CN111876432A - 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

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

technical field

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

Background technique

Adeno-associated virus (AAV) subtypes named by serotype so far are divided into AAV1-AAV12 (Gao Guangping et al., 2004, JVirolm 78:6381-6388; Mori S et al., 2004, Virology 330:375-383; Schmidt M et al., 2008 , JVirol 82:1399-1406), mainly in humans and primates as hosts, of which AAV1-6 are isolated from human tissues and have clear antibody reactivity, so they are more classic and recognized. AAV7 and AAV8 were rescued from macaque heart tissue by Gao Guangping through genetic engineering. They are suspected to be AAV subtypes that have disappeared in evolution. This strategy provides new opportunities for the discovery of new AAV subtypes and the design and modification of recombinant viruses. Ideas and paradigms (Gao Guang et al., 2002, ProcNatl Acad Sci USA 99: 11854-11859), and AAV9-12 were prepared from human and cynomolgus monkey tissues using the same technical route. Although viruses of different AAV serotypes have an icosahedral structure, the diversity of their capsid proteins in sequence and spatial conformation makes their cell surface binding receptors and cell infection tropisms significantly different ( Timpe J et al, 2005, Curr Gene Ther 5:273-284). In terms of infection tropism, AAV2 has a broad infection spectrum, especially for nerve cells; AAV1 and AAV7 have higher transduction efficiency in skeletal muscle; AAV3 is easy to transduce megakaryocytes; AAV5 and AAV6 infect respiratory epithelial cells. The advantage is significant; while AAV8 transduces hepatocytes more efficiently than other subtypes.

The AAV virus itself is replication-deficient and can only remain latent in host cells in the absence of a helper virus. The production of AAV viral vectors requires a helper plasmid (helper) to provide adenovirus (Ad) with key genes involved in AAV replication. These Ad genes include: the early gene E1A is responsible for the transcriptional activity of AAV, the early genes E1B and E4 are involved in 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).

Recombinant adeno-associated virus (rAAV) as a gene therapy vector 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), and has been used by Widely used in clinical experiments. At present, more than 100 gene therapy projects using AAV as a vector have entered clinical research, and the treatment range of diseases has expanded to tumors, retinal diseases, arthritis, AIDS, heart failure, muscular dystrophy, nervous system diseases and a series of other genes Defective disease.

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

Currently, there are more than 20 clinical studies on hemophilia gene therapy based on rAAV vector, among which hemophilia B is the most studied. AAV gene drugs with long-term expression ability are ideal for the treatment of hemophilia B. UniQure is conducting a clinical phase I/II study with AAV5 carrying the gene encoding human coagulation factor IX (hFIX) in the treatment of hemophilia B, and the 1-year follow-up data after administration shows the safety of the drug. The patient developed a humoral immune response 1 week after administration, but 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 were elevated but did not affect FIX activity, and no hepatotoxicity was found (Miesbach W et al., 2018, Blood 131:1022-1031). At present, there are some gene therapy projects using AAV8 vectors to treat hemophilia. These are clinical studies targeting the liver (Nathwani AC et al., 2006, Blood. 107:2653-2661). AAV8 is also currently recognized as the liver Targets optimal AAV serotypes.

Spinal muscular atrophy (SMA) refers to a class of diseases that cause muscle weakness and muscle atrophy due to the degeneration of cells in the anterior horn of the spinal cord. AveXis' AVXS-101 is currently on the market. Taking advantage of the advantages of AAV9 in nervous system infections, carrying the survivin motor neuron-encoding gene (SMN1) has achieved good results in the treatment of SMA, and all patients in the clinical trial did not have the carrier. 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), Treatment of Duchenne muscular dystrophy (DMD) with modified AAV2.5 carrying the gene encoding minidystrophin (Bowles DE et al., 2012, Mol Ther 20:443-455), the results of these clinical studies have shown that the drug effectiveness and safety.

Natural AAV targeting is limited, especially when using AAV vectors for systemic administration, depending on the serotype selection, the proportion of target cells and tissues that can be effectively infected varies greatly, which does not maximize the utilization of AAV; Other non-targeted tissue cells are potentially at risk of infection. In addition, because humans and other primates are naturally infected by AAV, neutralizing antibodies against natural AAV are produced, 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 the modification is: on the one hand, it can enhance the targeting of the viral vector, and on the other hand, it can reduce the immunogenic response of the viral vector.

For the carrier serotypes whose mechanism of viral cell receptors is relatively clear, small-scale or site-specific modifications can be carried out directly on the amino acids in the relevant regions of their cell receptors. The receptor of AAV2 on cells is relatively clear in current research. Heparan sulfate proteoglycan (HSPG) is the major cellular receptor for AAV2 and AAV3, and changes in amino acid sites R484, R487, K532, R585, and R588 on the coat protein of type 2 AAV 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).

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

In addition, when the cellular receptor mechanism of the virus is not clear, we can also use DNA shuffling (DNA shuffling) or error-prone PCR to obtain the coat protein of a novel AAV viral vector with optimized functions. For example, Grimm et al. used the shuffling library constructed by AAV to obtain a chimera AAV-DJ composed of AAV2, 8, and 9 under the strict screening of intravenous immunoglobulin. The vector is suitable for fibroblasts and lung cells. lines have higher transduction efficiencies (Grimm D et al., 2008, J Virol 82:5887-5911). Jang et al. screened a variant that can efficiently infect neural stem cells using a DNA shuffling library (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 necessary 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 traits of interest, such as targeting traits and/or neutralizing traits, obtained by in vivo screening methods for virus directed evolution (eg, the ability to evade neutralizing antibodies). The present invention also provides AAV capsids and virions comprising AAV capsids.

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

(a) 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 of nucleotides (a)-(i) but different from (a)-(i) due to the degeneracy of the genetic code.

In another aspect, the present invention provides an AAV capsid protein encoded by the nucleic acid of the present invention, wherein the amino acid sequence of the AAV capsid protein is selected from 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 recombinant virions 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 an AAV virus genome and a recombinant adeno-associated virus particle of 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 present invention provides a cell comprising the nucleic acid, AAV capsid protein, recombinant virus particle and/or recombinant adeno-associated virus particle of the present invention.

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

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 manufacture of a medicament for preventing or treating a disease.

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

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

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

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

The present invention also provides a method for identifying viral vectors, such as AAV vectors or AAV capsid proteins, having a certain tropism characteristic 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 AAV Rep protein interacting terminal repeats (eg, 5' and/or 3' terminal repeats) in which the viral vector genome is encapsulated in the AAV capsid protein.

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

(c) Recovery of multiple virions or viral vectors of viral genomes encoding AAV capsid proteins from the tissue of interest, 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 set of novel AAV viral vectors, AAV capsid proteins and virus particles comprising the AAV capsid proteins obtained by methods of directed evolution and in vivo screening, compared with the AAV viral vectors in the prior art , viruses with AAV capsids having traits of interest, eg, targeting traits (higher liver tissue targeting) and/or neutralizing traits (eg, ability to evade neutralizing antibodies).

The present disclosure will be further described below with reference to the accompanying drawings and specific embodiments, but is not intended to limit the present disclosure. Any equivalent replacement in the art based on the disclosure content of this patent application shall fall within the protection scope of this patent.

Description of drawings

Figure 1. The restriction map of randomly selected positive clones from the plasmid library. 1-12 correspond to randomly selected positive clone samples respectively, and M represents 5000bp DNA Marker.

Figure 2. Frequency of AAV mutants in liver, skeletal muscle, and heart after two rounds of in vivo screening. After the first in vivo screening, a total of 370 positive clones were obtained, and after the second in vivo screening, a total of 9 groups of novel AAV sequences targeting the liver with high frequency were obtained.

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 with MOI 500 in vitro, 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 the corresponding viruses carrying CAG-Luciferase. After 2 weeks of tail vein injection into mice at a dose of 1E+11vg, the number of vector genome copies 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 corresponding viruses carrying CAG-Luciferase were injected into mice by tail vein 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 with traits of interest, e.g., targeting traits and/or neutralizing traits (e.g., evading neutralizing antibodies, obtained by in vivo screening for directed evolution of viruses) Ability). The present invention also provides AAV capsids and virions comprising AAV capsids.

The present invention will be explained in more detail below. This description is not intended to enumerate in detail all the different ways to carry out the invention, and moreover, many variations of the various embodiments proposed by the invention will be apparent to those skilled in the art without departing from the invention. Accordingly, the following description is intended to illustrate some specific embodiments of the invention, but not to be exhaustive of all permutations, combinations and variations 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 to describe the present invention in the present invention are only used to describe specific embodiments, and are not used to limit the present invention.

Unless otherwise indicated, standard methods known to those skilled in the art can be used to produce recombinant and synthetic polypeptides, antibodies or antigen-binding fragments thereof, manipulate nucleic acid sequences, produce transformed cells, construct recombinant AAVs, modify capsid proteins, package AAV rep and/or cap coding sequences, and 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.

IDefinition

The assignment of all amino acid positions in the AAV capsid subunit in the present specification and its claims relates to the VP1 capsid subunit numbering.

In this specification and its claims, the singular forms "a," "an," and "the" include the plural forms as well, unless the context clearly dictates otherwise.

As used herein, "and/or" is meant to include any and all possible combinations of one or more of the stated items.

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

The term "adeno-associated virus (AAV)" in the context of the present invention includes, but is not limited to, AAV1, AAV2, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, avian AAV, bovine AAV, canine AAV AAV, equine AAV and sheep 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 have been identified and are also included in the term "AAV" of the present invention (Gao Guang et al., 2004, J. Virology 78:6381-6388).

The genomic sequences of various AAV and parvoviruses are known in the art, as well as the sequences of the ITRS, Rep protein and capsid protein subunits, and such sequences 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, AF028715, AF08 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. In addition, 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 Publications WO 00/28061, WO 99/61601, WO 98/11244; US Patent 6,156,303; these disclosures are also incorporated herein by reference in their entirety. For an early description of AAV1, AAV2, and AAV3 terminal repeats, see Xiao, X, 1996, "Characterization of Adeno-associated virus (AAV) DNA replication and integration," Ph.D. Dissertation, University of Pittsburgh, Pittsburgh, Pa. Incorporated herein by reference in its entirety.

"Shuffled" or "chimeric" AAV capsid coding sequences or AAV capsid proteins are nucleic acid and amino acid sequences produced by mixing two or more different AAV capsid protein sequences that combine two or more capsid sequences. 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 cells or tissue types and/or preferentially interacts with the cell surface to facilitate its entry into certain cells or tissue types, optionally and preferably in cells Sequences carried by the viral genome are expressed in a recombinant virus, for example, a recombinant virus expresses a heterologous nucleotide sequence. In the case of recombinant AAV genomes, the gene expression of the viral genome may be from stably integrated proviruses and/or non-integrated episomes, as well as any other form of viral nucleic acid that may occur intracellularly.

The term "targeting property" refers to the transduction pattern of one or more target cells, tissues and/or organs. For example, some shuffled AAV capsids may show efficient transduction of liver, gonads and/or germ cells, some shuffled AAV capsids have only low levels of transduction to skeletal, diaphragm and/or cardiac tissue, Typical shuffled AAV capsids have targeting features of high transduction in liver and low transduction in skeletal muscle.

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

As used herein, a "collection" or "plurality" of a virion, vector, capsid or capsid protein means two or more unless the context dictates otherwise.

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

Similarly, it can be determined whether a virus "ineffectively transduces" or "ineffectively targets" a target cell or tissue by reference to an appropriate control. In specific embodiments, the viral vector transduces skeletal muscle, cardiomyocytes inefficiently, and in specific embodiments, the ineffective transduction of tissue is 20% or less, 10% of the level of transduction of efficiently transduced tissue or less, 5% or less, 1% or less, 0.1% or less.

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

A "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 component of a naturally occurring organism or virus, eg, a cell with which the nucleic acid or nucleic acid sequence is normally associated or viral structural components or other polypeptides or nucleic acids.

Likewise, an "isolated" polypeptide refers to a polypeptide that is isolated from at least some other components 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 commonly associated.

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

As used herein, an "effective" dose is a dose sufficient to obtain some improvement or benefit in a subject. Alternatively, an "effective" dose is one that relieves or reduces at least one clinical symptom in a subject. Those skilled in the art will understand that the therapeutic effect need not be complete or curative so long as the subject experiences 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" can be a polypeptide that alleviates or reduces symptoms resulting from a deletion or deficiency of a protein in a cell or subject. In addition, a "therapeutic polypeptide" can be one that otherwise confers a benefit to the subject, such as an anticancer effect or an increase in transplant survival.

As used herein, "vector", "viral vector" and "delivery vector" generally refer to virions as nucleic acid delivery vehicles, which include viral nucleic acid packaged within the virion, ie, the vector genome. The viral vector according to the present invention comprises the chimeric AAV capsid of the present 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 vector" may be used to refer to the vector genome in the absence of virions and/or to the viral capsid as a transporter to deliver the Shell-attached or packaged molecules 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 to generate viruses; however, modified AAV-TRs and non-AAV-TRs can also be used for this purpose. All other viral sequences are dispensable and available in trans (Muzyczka, 1992, curr-topicsmicrobial. Immunol. 158:97). An rAAV vector may optionally contain two TRs, eg, AAV TRs, usually located 5' and 3' to the heterologous nucleotide sequence, but not necessarily adjacent to it. The 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 factors such as replication, viral packaging, integration and/or proviral rescue, etc. Function. TRs can be AAV TRs or non-AAV TRs. For example, non-AAV TR sequences 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 replication for SV40, and can be modified by truncation, substitution, deletion , Insert further modifications. In addition, TR can be partially or fully synthesized, as described in US Pat. No. 5,478,745 for the "double D sequence".

An "AAV-terminal repeat" or "AAV TR" can be from any AAV, including but not limited to serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or any other known or later Discovered AAV. It is not necessary to have native terminal repeats 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 insertion, deletion, truncation 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.

"Substantially retaining" a property means retaining at least about 75%, 85%, 90%, 95%, 97%, 98%, 99% or 100% of the property, eg, activity or other measurable property.

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

The inventors have identified "chimeric" or "shuffled" AAV capsid structures with characteristics of interest, eg, targeting properties and/or neutralizing properties. In specific embodiments, the chimeric AAV capsids exhibit efficient transduction of liver and/or inefficient transduction of skeletal and/or cardiac muscle.

Accordingly, in some embodiments, the present invention provides chimeric AAV capsids comprising or substantially comprising or consisting of the following amino acid sequences, and viruses comprising the chimeric AAV capsids, the amino acid sequences in Figure 3B , 3D, 3F, 3H, 3J, 3L, 3N, 3P or 3R.

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

Furthermore, in a non-limiting example, the chimeric AAV capsid proteins of the present invention may be encoded by nucleic acids comprising or substantially comprising the components shown in Figure 3A, Figure 3C, Figure 3E, Figure 3G, Figure 3I, Figure 3K, respectively , the nucleotide sequence shown in Figure 3M, Figure 3O or Figure 3Q, or consist of the nucleotide sequences shown in these Figures; or encode an AAV capsid or capsid protein encoded by any of the above nucleotide sequences , but differs from the above nucleotide sequence due to the degeneracy of codons. All assignments of amino acid positions in the present specification and in the appended claims relate to VP1 numbering. Those of skill in the art will appreciate that the modifications described herein may also result in changes to the VP2 and/or VP3 capsid subunits due to overlapping AAV capsid coding sequences.

The present invention also provides chimeric AAV capsid proteins comprising or consisting essentially of the following amino acid sequences, methods of assessing 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 specific embodiments, conservative amino acid substitutions include one or more of the following group substitutions: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; aspartic acid amide, glutamine; serine, threonine; lysine, arginine; and/or phenylalanine, tyrosine.

It will be obvious to those skilled in the art to apply the amino acid sequence of the chimeric AAV capsid protein shown in Figures 3B, 3D, 3F, 3H, 3J, 3L, 3N, 3P, 3R to any other modifications known in the art, by further Retouch 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 is modified to incorporate targeting sequences or sequences that facilitate purification and/or detection, e.g. the capsid protein can interact with glutathione-S-transferase, All or part of maltose binding protein, heparin/heparin sulfate binding domain, poly-HIS, ligand and/or reporter protein, immunoglobulin Fc fragment, single chain antibody, hemagglutinin, C-MYC, tag epitopes, etc. fused to form fusion proteins. Methods of inserting targeting peptides into AAV capsids are known in the art, eg, 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 dual viral genome as described in International Patent WO01/92551 and US Patent US7465583.

The present invention also provides a recombinant virus particle comprising the chimeric AAV capsid protein of the present invention, wherein the vector genome is encapsulated in the virus particle, 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. Thus, 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 present invention are used to deliver or transfer nucleic acids to animal cells, preferably mammalian cells.

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

Therapeutic polypeptides include, but are not limited to, insulin, glucagon, growth hormone, parathyroid hormone, growth hormone releasing factor, follicle stimulating hormone, progesterone, 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 bone morphogenetic proteins, nerve growth factor, brain-derived neurotrophic factor, neurotrophin NT-3 and NT-4/ 5. Ciliary neurotrophic factor, glial cell line-derived neurotrophic factor, aggregin, any of the semaphorin/disintegrin family, transin-1 and transin-2, hepatocyte growth factor, ephrin, Noggin, sonic hedgehog protein and tyrosine hydroxylase, thrombopoietin, interleukins IL-1 to IL-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, including but not limited to immunoglobulins IgG, IgM, IgA, IgD and IgE, chimeric immunoglobulins, humanized antibodies, single chain antibodies, T cell receptors , chimeric T cell receptors, single-chain T cell receptors, class I and class II MHC molecules, and engineered immunoglobulins, complement regulatory proteins, membrane cofactor proteins, decay accelerating factors, CR1, CF2, and CD59, low density Lipoprotein receptors, high density lipoprotein receptors, very low density lipoprotein receptors and clearance receptors, glucocorticoid receptors and estrogen receptors, vitamin D receptors and other nuclear receptors, jun/fos, max , mad, serum effector, AP-1, AP2, myb, MyoD and myogenin, TFE3, E2F, ATF1, ATF2, ATF3, ATF4, ZF5, NFAT, CREB, HNF-4, C/EBP, CCAAT- Forkhead protein family of box-binding proteins, interferon regulators, Wilms tumor proteins, ETS-binding proteins, STATs, GATA-box-binding proteins, and winged helical proteins, carbamoyl synthase 1, ornithine transcarbamylase , argininosuccinate synthase, argininosuccinate lyase, arginase, fumaric 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 metathesis Enzymes, Glutaryl-CoA Dehydrogenase, Insulin, Beta-Glucosidase, Pyruvate Carboxylate, Liver Phosphorylase, Phosphorylase Kinase, Glycine Decarboxylase, H-Protein, T-Protein, Cystic Fiber transmembrane conductance regulator proteins, dystrophin, alpha-galactosidase, beta-galactosidase, lysosomal enzymes, coagulation factors, and any other polypeptides that have therapeutic effects in an individual in need thereof.

A heterologous nucleotide sequence encoding a polypeptide includes a sequence encoding a reporter gene 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.

On the other hand, 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-translated functional RNAs, such as "guide" RNAS (Gorman et al., 1998, Proc. Nat. Acad. Sci. USA 95:4929; US Pat. No. 5,869,248) and the like.

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

Ribozymes are RNA-protein complexes that cleave nucleic acids in a site-specific manner. Ribozymes have specific catalytic domains with endonuclease activity (Kim et al., 1987, Proc. Natl. Acad. Sci. USA 84:8788; Gerlach et al., 1987, Nature 328:802; Forster and Symons, 1987, Cell 49: 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 in the treatment of cardiac and skeletal muscle diseases (Chen et al., 2006, Genet 38:228-233; van Rooij et al., 2008, Trends Genet. 24:159-166), and microRNAs can also be used in genes Modulation of the immune system after delivery (Brown et al., 2007, Blood 110:4144-4152).

The term "antisense oligonucleotide," including "antisense RNA," as used herein, 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 made according to conventional techniques.

It is understood by those of skill in the art that an antisense oligonucleotide does not have to be completely complementary to a target sequence, so long as the sequence similarity is sufficient to allow the antisense nucleotide sequence to specifically hybridize to the target sequence and reduce production of a protein product, eg, at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more. Hybridization of such oligonucleotides to target sequences can be performed under weak, moderate, 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 with the purpose of increasing the biological stability of the molecule and/or increasing the distance between the antisense and sense strands For the stability of the formed duplex, for example, phosphorothioate derivatives and acridine-substituted nucleotides can be used.

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-isoamyl 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 to another molecule. For example, antisense oligonucleotides can be conjugated to a molecule that facilitates delivery to cells of interest, enhances absorption from the nasal mucosa, provides a detectable label, and increases the 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 cellular receptor ligands. body.

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

RNA interference is a mechanism of post-transcriptional gene silencing that 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 persist across multiple cell divisions until gene expression is restored. Therefore, RNAi is an efficient 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). It has been demonstrated that short synthetic dsRNAs of approximately 21 nucleotides, also known as "short interfering RNAs", can mediate silencing in mammalian cells without triggering an antiviral response (Elbashir et al., 2001, Nature 411 : 494-498; Caplen et al., 2001, Proc. Nat Acad. Sci. 98:9742).

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

Methods of producing RNAi include chemical synthesis, in vitro transcription, Dicer digestion of long dsRNA in vitro or in vivo, delivery vector expression in vivo, 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, as long as it specifically hybridizes to the target sequence and reduces the production of protein products, such as at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more, it does not need to be fully complementary to the target sequence. In some embodiments, the hybridization of the oligonucleotide to the target sequence can be performed under conditions of weak, moderate or even high stringency as defined above.

In other embodiments, the antisense region of RNAi has at least about 60%, 70%, 80%, 90%, 95%, 97%, 98% or greater sequence identity to the target sequence and reduces the protein product Produce 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 at the central portion. RNAi molecules may contain modified sugars, modified nucleotides, backbone linkages, and other modifications of the antisense oligonucleotides described above.

The present invention also provides recombinant viral vectors expressing immunogenic polypeptides. The heterologous nucleic acid can encode any immunogen of interest known in the art, including, but not limited to, from human immunodeficiency virus, influenza virus, Gag proteins, tumor antigens, cancer antigens, bacterial antigens, viral antigens, and the like. Alternatively, the immunogen can be presented in the viral capsid, eg, bound to the viral coat 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 a 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 may be a protomyxovirus immunogen, eg, influenza virus immunogen, influenza virus hemagglutinin surface protein or influenza virus nucleoprotein gene; or a lentiviral immunogen, eg, equine infectious anemia virus immunogen, simian immunodeficiency Viral immunogens or human immunodeficiency virus immunogens; or arenavirus immunogens, e.g., Lassa fever virus immunogens; or poxvirus immunogens, flavivirus immunogens, filovirus immunogens, bunyavirus immunogens , coronavirus immunogens or severe acute respiratory syndrome immunogens. The immunogen may also be a polio immunogen, herpes immunogen, mumps immunogen, measles immunogen, rubella immunogen, diphtheria toxin or other diphtheria immunogen, pertussis antigen, hepatitis immunogen or any other known in the art Vaccine immunogens.

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, milk fat globulin, p53 tumor suppressor protein, mucin antigen, telomere Enzymes, nuclear matrix proteins, prostatic acid phosphatase, papilloma virus antigens, and related to 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, stomach cancer, esophageal cancer and head and neck cancer.

Alternatively, the heterologous nucleotide sequence can 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 isolated therefrom.

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

Those skilled in the art will further appreciate that various promoter/enhancer elements can 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 natural or foreign, and can be natural or synthetic sequences.

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

Inducible expression control elements are commonly used in applications that require 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, ocular (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, a specific initiation signal is 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 proteins of the present invention, as described in US Pat. No. 5,863,541. The chimeric AAV capsids of the invention can be used as "capsid carriers", molecules that can be covalently linked, bound or packaged and transferred into cells including DNA, RNA, lipids, carbohydrates, polypeptides, small organic molecules or a combination of these molecules. In addition, the molecule can be associated with the exterior of the viral capsid in order to transfer the molecule into the target cell 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 present invention can also be used to raise antibodies against novel capsid structures. Alternatively, exogenous amino acid sequences can be inserted into the viral capsid for antigen presentation to cells, eg, administration to a subject to generate an immune response to the exogenous amino acid sequence.

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

In an exemplary embodiment, the invention provides nucleic acid sequences encoding the AAV capsids shown in Figures 3B, 3D, 3F, 3H, 3J, 3L, 3N, 3P, or 3R. Representative nucleic acid sequences comprise or consist essentially of those shown in Figure 3A (L1) Figure 3C (L4), Figure 3E (L10), Figure 3G (L52), Figure 3I (L58), Figure 3K (L84), Figure 3M ( L37), Figure 3O (L107) or Figure 3Q (L57), or consist of the nucleotide sequences shown in these Figures, or encode an AAV coat encoded by any of the above nucleotide sequences Nucleotide sequence of a shell or capsid protein, but differs from the above-mentioned nucleotide sequence due to the degeneracy of codons, 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 AAV capsid proteins described above. In specific embodiments, the nucleic acid hybridizes to the complementary strand of the nucleic acid sequence specifically disclosed herein, encoding a variant capsid protein, under standard conditions known to those of skill in the art. See Figure 3A, Figure 3C, Figure 3E, Figure 3G, Figure 3I, Figure 3K, Figure 3M, Figure 3O, or Figure 3Q for nucleic acid sequences specifically disclosed herein, optionally, the variant capsid protein substantially retains the structure shown in Figure 3 At least one property of a capsid protein encoded by a nucleic acid sequence shown in 3A, 3C, 3E, 3G, 3I, 3K, 3M, 3O or 3Q. For example, a virion of a variant capsid protein may substantially retain a nucleic acid encoding sequence encoded by the nucleic acid shown in Figure 3A, Figure 3C, Figure 3E, Figure 3G, Figure 3I, Figure 3K, Figure 3M, Figure 3O, Figure 3Q Targeting characterization of virions with 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 known in the art, many different programs can be used to determine whether a nucleic acid or polypeptide has percent identity or similarity to a known sequence. Percent designation as used herein means that a nucleic acid or fragment thereof has a specified percentage of another nucleic acid when aligned to other nucleic acids using BLASTN, the program available from the National Center for Biotechnology Information (NCBI) via the Internet.

When referring to a polypeptide, percent identity or similarity indicates that the polypeptide exhibits a particular percent identity or similarity when compared to another protein, or a portion thereof, over a common length determined using BLASTP. This is also available from the National Center for Biotechnology Information (NCBI) over the Internet. Percent identity or similarity of polypeptides is typically determined using sequence analysis software, eg, see the sequence analysis software package of the Genetics Computer Group at the University of Wisconsin Biotechnology Center. Protein analysis software uses the homology of various substitutions, deletions and other modifications to match similar sequences. Conservative substitutions typically include substitutions in the following classes: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, Threonine; Lysine, Arginine; Phenylalanine, Tyrosine.

In certain embodiments, the 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, baculovirus vectors, or hybrid viral vectors.

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

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

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

Furthermore, the nucleic acid can be operably linked to a promoter element.

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

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

The cells are typically cells that allow the AAV virus to replicate. Any suitable cell known in the art can 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 , one or more of SF21 cell line, SF900 cell line and BHK cell line.

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

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

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

Those skilled in the art understand that it may be advantageous to provide the AAV replication and capsid sequences and the helper virus sequences on a single helper construct. The helper construct may be a non-viral or viral construct, or may be selected to be a hybrid adenovirus or a hybrid herpes virus comprising the AAVrep/cap gene sequence.

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

In another embodiment, the AAV rep and/or cap sequences and adenoviral helper sequences are provided by a single adenoviral helper vector. The rAAV genome template is provided by the 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 genome template is integrated into the cell as a precursor. Alternatively, the rAAV template is provided by an EBV vector maintained within the cell as an extrachromosomal element. In another exemplary embodiment, the AAV rep and/or cap sequences and adenoviral helper sequences are provided by a single adenoviral vector. The rAAV genome template is provided as a separate replicating viral vector. For example, the rAAV template can be provided by rAAV particles 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 by size. AAVs can also be separated from helper viruses based on their affinity for the heparin substrate. In a representative embodiment, a replication-deficient helper virus is used such that any contaminating helper virus cannot replicate. Alternatively, helper adenoviruses that lack late gene expression can be used, since only adenoviral early gene expression mediates packaging of AAV viruses. Adenovirus mutants deficient in 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, eg, for diagnostic or therapeutic use 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 types of immunoglobulins, including IgG, IgM, IgA, IgD and IgE. Antibodies may be monoclonal or polyclonal, and may be derived from any species, including mouse, rat, rabbit, horse, goat, sheep, chicken, monkey, alpaca, or human, or may be chimeric, human Antibodies, human antibodies. Antibodies can be recombinant monoclonal antibodies, or screened from phage libraries, yeast libraries, 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 by pepsin digestion of antibody molecules, and Fab fragments can be produced by reducing the disulfide bonds of F(ab') ₂ fragments. Alternatively, Fab expression libraries can be constructed for quick and easy identification of monoclonal Fab fragments with the desired specificity.

Polyclonal antibodies can be obtained by immunizing suitable animals, such as rabbits, goats, etc., with viruses, collecting immune serum from the animals, and separating them from the immune serum.

The present 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, eg, to produce polypeptides in vitro or for gene therapy in vitro. Vectors can also be used in methods of delivering nucleotide sequences to an individual in need thereof, eg, expressing immunogenic or therapeutic polypeptides.

In general, the viral vectors of the present invention can be used to deliver any exogenous nucleic acid that has a biological effect to treat or ameliorate any disease associated with gene expression. Furthermore, the present invention can be used to treat any disease that can be ameliorated by the 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 ( beta-globin), anemia (erythropoietin) and other blood disorders, Alzheimer's disease (GDF), multiple sclerosis (beta-interferon), Parkinson's disease (glial cell-derived neurotrophic factor) ), Huntington's disease (repeated inhibitory RNAs including but not limited to RNAi such as siRNA or shRNA, antisense RNA or microRNA), amyotrophic lateral sclerosis, epilepsy (galanin, neurotrophic factor) and others Neurological diseases, cancer (endostatin, angiostatin, TRAIL, FAS ligands, cytokines including interferons, inhibitory RNAs that inhibit VEGF including but not limited to siRNA, shRNA, antisense RNA, microRNA, multidrug resistance genes products, cancer immunogens), diabetes (insulin, PGC-α1, GLP-1, myostatin precursor peptide, glucose transporter), muscular dystrophies including Duchenne muscular dystrophy and Baker muscular dystrophy (e.g. dystrophin, mini-trophin, micro-dystrophin, insulin-like growth factor, etc.), glycogen storage diseases such as Fabry disease (alpha-galactosidase) and Pompe disease (lysosomal acid alpha-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 VEGF-inhibiting RNAi), 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, sarcoplasmic reticulum Ca2-ATPase, zinc finger proteins that regulate phospholipase genes, phospholipase inhibitors, 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 present 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 adjunctive therapy, eg, by administering immunosuppressive or inhibitory nucleic acids to block cytokine production.

Gene transfer has enormous potential use in understanding and treating disease. There are many genetic diseases where defective genes are known and have been cloned. Generally speaking, the above-mentioned disease states are divided into two categories: the first is a deficient state, usually an enzyme deficiency, and is usually inherited in a recessive manner; the second is an unbalanced state, which may involve regulatory or structural proteins, usually in a dominant manner. hereditary. For deficient state diseases, gene transfer can bring normal genes into affected tissues for replacement therapy, as well as create animal models of the disease using inhibitory RNAs, including siRNA or shRNA, microRNA or antisense RNA. For imbalanced disease states, gene transfer can be used to create disease states in model systems, which can then be used to treat disease states. Thus, the viral vectors according to the present invention allow the treatment of genetic diseases. As used herein, a disease state is treated by partially or fully remedying a disease or a deficiency 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 present invention can also be used to deliver nucleic acids for protein production, eg, for laboratory, industrial or commercial purposes.

Alternatively, viral vectors can be administered to cells and the altered cells administered to an individual. The heterologous nucleic acid is introduced into a cell, and the cell is administered to a 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, the 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 exposure to an immunogen." It involves the differentiation and proliferation of immunoregulatory cells in lymphoid tissue, leading to the synthesis of antibodies or a cell-mediated response, or both. Alternatively, after the host is exposed to the immunogen through infection or vaccination, a positive immune response occurs. Active immunity can be contrasted with passive immunity, which is obtained by "transferring preformed substances, such as antibodies, transfer factors, thymic grafts, interleukin-2, from an actively immunized host to a non-immunized host".

A "protective" immune response or "protective" immunity, as used herein, indicates that an immune response confers some benefit to a subject because it prevents or reduces the incidence of disease. Alternatively, a protective immune response or protective immunity can be used to treat a disease, particularly a cancer or tumor, eg, cause regression of the cancer or tumor and/or prevent metastasis and/or prevent the growth of metastatic nodules. Protection may 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 immunosimilar molecules or any other immunogen to generate an immune response against 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 the cancer cell antigen. As described herein, viral vectors can be administered to an individual 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 meaning as understood in the art, eg, having uncontrolled tissue growth that has spread or metastasized 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 cancer, head and neck cancer, esophageal cancer, thyroid cancer, etc. In embodiments of the present invention, the present invention is practiced in the treatment and/or prevention of tumor-forming cancers.

Cancer cell antigens have been described above. The term "treating cancer" is intended to reduce the severity of cancer, prevent or at least partially eliminate cancer. For example, in certain circumstances, these terms indicate that the treatment of cancer is to prevent or reduce, or at least partially eliminate. In yet another representative embodiment, these terms indicate preventing or reducing or at least partially eliminating the growth of metastatic nodules, eg, following surgical resection of the primary tumor. According to the term "cancer prevention", it is intended to at least partially eliminate or reduce the incidence or incidence of cancer. Alternatively, the onset or progression of the subject's cancer may be slowed, controlled, less likely or likely, or delayed.

In specific embodiments, 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 a subject, thereby eliciting an immune response to cancer cell antigens. This approach is particularly useful in immunocompromised subjects who do not develop an adequate immune response in the body, i.e. do not produce sufficient numbers of boosting antibodies.

Immunomodulatory cytokines are known in the art, such as alpha-interferon, beta-interferon, gamma-interferon, omega-interferon, tau-interferon, interleukin-1α, interleukin-1β, interleukin IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-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-inducible cytokines, can be administered to a subject with a viral vector.

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

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

In particular embodiments, the present invention provides pharmaceutical compositions comprising a viral vector of the present invention in a pharmaceutically acceptable carrier and optionally including other agents, stabilizers, buffers, carriers, adjuvants , diluents, etc., and the carrier for injection is usually a liquid. For other methods, the transport vehicle can be either solid or liquid. For administration by inhalation, the carrier will be respirable, preferably in the form of solid or liquid particulates.

"Pharmaceutically acceptable" refers to a material that is non-toxic or free of other undesirable properties, ie, the material can be administered to a subject without producing any undesirable biological effects.

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

The cells into which the viral vector is to be introduced may be of any type, including but not limited to neural cells, including cells of the peripheral and central nervous systems, especially brain cells, such as neurons, oligodendritic cells, glial cells, astrocytes Plasma cells, lung cells, ocular cells including retinal cells, retinal pigment epithelium and corneal cells, epithelial cells including intestinal and respiratory epithelial cells, skeletal muscle cells including myoblasts, myotubes and muscle fibers, diaphragmatic muscle cells, dendrites cells, pancreatic cells including islet cells, hepatocytes, 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, including, for example, cartilage, meniscus, synovium, and bone marrow joint cells, 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, as described above. Furthermore, as mentioned above, the cells can be derived from any species of origin.

Viral vectors can be introduced into cells ex vivo to administer the modified cells to an individual. In specific embodiments, the cells are removed from the subject, into which the viral vector is introduced, and the cells are then infused back into the subject. Methods for removing cells from a subject for in vitro treatment and then reintroducing 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, cultured cells, or cells from any other suitable source, and the cells are administered to an individual in need.

Cells suitable for in vitro gene therapy are as described above. The dose of cells administered to a subject will vary with the age, condition and species of the subject, cell type, nucleic acids expressed by the cells, mode of administration, and the like. Typically, at least 10 ² to 10 ⁸ or about 10 ³ to about 10 ⁶ 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 to a subject a viral vector or capsid of the invention. In specific embodiments, the method includes a method of delivering a nucleic acid of interest to an animal subject, the method comprising administering to the animal subject an effective amount of a viral vector of the invention. The viral vectors of the present invention can be administered to human subjects or animals in need thereof by any method known in the art. Optionally, the viral vector is delivered in an effective dose in a pharmaceutically acceptable carrier.

The viral vectors of the present invention can be further administered to an individual to elicit an immunogenic response, eg, as a vaccine. Typically, the vaccines of the present invention comprise an effective amount of virus in combination with a pharmaceutically acceptable carrier. Optionally, the dose is sufficient to generate a protective immune response. As long as the benefits of administering the immunogenic polypeptide outweigh any disadvantages thereof, the degree of protection imparted need not be complete or permanent. 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 to be treated, the circumstances of the individual, the particular viral vector and nucleic acid to be delivered, and can be determined in a conventional manner. Exemplary doses to achieve a therapeutic effect are viral titers of at least about ¹⁰⁵ , ¹⁰⁶ , ¹⁰⁷ , ¹⁰⁸ , ¹⁰⁹ , ¹⁰¹⁰ , ¹⁰¹¹ , ¹⁰¹² , ¹⁰¹³ , ¹⁰¹⁴ , ¹⁰¹⁵ Tu or more , preferably a virus titer of 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 administration may be used, eg, two, three, four or more administrations, 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 Intra, intrapleural, intracerebral and intraarticular, skin and mucosal surfaces, intralymphatic vessels, etc., as well as direct tissue or organ injection, such as direct injection into the liver, skeletal muscle, myocardium, diaphragm or brain. Tumors can also be administered, eg, injected in or near the tumor or lymph nodes. In any given situation, the most appropriate route will depend on the nature and severity of the condition being treated and the nature of the particular carrier used.

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

Pharmaceutical compositions suitable for oral administration may be presented in discrete units such as capsules, buffers, troches, or tablets, each unit containing a predetermined quantity of a composition of the present invention. As powders or granules, as solutions or suspensions in aqueous or non-aqueous liquids, or as oil-in-water or water-in-oil emulsions. Oral administration can be accomplished by complexing the viral vector of the present invention into a vector capable of resisting degradation by digestive enzymes in the intestinal tract of animals. 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 excipient ingredients as described above. In general, pharmaceutical compositions according to embodiments of the present invention are prepared by uniformly and intimately admixing the composition with a liquid or finely divided solid carrier, 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 excipient ingredients. Compressed tablets are prepared by compressing in a suitable machine a free-flowing form of the composition, such as powder or granules, optionally mixed with binders, lubricants, inert diluents and/or surface active/dispersing agents. Tablets are formed by moistening the powdered compound with an inert liquid binder in a suitable machine.

Pharmaceutical compositions suitable for oral administration include lozenges containing the ingredients of the present invention in a flavored base, typically sucrose and acacia or tragacanth, and inert base ingredients 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 the present invention, which preparations are optionally isotonic with the blood of the intended recipient. These formulations may contain antioxidants, buffers, bacteriostatic agents and solutes to render the components isotonic with the blood of the intended recipient. Aqueous and non-aqueous sterile suspensions, solutions and emulsions may include 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 drugs 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.

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

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

Compositions of the drug 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 then 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 glycols, alcohols, transdermal 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 present invention with a lipophilic agent capable of entering the skin.

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

The viral vectors disclosed herein can be administered to the lungs of an individual by any suitable method, such as administering a suspension of respirable particles consisting of the viral vector inhaled by the individual. Respirable particulate matter can be liquid or solid. Aerosols containing liquid particles of viral vectors can be produced by any suitable method, such as using a pressure driven aerosol nebulizer or an ultrasonic nebulizer known to those skilled in the art. Aerosols of solid particles containing viral vectors can likewise be produced by techniques known in the pharmaceutical art with any solid particle pharmaceutical 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 screening 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 present invention provides a method of identifying a viral vector for a property of interest, e.g., a method of identifying an AAV vector or AAV capsid for 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 previously shuffled AAV capsid protein coding sequence, an AAV Rep The coding sequence and at least one terminal repeat such as the 5' and/or 3' terminal repeats of AAV in which the viral vector genome is packaged in the AAV capsid.

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

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

The present invention can also be used to identify a chimeric AAV capsid or virus particle 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, a subject can be injected with a specific neutralizing antibody, and then a pool of chimeric viruses can be injected into the subject to select for viral genomes that enter the target tissue of interest (eg, heart, skeletal muscle, liver, etc.), Genomes isolated from target tissues correspond to capsids capable of evading neutralization.

Accordingly, in representative embodiments, the present invention provides a method of identifying a chimeric AAV capsid or viral vector capable of evading neutralizing IgGs, the method comprising:

First, administering IgGs to individual mammals;

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-encoding 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, comprising the AAV capsid protein coding sequence produced by the aforementioned shuffling, an AAV Rep and at least one terminal repeat, such as the 5' and/or 3' terminal repeat of AAV, wherein the viral vector genome is packaged in the AAV capsid.

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

Fourth, multiple viral vectors as virions or as viral vector genomes encoding AAV capsids are recovered from target tissues to identify viral vectors or AAV capsids with neutralizing antibody evasion properties.

"Escape" neutralizing antibodies, meaning that neutralization is at least partially reduced compared to an appropriate control, but as long as the degree of neutralization is reduced compared to the 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, diaphragm muscle, kidney, pancreas, spleen, gastrointestinal tract, lung, joint tissue, tongue, ovary, testis, germ cells, cancer cells, or the above combination.

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 the generation of chimeric capsids by shuffling from two or more of the following capsid sequences, the AAV capsids comprising: AAV1, AAV2, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, avian AAV, bovine AAV, canine AAV, equine AAV, ovine AAV, goose AAV or snake AAV. As 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 for introducing mutations into nucleic acid and/or amino acid sequences, eg, the use of chemical mutagens, error-prone PCR, cassette mutagenesis, and the like.

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

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

The mode of 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 present invention comprise recovering AAV particles or viral genomes encoding the same AAV particles from two or more target tissues, and identifying those two or more target tissues with desired properties chimeric virus or chimeric AAV capsid. For example, in specific embodiments, chimeric viruses or chimeric AAV capsids are identified that have inefficient targeting to skeletal muscle and/or cardiac muscle and/or have efficient targeting to liver.

The target cell or tissue, or one of them, can also be a cancer cell or 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. Sci 91:10747-10751, and Soong et al., 2000, Nature Genetics 25:436-439). This approach has also been applied to directed evolution of viruses (US Patent US6096548, US Patent US6596539). In a representative embodiment, a collection of AAV capsid protein coding sequences is fragmented and recombined in vitro by homologous and/or non-homologous recombination to form a collection of "chimeric" AAV capsid proteins. Each chimeric capsid encapsulates a nucleic acid, eg, an AAV genome, comprising the corresponding capsid-encoding sequence, thereby creating a chimeric virus. Chimeric virus pools 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 Embodiments

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

(a) the nucleotide sequence of SEQ ID NO: 1;

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

(c) the nucleotide sequence of SEQ ID NO: 5;

(d) the nucleotide sequence of SEQ ID NO: 7;

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

(f) the nucleotide sequence of SEQ ID NO: 11;

(g) the nucleotide sequence of SEQ ID NO: 13;

(h) the nucleotide sequence of 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 of the nucleotide sequences (a)-(i), which, due to the degeneracy of the genetic code, is different from the nucleotides of (a)-(i) The sequence is different.

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

In one aspect, the present invention also provides an AAV capsid protein encoded by the above-mentioned nucleic acid, the amino acid sequence of the AAV capsid protein comprising selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO: 6. Any 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 of DNA molecules, RNA molecules, polypeptides, carbohydrates, liposomes and small organic molecules or several.

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 an AAV vector genome and the aforementioned AAV capsid protein, wherein the AAV vector genome is encapsulated 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 is selected from insulin, glucagon, growth hormone, growth hormone releasing factor, erythropoietin Progenin, 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 comprising a pharmaceutically acceptable carrier or adjuvant and selected from the aforementioned nucleic acid, AAV capsid protein, recombinant virus particle, recombinant AAV virus particle and/or the aforementioned one or more of the cells.

In another aspect, the present invention also provides that one or more of the aforementioned nucleic acid, AAV capsid protein, recombinant virus particle, recombinant AAV virus particle, cell and/or the aforementioned pharmaceutical composition are prepared for prevention or treatment Use in a medicament for a disease selected from cystic fibrosis and other lung diseases, hemophilia A, hemophilia B, thalassemia, anemia and other blood diseases, Alzheimer's disease, multiple sclerosis, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, epilepsy, cancer, diabetes, muscular dystrophy, glycogen storage disease and other metabolic defects, congenital emphysema, Lesch-Nyhan syndrome, Niemann- One or more of the group consisting of Pick disease, AIDS, hepatitis, hyperammonemia and spinal cerebral ataxia.

In one aspect, the present invention provides a method for preparing recombinant AAV virions, the method comprising providing a cell with the aforementioned nucleic acid, a nucleic acid encoding sequence for an AAV Rep protein, a nucleic acid sequence carrying a heterologous nucleic acid in vitro AAV vector genome, cofactors that help to produce infectious AAV, and allow AAV vector genome to be wrapped in the AAV capsid encoded by the aforementioned nucleic acid, and realize the assembly of recombinant AAV virus particles, 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-drug.

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

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

Having described the present invention, the present invention will be illustrated in more detail in the following examples, which are 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 that are superior or superior to liver targeting. The AAV capsid gene was shuffled by DNA shuffling technology and an AAV capsid gene library was constructed for in vivo screening of mouse models. Mouse liver-enriched AAV mutants were isolated and their targeting was determined by testing for in vitro and in vivo activity of the AAV mutant coat.

Example 1 Construction of chimeric AAV plasmid library

In order to obtain the chimeric AAV coat gene Cap, firstly, using the upstream primer primerA and the downstream primer primerB to amplify the full-length coat gene from different serotype AAV mothers, respectively, the selected maternal AAV coat gene includes the AAV1 coat (NCBI SequenceID: AF063497 .1, Nucleic acid coding sequence 2223-4433 of Cap), AAV2 coat (NCBISequenceID: AF043303.1, Nucleic acid coding sequence 2203-4410 of Cap), AAV3B coat (NCBI SequenceID: AF028705.1, Nucleic acid coding sequence 2208-4418 of Cap) ), AAV7 shell (NCBI SequenceID: AF513851.1, nucleic acid coding sequence 2222-4435 of Cap), AAV8 shell (NCBI SequenceID: AF513852.1, 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 primer primerB is 5'-ATAAGAATGCGGCCGCAGAGACCAAAGTTCAACTGAAACGA-3'. PCR amplification was performed under the action of primeSTAR Max DNA polymerase (TaKaRa Company, Cat. No. R045A). Amplification conditions: 95℃ 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 amplified Cap genes were mixed with equal mass 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, after digestion at 22°C for 6-8 minutes, the DNaseI enzyme was inactivated at 75°C for 10 minutes. Dispersed DNA bands were obtained by agarose gel electrophoresis, most of which were concentrated in 500-1000 bp in length, and DNA fragments in this part of the length range were recovered.

The recovered DNA fragments were firstly subjected to random primerless amplification as primers for each other. In order to increase their diversity and amplification efficiency, an unconventional single annealing PCR mode was used (94°C for 60s, 65°C for 90s, 72°C for 90s, 10 cycles; 94℃ for 60s, 62℃ for 90s, 72℃ for 90s, 10 cycles; 94℃ for 60s, 59℃ for 90s, 72℃ for 90s, 10 cycles; 94℃ for 60s, 56℃ for 90s, 72℃ for 90s, 10 cycles; 94℃ 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, the upstream primer T-primerA (5'-ACGCCTGCCGTTCGACGATTCCCAAGCTTCGATCAACTACGCAGACAGGTACCAA-3') and the downstream primer T-primerB (5'-ACGCGCGGATCTTTCCAGAGATAAGAATGCGGCCGCAGAGACCAAAGTTCAACTGAAACGA-3') were used in HiFi DNA polymerase (TransGenBiotech, Cat. No. 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 30 s, 62 °C for 30 s, and 72 °C for 2.5 min, for a total of 40 cycles. A chimeric full-length Cap DNA fragment with a length of about 2500 bp was obtained, which was double digested with HindIII and NotI, and the pSNAV2.3 vector (containing the single-stranded AAV2 genome ITR and polymerase Rep gene sequences, which was subjected to the same double digestion treatment, was deleted). Cap gene) ligation, the ligation product was electroporated into E. coli HST08 cells, and a chimeric AAV plasmid library with a capacity of more than 1E+6 clones was prepared. The plasmids identified as positive clones by PCR were digested with PstI, HaeIII, and TaqI, respectively, and the differences in the shape of each plasmid were observed (Figure 1) to determine the diversity of the plasmid library.

Example 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 plasmid are used for transfection in 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 hybrid AAV, that is, the viral coat protein is not necessarily expressed by its packaged genome. After the harvest liquid is extracted and purified by chloroform, the genome titer is detected; the second step is to take the infection index MOI as 100 infected 293T cells to ensure that the number of copies of the virus genome infected by each cell is less than 10, and the final AAV virus library generated at this time is homozygous. After the harvest liquid is extracted and purified with chloroform, the genome titer is detected (see Muller et al., 2003 , Nature Biotechnology, 21:1040-1046). The library plasmids used in packaging are of very low quality to ensure that each novel AAV genome can be packaged in a shell formed by its own expressed Cap protein. The packaged virus particles can express the coat protein normally, which proves that they can form a complete viral coat and have the ability to infect.

Take an appropriate amount of purified AAV sample, prepare a DNase I digestion reaction mixture according to the following table (Table 1), incubate at 37 °C for 30 min, and incubate at 75 °C for 10 min 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 is diluted by an appropriate factor, the Q-PCR reaction system is configured with reference to the following table (Table 2), and the detection is performed according to the following procedure.

Table 2

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

table 3

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

The packaging yield results are shown in the table below (Table 4):

Table 4

viral 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 novel AAV vector was injected into 8-week-old C57BL/6J mice at a dose of 1.5E+11vg/vesicle via tail vein for liver targeting screening and enrichment. Mice were sacrificed three days after injection, and all liver tissues were collected, and total DNA was extracted after homogenization in liquid nitrogen. The primer pair T-primerA/T-primerB was used for amplification again, and the amplified DNA obtained by mixing the full-length Cap genes of the novel AAV was constructed into the pSNAV2.3 vector by the above method. 370 positive monoclones were obtained by PCR identification and mixed with equal mass to form the second plasmid library. It was repackaged into a virus library and injected into 8-week-old C57BL/6J mice with 1.5E+11vg/tail vein again. The mice were sacrificed three days after injection, and all liver tissue, heart tissue and skeletal muscle were collected. 100, 50 and 50 positive clones were randomly selected from the above tissues for sequencing. The sequenced positive clones were analyzed by BioEdit, VectorNTI, ClustalX2 and TreeViewX.

Novel AAV vectors with high frequency presence in the liver and low frequency presence or absence in the heart and skeletal muscle were selected. A total of 9 groups of high-frequency liver-targeted 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). Due to the low titers of the other four packages, they are not suitable for industrialization. Therefore, five new AAVs, namely L37, L57, L58, L107, and L10, were selected for subsequent infection activity testing experiments.

Example 4 Infectious activity of novel AAV on 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 novel AAV, the in vitro infection activity was first tested on 6 human normal liver or liver cancer cell lines. The obtained novel 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 foreign gene, and the packaged viruses were named AAV2/57, AAV2/ 58, AAV2/107, AAV2/10, and virus titers were detected (results not shown).

The above four recombinant novel AAV viruses carrying green fluorescent protein and AAV2/8 recombinant viruses carrying the same green fluorescent protein were simultaneously infected with various human hepatocyte cell lines. After 48 hours of infection, the infective activity was detected by flow cytometry. In order to avoid the infection efficiency of viral vector is too low or supersaturated on a certain cell line, each cell line was infected with at least two indices of infection (MOI). When the three infection indexes infect human liver cell lines 7721, HepG2, Huh7, and L02, the new AAV2/10, AAV2/57, AAV2/58, and AAV2/107 all showed a significant dose-dependent relationship to the infection of human liver cell lines. Next, the infection results of each cell at one of the MOIs are described, and the results of the remaining MOIs are shown in Figure 4.

When the above-mentioned four recombinant viruses infected human liver cell line 7402 with an infection index (MOI) of 2500, flow cytometry analysis was performed 48 hours after infection. The results (Fig. 4A) showed that at 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 AAV2/8. 1.6 times.

When the above four recombinant viruses infect human liver cell line 7721 with an infection index (MOI) of 2500, flow cytometry analysis was performed 48 hours after infection, the results (Fig. 4B) showed that at 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 higher than AAV2/8 infection efficiency, AAV2/107 infection efficiency is 21.8 times higher than AAV2/8 infection efficiency, AAV2/57 infection efficiency is AAV2/8 infection efficiency The infection efficiency of AAV2/58 was 15.9 times that of AAV2/8.

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. The results (Fig. 4C) showed that at this MOI, AAV2/10 had the highest infection efficiency , AAV2/107 infection efficiency is close to it, AAV2/10 infection efficiency is 14.8 times higher than AAV2/8 infection efficiency, AAV2/107 infection efficiency is 13.5 times higher than AAV2/8 infection efficiency. 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 human liver cell line Huh7 with an infection index (MOI) of 2500, flow cytometry analysis was performed 48 hours after infection. The results (Fig. 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. The results (Fig. 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 human liver cell line L02 with an infection index (MOI) of 50,000, flow cytometry analysis was performed 48 hours after infection. The results (Fig. 4F) showed that at this MOI, AAV2/10 had the highest infection efficiency , the infection efficiency of AAV2/8 is 42 times, the infection efficiency of AAV2/107 is 26.8 times that of AAV2/8, the infection efficiency of AAV2/57 is 2.5 times that of AAV2/8, and the infection efficiency of AAV2/58 is AAV2/8. 10.8 times the infection efficiency.

It can be seen from the above results that the infection activities of these four new recombinant AAVs on human hepatocytes or liver cancer cell lines are higher than that of the positive control AAV2/8.

4.2 The luciferase detection system was used to test the infection activity of the novel AAV on human liver 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 foreign gene, and the packaged virus was named AAV2/37 , AAV2/57, AAV2/58, AAV2/107, AAV2/10, and virus titers were detected (results not shown). The above recombinant novel AAV virus was used to infect 5 normal liver or liver cancer cell lines with an infection index MOI of 500. After 48 hours, the luciferase detection system was used to detect the infection activity. Comparisons of infectious activity were performed after subtracting NC background values from each assay value.

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

Comparison of the above-mentioned five recombinant novel AAV viruses on the infection activity of human hepatocyte cell line 7402, the results (Figure 5B) show that AAV2/10 has the highest activity, AAV2/107 is close to it, AAV2/58 is close to AAV2/37, and the infection activity is close to that of AAV2/58. The order from high to low is AAV2/10>AAV2/107>AAV2/58>AAV2/37>AAV2/57.

The comparison of the above-mentioned five recombinant novel AAV viruses on the infection activity of human hepatocyte cell line 7721, the results (Fig. 5C) show that AAV2/10 has the highest activity, AAV2/58 and AAV2/37 have close infection activities, and the infection activities are from high to low. AAV2/10>AAV2/107>AAV2/58>AAV2/37>AAV2/57.

Comparison of the above-mentioned five recombinant novel AAV viruses on the infection activity of human liver cell line HepG2, the results (Figure 5D) show that AAV2/10 has the highest activity, and the infection activity is AAV2/10>AAV2/37>AAV2/107 >AAV2/58>AAV2/57.

Comparison of the above-mentioned five recombinant novel AAV viruses in the human liver cell line L02 infection activity, the results (Figure 5E) show that AAV2/10 has the highest activity, and the infection activity from high to low is AAV2/10>AAV2/107>AAV2/37 >AAV2/58>AAV2/57.

It can be seen from the above results that AAV2/10 has the highest activity and AAV2/57 has relatively low activity among the above-mentioned novel AAVs.

Example 5 In vivo activity test of novel AAV

Five novel 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 CAG-Luciferase exogenous gene, and the packaged viruses were named AAV2/37, AAV2/57, AAV2/58, AAV2/107, AAV2/10, virus titers were detected (results not shown), and these novel AAV viruses were compared for in vivo infectious activity. C57BL/6J mice at 6-8 weeks were selected for tail vein injection at a dose of 1E+11vg/mice. The mice were sacrificed after 2 weeks, and the tissue DNA was extracted to test the number of copies of the AAV vector genome in the liver, heart, and skeletal muscle. The luciferase expression in these three tissues was tested.

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

These results show that these five novel AAV vectors all have excellent liver targeting, and AAV2/10 has the highest expression level of luciferase in liver compared with other novel AAV vectors, followed by AAV2/58, followed by AAV2/ 37 and AAV2/107 had similar luciferase expression levels in 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, the production of neutralizing antibodies against native AAV will greatly reduce the half-life of AAV. Neutralizing antibody levels of novel AAVs were tested here.

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

In this experiment, virus infection was used, 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. The serum virus mixture was found in a series of serum dilutions to infect cells with an efficiency of 50% of that of the virus without serum, and the inverse of this dilution was used as the amount of neutralizing antibody, which was evaluated against each viral vector (see Lochrie MA et al, 2006, Virology 353:68-82; Mori S et al, 2006, Jpn JInfect Dis 59:285-293 et al). A total of 10 cynomolgus monkey sera were tested in this experiment. After serial dilution of sera, the neutralizing antibodies of the novel AAV and the neutralizing antibodies 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 are respectively constructed on the RC plasmid vector containing type 2 AAV Rep, the plasmid carries the CAG-EGFP exogenous gene, and the packaged virus is named as AAV2/37, AAV2/57, AAV2/58, AAV2/107, AAV2/10, and virus titers were tested (results not shown). 7402 cells were seeded in 24-well plates, cynomolgus monkey serum samples were serially diluted, novel AAV viruses AAV2/37, AAV2/57, AAV2/58, AAV2/107, AAV2/10 and AAV2/8 recombinant viruses carrying the same green fluorescent protein The infection index MOI was 2000. The diluted serum sample and virus solution were mixed 1:1, and incubated at 37°C for 1 h, and then the mixture of serum sample and virus solution was added to the cells. Cells were harvested after 48 h, and the infection efficiency was detected by flow cytometry.

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

Therefore, through a comprehensive comparison of the new AAV and AAV2/8 neutralizing antibodies in cynomolgus monkey serum, the neutralizing antibody of the new AAV in the monkey population is significantly lower than that of AAV2/8. As a drug delivery carrier, the new AAV has lower immunogenicity.

Table 5 Detecting results of neutralizing antibodies in 10 serums of cynomolgus monkeys

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

In this experiment, a fixed MOI of viral infection index was used, and the serum was diluted to detect the level of neutralizing antibody of the new AAV and the level of AAV2/8 neutralizing antibody in individual human serum. The serum virus mixture was found in a series of serum dilutions to infect cells with an efficiency of 50% of the virus-free serum-infected cells, and the inverse of this dilution was used as the amount of neutralizing antibody, which was evaluated against each viral vector. A total of 10 serum samples were tested in this experiment, and the serum was 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 selected serum 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 respectively. The neutralizing antibody detection results are shown in Table 6, in which the 3# sample was negative for all virus neutralizing antibodies; the 2# sample was negative for the new AAV neutralizing antibodies, while the AAV2/8 neutralizing antibodies were positive; The neutralizing antibodies of samples 1#, 4#, 5#, 6#, 7#, 8#, 9#, and 10# against the new AAV were significantly lower than those of AAV2/8. The experimental results demonstrate that in the human population, the neutralizing antibody against the novel AAV is more suitable as a drug delivery vehicle than the neutralizing antibody against AAV2/8, which can reduce the reactogenicity.

Table 6 Test results of neutralizing antibodies in 10 human serums

Based on the above results, by comparing the serum neutralizing antibodies of the new AAV and AAV2/8, 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 Sannuo Jiayi Biotechnology Co., Ltd.

<120> Acquisition and application of a group of novel liver-targeted 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

ggaaagaaga ggcctgtaga gcagtctcct caggaaccgg actcctccgc gggtattggc 480

aaatcgggtg cacagcccgc taaaaagaga ctcaactttg gtcagactgg cgactcagag 540

tcagttccag accctcaacc tctcggagaa ccaccagcag cgccctctgg tgtgggacct 600

aatacaatgg cttcaggcgg tggcgcacca atggcagaca ataacgaagg cgccgacgga 660

gtgggtaatg cctcaggaaa ttggcattgc gattcccaat ggctgggcga cagagtcatc 720

accaccagca ccagaacctg ggccctgccc acttacaaca accatctcta caagcaaatc 780

tccagtgctt caacgggggc cagcaacgac aaccactact tcggctacag caccccctgg 840

gggtattttg actttaacag attccactgc cacttttcac cacgtgactg gcagcgactc 900

atcaacaaca attggggatt ccggcccaag agactcaact tcaagctctt caacatccaa 960

gtcaaggagg tcacgacgaa tgatggcgtc acgaccatcg ctaataacct taccagcacg 1020

gttcaagtct tctcggactc ggagtaccag ttgccgtacg tcctcggctc tgcgcaccag 1080

ggttgcctcc ctccgttccc ggcggacgtg ttcatgattc cgcaatacgg ctacctgacg 1140

ctcaacaatg gcagccaagc cgtgggacgt tcatcctttt accgcctgga atatttccct 1200

tctcagatgc tgagaacggg caacaacttt accttcagct acacctttga ggaagtgcct 1260

ttccacagca gctacgcgca cagccagagc ctggaccggc tgatgaatcc tctcatcgac 1320

cagtacctgt attacctgaa cagaactcag aatcagtccg gaagtgccca aaacaaggac 1380

ttgctgttta gccgtgggtc tccagctggc atgtctgttc agcccaaaaa ctggctacct 1440

ggaccctgtt accggcagca gcgcgtttct aaaacaaaaa cagacaacaa caacagcaac 1500

tttacctgga ctggtgcttc aaaatataac ctcaatgggc gtgaatccat catcaaccct 1560

ggcactgcta tggcctcaca caaagacgac aaagacaagt tctttcccat aagcggtgtc 1620

atgatttttg gaaaggagag cgccggagct tcaaacactg cattggacaa tgtcatgatc 1680

acagacgaag aggaaatcaa agccactaac cccgtggcca ccgaaagatt tgggactgtg 1740

gcagtcaatc tccagagcag cagcacagac cctgcgaccg gagatgtgca cgttatggga 1800

gccttacctg gaatggtgtg gcaagacaga gacgtatacc tgcagggtcc tatttgggcc 1860

aaaattcctc acacggatgg acactttcac ccgtctcctc tcatgggcgg ctttggactt 1920

aagcacccgc ctcctcagat cctcatcaaa aacacgcctg ttcctgcgaa tcctccggca 1980

gagttttcag ctacaaagtt tgcttcattc atcacccagt attccacagg acaagtgagc 2040

gtggagattg aatgggagct gcagaaagaa aacagcaagc gctggaatcc cgaagtgcag 2100

tacacatcca attatgcaaa atctgccaac gttgatttta ctgtggacaa caatggactt 2160

tatactgagc ctcgccccat tggcacccgt taccttaccc gtcccctgta a 2211

<210> 2

<211> 736

<212> PRT

<213> Artificial Sequence

<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 cagcaccccc 840

tgggggtatt ttgatttcaa cagattccac tgccatttct caccacgtga ctggcagcga 900

ctcatcaaca acaactgggg attccgaccc aagagactca acttcaaact cttcaacatc 960

caagtcaagg aggtcacgac gaatgacggc gttacgacca tcgctaataa ccttaccagc 1020

acgattcagg tattctcgga ctcggagtac cagcttccgt acgtcctcgg ctctgcgcac 1080

cagggctgcc tccctccgtt cccggcggac gtgttcatga ttccgcaata cggctacctg 1140

acgctcaaca atggcagcca agccgtggga cgttcatcct tttactgcct ggaatatttc 1200

ccttctcaga tgctgagaac gggcaacaac tttaccttca gctacacctt tgaggaagtg 1260

cctttccaca gcagctacgc gcacagccag agcctggacc ggctgatgaa tcctctcatc 1320

gaccagtacc tgtattacct gaacagaact cagaatcagt ccggaagtgc ccaaaacaag 1380

gacttgctgt ttagccgtgg gtcaccagct ggcatgtctg ttcagcccaa aaactggcta 1440

cctggaccct gttaccggca gcagcgcgtt tctaaaacaa aaacagacaa caacaacagc 1500

aactttacct ggactggtgc ttcaaaatat aacctcaatg ggcgtgaatc catcatcaac 1560

cctggcactg ctatggcctc acacaaagac gacgaagaca agttctttcc catgagcggt 1620

gtcatgattt ttggaaaaga gagcgccgga gcttcaaaca ctgcattgga caatgtcatg 1680

attacagacg aagaggaaat taaagccact aaccccgtgg ccaccgaaag atttgggacc 1740

gtggcagtca atttccagag cagcagcaca gaccctgcga ccggagatgt gcatgctatg 1800

ggagcattac ctggcatggt gtggcaagat agagacgtgt acctgcaggg tcccatttgg 1860

gccaaaattc ctcacacaga tggacacttt cacccgtctc ctcttatggg cggctttgga 1920

ctcaagaacc cgcctcctca gatcctcatc aaaaacacgc ctgttcctgc gaatcctccg 1980

gcagagtttt cggctacaaa gtttgcttca ttcatcaccc agtattccac aggacaagtg 2040

agcgtggaga ttgaatggga gctgcagaaa gaaaacagca aacgctggaa tcccgaagtg 2100

cagtatacat ctaactatgc aaaatctgcc aacgttgatt ttactgagga caacaatgga 2160

ctttatactg agcctcgccc cattggcacc cgttacctta cccgtcccct gtaa 2214

<210> 4

<211> 737

<212> PRT

<213> Artificial Sequence

<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

gtgggtagtt cctcgggaaa ttggcattgc gattccacat ggatgggcga cagagtcatc 720

accaccagca cccgaacctg ggccctgccc acctacaaca accacctcta caaacaaatt 780

tccagccaat caggagcctc gaacgacaat cactactttg gctacagcac cccttggggg 840

tattttgact ttaacagatt ccactgccac ttctcaccac gtgactggca gcgactcatt 900

aacaacaatt ggggattccg gcccaagaga ctcaacttca agctcttcaa catccaagtc 960

aaggaggtca cgacgaatga tggcgtcaca accatcgcta ataaccttac cagcacggtt 1020

caagtcttct cggactcgga gtaccagctt ccgtacgtcc tcggctctgc gcaccagggc 1080

tgcctgcctc cgttcccggc ggacgtcttc atgattcctc agtacggcta cctgactccc 1140

aacaatggca gtcagtctgt gggacgttcc tccttctact gcctggagta cttcccttct 1200

cagatgctga gaacgggcaa caactttacc ttcagctaca cctttgagga agtgcctttc 1260

cacagcagct acgcgcacag ccagagcctg gaccggctga tgaatcctct catcgaccag 1320

tacctgtatt acctgaacag aactcaaaat cagtccggaa gtgcccaaaa caaggacttg 1380

ctgtttagcc gtgggtctcc agctggcatg tctgttcagc ccaaaaactg gctacctgga 1440

ccctgttatc agcagcagcg cgtttctaaa acaaaaacag acaacaacaa cagcaacttt 1500

acctggactg gtgcttcaaa atataacctc aatgggcgtg aatccatcat caaccctggc 1560

actgctatgg cctcacacaa agacgacaaa gacaagttct ttcccatgag cggtgtcatg 1620

atttttggaa aggagagcgc cggagcttca aacactgcat tggacaatgt catgatcaca 1680

gacgaagagg aaatcaaagc cactaacccc gtggccaccg aaagatttgg gaccgtggca 1740

gtcaatctcc agagcagcag cacagaccct gcgaccggag atgtgcatgt tatgggagcc 1800

ttacctggaa tggtgtggca agacagagac gtatacctgc agggtcctat ttgggccaaa 1860

attcctcaca cggatggaca ctttcacccg tctcctctca tgggcggctt tggacttaag 1920

cacccgcctc ctcagatcct catcaaaaac acgcctgttc ctgcgaatcc tccggcagag 1980

ttttcggcta caaagtttgc ttcattcatc acccagtatt ccacaggacg agtgagcgtg 2040

gagattgaat gggagccgca gaaagaaaac agcaaacgct ggaatcccga agtgcagtat 2100

acatctaact atgcaaaatc tgccaacgtt gattttactg tggacaacaa tggactttat 2160

actgagcctc gccccattgg cacccgttac cttacccgtc ccctgtaa 2208

<210> 6

<211> 735

<212> PRT

<213> Artificial Sequence

<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 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 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 acaccaggac 120

aaccgtcggg gtcttgtgct tccgggttac aaatacctcg gacccggtaa cggactcgac 180

aaaggagagc cggtcaacga ggcagacgcc gcggccctcg agcacgacaa agcatacgac 240

cagcagctca aggccggtga caacccgtac ctcaagtaca accacgccga cgccgagttt 300

caggagcgtc tgcaagaaga tacgtctttt gggggcaacc tcgggcgagc agtcttccag 360

gccaaaaaga ggatccttga gcctcttggt ctggttgagg aagcagctaa gacggctcct 420

ggaaagaaga ggcctgtaga gcagtctcct caggaaccgg actcctccgc gggtattggc 480

aaatcgggtg cacagcccgc taaaaagaga ctcaatttcg gtcagactgg cgacacagag 540

tcagtcccag accctcaacc aatcggagaa cctccagcag cgccctctgg tgtgggacct 600

aatacaatgg ctgcgggcgg tggcgcacca atggcagaca ataacgaggg tgccgatgga 660

gtgggtaatt cctcaggaaa ttggcattgc gattcccaat ggctgggcga cagagtcatc 720

accaccagca ccagaacctg ggccctgccc acttacaaca accatctcta caagcaaatc 780

tccagtgctt caacgggggc cagcaacgac aaccactact tcggctacag caccccctgg 840

gggtattttg actttaacag attccactgc cacttttcac cacgtgactg gcagcgactc 900

atcaacaaca attggggatt ccggcccaag agactcaact tcaagctctt caacatccaa 960

gtcaaggagg tcacgacgaa tgatggcgtc acgaccatcg ctaataacct taccagcacg 1020

gttcaagtct tctcggactc ggagtaccag ttgccgtacg tcctcggctc tgcgcaccag 1080

ggttgcctcc ctccgttccc ggcggacgtg ttcatgattc cgcaatacgg ctacctgacg 1140

ctcaacaatg gcagccaagc cgtgggacgt tcatcctttt actgcctgga atatttccct 1200

tctcagatgc tgagaacggg caacaacttt accttcagct acacctttga ggaagtgcct 1260

ttccacagca gctacgcgca cagccagagc ctggaccggc tgatgaatcc tctcatcgac 1320

cagtacctgt attacctgaa cagaactcag aatcagtccg gaagtgccca aaacaaggac 1380

ttgctgttta gccgtgggtc tccagctggc atgtctgttc agcccaaaaa ctggctacct 1440

ggaccctgtt accggcagca gcgcgtttct aaaacaaaaa cagacaacaa caacagcaac 1500

tttacctgga ctggtgcttc aaaatataac ctcaatgggc gtgaatccat catcaaccct 1560

ggcactgcta tggcctcaca caaagacgac aaagacaagt tctttcccat aagcggtgtc 1620

atgatttttg gaaaggagag cgccggagct tcaaacactg cattggacaa tgtcacgatc 1680

acagacgaag aggaaatcaa agccactaac cccgtggcca ccgaaagatt tgggactgtg 1740

gcagtcaatc tccagagcag cagcacagac cctgcgaccg gagatgtgca tgttatggga 1800

gccttacctg gaatggtgtg gcaagacaga gacgtatacc tgcagggtcc tatttgggcc 1860

aagattcctc acacggatgg caactttcac ccgtctcctc tcatgggcgg ctttggactt 1920

aagcacccgc ctcctcagat cctcatcaaa aacacgcctg ttcctgcgaa tcctccggca 1980

gagttttcgg ctacaaagtt tgcttcattc atcacccaat actccacagg acaagtgagt 2040

gtggaaattg aatgggagct gcagaaagaa aacagcaaac gctggaatcc cgaagtgcag 2100

tacacatcca attatgcaaa atctgccaac gttgatttta ctgtggacaa caatggactt 2160

tatactgagc ctcgccccat tggcacccgt taccttaccc gtcccctgta a 2211

<210> 8

<211> 736

<212> PRT

<213> Artificial Sequence

<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 acaccaggac 120

aaccgtcggg gtcttgtgct tccgggttac aaatacctcg gacccggtaa cggactcgac 180

aaaggagagc cggtcaacga ggcagacgcc gcggccctcg agcacgacaa agcatacgac 240

cagcagctca aggccggtga caacccgtac ctcaagtaca accacgccga cgccgagttt 300

caggagcgtc tgcaagaaga tacgtctttt gggggcaacc tcgggcgagc agtcttccag 360

gccaaaaaga ggatccttga gcctcttggt ctggttgagg aagcagctaa gacggctcct 420

ggaaagaaga ggcctgtaga gcagtctcct caggaaccgg actcctccgc gggtattggc 480

aaatcgggtg cacagcccgc taaaaagaga ctcaatttcg gtcagactgg cgacacagag 540

tcagtcccag accctcaacc aatcggagaa cctccagcag cgccctctgg tgtgggacct 600

aatacaatgg ctgcgggcgg tggcgcacca atggcagaca ataacgaggg tgccgatgga 660

gtgggtaatt cctcaggaaa ttggcattgc gattcccaat ggctgggcga cagagtcatc 720

accaccagca ccagaacctg ggccctgccc acttacaaca accatctcta caagcaaatc 780

tccagtgctt caacgggggc cagcaacgac aaccactact tcggctacag caccccctgg 840

gggtattttg actttaacag attccactgc cacttttcac cacgtgactg gcagcgactc 900

atcaacaaca attggggatt ccggcccaag agactcaact tcaagctctt caacatccaa 960

gtcaaggagg tcacgacgaa tgatggcgtc acgaccatcg ctaataacct taccagcacg 1020

gttcaagtct tctcggactc ggagtaccag ttgccgtacg tcctcggctc tgcgcaccag 1080

ggttgcctcc ctccgttccc ggcggacgtg ttcatgattc cgcaatacgg ctacctgacg 1140

ctcaacaatg gcagccaagc cgtgggacgt tcatcctttt actgtctgga atatttccct 1200

tctcagatgc tgagaacggg caacaacttt accttcagct acacctttga ggaagtgcct 1260

ttccacagca gctacgcgca cagccagagc ctggaccggc tgatgaatcc tctcatcgac 1320

cagtacctgt attacctgaa cagaactcag aatcagtccg gaagtgccca aaacaaggac 1380

ttgctgttta gccgtgggtc tccagctggc atgtctgttc agcccaaaaa ctggctacct 1440

ggaccctgtt accggcagca gcgcgtttct aaaacaaaaa cagacaacaa caacagcaac 1500

tttacctgga ctggtgcttc aaaatataac ctcaatgggc gtgaatccat catcaaccct 1560

ggcactgcta tggcctcaca caaagacgac gaagacaagt tctttcccat gagcggtgtc 1620

ctgatttttg gaaaagagag cgccggagct tcaaacactg cattggacaa tgtcatgatt 1680

acagacgagg aggaaattaa agccactaac cccgtggcca ccgaaaaatt tgggactgtg 1740

gcagtcaatc tccagagcag cagcacagac cctgcgaccg gagatgtgca tgttatggga 1800

gccttacctg gaatggtgtg gcaagacaga gacgtatacc tgcagggtcc tatttgggcc 1860

aaaattcctc acacggatgg acactttcac ccgtctcctc tcatgggcgg ctttggactt 1920

aagcacccgc ctcctcagat cctcatcaaa aacacgcctg ttcctgcgaa tcctccggca 1980

gagttttcgg ctacaaagtt tgcttcattc atcacccagt attccacagg acaagtgagc 2040

gtggagattg aatgggagct gcagaaagaa aacagcaaac gctggaatcc cgaagtgcag 2100

tatacatcta actatgcaaa atctgccaac gttgatttta ctgtggacaa caatggactt 2160

tatactgagc ctcgccccat tggcacccgt taccttaccc gtcccctgta a 2211

<210> 10

<211> 736

<212> PRT

<213> Artificial Sequence

<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

ggaaagaaga ggcctgtaga gcagtctcct caggaaccgg actcctccgc gggtattagc 480

aaatcgggtg cacagcccgc taaaaagaga ctcaatttcg gtcagactgg cgacacagag 540

tcagtcccag accctcaacc aatcggagaa cctcccgcag ccccctcagg tgtgggatct 600

cttacaatgg cttcaggtgg tggcgcacca gtggcagaca ataacgaagg cgccgacgga 660

gtgggtaatg cctcaggaaa ttggcattgc gattccacat ggatgggcga cagagtcatc 720

accaccagca cccgaacttg ggccttgccc acctataaca accacctcta caagcaaatc 780

tccagtgctt caacgggggc cagcaacgat aaccactact tcggctacag caccccctgg 840

gggtattttg acttcaacag attccactgc cacttttcac cacgtgactg gcagcgactc 900

atcaacaaca actggggatt ccggcccaag agactcagct tcaagctctt caacatccag 960

gtcaaagagg ttacggacaa caatggagtc aagaccatcg ccaataatct taccagcacg 1020

gtccaggtct tcacggactc agactatcag ctcccgtacg ttctcggctc tgcccaccag 1080

ggctgcctgc ctccgttccc ggcggacgtg ttcatgattc cccagtacgg ctacctgaca 1140

ctcaacaacg gtagtcaggc cgtgggacgc tcctccttct actgcctgga atactttcct 1200

tctcagatgc tgagaacggg caacaacttt accttcagct acacctttga ggaagtgcct 1260

ttccacagca gctacgcgca cagccagagc ctggaccggc tgatgaatcc tctcatcgac 1320

cagtacctgt attacctgaa cagaactcag aatcagtccg gaagtgccca aaacaaggac 1380

ttgctgttta gccgtgggtc tccagctggc atgtctgttc agcccaaaaa ctggctacct 1440

ggaccctgtt accggcagca gcgcgtttct aaaacaaaaa cagacaacaa caacagcaac 1500

tttacctgga ctggtgcttc aaaatataac ctcaatgggc gtgaatccat catcaaccct 1560

ggcactgcta tggcctcaca caaagacgac aaagacaagt tctttcccat gagcggtgtc 1620

atgatttttg gaaaggagag cgccggagct tcaaacactg cattggacaa tgtcatgatc 1680

acagacgaag aggaaatcaa agccactaac cccgtggcca ccgaaagatt tgggactgtg 1740

gcagtcaatc tccagagcag cagcacagac cctgcgaccg gagatgtgca tgttatggga 1800

gccttacctg gaatggtgtg gcaagacaga gacgtatacc tgcagggtcc tatttgggcc 1860

aaaattcctc acacggatgg acactttcac ccgtctcctc tcatgggcgg ctttggactc 1920

aagcacccgc ctcctcagat cctcatcaaa aacacacccg ttcctgcgaa tcctccggca 1980

gagttttcgg ctacaaagtt tgcttcattc atcacccagt attccacagg acaagtgagc 2040

gtggagattg aatgggagct gcagaaagaa aacagcaaac gctggaatcc cgaagtgcag 2100

tacacatcca attatgcaaa atctgccgac gttgatttta ctgtggacaa caatggactt 2160

tatactgagc ctcgccccat tggcacccgt taccttaccc gtcccctgta a 2211

<210> 12

<211> 736

<212> PRT

<213> Artificial Sequence

<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 tgagaacggg caacaacttt accttcagct acacctttga ggaagtgcct 1260

ttccacagca gctacgcgca cagccagagc ctggaccggc tgatgaatcc tctcatcgac 1320

cagtacctgt attacctgaa cagaactcag aatcagtccg gaagtgccca aaacaaggac 1380

ttgctgttta gccgtgggtc tccagctggc atgtctgttc agcccaaaaa ctggctacct 1440

ggaccctgtt accggcagca gcgcgtttct aaaacaaaaa cagacaacaa caacagcaac 1500

tttacctgga ctggtgcttc aaaatataac ctcaatgggc gtgaatccat catcaaccct 1560

ggcactgcta tggcctcaca caaagacgac aaagacaagt tctttcccat gagcggtgtc 1620

atgatttttg gaaaggagag cgccggagct tcaaacactg cattggacaa tgtcatgatc 1680

acagacgaag aggaaatcaa agccactaac cccgtggcca ccgaaagatt tgggactgtg 1740

gcagtcaatc tccagagcag cagcacagac cctgcgaccg gagatgtgca tgttatggga 1800

gccttacctg gaatggtgtg gcaagacaga gacgtatacc tgcagggtcc catttgggcc 1860

aaaattcctc acacggatgg acactttcac ccgtctcctc tcatgggcgg ctttggactt 1920

aagcacccgc ctcctcagat cctcatcaaa aacacgcctg ttcctgcgaa tcctccggca 1980

gagttttcgg ctacaaagtt tgcttcattc atcacccagt attccacagg acaagtgagc 2040

gtggagattg aatgggagct gcagaaagaa aacagcaaac gctggaatcc cgaagtgcag 2100

tatacatcta actatgcaaa atctgccaac gttgatttta ctgtggacaa caatggactt 2160

tatactgagc ctcgccccat tggcacccgt taccttaccc gtcccctgta a 2211

<210> 14

<211> 736

<212> PRT

<213> Artificial Sequence

<400> 14

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

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

485 490 495

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

500 505 510

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

515 520 525

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

530 535 540

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

545 550 555 560

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

565 570 575

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

580 585 590

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

595 600 605

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

610 615 620

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

625 630 635 640

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

645 650 655

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

660 665 670

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

675 680 685

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

690 695 700

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

705 710 715 720

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

725 730 735

<210> 15

<211> 2211

<212> DNA

<213> Artificial Sequence

<400> 15

atggctgccg atggttatct tccagattgg ctcgaggaca acctctctga aggaataaga 60

cagtggtgga agctcaaacc tggcccacca ccaccaaagc ccgcagagcg gcataaggac 120

gacagcaggg gtcttgtgct tcctgggtac aagtacctcg gacccttcaa cggactcgac 180

aagggggagc cggtcaacgc ggcggacgca gcggccctcg agcacgacaa ggcctacgac 240

cagcagctca aagcgggtga caatccgtac ctgcggtata accacgccga cgccgagttt 300

caggagcgtc tgcaagaaga tacgtcattt gggggcaacc tcgggcgagc agtcttccag 360

gccaagaagc gggttctcga acctctcggt ctggttgagg aaggcgctaa gacggctcct 420

ggaaagaaac gtccggtaga gcagtctcct caggaaccgg actcctccgc gggtattggc 480

aaatcgggtg cacggcccgc taaaaagaga ctcaattttg gtcagactgg cgacacagag 540

tcagtcccag accctcaacc aatcggagaa cctcccgcag cccccacaag tttgggatct 600

aatacaatgg cttcaggcgg tggcgcacca atggcagaca ataacgaagg cgccgacgga 660

gtgggtaatg cctcaggaaa ttggcattgc gattccacat ggctgggcga cagagtcgtc 720

accaccagca cccgcacctg ggccttgccc acctataaca accacctcta caagcaaatc 780

tccagtgctt caacgggggc cagcaacgac aaccactact tcggctacag caccccctgg 840

gggtattttg atttcaacag attccactgc cacttttcac cacgtgactg gcagcgactc 900

atcaacaaca attggggatt ccggcccaag agactcaact tcaagctctt caacatccaa 960

gtcaaggagg tcacgacgaa tgatggcgtc acgaccatcg ctaataacct taccagcacg 1020

gtccaggtct tcacggactc agactatcag ctcccgtacg tgctcgggtc ggctcacgag 1080

ggctgcctcc ctccgttccc ggcggacgtg ttcatgattc cgcaatacgg ctacctgacg 1140

ctcaacaatg gcagccaagc cgtgggtcgt tcgtcctttt actgcctgga atatttcccg 1200

tcgcaaatgc taagaacagg caacaacttt accttcagct acacctttga ggaagtgcct 1260

ttccacagca gctacgcgca cagccagagc ctggaccggc tgatgaatcc tctcatcgac 1320

cagtacctgt attacctgaa cagaactcag aatcagtccg gaagtgccca aaacaaggac 1380

ttgctgttta gccgtgggtc tccagctggc atgtctgttc agcccaaaaa ctggctacct 1440

ggaccctgtt accggcagca gcgcgtttct aaaacaaaaa cagacaacaa caacagcaac 1500

tttacctgga ctggtgcttc aaaatataac ctcaatgggc gtgaatccat catcaaccct 1560

ggcactgcta tggcctcaca caaagacgac aaagacaagt tctttcccat gagcggtgtc 1620

atgatttttg gaaaggagag cgccggagct tcaaacactg cattggacaa tgtcatgatc 1680

acagacgaag aggaaatcaa agccactaac cccgtggcca ccgaaagatt tgggactgtg 1740

gcagtcaatc tccagagcag cagcacggac cctgcgactg gagatgtgca tgttatggga 1800

gccttacctg gaatggtgtg gcaagacaga gacgtatacc tgcagggtcc tatttgggcc 1860

aaaattcctc acacggatgg acactttcac ccgtctcctc tcatgggcgg ctttggactt 1920

aagcacccgc ctcctcagat cctcatcaaa aacacgcctg ttcctgcgaa tcctccggca 1980

gagttttcgg ctacaaagtt tgcttcattc atcacccagt attccacagg acaagtgagc 2040

gtggagattg aatgggagct gcagaaagaa aacagcaaac gctggaatcc cgaagtgcag 2100

tatacatcta actatgcaaa atctgccaac gttgatttta ctgtggacaa caatggactt 2160

tatactgagc ctcgccccat tggcacccgt taccttaccc gtcccctgta a 2211

<210> 16

<211> 736

<212> PRT

<213> Artificial Sequence

<400> 16

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 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 cagcaccccc 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 tttaccttca gctacacctt tgaggaagtg 1260

cctttccaca gcagctacgc gcacagccag agcctggacc ggctgatgaa tcctctcatc 1320

gaccaatacc tgtattacct gaacagaact caaaatcagt ccggaagtgc ccaaaacaag 1380

gacttgctgt ttagccgtgg gtctccagct ggcatgtctg ttcagcccaa aaactggcta 1440

cctggaccct gttatcggca gcagcgcgtt tctaaaacaa aaacagacaa caacaacagc 1500

aattttacct ggactggtgc ttcaaaatat aacctcaatg ggcgtgaatc catcatcaac 1560

cctggcactg ctatggcctc acacaaagac gacgaagaca agttctttcc catgagcggt 1620

gtcatgattt ttggaaaaga gagcgccgga gcttcaaaca ctgcattgga caatgtcatg 1680

attacagacg aagaggaaat taaagccact aaccctgtgg ccaccgaaag atttgggacc 1740

gtggcagtca atttccagag cagcagcaca gaccctgcga ccggagatgt gcatgctatg 1800

ggagcattac ctggcatggt gtggcaagat agagacgtgt acctgcaggg tcccatttgg 1860

gccaaaattc ctcacacaga tggacacttt cacccgtctc ctcttatggg cggctttgga 1920

ctcaagaacc cgcctcctca gatcctcatc aaaaacacgc ctgttcctgc gaatcctccg 1980

gcggagtttt cagctacaaa gtttgcttca ttcatcaccc aatactccac aggacaagtg 2040

agtgtggaaa ttgaatggga gctgcagaaa gaaaacagca agcgctggaa tcccgaagtg 2100

cagtacacat ccaattatgc aaaatctgcc aacgttgatt ttactgtgga caacaatgga 2160

ctttatactg agcctcgccc cattggcacc cgttacctta cccgtcccct gtaa 2214

<210> 18

<211> 737

<212> PRT

<213> Artificial Sequence

<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 (25)

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

(a) 1, nucleotide sequence SEQ ID NO;

(b) 3, nucleotide sequence SEQ ID NO;

(c) nucleotide sequence SEQ ID NO 5;

(d) nucleotide sequence SEQ ID NO 7;

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

(f) nucleotide sequence SEQ ID NO 11;

(g) 13 in nucleotide sequence SEQ ID NO;

(h) 15, nucleotide sequence SEQ ID NO;

(i) nucleotide sequence SEQ ID NO 17; or

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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