CN111876432A - Acquisition and application of novel liver-targeted adeno-associated viruses - Google Patents

Abstract

The present invention provides a set of adeno-associated viruses having properties of interest, e.g., targeting properties and/or neutralizing properties, obtained by directed evolution and in vivo screening methods. The present invention also provides adeno-associated virus capsid protein and viral particles comprising the adeno-associated virus capsid protein, which have excellent or superior targeting and/or neutralizing properties.

Description

Acquisition and application of novel liver-targeted adeno-associated viruses

Technical Field

The invention relates to a group of adeno-associated viruses obtained by directed evolution and in vivo screening methods, and optimized adeno-associated virus capsid proteins and viral vectors comprising the capsid proteins.

Background

The adeno-associated virus (AAV) subtypes, which are known by serotypes to date, are classified into AAV1-AAV12 (Gaoliping, et al, 2004, JVirol 78: 6381-6388; Mori S et al, 2004, Virology 330: 375-383; Schmidt M et al, 2008, JVirol 82:1399-1406), which mainly take human and primate as hosts, wherein the AAV1-6 is separated from human tissues and has definite antibody reactivity, so that the AAV is more classical and recognized. AAV7 and AAV8 are high light level obtained by rescue from heart tissue of macaque through genetic engineering means, and are suspected to be AAV subtypes which have been extincted in evolution, and the strategy provides new ideas and paradigms for discovery of new AAV subtypes and design and modification of recombinant viruses (high light level, 2002, ProcNatl Acad Sci USA 99: 11854-. Although viruses of different AAV serotypes have a regular icosahedral structure, the diversity in sequence and spatial conformation of their capsid proteins allows for significant differences in their cell surface binding to receptors and infection tropism for cells (Timpe J et al, 2005, Curr Gene Ther 5: 273-284). In the aspect of infection tropism, the infection spectrum of AAV2 is wide, and the effect on nerve cells is the best; AAV1 and AAV7 have higher transduction efficiency in skeletal muscle; AAV3 readily transduces megakaryocytes; the advantages of AAV5 and AAV6 for infecting respiratory epithelial cells are significant; the efficiency of transduction of hepatocytes by AAV8 is superior to that of other subtypes.

AAV viruses are replication-defective in nature and can only be latent in host cells in the absence of helper virus. Production of AAV viral vectors requires helper plasmids (helper) to provide key genes for adenovirus (Ad) to participate in AAV replication. These Ad genes include: the early gene E1A is responsible for the transcriptional activity of AAV, the early genes E1B and E4 are involved in maturation of AAVmRNA, and the early genes E2A and VA enhance translation of AAV RNA (Berns et al, 1984, Adeno-associatedvirus 563-592).

Recombinant adeno-associated virus (rAAV) has many advantages as a gene therapy vector, such as high infection efficiency, a wide infection range, long-term expression, and high safety (David AF et al, 2007, BMC Bio 7:75), and has been widely used in clinical trials. At present, the gene therapy project using AAV as carrier enters more than 100 clinical researches, and the disease range of the therapy is expanded to tumor, retina disease, arthritis, AIDS, heart failure, muscular dystrophy, nervous system disease and other series of gene defect diseases.

The clinical research of rAAV vector-based eye disease gene therapy is currently carried out, and most of the clinical research is directed to congenital amaurosis (LCA) caused by mutation of a retinal pigment epithelium specific 65kDa protein coding gene (RPE 65). The Luxturona drug developed by Spark corporation has been approved by FDA and marketed in 12 months of 2017, and is rAAV2 carrying hRPE65v2 gene for treating LCA and hereditary retinal dysplasia. The test results of the subjects after injection showed the effectiveness of their drugs and no significant side effects, especially vehicle-related ones, were found (Russell S et al, 2017, Lancet 390: 849-. Other clinical gene therapy ophthalmology diseases also include choroideremia, most of the research is in the clinical I, II stage, wherein the alberta university utilizes rAAV2 to carry a therapeutic project of Rab guard protein 1 coding gene (REP1), and the I stage research result published in 2018 shows the safety and the effectiveness of the medicine.

There are over twenty clinical studies on gene therapy for hemophilia based on rAAV vectors currently in progress, with hemophilia B being the most studied. AAV gene drugs with long-term expression ability are ideal candidates for treatment of hemophilia B. UniQure is conducting a phase I/II clinical study with AAV5 carrying the human coagulation factor IX encoding gene (hFIX) for hemophilia B, with drug safety shown by follow-up data 1 year after administration. The patient developed a humoral immune response 1 week after dosing, but did not affect the level of coagulation factor IX expression, and no T cell activation could be detected using the current "gold standard" system for T cell detection. Transaminase was elevated but did not affect FIX activity, nor was hepatotoxic response found (Miesbach W et al, 2018, Blood 131: 1022-. There are currently some gene therapy programs for treating hemophilia using AAV8 vector, which are liver-targeted clinical studies (Nathwani AC et al, 2006, blood.107:2653-2661), and AAV8 is currently recognized as the most liver-targeted AAV serotype.

Spinal Muscular Atrophy (SMA) refers to a group of diseases that result in muscle weakness and atrophy due to degeneration mainly of the anterior horn cells of the spinal cord. AVXS-101 from AveXis is currently on the market, and by taking advantage of AAV9 in nervous system infection, it has been shown to have a good effect in treating SMA with a survival motor neuron encoding gene (SMN1), and all patients in clinical trials have no clinical symptoms associated with vector side effects (Mendell JR et al, 2017, N Engl J Med 377: 1713-.

Other rare disease Gene therapy programs using AAV as a vector, for example, AAV1 carrying acid alpha-glucosidase coding Gene (GAA) is used for treating Pompe disease (Smith BK et al, 2013, Hum Gene Ther 24: 630-.

Natural AAV targeting is limited, especially when systemic administration is performed using AAV vectors, the proportion of target cell tissues that can be effectively infected varies greatly depending on the serotype, without maximizing AAV utilization; still other non-targeted tissue cells have the potential to be infected. In addition, since human and other primates are naturally infected with AAV to generate neutralizing antibodies against native AAV, the half-life of AAV is greatly reduced, and the activity of the drug is affected.

There is now an increasing search for the engineering of AAV coat proteins for the purposes of: on one hand, the targeting property of the virus vector can be enhanced, and on the other hand, the immunogenic response of the virus vector can be reduced.

For the carrier serotype with clear mechanism research of virus cell receptor, the small-range or site-specific modification can be directly carried out on the amino acid of the relevant region of the cell receptor. The receptor of AAV2 on cells is well-known in the present study. Heparan Sulfate Proteoglycans (HSPGs) are the major cellular receptors of AAV2 and AAV3 types, and alterations in the amino acid positions R484, R487, K532, R585, R588 on AAV coat protein type 2 affect binding to HSPG (Opie, S.R et al, 2003, J Virol77: 6995-7006; Summerford, C et al, 1999, Nat Med 5: 78-82).

The engineered novel AAV vectors have been used in clinical studies of gene therapy, such as AAV2.5 carrying minidstrophin gene therapy DMD (Bowles DE et al, 2012, Mol Ther 20:443-455), phase I clinical studies have been completed, and a great deal of research has been conducted on the safety of novel AAV. The AAV2.5 coat protein used in the project is a chimera, and 5 amino acids related to skeletal muscle targeting in the AAV1 coat protein are transplanted to the coat protein of AAV2. The chimera not only enhances targeting to skeletal muscle, but also has humoral immune response obviously lower than AAV2, and these clinical tests prove the safety of the modified virus vector.

In addition, under the condition that the cellular receptor mechanism of the virus is not clear, the coat protein of the novel AAV viral vector with optimized functions can be obtained by using a DNA shuffling (DNAsuffling) or error-prone PCR (polymerase chain reaction) method. For example, Grimm et al used AAV to construct a recombinant library, and obtained a chimeric AAV-DJ consisting of AAV2, 8, 9 under the condition of strict screening of intravenous immunoglobulin, and the vector has higher transduction efficiency on multiple cell lines such as fibroblasts and lungs (Grimm D et al, 2008, J Virol 82: 5887-. Jang et al screened a variant that efficiently infected neural stem cells using a DNA shuffling library (Jang J H, 2011, Mol Ther 19: 667-675).

At present, gene therapy medicines become hot spots of domestic and foreign research, and in order to enable the gene therapy medicines to play a role better and longer, a novel AAV vector with optimized functions is searched to better meet the requirement of serving as a gene therapy vector, so that the problem to be solved is urgent.

Disclosure of Invention

Based on the need to find novel AAV vectors, the present invention provides a panel of viruses comprising AAV capsids of traits of interest, e.g., targeting traits and/or neutralizing traits (e.g., the ability to evade neutralizing antibodies), obtained by methods for achieving directed evolution of the viruses by in vivo screening. The invention also provides AAV capsids and virions comprising AAV capsids.

In one aspect, the invention provides a nucleic acid encoding an AAV capsid protein, the nucleic acid comprising an AAV capsid protein coding sequence selected from the group consisting of:

(a) the nucleotide sequence of FIG. 3A (L1) (SEQ ID NO: 1);

(b) the nucleotide sequence of FIG. 3C (L4) (SEQ ID NO: 3);

(c) the nucleotide sequence of FIG. 3E (L10) (SEQ ID NO: 5);

(d) the nucleotide sequence of FIG. 3G (L52) (SEQ ID NO: 7);

(e) the nucleotide sequence of FIG. 3I (L58) (SEQ ID NO: 9);

(f) the nucleotide sequence of FIG. 3K (L84) (SEQ ID NO: 11);

(g) the nucleotide sequence of FIG. 3M (L37) (SEQ ID NO: 13);

(h) the nucleotide sequence of FIG. 3O (L107) (SEQ ID NO: 15);

(i) the nucleotide sequence of FIG. 3Q (L57) (SEQ ID NO: 17); or

(j) A nucleotide sequence that encodes an AAV capsid protein encoded by any one of (a) - (i) but differs from (a) - (i) due to the degeneracy of the genetic code.

In another aspect, the invention provides an AAV capsid protein encoded by a nucleic acid of the invention, the AAV capsid protein having an amino acid sequence selected from any one of SEQ ID NO 2, SEQ ID NO 4, SEQ ID NO 6, SEQ ID NO 8, SEQ ID NO 10, SEQ ID NO 12, SEQ ID NO 14, SEQ ID NO 16, or SEQ ID NO 18.

The invention further provides recombinant virions comprising a viral genome and an AAV capsid protein of the invention, wherein the viral genome is encapsidated in the AAV capsid protein. The invention further provides an AAV viral genome and a recombinant adeno-associated virion of an AAV capsid protein of the invention, wherein the viral genome is enveloped in the AAV capsid protein. In particular embodiments, the viral genome is a recombinant vector genome comprising a heterologous nucleic acid.

The invention provides a cell comprising a nucleic acid of the invention, an AAV capsid protein, a recombinant virion, and/or a recombinant adeno-associated virion.

The invention provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a nucleic acid, AAV capsid protein, recombinant virion, recombinant adeno-associated virion, and/or cell of the invention.

The invention provides uses of the nucleic acids, AAV capsid proteins, recombinant virions, recombinant adeno-associated virions, cells, and/or pharmaceutical compositions described herein in the preparation of a medicament for the prevention or treatment of a disease.

The invention provides a method of producing a recombinant virion comprising an AAV capsid protein, the method comprising: providing a nucleic acid of the invention, an AAV Rep protein coding sequence, a recombinant vector genome comprising a heterologous nucleic acid, and a helper for producing a productive infection to a cell in vitro allows assembly of a recombinant virion comprising AAV capsid proteins, and encapsidation of the recombinant vector genome.

The invention provides a method of producing a recombinant AAV particle comprising an AAV capsid protein, the method comprising: providing a nucleic acid of the invention, an AAV Rep protein coding sequence, a rAAV genome comprising a heterologous nucleic acid, and a helper for production of a productive infection to a cell in vitro allows assembly of a recombinant virion comprising AAV capsid proteins, and encapsidation of the recombinant vector genome.

The invention provides a method of delivering a nucleic acid of interest to a cell, the method comprising providing to the cell a nucleic acid, AAV capsid protein, recombinant virion, recombinant adeno-associated virion, and/or pharmaceutical composition of the invention.

The present invention provides a method of delivering a nucleic acid of interest to a mammalian subject, the method comprising: an effective amount of a nucleic acid, AAV capsid protein, recombinant virion, recombinant adeno-associated virion, cell, and/or pharmaceutical composition of the invention is administered to a mammal.

The invention also provides a method of identifying a viral vector, such as an AAV vector or an AAV capsid protein, having a tropism profile of interest, said method comprising:

(a) providing a collection of viral vectors, such as AAV vectors, wherein each viral vector in the collection comprises:

(i) an AAV capsid protein comprising a capsid protein produced by shuffling two or more different AAV capsid protein coding sequences, wherein the amino acid sequences of the two or more different AAV capsid proteins differ by at least two amino acids; and (ii) a viral vector genome, e.g., an AAV viral genome, comprising a coding sequence encoding (i) said AAV capsid proteins, an AAV Rep protein coding sequence, at least one terminal repeat (e.g., a 5 'and/or 3' terminal repeat) that interacts with AAV Rep proteins, wherein the viral vector genome is encapsidated in the AAV capsid proteins.

(b) Administering the collection of viral vectors to a mammalian subject, and

(c) recovering a plurality of virions or viral vectors encoding viral genomes of the AAV capsid proteins from the target tissue, thereby identifying viral vectors or AAV capsid proteins having a tropism of interest.

The invention has the positive effects that:

the present invention provides a novel set of AAV viral vectors obtained by directed evolution and in vivo screening methods, AAV capsid proteins and viral particles comprising said AAV capsid proteins, viruses having a characteristic AAV capsid of interest, e.g., a targeting characteristic (higher liver tissue targeting) and/or a neutralizing characteristic (e.g., the ability to evade neutralizing antibodies), relative to AAV viral vectors of the prior art.

The present disclosure is further described with reference to the following drawings and detailed description, but is not intended to be limiting. All technical equivalents which may be substituted for elements thereof according to the disclosure are intended to be encompassed by the present patent.

Drawings

FIG. 1 shows the random selection of positive cloning cleavage map from the plasmid library. 1-12 respectively corresponding to randomly selected positive clone samples, and M represents a 5000bp DNA Marker.

FIG. 2. number of AAV mutants in liver, skeletal muscle and heart after two rounds of in vivo screening. After the first in vivo screening, 370 positive clones are obtained, and after the second in vivo screening, 9 groups of novel AAV sequences with high-frequency target liver are obtained.

FIGS. 3A-3R. novel AAV-Cap sequences. Wherein the sequence diagram is divided into a nucleotide sequence and an encoded amino acid sequence.

FIGS. 4A-4F. comparison of infection of different in vitro cell lines (CAG-EGFP). Different AAV vectors are packaged into corresponding viruses carrying CAG-EGFP, different cell lines are infected in vitro through different MOI, and flow cytometry detection analysis is carried out after 48 hours. Values are mean ± sd.

FIGS. 5A-5E comparison of infection of different cell lines in vitro (CAG-Luciferase). Different AAV vectors are packaged into corresponding viruses carrying CAG-Luciferase, different cell lines are infected in vitro through MOI 500, and the Luciferase activity is detected after 48 h. Values are mean ± sd.

FIG. 6. vector genome copy number in different tissues of mice following systemic injection of AAV vectors. The different AAV vectors were packaged into corresponding viruses carrying CAG-Luciferase, and the vector genome copy number was compared in different tissues 2 weeks after tail vein injection of mice at 1E +11vg doses. Values are mean ± sd.

FIGS. 7A-7E. Luciferase activity in different tissues of mice following systemic injection of AAV vectors. The Luciferase activity in different tissues was compared 2 weeks after tail vein injection of mice with the 1E +11vg doses of the corresponding viruses packaged with different AAV vectors carrying CAG-Luciferase. Values are mean ± sd.

Detailed Description

The invention provides a set of viral vectors comprising AAV capsids of traits of interest, e.g., targeting traits and/or neutralizing traits (e.g., the ability to evade neutralizing antibodies), obtained by methods for achieving directed viral evolution by in vivo screening. The invention also provides AAV capsids and virions comprising AAV capsids.

The present invention will be explained in more detail hereinafter. This description is not intended to detail all of the various ways in which the invention may be practiced, and moreover, many variations of the various embodiments of the invention suggested will be apparent to those skilled in the art without departing from the invention. Accordingly, the following description is intended to illustrate some specific embodiments of the invention and not to exhaustively describe all permutations, combinations and variations thereof.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

Unless otherwise indicated, standard methods known to those skilled in the art can be used to produce recombinant and synthetic polypeptides, antibodies or antigen-binding fragments thereof, manipulate nucleic acid sequences, produce transformed cells, construct recombinant AAV, modify capsid proteins, package vectors comprising AAV rep and/or cap coding sequences, and transiently or stably transfect packaging cells. Such techniques are well known to those skilled in the art, see SAMBROOK et al, MOLECULAR CLONING: A LABORATORY MANUAL 2nd Ed. (1989, Cold Spring Harbor, N.Y); AUSUBEL et al, CURRENT PROTOCOLS IN MOLECULARBIOLOGY (Green Publishing Associates, Inc. and John Wiley & Sons, Inc., New York).

All publications, patent applications, patents, nucleotide sequences, amino acid sequences, and other references mentioned herein are incorporated by reference in their entirety.

Definition of I

The specification of all amino acid positions in the AAV capsid subunits in the description and claims herein are related to VP1 capsid subunit numbering.

In the description of the invention and in the claims hereof, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.

As used herein, "and/or" means any and all possible combinations that include one or more of the recited elements.

As used herein, the term "substantially comprises" in relation to a nucleic acid, protein, or capsid structure means that the nucleic acid, protein, or capsid structure comprises any element that can significantly alter the function of the nucleic acid, protein, or capsid structure of interest, e.g., the targeting or neutralizing properties of the protein or capsid or the protein or capsid encoded by the nucleic acid.

The term "adeno-associated virus (AAV)" in the context of the present invention includes, but is not limited to, AAV1, AAV2, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, avian AAV, bovine AAV, canine AAV, equine AAV and ovine AAV as well as any other AAV now known or later discovered (BERNARD n. fields et al, VIROLOGY, volume 2, chapter 69, 4th ed., Lippincott-Raven Publishers). Some additional AAV serotypes and branches have been identified and are also encompassed by the term "AAV" of the invention (highlight et al, 2004, J.virology 78: 6381-.

The genomic sequences of various AAV and parvoviruses are known in the art, as well as the sequences of ITRS, Rep proteins, and capsid protein subunits, and such sequences can be found in the literature or public databases. For example, in the GenBank database, GenBank accession numbers NC 002077, NC 001401, NC 001729, NC 001863, NC 001829, NC 001862, NC 000883, NC001701, NC 001510, AF063497, U89790, AF043303, AF028705, AF028704, J02275, J01901, J02275, X01457, AF288061, AH009962, AY028226, AY028223, NC 00135, NC 001540, AF513851, AF513852, AY530579, AY 631965, AY 631966, the disclosures of which are incorporated herein by reference. Additionally, srivistava et al, 1983, J.virology 45: 555; chiorini et al, 1998, J.virology 71: 6823; chiorini et al, 1999, J.virology 73: 1309; Bantel-Schaal et al, 1999, J.virology 73: 939; xiao et al, 1999, J.virology 73: 3994; muramatsu et al, 1996, Virology 221: 208; shade et al, 1986, J.Virol.58: 921; gao et al, 2002, Proc. Nat. Acad. Sci. USA 99: 11854; international publication Nos. WO00/28061, WO 99/61601, WO 98/11244; us patent 6156303; these disclosures are also incorporated herein by reference in their entirety. For an early description of the terminal repeats of AAV1, AAV2, and AAV3, see Xiao, X,1996, "Characterization of advanced-assisted virus (AAV) DNA replication and" ph.d. discovery, University of Pittsburgh, Pa, which are incorporated herein by reference in their entirety.

A "shuffled" or "chimeric" AAV capsid coding sequence or AAV capsid protein is a portion of nucleic acid sequences and amino acid sequences that are produced by mixing two or more different AAV capsid protein sequences to combine two or more capsid sequences. A "shuffled" or "chimeric" AAV virion comprises an AAV capsid protein that is "shuffled" or "chimeric".

The term "targeted" as used herein refers to the preferential entry of a virus into certain cell or tissue types and/or the preferential interaction with the cell surface to facilitate its entry into certain cell or tissue types, optionally and preferably the expression of sequences carried by the viral genome in the cell, e.g., the expression of heterologous nucleotide sequences by recombinant viruses. In the case of recombinant AAV genomes, gene expression of the viral genome can be from a stably integrated provirus and/or a non-integrated episome, as well as any other form in which viral nucleic acid may occur within a cell.

The term "targeting property" refers to a transduction pattern of one or more target cells, tissues and/or organs. For example, some shuffled AAV capsids may exhibit efficient transduction of liver, gonads, and/or germ cells, with some shuffled AAV capsids having only low levels of transduction of skeletal, diaphragm, and/or cardiac muscle tissue, with typical shuffled AAV capsids having targeting characteristics of high transduction of liver and low transduction of skeletal muscle.

As used herein, "transduction" of a cell by a viral vector refers to the transfer of genetic material into a cell by carrying nucleic acid by the viral vector and subsequently by the viral vector.

As used herein, unless the context indicates otherwise, a "set" or "plurality" of virions, vectors, capsids, or capsid proteins means two or more.

Unless otherwise indicated, "effective transduction" or "effective targeting" or similar terms may be determined with reference to a suitable control, e.g., at least 50%, 60%, 70%, 80%, 85%, 90%, 95% or more transduction or targeting relative to the control.

Similarly, it can be determined by reference to an appropriate control whether the virus is "non-efficiently transduced" or "not effectively targeted" to the target cell or tissue. In particular embodiments, the viral vector does not transduce efficiently skeletal muscle, cardiac myocytes, and in particular embodiments, the non-efficient transduction of the tissue is 20% or less, 10% or less, 5% or less, 1% or less, 0.1% or less of the level of efficient transduction of the tissue.

As used herein, unless otherwise indicated, the term "polypeptide" includes peptides and proteins.

A "nucleic acid" or "nucleotide sequence" is a nucleotide base sequence, which may be an RNA, DNA or DNA-RNA hybrid sequence, including naturally occurring and non-naturally occurring nucleotides, but is preferably a single-or double-stranded DNA sequence.

As used herein, an "isolated" nucleic acid or nucleic acid sequence refers to a nucleic acid or nucleic acid sequence that is separated from at least some other components of a naturally occurring organism or virus, such as, for example, cellular or viral structural components or other polypeptides or nucleic acids that are normally associated with the nucleic acid or nucleic acid sequence.

Likewise, an "isolated" polypeptide refers to a polypeptide that is isolated from at least some other component of a naturally occurring organism or virus, such as a cellular or viral structural component or other polypeptide or nucleic acid normally associated with the polypeptide.

The term "treatment" or grammatical equivalents, refers to a reduction in the severity or at least partial improvement or amelioration of the condition in a subject, and/or at least the achievement of a remission or reduction of one clinical symptom, and/or the delay in progression of the condition and/or the prevention or delay in the onset of a disease or disorder. The term "treating" as used herein also includes prophylactic treatment of a subject, e.g., to prevent the occurrence of an infection, cancer or disease. As used herein, the term "prevention" and grammatical equivalents thereof include any type of treatment in which prevention reduces the incidence of a condition, delays the onset and/or progression of a condition, and/or alleviates a symptom associated with a condition. Thus, unless the context indicates otherwise, the term "treatment" or grammatical equivalents refers to prophylactic and therapeutic methods or regimens.

An "effective" dose as used herein is a dose sufficient to obtain some improvement or benefit to the subject. Alternatively, an "effective" dose is a dose that provides relief or reduction of at least one clinical symptom in a subject. One skilled in the art will appreciate that the therapeutic effect need not be complete or curative so long as the subject gains improvement or benefit.

A "heterologous nucleotide sequence" or "heterologous nucleic acid" is generally not a sequence that occurs naturally in a virus. Typically, the heterologous nucleic acid or nucleotide sequence comprises an open reading frame encoding a polypeptide and/or an untranslated RNA.

A "therapeutic polypeptide" can be a polypeptide that can reduce or alleviate symptoms caused by a protein deletion or defect in a cell or subject. In addition, a "therapeutic polypeptide" can be a polypeptide that otherwise provides a benefit to a subject, such as an anti-cancer effect or an increase in transplant survival.

As used herein, "vector," "viral vector," "delivery vector" generally refers to a viral particle that is a nucleic acid delivery vector, which includes viral nucleic acid, i.e., a vector genome, packaged within the body of a virus. Viral vectors according to the invention include a chimeric AAV capsid of the invention, and may package an AAV or recombinant AAV genome or any other nucleic acid including viral nucleic acids. Alternatively, in certain instances, the terms "vector", "viral vector", "delivery vector" may be used to refer to the vector genome in the absence of viral particles and/or to the viral capsid as a transporter, for delivery of molecules associated with or packaged within the capsid.

A "recombinant AAV vector genome" or "rAAV genome" is an AAV genome comprising at least one inverted terminal repeat and one or more heterologous nucleotide sequences. rAAV vectors typically retain 145 base-Terminal Repeats (TRs) in cis structure to produce a virus; however, modified AAV-TRs and non-AAV-TRs may also be used for this purpose. All other viral sequences are optional and may be provided in trans (Muzyczka, 1992, curr-topics microbial. Immunol.158: 97). The rAAV vector optionally may comprise two TRs, e.g., AAV TRs, typically located at the 5 'and 3' ends of the heterologous nucleotide sequence, but not necessarily adjacent thereto. TRs may be the same or different. The vector genome may also comprise a TR at the 3 'or 5' end.

The term "terminal repeat" or "tr" includes any viral terminal repeat or synthetic sequence that forms a hairpin structure and functions as an inverted terminal repeat, i.e., mediates a desired function such as replication, viral packaging, integration, and/or proviral rescue. The TR may be an AAV TR or a non-AAV TR. For example, the non-AAV TR sequence may be other parvoviruses, e.g., canine parvovirus CPV, mouse parvovirus MVM, human parvovirus B-19, or SV40 hairpin as a replication source of SV40, and may be further modified by truncation, substitution, deletion, insertion. In addition, TR may be partially or fully synthesized, such as the "double D sequence" described in US 5478745.

The "AAV-terminal repeats" or "AAV TRs" may be from any AAV, including but not limited to serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or any other known or later discovered AAV. It is not necessary to have a native terminal repeat sequence, for example, the native AAV TR sequence may be altered by insertion, deletion, truncation and/or missense mutations, so long as the terminal repeat mediates the desired function, e.g., replication, viral packaging, integration, and/or proviral rescue, etc.

The terms "recombinant AAV particle" and "recombinant AAV particle" may be used interchangeably. A "recombinant AAV particle" or "recombinant AAV particle" comprises a recombinant AAV vector genome packaged within an AAV capsid.

"substantially retains" a property means that at least about 75%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% of the property, e.g., activity or other measurable property, is retained.

II. Chimeric AAV capsids identified by directed evolution and in vivo screening

The inventors have identified "chimeric" or "shuffled" AAV capsid structures having features of interest, e.g., targeting properties and/or neutralizing properties. In particular embodiments, the chimeric AAV capsid exhibits high transduction of liver and/or low transduction of skeletal and/or cardiac muscle.

Thus, in some embodiments, the invention provides chimeric AAV capsids comprising or consisting essentially of, and viruses comprising the following amino acid sequences shown in figures 3B, 3D, 3F, 3H, 3J, 3L, 3N, 3P, or 3R.

In particular embodiments, the chimeric AAV capsid protein may comprise or consist essentially of, or consist of, the amino acid sequences shown in fig. 3B, fig. 3D, fig. 3F, fig. 3H, fig. 3J, fig. 3L, fig. 3N, fig. 3P, or fig. 3R, respectively.

Furthermore, in non-limiting embodiments, a chimeric AAV capsid protein of the invention can be encoded by a nucleic acid comprising or consisting essentially of a nucleotide sequence set forth in, or consisting of, the nucleotide sequences set forth in fig. 3A, fig. 3C, fig. 3E, fig. 3G, fig. 3I, fig. 3K, fig. 3M, fig. 3O, or fig. 3Q, respectively; or a nucleotide sequence encoding an AAV capsid or capsid protein encoded by any of the nucleotide sequences described above, but which differs from the nucleotide sequences described above due to the degeneracy of the codons. All amino acid position designations in the present specification and appended claims are in relation to VP1 numbering. One skilled in the art will appreciate that the modifications described herein may also result in alterations of VP2 and/or VP3 capsid subunits due to overlapping AAV capsid coding sequences.

The invention also provides chimeric AAV capsid proteins comprising or consisting essentially of, and methods for evaluating biological properties such as viral transduction and/or antibody neutralization are well known in the art.

Conservative amino acid substitutions are known in the art. In particular embodiments, conservative amino acid substitutions include one or more substitutions in the following group: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; and/or phenylalanine, tyrosine.

It will be apparent to one skilled in the art that the amino acid sequences of the chimeric AAV capsid proteins represented by fig. 3B, 3D, 3F, 3H, 3J, 3L, 3N, 3P, 3R may be applied to any other modification known in the art, by further modification to obtain the desired properties. For example, mutations R484E and R585E in the AAV2 capsid sequence improve transduction of the AAV vector to the heart (Muller et al, 2006, Cardiovasular Research 70: 70-78). As a further non-limiting possibility of modification, the capsid proteins are modified to incorporate targeting sequences or sequences that facilitate purification and/or detection, e.g., the capsid proteins can be fused to all or part of glutathione-S-transferase, maltose binding protein, heparin/heparin sulfate binding domain, poly-HIS, ligands and/or reporters, immunoglobulin Fc fragment, single chain antibody, hemagglutinin, C-MYC, tag epitopes, etc. to form a fusion protein. Methods of inserting targeting peptides into AAV capsids are known in the art, for example, international patent WO 00/28004; nicklin et al, 2001, Molecular Therapy 474-; white et al, 2004, Circulation 109: 513-319; muller et al, 2003, Nature Biotech 21: 1040-.

The viruses of the present invention may further comprise dual viral genomes as described in international patent WO01/92551 and US 7465583.

The invention also provides recombinant virions comprising a chimeric AAV capsid protein of the invention, wherein the vector genome is enveloped in a virion, preferably an AAV vector genome. In particular embodiments, the invention provides a recombinant AAV particle comprising a chimeric AAV capsid protein of the invention, wherein the AAV vector genome is encapsidated in an AAV capsid.

In particular embodiments, the virus is a recombinant vector comprising a heterologous nucleic acid of interest. Thus, the invention is useful for delivering nucleic acids to cells in vitro and in vivo. In representative embodiments, the recombinant vectors of the invention are used to deliver or transfer nucleic acids to animal cells, preferably mammalian cells.

Any heterologous nucleotide sequence can be delivered by the viral vectors of the invention. Nucleic acids of interest include nucleic acids encoding polypeptides, optionally therapeutic polypeptides and/or immunogenic polypeptides.

Therapeutic polypeptides include, but are not limited to, insulin, glucagon, growth hormone, parathyroid hormone, growth hormone releasing factor, follicle stimulating hormone, luteinizing hormone, human chorionic gonadotropin, vascular endothelial growth factor, angiopoietin, angiostatin, granulocyte colony stimulating factor, erythropoietin, connective tissue growth factor, basic fibroblast growth factor, acidic fibroblast growth factor, epidermal growth factor, platelet-derived growth factor, insulin growth factors I and II, any of the transforming growth factor alpha superfamily, activin, inhibin, any of the bone morphogenetic proteins, nerve growth factor, brain-derived neurotrophic factor, neurotrophins NT-3 and NT-4/5, ciliary neurotrophic factor, glial cell line-derived neurotrophic factor, Agrin, any of the semaphorin/disorganin families, netrin-1 and netrin-2, hepatocyte growth factor, ephrin, noggin, sonic hedgehog protein and tyrosine hydroxylase, thrombopoietin, interleukins IL-1 to 1L-25, monocyte chemoattractant protein, leukemia inhibitory factor, granulocyte-macrophage colony stimulating factor, Fas ligand, tumor necrosis factors alpha and beta, interferons alpha, beta and gamma, stem cell factor, flk-2/flt3 ligand. Gene products produced by the immune system may also be used in the present invention, including, but not limited to, immunoglobulin 1gG, IgM, IgA, IgD and IgE, chimeric immunoglobulins, humanized antibodies, single chain antibodies, T cell receptors, chimeric T cell receptors, single chain T cell receptors, class I and II MHC molecules and engineered immunoglobulins, complement regulatory proteins, membrane cofactor proteins, decay accelerating factors, CR1, CF2 and CD59, low density lipoprotein receptors, high density lipoprotein receptors, very low density lipoprotein receptors and clearance receptors, glucocorticoid receptors and estrogen receptors, vitamin D receptors and other nuclear receptors, jun/fos, max, mad, serum effector, AP-1, AP2, myb, MyoD and myogenin, TFE3, E2F, ATF1, ATF2, ATF3, ATF4, ZF5, NFAT, CREB, HNF-4, HNF-EBP/EBP, CCAAT-cassette binding protein, interferon regulatory factor, Wilms tumor protein, ETS-binding protein, STAT, GATA-cassette binding protein and winged helix protein of the forkhead protein family, carbamoyl synthetase 1, ornithine transcarbamylase, argininosuccinate synthetase, argininosuccinate lyase, arginase, fumarylacetoacetate hydrolase, phenylalanine hydroxylase, alpha-1 antitrypsin, glucosidase, glucose-6-phosphatase, porphobilinogen deaminase, cystathionine B synthetase, branched chain ketoacid decarboxylase, isovaleryl-CoA dehydrogenase, propionyl-CoA carboxylase, methylmalonyl-CoA mutase, glutaryl-CoA dehydrogenase, insulin, beta-glucosidase, pyruvate carboxylate, liver phosphorylase, phosphorylase kinase, glycine decarboxylase, propionyl-CoA, and beta-glucosidase, H-protein, T-protein, cystic fibrosis transmembrane conductance regulator, dystrophin, alpha-galactosidase, beta-galactosidase, lysosomal enzyme, coagulation factors, and any other polypeptide having a therapeutic effect in an individual in need thereof.

Heterologous nucleotide sequences encoding polypeptides include sequences encoding reporter polypeptides. The reporter gene known in the art encodes a polypeptide, including but not limited to green fluorescent protein, beta-galactosidase, alkaline phosphatase, luciferase, chloramphenicol acetyltransferase, and the like.

In another aspect, the heterologous nucleic acid may encode an antisense oligonucleotide, including ribozymes, interfering RNAs, including small interfering RNAs that mediate gene silencing (Sharp et al, 2000, Science 287:2431), microRNAs, other untranslated functional RNAs, such as "guide" RNAS (Gorman et al, 1998, Proc. Nat. Acad. Sci. USA 95: 4929; U.S. Pat. No. 5,69248), and the like.

It is known in the art that antisense nucleic acids and inhibitory RNA sequences can induce "exon skipping". Thus, the heterologous nucleic acid may encode an antisense nucleic acid or an inhibitory RNA, inducing appropriate exon skipping.

Ribozymes are RNA protein complexes that cleave nucleic acids in a site-specific manner. Ribozymes have specific catalytic domains with endonuclease activity (Kim et al, 1987, Proc. Natl. Acad. Sci. USA 84: 8788; Gerlach et al, 1987, Nature328: 802; Forster and Symons, 1987, Cell 49: 211).

microRNAs are natural cellular RNA molecules that regulate the expression of multiple genes by controlling the stability of mRNA. Overexpression or reduction of specific micrornas can be used to treat dysfunction and has been shown to be effective in a number of disease states and animal models of disease (Couzin, 2008, Science 319: 1782-. Chimeric AAV can be used to introduce microRNAs into cells, tissues and subjects for the treatment of genetic and acquired diseases, or to enhance the function and promote growth of certain tissues, e.g., mir-1, mir-133, mir-206 and/or mir-208 can be used to treat cardiac and skeletal muscle diseases (Chen et al, 2006, Genet 38: 228-.

The term "antisense oligonucleotide," including "antisense RNA," as used herein, refers to a nucleic acid that is complementary to, and specifically hybridizes to, a particular DNA or RNA sequence. Antisense oligonucleotides and nucleic acids encoding the same can be made according to conventional techniques.

One of skill in the art understands that an antisense oligonucleotide need not be fully complementary to a target sequence, so long as the sequence is similar enough to specifically hybridize the antisense nucleotide sequence to the target sequence and reduce production of a protein product, e.g., at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more. To determine the specificity of hybridization, hybridization of such oligonucleotides to target sequences can be performed under weak, medium, or even stringent conditions.

Antisense oligonucleotides can be synthesized by chemical synthesis and enzyme-binding reactions by procedures known in the art. For example, an antisense oligonucleotide can be chemically synthesized using naturally occurring nucleotides or various modified nucleotides for the purpose of increasing the biological stability of the molecule and/or increasing the stability of the duplex formed between the antisense and sense strands, e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used.

Modified nucleotides useful in the production of antisense oligonucleotides include 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-carboxymethylaminomethyl-2-thiouracil, 5-carboxymethylaminomethyluracil, dihydrouracil, β -D-galactosylquinoline, inosine, N6-isopentenyllysine, 1-methylguanine, 1-methylinosine, 2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytidine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylquinoline, 5' -methoxycarboxymethyluracils, 5-methoxyuracils, 2-methylthio-N6-isopentenylamine, uracil-5-oxyacetic acid, quinoline, 2-thiocytosine, 5-methyl-2-thiouracils, 4-thiouracils, 5-methyluracils, uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid, 5-methyl-2-thiouracils, 2, 6-diaminopurine, and the like.

The antisense oligonucleotide may be chemically modified to covalently bind to another molecule. For example, the antisense oligonucleotide may be conjugated to a molecule that facilitates delivery to the cell of interest, enhances absorption through the nasal mucosa, provides a detectable label, increases the bioavailability of the oligonucleotide, increases the stability of the oligonucleotide, or improves formulation or pharmacokinetic properties, among others. Conjugated molecules include, but are not limited to, cholesterol, lipids, polyamines, polyamides, polyesters, reporter molecules, biotin, dyes, polyethylene glycol, human serum albumin, enzymes, antibodies or antibody fragments, or cell receptor ligands.

Other modifications to nucleic acids to improve stability, nuclease resistance, bioavailability, formulation characteristics, and/or pharmacokinetic properties are also known in the art.

RNA interference is a mechanism of post-transcriptional gene silencing by introducing double-stranded RNA (DsRNA) corresponding to a target sequence into a cell or organism, resulting in degradation of the corresponding mRNA. The mechanisms by which RNAi effects gene silencing have been reported in various review articles (Sharp et al, 2001, Genes Dcv 15: 485-490; Hammond et al, 2001, Nature Rev Gen 2: 110-119). RNAi effects persist through multiple cell divisions before gene expression is restored. Therefore, RNAi is an effective method for targeted knock-outs at the RNA level. RNAi has been shown to be successful in human cells, including human embryonic kidney and HeLa cells (Elbashir et al, 2001, Nature 411: 494-498). It has been demonstrated that short synthetic dsRNAs of approximately 21 nucleotides, also known as "short interfering RNAs", can mediate silencing in mammalian cells without triggering an antiviral response (Elbashir et al, 2001, Nature 411: 494-498; Caplen et al, 2001, Proc. Nat Acad. Sci.98: 9742).

The RNAi molecule can be a short hairpin RNA (Paddis et al, 2002, PNAS USA 99: 1443-. ShRNA typically has a stem-loop structure, i.e., two inverted repeats linked by a short spacer sequence.

Methods for generating RNAi include chemical synthesis, in vitro transcription, Dicer digestion of long dsRNA in vitro or in vivo, expression of delivery vectors in vivo, and expression of RNAi expression cassettes from PCR sources in vivo.

The antisense region of the RNAi molecule can be completely complementary to the target sequence, but need not be completely complementary to the target sequence so long as it specifically hybridizes to the target sequence and reduces production of the protein product, e.g., by at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more. In some embodiments, hybridization of the oligonucleotide to the target sequence can be performed under conditions of weak stringency, moderate stringency, or even high stringency as defined above.

In other embodiments, the antisense region of the RNAi has at least about 60%, 70%, 80%, 90%, 95%, 97%, 98% or more sequence identity to the target sequence and reduces production of the protein product by at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more. In some embodiments, the antisense region comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mismatches compared to the target sequence. Mismatches are generally more acceptable at the ends of the dsRNA than in the central portion. The RNAi molecules can comprise modified sugars, modified nucleotides, backbone linkages, and other modifications of the antisense oligonucleotides described above.

The invention also provides recombinant viral vectors expressing the immunogenic polypeptides. The heterologous nucleic acid can encode any immunogen of interest known in the art, including but not limited to from human immunodeficiency virus, influenza virus, Gag protein, tumor antigens, cancer antigens, bacterial antigens, viral antigens, and the like. Alternatively, the immunogen may be present in the viral capsid, for example, bound to the viral coat by covalent modification.

Parvoviruses are known in the art for use as vaccines (US5916563, US5905040, US 5882652). The antigen may be present in the viral capsid or the antigen may be expressed from a heterologous nucleic acid introduced into the recombinant vector genome.

The immunogenic polypeptide or immunogen may be any polypeptide suitable for protecting a subject against disease, including but not limited to microbial, bacterial, protozoan, parasitic, fungal and viral diseases. The immunogen may be a pro-myxovirus immunogen, for example, an influenza virus immunogen, an influenza virus hemagglutinin surface protein or an influenza virus nucleoprotein gene; or a lentiviral immunogen, e.g., an equine infectious anemia virus immunogen, a simian immunodeficiency virus immunogen, or a human immunodeficiency virus immunogen; or an arenavirus immunogen, e.g., a lassa fever virus immunogen; or a poxvirus immunogen, a flavivirus immunogen, a filovirus immunogen, a bunyavirus immunogen, a coronavirus immunogen or a severe acute respiratory syndrome immunogen. The immunogen may also be a polio immunogen, a herpes immunogen, a mumps immunogen, a measles immunogen, a rubella immunogen, a diphtheria toxin or other diphtheria immunogen, a pertussis antigen, a hepatitis immunogen or any other vaccine immunogen known in the art.

Alternatively, the immunogen may be any tumor or cancer cell antigen. Optionally, the tumor or cancer antigen is expressed on the surface of a cancer cell. Exemplary cancer and tumor antigens include, but are not limited to: BRCA1 gene product, BRCA2 gene product, GP100, tyrosinase, GAGE-1/2, RAGE, NY-ESO-1, CDK-4, 3-catenin, MUM-1, caspase-8, HPVE, SART-1, PRAME, p15, melanoma tumor antigen, HER-2/neu gene product, estrogen receptor, milk fat globulin, p53 tumor suppressor protein, mucin antigen, telomerase, nuclear matrix protein, prostatic acid phosphatase, papillomavirus antigens, and antigens associated with cancers including melanoma, adenocarcinoma, thymoma, sarcoma, lung cancer, liver cancer, colorectal cancer, non-hodgkin's lymphoma, leukemia, uterine cancer, breast cancer, prostate cancer, ovarian cancer, cervical cancer, bladder cancer, kidney cancer, pancreatic cancer, brain cancer, kidney cancer, stomach cancer, esophageal cancer, and head and neck cancer.

Alternatively, the heterologous nucleotide sequence may encode any polypeptide produced in a cell in vitro or in vivo. For example, the viral vector may be introduced into cultured cells and the expressed protein product isolated therefrom.

One skilled in the art understands that the heterologous nucleic acid of interest can be operably linked to appropriate control sequences. For example, the heterologous nucleic acid may be linked to expression control elements such as transcriptional and translational control signals, origin of replication, polyadenylation signals, internal ribosome entry sites, promoters, enhancers, and the like.

One skilled in the art will further appreciate that various promoter/enhancer elements may be used depending on the desired expression level and tissue-specific expression. Promoters/enhancers may be constitutive or inducible, depending on the desired expression pattern. Promoters/enhancers may be natural or foreign, and may be natural or synthetic sequences.

Promoter/enhancer elements may be native to the target cell or subject, or may be native to the heterologous nucleic acid sequence. The promoter/enhancer element is typically selected to function in the target cell of interest. In representative embodiments, the promoter/enhancer element is a mammalian promoter/enhancer element, which may be constitutive or inducible.

Inducible expression control elements are commonly used in applications where it is desirable to regulate the over-expression of a heterologous nucleic acid sequence. Inducible promoter/enhancer elements for gene delivery can be tissue specific or tissue preferred promoter/enhancer elements and include muscle specific or preferred, neural tissue specific or preferred, eye (including retina specific and corneal) specific or preferred, liver specific or preferred, bone marrow specific or preferred, pancreas specific or preferred, spleen specific or preferred, lung specific or preferred. Other inducible promoter/enhancer elements include hormone-inducible and metal-inducible elements. Exemplary inducible promoter/enhancer elements include, but are not limited to, the Tet on/off element, the RU 486-inducible promoter, the rapamycin-inducible promoter, and the metallothionein promoter.

In embodiments where the heterologous nucleic acid sequence is transcribed and translated in the target cell, specific initiation signals are typically used to effect translation of the inserted protein-coding sequence. These exogenous translational control sequences, which may include the ATG initiation codon and adjacent sequences, may be initiated in a variety of forms, including natural and synthetic.

The invention provides chimeric AAV particles comprising a chimeric AAV capsid and an AAV genome. The invention also provides a collection or library of said chimeric AAV particles, wherein said collection or library comprises 2 or more, 10 or more, 50 or more, 10²One or more, 10³One or more, 10⁴Or more, 10⁵Or more, or 10⁶Or more different sequences.

The invention also includes "empty" capsid particles comprising, consisting of, or consisting essentially of the chimeric AAV capsid proteins of the invention, as described in US 5863541. Chimeric AAV capsids of the invention can be used as "capsid carriers," molecules that can be covalently linked, bound, or packaged and transferred into cells include DNA, RNA, lipids, carbohydrates, polypeptides, small organic molecules, or combinations of these molecules. In addition, the molecule can be associated with the exterior of the viral capsid in order to transfer the molecule into a target cell of the host. In one embodiment of the invention, the molecule is covalently linked to the capsid protein. Methods for covalently linking molecules are well known to those skilled in the art.

The viral capsids of the invention can also be used to raise antibodies against new capsid structures. Alternatively, the exogenous amino acid sequence can be inserted into a viral capsid in order to present an antigen to a cell, e.g., administered to a subject to generate an immune response to the exogenous amino acid sequence.

The invention also provides nucleic acids encoding the chimeric capsid proteins of the invention. Further provided are vectors comprising said nucleic acids and cells comprising the nucleic acids and/or vectors of the invention. For example, the nucleic acids, vectors, and cells can be used as reagents to produce the viral vectors described herein.

In exemplary embodiments, the invention provides a nucleic acid sequence encoding an AAV capsid as depicted in figure 3B, 3D, 3F, 3H, 3J, 3L, 3N, 3P, or 3R. Representative nucleic acid sequences comprise or consist essentially of, or consist of, the nucleotide sequences shown in fig. 3A (L1) fig. 3C (L4), fig. 3E (L10), fig. 3G (L52), fig. 3I (L58), fig. 3K (L84), fig. 3M (L37), fig. 3O (L107), or fig. 3Q (L57), respectively, or the nucleotide sequences shown in these figures, or nucleotide sequences encoding an AAV capsid or capsid protein encoded by any of the above nucleotide sequences, but which differ from the above nucleotide sequences due to the degeneracy of the codons, which allow different nucleic acid sequences to encode the same AAV capsid.

The invention also provides nucleic acids encoding variants and fusion proteins of the above-described AAV capsid proteins. In particular embodiments, the nucleic acid hybridizes to the complementary strand of the nucleic acid sequence specifically disclosed herein under standard conditions known to those skilled in the art, encoding a variant capsid protein. The nucleic acid sequences specifically disclosed herein, with reference to fig. 3A, fig. 3C, fig. 3E, fig. 3G, fig. 3I, fig. 3K, fig. 3M, fig. 3O, or fig. 3Q, optionally, the variant capsid protein substantially retains at least one property of the capsid protein encoded by the nucleic acid sequences shown in fig. 3A, fig. 3C, fig. 3E, fig. 3G, fig. 3I, fig. 3K, fig. 3M, fig. 3O, or fig. 3Q. For example, a virion with a variant capsid protein can substantially retain the targeting characteristics of a virion comprising a capsid protein encoded by a nucleic acid coding sequence as shown in figure 3A, figure 3C, figure 3E, figure 3G, figure 3I, figure 3K, figure 3M, figure 3O, figure 3Q. Hybridization of such sequences can be carried out under weak, medium or even stringent conditions (Sambrook et al, 1989, Molecular Cloning, A Laboratory Manual 2dEd, Cold Spring Harbor Laboratory).

As is known in the art, many different procedures can be used to determine whether a nucleic acid or polypeptide has a percent identity or similarity to a known sequence. Percent identity, as used herein, refers to a nucleic acid or fragment thereof having a specified percentage of another nucleic acid when aligned with another nucleic acid using BLASTN, which is available from the National Center for Biotechnology Information (NCBI) via the internet.

When referring to a polypeptide, percent identity or similarity indicates that the polypeptide exhibits a particular percent identity or similarity when compared to another protein or portion thereof over a common length as determined using BLASTP. This is also available from the National Center for Biotechnology Information (NCBI) through the Internet. The percent identity or similarity of polypeptides is typically determined using sequence analysis software, for example, see the sequence analysis software package of the genetics computer group at the university of wisconsin biotechnology center. Protein analysis software uses the homology of various substitutions, deletions and other modifications to match similar sequences. Conservative substitutions typically include substitutions in the following classes: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; phenylalanine, tyrosine.

In particular embodiments, the nucleic acid may comprise, consist of, or consist essentially of, but is not limited to, a plasmid, a phage, a viral vector, a bacterial artificial chromosome, or a yeast artificial chromosome, among other vectors. Viral vectors include, but are not limited to, adeno-associated viral vectors, adenoviral vectors, herpes viral vectors, baculovirus vectors, or hybrid viral vectors.

In some embodiments, the nucleic acid encoding the chimeric AAV capsid protein further comprises an AAV Rep protein coding sequence.

The invention also provides cells stably comprising a nucleic acid of the invention. For example, the nucleic acid may be stably transformed into the genome of the cell, or may be stably maintained in an episomal form, e.g., an "EBV-based nuclear episome.

The nucleic acid can be inserted into a delivery vector, e.g., a viral delivery vector. For example, the nucleic acids of the invention may be encapsulated in AAV particles, adenovirus particles, herpesvirus particles, baculovirus particles, or any other suitable virus particles.

In addition, the nucleic acid may be operably linked to a promoter element.

The invention also provides a method for producing the viral vector of the invention. In one representative embodiment, the invention provides a method of producing a recombinant viral vector, the method comprising providing to a cell in vitro, comprising a heterologous nucleic acid and a signal sequence, e.g., an AAV terminal repeat, sufficient to package an AAV template into a virion; also included are AAV sequences sufficient to replicate and package the template into a virion, such as AAV Rep sequences, and sequences encoding the AAV capsids of the invention. The method may further comprise the step of collecting viral particles from the cells, which viral particles may be collected from the culture medium and/or lysed cells.

In an illustrative embodiment, the invention provides a method of making a recombinant AAV particle comprising an AAV capsid, the method comprising: providing a nucleic acid encoding a chimeric AAV capsid of the present invention, an AAV Rep coding sequence, an AAV vector genome comprising a heterologous nucleic acid, and a helper factor for production of infectious AAV to cells in vitro allows the AAV vector genome to be encapsulated in the AAV capsid and complete assembly of the AAV particle.

The cell is typically one that allows replication of the AAV virus. Any suitable cell known in the art may be used, including, but not limited to, one or more of the HEK293 cell line, HEK293T cell line, HEK293A cell line, HEK293S cell line, HEK293FT cell line, HEK293F cell line, HEK293H cell line, HeLa cell line, SF9 cell line, SF21 cell line, SF900 cell line, BHK cell line.

AAV replication and capsid sequences can be provided by any method known in the art. Current methods typically express AAV rep and cap genes on a single plasmid. AAV replication and packaging sequences need not be provided together. AAV rep and/or cap gene sequences may be provided by any viral or non-viral vector. For example, rep and/or cap gene sequences may be provided by hybrid adenovirus or herpes virus vectors. EBV vectors can also be used to express AAVcap and/or rep gene sequences. Alternatively, the rep and/or cap gene sequences may be stably present in the cell, in an episomal or integrated state.

Typically, AAV rep and/or cap gene sequences are not surrounded by AAV packaging sequences to prevent rescue and/or packaging of these sequences.

The template or vector genome may be provided to the cell using any method known in the art. The template or vector genome may be provided by a non-viral vector or a viral vector. In particular embodiments, the template or vector genome is provided by a herpesvirus or adenovirus vector. Baculovirus vectors, EBV vectors can also be used to deliver the template or vector genome. In another representative embodiment, the template or vector genome is provided by a replicating rAAV virus. In other embodiments, the AAV provirus is stably integrated into the chromosome of the cell.

To obtain maximal viral titers, the cells are usually supplied with helper viruses, such as adenovirus or herpes virus, which are necessary for the production of infectious AAV. Helper viral sequences, known in the art to be necessary for AAV replication, are typically provided by helper adenovirus or herpesvirus vectors. Alternatively, the adenoviral or herpesvirus sequences may be provided by another non-viral or viral vector (Ferrari et al, 1997, Nature Med.3: 1295). Furthermore, helper virus function can be provided by integration of the helper gene in the chromosome of the packaging cell or maintained as a stable extrachromosomal element.

It is understood by those skilled in the art that it may be advantageous to provide AAV replication and capsid sequences as well as helper viral sequences on a single helper construct. The helper construct may be a non-viral or viral construct, and may alternatively be a hybrid adenovirus or a hybrid herpesvirus comprising the AAVrep/cap gene sequence.

In a particular embodiment, the AAV rep and/or cap gene sequences and adenoviral helper sequences are provided by a single adenoviral helper vector. This vector further comprises a rAAV genomic template. AAV rep and/or cap sequences and/or rAAV templates may be inserted into deleted regions of the adenovirus, for example, the Ela or E3 regions.

In another embodiment, the AAV rep and/or cap sequences and adenoviral helper sequences are provided by a single adenoviral helper vector. The rAAV genome template is provided by a plasmid. In another illustrative embodiment, the AAV rep and/or cap sequences and adenoviral helper sequences are provided by a single adenoviral helper vector, and the rAAV genomic template is integrated into the cell as a precursor. Alternatively, the rAAV template is provided by an EBV vector maintained intracellularly as an extrachromosomal element. In another exemplary embodiment, the AAV rep and/or cap sequences and adenoviral helper sequences are provided by a single adenoviral vector. The rAAV genome template is provided as a separate replicating viral vector. For example, the rAAV template may be provided by a rAAV particle or a second recombinant adenovirus particle.

Herpes viruses are also used as helper viruses in AAV packaging methods.

As another alternative, the viral vectors of the invention may be used to deliver rep and/or cap genes and rAAV templates in insect cells using baculovirus vectors (Urabe et al, 2002, Human Gene Therapy 13: 1935-.

Other methods of producing AAV can also use stably transformed packaging cells (see U.S. Pat. No. 5,5658785).

AAV vectors free of helper virus contamination can be obtained by any method known in the art. For example, AAV and helper viruses can be readily distinguished by size. AAV can also be isolated from helper virus based on affinity for heparin substrates. In representative embodiments, replication-defective helper viruses are used such that any contaminating helper virus is unable to replicate. Alternatively, helper adenoviruses lacking late gene expression may be used, as only adenoviral early gene expression mediates packaging of AAV virus. Adenoviral mutants deficient in late gene expression are known in the art, e.g., TS100K and TS149 adenoviral mutants.

The packaging methods of the invention can be used to produce high titer viral particles. In particular embodiments, the titer of the viral stock is at least about 10⁵Tu/ml, at least about 10⁶Tu/ml, at least about 10⁷Tu/ml, at least about 10⁸Tu/ml, at least about 10⁹Tu/ml, at least about 10¹⁰Tu/ml。

The novel capsid proteins and capsid structures are useful for the production of antibodies, e.g., for diagnostic or therapeutic use or as research reagents. Accordingly, the invention also provides antibodies against the novel capsid proteins of the invention.

The term "antibody" or "antibody fragment" as used herein refers to all types of immunoglobulins, including IgG, IgM, IgA, IgD, and IgE. The antibody may be monoclonal or polyclonal, and may be derived from any species, including mouse, rat, rabbit, horse, goat, sheep, chicken, monkey, alpaca, or human, or may be a chimeric, humanized, human antibody. The antibody may be a recombinant monoclonal antibody, or screened from a phage library, a yeast library, a mammalian cell display library.

Antibody fragments encompassed within the scope of the invention include Fab, F (ab')₂And Fc fragments, as well as corresponding fragments obtained from antibodies other than IgG. Such fragments may be produced by known techniques. For example, F (ab')₂Fragments may be produced by pepsin digestion of an antibody molecule, and Fab fragments may be produced by reducing F (ab')₂Disulfide bonding of the fragments occurs. Alternatively, Fab expression libraries can be constructed to quickly and easily identify monoclonal Fab fragments with the desired specificity.

Polyclonal antibodies can be obtained by immunizing a suitable animal, such as a rabbit, a goat, etc., with a virus, collecting immune serum from the animal, and isolating the immune serum.

The invention also includes methods of delivering heterologous nucleotide sequences to a wide range of cells, including dividing and non-dividing cells. The viral vectors of the invention may be used to deliver nucleotide sequences of interest to cells in vitro, e.g., for the production of polypeptides in vitro or for in vitro gene therapy. The vectors may also be used in methods of delivering a nucleotide sequence to an individual in need thereof, e.g., to express an immunogenic or therapeutic polypeptide.

In general, the viral vectors of the invention can be used to deliver any exogenous nucleic acid with a biological effect to treat or ameliorate any disease associated with gene expression. Furthermore, the invention may be used to treat any disease for which delivery of a therapeutic polypeptide may be improved. Exemplary disease symptoms include, but are not limited to: cystic fibrosis (cystic fibrosis transmembrane regulator) and other diseases of the lung, hemophilia a (factor VIII), hemophilia B (factor IX), thalassemia (β -globin), anemia (erythropoietin) and other blood disorders, senile dementia (GDF), multiple sclerosis (β -interferon), parkinson's disease (glial cell line-derived neurotrophic factor), huntington's disease (abrogated inhibitory RNA including but not limited to RNAi, such as siRNA or shRNA, antisense RNA or microRNA), amyotrophic lateral sclerosis, epilepsy (galanin, neurotrophic factor) and other neurological disorders, cancer (endostatin, angiostatin, TRAIL, FAS ligand, cytokine including interferon, inhibitory RNA including but not limited to siRNA, shRNA, antisense RNA, microRNA, multiple drug-resistant gene products, VEGF-inhibiting inhibitory RNA, including but not limited to siRNA, shRNA, antisense RNA, microRNA, and the like, Cancer immunogens), diabetes (insulin, PGC-alpha 1, GLP-1, myostatin precursor peptide, glucose transporter), muscular dystrophy including Duchenne muscular dystrophy and Becker muscular dystrophy (e.g., dystrophin, mini-dystrophin, micro-dystrophin, insulin-like growth factor, etc.), glycogen storage cases such as Fabry's disease (alpha-galactosidase) and Pompe disease (lysosomal acid alpha-glucosidase), congenital emphysema (antitrypsin), Lesch-Nyhan syndrome (hypoxanthine guanine phosphoribosyltransferase), Niemann-Pick's disease (sphingomyelinase), retinal degenerative diseases and other diseases of the eye and retina (PDGF, endostatin and/or angiostatin to treat macular degeneration), astrocytomas (endostatin, angiostatin and/or RNAi to inhibit VEGF), Glioblastoma (endothelial growth factor, vascular endothelial growth factor and/or RNAi anti-vascular endothelial growth factor), liver (RNAi for hepatitis B and/or C genes, e.g., siRNA or shRNA, microRNA or antisense RNA), congestive heart failure or peripheral artery disease (phosphatase protein inhibitor I, phospholipase, intrasarcoplasmic Ca2-ATPase, zinc finger protein regulating phospholipase gene, phospholipase inhibitor, etc.), arthritis (insulin-like growth factor), AIDS (soluble CD4), muscle atrophy (insulin-like growth factor I, myostatin pro peptide, anti-apoptotic factors, etc.), limb ischemia (VEGF, FGF, PGC-I alpha, EC-SOD, HIF), erythropoietin), arthritis (anti-inflammatory factors such as IRAP and TNF alpha soluble receptors), hepatitis (alpha-interferon), kidney deficiency (kidney deficiency ), etc, Deficiency of low-density lipoprotein receptor (LDL receptor), hyperammonemia (ornithine aminotransferase), phenylketonuria (phenylalanine hydroxylase), autoimmune diseases, and the like.

The invention may also be used to increase the success of transplantation and/or reduce the side effects of organ transplantation or adjuvant therapy after organ transplantation, e.g., by blocking cytokine production by administration of immunosuppressive or inhibitory nucleic acids.

Gene transfer has great potential use in the recognition and treatment of disease. There are many genes that are defective in genetic diseases and have been cloned. In general, the above disease states fall into two categories: the first is a defective state, usually an enzyme defect, usually inherited in a recessive manner; the second is an unbalanced state, possibly involving regulatory or structural proteins, usually inherited in a dominant fashion. For defective conditions, gene transfer can bring normal genes into the affected tissues for replacement therapy, as well as create animal models for the disease using inhibitory RNAs, including siRNA or shRNA, microRNA, or antisense RNA. For unbalanced disease states, gene transfer can be used to create a disease state in the model system, which can then be used to treat the disease state. Thus, the viral vector according to the invention allows the treatment of genetic diseases. As used herein, a disease state is treated by a defect or imbalance that partially or wholly remedies or aggravates the disease.

In addition, the viral vectors of the invention have further utility in diagnostic and screening methods wherein the gene of interest is transiently or stably expressed in a cell culture system or transgenic animal model. The invention may also be used to deliver nucleic acids for protein production, e.g., for laboratory, industrial, or commercial purposes.

Alternatively, the viral vector can be administered to the cells and the altered cells to the subject. Introducing a heterologous nucleic acid into the cell, and administering the cell to the subject, wherein the heterologous nucleic acid encoding the immunogen is optionally expressed and induces an immune response in the subject to the immunogen. In particular embodiments, the cell is an antigen presenting cell, such as a dendritic cell.

An "active immune response" or "active immunity" is characterized by "involvement of host tissues and cells after contact with an immunogen". It involves the differentiation and proliferation of immunoregulatory cells in lymphoid tissues, resulting in antibody synthesis or cell-mediated responses, or both. Alternatively, a host may develop a positive immune response upon exposure to an immunogen, either through infection or vaccination. Active immunization can be contrasted with passive immunization, which is achieved by "transferring a preformed substance, such as an antibody, transfer factor, thymic graft, interleukin-2, from an actively immunized host to a non-immunized host".

As used herein, a "protective" immune response or "protective" immunity indicates that the immune response imparts some benefit to the subject in that it may prevent or reduce the incidence of disease. Alternatively, the protective immune response or protective immunity may be used to treat a disease, particularly a cancer or tumor, e.g., to cause regression of the cancer or tumor and/or to prevent metastasis and/or to prevent the growth of metastatic nodules. The protective effect may be complete or partial, as long as the therapeutic benefit is greater than the disadvantage.

The viral vectors of the invention may also be used in cancer immunotherapy by administering cancer cell antigens or immunologically similar molecules or any other immunogen to generate an immune response against cancer cells. For example, in treating a cancer patient, an immune response to a cancer cell antigen in the subject can be generated by administering a viral vector comprising a heterologous nucleotide sequence encoding the cancer cell antigen. As described herein, the viral vectors can be administered to a subject in vitro or by in vitro methods.

As used herein, the term "cancer" includes tumor-forming cancers. Similarly, the term "cancerous tissue" also includes tumors. "cancer cell antigens" include tumor antigens.

The term "cancer" has its understood meaning in the art, e.g., with uncontrolled tissue growth that spreads or metastasizes to remote sites in the body. Exemplary cancers include, but are not limited to, leukemia, lymphoma, colorectal cancer, renal cancer, liver cancer, breast cancer, lung cancer, prostate cancer, testicular cancer, ovarian cancer, uterine cancer, cervical cancer, brain cancer, bone cancer, sarcoma, melanoma, head and neck cancer, esophageal cancer, thyroid cancer, and the like. In embodiments of the invention, the invention is practiced to treat and/or prevent neoplasia cancer.

Cancer cell antigens have been described above. The term "treating cancer" is intended to reduce the severity of the cancer, prevent or at least partially eliminate the cancer. For example, in certain instances, these terms indicate that the treatment of cancer is prevented or reduced, or at least partially eliminated. In yet another representative embodiment, the terms indicate that the growth of metastatic nodules is prevented or reduced or at least partially eliminated, for example, after surgical resection of a primary tumor. According to the term "preventing cancer", it is intended to at least partially eliminate or reduce the incidence or incidence of cancer. In other words, the subject's cancer onset or progression may be slowed, controlled, reduced or delayed in likelihood or probability.

In particular embodiments, the cells can be removed from an individual having cancer and contacted with a viral vector of the invention. The modified cells are then administered to a subject, thereby eliciting an immune response to the cancer cell antigen. This method is particularly useful in immunocompromised subjects who are unable to produce an adequate immune response in vivo, i.e., produce an adequate amount of boosting antibodies.

Immunomodulatory cytokines are known in the art, such as alpha-interferon, beta-interferon, gamma-interferon, omega-interferon, tau-interferon, interleukin-l alpha, interleukin-1 beta, interleukin-2, interleukin-3, interleukin-4, interleukin 5, interleukin-6, interleukin-7, interleukin-8, interleukin-9, interleukin-10, interleukin-11, interleukin 12, interleukin-13, interleukin-14, interleukin-18, B cell growth factor, CD40 ligand, tumor necrosis factor-alpha, tumor necrosis factor-beta, monocyte chemotactic protein-1, granulocyte-macrophage colony stimulating factor, and lymphotoxin, and immunomodulatory cytokines, such as CTL-inducing cytokines, can be administered to the subject along with the viral vector.

The cytokine may be injected by any method known in the art. Exogenous cytokines can be injected into a subject or a nucleotide sequence encoding the cytokine can be delivered to the subject using a suitable carrier and the cytokine produced in vivo.

The recombinant viral vectors according to the invention can be used in veterinary and medical applications. Suitable subjects include birds and mammals. The term "avian" as used herein includes, but is not limited to, chickens, ducks, geese, quail, turkeys, pheasants, parrots. The term "mammal" as used herein includes, but is not limited to, humans, primates, non-human primates, cows, sheep, goats, pigs, horses, cats, dogs, rabbits, rodents and the like. Human subjects include neonates, infants, juveniles, and adults. Optionally, the individual "in need of the methods of the invention, for example, because the individual has or is thought to have a risk of, or would benefit from, delivery of nucleic acids including the invention. As a further alternative, the subject may be a laboratory animal and/or an animal model of disease.

In particular embodiments, the invention provides pharmaceutical compositions comprising a viral vector of the invention in a pharmaceutically acceptable carrier, and optionally including other agents, stabilizers, buffers, carriers, adjuvants, diluents, and the like, the carrier for injection being generally a liquid. For other methods, the transport carrier may be either solid or liquid. For administration by inhalation, the carrier will be respirable, preferably in the form of solid or liquid particles.

"pharmaceutically acceptable" refers to a material that is non-toxic or otherwise undesirable in character, i.e., the material can be administered to a subject without producing any undesirable biological effects.

One aspect of the invention is a method of transferring a nucleotide sequence to a cell in vitro. Viral vectors can be introduced into cells at appropriate fold infection according to standard transduction methods appropriate for the particular target cell. The titer of the viral vector or capsid to be administered can vary depending on the cell type and number of interest and the particular viral vector or capsid. In particular embodiments, at least about 10³Infectious unit, more preferably at least about 10⁵The infectious unit is introduced into the cell.

The cells to be introduced into the viral vector may be of any type, including, but not limited to, neural cells, including cells of the peripheral and central nervous system, particularly brain cells, such as neurons, oligodendrocytes, glial cells, astrocytes, lung cells, ocular cells including retinal cells, retinal pigment epithelium and corneal cells, epithelial cells including intestinal and respiratory epithelial cells, skeletal muscle cells including myoblasts, myotubes and muscle fibers, diaphragm muscle cells, dendritic cells, pancreatic cells including pancreatic islet cells, liver cells, gastrointestinal tract cells including smooth muscle cells and epithelial cells, cardiac cells including cardiac muscle cells, bone marrow cells, hematopoietic stem cells, spleen cells, keratinocytes, fibroblasts, endothelial cells, prostate cells, joint cells including, for example, cartilage, meniscus, synovium and bone marrow, Germ cells, and the like. Alternatively, the cell may be a stem cell, e.g., a neural stem cell, a hepatic stem cell. Alternatively, the cell may be a cancer or tumor cell, such as the cancers and tumors described above. Furthermore, as noted above, the cells may be from any species of origin.

The viral vector can be introduced into cells ex vivo to administer the modified cells to a subject. In particular embodiments, the cells are removed from the individual, into which the viral vector is introduced, and then returned to the individual. Methods of removing cells from a subject for in vitro therapy and then reintroducing into the subject are known in the art, see, e.g., U.S. patent No. 5399346. Alternatively, the recombinant viral vector is introduced into cells from another individual, cultured cells, or cells from any other suitable source, and the cells are administered to the individual in need thereof.

Cells suitable for in vitro gene therapy are described above. The dosage of cells administered to a subject will vary with the age, condition and species of the subject, the cell type, the nucleic acid expressed by the cell, the mode of administration, and the like. Typically, at least 10 doses per dose are administered in a pharmaceutically acceptable carrier²To 10⁸Or about 10³To about 10⁶And (4) cells. In particular embodiments, cells transduced with a viral vector are administered to an individual in an effective amount in combination with a pharmaceutical carrier.

Another aspect of the invention is a method of administering a viral vector or capsid of the invention to a subject. In particular embodiments, the methods include a method of delivering a nucleic acid of interest to an animal subject, the method comprising: an effective amount of a viral vector of the invention is administered to an animal subject. The viral vectors of the invention can be administered to a human subject or an animal in need thereof by any method known in the art. Optionally, the viral vector is delivered in an effective dose in a pharmaceutically acceptable carrier.

The viral vectors of the invention may further be administered to a subject to elicit an immunogenic response, e.g., as a vaccine. Typically, the vaccines of the present invention comprise an effective amount of the virus in combination with a pharmaceutically acceptable carrier. Optionally, the dose is sufficient to produce a protective immune response. The degree of protection afforded need not be complete or permanent, so long as the benefit of administering the immunogenic polypeptide outweighs any deficiency thereof. The subject and immunogen are as described above.

The dose of viral vector injected into a subject will depend on the mode of administration, the disease or condition to be treated, the condition of the individual, the particular viral vector and the nucleic acid to be delivered, and can be determined in a conventional manner. An exemplary dosage to achieve a therapeutic effect is at least about 10⁵，10⁶，10⁷，10⁸，10⁹，10¹⁰，10¹¹，10¹²，10¹³，10¹⁴，10¹⁵Tu or more, preferably about 10⁷Or 10⁸～10¹²，10¹³Or 10¹⁴Tu, more preferably about 10¹²Viral titer of Tu.

In particular embodiments, more than one administration may be used, e.g., two, three, four or more administrations, and the desired level of gene expression may be achieved at different time intervals.

Examples of modes of administration include oral, rectal, mucosal, topical, intranasal, inhalation, buccal, vaginal, intrathecal, intraocular, transdermal, intrauterine, parenteral, intravenous, subcutaneous, intradermal, intramuscular, intradermal, intrapleural, intracerebral and intraarticular, cutaneous and mucosal surfaces, intralymphatic, and the like, as well as direct tissue or organ injection, for example, in the liver, skeletal muscle, cardiac muscle, diaphragm muscle, or brain. The administration to a tumor can also be, for example, injection within or near a tumor or lymph node. The most suitable route in any given case will depend on the nature and severity of the condition being treated and on the nature of the particular vector used.

Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, or in solid forms suitable for liquid solutions or suspensions prior to injection, or as emulsions. Alternatively, the viral vector may be administered in a local rather than systemic manner, e.g., in a specific manner, such as a sustained release formulation. In addition, viral vectors can be delivered in dry form to surgical implant matrices, such as bone graft substitutes, sutures, stents, and the like.

Pharmaceutical compositions suitable for oral administration may be presented as discrete units, such as capsules, buffers, lozenges, or tablets, each unit containing a predetermined amount of a composition of the invention. As a powder or granules, as a solution or suspension in an aqueous liquid or a non-aqueous liquid, or as an oil-in-water or water-in-oil emulsion. Oral administration can be accomplished by incorporating the viral vectors of the present invention into a vector that is resistant to degradation by digestive enzymes in the intestinal tract of an animal. Examples of such carriers include capsules or tablets as known in the art. Such formulations are prepared by any suitable pharmaceutical method which includes the step of bringing the ingredients into association with a suitable carrier which may contain one or more accessory ingredients as described hereinbefore. In general, pharmaceutical compositions according to embodiments of the invention are prepared by uniformly and intimately admixing the compositions with liquid or finely divided solid carriers or both, and then shaping the resulting mixture. For example, tablets may be prepared by compressing or molding a powder or granules containing the composition, optionally with one or more accessory ingredients. Compressed tablets are prepared by compressing in a suitable machine the composition in a free-flowing form, such as a powder or granules, optionally mixed with a binder, lubricant, inert diluent and/or surface active/dispersing agent. Tablets are formed by wetting the powdered compound with an inert liquid binder in a suitable machine.

Pharmaceutical compositions suitable for oral administration include lozenges comprising the ingredients of the invention in a flavoured base, typically sucrose and gum arabic or tragacanth, and lozenges comprising inert base ingredients such as gelatin and glycerol or sucrose and gum arabic.

Pharmaceutical compositions suitable for parenteral administration may comprise sterile aqueous and nonaqueous injection solutions of the compositions of the invention, which formulations are optionally isotonic with the blood of the intended recipient. These formulations may contain antioxidants, buffers, bacteriostats and solutes that render the ingredients isotonic with the blood of the intended recipient. Aqueous and non-aqueous sterile suspensions, solutions and emulsions may include suspending agents and thickening agents. Examples of non-aqueous solvents include propylene glycol, polyethylene glycol, vegetable oils, and injectable organic esters. Aqueous carriers include water, alcohol/water solutions, emulsions or suspensions, including saline and buffered media. Parenteral drugs include sodium chloride solution, ringer's dextrose, dextrose and sodium chloride, lactated ringer's or fixed oils. Carriers for intravenous injection include liquid and nutritional supplements, electrolyte supplements such as ringer's dextrose based supplements, and the like. Preservatives and other additives such as antimicrobials, antioxidants, chelating agents, and inert gases and the like may also be present.

These ingredients may be presented in unit-dose or multi-dose containers, for example, in sealed ampoules and vials, and may be stored in a freeze-dried condition requiring only the addition of the sterile liquid carrier, for example, saline or water-for-injection, immediately prior to use.

Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind described above. For example, the injectable, stable, sterile compositions of the present invention may be provided in unit dosage form in a sealed container. The composition may be provided in the form of a lyophilizate which may be reconstituted with a suitable pharmaceutically acceptable carrier to form a liquid composition suitable for injection into an individual. The unit dosage form can be about 1 μ g to about 10g of the composition of the invention. When the composition is substantially water-insoluble, a sufficient amount of a physiologically acceptable emulsifier may be added to emulsify the composition in an aqueous carrier. One useful emulsifier is phosphatidylcholine.

Pharmaceutical compositions suitable for rectal administration may be presented as unit dose suppositories. These may be prepared by mixing the components with one or more conventional solid carriers and then forming the resulting mixture.

The pharmaceutical compositions of the present invention suitable for topical application to the skin may take the form of ointments, creams, lotions, pastes, gels, sprays, aerosols or oils. Carriers that can be used include, but are not limited to, petrolatum, lanolin, polyethylene glycols, alcohols, dermal penetration enhancers, and combinations of two or more thereof. For example, in some embodiments, topical delivery may be performed by mixing the pharmaceutical composition of the present invention with a lipophilic agent capable of entering the skin.

Pharmaceutical compositions suitable for transdermal administration may be in the form of discrete patches adapted to remain in intimate contact with the epidermis of the subject for an extended period of time. Compositions suitable for transdermal administration may also be delivered by iontophoresis and are generally in the form of an optionally buffered aqueous solution of the compositions of the present invention. Suitable formulations may comprise citrate or bis/tris buffers or ethanol/water and may contain 0.1 to 0.2M active ingredient.

The viral vectors disclosed herein can be administered to the lungs of a subject by any suitable method, such as by administering a suspension of respirable particles that consist of viral vectors inhaled by the subject. The inhalable particles may be liquid or solid. The aerosol of liquid particles comprising viral vectors may be generated by any suitable method, for example using a pressure driven aerosol nebulizer or an ultrasonic nebulizer known to those skilled in the art. Aerosols of solid particles comprising viral vectors may likewise be generated with any solid particle drug aerosol generator by techniques known in the medical arts.

III chimeric AAV capsid viral vector directed evolution and in vivo screening method

The invention also includes a method for preparing a viral bank comprising chimeric AAV capsids and then screening for chimeric AAV capsids or viruses having one or more desired properties in vivo. Non-limiting examples of desirable properties include targeting characteristics, the ability to evade neutralizing antibodies, and improved intracellular trafficking, among others.

In representative embodiments, the invention provides a method of identifying a viral vector, e.g., an AAV vector or AAV capsid, having a property of interest, the method comprising:

first, a collection of viral vectors, e.g., AAV particles, is provided, wherein each AAV vector within the collection comprises: comprising a capsid protein produced by shuffling the capsid coding sequences of two or more different AAVs, wherein the capsid amino acid sequences of the two or more different AAVs differ by at least two amino acids; and a viral vector genome, e.g., an AAV vector genome, comprising the AAV capsid protein coding sequence generated by the aforementioned shuffling, the coding sequence for one AAV Rep, and at least one terminal repeat, e.g., a 5 'and/or 3' terminal repeat of AAV, wherein the viral vector genome is encapsidated in an AAV capsid.

Second, administering to the subject a collection of viral vectors;

third, a plurality of viral vectors are recovered from the target tissue as virions or as viral vector genomes encoding AAV capsids, thereby identifying viral vectors or AAV capsids having the property of interest.

The invention can also be used to identify a chimeric AAV capsid or viral particle having the ability to evade neutralizing antibodies in vivo, e.g., neutralizing antibodies found in human serum. For example, in vivo screening for neutralizing antibody resistance can be performed by injecting human immunoglobulin into a subject. For example, IVIG is injected into a non-human mammalian subject. IVIG naturally contains a mixture of antibodies against all common AAV in humans. Alternatively, the subject can be injected with a specific neutralizing antibody, and then a library of chimeric viruses can be injected into the subject, and the viral genome can be selected for entry into a target tissue of interest (e.g., heart, skeletal muscle, liver, etc.), the genome isolated from the target tissue corresponding to a capsid capable of evading neutralization.

Thus, in representative embodiments, the invention provides a method of identifying a chimeric AAV capsid or viral vector having the ability to evade neutralizing IgGs, the method comprising:

first, administering IgGs to a mammalian subject;

second, a collection of viral vectors, e.g., AAV particles, is provided, wherein each AAV vector within the collection comprises: comprising a capsid protein produced by shuffling the capsid coding sequences of two or more different AAVs, wherein the capsid amino acid sequences of the two or more different AAVs differ by at least two amino acids; and a viral vector genome, e.g., an AAV vector genome, comprising the AAV capsid protein coding sequence generated by the aforementioned shuffling, the coding sequence for one AAV Rep, and at least one terminal repeat, e.g., a 5 'and/or 3' terminal repeat of AAV, wherein the viral vector genome is encapsidated in an AAV capsid.

Third, administering to the subject a collection of viral vectors;

fourth, a plurality of viral vectors are recovered from the target tissue as virions or as viral vector genomes encoding AAV capsids, thereby identifying viral vectors or AAV capsids having neutralizing antibody evading properties.

By "evading" the neutralizing antibody, it is meant that neutralization is at least partially reduced as compared to an appropriate control group, but as long as the degree of neutralization is reduced as compared to the control group, and as long as some of the vector is able to reach and transduce the target tissue, the degree of "evasion" need not be complete.

In particular embodiments, the target tissue is liver, skeletal muscle, cardiac muscle, diaphragm muscle, kidney, pancreas, spleen, gastrointestinal tract, lung, joint tissue, tongue, ovary, testis, germ cells, cancer cells, or a combination thereof.

The sequences of any combination of two or more AAV capsids, whether naturally occurring or modified, whether now known or later discovered, can be "shuffled" to produce a collection of AAV vectors comprising chimeric capsids. In representative embodiments, the collection of AAV vectors comprises chimeric capsids generated by shuffling from two or more capsid sequences comprising: AAV1, AAV2, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, avian AAV, bovine AAV, canine AAV, equine AAV, ovine AAV, goose AAV or snake AAV. As described above, the collection of AAV capsids may further comprise non-naturally occurring AAV capsids that are currently known or later discovered. Such modifications include substitutions, including substitutions of modified nucleic acids and/or amino acids, as well as deletions and/or insertions.

Further diversity of the chimeric capsids can be achieved by any method known in the art for introducing mutations into nucleic acid and/or amino acid sequences, e.g., using chemical mutagens, error-prone PCR, cassette mutations, and the like.

Optionally, the in vivo screening method can be combined with one or more rounds of in vitro screening to further optimize the vector. For example, in vivo selection can be performed to identify chimeric AAV capsids having desired properties, and then in vitro selection can be used to identify AAV capsids having the ability to evade antibody neutralization.

The collection of AAV particles can be administered to the subject by any suitable method. In particular embodiments, the assemblage is administered to the blood of a subject, e.g., intravenously or intraarticularly.

The mode of administration and the subject are as described elsewhere herein.

The present invention is useful for identifying chimeric viruses or viral capsids having desirable properties in vivo. Thus, in particular embodiments, the methods of the invention comprise recovering AAV particles or viral genomes encoding the same AAV particles from two or more target tissues and identifying a chimeric virus or chimeric AAV capsid having desired properties for the two or more target tissues. For example, in particular embodiments, chimeric viruses or chimeric AAV capsids are identified that have inefficient targeting to skeletal muscle and/or cardiac muscle and or efficient targeting to the liver.

The target cell or tissue or one of them may also be a cancer cell or a tumor tissue. For example, a chimeric virus can be administered to an animal model of cancer and the chimeric virus coat or viral genome encoding the virus isolated from cancer cells or tumors. In representative embodiments, the animal model may be a model with an increased likelihood of forming a cancer or tumor, or may be a xenograft model in which human tumor cells are transplanted into an animal.

Exemplary methods for "shuffling" or "chimerization" of DNA, also known as "molecular breeding", "rapid forced evolution", etc., are known in the art (US5605793, US6165793, US6117679, Stemmer,1994, Proc. Nati. Acad. Sci91: 10747-. This method is also applied to the directed evolution of viruses (US patent US6096548, US patent US 6596539). In one representative embodiment, the collection of AAV capsid protein coding sequences is fragmented and recombined in vitro by homologous and/or nonhomologous recombination to form a collection of "chimeric" AAV capsid proteins. Each chimeric capsid encapsidates a nucleic acid, e.g., an AAV genome, comprising the corresponding capsid coding sequence, thereby generating a chimeric virus. The chimeric virus pool is administered to a subject and selected in vivo according to a property of interest. For example, chimeric viruses can be isolated from one or more target tissues to identify optimized capsid proteins with desired properties.

IV detailed description of the invention

In one aspect, the invention provides a nucleic acid encoding an AAV capsid protein, the nucleic acid comprising a nucleotide sequence selected from any one of the group consisting of:

(a) 1, nucleotide sequence SEQ ID NO;

(b) 3, nucleotide sequence SEQ ID NO;

(c) nucleotide sequence SEQ ID NO 5;

(d) nucleotide sequence SEQ ID NO 7;

(e) the nucleotide sequence of SEQ ID NO 9;

(f) nucleotide sequence SEQ ID NO 11;

(g) 13 in nucleotide sequence SEQ ID NO;

(h) 15, nucleotide sequence SEQ ID NO;

(i) nucleotide sequence SEQ ID NO 17; or

(j) A nucleotide sequence of an AAV capsid protein encoded by any one of nucleotide sequences (a) - (i) that differs from the nucleotide sequences of (a) - (i) due to the degeneracy of the genetic code.

The nucleic acid is a plasmid, phage, viral vector, bacterial artificial chromosome, yeast artificial chromosome, preferably an AAV vector comprising a coding sequence, more preferably the nucleic acid further comprises a coding sequence for an AAV Rep protein.

In one aspect, the invention also provides an AAV capsid protein encoded by the nucleic acid described above, the AAV capsid protein having an amino acid sequence comprising any one selected from the group consisting of SEQ ID NO 2, SEQ ID NO 4, SEQ ID NO 6, SEQ ID NO 8, SEQ ID NO 10, SEQ ID NO 12, SEQ ID NO 14, SEQ ID NO 16, or SEQ ID NO 18. Preferably, the AAV capsid is covalently linked, associated or encapsulated with a composition selected from one or more of a DNA molecule, an RNA molecule, a polypeptide, a carbohydrate, a liposome and a small organic molecule.

In another aspect, the present invention provides a recombinant viral particle comprising a nucleic acid and/or a capsid protein as described above. The recombinant viral particle is selected from a recombinant AAV viral particle, a recombinant adenovirus particle, a recombinant herpes viral particle, a recombinant baculovirus particle, or a recombinant hybrid viral particle.

In another aspect, the invention provides a recombinant AAV virion comprising an AAV vector genome and an AAV capsid protein as described above, wherein the AAV vector genome is encapsidated in an AAV capsid. The AAV vector genome comprises a heterologous nucleic acid sequence. The heterologous nucleic acid sequence codes one or more of antisense RNA, microRNA, shRNA, polypeptide and immunogen. Preferably, the polypeptide encoded by the heterologous nucleic acid sequence is a therapeutic polypeptide or a reporter gene, wherein the therapeutic polypeptide encoded by the heterologous nucleic acid is selected from the group consisting of insulin, glucagon, growth hormone releasing factor, erythropoietin, insulin growth factor, transforming growth factor alpha, hepatocyte growth factor, tyrosine hydroxylase, thrombopoietin, interleukin 1-interleukin 25, low density lipoprotein receptor, glucocorticoid receptor, vitamin D receptor, interferon regulatory factor, factor VIII, factor IX, glucosidase, glucose-6-phosphatase, isovaleryl-CoA dehydrogenase, propionyl-CoA carboxylase, beta-glucosidase, liver phosphorylase, phosphorylase kinase, glycine decarboxylase, alpha-galactosidase, beta-galactosidase, and lysosomal enzyme.

In one aspect, the invention provides a cell comprising the aforementioned nucleic acid, AAV capsid protein, recombinant virion, and/or recombinant AAV virion.

In another aspect, the invention provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier or excipient and one or more selected from the group consisting of a nucleic acid as described in the preceding claims, an AAV capsid protein, a recombinant virion, a recombinant AAV virion and/or a cell as described above.

In a further aspect, the invention also provides the use of one or more of the aforementioned nucleic acids, AAV capsid proteins, recombinant virions, recombinant AAV virions, cells and/or the aforementioned pharmaceutical compositions for the preparation of a medicament for the prevention or treatment of a disease selected from the group consisting of cystic fibrosis and other diseases of the lung, hemophilia a, hemophilia B, thalassemia, anemia and other blood disorders, senile dementia, multiple sclerosis, parkinson's disease, huntington's disease, amyotrophic lateral sclerosis, epilepsy, cancer, diabetes, muscular dystrophy, glycogen storage disease and other metabolic defects, congenital emphysema, Lesch-Nyhan syndrome, Niemann-Pick disease, aids, hepatitis, hyperammonemia and spinocerebellar ataxia.

In one aspect, the invention provides a method of producing a recombinant AAV virion, comprising providing a cell with a nucleic acid as described above, a nucleic acid coding sequence for an AAV Rep protein, an AAV vector genome carrying a heterologous nucleic acid sequence, a helper factor that facilitates production of infectious AAV, and allowing the AAV vector genome to be enveloped in an AAV capsid encoded by the nucleic acid described above, in vitro, and effecting assembly of the recombinant AAV virion, the method being an AAV vector production system comprising a two plasmid packaging system, a three plasmid packaging system, a baculovirus packaging system, and an AAV packaging system adjuvanted with Ad or HSV, and the like.

In one aspect, the invention also provides a method of delivering a heterologous nucleic acid to a cell in vitro, the method comprising administering to the cell a nucleic acid, a viral capsid protein, a recombinant virion, a recombinant AAV virion and/or a pharmaceutical composition as described above, the cell being a mammalian cell, preferably a human stem cell or a liver cell.

In another aspect, the invention also provides a method of delivering a heterologous nucleic acid to a mammal, the method comprising administering to a mammalian subject an effective amount of the foregoing nucleic acids, viral capsid proteins, recombinant virions, recombinant AAV virions, the foregoing cells, and/or the foregoing pharmaceutical compositions, wherein the mammal is a human subject or a primate subject.

Having described the invention, the invention will be illustrated in greater detail in the following examples, which are intended to be illustrative only and are not intended to be limiting of the invention.

Examples

The following examples describe the shuffling of AAV capsid genes by directed evolution and in vivo screening methods to generate a set of vectors with superior or superior targeting to the liver. And (3) adopting a DNA shuffling technology to shuffle AAV capsid genes and construct an AAV capsid gene library for in vivo screening of a mouse model. AAV mutants enriched in mouse liver were isolated and their targeting was determined by in vitro and in vivo activity testing of AAV mutant capsids.

Example 1 chimeric AAV plasmid library construction

In order to obtain chimeric AAV capsid gene Cap, first, the capsid full-length gene was amplified from different serotype AAV parents using the upstream primer primerA and the downstream primer primerA, respectively, and the selected parental AAV capsid genes included AAV1 capsid (NCBI sequence ID: AF063497.1, nucleic acid coding sequence 2223-4433 of Cap), AAV2 capsid (NCBI sequence ID: AF043303.1, nucleic acid coding sequence 2203-4410 of Cap), AAV3B capsid (NCBI sequence ID: AF028705.1, nucleic acid coding sequence 2208-4418 of Cap), AAV7 capsid (NCBI sequence ID: AF513851.1, nucleic acid coding sequence 2222-4435 of Cap), AAV8 capsid (NCBI sequence ID: AF513852.1, nucleic acid coding sequence 2121-4337 of Cap), AAV9 capsid (NCBI sequence ID:530579.1, nucleic acid coding sequence 1-2211 of Cap). The upstream primer PrimerA was 5'-CCCAAGCTTCGATCAACTACGCAGACAGGTACCAA-3', the downstream primer PrimerB was 5'-ATAAGAATGCGGCCGCAGAGACCAAAGTTCAACTGAAACGA-3', and PCR amplification was carried out by the action of prime STAR Max DNA polymerase (TaKaRa Co., Ltd., cat # R045A) under the conditions: 5min at 95 ℃ for 1 cycle; 8s at 98 ℃, 5s at 60 ℃, 15s at 72 ℃ and 35 cycles; 5min at 72 ℃ for 1 cycle.

Mixing the amplified Cap gene and other substances to obtain a DNA template with the total mass of 4ug, randomly crushing and digesting with 0.04U DNaseI at 22 ℃ for 6-8min under normal conditions, and inactivating DNaseI enzyme at 75 ℃ for 10 min. Agarose gel electrophoresis was performed to obtain a diffuse DNA band, the length of which was mostly concentrated at 500-1000bp, and DNA fragments in this range were recovered.

The recovered DNA fragments are first subjected to random primer-free amplification with respect to each other, and in order to increase the diversity and amplification efficiency, an unconventional single annealing PCR mode (94 ℃ 60s, 65 ℃ 90s, 72 ℃ 90s, 10 cycles; 94 ℃ 60s, 62 ℃ 90s, 72 ℃ 90s, 10 cycles; 94 ℃ 60s, 59 ℃ 90s, 72 ℃ 90s, 10 cycles; 94 ℃ 60s, 56 ℃ 90s, 72 ℃ 90s, 10 cycles; 94 ℃ 60s, 53 ℃ 90s, 72 ℃ 90s, 10 cycles; 94 ℃ 60s, 50 ℃ 90s, 72 ℃ 90s, 10 cycles; 94 ℃ 60s, 47 ℃ 90s, 72 ℃ 90s, 10 cycles) is employed).

The full-length amplification of the chimeric Cap gene was carried out by using the primer-free PCR product AS a template and the forward primer T-primer A (5'-ACGCCTGCCGTTCGACGATTCCCAAGCTTCGATCAACTACGCAGACAGGTACCAA-3') and the reverse primer T-primer B (5'-ACGCGCGGATCTTCCAGAGATAAGAATGCGGCCGCAGAGACCAAAGTTCAACTGAAACGA-3') with HiFi DNA polymerase (TransGenBiotech, Inc., cat # AS 131-22). The amplification procedure was carried out at 94 ℃ for 30s, 62 ℃ for 30s, and 72 ℃ for 2.5min for 40 cycles. Obtaining a chimeric full-length Cap DNA fragment with the length of about 2500bp, carrying out double enzyme digestion by HindIII and NotI, connecting with a pSNAV2.3 vector (containing a single-chain AAV2 genome ITR and a polymerase Rep gene sequence, and lacking Cap genes) which is subjected to the same double enzyme digestion treatment, and carrying out electric shock transformation on a connecting product to E.coli HST08 cells to prepare a chimeric AAV plasmid library with the library capacity of more than 1E +6 clones. The plasmids identified as positive clones by PCR are subjected to enzyme cutting identification of PstI, HaeIII and TaqI respectively, and the enzyme cutting morphological difference of each plasmid is observed (figure 1) so as to judge the diversity of the plasmid library.

Example 2 packaging and titer detection of novel AAV viral libraries

The chimeric plasmid library is self-packaged and replicated under the assistance of a helper, the packaging method is a two-step method, firstly, library plasmids, R2C2 and the helper plasmids are transfected according to the mass ratio of 1:1:2 and packaged into an AAV intermediate virus library, the AAV forming the virus library is a hybrid AAV at the moment, namely virus coat protein is not necessarily expressed by packaged genomes, and the genome titer is detected after the harvest liquid is extracted and purified by chloroform; in the second step, 293T cells are infected with an infection index MOI of 100, which ensures that the copy number of viral genome infected by each cell is less than 10, the final AAV viral library generated at this time is homozygous, and the genome titer is determined after the harvest is purified by chloroform extraction (see Muller et al, 2003, Nature Biotechnology, 21: 1040-. The quality of the plasmids used in the packaging of the library is very low, and it is ensured that each novel AAV genome can be packaged in a coat formed by its own expressed Cap protein. The packaged virus particles can normally express coat protein, and the complete virus coat can be formed and has infection capacity.

Taking a proper amount of purified AAV samples, preparing DNase I digestion reaction mixed liquor according to the following table (Table 1), incubating for 30min at 37 ℃, incubating for 10min at 75 ℃, and inactivating DNase I.

TABLE 1

AAV sample	5μl
			10 XDnase I buffer	5μl
Dnase I	1μl
		RNase-free water	39μl
Total up to	50μl

After the treated purified AAV sample was diluted by an appropriate factor, the Q-PCR reaction system was prepared according to the following table (Table 2), and the detection was carried out according to the following procedure.

TABLE 2

The primers used therein are shown in the following table (table 3):

TABLE 3

Forward primer (5 '-3')	AAGGTGGTGGATGAGTGCTACA
		Reverse primer (5 '-3')	TGGAGCTCAGGCTGGGTTT
Probe primer	CCCCAATTACTTGCTCC

Packaging yield results are seen in the following table (table 4):

TABLE 4

Name of viral vector	Genome titer (vg/ml)
		Novel AAV virus library	2.5E+11

EXAMPLE 3 novel AAV vectors screened and enriched in mice

The virus packaged by the novel AAV vector is injected into a C57BL/6J mouse at 8 weeks old through tail veins at a dose of 1.5E +11vg, and liver targeted screening and enrichment are carried out. Three days after injection, mice were sacrificed and all liver tissues were collected and total DNA was extracted after liquid nitrogen homogenization. The primers were amplified again using T-primer A/T-primer B, and the amplified DNA obtained by mixing the full-length Cap genes of the novel AAV was constructed into pSNAV2.3 vector by the method described above. 370 positive single clones obtained by PCR identification were mixed in equal mass to form a second plasmid library. The cells were repackaged into a viral library, injected again into 8 week-old C57BL/6J mice at 1.5E +11 vg/tail vein, three days after injection, the mice were sacrificed and all liver, heart and skeletal muscle tissues were collected. 100, 50 positive clones were randomly selected from the above tissues, respectively, and sequenced. The positive clones after sequencing were subjected to sequence alignment analysis using BioEdit, VectorNTI, ClustalX2 and TreeViewX.

Novel AAV vectors were selected for high frequency liver, low frequency heart and skeletal muscle, or no frequency heart and skeletal muscle. A total of 9 groups of high-frequency liver-targeted novel AAV mutants were obtained (FIG. 2), and the nucleotide sequences and amino acid sequences of the above 9 novel AAV-Cap mutants were analyzed (FIG. 3). The other 4 packages are not suitable for industrial needs due to low titer. Therefore, 5 of the novel AAVs, i.e., L37, L57, L58, L107, L10, were selected for subsequent infection activity test experiments.

Example 4 Activity of novel AAV on infection of human liver cell lines in vitro

4.1 testing the Activity of novel AAV on infection of in vitro human liver cell lines by Green fluorescent protein detection System

To initially test the liver-targeting infectious activity of the novel AAV, an in vitro infectious activity test was first performed on 6 human normal livers or liver cancer cell lines. The obtained novel AAV Cap genes L57, L58, L107 and L10 are respectively constructed on an RC plasmid vector containing AAV type 2 Rep, the plasmid carries CAG-EGFP exogenous genes, the packaged viruses are named as AAV2/57, AAV2/58, AAV2/107 and AAV2/10, and the virus titer is detected (the result is not shown).

The 4 recombinant AAV viruses carrying green fluorescent protein and AAV2/8 recombinant virus carrying the same green fluorescent protein are used to infect several kinds of human liver cell lines simultaneously for 48 hr before the infection activity is detected in flow mode. In order to avoid the infection efficiency of the viral vectors on a certain cell line to be too low or too supersaturated, each cell line is infected by using at least two infection indexes (MOI), and as can be seen from the figures (4A-4F), when 2-3 infection indexes are used to infect human liver cell lines 7721, HepG2, Huh7 and L02, the novel AAV2/10, AAV2/57, AAV2/58 and AAV2/107 all show obvious dose-dependent relationship on the infection of human liver cell lines. The results of infection with one of the MOIs for each cell are described next, and the remaining MOIs are detailed in FIG. 4.

When the 4 recombinant viruses infected human liver cell line 7402 at an infection index (MOI) of 2500, and analyzed by flow cytometry 48h after infection, the results (FIG. 4A) show that: under the MOI, the AAV2/10 infection efficiency is 4.6 times of that of AAV2/8, the AAV2/57 infection efficiency is 2.2 times of that of AAV2/8, the AAV2/58 infection efficiency is 3.9 times of that of AAV2/8, and the AAV2/107 infection efficiency is 1.6 times of that of AAV 2/8.

When the 4 recombinant viruses infect a human liver cell line 7721 with an infection index (MOI) of 2500, and the test analysis is carried out by flow cytometry 48h after infection, the result (figure 4B) shows that the infection efficiency of AAV2/10 is the highest under the MOI, the infection efficiency of AAV2/107 is close to that of AAV, the infection efficiency of AAV2/10 is 23.4 times of that of AAV2/8, the infection efficiency of AAV2/107 is 21.8 times of that of AAV2/8, the infection efficiency of AAV2/57 is 4.6 times of that of AAV2/8, and the infection efficiency of AAV2/58 is 15.9 times of that of AAV 2/8.

When the 4 recombinant viruses infect a human liver cell line HepG2 with an infection index (MOI) of 2500, and the analysis is carried out by flow cytometry detection 48h after infection, the result (figure 4C) shows that the infection efficiency of AAV2/10 is the highest under the MOI, the infection efficiency of AAV2/107 is close to that, the infection efficiency of AAV2/10 is 14.8 times of that of AAV2/8, and the infection efficiency of AAV2/107 is 13.5 times of that of AAV 2/8. The AAV2/57 infection efficiency is 5.5 times of that of AAV2/8, and the AAV2/58 infection efficiency is 10.9 times of that of AAV 2/8.

When the 4 recombinant viruses infected human liver cell line Huh7 with an infection index (MOI) of 2500 and analyzed by flow cytometry 48h after infection, the results (fig. 4D) showed that the infection efficiency of AAV2/10 was the highest at this MOI, which was 31.8 times higher than AAV 2/8. The infection efficiency of AAV2/57 is 1.4 times that of AAV2/8, the infection efficiency of AAV2/58 is 6.6 times that of AAV2/8, and the infection efficiency of AAV2/107 is 19.5 times that of AAV 2/8.

When the 4 recombinant viruses infect a human liver cell line Huh6 with an infection index (MOI) of 10000, and flow cytometry detection analysis is carried out 48h after infection, the result (figure 4E) shows that the AAV2/10 infection efficiency is the highest under the MOI, the AAV2/107 infection efficiency is close to that, the AAV2/10 infection efficiency is 2.9 times of the AAV2/8 infection efficiency, and the AAV2/107 infection efficiency is 2.7 times of the AAV2/8 infection efficiency. The AAV2/57 infection efficiency is 1.8 times of that of AAV2/8, and the AAV2/58 infection efficiency is 2.6 times of that of AAV 2/8.

When the 4 recombinant viruses infect a human liver cell line L02 with an infection index (MOI) of 50000 and are subjected to flow cytometry detection analysis 48h after infection, the result (figure 4F) shows that the infection efficiency of AAV2/10 is highest at the MOI and is 42 times of the infection efficiency of AAV2/8, the infection efficiency of AAV2/107 is 26.8 times of the infection efficiency of AAV2/8, the infection efficiency of AAV2/57 is 2.5 times of the infection efficiency of AAV2/8, and the infection efficiency of AAV2/58 is 10.8 times of the infection efficiency of AAV 2/8.

From the above results, the infection activity of the 4 recombinant novel AAV on human hepatocytes or hepatoma cell lines is higher than that of the positive control AAV 2/8.

4.2 testing the Activity of novel AAV on infection by in vitro human liver cell lines Using luciferase assay System

The obtained novel AAV Cap genes L37, L57, L58, L107 and L10 are constructed on an RC plasmid vector containing AAV Rep type 2, the plasmid carries a CAG-Luciferase exogenous gene, the packaged viruses are named as AAV2/37, AAV2/57, AAV2/58, AAV2/107 and AAV2/10, and the virus titer is detected (the result is not shown). The recombinant AAV is used to infect 5 kinds of normal liver or liver cancer cell lines with infection index MOI of 500, and after 48 hr, the luciferase detection system is used to detect the infection activity. Comparison of infection activity was performed after subtraction of NC background values on each assay.

The results of the 5 recombinant novel AAV viruses on the human liver cell line Huh7 infection activity comparison (figure 5A) show that the infection activity is AAV2/10 > AAV2/107 > AAV2/58 > AAV2/37 > AAV2/57 from high to low.

The results of the comparison of the infection activities of the 5 recombinant novel AAV viruses on the human liver cell line 7402 (FIG. 5B) show that the activity of AAV2/10 is the highest, AAV2/107 is close to the activity of AAV, AAV2/58 is close to the infection activity of AAV2/37, and the infection activities are sequentially from high to low, AAV2/10 > AAV2/107 > AAV2/58 > AAV2/37 > AAV 2/57.

The results of the comparison of the infection activities of the 5 novel recombinant AAV viruses on the human liver cell line 7721 (FIG. 5C) show that the activity of AAV2/10 is the highest, the infection activity of AAV2/58 is close to that of AAV2/37, and the infection activities are sequentially AAV2/10 > AAV2/107 > AAV2/58 > AAV2/37 > AAV2/57 from high to low.

The results of the 5 recombinant novel AAV viruses on the human liver cell line HepG2 (FIG. 5D) show that the AAV2/10 has the highest activity, and the infection activities are sequentially AAV2/10 > AAV2/37 > AAV2/107 > AAV2/58 > AAV2/57 from high to low.

The results of the comparison of the infection activities of the 5 recombinant novel AAV viruses in the human liver cell line L02 (FIG. 5E) show that the AAV2/10 has the highest activity, and the infection activities are sequentially AAV2/10 > AAV2/107 > AAV2/37 > AAV2/58 > AAV2/57 from high to low.

From these results, it is clear that AAV2/10 is relatively the highest in activity and AAV2/57 is relatively low in activity among the above-mentioned novel AAV.

EXAMPLE 5 novel AAV Activity assay in vivo

5 novel AAV Cap coat genes L37, L57, L58, L107 and L10 are respectively constructed on an RC plasmid vector containing 2 type AAV Rep, the plasmid carries CAG-Luciferase exogenous genes, the packaged viruses are named as AAV2/37, AAV2/57, AAV2/58, AAV2/107 and AAV2/10, the virus titer is detected (the result is not shown), and the in vivo infection activities of the novel AAV viruses are compared. Mice at 6-8 weeks C57BL/6J were selected for tail vein injection at a dose of 1E +11 vg/mouse, sacrificed 2 weeks later, tissue DNA was extracted, AAV vector genome copy number in liver, heart, skeletal muscle was tested, and luciferase expression in these 3 tissues was tested separately.

The vector genome copy number test results (fig. 6) showed that AAV2/58 had the highest vector genome copy number in the liver compared to other novel AAV and the genome copy number in the liver was significantly increased compared to the heart and skeletal muscle of the same group; while AAV2/37, AAV2/57, AAV2/107, and AAV2/10 vector genomic copy numbers were lower than AAV2/58 vector genomic copy numbers, the genomic copy numbers were higher in the liver compared to gene copy numbers in the heart and skeletal muscle of the same group.

The results of luciferase expression assays (FIGS. 7A-E) showed that the expression levels of luciferase in liver were higher in AAV2/37, AAV2/57, AAV2/58, AAV2/107 and AAV2/10 than in heart and skeletal muscle of the same group.

These results indicate that all of the 5 novel AAV vectors have excellent liver targeting property, and compared with other novel AAV vectors, the level of luciferase expression in liver of AAV2/10 is highest, then AAV2/58, then AAV2/37 and AAV2/107 are close, and the level of luciferase expression in liver of AAV2/57 is relatively lowest.

Example 6 detection of novel AAV neutralizing antibodies in monkey serum or human serum

Because AAV is naturally infected by humans and other primates, neutralizing antibodies raised against native AAV will greatly reduce the half-life of AAV. Herein, the level of neutralizing antibodies of the novel AAV was tested.

6.1 detection and comparison of novel AAV and AAV2/8 neutralizing antibodies in monkey sera

In the experiment, virus infection is adopted, the MOI value is fixed, serum is diluted, and the levels of the novel AAV vector and the neutralizing antibody of AAV2/8 in the cynomolgus monkey serum are detected. The efficiency of infection of cells by the serum virus mixture was found to be 50% of the efficiency of infection of cells by the virus serum-free in a series of serum dilutions, and the reciprocal of this dilution was used as the amount of neutralizing antibody to evaluate the neutralizing antibody against each viral vector (see Lochie MA et al, 2006, Virology 353: 68-82; Mori S et al, 2006, Jpn JInfect Dis 59: 285-293 et al). In the experiment, 10 cynomolgus monkey serums are detected in total, and after serial dilution of the serums, the neutralizing antibody of the novel AAV and the neutralizing antibody of the AAV2/8 in each sample are respectively judged and compared.

The method comprises the following specific steps: the obtained novel AAV Cap genes L37, L57, L58, L107 and L10 are respectively constructed on an RC plasmid vector containing AAV type 2 Rep, the plasmid carries CAG-EGFP exogenous genes, and the packaged viruses are named as AAV2/37, AAV2/57, AAV2/58, AAV2/107 and AAV2/10, and the virus titer is detected (the result is not shown). 7402 cells are inoculated on a 24-well plate, a cynomolgus monkey serum sample is serially diluted, the infection index MOI of the novel AAV virus AAV2/37, AAV2/57, AAV2/58, AAV2/107, AAV2/10 and AAV2/8 recombinant virus carrying the same green fluorescent protein is 2000, the diluted serum sample is mixed with virus solution 1:1, the mixture is incubated for 1h at 37 ℃, and then the mixed solution of the serum sample and the virus solution is added into the cells. Cells were harvested after 48h and the infection efficiency was examined by flow cytometry.

The results of detection of the neutralizing antibody are shown in Table 5, and when the amount of the neutralizing antibody is less than 5, the neutralizing antibody against the virus is considered negative. 10 serum samples are numbered 1#, 2#, 3#, 4#, 5#, 6#, 7#, 8#, 9# and 10# respectively, wherein 7# is negative for all virus vector neutralizing antibodies; 1# and 9# are negative for the neutralizing antibodies of the novel AAV vector, and positive for the neutralizing antibodies of AAV 2/8; the novel AAV neutralizing antibodies of the 2#, 3#, 4#, 5#, 6#, 8#, and 10# samples are all obviously lower than that of AAV 2/8.

Therefore, through comprehensive comparison of the novel AAV and the AAV2/8 neutralizing antibody in the cynomolgus monkey serum, the neutralizing antibody of the novel AAV in a monkey population is obviously lower than that of the AAV2/8, and the novel AAV is used as a drug delivery vector and has lower immunogenicity.

TABLE 5 neutralizing antibody assay results for cynomolgus monkey 10 sera

6.2 detection and comparison of novel AAV and AAV2/8 neutralizing antibodies in human serum

The experiment adopts fixed virus infection index MOI, and serum is diluted to detect the level of the novel AAV neutralizing antibody and the level of the AAV2/8 neutralizing antibody in individual human serum. The efficiency of infection of cells by the serum virus mixture was found to be 50% of the efficiency of infection of cells by the virus serum-free in a series of serum dilutions, and the amount of neutralizing antibody was evaluated for each viral vector using the reciprocal of this dilution as the amount of neutralizing antibody. In this experiment, 10 sera were tested, and a series of dilutions were made to compare the neutralizing antibodies of the novel AAV to those of AAV2/8 in each sample.

The specific procedure was as in 6.1, except that the serum selected was normal human serum. In this experiment, 10 normal human sera were randomly selected for neutralizing antibody experiments, and the novel AAV neutralizing antibody and AAV2/8 neutralizing antibody were compared in each sample. The neutralizing antibody test results are shown in table 6, where sample # 3 showed negative for all virus neutralizing antibodies; sample # 2 was negative for the novel AAV neutralizing antibodies, and positive for AAV2/8 neutralizing antibodies; the neutralizing antibodies of the 1#, 4#, 5#, 6#, 7#, 8#, 9#, and 10# samples to the novel AAV are all significantly lower than that of AAV 2/8. The experimental results demonstrate that in the human population, neutralizing antibodies against the novel AAV are more suitable as drug delivery vehicles, enabling reduced reactogenicity, compared to neutralizing antibodies to AAV 2/8.

TABLE 610 parts human serum neutralizing antibody test results

Combining the above results, the novel AAV has lower immunogenicity than AAV2/8 vector by comparing the serum neutralizing antibody of the novel AAV with AAV2/8, and is more suitable for gene therapy vector.

Sequence listing

<110> Shutaishen (Beijing) biopharmaceutical corporation; beijing Sanokui-Yi Biotechnology Limited liability company

<120> acquisition of a group of novel liver-targeted adeno-associated viruses and application thereof

<130>2020070729

<160>18

<170>SIPOSequenceListing 1.0

<210>1

<211>2211

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>1

atggctgccg atggttatct tccagattgg ctcgaggaca acctctctga gggcattcgc 60

gagtggtggg acctgaaacc tggagccccg aaacccaaag ccaaccagca aaagcaggac 120

aacggccggg gtctggtgct tcctggctac aagtacctcg gacccttcaa cggactcgac 180

aagggggagc ccgtcaacgc ggcggacgca gcggccctcg agcacgacaa ggcctacgac 240

cagcagctgc aggcgggtga caatccgtac ctgcggtata accacgccga cgccgagttt 300

caggagcgtc tgcaagaaga tacgtctttt gggggcaacc tcgggcgagc agtcttccag 360

gcgaaaaaga ggatccttga gcctcttggt ctggttgagg aagcggctaa gacggctcct 420

ggaaagaaga ggcctgtaga gcagtctcct caggaaccgg actcctccgc gggtattggc 480

aaatcgggtg cacagcccgc taaaaagaga ctcaactttg gtcagactgg cgactcagag 540

tcagttccag accctcaacc tctcggagaa ccaccagcag cgccctctgg tgtgggacct 600

aatacaatgg cttcaggcgg tggcgcacca atggcagaca ataacgaagg cgccgacgga 660

gtgggtaatg cctcaggaaa ttggcattgc gattcccaat ggctgggcga cagagtcatc 720

accaccagca ccagaacctg ggccctgccc acttacaaca accatctcta caagcaaatc 780

tccagtgctt caacgggggc cagcaacgac aaccactact tcggctacag caccccctgg 840

gggtattttg actttaacag attccactgc cacttttcac cacgtgactg gcagcgactc 900

atcaacaaca attggggatt ccggcccaag agactcaact tcaagctctt caacatccaa 960

gtcaaggagg tcacgacgaa tgatggcgtc acgaccatcg ctaataacct taccagcacg 1020

gttcaagtct tctcggactc ggagtaccag ttgccgtacg tcctcggctc tgcgcaccag 1080

ggttgcctcc ctccgttccc ggcggacgtg ttcatgattc cgcaatacgg ctacctgacg 1140

ctcaacaatg gcagccaagc cgtgggacgt tcatcctttt accgcctgga atatttccct 1200

tctcagatgc tgagaacggg caacaacttt accttcagct acacctttga ggaagtgcct 1260

ttccacagca gctacgcgca cagccagagc ctggaccggc tgatgaatcc tctcatcgac 1320

cagtacctgt attacctgaa cagaactcag aatcagtccg gaagtgccca aaacaaggac 1380

ttgctgttta gccgtgggtc tccagctggc atgtctgttc agcccaaaaa ctggctacct 1440

ggaccctgtt accggcagca gcgcgtttct aaaacaaaaa cagacaacaa caacagcaac 1500

tttacctgga ctggtgcttc aaaatataac ctcaatgggc gtgaatccat catcaaccct 1560

ggcactgcta tggcctcaca caaagacgac aaagacaagt tctttcccat aagcggtgtc 1620

atgatttttg gaaaggagag cgccggagct tcaaacactg cattggacaa tgtcatgatc 1680

acagacgaag aggaaatcaa agccactaac cccgtggcca ccgaaagatt tgggactgtg 1740

gcagtcaatc tccagagcag cagcacagac cctgcgaccg gagatgtgca cgttatggga 1800

gccttacctg gaatggtgtg gcaagacaga gacgtatacc tgcagggtcc tatttgggcc 1860

aaaattcctc acacggatgg acactttcac ccgtctcctc tcatgggcgg ctttggactt 1920

aagcacccgc ctcctcagat cctcatcaaa aacacgcctg ttcctgcgaa tcctccggca 1980

gagttttcag ctacaaagtt tgcttcattc atcacccagt attccacagg acaagtgagc 2040

gtggagattg aatgggagct gcagaaagaa aacagcaagc gctggaatcc cgaagtgcag 2100

tacacatcca attatgcaaa atctgccaac gttgatttta ctgtggacaa caatggactt 2160

tatactgagc ctcgccccat tggcacccgt taccttaccc gtcccctgta a 2211

<210>2

<211>736

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>2

Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn Leu Ser

1 5 10 15

Glu Gly Ile Arg Glu Trp Trp Asp Leu Lys Pro Gly Ala Pro Lys Pro

20 25 30

Lys Ala Asn Gln Gln Lys Gln Asp Asn Gly Arg Gly Leu Val Leu Pro

35 40 45

Gly Tyr Lys Tyr Leu Gly Pro Phe Asn Gly Leu Asp Lys Gly Glu Pro

50 55 60

Val Asn Ala Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp

65 70 75 80

Gln Gln Leu Gln Ala Gly Asp Asn Pro Tyr Leu Arg Tyr Asn His Ala

85 90 95

Asp Ala Glu Phe Gln Glu Arg Leu Gln Glu Asp Thr Ser Phe Gly Gly

100 105 110

Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Ile Leu Glu Pro

115 120 125

Leu Gly Leu Val Glu Glu Ala Ala Lys Thr Ala Pro Gly Lys Lys Arg

130 135 140

Pro Val Glu Gln Ser Pro Gln Glu Pro Asp Ser Ser Ala Gly Ile Gly

145 150 155 160

Lys Ser Gly Ala Gln Pro Ala Lys Lys Arg Leu Asn Phe Gly Gln Thr

165 170 175

Gly Asp Ser Glu Ser Val Pro Asp Pro Gln Pro Leu Gly Glu Pro Pro

180 185 190

Ala Ala Pro Ser Gly Val Gly Pro Asn Thr Met Ala Ser Gly Gly Gly

195 200 205

Ala Pro Met Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Asn Ala

210 215 220

Ser Gly Asn Trp His Cys Asp Ser Gln Trp Leu Gly Asp Arg Val Ile

225 230 235 240

Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His Leu

245 250 255

Tyr Lys Gln Ile Ser Ser Ala Ser Thr Gly Ala Ser Asn Asp Asn His

260 265 270

Tyr Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg Phe

275 280 285

His Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn Asn

290 295 300

Trp Gly Phe Arg Pro Lys Arg Leu Asn Phe Lys Leu Phe Asn Ile Gln

305 310 315 320

Val Lys Glu Val Thr Thr Asn Asp Gly Val Thr Thr Ile Ala Asn Asn

325 330 335

Leu Thr Ser Thr Val Gln Val Phe Ser Asp Ser Glu Tyr Gln Leu Pro

340 345 350

Tyr Val Leu Gly Ser Ala His Gln Gly Cys Leu Pro Pro Phe Pro Ala

355 360 365

Asp Val Phe Met Ile Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asn Gly

370 375 380

Ser Gln Ala Val Gly Arg Ser Ser Phe Tyr Arg Leu Glu Tyr Phe Pro

385 390 395 400

Ser Gln Met Leu Arg Thr Gly Asn Asn Phe Thr Phe Ser Tyr Thr Phe

405 410 415

Glu Glu Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu Asp

420 425 430

Arg Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Asn Arg

435 440 445

Thr Gln Asn Gln Ser Gly Ser Ala Gln Asn Lys Asp Leu Leu Phe Ser

450 455 460

Arg Gly Ser Pro Ala Gly Met Ser Val Gln Pro Lys Asn Trp Leu Pro

465 470 475 480

Gly Pro Cys Tyr Arg Gln Gln Arg Val Ser Lys Thr Lys Thr Asp Asn

485 490 495

Asn Asn Ser Asn Phe Thr Trp Thr Gly Ala Ser Lys Tyr Asn Leu Asn

500 505 510

Gly Arg Glu Ser Ile Ile Asn Pro Gly Thr Ala Met Ala Ser His Lys

515 520 525

Asp Asp Lys Asp Lys Phe Phe Pro Ile Ser Gly Val Met Ile Phe Gly

530 535 540

Lys Glu Ser Ala Gly Ala Ser Asn Thr Ala Leu Asp Asn Val Met Ile

545 550 555 560

Thr Asp Glu Glu Glu Ile Lys Ala Thr Asn Pro Val Ala Thr Glu Arg

565 570 575

Phe Gly Thr Val Ala Val Asn Leu Gln Ser Ser Ser Thr Asp Pro Ala

580 585 590

Thr Gly Asp Val His Val Met Gly Ala Leu Pro Gly Met Val Trp Gln

595 600 605

Asp Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile Pro His

610 615 620

Thr Asp Gly His Phe His Pro Ser Pro Leu Met Gly Gly Phe Gly Leu

625 630 635 640

Lys His Pro Pro Pro Gln Ile Leu Ile Lys Asn Thr Pro Val Pro Ala

645 650 655

Asn Pro Pro Ala Glu Phe Ser Ala Thr Lys Phe Ala Ser Phe Ile Thr

660 665 670

Gln Tyr Ser Thr Gly Gln Val Ser Val Glu Ile Glu Trp Glu Leu Gln

675 680 685

Lys Glu Asn Ser Lys Arg Trp Asn Pro Glu Val Gln Tyr Thr Ser Asn

690 695 700

Tyr Ala Lys Ser Ala Asn Val Asp Phe Thr Val Asp Asn Asn Gly Leu

705 710 715 720

Tyr Thr Glu Pro Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg Pro Leu

725 730 735

<210>3

<211>2214

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>3

atggctgccg atggttatct tccagattgg ctcgaggaca acctctctga gggcattcgc 60

gagtggtggg acctgaaacc tggagccccg aaacccaaag ccaaccagca aaagcaggac 120

aacggccggg gtctggtgct tcctggctac aagtacctcg gacccttcaa cggactcgac 180

aagggggagc ccgtcaacgc ggcggacgca gcggccctcg agcacgacaa ggcctacgac 240

cagcagctca aagcgggtga caatccgtac ctgcggtata accacgccga cgccgagttt 300

caggggcgtc tgcaagaaga tacgtctttt gggggcaacc tcgggcgagc agtcttccag 360

gcgaaaaaga gggttcttga acctctgggc ctggttgagg aacctgttaa gacggctccg 420

ggaaaaaaga ggccggtaga gccgccacct cagcgttccc ccgactcctc cacgggcatc 480

ggcaagaaag gccagcagcc cgccaaaaag agactcaatt ttggtcagac tggcgactca 540

gagtcagtcc ccgacccaca acctctcgga gaacctccag caacccccgc tgctgtggga 600

cctactacaa tggcttcagg cggtggcgca ccaatggcag acaataacga aggcgccgac 660

ggagtgggta atgcctcagg aaattggcat tgcgattcca catggctggg cgacagagtc 720

atcaccacca gcacccgcac ctgggccttg cccacctaca ataaccacct ctacaagcaa 780

atctccagtg cttcaacggg ggccagcaac gacaaccact acttcggcta cagcaccccc 840

tgggggtatt ttgatttcaa cagattccac tgccatttct caccacgtga ctggcagcga 900

ctcatcaaca acaactgggg attccgaccc aagagactca acttcaaact cttcaacatc 960

caagtcaagg aggtcacgac gaatgacggc gttacgacca tcgctaataa ccttaccagc 1020

acgattcagg tattctcgga ctcggagtac cagcttccgt acgtcctcgg ctctgcgcac 1080

cagggctgcc tccctccgtt cccggcggac gtgttcatga ttccgcaata cggctacctg 1140

acgctcaaca atggcagcca agccgtggga cgttcatcct tttactgcct ggaatatttc 1200

ccttctcaga tgctgagaac gggcaacaac tttaccttca gctacacctt tgaggaagtg 1260

cctttccaca gcagctacgc gcacagccag agcctggacc ggctgatgaa tcctctcatc 1320

gaccagtacc tgtattacct gaacagaact cagaatcagt ccggaagtgc ccaaaacaag 1380

gacttgctgt ttagccgtgg gtcaccagct ggcatgtctg ttcagcccaa aaactggcta 1440

cctggaccct gttaccggca gcagcgcgtt tctaaaacaa aaacagacaa caacaacagc 1500

aactttacct ggactggtgc ttcaaaatat aacctcaatg ggcgtgaatc catcatcaac 1560

cctggcactg ctatggcctc acacaaagac gacgaagaca agttctttcc catgagcggt 1620

gtcatgattt ttggaaaaga gagcgccgga gcttcaaaca ctgcattgga caatgtcatg 1680

attacagacg aagaggaaat taaagccact aaccccgtgg ccaccgaaag atttgggacc 1740

gtggcagtca atttccagag cagcagcaca gaccctgcga ccggagatgt gcatgctatg 1800

ggagcattac ctggcatggt gtggcaagat agagacgtgt acctgcaggg tcccatttgg 1860

gccaaaattc ctcacacaga tggacacttt cacccgtctc ctcttatggg cggctttgga 1920

ctcaagaacc cgcctcctca gatcctcatc aaaaacacgc ctgttcctgc gaatcctccg 1980

gcagagtttt cggctacaaa gtttgcttca ttcatcaccc agtattccac aggacaagtg 2040

agcgtggaga ttgaatggga gctgcagaaa gaaaacagca aacgctggaa tcccgaagtg 2100

cagtatacat ctaactatgc aaaatctgcc aacgttgatt ttactgagga caacaatgga 2160

ctttatactg agcctcgccc cattggcacc cgttacctta cccgtcccct gtaa 2214

<210>4

<211>737

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>4

Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn Leu Ser

1 5 10 15

Glu Gly Ile Arg Glu Trp Trp Asp Leu Lys Pro Gly Ala Pro Lys Pro

20 25 30

Lys Ala Asn Gln Gln Lys Gln Asp Asn Gly Arg Gly Leu Val Leu Pro

35 40 45

Gly Tyr Lys Tyr Leu Gly Pro Phe Asn Gly Leu Asp Lys Gly Glu Pro

50 55 60

Val Asn Ala Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp

65 70 75 80

Gln Gln Leu Lys Ala Gly Asp Asn Pro Tyr Leu Arg Tyr Asn His Ala

85 90 95

Asp Ala Glu Phe Gln Gly Arg Leu Gln Glu Asp Thr Ser Phe Gly Gly

100 105 110

Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Val Leu Glu Pro

115 120 125

Leu Gly Leu Val Glu Glu Pro Val Lys Thr Ala Pro Gly Lys Lys Arg

130 135 140

Pro Val Glu Pro Pro Pro Gln Arg Ser Pro Asp Ser Ser Thr Gly Ile

145 150 155 160

Gly Lys Lys Gly Gln Gln Pro Ala Lys Lys Arg Leu Asn Phe Gly Gln

165 170 175

Thr Gly Asp Ser Glu Ser Val Pro Asp ProGln Pro Leu Gly Glu Pro

180 185 190

Pro Ala Thr Pro Ala Ala Val Gly Pro Thr Thr Met Ala Ser Gly Gly

195 200 205

Gly Ala Pro Met Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Asn

210 215 220

Ala Ser Gly Asn Trp His Cys Asp Ser Thr Trp Leu Gly Asp Arg Val

225 230 235 240

Ile Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His

245 250 255

Leu Tyr Lys Gln Ile Ser Ser Ala Ser Thr Gly Ala Ser Asn Asp Asn

260 265 270

His Tyr Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg

275 280 285

Phe His Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn

290 295 300

Asn Trp Gly Phe Arg Pro Lys Arg Leu Asn Phe Lys Leu Phe Asn Ile

305 310 315 320

Gln Val Lys Glu Val Thr Thr Asn Asp Gly Val Thr Thr Ile Ala Asn

325 330 335

Asn Leu Thr Ser Thr Ile Gln Val Phe Ser Asp SerGlu Tyr Gln Leu

340 345 350

Pro Tyr Val Leu Gly Ser Ala His Gln Gly Cys Leu Pro Pro Phe Pro

355 360 365

Ala Asp Val Phe Met Ile Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asn

370 375 380

Gly Ser Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe

385 390 395 400

Pro Ser Gln Met Leu Arg Thr Gly Asn Asn Phe Thr Phe Ser Tyr Thr

405 410 415

Phe Glu Glu Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu

420 425 430

Asp Arg Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Asn

435 440 445

Arg Thr Gln Asn Gln Ser Gly Ser Ala Gln Asn Lys Asp Leu Leu Phe

450 455 460

Ser Arg Gly Ser Pro Ala Gly Met Ser Val Gln Pro Lys Asn Trp Leu

465 470 475 480

Pro Gly Pro Cys Tyr Arg Gln Gln Arg Val Ser Lys Thr Lys Thr Asp

485 490 495

Asn Asn Asn Ser Asn Phe Thr Trp Thr Gly Ala Ser Lys TyrAsn Leu

500 505 510

Asn Gly Arg Glu Ser Ile Ile Asn Pro Gly Thr Ala Met Ala Ser His

515 520 525

Lys Asp Asp Glu Asp Lys Phe Phe Pro Met Ser Gly Val Met Ile Phe

530 535 540

Gly Lys Glu Ser Ala Gly Ala Ser Asn Thr Ala Leu Asp Asn Val Met

545 550 555 560

Ile Thr Asp Glu Glu Glu Ile Lys Ala Thr Asn Pro Val Ala Thr Glu

565 570 575

Arg Phe Gly Thr Val Ala Val Asn Phe Gln Ser Ser Ser Thr Asp Pro

580 585 590

Ala Thr Gly Asp Val His Ala Met Gly Ala Leu Pro Gly Met Val Trp

595 600 605

Gln Asp Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile Pro

610 615 620

His Thr Asp Gly His Phe His Pro Ser Pro Leu Met Gly Gly Phe Gly

625 630 635 640

Leu Lys Asn Pro Pro Pro Gln Ile Leu Ile Lys Asn Thr Pro Val Pro

645 650 655

Ala Asn Pro Pro Ala Glu Phe Ser Ala Thr Lys Phe Ala Ser Phe Ile

660 665 670

Thr Gln Tyr Ser Thr Gly Gln Val Ser Val Glu Ile Glu Trp Glu Leu

675 680 685

Gln Lys Glu Asn Ser Lys Arg Trp Asn Pro Glu Val Gln Tyr Thr Ser

690 695 700

Asn Tyr Ala Lys Ser Ala Asn Val Asp Phe Thr Glu Asp Asn Asn Gly

705 710 715 720

Leu Tyr Thr Glu Pro Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg Pro

725 730 735

Leu

<210>5

<211>2208

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>5

atggctgccg atggttatct tccagattgg ctcgaggaca acctctctga gggcattcgc 60

gagtggtggg cgctgaaacc tggagccccg aagcccaaag ccaaccagca aaagcaggac 120

gacggccggg gtctggtgct tcctggctac aagtacctcg gacccttcaa cggactcgac 180

aagggggagc ccgtcaacgc ggcggacgca gcggccctcg agcacgacaa ggcctacgac 240

cagcagctca aagccggaga caacccgtac ctcaagtaca accacgccga cgccgagttc 300

caggagcggc tcaaagaaga tacgtctttt gggggcaacc tcgggcgagc agtcttccag 360

gcgaagaagc gggttctcga acctctcggt ctggttgagg aaggcgctaa gacggctcct 420

ggaaagaaac gtccggtaga gcagtcgcca caagagccag actcctcctc gggcatcggc 480

aagacaggcc agcagcccgc taaaaaaaga ctcaattttg gtcagactgg cgactcagag 540

tcagttccag accctcaacc tctcggagaa cctccagcag cgccctctgg tgtgggacct 600

aatacaatgg ctgcaggcgg tggcgcacca atggcagaca ataacgaagg cgccgacgga 660

gtgggtagtt cctcgggaaa ttggcattgc gattccacat ggatgggcga cagagtcatc 720

accaccagca cccgaacctg ggccctgccc acctacaaca accacctcta caaacaaatt 780

tccagccaat caggagcctc gaacgacaat cactactttg gctacagcac cccttggggg 840

tattttgact ttaacagatt ccactgccac ttctcaccac gtgactggca gcgactcatt 900

aacaacaatt ggggattccg gcccaagaga ctcaacttca agctcttcaa catccaagtc 960

aaggaggtca cgacgaatga tggcgtcaca accatcgcta ataaccttac cagcacggtt 1020

caagtcttct cggactcgga gtaccagctt ccgtacgtcc tcggctctgc gcaccagggc 1080

tgcctgcctc cgttcccggc ggacgtcttc atgattcctc agtacggcta cctgactccc 1140

aacaatggca gtcagtctgt gggacgttcc tccttctact gcctggagta cttcccttct 1200

cagatgctga gaacgggcaa caactttacc ttcagctaca cctttgagga agtgcctttc 1260

cacagcagct acgcgcacag ccagagcctg gaccggctga tgaatcctct catcgaccag 1320

tacctgtatt acctgaacag aactcaaaat cagtccggaa gtgcccaaaa caaggacttg 1380

ctgtttagcc gtgggtctcc agctggcatg tctgttcagc ccaaaaactg gctacctgga 1440

ccctgttatc agcagcagcg cgtttctaaa acaaaaacag acaacaacaa cagcaacttt 1500

acctggactg gtgcttcaaa atataacctc aatgggcgtg aatccatcat caaccctggc 1560

actgctatgg cctcacacaa agacgacaaa gacaagttct ttcccatgag cggtgtcatg 1620

atttttggaa aggagagcgc cggagcttca aacactgcat tggacaatgt catgatcaca 1680

gacgaagagg aaatcaaagc cactaacccc gtggccaccg aaagatttgg gaccgtggca 1740

gtcaatctcc agagcagcag cacagaccct gcgaccggag atgtgcatgt tatgggagcc 1800

ttacctggaa tggtgtggca agacagagac gtatacctgc agggtcctat ttgggccaaa 1860

attcctcaca cggatggaca ctttcacccg tctcctctca tgggcggctt tggacttaag 1920

cacccgcctc ctcagatcct catcaaaaac acgcctgttc ctgcgaatcc tccggcagag 1980

ttttcggcta caaagtttgc ttcattcatc acccagtatt ccacaggacg agtgagcgtg 2040

gagattgaat gggagccgca gaaagaaaac agcaaacgct ggaatcccga agtgcagtat 2100

acatctaact atgcaaaatc tgccaacgtt gattttactg tggacaacaa tggactttat 2160

actgagcctc gccccattgg cacccgttac cttacccgtc ccctgtaa 2208

<210>6

<211>735

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>6

Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn Leu Ser

1 5 10 15

Glu Gly Ile Arg Glu Trp Trp Ala Leu Lys Pro Gly Ala Pro Lys Pro

20 2530

Lys Ala Asn Gln Gln Lys Gln Asp Asp Gly Arg Gly Leu Val Leu Pro

35 40 45

Gly Tyr Lys Tyr Leu Gly Pro Phe Asn Gly Leu Asp Lys Gly Glu Pro

50 55 60

Val Asn Ala Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp

65 70 75 80

Gln Gln Leu Lys Ala Gly Asp Asn Pro Tyr Leu Lys Tyr Asn His Ala

85 90 95

Asp Ala Glu Phe Gln Glu Arg Leu Lys Glu Asp Thr Ser Phe Gly Gly

100 105 110

Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Val Leu Glu Pro

115 120 125

Leu Gly Leu Val Glu Glu Gly Ala Lys Thr Ala Pro Gly Lys Lys Arg

130 135 140

Pro Val Glu Gln Ser Pro Gln Glu Pro Asp Ser Ser Ser Gly Ile Gly

145 150 155 160

Lys Thr Gly Gln Gln Pro Ala Lys Lys Arg Leu Asn Phe Gly Gln Thr

165 170 175

Gly Asp Ser Glu Ser Val Pro Asp Pro Gln Pro Leu Gly Glu Pro Pro

180 185 190

Ala Ala Pro Ser Gly Val Gly Pro Asn Thr Met Ala Ala Gly Gly Gly

195 200 205

Ala Pro Met Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Ser Ser

210 215 220

Ser Gly Asn Trp His Cys Asp Ser Thr Trp Met Gly Asp Arg Val Ile

225 230 235 240

Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His Leu

245 250 255

Tyr Lys Gln Ile Ser Ser Gln Ser Gly Ala Ser Asn Asp Asn His Tyr

260 265 270

Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg Phe His

275 280 285

Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn Asn Trp

290 295 300

Gly Phe Arg Pro Lys Arg Leu Asn Phe Lys Leu Phe Asn Ile Gln Val

305 310 315 320

Lys Glu Val Thr Thr Asn Asp Gly Val Thr Thr Ile Ala Asn Asn Leu

325 330 335

Thr Ser Thr Val Gln Val Phe Ser Asp Ser Glu Tyr Gln Leu Pro Tyr

340 345 350

Val Leu Gly Ser Ala His Gln Gly Cys Leu Pro Pro Phe Pro Ala Asp

355 360 365

Val Phe Met Ile Pro Gln Tyr Gly Tyr Leu Thr Pro Asn Asn Gly Ser

370 375 380

Gln Ser Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe Pro Ser

385 390 395 400

Gln Met Leu Arg Thr Gly Asn Asn Phe Thr Phe Ser Tyr Thr Phe Glu

405 410 415

Glu Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu Asp Arg

420 425 430

Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Asn Arg Thr

435 440 445

Gln Asn Gln Ser Gly Ser Ala Gln Asn Lys Asp Leu Leu Phe Ser Arg

450 455 460

Gly Ser Pro Ala Gly Met Ser Val Gln Pro Lys Asn Trp Leu Pro Gly

465 470 475 480

Pro Cys Tyr Gln Gln Gln Arg Val Ser Lys Thr Lys Thr Asp Asn Asn

485 490 495

Asn Ser Asn Phe Thr Trp Thr Gly Ala Ser Lys Tyr Asn Leu Asn Gly

500 505 510

Arg Glu Ser Ile Ile Asn Pro Gly Thr Ala Met Ala Ser His Lys Asp

515 520 525

Asp Lys Asp Lys Phe Phe Pro Met Ser Gly Val Met Ile Phe Gly Lys

530 535 540

Glu Ser Ala Gly Ala Ser Asn Thr Ala Leu Asp Asn Val Met Ile Thr

545 550 555 560

Asp Glu Glu Glu Ile Lys Ala Thr Asn Pro Val Ala Thr Glu Arg Phe

565 570 575

Gly Thr Val Ala Val Asn Leu Gln Ser Ser Ser Thr Asp Pro Ala Thr

580 585 590

Gly Asp Val His Val Met Gly Ala Leu Pro Gly Met Val Trp Gln Asp

595 600 605

Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile Pro His Thr

610 615 620

Asp Gly His Phe His Pro Ser Pro Leu Met Gly Gly Phe Gly Leu Lys

625 630 635 640

His Pro Pro Pro Gln Ile Leu Ile Lys Asn Thr Pro Val Pro Ala Asn

645 650 655

Pro Pro Ala Glu Phe Ser Ala Thr Lys Phe Ala Ser Phe Ile Thr Gln

660 665 670

Tyr Ser Thr Gly Arg Val Ser Val Glu Ile Glu Trp Glu Pro Gln Lys

675 680 685

Glu Asn Ser Lys Arg Trp Asn Pro Glu Val Gln Tyr Thr Ser Asn Tyr

690 695 700

Ala Lys Ser Ala Asn Val Asp Phe Thr Val Asp Asn Asn Gly Leu Tyr

705 710 715 720

Thr Glu Pro Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg Pro Leu

725 730 735

<210>7

<211>2211

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>7

atggctgccg atggttatct tccagattgg ctcgaggaca acctttctga aggcattcgt 60

gagtggtggg ctctgaaacc tggagtccct caacccaaag cgaaccaaca acaccaggac 120

aaccgtcggg gtcttgtgct tccgggttac aaatacctcg gacccggtaa cggactcgac 180

aaaggagagc cggtcaacga ggcagacgcc gcggccctcg agcacgacaa agcatacgac 240

cagcagctca aggccggtga caacccgtac ctcaagtaca accacgccga cgccgagttt 300

caggagcgtc tgcaagaaga tacgtctttt gggggcaacc tcgggcgagc agtcttccag 360

gccaaaaaga ggatccttga gcctcttggt ctggttgagg aagcagctaa gacggctcct 420

ggaaagaaga ggcctgtaga gcagtctcct caggaaccgg actcctccgc gggtattggc 480

aaatcgggtg cacagcccgc taaaaagaga ctcaatttcg gtcagactgg cgacacagag 540

tcagtcccag accctcaacc aatcggagaa cctccagcag cgccctctgg tgtgggacct 600

aatacaatgg ctgcgggcgg tggcgcacca atggcagaca ataacgaggg tgccgatgga 660

gtgggtaatt cctcaggaaa ttggcattgc gattcccaat ggctgggcga cagagtcatc 720

accaccagca ccagaacctg ggccctgccc acttacaaca accatctcta caagcaaatc 780

tccagtgctt caacgggggc cagcaacgac aaccactact tcggctacag caccccctgg 840

gggtattttg actttaacag attccactgc cacttttcac cacgtgactg gcagcgactc 900

atcaacaaca attggggatt ccggcccaag agactcaact tcaagctctt caacatccaa 960

gtcaaggagg tcacgacgaa tgatggcgtc acgaccatcg ctaataacct taccagcacg 1020

gttcaagtct tctcggactc ggagtaccag ttgccgtacg tcctcggctc tgcgcaccag 1080

ggttgcctcc ctccgttccc ggcggacgtg ttcatgattc cgcaatacgg ctacctgacg 1140

ctcaacaatg gcagccaagc cgtgggacgt tcatcctttt actgcctgga atatttccct 1200

tctcagatgc tgagaacggg caacaacttt accttcagct acacctttga ggaagtgcct 1260

ttccacagca gctacgcgca cagccagagc ctggaccggc tgatgaatcc tctcatcgac 1320

cagtacctgt attacctgaa cagaactcag aatcagtccg gaagtgccca aaacaaggac 1380

ttgctgttta gccgtgggtc tccagctggc atgtctgttc agcccaaaaa ctggctacct 1440

ggaccctgtt accggcagca gcgcgtttct aaaacaaaaa cagacaacaa caacagcaac 1500

tttacctgga ctggtgcttc aaaatataac ctcaatgggc gtgaatccat catcaaccct 1560

ggcactgcta tggcctcaca caaagacgac aaagacaagt tctttcccat aagcggtgtc 1620

atgatttttg gaaaggagag cgccggagct tcaaacactg cattggacaa tgtcacgatc 1680

acagacgaag aggaaatcaa agccactaac cccgtggcca ccgaaagatt tgggactgtg 1740

gcagtcaatc tccagagcag cagcacagac cctgcgaccg gagatgtgca tgttatggga 1800

gccttacctg gaatggtgtg gcaagacaga gacgtatacc tgcagggtcc tatttgggcc 1860

aagattcctc acacggatgg caactttcac ccgtctcctc tcatgggcgg ctttggactt 1920

aagcacccgc ctcctcagat cctcatcaaa aacacgcctg ttcctgcgaa tcctccggca 1980

gagttttcgg ctacaaagtt tgcttcattc atcacccaat actccacagg acaagtgagt 2040

gtggaaattg aatgggagct gcagaaagaa aacagcaaac gctggaatcc cgaagtgcag 2100

tacacatcca attatgcaaa atctgccaac gttgatttta ctgtggacaa caatggactt 2160

tatactgagc ctcgccccat tggcacccgt taccttaccc gtcccctgta a 2211

<210>8

<211>736

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>8

Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn Leu Ser

1 5 10 15

Glu Gly Ile Arg Glu Trp Trp Ala Leu Lys Pro Gly Val Pro Gln Pro

20 25 30

Lys Ala Asn Gln Gln His Gln Asp Asn Arg Arg Gly Leu Val Leu Pro

3540 45

Gly Tyr Lys Tyr Leu Gly Pro Gly Asn Gly Leu Asp Lys Gly Glu Pro

50 55 60

Val Asn Glu Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp

65 70 75 80

Gln Gln Leu Lys Ala Gly Asp Asn Pro Tyr Leu Lys Tyr Asn His Ala

85 90 95

Asp Ala Glu Phe Gln Glu Arg Leu Gln Glu Asp Thr Ser Phe Gly Gly

100 105 110

Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Ile Leu Glu Pro

115 120 125

Leu Gly Leu Val Glu Glu Ala Ala Lys Thr Ala Pro Gly Lys Lys Arg

130 135 140

Pro Val Glu Gln Ser Pro Gln Glu Pro Asp Ser Ser Ala Gly Ile Gly

145 150 155 160

Lys Ser Gly Ala Gln Pro Ala Lys Lys Arg Leu Asn Phe Gly Gln Thr

165 170 175

Gly Asp Thr Glu Ser Val Pro Asp Pro Gln Pro Ile Gly Glu Pro Pro

180 185 190

Ala Ala Pro Ser Gly Val Gly Pro Asn Thr Met Ala Ala Gly Gly Gly

195200 205

Ala Pro Met Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Asn Ser

210 215 220

Ser Gly Asn Trp His Cys Asp Ser Gln Trp Leu Gly Asp Arg Val Ile

225 230 235 240

Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His Leu

245 250 255

Tyr Lys Gln Ile Ser Ser Ala Ser Thr Gly Ala Ser Asn Asp Asn His

260 265 270

Tyr Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg Phe

275 280 285

His Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn Asn

290 295 300

Trp Gly Phe Arg Pro Lys Arg Leu Asn Phe Lys Leu Phe Asn Ile Gln

305 310 315 320

Val Lys Glu Val Thr Thr Asn Asp Gly Val Thr Thr Ile Ala Asn Asn

325 330 335

Leu Thr Ser Thr Val Gln Val Phe Ser Asp Ser Glu Tyr Gln Leu Pro

340 345 350

Tyr Val Leu Gly Ser Ala His Gln Gly Cys Leu Pro Pro Phe Pro Ala

355 360 365

Asp Val Phe Met Ile Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asn Gly

370 375 380

Ser Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe Pro

385 390 395 400

Ser Gln Met Leu Arg Thr Gly Asn Asn Phe Thr Phe Ser Tyr Thr Phe

405 410 415

Glu Glu Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu Asp

420 425 430

Arg Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Asn Arg

435 440 445

Thr Gln Asn Gln Ser Gly Ser Ala Gln Asn Lys Asp Leu Leu Phe Ser

450 455 460

Arg Gly Ser Pro Ala Gly Met Ser Val Gln Pro Lys Asn Trp Leu Pro

465 470 475 480

Gly Pro Cys Tyr Arg Gln Gln Arg Val Ser Lys Thr Lys Thr Asp Asn

485 490 495

Asn Asn Ser Asn Phe Thr Trp Thr Gly Ala Ser Lys Tyr Asn Leu Asn

500 505 510

Gly Arg Glu Ser Ile Ile Asn Pro Gly Thr Ala Met Ala Ser His Lys

515 520525

Asp Asp Lys Asp Lys Phe Phe Pro Ile Ser Gly Val Met Ile Phe Gly

530 535 540

Lys Glu Ser Ala Gly Ala Ser Asn Thr Ala Leu Asp Asn Val Thr Ile

545 550 555 560

Thr Asp Glu Glu Glu Ile Lys Ala Thr Asn Pro Val Ala Thr Glu Arg

565 570 575

Phe Gly Thr Val Ala Val Asn Leu Gln Ser Ser Ser Thr Asp Pro Ala

580 585 590

Thr Gly Asp Val His Val Met Gly Ala Leu Pro Gly Met Val Trp Gln

595 600 605

Asp Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile Pro His

610 615 620

Thr Asp Gly Asn Phe His Pro Ser Pro Leu Met Gly Gly Phe Gly Leu

625 630 635 640

Lys His Pro Pro Pro Gln Ile Leu Ile Lys Asn Thr Pro Val Pro Ala

645 650 655

Asn Pro Pro Ala Glu Phe Ser Ala Thr Lys Phe Ala Ser Phe Ile Thr

660 665 670

Gln Tyr Ser Thr Gly Gln Val Ser Val Glu Ile Glu Trp Glu Leu Gln

675 680685

Lys Glu Asn Ser Lys Arg Trp Asn Pro Glu Val Gln Tyr Thr Ser Asn

690 695 700

Tyr Ala Lys Ser Ala Asn Val Asp Phe Thr Val Asp Asn Asn Gly Leu

705 710 715 720

Tyr Thr Glu Pro Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg Pro Leu

725 730 735

<210>9

<211>2211

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>9

atggctgccg atggttatct tccagattgg ctcgaggaca acctttctga aggcattcgt 60

gagtggtggg ctctgaaacc tggagtccct caacccaaag cgaaccaaca acaccaggac 120

aaccgtcggg gtcttgtgct tccgggttac aaatacctcg gacccggtaa cggactcgac 180

aaaggagagc cggtcaacga ggcagacgcc gcggccctcg agcacgacaa agcatacgac 240

cagcagctca aggccggtga caacccgtac ctcaagtaca accacgccga cgccgagttt 300

caggagcgtc tgcaagaaga tacgtctttt gggggcaacc tcgggcgagc agtcttccag 360

gccaaaaaga ggatccttga gcctcttggt ctggttgagg aagcagctaa gacggctcct 420

ggaaagaaga ggcctgtaga gcagtctcct caggaaccgg actcctccgc gggtattggc 480

aaatcgggtg cacagcccgc taaaaagaga ctcaatttcg gtcagactgg cgacacagag 540

tcagtcccag accctcaacc aatcggagaa cctccagcag cgccctctgg tgtgggacct 600

aatacaatgg ctgcgggcgg tggcgcacca atggcagaca ataacgaggg tgccgatgga 660

gtgggtaatt cctcaggaaa ttggcattgc gattcccaat ggctgggcga cagagtcatc 720

accaccagca ccagaacctg ggccctgccc acttacaaca accatctcta caagcaaatc 780

tccagtgctt caacgggggc cagcaacgac aaccactact tcggctacag caccccctgg 840

gggtattttg actttaacag attccactgc cacttttcac cacgtgactg gcagcgactc 900

atcaacaaca attggggatt ccggcccaag agactcaact tcaagctctt caacatccaa 960

gtcaaggagg tcacgacgaa tgatggcgtc acgaccatcg ctaataacct taccagcacg 1020

gttcaagtct tctcggactc ggagtaccag ttgccgtacg tcctcggctc tgcgcaccag 1080

ggttgcctcc ctccgttccc ggcggacgtg ttcatgattc cgcaatacgg ctacctgacg 1140

ctcaacaatg gcagccaagc cgtgggacgt tcatcctttt actgtctgga atatttccct 1200

tctcagatgc tgagaacggg caacaacttt accttcagct acacctttga ggaagtgcct 1260

ttccacagca gctacgcgca cagccagagc ctggaccggc tgatgaatcc tctcatcgac 1320

cagtacctgt attacctgaa cagaactcag aatcagtccg gaagtgccca aaacaaggac 1380

ttgctgttta gccgtgggtc tccagctggc atgtctgttc agcccaaaaa ctggctacct 1440

ggaccctgtt accggcagca gcgcgtttct aaaacaaaaa cagacaacaa caacagcaac 1500

tttacctgga ctggtgcttc aaaatataac ctcaatgggc gtgaatccat catcaaccct 1560

ggcactgcta tggcctcaca caaagacgac gaagacaagt tctttcccat gagcggtgtc 1620

ctgatttttg gaaaagagag cgccggagct tcaaacactg cattggacaa tgtcatgatt 1680

acagacgagg aggaaattaa agccactaac cccgtggcca ccgaaaaatt tgggactgtg 1740

gcagtcaatc tccagagcag cagcacagac cctgcgaccg gagatgtgca tgttatggga 1800

gccttacctg gaatggtgtg gcaagacaga gacgtatacc tgcagggtcc tatttgggcc 1860

aaaattcctc acacggatgg acactttcac ccgtctcctc tcatgggcgg ctttggactt 1920

aagcacccgc ctcctcagat cctcatcaaa aacacgcctg ttcctgcgaa tcctccggca 1980

gagttttcgg ctacaaagtt tgcttcattc atcacccagt attccacagg acaagtgagc 2040

gtggagattg aatgggagct gcagaaagaa aacagcaaac gctggaatcc cgaagtgcag 2100

tatacatcta actatgcaaa atctgccaac gttgatttta ctgtggacaa caatggactt 2160

tatactgagc ctcgccccat tggcacccgt taccttaccc gtcccctgta a 2211

<210>10

<211>736

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>10

Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn Leu Ser

1 5 10 15

Glu Gly Ile Arg Glu Trp Trp Ala Leu Lys Pro Gly Val Pro Gln Pro

20 25 30

Lys Ala Asn Gln Gln His Gln Asp Asn Arg Arg Gly Leu Val Leu Pro

35 40 45

Gly Tyr Lys Tyr Leu Gly Pro Gly Asn Gly Leu Asp Lys Gly Glu Pro

50 55 60

Val Asn Glu Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp

65 70 75 80

Gln Gln Leu Lys Ala Gly Asp Asn Pro Tyr Leu Lys Tyr Asn His Ala

85 90 95

Asp Ala Glu Phe Gln Glu Arg Leu Gln Glu Asp Thr Ser Phe Gly Gly

100 105 110

Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Ile Leu Glu Pro

115 120 125

Leu Gly Leu Val Glu Glu Ala Ala Lys Thr Ala Pro Gly Lys Lys Arg

130 135 140

Pro Val Glu Gln Ser Pro Gln Glu Pro Asp Ser Ser Ala Gly Ile Gly

145 150 155 160

Lys Ser Gly Ala Gln Pro Ala Lys Lys Arg Leu Asn Phe Gly Gln Thr

165 170 175

Gly Asp Thr Glu Ser Val Pro Asp Pro Gln Pro Ile Gly Glu Pro Pro

180 185 190

Ala Ala Pro Ser Gly Val Gly Pro Asn Thr Met Ala Ala Gly Gly Gly

195 200 205

Ala Pro Met Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Asn Ser

210 215 220

Ser Gly Asn Trp His Cys Asp Ser Gln Trp Leu Gly Asp Arg Val Ile

225 230 235 240

Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His Leu

245 250 255

Tyr Lys Gln Ile Ser Ser Ala Ser Thr Gly Ala Ser Asn Asp Asn His

260 265 270

Tyr Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg Phe

275 280 285

His Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn Asn

290 295 300

Trp Gly Phe Arg Pro Lys Arg Leu Asn Phe Lys Leu Phe Asn Ile Gln

305 310 315 320

Val Lys Glu Val Thr Thr Asn Asp Gly Val Thr Thr Ile Ala Asn Asn

325 330 335

Leu Thr Ser Thr Val Gln Val Phe Ser Asp Ser Glu Tyr Gln Leu Pro

340 345 350

Tyr Val Leu Gly Ser Ala His Gln Gly Cys Leu Pro Pro Phe Pro Ala

355 360 365

Asp Val Phe Met Ile Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asn Gly

370 375 380

Ser Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe Pro

385 390 395 400

Ser Gln Met Leu Arg Thr Gly Asn Asn Phe Thr Phe Ser Tyr Thr Phe

405 410 415

Glu Glu Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu Asp

420 425 430

Arg Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Asn Arg

435 440 445

Thr Gln Asn Gln Ser Gly Ser Ala Gln Asn Lys Asp Leu Leu Phe Ser

450 455 460

Arg Gly Ser Pro Ala Gly Met Ser Val Gln Pro Lys Asn Trp Leu Pro

465 470 475 480

Gly Pro Cys Tyr Arg Gln Gln Arg Val Ser Lys Thr Lys Thr Asp Asn

485 490 495

Asn Asn Ser Asn Phe Thr Trp Thr Gly Ala Ser Lys Tyr Asn Leu Asn

500 505 510

Gly Arg Glu Ser Ile Ile Asn Pro Gly Thr Ala Met Ala Ser His Lys

515 520 525

Asp Asp Glu Asp Lys Phe Phe Pro Met Ser Gly Val Leu Ile Phe Gly

530 535 540

Lys Glu Ser Ala Gly Ala Ser Asn Thr Ala Leu Asp Asn Val Met Ile

545 550 555 560

Thr Asp Glu Glu Glu Ile Lys Ala Thr Asn Pro Val Ala Thr Glu Lys

565 570 575

Phe Gly Thr Val Ala Val Asn Leu Gln Ser Ser Ser Thr Asp Pro Ala

580 585 590

Thr Gly Asp Val His Val Met Gly Ala Leu Pro Gly Met Val Trp Gln

595 600 605

Asp Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile Pro His

610 615 620

Thr Asp Gly His Phe His Pro Ser Pro Leu Met Gly Gly Phe Gly Leu

625 630 635 640

Lys His Pro Pro Pro Gln Ile Leu Ile Lys Asn Thr Pro Val Pro Ala

645 650 655

Asn Pro Pro Ala Glu Phe Ser Ala Thr Lys Phe Ala Ser Phe Ile Thr

660 665 670

Gln Tyr Ser Thr Gly Gln Val Ser Val Glu Ile Glu Trp Glu Leu Gln

675 680 685

Lys Glu Asn Ser Lys Arg Trp Asn Pro Glu Val Gln Tyr Thr Ser Asn

690695 700

Tyr Ala Lys Ser Ala Asn Val Asp Phe Thr Val Asp Asn Asn Gly Leu

705 710 715 720

Tyr Thr Glu Pro Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg Pro Leu

725 730 735

<210>11

<211>2211

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>11

atggctggcg atggttatct tccagattgg ctcgaggaca acctctctga gggcattcgc 60

gagtggtggg acttgaaacc tggagccccg aagcccaaag ccaaccagca aaagcaggac 120

aacggccggg gtctggtgct tcctggctac aagtacctcg gacccttcaa cggactcgac 180

aagggggagc cggtcaacgc agcagacgca gcggccctcg agcacgacaa ggcctacgac 240

cagcagctca aagcgggtga caatccgtac ctgcggtata accacgccga cgccgagttt 300

caggagcgtc tgcaagaaga tacgtctttt gggggcaacc tcgggcgagc agtcttccag 360

gcgaaaaagc ggcttcttga acctcttggt ctggttgagg aagcggctaa gacggctcct 420

ggaaagaaga ggcctgtaga gcagtctcct caggaaccgg actcctccgc gggtattagc 480

aaatcgggtg cacagcccgc taaaaagaga ctcaatttcg gtcagactgg cgacacagag 540

tcagtcccag accctcaacc aatcggagaa cctcccgcag ccccctcagg tgtgggatct 600

cttacaatgg cttcaggtgg tggcgcacca gtggcagaca ataacgaagg cgccgacgga 660

gtgggtaatg cctcaggaaa ttggcattgc gattccacat ggatgggcga cagagtcatc 720

accaccagca cccgaacttg ggccttgccc acctataaca accacctcta caagcaaatc 780

tccagtgctt caacgggggc cagcaacgat aaccactact tcggctacag caccccctgg 840

gggtattttg acttcaacag attccactgc cacttttcac cacgtgactg gcagcgactc 900

atcaacaaca actggggatt ccggcccaag agactcagct tcaagctctt caacatccag 960

gtcaaagagg ttacggacaa caatggagtc aagaccatcg ccaataatct taccagcacg 1020

gtccaggtct tcacggactc agactatcag ctcccgtacg ttctcggctc tgcccaccag 1080

ggctgcctgc ctccgttccc ggcggacgtg ttcatgattc cccagtacgg ctacctgaca 1140

ctcaacaacg gtagtcaggc cgtgggacgc tcctccttct actgcctgga atactttcct 1200

tctcagatgc tgagaacggg caacaacttt accttcagct acacctttga ggaagtgcct 1260

ttccacagca gctacgcgca cagccagagc ctggaccggc tgatgaatcc tctcatcgac 1320

cagtacctgt attacctgaa cagaactcag aatcagtccg gaagtgccca aaacaaggac 1380

ttgctgttta gccgtgggtc tccagctggc atgtctgttc agcccaaaaa ctggctacct 1440

ggaccctgtt accggcagca gcgcgtttct aaaacaaaaa cagacaacaa caacagcaac 1500

tttacctgga ctggtgcttc aaaatataac ctcaatgggc gtgaatccat catcaaccct 1560

ggcactgcta tggcctcaca caaagacgac aaagacaagt tctttcccat gagcggtgtc 1620

atgatttttg gaaaggagag cgccggagct tcaaacactg cattggacaa tgtcatgatc 1680

acagacgaag aggaaatcaa agccactaac cccgtggcca ccgaaagatt tgggactgtg 1740

gcagtcaatc tccagagcag cagcacagac cctgcgaccg gagatgtgca tgttatggga 1800

gccttacctg gaatggtgtg gcaagacaga gacgtatacc tgcagggtcc tatttgggcc 1860

aaaattcctc acacggatgg acactttcac ccgtctcctc tcatgggcgg ctttggactc 1920

aagcacccgc ctcctcagat cctcatcaaa aacacacccg ttcctgcgaa tcctccggca 1980

gagttttcgg ctacaaagtt tgcttcattc atcacccagt attccacagg acaagtgagc 2040

gtggagattg aatgggagct gcagaaagaa aacagcaaac gctggaatcc cgaagtgcag 2100

tacacatcca attatgcaaa atctgccgac gttgatttta ctgtggacaa caatggactt 2160

tatactgagc ctcgccccat tggcacccgt taccttaccc gtcccctgta a 2211

<210>12

<211>736

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>12

Met Ala Gly Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn Leu Ser

1 5 10 15

Glu Gly Ile Arg Glu Trp Trp Asp Leu Lys Pro Gly Ala Pro Lys Pro

20 25 30

Lys Ala Asn Gln Gln Lys Gln Asp Asn Gly Arg Gly Leu Val Leu Pro

35 40 45

Gly Tyr Lys Tyr Leu Gly Pro Phe Asn Gly Leu Asp Lys Gly Glu Pro

50 55 60

Val Asn Ala Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp

65 70 75 80

Gln Gln Leu Lys Ala Gly Asp Asn Pro Tyr Leu Arg Tyr Asn His Ala

85 90 95

Asp Ala Glu Phe Gln Glu Arg Leu Gln Glu Asp Thr Ser Phe Gly Gly

100 105 110

Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Leu Leu Glu Pro

115 120 125

Leu Gly Leu Val Glu Glu Ala Ala Lys Thr Ala Pro Gly Lys Lys Arg

130 135 140

Pro Val Glu Gln Ser Pro Gln Glu Pro Asp Ser Ser Ala Gly Ile Ser

145 150 155 160

Lys Ser Gly Ala Gln Pro Ala Lys Lys Arg Leu Asn Phe Gly Gln Thr

165 170 175

Gly Asp Thr Glu Ser Val Pro Asp Pro Gln Pro Ile Gly Glu Pro Pro

180 185 190

Ala Ala Pro Ser Gly Val Gly Ser Leu Thr Met Ala Ser Gly Gly Gly

195 200 205

Ala Pro Val Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Asn Ala

210 215 220

Ser Gly Asn Trp His Cys Asp Ser Thr Trp Met Gly Asp ArgVal Ile

225 230 235 240

Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His Leu

245 250 255

Tyr Lys Gln Ile Ser Ser Ala Ser Thr Gly Ala Ser Asn Asp Asn His

260 265 270

Tyr Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg Phe

275 280 285

His Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn Asn

290 295 300

Trp Gly Phe Arg Pro Lys Arg Leu Ser Phe Lys Leu Phe Asn Ile Gln

305 310 315 320

Val Lys Glu Val Thr Asp Asn Asn Gly Val Lys Thr Ile Ala Asn Asn

325 330 335

Leu Thr Ser Thr Val Gln Val Phe Thr Asp Ser Asp Tyr Gln Leu Pro

340 345 350

Tyr Val Leu Gly Ser Ala His Gln Gly Cys Leu Pro Pro Phe Pro Ala

355 360 365

Asp Val Phe Met Ile Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asn Gly

370 375 380

Ser Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe Pro

385 390 395 400

Ser Gln Met Leu Arg Thr Gly Asn Asn Phe Thr Phe Ser Tyr Thr Phe

405 410 415

Glu Glu Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu Asp

420 425 430

Arg Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Asn Arg

435 440 445

Thr Gln Asn Gln Ser Gly Ser Ala Gln Asn Lys Asp Leu Leu Phe Ser

450 455 460

Arg Gly Ser Pro Ala Gly Met Ser Val Gln Pro Lys Asn Trp Leu Pro

465 470 475 480

Gly Pro Cys Tyr Arg Gln Gln Arg Val Ser Lys Thr Lys Thr Asp Asn

485 490 495

Asn Asn Ser Asn Phe Thr Trp Thr Gly Ala Ser Lys Tyr Asn Leu Asn

500 505 510

Gly Arg Glu Ser Ile Ile Asn Pro Gly Thr Ala Met Ala Ser His Lys

515 520 525

Asp Asp Lys Asp Lys Phe Phe Pro Met Ser Gly Val Met Ile Phe Gly

530 535 540

Lys Glu Ser Ala Gly Ala Ser Asn Thr Ala Leu Asp Asn Val Met Ile

545 550 555 560

Thr Asp Glu Glu Glu Ile Lys Ala Thr Asn Pro Val Ala Thr Glu Arg

565 570 575

Phe Gly Thr Val Ala Val Asn Leu Gln Ser Ser Ser Thr Asp Pro Ala

580 585 590

Thr Gly Asp Val His Val Met Gly Ala Leu Pro Gly Met Val Trp Gln

595 600 605

Asp Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile Pro His

610 615 620

Thr Asp Gly His Phe His Pro Ser Pro Leu Met Gly Gly Phe Gly Leu

625 630 635 640

Lys His Pro Pro Pro Gln Ile Leu Ile Lys Asn Thr Pro Val Pro Ala

645 650 655

Asn Pro Pro Ala Glu Phe Ser Ala Thr Lys Phe Ala Ser Phe Ile Thr

660 665 670

Gln Tyr Ser Thr Gly Gln Val Ser Val Glu Ile Glu Trp Glu Leu Gln

675 680 685

Lys Glu Asn Ser Lys Arg Trp Asn Pro Glu Val Gln Tyr Thr Ser Asn

690 695 700

Tyr Ala Lys Ser Ala Asp Val Asp Phe Thr Val Asp Asn Asn Gly Leu

705 710 715 720

Tyr Thr Glu Pro Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg Pro Leu

725 730 735

<210>13

<211>2211

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>13

atggctgccg atggttatct tccagattgg ctcgaggaca accttagtga gggcattcgc 60

gagtggtggg acttgaaacc tggagccccg aagcccaaag ccaaccagca aaagcaggac 120

aacggccggg gtctggtgct tcctggctac aagtacctcg gacccttcaa cggactcgac 180

aggggggagc ccgtcaacgc ggcggacgca gcggccctcg agcacgacaa ggcctacgac 240

cagcagctca aagcgggtga caatccgtac ctgcggtata accacgccga cgccgagttt 300

caggagcgtc tgcaagaaga tacgtctttt gggggcaacc tcgggcgagc agtcttccgg 360

gccaagaagc gggttctcga acctctcggt ctggttgagg aagcagctaa aacggctcct 420

ggaaagaaga ggcctgtaga tcagtctcct caggaaccgg actcatcatc tggtgttggc 480

aaatcgggca aacagcctgc cagaaaaaga ctcaatttcg gtcagactgg cgactcagag 540

tcagtccccg accctcaacc tctcggagaa cctccagcag cgccctctag tgtgggatct 600

ggtacagtgg ctgcaggcgg tggcgcacca atggcagaca ataacgaagg cgccgacgga 660

gtgggtaatg cctcaggaaa ttggcattgc gattccacat ggctgggcga cagagtcatc 720

accaccagca cccgaacatg ggccttgccc acctataaca accacctcta caagcaaatc 780

tccagtgctt caacgggggc cagcaacgac aaccactact tcggctacag caccccctgg 840

gggtattttg atttcaacag attccactgc catttctcac cacgtgactg gcagcgactc 900

atcaacaaca attggggatt ccggcccaag agactcaact tcaagctctt caacatccaa 960

gtcaaggagg tcacgacgaa tgatggcgtc acgaccatcg ctaataacct taccagcacg 1020

gttcaagtct tctcggactc ggagtaccag ttgccgtacg tcctcggctc tgcgcaccag 1080

ggctgcctcc ctccgttccc ggcggacgtg ttcatgattc cgcaatacgg ctacctgacg 1140

ctcaacaatg gcagccaagc cgtgggacgt tcatcctttt actgcctgga atatttccct 1200

tctcagatgc tgagaacggg caacaacttt accttcagct acacctttga ggaagtgcct 1260

ttccacagca gctacgcgca cagccagagc ctggaccggc tgatgaatcc tctcatcgac 1320

cagtacctgt attacctgaa cagaactcag aatcagtccg gaagtgccca aaacaaggac 1380

ttgctgttta gccgtgggtc tccagctggc atgtctgttc agcccaaaaa ctggctacct 1440

ggaccctgtt accggcagca gcgcgtttct aaaacaaaaa cagacaacaa caacagcaac 1500

tttacctgga ctggtgcttc aaaatataac ctcaatgggc gtgaatccat catcaaccct 1560

ggcactgcta tggcctcaca caaagacgac aaagacaagt tctttcccat gagcggtgtc 1620

atgatttttg gaaaggagag cgccggagct tcaaacactg cattggacaa tgtcatgatc 1680

acagacgaag aggaaatcaa agccactaac cccgtggcca ccgaaagatt tgggactgtg 1740

gcagtcaatc tccagagcag cagcacagac cctgcgaccg gagatgtgca tgttatggga 1800

gccttacctg gaatggtgtg gcaagacaga gacgtatacc tgcagggtcc catttgggcc 1860

aaaattcctc acacggatgg acactttcac ccgtctcctc tcatgggcgg ctttggactt 1920

aagcacccgc ctcctcagat cctcatcaaa aacacgcctg ttcctgcgaa tcctccggca 1980

gagttttcgg ctacaaagtt tgcttcattc atcacccagt attccacagg acaagtgagc 2040

gtggagattg aatgggagct gcagaaagaa aacagcaaac gctggaatcc cgaagtgcag 2100

tatacatcta actatgcaaa atctgccaac gttgatttta ctgtggacaa caatggactt 2160

tatactgagc ctcgccccat tggcacccgt taccttaccc gtcccctgta a 2211

<210>14

<211>736

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>14

Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn Leu Ser

1 5 10 15

Glu Gly Ile Arg Glu Trp Trp Asp Leu Lys Pro Gly Ala Pro Lys Pro

20 25 30

Lys Ala Asn Gln Gln Lys Gln Asp Asn Gly Arg Gly Leu Val Leu Pro

35 40 45

Gly Tyr Lys Tyr Leu Gly Pro Phe Asn Gly Leu Asp Arg Gly Glu Pro

50 55 60

Val Asn Ala Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp

65 70 75 80

Gln Gln Leu Lys Ala Gly Asp Asn Pro Tyr Leu Arg Tyr Asn His Ala

85 90 95

Asp Ala Glu Phe Gln Glu Arg Leu Gln Glu Asp Thr Ser Phe Gly Gly

100 105 110

Asn Leu Gly Arg Ala Val Phe Arg Ala Lys Lys Arg Val Leu Glu Pro

115 120 125

Leu Gly Leu Val Glu Glu Ala Ala Lys Thr Ala Pro Gly Lys Lys Arg

130 135 140

Pro Val Asp Gln Ser Pro Gln Glu Pro Asp Ser Ser Ser Gly Val Gly

145 150 155 160

Lys Ser Gly Lys Gln Pro Ala Arg Lys Arg Leu Asn Phe Gly Gln Thr

165 170 175

Gly Asp Ser Glu Ser Val Pro Asp Pro Gln Pro Leu Gly Glu Pro Pro

180 185 190

Ala Ala Pro Ser Ser Val Gly Ser Gly Thr Val Ala Ala Gly Gly Gly

195 200 205

Ala Pro Met Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Asn Ala

210 215 220

Ser Gly Asn Trp His Cys Asp Ser Thr Trp Leu Gly Asp Arg Val Ile

225 230 235 240

Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His Leu

245 250 255

Tyr Lys Gln Ile Ser Ser Ala Ser Thr Gly Ala Ser Asn Asp Asn His

260 265 270

Tyr Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg Phe

275 280 285

His Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn Asn

290 295 300

Trp Gly Phe Arg Pro Lys Arg Leu Asn Phe Lys Leu Phe Asn Ile Gln

305 310 315 320

Val Lys Glu Val Thr Thr Asn Asp Gly Val Thr Thr Ile Ala Asn Asn

325 330 335

Leu Thr Ser Thr Val Gln Val Phe Ser Asp Ser Glu Tyr Gln Leu Pro

340 345 350

Tyr Val Leu Gly Ser Ala His Gln Gly Cys Leu Pro Pro Phe Pro Ala

355 360 365

Asp Val Phe Met Ile Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asn Gly

370 375 380

Ser Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe Pro

385 390 395 400

Ser Gln Met Leu Arg Thr Gly Asn Asn Phe Thr Phe Ser Tyr Thr Phe

405 410 415

Glu Glu Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu Asp

420 425 430

Arg Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Asn Arg

435 440 445

Thr Gln Asn Gln Ser Gly Ser Ala Gln Asn Lys Asp Leu Leu Phe Ser

450 455 460

Arg Gly Ser Pro Ala Gly Met Ser Val Gln Pro Lys Asn Trp Leu Pro

465 470 475 480

Gly Pro Cys Tyr Arg Gln Gln Arg Val Ser Lys Thr Lys Thr Asp Asn

485 490 495

Asn Asn Ser Asn Phe Thr Trp Thr Gly Ala Ser Lys Tyr Asn Leu Asn

500 505 510

Gly Arg Glu Ser Ile Ile Asn Pro Gly Thr Ala Met Ala Ser His Lys

515 520 525

Asp Asp Lys Asp Lys Phe Phe Pro Met Ser Gly Val Met Ile Phe Gly

530 535 540

Lys Glu Ser Ala Gly Ala Ser Asn Thr Ala Leu Asp Asn Val Met Ile

545 550 555 560

Thr Asp Glu Glu Glu Ile Lys Ala Thr Asn Pro Val Ala Thr Glu Arg

565 570 575

Phe Gly Thr Val Ala Val Asn Leu Gln Ser Ser Ser Thr Asp Pro Ala

580 585 590

Thr Gly Asp Val His Val Met Gly Ala Leu Pro Gly Met Val Trp Gln

595 600 605

Asp Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile Pro His

610 615 620

Thr Asp Gly His Phe His Pro Ser Pro Leu Met Gly Gly Phe Gly Leu

625 630 635 640

Lys His Pro Pro Pro Gln Ile Leu Ile Lys Asn Thr Pro Val Pro Ala

645 650 655

Asn Pro Pro Ala Glu Phe Ser Ala Thr Lys Phe Ala Ser Phe Ile Thr

660 665 670

Gln Tyr Ser Thr Gly Gln Val Ser Val Glu Ile Glu Trp Glu Leu Gln

675 680 685

Lys Glu Asn Ser Lys Arg Trp Asn Pro Glu Val Gln Tyr Thr Ser Asn

690 695 700

Tyr Ala Lys Ser Ala Asn Val Asp Phe Thr Val Asp Asn Asn Gly Leu

705 710 715 720

Tyr Thr Glu Pro Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg Pro Leu

725 730 735

<210>15

<211>2211

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>15

atggctgccg atggttatct tccagattgg ctcgaggaca acctctctga aggaataaga 60

cagtggtgga agctcaaacc tggcccacca ccaccaaagc ccgcagagcg gcataaggac 120

gacagcaggg gtcttgtgct tcctgggtac aagtacctcg gacccttcaa cggactcgac 180

aagggggagc cggtcaacgc ggcggacgca gcggccctcg agcacgacaa ggcctacgac 240

cagcagctca aagcgggtga caatccgtac ctgcggtata accacgccga cgccgagttt 300

caggagcgtc tgcaagaaga tacgtcattt gggggcaacc tcgggcgagc agtcttccag 360

gccaagaagc gggttctcga acctctcggt ctggttgagg aaggcgctaa gacggctcct 420

ggaaagaaac gtccggtaga gcagtctcct caggaaccgg actcctccgc gggtattggc 480

aaatcgggtg cacggcccgc taaaaagaga ctcaattttg gtcagactgg cgacacagag 540

tcagtcccag accctcaacc aatcggagaa cctcccgcag cccccacaag tttgggatct 600

aatacaatgg cttcaggcgg tggcgcacca atggcagaca ataacgaagg cgccgacgga 660

gtgggtaatg cctcaggaaa ttggcattgc gattccacat ggctgggcga cagagtcgtc 720

accaccagca cccgcacctg ggccttgccc acctataaca accacctcta caagcaaatc 780

tccagtgctt caacgggggc cagcaacgac aaccactact tcggctacag caccccctgg 840

gggtattttg atttcaacag attccactgc cacttttcac cacgtgactg gcagcgactc 900

atcaacaaca attggggatt ccggcccaag agactcaact tcaagctctt caacatccaa 960

gtcaaggagg tcacgacgaa tgatggcgtc acgaccatcg ctaataacct taccagcacg 1020

gtccaggtct tcacggactc agactatcag ctcccgtacg tgctcgggtc ggctcacgag 1080

ggctgcctcc ctccgttccc ggcggacgtg ttcatgattc cgcaatacgg ctacctgacg 1140

ctcaacaatg gcagccaagc cgtgggtcgt tcgtcctttt actgcctgga atatttcccg 1200

tcgcaaatgc taagaacagg caacaacttt accttcagct acacctttga ggaagtgcct 1260

ttccacagca gctacgcgca cagccagagc ctggaccggc tgatgaatcc tctcatcgac 1320

cagtacctgt attacctgaa cagaactcag aatcagtccg gaagtgccca aaacaaggac 1380

ttgctgttta gccgtgggtc tccagctggc atgtctgttc agcccaaaaa ctggctacct 1440

ggaccctgtt accggcagca gcgcgtttct aaaacaaaaa cagacaacaa caacagcaac 1500

tttacctgga ctggtgcttc aaaatataac ctcaatgggc gtgaatccat catcaaccct 1560

ggcactgcta tggcctcaca caaagacgac aaagacaagt tctttcccat gagcggtgtc 1620

atgatttttg gaaaggagag cgccggagct tcaaacactg cattggacaa tgtcatgatc 1680

acagacgaag aggaaatcaa agccactaac cccgtggcca ccgaaagatt tgggactgtg 1740

gcagtcaatc tccagagcag cagcacggac cctgcgactg gagatgtgca tgttatggga 1800

gccttacctg gaatggtgtg gcaagacaga gacgtatacc tgcagggtcc tatttgggcc 1860

aaaattcctc acacggatgg acactttcac ccgtctcctc tcatgggcgg ctttggactt 1920

aagcacccgc ctcctcagat cctcatcaaa aacacgcctg ttcctgcgaa tcctccggca 1980

gagttttcgg ctacaaagtt tgcttcattc atcacccagt attccacagg acaagtgagc 2040

gtggagattg aatgggagct gcagaaagaa aacagcaaac gctggaatcc cgaagtgcag 2100

tatacatcta actatgcaaa atctgccaac gttgatttta ctgtggacaa caatggactt 2160

tatactgagc ctcgccccat tggcacccgt taccttaccc gtcccctgta a 2211

<210>16

<211>736

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>16

Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn Leu Ser

1 5 10 15

Glu Gly Ile Arg Gln Trp Trp Lys Leu Lys Pro Gly Pro Pro Pro Pro

20 25 30

Lys Pro Ala Glu Arg His Lys Asp Asp Ser Arg Gly Leu Val Leu Pro

35 40 45

Gly Tyr Lys Tyr Leu Gly Pro Phe Asn Gly Leu Asp Lys Gly Glu Pro

50 55 60

Val Asn Ala Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp

65 70 75 80

Gln Gln Leu Lys Ala Gly Asp Asn Pro Tyr Leu Arg Tyr Asn His Ala

85 9095

Asp Ala Glu Phe Gln Glu Arg Leu Gln Glu Asp Thr Ser Phe Gly Gly

100 105 110

Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Val Leu Glu Pro

115 120 125

Leu Gly Leu Val Glu Glu Gly Ala Lys Thr Ala Pro Gly Lys Lys Arg

130 135 140

Pro Val Glu Gln Ser Pro Gln Glu Pro Asp Ser Ser Ala Gly Ile Gly

145 150 155 160

Lys Ser Gly Ala Arg Pro Ala Lys Lys Arg Leu Asn Phe Gly Gln Thr

165 170 175

Gly Asp Thr Glu Ser Val Pro Asp Pro Gln Pro Ile Gly Glu Pro Pro

180 185 190

Ala Ala Pro Thr Ser Leu Gly Ser Asn Thr Met Ala Ser Gly Gly Gly

195 200 205

Ala Pro Met Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Asn Ala

210 215 220

Ser Gly Asn Trp His Cys Asp Ser Thr Trp Leu Gly Asp Arg Val Val

225 230 235 240

Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His Leu

245 250 255

Tyr Lys Gln Ile Ser Ser Ala Ser Thr Gly Ala Ser Asn Asp Asn His

260 265 270

Tyr Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg Phe

275 280 285

His Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn Asn

290 295 300

Trp Gly Phe Arg Pro Lys Arg Leu Asn Phe Lys Leu Phe Asn Ile Gln

305 310 315 320

Val Lys Glu Val Thr Thr Asn Asp Gly Val Thr Thr Ile Ala Asn Asn

325 330 335

Leu Thr Ser Thr Val Gln Val Phe Thr Asp Ser Asp Tyr Gln Leu Pro

340 345 350

Tyr Val Leu Gly Ser Ala His Glu Gly Cys Leu Pro Pro Phe Pro Ala

355 360 365

Asp Val Phe Met Ile Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asn Gly

370 375 380

Ser Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe Pro

385 390 395 400

Ser Gln Met Leu Arg Thr Gly Asn Asn Phe Thr Phe Ser Tyr Thr Phe

405 410 415

Glu Glu Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu Asp

420 425 430

Arg Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Asn Arg

435 440 445

Thr Gln Asn Gln Ser Gly Ser Ala Gln Asn Lys Asp Leu Leu Phe Ser

450 455 460

Arg Gly Ser Pro Ala Gly Met Ser Val Gln Pro Lys Asn Trp Leu Pro

465 470 475 480

Gly Pro Cys Tyr Arg Gln Gln Arg Val Ser Lys Thr Lys Thr Asp Asn

485 490 495

Asn Asn Ser Asn Phe Thr Trp Thr Gly Ala Ser Lys Tyr Asn Leu Asn

500 505 510

Gly Arg Glu Ser Ile Ile Asn Pro Gly Thr Ala Met Ala Ser His Lys

515 520 525

Asp Asp Lys Asp Lys Phe Phe Pro Met Ser Gly Val Met Ile Phe Gly

530 535 540

Lys Glu Ser Ala Gly Ala Ser Asn Thr Ala Leu Asp Asn Val Met Ile

545 550 555 560

Thr Asp Glu Glu Glu Ile Lys Ala Thr Asn Pro Val Ala Thr Glu Arg

565 570 575

Phe Gly Thr Val Ala Val Asn Leu Gln Ser Ser Ser Thr Asp Pro Ala

580 585 590

Thr Gly Asp Val His Val Met Gly Ala Leu Pro Gly Met Val Trp Gln

595 600 605

Asp Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile Pro His

610 615 620

Thr Asp Gly His Phe His Pro Ser Pro Leu Met Gly Gly Phe Gly Leu

625 630 635 640

Lys His Pro Pro Pro Gln Ile Leu Ile Lys Asn Thr Pro Val Pro Ala

645 650 655

Asn Pro Pro Ala Glu Phe Ser Ala Thr Lys Phe Ala Ser Phe Ile Thr

660 665 670

Gln Tyr Ser Thr Gly Gln Val Ser Val Glu Ile Glu Trp Glu Leu Gln

675 680 685

Lys Glu Asn Ser Lys Arg Trp Asn Pro Glu Val Gln Tyr Thr Ser Asn

690 695 700

Tyr Ala Lys Ser Ala Asn Val Asp Phe Thr Val Asp Asn Asn Gly Leu

705 710 715 720

Tyr Thr Glu Pro Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg Pro Leu

725 730 735

<210>17

<211>2214

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>17

atggctgccg atggttatct tccagattgg ctcgaggaca acctctctga aggcattcgt 60

gagtggtggg acctgaaacc tggagccccg aaacccaaag ccaaccagca aaagcaggac 120

aacggccggg gtctggtgct tcctggctac aagtacctcg gacccttcaa cggactcgac 180

aagggggagc ccgtcaacgc ggcggacgca gcggccctcg agcacgacaa ggcctacgac 240

cagcagctca aagcgggtga caatccgtac ctgcggtata accacgccga cgcggagttt 300

caggagcgcc ttaaagaaga tacgtctttt gggggcaacc tcggacgagc agtcttccag 360

gccaagaagc gggttctcga acctctcggt ctggttgagg aaggcgctaa gacggctcct 420

ggaaagaaga gaccggtaga gccatcaccc cagcgttctc cagactcctc tacgggcatc 480

ggcaagaaag gccaacagcc cgccagaaaa agactcaatt tcggtcagac tggcgacaca 540

gagtcagtcc cagaccctca accaatcgga gaacctcccg cagccccctc aggtgtggaa 600

tctcttacaa tggcttcagg tggtggcgca ccagtggcag acagtaacga aggcgccgac 660

ggagtgggta atgcctcagg aaattggcat tgcgattcca catggctggg cgacagagtc 720

atcaccaaca gcacccgaac atgggccttg cccacctata acaaccacct ctacaagcaa 780

atctccagtg cttcaacggg ggccagcaac gacaaccact acttcggcta cagcaccccc 840

tgggggtatt ttgatttcaa cagattccac tgccactttt caccacgtga ctggcagcga 900

ctcatcaaca acaattgggg attccggcccaagagactca acttcaaact cttcaacatc 960

caagtcaagg aggtcacgac gaatgatggc gtcacaacca tcgctaataa ccttaccagc 1020

acggttcaag tcttctcgga ctcggagtac cagcttccgt acgtcctcgg ctctgcgcac 1080

cagggctgcc tccctccgtt cccggcggac gtgttcatga ttccgcaata cggctacctg 1140

acgctcaaca atggcagcca agccgtggga cgttcatcct tttactgcct ggaatatttc 1200

ccttctcaga tgctgagaac gggcaacaac tttaccttca gctacacctt tgaggaagtg 1260

cctttccaca gcagctacgc gcacagccag agcctggacc ggctgatgaa tcctctcatc 1320

gaccaatacc tgtattacct gaacagaact caaaatcagt ccggaagtgc ccaaaacaag 1380

gacttgctgt ttagccgtgg gtctccagct ggcatgtctg ttcagcccaa aaactggcta 1440

cctggaccct gttatcggca gcagcgcgtt tctaaaacaa aaacagacaa caacaacagc 1500

aattttacct ggactggtgc ttcaaaatat aacctcaatg ggcgtgaatc catcatcaac 1560

cctggcactg ctatggcctc acacaaagac gacgaagaca agttctttcc catgagcggt 1620

gtcatgattt ttggaaaaga gagcgccgga gcttcaaaca ctgcattgga caatgtcatg 1680

attacagacg aagaggaaat taaagccact aaccctgtgg ccaccgaaag atttgggacc 1740

gtggcagtca atttccagag cagcagcaca gaccctgcga ccggagatgt gcatgctatg 1800

ggagcattac ctggcatggt gtggcaagat agagacgtgt acctgcaggg tcccatttgg 1860

gccaaaattc ctcacacaga tggacacttt cacccgtctc ctcttatggg cggctttgga 1920

ctcaagaacc cgcctcctca gatcctcatc aaaaacacgc ctgttcctgc gaatcctccg 1980

gcggagtttt cagctacaaa gtttgcttca ttcatcaccc aatactccac aggacaagtg 2040

agtgtggaaa ttgaatggga gctgcagaaa gaaaacagca agcgctggaa tcccgaagtg 2100

cagtacacat ccaattatgc aaaatctgcc aacgttgatt ttactgtgga caacaatgga 2160

ctttatactg agcctcgccc cattggcacc cgttacctta cccgtcccct gtaa 2214

<210>18

<211>737

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>18

Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn Leu Ser

1 5 10 15

Glu Gly Ile Arg Glu Trp Trp Asp Leu Lys Pro Gly Ala Pro Lys Pro

20 25 30

Lys Ala Asn Gln Gln Lys Gln Asp Asn Gly Arg Gly Leu Val Leu Pro

35 40 45

Gly Tyr Lys Tyr Leu Gly Pro Phe Asn Gly Leu Asp Lys Gly Glu Pro

50 55 60

Val Asn Ala Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp

65 70 75 80

Gln Gln Leu Lys Ala Gly Asp Asn Pro Tyr Leu Arg Tyr Asn His Ala

85 90 95

Asp Ala Glu Phe Gln Glu Arg Leu Lys Glu Asp Thr Ser Phe Gly Gly

100 105 110

Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Val Leu Glu Pro

115 120 125

Leu Gly Leu Val Glu Glu Gly Ala Lys Thr Ala Pro Gly Lys Lys Arg

130 135 140

Pro Val Glu Pro Ser Pro Gln Arg Ser Pro Asp Ser Ser Thr Gly Ile

145 150 155 160

Gly Lys Lys Gly Gln Gln Pro Ala Arg Lys Arg Leu Asn Phe Gly Gln

165 170 175

Thr Gly Asp Thr Glu Ser Val Pro Asp Pro Gln Pro Ile Gly Glu Pro

180 185 190

Pro Ala Ala Pro Ser Gly Val Glu Ser Leu Thr Met Ala Ser Gly Gly

195 200 205

Gly Ala Pro Val Ala Asp Ser Asn Glu Gly Ala Asp Gly Val Gly Asn

210 215 220

Ala Ser Gly Asn Trp His Cys Asp Ser Thr Trp Leu Gly Asp Arg Val

225 230 235 240

Ile Thr Asn Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His

245 250 255

Leu Tyr Lys Gln Ile Ser Ser Ala Ser Thr Gly Ala Ser Asn Asp Asn

260 265 270

His Tyr Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg

275 280 285

Phe His Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn

290 295 300

Asn Trp Gly Phe Arg Pro Lys Arg Leu Asn Phe Lys Leu Phe Asn Ile

305 310 315 320

Gln Val Lys Glu Val Thr Thr Asn Asp Gly Val Thr Thr Ile Ala Asn

325 330 335

Asn Leu Thr Ser Thr Val Gln Val Phe Ser Asp Ser Glu Tyr Gln Leu

340 345 350

Pro Tyr Val Leu Gly Ser Ala His Gln Gly Cys Leu Pro Pro Phe Pro

355 360 365

Ala Asp Val Phe Met Ile Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asn

370 375 380

Gly Ser Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe

385 390 395 400

Pro Ser Gln Met Leu Arg Thr Gly Asn Asn Phe Thr Phe Ser Tyr Thr

405 410 415

Phe Glu Glu Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu

420425 430

Asp Arg Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Asn

435 440 445

Arg Thr Gln Asn Gln Ser Gly Ser Ala Gln Asn Lys Asp Leu Leu Phe

450 455 460

Ser Arg Gly Ser Pro Ala Gly Met Ser Val Gln Pro Lys Asn Trp Leu

465 470 475 480

Pro Gly Pro Cys Tyr Arg Gln Gln Arg Val Ser Lys Thr Lys Thr Asp

485 490 495

Asn Asn Asn Ser Asn Phe Thr Trp Thr Gly Ala Ser Lys Tyr Asn Leu

500 505 510

Asn Gly Arg Glu Ser Ile Ile Asn Pro Gly Thr Ala Met Ala Ser His

515 520 525

Lys Asp Asp Glu Asp Lys Phe Phe Pro Met Ser Gly Val Met Ile Phe

530 535 540

Gly Lys Glu Ser Ala Gly Ala Ser Asn Thr Ala Leu Asp Asn Val Met

545 550 555 560

Ile Thr Asp Glu Glu Glu Ile Lys Ala Thr Asn Pro Val Ala Thr Glu

565 570 575

Arg Phe Gly Thr Val Ala Val Asn Phe Gln Ser Ser Ser Thr Asp Pro

580585 590

Ala Thr Gly Asp Val His Ala Met Gly Ala Leu Pro Gly Met Val Trp

595 600 605

Gln Asp Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile Pro

610 615 620

His Thr Asp Gly His Phe His Pro Ser Pro Leu Met Gly Gly Phe Gly

625 630 635 640

Leu Lys Asn Pro Pro Pro Gln Ile Leu Ile Lys Asn Thr Pro Val Pro

645 650 655

Ala Asn Pro Pro Ala Glu Phe Ser Ala Thr Lys Phe Ala Ser Phe Ile

660 665 670

Thr Gln Tyr Ser Thr Gly Gln Val Ser Val Glu Ile Glu Trp Glu Leu

675 680 685

Gln Lys Glu Asn Ser Lys Arg Trp Asn Pro Glu Val Gln Tyr Thr Ser

690 695 700

Asn Tyr Ala Lys Ser Ala Asn Val Asp Phe Thr Val Asp Asn Asn Gly

705 710 715 720

Leu Tyr Thr Glu Pro Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg Pro

725 730 735

Leu

Claims (25)

1. A nucleic acid encoding an adeno-associated virus capsid protein, wherein the nucleic acid comprises a nucleotide sequence selected from any one of the group consisting of:

(a) 1, nucleotide sequence SEQ ID NO;

(b) 3, nucleotide sequence SEQ ID NO;

(c) nucleotide sequence SEQ ID NO 5;

(d) nucleotide sequence SEQ ID NO 7;

(e) the nucleotide sequence of SEQ ID NO 9;

(f) nucleotide sequence SEQ ID NO 11;

(g) 13 in nucleotide sequence SEQ ID NO;

(h) 15, nucleotide sequence SEQ ID NO;

(i) nucleotide sequence SEQ ID NO 17; or

(j) A nucleotide sequence of an adeno-associated virus capsid protein encoded by a nucleotide sequence of any one of (a) - (i) which differs from the nucleotide sequence of (a) - (i) due to the degeneracy of the genetic code.

2. The nucleic acid of claim 1, wherein the nucleic acid is a plasmid, a bacteriophage, a viral vector, a bacterial artificial chromosome, or a yeast artificial chromosome.

3. The nucleic acid of claim 2, wherein the nucleic acid is an adeno-associated viral vector comprising a coding sequence.

4. The nucleic acid of claim 3, further comprising a coding sequence for an adeno-associated viral Rep protein.

5. An adeno-associated virus capsid protein encoded by the nucleic acid of claim 1.

6. The adeno-associated virus capsid protein according to claim 5, wherein the amino acid sequence of the adeno-associated virus capsid protein comprises any one of SEQ ID NO 2, SEQ ID NO 4, SEQ ID NO 6, SEQ ID NO 8, SEQ ID NO 10, SEQ ID NO 12, SEQ ID NO 14, SEQ ID NO 16, or SEQ ID NO 18.

7. The adeno-associated virus capsid protein according to claim 5 or 6, wherein said adeno-associated virus capsid protein is covalently linked to, associated with or encapsulating a composition selected from the group consisting of one or more of a DNA molecule, an RNA molecule, a polypeptide, a carbohydrate, a liposome and a small organic molecule.

8. A recombinant viral particle comprising a nucleic acid according to any one of claims 1 to 4 and/or a capsid protein according to any one of claims 5 to 7.

9. The recombinant viral particle of claim 8, wherein the recombinant viral particle is a recombinant adeno-associated viral particle, a recombinant adenoviral particle, a recombinant herpes viral particle, a recombinant baculovirus particle, or a recombinant hybrid viral particle.

10. A recombinant adeno-associated viral particle comprising an adeno-associated viral vector genome and the adeno-associated viral capsid protein of claim 5 or 6, wherein the adeno-associated viral vector genome is enveloped in the adeno-associated viral capsid protein.

11. The recombinant adeno-associated viral particle of claim 10, wherein the genome of the adeno-associated viral vector comprises a heterologous nucleic acid sequence.

12. The recombinant adeno-associated viral particle according to claim 11 wherein the heterologous nucleic acid sequence encodes one or more selected from the group consisting of antisense RNA, microRNA, shRNA, polypeptides and immunogens.

13. The recombinant adeno-associated viral particle of claim 12, wherein the heterologous nucleic acid sequence encodes a polypeptide that is a therapeutic polypeptide or a reporter gene.

14. The recombinant adeno-associated viral particle according to claim 13, the therapeutic polypeptide encoded by the heterologous nucleic acid is selected from the group consisting of insulin, glucagon, growth hormone releasing factor, erythropoietin, insulin growth factor, transforming growth factor alpha, hepatocyte growth factor, tyrosine hydroxylase, thrombopoietin, interleukin 1-interleukin 25, low density lipoprotein receptor, glucocorticoid receptor, vitamin D receptor, interferon regulatory factor, factor viii, factor ix, glucosidase, glucose-6-phosphatase, isovaleryl-CoA dehydrogenase, propionyl-CoA carboxylase, beta-glucosidase, liver phosphorylase, phosphorylase kinase, glycine decarboxylase, alpha-galactosidase, beta-galactosidase, and lysosomal enzyme.

15. A cell comprising the nucleic acid of any one of claims 1 to 4, the adeno-associated virus capsid protein of any one of claims 5 to 7, the recombinant virion of any one of claims 8 to 9 and/or the recombinant adeno-associated virion of any one of claims 10 to 14.

16. The cell of claim 15, wherein the cell is selected from the group consisting of e.coli, a HEK293 cell line, a HEK293T cell line, a HEK293A cell line, a HEK293S cell line, a HEK293FT cell line, a HEK293F cell line, a HEK293H cell line, a HeLa cell line, a SF9 cell line, a SF21 cell line, a SF900 cell line, and a BHK cell line.

17. A pharmaceutical composition comprising a pharmaceutically acceptable carrier or excipient and one or more selected from the group consisting of the nucleic acid of any one of claims 1 to 4, the adeno-associated virus capsid protein of any one of claims 5 to 7, the recombinant virion of any one of claims 8 to 9, the recombinant adeno-associated virion of any one of claims 10 to 14 and/or the cell of claim 15.

18. Use of one or more of the nucleic acid of any one of claims 1-4, the adeno-associated virus capsid protein of any one of claims 5-7, the recombinant viral particle of any one of claims 8-9, the recombinant adeno-associated viral particle of any one of claims 10-14, the cell of claim 15 and/or the pharmaceutical composition of claim 17 in the manufacture of a medicament for the prevention or treatment of a disease.

19. The disease of claim 18, wherein the disease is selected from the group consisting of cystic fibrosis and other diseases of the lung, hemophilia a, hemophilia B, thalassemia, anemia and other blood disorders, senile dementia, multiple sclerosis, parkinson's disease, huntington's disease, amyotrophic lateral sclerosis, epilepsy, cancer, diabetes, muscular dystrophy, glycogen storage disease and other metabolic defects, congenital emphysema, Lesch-Nyhan syndrome, Niemann-Pick disease, aids, hepatitis, hyperammonemia, and spinocerebral ataxia.

20. A method of producing recombinant adeno-associated virions, the method comprising providing a nucleic acid of claim 1, a nucleic acid encoding a Rep protein of an adeno-associated virus, an adeno-associated viral vector genome carrying heterologous nucleic acid sequences, a helper factor that facilitates production of infectious gonadal-associated virus, and allowing the adeno-associated viral vector genome to be enveloped in adeno-associated viral capsid proteins encoded by the nucleic acid of claim 1 and allowing assembly of the recombinant adeno-associated virions in vitro.

21. The method of claim 20, wherein the method is an adeno-associated virus vector production system, comprising a two-plasmid packaging system, a three-plasmid packaging system, a baculovirus packaging system, and an adeno-associated virus packaging system using adenovirus or herpes simplex virus as a helper virus.

22. A method of delivering a heterologous nucleic acid to a cell in vitro, comprising administering to the cell the nucleic acid of any one of claims 1-4, the adeno-associated virus capsid protein of any one of claims 5-7, the recombinant virion of claim 8 or 9, the recombinant adeno-associated virion of any one of claims 10-14, and/or the pharmaceutical composition of claim 17.

23. The method of delivering a heterologous nucleic acid to a cell according to claim 22, wherein the cell is a mammalian cell, preferably a human stem cell or a liver cell.

24. A method of delivering a heterologous nucleic acid to a mammal, the method comprising administering to a mammalian subject an effective amount of the nucleic acid of any one of claims 1 to 4, the adeno-associated virus capsid protein of any one of claims 5 to 7, the recombinant virion of claim 8 or 9, the recombinant adeno-associated virion of any one of claims 10 to 14, the cell of claim 15, and/or the pharmaceutical composition of claim 17.

25. The method of claim 24, wherein the mammal is a human subject or a primate subject.

Images