CN114250227A

CN114250227A - Expression vector for high-level expression of foreign gene

Info

Publication number: CN114250227A
Application number: CN202011026481.5A
Authority: CN
Inventors: 谢兴旺; 刘海燕
Original assignee: Beijing Anlong Gene Pharmaceutical Technology Co ltd
Current assignee: Beijing Anlong Gene Pharmaceutical Technology Co ltd
Priority date: 2020-09-25
Filing date: 2020-09-25
Publication date: 2022-03-29

Abstract

The present disclosure relates to an expression vector for high level expression of a foreign gene, the expression vector comprising a regulatory nucleic acid molecule that enhances gene expression, the regulatory nucleic acid molecule comprising, in 5 'to 3' order, a tripartite leader sequence (TPL) of an adenovirus and an enhancer element (eMLP) of the major late promoter of the adenovirus. The expression vector can obviously improve the expression level of the exogenous gene, can improve the protein expression level coded by the intracellular exogenous gene by more than tens of times, and can improve the protein level coded by the secretory exogenous gene by several times.

Description

Expression vector for high-level expression of foreign gene

Technical Field

The present disclosure relates to a gene expression vector, and more particularly to a vector mediating the high expression of exogenous genes in cells and applications thereof.

Background

Exogenous genes are delivered to specific tissues or cells by using the vector, and diseases such as genetic diseases, tumors, degenerative diseases and the like are treated or prevented by using expressed exogenous proteins or non-coding RNA molecules, so that the method is a treatment mode with wide prospect.

Viral vectors are a gene delivery vector that has been rapidly developed in recent years, and among them, vectors of adeno-associated virus (AAV) are becoming the best vector choice for gene therapy due to their low immunogenicity, non-integration, and properties that mediate long-term expression of foreign genes.

Researchers have developed promoters with high transcriptional activity such as CMV and CAG, and have improved the expression efficiency of foreign genes. However, gene expression levels in mammalian cells are regulated at multiple levels, including transcriptional, post-transcriptional mRNA stability, translational, and post-translational protein stability. Thus, where delivery of a foreign gene into a mammalian cell is desired, for example in gene therapy, optimization can be made from a number of perspectives as described above, such as gene transcription and translation regulatory elements, copy number of the foreign gene, integration site of the foreign gene in the host cell genome, RNA processing and mRNA stability, translational modification of proteins by the host cell itself, and the like.

Thus, there is a need for improved methods and optimized expression vectors for expressing genes in cells, particularly mammalian cells, to ensure long-term high-level expression of exogenous genes.

Disclosure of Invention

In order to solve the problems in the prior art, the present disclosure provides a method of the present invention that includes various sequence elements that promote and maintain the expression of a foreign gene, so as to ensure long-term high-level expression of the foreign gene.

Accordingly, in one aspect, the present disclosure provides a regulatory nucleic acid molecule for enhancing gene expression, the regulatory nucleic acid molecule comprising, in 5 'to 3' order, a tripartite leader sequence (TPL) of an adenovirus and an enhancer element (eMLP) of a major late promoter of the adenovirus, wherein:

the tripartite leader sequence (TPL) of the adenovirus has a TPL sequence selected from the group consisting of SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4 or a sequence at least 85% identical thereto;

the enhancer element (eMLP) of the major late promoter of the adenovirus has an eMLP sequence selected from the group consisting of SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8 or a sequence having at least 85% identity thereto.

In one aspect, the present disclosure provides an expression vector comprising, in 5 'to 3' order:

(a) a promoter region;

(b) a 5' UTR region;

(c) a coding sequence encoding a polypeptide gene product;

(d) a polyadenylation region (polyA);

wherein the 5' UTR region comprises the aforementioned regulatory nucleic acid molecule;

the coding sequence is operably linked to the promoter region.

In one aspect, the present disclosure provides a recombinant virus comprising:

a) a capsid protein; and

b) the aforementioned expression vector.

In one aspect, the present disclosure provides a pharmaceutical composition comprising the aforementioned expression vector and/or recombinant virus and a pharmaceutically acceptable excipient.

In one aspect, the present disclosure provides an isolated host cell transfected or transduced with the aforementioned expression vector.

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

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

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

The invention can obviously improve the expression level of the aflibercept, and can improve the expression level of intracellular aflibercept protein by more than ten times and improve the protein level of secreted aflibercept by several times compared with a vector which uses a pure CMV promoter/enhancer to start an aflibercept expression frame.

Drawings

Figure 1 shows the structure of the AO expression vector of the present disclosure.

FIG. 2 shows the levels of Aflibercept protein in cells after transfection of the Aflibercept expression vector into 293T cells (FIG. 2A) and ARPE-19 cells (FIG. 2B).

FIG. 3 shows the levels of Aflibercept protein in the supernatant after transfection of Aflibercept expression vectors into 293T cells (FIG. 2A) and ARPE-19 cells (FIG. 2B).

FIG. 4 shows the levels of aflibercept protein in cells following AAV virus infection of ARPE-19 cells.

FIG. 5A shows the average AAV genomic copy number in a cell after AAV virus infection of ARPE-19 cells; FIG. 5B shows secreted Aflibercept protein levels in the supernatant for each AAV genomic copy in cells following AAV infection in ARPE-19 cells.

FIG. 6 shows the correlation analysis of intracellular and secreted Aflibercepts following transfection of Aflibercept expression vectors or infection of 293T cells and ARPE-19 cells with AAV virus.

FIG. 7 shows intracellular and secreted Aflibercept protein levels following injection of AO virus into BN rat retinas, where BLK is a control rat eyeball draw sample without AO virus injection; b2_20R, B2_21R and B2_22R are obtained from the eyeball of a rat injected with AO-1 virus; b2_26R, B2_26L and B2_29L are obtained from the eyeball of a rat injected with AO-2 virus; b2_30L, B2_31L and B2_32R are obtained from the eyeball of a rat injected with AO-3 virus; b2_37R, B2_36L, B2_38R and B2_39L are obtained from the eyeball of a rat injected with AO-4 virus.

Detailed Description

Definition of

In the present invention, unless otherwise specified, scientific and technical terms used herein have the meanings that are commonly understood by those skilled in the art. Also, protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology related terms, and laboratory procedures used herein are all terms and conventional procedures used extensively in the relevant art. Meanwhile, in order to better understand the present invention, the definitions and explanations of related terms are provided below.

As used herein, "carrier" refers to a macromolecule or association of macromolecules that includes or is associated with a polynucleotide and can be used to mediate 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 vehicles.

The term "AAV" is an abbreviation for adeno-associated virus, and can be used to refer to the virus itself or derivatives thereof. The term encompasses all subtypes as well as naturally occurring and recombinant forms, unless otherwise required. The term "AAV" encompasses 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 ovine AAV. "primate AAV" refers to AAV infecting a primate, "non-primate AAV" refers to AAV infecting a non-primate mammal, and "bovine AAV" refers to AAV infecting a bovine mammal, and the like.

An "AAV virus" or "AAV viral 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 a wild-type AAV) and an encapsidation polynucleotide. If the particle includes a heterologous polynucleotide (i.e., a polynucleotide other than the wild-type AAV genome, such as a transgene to be delivered to a mammalian cell), it is often referred to as a recombinant AAV vector or rAAV. Typically, the heterologous polynucleotide is flanked by AAV Inverted Terminal Repeats (ITRs).

As used herein, the term "replication defective" with respect to an AAV viral vector of the present invention means that the AAV vector is unable to independently replicate and package its genome. For example, when a subject's cells are infected with rAAV virions, the heterologous gene is expressed in the infected cells, however, rAAV cannot replicate further due to the fact that the infected cells lack AAV rep and cap genes and helper function genes.

As used herein, an "AAV variant" or "AAV mutant" refers to a viral particle comprised of a variant AAV capsid protein, wherein the variant AAV capsid protein comprises at least one amino acid difference (e.g., amino acid substitution, amino acid insertion, amino acid deletion) relative to a corresponding parental AAV capsid protein, and wherein the variant capsid protein confers increased infectivity to retinal cells compared to the infectivity of retinal cells of an AAV virion comprising the corresponding parental AAV capsid protein, wherein the AAV capsid protein does not comprise the amino acid sequences present in naturally occurring AAV capsid proteins. The polynucleotide expression vectors of the present disclosure can be packaged in variant AAV particles to facilitate delivery of the expression vectors to specific cell types (e.g., retinal cells) in a target tissue.

As used herein, the term "packaging" refers to a series of intracellular events that result in the assembly and encapsidation of AAV particles.

As used herein, the terms AAV "rep" and "cap" gene refer to polynucleotide sequences encoding replication and encapsidation proteins of adeno-associated virus. AAV rep and cap are referred to herein as AAV "packaging genes".

As used herein, the term "helper virus" of an AAV refers to a virus that allows mammalian cells to replicate and package the AAV (e.g., a wild-type AAV). A variety of such helper viruses for AAV are known in the art, including adenovirus, herpesvirus, and poxvirus (e.g., vaccinia). Although adenovirus type 5 of subgroup C is most commonly used, adenoviruses encompass many different subgroups. Many adenoviruses of human, non-human mammalian and avian origin are known and available from stores such as the ATCC. Viruses of the herpes family include, for example, Herpes Simplex Virus (HSV) and Epstein-Barr virus (EBV) as well as Cytomegalovirus (CMV) and pseudorabies virus (PRV); also available from depositories such as ATCC.

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

As used herein, the term "gene" or "coding sequence" refers to a nucleotide sequence that encodes a gene product in vitro or in vivo. The term "transgene" refers to a coding sequence or gene that is delivered into a cell by a vector. The coding sequence or gene may encode a peptide or polypeptide molecule.

As used herein, "therapeutic gene" and "therapeutic protein" refer to a gene or protein that, when expressed, confers a beneficial effect on the cell or tissue or mammal in which it is present, in which it is expressed. Examples of beneficial effects can be alleviation or amelioration of signs or symptoms of a condition or disease, prevention or inhibition of a condition or disease, or imparting a desired characteristic. Therapeutic genes and proteins include genes and proteins that correct genetic defects in a cell or mammal.

A "therapeutically effective amount" or "effective amount" of an expression vector, recombinant virus, or pharmaceutical composition of the 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 can be a human or non-human mammal.

As used herein, the term "gene product" refers to the desired expression product of a polynucleotide sequence (e.g., a peptide or protein).

As used herein, the terms "polypeptide" and "protein" refer to polymers of amino acids of any length. The term "peptide" refers to a polymer of amino acids of about 50 amino acids or less. The term also encompasses amino acid polymers that have been modified as by, for example, disulfide bond formation, glycosylation, lipidation, or phosphorylation. In some examples, the polypeptide can have a length of greater than 50 amino acids.

As used herein, a "secreted protein" or "secreted polypeptide" of a "secreted protein" is any protein secreted by or exported from a living cell. One non-limiting example of a secreted protein for use with the presently described expression vectors is sFLT-1.

"comprising" means that the listed elements are, for example, essential in the compositions, methods, kits, etc., but that other elements may be included to form, for example, the compositions, methods, kits, etc., within the scope of the claims. For example, an expression vector that "comprises" a gene encoding a therapeutic polypeptide operably linked to a promoter is one that may contain elements other than the gene and promoter (e.g., polyadenylation sequences, enhancer elements, other genes, linker domains, etc.).

"consisting essentially of … …" is intended to mean a limitation on the scope of a particular material or step described, e.g., as a composition, method, kit, etc., that does not materially affect one or more of the basic and novel characteristics of the composition, method, kit, etc. For example, an expression vector "consisting essentially of" a gene encoding a therapeutic polypeptide operably linked to a promoter and polyadenylation sequence may comprise additional sequences, e.g., linker sequences, so long as they do not substantially affect the transcription or translation of the gene. As another example, a variant or mutant polypeptide fragment "consisting essentially of" the recited sequence has an amino acid sequence of the sequence plus or minus about 10 amino acid residues at the boundaries of the sequence based on the full-length untreated polypeptide from which it is derived, e.g., 10, 9, 8, 7,6, 5,4, 3,2, or 1 fewer than the binding amino acid residues or 1, 2,3, 4,5, 6, 7,8, 9, or 10 more residues than the binding amino acid residues.

"consisting of … …" means that any element, step, or ingredient not specified in the claims is excluded from the composition, method, or kit. For example, an expression vector "consisting of" a gene encoding a therapeutic polypeptide operably linked to a promoter and a polyadenylation sequence "consists of only the promoter, the polynucleotide sequence encoding the therapeutic polypeptide, and the polyadenylation sequence. As another example, a polypeptide "consisting of" a recited sequence contains only the recited sequence.

As used herein, "expression vector" is intended to encompass vectors, e.g., plasmids, minicircles, viral vectors, liposomes, etc., that encode a gene product of interest and are used to deliver the polynucleotide to the intended target cell.

As used herein, "promoter" encompasses a DNA sequence that directs RNA polymerase to bind and thereby promote RNA synthesis. Promoters and corresponding protein or polypeptide expression may be ubiquitous (meaning having strong activity in a wide range of cells, tissues and species) or cell type specific, tissue specific or species specific. Promoters may be "constitutive" (meaning persistently active) or "inducible" (meaning that the promoter may be activated or inactivated by the presence or absence of a biological or non-biological agent). Also included in the nucleic acid constructs or vectors of the invention are enhancer sequences, which may or may not be contiguous with the promoter sequence. Enhancer sequences affect promoter-dependent gene expression and may be located in the 5 'region or the 3' region of the native gene.

As used herein, "enhancer" encompasses cis-acting elements that stimulate or inhibit transcription of adjacent genes. Enhancers that inhibit transcription are also referred to as "silencers". Enhancers can function in either orientation at a distance of a few thousand base pairs (kb) from the coding sequence and a position downstream of the transcribed region (i.e., can be related to the coding sequence).

As used herein, "polyadenylation signal sequence" encompasses the recognition region required for endonuclease cleavage of an RNA transcript, followed by the polyadenylation consensus sequence AATAAA. The polyadenylation signal sequence provides a "polyA site", i.e., a site on the RNA transcript to which adenine residues will be added by post-transcriptional polyadenylation.

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 permitting them to operate in the intended manner. For example, a promoter is operably linked to a coding region if it helps to initiate transcription of the coding sequence. Intervening residues may be present between the promoter and the coding region so long as this functional relationship is maintained.

As used herein, the term "heterologous" refers to an entity that is of a different genotype from the rest of the entity to which it is compared. For example, polynucleotides introduced into plasmids or vectors derived from different species by genetic engineering techniques are heterologous polynucleotides. As another example, a promoter removed from its native coding sequence and operably linked to a coding sequence not found in nature is a heterologous promoter. Thus, for example, a rAAV comprising a heterologous nucleic acid encoding a heterologous gene product is a rAAV comprising nucleic acid not normally comprised in a naturally-occurring wild-type AAV, and the encoded heterologous gene product is a gene product not normally encoded by a naturally-occurring wild-type AAV.

As used herein, the term "endogenous" with respect to a nucleotide molecule or gene product refers to a nucleic acid sequence (e.g., a gene or genetic element) or gene product (e.g., RNA, protein) that naturally occurs in or is associated with a host virus or cell.

As used herein, the term "native" refers to a nucleotide sequence (e.g., a gene) or a gene product (e.g., RNA, protein) that is present in a wild-type virus or cell.

As used herein, the term "variant" refers to a mutant of a reference polynucleotide or polypeptide sequence, e.g., a native polynucleotide or polypeptide sequence, i.e., having less than 100% sequence identity to 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).

As used herein, the term "sequence identity" or "percent identity" refers 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, the term "identical" or percent "identity," when used in the context of two or more nucleotides or amino acid sequences, refers to two sequences that are the same or have a particular percentage of amino acid residues or nucleotides when compared and aligned for maximum correspondence, e.g., as measured using a sequence comparison algorithm (e.g., Smith-Waterman algorithm, etc.) or by visual inspection. For example, percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (1970, journal of molecular biology (J.mol. biol.), 48:444-453) algorithm, which has been incorporated into the GAP program in the GCG software package, using either the Blossum62 matrix or the PAM250 matrix with GAP weights of 16, 14, 12, 10, 8, 6, or 4 and length weights of 1, 2,3, 4,5, or 6. As another example, the percent identity between two nucleotide sequences can be determined using the GAP program in the GCG software package, using the nwsgapdna. cmp matrix and GAP weights of 40, 50, 60, 70, or 80 and length weights of 1, 2,3, 4,5, or 6. A particularly preferred set of parameters (which should be used unless otherwise specified) is the Blossum62 scoring matrix, a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5. Percent 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 bioscience (cabaos), 4:11-17), which has been incorporated into the ALIGN program (version 2.0), using the PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. The nucleic acid and protein sequences described herein can be used as "query sequences" to search public databases to, for example, identify other family members or related sequences. This search 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 NBLAST program (score 100, word length 12) to obtain nucleotide sequences homologous to the nucleic acid molecules of the invention. BLAST protein searches can be performed using the XBLAST program (score 50, word length 3) to obtain amino acid sequences homologous to the protein molecules of the invention. To obtain gapped alignments for comparison purposes, gapped BLAST as described in Altschul et al (1997, Nucleic Acids Res., 25: 3389-. When utilizing BLAST and gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.

As used herein, the terms "biological activity" and "biological activity" refer to an activity attributed to a particular biological element in a cell. For example, "biological activity" of an "immunoglobulin", "antibody" or fragment or variant thereof refers to the ability to bind an antigenic determinant and thereby promote immune function. As another example, the biological activity of a polypeptide or functional fragment or variant thereof refers to the ability of the polypeptide or functional fragment or variant thereof to perform its native function, e.g., 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, respectively, to regulate the expression of (i.e., promote, enhance, or activate translation of) a gene to which it is operably linked.

As used herein, the term "administration" or "introducing" refers to the delivery of a vector for recombinant protein expression to a cell, a cell and/or an organ of a subject, or a subject. Such administration or introduction may occur in vivo, in vitro, or ex vivo. The vector for expressing the gene product can be introduced into the cell by transfection, which generally means insertion of heterologous DNA into the cell by physical means (e.g., calcium phosphate transfection, electroporation, microinjection, or lipofection); or by infection or transduction, which generally refers to the introduction of a nucleic acid molecule into a cell by an infectious agent (i.e., a virus or viral vector).

In general, a cell is referred to as "transduced", "infected", "transfected" or "transformed" according to the method used to administer, introduce or insert the heterologous DNA (i.e., vector) to the cell. When DNA is introduced into a cell by a virus or viral vector, the cell is transduced with exogenous or heterologous DNA. When DNA is introduced into a cell by non-viral methods, the cell is transfected with exogenous or heterologous DNA. Non-viral methods include chemical methods (e.g., lipofection) and non-chemical methods. The terms "transduced" and "infected" are used interchangeably herein to refer to a cell that has received heterologous DNA or a heterologous polynucleotide from a virus or viral vector.

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, a viral particle, a phage, or the like. Culture conditions (e.g., temperature, pH, etc.) are those previously used with the host cell selected for expression, and will be apparent to those skilled in the art. It is understood that the term "host cell" refers to the originally transduced, infected, transfected or transformed cell and its progeny.

The terms "treatment" and "treating" refer to the alleviation of one or more signs or symptoms of a disease or disorder.

The terms "disease," "disorder," and "medical condition" are synonymous and are used interchangeably herein.

"ocular disease" refers to a disease, illness, or condition that affects or involves the eye or one or more portions or regions of the eye. Thus, ocular diseases include retinal diseases or diseases that affect the light sensitive layer of the posterior tissues of the eye. The eye contains the eyeball and the tissues and fluids that make up the eyeball, the periocular muscles (e.g., the oblique and rectus muscles), and the portion of the optic nerve within or near the eyeball.

A tissue "explant" is a piece of tissue that has been transferred from an animal to a nutrient medium.

The terms "individual", "host", "subject" and "patient" are used interchangeably herein and refer to a mammal, including but not limited to: human and non-human primates, including apes and humans; mammalian sport animals (e.g., horses); mammalian farm animals (e.g., sheep, goats, etc.); mammalian pets (dogs, cats, etc.); and rodents (e.g., mice, rats, etc.).

Various compositions and methods of the invention are described below. Although specific compositions and methods are exemplified herein, it should be understood that any of a number of alternative compositions and methods are suitable and suitable for practicing the present invention. It will also be appreciated that the expression constructs and methods of the invention can be evaluated using standard procedures in the art.

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry and immunology, which are within the skill of the art. Such techniques are well described in the literature, e.g., molecular cloning: a laboratory Manual (Molecular Cloning: A laboratory Manual), second edition (Sambrook et al, 1989); oligonucleotide Synthesis (oligo Synthesis) (edited by m.j. gate, 1984); animal Cell Culture (Animal Cell Culture), edited by r.i. freshney, 1987; methods in Enzymology (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 (Gene Transfer Vectors for Mammarian Cells) (edited by J.M.Miller and M.P.Calos, 1987); current Protocols in molecular Biology (Current Protocols in molecular Biology) (edited by F.M. Ausubel et al, 1987); PCR: polymerase Chain Reaction (PCR: the polymerase Chain Reaction), ed (edited by Mullis et al, 1994); and Current protocols in Immunology (J.E. Coligan et al, 1991), each of which is expressly incorporated herein by reference.

Several aspects of the invention are described below with reference to example applications for illustration. It should be understood that numerous specific details, relationships, and methods are set forth to provide a full understanding of the invention. One skilled in the relevant art will readily recognize, however, that the invention can be practiced without one or more of the specific details, or with other methods. The present invention is not limited by the illustrated ordering of acts or events, as some acts may occur in different orders and/or concurrently with other acts or events. Moreover, not all illustrated acts or events are required to implement a methodology in accordance with the present invention.

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

The terms "about" or "approximately" mean that the particular value determined by one of ordinary skill in the art is within an acceptable error range, which will depend in part on how the value is measured or determined, i.e., limited by the measurement system. For example, "about" can mean within 1 or a standard deviation of greater than 1, according to practice in the art. Alternatively, "about" or "approximately" may mean a range of up to 20%, preferably up to 10%, more preferably up to 5% and more preferably still 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-fold and more preferably within 2-fold of the value. When particular values are described in the present application and claims, unless otherwise specified, it should be assumed that the term "about" means within an acceptable error range for the particular value.

All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. It should be understood that in case of conflict, the present disclosure supersedes any disclosure incorporated herein.

It is further noted that the claims may be drafted to exclude any optional element. Accordingly, this statement is intended to serve as antecedent basis for use of such exclusive terminology as "solely," "only," etc. in connection with the recitation of claim elements, or use of a "negative type" limitation.

The disclosure in the publications discussed herein is provided solely for their purpose of illustration prior to the filing date of the present application. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such publication by virtue of prior invention. In addition, the dates of publication provided may be different from the actual publication dates which may need to be independently determined.

Unless otherwise indicated, all terms used herein have the same meaning as they would to one skilled in the art and the practice of the present invention will employ conventional techniques of microbiology and recombinant DNA technology, which are within the knowledge of one skilled in the art.

Detailed description of the preferred embodiments

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

In some embodiments of the present disclosure, the regulatory nucleic acid molecule has a sequence selected from the group consisting of the sequences set forth in SEQ ID NOS 19-34 or a sequence having at least 85% identity thereto.

In some preferred embodiments of the present 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 thereto.

In some preferred embodiments of the present disclosure, the regulatory nucleic acid molecule has the sequence shown in SEQ ID NO. 26 or a sequence having at least 85% identity thereto.

(a) a promoter region;

(b) a 5' UTR region;

(c) a coding sequence encoding a polypeptide gene product;

(d) a polyadenylation region (polyA);

the coding sequence is operably linked to the promoter region.

In some embodiments of the disclosure, the (a) promoter region is selected from the group consisting of Cytomegalovirus (CMV) promoter, actin promoter, elongation factor 1 α (EF1 α) promoter, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) promoter; the promoter region of (a) comprises the Cytomegalovirus (CMV) promoter sequence shown in SEQ ID NO:9 or a sequence having at least 85% identity therewith.

In some embodiments of the disclosure, the polypeptide gene product is a therapeutic protein.

In some preferred embodiments of the present disclosure, the therapeutic protein is selected from an anti-angiogenic polypeptide or alpha-1 antitrypsin.

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

In some preferred embodiments of the present disclosure, the anti-angiogenic polypeptide is aflibercept; preferably, the amino acid of aflibercept is shown in SEQ ID NO 10; more preferably, the nucleic acid encoding aflibercept is shown in SEQ ID NO 11.

In some embodiments of the disclosure, the polyadenylation region is selected from the human growth hormone (HGH or HGH), bovine growth hormone (BGH or BGH), or β -globin (β -globin) polyA sequences.

In some preferred embodiments of the present 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 thereto.

In some embodiments of the present disclosure, the expression vector further comprises (i) a first enhancer region, said (i) first enhancer region being located upstream of said (a) promoter region.

In some preferred embodiments of the present disclosure, the (i) first enhancer region comprises a sequence selected from the CMV enhancer or the EF 1a enhancer.

In some preferred embodiments of the present disclosure, the (i) first enhancer region comprises the CMV enhancer sequence shown in SEQ ID NO. 13 or a sequence having at least 85% identity thereto.

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

In some preferred embodiments of the present disclosure, the (ii) intron region comprises a sequence selected from the SV40 intron, the elongation factor 1 α (EF1 α) intron, the actin intron, or the CMVc intron.

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

In some embodiments of the present disclosure, the expression vector further comprises (iii) a second enhancer region located downstream of the (c) coding sequence encoding the polypeptide gene product and upstream of the (d) polyadenylation region.

In some preferred embodiments of the present disclosure, the (iii) second enhancer region comprises an Expression Enhancer Sequence (EES).

In some preferred embodiments of the present disclosure, the (iii) second enhancer region comprises a Scaffold Attachment Region (SAR) of interferon.

In some preferred embodiments of the present disclosure, the scaffold attachment region Sequence (SAR) is the human scaffold attachment region of human interferon-beta (IFNB SAR).

In some preferred embodiments of the present disclosure, the human interferon-beta human scaffold attachment region (IFNB SAR) comprises the SAR sequence shown in SEQ ID NO. 15 or a sequence having at least 85% identity thereto.

In some embodiments of the disclosure, the expression vector further comprises an Inverted Terminal Repeat (ITR) at the 5 'end of (iv) upstream of the (i) first enhancer and an Inverted Terminal Repeat (ITR) at the 3' end of (v) downstream of the (d) polyadenylation region.

In some preferred embodiments of the present disclosure, the (iv) Inverted Terminal Repeat (ITR) at the 5 'end or the (v) Inverted Terminal Repeat (ITR) at the 3' end comprises an Inverted Terminal Repeat (ITR) selected from Adenovirus (AV) or adeno-associated virus (AAV);

in some preferred embodiments of the present disclosure, the (iv)5' Inverted Terminal Repeat (ITR) comprises the AAV ITR shown in SEQ ID NO 16 or a sequence having at least 85% identity thereto.

In some preferred embodiments of the present disclosure, the (v) Inverted Terminal Repeat (ITR) at the 3' terminus comprises the AAV ITR shown in SEQ ID NO:17 or a sequence having at least 85% identity thereto.

In some embodiments of the present disclosure, the expression vector further comprises (vi) a selectable marker gene.

In some preferred embodiments of the present disclosure, the (vi) selectable marker gene is located downstream of the Inverted Terminal Repeat (ITR) at the 3' end of (v).

In some preferred embodiments of the present disclosure, the (vi) selectable marker gene is selected from the group consisting of an ampicillin resistance gene, a hygromycin resistance gene, a neomycin resistance gene, a bialaphos resistance gene, and a dihydrofolate reductase gene.

In some preferred embodiments of the present disclosure, the ampicillin resistance gene comprises the sequence shown as SEQ ID NO 18 or a sequence having at least 85% identity thereto.

In some embodiments of the disclosure, the expression vector comprises, 5 'to 3':

(i) a first enhancer region comprising the CMV enhancer sequence shown in SEQ ID NO 13 or a sequence having at least 85% identity thereto;

(a) a promoter region comprising the Cytomegalovirus (CMV) promoter sequence shown in SEQ ID NO:9 or a sequence having at least 85% identity thereto;

(b) a 5'UTR region, the 5' UTR region comprising a regulatory nucleic acid molecule consisting of the TPL sequence shown in SEQ ID NO. 2 and the eMLP sequence shown in SEQ ID NO. 8 or a sequence having at least 85% identity thereto;

(c) a coding sequence encoding a polypeptide gene product;

(iii) a second enhancer region comprising the SAR sequence shown in SEQ ID NO. 15 or a sequence having at least 85% identity thereto; and

(d) a polyadenylation 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 thereto, and

optionally wherein the expression vector does not comprise an RNA export signal.

(ii) an intron region comprising the SV40 intron sequence shown as SEQ ID NO. 14 or a sequence having at least 85% identity thereto;

(c) a coding sequence encoding a polypeptide gene product, wherein the coding sequence is operably linked to the promoter region;

(iii) a second enhancer region comprising the SAR sequence shown in SEQ ID NO. 15 or a sequence having at least 85% identity thereto;

(d) a polyadenylation 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 thereto.

(iv) an Inverted Terminal Repeat (ITR) at the 5 'end, the Inverted Terminal Repeat (ITR) at the 5' end comprising the AAV ITR set forth in SEQ ID NO 16 or a sequence having at least 85% identity thereto;

(c) a coding sequence encoding a polypeptide gene product;

In some embodiments of the disclosure, it comprises, 5 'to 3':

(c) a coding sequence encoding a polypeptide gene product;

(d) a polyadenylation 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 thereto;

(v) (iv) a 3 'terminal Inverted Terminal Repeat (ITR), said (v)3' terminal Inverted Terminal Repeat (ITR) comprising the AAV ITR set forth in SEQ ID NO:17 or a sequence having at least 85% identity thereto.

In some embodiments of the disclosure, it comprises, 5 'to 3':

(c) a coding sequence encoding a polypeptide gene product, which is aflibercept; preferably, the amino acid of aflibercept is shown in SEQ ID NO 10; more preferably, the nucleic acid encoding aflibercept (aflibercept) is shown in SEQ ID NO 11;

In some preferred embodiments of the present disclosure, the expression vector comprises the sequence shown as SEQ ID NO 35, 36, 37 or 38 or a sequence having at least 85% identity thereto.

In some preferred embodiments of the present disclosure, the expression vector comprises the sequence shown as SEQ ID No. 36 or a sequence having at least 85% identity thereto.

In one aspect, the present disclosure provides a recombinant virus comprising:

a) a capsid protein; and

b) the aforementioned expression vector.

In some embodiments of the present disclosure, the recombinant virus is a recombinant adeno-associated virus; preferably, the adeno-associated virus is selected from the group consisting of 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 ovine AAV.

In some preferred embodiments of the present disclosure, the adeno-associated virus is selected from AAV type 5 (AAV-5) or AAV type 6 (AAV-6).

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

In some preferred embodiments of the present disclosure, the disease is an ocular disease and the pharmaceutical composition is administered to the eye of the mammal.

In some preferred embodiments of the present disclosure, the pharmaceutical composition is administered to the eye of the mammal by intraocular injection or intravitreal injection.

In some preferred embodiments of the present disclosure, the ocular disease is selected from the group consisting of age-related macular degeneration (AMD), wet AMD, dry AMD, retinal neovascularization, choroidal neovascularization, and diabetic retinopathy.

In one aspect, the present disclosure provides the use of the aforementioned expression vectors, recombinant viruses, pharmaceutical compositions, and/or host cells in the preparation of a medicament for treating or preventing a disease in a mammal in need of treatment or prevention of the disease.

In some aspects of the disclosure, compositions for expressing a transgene in one or more eukaryotic cells are provided. In some aspects, the eukaryotic cell is a mammalian cell. In some aspects, the mammalian cell is a retinal cell, such as a retinal ganglion cell, amacrine cell, horizontal cell, bipolar cell, photoreceptor cell, cone cell, rod cell, muller glial cell, or retinal pigment epithelial cell.

In some embodiments of the disclosure, the composition is an expression vector. "expression vector" refers to a polynucleotide sequence, e.g., regulatory elements, translation initiation sequences, coding sequences, termination sequences, and the like, typically operably linked to each other, comprising two or more functional polynucleotide sequences. Typically, the polynucleotide sequence is comprised of DNA. Likewise, "expression vector for expressing a transgene in a mammalian cell" refers to a combination of two or more functional polynucleotide sequences (e.g., promoters, enhancers, 5' UTRs, switch initiation sequences, coding sequences, termination sequences, etc.) that facilitate expression of the transgene in the cell.

In some embodiments, the expression vectors of the present disclosure provide for enhanced expression of a transgene in a mammalian cell. In certain embodiments, the arrangement of two or more functional polynucleotide sequences within an expression vector of the present disclosure provides for enhanced expression of a transgene in a mammalian cell. By "enhanced" is meant that expression of the transgene is increased, enhanced, or greater in a cell carrying an expression vector of the present disclosure relative to a cell carrying a transgene operably linked to a comparable regulatory element. In other words, transgene expression by an expression vector of the present disclosure is increased, enhanced, or stronger relative to expression by an expression vector that does not include one or more optimized elements of the present disclosure (i.e., a reference control vector, such as a CMV reference control vector as described herein). In certain embodiments, the expression enhancement is specific for or limited to one or more desired cell types. In one embodiment, the transgene encodes a protein that is secreted by the cell into the aqueous environment surrounding the cell.

For example, expression of a transgene in a cell comprising an expression vector disclosed herein comprising a promoter may be enhanced, or stronger than expression of the transgene in a cell carrying a transgene operably linked to a different promoter. As another example, expression of a transgene in a cell comprising an expression vector disclosed herein including an enhancer sequence may be enhanced, or increased, enhanced, or stronger than expression of the transgene in a cell carrying a transgene operably linked to a different enhancer sequence. As another example, expression of a transgene in a cell comprising an expression vector encoding a 5'UTR disclosed herein can be enhanced, or increased, enhanced, or stronger than expression of the transgene in a cell carrying a transgene operably linked to a different 5' UTR coding sequence. As another example, expression of a transgene in a cell comprising an expression vector disclosed herein comprising an intron can be enhanced, or increased, enhanced, or stronger than expression of the transgene in a cell carrying a transgene operably linked to a different intron sequence. In yet another example, expression of the transgene in a cell comprising an expression vector comprising an intron disclosed herein can be enhanced, or increased, potentiated, or stronger than expression of the transgene in a cell carrying a transgene operably linked to a reference control vector (such as a CMV reference control vector disclosed herein).

In preferred embodiments, the polynucleotide expression vector promotes expression of the transgene (or a higher level of expression compared to a reference vector) in one or more specific cell or tissue types 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, Muller glia cells, and retinal pigment epithelial cells. In another embodiment, increased expression is observed in cells of a retinal tissue explant.

In some embodiments, the expression of the secreted polypeptide by the expression vector in the mammalian cell is at least about 2-fold, 3-fold, 5-fold, 9-fold, 10-fold, 20-fold, or 50-fold greater than the expression of the secreted polypeptide by the reference vector in the mammalian cell in vitro or in vivo. More typically, the expression of the secreted polypeptide is 2 to 10 fold, 5 to 10 fold, 9 to 10 fold, at least 2 fold, at least 5 fold, or at least 10 fold greater than the expression of the polypeptide in a mammalian cell by a reference vector. In other words, the expression vector expresses the secreted protein in mammalian cell culture at a level that is at least or greater than 2-fold, 5-fold, 10-fold, 50-fold, or about 5-fold to about 10-fold greater than the level of expression obtained by the reference vector in mammalian cell culture.

Without wishing to be bound by theory, it is believed that enhanced expression of the transgene in the intracellular or extracellular environment (e.g., culture supernatant or tissue matrix) results from faster accumulation of the gene product in the cell or a more stable gene product in the cell. Thus, enhanced transgene expression by the expression vectors of the present disclosure can be observed in a variety of ways. For example, if a transgene is operably linked to comparable regulatory elements (such as those in a CMV reference control vector as described herein), enhanced expression can be observed by detecting expression of the transgene, followed by detecting exposure of the expression vector to the cell earlier than expressed (e.g., 7 days, 2 weeks, 3 weeks, 4 weeks, 8 weeks, 12 weeks or more earlier than expressed). Enhanced expression with increasing amounts of gene product per cell can also be observed. For example, the amount of gene product per mammalian cell may be increased 2-fold or more, e.g., increased 3-fold or more, increased 4-fold or more, increased 5-fold or more, or increased 10-fold or more. Enhanced expression may 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 be increased 2-fold or more, e.g., increased 3-fold or more, increased 4-fold or more, increased 5-fold or more, or increased 10-fold or more. As another example, the polynucleotides of the invention can promote detectable levels of transgenes in a greater percentage of cells compared to conventional expression vectors; for example, while a conventional vector can promote detectable levels of transgene expression, e.g., in less than 5% of the cells in a region, a polynucleotide of the invention promotes detectable levels of expression in 5% or more of the cells in the 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, in some cases 50% or more, 55% or more of the contact; 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 a detectable level of the gene product. Enhanced expression with altered cell viability and/or function may also be observed.

Expression vectors of the present disclosure typically include a promoter region. In certain embodiments, the promoter region promotes expression of the coding sequence in mammalian cells. In some cases, the promoter is a ubiquitous promoter, i.e., it is a promoter that is active in a wide range of cells, tissues and species. Suitable examples include actin, Cytomegalovirus (CMV), elongation factor 1 α (EF1 α), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) promoters.

In some embodiments, the polynucleotide comprises one or more enhancers. Enhancers are nucleic acid elements that enhance 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, EF1 α, CMV, intact EES or portions thereof, such as 410-564EES or 511-810 EES. EES (expression enhancer sequence) corresponds to the human scaffold attachment region of human interferon-beta or SAR (Agarwal, M et al (1998) "enhancement of retroviral vector expression in primary T cells mediated by the scaffold attachment region" [ J. virol.) ] 72 (5: 3720-. The nuclear scaffold binding region (SAR) is an AT-rich stretch of DNA sequence, also known as the Matrix Attachment Region (MAR). Researches show that the SAR region sequence of human interferon gene is added in the expression frame of exogenous gene, which is beneficial to preventing methylation silencing of exogenous gene and maintaining long-term stable expression of exogenous gene.

The Tripartite leader sequence (TPL) of adenovirus is the UTR sequence at the 5' end of the major late transcribed mRNA of adenovirus, and research shows that the TPL sequence can promote translation of mRNA, and the translation level of the mRNA containing the TPL sequence at the 5' end is obviously higher than that of the mRNA without the TPL sequence at the 5' end.

The Major Late Promoter (MLP) of adenovirus is a common Promoter for controlling the transcription of most Late genes after the replication of adenovirus genome begins, and the activity of MLP is obviously enhanced once the replication of adenovirus genome begins. An enhancer sequence (eMLP) of adenovirus late gene promoter is added near the promoter, so that the expression level of the exogenous gene can be obviously improved.

In some embodiments, the expression vector includes a sequence encoding a 5' untranslated region, i.e., a polynucleotide sequence encoding 5' of the untranslated region of the coding sequence, also referred to as a 5' UTR. In some embodiments, the 5' UTR does not contain a polynucleotide ATG. Suitable exemplary 5' UTR sequences include, but are not limited to, sequences selected from: i) triple leader sequence (TPL) from Adenovirus (Logan, J et al (6 1984) "triple leader sequence of Adenovirus enhances translation of mRNA in the late stage of infection (Adenoviral leader sequences of mRNA laterovefter infection)", (Proc. Natl. Acad. Sci. USA) 81: 3655-); ii) enhancer element sequences from the adenovirus major late promoter (eMLP) (Durocher, Y et al (2002) "High-level and High-throughput recombinant proteins produced by transient transfection of human 293-EBNA1cells grown in suspension by High-level and High-throughput recombinant proteins (High-level and High-throughput recombinant protein production by transformed transformation of sub-growth human 293-EBNA1 cells)", "nucleic acid research (Nucl. acids. Res., (30 (2): e 9); (iii) UTR 1; and (iv) UTR 2. In a preferred embodiment, the 5' UTR comprises in 5' to 3' order TPL and eMLP sequences.

In some embodiments, the expression vector further comprises an intron comprising a splice donor/acceptor region. In some embodiments, the intron is 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 by intron splicing and are removed during mRNA processing. Expression vectors containing introns are generally expressed more highly than those without introns. Introns can stimulate expression between 2-fold and 500-fold (Buchman and Berg,1988, Mol cell biology (Mol CellBio), 8(10): 4395). The efficiently spliced intron contains a pre-splice donor, a branch point and a Py-rich region (Senapathy et al, 1990; "methods in enzymology" (meth. enzymol.) 183,252-78; Wu and Krainer, 1999; "molecular cell biology", 19(5): 3225-36). The 5 'intron is generally more effective than the intron at the 3' end (Huang and Gorman, 1990; "molecular cell biology", 10: 1805). While introns are known to generally increase gene expression levels, the specific increase (if any) of a given cDNA is empirical and must be tested; for example, chimeric introns in the pSI vector increased CAT expression 21-fold, but luciferase expression only 3-fold. Exemplary intron sequences include, but are not limited to, sequences from actin, elongation factor 1 α (EF1 α), enhancer element from adenovirus major late promoter (eMLP), and CMVc.

The coding sequence to be expressed in a cell may be any polynucleotide sequence, for example, a gene or cDNA encoding a gene product, such as a polypeptide. The coding sequence may be heterologous to the promoter sequence and/or 5'UTR sequence to which said coding sequence is operably linked, i.e. said coding sequence is not naturally operably associated with said promoter or 5' UTR. Alternatively, the coding sequence may be endogenous to a promoter sequence and/or 5'UTR sequence to which the coding sequence is operably linked, i.e. the coding sequence is naturally associated with the promoter or 5' UTR. The gene product may act internally on the mammalian cell, or it may act externally, e.g., it may be secreted. For example, when the transgene is a therapeutic gene, the coding sequence can be any gene that encodes a desired gene product, or a functional fragment or variant thereof, which can be used as a therapeutic agent for treating a disease or disorder. Thus, the coding sequence in the expression vector may encode, for example, an opsin protein or a protein that inhibits VEGF, or the polynucleotide may encode a protein or enzyme effective to reduce one or more signs or symptoms of a disease.

In various preferred embodiments, the transgene encodes a peptide or protein that is secreted from the cell. In some embodiments, the secreted protein is a therapeutic protein or a protein effective to treat a disease in a subject. In some embodiments, the therapeutic protein is an anti-angiogenic polypeptide or a polypeptide that inhibits the growth of new blood vessels (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 (ranibizumab), bevacizumab (bevacizumab), and aflibercept. Another example of an anti-VEGF polypeptide is soluble fms-like tyrosine kinase-1 (sFLT-1). In other cases, the secreted protein includes or consists of a VEGF-binding protein or a functional fragment thereof (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 as disclosed, for example, in U.S. patent No. 7,635,474. In some forms, the secreted protein comprises or consists of a single chain antibody (such as, for example, a single chain anti-VEGF antibody). 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 comprise a sequence encoding a functional VEGF-binding fragment of sFLT-1 (Wiesmann et al, 1997; Cell (Cell), 91: 695-. According to another example, the transgene encodes A1AT or alpha-1 antitrypsin (Chiuchiolo et al, 2013,24(4): 161-.

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 and inhibits VEGF (Kendall and Thomas, 1993; Proc. Natl. Acad. Sci. USA 90(22): 10705-10709). In nature, recombinant sFLT-1 is produced by alternative mRNA splicing and lacks the membrane proximal immunoglobulin-like domain, transmembrane region (transmembrane spanning region), and intracellular tyrosine kinase domain. As described herein, "soluble" FLT-1 or sFLT-1 refers to FLT-1, said FLT-1 is not limited to a cell membrane. Unbound sFLT-1 can diffuse freely in the extracellular space or in solution.

In one embodiment of the invention, the transgene coding sequence is modified or "codon optimized" to enhance expression by replacing infrequently represented codons with more frequently represented codons. The coding sequence is a portion of the mRNA sequence that encodes the amino acids 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 will utilize trnas that encode the same amino acid at different frequencies (each carrying an anticodon). When a gene sequence contains codons that are infrequently represented by the corresponding tRNA, the ribosomal translation mechanism may slow, thereby preventing efficient translation. Expression may be improved by "codon optimisation" of a particular species in which the coding sequence is altered to encode the same protein sequence using codons highly expressed and/or used by highly expressed human proteins (Cid-Arregui et al, 2003; "journal of virology" 77: 4928). In one aspect, 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 having at least 85% sequence identity, e.g., at least 90% sequence identity, e.g., at least 95% sequence identity, at least 98% identity, at least 99% identity, to a polypeptide encoded by a sequence disclosed above or herein, 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.

In some embodiments, the expression vector of the invention further comprises an RNA export signal. RNA export signals are cis-acting post-transcriptional regulatory elements that enhance the export of RNA from the nucleus. Exemplary RNA export sequences include, but are not limited to, sequences from The hepatitis b virus post-transcriptional regulatory element (HPRE) and woodchuck hepatitis virus post-transcriptional element (WPRE) (Higashimoto, T et al, "woodchuck hepatitis virus post-transcriptional regulatory element reduces read-through transcription from retroviral vectors (The wood hepatitis virus site-transcriptional regulatory elements)," gene therapy (GeneTher.) (2007, 9 months, 14(17): 1298-.

In some embodiments, the expression vector of the invention further comprises a polyadenylation region. As understood in the art, RNA polymerase II transcripts terminate by cleavage and addition of a polyadenylation region, which may also be referred to as the poly (a) signal, poly (a) region, or poly (a) tail. The polyA region contains multiple consecutive adenosine monophosphate, which typically has a repeat of the motif AAUAAA. Several effective polyadenylation sites have been identified, including those from SV40, bovine growth hormone, human growth hormone, and rabbit beta globin (Xu et al, 2001; "Gene (Gene) 272(1-2): 149-. The most effective polyA signal for expression of a transgene in a mammalian cell may depend on the cell type and species of interest and the particular vector used. In some embodiments of the invention, the expression vector comprises a polyA region selected from the group consisting of: bovine Growth Hormone (BGH), Human Growth Hormone (HGH), and beta-globin (beta globin).

As understood by one of ordinary skill, two or more of the above-mentioned polynucleotide elements may be combined to produce the expression vectors of the present disclosure. Thus, for example, an expression vector may comprise, in 5' to 3' order, in operable linkage, a CMV enhancer, CMV or EF1 α promoter, optionally a CMVc or EF1 α intron, UTR1, UTR2 or TPL and eMLP 5' UTR, the coding sequence or secreted polypeptide of sFLT1, the entire EES, 410-and 564-EES or 511-and 810-EES enhancer, optionally a HPRE or WPRE RNA export sequence, and a BGH, HGH or β globin polyadenylation signal sequence.

Another expression vector may include, in 5' to 3' order, a 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 polypeptide), a full length EES enhancer, and an HGH polyA signal sequence in operable linkage. In particular embodiments, the coding sequence encodes an anti-angiogenic polypeptide. In particular embodiments, the coding sequence is codon optimized. In certain of these embodiments, the expression vector includes one or more sequences selected from SEQ ID NOS 35-38.

As one of ordinary skill in the art will recognize, the expression vector may optionally contain other elements, including but not limited to restriction sites to facilitate cloning and regulatory elements for the particular gene expression vector. Examples of regulatory sequences include ITRs of AAV vectors, bacterial sequences of plasmid vectors, attP sites or attB sites of phage integrase vectors, and transposable elements of transposons.

As disclosed herein, in some aspects of the invention, expression vectors are used to deliver genes to animal cells, e.g., to determine the effect of genes on cell viability and/or function, to treat cellular disorders, and the like. Thus, in some aspects of the invention, compositions for expressing a transgene in a mammalian cell are provided as gene delivery vectors, wherein the gene delivery vectors include the expression vectors of the present disclosure.

The gene delivery vectors of the present disclosure encompass any convenient gene delivery vector for delivering a polynucleotide sequence to a mammalian cell. For example, the vector may comprise single-stranded nucleic acid or double-stranded nucleic acid, such as single-stranded DNA or double-stranded DNA. For example, the gene delivery vector can be DNA, e.g., naked DNA, such as a plasmid or minicircle, and the like. The vector may comprise single-stranded RNA or double-stranded RNA, including modified forms of RNA. In another example, the gene delivery vector may be an RNA, such as an mRNA or a modified mRNA.

As another example, the gene delivery vector may be a viral vector derived from a virus, such as adenovirus, adeno-associated virus (AAV), lentivirus, herpes virus, alphavirus or retrovirus, such as Moloney (Moloney) murine leukemia virus (M-MuLV), Moloney murine sarcoma virus (MoMSV), havey (Harvey) murine sarcoma virus (hamsv), murine mammary tumor virus (MuMTV), gibbon ape leukemia virus (GaLV), Feline Leukemia Virus (FLV), foamy virus, Friend (Friend) murine leukemia virus, Murine Stem Cell Virus (MSCV) and Rous (Rous) sarcoma virus (RSV) or lentivirus. Although the examples encompassing the use of adeno-associated viruses are described in more detail below, it is contemplated that one of ordinary skill will recognize that similar knowledge and skills in the art may also be applied to non-AAV gene delivery vectors. See, e.g., the discussion of retroviral vectors in, e.g., U.S. patent No. 7,585,676 and U.S. patent No. 8,900,858, and adenoviral vectors in, e.g., U.S. patent No. 7,858,367, the entire disclosures of which are incorporated herein by reference.

In some embodiments, the gene delivery vector is a recombinant adeno-associated virus (rAAV). In this example, the expression vector is flanked at the 5 'and 3' ends by functional AAV Inverted Terminal Repeat (ITR) sequences. By "functional AAV ITR sequence" is meant an ITR sequence that is used as intended to rescue, replicate and package AAV virions. Thus, the AAV ITRs for use in the gene delivery vectors of the invention need not have a wild-type nucleotide sequence and may be altered by insertion, deletion or substitution of nucleotides, or the AAV ITRs may be derived from any of several AAV serotypes, e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV 10. Preferred AAV vectors have all or part of the wild-type Rep and Cap genes deleted, but retain functional flanking ITR sequences. In particular embodiments, the AAV viral vector is AAV2 variant 7m8.

In some embodiments, the expression vector is encapsidated within an AAV capsid, which may be derived from any adeno-associated virus serotype, including but not limited to AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, etc., any of which may serve as a gene delivery vector. For example, the AAV capsid may be a wild-type capsid or a native capsid. Wild type AAV capsids of particular interest comprise AAV2, AAV5, and AAV 9. However, as with the ITRs, the capsid need not have a wild-type nucleotide sequence, but rather can be altered relative to the wild-type sequence by insertion, deletion or substitution of nucleotides in the VP1, VP2 or VP3 sequences so long as the capsid is capable of transducing mammalian cells. In other words, the AAV capsid may be a variant AAV capsid comprising one or more amino acid substitutions, deletions, or insertions relative to the parental capsid protein or AAV capsid protein from which it is derived. Variant AAVs of particular interest include those disclosed in U.S. patent 9,193,956, the entire disclosure of which is incorporated herein by reference. In some embodiments, the variant AAV comprises a 7m8 variant capsid protein (which may be referred to herein as aav2.7m8 or 7m8.aav2) disclosed in U.S. patent 9,193,956. .

Preferably, the rAAV is replication-defective, in that the AAV vector is unable to independently replicate and package its genome further. For example, when cones are transduced with rAAV virions, the gene is expressed in the transduced cones, however, rAAV cannot replicate due to the fact that the transduced cones lack AAV rep and cap genes as well as helper functions.

Standard methods can be used to generate gene delivery vectors (e.g., rAAV virions) that encapsidate the expression vectors of the disclosure. For example, in the case of rAAV virions, an AAV expression vector according to the present invention can be introduced into a producer cell, followed by introduction of an AAV helper construct, wherein the helper construct comprises an AAV coding region capable of expression in the producer cell and which AAV coding region can complement AAV helper functions not present in the AAV vector. A helper virus and/or additional vector is then introduced into the producer cell, wherein the helper virus and/or additional vector provides helper functions capable of supporting efficient rAAV virus production. The producer cells are then cultured to produce rAAV. These steps are performed using standard methods. AAV packaging cells and packaging techniques are used to prepare replication-defective AAV virions that encapsidate the recombinant AAV vectors of the invention by standard 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 No. 5,753,500, U.S. patent No. 6,040,183, U.S. patent No. 6,093,570, and U.S. patent No. 6,548,286, 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.

Any concentration of viral particles suitable for efficient transduction of mammalian cells can be prepared for contacting mammalian cells in vitro or in vivo. For example, it may be at 10 per ml⁸Viral particles are formulated at a concentration of one or more vector genomes (vg/mL), e.g., 5X 10 per mL⁸A vector genome; 10 per ml⁹A vector genome; 5X 10 per ml ⁹10 per ml of vector genome¹⁰5X 10 vector genomes per ml¹⁰A vector genome; 10 per ml¹¹A vector genome; 5X 10 per ml¹¹A vector genome; 10 per ml¹²A vector genome; 5X 10 per ml¹²A vector genome; 10 per ml¹³A vector genome; 1.5X 10 per ml¹³A vector genome; 3X 10 per ml¹³A vector genome; 5X 10 per ml¹³A vector genome; 7.5X 10 per ml¹³A vector genome; 9X 10 per ml¹³A vector genome; 1X 10 per ml¹⁴5X 10 vector genomes per ml¹⁴One or more vector genomes, but usually not more than 1X 10 per ml¹⁵And (3) a vector genome. Similarly, any total number of viral particles suitable to provide appropriate cell transduction to confer a desired effect or treat a disease may be administered to a mammal. In various preferred embodiments, at least 10⁸A plurality of; 5X 10⁸A plurality of; 10⁹A plurality of; 5X 10⁹1, 10¹⁰5 x 10 pieces of¹⁰A plurality of; 10¹¹A plurality of; 5X 10¹¹A plurality of; 10¹²A plurality of; 5 is prepared from10¹²A plurality of; 10¹³A plurality of; 1.5X 10¹³A plurality of; 3X 10¹³A plurality of; 5X 10¹³A plurality of; 7.5X 10¹³A plurality of; 9X 10¹³1,1 × 10¹⁴Or 5X 10¹⁴One or more virus particles, but generally not more than 1X 10 virus particles per eye¹⁵And (4) respectively. Any suitable number of vectors can be administered to the mammalian or primate eye. In one embodiment, the method comprises a single administration; in other embodiments, multiple administrations may be performed over time as deemed appropriate by the attending clinician.

The subject viral vectors can be formulated into pharmaceutical compositions comprising any suitable unit dose of the vector, which can be administered to a subject to produce a change in the subject or to treat a disease in the subject. In some embodiments, a unit dose includes, but is not limited to, 1 × 10 of a viral vector⁸One or more vector genomes, e.g., at least about 1X 10⁹1,1 × 10¹⁰1,1 × 10¹¹1,1 × 10¹²1,1 × 10¹³1,1 × 10¹⁴Or at least about 3 x 10¹⁴One or more vector genomes, in some cases, at least about 1X 10¹⁴A vector genome, but usually not more than 4X 10¹⁵And (3) a vector genome. In some cases, the unit dose comprises up to about 5 x 10¹⁵A vector genome, e.g. 1X 10¹⁴Or 5X 10¹⁴With less or no vector genome, e.g. 1X 10¹³1,1 × 10¹²1,1 × 10¹¹1,1 × 10¹⁰Or 1 x 10⁹One or less, and in some cases, 1X 10, vector genomes⁸A vector genome of 1X 10 or less, and usually not less than⁸And (3) a vector genome. In some cases, the unit dose comprises 1 × 10¹⁰1 to 10¹¹And (3) a vector genome. In some cases, the unit dose comprises 1 × 10¹⁰To 3 x 10¹²And (3) a vector genome. In some cases, the unit dose comprises 1 × 10⁹To 3 x 10¹³And (3) a vector genome. In some cases, a unit doseComprises 1 × 10⁸To 3 x 10¹⁴And (3) a vector genome. In some cases, the unit dose comprises about 1 × 10¹⁰From one to about 5X 10¹⁴And (3) a vector genome.

In some cases, the multiplicity of infection (MOI) can be used to measure the unit dose of a pharmaceutical composition. MOI refers to the ratio or fold of vector or viral genome to cells into which nucleic acid can be delivered. In some cases, the MOI may be 1 × 10⁶And (4) respectively. In some cases, the MOI may be 1 × 10⁵-1×10⁷And (4) respectively. In some cases, the MOI may be 1 × 10⁴-1×10⁸And (4) respectively. In some cases, a recombinant virus of the disclosure is at least about 1 × 10¹1,1 × 10²1,1 × 10³、1×10⁴1,1 × 10⁵1,1 × 10⁶1,1 × 10⁷1,1 × 10⁸1,1 × 10⁹1,1 × 10¹⁰1,1 × 10¹¹1,1 × 10¹²1,1 × 10¹³1,1 × 10¹⁴1,1 × 10¹⁵1,1 × 10¹⁶1,1 × 10¹⁷And 1X 10¹⁸And (4) the MOI. In some cases, the recombinant virus of the disclosure is 1 × 10⁸To 3 x 10¹⁴And (4) the MOI. In some cases, recombinant viruses of the present disclosure are up to about 1 × 10¹1,1 × 10²1,1 × 10³、1×10⁴1,1 × 10⁵1,1 × 10⁶1,1 × 10⁷1,1 × 10⁸1,1 × 10⁹1,1 × 10¹⁰1,1 × 10¹¹1,1 × 10¹²1,1 × 10¹³1,1 × 10¹⁴1,1 × 10¹⁵1,1 × 10¹⁶1,1 × 10¹⁷And 1X 10¹⁸And (4) the MOI.

In some aspects, the amount of the pharmaceutical composition comprises about 1 x 10⁸From one to about 1X 10¹⁵ About 1X 10 of recombinant virus⁹From one to about 1X 10¹⁴ About 1X 10 of recombinant virus¹⁰From one to about 1X 10¹³Recombinant virus or about 1X 10¹¹From one to about 3X 10¹²And (3) recombinant viruses.

In preparing the rAAV compositions, any host cell for producing rAAV virions can be employed, including, but not limited to, e.g., mammalian cells (e.g., 293 cells), insect cells (e.g., SF9 cells), microorganisms, and yeast. The host cell may also be a packaging cell in which the AAV rep and cap genes are stably maintained in the host cell or a producer cell in which the AAV vector genome is stably maintained and packaged. Exemplary packaging and production cells are derived from SF-9, 293, A549, or HeLa cells. AAV vectors are purified and formulated using standard techniques known in the art.

The invention encompasses pharmaceutical compositions comprising an expression vector or gene delivery vector described herein and a pharmaceutically acceptable carrier, diluent, or excipient. For example, one embodiment is a pharmaceutical composition comprising a recombinant virus comprising a polynucleotide of the present disclosure and a pharmaceutically acceptable excipient. In particular embodiments, the recombinant virus is a recombinant adeno-associated virus (AAV). The expression vector or gene delivery vector can be combined with pharmaceutically acceptable carriers, diluents, and agents that can be used to prepare formulations that are generally safe, non-toxic, and desirable and include acceptable excipients for primate use. Such excipients may 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, dextrose solution, and 5% human serum albumin. Supplementary active compounds may also be incorporated into these formulations. The solution or suspension for the formulation may comprise: sterile diluents such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents; antibacterial compounds such as benzyl alcohol or methyl paraben; antioxidants, such as ascorbic acid or sodium bisulfite; chelating compounds such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetate, citrate or phosphate; detergents, such as Tween (Tween)20 to prevent aggregation; and compounds for regulating tonicity, such as sodium chloride or dextrose. The pH can be adjusted with an acid or base (e.g., hydrochloric acid or sodium hydroxide). In particular embodiments, the pharmaceutical composition is sterile.

In the case of contacting cone cells in vivo, the expression vector or a gene delivery vector comprising the expression vector may be considered suitable for delivery to the eye.

Pharmaceutical compositions suitable for use in the present invention further comprise sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. Thus, the pharmaceutical compositions may be in the form of sterile injectable solutions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, or Phosphate Buffered Saline (PBS). In some cases, the composition is sterile and should have fluidity to the extent that it is easy to inject. In certain embodiments, the compositions are stable under the conditions of manufacture and storage, and are preserved against the contaminating action of microorganisms (e.g., bacteria and fungi). The carrier can be, for example, a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents (e.g., parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like). In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of the internal composition can be brought about by including in the composition an agent that delays absorption (e.g., aluminum monostearate and gelatin).

If desired, sterile solutions may be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a base dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

In one embodiment, the composition is prepared with a carrier that protects the expression vector from rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid may be used. Methods for preparing such formulations will be apparent to those skilled in the art. The materials are also commercially available. Liposomal suspensions (comprising liposomes targeted to infected cells with monoclonal antibodies to 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, for example, as described in U.S. Pat. No. 4,522,811.

It is particularly advantageous to formulate oral, ophthalmic or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suitable as unitary dosages for the subjects to be treated; each unit containing a predetermined amount of gene delivery or expression vector calculated to produce the desired therapeutic effect in association with the desired pharmaceutical carrier. The specification for the dosage unit form of the invention is dictated by the gene delivery vector, the unique characteristics of the expression vector and the particular therapeutic effect to be achieved.

The pharmaceutical composition can be contained in a container, package, or dispenser (e.g., a syringe, such as a prefilled syringe) with instructions for administration.

By "pharmaceutically acceptable excipient" is meant a material, substance, diluent or carrier that is substantially non-toxic to the cells or subject to which it is to be administered. That is, a pharmaceutically acceptable excipient may be incorporated into a pharmaceutical composition and administered to a cell or patient without substantially causing an undesirable biological effect or interacting in a deleterious manner with any of the other components of the composition in which the pharmaceutically acceptable excipient is contained.

Expression vectors or gene delivery vectors (e.g., recombinant viruses (virions)) can be incorporated into pharmaceutical compositions for administration to mammalian patients, particularly primates and more particularly humans. The expression vector or gene delivery vector (e.g., viral particle) may be formulated in a non-toxic, inert, pharmaceutically acceptable aqueous carrier having a pH preferably 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 include vectors or viral particles containing a nucleic acid encoding a therapeutic molecule dissolved in an aqueous buffer having an acceptable pH upon reconstitution.

In some embodiments, the pharmaceutical compositions provided herein comprise a therapeutically effective amount of the carrier or viral particle in admixture with a pharmaceutically acceptable carrier and/or excipient, e.g., 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 and the like are octylphenoxy polyethoxyethanol 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 (Hank's) solution, cysteine, arginine, carnitine, alanine, glycine, lysine, valine, leucine, polyvinylpyrrolidone, polyethylene, and ethylene glycol. Preferably, such formulations are stable at 4 ℃ for at least six months.

In some embodiments, the pharmaceutical compositions provided herein include buffers such as Phosphate Buffered Saline (PBS) or sodium phosphate/sulfate, tris buffers, glycine buffers, sterile water, and other buffers known to those of ordinary skill, such as those described by 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 can include an adenovirus or adeno-associated adenovirus vector delivery system comprising an expression vector of the disclosure.

The ability to deliver the expression vectors of the invention to selected target cells in vivo and obtain a therapeutically effective amount of the gene product in the cells and in the extracellular environment following a transduction event may be beneficial for the treatment of a number of different diseases, including those that are dependent on the growth of new blood vessels, where the goal of treatment may be to provide the target cells with the ability to secrete a therapeutically effective amount of an anti-angiogenic protein. While not wishing to be bound by any theory, even when the gene delivery vector is able to successfully transduce only a fraction of the target cells, an expression vector that is capable of enhancing expression and ultimately therapeutic protein secretion may help provide a clinically significant benefit to the patient. High levels of secretion of therapeutic proteins by transduced cells may help balance the infectivity or transduction efficiency achieved with any given dose or gene therapy round.

Thus, 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 to study, e.g., determine the effect of a gene on cell viability and/or function. As another example, the compositions may be used in medicine, for example, to treat a disorder. The methods and compositions of the present disclosure may be used to treat any condition that can be at least partially addressed by gene therapy of cells. Cells include, but are not limited to, blood, eye, liver, kidney, heart, muscle, stomach, intestine, pancreas, and skin.

Accordingly, the present invention provides a method for treating or preventing a disease or disorder (e.g., an ocular disease or disorder) in a subject in need thereof, the method comprising administering to the subject in need thereof a viral vector or viral particle comprising an expression vector of the invention encoding a therapeutic gene product. In a preferred embodiment, the therapeutic gene product is a secreted polypeptide or a protein that is secreted or exported from the 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 a human IFNB SAR (SEQ ID NO: 15); and (d) an HGH polyadenylation site (SEQ ID NO: 12).

In related embodiments, some methods provide for expression of a gene in a cell in vitro or in vivo, the method comprising contacting the cell with a composition of the disclosure. In some embodiments, the contacting occurs in vitro. In some embodiments, the contacting occurs in vivo, i.e., the composition is administered to a subject. The composition may be administered parenterally by intravenous injection or oral infusion. In certain embodiments, the composition is administered to the eye by injection, for example to the retina, lower retina, or vitreous. In certain embodiments, the composition is administered by retinal injection, subretinal injection, or intravitreal injection. In certain embodiments, the composition is administered topically or directly to a tissue or organ of interest, for example by injection into the liver.

The subject can be a mammal, including, for example, a human subject in need of treatment for a particular disease or condition.

For the case where mammalian cells are contacted in vitro with an expression vector or gene delivery vector comprising an expression vector, the cells can be from any mammalian species, e.g., rodent (e.g., mouse, rat, gerbil, squirrel), rabbit, cat, dog, goat, sheep, pig, horse, cow, primate, human. The cells may be from an established cell line or may be primary cells, which

The terms "primary cell," "primary cell line," and "primary culture" are used interchangeably herein to refer to cells and cell cultures derived from a subject and allow for a limited number of passages, i.e., divisions, of a growing culture 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 not enough to undergo a crisis stage. Typically, the primary cell lines of the invention are maintained in vitro for less than 10 passages.

If the cells are primary cells, the cells may be harvested from the mammal by any convenient method, e.g., whole explants, biopsies, etc. The collected cells may be dispersed or suspended with a suitable solution. Such a solution will typically be a balanced salt solution, such as physiological saline, PBS, hank's balanced salt solution, or the like, conveniently supplemented with fetal bovine serum or other naturally occurring factors at low concentrations (typically 5-25mM) in combination with an acceptable buffer. Convenient buffers include HEPES, phosphate buffer, lactate buffer, and the like. The cells may be used immediately or may be stored, frozen for extended periods of time, thawed and capable of being reused. In this case, cells are typically frozen in 10% DMSO, 50% serum, 40% buffered medium, or some other such solution as is commonly used in the art, to preserve the cells at such freezing temperatures and to be thawed in a manner as is commonly known in the art for thawing frozen cultured cells.

To facilitate expression of the transgene, the expression vector or the gene delivery vector including the expression vector is contacted with the cell for about 30 minutes to 24 hours or more, e.g., 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 comprising the expression vector can be provided to the subject cells one or more times (e.g., once, twice, three times, or more than three times) and the cells are allowed to incubate with the agent(s) for a 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. Contacting the cells can occur in any medium and under any culture conditions that promote cell survival. For example, the cells may be suspended in any suitable convenient nutrient medium, such as Iscove's modified DMEM or RPMI 1640, supplemented with foetal calf serum or heat-inactivated goat serum (about 5-10%), L-glutamine, thiols, especially 2-mercaptoethanol, and antibiotics, such as penicillin and streptomycin. The culture may contain growth factors to which the cells respond. As defined herein, a growth factor is a molecule capable of promoting the survival, growth and/or differentiation of cells in culture or in intact tissues through specific action on transmembrane receptors. Growth factors include polypeptide factors and non-polypeptide factors.

Typically, an effective amount of an expression vector or gene delivery vector comprising an expression vector is provided to produce transgene expression in a cell. As discussed elsewhere herein, an effective amount can be readily determined empirically, e.g., by detecting the presence or level of a transgene product, by detecting an effect on cell viability or function, etc. Typically, expression will be enhanced 2-fold or more, e.g., 3-fold, 4-fold or 5-fold or more, in some cases 10-fold, 20-fold or 50-fold or more, e.g., 100-fold, relative to expression from a reference or control expression vector. One example of a reference expression vector for comparison purposes is the CMV reference expression vector described herein. In particular embodiments, the transgene encodes a secreted protein, and the expression vector expresses the secreted protein in the mammalian cell at a level that is at least 2-fold, 5-fold, 10-fold, 5-fold to 15-fold, or 10-fold to 15-fold greater than the level of expression of the secreted protein in the mammalian cell by the CMV reference control expression vector. According to some embodiments, when the transgene is a transgene encoding a non-secreted protein, the expression vector of the invention expresses the non-secreted protein in the mammalian cell at a level that is about the same as, within 10-20%, about 1.5-fold or about 2-fold less than the level of expression of the non-secreted protein in the mammalian cell by the CMV reference control expression vector. The expression level of secreted protein for each expression vector can be measured by an immunoassay or an antigen capture assay, and 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.

Immunoassays for measuring The presence and amount (and thus The expression level) of proteins in biological or cellular samples are known in The art (e.g., Hage, d.s. (1999) "Immunoassays" (Analytical Chemistry), "71 (12) in Analytical Chemistry 294-. 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 carried out in liquid phase or solid phase systems, but for ease of detection, a solid phase may be preferred. Suitable immunoassays include, but are not limited to, sandwich and competition assays, western blots, ELISA (enzyme linked immunosorbent assays), Radioimmunoassays (RIA), Fluorescent Immunoassays (FIA), and the like. The biological sample may be cell culture medium or supernatant (a sample taken from a culture without lysing the cells), cell lysate, whole cells, blood, serum, plasma, or other bodily fluids or tissues. In some embodiments, such as when the transgene is a selectable marker, the cell population can be enriched for those comprising the expression vector by separating the modified cells from the remaining population. The separation may be carried out by any convenient separation technique appropriate for the selectable marker used. For example, if the transgene is a fluorescent label, the cells may be isolated by fluorescence activated cell sorting, whereas if the transgene is a cell surface label, the cells may be separated from the heterogeneous population by affinity separation techniques, such as magnetic separation, affinity chromatography, "panning" with affinity reagents attached to a solid matrix, or other convenient techniques. Techniques for providing accurate separation include fluorescence activated cell sorters, which may have varying degrees of complexity, such as multi-color channels, low and obtuse angle light scatter detection channels, impedance channels, and the like. Cells can be selected for dead cells by using a dye (e.g., propidium iodide) associated with the dead cells. Any technique that is not unduly detrimental to cell viability may be employed. In this way a highly enriched cell composition of cells comprising the polynucleotide is achieved. By "highly enriched" is meant that the genetically modified cells will be 70% or more, 75% or more, 80% or more, 85% or more, 90% or more of the cellular composition, for example about 95% or more or 98% or more of the cellular composition.

For the case where the cell is contacted with the expression vector or gene delivery vector comprising the expression vector in vivo, the subject can be any mammal, e.g., a rodent (e.g., mouse, rat, gerbil), rabbit, cat, dog, goat, sheep, pig, horse, cow, human, or non-human primate.

The methods and compositions of the invention may be used to treat any condition that can be at least partially addressed by gene therapy of cells. Cells include, but are not limited to, blood, eye, liver, kidney, heart, muscle, stomach, intestine, pancreas, and skin. One embodiment is a method for treating a medical condition in a subject in need of treatment, 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 effective to reduce 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 a cell transduced by the vector. In one embodiment, the 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. Particular forms of macular degeneration may include acute macular degeneration, non-exudative age-related macular degeneration, and exudative age-related macular degeneration. Administration may be 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.

Age related macular degeneration (AMD) is a common eye disease and is a significant cause of vision loss in the elderly. Advanced AMD can be classified into dry AMD and wet AMD, depending on the ocular fundus manifestations. VEGF-induced pathologic choroidal neovascularization is a central mechanism for the onset and progression of wet AMD, which can lead to exudation, hemorrhage, and fibrous scarring. anti-VEGF therapy is the most effective way to treat wet AMD, but the efficacy of anti-VEGF antibody injections is only sustained for a short period of time, thus requiring repeated ocular injections. The protein coding sequence capable of expressing and blocking the biological activity of VEGF is delivered to the fundus by using a gene therapy vector such as adeno-associated virus (AAV), so that the continuous expression of the protein capable of blocking the biological activity of VEGF can be realized, and the continuous inhibition of pathological choroid neovascularization by single administration can be realized.

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 by intraocular injection, by intravitreal injection, or by any other convenient mode or route of administration. In some embodiments, the subject is a human subject having or at risk of developing macular degeneration or ocular neovascularization.

In some embodiments, the methods result in a therapeutic benefit, such as preventing the development of a disorder, halting the progression of a disorder, reversing the progression of a disorder, and the like. In some embodiments, the method includes the step of detecting whether a therapeutic benefit is achieved. The ordinarily skilled artisan will understand that such measurement of treatment efficacy will be appropriate for the particular disease being modified, and will recognize appropriate detection methods for measuring treatment efficacy.

It is desirable that transgene expression obtained using a transgene is robust. Thus, in some cases, transgene expression (e.g., as detected by measuring the level of a gene product) can be observed two months or less after administration (e.g., 4 weeks, 3 weeks, or 2 weeks or less after administration (e.g., 1 week after administration of the composition)) by measuring therapeutic efficacy, or the like. It is expected that transgene expression will also persist over time. Thus, in some cases, transgene expression (e.g., as detected by measuring levels of gene products) can be observed at 2 months or more (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, in some cases more than 5 years) after administration of the composition, by measuring therapeutic efficacy, or the like.

In certain embodiments, the methods comprise the step of detecting transgene expression in a cell, wherein expression is enhanced relative to expression from an expression vector that does not include one or more improved elements of the present disclosure (i.e., a reference control). Typically, expression will be enhanced by 2-fold or more, e.g., 3-fold, 4-fold or 5-fold or more, in some cases 10-fold, 20-fold or 50-fold or more (e.g., 100-fold) relative to expression by the reference (i.e., control expression vector), as evidenced by, e.g., early detection, higher levels of gene product, greater functional impact on the cell, etc. In one aspect, the transgene encodes a secreted polypeptide (e.g., sFLT 1).

Generally, if the subject composition is a virus (e.g., a rAAV comprising a polynucleotide expression vector of the disclosure), an effective amount for achieving a change or producing a therapeutic effect in a subject will be about 1 x 10⁸The vector genome is one or more, in some cases, 1X 10⁹1,1 × 10¹⁰1,1 × 10¹¹1,1 × 10¹²Or 1X 10¹³One or more, in some cases, 1X 10, genomes¹⁴The number of vector genomes is one or more, and usually not more than 1X 10¹⁶And (3) a vector genome. In some cases, the amount of vector genome delivered is up to about 1 × 10¹⁶A vector genome, e.g. 1X 10¹⁵Less than one vector genome, e.g. 1X 10¹³1,1 × 10¹²1,1 × 10¹¹1,1 × 10¹⁰Or 1X 10⁹A vector genome or less, in some cases, 1X 10⁸A vector genome, and usually not less than 1X 10⁸Individual vector geneAnd (4) grouping. In some cases, the amount of vector genome delivered is 1 × 10¹⁰1 to 10¹¹And (3) a vector genome. In some cases, the amount of vector genome delivered is 1 × 10¹⁰To 3 x 10¹²And (3) a vector genome. In some cases, the amount of vector genome delivered is 1 × 10⁹To 3 x 10¹³And (3) a vector genome. In some cases, the amount of vector genome delivered is 1 × 10⁸To 3 x 10¹⁴And (3) a vector genome.

In some cases, the multiplicity of infection (MOI) can be used to measure the amount of the pharmaceutical composition to be administered. In some cases, MOI may refer to the ratio or fold of vector particle or viral genome to cells to which the polynucleotide expression vector may be delivered. In some cases, the MOI may be 1 × 10⁶And (4) respectively. In some cases, the MOI may be 1 × 10⁵1 to 10⁷And (4) respectively. In some cases, the MOI may be 1 × 10⁴1 to 10⁸And (4) respectively. In some cases, the recombinant viruses of the present disclosure are about 1 × 10¹1,1 × 10²1,1 × 10³1,1 × 10⁴1,1 × 10⁵1,1 × 10⁶Or 1 x 10⁷And (4) the MOI.

In some aspects, the individual dose is generally no less than the amount required to produce a measurable effect on the subject, and may be determined based on the pharmacokinetics and pharmacology of absorption, distribution, metabolism, and excretion ("ADME") of the composition or its byproducts, and thus based on the disposition of the composition within the subject. This includes consideration of the route of administration and dosage. Effective dosages and/or dosage regimens can be readily determined empirically, by preclinical determination, by safety and escalation and dose ranging trials, by individual clinician-patient relationships, and by in vitro and in vivo assays.

In many embodiments, the recombinant virus can be administered in conjunction with one or more additional compounds or therapies, including a second recombinant virus, a chemotherapeutic agent, surgery, a catheter device, and radiation. Combination therapy includes administration of a single pharmaceutical dosage formulation comprising a recombinant virus and one or more additional agents; also included is the administration of the recombinant virus and one or more additional agents in its own separate pharmaceutical dosage formulation. For example, the recombinant virus and the cytotoxic, chemotherapeutic or growth inhibitory agent may be administered to the patient together in a single dosage composition, e.g., as a combined preparation, or each agent may be administered as a separate dosage formulation. When separate dosage formulations are used, the VEGF-specific fusion polypeptide of the invention and one or more additional agents may be administered simultaneously or at separate staggered times, i.e., sequentially.

The term "cytotoxic agent" as used herein refers to a substance that inhibits or hinders the function of cells and/or causes cell destruction. The term is meant to include 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.

A "chemotherapeutic agent" is a chemical compound used to treat cancer. Examples of chemotherapeutic agents include alkylating agents such as thiotepa, cyclophosphamide formulations; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa and uredopa; the class of ethyleneimine and methylenemelamine includes hexamethylmelamine, tritylamine, triethylenephosphoramide (triethylenephosphoramide), triethylenethiophosphoramide, trimethylolomelamine; nitrogen mustards such as chlorambucil, naphazel, cholorophosphamide (cholophosphamide), estramustine, ifosfamide, mechlorethamine hydrochloride, melphalan, neomustard, mechlorethamine cholesterol, prednimustine, trofosfamide, uramustine, nitrosoureas (nitrosoureas) such as carmustine, chlorouramicin, fotemustine, lomustine, nimustine, ramustine; antibiotics such as aclacinomysins, actinomycin, authramycin, azaserine, bleomycin, actinomycin C, calicheamicin (calicheamicin), carabicin, carminomycin, carcinomycin, chromomycin, actinomycin D, daunorubicin, ditobicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, marisulosin, mitomycin, mycophenolic acid, norubicin, olivomycin, pelomycin, potfiromycin, puromycin, trirubicin, roxobicin, streptonigromycin, streptonigrin, tubercidin, ubenicillin, ulinastatin (neocarzinostatin), zorubicin, antimetabolites such as methotrexate, 5-fluorouracil, folic acid analogs such as denopterin, methotrexate, pterin, triptorelbine, purine analogs such as fludarabine, 6-mercaptopurine analogs such as fludarabine, and mercaptopurine analogs such as, Thiamiprine, thioguanine, pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine;

androgens such as carposterone, drotanolone propionate, epithioandrostanol, meiandrane, testolactone, adrenal suppressing drugs such as aminoglutethimide, mitotane, trostane, folic acid supplementing drugs such as frolinic acid; acetic acid glucurolactone; (ii) an aldophosphamide glycoside; 5-aminolevulinic acid; amsacrine; bestrabuucil; a bisantrene group; edatraxate; defofamine; colchicine, diazaquinone, elfornitine; ammonium etiolate; etoglut, gallium nitrate, hydroxyurea; lentinan; lonidamine; mitoguazone, mitoxantrone; mopidanol; nisridine; pentostatin; phenamett; pirarubicin, podophyllic acid; 2-ethyl hydrazide; procarbazine; lezoxan; a texaphyrin; a germanium spiroamine; geobacillus azavor; a tri-imine quinone; 2, 2' -trichlorotriethylamine; urethan; vindesine; dacarbazine; mannomustine; dibromomannitol; dibromodulcitol; (ii) a paraben; gacytosine; adenine arabinoside (arabinoside); cyclophosphamide; thiotepa; paclitaxel drugs (Taxanes), such as paclitaxel (Bristol-Myers squibbonology, Princeton, n.j.); docetaxel (Aventis antonyx, france); chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum drugs such as cisplatin and Carboplatin; vinblastine; platinum; etoposide (VP16) (etoposide (VP-16)); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; novoxil; noxiaoling; (ii) teniposide; daunomycin; aminopterin; (ii) an aptamer; ibandronate; CPT-11; topoisomerase inhibitor RFS 2000; difluoromethyl ornithine (DMFO); tretinoin; an esperamicin class; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the foregoing. Also included in this definition are anti-hormonal agents used to modulate or inhibit the action of hormones on tumors, including, for example, anti-estrogens; such as tamoxifen, raloxifene, aromatase inhibiting 4(5) -imidazole, 4-hydroxytrytoxifen, travoxifen, keoxifene, LY 117018, onapristone, and toremifene (Faleton); and antiandrogens such as flutamide, nilutamide, bicalutamide, leuprorelin and goserelin; and pharmaceutically acceptable salts, acids or derivatives of any of the foregoing.

"growth inhibitory agent" when used herein refers to a compound or composition that inhibits cell growth, particularly cancer cell growth, in vitro or in vivo. Examples of growth inhibitory agents include agents that block the cell cycle (at other stages besides S phase), such as agents that induce G1 phase block and M phase block. Classical M-phase blockers include Vincas (vincristine and vinblastine), and topoisomerase II inhibitors such as doxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin. Those agents that block G1 also prolong blocking to S phase, for example, DNA alkylating agents such as tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin, methotrexate, 5-fluorouracil, and cytarabine.

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 referred to in this specification are incorporated herein by reference in their entirety.

From the foregoing it will be appreciated that, although specific embodiments of the invention have been described herein for purposes of illustration, various modifications may be made without deviating from the spirit and scope of the invention. Accordingly, the invention is not limited except as by the appended claims.

Examples

The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed.

EXAMPLE 1 construction of expression vectors

To investigate the effect of the tripartite leader sequence (TPL) sequence from adenovirus and the enhancer element sequence (eMLP) from the major late promoter of adenovirus on gene expression, regulatory nucleic acid molecules consisting of the TPL sequence and the eMLP sequence were designed to regulate gene expression. The following 4 TPL sequences were selected: TPL1, TPL2, TPL3 and TPL4, the nucleotide sequences of which are respectively shown as SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3 and SEQ ID NO 4. The following 4 eMLP sequences were selected: eMLP1, eMLP2, eMLP3 and eMLP4, the nucleotide sequences of which are respectively shown in SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7 and SEQ ID NO. 8. Based on the combination of the above 4 TPL sequences and 4 eMLP sequences, the following 16 regulatory nucleic acid molecules were designed:

the regulatory nucleic acid molecule 11 consists of TPL1 and eMLP1, and the nucleotide sequence of the regulatory nucleic acid molecule is shown as SEQ ID NO. 19;

the regulatory nucleic acid molecule 12 consists of TPL1 and eMLP2, and the nucleotide sequence of the regulatory nucleic acid molecule is shown as SEQ ID NO. 20;

the regulatory nucleic acid molecule 13 consists of TPL1 and eMLP3, and the nucleotide sequence of the regulatory nucleic acid molecule is shown as SEQ ID NO. 21;

a regulatory nucleic acid molecule 14 which consists of TPL1 and eMLP4, and the nucleotide sequence of the regulatory nucleic acid molecule is shown as SEQ ID NO. 22;

the regulatory nucleic acid molecule 21 consists of TPL2 and eMLP1, and the nucleotide sequence of the regulatory nucleic acid molecule is shown as SEQ ID NO. 23;

the regulatory nucleic acid molecule 22 consists of TPL2 and eMLP2, and the nucleotide sequence of the regulatory nucleic acid molecule is shown as SEQ ID NO. 24;

the regulatory nucleic acid molecule 23 consists of TPL2 and eMLP3, and the nucleotide sequence of the regulatory nucleic acid molecule is shown as SEQ ID NO. 25;

the regulatory nucleic acid molecule 24 consists of TPL2 and eMLP4, and the nucleotide sequence of the regulatory nucleic acid molecule is shown as SEQ ID NO. 26;

the regulatory nucleic acid molecule 31 consists of TPL3 and eMLP1, and the nucleotide sequence of the regulatory nucleic acid molecule is shown as SEQ ID NO. 27;

a regulatory nucleic acid molecule 32, which consists of TPL3 and eMLP2, and the nucleotide sequence of the regulatory nucleic acid molecule is shown as SEQ ID NO. 28;

the regulatory nucleic acid molecule 33 consists of TPL3 and eMLP3, and the nucleotide sequence of the regulatory nucleic acid molecule is shown as SEQ ID NO. 29;

a regulatory nucleic acid molecule 34 consisting of TPL3 and eMLP4, the nucleotide sequence of which is shown as SEQ ID NO. 30;

the regulatory nucleic acid molecule 41 consists of TPL4 and eMLP1, and the nucleotide sequence of the regulatory nucleic acid molecule is shown as SEQ ID NO. 31;

a regulatory nucleic acid molecule 42 consisting of TPL4 and eMLP2, the nucleotide sequence of which is shown in SEQ ID NO: 32;

a regulatory nucleic acid molecule 43 consisting of TPL4 and eMLP3, the nucleotide sequence of which is shown as SEQ ID NO. 33;

the regulatory nucleic acid molecule 44 consists of TPL4 and eMLP4, and the nucleotide sequence is shown in SEQ ID NO. 34.

An expression vector of aflibercept is constructed, and the structure of the expression vector is shown in figure 1. The expression vector comprises, in 5 'to 3' order: the CMV enhancer (the nucleotide sequence of which is shown in SEQ ID NO:13), the CMV promoter (the nucleotide sequence of which is shown in SEQ ID NO:9), the TPL sequence, the eMLP sequence, the intron sequence (the nucleotide sequence of which is shown in SEQ ID NO: 14), the Kozak sequence, the aflibercept coding sequence (the coding protein of which is shown in SEQ ID NO: 10; the nucleotide sequence of which is shown in SEQ ID NO: 11), the human IFNB SAR sequence (the nucleotide sequence of which is shown in SEQ ID NO:15) and the bGH polyA sequence (the nucleotide sequence of which is shown in SEQ ID NO: 12).

The following 16 AAV-aflibercept expression vectors were constructed containing different combinations of TPL and eMLP (see table 1), numbered 11, 12, 13, 14, 21, 22, 23, 24, 31, 32, 33, 34, 41, 42, 43, 44, respectively. The 16 AAV-aflibercept expression vectors described above are identical in sequence except for the regulatory nucleic acid molecule consisting of TPL and eMLP.

A baseline control vector AAV-Afibercept-basic, numbered basic (hereinafter referred to as basic vector), comprising only the CMV enhancer and CMV promoter, the aflibercept coding sequence and the bGH polyA sequence was also constructed. The basic vector was identical to the 16 AAV-aflibercept expression vectors described above, except that it did not have TPL, eMLP and Intron sequences.

TABLE 1 combination of sequence elements of AAV vectors

Example 2 cell transfection experiment

1.293T cell transfection model

The 16 AAV-aflibercept expression vectors and the basic vector, which contained regulatory nucleic acid molecules consisting of different TPLs and emlps, constructed in example 1, were transfected into 293T cells using PEI, respectively. 48 hours after transfection, cell culture supernatant and total cell protein were harvested, respectively. Expression levels of aflibercept protein in 293T cells were analyzed using western blot, captured and detected using HRP anti-human IgG (recognizing Fc region of aflibercept) (fig. 2A), and GAPDH protein was detected using HRP anti-human GAPDH as an internal control. Secreted aflibercept proteins in 293T supernatants were analyzed by ELISA, captured and detected using anti-human IgG (Fc fragment recognizing aflibercept) antibody (fig. 3A). As a result, a number of AAV-Aflibercept vectors containing TPL and eMLP elements were found to have significantly increased intracellular and supernatant Aflibercept levels compared to the basic vector, including vectors numbered 11, 13, 14, 21, 22, 23, 24, 31, 41, 42, 43 and 44.

ARPE-19 cell transfection model

The 16 AAV-aflibercept expression vectors and basic vectors comprising regulatory nucleic acid molecules consisting of different TPL and eMLP constructed in example 1 were transfected into ARPE-19 cells using PEI, respectively. 48 hours after transfection, cell culture supernatant and total cell protein were harvested, respectively. Expression levels of aflibercept protein in ARPE-19 cells were analyzed using western blot, captured and detected using HRP anti-human IgG (recognizing Fc region of aflibercept) (fig. 2B), and GAPDH protein was detected using HRP anti-human GAPDH as an internal control. Secreted aflibercept proteins in 293T supernatants were analyzed by ELISA, captured and detected using anti-human IgG (Fc fragment recognizing aflibercept) antibody (fig. 3B). As a result, a number of AAV-Aflibercept vectors containing TPL and eMLP elements were found to have significantly increased intracellular levels of Aflibercept compared to basic vectors, including vectors numbered 11, 13, 14, 21, 22, 23, 24, 31, 41, 42, 43, and 44.

Example 3 cell infection experiments

Considering that the DNA state containing exogenous Aflibercept coding sequence has obvious difference after plasmid transfected cells and AAV virus particles infected cells, 293T cells (A) are used for further simulating the application scene of AAV gene therapy

CRL-3216TM), AAV-DJ, AAV Helper packaging system (Cell Biolabs, VPK-400-DJ), and any one of the Afibercep expression vectors, for packaging to produce AAV viral particles.

The AAV virus packaging and purifying steps are as follows:

1) day 0 (Day 0): cell seeding

The 293T was inoculated into 15cm dishes (the amount of inoculation was determined by the amount of virus expected)

2) Day 1 (Day 1): plasmid transfection

15cm dish transfection system: 15 ug AAV expression plasmid +15 ug AAV Helper +15 ug g g envelope

3) Day 3 (Day 3): 48h after transfection

A. Preparation of liquid Nitrogen

B. AAV293 was digested and collected into 50ml centrifuge tubes (Corning tubes are relatively freeze tolerant), washed twice with PBS, centrifuged at 1200rpm for 5min, and PBS was blotted dry;

C. resuspending the cells in 2ml cell lysis buffer per 15cm dish (total lysate volume of each virus is no more than 6ml), freezing the cells with liquid nitrogen, thawing immediately with 37 ℃ water bath, repeating for 3 times to fully lyse the cells; freezing the cells with liquid nitrogen again, and storing at-80 deg.C, or continuing the purification process;

4) after thawing the cell lysate in a 37 ℃ water bath, adding Benzonase to a final concentration of 50U/ml (25U/ul Benzonase stock solution added with 8. mu.l to 4ml), and adding sodium deoxycholate to a final concentration of 0.5% in a 37 ℃ water bath for 30 min;

5) iodixanol (Iodixanol) density gradient solution configuration (OptPrep is 60% Iodixanol, 361625 centrifuge tube capacity is 32.4)

TABLE 2 Iodixanol (Iodixanol) Density gradient solution configuration

6) Density gradient was perfused with a 10ml syringe: sequentially filling 9ml of 15% iodixanol layer, 6ml of 25% iodixanol layer, 5ml of 40% iodixanol layer and 2ml of 60% iodixanol layer, filling from low density to high density, inserting a syringe needle to the bottom of a centrifuge tube during each filling, and avoiding generating bubbles during filling;

7) centrifuging at 4500rpm at 4 deg.C for 30 min;

8) cell lysis supernatant (about 6ml) containing virus, plated on the uppermost layer of density gradient, capped with cell lysate and trimmed;

9) centrifugation was carried out using a Beckman Ti70 rotor at 67,000rpm for 1h at 18 ℃ with maximum acceleration and braking;

10) using a 10ml syringe and 18g needle, 3-5 mm puncture (needle bevel up) at 60-40% interface, collect 3-4 ml of a layer of virus-containing 40% iodixanol liquid (avoid protein contamination at 40-25% interface); can collect about 500ul per tube;

11) collecting 1-2ul of each tube, diluting by 20 times, and measuring absorbance at 340nm, wherein the collection tubes before the highest absorbance can be retained and mixed;

12) centrifuging at the highest rotation speed by using a 100K protein concentration tube, concentrating virus supernatant, adding 20ml of PBS for rinsing once, and centrifuging again for concentration to the desired volume;

13) the virus can be further purified using a HiTrap heparin affinity column or further concentrated using a 100K low volume protein concentration tube.

After purification and quantification of AAV viruses, ARPE-19 cells were infected in vitro with viruses carrying different AAV-Aflibercept vectors. Cell culture supernatants and total cellular proteins were harvested 72 hours after infection, respectively. Expression levels of intracellular aflibercept proteins were analyzed using western blots, captured and detected using anti-human IgG (recognizing the Fc-domain of aflibercept) (fig. 4). Analysis of secreted aflibercept proteins in culture supernatants by ELISA anti-human IgG (recognizing the Fc fragment of aflibercept) was used for capture and detection (fig. 5B). As a result, it was found that a number of AAV-Aflibercept vectors containing TPL and eMLP elements had significantly increased intracellular and supernatant Aflibercept levels compared to the basic vector.

Meanwhile, DNA is extracted from a part of ARPE-19 cells infected for 72 hours, then AAV genome ITR specific primers are used for real-time PCR, the average AAV genome copy number in each experimental group of cells is determined, the virus copy number in the cells after virus infection is quantitatively analyzed (figure 5A), after the ARPE-19 cells are infected by corresponding Aflibercept expression plasmids and packaged into the obtained AAV viruses, the Aflibercept expression level corresponding to each virus copy in the cells is calculated, and the Aflibercept protein amount in the supernatant corresponding to each virus genome in each cell is averaged, so that the expression levels of various AAV-Aflibercept vectors are further accurately evaluated (figure 5B).

Example 4 Integrated analysis of different cell model data

To integrate the expression levels of the vectors in the three cell models described above in example 2 and example 3, the most potential vectors were selected for in vivo evaluation and correlation analysis was performed on the expression levels of the aflibercept proteins in the intracellular and supernatant of the cells after transfection and infection with the different AO vectors (fig. 6). Air bubble maps were generated using Aflibercept protein levels in three different cell models. The X axis represents the level of secreted Aflibercept protein in the supernatant of ARPE-19 cell transfection model, and the Y axis represents the level of secreted Aflibercept protein in the supernatant of 293T cell transfection model; the size of the air bubbles represents the level of secreted Aflibercept protein in the supernatant of the ARPE-19 cell infection model, and the depth of air bubble coloration represents the expression level of the Aflibercept protein in the ARPE-19 cell infection model cells; the font size of the numerical number above the bubble represents the expression level of the Aflibercept protein in the cell in the 293T cell transfection model; the font size of the numerical numbers on the right side of the bubbles represents the intracellular expression level of Aflibercept protein in the 293T cell transfection model.

As a result, the AAV-Aflibercept-23 and AAV-Aflibercept-24 vectors have higher protein expression levels in 293T cells and ARPE-19 cells, AAV-Aflibercept-14 has the highest protein expression in 293T cells, and AAV-Aflibercept-43 has the highest protein expression in ARPE-19 cells.

EXAMPLE 5 evaluation of the expression level of AAV-Aflibercept vector in rat retina

AAV-Aflibercept-23, AAV-Aflibercept-24, AAV-Aflibercept-43 and AAV-Aflibercept-14 are selected for subsequent development, and are respectively named as AO-1 (the nucleotide sequence of which is shown in SEQ ID NO: 35), AO-2 (the nucleotide sequence of which is shown in SEQ ID NO: 36), AO-3 (the nucleotide sequence of which is shown in SEQ ID NO: 37) and AO-4 (the nucleotide sequence of which is shown in SEQ ID NO: 38). AAV viral particles were produced by packaging using AAV8 packaging system, 293T transfected AAV8, AAV Helper and any one of Afibercep expression vectors.

TABLE 3 AAV vector name comparison Table

BN rat is taken, four viruses of AO-1, AO-2, AO-3 and AO-4 are respectively injected under retina, and 4E10 GC (genome Copies) virus is injected into eyeball of each rat. Four weeks after injection, aqueous humor was isolated for ELISA analysis of levels of secreted aflibercept protein (fig. 7A); total retinal and choroidal tissue proteins were separately extracted and used for western blot analysis of intracellular levels of aflibercept protein expression (fig. 7B, 7C).

Simultaneously, extracting retinal tissue DNA, detecting AAV genome copy number in the retinal tissue by using real-time PCR, quantitatively analyzing virus copy number in cells after virus infection (figure 7D), and calculating Aflibercept expression level corresponding to each virus copy in the cells so as to further accurately evaluate the level of secretory Aflibercept protein corresponding to each virus genome in rat retinal aqueous humor (figure 7E) after injecting four viruses of AO-1, AO-2, AO-3 and AO-4; and the level of intracellular aflibercept protein per viral genome on average in rat retina and choroid. As a result, the average AAV genome copy number in the cells is the highest in the rats injected with AO-1 virus; in rats injected with AO-2 virus, secreted aflibercept levels in aqueous humor, and intracellular mean aflibercept levels in retina and choroid were all highest (FIG. 7F, G).

Sequence listing

<110> Beijing Anlong Gene medicine technology Co., Ltd

<120> expression vector for high-level expression of foreign gene

<130> MTI20136

<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

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 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

cgttcgtcct 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

agacgaaggc tcgcgtccag gccagcacga aggaggctaa 400

<210> 7

<211> 400

<212> DNA

<213> eMLP3(Artificial Sequence)

<400> 7

cagacgggcc agggtcatgt ctttccacgg gcgcagggtc ctcgtcagcg tagtctgggt 60

cacggtgaag gggtgcgctc cgggctgcgc gctggccagg gtgcgcttga ggctggtcct 120

gctggtgctg aagcgctgcc ggtcttcgcc ctgcgcgtcg gccaggtagc atttgaccat 180

ggtgtcatag tccagcccct ccgcggcatg gcccttggcg cgcagcttgc ccttggagga 240

ggcgccgcac gaggggcagt gcagactttt gagggcgtag agcttgggcg cgagaaatac 300

cgattccggg gagtaggcat ccgcgccgca ggccccgcag acggtctcgc attccacgag 360

ccaggtgagc tctggccgtt cggggtcaaa aaccaggttt 400

<210> 8

<211> 400

<212> DNA

<213> eMLP4(Artificial Sequence)

<400> 8

cgatagcagt tcttgcaagg aagcaaagtt tttcaacggt ttgaggccgt ccgccgtagg 60

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

<210> 10

<211> 431

<212> PRT

<213> Abbericept (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> Abbericept-encoding nucleic acid (Artificial Sequence)

<400> 11

atggtctctt attgggacac gggagttctc ctgtgtgcac tgctgagctg ccttctcctc 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 interferon-beta 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 ttctatatac attttagaat gaggttggca agttaacaaa 420

cagctttttt 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

ctagatttat 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 ctgaggccgc ccgggcgtcg ggcgaccttt 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 300

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 600

aa 602

<210> 21

<211> 602

<212> DNA

<213> regulatory nucleic acid molecule 13(Artificial Sequence)

<400> 21

actctcttcc gcatcgctgt ctgcgagggc cagctgttgg gctcgcggtt gaggacaaac 60

tcttcgcggt ctttccagta ctcttggatc ggaaacccgt cggcctccga acaggtactc 120

cgccgccgag ggacctgagc gagtccgcat cgaccggatc ggaaaacctc tcgagaaagg 180

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 300

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

<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

tctaaccagt cacagtcgca 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> 28

<211> 600

<212> DNA

<213> regulatory nucleic acid molecule 32(Artificial Sequence)

<400> 28

actctcttcc gcatcgctgt ctgcgagggc cagctgttgg gctcgcggtt gaggacaaac 60

tcttcgcggt ctttccagta ctcttggatc ggaaacccgt cggcctccga acgtactccg 120

ccaccgaggg acctgagcga gtccgcatcg accggatcgg aaaacctctc gagaaaggcg 180

tctaaccagt cacagtcgca ccgcggcatg gcccttggcg cgcagcttgc ccttggagga 240

ggcgccgcac gaggggcagt gcagactttt gagggcgtag agcttgggcg cgagaaatac 300

cgattccggg gagtaggcat ccgcgccgca ggccccgcag acggtctcgc attccacgag 360

ccaggtgagc tctggccgtt cggggtcaaa aaccaggttt cccccatgct ttttgatgcg 420

tttcttacct ctggtttcca tgagccggtg tccacgctcg gtgacgaaaa ggctgtccgt 480

gtccccgtat acagacttga gaggcctgtc ctcgagcggt gttccgcggt cctcctcgta 540

tagaaactcg gaccactctg agacgaaggc tcgcgtccag gccagcacga aggaggctaa 600

<210> 29

<211> 600

<212> DNA

<213> regulatory nucleic acid molecule 33(Artificial Sequence)

<400> 29

actctcttcc gcatcgctgt ctgcgagggc cagctgttgg gctcgcggtt gaggacaaac 60

tcttcgcggt ctttccagta ctcttggatc ggaaacccgt cggcctccga acgtactccg 120

ccaccgaggg acctgagcga gtccgcatcg accggatcgg aaaacctctc gagaaaggcg 180

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 600

<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

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 240

ggcgccgcac gaggggcagt gcagactttt gagggcgtag agcttgggcg cgagaaatac 300

cgattccggg gagtaggcat ccgcgccgca ggccccgcag acggtctcgc attccacgag 360

ccaggtgagc tctggccgtt cggggtcaaa aaccaggttt cccccatgct ttttgatgcg 420

tttcttacct ctggtttcca tgagccggtg tccacgctcg gtgacgaaaa ggctgtccgt 480

gtccccgtat acagacttga gaggcctgtc ctcgagcggt gttccgcggt cctcctcgta 540

tagaaactcg gaccactctg agacgaaggc tcgcgtccag gccagcacga aggaggctaa 600

<210> 33

<211> 600

<212> DNA

<213> regulatory nucleic acid molecule 43(Artificial Sequence)

<400> 33

actgtcttcc ggatcgctgt ccaggagcgc cagctgttgg gctcgcggtt gagaaggtat 60

tcttcgcgat ccttccagta ctcttcgagg ggaaacccgt ctttttctgc acggtactcc 120

gcgcaaggac ctgatcgtct caagatccac gggatctgaa aacctttcga cgaaagcgtc 180

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

acggtctcgc attccacgag ccaggtgagc tctggccgtt cggggtcaaa aaccaggttt 600

<210> 34

<211> 600

<212> DNA

<213> regulatory nucleic acid molecule 44(Artificial Sequence)

<400> 34

actgtcttcc ggatcgctgt ccaggagcgc cagctgttgg gctcgcggtt gagaaggtat 60

tcttcgcgat ccttccagta ctcttcgagg ggaaacccgt ctttttctgc acggtactcc 120

gcgcaaggac ctgatcgtct caagatccac gggatctgaa aacctttcga cgaaagcgtc 180

taaccagtcg caatcgcaag 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 600

<210> 35

<211> 6844

<212> DNA

<213> AO-1(Artificial Sequence)

<400> 35

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

aatgtcgtaa caactccgcc ccattgacgc aaatgggcgg taggcgtgta cggtgggagg 720

tctatataag cagagctctc tggctaacta ccggtcttcc 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 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 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 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

gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg accacttctg 5820

cgctcggccc ttccggctgg ctggtttatt 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

aatgtcgtaa caactccgcc ccattgacgc aaatgggcgg taggcgtgta cggtgggagg 720

tctatataag cagagctctc tggctaacta ccggtcttcc 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 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 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 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

gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg accacttctg 5820

cgctcggccc ttccggctgg ctggtttatt 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> 37

<211> 6671

<212> DNA

<213> AO-3(Artificial Sequence)

<400> 37

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

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 tttgtctttt 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 atggcaacag ggaaacgttt tttcctgctc tgtgatgcat gaagcacttc 2880

ataatcacta cacgcagaag tcactttcac tgtcaccggg aaagtaaact gaagtcatga 2940

tggcatgctt ctatattatt ttctaaaaga tttaaagttt tgccttctcc atttagactt 3000

ataattcact ggaatttttt tgtgtgtatg gtatgacata tgggttccct tttatttttt 3060

acatataaat atatttccct gtttttctaa aaaagaaaaa gatcatcatt ttcccattgt 3120

aaaatgccat atttttttca taggtcactt acatatatca atgggtctgt ttctgagctc 3180

tactctattt tatcagcctc actgtctatc cccacacatc tcatgctttg ctctaaatct 3240

tgatatttag tggaacattc tttcccattt tgttctacaa gaatattttt gttattgtct 3300

tttgggcttc tatatacatt ttagaatgag gttggcaagt taacaaacag cttttttggg 3360

gtgaacatat tgactacaaa tttatgtgga aagaaagtat accttcacaa tattaagtct 3420

tttagttcat gaatatagta tgtctctccg tttctgcatt aacttagaca ttcattaatt 3480

tctctcacaa tttataagtt tatttagatc ttcattcatt taaatcttca ctaacctctc 3540

atttacaatt tgtaagtttt ctgggtaaca gtcttgcact tctttgccta gatttatttc 3600

caagtagatt attttcatac atcgtctatg gtgtcatttt taaaatgtaa tttttcacct 3660

ttttattgct aaagagagat gactgattgt taatattgat 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 ttttctcctt 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

aaggaagagt 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 cacaacatgg gggatcatgt 5460

aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga 5520

caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact 5580

tactctagct tcccggcaac aattaataga ctggatggag gcggataaag 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 tttttttctg 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 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

aatgtcgtaa caactccgcc ccattgacgc aaatgggcgg taggcgtgta cggtgggagg 720

tctatataag cagagctctc tggctaacta ccggtactct cttccgcatc gctgtctgcg 780

agggccagct gttgggctcg cggttgagga caaactcttc gcggtctttc cagtactctt 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 tccacgggcg cagggtcctc 1200

gtcagcgtag tctgggtcac ggtgaagggg tgcgctccgg gctgcgcgct ggccagggtg 1260

cgcttgaggc tggtcctgct ggtgctgaag cgctgccggt cttcgccctg cgcgtcggcc 1320

aggtagcatt tgaccatggt gtcatagtcc agcccctgat ccggtactcg aggaactgaa 1380

aaaccagaaa gttaactggt aagtttagtc tttttgtctt ttatttcagg tcccggatcc 1440

ggtggtggtg caaatcaaag aactgctcct cagtggatgt tgcctttact tctaggcctg 1500

tacggaagtg ttacttctgc tctaaaagct gcggaattgt acccgcgcca ccatggtctc 1560

ttattgggac acgggagttc tcctgtgtgc actgctgagc tgccttctcc tcaccggaag 1620

ttcaagtggt tccgatacag ggcggccgtt cgttgagatg tactccgaaa ttccggaaat 1680

tattcatatg acagaaggtc gcgaactcgt tattccgtgt cgcgtaacgt ctccaaacat 1740

cacggtaaca ctcaaaaaat tcccacttga cacgttgatc ccggacggca aacggattat 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 ggaaaggagt acaagtgcaa ggtatccaat aaggccctcc cggctcccat 2580

cgaaaagacc atatcaaagg cgaaaggcca gccgagggag ccgcaggttt atactctccc 2640

cccgtccagg gacgaattga caaagaatca ggtgagcctc acatgccttg tgaaggggtt 2700

ctaccccagt gatattgcag tggagtggga gtctaacggt caacccgaaa ataattacaa 2760

gacgacacct ccggtcttgg atagcgatgg gtctttcttc ctctattcaa agctcacggt 2820

agataagtcc agatggcaac agggaaacgt tttttcctgc tctgtgatgc atgaagcact 2880

tcataatcac tacacgcaga agtcactttc actgtcaccg ggaaagtaaa ctgaagtcat 2940

gatggcatgc ttctatatta ttttctaaaa gatttaaagt tttgccttct ccatttagac 3000

ttataattca ctggaatttt tttgtgtgta tggtatgaca tatgggttcc cttttatttt 3060

ttacatataa atatatttcc ctgtttttct aaaaaagaaa aagatcatca ttttcccatt 3120

gtaaaatgcc atattttttt cataggtcac ttacatatat caatgggtct gtttctgagc 3180

tctactctat tttatcagcc tcactgtcta tccccacaca tctcatgctt tgctctaaat 3240

cttgatattt agtggaacat tctttcccat tttgttctac aagaatattt ttgttattgt 3300

cttttgggct tctatataca ttttagaatg 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 ttattttcat acatcgtcta tggtgtcatt tttaaaatgt aatttttcac 3660

ctttttattg ctaaagagag atgactgatt gttaatattg atcttgtgcg tggcgacctc 3720

tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct 3780

ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct 3840

gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg 3900

ggaagagaat agcaggcatg ctggggagcg gccgcaggaa cccctagtga tggagttggc 3960

cactccctct ctgcgcgctc gctcgctcac tgaggccggg cgaccaaagg tcgcccgacg 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 atcgggggct 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 gctgatttaa 4560

caaaaattta acgcgaattt taacaaaata 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

cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga 4980

aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca 5040

ttttgccttc ctgtttttgc 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, said regulatory nucleic acid molecule comprising in 5 'to 3' order a tripartite leader sequence (TPL) of an adenovirus and an enhancer element (eMLP) of the major late promoter of an adenovirus, wherein:

2. The regulatory nucleic acid molecule of claim 1, wherein said regulatory nucleic acid molecule has a sequence selected from the group consisting of the sequences set forth in SEQ ID Nos. 19-34 or a sequence having at least 85% identity thereto;

preferably, the regulatory nucleic acid molecule has the sequence shown in SEQ ID NO. 22, 25, 26 or 33 or a sequence with at least 85% identity thereto.

3. The regulatory nucleic acid molecule of claim 1 or 2, wherein the regulatory nucleic acid molecule has the sequence of SEQ ID NO. 26 or a sequence having at least 85% identity thereto.

4. An expression vector comprising, in 5 'to 3' order:

(a) a promoter region;

(b) a 5' UTR region;

(c) a coding sequence encoding a polypeptide gene product;

(d) a polyadenylation region (polyA);

wherein the 5' UTR region comprises a regulatory nucleic acid molecule of any one of claims 1-3;

the coding sequence is operably linked to the promoter region.

5. The expression vector of claim 4, wherein the (a) promoter region is selected from the group consisting of Cytomegalovirus (CMV) promoter, actin promoter, elongation factor 1 α (EF1 α) promoter, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) promoter;

the promoter region of (a) comprises the Cytomegalovirus (CMV) promoter sequence shown in SEQ ID NO:9 or a sequence having at least 85% identity therewith.

6. The expression vector of claim 4 or 5, wherein the polypeptide gene product is a therapeutic protein;

preferably, the therapeutic protein is selected from an anti-angiogenic polypeptide or alpha-1 antitrypsin;

preferably, the anti-angiogenic polypeptide comprises soluble fms-like tyrosine kinase-1 (sFLT-1) or a VEGF-binding fragment of sFLT-1;

more preferably, the anti-angiogenic polypeptide is aflibercept; preferably, the amino acid of aflibercept is shown in SEQ ID NO 10; more preferably, the nucleic acid encoding aflibercept is shown in SEQ ID NO 11.

7. The expression vector of any one of claims 4-6, wherein the polyadenylation region is selected from the human growth hormone (HGH or hGH), bovine growth hormone (BGH or bGH), or β -globin (β -globin) polyA sequence;

more preferably, 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 thereto;

preferably, the expression vector comprises the sequence shown as SEQ ID NO 35, 36, 37 or 38 or a sequence having at least 85% identity thereto.

8. The expression vector of any one of claims 4-7, wherein said expression vector further comprises (i) a first enhancer region, said (i) first enhancer region being located upstream of said (a) promoter region;

preferably, the (i) first enhancer region comprises a sequence selected from the CMV enhancer or the EF 1a enhancer;

more preferably, the (i) first enhancer region comprises the CMV enhancer sequence shown in SEQ ID NO 13 or a sequence having at least 85% identity thereto.

9. The expression vector of any one of claims 4-8, wherein said expression vector further comprises (ii) an intron region downstream of said (b)5' UTR region and upstream of said (c) coding sequence encoding a polypeptide gene product;

preferably, said (ii) intron region comprises a sequence selected from the SV40 intron, the elongation factor 1 α (EF1 α) intron, the actin intron, or the CMVc intron;

more preferably, the (ii) intron region comprises the SV40 intron sequence shown in SEQ ID NO:14 or a sequence having at least 85% identity thereto.

10. The expression vector according to any one of claims 4-wherein said expression vector further comprises (iii) a second enhancer region located downstream of said (c) coding sequence encoding a polypeptide gene product and upstream of said (d) polyadenylation region;

preferably, the (iii) second enhancer region comprises an Expression Enhancer Sequence (EES);

preferably, the (iii) second enhancer region comprises a Scaffold Attachment Region (SAR) for interferon;

preferably, the scaffold attachment region Sequence (SAR) is a human scaffold attachment region of human interferon-beta (IFNB SAR);

more preferably, the human interferon-beta human scaffold attachment region (IFNB SAR) comprises the SAR sequence shown in SEQ ID NO. 15 or a sequence having at least 85% identity thereto.

11. The expression vector of any one of claims 4-10, wherein said expression vector further comprises (iv) an Inverted Terminal Repeat (ITR) at the 5 'end upstream of said (i) first enhancer and (v) an Inverted Terminal Repeat (ITR) at the 3' end downstream of said (d) polyadenylation region;

preferably, said (iv) Inverted Terminal Repeat (ITR) at the 5 'end or said (v) Inverted Terminal Repeat (ITR) at the 3' end comprises an Inverted Terminal Repeat (ITR) selected from Adenovirus (AV) or adeno-associated virus (AAV);

more preferably, the (iv) Inverted Terminal Repeat (ITR) at the 5' end comprises an AAV ITR as set forth in SEQ ID NO 16 or a sequence having at least 85% identity thereto;

more preferably, the Inverted Terminal Repeat (ITR) at the 3' terminus of (v) comprises the AAV ITR shown in SEQ ID NO 17 or a sequence having at least 85% identity thereto.

12. The expression vector of any one of claims 4-11, wherein the expression vector further comprises (vi) a selectable marker gene;

preferably, said (vi) selectable marker gene is located downstream of the Inverted Terminal Repeat (ITR) at the 3' end of (v);

preferably, said (vi) selectable marker gene is selected from the group consisting of ampicillin resistance gene, hygromycin resistance gene, neomycin resistance gene, bialaphos resistance gene, and dihydrofolate reductase gene;

preferably, the ampicillin resistance gene comprises the sequence shown as SEQ ID NO 18 or a sequence having at least 85% identity thereto.

13. The expression vector of any one of claims 4-12, wherein the expression vector comprises, 5 'to 3':

(c) a coding sequence encoding a polypeptide gene product;

14. The expression vector of any one of claims 4-13, wherein the expression vector comprises, 5 'to 3':

15. The expression vector of any one of claims 4-14, wherein the expression vector comprises, 5 'to 3':

(c) a coding sequence encoding a polypeptide gene product;

16. The expression vector according to any one of claims 4-15, comprising 5 'to 3':

(c) a coding sequence encoding a polypeptide gene product;

17. The expression vector according to any one of claims 4-16, comprising 5 'to 3':

(v) (iv) a 3' terminal Inverted Terminal Repeat (ITR) comprising the AAV ITR of SEQ ID No. 17 or a sequence having at least 85% identity thereto;

preferably, the expression vector comprises the sequence shown as SEQ ID NO. 36 or a sequence having at least 85% identity thereto.

18. A recombinant virus, comprising:

a) a capsid protein; and

b) an expression vector according to any one of claims 4 to 17.

19. The recombinant virus of claim 18, wherein the recombinant virus is a recombinant adeno-associated virus; preferably, the adeno-associated virus is selected from the group consisting of 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 ovine AAV;

more preferably, the adeno-associated virus is selected from AAV type 5 (AAV-5) or AAV type 6 (AAV-6).

20. The recombinant virus of claim 19, wherein the capsid protein is an AAV variant 7m8 capsid protein or is derived from said AAV variant 7m8 capsid protein.

21. A pharmaceutical composition comprising an expression vector according to any one of claims 4-17 and/or a recombinant virus according to any one of claims 18-20 and a pharmaceutically acceptable excipient.

22. An isolated host cell transfected or transduced with the expression vector according to any one of claims 4-17.

23. An isolated host cell infected with the recombinant virus of any one of claims 18-20.

24. A method for expressing a transgene in a mammalian cell, the method comprising contacting one or more mammalian cells with an amount of the expression vector of any one of claims 4-17 and/or the recombinant virus of any one of claims 18 to 20, wherein the secreted polypeptide is expressed in the one or more mammalian cells at a level.

25. A method for treating or preventing a disease in a mammal in need of such treatment or prevention, the method comprising administering to the mammal an effective amount of the expression vector of any one of claims 4-17, the recombinant virus of any one of claims 18-20, the pharmaceutical composition of claim 21, and/or the host cell of claim 22 or 23;

preferably, the disease is an ocular disease and the pharmaceutical composition is administered to the eye of the mammal;

preferably, the pharmaceutical composition is administered to the eye of the mammal by intraocular injection or intravitreal injection;

preferably, the ocular disease is selected from the group consisting of age-related macular degeneration (AMD), wet AMD, dry AMD, retinal neovascularization, choroidal neovascularization, and diabetic retinopathy.

26. Use of an expression vector according to any one of claims 4 to 17, a recombinant virus according to any one of claims 18 to 20, a pharmaceutical composition according to claim 21 and/or a host cell according to claim 22 or 23 for the preparation of a medicament for the treatment or prevention of a disease in a mammal in need of such treatment or prevention;