CN111876432B - Acquisition and application of a group 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

The acquisition and application of a group of liver-targeted novel adeno-associated viruses

technical field

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

Background technique

Adeno-associated virus (AAV) subtypes named by serotypes are divided into AAV1-AAV12 (Gao Guangping, 2004, JVirolm 78:6381-6388; Mori S et al., 2004, Virology 330:375-383; Schmidt M et al., 2008 , JVirol 82:1399-1406), which mainly use humans and primates as hosts, among which AAV1-6 is isolated from human tissues and has clear antibody reactivity, so it is more classic and recognized. AAV7 and AAV8 were rescued by Gao Guangping and others from macaque heart tissue through genetic engineering. They are suspected to be AAV subtypes that have been extinct in evolution. This strategy provides a new way for the discovery of new AAV subtypes and the design and transformation of recombinant viruses. Ideas and examples (Gao Guangping, 2002, ProcNatl Acad Sci USA 99:11854-11859), and AAV9-12 was prepared from human and macaque tissues using similar technical routes. Although viruses of different AAV serotypes all have icosahedral structures, the diversity of their capsid proteins in sequence and spatial conformation makes their cell surface binding receptors and infection tropism to cells significantly different ( Timpe J et al., 2005, Curr Gene Ther 5:273-284). In terms of infection tropism, AAV2 has a wider infection spectrum, especially for nerve cells; AAV1 and AAV7 have higher transduction efficiency in skeletal muscle; AAV3 is easy to transduce megakaryocytes; The advantages are significant; and the efficiency of AAV8 transducing hepatocytes is better than other subtypes.

The AAV virus itself is replication-deficient and can only be latent in the host cell in the absence of a helper virus. The production of AAV viral vectors requires a helper plasmid (helper) to provide key genes involved in AAV replication from adenovirus (Ad). 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 the maturation of AAV mRNA, and the early genes E2A and VA enhance AAV RNA translation (Berns et al., 1984, Adeno-associated virus 563–592).

As a gene therapy vector, recombinant adeno-associated virus (rAAV) has many advantages, such as high infection efficiency, wide infection range, long-term expression and high safety (David AF et al., 2007, BMC Bio 7:75), has been Widely used in clinical trials. At present, more than 100 gene therapy projects using AAV as vectors have entered clinical research, and the range of diseases treated has expanded to tumors, retinal diseases, arthritis, AIDS, heart failure, muscular dystrophy, nervous system diseases and a series of other genes deficiency disease.

Most of the ongoing clinical research on eye disease gene therapy based on rAAV vectors is aimed at congenital amaurosis (LCA, Leber’s congenital amaurosis) caused by mutations in the retinal pigment epithelium-specific 65kDa protein coding gene (RPE65). The Luxturna drug developed by Spark has been approved by the FDA for marketing in December 2017. This drug is rAAV2 carrying the hRPE65v2 gene to treat LCA and hereditary retinal dysplasia. The test results of the subjects after injection showed the effectiveness of the drug, and no obvious side effects were found, especially vehicle-related side effects (Russell S et al., 2017, Lancet 390:849-860). Other gene therapy eye diseases that have entered the clinic include choroideremia, most of which are in clinical phase I and II. Among them, the University of Alberta uses rAAV2 to carry the gene encoding Rab guard protein 1 (REP1) treatment project, which was announced in phase I in 2018. The results of the study showed the safety and effectiveness of the drug.

Currently, there are more than 20 clinical studies on gene therapy for hemophilia based on rAAV vectors, among which hemophilia B is the most researched. AAV gene medicine with long-term expression ability is an ideal choice for the treatment of hemophilia B. UniQure is conducting phase I/II clinical research, using AAV5 carrying the gene encoding human coagulation factor IX (hFIX) to treat hemophilia B, and the follow-up data of 1 year after administration shows the safety of the drug. The patient developed a humoral immune response 1 week after administration, but it did not affect the expression level of coagulation factor IX, and T cell activation could not be detected using the current "gold standard" system for T cell detection. Transaminases increased but did not affect FIX activity, and no hepatotoxicity was found (Miesbach W et al., 2018, Blood 131:1022-1031). At present, there are still some gene therapy projects using AAV8 vectors to treat hemophilia, and these are liver-targeted clinical studies (Nathwani AC et al., 2006, Blood.107:2653-2661), and AAV8 is also currently recognized as a liver-targeted gene therapy project. Targets optimal AAV serotypes.

Spinal muscular atrophy (SMA) refers to a group of diseases that cause muscle weakness and atrophy due to the degeneration of the anterior horn cells of the spinal cord. AveXis’s AVXS-101 is currently on the market. Taking advantage of AAV9’s advantages in nervous system infection, carrying the gene encoding surviving motor neuron (SMN1) in the treatment of SMA has achieved good results, and no carrier appeared in all patients in clinical trials Clinical symptoms associated with side effects (Mendell JR et al., 2017, N Engl J Med 377:1713-1722).

Other rare disease gene therapy projects using AAV as a vector, for example, using AAV1 to carry the gene encoding acid α-glucosidase (GAA) to treat Pompe disease (Smith BK et al., 2013, Hum Gene Ther 24:630-640), Duchenne muscular dystrophy (DMD) was treated with the modified AAV2.5 carrying the minidystrophin gene (minidystrophin) (Bowles DE et al., 2012, Mol Ther 20:443-455). The results of these clinical studies all showed that the drug effectiveness and safety.

Natural AAV targeting is limited, especially when using AAV vectors for systemic administration, depending on the selection of serotypes, the proportion of cells and tissues that can effectively infect target cells varies greatly, which does not maximize the utilization of AAV; in addition Other non-targeted tissue cells are potentially at risk of infection. In addition, because humans and other primates are naturally infected by AAV, they produce neutralizing antibodies against natural AAV, which will greatly reduce the half-life of AAV and affect its drug activity.

At present, there are more and more studies on the modification of AAV coat protein. The purpose of modification is: on the one hand, it can enhance the targeting of viral vectors, and on the other hand, it can reduce the immunogenic response of viral vectors.

For the carrier serotypes with relatively clear research on the mechanism of virus cell receptors, small-scale or fixed-point modification can be directly targeted at the amino acids in the relevant regions of the cell receptors. The receptors of AAV2 on cells are relatively clear in current research. Heparan sulfate proteoglycan (HSPG) is the main cellular receptor of AAV2 and AAV3 types, and the changes of R484, R487, K532, R585, R588 amino acid sites on the type 2 AAV coat protein will affect its binding to HSPG (Opie, S.R et al., 2003, J Virol 77:6995–7006; Summerford, C et al., 1999, Nat Med 5:78–82).

The modified new AAV vector has been used in gene therapy clinical research, such as AAV2.5 carrying minidystrophin gene therapy for DMD (Bowles DE et al., 2012, Mol Ther 20:443-455), its phase I clinical research has been completed, and Numerous studies have been conducted on the safety of novel AAVs. The AAV2.5 coat protein used in this project is a chimera, and the 5 amino acids related to skeletal muscle targeting in the AAV1 coat protein are grafted onto the AAV2 coat protein. This chimera not only enhances the targeting of skeletal muscle, but also the humoral immune response is significantly lower than that of AAV2. These clinical trials have proved the safety of the modified viral vector.

In addition, when the cell receptor mechanism of the virus is not clear, we can also use DNA shuffling (DNA shuffling) or error-prone PCR methods to obtain a function-optimized coat protein of a new AAV viral vector. For example, Grimm et al. used the shuffled library constructed by AAV to obtain a chimeric AAV-DJ composed of AAV2, 8, and 9 under the strict screening of intravenous immunoglobulin. lines have higher transduction efficiencies (Grimm D et al., 2008, J Virol 82:5887-5911). Jang et al. used a DNA shuffling library to screen a variant that can efficiently infect neural stem cells (Jang J H, 2011, Mol Ther 19:667-675).

At present, gene therapy drugs have increasingly become a research hotspot at home and abroad, and in order to make gene therapy drugs work better and longer, it is important to find a new type of AAV vector with optimized functions to better meet the needs of gene therapy vectors. Problems to be solved.

SUMMARY OF THE INVENTION

Based on the need to find novel AAV vectors, the present invention provides a set of viruses containing AAV capsids with interesting traits, such as targeting and/or neutralizing traits, obtained through in vivo screening to achieve directed evolution of viruses (eg, the ability to evade neutralizing antibodies). 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, said nucleic acid comprising an AAV capsid protein coding sequence selected from the group consisting of:

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

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

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

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

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

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

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

(h) the nucleotide sequence (L107) of Figure 30 (SEQ ID NO: 15);

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

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

In another aspect, the present invention provides the AAV capsid protein encoded by the nucleic acid of the present invention, the amino acid sequence of the AAV capsid protein is selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID Any of NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, or SEQ ID NO:18.

The present invention further provides a recombinant virion comprising a viral genome and the AAV capsid protein of the present invention, wherein the viral genome is encapsulated in the AAV capsid protein. The present invention further provides a recombinant adeno-associated virus particle of an AAV viral genome and the AAV capsid protein of the present invention, wherein the viral genome is encapsulated in the AAV capsid protein. In specific embodiments, the viral genome is a recombinant vector genome comprising heterologous nucleic acid.

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

The present invention provides a pharmaceutical composition, which comprises a pharmaceutically acceptable carrier and the nucleic acid of the present invention, AAV capsid protein, recombinant virus particles, recombinant adeno-associated virus particles and/or cells.

The present invention provides the use of the nucleic acid, AAV capsid protein, recombinant virus particle, recombinant adeno-associated virus particle, cell and/or pharmaceutical composition described herein in the preparation of medicines for preventing or treating diseases.

The present invention provides a method for producing recombinant virion comprising AAV capsid protein, said method comprising: providing nucleic acid of the present invention, AAV Rep protein coding sequence, recombinant vector genome comprising heterologous nucleic acid to cells in vitro, and Produces helpers for productive infection, allowing assembly of recombinant virions containing AAV capsid proteins, and encapsidation of recombinant vector genomes.

The present invention provides a method for producing a recombinant AAV particle comprising an AAV capsid protein, the method comprising: providing the nucleic acid of the present invention, an AAV Rep protein coding sequence, an rAAV genome comprising a heterologous nucleic acid to cells in vitro, and producing Helper for productive infection, allowing assembly of recombinant virions containing AAV capsid proteins, and encapsidation of recombinant vector genomes.

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

The present invention provides a method for delivering a nucleic acid of interest to a mammalian individual, the method comprising: administering an effective dose of the nucleic acid of the present invention, AAV capsid protein, recombinant virion, recombinant adeno-associated virion, cell and/or drug The compositions are administered to mammals.

The present invention also provides a method of identifying a viral vector such as an AAV vector or an AAV capsid protein having a certain tropism property of interest, the 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 have at least two and (ii) a viral vector genome such as an AAV viral genome comprising a coding sequence encoding (i) the AAV capsid protein, an AAV Rep protein coding sequence, at least one and AAV Rep protein interacting terminal repeats (eg 5' and/or 3' terminal repeats) where the viral vector genome is encapsulated in the AAV capsid protein.

(b) administering the collection of viral vectors to a mammalian individual, and

(c) recovering multiple virions or viral vectors encoding the viral genome of the AAV capsid protein from the target tissue, thereby identifying viral vectors or AAV capsid proteins with a tropism of interest.

The positive effects of the present invention are:

The present invention provides a group of novel AAV viral vectors, AAV capsid proteins and virus particles comprising the AAV capsid proteins obtained by directed evolution and in vivo screening methods, compared to the AAV viral vectors in the prior art , viruses with AAV capsids with properties of interest, eg, targeting properties (higher liver tissue targeting) and/or neutralizing properties (eg, ability to evade neutralizing antibodies).

The present disclosure will be further described below in conjunction with the accompanying drawings and specific embodiments, which are not intended to limit the present disclosure. Any equivalent replacement in the field based on the disclosure content of this patent application falls within the scope of protection of this patent.

Description of drawings

Figure 1. Restriction pattern of positive clones randomly selected from the plasmid library. 1-12 respectively correspond to randomly selected positive clone samples, and M stands for 5000bp DNA Marker.

Figure 2. Number of occurrences of AAV mutants in liver, skeletal muscle, and heart after two rounds of in vivo selection. A total of 370 positive clones were obtained after the first in vivo screening, and a total of 9 groups of novel AAV sequences targeting the liver were obtained after the second in vivo screening.

Figures 3A-3R. Novel AAV-Cap sequences. The sequence diagram is divided into nucleotide sequence and encoded amino acid sequence.

Figures 4A-4F. Comparison of infection of different in vitro cell lines (CAG-EGFP). Different AAV vectors were packaged into corresponding viruses carrying CAG-EGFP, and different cell lines were infected with different MOIs in vitro, and flow cytometry analysis was performed 48 hours later. Values are mean ± standard deviation.

Figures 5A-5E. Comparison of in vitro infection of different cell lines (CAG-Luciferase). Different AAV vectors were packaged into corresponding viruses carrying CAG-Luciferase, and different cell lines were infected in vitro by MOI 500, and Luciferase activity was detected after 48 hours. Values are mean ± standard deviation.

Figure 6. Vector genome copy number in different tissues of mice after systemic injection of AAV vector. Different AAV vectors were packaged into corresponding viruses carrying CAG-Luciferase, and after 2 weeks of tail vein injection into mice at a dose of 1E+11vg, the copy number of the vector genome in different tissues was compared. Values are mean ± standard deviation.

Figures 7A-7E. Luciferase activity in different tissues of mice after systemic injection of AAV vector. Different AAV vectors packaged into the corresponding virus carrying CAG-Luciferase were injected into the tail vein of mice at a dose of 1E+11vg for 2 weeks, and the Luciferase activity in different tissues was compared. Values are mean ± standard deviation.

Detailed ways

The present invention provides a set of viral vectors comprising AAV capsids for traits of interest, e.g., targeting traits and/or neutralizing traits (e.g., evasion of neutralizing antibodies) obtained by in vivo screening for directed evolution of viruses Ability). 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 be an exhaustive list of all the different ways of carrying out the invention; moreover, many variations of the various embodiments suggested by the invention will be apparent to those skilled in the art without departing from the invention. Therefore, the following description is intended to illustrate some specific embodiments of the present invention, rather than exhaustively explain all permutations, combinations and changes thereof.

Unless otherwise defined, 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 terms used in the present invention to describe the present invention are only used to describe specific embodiments, and are not used to limit the present invention.

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 containing Vectors for AAV rep and/or cap coding sequences, and for transient or stable transfection of packaging cells. These 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); F.M. 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.

I define

The designation of all amino acid positions in the AAV capsid subunits in the specification and claims of the present invention is related to the numbering of the VP1 capsid subunits.

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

In the present invention, "and/or" refers to any and all possible combinations including one or more of the stated contents.

As used herein, the term "substantially comprising" in relation to a nucleic acid, protein or capsid structure means that said nucleic acid, protein or capsid structure comprises any element that significantly alters the function of said nucleic acid, protein or capsid structure of interest , for example, the targeting or neutralizing properties of the protein or capsid or of 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, AAV, equine AAV and ovine AAV and 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 clades that have been identified are also included in the term "AAV" of the present invention (Gao Guangping, 2004, J. Virology 78:6381-6388).

The genome sequences of various AAV and parvoviruses, as well as the sequences of ITRS, Rep proteins and capsid protein subunits are known in the art and can be found in the literature or in 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, J01, AF0282705, 74 J02275, X01457, AF288061, AH009962, AY028226, AY028223, NC 00135, NC 001540, AF513851, AF5 13852, AY530579, AY63 1965, AY63 1966, 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 Published Patents WO00/28061, WO 99/61601, WO 98/11244; US Patent 6156303; the disclosures of which are also incorporated herein by reference in their entirety. For the early description of AAV1, AAV2 and AAV3 terminal repeat sequences, see Xiao, X, 1996, "Characterization of Adeno-associated virus (AAV) DNA replication and integration," Ph.D. Dissertation, University of Pittsburgh, Pittsburgh, Pa, these contents Incorporated herein by reference in its entirety.

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

The term "targeted" as used herein means that the virus preferentially enters certain cell or tissue types and/or preferentially interacts with the cell surface to facilitate its entry into certain cell or tissue types, optionally and preferably in cells A sequence carried by a viral genome is expressed in a recombinant virus, for example, a heterologous nucleotide sequence is expressed in a recombinant virus. In the case of recombinant AAV genomes, gene expression of the viral genome may be from stably integrated proviruses and/or non-integrating episomes, as well as any other form in which viral nucleic acid may occur intracellularly.

The term "targeting properties" refers to the mode of transduction of one or more target cells, tissues and/or organs. For example, some shuffled AAV capsids may show efficient transduction of liver, gonad, and/or germ cells, some shuffled AAV capsids have only low levels of transduction of skeletal muscle, diaphragmatic muscle, and/or cardiac tissue, The targeting characteristics of typical shuffled AAV capsids are high transduction to liver and low transduction to skeletal muscle.

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

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

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

Similarly, whether a virus "does not efficiently transduce" or "does not effectively target" a target cell or tissue can be determined by reference to an appropriate control. In a specific embodiment, the viral vector ineffectively transduces skeletal muscle and cardiomyocytes. In a specific embodiment, the non-effective transduction of the tissue is 20% or less, 10% of the transduction level of the effective transduction tissue or less, 5% or less, 1% or less, 0.1% or less.

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

"Nucleic acid" or "nucleotide sequence" is a sequence of nucleotide bases, which may be RNA, DNA, or DNA-RNA hybrid sequences, including naturally occurring and non-naturally occurring nucleotides, but preferably single-stranded or double-stranded strand 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, e.g., the cells with which the nucleic acid or nucleic acid sequence is ordinarily associated Or viral structural components or other polypeptides or nucleic acids.

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

The term "treatment" or grammatically equivalent terms means a reduction in the severity or at least partial amelioration or amelioration of a subject's condition, and/or achieving remission or reduction of at least one clinical symptom, and/or a delay in the progression of the condition and and/or prevent or delay the onset of a disease or disorder. The term "treatment" herein also includes prophylactic treatment of a subject, eg, preventing the development of infection, cancer or disease. As used herein, the term "prevention" and its grammatical equivalents include any type of preventive treatment that reduces the incidence of a condition, delays the onset and/or progression of a condition, and/or alleviates symptoms associated with a condition. Accordingly, unless the context dictates otherwise, the term "treatment" or grammatical equivalents refers to prophylactic and therapeutic methods or regimens.

As used herein, an "effective" dose is a dose sufficient to achieve some improvement or benefit in a subject. Alternatively, an "effective" dose is that dose that alleviates or reduces at least one clinical symptom in a subject. Those skilled in the art will appreciate that the effect of the treatment need not be complete or curative as long as the subject obtains an improvement or benefit.

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

A "therapeutic polypeptide" may be a polypeptide that alleviates or reduces symptoms resulting from a missing or defective protein in a cell or subject. In addition, a "therapeutic polypeptide" may be a polypeptide that otherwise benefits the subject, such as an anti-cancer effect or an increase in transplant survival.

As used herein, "vector", "viral vector", and "delivery vector" generally refer to virus particles as nucleic acid delivery vehicles, which include viral nucleic acid packaged in the virus body, that is, the vector genome. Viral vectors according to the invention comprise chimeric AAV capsids of the invention and can package AAV or recombinant AAV genome or any other nucleic acid including viral nucleic acid. Alternatively, in some cases, the terms "vector", "viral vector", "delivery vehicle" may be used to refer to the vector genome in the absence of the virion and/or to the viral capsid as a transporter to deliver and Shell-attached or packaged molecule within a 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 for virus production; however, modified AAV-TRs and non-AAV-TRs can also be used for this purpose. All other viral sequences are dispensable and can be provided in trans (Muzyczka, 1992, curr-topicsmicrobial. Immunol. 158:97). rAAV vectors optionally contain two TRs, such as AAV TRs, usually located 5' and 3' to, but not necessarily adjacent to, the heterologous nucleotide sequence. TRs can be the same or different. The vector genome may also contain 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., required to mediate, for example, replication, viral packaging, integration and/or proviral rescue Function. TR can be AAV TR or non-AAV TR. For example, the non-AAV TR sequence can be other parvoviruses, e.g., canine parvovirus CPV, mouse parvovirus MVM, human parvovirus B-19, or the SV40 hairpin that is the source of SV40 replication, and can be modified by truncation, substitution, deletion , insert further modifications. In addition, TR can be partially or fully synthesized, such as the "double D sequence" described in US Patent No. 5,478,745.

"AAV-terminal repeat" or "AAV TR" 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 Discovery of AAV. It is not necessary to have native terminal repeat sequences as long as the terminal repeats mediate the desired function, such as replication, viral packaging, integration, and/or proviral rescue, etc., e.g., native AAV TR sequences can be modified by insertions, deletions, truncations, and/or missense change by mutation.

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

By "substantially retaining" a property is meant that at least about 75%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% of said property, eg, 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 with interesting characteristics, eg, targeting properties and/or neutralizing properties. In specific embodiments, chimeric AAV capsids exhibit efficient transduction of liver and/or inefficient transduction of skeletal and/or cardiac muscle.

Accordingly, in some embodiments, the invention provides chimeric AAV capsids comprising or consisting essentially of or consisting of the amino acid sequence shown in Figure 3B and viruses comprising said chimeric AAV capsids , 3D, 3F, 3H, 3J, 3L, 3N, 3P or 3R.

In specific embodiments, the chimeric AAV capsid protein may comprise or substantially comprise the expression shown in Figure 3B, Figure 3D, Figure 3F, Figure 3H, Figure 3J, Figure 3L, Figure 3N, Figure 3P or Figure 3R, respectively. or consisting of the amino acid sequences shown in these figures.

Furthermore, in a non-limiting example, a chimeric AAV capsid protein of the invention may be encoded by a nucleic acid comprising or essentially comprising the , the nucleotide sequence shown in Figure 3M, Figure 3O or Figure 3Q, or consist of the nucleotide sequence shown in these Figures; or encode the AAV capsid or capsid protein encoded by any of the above-mentioned nucleotide sequences nucleotide sequence, but differs from the above nucleotide sequence due to codon degeneracy. All designations of amino acid positions in the present specification and appended claims are relative to VP1 numbering. Those skilled in the art will appreciate that the modifications described herein may also result in changes in the VP2 and/or VP3 capsid subunits due to overlapping AAV capsid coding sequences.

The present invention also provides a chimeric AAV capsid protein comprising or substantially comprising or consisting of the following amino acid sequence, methods for evaluating biological properties such as viral transduction and/or antibody neutralization are well known in the art of.

Conservative amino acid substitutions are known in the art. In particular embodiments, conservative amino acid substitutions include one or more of the following group: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; aspartic acid Amide, Glutamine; Serine, Threonine; Lysine, Arginine; and/or Phenylalanine, Tyrosine.

Those skilled in the art will obviously apply the amino acid sequence of the chimeric AAV capsid protein shown in Figure 3B, 3D, 3F, 3H, 3J, 3L, 3N, 3P, 3R to any other modification known in the art, by further Modified to obtain desired properties. For example, R484E and R585E mutations in the AAV2 capsid sequence improved transduction of the heart by AAV vectors (Muller et al., 2006, Cardiovascular Research 70:70-78). As a further non-limiting modification possibility, the capsid protein can be modified to include targeting sequences or sequences that facilitate purification and/or detection, e.g. capsid protein can interact with glutathione-S-transferase, All or part of maltose binding protein, heparin/heparan sulfate binding domain, poly-HIS, ligand and/or reporter protein, immunoglobulin Fc fragment, single chain antibody, hemagglutinin, C-MYC, tag epitope, etc. fused to form a fusion protein. Methods known in the art for inserting targeting peptides into AAV capsids, for example, International Patent WO 00/28004; Nicklin et al., 2001, Molecular Therapy 474-181; White et al., 2004, Circulation 109:513-319; Muller et al. , 2003, Nature Biotech 21:1040-1046.

The virus of the present invention may further comprise a double viral genome as described in International Patent WO01/92551 and US Patent US7465583.

The present invention also provides a recombinant virion comprising the chimeric AAV capsid protein of the present invention, wherein the vector genome is encapsulated in the virion, preferably the AAV vector genome. In a specific embodiment, the present invention provides a recombinant AAV particle comprising the chimeric AAV capsid protein of the present invention, wherein the AAV vector genome is encapsulated in the AAV capsid.

In specific embodiments, the virus is a recombinant vector comprising a heterologous nucleic acid of interest. Accordingly, the present invention can be used to deliver 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 into 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 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 factor 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, aggregation protein, any one of the semaphorin/disintegration protein family, 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 Ligands, tumor necrosis factor alpha and beta, interferon alpha, beta and gamma, stem cell factor, flk-2/flt3 ligand. Gene products produced by the immune system can also be used in the present invention, these products include but are not limited to immunoglobulins IgG, IgM, IgA, IgD and IgE, chimeric immunoglobulins, humanized antibodies, single chain antibodies, T cell receptors , chimeric T cell receptor, single chain T cell receptor, MHC class I and class II 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 effectors, AP-1, AP2, myb, MyoD and myogenin, TFE3, E2F, ATF1, ATF2, ATF3, ATF4, ZF5, NFAT, CREB, HNF-4, C/EBP, CCAAT- Box-binding proteins, interferon regulators, Wilms tumor protein, ETS-binding proteins, STAT, GATA-box-binding proteins and the forkhead protein family of winged helix proteins, carbamyl synthetase 1, ornithine transcarbamylase , argininosuccinate synthase, argininosuccinate lyase, arginase, fumaryl acetoacetate hydrolase, phenylalanine hydroxylase, alpha-1 antitrypsin, glucosidase, glucose-6 -Phosphatase, porphobilinogen deaminase, cystathionine B synthase, branched-chain ketoacid decarboxylase, isovaleryl-CoA dehydrogenase, propionyl-CoA carboxylase, methylmalonyl-CoA transmutation Enzyme, glutaryl-CoA dehydrogenase, insulin, beta-glucosidase, pyruvate carboxylate, liver phosphorylase, phosphorylase kinase, glycine decarboxylase, H-protein, T-protein, cystic fibrosis Transmembrane conductance regulator protein, dystrophin, α-galactosidase, β-galactosidase, lysosomal enzymes, coagulation factors, and any other polypeptide having a therapeutic effect in an individual in need thereof.

A heterologous nucleotide sequence encoding a polypeptide includes a sequence encoding a reporter polypeptide. Polypeptides encoded by reporter genes known in the art include, but are not limited to, green fluorescent protein, β-galactosidase, alkaline phosphatase, luciferase, and chloramphenicol acetyltransferase.

In another aspect, the heterologous nucleic acid can 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 non-translational functional RNAs, such as "guide" RNAS (Gorman et al., 1998, Proc. Nat. Acad. Sci. USA 95:4929; US Patent US5869248) and the like.

Antisense nucleic acids and inhibitory RNA sequences are known in the art to induce "exon skipping". Thus, the heterologous nucleic acid can encode an antisense nucleic acid or suppressor RNA, inducing proper exon skipping.

Ribozymes are RNA-protein complexes that cleave nucleic acids in a site-specific manner. Ribozyme has a specific catalytic domain and has endonuclease activity (Kim et al., 1987, Proc. 211).

microRNAs are natural cellular RNA molecules that regulate the expression of multiple genes by controlling mRNA stability. Overexpression or reduction of specific microRNAs can be used to treat dysfunction and has been shown to be effective in many disease states and animal models of disease (Couzin, 2008, Science 319: 1782-1784). Chimeric AAVs 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 growth of certain tissues, e.g., mir-1, mir-133, mir -206 and/or mir-208 can be used to treat heart and skeletal muscle diseases (Chen et al., 2006, Genet 38: 228-233; van Rooij et al., 2008, Trends Genet. 24: 159-166), microRNA can also be used in gene Post-transmission modulation of the immune system (Brown et al., 2007, Blood 110:4144-4152).

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

Those skilled in the art understand that the antisense oligonucleotide need not be completely complementary to the target sequence, as long as the sequence similarity is sufficient to allow the antisense nucleotide sequence to specifically hybridize to the target sequence and reduce the production of protein products, for example, the similarity is at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more. Hybridization of such oligonucleotides to target sequences can be performed under mildly, moderately or even stringent conditions in order to determine the specificity of hybridization.

Antisense oligonucleotides can be synthesized by chemical synthesis and enzymatic conjugation reactions by procedures known in the art. For example, an antisense oligonucleotide can be chemically synthesized using naturally occurring nucleotides or various modified nucleotides in order to increase the biological stability of the molecule and/or to increase the distance between the antisense and sense strands. The duplex formed is stabilized, for example, using phosphorothioate derivatives and acridine-substituted nucleotides.

Modified nucleotides that can be used to generate antisense oligonucleotides, including 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine , 5-carboxymethylaminomethyl-2-thiouracil, 5-carboxymethylaminomethyluracil, dihydrouracil, β-D-galactosylquinoline, inosine, N6-isopentene lysine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytidine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, β-D-mannose Quinoline, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-prenylamine, uracil-5-oxyacetic acid, quinoline, 2 -thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxoacetate methyl ester, uracil-5- Oxyacetic acid, 5-methyl-2-thiouracil, and 2,6-diaminopurine, etc.

Antisense oligonucleotides can be chemically modified to covalently bind another molecule. For example, antisense oligonucleotides can be conjugated to a molecule that facilitates delivery to cells of interest, enhances uptake in the nasal mucosa, provides a detectable marker, increases bioavailability of the oligonucleotide , Improve the stability of oligonucleotides, or improve formulation or pharmacokinetic properties, etc. 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 body.

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, which introduces double-stranded RNA (DsRNA) corresponding to the target sequence into cells or organisms, resulting in the degradation of the corresponding mRNA. The mechanism by which RNAi achieves gene silencing has been reported in several review articles (Sharp et al., 2001, Genes Dcv 15:485-490; Hammond et al., 2001, Nature Rev Gen 2:110-119). RNAi effects persisted across multiple cell divisions before gene expression was restored. Therefore, RNAi is an effective method for targeted knockout 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). Short synthetic dsRNAs of approximately 21 nucleotides, also known as "short interfering RNAs", have been shown to mediate silencing in mammalian cells without triggering antiviral responses (Elbashir et al., 2001, Nature 411 :494-498; Caplen et al., 2001, Proc. Nat Acad. Sci. 98:9742).

The RNAi molecule can be short hairpin RNA (Paddison et al., 2002, PNAS USA 99:1443-1448), which is processed into 20-25 siRNA molecules in cells by RNaseIII enzyme cleavage. ShRNA usually has a stem-loop structure, that is, two inverted repeats connected by a short spacer.

Methods to generate RNAi include chemical synthesis, in vitro transcription, Dicer digestion of long dsRNA in vitro or in vivo, in vivo expression from delivery vectors, and in vivo expression from PCR-derived RNAi expression cassettes.

The antisense region of the RNAi molecule can be completely complementary to the target sequence, but as long as it specifically hybridizes to the target sequence and reduces the production of protein product, such as at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more, it does not need to be completely complementary to the target sequence. In some embodiments, the hybridization of the oligonucleotide to the target sequence may be performed under conditions of low stringency, medium 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 the protein product. Produces 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 center. RNAi molecules may contain modified sugars, modified nucleotides, backbone linkages, and other modifications of the antisense oligonucleotides described above.

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

The use of parvoviruses as vaccines is well known in the art (US5916563, US5905040, US5882652). The antigen may be present in the viral capsid, or the antigen may be expressed from heterologous nucleic acid introduced into the recombinant vector genome.

An immunogenic polypeptide or immunogen can be any polypeptide suitable for protecting a subject from disease, including but not limited to microbial, bacterial, protozoan, parasitic, fungal, and viral diseases. The immunogen can be a native myxovirus immunogen, e.g., influenza virus immunogen, influenza virus hemagglutinin surface protein, or influenza virus nucleoprotein gene; or a lentiviral immunogen, e.g., equine infectious anemia virus immunogen, simian immunodeficiency Viral immunogen or human immunodeficiency virus immunogen; or arenavirus immunogen, e.g., Lassa fever virus immunogen; or poxvirus immunogen, flavivirus immunogen, filovirus immunogen, bunyavirus immunogen , coronavirus immunogen or severe acute respiratory syndrome immunogen. The immunogen can 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 immunogen known in the art. Vaccine immunogen.

Alternatively, the immunogen can be any tumor or cancer cell antigen. Optionally, tumor or cancer antigens are expressed on the surface of cancer cells. 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, lactoglobulin, p53 tumor suppressor protein, mucin antigen, telomere Enzyme, nuclear matrix protein, prostatic acid phosphatase, papillomavirus antigen, and the following cancers including melanoma, adenocarcinoma, thymoma, sarcoma, lung cancer, liver cancer, colorectal cancer, non-Hodgkin lymphoma, Hodgkin Antigens related to lymphoma, leukemia, uterine cancer, breast cancer, prostate cancer, ovarian cancer, cervical cancer, bladder cancer, kidney cancer, pancreatic cancer, brain cancer, kidney cancer, gastric 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, viral vectors can be introduced into cultured cells and the expressed protein product can be isolated therefrom.

Those skilled in the art understand that a heterologous nucleic acid of interest may be operably linked to appropriate control sequences. For example, a heterologous nucleic acid may be linked to transcriptional translational control signals, replication origins, polyadenylation signals, endosomal entry sites, promoters, enhancers, and other expression control elements.

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

The promoter/enhancer element can be native to the target cell or subject, or it can be native to the heterologous nucleic acid sequence. Promoter/enhancer elements are generally chosen such that they function in the target cell of interest. In representative embodiments, the promoter/enhancer element is a mammalian promoter/enhancer element, which can be constitutive or inducible.

Inducible expression control elements are commonly used in those applications requiring regulation of overexpression of heterologous nucleic acid sequences. 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, Tet on/off elements, RU486 inducible promoters, rapamycin inducible promoters, and metallothionein promoters.

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

The present invention provides chimeric AAV particles comprising a chimeric AAV capsid and an AAV genome. The present 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, 102 or more, ¹⁰³ or more, 10 ⁴ or more, 10 ⁵ or more, or 10 ⁶ or more different sequences.

The present invention also includes "empty" capsid particles comprising, consisting of, or consisting essentially of the chimeric AAV capsid protein of the present invention, as described in US Pat. No. 5,863,541. The chimeric AAV capsids of the present invention can be used as "capsid vehicles", 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, molecules can be associated with the exterior of the viral capsid in order to transfer the molecule into target cells of the host. In one embodiment of the invention, the molecule is covalently linked to the capsid protein. Methods of 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 novel capsid structures. Alternatively, exogenous amino acid sequences can be inserted into viral capsids for presentation of antigens to cells, eg, administered to a subject to generate an immune response to the exogenous amino acid sequences.

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 nucleic acids and/or vectors of the invention. For example, the nucleic acids, vectors, and cells are useful as reagents for producing the viral vectors described herein.

In exemplary embodiments, the present invention provides a nucleic acid sequence encoding an AAV capsid as shown in Figure 3B, 3D, 3F, 3H, 3J, 3L, 3N, 3P, or 3R. The representative nucleic acid sequence comprises or comprises substantially respectively by Fig. 3A (L1) Fig. 3C (L4), Fig. 3E (L10), Fig. 3G (L52), Fig. 3I (L58), Fig. 3K (L84), Fig. 3M ( L37), the nucleotide sequence shown in Figure 3O (L107) or Figure 3Q (L57), or consists of the nucleotide sequence shown in these figures, or encodes an AAV coat encoded by any of the above-mentioned nucleotide sequences The nucleotide sequence of the shell or capsid protein, but differs from the above nucleotide sequences due to codon degeneracy, which allows different nucleic acid sequences to encode the same AAV capsid.

The present invention also provides nucleic acids encoding variants and fusion proteins of the above-mentioned AAV capsid proteins. In specific embodiments, the nucleic acid encodes a variant capsid protein by hybridizing to the complement of a nucleic acid sequence specifically disclosed herein under standard conditions known to those skilled in the art. See Figure 3A, Figure 3C, Figure 3E, Figure 3G, Figure 3I, Figure 3K, Figure 3M, Figure 3O or Figure 3Q for the nucleic acid sequences disclosed herein. At least one property of the capsid protein encoded by the nucleic acid sequence shown in Figure 3A, Figure 3C, Figure 3E, Figure 3G, Figure 3I, Figure 3K, Figure 3M, Figure 3O or Figure 3Q. For example, the virion of the variant capsid protein can substantially retain and comprise the nucleic acid coding sequence coding shown in Figure 3A, Figure 3C, Figure 3E, Figure 3G, Figure 3I, Figure 3K, Figure 3M, Figure 3O, Figure 3Q. Virion targeting characteristics of capsid proteins. Hybridization of such sequences can be performed under weak, moderate or even stringent conditions (Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual 2dEd, Cold Spring Harbor Laboratory).

As is known in the art, a number of different programs can be used to determine whether a nucleic acid or polypeptide has a percent identity or similarity to a known sequence. A percentage designation as used herein refers to a nucleic acid or fragment thereof having a specified percentage to another nucleic acid when aligned to another nucleic acid using BLASTN, the program available on the Internet from the National Center for Biotechnology Information (NCBI).

When referring to polypeptides, percent identity or similarity indicates that the polypeptide exhibits the specified percent identity or similarity when compared to another protein or portion thereof over a common length as determined using BLASTP. This is also available via the Internet from the National Center for Biotechnology Information (NCBI). The percent identity or similarity of polypeptides is typically determined using sequence analysis software, eg, see the Sequence Analysis 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 categories: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine; Threonine; Lysine, Arginine; Phenylalanine, Tyrosine.

In certain embodiments, a nucleic acid may comprise, consist of, or consist essentially of, but is not limited to, vectors such as plasmids, bacteriophages, viral vectors, bacterial artificial chromosomes, or yeast artificial chromosomes. Viral vectors include, but are not limited to, adeno-associated viral vectors, adenoviral vectors, herpes viral vectors, baculoviral vectors, or hybrid viral vectors.

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

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

Nucleic acids can be inserted into delivery vehicles, eg, viral delivery vehicles. For example, nucleic acids of the invention may be encapsulated in AAV particles, adenovirus particles, herpes virus particles, baculovirus particles, or any other suitable virus particles.

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

The present invention also provides a method for producing the viral vector of the present invention. In an exemplary embodiment, the invention provides a method of producing a recombinant viral vector comprising providing, in vitro to a cell, a signal sequence comprising a heterologous nucleic acid and sufficient to package an AAV template into a virion, e.g. , AAV terminal repeat sequence; also includes AAV sequences sufficient for template replication and packaging into virions, such as AAV Rep sequences, and sequences encoding AAV capsids of the present invention. The method further comprises the step of collecting the virus particles from the cells, the virus particles can be collected from the culture medium and/or lysed cells.

In an illustrative embodiment, the invention provides a method of producing a recombinant AAV particle comprising an AAV capsid, the method comprising: providing to a cell in vitro a nucleic acid encoding a chimeric AAV capsid of the invention, an AAV Rep The coding sequence, AAV vector genome comprising heterologous nucleic acid, and cofactors for production of infectious AAV allow encapsulation of the AAV vector genome into an AAV capsid and complete assembly of the AAV particle.

The cells are typically cells that allow replication of the AAV virus. Any suitable cell known in the art may be used, including but not limited to 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 or one or more.

AAV replication and capsid sequences can be provided by any method known in the art. Current methods typically express the 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 can be provided by any viral or non-viral vector. For example, rep and/or cap gene sequences can be provided by hybrid adenoviral or herpes viral vectors. EBV vectors can also be used to express AAVcap and/or rep gene sequences. Alternatively, the rep and/or cap gene sequences can be stably present in the cell, either 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.

Template or vector genomes can be provided to cells using any method known in the art. The template or vector genome can be provided by a non-viral vector or a viral vector. In specific embodiments, the template or vector genome is provided by a herpesvirus or adenovirus vector. Baculovirus vectors, EBV vectors can also be used to deliver templates or vector genomes. 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 achieve maximum viral titers, cells are often provided with helper viruses, such as adenoviruses or herpesviruses, which are required for the production of infectious AAV. The necessary helper viral sequences known in the art for AAV replication are typically provided by helper adenoviral or herpes viral vectors. Alternatively, the adenoviral or herpes viral sequences can be provided by another non-viral or viral vector (Ferrari et al., 1997, Nature Med. 3:1295). In addition, helper viral function can be provided by integration of the helper gene in the chromosome of the packaging cell or maintained as a stable extrachromosomal element.

Those skilled in the art appreciate that it may be advantageous to provide AAV replication and capsid sequences as well as helper virus sequences on a single helper construct. The auxiliary construct can be a non-viral or viral construct, and can also optionally be a hybrid adenovirus or a hybrid herpes virus containing the AAVrep/cap gene sequence.

In a specific embodiment, the AAV rep and/or cap gene sequences and the adenoviral helper sequences are provided by a single adenoviral helper vector. This vector further contains the rAAV genome template. AAV rep and/or cap sequences and/or rAAV templates can be inserted into deleted regions of the adenovirus, eg, the Ela or E3 regions.

In another embodiment, the AAV rep and/or cap sequences and the 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 inside the cell as an extrachromosomal element. In another exemplary embodiment, the AAV rep and/or cap sequences and the 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 can be provided by an rAAV particle or a second recombinant adenoviral particle.

Herpes viruses can also be used as helper viruses in AAV packaging methods.

As another alternative, the viral vectors of the present invention can use baculovirus vectors to deliver rep and/or cap genes and rAAV templates in insect cells (Urabe et al., 2002, Human Gene Therapy 13:1935-1943).

Other methods of producing AAV may also use stably transformed packaging cells (see US Pat. No. 5,658,785).

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 easily distinguished based on size. AAV can also be separated from the helper virus based on its affinity for the heparin substrate. In representative embodiments, a replication deficient helper virus is used such that any contaminating helper virus cannot replicate. Alternatively, a helper adenovirus lacking late gene expression can be used, since only adenoviral early gene expression mediates packaging of AAV virus. Adenovirus mutants that are defective for late gene expression are known in the art, eg, TS100K and TS149 adenovirus mutants.

The packaging method of the present invention can be used to produce high titer virus particles. In specific embodiments, the viral stock has a titer of at least about 10 ⁵ Tu/ml, at least about 10 ⁶ Tu/ml, at least about 10 ⁷ Tu/ml, at least about 10 ⁸ Tu/ml, at least about 10 ⁹ Tu /ml, at least about 10 ¹⁰ Tu/ml.

Novel capsid proteins and capsid structures can be used to generate antibodies, for example, for diagnostic or therapeutic applications or as research reagents. Accordingly, the present invention also provides antibodies against the novel capsid proteins of the present invention.

The term "antibody" or "antibody fragment" as used herein refers to all classes of immunoglobulins, including IgG, IgM, IgA, IgD and IgE. Antibodies can be monoclonal or polyclonal, and can be of any species origin, including mouse, rat, rabbit, horse, goat, sheep, chicken, monkey, alpaca, or human, or can be chimeric, human Antibodies, human antibodies. Antibodies can be recombinant monoclonal antibodies, or screened from phage libraries, yeast libraries, and mammalian cell display libraries.

Antibody fragments included within the scope of the present invention include Fab, F(ab') ₂ and Fc fragments, as well as corresponding fragments obtained from antibodies other than IgG. Such fragments can be produced by known techniques. For example, F(ab') ₂ fragments can be produced from antibody molecules by pepsin digestion, and Fab fragments can be generated by reducing the disulfide bonds of F(ab') ₂ fragments. Alternatively, Fab expression libraries can be constructed to quickly and easily identify monoclonal Fab fragments with the desired specificity.

Polyclonal antibodies can be immunized with viruses to suitable animals, such as rabbits, goats, etc., and the immune serum is collected from the animals and isolated from 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 present invention can be used to deliver nucleotide sequences of interest to cells in vitro, for example, to produce polypeptides in vitro or for gene therapy in vitro. Vectors can also be used in methods of delivering a nucleotide sequence to an individual in need thereof, eg, to express an immunogenic or therapeutic polypeptide.

In general, the viral vectors of the present invention can be used to deliver any foreign nucleic acid with biological effects to treat or improve any disease related to gene expression. In addition, the present invention can be used to treat any disease that can be ameliorated by delivery of a therapeutic polypeptide. Exemplary disease symptoms include, but are not limited to: cystic fibrosis (cystic fibrosis transmembrane regulatory protein) and other diseases of the lung, hemophilia A (factor VIII), hemophilia B (factor IX), thalassemia ( β-globin), anemia (erythropoietin) and other blood disorders, Alzheimer’s disease (GDF), multiple sclerosis (β-interferon), Parkinson’s disease (glial cell-derived neurotrophic factor ), Huntington's disease (elimination of repetitive inhibitory RNA, including but not limited to RNAi, such as siRNA or shRNA, antisense RNA or microRNA), ALS, epilepsy (galanin, neurotrophic factor) and others Neurological diseases, cancer (endostatin, angiostatin, TRAIL, FAS ligand, cytokines including interferon, inhibitory RNA that inhibits VEGF including but not limited to siRNA, shRNA, antisense RNA, microRNA, multidrug resistance gene products, cancer immunogens), diabetes (insulin, PGC-α1, GLP-1, myostatin pro-peptide, glucose transporter), muscular dystrophies including Duchenne muscular dystrophy and Becker muscular dystrophy (e.g. dystrophin, mini-dystrophin, micro-dystrophin, insulin-like growth factor, etc.), glycogen storage diseases such as Fabry disease (α-galactosidase) and Pompe disease (lysosomal acid α-glucosidase), congenital emphysema (antitrypsin), Lesch-Nyhan syndrome (hypoxanthine-guanine phosphoribosyltransferase), Niemann-Pick disease (sphingomyelinase), retinal degenerative diseases, and other diseases of the eye and retina (PDGF, endostatin, and/or angiostatin for macular degeneration), astrocytoma (endostatin, angiostatin, and/or RNAi that inhibits VEGF), glioblastoma (endothelial growth factor, angiostatin endothelial growth factor and/or RNAi anti-vascular endothelial growth factor), liver (RNAi for hepatitis B and/or hepatitis C genes, such as siRNA or shRNA, microRNA or antisense RNA), congestive heart failure or peripheral arterial disease (phosphatase protein inhibitor I, phospholipase, intrasarcoplasmic reticulum 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 propeptide, anti-apoptotic factors, etc.), limb ischemia (VEGF, FGF, PGC-Iα, EC-SOD, HIF), kidney deficiency (erythropoietin), arthritis ( Anti-inflammatory factors such as soluble receptors such as IRAP and TNFα), hepatitis (α-interferon), low-density lipoprotein receptor deficiency (LDL receptor), hyperammonemia (ornithine aminotransferase), phenylketonuria ( phenylalanine hydroxylase), autoimmune diseases, etc.

The invention may also be used after organ transplantation to increase the success rate of transplantation and/or reduce the side effects of organ transplantation or adjuvant therapy, for example, by administering immunosuppressants or inhibitory nucleic acids to block cytokine production.

Gene transfer has enormous potential utility in understanding and treating disease. There are many genetic disorders in which defective genes are known and have been cloned. In general, the above disease states fall into two categories: first, deficient states, usually enzyme deficient, usually inherited in a recessive manner; second, imbalanced states, which may involve regulatory or structural proteins, usually in a dominant manner genetic. For deficient state diseases, gene transfer can bring normal genes into affected tissues for replacement therapy, as well as create animal models of disease using inhibitory RNA, including siRNA or shRNA, microRNA or antisense RNA. For unbalanced disease states, gene transfer can be used to create the disease state in a model system, which can then be used to treat the disease state. Thus, the viral vectors according to the invention allow the treatment of genetic diseases. As used herein, a disease state is treated by partially or fully remediating the disease or a defect or imbalance that makes the disease worse.

In addition, the viral vectors of the present invention have further utility in diagnostic and screening methods in which a gene of interest is transiently or stably expressed in cell culture systems or transgenic animal models. The invention can also be used to deliver nucleic acids for protein production, eg for laboratory, industrial or commercial purposes.

Alternatively, the viral vector can be administered to the cells and the altered cells administered to the individual. The heterologous nucleic acid is introduced into the cell, and the cell is administered to the subject, wherein the heterologous nucleic acid encoding the immunogen is optionally expressed and an immune response to the immunogen is induced in the subject. In specific embodiments, said cells are antigen presenting cells, such as dendritic cells.

An "active immune response" or "active immunity" is characterized by the "involvement of host tissues and cells following contact with the immunogen". It involves the differentiation and proliferation of immunoregulatory cells in lymphoid tissues, leading to antibody synthesis or cell-mediated responses, or both. Alternatively, the host mounts an aggressive immune response following exposure to the immunogen through infection or vaccination. Active immunity can be contrasted with passive immunity, which is obtained by "transferring preformed substances, such as antibodies, transfer factors, thymus grafts, interleukin-2, from an actively immunized host to a non-immune host".

A "protective" immune response or "protective" immunity as used herein means that the immune response confers some benefit on the subject in that it prevents or reduces the incidence of disease. Alternatively, a protective immune response or protective immunity can be used to treat a disease, in particular a cancer or a tumor, for example, to cause regression of the cancer or tumor and/or to prevent metastasis and/or to prevent the growth of metastatic nodules. Protection can be complete or partial as long as the benefits of the treatment outweigh the disadvantages.

The viral vectors of the present invention can also be used in cancer immunotherapy by administering cancer cell antigens or immunologically similar molecules or any other immunogen to generate an immune response to cancer cells. For example, in treating a cancer patient, an immune response against a cancer cell antigen in the individual can be generated by administering a viral vector comprising a heterologous nucleotide sequence encoding a cancer cell antigen. As described herein, 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. Likewise, the term "cancerous tissue" includes tumors. "Cancer cell antigen" includes tumor antigens.

The term "cancer" has its understood meaning in the art, eg, uncontrolled tissue growth with spread or metastasis to distant parts of the body. Exemplary cancers include, but are not limited to, leukemia, lymphoma, colorectal cancer, kidney cancer, liver cancer, breast cancer, lung cancer, prostate cancer, testicular cancer, ovarian cancer, uterine cancer, cervical cancer, brain cancer, bone cancer, sarcoma, melanoma tumor, head and neck cancer, esophageal cancer, thyroid cancer, etc. In an embodiment of the invention, the invention is practiced in the treatment and/or prevention of neoplastic cancer.

Cancer cell antigens have been described above. The term "treating cancer" aims at reducing the severity of, preventing or at least partially eliminating cancer. For example, these terms indicate that the treatment of cancer prevents or reduces, or at least partially eliminates, under certain circumstances. In yet another representative embodiment, these terms indicate arresting or reducing or at least partially eliminating the growth of metastatic nodules, eg, following surgical resection of the primary tumor. According to the term "prevention of cancer", the aim is to at least partially eliminate or reduce the occurrence or incidence of cancer. In other words, the onset or progression of the subject's cancer may be slowed, controlled, reduced or delayed in likelihood or probability.

In a specific example, cells can be removed from an individual with cancer and contacted with a viral vector of the invention. The modified cells are then administered to the subject, thereby eliciting an immune response to the cancer cell antigen. This approach is particularly useful in immunocompromised subjects who are unable to mount an adequate immune response in vivo, ie, do not produce sufficient quantities of boosting antibodies.

Immunomodulatory cytokines are known in the art, such as α-interferon, β-interferon, γ-interferon, ω-interferon, τ-interferon, interleukin-1α, interleukin-1β, interleukin 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 chemoattractant protein-1, granulocyte-macrophage colony-stimulating factor, and lymphotoxin, and immunomodulatory cytokines, such as CTL-inducing cytokines, can be administered to a subject together with a viral vector.

Cytokines can be injected by any method known in the art. The exogenous cytokine can be injected into the subject, or the nucleotide sequence encoding the cytokine can be delivered to the subject using a suitable vector, and the cytokine can be produced in vivo.

The recombinant viral vector according to the present invention can be used in veterinary medicine and medicine. Suitable subjects include birds and mammals. The term "bird" as used herein includes, but is not limited to, chicken, duck, goose, quail, turkey, pheasant, parrot. The term "mammal" as used herein includes, but is not limited to, humans, primates, non-human primates, cattle, sheep, goats, pigs, horses, cats, dogs, rabbits, rodents, and the like. Human subjects include neonates, infants, juveniles and adults. Optionally, the individual is "in need of" the method of the invention, for example, because the individual has or is believed to be at risk of a disease comprising the invention, or would benefit from delivery of a nucleic acid comprising the invention. As a further option, the subject can be a laboratory animal and/or an animal model of disease.

In a specific embodiment, the present invention provides a pharmaceutical composition comprising the viral vector of the present invention, the viral vector is located in a pharmaceutically acceptable carrier, and optionally includes other agents, stabilizers, buffers, carriers, adjuvants , diluent, etc., the carrier for injection is usually a liquid. For other methods, the delivery vehicle can be either solid or liquid. For administration by inhalation, the carrier will be respirable, preferably in solid or liquid particulate form.

"Pharmaceutically acceptable" means a material that is non-toxic or otherwise undesired, ie, the material can be administered to a subject without producing any undesired 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 multiplies of infection according to standard transduction methods appropriate for the particular target cell. The titer of the viral vector or capsid to be administered will vary depending on the target cell type and number and the particular viral vector or capsid. In specific embodiments, at least about 10 ³ infectious units, more preferably at least about 10 ⁵ infectious units, are introduced into the cells.

The cells into which the viral vector is to be introduced can be of any type, including but not limited to nerve cells, including cells of the peripheral and central nervous system, especially brain cells, such as neurons, oligodendrocytes, glial cells, astrocytes Pulmonary 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 cells, dendrites cells, pancreatic cells including islet cells, liver cells, gastrointestinal cells including smooth muscle cells and epithelial cells, cardiac cells including cardiomyocytes, 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 cells may be stem cells, eg, neural stem cells, hepatic stem cells. Alternatively, the cells may be cancer or tumor cells, cancers and tumors as described above. Furthermore, as noted above, cells can be derived from any species of source.

Viral vectors can be introduced into cells ex vivo to administer the modified cells to an individual. In specific embodiments, cells are removed from the individual, a viral vector is introduced therein, and the cells are then returned to the individual. Methods of removing cells from a subject for ex vivo therapy and then reintroducing them into the subject are known in the art, see eg US Pat. No. 5,399,346. Alternatively, the recombinant viral vector is introduced into cells from another individual, in culture, or from any other suitable source, and the cells are administered to the individual in need.

Cells suitable for in vitro gene therapy are described above. The dose of cells administered to a subject will vary with the subject's age, condition and species, cell type, nucleic acid expressed by the cells, mode of administration, and the like. Typically, at least ¹⁰² to ¹⁰⁸ or about ¹⁰³ to about ¹⁰⁶ cells are administered per dose in a pharmaceutically acceptable carrier. In specific 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 specific embodiments, the method includes a method of delivering a nucleic acid of interest to an individual animal, the method comprising: administering to the individual animal an effective amount of the viral vector of the present invention. Administration of the viral vectors of the invention to a human subject or animal in need thereof can be by any method known in the art. Optionally, the viral vector is delivered in a pharmaceutically acceptable carrier in an effective dose.

The viral vectors of the invention can further be administered to an individual to elicit an immunogenic response, eg, as a vaccine. Typically, the vaccines of the invention comprise an effective amount of virus in combination with a pharmaceutically acceptable carrier. Optionally, the dose is sufficient to produce a protective immune response. The degree of protection conferred need not be complete or permanent as long as the benefits of conferring the immunogenic polypeptide outweigh any disadvantages. Subjects and immunogens are as described above.

The dose of viral vector injected into a subject will depend on the mode of administration, the disease or condition being treated, the individual's condition, the particular viral vector and nucleic acid to be delivered, and can be determined in a routine manner. Exemplary doses to achieve a therapeutic effect are viral titers of at least about 10 ⁵ , 10 ⁶ , 10 ⁷ , 10 ⁸ , 10 ⁹ , 10 ¹⁰ , 10 ¹¹ , 10 ¹² , 10 ¹³ , 10 ¹⁴ , 10 ¹⁵ Tu or more , preferably about 10 ⁷ or 10 ⁸ to 10 ¹² , 10 ¹³ or 10 ¹⁴ Tu, more preferably about 10 ¹² Tu.

In particular embodiments, more than one dose may be used, eg, two, three, four or more doses, may be at different time intervals to achieve the desired level of gene expression.

Examples of modes of administration include oral, rectal, mucosal, topical, intranasal, inhalation, buccal, vaginal, intrathecal, intraocular, transdermal, intrauterine, parenteral, intravenous, subcutaneous, intradermal, intramuscular, dermal Intrapleural, intracerebral and intraarticular, skin and mucosal surfaces, intralymphatic vessels, etc., and direct tissue or organ injections, such as direct injection in the liver, skeletal muscle, cardiac muscle, diaphragm or brain. Administration to tumors can also be performed, for example, by injection in or near the tumor or lymph nodes. The most appropriate route in any given case will depend upon the nature and severity of the condition being treated and the nature of the particular vector employed.

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

Pharmaceutical compositions suitable for oral administration may be presented as discrete units such as capsules, cachets, troches 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 or non-aqueous liquid, or as an oil-in-water or water-in-oil emulsion. Oral administration can be accomplished by complexing the viral vector of the present invention to a vector that is resistant to degradation by digestive enzymes in the gut of the animal. Examples of such carriers include capsules or tablets known in the art. Such formulations are prepared by any suitable method of pharmacy which includes the step of bringing into association the ingredients with a suitable carrier which may contain one or more accessory ingredients as hereinbefore described. In general, pharmaceutical compositions according to embodiments of the present invention are prepared by uniformly and intimately mixing said composition with liquid or finely divided solid carriers or both, and then shaping the resulting mixture. For example, a tablet 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 agents. 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 flavored base, typically sucrose and acacia or tragacanth, and an inert base such as gelatin and glycerin or sucrose and acacia. lozenges.

Pharmaceutical compositions suitable for parenteral administration may comprise sterile aqueous and non-aqueous injectable solutions of the compositions of this invention, the formulations being optionally isotonic with the blood of the intended recipient. These formulations may contain antioxidants, buffers, bacteriostats, and solutes to render the ingredients isotonic with the blood of the intended recipient. Aqueous and non-aqueous sterile suspensions, solutions and emulsions may contain suspending and thickening agents. Examples of non-aqueous solvents include propylene glycol, polyethylene glycol, vegetable oils, and injectable organic esters. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral agents include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers such as those based on Ringer's dextrose, and the like. Preservatives and other additives such as antimicrobials, antioxidants, chelating agents and inert gases and the like may also be present.

The compositions can be presented in unit-dose or multi-dose containers, for example, in sealed ampoules and vials, and can be stored in a freeze-dried condition requiring only the addition of a sterile liquid carrier, for example, saline or water for injection, immediately before use.

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

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

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 glycol, alcohols, skin penetration enhancers and combinations of two or more thereof. For example, in some embodiments, topical delivery can be performed by admixing a pharmaceutical composition of the invention with a lipophilic agent capable of entering the skin.

A pharmaceutical composition suitable for transdermal administration may be in the form of a discrete patch, adapted to remain in close contact with the epidermis of a subject for an extended period of time. Compositions suitable for transdermal administration may also be delivered by iontophoresis, and generally take the form of optionally buffered aqueous solutions of compositions of the invention. Suitable formulations may comprise citrate or bis/tris buffer or ethanol/water and may contain from 0.1 to 0.2M active ingredient.

The viral vectors disclosed herein may be administered to the lungs of a subject by any suitable method, such as administration of a suspension of respirable particles consisting of the viral vectors inhaled by the subject. Respirable particulate matter can be liquid or solid. Aerosols of liquid particles comprising viral vectors may be generated by any suitable method, for example using pressure-driven aerosol nebulizers or ultrasonic nebulizers known to those skilled in the art. Aerosols of solid particles comprising viral vectors can likewise be generated by techniques known in the medical art with any solid particle drug aerosol generator.

III Directed evolution and in vivo screening methods of chimeric AAV capsid virus vectors

The present invention also includes a method for preparing a viral library comprising chimeric AAV capsids and then selecting in vivo for chimeric AAV capsids or viruses having one or more desired properties. 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 with a property of interest, e.g., a method of identifying an AAV vector or an AAV capsid with a property of interest, the method comprising:

First, a collection of viral vectors, such as AAV particles, is provided, wherein each AAV vector within the collection comprises a capsid protein produced by shuffling the capsid coding sequences of two or more different AAVs, wherein the The capsid amino acid sequences of two or more different AAVs differ by at least two amino acids; and a viral vector genome, such as an AAV vector genome, comprising the aforementioned shuffled AAV capsid protein coding sequence, an AAV Rep A coding sequence and at least one terminal repeat such as the 5' and/or 3' terminal repeats of AAV, wherein the viral vector genome is encapsulated in an AAV capsid.

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

Third, multiple viral vectors are recovered from target tissues as virions or as viral vector genomes encoding AAV capsids, thereby identifying viral vectors or AAV capsids with properties of interest.

The invention can also be used to identify a chimeric AAV capsid or virion that has the ability to evade neutralizing antibodies in vivo, eg, neutralizing antibodies found in human serum. For example, in vivo screening for neutralizing antibody resistance can be performed by injecting a subject with human immunoglobulin. For example, IVIG is injected into a non-human mammalian subject. IVIG naturally contains a cocktail of antibodies against all common AAVs in humans. Alternatively, the subject can be injected with specific neutralizing antibodies, and then the chimeric virus library can be injected into the subject to select for viral genomes that enter the target tissue of interest (e.g., heart, skeletal muscle, liver, etc.), Genomes isolated from target tissues correspond to capsids that escape neutralization.

Accordingly, in an exemplary embodiment, the invention provides a method of identifying chimeric AAV capsids or viral vectors that have the ability to evade neutralizing IgGs, the method comprising:

First, administering IgGs to a mammalian individual;

Second, a collection of viral vectors, such as AAV particles, is provided, wherein each AAV vector within the collection comprises a capsid protein produced by shuffling the capsid coding sequences of two or more different AAVs, wherein the The capsid amino acid sequences of the two or more different AAVs differ by at least two amino acids; and a viral vector genome, such as an AAV vector genome, which comprises the AAV capsid protein coding sequence produced by the aforementioned shuffling, an AAV Rep The coding sequence and at least one terminal repeat such as the 5' and/or 3' terminal repeat of AAV, wherein the viral vector genome is wrapped in the AAV capsid.

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

Fourth, multiple viral vectors are recovered from target tissues as virions or as viral vector genomes encoding AAV capsids, thereby identifying viral vectors or AAV capsids with properties to evade neutralizing antibodies.

"Evading" neutralizing antibodies, meaning neutralization is at least partially reduced compared to an appropriate control, but as long as the degree of neutralization is reduced compared to a control, and as long as some vector is able to reach and transduce the target tissue, then The degree of "escape" does not need to be complete.

In specific embodiments, the target tissue is liver, skeletal muscle, cardiac muscle, diaphragmatic muscle, kidney, pancreas, spleen, gastrointestinal tract, lung, joint tissue, tongue, ovary, testis, germ cells, cancer cells, or any of the above combination.

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 generate AAV vectors containing chimeric capsids gather. 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, Sheep AAV, Goose AAV or Snake AAV. As noted above, the collection of AAV capsids may further comprise currently known or later discovered non-naturally occurring AAV capsids. Such modifications include substitutions, including substitutions of modified nucleic acids and/or amino acids, as well as deletions and/or insertions.

Further diversity of chimeric capsids can be achieved by any method known in the art to introduce mutations into nucleic acid and/or amino acid sequences, eg, use of chemical mutagens, error-prone PCR, cassette mutagenesis, and the like.

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

The collection of AAV particles can be administered to a subject by any suitable method. In specific embodiments, the collection is administered into the blood of a subject, eg, intravenously or intra-articularly.

Administration and subjects are as described elsewhere herein.

The present invention can be used to identify chimeric viruses or viral capsids with desired properties in vivo. Thus, in specific 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 protein having desired properties for said two or more target tissues. chimeric virus or chimeric AAV capsid. For example, in particular embodiments, chimeric viruses or chimeric AAV capsids with inefficient targeting of skeletal muscle and/or cardiac muscle and or efficient targeting of the liver are identified.

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 viral coat or viral genome encoding the virus isolated from cancer cells or tumors. In representative embodiments, the animal model can be a model with an increased likelihood of developing a cancer or tumor, or can be a xenograft model in which human tumor cells are transplanted into an animal.

Exemplary methods of DNA "shuffling" or "chimerism", also known as "molecular breeding", "rapid forced evolution", etc., are known in the art (US5605793, US6165793, US6117679, Stemmer, 1994, Proc. Nati Acad. Sci91:10747-10751, and Soong et al., 2000, Nature Genetics 25:436-439). This method has also been applied to directed evolution of viruses (US Patent US6096548, US Patent US6596539). In a representative embodiment, collections of AAV capsid protein coding sequences are fragmented and recombined in vitro by homologous and/or non-homologous recombination to form collections of "chimeric" AAV capsid proteins. Each chimeric capsid encapsulates a nucleic acid, such as an AAV genome, comprising the corresponding capsid coding sequence, thereby generating a chimeric virus. Collections of chimeric viruses are administered to subjects and selected in vivo for properties of interest. For example, chimeric viruses can be isolated from one or more target tissues to identify optimized capsid proteins with desired properties.

Ⅳ specific implementation

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

(a) nucleotide sequence SEQ ID NO: 1;

(b) nucleotide sequence SEQ ID NO: 3;

(c) nucleotide sequence SEQ ID NO:5;

(d) nucleotide sequence SEQ ID NO:7;

(e) nucleotide sequence SEQ ID NO:9;

(f) nucleotide sequence SEQ ID NO: 11;

(g) nucleotide sequence SEQ ID NO: 13;

(h) nucleotide sequence SEQ ID NO: 15;

(i) the nucleotide sequence of SEQ ID NO: 17; or

(j) the nucleotide sequence of the AAV capsid protein encoded by any nucleotide sequence of (a)-(i), which is different from the nucleotide of (a)-(i) due to the degeneracy of the genetic code The sequence is different.

The nucleic acid is a plasmid, phage, virus vector, bacterial artificial chromosome, yeast artificial chromosome, preferably an AAV vector containing a coding sequence, more preferably the nucleic acid also contains a coding sequence of an AAV Rep protein.

In one aspect, the present invention also provides an AAV capsid protein encoded by the nucleic acid described above, the amino acid sequence of the AAV capsid protein comprises a sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO: 6. Any one of 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 covalently links, binds or encapsulates a composition selected from one or more of DNA molecules, RNA molecules, polypeptides, carbohydrates, liposomes and small organic molecules Several kinds.

In another aspect, the present invention provides a recombinant virus particle comprising the aforementioned nucleic acid and/or capsid protein. The recombinant virus particles are selected from recombinant AAV virus particles, recombinant adenovirus particles, recombinant herpes virus particles, recombinant baculovirus particles, or recombinant hybrid virus particles.

In another aspect, the present invention provides a recombinant AAV virion comprising the AAV vector genome and the aforementioned AAV capsid protein, wherein the AAV vector genome is wrapped in the AAV capsid. The AAV vector genome contains heterologous nucleic acid sequences. The heterologous nucleic acid sequence encodes one or more selected from 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 sequence is selected from insulin, glucagon, growth hormone, growth hormone releasing factor, erythrocyte-stimulating Propoietin, 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 body, interferon regulatory factor, factor VIII, factor IX, glucosidase, glucose-6-phosphatase, isovaleryl-CoA dehydrogenase, propionyl-CoA carboxylase, beta-glucosidase, liver phosphorylation One or more of the group consisting of enzyme, phosphorylase kinase, glycine decarboxylase, α-galactosidase, β-galactosidase and lysosomal enzyme.

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

In another aspect, the present invention provides a pharmaceutical composition, which comprises a pharmaceutically acceptable carrier or adjuvant and selected from the aforementioned nucleic acid, AAV capsid protein, recombinant virion, recombinant AAV virion and/or the aforementioned one or more types of cells.

In another aspect, the present invention also provides one or more of the aforementioned nucleic acid, AAV capsid protein, recombinant virion, recombinant AAV virion, cell and/or the aforementioned pharmaceutical composition in preparation for prevention or treatment Use in medicine for 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 diseases, Alzheimer's disease, multiple sclerosis, Parkinson's disease, Huntington's disease, ALS, epilepsy, cancer, diabetes, muscular dystrophy, glycogen storage disease and other metabolic defects, congenital emphysema, Lesch-Nyhan syndrome, Niemann- One or more of the group consisting of Pick's disease, AIDS, hepatitis, hyperammonemia, and spinal ataxia.

In one aspect, the present invention provides a method for preparing recombinant AAV virions, the method comprising providing cells with the aforementioned nucleic acid, a nucleic acid coding sequence of AAV Rep protein, a heterologous nucleic acid sequence carrying The AAV vector genome, the auxiliary factor that helps to produce infectious AAV, and allows the AAV vector genome to be wrapped in the AAV capsid encoded by the aforementioned nucleic acid, and realizes the assembly of the recombinant AAV virion, the method is an AAV vector preparation system, Including two-plasmid packaging system, three-plasmid packaging system, baculovirus packaging system and AAV packaging system with Ad or HSV as co-virus, etc.

In one aspect, the present invention also provides a method for delivering heterologous nucleic acid to cells in vitro, said method comprising administering the aforementioned nucleic acid, viral capsid protein, recombinant virion, recombinant AAV virion and/or drug to the cell Compositions, the cells are mammalian cells, preferably human cells, preferably human stem cells or hepatocytes.

In another aspect, the present invention also provides a method for delivering heterologous nucleic acid to mammals, said method comprising administering an effective dose of the aforementioned nucleic acid, viral capsid protein, recombinant virion, or recombinant AAV virion to a mammalian individual . The aforementioned cell and/or the aforementioned pharmaceutical composition, wherein the mammal is a human individual or a primate individual.

After describing the present invention, it will be illustrated in more detail in the following examples, which are provided for illustrative purposes only and are not intended to limit the present invention.

Example

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 good or excellent liver targeting. The AAV capsid gene was shuffled by DNA shuffling technology and an AAV capsid gene library was constructed for in vivo screening in mouse models. Mouse liver-enriched AAV mutants were isolated and their targeting determined by in vitro and in vivo activity testing of the AAV mutant coat.

Example 1 Chimeric AAV plasmid library construction

In order to obtain the chimeric AAV coat gene Cap, first use the upstream primer primerA and the downstream primer primerB to amplify the full-length coat gene from different serotype AAV mothers, and the selected maternal AAV coat gene includes the AAV1 coat (NCBI SequenceID: AF063497 .1, Cap's nucleic acid coding sequence 2223-4433), AAV2 shell (NCBI SequenceID: AF043303.1, Cap's nucleic acid coding sequence 2203-4410), AAV3B shell (NCBI SequenceID: AF028705.1, Cap's nucleic acid coding sequence 2208-4418 ), AAV7 shell (NCBI SequenceID: AF513851.1, the nucleic acid coding sequence 2222-4435 of Cap), AAV8 shell (NCBI SequenceID: AF513852.1, the nucleic acid coding sequence 2121-4337 of Cap), AAV9 shell (NCBI SequenceID: 530579.1, Cap's nucleic acid coding sequence 1-2211). The upstream primer primerA is 5'-CCCAAGCTTCGATCAACTACGCAGACAGGTACCAA-3', and the downstream primerB is 5'-ATAAGAATGCGGCCGCAGAGACCAAAGTTCAACTGAAACGA-3'. PCR amplification is performed under the action of primeSTAR Max DNA polymerase (TaKaRa Company, product number R045A). Amplification conditions: 95°C for 5min 1 cycle; 98°C for 8s, 60°C for 5s, 72°C for 15s, 35 cycles; 72°C for 5min, 1 cycle.

The equal masses of the amplified Cap genes were mixed to obtain a DNA template with a total mass of 4ug, which was randomly fragmented and digested with 0.04U of DNaseI. Under normal conditions, the DNaseI enzyme was inactivated at 75°C for 10 minutes after digestion at 22°C for 6-8min. Diffuse DNA bands were obtained by agarose gel electrophoresis, most of which were concentrated in the length of 500-1000bp, and the DNA fragments in this part of the length range were recovered.

The recovered DNA fragments were first used as primers for random amplification without primers. In order to increase its diversity and amplification efficiency, an unconventional single annealing PCR mode was adopted (94°C for 60s, 65°C for 90s, 72°C for 90s, 10 cycles; 94°C for 60s, 62°C for 90s, 72°C for 90s, 10 cycles; 94°C for 60s, 59°C for 90s, 72°C for 90s, 10 cycles; 94°C for 60s, 56°C for 90s, 72°C for 90s, 10 cycles; 94°C 60s, 53°C 90s, 72°C 90s, 10 cycles; 94°C 60s, 50°C 90s, 72°C 90s, 10 cycles; 94°C 60s, 47°C 90s, 72°C 90s, 10 cycles).

Using the primerless PCR product as a template, using the upstream primer T-primerA (5'-ACGCCTGCCGTTCGACGATTCCCAAGCTTCGATCAACTACGCAGACAGGTACCAA-3') and the downstream primer T-primerB (5'-ACGCGCGGATCTTCCAGAGATAAGAATGCGGCCGCAGAGACCAAAGTTCAACTGAAACGA-3'), HiFi DNA polymerase (TransGenBiotech, product number AS131 Under the action of -22), the full-length amplification of the chimeric Cap gene is carried out. The amplification program was 94°C for 30s, 62°C for 30s, and 72°C for 2.5min, a total of 40 cycles. A chimeric full-length Cap DNA fragment with a length of about 2500bp was obtained, which was double-digested with HindIII and NotI, and pSNAV2.3 vector (containing single-stranded AAV2 genome ITR and polymerase Rep gene sequence, deleted Cap gene) ligation, the ligation product was transformed into E.coli HST08 cells by electric shock, and a chimeric AAV plasmid library with a library capacity greater than 1E+6 clones was prepared. Plasmids identified as positive clones by PCR were identified by PstI, HaeIII, and TaqI digestion, and the differences in the shape of each plasmid were observed (Figure 1) to determine the diversity of the plasmid library.

Embodiment 2 Packaging and titer detection of novel AAV virus library

The above-mentioned chimeric plasmid library is self-packaged and replicated with helper assistance. The packaging method is a two-step method. First, the library plasmid, R2C2, and helper plasmids are transfected at a mass ratio of 1:1:2, and packaged into an AAV intermediate virus library. At this time, the AAV that constitutes the virus library is a heterozygous AAV, that is, the viral coat protein is not necessarily expressed by the genome packaged by it, and the titer of the genome is detected after the harvest solution is extracted and purified with chloroform; the second step is to use the infection index MOI as 100 infected 293T cells to ensure that the genome copy number of virus infected by each cell was less than 10, and the final AAV virus pool produced at this time was homozygous. , Nature Biotechnology, 21:1040-1046). The quality of library plasmids used in packaging is very low to ensure that each novel AAV genome can be packaged in the shell formed by its own expressed Cap protein. The packaged virus particles can normally express the coat protein, which proves that it can form a complete virus coat and has the ability to infect.

Take an appropriate amount of purified AAV sample, prepare a DNase I digestion reaction mixture as shown in the following table (Table 1), incubate at 37°C for 30 minutes, and incubate at 75°C for 10 minutes to inactivate DNase I.

Table 1

AAV samples 5μl 10×DNase I buffer 5μl DNase I 1μl RNase-free water 39μl total 50μl

After the treated AAV purified sample was diluted to an appropriate multiple, the Q-PCR reaction system was configured with reference to the following table (Table 2), and the detection was performed according to the following procedure.

Table 2

The primers used wherein refer to the following table (Table 3):

table 3

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

See the following table (Table 4) for the packaging output results:

Table 4

Virus vector name Genome titer (vg/ml) Novel AAV Virus Library 2.5E+11

Example 3 Screening and enrichment of novel AAV vectors in mice

The virus packaged by the new AAV vector was injected into 8-week-old C57BL/6J mice at a dose of 1.5E+11vg/mouse through the tail vein for liver-targeted screening and enrichment. Three days after the injection, the mice were sacrificed, all the liver tissues were collected, and the total DNA was extracted after liquid nitrogen homogenization. The pair of primers T-primerA/T-primerB was used to amplify again, and the DNA obtained by mixing the amplified novel AAV full-length Cap gene was constructed on the pSNAV2.3 vector by the above method. 370 positive single clones obtained through PCR identification were mixed with equal mass to form the second plasmid library. Repackage into a virus library, inject 1.5E+11vg/tail vein into 8-week-old C57BL/6J mice again, kill the mice three days after injection, and collect all liver tissues, heart tissues and skeletal muscles. 100, 50, and 50 positive clones were randomly selected from the above tissues for sequencing. The positive clones after sequencing were compared and analyzed by BioEdit, VectorNTI, ClustalX2 and TreeViewX.

Select novel AAV vectors with high frequency in the liver and low or no heart and skeletal muscle. A total of 9 groups of high-frequency liver-targeting novel AAV mutants were obtained (Fig. 2), and the nucleotide and amino acid sequences of the above 9 novel AAV-Cap mutants were analyzed (Fig. 3). Because the titer of other 4 kinds of packages is low, it is not suitable for industrialization needs. Therefore, five of the novel AAVs, namely L37, L57, L58, L107, and L10, were selected for subsequent infection activity test experiments.

Example 4 Novel AAV Infection Activity of Human Liver Cell Lines in Vitro

4.1 Using the green fluorescent protein detection system to test the infection activity of the new AAV on human liver cell lines in vitro

In order to preliminarily test the liver-targeted infection activity of the new AAV, the in vitro infection activity test was first performed on six human normal liver or liver cancer cell lines. The obtained new AAV Cap genes L57, L58, L107, and L10 were respectively constructed on the RC plasmid vector containing type 2 AAV Rep, which carried the CAG-EGFP exogenous gene, and the packaged viruses were named AAV2/57, AAV2/ 58, AAV2/107, AAV2/10, and virus titer was detected (results not shown).

The above four recombinant AAV viruses carrying the green fluorescent protein and the AAV2/8 recombinant virus carrying the same green fluorescent protein were simultaneously infected with various human liver cell lines. After 48 hours of infection, the infection activity was detected by flow cytometry. In order to avoid low infection efficiency or oversaturation of viral vectors on a certain cell line, each cell line is infected with at least two infection indices (MOI). It can be seen from Figures (4A-4F) that when using 2- When the three infection indices infected human liver cell lines 7721, HepG2, Huh7, and L02, the infection of the new AAV2/10, AAV2/57, AAV2/58, and AAV2/107 on human liver cell lines showed a significant dose-dependent relationship. Next, the infection results of each type of cell are described with one of the MOIs, and the results of other MOIs are shown in Figure 4.

When the above four recombinant viruses infected the human liver cell line 7402 with an infection index (MOI) of 2500, flow cytometry analysis was performed 48 hours after infection, and the results (Figure 4A) showed that: under this MOI, the infection efficiency of AAV2/10 was The infection efficiency of AAV2/8 is 4.6 times, the infection efficiency of AAV2/57 is 2.2 times that of AAV2/8, the infection efficiency of AAV2/58 is 3.9 times that of AAV2/8, and the infection efficiency of AAV2/107 is that of AAV2/8 1.6 times.

When the above four recombinant viruses infected human liver cell line 7721 with an infection index (MOI) of 2500, flow cytometry analysis was performed 48 hours after infection, and the results (Figure 4B) showed that under this MOI, AAV2/10 had the highest infection efficiency , AAV2/107 infection efficiency is close to it, AAV2/10 infection efficiency is 23.4 times of AAV2/8 infection efficiency, AAV2/107 infection efficiency is 21.8 times of AAV2/8 infection efficiency, AAV2/57 infection efficiency is AAV2/8 infection efficiency 4.6 times of AAV2/58 infection efficiency is 15.9 times of AAV2/8 infection efficiency.

When the above four recombinant viruses infected the human liver cell line HepG2 with an infection index (MOI) of 2500, flow cytometry analysis was performed 48 hours after infection, and the results (Figure 4C) showed that AAV2/10 had the highest infection efficiency at this MOI , the infection efficiency of AAV2/107 is close to it, the infection efficiency of AAV2/10 is 14.8 times that of AAV2/8, and the infection efficiency of AAV2/107 is 13.5 times that of AAV2/8. The infection efficiency of AAV2/57 was 5.5 times that of AAV2/8, and the infection efficiency of AAV2/58 was 10.9 times that of AAV2/8.

When the above four recombinant viruses infected the human liver cell line Huh7 with an infection index (MOI) of 2500, flow cytometry analysis was performed 48 hours after infection, and the results (Figure 4D) showed that AAV2/10 had the highest infection efficiency at this MOI, It is 31.8 times that of AAV2/8. The infection efficiency of AAV2/57 was 1.4 times that of AAV2/8, the infection efficiency of AAV2/58 was 6.6 times that of AAV2/8, and the infection efficiency of AAV2/107 was 19.5 times that of AAV2/8.

When the above four recombinant viruses infected the human liver cell line Huh6 with an infection index (MOI) of 10,000, flow cytometry analysis was performed 48 hours after infection, and the results (Figure 4E) showed that AAV2/10 had the highest infection efficiency at this MOI, The infection efficiency of AAV2/107 was close to it, the infection efficiency of AAV2/10 was 2.9 times that of AAV2/8, and the infection efficiency of AAV2/107 was 2.7 times that of AAV2/8. The infection efficiency of AAV2/57 was 1.8 times that of AAV2/8, and the infection efficiency of AAV2/58 was 2.6 times that of AAV2/8.

When the above four recombinant viruses infected the human liver cell line L02 with an infection index (MOI) of 50,000, flow cytometry analysis was performed 48 hours after infection, and the results (Figure 4F) showed that AAV2/10 had the highest infection efficiency at this MOI , is 42 times the infection efficiency of AAV2/8, the infection efficiency of AAV2/107 is 26.8 times the infection efficiency of AAV2/8, the infection efficiency of AAV2/57 is 2.5 times the infection efficiency of AAV2/8, and the infection efficiency of AAV2/58 is AAV2/8 10.8 times the infection efficiency.

From the above results, it can be seen that the infection activity of these four recombinant novel AAVs on human hepatocytes or liver cancer cell lines is higher than that of the positive control AAV2/8.

4.2 Using the luciferase detection system to test the infection activity of the novel AAV on human hepatic cell lines in vitro

The obtained new AAV Cap genes L37, L57, L58, L107, L10 were constructed on the RC plasmid vector containing type 2 AAV Rep, which carried the CAG-Luciferase exogenous gene, and the packaged virus was named AAV2/37 , AAV2/57, AAV2/58, AAV2/107, AAV2/10, and detect virus titers (results not shown). The above-mentioned recombinant novel AAV virus was used to infect five normal liver or liver cancer cell lines with an MOI of 500. After 48 hours, the infection activity was detected by a luciferase detection system. Comparisons of infective activity were performed after subtracting the NC background value from each assay.

The comparison of the infective activity of the above five recombinant novel AAV viruses on the human liver cell line Huh7 showed that the infective activity from high to low was AAV2/10>AAV2/107>AAV2/58>AAV2/37>AAV2/ 57.

The comparison of the infection activity of the above five recombinant new AAV viruses on the human liver cell line 7402 showed that AAV2/10 had the highest activity, AAV2/107 was close to it, AAV2/58 was close to AAV2/37, and the infection activity was close to that of AAV2/37. The order from high to low is AAV2/10>AAV2/107>AAV2/58>AAV2/37>AAV2/57.

The comparison of the infection activity of the above five recombinant new AAV viruses on the human liver cell line 7721 showed that AAV2/10 had the highest activity, AAV2/58 was close to AAV2/37, and the order of infection activity from high to low was AAV2/10>AAV2/107>AAV2/58>AAV2/37>AAV2/57.

The comparison of the infection activity of the above five recombinant new AAV viruses on the human liver cell line HepG2 showed that AAV2/10 had the highest activity, and the order of infection activity from high to low was AAV2/10>AAV2/37>AAV2/107 > AAV2/58 > AAV2/57.

The comparison of the infection activity of the above five recombinant new AAV viruses in the human liver cell line L02 showed that AAV2/10 had the highest activity, and the order of infection activity from high to low was AAV2/10>AAV2/107>AAV2/37 > AAV2/58 > AAV2/57.

From the above results, it can be seen that among the above-mentioned several novel AAVs, AAV2/10 has the highest activity and AAV2/57 has the lowest activity.

Example 5 In vivo activity test of novel AAV

Five new AAV Cap coat genes L37, L57, L58, L107, and L10 were respectively constructed on the RC plasmid vector containing type 2 AAV Rep, which carried the exogenous CAG-Luciferase gene, and the packaged virus was named AAV2/37, AAV2/57, AAV2/58, AAV2/107, AAV2/10, the virus titers were detected (results not shown), and the in vivo infection activities of these novel AAV viruses were compared. Select 6-8 week old C57BL/6J mice with a dose of 1E+11vg/mouse for tail vein injection, kill the mice 2 weeks later, extract tissue DNA, test the copy number of AAV vector genome in liver, heart, and skeletal muscle, and respectively The expression of luciferase in these three tissues was tested.

The detection results of the vector genome copy number (Figure 6) showed that AAV2/58 had the highest vector genome copy number in the liver compared with other new AAVs, and compared with the heart and skeletal muscle of the same group, the genome copy number in the liver was significantly Elevated; although AAV2/37, AAV2/57, AAV2/107 and AAV2/10 vector genome copy number is lower than AAV2/58 vector genome copy number, compared with the gene copy number in heart and skeletal muscle of the same group Genome copy number was higher in the liver than in the liver.

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

These results indicated that these five novel AAV vectors all had excellent liver targeting, and AAV2/10 had the highest luciferase expression level in the liver compared with other novel AAV vectors, followed by AAV2/58, followed by AAV2/ 37 and AAV2/107 had similar levels of luciferase expression in the liver, and AAV2/57 was relatively the 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 against natural AAV will greatly reduce the half-life of AAV. The level of neutralizing antibodies to novel AAV was detected here.

6.1 Detection and comparison of novel AAV and AAV2/8 neutralizing antibodies in monkey serum

In this experiment, the virus was used to infect, the MOI value was fixed, and the serum was diluted to detect the neutralizing antibody levels of the new AAV vector and AAV2/8 in the serum of cynomolgus monkeys. In a series of serum dilutions, the efficiency of infecting cells with serum-virus mixture was found to be 50% of the efficiency of virus-free serum infecting cells, and the reciprocal of this dilution was used as the amount of neutralizing antibodies, and neutralizing antibodies were evaluated for each viral vector (see Lochrie MA et al., 2006, Virology 353:68-82; Mori S et al., 2006, Jpn JInfect Dis 59:285-293, etc.). In this experiment, the serum of 10 cynomolgus monkeys was tested. After the serum was serially diluted, the neutralizing antibody of the new AAV and the neutralizing antibody of AAV2/8 in each sample were determined and compared.

The specific steps are as follows: the obtained new AAV Cap genes L37, L57, L58, L107, and L10 were respectively constructed on the RC plasmid vector containing type 2 AAV Rep, which carried the CAG-EGFP exogenous gene, and the packaged virus was named as AAV2/37, AAV2/57, AAV2/58, AAV2/107, AAV2/10, and virus titers were detected (results not shown). 7402 cells were inoculated into 24-well plates, cynomolgus monkey serum samples were serially diluted, new AAV viruses AAV2/37, AAV2/57, AAV2/58, AAV2/107, AAV2/10 and AAV2/8 recombinant viruses carrying the same green fluorescent protein The MOI of infection index is 2000, and the diluted serum sample is mixed with the virus solution at 1:1, and incubated at 37°C for 1 hour after mixing, and then the mixture of the serum sample and the virus solution is added to the cells. The cells were harvested after 48 h, and the infection efficiency was detected by flow cytometry.

The results of the neutralizing antibody test are shown in Table 5. When the amount of neutralizing antibody is less than 5, it is considered negative for the neutralizing antibody against the virus. The 10 serum samples were numbered 1#, 2#, 3#, 4#, 5#, 6#, 7#, 8#, 9#, 10#, of which 7# was negative for all virus vector neutralizing antibodies ; 1#, 9# were negative for the neutralizing antibody against the new AAV vector, but were positive for the neutralizing antibody against AAV2/8; 2#, 3#, 4#, 5#, 6#, 8#, 10# The new AAV neutralizing antibodies of the samples were significantly lower than those of AAV2/8.

Therefore, through a comprehensive comparison of the new AAV and AAV2/8 neutralizing antibodies in the serum of cynomolgus monkeys, the neutralizing antibodies of the new AAV in monkey populations are significantly lower than those of AAV2/8, and the new AAV, as a drug delivery carrier, has Lower immunogenicity.

Table 5 Test results of neutralizing antibody in 10 sera of cynomolgus monkeys

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

In this experiment, fixed virus infection index MOI was used to dilute the serum to detect the level of neutralizing antibody to new AAV and the level of neutralizing antibody to AAV2/8 in individual human serum. In a series of serum dilutions, find that the cell infection efficiency of the serum-virus mixture is 50% of the cell infection efficiency of the virus-free serum, and the reciprocal of this dilution is used as the amount of neutralizing antibodies, and the neutralizing antibodies are evaluated for each virus vector. In this experiment, a total of 10 human sera were tested, and the sera were serially diluted to compare the neutralizing antibodies of the novel AAV and the neutralizing antibodies of AAV2/8 in each sample.

The specific steps are as shown in the specific steps in 6.1, the difference is that the serum used is normal human serum. In this experiment, 10 normal human sera were randomly selected for neutralizing antibody experiments, and the new AAV neutralizing antibodies and AAV2/8 neutralizing antibodies in each sample were compared. The neutralizing antibody detection results are shown in Table 6, wherein the 3# sample is negative for all virus neutralizing antibodies; the 2# sample is negative for the new AAV neutralizing antibody, and positive for the AAV2/8 neutralizing antibody; 1#, 4#, 5#, 6#, 7#, 8#, 9#, 10# samples had significantly lower neutralizing antibodies against the new AAV than AAV2/8. The experimental results prove that in the human population, compared with neutralizing antibodies against AAV2/8, the new AAV is more suitable as a drug delivery carrier and can reduce reactogenicity.

Table 6 Test results of neutralizing antibodies in 10 human sera

Based on the above results, the comparison of serum neutralizing antibodies between the new AAV and AAV2/8 shows that the new AAV has lower immunogenicity than the AAV2/8 vector and is more suitable as a gene therapy vector.

sequence listing

<110> Shutaishen (Beijing) Biopharmaceutical Co., Ltd.; Beijing Sannuojiayi Biotechnology Co., Ltd.

<120> Obtainment and application of a group of liver-targeting novel adeno-associated viruses

<130> 2020070729

<160> 18

<170> SIPOSequenceListing 1.0

<210> 1

<211> 2211

<212> DNA

<213> 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

ggaaagaagaaga 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 tgagaacgggg 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

<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

<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 cagcacccccc 840

tggggggtatt 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 tttacccttca 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

<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 Pro Gln 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 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

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 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

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

<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

gtggggtagtt 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

attttggaa aggagagcgc cggagcttca aacactgcat tggacaatgt catgatcaca 1680

gacgaagagg aaatcaaagc cactaaccccc gtggccaccg aaagatttgg gaccgtggca 1740

gtcaatctcc agagcagcag cacagaccct gcgaccggag atgtgcatgt tatggggagcc 1800

ttacctggaa tggtgtggca aagacagagac gtatacctgc agggtcctat ttgggccaaa 1860

attcctcaca cggatggaca ctttcacccg tctcctctca tgggcggctt tggacttaag 1920

cacccgcctc ctcagatcct catcaaaaac acgcctgttc ctgcgaatcc tccggcagag 1980

ttttcggcta caaagtttgc ttcattcatc accccagtatt 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

<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 25 30

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 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

<400> 7

atggctgccg atggttatct tccagattgg ctcgaggaca acctttctga aggcattcgt 60

gagtggtggg ctctgaaacc tggagtccct caacccaaag cgaaccaaca acacaggac 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

ggaaagaagaaga 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 tgagaacgggg 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

<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

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 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

675 680 685

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> 9

<211> 2211

<212> DNA

<213> Artificial Sequence

<400> 9

atggctgccg atggttatct tccagattgg ctcgaggaca acctttctga aggcattcgt 60

gagtggtggg ctctgaaacc tggagtccct caacccaaag cgaaccaaca acacaggac 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

ggaaagaagaaga 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 tgagaacgggg 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 cattggaca 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 ctgtggaca caatggactt 2160

tatactgagc ctcgccccat tggcacccgt taccttaccc gtcccctgta a 2211

<210> 10

<211> 736

<212> PRT

<213> 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

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> 11

<211> 2211

<212> DNA

<213> 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

ggaaagaagaaga 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

gtccagggtct tcacggactc agactatcag ctcccgtacg ttctcggctc tgcccaccag 1080

ggctgcctgc ctccgttccc ggcggacgtg ttcatgattc cccagtacgg ctacctgaca 1140

ctcaacaacg gtagtcaggc cgtgggacgc tcctccttct actgcctgga atactttcct 1200

tctcagatgc tgagaacgggg 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

<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 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 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

<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 tgagaacgggg 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 ctgtggaca caatggactt 2160

tatactgagc ctcgccccat tggcacccgt taccttaccc gtcccctgta a 2211

<210> 14

<211> 736

<212> PRT

<213> 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 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

<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

gtccagggtct 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 ctgtggaca caatggactt 2160

tatactgagc ctcgccccat tggcacccgt taccttaccc gtcccctgta a 2211

<210> 16

<211> 736

<212> PRT

<213> 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 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 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 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

<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 cagcacccccc 840

tgggggtatt ttgatttcaa cagattccac tgccactttt caccacgtga ctggcagcga 900

ctcatcaaca acaattgggg attccggccc aagagactca 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 tttacccttca 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

<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

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 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

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 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 (22)

1. A nucleic acid, characterized in that, the nucleic acid comprises: A nucleotide sequence shown by SEQ ID NO: 5 for encoding an adeno-associated virus capsid protein; or The nucleotide sequence of the adeno-associated virus capsid protein encoded by the nucleotide sequence shown in SEQ ID NO:5, which is different from the nucleotide sequence shown in SEQ ID NO:5 due to the degeneracy of the genetic code . 2. The nucleic acid according to claim 1, wherein the nucleic acid is a plasmid, phage, viral vector, bacterial artificial chromosome or yeast artificial chromosome. 3. The nucleic acid according to claim 2, characterized in that the nucleic acid is an adeno-associated virus vector containing a coding sequence. 4. The nucleic acid of claim 3, further comprising a coding sequence of an adeno-associated virus Rep protein. 5. The adeno-associated virus capsid protein encoded by the nucleotide sequence shown in SEQ ID NO:5 according to claim 1. 6. The adeno-associated virus capsid protein of claim 5, wherein the amino acid sequence of the adeno-associated virus capsid protein is the amino acid sequence shown in SEQ ID NO:6. 7. The adeno-associated virus capsid protein according to claim 5 or 6, characterized in that, the adeno-associated virus capsid protein is covalently linked, bound or encapsulated with a composition selected from the group consisting of DNA, RNA One or more of peptides, carbohydrates, liposomes and small organic molecules. 8. A recombinant virion, characterized in that the recombinant virion comprises the nucleic acid according to any one of claims 1-4 and/or the capsid protein according to any one of claims 5-7. 9. The recombinant virus particle according to claim 8, characterized in that, the recombinant virus particle is a recombinant adeno-associated virus particle, a recombinant adenovirus particle, a recombinant herpes virus particle, a recombinant baculovirus particle, or a recombinant hybrid virus particle . 10. A recombinant adeno-associated virus particle, characterized in that, the recombinant adeno-associated virus particle comprises an adeno-associated virus vector genome and the adeno-associated virus capsid protein according to claim 5 or 6, wherein the adeno-associated virus vector genome wraps in the adeno-associated virus capsid protein. 11. The recombinant adeno-associated virus particle according to claim 10, characterized in that the genome of the adeno-associated virus vector contains heterologous nucleic acid sequences. 12. The recombinant adeno-associated virus particle according to claim 11, characterized in that, the heterologous nucleic acid sequence encodes one or more selected from antisense RNA, microRNA, shRNA, polypeptide and immunogen. 13. The recombinant adeno-associated virus particle according to claim 12, characterized in that, the polypeptide encoded by the heterologous nucleic acid sequence is a therapeutic polypeptide or a reporter gene. 14. The recombinant adeno-associated virus particle according to claim 13, wherein the therapeutic polypeptide encoded by the heterologous nucleic acid is selected from insulin, glucagon, growth hormone, 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, interference Factor Ⅷ, factor Ⅸ, glucosidase, glucose-6-phosphatase, isovaleryl-CoA dehydrogenase, propionyl-CoA carboxylase, β-glucosidase, liver phosphorylase, phosphate One or more of the group consisting of karase kinase, glycine decarboxylase, α-galactosidase, β-galactosidase and lysosomal enzymes. 15. A cell, characterized in that, the cell comprises the nucleic acid according to any one of claims 1-4, the adeno-associated virus capsid protein according to any one of claims 5-7, the adeno-associated virus capsid protein according to any one of claims 8-9 The recombinant virus particle described in any one of claims and/or the recombinant adeno-associated virus particle described in any one of claims 10-14. 16. The cell according to claim 15, wherein the cell is selected from Escherichia coli, HEK293 cell line, HEK293T cell line, HEK293A cell line, HEK293S cell line, HEK293FT cell line, HEK293F cell line, HEK293H cell line, One or more of HeLa cell line, SF9 cell line, SF21 cell line, SF900 cell line, and BHK cell line. 17. A pharmaceutical composition, characterized in that the pharmaceutical composition comprises a pharmaceutically acceptable carrier or adjuvant and a nucleic acid selected from any of claims 1-4, any of claims 5-7, A described adeno-associated virus capsid protein, a recombinant adeno-associated virus particle described in any one of claims 8-9, a recombinant adeno-associated virus particle described in any one of claims 10-14 and/or a described recombinant adeno-associated virus particle described in claim 15 one or more of the cells. 18. The nucleic acid according to any one of claims 1-4, the adeno-associated virus capsid protein according to any one of claims 5-7, the recombinant virus particle according to any one of claims 8-9, any one of the claims One or more of the recombinant adeno-associated virus particles described in any one of 10-14, the cells described in claim 15 and/or the pharmaceutical composition described in claim 17 are prepared for the prevention or treatment of diseases Use in medicine, wherein the disease is one or more selected from the group consisting of hemophilia A, hemophilia B, diabetes, glycogen storage disease, Niemann Pick disease, hepatitis and hyperammonemia. 19. A method for preparing recombinant adeno-associated virus particles, characterized in that the method comprises providing cells with a nucleic acid according to claim 1, a nucleic acid coding sequence of an adeno-associated virus Rep protein, a an adeno-associated virus vector genome carrying a heterologous nucleic acid sequence, an auxiliary factor that helps produce an infectious adeno-associated virus, and allows the adeno-associated virus vector genome to be wrapped in the adeno-associated virus capsid protein encoded by the nucleic acid described in claim 1, And realize the assembly of recombinant adeno-associated virus particles. 20. The method for preparing recombinant adeno-associated virus particles according to claim 19, characterized in that, the method is an adeno-associated virus vector preparation system, comprising a two-plasmid packaging system, a three-plasmid packaging system, a baculovirus packaging system and Adeno-associated virus packaging system with adenovirus or herpes simplex virus as the auxiliary virus, etc. 21. A method for delivering heterologous nucleic acid to cells in vitro, characterized in that the method comprises administering the nucleic acid according to any one of claims 1-4, the nucleic acid according to any one of claims 5-7 to cells Adeno-associated virus capsid protein, the recombinant virus particle described in claim 8 or 9, the recombinant adeno-associated virus particle described in any one of claims 10-14 and/or the pharmaceutical composition described in claim 17, wherein the Said cells are mammalian hepatocytes. 22. The method of delivering heterologous nucleic acid to a cell of claim 21, wherein the cell is a human hepatocyte.

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