccc ctccgcggca tggcccttgg 420 cgcgcagctt gcccttggag gaggcgccgc acgaggggca gtgcagactt ttgagggcgt 480 agagcttggg cgcgagaaat accgattccg gggagtaggc atccgcgccg caggccccgc 540 agacggtctc gcattccacg agccaggtga gctctggccg ttcggggtca aaaaccaggt 600 tt 602 <210> 22 <211> 602 <212> DNA <213> Regulatory nucleic acid molecule 14 (Artificial Sequence) <400> 22 actctcttcc gcatcgctgt ctgcgagggc cagctgttgg gctcgcggtt gaggacaaac 60 tcttcgcggt ctttccagta ctcttggatc ggaaacccgt cggcctccga acaggtactc 120 cgccgccgag ggacctgagc gagtccgcat cgaccggatc ggaaaacctc tcgagaaagg 180 cgtctaacca gtcacagtcg cacgatagca gttcttgcaa ggaagcaaag tttttcaacg 240 gtttgaggcc gtccgccgta ggcatgcttt tgagcgtttg accaagcagt tccaggcggt 300 cccacagctc ggtcacgtgc tctacggcat ctcgatccag catatctcct cgtttcgcgg 360 gttggggcgg ctttcgctgt acggcagtag tcggtgctcg tccagacggg ccagggtcat 420 gtctttccac gggcgcaggg tcctcgtcag cgtagtctgg gtcacggtga aggggtgcgc 480 tccgggctgc gcgctggcca gggtgcgctt gaggctggtc ctgctggtgc tgaagcgctg 540 ccggtcttcg ccctgcgcgt cggccaggta gcatttgacc atggtgtcat agtccagccc 600 ct 602 <210> 23 <211> 773 <212> DNA <213> Regulatory nucleic acid molecule 21 (Artificial Sequence) <400> 23 cttccgcatc gctgtctgcg agggccagct gttggggtga gtactccctc tcaaaagcgg 60 gcatgacttc tgcgctaaga ttgtcagttt ccaaaaacga ggaggatttg atattcactg 120 gcccgcggtg atgcctttga gggtggccgc gtccatctgg tcagaaaaga caatcttttt 180 gttgtcaagc ttccttgatg atgtcatact tatcctgtcc cttttttttc cacagctcgc 240 ggttgaggac aaactcttcg cggtctttcc agtactcttg gatcggaaac ccgtcggcct 300 ccgaacggta ctccgccacc gagggacctg agcgagtccg catcgaccgg atcggaaaac 360 ctctcgaggt acccccccat gctttttgat gcgtttctta cctctggttt ccatgagccg 420 gtgtccacgc tcggtgacga aaaggctgtc cgtgtccccg tatacagact tgagaggcct 480 gtcctcgagc ggtgttccgc ggtcctcctc gtatagaaac tcggaccact ctgagacgaa 540 ggctcgcgtc caggccagca cgaaggaggc taagtgggag gggtagcggt cgttgtccac 600 tagggggtcc actcgctcca gggtgtgaag acacatgtcc ccctcttcgg catcaaggaa 660 ggtgattggt ttataggtgt atgccacgtg accgggtgtt cctgaagggg gggtataaaa 720 gggggtgggg gcgcgttcgt cctcactctc ttccgcatcg ctgtctgcga ggg 773 <210> 24 <211> 773 <212> DNA <213> Regulatory nucleic acid molecule 22 (Artificial Sequence) <400> 24 cttccgcatc gctgtctgcg agggccagct gttggggtga gtactccctc tcaaaagcgg 60 gcatgacttc tgcgctaaga ttgtcagttt ccaaaaacga ggaggatttg atattcactg 120 gcccgcggtg atgcctttga gggtggccgc gtccatctgg tcagaaaaga caatcttttt 180 gttgtcaagc ttccttgatg atgtcatact tatcctgtcc cttttttttc cacagctcgc 240 ggttgaggac aaactcttcg cggtctttcc agtactcttg gatcggaaac ccgtcggcct 300 ccgaacggta ctccgccacc gagggacctg agcgagtccg catcgaccgg atcggaaaac 360 ctctcgaggt accccgcggc atggcccttg gcgcgcagct tgcccttgga ggaggcgccg 420 cacgaggggc agtgcagact tttgagggcg tagagcttgg gcgcgagaaa taccgattcc 480 ggggagtagg catccgcgcc gcaggccccg cagacggtct cgcattccac gagccaggtg 540 agctctggcc gttcggggtc aaaaaccagg tttcccccat gctttttgat gcgtttctta 600 cctctggttt ccatgagccg gtgtccacgc tcggtgacga aaaggctgtc cgtgtccccg 660 tatacagact tgagaggcct gtcctcgagc ggtgttccgc ggtcctcctc gtatagaaac 720 tcggaccact ctgagacgaa ggctcgcgtc caggccagca cgaaggaggc taa 773 <210> 25 <211> 773 <212> DNA <213> Regulatory nucleic acid molecule 23 (Artificial Sequence) <400> 25 cttccgcatc gctgtctgcg agggccagct gttggggtga gtactccctc tcaaaagcgg 60 gcatgacttc tgcgctaaga ttgtcagttt ccaaaaacga ggaggatttg atattcactg 120 gcccgcggtg atgcctttga gggtggccgc gtccatctgg tcagaaaaga caatcttttt 180 gttgtcaagc ttccttgatg atgtcatact tatcctgtcc cttttttttc cacagctcgc 240 ggttgaggac aaactcttcg cggtctttcc agtactcttg gatcggaaac ccgtcggcct 3并返回 ccgaacggta ctccgccacc gagggacctg agcgagtccg catcgaccgg atcggaaaac 360 ctctcgaggt acccagacgg gccagggtca tgtctttcca cgggcgcagg gtcctcgtca 420 gcgtagtctg ggtcacggtg aaggggtgcg ctccgggctg cgcgctggcc agggtgcgct 480 tgaggctggt cctgctggtg ctgaagcgct gccggtcttc gccctgcgcg tcggccaggt 540 agcatttgac catggtgtca tagtccagcc cctccgcggc atggcccttg gcgcgcagct 600 tgcccttgga ggaggcgccg cacgaggggc agtgcagact tttgagggcg tagagcttgg 660 gcgcgagaaa taccgattcc ggggagtagg catccgcgcc gcaggccccg cagacggtct 720 cgcattccac gagccaggtg agctctggcc gttcggggtc aaaaaccagg ttt 773 <210> 26 <211> 773 <212> DNA <213> Regulatory nucleic acid molecule 24 (Artificial Sequence) <400> 26 cttccgcatc gctgtctgcg agggccagct gttggggtga gtactccctc tcaaaagcgg 60 gcatgacttc tgcgctaaga ttgtcagttt ccaaaaacga ggaggatttg atattcactg 120 gcccgcggtg atgcctttga gggtggccgc gtccatctgg tcagaaaaga caatcttttt 180 gttgtcaagc ttccttgatg atgtcatact tatcctgtcc cttttttttc cacagctcgc 240 ggttgaggac aaactcttcg cggtctttcc agtactcttg gatcggaaac ccgtcggcct 300 ccgaacggta ctccgccacc gagggacctg agcgagtccg catcgaccgg atcggaaaac 360 ctctcgaggt acccgatagc agttcttgca aggaagcaaa gtttttcaac ggtttgaggc 420 cgtccgccgt aggcatgctt ttgagcgttt gaccaagcag ttccaggcgg tcccacagct 480 cggtcacgtg ctctacggca tctcgatcca gcatatctcc tcgtttcgcg ggttggggcg 540 gctttcgctg tacggcagta gtcggtgctc gtccagacgg gccagggtca tgtctttcca 600 cgggcgcagg gtcctcgtca gcgtagtctg ggtcacggtg aaggggtgcg ctccgggctg 660 cgcgctggcc agggtgcgct tgaggctggt cctgctggtg ctgaagcgct gccggtcttc 720 gccctgcgcg tcggccaggt agcatttgac catggtgtca tagtccagcc cct 773 <210> 27[[ID=1 >] <211> 600 <212> DNA <213> Regulatory nucleic acid molecule 31 (Artificial Sequence) <--400> 27 actctcttcc gcatcgctgt ctgcgagggc cagctgttgg gctcgcggtt gaggacaaac 60 tcttcgcggt ctttccagta ctcttggatc ggaaacccgt cggcctccga acgtactccg 120 ccaccgaggg acctgagcga gtccgcatcg accggatcgg aaaacctctc gagaaaggcg 180 240. tctaaccagt cacagtcgca cccccatgct ttttgatgcg tttcttacct ctggtttcca tgagccggtg tccacgctcg gtgacgaaaa ggctgtccgt gtccccgtat acagacttga gaggcctgtc ctcgagcggt gttccgcggt cctcctcgta tagaaactcg gaccactctg 360 agacgaaggc tcgcgtccag gccagcacga aggaggctaa gtgggagggg tagcggtcgt 420 tgtccactag ggggtccact cgctccaggg tgtgaagaca catgtccccc tcttcggcat 480 caaggaaggt gattggttta taggtgtatg ccacgtgacc gggtgttcct gaaggggggg 540 tataaaaggg ggtggggggcg cgttcgtcct cactctcttc cgcatcgctg tctgcgaggg 600 <210> 28 <211> 600 <212> DNA <213> Entertainment Sequence 32(Artificial Sequence) <400> 28 actctcttcc gcatcgctgt ctgcgagggc cagctgttgg gctcgcggtt gaggacaaac tcttcgcggt ctttccagta ctcttggatc ggaaacccgt cggcctccga acgtactccg 120 180. ccaccgaggg acctgagcg gtccgcatcg accggatcgg aaaacctctc gagaaaggcg tctaaccagt cacagtcgca ccgcggcatg gcccttggcg cgcagcttgc ccttggagga 240 ggcgccgcac gaggggcagt gcagactttt gaggcgtag agcttgggcg cgagaaatac 300. cgattccggg gagtaggcat ccgcgccgca ggccccgcag acggtctcgc attccacgag 360 420. ccaggtgagc tctggccgtt cggggtcaaa aaccaggttt cccccatgct ttttgatgcg tttcttacct ctggtttcca tgagccggtg tccacgctcg gtgacgaaaa ggctgtccgt 480 gtccccgtat acagacttga gaggcctgtc ctcgagcggt gttccgcggt cctcctcgta 540 tagaaactcg gaccactctg agacgaaggc tcgcgtccag gccagcacga aggaggctaa <210> 29 <211> 600 <212> DNA <213> Entertainment Sequence 33(Artificial Sequence) <400> 29 actctcttcc gcatcgctgt ctgcgagggc cagctgttgg gctcgcggtt gaggacaaac tcttcgcggt ctttccagta ctcttggatc ggaaacccgt cggcctccga acgtactccg 120 180. ccaccgaggg acctgagcg gtccgcatcg accggatcgg aaaacctctc gagaaaggcg tctaaccagt cacagtcgca cagacgggcc agggtcatgt ctttccacgg gcgcagggtc 240 ctcgtcagcg tagtctgggt cacggtgaag gggtgcgctc cgggctgcgc gctggccagg 300 gtgcgcttga ggctggtcct gctggtgctg aagcgctgcc ggtcttcgcc ctgcgcgtcg 360 gccaggtagc atttgaccat ggtgtcatag tccagcccct ccgcggcatg gcccttggcg 420 cgcagcttgc ccttggagga ggcgccgcac gaggggcagt gcagactttt gagggcgtag 480 agcttgggcg cgagaaatac cgattccggg gagtaggcat ccgcgccgca ggccccgcag 540 acggtctcgc attccacgag ccaggtgagc tctggccgtt cggggtcaaa aaccaggttt 600 <210> 30 <211> 600 <212> DNA <213> Regulatory nucleic acid molecule 34 (Artificial Sequence) <400> 30 actctcttcc gcatcgctgt ctgcgagggc cagctgttgg gctcgcggtt gaggacaaac 60 tcttcgcggt ctttccagta ctcttggatc ggaaacccgt cggcctccga acgtactccg 120 ccaccgaggg acctgagcga gtccgcatcg accggatcgg aaaacctctc gagaaaggcg 180 tctaaccagt cacagtcgca cgatagcagt tcttgcaagg aagcaaagtt tttcaacggt 240 ttgaggccgt ccgccgtagg catgcttttg agcgtttgac caagcagttc caggcggtcc 300 cacagctcgg tcacgtgctc tacggcatct cgatccagca tatctcctcg tttcgcgggt 360 tggggcggct ttcgctgtac ggcagtagtc ggtgctcgtc cagacgggcc agggtcatgt 420 ctttccacgg gcgcagggtc ctcgtcagcg tagtctgggt cacggtgaag gggtgcgctc 480 cgggctgcgc gctggccagg gtgcgcttga ggctggtcct gctggtgctg aagcgctgcc 540 ggtcttcgcc ctgcgcgtcg gccaggtagc atttgaccat ggtgtcatag tccagcccct 6oo <210> 31 <211> 600 <212> DNA <213> Regulatory nucleic acid molecule 41 (Artificial Sequence) <400> 31 actgtcttcc ggatcgctgt ccaggagcgc cagctgttgg gctcgcggtt gagaaggtat 60 tcttcgcgat ccttccagta ctcttcgagg ggaaacccgt ctttttctgc acggtactcc 120 gcgcaaggac ctgatcgtct caagatccac gggatctgaa aacctttcga cgaaagcgtc 180 It should be noted that there may be an error in the original text where "6oo" in line 12 should probably be "600". The translation has been done based on the provided text as accurately as possible.taaccagtcg caatcgcaag cccccatgct ttttgatgcg tttcttacct ctggtttcca 240 tgagccggtg tccacgctcg gtgacgaaaa ggctgtccgt gtccccgtat acagacttga 300 gaggcctgtc ctcgagcggt gttccgcggt cctcctcgta tagaaactcg gaccactctg 360 agacgaaggc tcgcgtccag gccagcacga aggaggctaa gtgggagggg tagcggtcgt 420 tgtccactag ggggtccact cgctccaggg tgtgaagaca catgtccccc tcttcggcat 480 caaggaaggt gattggttta taggtgtatg ccacgtgacc gggtgttcct gaaggggggg 540 tataaaaggg ggtgggggcg cgttcgtcct cactctcttc cgcatcgctg tctgcgaggg 600 <210> 32 <211> 600 <212> DNA <213> Regulatory nucleic acid molecule 42 (Artificial Sequence) <400> 32 actgtcttcc ggatcgctgt ccaggagcgc cagctgttgg gctcgcggtt gagaaggtat 60 tcttcgcgat ccttccagta ctcttcgagg ggaaacccgt ctttttctgc acggtactcc 120 gcgcaaggac ctgatcgtct caagatccac gggatctgaa aacctttcga cgaaagcgtc 180 taaccagtcg caatcgcaag ccgcggcatg gcccttggcg cgcagcttgc ccttggagga ggcgccgcac gaggggcagt gcagactttt gaggcgtag agcttgggcg cgagaaatac 300. cgattccggg gagtaggcat ccgcgccgca ggccccgcag acggtctcgc attccacgag 360 420. ccaggtgagc tctggccgtt cggggtcaaa aaccaggttt cccccatgct ttttgatgcg tttcttacct ctggtttcca tgagccggtg tccacgctcg gtgacgaaaa ggctgtccgt 480 gtccccgtat acagacttga gaggcctgtc ctcgagcggt gttccgcggt cctcctcgta 540 tagaaactcg gaccactctg agacgaaggc tcgcgtccag gccagcacga aggaggctaa <210> 33 <211> 600 <212> DNA <213> Episode 43(Artificial Sequence) <400> 33 actgtcttcc ggatcgctgt ccaggagcgc cagctgttgg gctcgcggtt gagaaggtat tcttcgcgat ccttccagta ctcttcgagg ggaaacccgt ctttttctgc acggtactcc 120 gcgcaaggac ctgatcgtct caagatccac gggatctgaa aacctttcga cgaaagcgtc taaccagtcg caatcgcaag cagacgggcc agggtcatgt ctttccacgg gcgcagggtc 240 ctcgtcagcg tagtctgggt cacggtgaag gggtgcgctc cgggctgcgc gctggccagg 300 gtgcgcttga ggctggtcct gctggtgctg aagcgctgcc ggtcttcgcc ctgcgcgtcg 360 gccaggtagc atttgaccat ggtgtcatag tccagcccct ccgcggcatg gcccttggcg 420 cgcagcttgc ccttggagga ggcgccgcac gaggggcagt gcagactttt gagggcgtag 480 agcttgggcg cgagaaatac cgattccggg gagtaggcat ccgcgccgca ggccccgcag 540​​​​​​​​​​​​​​​​​​​taaccagtcg caatcgcaag cgatagcagt tcttgcaagg aagcaaagtt tttcaacggt 240 ttgaggccgt ccgccgtagg catgcttttg agcgtttgac caagcagttc caggcggtcc 300 cacagctcgg tcacgtgctc tacggcatct cgatccagca tatctcctcg ttcgcgggt 360 tggggcggct ttcgctgtac ggcagtagtc ggtgctcgtc cagacgggcc agggtcatgt 420 ctttccacgg gcgcagggtc ctcgtcagcg tagtctgggt cacggtgaag gggtgcgctc 480 cgggctgcgc gctggccagg gtgcgcttga ggctggtcct gctggtgctg aagcgctgcc 540 ggtcttcgcc ctgcgcgtcg gccaggtagc atttgaccat ggtgtcatag tccagcccct 600 <210> 35 <211> 6844 <212> DNA <213> AO‐1(Artificial Sequence) <400> 35 cctgcaggca gctgcgcgct cgctcgctca ctgaggcgc cgggcgtcg ggcgacctt 60 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact 120 aggggttcct gcggcctcta gactcgaggc gttgacattg attattgact agttattaat 180 agtaatcaat tacggggtca ttagttcata gcccatatat ggagttccgc gttacataac 240 ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa 300 tgacgtatgt tcccatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt 360 atttacggta aactgcccac ttggcagtac atcaagtgta tcatatgcca agtacgcccc 420 ctattgacgt caatgacggt aaatggcccg cctggcatta tgcccagtac atgaccttat 480 gggactttcc tacttggcag tacatctacg tattagtcat cgctattacc atggtgatgc 540 ggttttggca gtacatcaat gggcgtggat agcggtttga ctcacgggga tttccaagtc 600 tccaccccat tgacgtcaat gggagtttgt tttggcacca aaatcaacgg gactttccaa 660 aatgcgtaa caactccgcc ccattgacgc aaatgggcgg taggcgtgta cggtgggagg 720 tctatatag cagagctctc tggctaacta cggtcttc gcatcgctgt ctgcgagggc 780 cagctgttgg ggtgagtact ccctctcaaa agcgggcatg acttctgcgc taagattgtc 840 agtttccaaa aacgaggagg atttgatatt cactggcccg cggtgatgcc tttgagggtg 900 gccgcgtcca tctggtcaga aaagacaatc tttttgttgt caagcttcct tgatgatgtc 960 atacttatcc tgtccctttt ttttccacag ctcgcggttg aggacaaact cttcgcggtc 1020 tttccagtac tcttggatcg gaaacccgtc ggcctccgaa cggtactccg ccaccgaggg 1080 acctgagcga gtccgcatcg accggatcgg aaaacctctc gaggtaccca gacgggccag 1140 ggtcatgtct ttccacgggc gcagggtcct cgtcagcgta gtctgggtca cggtgaaggg 1200 gtgcgctccg ggctgcgcgc tggccagggt gcgcttgagg ctggtcctgc tggtgctgaa 1260 gcgctgccgg tcttcgccct gcgcgtcggc caggtagcat ttgaccatgg tgtcatagtc 1320 cagcccctcc gcggcatggc ccttggcgcg cagcttgccc ttggaggagg cgccgcacga 1380 ggggcagtgc agacttttga gggcgtagag cttgggcgcg agaaataccg attccgggga 1440 gtaggcatcc gcgccgcagg ccccgcagac ggtctcgcat tccacgagcc aggtgagctc 1500 tggccgttcg gggtcaaaaa ccaggtttga tccggtactc gaggaactga aaaaccagaa 1560 agttaactgg taagtttagt ctttttgtct tttatttcag gtcccggatc cggtggtggt 1620 gcaaatcaaa gaactgctcc tcagtggatg ttgcctttac ttctaggcct gtacggaagt 1680 gttacttctg ctctaaaagc tgcggaattg tacccgcgcc accatggtct cttattggga 1740 cacgggagtt ctcctgtgtg cactgctgag ctgccttctc ctcaccggaa gttcaagtgg 1800 ttccgataca gggcggccgt tcgttgagat gtactccgaa attccggaaa ttattcatat 1860 gacagaaggt cgcgaactcg ttattccgtg tcgcgtaacg tctccaaaca tcacggtaac 1920 actcaaaaaa ttcccacttg acacgttgat cccggacggc aaacggatta tctgggatag 1980 caggaaaggt tttatcattt ctaacgcgac gtataaagaa atcgggctcc tgacatgcga 2040 agctactgta aatggccact tgtataaaac caattacctg acgcatcggc agacgaacac 2100 cattatagac gtagtcctga gtccgagcca cggcattgaa cttagtgttg gcgagaaact 2160 tgtattgaac tgtacggctc ggactgagct gaacgtcggc atagatttta attgggagta 2220 tcctagttca aaacatcagc ataagaaact cgtcaatagg gacctcaaaa cccagagtgg 2280 ttctgagatg aagaagtttt tgtcaaccct gacgatcgat ggtgttacgc gctcagatca 2340 agggctctat acgtgtgccg cgtcttcagg gctcatgacc aaaaagaact ccacgtttgt 2400 acgcgtgcac gaaaaagaca agactcatac atgcccacct tgccccgcc ctgaactgct 2460 tggcggtccc tctgtatttc ttttccctcc taaaccgaaa gatactttga tgatatcccg 2520 gaccccgaa gtgacatgtg tagttgtcga cgtatcacat gaagatccgg aggttaaatt 2580 taactggtac gttgatggcg ttgaagttca caatgctaag actaaaccga gggaagagca 2640 atataacagt acatatcgag tcgtatccgt attgactgtg ctccaccagg actggctgaa 2700 cggaaaggag tacaagtgca aggtatccaa taaggccctc ccggctccca tcgaaaagac 2760 catatcaaag gcgaaaggcc agccgaggga gccgcaggtt tatactctcc ccccgtccag 2820 ggacgaattg acaaagaatc aggtgagcct cacatgcctt gtgaaggggt tctaccccag 2880 tgatattgca gtggagtggg agtctaacgg tcaacccgaa aataattaca agacgacacc 2940 tccggtcttg gatagcgatg ggtctttctt cctctattca aagctcacgg tagataagtc 3000 cagatggcaa cagggaaacg ttttttcctg ctctgtgatg catgaagcac ttcataatca 3060 ctacacgcag aagtcacttt cactgtcacc gggaaagtaa actgaagtca tgatggcatg 3120 cttctatatt attttctaaa agatttaaag ttttgccttc tccatttaga cttataattc 3180 actggaattt ttttgtgtgt atggtatgac atatgggttc ccttttattt tttacatata 3240 aatatatttc cctgtttttc taaaaaagaa aaagatcatc attttcccat tgtaaaatgc 3300 catatttttt tcataggtca cttacatata tcaatgggtc tgtttctgag ctctactcta 3360 ttttatcagc ctcactgtct atccccacac atctcatgct ttgctctaaa tcttgatatt 3420 tagtggaaca ttctttccca ttttgttcta caagaatatt tttgttattg tcttttgggc 3480 ttctatatac attttagaat gaggttggca agttaacaaa cagctttttt ggggtgaaca 3540 tattgactac aaatttatgt ggaaagaaag tataccttca caatattaag tcttttagtt 3600 catgaatata gtatgtctct ccgtttctgc attaacttag acattcatta atttctctca 3660 caatttataa gtttatttag atcttcattc atttaaatct tcactaacct ctcatttaca 3720 atttgtaagt tttctgggta acagtcttgc acttctttgc ctagatttat ttccaagtag 3780 attattttca tacatcgtct atggtgtcat ttttaaaatg taatttttca cctttttatt 3840 gctaaagaga gatgactgat tgttaatatt gatcttgtgc gtggcgacct ctgtgccttc 3900 tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc 3960 cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgagtaggtg 4020 tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt gggaagagaa 4080 tagcaggcat gctggggagc ggccgcagga acccctagtg atggagttgg ccactccctc 4140 tctgcgcgct cgctcgctca ctgaggccgg gcgaccaaag gtcgcccgac gcccgggctt 4200 tgcccgggcg gcctcagtga gcgagcgagc gcgcagctgc ctgcaggggc gcctgatgcg 4260 gtattttctc cttacgcatc tgtgcggtat ttcacaccgc atacgtcaaa gcaaccatag 4320 tacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc 4380 gctacacttg ccagcgcctt agcgcccgct cctttcgctt tcttcccttc ctttctcgcc 4440 acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt 4500 agtgctttac ggcacctcga ccccaaaaaa cttgatttgg gtgatggttc acgtagtggg 4560 ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt 4620 ggactcttgt tccaaactgg aacaacactc aactctatct cgggctattc ttttgattta 4680 taagggattt tgccgatttc ggtctattgg ttaaaaaatg agctgattta acaaaaattt 4740 aacgcgaatt ttaacaaaat attaacgttt acaattttat ggtgcactct cagtacaatc 4800 tgctctgatg ccgcatagtt aagccagccc cgacacccgc caacacccgc tgacgcgccc 4860 tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc 4920 tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgagacgaaa gggcctcgtg 4980 atacgcctat ttttataggt taatgtcatg ataataatgg tttcttagac gtcaggtggc 5040 acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat 5100 atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag 5160 agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt 5220 cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt 5280 gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga gagtttttcg 5340 cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta 5400 tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac 5460 ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa 5520 ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg 5580 atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc 5640 cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg 5700 atgcctgtag caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta 5760 gcttccccggc aaaattaat agactggatg gaggcggata aagttgcagg accacttctg 5820 cgctcggccc ttccggctgg ctggttatt gctgataaat ctggagccgg tgagcgtgga 5880 agccgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc 5940 tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt 6000 gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt 6060 gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc 6120 atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag 6180 atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa 6240 aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg 6300 aaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag 6360 ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg 6420 ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga 6480 tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc 6540 ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc 6600 acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga 6660 gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt 6720 cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg 6780 aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac 6840 atgt 6844 <210> 36 <211> 6844 <212> DNA <213> AO‑2(Artificial Sequence) <400> 36 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt 60 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact 120 aggggttcct gcggcctcta gactcgaggc gttgacattg attattgact agttattaat 180 agtaatcaat tacggggtca ttagttcata gcccatatat ggagttccgc gttacataac 240 ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa 300 tgacgtatgt tcccatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt 360 atttacggta aactgcccac ttggcagtac atcaagtgta tcatatgcca agtacgcccc 420 ctattgacgt caatgacggt aaatggcccg cctggcatta tgcccagtac atgaccttat 480 gggactttcc tacttggcag tacatctacg tattagtcat cgctattacc atggtgatgc 540 ggttttggca gtacatcaat gggcgtggat agcggtttga ctcacgggga tttccaagtc 600 tccaccccat tgacgtcaat gggagtttgt tttggcacca aaatcaacgg gactttccaa 660 aatgcgtaa caactccgcc ccattgacgc aaatgggcgg taggcgtgta cggtgggagg 720 tctatatag cagagctctc tggctaacta cggtcttc gcatcgctgt ctgcgagggc 780 cagctgttgg ggtgagtact ccctctcaaa agcgggcatg acttctgcgc taagattgtc 840 agtttccaaa aacgaggagg atttgatatt cactggcccg cggtgatgcc tttgagggtg 900 gccgcgtcca tctggtcaga aaagacaatc tttttgttgt caagcttcct tgatgatgtc 960 atacttatcc tgtcccttttt tttccacag ctcgcggttg aggacaaact cttcgcggtc 1020 tttccagtac tcttggatcg gaaacccgtc ggcctccgaa cggtactccg ccaccgaggg 1080 acctgagcga gtccgcatcg accggatcgg aaaacctctc gaggtacccg atagcagttc 1140 ttgcaaggaa gcaaagtttt tcaacggttt gaggccgtcc gccgtaggca tgcttttgag 1200 cgtttgacca agcagttcca ggcggtccca cagctcggtc acgtgctcta cggcatctcg 1260 atccagcata tctcctcgtt tcgcgggttg gggcggcttt cgctgtacgg cagtagtcgg 1320 tgctcgtcca gacgggccag ggtcatgtct ttccacgggc gcagggtcct cgtcagcgta 1380 gtctgggtca cggtgaaggg gtgcgctccg ggctgcgcgc tggccagggt gcgcttgagg 1440 ctggtcctgc tggtgctgaa gcgctgccgg tcttcgccct gcgcgtcggc caggtagcat 1500 ttgaccatgg tgtcatagtc cagcccctga tccggtactc gaggaactga aaaaccagaa 1560 agttaactgg taagtttagt ctttttgtct tttatttcag gtcccggatc cggtggtggt 1620 gcaaatcaaa gaactgctcc tcagtggatg ttgcctttac ttctaggcct gtacggaagt 1680 gttacttctg ctctaaaagc tgcggaattg tacccgcgcc accatggtct cttattggga 1740 cacgggagtt ctcctgtgtg cactgctgag ctgccttctc ctcaccggaa gttcaagtgg 1800 ttccgataca gggcggccgt tcgttgagat gtactccgaa attccggaaa ttattcatat 1860 gacagaaggt cgcgaactcg ttattccgtg tcgcgtaacg tctccaaaca tcacggtaac 1920 actcaaaaaa ttcccacttg acacgttgat cccggacggc aaacggatta tctgggatag 1980 caggaaaggt tttatcattt ctaacgcgac gtataaagaa atcgggctcc tgacatgcga 2040 agctactgta aatggccact tgtataaaac caattacctg acgcatcggc agacgaacac 2100 cattatagac gtagtcctga gtccgagcca cggcattgaa cttagtgttg gcgagaaact 2160 tgtattgaac tgtacggctc ggactgagct gaacgtcggc atagatttta attgggagta 2220 tcctagttca aaacatcagc ataagaaact cgtcaatagg gacctcaaaa cccagagtgg 2280 ttctgagatg aagaagtttt tgtcaaccct gacgatcgat ggtgttacgc gctcagatca 2340 agggctctat acgtgtgccg cgtcttcagg gctcatgacc aaaaagaact ccacgtttgt 2400 acgcgtgcac gaaaaagaca agactcatac atgcccacct tgccccgccc ctgaactgct 2460 tggcggtccc tctgtatttc ttttccctcc taaaccgaaa gatactttga tgatatcccg 2520 gacccccgaa gtgacatgtg tagttgtcga cgtatcacat gaagatccgg aggttaaatt 2580 taactggtac gttgatggcg ttgaagttca caatgctaag actaaaccga gggaagagca 2640 atataacagt acatatcgag tcgtatccgt attgactgtg ctccaccagg actggctgaa 2700 cggaaaggag tacaagtgca aggtatccaa taaggccctc ccggctccca tcgaaaagac 2760 catatcaaag gcgaaaggcc agccgaggga gccgcaggtt tatactctcc ccccgtccag 2820 ggacgaattg acaaagaatc aggtgagcct cacatgcctt gtgaaggggt tctaccccag 2880 tgatattgca gtggagtggg agtctaacgg tcaacccgaa aataattaca agacgacacc 2940 tccggtcttg gatagcgatg ggtctttctt cctctattca aagctcacgg tagataagtc 3000 cagatggcaa cagggaaacg ttttttcctg ctctgtgatg catgaagcac ttcataatca 3060 ctacacgcag aagtcacttt cactgtcacc gggaaagtaa actgaagtca tgatggcatg 3120 cttctatatt attttctaaa agatttaaag ttttgccttc tccattaga cttataattc 3180 actggaattt ttttgtgtgt atggtatgac atatgggttc ccttttattt tttacatata 3240 aatatatttc cctgtttttc taaaaaagaa aaagatcatc atttcccat tgtaaaatgc 3300 catatttttt tcataggtca cttacatata tcaatgggtc tgtttctgag ctctactcta 3360 tttatcagc ctcactgtct atccccacac atctcatgct ttgctctaaa tcttgatatt 3420 tagtggaaca ttctttccca ttttgttcta caagaatatt tttgttattg tcttttgggc 3480 ttctatac attttagaat gaggttggca agttaacaaa cagcttttttt ggggtgaaca 3540 tattgactac aaatttatgt ggaaagaaag tataccttca caatattaag tcttttagtt 3600 catgaatata gtatgtctct ccgtttctgc attaacttag acattcatta atttctctca 3660 caatttataa gttatttag atcttcattc atttaaatct tcactaacct ctcatttaca 3720 atttgtaagt tttctgggta acagtcttgc acttctttgc ctagatttat ttccaagtag 3780 attattttca tacatcgtct atggtgtcat ttttaaaatg taatttttca cctttttatt 3840 gctaaagaga gatgactgat tgttaatatt gatcttgtgc gtggcgacct ctgtgccttc 3900 tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc 3960 cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgagtaggtg 4020 tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt gggaagagaa 4080 tagcaggcat gctggggagc ggccgcagga acccctagtg atggagttgg ccactccctc 4140 tctgcgcgct cgctcgctca ctgaggccgg gcgaccaaag gtcgcccgac gcccgggctt 4200 tgcccgggcg gcctcagtga gcgagcgagc gcgcagctgc ctgcaggggc gcctgatgcg 4260 gtattttctc cttacgcatc tgtgcggtat ttcacaccgc atacgtcaaa gcaaccatag 4320 tacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc 4380 gctacacttg ccagcgcctt agcgcccgct cctttcgctt tcttcccttc ctttctcgcc 4440 acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt 4500 agtgctttac ggcacctcga ccccaaaaaa cttgatttgg gtgatggttc acgtagtggg 4560 ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt 4620 ggactcttgt tccaaactgg aacaacactc aactctatct cgggctattc ttttgattta 4680 taagggattt tgccgatttc ggtctattgg ttaaaaaatg agctgattta acaaaaattt 4740 aacgcgaatt ttaacaaaat attaacgttt acaattttat ggtgcactct cagtacaatc 4800 tgctctgatg ccgcatagtt aagccagccc cgacacccgc caacacccgc tgacgcgccc 4860 tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc 4920 tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgagacgaaa gggcctcgtg 4980 atacgcctat ttttataggt taatgtcatg ataataatgg tttcttagac gtcaggtggc 5040 acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat 5100 atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag 5160 agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt 5220 cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt 5280 gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga gagttttcgc 5340 cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta 5400 tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac 5460 ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa 5520 ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg 5580 atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc 5640 cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg 5700 atgcctgtag caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta 5760 gcttccccggc aaaattaat agactggatg gaggcggata aagttgcagg accacttctg 5820 cgctcggccc ttccggctgg ctggttatt gctgataaat ctggagccgg tgagcgtgga 5880 agccgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc 5940 tacacgacgg ggagtcaggc aactatggat gacgaata gacagatcgc tgagataggt 6000 gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt 6060 gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc 6120 atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag 6180 atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa 6240 aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg 6300 aaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag 6360 ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg 6420 ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga 6480 tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc 6540 ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc 6600 acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga 6660 gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt 6720 cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg 6780 aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac 6840 atgt 6844 <210> 37 <211> 6671 <212> DNA <213> AO‑3(Artificial Sequence) <400> 37 cctgcaggca gctgcgcgct cgctcgctca ctgaggcgc cgggcgtcg ggcgacctt 60 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact 120 aggggttcct gcggcctcta gactcgaggc gttgacattg attattgact agttattaat 180 agtaatcaat tacggggtca ttagttcata gcccatatat ggagttccgc gttacataac 240 ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa 300 tgacgtatgt tcccatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt 360 atttacggta aactgcccac ttggcagtac atcaagtgta tcatatgcca agtacgcccc 420 ctattgacgt caatgacggt aaatggcccg cctggcatta tgcccagtac atgaccttat 480 gggactttcc tacttggcag tacatctacg tattagtcat cgctattacc atggtgatgc 540 ggttttggca gtacatcaat gggcgtggat agcggtttga ctcacgggga tttccaagtc 600 tccaccccat tgacgtcaat gggagtttgt tttggcacca aaatcaacgg gactttccaa 660 aatgtcgtaa caactccgcc ccattgacgc aaatgggcgg taggcgtgta cggtgggagg 720 tctatataag cagagctctc tggctaacta ccggtactgt cttccggatc gctgtccagg 780 agcgccagct gttgggctcg cggttgagaa ggtattcttc gcgatccttc cagtactctt 840 cgaggggaaa cccgtctttt tctgcacggt actccgcgca aggacctgat cgtctcaaga 900 tccacgggat ctgaaaacct ttcgacgaaa gcgtctaacc agtcgcaatc gcaagcagac 960 gggccagggt catgtctttc cacgggcgca gggtcctcgt cagcgtagtc tgggtcacgg 1020 tgaaggggtg cgctccgggc tgcgcgctgg ccagggtgcg cttgaggctg gtcctgctgg 1080 tgctgaagcg ctgccggtct tcgccctgcg cgtcggccag gtagcatttg accatggtgt 1140 catagtccag cccctccgcg gcatggccct tggcgcgcag cttgcccttg gaggaggcgc 1200 cgcacgaggg gcagtgcaga cttttgaggg cgtagagctt gggcgcgaga aataccgatt 1260 ccggggagta ggcatccgcg ccgcaggccc cgcagacggt ctcgcattcc acgagccagg 1320 tgagctctgg ccgttcgggg tcaaaaacca ggtttgatcc ggtactcgag gaactgaaaa 1380 accagaaagt taactggtaa gtttagtctt tttgtcttt atttcaggtc ccggatccgg 1440 tggtggtgca aatcaaagaa ctgctcctca gtggatgttg cctttacttc taggcctgta 1500 cggaagtgtt acttctgctc taaaagctgc ggaattgtac ccgcgccacc atggtctctt 1560 attgggacac gggagttctc ctgtgtgcac tgctgagctg ccttctcctc accggaagtt 1620 caagtggttc cgatacaggg cggccgttcg ttgagatgta ctccgaaatt ccggaaatta 1680 ttcatatgac agaaggtcgc gaactcgtta ttccgtgtcg cgtaacgtct ccaaacatca 1740 cggtaacact caaaaaattc ccacttgaca cgttgatccc ggacggcaaa cggattatct 1800 gggatagcag gaaaggtttt atcatttcta acgcgacgta taaagaaatc gggctcctga 1860 catgcgaagc tactgtaaat ggccacttgt ataaaaccaa ttacctgacg catcggcaga 1920 cgaacaccat tatagacgta gtcctgagtc cgagccacgg cattgaactt agtgttggcg 1980 agaaacttgt attgaactgt acggctcgga ctgagctgaa cgtcggcata gattttaatt 2040 gggagtatcc tagttcaaaa catcagcata agaaactcgt caatagggac ctcaaaaccc 2100 agagtggttc tgagatgaag aagtttttgt caaccctgac gatcgatggt gttacgcgct 2160 cagatcaagg gctctatacg tgtgccgcgt cttcagggct catgaccaaa aagaactcca 2220 cgtttgtacg cgtgcacgaa aaagacaaga ctcatacatg cccaccttgc cccgcccctg 2280 aactgcttgg cggtccctct gtatttcttt tccctcctaa accgaaagat actttgatga 2340 tatcccggac ccccgaagtg acatgtgtag ttgtcgacgt atcacatgaa gatccggagg 2400 ttaaatttaa ctggtacgtt gatggcgttg aagttcacaa tgctaagact aaaccgaggg 2460 aagagcaata taacagtaca tatcgagtcg tatccgtatt gactgtgctc caccaggact 2520 ggctgaacgg aaaggagtac aagtgcaagg tatccaataa ggccctcccg gctcccatcg 2580 aaaagaccat atcaaaggcg aaaggccagc cgagggagcc gcaggtttat actctccccc 2640 cgtccaggga cgaattgaca aagaatcagg tgagcctcac atgccttgtg aaggggttct 2700 accccagtga tattgcagtg gagtgggagt ctaacggtca acccgaaaat aattacaaga 2760 cgacacctcc ggtcttggat agcgatgggt ctttcttcct ctattcaaag ctcacggtag 2820 ataagtccag atggcacag ggaaacgttt ttcctgctc tgtgatgcat gaagcacttc 2880 ataatcacta cacgcagaag tcacttcac tgtcaccggg aaagtaact gaagtcatga 2940 tggcatgctt ctatattatt ttctaaaaga tttaaagttt tgccttctcc atttagactt 3000 atattcact ggaattttt tgtgtgtag gtatgacata tgggttccct tttattttt 3060 acatataaat atatttccct gttttctaa aaaagaaaa gatcatcatt ttcccattgt 3120 aaaatgccat atttttca taggtcactt acatatatca atgggtctt ttctgagctc 3180 tacttattt tatcagcctc actgtctatc cccacacatc tcatgctttg ctctaatct 3240 tgatatttag tggacattc ttcccattt tgttcaca gatattttt gttattgtct 3300 tttggcttc tatatacatt ttagaatgag gttggcaagt taacaacag cttttttggg 3360 gtgaacatat tgactacaa ttatgtgga aagaaagtat accttcaca tattaagtct 3420 tttagttcat gatatagta tgtctctccg tttctgcatt aacttagaca ttcattaatt 3480 tctctcacaa ttataagtt tatttagatc ttcattcatt taaatcttca ctaacctctc 3540 attacaatt tgtaagtttt ctgggtaaca gtcttgcact tctttgccta gatttatttc 3600 caagtagatt atttcatac atcgtctatg gtgtcattttt taaaatgtaa ttttcacct 3660 ttttattgct aaagagagat gactgattgt tatattgat cttgtgcgtg gcgacctctg 3720 tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg 3780 aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga 3840 gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg 3900 aagagaatag caggcatgct ggggagcggc cgcaggaacc cctagtgatg gagttggcca 3960 ctccctctct gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgacgcc 4020 cgggctttgc ccgggcggcc tcagtgagcg agcgagcgcg cagctgcctg caggggcgcc 4080 tgatgcggta tttctcctt acgcatctgt gcggtatttc acaccgcata cgtcaaagca 4140 accatagtac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag 4200 cgtgaccgct acacttgcca gcgccttagc gcccgctcct ttcgctttct tcccttcctt 4260 tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt 4320 ccgatttagt gctttacggc acctcgaccc caaaaaactt gatttgggtg atggttcacg 4380 tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt 4440 taatagtgga ctcttgttcc aaactggaac aacactcaac tctatctcgg gctattcttt 4500 tgatttataa gggattttgc cgatttcggt ctattggtta aaaaatgagc tgatttaaca 4560 aaaatttaac gcgaatttta acaaaatatt aacgtttaca attttatggt gcactctcag 4620 tacaatctgc tctgatgccg catagttaag ccagccccga cacccgccaa cacccgctga 4680 cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc 4740 cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga gacgaaaggg 4800 cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt cttagacgtc 4860 aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca 4920 ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa 4980 aaagaagt atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt 5040 ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca 5100 gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag 5160 ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc 5220 ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca 5280 gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt 5340 aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct 5400 gacaacgatc ggaggaccga aggagctaac cgcttttttg caacacatgg gggatcatgt 5460 aactcgcctt gatcgttgg aaccggagct gaatgaagcc ataccaaacg acgagcgtga 5520 caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact 5580 tactctagct tcccggcaac aattaataga ctggatggag gcggataag ttgcaggacc 5640 acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga 5700 gcgtggaagc cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt 5760 agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga 5820 gataggtgcc tcactgatta agcattggta actgtcagac caagtttact catatatact 5880 ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga 5940 taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt 6000 agaaaagatc aaaggatctt cttgagatcc ttttttctg cgcgtaatct gctgcttgca 6060 aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct 6120 ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta 6180 gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct 6240 aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc 6300 aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca 6360 gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga 6420 aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg 6480 aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt 6540 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag 6600 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt 6660 tgctcacatg t 6671 <210> 38 <211> 6673 <212> DNA <213> AO‑4(Artificial Sequence) <400> 38 cctgcaggca gctgcgcgct cgctcgctca ctgaggcgc cgggcgtcg ggcgacctt 60 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact 120 aggggttcct gcggcctcta gactcgaggc gttgacattg attattgact agttattaat 180 agtaatcaat tacggggtca ttagttcata gcccatatat ggagttccgc gttacataac 240 ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa 300 tgacgtatgt tcccatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt 360 atttacggta aactgcccac ttggcagtac atcaagtgta tcatatgcca agtacgcccc 420 ctattgacgt caatgacggt aaatggcccg cctggcatta tgcccagtac atgaccttat 480 gggactttcc tacttggcag tacatctacg tattagtcat cgctattacc atggtgatgc 540 ggttttggca gtacatcaat gggcgtggat agcggtttga ctcacgggga tttccaagtc 600 tccacccat tgacgtcaat gggagtttgt tttggcacca aaatcaacgg gactttccaa 660 aatgtcgtaa caactccgcc ccattgacgc aaatgggcgg taggcgtgta cggtgggagg 720 tctatataag cagagctctc tggctaacta ccggtactct cttccgcatc gctgtctgcg 780 agggccagct gttgggctcg cggttgagga caaactcttc gcggtctttc fòtctctt 840 ggatcggaaa cccgtcggcc tccgaacagg tactccgccg ccgagggacc tgagcgagtc 900 cgcatcgacc ggatcggaaa acctctcgag aaaggcgtct aaccagtcac agtcgcacga 960 tagcagttct tgcaaggaag caaagttttt caacggtttg aggccgtccg ccgtaggcat 1020 gcttttgagc gtttgaccaa gcagttccag gcggtcccac agctcggtca cgtgctctac 1080 ggcatctcga tccagcatat ctcctcgttt cgcgggttgg ggcggctttc gctgtacggc 1140 agtagtcggt gctcgtccag acgggccagg gtcatgtctt tccacggggcg cagggtcctc 1200 gtcagcgtag tctgggtcac ggtgaagggg tgcgctccgg gctgcgcgct ggccagggtg 1260 cgcttgaggc tggtcctgct ggtgctgaag cgctgccggt cttcgccctg cgcgtcggcc 1320 aggtagcatt tgaccatggt gtcatagtcc agcccctgat ccggtactcg aggaactgaa 1440. aaaccagaaa gttaactggt aagtttagtc tttttgtctt ttatttcagg tcccggatcc ggtggtggtg caaatcaaag aactgctcct cagtggatgt tgcctttact tctaggcctg tacggaagtg ttacttctgc tctaaagct gcggaattgt acccgcgcca ccatggtctc ttattgggac acgggagttc tcctgtgtgc actgctgagc tgccttctcc tcaccggaag 1620 ttcaagtggt tccgatacag ggcggccgtt cgttgagatg tactccgaaa ttccggaaat 1680 tattcatatg acagaaggtc gcgaactcgt tattccgtgt cgcgtaacgt ctccaaacat 1800. 1800. 1800. 1800. 1800. 1800. 1800. 1800. 1800. 1800 ctgggatagc aggaaaggtt ttatcatttc taacgcgacg tataaagaaa tcgggctcct 1860 gacatgcgaa gctactgtaa atggccactt gtataaaacc aattacctga cgcatcggca 1920 gacgaacacc attatagacg tagtcctgag tccgagccac ggcattgaac ttagtgttgg 1980 cgagaaactt gtattgaact gtacggctcg gactgagctg aacgtcggca tagattttaa 2040 ttgggagtat cctagttcaa aacatcagca taagaaactc gtcaataggg acctcaaaac 2100 ccagagtggt tctgagatga agaagttttt gtcaaccctg acgatcgatg gtgttacgcg 2160 ctcagatcaa gggctctata cgtgtgccgc gtcttcaggg ctcatgacca aaaagaactc 2220 cacgtttgta cgcgtgcacg aaaaagacaa gactcataca tgcccacctt gccccgcccc 2280 tgaactgctt ggcggtccct ctgtatttct tttccctcct aaaccgaaag atactttgat 2340 gatatcccgg acccccgaag tgacatgtgt agttgtcgac gtatcacatg aagatccgga 2400 ggttaaattt aactggtacg ttgatggcgt tgaagttcac aatgctaaga ctaaaccgag 2460 ggaagagcaa tataacagta catatcgagt cgtatccgta ttgactgtgc tccaccagga 2520 ctggctgaac ggaaggagt acagtgcaa ggtatccaat aaggccctcc cggctcccat 2580 cgaaaagacc atatcaagg cgaaaggcca gccgagggag ccgcaggtttt attackccc 2640 cccgtccagg gacgaattga caagatca ggtgagccctc acatgccttg tgaaggggtt 2700 ctaccccagt gatattgcag tggagtggga gtctaacggt caacccgaaa attackacaa 2760 gacgacacct ccggtcttgg atagcgatgg gtctttctc ctctattcaa agctcacggt 2820 agataagtcc agatggcac agggaacgt ttttcctgc tctgtgatgc atgaagcact 2880 tcataatcac tacacgcaga agtcactttc actgtcaccg ggaagtaaa ctgaagtcat 2940 gatggcatgc ttctatatta tttctaaaa gatttaaagt ttgccttct ccatttagac 3000 ttataattca ctggaattttt ttgtgtgta tggtatgaca tattggttcc cttttatttt 3060 ttacatataa atatatttcc ctgttttttct aaaaaagaa aagatcatca tttcccatt 3120 gtaaaatgcc atatttttt cataggtcac ttacatatat caatgggtct gtttctgagc 3180 3240 cttgatatt agtggaacat tctttcccat tttgttctac aagaatattt ttgttattgt 3300 cttttgggct tctatataca tttagaatg aggttggcaa gttaacaaac agcttttttg 3360 gggtgaacat attgactaca aatttatgtg gaaagaaagt ataccttcac aatattaagt 3420 cttttagttc atgaatatag tatgtctctc cgtttctgca ttaacttaga cattcattaa 3480 tttctctcac aatttataag tttatttaga tcttcattca tttaaatctt cactaacctc 3540 tcatttacaa tttgtaagtt ttctgggtaa cagtcttgca cttctttgcc tagatttatt 3600 tccaagtaga ttatttcat acatcgtcta tggtgtcatt tttaaaatgt aatttttcac 3660 ctttttattg ctaaaagagag atgactgatt gttaatattg atcttgtgcg tggcgacctc 3720 tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct 3780 ggaaggtgcc actcccactg tcctttccta ataaatgag gaaattgcat cgcattgtct 3840 gagtaggtgt cattctattc tgggggtgg ggtggggcag gacagcaagg gggaggattg 3900 ggaagagaat agcaggcatg ctggggagcg gccgcaggaa cccctagtga tggagttggc 3960 cactccctct ctgcgcgctc gctcgctcac tgaggccggg cgaccaaagg tcgccccgacg 4020 cccgggcttt gcccgggcgg cctcagtgag cgagcgagcg cgcagctgcc tgcaggggcg 4080 cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca tacgtcaaag 4140 caaccatagt acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc 4200 agcgtgaccg ctacacttgc cagcgcctta gcgcccgctc ctttcgcttt cttcccttcc 4260 tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcggggct ccctttaggg 4320 ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgatttggg tgatggttca 4380 cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc 4440 tttaatagtg gactcttgtt ccaaactgga acaacactca actctatctc gggctattct 4500 tttgatttat aagggatttt gccgatttcg gtctattggt taaaaaatga gctgattttaa 4560 caaaaattta acgcgaattt taacaaata ttaacgttta caattttatg gtgcactctc 4620 agtacaatct gctctgatgc cgcatagtta agccagcccc gacacccgcc aacacccgct 4680 gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc 4740 tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gagacgaaag 4800 ggcctcgtga tacgcctatt tttataggtt aatgtcatga taataatggt ttcttagacg 4860 tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata 4920 4980 aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttatccctt ttttgcggca 5040 ttttgccttc ctgttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat 5100 cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag 5160 agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc 5220 gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct 5280 cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca 5340 gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt 5400 ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat 5460 gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt 5520 gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta 5580 cttactctag cttcccggca acaattaata gactggatgg aggcggataa agttgcagga 5640 ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt 5700 gagcgtggaa gccgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc 5760 gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct 5820 gagataggtg cctcactgat taagcattgg taactgtcag accaagttta ctcatatata 5880 ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt 5940 gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc 6000 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg 6060 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact 6120 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg 6180 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg 6240 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac 6300 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca 6360 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga 6420 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc 6480 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct 6540 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg 6600 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct 6660 tttgctcaca tgt 6673

Claims

1. A regulatory nucleic acid molecule for enhancing gene expression, the sequence of which is shown in SEQ ID NO:

25.

2. An expression vector, said expression vector comprising, in 5' to 3' order: (a) Start the sub-region; (b) 5'UTR region; (c) The coding sequence that encodes the polypeptide gene product; (d) Polyadenosine monophosphate region (polyA); wherein The 5'UTR region contains the regulatory nucleic acid molecule as described in claim 1; The encoded sequence is operatively linked to the promoter region.

3. The expression vector of claim 2, wherein, The promoter region of (a) is selected from the cytomegalovirus (CMV) promoter, actin promoter, elongation factor 1αEF1α promoter, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter.

4. The expression vector of claim 3, wherein, The promoter region (a) contains the cytomegalovirus (CMV) promoter sequence shown in SEQ ID NO:

9.

5. The expression vector of claim 2, wherein, The polypeptide gene product is a therapeutic protein.

6. The expression vector of claim 5, wherein, The therapeutic protein is selected from anti-angiogenic peptides or α-1 antitrypsin.

7. The expression vector of claim 6, wherein, The anti-angiogenic peptide contains a VEGF-binding fragment of soluble fms-like tyrosine kinase-1 sFLT-1 or sFLT-1.

8. The expression vector of claim 6, wherein, The anti-angiogenic polypeptide is aflibercept.

9. The expression vector of claim 8, wherein, The amino acids of aflibercept are shown in SEQ ID NO:

10.

10. The expression vector of claim 8, wherein, The coding nucleic acid for aflibercept is shown in SEQ ID NO:

11.

11. The expression vector of claim 2, wherein, The polyadenylated region is selected from the polyA sequence of human growth hormone, bovine growth hormone, or β-globin.

12. The expression vector of claim 11, wherein, The polyadenylation region contains the bovine growth hormone polyA sequence shown in SEQ ID NO:

12.

13. The expression vector of claim 2, wherein, The expression vector contains the sequence shown in SEQ ID NO:

35.

14. The expression vector of claim 2, wherein, The expression vector further includes (i) a first enhancer region, which is located upstream of the (a) promoter region.

15. The expression vector of claim 14, wherein, The (i) first enhancer region contains a sequence selected from CMV enhancers or EF1α enhancers.

16. The expression vector of claim 15, wherein, The first enhancer region (i) contains the CMV enhancer sequence shown in SEQ ID NO:

13.

17. The expression vector of claim 2, wherein, The expression vector further includes (ii) an intron region located downstream of the (b) 5'UTR region and upstream of the (c) coding sequence encoding the polypeptide gene product.

18. The expression vector of claim 17, wherein, The (ii) intron region contains a sequence selected from the SV40 intron, the elongation factor 1αEF1α intron, the actin intron, or the CMV intron.

19. The expression vector of claim 18, wherein, The (ii) intron region contains the SV40 intron sequence shown in SEQ ID NO:

14.

20. The expression vector of claim 2, wherein, The expression vector further includes (iii) a second enhancer region, which is located downstream of the coding sequence encoding the polypeptide gene product in (c) and upstream of the polyadenylation region in (d).

21. The expression vector of claim 20, wherein, The second enhancer region (iii) contains the expression enhancer sequence EES.

22. The expression vector of claim 20, wherein, The second enhancement sub-region (iii) contains the interferon scaffold attachment region SAR.

23. The expression vector of claim 22, wherein, The stent attachment region sequence SAR is the human stent attachment region IFNB SAR of human β-interferon.

24. The expression vector of claim 23, wherein, The human β-interfering stenosis attachment region IFNB SAR contains the SAR sequence shown in SEQ ID NO:

15.

25. The expression vector of claim 14, wherein, The expression vector further comprises an inverted terminal repeat (ITR) at the 5' end upstream of the (i) first enhancer and an inverted terminal repeat (ITR) at the 3' end downstream of the (d) polyadenylation region.

26. The expression vector of claim 25, wherein, The inverted terminal repeat sequence ITR at the 5' end of (iv) or the inverted terminal repeat sequence ITR at the 3' end of (v) comprises an inverted terminal repeat sequence ITR selected from adenovirus AV or adeno-associated virus AAV.

27. The expression vector of claim 25, wherein, The inverted terminal repeat sequence ITR at the 5' end of (iv) contains the AAV ITR shown in SEQ ID NO:

16.

28. The expression vector of claim 25, wherein, The inverted terminal repeat sequence ITR at the end of (v)3' contains the AAV ITR shown in SEQ ID NO:

17.

29. The expression vector of claim 25, wherein, The expression vector also contains (vi) a selection marker gene.

30. The expression vector of claim 29, wherein, The (vi) selectable marker gene is located downstream of the inverted terminal repeat (ITR) at the 3' end of (v).

31. The expression vector of claim 30, wherein, The (vi) selected marker genes are selected from ampicillin resistance genes, hygromycin resistance genes, neomycin resistance genes, diammonium phosphate resistance genes, and dihydrofolate reductase genes.

32. The expression vector of claim 31, wherein, The ampicillin resistance gene contains the sequence shown in SEQ ID NO:

18.

33. The expression vector according to claim 2, comprising, from 5' to 3': (iv) An inverted terminal repeat sequence ITR at the 5' end, wherein the inverted terminal repeat sequence ITR at the 5' end comprises the AAV ITR shown in SEQ ID NO: 16; (i) A first enhancer region, wherein the first enhancer region contains the CMV enhancer sequence shown in SEQ ID NO:13; (a) A promoter region containing the cytomegalovirus (CMV) promoter sequence shown in SEQ ID NO:9; (b) A 5'UTR region comprising the regulatory nucleic acid molecule of claim 1; (ii) an intron region comprising the SV40 intron sequence shown in SEQ ID NO:14; (c) The coding sequence that encodes the polypeptide gene product; (iii) A second enhancement sub-region, the second enhancement sub-region containing the SAR sequence shown in SEQ ID NO:15; (d) Polyadenylation region, wherein the polyadenylation region contains the bovine growth hormone polyA sequence shown in SEQ ID NO:12; (v)3' end reversed terminal repeat sequence ITR, the (v)3' end reversed terminal repeat sequence ITR comprising the AAV ITR shown in SEQ ID NO:

17.

34. The expression vector according to claim 2, comprising, from 5' to 3': (iv) An inverted terminal repeat sequence ITR at the 5' end, wherein the inverted terminal repeat sequence ITR at the 5' end comprises the AAV ITR shown in SEQ ID NO: 16; (i) A first enhancer region, wherein the first enhancer region contains the CMV enhancer sequence shown in SEQ ID NO:13; (a) A promoter region containing the cytomegalovirus (CMV) promoter sequence shown in SEQ ID NO:9; (b) A 5'UTR region comprising the regulatory nucleic acid molecule of claim 1; (ii) an intron region comprising the SV40 intron sequence shown in SEQ ID NO:14; (c) The coding sequence encoding a polypeptide gene product, wherein the polypeptide gene product is aflibercept, the amino acid of which is shown in SEQ ID NO:10; and the coding nucleic acid of which is shown in SEQ ID NO:11; (iii) A second enhancement sub-region, the second enhancement sub-region comprising the SAR sequence shown in SEQ ID NO:15 or; (d) Polyadenylation region, wherein the polyadenylation region contains the bovine growth hormone polyA sequence shown in SEQ ID NO:12; (v)3' end reversed terminal repeat sequence ITR, the (v)3' end reversed terminal repeat sequence ITR comprising the AAV ITR shown in SEQ ID NO:17; The expression vector contains the sequence shown in SEQ ID NO:

35.

35. A recombinant virus comprising: a) Capsid proteins; and b) The expression vector according to any one of claims 2-34.

36. The recombinant virus according to claim 35, wherein, The recombinant virus is a recombinant adeno-associated virus.

37. The recombinant virus according to claim 36, wherein, The recombinant adeno-associated virus is selected from AAV 1, AAV 2, AAV 3, AAV 4, AAV 5, AAV 6, AAV 7, and AAV 8.

38. The recombinant virus according to claim 36, wherein, The recombinant adeno-associated virus was selected from avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, and sheep AAV.

39. The recombinant virus according to claim 36, wherein, The recombinant adeno-associated virus is selected from AAV 5 or AAV 6.

40. The recombinant virus according to claim 35, wherein, The capsid protein is the AAV variant 7m8 capsid protein.

41. A pharmaceutical composition comprising an expression vector according to any one of claims 2-34 and / or a recombinant virus according to any one of claims 35-40 and a pharmaceutically acceptable excipient.

42. An isolated host cell transfected or transduced using an expression vector according to any one of claims 2-34.

43. An isolated host cell infected with a recombinant virus according to any one of claims 35-40.

44. A method for expressing a transgene in mammalian cells, the method comprising contacting one or more mammalian cells with an expression vector according to any one of claims 2-34 and / or a recombinant virus according to any one of claims 35-40, wherein, The secreted polypeptide is expressed at a certain level in one or more mammalian cells.

45. Use of the expression vector according to any one of claims 8-10 or 34 in the preparation of a medicament for the treatment or prevention of an eye disease in a mammal selected from wet age-related macular degeneration (AMD), retinal neovascularization, choroidal neovascularization, and diabetic retinopathy.

46. ​​The use according to claim 45, wherein, The drug is administered to the eye of the mammal via intraocular injection or intravitreal injection.

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