Method for producing a recombinant viral vector

Abstract

The present invention relates to methods for producing high titer recombinant viral vectors, more specifically recombinant AAV vectors, so that the methods can be effectively used on a scale suitable for practical application of gene therapy technology.

Description

Technical Field

[0001] The present invention relates to methods for producing high titer recombinant viral vectors, more specifically recombinant AAV vectors, so that the methods can be effectively used on a scale suitable for practical application of gene therapy technology. Background Art

[0002] Gene delivery is a promising approach for the treatment of acquired and inherited diseases. Many viral-based systems have been described for gene transfer purposes, including adeno-associated virus (AAV)-based systems.

[0003] AAV has unique features that make it attractive as a vector for gene therapy. AAV infects a wide variety of cell types. However, they are non-transforming and have not been implicated as the cause of any human disease. Introduction of DNA into recipient host cells generally results in long-term persistence and expression of the DNA without interfering with the normal metabolism of the cell.

[0004] AAV particles consist of a protein capsid with three capsid proteins, VP1, VP2, and VP3, which contain a linear single-stranded DNA genome of approximately 4.7 kb, which includes the Rep and Cap genes, flanked by viral inverted terminal repeats (ITRs). A single particle encapsidates only one strand of the DNA molecule, but this may be the plus or minus strand. Particles containing either strand are infectious, and replication occurs by converting the parental infectious single strand into a duplex form and subsequently amplifying it, with the progeny single strands being displaced and packaged into capsids.

[0005] AAV vectors can be designed to carry heterologous nucleotide sequences of interest (e.g., selected genes, antisense nucleic acid molecules, ribozymes, etc.) by deleting the internal portion of the AAV genome and inserting the DNA sequence of interest between the ITRs. ITRs are the only sequences required for cis-replication and packaging of the vector genome containing the heterologous nucleotide sequence of interest. The heterologous nucleotide sequence is usually also linked to a promoter sequence that can drive gene expression in the patient's target cells under certain conditions. Termination signals, such as polyadenylation sites, are usually included in the vector.

[0006] The rep and cap AAV gene products provide functions for replication and encapsidation of the vector genome, respectively, and their presence in trans is sufficient.

[0007] Despite the potential benefits of gene therapy as a treatment for human genetic diseases, serious practical limitations have hindered its widespread use in the clinic. In this sense, it is necessary to produce large quantities of rAAV particles to produce clinically effective doses. Producing large quantities of particles using current technologies requires large numbers of producer cells. At laboratory scale, it requires thousands of tissue culture flasks. At commercial scale, more efficient and intensive production platforms are required to reach clinical application. From a commercial production perspective, the benefits of increasing the number of producer cells and the particle yield per cell would be very significant.

[0008] Therefore, in the development of recombinant AAV vectors, such as those used in gene therapy, strategies to obtain high titers of AAV are needed so that these approaches can be used effectively on a scale suitable for practical application of gene therapy techniques.

[0009] The present disclosure provides methods to achieve these competing goals and demonstrates that such techniques can be used to produce recombinant AAV vector preparations on a large scale. SUMMARY OF THE INVENTION

[0011] The inventors of the present invention have found that by performing repeated transfections, recombinant virus production, more particularly rAAV production, can be significantly improved. As shown in the following examples, in contrast to conventional methods requiring a single round of transfection, when repeated transfection rounds are performed, sustained levels of gene expression are obtained over time.

[0012] Therefore, in a first aspect, the present invention relates to a method for producing a recombinant viral vector, the method comprising the following steps:

[0013] a) co-transfecting a suitable cell culture with at least two plasmid vectors comprising a heterologous nucleotide sequence and replication and packaging gene sequences;

[0014] b) culturing the cells under conditions that allow viral replication and packaging;

[0015] c) recovering the viral vector produced in step b) and maintaining the cells in said cell culture under conditions that allow further division and growth;

[0016] d) re-transfecting the cells of step c) with the plasmid vector of step a); and

[0017] e) Repeat steps b) to c).

[0018] In addition, the inventors have found that the combination of optimized plasmids used for transfection results in higher rAAV yields than production using standard plasmids commonly used for AAV vector production. In addition, as shown in the examples below, the inventors surprisingly found that the use of optimized plasmids according to the present invention results in reduced reverse packaging of bacterial sequences.

[0019] Therefore, another aspect of the present invention is a method for producing a recombinant AAV, the method comprising the steps of:

[0020] a) Co-transfect appropriate cells with

[0021] i) a first plasmid vector comprising a heterologous nucleotide sequence flanked by ITRs;

[0022] ii) a second plasmid vector comprising a nucleotide sequence from 5' to 3' AAV rep coding region, an AAV cap coding region and comprising an AAV p5 promoter region, and

[0023] iii) a third plasmid vector comprising adenovirus helper functions, including VA-RNA, E2A and E4 sequences, wherein the plasmid does not comprise E3, pTB (E2B) and Ad ITR protease sequences;

[0024] b) culturing the cells under conditions that allow AAV replication and packaging; and

[0025] c) recovering the AAV produced in step b).

[0026] The present inventors surprisingly found that by using a combination of optimized plasmids in transfection, reverse packaging was greatly reduced and higher vector genome yields were obtained.

[0027] Therefore, in another aspect, the present invention relates to a plasmid vector comprising:

[0028] a) a heterologous nucleotide sequence flanked by ITRs; and

[0029] b) a stuffer DNA sequence located outside the ITR and adjacent to one ITR, wherein the length of the stuffer sequence is between 4400 Kb and 4800 Kb, so that the plasmid backbone size is above 5 Kb, preferably between 7000 bp and 7500 bp;

[0030] The plasmid vector does not contain F1Ori nucleotide sequence in the main chain sequence.

[0031] In another aspect, the present invention relates to a plasmid vector comprising adenovirus helper functions, including VA-RNA, E2A and E4 sequences, wherein the plasmid does not comprise E3, pTB (E2B) and Ad ITR protease sequences. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 is the yield of AAV9-CAG-cohSgsh expressed in total VGs generated by triple transfection with a set of standard plasmids or a set of optimized plasmids.

[0033] Figure 2 is a copy of the bacterial sequence present in AAV9-CAG-cohSgsh generated by triple transfection of a set of standard plasmids or a set of optimized plasmids.

[0034] Figure 3 is the effect of the optimized plasmid pcohSgsh-900 alone or in combination with other optimized helper plasmids (pRepCap9-809 and pAdhelper861) on AAV vector yield (total vg).

[0035] Figure 4 It is the reverse packaging of bacterial sequences. The effect of the optimized mega-plasmid pcohSgsh-900 alone or in combination with other optimized helper plasmids (pRepCap9-809 and pAdhelper861).

[0036] Figure 5 It is the reverse packaging of bacterial sequences. The effect of the optimized mega-plasmid pcohSgsh-900 when combined with other optimized helper plasmids (pRepCap9-809 and pAdhelper861).

[0037] Figure 6 is the total vg recovered after transient transfection with pohIDS-874 plasmid. Cell lysis and total vg recovered in culture supernatant after performing several rounds of re-transfection. Time is expressed in hours post-transfection (hpt).

[0038] Figure 7 is the total VG recovered after transient transfection with the pohSGSH-900 plasmid. Cell lysis and total VG recovered in the culture supernatant after performing several rounds of re-transfection. Time is expressed in hours post-transfection (hpt).

[0039] Microbial deposition

[0040] Plasmid pAdHelper861 was deposited on December 5, 2018 at DSMZ–Deutsche Sammlung von Mikroorganismen und Zellkulturen, Inhoffenstraβe 7B, D-38124 Braunschweig, Federal Republic of Germany, with the deposit number DSM 32965.

[0041] Plasmid pcohSgsh-827 was deposited on December 5, 2018 at DSMZ–Deutsche Sammlung von Mikroorganismen und Zellkulturen, Inhoffenstraβe 7B, D-38124 Braunschweig, Federal Republic of Germany, with the deposit number DSM 32966.

[0042] Plasmid pcohSgsh-900 was deposited on December 5, 2018 at DSMZ–Deutsche Sammlung von Mikroorganismen und Zellkulturen, Inhoffenstraβe 7B, D-38124 Braunschweig, Federal Republic of Germany, with the accession number DSM 32967.

[0043] Plasmid pohIDS-874 was deposited on December 5, 2018 at DSMZ–Deutsche Sammlung von Mikroorganismen und Zellkulturen, Inhoffenstraβe 7B, D-38124 Braunschweig, Federal Republic of Germany, with the deposit number DSM 32968.

[0044] Plasmid pRepCap9-809 was deposited on December 5, 2018 at DSMZ–Deutsche Sammlung von Mikroorganismen und Zellkulturen, Inhoffenstraβe 7B, D-38124 Braunschweig, Federal Republic of Germany, with the deposit number DSM 32969.

[0045] definition

[0046] As used herein, "vector" refers to a macromolecule or combination of macromolecules that contains or is associated with a polynucleotide and can be used to mediate the delivery of the polynucleotide to a cell. Exemplary vectors include, for example, plasmids, viral vectors, liposomes, and other gene delivery vectors.

[0047] The terms "adeno-associated virus", "AAV virus", "AAV virion", "AAV virion" and "AAV particle" are used as synonyms herein and refer to a virus particle composed of at least one AAV capsid protein (preferably composed of all capsid proteins of a specific AAV serotype) and an encapsulated polynucleotide corresponding to the AAV genome. Wild-type AAV refers to a virus belonging to the genus Dependovirus in the family Parvoviridae. The wild-type AAV genome is approximately 4.7 Kb long, consists of single-stranded deoxyribonucleic acid (ssDNA), and can be positive or negative. The wild-type genome includes inverted terminal repeats (ITRs) at both ends of the DNA strand and three open reading frames (ORFs). ORF rep encodes the four Rep proteins required for the AAV life cycle. ORF cap contains nucleotide sequences encoding capsid proteins: VP1, VP2 and VP3, which interact to form an icosahedral symmetrical capsid. Finally, the AAP ORF, which overlaps with the Cap ORF, encodes the AAP protein that appears to promote capsid assembly. If the particle comprises a heterologous polynucleotide (i.e., a polynucleotide different from the wild-type AAV genome, such as a transgene to be delivered to a mammalian cell) flanked by AAV ITRs, it is often referred to as an "AAV vector particle" or "AAV viral vector" or "AAV vector" or "recombinant AAV vector." The invention also encompasses the use of double-stranded AAV or self-complementary AAV, also referred to as dsAAV or scAAV.

[0048] As used herein, the term "adeno-associated virus ITR" or "AAV ITR" refers to the reverse terminal repeat sequence present at both ends of the AAV genomic DNA chain. The ITR sequence is required for the efficient propagation of the AAV genome. Another characteristic of these sequences is their ability to form hairpins. This characteristic contributes to their self-priming, thereby allowing the synthesis of the second DNA chain independent of primer enzymes. ITR has also been shown to be necessary for integrating wild-type AAV DNA into the host cell genome (for example, in human chromosome 19 for serotype 2AAV) and rescuing it and effectively packaging it into fully assembled, DNA-resistant AAV particles. The ITR sequence is about 145bp long. Preferably, the complete sequence of ITR is used in the genome of the AAV viral vector, although some degree of minor modification of these sequences is allowed. The wild-type ITR sequence can be changed by insertion, deletion or truncation, as long as the ITR mediates the desired function, such as replication, nicking, viral packaging, integration and / or proviral rescue. The procedures for modifying these ITR sequences are well known in the art. The ITR can be from any wild-type AAV, including but not limited to serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 or any other known or later discovered AAV. AAV includes two ITRs that may be the same or different. In addition, the two AAV ITRs can be from the same AAV serotype as the AAV capsid, or they can be different. In a preferred embodiment, the 5' and 3' AAV ITRs are derived from AAV1, AAV2, AAV4, AAV5, AAV7, AAV8 and / or AAV9. Preferably, the ITRs are from AAV2, AAV8 and / or AAV9, most preferably AAV2. In one embodiment, AAV2 ITRs are selected to produce a pseudotyped AAV (i.e., an AAV having a capsid and ITRs derived from a different serotype).

[0049] As used herein, the expression "recombinant viral genome" refers to an AAV genome in which at least one foreign polynucleotide is inserted into a naturally occurring AAV genome. The genome of the AAV according to the present invention typically comprises cis-acting 5' and 3' inverted terminal repeats (ITRs) and an expression cassette. As used herein, a "rAAV vector" refers to an AAV vector comprising a polynucleotide sequence from a non-AAV source (i.e., a polynucleotide heterologous to AAV), typically a sequence of interest for genetic transformation of cells. In a preferred vector construct of the present invention, the heterologous polynucleotide is flanked by two AAV inverted terminal repeats (ITRs).

[0050] "AAV virus" or "AAV virus particle" refers to a virus particle composed of at least one AAV capsid protein (preferably all capsid proteins of wild-type AAV) and an encapsidated polynucleotide. If the particle contains 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 usually referred to as a "rAAV vector particle" or simply "rAAV vector".

[0051] "Packaging" refers to the series of intracellular events that lead to the assembly of capsid proteins and encapsidation of the vector genome to form AAV particles.

[0052] The AAV "rep" and "cap" genes refer to polynucleotide sequences encoding adeno-associated virus replication and encapsidation proteins. They have been found in all AAV serotypes examined and are described below and in the art. AAV rep and cap are referred to herein as AAV "packaging genes".

[0053] The term "CAG promoter" refers to a combination of the cytomegalovirus early enhancer element, the chicken β-actin promoter and the 3' splice sequence from the rabbit β-globin gene (see Alexopoulou A et al., BMC Cell Biology 2008; 9(2): 1-11, Niwa et al., Gene. 1991 Dec 15; 108(2): 193-9).

[0054] The term "polynucleotide" refers to a polymeric form of nucleotides of any length, including deoxyribonucleotides or ribonucleotides, or analogs thereof. A polynucleotide may contain modified nucleotides, such as methylated nucleotides and nucleotide analogs, and may be interrupted by non-nucleotide components. If present, modifications to the nucleotide structure may be imparted before or after polymer assembly. As used herein, the term polynucleotide refers interchangeably to double-stranded and single-stranded molecules. Unless otherwise specified or required, any embodiment of the invention described herein is a polynucleotide, including each of the double-stranded form and the two complementary single-stranded forms known or predicted to constitute the double-stranded form.

[0055] "Gene" refers to a polynucleotide containing at least one open reading frame that can encode a specific protein after transcription and translation, or a polynucleotide containing at least one non-coding RNA.

[0056] The term "nucleotide sequence" refers to a nucleic acid molecule, DNA or RNA, comprising deoxyribonucleotides or ribonucleotides. A nucleic acid may be double-stranded, single-stranded, or contain a portion of a double-stranded or single-stranded sequence.

[0057] The term "coding" refers to the genetic code that determines how a nucleotide sequence is translated into a polypeptide or protein. The order of nucleotides in the sequence determines the order of amino acids in the polypeptide or protein.

[0058] "Recombinant", when applied to polynucleotides, means that the polynucleotide is the product of various combinations of cloning, restriction or ligation steps, and other procedures that result in a construct that is different from the polynucleotide found in nature. A recombinant virus is a viral particle that contains a recombinant polynucleotide. The term includes copies of the original polynucleotide construct and progeny of the original viral construct, respectively.

[0059] "Control elements" or "control sequences" are nucleotide sequences that participate in molecular interactions that contribute to the regulation of the functions of a polynucleotide, including replication, duplication, transcription, splicing, translation, or degradation of the polynucleotide. The regulation may affect the frequency, speed, or specificity of the process, and may be enhancing or inhibitory in nature. Control elements known in the art include, for example, transcriptional regulatory sequences, such as promoters and enhancers. A promoter is a region of DNA that is capable of binding RNA polymerase under certain conditions and initiates transcription of a coding region that is usually located downstream (in the 3' direction) of the promoter.

[0060] "Operationally linked" or "operably linked" refers to a juxtaposition of genetic elements wherein the elements are in a relationship that permits them to operate in their intended manner. For example, a promoter is operably linked to a coding region if the promoter helps initiate transcription of the coding sequence. Intervening residues may exist between the promoter and the coding region as long as this functional relationship is maintained.

[0061] An "expression vector" is a vector that contains a region encoding a polypeptide of interest, and is used to achieve expression of the protein in the intended target cell. The expression vector also contains control elements operably linked to the coding region to promote expression of the protein in the target. The combination of control elements and one or more genes to which they are operably linked for expression is sometimes referred to as an "expression cassette," many of which are known and available in the art, or can be readily constructed from components available in the art.

[0062] "Heterologous" means derived from a genotypically different entity that is genotypically different from the rest of the entity to which it is compared. For example, a polynucleotide introduced by genetic engineering techniques into a plasmid or vector derived from a different species is a heterologous polynucleotide. A promoter removed from its native coding sequence and operably linked to a coding sequence not found naturally linked thereto is a heterologous promoter. In particular, the term "heterologous nucleotide sequence" as used herein includes coding as well as non-coding nucleotide sequences.

[0063] "Genetic alteration" refers to the process by which a genetic element is introduced into a cell other than by mitosis or meiosis. The element may be foreign to the cell, or it may be an additional copy or improved version of an element already present in the cell. For example, genetic alteration can be achieved by transfecting cells with a recombinant plasmid or other polynucleotide by any method known in the art, such as electroporation, calcium phosphate precipitation, or contact with a polynucleotide-liposome complex. Genetic alteration can also be achieved by transduction or infection with, for example, a DNA or RNA virus or viral vector. Preferably, the genetic element is introduced into a chromosome or mini-chromosome in the cell; however, any alteration that changes the phenotype and / or genotype of the cell and its progeny is included in the term.

[0064] The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to polymers of amino acids of any length. The term also includes amino acid polymers that have been modified; for example, disulfide bond formation, glycosylation, lipidation, or conjugation with a labeling component.

[0065] "Isolated" plasmid, virus or other material refers to a preparation of material without at least some other components, which components may also be present in naturally occurring or initially prepared materials or similar materials. Thus, for example, isolated material can be prepared by enriching the isolated material from a source mixture using purification techniques. Enrichment can be measured on an absolute basis, such as weight per volume of solution, or a material related to a second potential interfering substance present in the source mixture can be measured. Increased enrichment of embodiments of the present invention is increasingly preferred. Thus, for example, 2-fold enrichment is preferred, 10-fold enrichment is more preferred, 100-fold enrichment is more preferred, and even 1000-fold enrichment is more preferred.

[0066] An "individual" or "subject" treated according to the present invention refers to a vertebrate, particularly a member of a mammalian species, and includes, but is not limited to, domestic animals, sport animals, and primates, including humans.

[0067] "Treatment" of an individual or cell is any type of intervention that attempts to alter the natural course of the individual or cell at the time treatment is initiated. For example, an individual may be treated to reduce or limit pathology caused by any pathological condition, including (but not limited to) inherited or induced genetic defects, viral, bacterial or parasitic infections, tumors or aplastic disorders, or immune system dysfunction, such as autoimmunity or immunosuppression. Treatment includes (but is not limited to) administration of a composition, such as a pharmaceutical composition, and administration of compatible cells that have been treated with a composition. Treatment may be performed prophylactically or therapeutically; that is, before or after the onset of a pathological event or before or after contact with a pathogen.

[0068] The term "effective amount" refers to the amount of a substance that is sufficient to achieve the intended purpose. For example, the effective amount of an expression vector that increases sulfamidase activity is an amount that is sufficient to reduce the accumulation of glycosaminoglycans. A "therapeutically effective amount" of an expression vector used to treat a disease or condition is an amount of the expression vector that is sufficient to alleviate or eliminate the symptoms of the disease or condition. The effective amount of a given substance will vary depending on factors such as the nature of the substance, the route of administration, the size and type of the animal receiving the substance, and the purpose for which the substance is administered. The technician can determine the effective amount in each individual case empirically according to established methods in the art.

[0069] The term "gene therapy" refers to the transfer of genetic material of interest (e.g., DNA or RNA) into a host to treat or prevent a genetic or acquired disease or condition. The genetic material of interest encodes a product (e.g., a protein polypeptide, a peptide, or a functional RNA) that needs to be produced in vivo. For example, the genetic material of interest can encode an enzyme, hormone, receptor, or polypeptide with therapeutic value. DETAILED DESCRIPTION OF THE INVENTION

[0071] It is an object of the present invention to provide a method for producing high titer preparations of recombinant viral vectors, particularly recombinant adeno-associated viral vectors (rAAV), so that the method can be effectively used on a scale suitable for practical application in gene therapy procedures.

[0072] As shown in the accompanying embodiments of the present invention, the inventors have surprisingly found that rAAV production can be significantly improved by repeated rotations of cell cultures using plasmid vectors for triple or double transfection, ie by re-transfection.

[0073] Therefore, in a first aspect, the present invention relates to a method for producing a recombinant viral vector, the method comprising the following steps:

[0074] a) co-transfecting a suitable cell culture with at least two plasmid vectors comprising a heterologous nucleotide sequence and replication and packaging gene sequences;

[0075] b) culturing the cells under conditions that allow viral replication and packaging;

[0076] c) recovering the viral vector produced in step b) and maintaining the cells in said cell culture under conditions that allow further division and growth;

[0077] d) re-transfecting the cells of step c) with the plasmid vector of step a); and

[0078] e) Repeat steps b) to c).

[0079] In a specific embodiment of the present invention, the recombinant viral vector is selected from adenovirus, adeno-associated virus (AAV), alphavirus, flavivirus, herpes simplex virus (HSV), measles virus, rhabdovirus, retrovirus, lentivirus, Newcastle disease virus (NDV), poxvirus and picornavirus. In a specific embodiment, the viral vector is selected from retroviral vector, adenoviral vector or AAV vector.

[0080] In a preferred embodiment, the viral vector is an AAV vector.

[0081] AAV according to the present invention includes any serotype of AAV known serotypes. In general, AAVs of different serotypes have genome sequences with significant homology, provide a series of identical genetic functions, produce virions that are substantially identical in physical and functional aspects, and replicate and assemble by almost the same mechanism. Specifically, the AAV of the present invention may belong to serotype 1 of AAV (AAV1), AAV2, AAV3 (including 3A and 3B types), AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, avian AAV, bovine AAV, canine AAV, horse AAV, sheep AAV, and any other AAV. Examples of genome sequences of different AAV serotypes can be found in the literature or in public databases such as GenBank. Please refer to GenBank accession numbers AF028704.1 (AAV6), NC006260 (AAV7), NC006261 (AAV8), and AX753250.1 (AAV9). In a preferred embodiment, the AAV vector of the present invention is a serotype selected from the group consisting of AAV2, AAV5, AAV7, AAV8, AAV9, AAV10 and AAVrh10 serotypes. In a preferred embodiment, the AAV vector of the present invention is serotype 9.

[0082] In a specific embodiment, the present invention relates to a method for producing a recombinant AAV, the method comprising the steps of:

[0083] a) co-transfecting a suitable cell culture with at least two plasmid vectors comprising a heterologous nucleotide sequence flanked by ITR, AAV rep and AAV cap gene sequences and adenovirus helper function sequences;

[0084] b) culturing the cells under conditions that allow AAV replication and packaging;

[0085] c) recovering the AAV produced in step b) and maintaining the cells in said cell culture under conditions that permit further division and growth;

[0086] d) re-transfecting the cells of step c) with the plasmid vector of step a); and

[0087] e) Repeat steps b) to c).

[0088] As described in the art, any one of a variety of methods can be used to introduce genetic material into cells to transform or transduce such cells. For example, such technology includes, for example, using bacterial plasmid transfection. In fact, a variety of transfection techniques can be used to introduce the plasmid vector according to the step a) of the inventive method into cells. Such transfection methods have been described in the art, including, for example, calcium phosphate coprecipitation, direct microinjection into cultured cells, electroporation, liposome-mediated gene transfer, lipid-mediated transfection or the use of high-speed microparticles for nucleic acid delivery. Other suitable transfection media include strontium phosphate, polycationic polymers such as Superfect (QIAGEN TM)), liposomes and cationic polymers such as polyethyleneimine (PEI). In a preferred embodiment, PEI is used to transfect the plasmid vector according to the inventive method. In this case, the PEI / DNA complex is formed by adding PEI to the plasmid DNA and then adding it to the cell culture.

[0089] Any of these techniques can be used to introduce one or more foreign DNA components, such as a vector construct, into a suitable host cell. Typically, the foreign DNA must cross the host cell plasma membrane to be exposed to the cell's transcription and replication machinery.

[0090] The resulting cells can be transiently transfected with exogenous nucleic acid molecules, i.e., the exogenous DNA will not be integrated into the genome of the transfected cells, but will exist freely. Alternatively, the resulting cells can be stably transfected, i.e., the nucleic acid molecules will be covalently linked to the host cell genome or will be maintained and replicated as episomal units, which can be passed on to progeny cells (e.g., capable of chromosome replication at a sufficient rate).

[0091] According to the method of the present invention, the plasmid vector to be transfected comprises a heterologous nucleotide sequence flanked by ITR, AAV rep and AAV cap gene sequences and an adenovirus helper function sequence.

[0092] A heterologous nucleotide sequence or polynucleotide is often of interest because it can provide a function to a target cell in the context of gene therapy, such as upregulating or downregulating the expression of a certain phenotype. Such a heterologous polynucleotide or "transgene" is often of sufficient length to provide the desired function or coding sequence.

[0093] In the case where transcription of a heterologous polynucleotide is desired in the intended target cell, it can be operably linked to its own or a heterologous promoter, depending on, for example, the level of transcription and / or specificity desired in the target cell, as known in the art. Various types of promoters and enhancers are suitable for this situation. Constitutive promoters provide continuous levels of gene transcription and are preferred when continuous expression of therapeutic polynucleotides is desired. Inducible promoters generally exhibit low activity in the absence of an inducer and are upregulated in the presence of an inducer. They may be preferred when expression is required only at a specific time or a specific location, or when expression levels need to be titrated using an inducer. Promoters and enhancers may also be tissue-specific, that is, they are active only in certain cell types, which may be due to the uniqueness of the gene regulatory elements found in these cells.

[0094] Illustrative examples of promoters are the SV40 late promoter from simian virus 40, the baculovirus polyhedral enhancer / promoter element, herpes simplex virus thymidine kinase (HSV tk), the immediate early promoter from cytomegalovirus (CMV), the CMV early enhancer / chicken beta actin (CAG) promoter, and various retroviral promoters, including LTR elements. Inducible promoters include heavy metal ion inducible promoters (such as the mouse mammary tumor virus (mMTV) promoter or various growth hormone promoters), and T7 phage promoters active in the presence of T7 RNA polymerase. For example, examples of tissue-specific promoters include various surfactant protein promoters (for expression in the lung), myosin promoters (for expression in muscle), and albumin promoters or human α1-antitrypsin hAAT (for expression in the liver). A large number of other promoters are known in the art and are generally available, and the sequences of many such promoters are available in sequence databases such as the GenBank database. In a preferred embodiment, the CAG promoter is used.

[0095] In the case where translation is also required in the expected target cell, the heterologous polynucleotide preferably also comprises a control element (e.g., a ribosome binding site or "RBS" and / or a polyadenylation signal) that promotes translation. Therefore, the heterologous polynucleotide generally comprises at least one coding region operably linked to a suitable promoter, and may also comprise, for example, an enhancer, a ribosome binding site, and / or a poly-A signal that is operably linked. The heterologous polynucleotide may comprise one coding region, or more than one coding region under the control of the same or different promoters. The entire unit comprising a combination of control elements and coding or non-coding regions is generally referred to as an expression cassette. In a specific embodiment, the heterologous polynucleotide according to the method of the present invention contains a poly-A signal.

[0096] The heterologous polynucleotide is integrated into or preferably replaces the AAV genome coding region by recombinant technology, and is usually flanked by AAV inverted terminal repeat (ITR) regions on both sides. Alternatively, a vector construct with only one ITR can be used. In a specific embodiment, the flank of the heterologous nucleotide sequence is two ITRs.

[0097] In view of the encapsidation size limit of AAV particles, the insertion of large heterologous polynucleotides into the genome requires the removal of a portion of the AAV sequence. In any case, it is necessary to remove one or more AAV genes to reduce the possibility of generating replication-competent AAV ("RCA"). Therefore, it is preferred to remove the coding or promoter sequences of rep, cap, or both, because the functions provided by these genes can be provided in trans.

[0098] In one embodiment, the heterologous nucleotide sequence is flanked by AAV ITRs and the AAV packaging genes provided in trans are introduced into the host cell in a separate vector plasmid.

[0099] The rep gene is expressed from two promoters, p5 and p19, and produces four proteins, called Rep78, Rep68, Rep52, and Rep40. Only Rep78 and Rep68 are required for AAV double-stranded DNA replication, but Rep52 and Rep40 appear to be required for progeny, single-stranded DNA accumulation. Rep68 and Rep78 bind specifically to the hairpin conformation of the AAV ITR and have several enzymatic activities required to resolve AAV terminal replication. Rep78 and Rep68 also exhibit pleiotropic regulatory activities, including positive and negative regulation of AAV genes and expression from some heterologous promoters, as well as inhibitory effects on cell growth.

[0100] The cap gene encodes the capsid proteins VP1, VP2, and VP3. These proteins share a common overlapping sequence, but VP1 and VP2 contain additional amino-terminal sequences that are transcribed from the p40 promoter using alternative start codons. All three proteins are required for efficient capsid production.

[0101] The packaging of AAV vectors into viral particles still relies on the presence of a suitable helper virus for AAV or the provision of helper virus function. According to the method of the present invention, the helper virus function gene sequence is provided in a plasmid vector. The presence of a large amount of infectious helper virus in AAV vector preparations is problematic because the preparations are intended for human administration.

[0102] In a specific embodiment of the method of the present invention, two plasmid vectors are used for transfection, wherein one of the plasmid vectors comprises a heterologous nucleotide sequence flanking the ITRs, and the second plasmid vector comprises the AAV rep and AAV cap gene sequences and adenovirus helper function sequences. In a preferred embodiment, the second plasmid vector comprises, from 5' to 3', the AAV rep coding region, the AAV cap coding region, and a nucleotide sequence comprising the AAV p5 promoter region.

[0103] In a preferred embodiment of the method of the present invention, three plasmid vectors are used for transfection, i.e., the host cells are triple transfected with at least one vector encoding a heterologous nucleotide sequence of interest, at least one vector encoding AAV rep and cap genes, and at least one vector encoding adenoviral helper functions.

[0104] The selection of appropriately altered cells can be performed by any technique in the art. For example, the polynucleotide sequence used to alter the cell can be introduced or operably linked simultaneously with one or more detectable or selectable markers known in the art. For example, a drug resistance gene can be used as a selection marker. The drug resistant cells can then be selected and cultured, and then the expression of the desired sequence can be tested-i.e., the product of the packaging gene product or the product of the heterologous polynucleotide, as the case may be. DNA hybridization-based techniques (e.g., Southern blotting and other procedures known in the art) can be used to test the acquisition, location and / or maintenance of the introduced polynucleotides. Expression testing can be easily performed by Northern analysis of RNA extracted from genetically altered cells, or by indirect immunofluorescence of the corresponding gene products.

[0105] According to step b) of the method of the invention, the transfected cells are thus cultured under conditions that allow replication and packaging of the viral vector. In a particular embodiment, the cells are cultured under conditions that allow replication and packaging of rAAV.

[0106] Several criteria influence the selection of cells for producing the rAAV particles described herein. Preferred cells and cell lines are those that can be easily grown in culture to facilitate large-scale production of recombinant AAV vector preparations. If large-scale production is required, the choice of production method will also influence the choice of host cells. For example, some production techniques and culture vessels or chambers are designed for the growth of adherent or attached cells, while others are designed for the growth of suspended cells. In the latter case, the host cells will therefore preferably be adapted or capable of adapting to suspension growth. According to the present invention, large-scale production of rAAV is required.

[0107] A variety of cell lines are considered for large-scale production of rAAV. Particularly suitable for rAAV cell culture is the human embryonic kidney (HEK) 293 cell line, which can be an adherent cell line or a cell line selected for suspension growth. Other examples of cell lines suitable for producing rAAV include: Vero cells, HeLa cells and CHO cell lines. As used herein, the term "cell line" refers to a cell population capable of continuous or prolonged growth and division in vitro. Typically, a cell line is a clonal population derived from a single progenitor cell. It is known in the art that spontaneous or induced changes in the karyotype may occur during storage or transfer of such clonal populations. Therefore, cells derived from the referred cell line may not be exactly the same as the ancestral cell or culture. However, the term "cell line" includes such variants.

[0108] Once a cell line is selected, the selected cell culture device is seeded with cells at a density suitable for supporting cell culture. The cell density used to seed a particular device will depend on the size of the device. A variety of volumes can be used to culture cells in the selected culture device. The volume of culture medium used will vary depending on the size of the culture device used to culture the cells. Large-scale production methods such as suspension culture can be used. The AAV particles are then collected and isolated from the cells used to prepare them.

[0109] The cells are cultured under conditions that allow replication of AAV and packaging of rAAV vectors. The culture time is preferably adjusted to correspond to the peak production level. Preferably, at least 100 virus particles are produced per cell; more preferably, at least about 1000 virus particles are produced per cell, and even more preferably, at least about 10,000 virus particles are produced per cell. Preferably, every 2×10 5 cells produce at least 0.5×10 6 , more preferably at least about 1×10 6 , even more preferably at least about 2×10 6 RU / ml of AAV vector.

[0110] Various growth media can be used in the disclosed invention to achieve large-scale cell growth and AAV production. The choice of growth medium depends on the cell type being cultured. In a specific embodiment, the cells are mammalian cells. In a preferred embodiment, the cells are human embryonic kidney (HEK) 293 cell line. More preferably, the cells are cells suitable for suspension growth.

[0111] For the culture of adherent cells, roller bottles (cylindrical tissue culture flasks that rotate at a given speed) are used to provide the surface required for cell attachment. Alternatively, microcarriers are also a suitable system to support the culture of adherent cells. For the culture of suspension cells, stirred tank bioreactors are the most suitable system because they can achieve high cell density cultures, which can provide large amounts of rAAV after purification.

[0112] In a specific embodiment of the invention, a rocking motion bioreactor is used. In another specific embodiment, a stirred tank bioreactor may also be used. Once the cell culture in the reactor reaches a given point, cell transfection is performed as described above.

[0113] According to the method of the present invention, step c) comprises recovering the AAV produced in step b) and maintaining the cells in the cell culture under conditions that allow further division and growth. In a preferred embodiment of the method, step b) is performed by culturing the cells in suspension in a stirred liquid culture medium.

[0114] According to a specific embodiment of the method of the present invention, in step b), the AAV produced is secreted into the supernatant of the cell culture. Therefore, according to step c) of the method of the present invention, rAAV is harvested from the supernatant and the cells are maintained in the cell culture for further division and growth. The spent culture medium or supernatant is then collected together with the rAAV thus produced, because as shown in the following examples, AAV is secreted into the supernatant of the cell culture without cell lysis.

[0115] In a specific embodiment, the cell culture medium of the cell culture is exchanged before re-transfection, i.e., before step d). In a specific embodiment, the cell culture medium exchange is performed by centrifugation. In a preferred embodiment, the culture medium exchange is performed by perfusion. In a more preferred embodiment, continuous culture medium exchange is performed by perfusion. More preferably, the culture medium is automatically exchanged by a perfusion system. Through this system, the culture is supplemented with fresh culture medium and the cell-free supernatant is removed using a cell retention device. The process is preferably performed at a constant harvest flow rate. In this regard, in a preferred embodiment, new, fresh culture medium is added to the retained cells to allow them to further divide and grow. The continuous addition of nutrients and the removal of toxic metabolites enable perfusion cultures to reach and maintain high cell densities within weeks.

[0116] The inventors have surprisingly found that the production of AAV can be extended by re-transfecting the cells retained in the cell culture with the plasmid vector described in step a) above. In this sense, more rAAV is produced using cells retained in the cell culture device, which are allowed to further divide and grow. Therefore, the inventors have found that by performing repeated rounds of transfection, the production of rAAV can be extended over time using the same cell culture. In addition, as can be seen in the examples attached to the present invention, the inventors found that by performing these culture medium exchanges, AAV is secreted into the supernatant of the cell culture without cell lysis, and AAV can be harvested each time the culture medium is exchanged before each round of re-transfection. Another advantage of the proposed method is that rAAV is recovered from the supernatant, which is an advantage from a purification point of view because cells do not need to be lysed to harvest AAV, thereby reducing the occurrence of contamination with host cell DNA and host cell proteins.

[0117] Therefore, the method of the present invention comprises the step d) re-transfecting the remaining cells according to step c) with the plasmid vector according to step a).

[0118] According to step d) of the method of the invention, re-transfection or repeated transfection rounds are performed in cell culture, in contrast to conventional transient gene expression (TGE) methods which require a single round of transfection without medium exchange.

[0119] Furthermore, the inventors have surprisingly found that the re-transfection and recovery steps can be repeated more than twice using the same cell culture. In a particular embodiment of the method of the present invention, steps d), b) and c) are repeated at least once more after the recovery step c). In another particular embodiment, after the recovery step c), steps d), b) and c) are repeated at least twice. In a more specific embodiment, after the recovery step c), steps d), b) and c) are repeated at least 3 times. In a preferred embodiment, after the recovery step c), steps d), b) and c) are repeated at least four times. In a more specific embodiment, after the recovery step c), steps d), b) and c) are repeated 1 to 3 times. In a more specific embodiment, after the recovery step c), steps d), b) and c) are repeated 1 to 2 times. In a preferred embodiment, the method of the present invention comprises repeating steps d), b) and c) after the recovery step c). In a preferred embodiment of the method of the present invention, steps d), b) and c) are repeated once more after the recovery step c).

[0120] As previously mentioned, in a specific embodiment of the method of the invention, continuous medium exchange is performed by perfusion. In a preferred embodiment, perfusion remains unchanged each time transfection is performed. This allows the plasmid vector to enter the cell. In a more preferred embodiment, perfusion continues for at least 1.5 hours, more preferably at least 2 hours, more preferably at least 3 hours, and even more preferably at least 4 hours. In a preferred embodiment, there is a minimum time interval between each round of re-transfection. In a more preferred embodiment, the re-transfection is performed after a time interval of at least 36 hours, more preferably at least 48 hours.

[0121] In another specific embodiment, the method comprises the additional step of lysing the cells in the supernatant after all rounds of re-transfection and recovery are completed.

[0122] There are several well-known techniques in the prior art that can be used for cell disruption or cell lysis. Although freeze-thaw and / or ultrasonic treatment can be used to destroy cells, such techniques are not suitable for large-scale preparation. Therefore, mechanical lysis techniques are preferred in these aspects. Detergents and other chemical reagents can also be used to mediate or promote lysis. It has been found that treating the lysate with a nuclease (such as benzonase) helps to reduce viscosity and improve filterability. Clarification, for example, separating the carrier from at least part of the cell debris by microfiltration, also helps to promote recovery and purification. In a specific embodiment of the present invention, the lysis step according to the method of the present invention comprises the use of a nuclease, and more preferably, the nuclease is benzonase.

[0123] In a specific embodiment of the method of the present invention, the collected supernatant containing the above-mentioned rAAV of the present invention is further processed to purify the rAVV. In this regard, in another specific embodiment, the process is carried out in a bioreactor coupled to a cell retention membrane, so that the cells are retained in the reactor system, and the AAV is collected through the membrane in a clean supernatant, free of cell debris, thereby making purification easier.

[0124] To be particularly useful for the production of AAV for gene therapy, the technology would ideally be "scalable," meaning it could be used in conjunction with large-scale manufacturing equipment and procedures.

[0125] For example, AAV can be loaded onto a positively charged anion exchange column, such as an N-charged amino or imino resin (e.g., POROS or any DEAE, TMAE, tertiary or quaternary amine, or PEI-based resin) or a negatively charged cation exchange column (e.g., HS, SP, CM, or any sulfonyl, phospho, or carboxyl-based cationic resin). The column can be washed with a buffer. The column can be eluted with a gradient of increasing NaCl concentration or a gradient of decreasing pH, and fractions can be collected and analyzed for the presence of AAV and / or contaminants.

[0126] Other procedures may be used instead of, or preferably in addition to, the above-described anion and cation exchange procedures based on intermolecular associations mediated by features other than charge as known in the art. Such other procedures include intermolecular associations based on ligand-receptor pairs (e.g., antibody-antigen or lectin-carbohydrate interactions), and separations based on other properties of molecules, such as molecular sieving chromatography based on size and / or shape.

[0127] The pool of AAV-containing fractions eluted from the column as described above can be concentrated and purified by tangential flow filtration (TFF). The preparation is filtered through a membrane and the product is retained. The retained material can be diafiltered using a membrane and washed successively with appropriate buffers. The final sample is highly concentrated for the product and can be filtered and stored for use.

[0128] Vector plasmid transfection can be performed on the same day as cell seeding, or on the first, second, third, fourth, or fifth day after cell seeding, depending on the cell seeding density.

[0129] In one embodiment of the invention, the selected host cells are triple transfected with at least one vector encoding a heterologous nucleotide sequence of interest, at least one vector encoding AAV rep and cap genes, and at least one vector encoding adenovirus helper functions. In a specific embodiment, the triple transfection is performed using PEI transfection.

[0130] Packaging of plasmid backbone sequences (reverse packaging) during the production of rAAV by transient transfection is a common problem well known in the art. As used herein, the term "reverse packaging" refers to the encapsidation of the backbone of the plasmid containing ITRs rather than heterologous sequences. In fact, for AAV vectors, the main types of packaging DNA impurities are plasmid sequences adjacent to the ITRs, which are located on the flanks of the expression cassette and may be generated by the reverse packaging of the ITRs. The presence of these particles in rAAV raw materials may represent a safety issue for clinical applications.

[0131] As shown in Example 1 below, the combination of optimized plasmids used for transfection results in higher yields and lower reverse packaging of bacterial sequences than production using standard plasmids commonly used for AAV vector production. In particular, the inventors have found that when cells are transfected with three plasmid vectors, wherein the first plasmid vector i) comprises a heterologous nucleotide sequence flanking the ITR and a stuffer DNA sequence located outside the ITR, wherein the length of the stuffer sequence is between 4400 Kb and 4800 Kb, making the plasmid backbone size above 5 Kb; the second plasmid vector ii) comprises the AAV rep coding region from 5' to 3', the AAV cap coding region and a nucleotide sequence comprising the AAV p5 promoter region, and the third plasmid vector iii) comprises adenovirus helper functions including VA-RNA, E2Ay E4 sequences, wherein the plasmid does not contain E3, pTB (E2B) and Ad ITR, protease sequences, rAAV yield is significantly increased. In this regard, according to the results shown below, when a combination of these three optimized plasmids is used instead of a combination of standard plasmids, AAV yield is increased threefold. In addition, reverse packaging is also significantly reduced compared to triple transfection using standard plasmids well known to those skilled in the art, i.e., non-super plasmids containing heterologous nucleotide sequences, pRepCap vectors such as pRep2Cap9 vectors, and plasmids containing adenoviral sequences required for AAV production (VA-RNA, E2A y E4). As previously described, the optimized helper plasmid used in the present invention contains adenoviral helper functions, including VA-RNA, E2A y E4 sequences required for AAV production. The plasmid has been modified so that it does not contain certain sequences, including E3, pTB (E2B), and Ad ITR sequences.

[0132] like Figure 3 It is clearly shown that the use of an optimized plasmid containing a heterologous nucleotide sequence, wherein the plasmid is a super-plasmid containing a stuffer DNA sequence, does not increase vector genome yield when used in combination with a non-optimized plasmid, i.e., in combination with a standard vector for triple transfection. Thus, the use of an optimized super-plasmid alone does not increase vector genome yield by triple transfection.

[0133] However, the combination of this optimized superplasmid with an optimized plasmid containing adenoviral helper functions (pAdhelper861) or an optimized plasmid containing both the AAV rep coding region and the AAV cap coding region (pRepCap9-809) increased vector genome yield (see Figure 3 ). In addition, when three optimized vectors were combined, the vector genome yield was greatly improved (see Figure 3 ).

[0134] Furthermore, the inventors surprisingly found that when the optimized super plasmid (pcohSgsh-900) was co-transfected with one or both of the optimized helper plasmids pRepCap9-809 and / or pAdhelper861, reverse packaging of bacterial sequences was reduced ( Figure 4 In particular, the use of triple combinations of these optimized plasmids can greatly reduce reverse packaging ( Figure 4 and Figure 5 In fact, the reduction in the percentage of bacterial sequences due to the use of optimized helper plasmids (pAdhelper861) and / or (pRepCap9-809) was more pronounced when co-transfecting an optimized plasmid containing an oversized backbone (pcohSgsh-900) ( Figure 5 ).

[0135] As used herein, the term "super-large backbone" refers to a plasmid backbone whose base pair size is above the encapsidation limit of AAV vectors (approximately 4700 bp).

[0136] In a preferred embodiment, the second plasmid vector used in the method of the present invention comprises AAV Rep2 and AAV Cap9 coding regions. In a preferred embodiment, the plasmid vector is pRepCap-809, which has the accession number DSM 32969, as shown in SEQ ID NO:5.

[0137] Therefore, in a particular embodiment of the method of the invention, the following three-plasmid vector is used in step a):

[0138] i) a first plasmid vector comprising a heterologous nucleotide sequence flanked by ITRs and a stuffer DNA sequence located outside the ITRs, wherein the length of the stuffer sequence is between 4400 Kb and 4800 Kb, such that the plasmid backbone size is greater than 5 Kb;

[0139] ii) a second plasmid vector comprising the AAV rep coding region, the AAV cap coding region and a nucleotide sequence comprising the AAVp5 promoter region from 5' to 3'; and

[0140] iii) a third plasmid vector comprising adenovirus helper functions, including VA-RNA, E2Ay E4 sequences, wherein the plasmid does not comprise E3, pTB (E2B) and Ad ITR protease sequences.

[0141] In a specific embodiment, the stuffer DNA sequence is located adjacent to an ITR in the first plasmid vector comprising the heterologous nucleotide sequence.

[0142] In another specific embodiment, the plasmid backbone size is between 7000 bp and 7500 bp. In a more specific embodiment, the plasmid backbone size is between 7000 bp and 7200 bp.

[0143] In a preferred embodiment, the first plasmid vector does not contain the F1Ori nucleotide sequence in the backbone sequence.

[0144] In a more preferred embodiment, the first plasmid vector is pcohSgsh-900 with the deposit number of DSM 32967, as shown in SEQ ID NO:2.

[0145] According to another embodiment of the method of the present invention, the remaining cells in the cell culture can be re-transfected with the two plasmid vectors to extend the production of AAV as described above.

[0146] Therefore, the present invention also relates to the method according to the invention, wherein in step a) the following two plasmid vectors are used:

[0147] i) a first plasmid vector comprising a heterologous nucleotide sequence flanked by ITRs and a stuffer DNA sequence located outside the ITRs, preferably adjacent to one ITR, wherein the stuffer sequence has a length between 4400 Kb and 4800 Kb, such that the plasmid backbone size is greater than 5 Kb, preferably between 7000 bp and 7500 bp, more preferably between 7000 bp and 7200 bp; and

[0148] ii) a second plasmid vector comprising the AAV rep coding region, the AAV cap coding region, a nucleotide sequence comprising the AAV p5 promoter region, and adenovirus helper functions including VA-RNA, E2A and E4 sequences, wherein the plasmid does not comprise E3, pTB (E2B) and Ad ITR protease sequences.

[0149] In another specific embodiment, the second plasmid vector comprises adenovirus helper functions from 5' to 3', including VA-RNA, E2A and E4 sequences, AAV rep coding region, AAV cap coding region, and a nucleotide sequence comprising an AAV p5 promoter region.

[0150] In a specific embodiment, the first plasmid vector does not contain a F1Ori nucleotide sequence in the backbone sequence.

[0151] In another specific embodiment, the second plasmid vector comprises AAV Rep2 and AAV Cap9 coding regions.

[0152] As mentioned previously, the combination of optimized plasmids used for transfection resulted in higher yields and lower reverse packaging of bacterial sequences than using standard plasmids commonly used for AAV vector production.

[0153] In the following examples, it is shown that, compared to standard plasmids used for producing AAV by triple transfection, when rAAV is produced using: i) a plasmid vector comprising a heterologous nucleotide sequence flanked by ITRs; ii) a plasmid vector comprising the AAV rep coding region from 5' to 3', the AAV cap coding region and a nucleotide sequence comprising the AAV p5 promoter region; iii) a plasmid vector comprising adenovirus helper functions, including VA-RNA, E2Ay E4 sequences, wherein the plasmid does not contain E3, pTB (E2B) and Ad ITR protease sequences, reverse packaging is significantly reduced and productivity is improved.

[0154] Thus, in another aspect, the present invention relates to a method for producing a recombinant AAV, the method comprising the steps of:

[0155] a) Co-transfect appropriate cells with

[0156] i) a first plasmid vector comprising a heterologous nucleotide sequence flanked by ITRs;

[0157] ii) a second plasmid vector comprising a nucleotide sequence from 5' to 3' AAV rep coding region, an AAV cap coding region and comprising an AAV p5 promoter region, and

[0158] iii) a third plasmid vector comprising adenovirus helper functions, including VA-RNA, E2Ay E4 sequences, wherein the plasmid does not comprise E3, pTB (E2B) and Ad ITR protease sequences;

[0159] b) culturing the cells under conditions that allow AAV replication and packaging; and

[0160] c) recovering the AAV produced in step b).

[0161] In a preferred embodiment, the first plasmid vector i) is characterized in that the plasmid backbone size is above 5000pb, preferably between 7000bp and 7500bp, more preferably between 7000bp and 7200bp, and it comprises a stuffer DNA sequence located outside the ITR, preferably adjacent to one of the ITRs, and wherein the length of the stuffer sequence is between 4400pb and 4800. In a more preferred embodiment, the first plasmid vector i) does not comprise a F1Ori nucleotide sequence in the backbone sequence. In a more preferred embodiment of the present invention, the first plasmid is pcohSgsh-900 with a deposit number of DSM32967, as shown in SEQ ID NO:2.

[0162] In another preferred embodiment, the second plasmid vector ii) comprises AAV Rep2 and AAV Cap9 coding regions. In a preferred embodiment, the plasmid vector is pRepCap-809, which has the deposit number of DSM 32969, as shown in SEQ ID NO:5.

[0163] In another preferred embodiment, the third plasmid vector iii) is pAdHelper861, deposited as DSM 32965, as shown in SEQ ID NO:6.

[0164] As mentioned above, in another specific embodiment of the present invention, step b) is performed by culturing the cells in suspension in a stirred liquid culture medium.

[0165] In another aspect, the present invention also relates to a plasmid vector comprising:

[0166] a) heterologous nucleotide sequences flanking the ITRs; and

[0167] b) a stuffer DNA sequence located outside the ITR and adjacent to one ITR, wherein the length of the stuffer sequence is between 4400 Kb and 4800 Kb, more preferably between 4500 Kb and 4700 Kb, even more preferably between 4600 Kb and 4700 Kb, so that the plasmid backbone size is above 5 Kb, preferably between 7000 bp and 7500 bp; more preferably, between 7000 bp and 7200 bp;

[0168] The plasmid vector does not contain the F1Ori nucleotide sequence in the main chain sequence. In a more preferred embodiment of the present invention, the plasmid is pcohSgsh-900, with the deposit number of DSM 32967, as shown in SEQ ID NO:2.

[0169] In another specific aspect, the present invention relates to a helper plasmid vector comprising adenovirus sequences E2, E4 and VA-RNA, wherein the plasmid vector does not comprise E3, pTB (E2B) and Ad ITR protease sequences. More specifically, the plasmid contains a 731 bp region including the VA gene, a 5346 bp region including the E2A gene and the E4 gene, and their regulatory regions are included in a 3181 bp fragment. The plasmid backbone comprises a high copy number bacterial origin of replication. In a more preferred embodiment, the present invention relates to a plasmid vector of SEQ ID NO: 6, which corresponds to pAdHelper861 with a deposit number of DSM 32965.

[0170] Application of rAAV in gene therapy

[0171] For the purpose of gene therapy, the rAAV viral vector of the present invention can be used for administration to an individual. Diseases suitable for gene therapy include, but are not limited to, diseases caused by viral, bacterial or parasitic infections, various malignancies and hyperproliferative diseases, autoimmune diseases and congenital defects. In a preferred embodiment, the rAAV vector produced according to the method of the present invention is used to treat lysosomal storage diseases (LSD). More preferably, the vector contributes to the treatment of mucopolysaccharidosis (MPS). In a preferred embodiment, the heterologous nucleotide sequence is a sequence encoding an enzyme for the treatment of MPS. In particular, the sequence is selected from human α-L-iduronidase, human heparan sulfamidase, human N-sulfoglucosamine sulfohydrolase, human N-acetylglucosaminidase, α, human acetylglucosamine, human N-acetyltransferase, human iduronide 2-sulfatase (IDS), human N-acetylglucosamine 6-sulfatase, human galactose-6-sulfate sulfatase, human β-galactosidase, human N-acetylgalactosamine-4-sulfatase, β-glucuronidase and human hyaluronidase.

[0172] Gene therapy can be performed to increase the expression level of a specific protein in a cell or in a cell secretion. The carrier of the present invention can be used for genetically altered cells to carry out gene tagging, replacement of missing or defective genes or insertion of therapeutic genes. Alternatively, polynucleotides that reduce expression levels can be provided to cells. This can be used to suppress unwanted phenotypes, such as products of genes that are amplified or overexpressed during malignant tumors, or genes that are introduced or overexpressed during microbial infection. Expression levels can be reduced by providing therapeutic polynucleotides comprising sequences that can, for example, form stable hybrids (antisense therapy) with target genes or RNA transcripts, can be used as ribozymes to cut related mRNAs or can serve as baits for target gene products.

[0173] The pharmaceutical composition may be provided as a liquid solution or suspension, as an emulsion or as a solid form suitable for solution or suspension in liquid prior to use. The carrier is, for example, water or a buffered saline solution, with or without a preservative.

[0174] The pharmaceutical composition can be lyophilized at the time of administration or in solution to be resuspended. In a preferred embodiment, the pharmaceutical composition of the present invention is a suspension for injection. The final product is then formulated in a suitable buffer, filled into vials and stored until use.

[0175] Although not required, the pharmaceutical compositions may optionally be presented in unit dosage form suitable for administration of precise quantities.

[0176] Having generally described the present invention, the same will be more readily understood by reference to the following examples which are intended as illustrations only and are not intended to limit the present invention in any way. Example

[0177] Unless otherwise indicated, the practice of the present invention will employ conventional techniques of molecular biology, virology, animal cell culture, and biochemistry, which are within the skill of the art and are fully explained in the literature.

[0178] Example 1: Triple transfection using standard plasmid vector and optimized vector

[0179] 1.1. Plasmid description

[0180] Plasmid pcohSgsh-827 (SEQ ID NO: 1)

[0181] A plasmid containing a 1512 bp codon-optimized cDNA encoding human N-sulfoglucosamine sulfohydrolase under the control of a 1756 bp CAG promoter and a 519 bp fragment corresponding to rabbit b-globin pA. These three elements are flanked by AAV2 ITR sequences. The plasmid backbone size is 3156 bp (standard backbone size) and contains a 966 bp ampicillin resistance gene, a 514 bp phage replication origin, and a 589 bp high copy number bacterial replication origin.

[0182] Plasmid pcohSgsh-900 (SEQ ID NO: 2)

[0183] A super plasmid containing a 1512 bp codon-optimized cDNA encoding human N-sulfoglucosamine sulfohydrolase under the control of a 1756 bp CAG promoter and a 519 bp fragment corresponding to rabbit b-globin pA. These three elements are flanked by AAV2 ITR sequences. The plasmid backbone is 7073 bp and contains a 1043 bp kanamycin resistance gene, a 589 bp high copy number bacterial origin of replication, and an additional 4657 bp of random sequence.

[0184] Plasmid pohIDS-874 (SEQ ID NO: 3)

[0185] A super plasmid containing a 1653 bp codon-optimized cDNA encoding human iduronic acid 2-sulfatase under the control of a 1756 bp CAG promoter and a 519 bp fragment corresponding to rabbit b-globin pA. These three elements are flanked by AAV 2 ITR sequences. The plasmid backbone is 7073 bp and contains a 1043 bp kanamycin resistance gene, a 589 bp high copy number bacterial origin of replication, and an additional 4657 bp of random sequence.

[0186] Plasmid pRepCap9-808 (SEQ ID NO: 4)

[0187] Plasmid encoding rep2 and cap9 proteins with the P5 promoter in its native position. This plasmid contains a 1866 bp fragment corresponding to the AAV2 Rep gene followed by a 2211 bp sequence encoding the AAV9 Cap gene. The 130 bp AAV2 P5 promoter is located in its native position upstream of the Rep gene. The plasmid backbone contains an ampicillin resistance gene and a high copy number bacterial origin of replication.

[0188] Plasmid pRepCap9-809 (SEQ ID NO: 5)

[0189] Plasmid encoding rep2 and cap9 proteins with P5 promoter following the Cap9 gene. This plasmid contains a 1866 bp fragment corresponding to the AAV2 Rep gene followed by a 2211 bp sequence encoding the AAV9 Cap gene. A 130 bp sequence corresponding to the AAV2 P5 promoter is located downstream of the Cap gene. The plasmid backbone contains a kanamycin resistance gene and a high copy number bacterial origin of replication.

[0190] Plasmid pXX6

[0191] A plasmid containing the adenoviral sequences (E2, E4 and VA-RNA) required for AAV production. The plasmid backbone contains an ampicillin resistance gene and a high copy number bacterial replication origin (Xiao et al., J Virol. 1998 Mar; 72(3): 2224–2232). Plasmid pXX6-80 is derived from pXX6, in which only the backbone is changed. The pXX6-80 map and sequence are well described in the literature.

[0192] Plasmid pAdHelper861 (SEQ ID NO: 6)

[0193] A plasmid containing the adenoviral sequences (E2, E4 and VA-RNA) required for AAV production. It contains a 731 bp region including the VA gene, a 5346 bp region including the E2A gene and the E4 gene, and their regulatory regions are included in a 3181 bp fragment. The plasmid backbone contains the kanamycin resistance gene and a high copy number bacterial origin of replication.

[0194] 1.2. Generation of AAV9-CAG-cohSgsh vector by triple transfection

[0195] Materials and methods

[0196] The AAV9-CAG-cohSgsh vector was generated by transient triple transfection using PEI-MAX as a transfection agent, as previously described (Ayuso E et al., Gene Ther. 2010 Apr; 17(4): 503-10). Briefly, confluent HEK293 adherent cells were transfected with equimolar amounts of three plasmids: pcohSgsh-827, pRepCap9-808 and pXX6 (standard plasmid) or pcohSgsh-900, pRepCap9-809 and pAdHelper861 (optimized plasmid). PEI-MAX was used at a PEI:DNA ratio of 2:1. Cells were harvested 72 hours after transfection and lysed by three cycles of freezing and thawing to release the rAAV vector from the interior of the cells (see Example 2 below). The production of rAAV vectors using suspension HEK293 cells has also been described (Joshua C Grieger et al. Mol Ther. 2016 Feb;24(2):287-297).

[0197] Vector genomes were quantified by Taqman qPCR as previously described (Ayuso et al., cited above) using primers and probes specific for the rabbit β-globin polyadenylation sequence present in the expression cassette. In addition, reverse packaging was quantified by Taqman qPCR with primers and probes specific for the antibiotic resistance promoter sequence present in all plasmid backbones used.

[0198] Statistical analysis was performed using GraphPad Prism 7.00.

[0199] Table 1. List of plasmids used for triple transfection.

[0200]

[0201]

[0202] result

[0203] As described above, the recombinant AAV9 vector AAV9-CAG-cohSgsh was produced by triple transfection using the optimized plasmid, with a higher yield than that using the standard plasmid commonly used for AAV vector production ( Figure 1 ).

[0204] The reverse packaging of bacterial sequences generated by triple transfection of the AAV9-CAG-cohSgsh vector with the optimized plasmid was lower than that obtained with the standard plasmid commonly used for AAV production ( Figure 2 ).

[0205] The use of an optimized plasmid comprising a heterologous nucleotide sequence, wherein the plasmid is a super-plasmid comprising a stuffer DNA sequence (pcohSgsh-900), does not increase vector genome yield when used in triple transfection in combination with a non-optimized plasmid (i.e., with a standard vector for triple transfection). Thus, the use of an optimized super-plasmid alone does not increase vector genome yield by triple transfection ( Figure 3 ).

[0206] However, the combination of this optimized superplasmid with an optimized plasmid containing adenoviral helper functions (pAdhelper861) or an optimized plasmid containing both the AAV rep coding region and the AAV cap coding region (pRepCap9-809) increased vector genome yield (see Figure 3 ). In addition, when three optimized vectors were combined, the vector genome yield was greatly improved (see Figure 3 ).

[0207] Furthermore, the inventors surprisingly found that when the optimized super plasmid (pcohSgsh-900) was co-transfected with one or both of the optimized helper plasmids pRepCap9-809 and / or pAdhelper861, reverse packaging of bacterial sequences was reduced ( Figure 4 In particular, the use of triple combinations of these optimized plasmids can greatly reduce reverse packaging ( Figure 4 and Figure 5 In fact, the reduction in the percentage of bacterial sequences due to the use of optimized helper plasmids (pAdhelper861) and / or (pRepCap9-809) was more pronounced when co-transfecting an optimized plasmid containing an oversized backbone (pcohSgsh-900) ( Figure 5 ).

[0208] Example 2: Triple transfection and extended gene expression (EGE)

[0209] Cell lines, culture media, and culture conditions

[0210] The cell line used is a serum-free suspension adaptation HEK 293 cell line (HEK 293SF-3F6) provided by the Institute of Biotechnology, National Research Council of Canada (Montreal, Canada). The cell is cultured in Freestyle F17 culture medium (Invitrogen, Carlsbad, CA) supplemented with Glutamax 8mM, 0.1% Pluronic1 (Invitrogen) and 0.05ng / L IGF. Cells are usually stored in 125mL disposable polycarbonate conical flasks (Corning, Steuben, NY) in 20mL culture medium. The flask is shaken at a speed of 130rpm using an orbital shaker (Kuhner shaker, Switzerland), which is placed in an incubator maintained at 37°C, containing 5% CO in the air in a humidified atmosphere. Cell count and viability are determined using Nucleocounter NC-3000 (Chemometec, Denmark).

[0211] 2.2. Transient transfection (TGE)

[0212] HEK 293 suspension cells were transiently transfected using PEIPro (Polysciences, Warrington, PA). 6 The cells were inoculated at a concentration of 10 cells / mL in a 125-mL disposable flask and grown to 2.10 6 Cells / mL were transfected with 0.76 μg pAdHelper861 / mL culture, 0.77 μg pRepCap9-809 / mL culture, and 0.38 μg pohIDS-874 / mL or 0.38 μg pcohSgsh-900 / mL culture, with a DNA to PEI mass ratio of 1:2. PEI / DNA complexes were formed by rapidly adding PEI to DNA, both diluted in fresh culture medium to reach the same volume (complex mixture volume was 5% of the total volume of the culture to be transfected). The mixture was incubated at room temperature for 15 minutes to form the complex before adding it to the cell culture.

[0213] 2.3. Extended gene expression method of coh-IDS gene

[0214] In contrast to the traditional TGE method that requires a single round of transfection, the extended gene expression (EGE) production strategy involves performing repeated transfection rounds to achieve sustained gene expression levels over time. After the first transfection, the same plasmid and conditions as in 2.2. above are used, and after complete medium exchange (300xg, 5 minutes) by centrifugation, re-transfection rounds are performed every 48 hours using the same plasmid and PEI concentration. By performing these medium exchanges, AAV is secreted into the supernatant of the culture, and AAV can be harvested at each medium exchange before each re-transfection.

[0215] The results showed (see Figure 3 ), the total vg harvested from the supernatant increased 3-fold when the EGE method was performed. The total vg recovered after batch (single transfection) was 8E11 vg obtained after cell lysis. The total vg recovered from the supernatant after the EGE strategy was 2,5E12 vg, which is an advantage from a purification point of view, as cells do not need to be lysed to harvest the AAV, so less contamination with host cell DNA and host cell proteins is expected.

[0216] 2.4. Extended gene expression method of coh-Sgsh gene

[0217] The EGE method has also been tested for the gene coh-Sgsh, as described above in 2.3.

[0218] The results showed that (see Figure 4 ), the total vg harvested from the supernatant increased 1.7-fold when the EGE approach was performed. The total vg recovered after batch (single transfection) was 1.9E12 vg obtained after cell lysis. The total vg recovered from the supernatant after the EGE strategy was 3.18E12 vg. AAV production followed a similar pattern as phIDS (see 2.3 above), thus supporting the general applicability of the EGE strategy. AAVP Sequence Listing <110> Esteve Pharmaceuticals Ltd. Autonomous University of Barcelona <120> Method for producing a recombinant viral vector <130> ESTA-1818-WO <150> EP19382220.2 <151> 2019-03-28 <160> 6 <170> PatentIn version 3.5 <210> 1 <211> 7771 <212> DNA <213> Artificial Sequence <220> <221> <222> <223> Plasmid pcohSgsh-827 <400> 1 gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc gggcgacctt 60 tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca actccatcac 120 taggggttcc ttgtagttaa tgattaaccc gccatgctac ttatctactc gacattgatt 180 attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc catatatgga 240 gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca acgacccccg 300 cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga ctttccattg 360 acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc aagtgtatca 420 tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc 480 ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat tagtcatcgc 540 tattaccatg gtcgaggtga gccccacgtt ctgcttcact ctccccatct cccccccctc 600 cccaccccca attttgtatt tatttatttt ttaattattt tgtgcagcga tgggggcggg 660 gggggggggg gggcgcgcgc caggcggggc ggggcggggc gaggggcggg gcggggcgag 720 gcggagaggt gcggcggcag ccaatcagag cggcgcgctc cgaaagtttc cttttatggc 780 gaggcggcgg cggcggcggc cctataaaaa gcgaagcgcg cggcgggcgg gagtcgctgc 840 gcgctgcctt cgccccgtgc cccgctccgc cgccgcctcg cgccgcccgc cccggctctg 900 actgaccgcg ttactcccac aggtgagcgg gcgggacggc ccttctcctc cgggctgtaa 960 ttagcgcttg gtttaatgac ggcttgtttc ttttctgtgg ctgcgtgaaa gccttgaggg 1020 gctccgggag ggccctttgt gcggggggag cggctcgggg ggtgcgtgcg tgtgtgtgtg 1080 cgtggggagc gccgcgtgcg gctccgcgct gcccggcggc tgtgagcgct gcgggcgcgg 1140 cgcggggctt tgtgcgctcc gcagtgtgcg cgaggggagc gcggccgggg gcggtgcccc 1200 gcggtgcggg gggggctgcg aggggaacaa aggctgcgtg cggggtgtgt gcgtgggggg 1260 gtgagcaggg ggtgtgggcg cgtcggtcgg gctgcaaccc cccctgcacc cccctccccg 1320 agttgctgag cacggcccgg cttcgggtgc ggggctccgt acggggcgtg gcgcggggct 1380 cgccgtgccg ggcggggggt ggcggcaggt gggggtgccg ggcggggcgg ggccgcctcg 1440 ggccggggag ggctcggggg aggggcgcgg cggcccccgg agcgccggcg gctgtcgagg 1500 cgcggcgagc cgcagccatt gccttttatg gtaatcgtgc gagagggcgc agggacttcc 1560 tttgtcccaa atctgtgcgg agccgaaatc tgggaggcgc cgccgcaccc cctctagcgg 1620 gcgcggggcg aagcggtgcg gcgccggcag gaaggaaatg ggcggggagg gccttcgtgc 1680 gtcgccgcgc cgccgtcccc ttctccctct ccagcctcgg ggctgtccgc ggggggacgg 1740 ctgccttcgg gggggacggg gcagggcggg gttcggcttc tggcgtgtga ccggcggctc 1800 tagagcctct gctaaccatg ttcatgcctt cttctttttc ctacagctcc tgggcaacgt 1860 gctggttatt gtgctgtctc atcattttgg caaagaattg attaattcga gcgaacgcgt 1920 gccaccatga gctgccctgt gcccgcctgt tgtgccctgc tgctggtgct gggactgtgc 1980 agagccagac cccggaacgc tctgctgctg ctggccgacg atggcggatt tgagagcggc 2040 gcctacaaca acagcgccat tgccacccct catctggacg ccctggccag aagaagcctg 2100 ctgttccgga acgccttcac cagcgtgtcc agctgcagcc ctagcagagc ttccctgctg 2160 acaggcctgc cccagcatca gaatggcatg tacggcctgc accaggatgt gcatcacttc 2220 aacagcttcg acaaagtgcg gagcctgcca ctgctcctgt cacaggctgg cgtgagaacc 2280 ggcatcatcg gcaagaaaca cgtgggcccc gagacagtgt accccttcga cttcgcctac 2340 accgaagaga acggcagcgt gctgcaggtc ggccggaaca tcacccggat caagctgctc 2400 gtgcggaagt ttctccagac ccaggacgac cggcccttct tcctgtacgt ggccttccac 2460 gaccctcaca gatgcggcca cagccagccc cagtacggca ccttctgcga gaagttcggc 2520 aacggcgaga gcggcatggg cagaatcccc gactggaccc cccaggcata cgaccctctg 2580 gacgtgctgg tgccctactt cgtgcccaac acccctgccg ccagagctga tctggccgcc 2640 cagtacacca ccgtgggcag aatggatcag ggcgtgggcc tggtgctgca ggaactgagg 2700 gacgctggcg tgctgaacga caccctggtc atcttcacct ccgacaacgg catcccattc 2760 cccagcggcc ggaccaatct gtactggccc ggcacagccg aacctctgct ggtgtccagc 2820 cccgagcacc ctaagagatg gggccaggtg tccgaggcct acgtgtccct gctggacctg 2880 acccccacca tcctggactg gttcagcatc ccctacccca gctacgccat ctttggaagc 2940 aagaccatcc acctgaccgg cagatctctg ctgcctgccc tggaagctga gcctctgtgg 3000 gccaccgtgt tcggcagcca gagccaccac gaagtgacca tgagctaccc catgcggagc 3060 gtgcagcacc ggcacttccg gctggtgcac aacctgaact tcaagatgcc cttcccaatc 3120 gaccaggact tttacgtgtc ccccaccttc caggacctgc tgaacagaac cacagccggc 3180 cagcccaccg gctggtacaa ggacctgcgg cactactact accgggccag atgggagctg 3240 tacgacagaa gccgggaccc ccacgagaca cagaacctgg ccaccgaccc cagattcgcc 3300 cagctcctgg aaatgctgcg ggaccagctg gccaagtggc agtgggagac acacgaccct 3360 tgggtctgcg ctcccgacgg cgtgctggaa gagaagctgt ccccccagtg ccagccactg 3420 cacaacgagc tgtgatgaga attcgagctc ggtacccggg aatcaattca ctcctcaggt 3480 gcaggctgcc tatcagaagg tggtggctgg tgtggccaat gccctggctc acaaatacca 3540 ctgagatctt tttccctctg caaaaatta tggggacatc atgaagcccc ttgagcatct 3600 gacttctggc tataaagga aatttatttt cattgcaata gtgtgttgga atttttgtg 3660 tctctcactc ggaaggacat atgggagggc aaatcattta aaacatcaga atgagtattt 3720 ggtttagagt ttggcaacat atgcccatat gctggctgcc atgaacaaag gttggctata 3780 aagaggtcat cagtatatga aacagccccc tgctgtccat tccttattcc atagaaaagc 3840 cttgacttga ggttagattt tttttatatt ttgttttgtg ttattttttt cttaacatc 3900 cctaaaattt tccttacatg ttttactagc cagatttttc ctcctctcct gactactccc 3960 agtcatagct gtccctcttc tcttatggag atccctcgac ctgcagccca agctgtagat 4020 aagtagcatg gcgggttaat cattaactac aaggaacccc tagtgatgga gttggccact 4080 cccctctgc gcgctcgctc gctcactgag gccgggcgac caaaggtcgc ccgacgccg 4140 ggctttgccc gggcggcctc agtgagcgag cgagcgcgca gctgcattaa tgaatcggcc 4200 aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact 4260 cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac 4320 ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa 4380 aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg 4440 acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa 4500 gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc 4560 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac 4620 gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac 4680 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg 4740 taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt 4800 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa 4860 cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct 4920 cttgatccgg caacaaacc accgctgta gcggtggtt tttgttgc aagcagcaga 4980 ttacgcgcag aaaaaaagga tctcagaag atcctttgat ctttctacg gggtctgacg 5040 ctcagtggaa cgaaaactca cgttaaggga ttttgtcat gagattatca aaaggatct 5100 tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt 5160 aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctactca gcgatctgtc 5220 tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg 5280 gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag 5340 atttatcagc ataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt 5400 tatccgcctc catccagtct atttattgtt gccgggaagc tagtagt agttcgccag 5460 ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt 5520 ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca 5580 tgttgtgcaa aaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg 5640 ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat 5700 ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta 5760 tgcggcgacc gagttgctct tgcccggcgt caatacggga tataccgcg ccacatagca 5820 gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct 5880 taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat 5940 cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa 6000 agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt 6060 gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa 6120 ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac gtctaagaaa 6180 ccattattat catgacatta acctataaaa ataggcgtat cacgaggccc tttcgtctcg 6240 cgcgtttcgg tgatgacggt gaaaacctct gacacatgca gctcccggag acggtcacag 6300 cttgtctgta agcggatgcc gggagcagac aagcccgtca gggcgcgtca gcgggtgttg 6360 gcgggtgtcg gggctggctt aactatgcgg catcagagca gattgtactg agagtgcacc 6420 atatgcggtg tgaaataccg cacagatgcg tagggaagaaa ataccgcatc aggcgattcc 6480 aacatccaat aaatcataca ggcaaggcaa agaattagca aaattaagca ataaagcctc 6540 agagcataaa gctaaatcgg ttgtaccaaa aacattatga ccctgtaata cttttgcggg 6600 agaagccttt atttcaacgc areataaa atttttagaa ccctcatata ttttaaatgc 6660 aatgcctgag taatgtgtag gtaaagattc aaacgggtga gaaggccgg agacagtcaa 6720 atcaccatca atatgatatt caaccgttct agctgataaa ttcatgccgg agaggggtagc 6780 tatttttgag aggtctctac aaaggctatc aggtcattgc ctgagagtct ggagcaaaca 6840 agagaatcga tgaacggtaa tcgtaaact agcatgtcaa tcatatgtac cccggttgat 6900 aatcagaaaa gccccaaaaa caggagaatt gtataagcaa atatttaaat tgtaagcgtt 6960 aatattttgt taaaattcgc gttaaatttt tgttaaatca gctcatttt taaccaatag 7020 gccgaaatcg gcaaaatccc ttataaatca aaagaataga ccgagatagg gttgagtgtt 7080 gttccagttt ggaacaagag tccactatta aagaacgtgg actccaacgt caaaggcga 7140 aaaaccgtct atcagggcga tggcccacta cgtgaaccat caccctaatc aagttttttg 7200 gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag ggagccccccg atttagagct 7260 tgacggggaa agccggcgaa cgtggcgaga aaagaaggga agaaagcgaa aggagcggc 7320 gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc cgccgcgctt 7380 aatgcgccgc tacagggcgc gtactatggt tgctttgacg agcacgtata acgtgctttc 7440 ctcgttagaa tcagagcggg agctaaacag gaggccgatt aaagggattt tagacaggaa 7500 cggtacgcca gaatcctgag aagtgtttt ataatcagtg aggccaccga gtaaaagagt 7560 ctgtccatca cgcaaattaa ccgttgtcgc aatacttctt tgattagtaa taacatcact 7620 tgcctgagta gaagaactca aactatcggc cttgctggta atatccagaa caatattacc 7680 gccagccatt gcaacggaat cgccattcgc cattcaggct gcgcaactgt tgggaagggc 7740 gatcggtgcg ggcctcttcg ctattacgcc a 7771 <210> 2 <211> 11255 <212> DNA <213> Artificial Sequence <220> <221> <222> <223> Plasmid pcohSgsh-900 <400> 2 attacgccag ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg 60 ggcgaccttt ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa 120 ctccatcact aggggttcct tgtagttaat gattaacccg ccatgctact tatctactcg 180 acattgatta ttgactagtt attaatagta atcaattacg gggtcattag ttcatagccc 240 atatatggag ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa 300 cgacccccgc ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac 360 tttccattga cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca 420 agtgtatcat atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg 480 gcattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt 540 agtcatcgct attaccatgg tcgaggtgag ccccacgttc tgcttcactc tccccatctc 600 ccccccctcc ccacccccaa ttttgtattt attattttt taattatttt gtgcagcgat 660 gggggcgggg gggggggggg ggcgcgcgcc aggcggggcg gggcggggcg aggggcgggg 720 cggggcgagg cggagaggtg cggcggcagc caatcagagc ggcgcgctcc gaaagttttcc 780 tttatggcg aggcggcggc ggcggcggcc ctataaaaag cgaagcgcgc ggcgggcggg 840 agtcgctgcg cgctgccttc gcccgtgc ccgctccgc gcgcctcgc gcgcccgcc 900 ccggctctga ctgaccgcgt tactcccaca ggtgagcggg cgggacggcc cttctcctcc 960 gggctgtaat tagcgcttgg tttaatgacg gcttgtttct tttctgtggc tgcgtgaaag 1020 ccttgagggg ctccgggagg gccctttgtg cggggggagc ggctcggggg gtgcgtgcgt 1080 gtgtgtgtgc gtggggagcg ccgcgtgcgg ctccgcgctg cccggcggct gtgagcgctg 1140 cgggcgcggc gcggggcttt gtgcgctccg cagtgtgcgc gaggggagcg cggccggggg 1200 cggtgccccg cggtgcgggg ggggctgcga ggggaacaaa ggctgcgtgc ggggtgtgtg 1260 cgtgggggg tgagcagggg gtgtgggcgc gtcggtcggg ctgcaacccc ccctgcaccc 1320 ccctccccga gttgctgagc acggcccggc ttcgggtgcg gggctccgta cggggcgtgg 1380 cgcggggctc gccgtgccgg gcggggggtg gcggcaggtg ggggtgccgg gcggggcggg 1440 gccgcctcgg gccgggggagg gctcggggga ggggcgcggc ggccccccgga gcgccggcgg 1500 ctgtcgaggc gcggcgagcc gcagccattg ccttttatgg taatcgtgcg agagggcgca 1560 gggacttcct ttgtcccaaa tctgtgcgga gccgaaatct gggaggcgcc gccgcacccc 1620 ctctagcggg cgcggggcga agcggtgcgg cgccggcagg aaggaaatgg gcggggaggg 1680 ccttcgtgcg tcgccgcgcc gccgtcccct tctccctctc cagcctcggg gctctccgcg 1740 ggggacggc tgccttggg ggggacgggg cagggcgggg ttcggcttct ggcgtgtgac 1800 cggcggctct agagcctctg ctaaccatgt tcatgccttt ttctttttcc tacagctcct 1860 gggcaacgtg ctggttattg tgctgtctca tcattttggc aaagaattga ttaattcgag 1920 cgaacgcgtg ccaccatgag ctgccctgtg cccgcctgtt gtgccctgct gctggtgctg 1980 ggactgtgca gagccagacc ccggaacgct ctgctgctgc tggccgacga tggcggattt 2040 gagagcggcg cctacaacaa cagcgccatt gccacccctc atctggacgc cctggccaga 2100 agaagcctgc tgttccggaa cgccttcacc agcgtgtcca gctgcagccc tagcagagct 2160 tccctgctga caggcctgcc ccagcatcag aatggcatgt acggcctgca ccaggatgtg 2220 catcacttca acagcttcga caaagtgcgg agcctgccac tgctcctgtc acaggctggc 2280 gtgagaaccg gcatcatcgg caagaaacac gtgggccccg agacagtgta ccccttcgac 2340 ttcgcctaca ccgaagagaa cggcagcgtg ctgcaggtcg gccggaacat cacccggatc 2400 aagctgctcg tgcggaagtt tctccagacc caggacgacc ggcccttctt cctgtacgtg 2460 gccttccacg accctcacag atgcggccac agccagcccc agtacggcac cttctgcgag 2520 aagttcggca acggcgagag cggcatgggc agaatccccg actggacccc ccaggcatac 2580 gaccctctgg acgtgctggt gccctacttc gtgcccaaca cccctgccgc cagagctgat 2640 ctggccgccc agtacaccac cgtgggcaga atggatcagg gcgtgggcct ggtgctgcag 2700 gaactgaggg acgctggcgt gctgaacgac accctggtca tcttcacctc cgacaacggc 2760 atcccattcc ccagcggccg gaccaatctg tactggcccg gcacagccga acctctgctg 2820 gtgtccagcc ccgagcaccc taagagatgg ggccaggtgt ccgaggccta cgtgtccctg 2880 ctggacctga cccccaccat cctggactgg ttcagcatcc cctaccccag ctacgccatc 2940 tttggaagca agaccatcca cctgaccggc agatctctgc tgcctgccct ggaagctgag 3000 cctctgtggg ccaccgtgtt cggcagccag agccaccacg aagtgaccat gagctacccc 3060 atgcggagcg tgcagcaccg gcacttccgg ctggtgcaca acctgaactt caagatgccc 3120 ttcccaatcg accaggactt ttacgtgtcc cccaccttcc aggacctgct gaacagaacc 3180 acagccggcc agcccaccgg ctggtacaag gacctgcggc actactacta ccgggccaga 3240 tgggagctgt acgacagaag ccgggacccc cacgagacac agaacctggc caccgacccc 3300 agattcgccc agctcctgga aatgctgcgg gaccagctgg ccaagtggca gtgggagaca 3360 cacgaccctt gggtctgcgc tcccgacggc gtgctggaag agaagctgtc cccccagtgc 3420 cagccactgc acaacgagct gtgatgagaa ttcgagctcg gtacccggga atcaattcac 3480 tcctcaggtg caggctgcct atcagaaggt ggtggctggt gtggccaatg ccctggctca 3540 caaataccac tgagatcttt ttccctctgc caaaaattat ggggacatca tgaagcccct 3600 tgagcatctg acttctggct aataaaggaa atttattttc attgcaatag tgtgttggaa 3660 ttttttgtgt ctctcactcg gaaggacata tgggagggca aatcatttaa aacatcagaa 3720 tgagtatttg gtttagagtt tggcaacata tgcccatatg ctggctgcca tgaacaaagg 3780 ttggctataa agaggtcatc agtatatgaa acagccccct gctgtccatt ccttattcca 3840 tagaaaagcc ttgacttgag gttagatttt ttttatattt tgttttgtgt tatttttttc 3900 tttaacatcc ctaaaatttt ccttacatgt tttactagcc agatttttcc tcctctcctg 3960 actactccca gtcatagctg tccctcttct cttatggaga tccctcgacc tgcagcccaa 4020 gctgtagata agtagcatgg cgggttaatc attaactaca aggaacccct agtgatggag 4080 ttggccactc cctctctgcg cgctcgctcg ctcactgagg ccgggcgacc aaaggtcgcc 4140 cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc gagcgcgcag ctgcattaat 4200 gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc 4260 tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg 4320 cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag 4380 gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc 4440 gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag 4500 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga 4560 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc 4620 atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg 4680 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt 4740 ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca 4800 gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca 4860 ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaagag 4920 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt ttgtttgca 4980 agcagcagat tacgcgcaga aaaaaaggat ctcaagaga tcctttgatc tttctacgg 5040 ggtctgacgc tcagtggaac gaaaactcac gttaaggt ttggtcatg agatttaca 5100 aaaggatctt cacctagatc cttttaaattt aaaaatgaag ttttaatca atctaagta 5160 tatatgagta aacttggtct vakagttacc aatgctat cagtgaggca cctatctctcag 5220 cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga 5280 tacgggaggg cttaccatct ggccccagtg ctgcaatgat accggggtta acgcgaattt 5340 taacaaata ttaacgctta caatttaggt ggcactttc ggggaatgt gcgcggaacc 5400 cctatttgtt tattttcta atacattca atatgtatc cgctcatgag acaataaccc 5460 tgataaatgc ttcaataata tgaaaaagg agagtag tgaacagga tggcctgcat 5520 gcgggtagcc cggcagcgtg ggtggaacgt ctgttttggct atgattgggc gcagcagacc 5580 attggctgct ctgatgcggc ggtgtttcgt ctgagcgcgc agggtcgtcc ggtgctgttt 5640 gtgaaaaccg atctgagcgg tgcgctgaac gagctgcagg atgaagcggc gcgtctgagc 5700 tggctggcca ccaccggtgt tccgtgtgcg gcggtgctgg atgtggtgac cgaagcgggc 5760 cgtgattggc tgctgctggg cgaagtgccg ggtcaggatc tgctgtctag ccatctggcg 5820 ccggcagaaa aagtgagcat tatggcggat gccatgcgtc gtctgcatac cctggacccg 5880 gcgacctgtc cgtttgatca tcaggcgaaa catcgtattg aacgtgcgcg tacccgtatg 5940 gaagcgggcc tggtggatca ggatgatctg gatgaagaac atcagggcct ggcaccggca 6000 gagctgtttg cgcgtctgaa agcgagcatg ccggatggcg aagatctggt ggtgacccat 6060 ggtgatgcgt gcctgccgaa cattatggtg gaaaatggcc gttttagcgg ctttattgat 6120 tgcggccgtc tgggcgtggc ggatcgttat caggatattg cgctggccac ccgtgatatt 6180 gcggaagaac tgggcggcga atgggcggat cgttttctgg tgctgtatgg cattgcggca 6240 ccggatagcc agcgtattgc gttttatcgt ctgctggatg aatttttcta ataactgtca 6300 gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta atttaaaaca 6360 attggatggg caattggata tcgtcatctt tgagacacaa tctcccacct cactggaatt 6420 tagttcctgc tataattagc cttcctcata agttgcacta cttcagcgtc ccatatgcac 6480 ccttaccacg aagacaggtt tgtccaatcc catattgcga ccttggcagg gggttcgcaa 6540 gtcccacccg aaacgttgct gaaggctcag gtttctgagc gacaaaaggt taatacgcga 6600 gttcccgctc ataacctgga ccgaatgcgg aatcatgcat cgttccactg tgtttgtctc 6660 atgtaggacg ggcgcaaagc atacttagtt caatcttgaa taccttatat tattgtacac 6720 ctaccggtca ccagccaaca atgtgcggac ggcgttgcaa ctttcagggc ctaatctgac 6780 cgttgtagat accgcactct gggcaatacg aggtaatgcc agtcacccag tgtcgaacaa 6840 cacctgacct aacggtaaga ggctcacata atggctctgc cggcgtgccc agggtatatt 6900 aggtcagcat cagatggact gacatgaatc tttacaccga agcggaaacg ggtgcgtgga 6960 ctagcgagga gcaaacgaaa attcctggcc tgcttgatgt ctcgtaatct tcttagagat 7020 ggacgaaatg tttcacgacc taggaaaagg tcgccctaca aaatagattt gcgttactct 7080 cttcatagga tccccggtgt agcgaaagat caaggcgacc ctaggtagca accgccggct 7140 tcggcggtaa ggtatcactc aagaagcaga ctcagtaaga cacggtctag ctgactgtct 7200 atcgcctagg tcaaataggg agctttgatt ctgcatgtcc agctttagat tcactttagc 7260 gcgcagatct gggtcgagat aaaatcacca gtacccaaga ccaggggggc tcgccgcgtt 7320 ggctaatcct ggtacatctt gtaatgaata ttcagtagaa aatttgtgtt agaaggacga 7380 gtcaccatgt accaaaagcg ataacgatcg gtgggagtat tcattgtggt gaagacgctg 7440 ggttacgtg ggaaaggtgc ttgtgtccca acaggctagg atataatgct gaagcccttc 7500 cccaagcgtt cagggtggga tttgctacaa cttccgagtc caacgtgtcc gtgttcatgt 7560 tatatatgca caaggccgag aattggacgt agctttcgtg ttagtacgta gcatggtcac 7620 acaagcacag tagatcctgc ccgcgcatcc tatatattaa gttaattcta atggaatacg 7680 atgacatgtg gatgggcagt ggccggttgt tacacgccta ccgcgatgct gaatgacccg 7740 gactaaagtg gcgaaaatta tggcgtgtga cccgttatgc tccagttcgg tcagtgggtc 7800 attgcaagta gtcgattgca ttgtcaatct ccgagtgatt tagcgtgaca gccgcaggga 7860 acccataaaa tgcgatcgta gtccatccga tcgtacatag aaatgagggt ccccatacgc 7920 ccacgcacct gttcactcgt cgtttgcatt taagagccgc acgaaccaca gagcataaag 7980 aggacctcta gctcctttac aaagtggggt cgaccgatcg cttgcgcaac ttgtgaagtg 8040 tctaccatcc ctaagcccat ttcccgcata ttaacccctg attgtatccg catctgatgc 8100 taccgtggtt gagttagcgt cgagcacgcg ggacttattg catgagtaga gttgactaag 8160 agccgttaga tggctcgctg agctaatagt tgccgacaga tcgtcaagat tagaaaacgg 8220 ttgtagcatt atcggaggtt ctctaactag tatcgatagc cgtgtcttca ctgtgccgcg 8280 gctacctatc gcctgaaaac cagttggtgt taaggggtcc cctgtccagg acgccaccgg 8340 tagtgagaca tacacgttcg ttgggttcac cgcggtcgga cctgagtgca ccaaggacac 8400 actgcagctc cgacccctac tgtcgagaaa tttgtatccc gcccccgcag cttgccagct 8460 ctttcagtat catggagccc atggttgaat gagtccaata acgaacttcg acatgataaa 8520 atccccccct cgcgacttcc agagaagaag actactgact tgagcgttcc cagcacttca 8580 gccaaggaag ttaccaattt tttgtttccg aatgacaccg gtctccttgc gggtagatcg 8640 ccgaccgcag aacttacgag ccaggggaa cattaaggcc taattaggta aagggagtaa 8700 gtgctcgaac gcttcagatg taaccatata cttacgctgg atcttctccc gcgaatttta 8760 accctcacca actacgagat ttgaggtaaa ccaaataagc acgtagtggc gctatccgac 8820 tgttcccaaa ttgtaactta tcgttccgtg aaggccagag ttacttcccg gccctttcca 8880 tgcgcgcacc ataccctcct agttccccgg ttatctctcc gaggagggag tgagcgatcc 8940 tccgtttacg ttttgttacc aatgacgtag ctatgtattt tgtacaggtt gccaacgggt 9000 ttcacaattc acagatagtg gggtacccgg caaagggcct atatttgcgg tccaacttag 9060 gcgtaacta cgatggtacc tactcagacc cagctcgcgc ggcgtaaata acgcactcat 9120 cccagctgat tctcggcgat ctacgcagcg acatgattat caacagctgt ctggcagctc 9180 taatctttta ccatggtcgt aaaagcctcc aagagttaga tcatacctaa cgccacaaaa 9240 gtgacacgac gccgatgggt accggacttt aggtgcacca cagttcggta agggagaggc 9300 cctgcggcgt acttcatttt gtatatgcaa cgtgcccaag tggcgccagg caagtctcag 9360 ctggttcctg tgttagctgc aggctaggca tgggagctga ttgaacatgg gttggggggcc 9420 tcgaaccgtc gaggacccca tagtacgttt aaacccaagt agggcagcct atagtttgaa 9480 gcagtactat ttcagggggg gagccctcat ggtctcttct actgatgact caacacgcta 9540 gggacgtgaa gtcgattcct tcgatggtta taaatcaaag gctcagagtg cagtctggag 9600 cgcccatcta acggtacgca tctcgattgc tcggtcgcct ttcacactcc gcgaaaattc 9660 ataccgctca ttcactaggt tgcgaagcct acactgatat atgaatccaa gctagagcag 9720 ggctcttaaa attcggagtt gtagatgctc aatactccaa tcggtttttt cgtgcaccac 9780 cgcgggtggc tgacaagggt ttgacatcga gaaacaaggc agttccgggc tgaaagtagc 9840 gccgggtaag gtacgcgcct ggtatggcag gactatgaag ccaatacaaa ggctacatcc 9900 tcactcgggt ggacggaaac gcagaattat ggttactttt tggatacgtg aaacatgtcc 9960 catggtagcc caaagacttg ggagtctatc accctaggg cccatttctg gatatagacg 10020 ccaggttgaa tccgtatttg gaggtacgat ggatcagtct gggtgggacg tgctccattt 10080 ataccctgcg caggctggac cgaggaccgc aagatgcgac ggtgcacaag taattgacaa 10140 caaaccatcg tgttttcatt atggtaccag gatcttcaag ccgagtcaat caagctcgga 10200 ttacagtgtt taccgcgtct tgcggttact cacaaactgt aatccaccac aagtcaagcc 10260 attgcctctc tgagacgccg tatgaattaa tatgtaaact ttgcgcgggt tcactgcgat 10320 ccgttcagtc tcgtccaagg gcacaatcga attcccattt gtatgttcgg ctaacttcta 10380 cccatcccccc gaagtttagc aggtcgtgag gtgtcatgga ggctctcgtt catcccgtgg 10440 gacatcgttt aaacgccttg ataaagcacc ccgctcgggt gtagcagaga agacgcctac 10500 tgaattgtgc gatccctcca cctcagctaa ggtagctacc aatatttagt tttttagcct 10560 tgcgacagac ctcctactta gattgccacg cattgagcta gcgagtcagc gataagcatg 10620 acgcgctttc aagcgtcgcg agtatgtgaa ccaaggctcc ggacaggact atatacttgg 10680 gtttgatctc gccccgacaa ctgcaaacct caacatttat agattataag gttagccgaa 10740 attgcacgtg gtggcgcccg ccgactgctc ccgagtgtg gctctttgat ctgacaacgc 10800 gcgacctcca tcgcggccga ttgtttctgc ggaccatgtc gtcctcatag tttgggcatg 10860 tttccgttgt aggagtgaag ccacttagct ttgcgccgta gtcccaatga aaaacctatg 10920 gactttgttt tgggtagcat caggaatctg aaccctgtga atgtgggggt cgcgcgcata 10980 gacctttatc tccggttcaa gttaggcatg aggctgtcga cccctaatca gtgaggccac 11040 cgagtaaaag agtctgtcca tcacgcaaat taaccgttgt cgcaatactt ctttgattag 11100 tataacatc acttgcctga gtagaagaac tcaaactatc ggccttgctg gtaatatcca 11160 gaacaatatt accgccagcc attgcaacgg aatcgccatt cgccattcag gctgcgcaac 11220 tgttgggaag ggcgatcggt gcgggcctct tcgct 11255 <210> 3 <211> 11396 <212> DNA <213> Artificial Sequence <220> <221> <222> <223> Plasmid pohIDS-874 <400> 3 attacgccag ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg 60 ggcgaccttt ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa 120 ctccatcact aggggttcct tgtagttaat gattaacccg ccatgctact tatctactcg 180 acattgatta ttgactagtt attaatagta atcaattacg gggtcattag ttcatagccc 240 atatatggag ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa 300 cgacccccgc ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac 360 tttccattga cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca 420 agtgtatcat atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg 480 gcattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt 540 agtcatcgct attaccatgg tcgaggtgag ccccacgttc tgcttcactc tccccatctc 600 ccccccctcc ccacccccaa ttttgtattt atttattttt taattatttt gtgcagcgat 660 gggggcgggg gggggggggg ggcgcgcgcc aggcggggcg gggcggggcg aggggcgggg 720 cggggcgagg cggagaggtg cggcggcagc caatcagagc ggcgcgctcc gaaagtttcc 780 ttttatggcg aggcggcggc ggcggcggcc ctataaaaag cgaagcgcgc ggcgggcggg 840 agtcgctgcg cgctgccttc gccccgtgcc ccgctccgcc gccgcctcgc gccgcccgcc 900 ccggctctga ctgaccgcgt tactcccaca ggtgagcggg cgggacggcc cttctcctcc 960 gggctgtaat tagcgcttgg tttaatgacg gcttgtttct tttctgtggc tgcgtgaaag 1020 ccttgagggg ctccgggagg gccctttgtg cggggggagc ggctcggggg gtgcgtgcgt 1080 gtgtgtgtgc gtggggagcg ccgcgtgcgg ctccgcgctg cccggcggct gtgagcgctg 1140 cgggcgcggc gcggggcttt gtgcgctccg cagtgtgcgc gaggggagcg cggccggggg 1200 cggtgccccg cggtgcgggg ggggctgcga ggggaacaaa ggctgcgtgc ggggtgtgtg 1260 cgtggggggg tgagcagggg gtgtgggcgc gtcggtcggg ctgcaacccc ccctgcaccc 1320 ccctccccga gttgctgagc acggcccggc ttcgggtgcg gggctccgta cggggcgtgg 1380 cgcggggctc gccgtgccgg gcggggggtg gcggcaggtg ggggtgccgg gcggggcggg 1440 gccgcctcgg gccgggggagg gctcggggga ggggcgcggc ggccccccgga gcgccggcgg 1500 ctgtcgaggc gcggcgagcc gcagccattg ccttttatgg taatcgtgcg agagggcgca 1560 gggacttcct ttgtcccaaa tctgtgcgga gccgaaatct gggaggcgcc gccgcacccc 1620 ctctagcggg cgcggggcga agcggtgcgg cgccggcagg aaggaaatgg gcggggaggg 1680 ccttcgtgcg tcgccgcgcc gccgtcccct tctccctctc cagcctcggg gctctccgcg 1740 ggggacggc tgccttggg ggggacgggg cagggcgggg ttcggcttct ggcgtgtgac 1800 cggcggctct agagcctctg ctaaccatgt tcatgccttt ttctttttcc tacagctcct 1860 gggcaacgtg ctggttattg tgctgtctca tcattttggc aaagaattga ttaattcgag 1920 cgaacgcgtg ccaccatgcc cccacctaga accggaagag gattgctctg gctcggactt 1980 gtgctgtcca gcgtgtgtgt ggccctgggc tcggaaaccc aggccaacag caccaccgac 2040 gcctgaatg tgctgctgat tatcgtggac gatctccggc cttcgctggg ctgctacggg 2100 gataagctgg tccgctcccc gaatatcgac caactggctt cacatagcct gcttttccaa 2160 aacgcattcg cccaacaagc cgtgtgcgcc ccgagccgcg tgtctttcct caccggcgg 2220 cgccctgata ctacccggct ctacgacttc aacagctact ggagagtgca cgcaggaaac 2280 ttctccacca ttcctcagta cttaaggag aacggttacg tcaccatgag cgtggggaag 2340 gtgttccacc ctggaatttc ctccaaccac accgacgact cgccatactc ctggtccttt 2400 cccccttacc acccatcatc cgagaagtac gagaacacca agacgtgcag gggcccagac 2460 ggggaactgc acgcgaacct cctctgccg gtcgatgtgc tggatgtgcc cgaaggcacc 2520 ctccctgaca aacagagcac cgaacaggcc atccagctcc tcgagaagat gaaaacttca 2580 gcctccccgt tctttctggc cgtgggatac cacaagccgc atatcccctt cggtaccca 2640 aaggagttcc agaagctgta cccgctggag aacattaccc tggctcctga tcccgaagtg 2700 ccggacggcc tgccgcccgt ggcatacaac ccttggatgg acatccgcca gagggaggat 2760 gtgcaagccc tgaacatctc cgtgccatac ggtccgatcc cggtcgactt ccagcggaag 2820 attaggcagt catatttcgc gtccgtgtcc tacttggaca ctcaggtcgg acgcctcctc 2880 tccgctctcg acgatctgca gctggccaac tcgaccatta tcgcgttcac ctcggaccat 2940 ggttgggctc tgggcgaaca cggagaatgg gccaagtaca gcaatttcga tgtcgcgact 3000 cacgtgcccc tgatcttcta cgtgcccgga cgcacagcca gcttgcctga agcgggggaa 3060 aagctgttcc cttacctgga tcccttcgac tccgcctctc aacttatgga gccaggcaga 3120 cagtcgatgg acctggtgga actcgtgtca ctgttcccta ccctcgccgg tctggccgga 3180 cttcaggtcc cgcctcggtg cccggtgccg tccttccacg tggagctgtg tcgcgaggga 3240 aagaacctcc tgaaacactt ccggttccgc gacctggagg aagatcccta cttgccgggc 3300 aacccgagag aacttatcgc atactcccag taccctcgcc cctccgacat cccgcagtgg 3360 aactccgaca agccgagcct gaaggacatt aagatcatgg ggtactccat ccggactatt 3420 gactatcggt acactgtgtg ggtcgggttc aacccagatg agtttctggc caacttctcc 3480 gatatccatg ccggagagct gtacttcgtg gactcggacc cgctgcagga ccacaacatg 3540 tacaacgact cacagggcgg cgacctgttc cagttgctga tgccctgaga attcgagctc 3600 ggtacccggg aatcaattca ctcctcaggt gcaggctgcc tatcagaagg tggtggctgg 3660 tgtggccaat gccctggctc acaaatacca ctgagatctt tttccctctg ccaaaaatta 3720 tggggacatc atgaagcccc ttgagcatct gacttctggc tataaagga aatttatttt 3780 cattgcaata gtgtgttgga atttttgtg tctctcactc ggaaggacat atgggagggc 3840 aaatcattta aaacatcaga atgagtattt ggtttagagt ttggcaacat atgcccatat 3900 gctggctgcc atgaacaaag gttggctata aagaggtcat cagtatatga aacagccccc 3960 tgctgtccat tccttattcc atagaaaagc cttgacttga ggttagattt tttttatatt 4020 ttgttttgtg ttattttttt cttaacatc cctaaaattt tccttacatg ttttactagc 4080 cagattttc ctcctctcct gactactccc agtcatagct gtccctcttc tcttatggag 4140 atccctcgac ctgcagccca agctgtagat aagtagcatg gcgggttaat cattaactac 4200 aaggaacccc tagtgatgga gttggccact ccctctctgc gcgctcgctc gctcactgag 4260 gccgggcgac caaaggtcgc ccgacgccccg ggctttgccc gggcggcctc agtgagcgag 4320 cgagcgcgca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg 4380 ggcgctcttc cgcttctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag 4440 cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag 4500 gaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc 4560 tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc 4620 agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc 4680 tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt 4740 cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg 4800 ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat 4860 ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag 4920 ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt 4980 ggtggcctaa ctacggctac actagaagaa foottttgg tatctgcgct ctgctgaagc 5040 cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta 5100 gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag 5160 atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga 5220 ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa 5280 gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa 5340 tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc 5400 ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga 5460 taccggggtt aacgcgaatt ttaacaaaat attaacgctt acaattttagg tggcactttt 5520 cggggaaatg tgcgcggaac ccctatttgt ttattttct aaatacattc aaatatgtat 5580 ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg 5640 attgaacagg atggcctgca tgcgggtagc ccggcagcgt gggtggaacg tctgtttggc 5700. tatgattggg cgcagcagac cattggctgc tctgatgcgg cggtgtttcg tctgagcgcg 5760 cagggtcgtc cggtgctgtt tgtgaaaacc gatctgagcg gtgcgctgaa cgagctgcag 5820 gatgaagcgg cgcgtctgag ctggctggcc accaccggtg ttccgtgtgc ggcggtgctg 5880 gatgtggtga ccgaagcgggg ccgtgattgg ctgctgctgg gcgaagtgcc gggtcaggat 5940 ctgctgtcta gccatctggc gccggcagaa aaagtgagca ttatggcgga tgccatgcgt cgtctgcata ccctggaccc ggcgacctgt ccgtttgatc atcaggcgaa acatcgtatt gaacgtgcgc gtacccgtat ggagcgggc ctggtggatc aggatgatct ggatgaagaa 6120. catcagggcc tggcaccggc agagctgttt gcgcgtctga aagcgagcat gccggatggc 6180. gaagatctgg tggtgaccca tggtgatgcg tgcctgccga acattatggt ggaaaatggc 6240 cgttttagcg gctttattga ttgcggccgt ctggggcgtgg cggatcgtta tcaggatatt 6300. gcgctggcca cccgtgatat tgcggaagaa ctgggcggcg aatgggcgga tcgttttctg gtgctgtatg gcattgcggc accggatagc cagcgtattg cgttttatcg tctgctggat 6420 gaatttttct aataactgtc agaccaagtt tactcatata tactttagat tgatttaaaa 6480 cttcattttt aatttaaaac aattggatgg gcaattggat atcgtcatct ttgagacaca 6540 atctcccacc tcactggaat ttagttcctg ctataattag ccttcctcat aagttgcact 6600 acttcagcgt cccatatgca cccttaccac gaagacaggt ttgtccaatc ccatattgcg 6660 accttggcag ggggttcgca agtcccaccc gaaacgttgc tgaaggctca ggtttctgag 6720 cgacaaaagg ttaatacgcg agttcccgct cataacctgg accgaatgcg gaatcatgca 6780 tcgttccact gtgtttgtct catgtaggac gggcgcaaag catacttagt tcaatcttga 6840 ataccttata ttattgtaca cctaccggtc accagccaac aatgtgcgga cggcgttgca 6900 actttcaggg cctaatctga ccgttgtaga taccgcactc tgggcaatac gaggtaatgc 6960 cagtcaccca gtgtcgaaca acacctgacc taacggtaag aggctcacat aatggctctg 7020 ccggcgtgcc cagggtatat taggtcagca tcagatggac tgacatgaat ctttacaccg 7080 aagcggaaac gggtgcgtgg actagcgagg agcaaacgaa aattcctggc ctgcttgatg 7140 tctcgtaatc ttcttagaga tggacgaaat gtttcacgac ctaggaaaag gtcgccctac 7200 aaaatagatt tgcgttactc tcttcatagg atccccggtg tagcgaaaga tcaaggcgac 7260 cctaggtagc aaccgccggc ttcggcggta aggtatcact caagaagcag actcagtaag 7320 acacggtcta gctgactgtc tatcgcctag gtcaaatagg gagctttgat tctgcatgtc 7380 cagctttaga ttcactttag cgcgcagatc tgggtcgaga taaaatcacc agtacccaag 7440 accagggggg ctcgccgcgt tggctaatcc tggtacatct tgtaatgaat attcagtaga 7500 aaatttgtgt tagaaggacg agtcaccatg taccaaaagc gataacgatc ggtgggagta 7560 ttcattgtgg tgaagacgct gggtttacgt gggaaaggtg cttgtgtccc aacaggctag 7620 gatataatgc tgaagccctt ccccaagcgt tcagggtggg atttgctaca acttccgagt 7680 ccaacgtgtc cgtgttcatg ttatatatgc acaaggccga gaattggacg tagctttcgt 7740 gttagtacgt agcatggtca cacaagcaca gtagatcctg cccgcgcatc ctatatatta 7800 agttaattct aatggaatac gatgacatgt ggatgggcag tggccggttg ttacacgcct 7860 accgcgatgc tgaatgaccc ggactaaagt ggcgaaaatt atggcgtgtg acccgttatg 7920 ctccagttcg gtcagtgggt cattgcaagt agtcgattgc attgtcaatc tccgagtgat 7980 ttagcgtgac agccgcaggg aacccataaa atgcgatcgt agtccatccg atcgtacata 8040 gaaatgaggg tccccatacg cccacgcacc tgttcactcg tcgtttgcat ttaagagccg 8100 cacgaaccac agagcataaa gaggacctct agctccttta caaagtgggg tcgaccgatc 8160 gcttgcgcaa cttgtgaagt gtctaccatc cctaagccca tttcccgcat attaacccct 8220 gattgtatcc gcatctgatg ctaccgtggt tgagttagcg tcgagcacgc gggacttatt 8280 gcatgagtag agttgactaa gagccgttag atggctcgct gagctaatag ttgccgacag 8340 atcgtcaaga ttagaaaacg gttgtagcat tatcggaggt tctctaacta gtatcgatag 8400 ccgtgtcttc actgtgccgc ggctacctat cgcctgaaaa ccagttggtg ttaaggggtc 8460 ccctgtccag gacgccaccg gtagtgagac atacacgttc gttgggttca ccgcggtcgg 8520 acctgagtgc accaaggaca cactgcagct ccgaccccta ctgtcgagaa atttgtatcc 8580 cgcccccgca gcttgccagc tctttcagta tcatggagcc catggttgaa tgagtccaat 8640 aacgaacttc gacatgataa aatcccccccc tcgcgacttc cagagaagaa gactactgac 8700 ttgagcgttc ccagcacttc agccaaggaa gttaccaatt ttttgtttcc gaatgacacc 8760 ggtctccttg cgggtagatc gccgaccgca gaacttacga gccagggggaa acagtaaggc 8820 ctaattaggt aaagggagta agtgctcgaa cgcttcagat gtaaccatat acttacgctg 8880 gatcttctcc cgcgaatttt aaccctcacc aactacgaga tttgaggtaa accaaataag 8940 cacgtagtgg cgctatccga ctgttcccaa attgtaactt atcgttccgt gaaggccaga 9000 gttattccc ggccctttcc atgcgcgcac cataccctcc tagttccccg gttatctctc 9060 cgaggaggga gtgagcgatc ctccgtttac gttttgttac caatgacgta gctatgtatt 9120 ttgtacaggt tgccaacggg tttcacaatt cacagatagt ggggtacccg gcaaagggcc 9180 tatatttgcg gtccaactta ggcgtaaact acgatggtac ctactcagac ccagctcgcg 9240 cggcgtaaat aacgcactca tcccagctga ttctcggcga tctacgcagc gacatgatta 9300 tcaacagctg tctggcagct ctaatctttt accatggtcg taaaagcctc caagagttag 9360 atcataccta acgccacaaa agtgacacga cgccgatggg taccggactt taggtgcacc 9420 acagttcggt aagggagagg ccctgcggcg tacttcattt tgtatatgca acgtgcccaa 9480 gtggcgccag gcaagtctca gctggttcct gtgttagctg caggctaggc atgggagctg 9540 attgaacatg ggttggggc ctcgaaccgt cgaggacccc atagtacgtt taaacccaag 9600 tagggcagcc tatagtttga agcagtacta tttcaggggg ggagccctca tggtctcttc 9660 tactgatgac tcaacacgct agggacgtga agtcgattcc ttcgatggtt ataaatcaaa 9720 ggctcagagt gcagtctgga gcgcccatct aacggtacgc atctcgattg ctcggtcgcc 9780 tttcacactc cgcgaaaatt cataccgctc attcactagg ttgcgaagcc tacactgata 9840 tatgaatcca agctagagca gggctcttaa aattcggagt tgtagatgct caatactcca 9900 atcggttttt tcgtgcacca ccgcgggtgg ctgacaaggg tttgacatcg agaaaagg 9960 cagttccggg ctgaaagtag cgccgggtaa ggtacgcgcc tggtatggca ggactatgaa 10020 gccaatacaa aggctacatc ctcactcggg tggacggaaa cgcagaatta tggttacttt 10080 ttggatacgt gaaacatgtc ccatggtagc ccaaagactt gggagtctat cacccctagg 10140 gcccatttct ggatatagac gccaggttga atccgtattt ggaggtacga tggatcagtc 10200 tgggtgggac gtgctccatt tataccctgc gcaggctgga ccgaggaccg caagatgcga 10260 cggtgcacaa gtaattgaca acaaaccatc gtgttttcat tatggtacca ggatcttcaa 10320 gccgagtcaa tcaagctcgg attacagtgt ttaccgcgtc ttgcggttac tcacaaactg 10380 taatccacca caagtcaagc cattgcctct ctgagacgcc gtatgaatta atatgtaaac 10440 tttgcgcggg ttcactgcga tccgttcagt ctcgtccaag ggcacaatcg aattcccatt 10500 tgtatgttcg gctaacttct acccatcccc cgaagtttag caggtcgtga ggtgtcatgg 10560 aggctctcgt tcatcccgtg ggacatcgtt taaacgcctt gataaagcac cccgctcggg 10620 tgtagcagag aagacgccta ctgaattgtg cgatccctcc acctcagcta aggtagctac 10680 caatattag tttttagcc ttgcgacaga cctctactt agatgccac gcattgagct 10740 agcgagtcag cgataagcat gacgcgcttt caagcgtcgc gagtagtga accaaggctc 10800 cggacaggac tatatacttg ggttgatct cgccccgaca actgcaacc tcacaattta 10860 tagattataa ggttagccga aattgcacgt ggtggcgccc gccgactgct ccccgagtgt 10920 ggctctttga tctgacaacg cgcgacctcc atcgcggccg attgttctg cggaccatgt 10980 cgtcctcata gtttgggcat gtttccgttg taggagtgaa gccacttagc ttgcgccgt 11040 agtcccaatg aaaaacctat ggacttgtt tgggtagca tcaggaatct gaaccctgtg 11100 aatgtggggg tcgcggcat agacctttat ctccggttca agttaggcat gaggctgtcg 11160 acccctaatc agtgaggcca ccgagtaaaa gagtctgtcc atcacccaaa ttaaccgttg 11220 tcgcaatact tctttgatta gtaatacat cacttgcctg agtagagaa ctcaactat 11280 cggccttgct gtatatatcc agaacaat taccgccagc cattgcaacg gatcgccat 11340 tcgccattca ggctgcgcaa ctgttgggaa gggcgatcgg tgcgggcctc ttcgct 11396 <210> 4 <211> 6630 <212> DNA <213> Artificial Sequence <220> <221> <222> <223> Plasmid pRepCap9-808 <400> 4 ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc 60 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga 120 gatagggttg agtggccgct acagggcgct cccattcgcc attcaggctg cgcaactgtt 180 gggaagggcg tttcggtgcg ggcctcttcg ctattacgcc agctggcgaa agggggatgt 240 gctgcaaggc gattaagttg ggtaacgcca gggttttccc agtcacgacg ttgtaaaacg 300 acggccagtg agcgcgacgt aatacgactc actatagggc gaattggcgg aaggccgtca 360 aggccgcatc ccggggcggc cgcgaggtcc tgtattagag gtcacgtgag tgttttgcga 420 cattttgcga caccatgtgg tcacgctggg tatttaagcc cgagtgagca cgcagggtct 480 ccattttgaa gcgggaggtt tgaacgcgca gccgccatgc cggggtttta cgagattgtg 540 attaaggtcc ccagcgacct tgacgagcat ctgcccggca tttctgacag ctttgtgaac 600 tgggtggccg agaaggaatg ggagttgccg ccagattctg acatggatct gaatctgatt 660 gagcaggcac ccctgaccgt ggccgagaag ctgcagcgcg actttctgac ggaatggcgc 720 cgtgtgagta aggccccgga ggcccttttc tttgtgcaat ttgagaaggg agagagctac 780 ttccacatgc acgtgctcgt ggaaaccacc ggggtgaaat ccatggtttt gggacgtttc 840 ctgagtcaga ttcgcgaaaa actgattcag agaatttacc gcgggatcga gccgactttg 900 ccaaactggt tcgcggtcac aaagaccaga aatggcgccg gaggcgggaa caaggtggtg 960 gatgagtgct acatccccaa ttacttgctc cccaaaaccc agcctgagct ccagtgggcg 1020 tggactaata tggaacagta tttaagcgcc tgtttgaatc tcacggagcg taaacggttg 1080 gtggcgcagc atctgacgca cgtgtcgcag acgcaggagc agaacaaaga gaatcagaat 1140 cccaattctg atgcgccggt gatcagatca aaaacttcag ccaggtacat ggagctggtc 1200 gggtggctcg tggacaaggg gattacctcg gagaagcagt ggatccagga ggaccaggcc 1260 tcatacatct ccttcaatgc gggctccac tcgcggtccc aaatcaagc tgccttggac 1320 aatgcgggaa agattgag cctgactaaa accgcccccg actacctggt gggccagcag 1380 cccgtggagg acatttccag caatcggatt tataaaattt tggaactaa cgggtacgat 1440 cccaatatg cggcttccgt ctttctggga tgggccacga aaagttcgg cagaggaac 1500 accatctggc tgttgggcc tgcactacc gggaagacca accatcgcgga ggccatagcc 1560 cacactgtgc cctttacgg gtgcgtaac tggaccaatg agaactttcc cttcacgac 1620 tgtgtcgaca agatggtgat ctggtgggag gaggggaga tgaccgccaa ggtcgtggag 1680 tcggccaag ccattctcgg aggaagcaag gtgcgcgtgg accagaatg caagtccctcg 1740 gccagatag acccgactcc cgtgatcgtc acctccaaca ccacatgtg cgccgtgatt 1800 gacgggaact caacgacctt cgacaccag cagccgttgc agaccggatt gttcaattt 1860 gaactcaccc gccgtctgga tcatgacttt gggaaggtca ccaagcagga agtcaagac 1920 tttttccggt gggcaaagga tcacgtggtt gaggtggagc atgaattcta cgtcaaaaag 1980 ggtggagcca agaaaagacc cgcccccagt gacgcagata taagtgagcc caaacgggtg 2040 cgcgagtcag ttgcgcagcc atcgacgtca gacgcggaag cttcgatcaa ctacgcagac 2100 aggtaccaaa acaaatgttc tcgtcacgtg ggcatgaatc tgatgctgtt tccctgcaga 2160 caatgcgaga gaatgaatca gaattcaaat atctgcttca ctcacggaca gaaagactgt 2220 ttagagtgct ttcccgtgtc agaatctcaa cccgtttctg tcgtcaaaaa ggcgtatcag 2280 aaactgtgct acattcatca tatcatggga aaggtgccag acgcttgcac tgcctgcgat 2340 ctggtcaatg tggatttgga tgactgcatc tttgaacaat aaatgactta aaccaggtat 2400 ggctgccgat ggttatcttc cagattggct cgaggacaac cttagtgaag gaattcgcga 2460 gtggtgggct ttgaaacctg gagcccctca acccaaggca aatcaacaac atcaagacaa 2520 cgctcgaggt cttgtgcttc cgggttacaa ataccttgga cccggcaacg gactcgacaa 2580 gggggagccg gtcaacgcag cagacgcggc ggccctcgag cacgacaagg cctacgacca 2640 gcagctcaag gccggagaca acccgtacct caagtacaac cacgccgacg ccgagttcca 2700 ggagcggctc aaagaagata cgtcttttgg gggcaacctc gggcgagcag tcttccaggc 2760 caaaaagagg cttcttgaac ctcttggtct ggttgaggaa gcggctaaga cggctcctgg 2820 aaagaagagg cctgtagagc agtctcctca ggaaccggac tcctccgcgg gtattggcaa 2880 atcgggtgca cagcccgcta aaaagagact caatttcggt cagactggcg acacagagtc 2940 agtcccagac cctcaaccaa tcggagaacc tcccgcagcc ccctcaggtg tgggatctct 3000 tacaatggct tcaggtggtg gcgcaccagt ggcagacaat aacgaaggtg ccgatggagt 3060 gggtagttcc tcgggaaatt ggcattgcga ttcccaatgg ctgggggaca gagtcatcac 3120 caccagcacc cgaacctggg ccctgcccac ctacaacaat cacctctaca agcaaatctc 3180 caacagcaca tctggaggat cttcaaatga caacgcctac ttcggctaca gcaccccctg 3240 ggggtatttt gacttcaaca gattccactg ccacttctca ccacgtgact ggcagcgact 3300 catcaacaac aactggggat tccggcctaa gcgactcaac ttcaagctct tcaacattca 3360 ggtcaaagag gttacggaca acaatggagt caagaccatc gccaataacc ttaccagcac 3420 ggtccaggtc ttcacggact cagactatca gctcccgtac gtgctcgggt cggctcacga 3480 gggctgcctc ccgccgttcc cagcggacgt tttcatgatt cctcagtacg ggtatctgac 3540 gcttaatgat ggaagccagg ccgtgggtcg ttcgtcctttt tactgcctgg aatatttccc 3600 gtcgcaaatg ctaagaacgg gtaacaactt ccagttcagc tacgagtttg agaacgtacc 3660 tttccatagc agctacgctc acagccaaag cctggaccga ctaatgaatc cactcatcga 3720 ccaatacttg tactatctt caaagactat taacggttct ggacagaatc aaaacgct 3780 aaaattcagt gtggccggac ccagcaacat ggctgtccag ggaagaaact acatacctgg 3840 acccagctac cgacaacaac gtgtctcaac cactgtgact caaaacaaca acagcgaatt 3900 tgcttggcct ggagcttctt cttgggctct caatggacgt aatagcttga tgaatcctgg 3960 acctgctatg gccagccaca aagaaggaga ggaccgtttc tttcctttgt ctggatcttt 4020 aatttttggc aaacaaggaa ctggaagaga caacgtggat gcggacaaag tcatgataac 4080 4140 cacaaaccac cagagtgccc aagcacaggc gcagaccggc tgggttcaaa accaaggaat 4200 acttccgggt atggtttggc aggacagaga tgtgtacctg caaggaccca tttgggccaa 4260 aattcctcac acggacggca actttcaccc ttctccgctg atgggagggt ttggaatgaa 4320 gcacccgcct cctcagatcc tcatcaaaaa cacacctgta cctgcggatc ctccaacggc 4380 cttcaacaag gaaagctga actctttcat cacccagtat tctactggcc aagtcagcgt 4440 ggagatcgag tgggagctgc agaaggaaaa cagcaagcgc tggaacccgg agatccagta 4500 cacttccaac tattacaagt ctaataatgt tgaatttgct gttaatactg aaggtgtata 4560 tagtgaaccc cgccccattg gcaccagata cctgactcgt aatctgtaat tgcttgttaa 4620 tcaataaacc gtttaatttc tagagcggcc gccccgggct gggcctcatg ggccttccgc 4680 tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaaca tggtcatagc 4740 tgtttccttg cgtattgggc gctctccgct tcctcgctca ctgactcgct gcgctcggtc 4800 gttcgggtaa agcctggggt gcctaatgag caaaaggcca gcaaaaggcc aggaaccgta 4860 aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa 4920 atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc 4980 cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt 5040 ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca 5100 gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg 5160 accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat 5220 cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta 5280 cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct 5340 gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac 5400 aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa 5460 aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa 5520 actcacgtta agggatttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt 5580 5640 gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca 5700 tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc 5760 ccagtgctgc aatgataccg cgagaaccac gctcaccggc tccagattta tcagcaataa 5820 accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc 5880 agtctattaa ttgttgccgg gaagctagag tagtagttc gccagttaat agtttgcgca 5940 acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat 6000 tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag 6060 cggttagctc cttcggtcct ccgatcgttg tcaagtaa gttggccgca gtgttatcac 6120 tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt 6180 ctgtgactgg tgagtactca accaagtcat tctgagata gtgtatgcgg cgaccgagtt 6240 gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccgttcgat gtaacccact cgtgcaccca actgatcttc agcatcttt actttcacca 6420. gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggg ataaggggcga 6480. cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa caatagggg ttccgcgcac atttccccga aaagtgccac <210> 5 <211> 6684 <212> DNA <213> Private Sequence(Artificial Sequence) <220> <221> <222> <223> Project pRepCap9-809(Plasmid pRepCap9-809) <400> 5 ctaaattgta agcgttaata ttttgttaa attcgcgtta aatttttgtt aaatcagctc attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga gatagggttg agtggccgct acagggcgct cccattcgcc attcaggctg cgcaactgtt 180 gggaagggcg tttcggtgcg ggcctcttcg ctattacgcc agctggcgaa agggggatgt 240 gctgcaaggc gattaagttg ggtaacgcca gggttttccc agtcacgacg ttgtaaaacg 300 acggccagtg agcgcgacgt aatacgactc actatagggc gaattgaagg aaggccgtca 360 aggccgcatg atatctgagc gcgcgtaata cgactcacta tagggcgaat tgatatcggg 420 tcccggggcg gccgccgcag ggtctccatt ttgaagcggg aggtttgaac gcgcagccgc 480 catgccgggg ttttacgaga ttgtgattaa ggtccccagc gaccttgacg agcatctgcc 540 cggcatttct gacagctttg tgaactgggt ggccgagaag gaatgggagt tgccgccaga 600 ttctgacatg gatctgaatc tgattgagca ggcacccctg accgtggccg agaagctgca 660 gcgcgacttt ctgacggaat ggcgccgtgt gagtaaggcc ccggaggccc ttttctttgt 720 gcaatttgag aagggagaga gctacttcca catgcacgtg ctcgtggaaa ccaccggggt 780 gaaatccatg gttttgggac gtttcctgag tcagattcgc gaaaaactga ttcagagaat 840 ttaccgcggg atcgagccga ctttgccaaa ctggttcgcg gtcacaaga ccagaatgg 900 cgccggaggc gggaacaag tggtggatga gtgctacatc cccaattact tgctccccaa 960 aacccagcct gagctccagt gggcgtggac taatatggaa cagtatttaa gcgcctgttt 1020 gatctcacg gagcgtaaac gttggtggc gcagcatctg acgcacgtgt cgcagacgca 1080 ggagcagaac aaagagaatc agaatccca ttctgatgcg ccggtca gatcaaaac 1140 ttcagccagg tacatggagc tggtcgggtg gctcgtggac aagggatta cctcggagaa 1200 gcagtggatc caggaggacc aggctcata catctccttc atgcggcct ccaaccgcg 1260 gtcccaaatc aaggctgcct tggacaatgc gggaagatt atgagcctga ctaaaaccgc 1320 ccccgactac ctggtgggcc agcagccgt ggaggacatt tccagcaatc ggattatataa 1380 aattttggaa ctaaacggggt acgatcccca atatgcggct tccgtttc tgggatgggc 1440 cacgaaaaag ttcggcaaga ggacaccat ctggctgttt gggcctgcaa ctaccgggaa 1500 gaccacatc gcggaggcca tagcccac tgtgccctc tacgggtgcg taaactggac 1560 caatgagaac tttcccttca acgactgtgt cgacaagatg gtgatctggt gggaggaggg 1620 gaagatgacc gccaaggtcg tggagtcggc caaagccatt ctcggaggaa gcaaggtgcg 1680 cgtggaccag aaatgcaagt cctcggccca gatagacccg actcccgtga tcgtcacctc 1740 caacaccaac atgtgcgccg tgattgacgg gaactcaacg accttcgaac accagcagcc 1800 gttgcaagac cggatgttca aatttgaact cacccgccgt ctggatcatg actttgggaa 1860 ggtcaccaag caggaagtca aagacttttt ccggtgggca aaggatcacg tggttgaggt 1920 ggagcatgaa ttctacgtca aaaagggtgg agccaagaaa agacccgccc ccagtgacgc 1980 agatataagt gagcccaaac gggtgcgcga gtcagttgcg cagccatcga cgtcagacgc 2040 ggaagcttcg atcaactacg cagacaggta ccaaaacaaa tgttctcgtc acgtgggcat 2100 gaatctgatg ctgtttccct gcagacaatg cgagagaatg aatcagaatt caaatatctg 2160 cttcactcac ggacagaaag actgtttaga gtgctttccc gtgtcagaat ctcaacccgt 2220 ttctgtcgtc aaaaaggcgt atcagaaact gtgctacatt catcatatca tgggaaaggt 2280 gccagacgct tgcactgcct gcgatctggt caatgtggat ttggatgact gcatctttga 2340 acaataaatg acttaaacca ggtatggctg ccgatggtta tcttccagat tggctcgagg 2400 acaaccttag tgaaggaatt cgcgagtggt gggctttgaa acctggagcc cctcaaccca 2460 aggcaaatca acaacatcaa gacaacgctc gaggtcttgt gcttccgggt tacaaatacc 2520 ttggacccgg caacggactc gacaaggggg agccggtcaa cgcagcagac gcggcggccc 2580 tcgagcacga caaggcctac gaccagcagc tcaaggccgg agacaacccg tacctcaagt 2640 acaaccacgc cgacgccgag ttccaggagc ggctcaaaga agatacgtct tttgggggca 2700 acctcgggcg agcagtcttc caggccaaaa agaggcttct tgaacctctt ggtctggttg 2760 aggaagcggc taagacggct cctggaaaga agaggcctgt agagcagtct cctcaggaac 2820 cggactcctc cgcgggtatt ggcaaatcgg gtgcacagcc cgctaaaaag agactcaatt 2880 tcggtcagac tggcgacaca gagtcagtcc cagaccctca accaatcgga gaacctcccg 2940 cagccccctc aggtgtggga tctcttacaa tggcttcagg tggtggcgca ccagtggcag 3000 acaataacga aggtgccgat ggagtgggta gttcctcggg aaattggcat tgcgattccc 3060 aatggctggg ggacagagtc atcaccacca gcacccgaac ctgggccctg cccacctaca 3120 acaatcacct ctacaagcaa atctccaaca gcacatctgg aggatcttca aatgacaacg 3180 cctacttcgg ctacagcacc ccctgggggt attttgactt caacagattc cactgccact 3240 tctcaccacg tgactggcag cgactcatca acaacaactg gggattccgg cctaagcgac 3300 tcaacttcaa gctcttcaac attcaggtca aagaggttac ggacaacaat ggagtcaaga 3360 ccatcgccaa taaccttacc agcacggtcc aggtcttcac ggactcagac tatcagctcc 3420 cgtacgtgct cgggtcggct cacgagggct gcctcccgcc gttcccagcg gacgttttca 3480 tgattcctca gtacgggtat ctgacgctta atgatggaag ccaggccgtg ggtcgttcgt 3540 ccttttactg cctggaatat ttcccgtcgc aaatgctaag aacgggtaac aacttccagt 3600 tcagctacga gtttgagaac gtacctttcc atagcagcta cgctcacagc caaagcctgg 3660 accgactaat gaatccactc atcgaccaat acttgtacta tctctcaaag actattaacg 3720 gttctggaca gaatcaacaa acgctaaaat tcagtgtggc cggacccagc aacatggctg 3780 tccagggaag aaactacata cctggaccca gctaccgaca acaacgtgtc tcaaccactg 3840 tgactcaaaa caacaacagc gaatttgctt ggcctggagc ttcttcttgg gctctcaatg 3900 gacgtaatag cttgatgaat cctggacctg ctatggccag ccacaaagaa ggagaggacc 3960 gtttctttcc tttgtctgga tctttaattt ttggcaaaca aggaactgga agagacaacg 4020 tggatgcgga caaagtcatg ataaccaacg aagaagaaat taaaactact aacccggtag 4080 caacggagtc ctatggacaa gtggccacaa accaccagag tgcccaagca caggcgcaga 4140 ccggctgggt tcaaaaccaa ggaatacttc cgggtatggt ttggcaggac agagatgtgt 4200 acctgcaagg acccatttgg gccaaaattc ctcacacgga cggcaacttt cacccttctc 4260 cgctgatggg agggtttgga atgaagcacc cgcctcctca gatcctcatc aaaaacacac 4320 ctgtacctgc ggatcctcca acggccttca acaaggacaa gctgaactct ttcatcaccc 4380 agtattctac tggccaagtc agcgtggaga tcgagtggga gctgcagaag gaaaacagca 4440 agcgctggaa cccggagatc footcactt ccaactatta caagtctaat aatgttgaat 4500 ttgctgttaa tactgaaggt gtatatagtg aaccccgccc cattggcacc agatacctga 4560 ctcgtaatct gtaattgctt gttaatcaat aaaccgttta attaggacta gtgaggtcct 4620 gtattagagg tcacgtgagt gttttgcgac attttgcgac accatgtggt cacgctgggt 4680 atttaagccc gagtgagcac gcagggtctc cattttgaag cgggaggttt gaacgcgcag 4740 ccgccctcca ttttgtctag agcggccgcc ccgggctggg cctcatgggc cttcctttca 4800 ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaacatgg tcatagctgt 4860 ttccttgcgt attgggcgct ctccgcttcc tcgctcactg actcgctgcg ctcggtcgtt 4920 cgggtaaagc ctggggtgcc taatgagcaa aaggccagca aaaggccagg aaccgtaaaa 4980 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaaatc 5040 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc 5100 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg 5160 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt 5220 cggtgtaggt cgtcgctcc aagctgggct gtgtgcacga accccgtt cagcccgacc 5280 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc 5340 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag 5400 agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg 5460 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa 5520 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag 5580 gatctcaaga agatccttg atcttttcta cggggtctga cgctcagtgg aacgaaaact 5640 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa 5700 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt 5760 attagaaaaa ttcatccagc agacgataaa acgcaatacg ctggctatcc ggtgccgcaa 5820 tgccatacag caccagaaaa cgatccgccc attcgccgcc cagttcttcc gcaatatcac 5880 gggtggccag cgcaatatcc tgataacgat ccgccacgcc cagacggccg caatcaata agccgctaaa acggccattt tccaccataa tgttcggcag gcacgcatca ccatgggtca ccaccagatc ttcgccatcc ggcatgctcg ctttcagacg cgcaaacagc tctgccggtg ccaggccctg atgttcttca tccagatcat cctgatccac caggcccgct tccatacggg 6120. 6180. tacgcgcacg ttcaatacga tgtttcgcct gatgatcaaa cggacaggtc gccgggtcca gggtatgcag acgacgcatg gcatccgcca taatgctcac tttttctgcc ggcgccagat 6240 ggctagacag cagatcctga cccggcactt cgcccagcag cagccaatca cggcccgctt cggtcaccac atccagcacc gccgcacacg gaacaccggt ggtggccagc cagctcagac gcgccgcttc atcctgcagc tcgttcagcg caccgctcag atcggttttc acaaacagca ccggacgacc ctgcgcgctc agacgaaaca ccgccgcatc agagcagcca atggtctgct 6480 gcgcccaatc atagccaaac agacgttcca cccacgctgc cgggctaccc gcatgcaggc catcctgttc aatcatactc ttcctttttc aatattattg aagcatttt cagggttatt 6600. gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc 6660 gcacatttcc ccgaaaagtg ccac 6684 <210> 6 <211> 11584 <212> DNA <213> Artificial Sequence <220> <221> <222> <223> Plasmid pAdHelper861 <400> 6 ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc 60 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga 120 gatagggttg agtggccgct acagggcgct cccattcgcc attcaggctg cgcaactgtt 180 gggaagggcg tttcggtgcg ggcctcttcg ctattacgcc agctggcgaa agggggatgt 240 gctgcaaggc gattaagttg ggtaacgcca gggttttccc agtcacgacg ttgtaaaacg 300 acggccagtg agcgcgacgt aatacgactc actatagggc gaattgaagg aaggccgtca 360 aggccgcatg cggccgcagt actatccgta gatgtacctg gacatccagg tgatgccggc 420 ggcggtggtg gaggcgcgcg gaaagtcgcg gacgcggttc cagatgttgc gcagcggcaa 480 aaagtgctcc atggtcggga cgctctggcc ggtcaggcgc gcgcaatcgt tgacgctcta 540 gaccgtgcaa aaggagagcc tgtaagcggg cactcttccg tggtctggtg gataaattcg 600 caagggtatc atggcggacg accggggttc gagccccgta tccggccgtc cgccgtgatc 660 catgcggtta ccgcccgcgt gtcgaaccca ggtgtgcgac gtcagacaac gggggagtgc 720 tccttttggc ttccttccag gcgcggcggc tgctgcgcta gctttttgg ccactggccg 780 cgcgcagcgt aagcggttag gctggaaagc gaaagcatta agtggctcgc tccctgtagc 840 cggagggtta ttttccaagg gttgagtcgc gggacccccg gttcgagtct cggaccggcc 900 ggactgcggc gaacggggt ttgcctcccc gtcatgcaag accccgcttg caaattcctc 960 cggaaaagg gacgagcccc ttttttgctt ttcccagatg catccggtgc tgcggcagat 1020 gcgcccccct cctcagcagc ggcaagagca agagcagcgg cagacatgca gggcaccctc 1080 ccctcctcct accgcgtcag gaggggcgac atcctcgcga ggtacccaac tccatgctca 1140 acagtcccca ggtacagccc accctgcgtc gcaaccagga acagctctac agcttcctgg 1200 agcgccactc gccctacttc cgcagccaca gtgcgcagat taggagcgcc acttcttttt 1260 gtcacttgaa aaacatgtaa aaataatgta ctagagacac tttcaataaa ggcaaatgct 1320 tttatttgta cactctcggg tgattattta cccccaccct tgccgtctgc gccgtttaaa 1380 aatcaaaggg gttctgccgc gcatcgctat gcgccactgg cagggacacg ttgcgatact 1440 ggtgtttagt gctccactta aactcaggca caaccatccg cggcagctcg gtgaagtttt 1500 cactccacag gctgcgcacc atcaccaacg cgtttagcag gtcgggcgcc gatatcttga 1560 agtcgcagtt ggggcctccg ccctgcgcgc gcgagttgcg atacacaggg ttgcagcact 1620 ggaacactat cagcgccggg tggtgcacgc tggccagcac gctcttgtcg gagatcagat 1680 ccgcgtccag gtcctccgcg ttgctcaggg cgaacggagt caactttggt agctgccttc 1740 ccaaaaaggg cgcgtgccca ggctttgagt tgcactcgca ccgtagtggc atcaaaaggt 1800 gaccgtgccc ggtctgggcg ttaggataca gcgcctgcat aaaagccttg atctgcttaa 1860 aagccacctg agcctttgcg ccttcagaga agaacatgcc gcaagacttg ccggaaaact 1920 gattggccgg acaggccgcg tcgtgcacgc agcaccttgc gtcggtgttg gagatctgca 1980 ccacatttcg gccccaccgg ttcttcacga tcttggcctt gctagactgc tccttcagcg 2040 cgcgctgccc gttttcgctc gtcacatcca tttcaatcac gtgctcctta tttatcataa 2100 tgcttccgtg tagacactta agctcgcctt cgatctcagc gcagcggtgc agccacaacg 2160 cgcagcccgt gggctcgtga tgcttgtagg tcacctctgc aaacgactgc aggtacgcct 2220 gcaggaatcg ccccatcatc gtcacaaagg tcttgttgct ggtgaaggtc agctgcaacc 2280 cgcggtgctc ctcgttcagc caggtcttgc atacggccgc cagagcttcc acttggtcag 2340 gcagtagttt gaagttcgcc tttagatcgt tatccacgtg gtacttgtcc atcagcgcgc 2400 gcgcagcctc catgcccttc tcccacgcag acacgatcgg cacactcagc gggttcatca 2460 ccgtaatttc actttccgct tcgctgggct cttcctcttc ctcttgcgtc cgcataccac 2520 gcgccactgg gtcgtcttca ttcagccgcc gcactgtgcg cttacctcct ttgccatgct 2580 tgattagcac cggtgggttg ctgaaaccca ccatttgtag cgccacatct tctctttctt 2640 cctcgctgtc cacgattacc tctggtgatg gcgggcgctc gggcttggga gaagggcgct 2700 tctttttctt cttgggcgca atggccaaat ccgccgccga ggtcgatggc cgcgggctgg 2760 gtgtgcgcgg caccagcgcg tcttgtgatg agtcttcctc gtcctcggac tcgatacgcc 2820 gcctcatccg cttttttggg ggcgcccggg gaggcggcgg cgacggggac ggggacgaca 2880 cgtcctccat ggttggggga cgtcgcgccg caccgcgtcc gcgctcgggg gtggtttcgc 2940 gctgctcctc ttcccgactg gccatttcct tctcctatag gcagaaaaag atcatggagt 3000 cagtcgagaa gaaggacagc ctaaccgccc cctctgagtt cgccaccacc gcctccaccg 3060 atgccgccaa cgcgcctacc accttccccg tcgaggcacc cccgcttgag gaggaggaag 3120 tgattatcga gcaggaccca ggttttgtaa gcgaagacga cgaggaccgc tcagtaccaa 3180 cagaggataa aaagcaagac caggacaacg cagaggcaaa cgaggaacaa gtcgggcggg 3240 gggacgaaag gcatggcgac tacctagatg tgggagacga cgtgctgttg aagcatctgc 3300 agcgccagtg cgccattatc tgcgacgcgt tgcaagagcg cagcgatgtg cccctcgcca 3360 tagcggatgt cagccttgcc tacgaacgcc acctattctc accgcgcgta ccccccaaac 3420 gccaagaaaa cggcacatgc gagcccaacc cgcgcctcaa cttctacccc gtatttgccg 3480 tgccagaggt gcttgccacc tatcacatct ttttccaaaa ctgcaagata cccctatcct 3540 gccgtgccaa ccgcagccga gcggacaagc agctggcctt gcggcagggc gctgtcatac 3600 ctgatatcgc ctcgctcaac gaagtgccaa aaatctttga gggtcttgga cgcgacgaga 3660 agcgcgcggc aaacgctctg caacaggaaa acagcgaaaa tgaaagtcac tctggagtgt 3720 tggtggaact cgagggtgac aacgcgcgcc tagccgtact aaaacgcagc atcgaggtca 3780 cccactttgc ctacccggca cttaacctac cccccaaggt catgagcaca gtcatgagtg 3840 agctgatcgt gcgccgtgcg cagcccctgg agagggatgc aaatttgcaa gaacaaacag 3900 aggagggcct acccgcagtt ggcgacgagc agctagcgcg ctggcttcaa acgcgcgagc 3960 ctgccgactt ggaggagcga cgcaaactaa tgatggccgc agtgctcgtt accgtggagc 4020 ttgagtgcat gcagcggttc tttgctgacc cggagatgca gcgcaagcta gaggaaacat 4080 tgcactacac ctttcgacag ggctacgtac gccaggcctg caagatctcc aacgtggagc 4140 tctgcaacct ggtctcctac cttggaattt tgcacgaaaa ccgccttggg caaaacgtgc 4200 ttcattccac gctcaagggc gaggcgcgcc gcgactacgt ccgcgactgc gtttacttat 4260 ttctatgcta cacctggcag acggccatgg gcgtttggca gcagtgcttg gaggagtgca 4320 acctcaagga gctgcagaaa ctgctaaagc aaaacttgaa ggacctatgg acggccttca 4380 acgagcgctc cgtggccgcg cacctggcgg acatcatttt ccccgaacgc ctgcttaaaa 4440 ccctgcaaca gggtctgcca gacttcacca gtcaaagcat gttgcagaac tttaggaact 4500 ttatcctaga gcgctcagga atcttgcccg ccacctgctg tgcacttcct agcgactttg 4560 tgcccattaa gtaccgcgaa tgccctccgc cgctttgggg ccactgctac cttctgcagc 4620 tagccaacta ccttgcctac cactctgaca taatggaaga cgtgagcggt gacggtctac 4680 tggagtgtca ctgtcgctgc aacctatgca ccccgcaccg ctccctggtt tgcaattcgc 4740 agctgcttaa cgaaagtcaa attatcggta cctttgagct gcagggtccc tcgcctgacg aaaagtccgc ggctccgggg ttgaaactca ctccggggct gtggacgtcg gcttaccttc 4860 gcaaatttgt acctgaggac taccacgccc accgagattag gttctacgaa gaccaatccc gcccgccaaa tgcggagctt accgcctgcg tcattaccca gggccacatt cttggccaat tgcaagccat caacaaagcc cgccaagagt ttctgctacg aaagggacgg ggggtttact tggaccccca gtccggcgag gagctcaacc caatcccccc gccgccgcag ccctatcagc agcagccgcg ggcccttgct tcccaggatg gcacccaaaa agaagctgca gctgccgccg 5160. ccacccacgg acgagga atactgggac agtcaggcag aggaggtttt ggacgaggag 5220. gaggagaca tgatggaga ctgggagagc ctagacgagg aagcttccga ggtcgaagag gtgtcagacg aaacaccgtc accctcggtc gcattcccct cgccggcgcc ccagaaatcg gcaaccggtt ccagcatggc tacaacctcc gctcctcagg cgccgccggc actgcccgtt 5400 cgccgaccca accgtagtg ggacaccact ggaccaggg ccggtaagtc caagcagccg ccgccgttag cccaagagca acaacagcgc caaggctacc gctcatggcg cgggcacaag 5520 aacgccatag ttgcttgctt gcaagactgt gggggcaaca tctccttcgc ccgccgcttt 5580 cttctctacc atcacggcgt ggccttcccc cgtaacatcc tgcattacta ccgtcatctc 5640 tacagcccat actgcaccgg cggcagcggc agcggcagca acagcagcgg ccacacagaa 5700 gcaaaggcga ccggatagca agactctgac aaagcccaag aaatccacag cggcggcagc 5760 agcaggagga ggagcgctgc gtctggcgcc caacgaaccc gtatcgaccc gcgagcttag 5820 aaacaggatt tttcccactc tgtatgctat atttcaacag agcaggggcc aagaacaaga 5880 gctgaaaata aaaaacaggt ctctgcgatc cctcacccgc agctgcctgt atcacaaaag 5940 cgaagatcag cttcggcgca cgctggaaga cgcggaggct ctcttcagta aatactgcgc 6000 gctgactctt aaggactagt ttcgcgccct ttctcaaatt taagcgcgaa aactacgtca 6060 tctccagcgg ccacacccgg cgccagcacc tgtcgtcagc gccattatga gcaaggaaat 6120 tcccacgccc tacatgtgga gttaccagcc acaaatggga cttgcggctg gagctgccca 6180 agactactca acccgaataa actacatgag cgcgggaccc cacatgatat cccgggtcaa 6240 cggaatccgc gcccaccgaa accgaattct cttggaacag gcggctatta ccaccacacc 6300 tcgtaataac cttaatcccc gtagttggcc cgctgccctg gtgtaccagg aaagtcccgc 6360 tcccaccact gtggtacttc ccagagacgc ccaggccgaa gttcagatga ctaactcagg 6420 ggcgcagctt gcgggcggct ttcgtcacag ggtgcggtcg cccgggtcgc gagtttaaac 6480 tacacaggaa acaggagaca caactccaag tgcatactct atgtcatttt catgggactg 6540 gtctggccac aactacatta atgaaatatt tgccacatcc tcttacactt tttcatacat 6600 tgcccaagaa taaagaatcg tttgtgttat gtttcaacgt gtttattttt caattgcaga 6660 aaatttcaag tcatttttca ttcagtagta tagccccacc accacatagc ttatacagat 6720 caccgtacct taatcaaact cacagaaccc tagtattcaa cctgccacct ccctcccaac 6780 acacagagta cacagtcctt tctccccggc tggccttaaa aagcatcata tcatgggtaa 6840 cagacatatt cttaggtgtt atattccaca cggtttcctg tcgagccaaa cgctcatcag 6900 tgatattaat aaactccccg ggcagctcac ttaagttcat gtcgctgtcc agctgctgag 6960 ccacaggctg ctgtccaact tgcggttgct taacgggcgg cgaaggagaa gtccacgcct 7020 acatggggt agagtcataa tcgtgcatca ggatagggcg gtggtgctgc agcagcgcgc 7080 gaataaactg ctgccgccgc cgctccgtcc tgcaggaata caacatggca gtggtctcct 7140 cagcgatgat tcgcaccgcc cgcagcataa ggcgccttgt cctccgggca cagcagcgca 7200 ccctgatctc acttaaatca gcacagtaac tgcagcacag caccacaata ttgttcaaaa 7260 tcccacagtg caaggcgctg tatccaaagc tcatggcggg gaccacagaa cccacgtggc 7320 catcatacca caagcgcagg tagattaagt ggcgacccct cataaacagg ctggacataa 7380 acattacctc ttttggcatg ttgtaattca ccacctcccg gtaccatata aacctctgat 7440 taaacatggc gccatccacc accatcctaa accagctggc caaaacctgc ccgccggcta 7500 tacactgcag ggaaccggga ctggaacaat gacagtggag agcccaggac tcgtaaccat 7560 ggatcatcat gctcgtcatg atatcaatgt tggcacaaca caggcacacg tgcatacact 7620 tcctcaggat tacaagctcc tcccgcgtta gaccatatc ccaggaca acccattcct 7740. gaatcagcgt aaatcccaca ctgcagggaa gacctcgcac gtaactcacg ttgtgcattg tcaaagtgtt acattcgggc agcagcggat gatcctccag tatggtagcg cggggtttctg 7800. tctcaaaagg aggtagcga tccctactgt acggagtgcg ccgagacaac cgagatcgtg ttggtcgtag tgtcatgcca aatggaacgc cggacgtagt catatttcct gaagcaaaac caggtgcggg cgtgacaaac agatctgcgt ctccggtctc gccgcttaga tcgctctgtg 7980 tagtagttgt agtatatcca ctctctcaaa gcatccaggc gccccctggc ttcggggttct 8040 atgtaaactc cttcatgcgc cgctgccctg 8100. atgtaaactc cttcatgcgc cgctgccctg cccagccaac ctacacattc gttctgcgag tcacacacgg gaggagcggg aagagctgga agaaccatgt tttttttttt attccaaaag attatccaaa acctcaaaat gaagatctat tagtgaacg cgctcccctc cggtggcgtg gtcaaactct acagccaaag aacagataat ggcatttgta agatgttgca caatggcttc caaaaggcaa acggccctca cgtccaagtg gacgtaaagg ctaaaccctt cagggtgaat ctcctctata aacattccag caccttcaac 8400 catgcccaaa taattctcat ctcgccacct tctcaatata tctctaagca aatcccgaat 8460 attaagtccg gccattgtaa aaatctgctc cagagcgccc tccaccttca gcctcaagca 8520 gcgaatcatg attgcaaaaa ttcaggttcc tcacagacct gtataagatt caaaagcgga 8580 acattaacaa aaataccgcg atcccgtagg tcccttcgca gggccagctg aacataatcg 8640 tgcaggtctg cacggaccag cgcggccact tccccgccag gaaccatgac aaaagaaccc 8700 acactgatta tgacacgcat actcggagct atgctaacca gcgtagcccc gatgtaagct 8760 tgttgcatgg gcggcgatat aaaatgcaag gtgctgctca aaaaatcagg caaagcctcg 8820 cgcaaaaaag aaagcacatc gtagtcatgc tcatgcagat aaaggcaggt aagctccgga 8880 accaccacag aaaaagacac catttttctc tcaaacatgt ctgcgggttt ctgcataaac 8940 acaaaataaa ataacaaaaa aacatttaaa cattagaagc ctgtcttaca acaggaaaaa 9000 caacccttat aagcataaga cggactacgg ccatgccggc gtgaccgtaa aaaaactggt 9060 caccgtgatt aaaaagcacc accgacagct cctcggtcat gtccggagtc atatgtaag 9120 actcggtaaa cacatcaggt tgatcacat cggtcagtgc taaaaagcga ccgaatagc 9180 ccgggggaat acatacccgc aggcgtag acacattac agccccata ggaggtataa 9240 caaaattaat aggagaaa aacacataaaaccctgaaa accctccctgc ctaggcaaaa 9300 tagcaccctc ccgctccaga acacataca gcgcttccac agcggcagcc atacagtca 9360 gccttaccag taaaaaagaa aacctattaa aaaaacacca ctcgacacgg caccagctca 9420 atcagtcaca gtgtaaaaaa gggccaagtg cagagcgagt atatatagga ctaaaaaatg 9480 acgtaacggt taaagtccac aaaaaaacacc cagaaaccg cacgcgaacc tacgcccaga 9540 aacgaaagcc aaaaaaccca caacctcctc aaatcgtcac ttccgttttc ccacgttacg 9600 taacttccca ttttagaaa actacaatc ccacacata caagttactc cgccctaaaa 9660 cgttaacgcg gccgcctggg cctcatgggc cttcctttca ctgcccgctt tccagtcggg 9720 aaacctgtcg tgccagctgc attackacatgg tcatagctgt ttccttgcgt attgggcgct 9780 ctccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cgggtaaagc ctggggtgcc 9840 taatgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt 9900 ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg 9960 cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc 10020 tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc 10080 gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc 10140 aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac 10200 tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt 10260 aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct 10320 aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc 10380 ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt 10440 ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg 10500 atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa attaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt attagaaaaa ttcatccagc agcgataaa acgcaatacg ctggctatcc ggtgccgcaa tgccatacag caccagaaaa cgatccgccc attcgccgcc cagttcttcc gcaatatcac gggtggccag cgcaatatcc 10800 tgataacgat ccgccacgcc cagacggccg caatcaata agccgctaa acggccattt tccaccataa tgttcggcag gcacgcatca ccatgggtca ccaccagatc ttcgccatcc ggcatgctcg ctttcagacg cgcaaacagc tctgccggtg ccaggccctg atgttcttca 10980 tccagatcat cctgatccac caggcccgct tccatacggg tacgcgcacg ttcaatacga tgtttcgcct gatgatcaaa cggacaggtc gccgggtcca gggtatgcag acgacgcatg gcatccgcca taatgctcac tttttctgcc ggcgccagat ggctagacag cagatcctga cccggcactt cgcccagcag cagccaatca cggcccgctt cggtcaccac atccagcacc 11220 gccgcacacg gaacaccggt ggtggccagc cagctcagac gcgccgcttc atcctgcagc 11280 tcgttcagcg caccgctcag atcggttttc acaaacagca ccggacgacc ctgcgcgctc 11340 agacgaaaca ccgccgcatc agagcagcca atggtctgct gcgcccaatc atagccaaac 11400 agacgttcca cccacgctgc cgggctaccc gcatgcaggc catcctgttc aatcatactc 11460 ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata 11520 tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg 11580 ccac 11584

Claims

1. A method for producing a recombinant AAV vector, the method comprising the following steps: a) co-transfecting a suitable cell culture with at least two vectors: i) a first plasmid vector characterized in that the plasmid backbone size is above 5000 bp and comprises a heterologous nucleotide sequence flanked by ITRs and a stuffer DNA sequence located outside the ITRs, wherein the length of the stuffer sequence is between 4400 bp and 4800 bp; wherein the first plasmid vector is the plasmid pcohSgsh-900 set forth in SEQ ID NO: 2; and ii) a second plasmid vector comprising a 5' to 3' AAV rep coding region, an AAV cap coding region and a nucleotide sequence comprising an AAV p5 promoter region; wherein the second plasmid vector is the plasmid pRepCap9-809 set forth in SEQ ID NO:5 or the plasmid pAdHelper-861 set forth in SEQ ID NO:6; b) culturing the cells under conditions that allow AAV replication and packaging; c) recovering the AAV produced in step b) and maintaining the cells in said cell culture under conditions that permit further division and growth; d) re-transfecting the cells of step c) with the plasmid vector of step a); and e) Repeat steps b) to c).

2. The method of claim 1, wherein in step b), the AAV is secreted into the supernatant of the cell culture.

3. The method according to claim 1, wherein: Prior to step d) the cell culture medium of the cell culture is exchanged.

4. The method according to claim 3, wherein: Cell culture medium exchange was performed by perfusion.

5. The method according to claim 1, wherein: After resuming step c), steps d), b) and c) are repeated at least once more.

6. The method according to claim 1, wherein: Step b) is performed by culturing the cells in suspension in a stirred liquid culture medium.

7. The method according to claim 1, wherein: The first plasmid vector does not contain the F1Ori nucleotide sequence in the main chain sequence.

8. A method for producing a recombinant AAV vector, the method comprising the steps of: a) Co-transfect appropriate cells with i) a first plasmid vector comprising a heterologous nucleotide sequence flanked by ITRs, wherein the first plasmid vector is characterized in that the plasmid backbone size is greater than 5000 bp and it comprises a stuffer DNA sequence located outside the ITRs, wherein the length of the stuffer sequence is between 4400 bp and 4800 bp; wherein the first plasmid vector is the plasmid pcohSgsh-900 set forth in SEQ ID NO: 2; ii) a second plasmid vector comprising a nucleotide sequence from 5' to 3' AAV rep coding region, an AAV cap coding region and comprising an AAV p5 promoter region, wherein the second plasmid vector is plasmid pRepCap9-809 set forth in SEQ ID NO:5, and iii) a third plasmid vector comprising adenovirus helper functions, including VA-RNA, E2A and E4 sequences, wherein the plasmid does not comprise E3, pTP (E2B), Ad ITR and protease sequences; wherein the third plasmid vector is the plasmid pAdHelper-861 set forth in SEQ ID NO:6; b) culturing the cells under conditions that allow AAV replication and packaging; and c) recovering the AAV produced in step b).

9. The method according to claim 8, wherein: The first plasmid vector i) does not contain the F1Ori nucleotide sequence in the main chain sequence.

10. The method according to claim 8, wherein: The second plasmid vector (ii) comprises an AAV Rep2 coding region and an AAVCap9 coding region.

11. A plasmid vector, comprising: a) a heterologous nucleotide sequence flanked by ITRs; as well as b) a stuffer DNA sequence located outside the ITR and adjacent to one ITR, wherein the length of the stuffer sequence is between 4400 bp and 4800 bp, so that the plasmid backbone size is above 5000 bp; The plasmid vector does not contain the F1Ori nucleotide sequence in the main chain sequence; wherein the plasmid vector is pcohSgsh-900, as shown in SEQ ID NO:

2.

12. A plasmid vector comprising adenovirus helper functions, including VA-RNA, E2A and E4 sequences, wherein the plasmid does not comprise E3, pTP (E2B), Ad ITR and protease sequences; wherein the plasmid vector is pAdHelper-861, as shown in SEQ ID NO:6.

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