WO2019218983A1 - Recombinant adeno-associated virus vector and use thereof - Google Patents

Abstract

Disclosed are a recombinant adeno-associated virus vector and a use thereof. The recombinant adeno-associated virus vector is only expressed in dopaminergic neurons in the region of the Substantia nigra, and is not expressed in astrocytes and oligodendrocytes. In addition, an injection of AAV-MANFHIS in the Substantia nigra can effectively improve the behavioral function of rats and can specifically treat neurodegenerative diseases in animals.

Description

Recombinant adeno-associated virus vector and application thereof

The present application claims priority to Chinese Patent Application No. 201101, 468, 718, 181, entitled "Recombinant Adeno-associated Virus Vectors and Applications" on May 16, 2018, the entire contents of which are incorporated herein by reference. in.

Technical field

The present invention relates to the field of biotechnology, and in particular to recombinant adeno-associated virus vectors and uses thereof.

Background technique

Parkinson's disease (PD) is a neurodegenerative disease caused by degeneration and necrosis of dopaminergic neurons in the substantia nigra of the midbrain, which leads to a decrease in dopamine transmitters in the substantia nigra and striatum. The main symptoms include bradykinesia and rest. Sexual tremor, muscle stiffness, unstable posture, etc., seriously affect the quality of life of patients. Currently, PD clinical treatment relies mainly on dopamine replacement therapy, including dopa preparations or dopamine receptor agonists. This method does not fundamentally save the progressively lost dopaminergic neurons, and its efficacy is gradually lost with the progressive loss of dopamine neurons, and may also lead to serious adverse reactions such as dyskinesia. Neurotrophic factors play an important role in the survival and differentiation of vertebrate neurons, and have long been highly anticipated in neuroprotection and nerve regeneration studies. Among them, Glial Cell Derived Neurotrophic Factor (GDNF) not only has neuroprotective effects but also promotes repair of dopaminergic neurons after injury. Mesencephalic Astropcyte-derived Neurotrophic Factor (MANF) is a highly conserved neurotrophic factor found in 2003 that promotes the survival of dopamine neurons. However, these nerve growth factors have not shown good prospects for drug development in drug research, and the disadvantages mainly include: 1) difficulty in administration. As a macromolecular protein, neurotrophic factor can't pass the blood-brain barrier. Stereotactic intracerebral injection technology requires high risk and high risk; 2) short metabolic time and poor bioavailability; 3) cause complications; 4) non-specific diffusion exists. Potential to tumor. It can be seen that finding a lead with higher selectivity and biocompatibility, establishing a convenient and safe route of administration, and real-time control of the dose, reducing tumorigenicity and complications are the core issues in the development of PD drugs.

The rise of gene therapy and the emergence of new transgenic vectors have opened up new avenues for the application of neurotrophic factors in neurodegenerative diseases. Through the integration of the target gene with the genome, the target protein forms a continuous expression in the brain, thereby avoiding the damage and risk caused by frequent brain stereotactic injection.

Adeno-associated virus (AAV) is the preferred vector for gene therapy of central nervous system diseases. It does not affect the function of normal human genes when delivering target genes, and can efficiently express heterologous proteins. At present, there are more than 40 clinical trials in Europe and America using AAV as a carrier. The use of AAV as a carrier to introduce glutamate decarboxylase and amino acid decarboxylase genes into the subthalamic nucleus for Parkinson's disease has shown good safety. After 3 months of treatment, the patient's motor function improved by 25% to 30%. There have been reports of the use of adeno-associated virus (AAV)-based gene delivery systems to deliver glial cell-derived neurotrophic factor (GDNF) to neurodegenerative disorders such as Parkinson's disease. Patient's method. There have also been reports of a method for expressing GDNF in vivo using tetracycline-regulated adeno-associated viral vectors. In addition, a method of expressing neruturin in vivo using a viral expression construct is also disclosed. The targeted injection of gene therapy methods with respect to proteins has the advantage of long lasting efficacy, but biosafety, especially non-specific tissue distribution, remains a potential risk. Therefore, the selection of appropriate genes as well as viral vectors is a key factor in improving drug availability.

Summary of the invention

In view of this, the present invention provides a recombinant adeno-associated virus vector and use thereof. The recombinant adeno-associated virus vector was only expressed in dopaminergic neurons in the substantia nigra region, and was not expressed in astrocytes and oligodendrocytes; and the substantia nigra injection of AAV-MANFHIS could effectively improve the behavior of rats. Learning function, can specifically treat animal neurodegenerative diseases.

In order to achieve the above object, the present invention provides the following technical solutions:

The invention provides a recombinant adeno-associated virus vector comprising:

I. A nucleotide sequence having a polypeptide encoding astrocyte-derived neurotrophic factor (MANF);

II. A nucleotide sequence obtained by modifying, substituting, deleting or adding one or more bases of a nucleotide sequence as shown in I;

III. A sequence having at least 80% homology with a nucleotide sequence as shown in I or a polypeptide sequence obtained after translation is a nucleotide sequence corresponding to an amino acid sequence of an astrocyte-derived neurotrophic factor;

IV, a complement of a sequence as shown by I, II or III.

In some specific embodiments of the invention, the adeno-associated viral vector comprises: AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8 or AAV9.

In some embodiments of the invention, the nucleotide sequence further comprises a secretion sequence located at the 5' position and in accordance with the reading frame encoding the MANF polypeptide sequence.

In some specific embodiments of the invention, the secretory sequence is a viral promoter, and the viral promoter comprises MLP, CMV or RSVLTR.

In some embodiments of the invention, the recombinant adeno-associated viral vector comprises an expression vector comprising, in order from 5' to 3'

V, upstream ITR, CMV promoter, MANF gene and downstream ITR; or

VI, upstream ITR, CMV promoter, MANF gene, HIS sequence, polyA and downstream ITR.

In some specific embodiments of the invention, the recombinant adeno-associated virus vector further comprises an auxiliary function vector and/or an accessory function vector;

The helper function carrier comprises trans providing a protein Rep and/or Cap having AAV replication and/or packaging function;

The accessory function vector comprises an adenovirus VA RNA coding region, an adenovirus E4ORF6 coding region, an adenovirus E2A 72kD coding region, an adenovirus E1A coding region, an adenoviral E1B region lacking a complete E1B 55k coding region, or a helper virus Ad5. Important element E1a, E1b, E2a or E4.

In some specific embodiments of the invention, the nucleotide sequence has any one of the nucleotide sequences set forth in VII, VIII, IX or X:

VII, having the nucleotide sequence shown in SEQ ID NO: 1;

VIII. A nucleotide sequence having the nucleotide sequence of SEQ ID NO: 1 modified, substituted, deleted or added with one or more bases;

IX, a sequence having at least 80% homology with the nucleotide sequence shown in SEQ ID NO: 1 or a polypeptide sequence obtained after translation is a nucleotide sequence corresponding to the amino acid sequence shown in SEQ ID NO: 2;

X. The complement of the sequence as shown in VII, VIII or IX.

In some specific embodiments of the invention, the expression vector has any one of the nucleotide sequences set forth in XI, XII, XIII or XIV:

XI, having the nucleotide sequence shown in SEQ ID NO: 4;

XII, a nucleotide sequence obtained by modifying, substituting, deleting or adding one or more bases of the nucleotide sequence shown by SEQ ID NO: 4;

XIII, a sequence having at least 80% homology to the nucleotide sequence set forth in SEQ ID NO: 4;

XIV, the complement of the sequence as shown by XI, XII or XIII.

The present invention also provides the use of the recombinant adeno-associated virus vector for the preparation of a medicament for preventing and/or treating a neurodegenerative disease.

In some embodiments of the invention, the neurodegenerative disease is Parkinson's disease.

In some embodiments of the invention, the Parkinson's disease comprises mild nigrostriatal dopamine (DA) damage and/or moderate nigrostriatal dopamine (DA) damage.

In some embodiments of the invention, the recombinant adeno-associated viral vector specifically acts on a neuron of an animal.

In some embodiments of the invention, the recombinant adeno-associated viral vector specifically acts on the striatum and/or substantia nigra of an animal.

The invention provides a recombinant adeno-associated virus vector comprising:

I. A nucleotide sequence having a polypeptide encoding astrocyte-derived neurotrophic factor (MANF);

II. A nucleotide sequence obtained by modifying, substituting, deleting or adding one or more bases of a nucleotide sequence as shown in I;

III. A sequence having at least 80% homology with a nucleotide sequence as shown in I or a polypeptide sequence obtained after translation is a nucleotide sequence corresponding to an amino acid sequence of an astrocyte-derived neurotrophic factor;

IV, a complement of a sequence as shown by I, II or III.

The recombinant adeno-associated virus vector provided by the invention is only expressed in dopaminergic neurons in the substantia nigra region, and is not expressed in astrocytes and oligodendrocytes; and the substantia nigra injection of AAV-MANFHIS can effectively increase the size. The behavioral function of the mouse is capable of specifically treating neurodegenerative diseases in animals.

DRAWINGS

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the embodiments or the prior art description will be briefly described below.

Figure 1 shows the AAV vector plasmid pAAV2-MANF-HIS map;

Figure 2 (A) shows dot-hybridization detection of AAV8-regulated-MANF virus titer, diluted with the target gene plasmid, as a standard curve, sample conversion titer 1 × 1011v.g / ml;

Figure 2 (B) shows the AAV8-regulated-MANF virus titer by real-time PCR, diluted with the target gene plasmid as a standard curve, and the sample titer is converted to 1.33×1011 v.g/ml;

Figure 3 shows the transcription level of MANF gene mRNA in AAV8-MANF virus-infected cells by real-time PCR to compare the mRNA transcription level of MANF in uninfected virus cells as 1.0, and the mRNA transcription levels of MANF mRNA in other samples were compared with those in uninfected virus cells;

Figure 4 shows the AAV8-MANF-his target gene targeting study;

Figure 5 shows Amorphism of TH in rat PD model induced by 6-OHDA by AAV8-MANF;

Figure 6 shows AAV8-MANF on 6-OHDA-induced rat behavioral experiments;

Figure 7 shows that the target gene is mainly expressed in dopamine neurons, and no obvious expression is observed in astrocytes and oligodendrocytes;

Figure 8 shows the distribution of AAV8 capsid protein in various organs.

Detailed ways

The invention discloses a recombinant adeno-associated virus vector and an application thereof, and those skilled in the art can learn from the contents of the paper and appropriately improve the process parameters. It is to be understood that all such alternatives and modifications are obvious to those skilled in the art and are considered to be included in the present invention. The method and the application of the present invention have been described by the preferred embodiments, and it is obvious that the method and application described herein may be modified or appropriately modified and combined without departing from the scope of the present invention. The technique of the present invention is applied.

The present invention provides a recombinant adeno-associated virus (AAV) vector for treating a neurodegenerative disease, comprising a recombinant adeno-associated virus (AAV) vector for treating a neurodegenerative disease,

(a) The viral vector comprises a polynucleotide encoding an astrocyte-derived neurotrophic factor (MANF) polypeptide operably linked to an expression control element comprising a promoter.

(b). The viral vector is used to specifically infect mammalian neurons or to specifically express a MANF polypeptide in mammalian neurons.

Preferably, the recombinant adeno-associated virus is AAV8.

Advantageously, said polynucleotide further comprises a secretion sequence located at the 5' position and which corresponds to the reading frame encoding the MANF polypeptide sequence.

Preferably, the nucleotide encodes human MANF.

Preferably, the promoter of the recombinant adeno-associated virus (AAV) vector is a viral promoter; more preferably, the promoter is an MLP, CMV or RSV LTR.

Preferably, the virus is prepared using three vector systems, the three vectors being constructed as follows:

(a) AAV helper vector: trans provides the proteins Rep and Cap with AAV replication and packaging functions

(b) AAV expression vector: upstream ITR, CMV promoter, human MANF gene sequence, downstream ITR or upstream ITR, CMV promoter, human MANF gene sequence, HIS sequence, composed of AAV2 type from 5' to 3' end polyA, downstream ITR

(c). Affiliated functional carrier: important components E1a, E1b, E2a, E4, etc. containing auxiliary toxic Ad5

Preferably, the nucleotide sequence of the AAV expression vector is as shown in Seq NO.

The invention also provides the use of the viral vector for the treatment of a medicament for a neurodegenerative disease.

Preferably, the neurodegenerative disease is Parkinson's disease.

Preferably, the Parkinson's disease comprises mild to moderate nigrostriatal dopamine (DA) damage.

Preferably, the virus is administered to the striatum and/or substantia nigra of the patient.

Preferably, the AAV virion is administered to the striatum using a strong delivery system (CED).

Preferably, the recombinant adeno-associated virus (AAV) vector is administered using an osmotic pump.

Preferably, the recombinant adeno-associated virus (AAV) vector is administered using an infusion pump.

The practice of the present invention is carried out using conventional methods of virology, microbiology, molecular biology, and recombinant DNA techniques within the skill of the art, unless otherwise indicated. The technique is fully explained in the literature. See, for example, Sambrook et al. Molecular Cloning: A Laboratory Manual (recent version); DNA Cloning: A Practical Approach, Volume I & II (D. Glover, ed.); Oligonucleotide Synthesis (N. Gait, edited, most recent edition); Nucleic Acid Hybridization (B. Hames & S. Higgins, editor, recent version); Transcription and Translation (B. Hames & S. Higgins, editor, recent version); CRC Handbook of Parvoviruses, Volume I & II (P Tijssen, ed.); Fundamental Virology, 2nd Edition , Volume I & II (BN Fields and DMKnipe, ed.); Freshney Culture of Animal Cells, A Manual of Basic Technique (Wiley-Liss, 3rd edition); and Ausubel et al. (1991) Current Protocols in Moleular Biology (Wiley Interscience, NY).

The singular forms "a", "the", and "the"

definition

In the description of the present invention, the following terms will be used, and the brief description is hereby defined as follows:

The term "Mesencephalic Astropcyte-derived Neurotrophic Factor (MANF)" or "MANF" as used herein refers to a neurotrophic factor of any origin, which is basic to any of a variety of known MANFs. The homology and function are equivalent. The degree of homology between mammalian representative MANF proteins is approximately 93%, and all mammalian MANFs have similar high homology. The MANF is present in a biologically active form of a monomer, dimer or other multimer. Accordingly, the term "MANF polypeptide" as used herein includes the active monomer MANF as well as the active multimeric MANF, the active glycosylated and non-glycosylated forms of the MANF and the active truncated form of the molecule.

As used herein, "functionally equivalent" means that the MANF polypeptide retains some or all of the neurotrophic properties, but is not necessarily the same degree as the native MANF molecule.

"Homologous" refers to a percentage of similarity between two polynucleotides or two polypeptide portions. When two polynucleotides or two polypeptide sequences are at least about 50% similar, preferably at least 75%, more preferably at least 80-85%, preferably at least 90%, more preferably at least 95-99% or more than the specified molecular length Where the multiple sequences are identical or the sequences are identical, the sequences are "substantially homologous" to each other. Substantial homology as used herein also refers to sequences that are identical to a particular polynucleotide or polypeptide sequence.

"Identity" generally refers to the exact nucleotide and nucleotide or amino acid to amino acid correspondence of two polynucleotide or polypeptide sequences, respectively. The percent identity can be determined by direct comparison of the sequence information between two molecules. By arranging the sequences, the exact number of matches between the two columns is calculated, divided by the shortest sequence, and the result is multiplied by 100.

Furthermore, homology can be determined by hybridization of a polynucleotide under conditions in which a stable double strand is formed in a homologous region, followed by digestion with a single-strand specific nuclease, and the size of the digested fragment is determined. DNA sequences are determined to be substantially homologous by Southern blot hybridization experiments under stringent conditions as defined for particular systems, for example. Suitable hybridization conditions as defined are techniques in the art. See, for example, Sambrook et al., supra; DNA Cloning, supra; Nucleic Acid hybridization, supra.

By "MANF variant" is meant a reference to a biologically active derivative of a MANF molecule, or the derivative retains a desired active fragment, such as neurotrophic activity in the experiments described herein. Generally, the term "variant" refers to a compound having a native polypeptide sequence and structure, with one or more amino acid insertions, substitutions (usually conserved) and/or deletions relative to the native molecule, so long as the alteration does not destroy the nerve. Nutritional activity. Preferably, the variant has at least the same neurotrophic activity as the native molecule. Methods for making polynucleotides encoding MANF variants are known in the art and are described further below. For MANF deletion variants, typically the deletion range is from about 1 to 30 residues, more typically from about 1 to 10 residues, typically from about 1 to 5 adjacent residues, or any within the range Integer. N-terminal, c-terminal and internal deletions are envisaged. In order to maintain maximum biological activity, deletions are typically selected to maintain the tertiary structure of the MANF protein product in the affected region, for example, cysteine cross-linking. Non-limiting examples of deletion variants include a truncated MANF protein product lacking a MANF 1-40 N-terminal amino acid, or a variant lacking a MANF C-terminal residue or a combination thereof.

For MANF insertion variants, amino acid sequence insertions typically include N- and/or C-terminal fusions ranging from one residue to the length of a polypeptide comprising one hundred or more residues, and the addition of single or multiple internals Amino acid residues. Internal insertions typically range from about 1 to 10 residues, typically from about 1 to 5 residues, often from 1 to 3 amino acid residues, or any integer within the range. Examples of N-terminal insertion variants include fusion of a heterologous N-terminal signal sequence with the N-terminus of MANF, and fusion of amino acid sequences derived from other neurotrophin sequences.

The MANF substitution variant has at least one amino acid residue removed from the MANF amino acid sequence and is inserted into its position by a different residue. The substitution variants include variants of the allele characterized by a change in the nucleotide sequence that occurs naturally in a single population, which may or may not result in an amino acid change. Particularly preferred substitutions are conservative, i.e., these substitutions are substitutions within a family of amino acids associated with their side chains. Specifically, amino acids are generally classified into four types: (1) acidic-aspartic acid, glutamic acid; (2) basic-lysine, arginine, histidine; (3) non-polar -Glycine, alanine, valine, leucine, isoleucine, phenylalanine, valine; and (4) uncharged polar-tryptophan, tyrosine, Serine, cysteine, methionine, asparagine, glutamine, threonine; phenylalanine, tryptophan and tyrosine are sometimes classified as aromatic amino acids. For example, it is foreseen to replace leucine with isoleucine or valine, aspartic acid with glutamic acid, threonine with serine, or replace a similarly conserved amino acid with a structurally related amino acid, These substitutions do not have a large effect on biological activity. For example, the MANF molecule can include up to about 5-10 conservative or non-conservative amino acid substitutions, or even about 15-25 conservative or non-conservative amino acid substitutions, or any integer between 5-25, as long as you keep thinking The desired molecule is functionally intact. Those skilled in the art can readily determine the regions of the molecule of interest that are permitted to be altered using techniques well known in the art. Specific mutations in the MANF amino acid sequence can include modifications of the glycosylation site (eg, serine, threonine, or aspartic acid). Loss of glycosylation or partial glycosylation is caused by any aspartate-linked glycosylation recognition site or by amino acid substitutions or deletions at any position of the molecule inserted into the O-linked carbohydrate modification. The aspartate-linked glycosylation recognition site includes a tripeptide sequence specifically recognized by a suitable cell glycosylase. These tripeptide sequences are Asn-Xaa-Thr or Asn-Xaa-Ser, wherein Xaa can be any amino acid other than Pro. Alteration of an amino acid substitution or deletion at one or both of the first or third amino acid positions of the glycosylation recognition site (and/or deletion of the amino acid in the second position) results in a modified tripeptide sequence Non-glycosylation. Thus expression of a suitably altered nucleotide sequence results in a variant that is not glycosylated at this site. Alternatively, the MANF amino acid sequence can be modified to add a glycosylation site. Methods for identifying mutant MANF amino acid residues or regions are well known in the art. One known method is the "alanine scanning mutation". See, for example, Cunningham and Wells, Science (1989) 244: 1081-10850. In this method, amino acid residues or groups of target residues (eg, charged residues such as Arg, Asp, His, Lys, and Glu) are identified and used. Substituting or negatively charged amino acids (most preferably alanine and polyalanine) to affect the interaction between the amino acid and the surrounding water environment inside and outside the cell. The domain of the substitution-sensitive function is precisely located by introducing additional or alternative residues at the substituted site. Therefore, the target site in which the amino acid sequence variation is introduced is determined, alanine scanning or random mutagenesis is performed on the corresponding target codon or DNA sequence region, and the expressed MANF variant is screened to maximize the required activity and activity level. Excellent combination.

The most critical sites for mutagenesis include sites in which the amino acids of the MANF proteins of different species differ significantly in terms of side chain volume, charge, and/or hydrophobicity. Other target sites are those from different species that are identical at the particular residue of the MANF-like protein. The position is generally important for the biological activity of the protein. These sites were initially replaced in a related conservative manner. If the substitution results in a change in biological activity, then a greater change (typical substitution) is introduced, and/or other insertions or deletions are made and the activity of the resulting product is screened. Determination of MANF activity is known in the art and includes the ability to enhance the absorption of dopamine in cultured midbrain neurons and the ability to enhance the survival of sympathetic ganglion neurons. See, for example, U.S. Patent No. 6,362,319.

"Parkinson's disease-like symptoms" include muscle tremors, muscle weakness, stiffness, bradykinesia, changes in posture and balance, dementia, and similar symptoms, usually associated with Parkinson's disease or other neurodegenerative diseases.

"Vector" refers to any genetic element, such as a plasmid, bacteriophage, transposon, cosmid, chromosome, virus, virion, etc., which, when linked to appropriate control factors, are capable of replicating and capable of transporting a gene sequence between cells. . Thus, the term includes both cloning and expression vectors, as well as viral vectors. "AAV vector" refers to a vector derived from an adeno-associated virus serotype, including, without limitation, AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV. -8 and AAV9. The AAV vector may have one or more AAV wild-type genes, preferably rep and/or cap genes, deleted in whole or in part, but retains a functional flanking ITR sequence. Functional ITR sequences are required for rescue, replication and packaging of AAV virions. Thus, AAV vectors as defined herein include those sequences (eg, functional ITRs) that are at least required for viral replication and packaging cis. The ITR need not be a wild-type nucleotide sequence and may be altered, for example, by insertion, deletion or substitution of a nucleotide, as long as the sequence provides for functional rescue, replication and packaging.

"AAV helper function" refers to an AAV-derived coding sequence that can express an AAV gene product, which product then acts on the productive AAV replication trans. Therefore, the AAV helper function includes two main AAV open reading frames (ORFs), namely rep and cap. The Rep expression product has been shown to have a variety of functions including: recognition, binding and cleavage of AAV DNA origin of replication; DNA helicase activity; and regulation of transcription by AAV (or other heterologous) promoter. The Cap expression product provides the necessary packaging functionality. The AAV helper function is used herein to complement the trans AAV function lost by the AAV vector.

The term "AAV helper construct" generally refers to a nucleic acid molecule comprising a nucleotide sequence that provides AAV function deletion of an AAV vector, which is used to produce a transduction vector for delivery of a nucleotide sequence of interest. AAV helper constructs are typically used to provide transient expression of the AAV rep and/or cap genes to complement the AAV function necessary for lost cell lytic AAV replication; however, the helper construct lacks AAV ITR and is not self-replicating and self-packaging. The AAV helper construct can be a plasmid, phage, transposon, cosmid, virus or virion. A number of AAV helper constructs are described, such as the commonly used plasmids pAAV/Ad and pIM29+45 encoding Rep and Cap expression products. See, for example, Samulski et al. (1989) J. Virol. 63:3822-3828; and McCarty et al. (1991) J. Virol. 65: 2936-2945. There are many other vectors encoding Rep and Cap expression products. See, for example, U.S. Patent Nos. 5,139,941 and 6,376,237.

The term "adjunct function" refers to the function of a non-AAV-derived virus and/or cell (AAV relies on it for replication). Thus, the term encompasses proteins and RNAs required for AAV replication including those involved in AAV gene transcriptional activation, time-specific AAV mRNA splicing, AAV DNA replication, synthesis of Cap expression products, and AAV capsid assembly. The virus-based accessory function can be derived from any known helper virus such as adenovirus, herpes virus (except herpes simplex virus type 1) and vaccinia virus.

The term "adjunct functional vector" generally refers to a nucleic acid molecule comprising a nucleotide that provides an accessory function. The accessory function vector can be transfected into a suitable host cell, and the vector can then support AAV virion production in the host cell. Particularly excluded from this term is a naturally occurring infectious virus, such as an adenovirus, herpes virus or vaccinia virus. Thus, the accessory function vector can be in the form of a plasmid, phage, transposon or cosmid.

It has been particularly demonstrated that a fully complemented adenoviral gene does not require ancillary ancillary functions. Specifically, adenoviral mutants that are incapable of DNA replication and late gene synthesis indicate that AAV replication permits.

Particularly preferred accessory functional vectors include the adenoviral VA RNA coding region, the adenovirus E4 ORF6 coding region, the adenovirus E2A 72kD coding region, the adenoviral E1A coding region, and the adenoviral E1B coding region lacking the complete E1B 55k region. "Able to support efficient production of rAAV virions" means that the accessory function vector or system, in a particular host cell, is capable of providing substantially comparable or higher levels of rAAV virion production that can be obtained by infection of the host cell with an adenovirus helper virus. Accessibility. Thus, the ability of an accessory functional vector or system to support efficient rAAV virion production can be determined by comparing the rAAV virion titer obtained with the additional vector or system to the titer obtained using infectious adenovirus infection. More specifically, if the same number of host cells obtain less than 200 times more virions than the amount infected with adenovirus, more preferably no more than 100 times, most preferably equal or higher than the amount obtained by infection with an adenovirus. The accessory functional vector or system supports efficient rAAV virion production that is approximately equal or higher than that obtained with infectious adenovirus.

"Recombinant virus" refers to a genetically altered virus, for example, by the addition or insertion of a heterologous nucleic acid construct into a particle.

"AAV virion" refers to a complete virion, such as a wild-type (wt) AAV virion (including a linear single-chain AAV nucleic acid genome that binds to the AAV capsid protein coat). In this regard, any complementary single-chain AAV nucleic acid molecule, such as a sense strand or a non-meaning strand, can be packaged into any AAV virion, and both strands are equally infectious.

"Recombinant AAV virion" or "rAAV virion" is defined herein as an infectious replication-defective virus, including an AAV protein capsid, encapsulating a heterologous nucleotide sequence of interest flanked by AAVITR. The rAAV virion is produced in a host cell having an AAV helper function, an AAV-assisted function, and an accessory function introduced therein. Thus, the host cell has the ability to encode an AAV polypeptide which, for later gene delivery, entails packaging the AAV vector (including the recombinant nucleotide sequence of interest) into an infectious recombinant virion.

The term "transfection" is used to mean that a cell absorbs foreign DNA, which is transfected when the foreign DNA is introduced into the cell membrane. Many transfection techniques are generally known in the art. The techniques can be used to introduce one or more exogenous DNA moieties, such as nucleotide integration vectors and other nucleic acid molecules, into a suitable host cell.

The term "host cell" means, for example, a microorganism, a yeast cell, an insect cell, and a mammalian cell that can or has been used to receive an AAV helper construct, an AAV vector plasmid, an accessory function vector, or other transport DNA. The term includes progeny of the transfected original cells. Thus, as used herein, "host cell" generally refers to a cell that has been transfected with a foreign DNA sequence. It will be appreciated that due to natural, accidental or purposeful mutations, the progeny of a single parental cell need not be identical to the original parent in morphological or genetic or total DNA complementation.

The term "cell line" as used herein refers to a population of cells capable of continuing or prolonging growth and division in vitro. Typically, a cell line is a population of asexual (breeding) lines derived from a single progenitor cell. It is well known in the art that karyotypes may undergo self-friendly or induced changes in the preservation or transport of the population of the asexual (breeding) line. Thus, a cell line derived cell means that it may not be exactly identical to a progenitor cell or culture, said cell line including said variant.

The term "heterologous" relates to nucleic acid sequences, such as coding sequences and control sequences, to sequences that are not normally joined together, and/or which are typically not of the particular cell. Thus, a "heterologous" region of a nucleic acid construct or vector is a nucleic acid segment that is in or associated with another nucleic acid molecule, and is naturally independent of the additional molecule. For example, a heterologous region of a nucleic acid construct can include a coding sequence and a non-native coding sequence flanking sequence. Another example of a heterologous coding sequence is that the coding sequence itself is a construct that is not found in nature (e.g., a synthetic sequence having a codon different from the native gene). Likewise, it is the object of the present invention to recognize that cells transformed with constructs that are not normally present in the cell are heterologous. Allelic variation or naturally occurring mutation events do not result in the heterologous DNA used herein.

A "coding sequence" or a sequence encoding a particular protein is a nucleic acid sequence which, when under the control of a suitable regulatory sequence, is transcribed in vitro or in vivo (in the case of DNA) and translated (in the case of mRNA) into a polypeptide. The boundaries of the coding sequence are determined by the 5' late (amino) start codon and the 3' end (carboxyl) stop codon. A coding sequence can include, but is not limited to, cDNA from prokaryotic or eukaryotic mRNA, genomic DNA sequences from prokaryotic or eukaryotic DNA, and even synthetic DNA sequences. The transcription termination sequence is usually located at the 3' end of the coding sequence. A "nucleic acid" sequence refers to a DNA or RNA sequence. The term includes sequences that combine any known DNA and RNA base analogs.

The term DNA "control sequence" refers to a promoter sequence, a polyadenylation signal, a transcription termination sequence, an upstream regulatory region, an origin of replication, an internal ribosome entry site, a collection of ("IRES"), enhancers, and the like, which Together, it provides for the replication, transcription and translation of the coding sequence in the recipient cell. Not all of these control sequences need to be present as long as the selected coding sequence is capable of replicating, transcription and translation in a suitable host cell.

The term "promoter" as used herein generally refers to a region of a nucleotide comprising a DNA regulatory sequence, wherein the regulatory sequence is a gene that is capable of binding to RNA polymerase and the downstream (3'-direction) coding sequence of the promoter. Derived. Transcription promoters include "inducible promoters" (polynucleotide sequence expression operably linked to a promoter is induced by an analyte, cofactor, regulatory protein, etc.) and a "constitutive promoter".

"Effectively connected" refers to an arrangement of elements in which the components described are configured to perform their usual functions. Thus, operably linking a control sequence to a coding sequence can affect the expression of the coding sequence. The control sequences need not be adjacent to the coding sequence as long as they control the expression of the coding sequence. Thus, for example, intervening non-translatable but transcribed sequences may be present between the promoter sequence and the coding sequence, and the promoter sequence may still be considered "operably linked" to the coding sequence.

By "isolated" when referring to a nucleotide sequence is meant that the molecule is present in the substantial absence of other biological macromolecules of the same type. Thus, "an isolated nucleic acid molecule encoding a particular polypeptide" refers to a nucleic acid molecule substantially free of other nucleic acid molecules that do not encode the polypeptide; however, the molecule may include other bases that do not deleteriously affect the essential characteristics of the component. Base or part. Where the corresponding position of a nucleotide sequence in a particular nucleic acid molecule is described throughout the application, for example, when a particular nucleotide sequence is described as being located "upstream", "downstream", "3" or "5" relative to another sequence "Or" encodes the position in the chain, which is common in the art.

A "functional homolog" or "functional equivalent" of a particular AAV polypeptide includes a molecule derived from a native polypeptide sequence, as well as a recombinantly produced or chemically synthesized polypeptide that functions in a manner similar to a reference AAV molecule to achieve the desired result. Thus, AAV Rep68 or Rep78 functional homologs include derivatives and analogs of those polypeptides - single or multiple amino acid additions, substitutions and/or deletions containing internal or amino or carboxy terminus thereof, as long as the intact activity is retained.

A specific adenoviral nucleotide region "functional homolog" or "functional equivalent" includes a similar region derived from a heterologous adenovirus serotype, a nucleotide region derived from another virus or cell, and a reference nucleus A nucleotide-like manner that functions in a similar manner to achieve the desired result of recombinantly produced or chemically synthesized polynucleotides. Thus, a functional homolog of the adenoviral VA RNA gene region or the adenoviral E2a gene region comprises derivatives and analogs of the gene region - including the addition, substitution and substitution of single or multiple nucleotide bases in the region / or deletion, as long as the homolog remains to provide its intrinsic support for the AAV virion-producing accessory function at a level that is detectable at the background.

"Strong delivery system" refers to any non-manual delivery of a drug. In the present invention, an example of a powerful delivery system (CED) of AAV can be realized by an infusion pump or an osmotic pump. Below is a more detailed description of the CED.

The term "central nervous system" or "CNS" encompasses all cells and tissues of the vertebrate brain and spinal cord. Thus, the term includes, but is not limited to, neuronal cells, glial cells, astrocytes, cerebrospinal fluid (CSF), interstitial spaces, bone, cartilage, and the like. Each area of the CNS is associated with different behaviors and/or functions. For example, the basal ganglia of the brain is associated with motor function, especially autonomic movement. The basal ganglia is composed of 6 pairs of nucleus composed of caudate nucleus, putamen, globus pallidus, nucleus accumbens, nucleus at the base of the thalamus, and substantia nigra. Although separated by the inner capsule, the caudate nucleus and the putamen share a combination of cellular structural, chemical, and physiological properties, commonly referred to as the striatum. Degenerative substantia nigra in patients with Parkinson's disease provides the primary dopaminergic input to the basal ganglia.

The terms "subject", "individual" or "patient" are used interchangeably herein to refer to a vertebrate, preferably a mammal. Mammals include, but are not limited to, murines, baboons, humans, livestock animals, sport animals, and pets. An "effective amount" is a dose sufficient to serve a beneficial or desired effect. An effective amount can be administered in one or more doses, applications, or dosages.

normal method

The basis of the present invention is the unexpected discovery that AAV vector-mediated MANF gene delivery reverses the progressive degradation of dopaminergic (DA) neurons in a delayed manner while preserving the pathway of functional nigrostriatal striatum. As specifically described in the Examples, an acceptable animal model of Parkinson's disease was injected into the diseased striatum by an AAV vector expressing HIS peptide-tagged MANF (AAV-MANFhis) or GFP (AAV-EGFP). HIS immunostaining demonstrated that MANFhis can be significantly expressed in the midbrain. In the AAV-MANFhis group, the density of tyrosine hydroxylase (TH)-positive DA fibers in the striatum and the number of TH-positive neurons in SN were significantly increased compared with the sham operation group. Compared with the sham operation group, the level of dopamine and its metabolites in the AAV-MANFhis group were significantly higher than those in the sham operation group. Consistent anatomical and biochemical changes were observed after AAV-MANFhis injection, with significant behavioral recovery. These data unexpectedly show that the MANF gene that is delayed in the delivery of AAV-based delivery systems is effective even after the onset of progressive degradation.

Accordingly, the present invention provides methods of treating neurological diseases after neurodegenerative diseases and other neurological damage have occurred. In a preferred embodiment, the nerve injury is a dopaminergic neuron due to Parkinson's disease or impaired or abnormal function. Thus, the invention may be used to deliver a polynucleotide encoding a MANF polypeptide to a patient suffering from any severe neurodegenerative disease including, but not limited to, Parkinson's disease (PD), amyotrophic (spinal) lateral sclerosis ( ALS), epilepsy, Alzheimer's disease, etc. Furthermore, the present invention can be used to deliver MANF to patients suffering from a neurological injury caused by a temporary or permanent blood flow to the cessation of a part of the nervous system, such as a stroke; or a nerve injury caused by exposure to a neurotoxin, such as cancer. The chemotherapeutic agent Taxus, cisplatin or vinblastine and the AIDS chemotherapeutic agent ddI or ddC, and nerve damage caused by chronic metabolic diseases such as diabetes or renal function diseases.

As explained above, MANF is a protein that can be identified or obtained by glial cells and has neurotrophic activity. In more detail, MANF is a dopaminergic neurotrophic protein characterized in that it increases the absorption of dopamine by embryonic precursors of substantia nigra dopaminergic neurons and, in addition, promotes the survival of parasympathetic and sympathetic nerve cells.

Mesencephalic astrocyte-derived neurotrophic factor (MANF) is a new type of conserved neurotrophic factor, and its structure and action form are different from traditional neurotrophic factors. MANF is an approximately 20 kDa protein consisting of 182 amino acids, including a signal peptide consisting of 24 amino acids. MANF has a unique three-dimensional structure, including an N-terminal saposin domain and a C-terminal helix-loop-helix structure. MANF is widely expressed in mammals, especially in the nervous system. MANF is relatively highly expressed in the cerebral cortex, hippocampus and cerebellar Purkinje cells. At the same time, MANF, as a secreted protein, not only has the function of protecting and promoting the repair of dopaminergic neurons, but also has protective effects on other cell types including islet β cells, cardiomyocytes and retinal ganglion cells.

The human MANF polynucleotide and amino acid sequences are known as SEQ ID NO: 1 and SEQ ID NO: 2.

The efficacy of AAV-delivering MANF polynucleotides can be tested using any animal model of the above mentioned diseases known in the art. For example, the most widely used animal model of Parkinson's disease typically replicates neurodegenerative lesions of dopaminergic neurons by administering toxins. Unilateral injection of 6-hydroxydopa (6-OHDA) into the striatum of mice or rats resulted in loss of neurons in the ipsilateral striatum and substantia nigra parsing, with little change in the contralateral hemisphere. Similarly, deoxyephedrine-induced neurotoxins cause neurodegenerative lesions of dopaminergic neurons and serotonergic neurons, which are considered by those skilled in the art to be closely linked to human disease. The efficacy of the therapeutic drug can be evaluated by the behavioral results of the application of dehydrated morphine to induce rotational behavior. Another Parkinson's disease model was constructed using the neurotoxin N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). MPTP is administered to mammals such as mice, rats and monkeys. The result of giving MPTP to monkeys is not only the loss of dopaminergic neurons and serotonergic neurons in the dense parts of the substantia nigra and striatum, but also behaves similarly to patients with Parkinson's disease, such as exercise and posture stiffness. . See, for example, U.S. Patent No. 6,362,319.

In comparison to the above animal model of Parkinson's disease, the acquisition of many inbred strains of mice demonstrated a gradual decline in the number of dopaminergic cells. For example, D2 receptor deficient mice can be produced by homologous recombination with behavioral characteristics similar to those of patients with Parkinson's disease.

An animal model of other neurodegenerative diseases that can be used to evaluate the therapeutic efficacy of AAV-delivered MANF polynucleotides in the treatment of neurodegenerative diseases other than PD.

Recombinant AAV virions comprising the MANF coding sequence can be produced using art-recognized techniques well described below. Wild-type AAV and helper viruses can be used to provide the requisite replication function to produce rAAV virions. Alternatively, a plasmid containing a helper function gene can be used as a source of replication function in conjunction with a well-known helper virus infection. Likewise, a plasmid containing an accessory function gene can be used in conjunction with wild-type AAV infection to provide the requisite replication function. When used in combination with rAAV virions, all three methods are sufficient to produce rAAV virions. Other methods well known in the art can also be used by the skilled artisan to generate rAAV virions.

In a preferred embodiment of the invention, the triple transfection method is used to generate rAAV virions, since the method does not require the use of infectious helper viruses to initiate rAAV virion production in the absence of any detectable helper virus. This was achieved by the production of three vectors for the production of rAAV virions: AAV helper vector, AAV accessory function vector, and AAV expression vector. However, those skilled in the art recognize that the nucleic acid sequences encoded by these vectors can be provided in two or more vectors that are differently bound. As explained herein, the AAV helper vector encodes an "AAV helper" sequence (ie, rep and cap) whose trans-acting action is used for productive AAV replication and coats. Preferably, the AAV helper vector supports efficient AAV vector production without producing any detectable wtAAV virions (i.e., AAV virions including functional rep and cap genes). The rep and cap genes of the AAV helper function can be derived from any known AAV serotype, as explained above. For example, an AAV helper vector can have a rep gene derived from AAV-2 and a cap gene derived from AAV-6; those skilled in the art will recognize that there may be other combinations of rep and cap genes, the defining feature being capable of supporting rAAV Virosomes are produced.

The accessory function vector encodes a nucleotide sequence for non-AAV-derived virus and/or cellular function (i.e., "adjunct function") to which AAV is based. Affiliated functions include functions required for AAV replication including, but not limited to, portions associated with transcriptional activation of the AAV gene, time-specific AAV mRNA splicing, AAV DNA replication, synthesis of cap expression products, and assembly of AAV capsids. The virus-based accessory function can be derived from any well-known helper virus such as adenovirus, herpes virus (except herpes simplex virus type 1) and vaccinia virus. In a preferred embodiment, the accessory functional plasmid pLadeno5 is used. This plasmid provides a full set of adenoviral accessory functions for AAV vector production, but lacks the necessary components to form a replicating adenovirus.

To further understand the present invention, a more detailed discussion of recombinant AAV expression vectors, AAV helper and accessory functions, compositions comprising AAV virions, and delivery of virions is provided below.

Recombinant AAV expression vector

Recombinant AAV (rAAV) expression vectors are constructed using known techniques to provide at least operably linked components in the transcriptional direction, control elements (including transcription initiation regions), MANF polynucleotides of interest, and transcription termination regions. Control element selection is functional in mammalian muscle cells. The final construct containing the operably linked components is bordered by a functional AAV ITR sequence (5' and 3').

The nucleotide sequence of the AAV ITR region is known. . The AAV ITR used in the vectors of the present invention does not require a wild-type nucleotide sequence and can be altered, for example, insertions, deletions or substitutions of nucleotides. In addition, AAV ITR can be derived from any of a variety of AAV serotypes including, but not limited to, AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, and AAV. -9 and so on. Furthermore, the AAV expression vector flanking the 5' and 3' ITRs of the selected nucleotide sequence need not be identical or derived from the same AAV serotype isolate, as long as their function is desired, ie, allowed to be excised by the host cell genome or vector And salvaging in the sequence of interest, and allowing the integration of DNA molecules into the recipient cell genome when the AAV Rep gene product is present in the cell.

A suitable MANF polynucleotide molecule for use in an AAV vector is 546 bp in size. The selected polynucleotide sequence is operably linked to a control element that directs transcription or expression thereof in the patient. The control element can include a control sequence that is typically linked to a selection gene. Alternatively, a heterologous control sequence can be used. Useful heterologous control sequences typically include those derived from the coding sequence of a mammalian or viral gene. Examples include, but are not limited to, a neuron-specific enolase promoter, a GFAP promoter, an SV40 early promoter, a mouse mammary tumor virus LTR promoter, an adenovirus major late promoter (from Ad MLP); herpes simplex Virus (HSV) promoter, cytomegalovirus (CMV) promoter such as CMV immediate early promoter region (CMVIE), Rous sarcoma virus (RSV) promoter, synthetic promoter, hybrid promoter, and the like. In addition, sequences derived from non-viral genes, such as the metallothionein gene of the murine family, are also used herein. The promoter sequence is commercially available, for example, from Stratagene (San Diego, CA).

An AAV expression vector having a MANF polynucleotide molecule of interest bounded by AAV ITR can be inserted directly into the AAV genome from which the AAV major open reading frame (ORF) is cleaved. Other parts of the AAV genome can also be deleted as long as a sufficient portion of the ITR is retained to allow for replication and packaging functions. The constructs can be designed using techniques well known in the art.

Alternatively, the AAV ITR can be obtained by cleavage of the viral genome or by an AAV vector comprising the same, and the AAV vector comprises the same and 5' and 3 of the selected nucleic acid construct present in another vector using standard ligation techniques. 'End fusions, such as those described in Sambrook et al., supra. For example, 20 mM Tris-Cl pH 7.5, 10 mM MgCl ₂ , 10 mM DDT, 33 μg/ml BSA, 10 mM-50 mM NaCl, and 40 μM ATP, 0.01-0.02 (Weiss) unit T4 DNA ligase at 0 ° C (for "Adhesive ends" ligated) or 1 μM ATP, 0.3-0.6 (Weiss) unit T4 DNA ligase was ligated at 14 ° C (for "blunt" linkage). Intermolecular "sticky end" ligation is typically carried out at a total DNA concentration of 30-100 [mu]g/ml (5-10 nM total final concentration). Several AAV vectors are specifically described therein, which are available from the American Type Culture Collection ("ATCC") under accession numbers 53222, 53223, 53224, 53225, and 53226.

In addition, chimeric genes can be produced synthetically, including arranging AAV ITR sequences at the 5' and 3' ends of one or more selected nucleic acid sequences. Preferred codons for expression of chimeric gene sequences in mammalian muscle cells can be used. The fully chimeric sequence is assembled from overlapping oligonucleotides prepared by standard methods.

For the purposes of the present invention, suitable host cells for the production of rAAV virions from AAV expression vectors include microorganisms, yeast cells, insect cells and mammalian cells, which may or may have been used as receptors for heterologous DNA molecules. The term includes progeny of the original cells that have been transfected. Thus, as used herein, "host cell" generally refers to a cell that has been transfected with a foreign DNA sequence. The HEK293 cell of a stable human cell line (available, for example, by the American Type Culture Collection, ATCC CRL 1573) is preferred in the practice of the present invention. In detail, the human cell line HEK293 is a human embryonic kidney cell line transformed with an adenovirus type 5 DNA fragment, and expresses the adenovirus E1a and E1b genes. The HEK293 cell line is readily transfected and provides a particularly convenient platform for the production of rAAV virions.

AAV accessibility

Host cells comprising the above AAV expression vectors must be capable of providing AAV helper functions in order to replicate and coat the nucleotide sequence flanking the AAV ITR to produce rAAV virions. The AAV helper function is usually an AAV-derived coding sequence that can express the AAV gene product, and the AAV gene product is trans-acting for productive AAV replication. AAV helper functions are used herein to complement the necessary AAV function lost by the AAV expression vector. Thus, AAV helper functions include one or two AAV major ORFs, termed Rep and cap coding regions, or functional homologs thereof. "AAV Rep coding region" refers to a region recognized in the art to encode the AAV genome of the replication proteins Rep78, Rep68, Rep52 and Rep40. These Rep expression products possess a variety of functions including recognition, binding and cleavage of the origin of AAV DNA replication, DNA helicase activity, and regulation of AAV (or other heterologous) promoter transcription. Rep expression products are commonly required for replication of the AAV genome.

"AAV cap coding region" refers to a region of the AAV genome encoding the capsid proteins VP1, VP2 and VP3 or functional homologs thereof, which are art-recognized. These Cap expression products provide packaging functions that are commonly required for packaging viral genomes. For a description of the AAV cap coding region, see, for example, Muzyczka, N. and Kotin, R.M. (ibid.).

The AAV helper function is introduced into the host cell by using the AAV helper construct before or simultaneously transfecting the host cell with the AAV expression vector. Thus, the AAV helper construct provides at least transient expression of the AAV Rep and/or cap genes to complement the AAV function necessary for loss of productive AAV infection. AAV helper constructs lack AAVITR and are not self-replicating and packaging.

These constructs may be in the form of plasmids, phage, transposons, cosmids, viruses or virions.

The AAV expression vector and the AAV helper construct can be constructed to contain one or more selectable markers. Suitable markers include genes that confer antibiotic resistance or sensitivity, impart color, fluorescence, or alter their antigenic properties when cells are cultured in a suitable selective medium using cells transfected with a nucleic acid construct comprising a selectable marker. A variety of selectable marker genes useful in the practice of the invention include the hygromycin B resistance gene (encoding aminoglycoside phosphotransferase (APH)), which can be screened by conferring anti-G418 resistance (obtained by Sigma) , St.Louis, Mo.). Those skilled in the art are aware of other suitable labels.

AAV accessory function

The host cell (or packaging cell) must also have the ability to provide a non-AAV-derived function or "adjunct function" to produce the rAAV virion. The accessory function is a function of a non-AAV-derived virus and/or cell, and AAV can be replicated depending on the function. Thus, accessory functions include at least those non-AAV proteins and RNA required for AAV replication, including those involved in AAV gene transcription activation, time-specific AAV mRNA splicing, AAV DNA replication, Cap expression product synthesis, and AAV capsid assembly. Virus-based accessory functions can be derived from any known helper virus. In particular, accessory functions can be introduced by methods known to those skilled in the art and subsequently expressed in host cells. Typically, accessory functions are provided by infecting host cells with an unrelated helper virus. Many suitable helper viruses are known, including adenoviruses; herpes viruses such as herpes simplex virus types 1 and 2; and vaccinia virus. Non-viral accessory functions are also used herein, such as those provided by synchronizing cells by applying any of the different known factors.

Alternatively, ancillary functions may be provided using the accessory function carrier defined above. See, for example, U.S. Patent No. 6,004,797 and International Publication No. WO 01/83797. Nucleic acid sequences that provide ancillary functions can be obtained from natural sources, such as from the genome of an adenovirus, or constructed using recombinant or synthetic methods known in the art. As explained above, it was demonstrated that the accessory helper function does not require complete complementation of the adenoviral gene. Specifically, adenoviral mutants without DNA replication and late gene synthesis capabilities have demonstrated AAV replication. Similarly, mutations in the E2B and E3 regions demonstrated support for AAV replication, indicating that the E2B and E3 regions may not be involved in providing accessory functions.

Particularly preferred accessory functional vectors include the adenoviral VA RNA coding region, the adenovirus E4ORF6 coding region, the adenovirus E2A 72kD coding region, the adenoviral E1A coding region, and the adenoviral E1B region lacking the entire E1B 55k coding region. The vector is described in International Publication No. WO 01/83797.

As a result of infecting a host cell with a helper virus or transfecting a host cell with an accessory function vector, the accessory function expresses a transactivation of the AAV helper construct to produce an AAV Rep or Cap protein. The Rep expression product removes recombinant DNA (including the DNA of interest) from the AAV expression vector. Rep proteins are also used to replicate the AAV genome. The expressed Cap protein assembles into a capsid and the recombinant AAV genome is packaged into the capsid. Therefore, productive AAV replication is followed and the DNA is packaged into rAAV virions.

After recombinant AAV replication, the rAAV virions can be purified from host cells using a variety of conventional purification methods, for example, column chromatography, CsCl gradients, and the like. For example, multiple column purification steps can be used, such as purification by an anion exchange column, an affinity column, and/or a cation exchange column. For example, the adenovirus can be inactivated by heating to about 60 ° C, such as 20 minutes or longer. Since AAV is very stable to heat and the adenovirus is heat labile, such treatment is only effective inactivation of the helper virus. The resulting rAAV virion comprising the nucleotide sequence of the desired MANF can then be utilized for gene delivery following this technique.

combination

The composition includes sufficient genetic material to produce a therapeutically effective amount of the desired MANF, i.e., an amount sufficient to reduce or ameliorate the symptoms of the disease or an amount sufficient to achieve the desired benefit. The composition also includes a pharmaceutically acceptable excipient. The excipients include any pharmaceutically acceptable substance that does not itself produce deleterious antibodies to an individual receiving the composition, and may be devoid of undue toxicity when used. Pharmaceutically acceptable excipients include, but are not limited to, sorbitol, any of the various TWEEN compounds, liquids such as water, saline, glycerol, and ethanol. Pharmaceutically acceptable salts may include, for example, mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like; organic acid salts such as acetates, propionates, malonates, benzoic acids Salt and so on. In addition, auxiliary substances such as wetting or emulsifying agents, pH buffers and the like may be present in the medium.

A particularly useful formulation includes recombinant AAV virions and one or more dihydroxy or polyhydric alcohols, and optionally a detergent such as sorbitan vinegar. It will be apparent to those skilled in the art from the teachings of this specification that an effective amount of viral vector that must be added can be determined empirically. Representative dosages are detailed below. It can be administered in one dose, continuously or intermittently during the course of treatment. Methods for determining the most effective mean and dose are well known to those skilled in the art and will vary with viral vectors, therapeutic compositions, target cells, and patients. Single or multiple administrations can be accomplished with dosage levels and modes selected by the treating physician.

It is understood that more than one transgene is expressed by delivery of a recombinant virion. Alternatively, vectors expressing one or more different transgenes, respectively, can also be delivered to the CNS as described herein. In addition, it is also contemplated to deliver the viral vector in combination with other suitable compositions and therapies by the methods of the invention. For example, recombinant AAV virions and other drugs such as AADC, dopamine precursor (eg, levodopa), dopamine synthesis inhibition can be co-administered to express MANF to the CNS (eg, to the striatum caudate nucleus or putamen) Agent (eg, carbidopa), dopamine metabolism inhibitor (eg, MaOB inhibitor), dopamine agonist or antagonist may be administered prior to or subsequent to or concurrently with a recombinant virion encoding MANF to treat Parkinson's disease . For example, a gene encoding AADC can be administered to the CNS along with a gene encoding MANF. Likewise, levodopa and optionally carbidopa can be administered systemically. Thus, it is apparent that the naturally lost dopamine in PD patients can be restored by AADC which converts levodopa to dopamine. The transgene, under the control of an inducible promoter, can be administered to certain systems such as ceramide, porphyrin, tetracycline or aufin in order to regulate expression of the transgene.

Delivery of AAV virions

Recombinant AAV virions can be introduced into CNS cells to treat pre-existing neuronal damage using in vivo or in vitro (also known as in vitro) transduction techniques. If transduced in vitro, the desired recipient cells can be removed from the patient, transduced with rAAV virions and reintroduced into the patient. Alternatively, homologous or xenogeneic cells can be used, and those cells do not produce an inappropriate immune response in the patient. In addition, neural progenitor cells can be transduced in vitro and subsequently delivered to the CNS.

Methods for the delivery and introduction of suitable transduced cells into a patient are described. For example, by mixing recombinant AAV virions with cells for transduction in a suitable medium, the cells can be transduced in vitro, and those cells containing the DNA of interest can be applied using conventional techniques such as Southern blotting and/or PCR, or applying detectable labels. To filter. The transduced cells can then be formulated into a pharmaceutical composition which, as described above, is introduced into the patient in one or more doses by a variety of techniques as described below.

For in vivo delivery, the rAAV virion can be formulated into a pharmaceutical composition and can be administered in one or more doses directly in a prescribed manner. A therapeutically effective amount can include from about 10 ⁶ to 10 ¹⁵ of rAAV virions, more preferably from 10 ⁷ to 10 ¹² , even more preferably from about 10 ⁸ to 10 ¹⁰ of rAAV virions (or viral genomes, also referred to as "vg"), Or any value within these ranges. Generally, from 0.01 to 1 ml of the composition, preferably from 0.01 to about 0.05 ml, preferably from about 0.05 to 0.3 ml, such as 0.08, 0.09, 0.1, 0.2, etc., can be delivered, and any integer composition within these ranges can be delivered.

In vitro transduced recombinant AAV virions or cells can be delivered directly to the CNS or brain, such as the ventricular zone, and by lines using needles, catheters, or related devices by applying neurosurgical techniques known in the art, such as stereotactic injections. The corpus (eg, the caudate or putamen of the striatum), the spinal cord and neuromuscular junctions, or the cerebellar lobule. Cerebellar injections are complex because the stereotactic coordinates are not exactly aligned with the injection site; there are animal and animal variations in the size of the cerebellar lobules and their full three-dimensional orientation. Thus, the cholera toxin subunit b (CTb) can be used to determine the precise location of the injection and to reveal a collection of neurons that can be transduced at the injection site. It can be injected into the molecular layer, the Purkinje cell layer, the granule cell layer and the white matter of the cerebellar living tree, but does not extend to the deep cerebellar nucleus.

One mode of administration to the CNS uses a powerful delivery system (CED). In this way, recombinant virions can be delivered to many cells in large areas of the brain. In addition, the delivered vector efficiently expresses the transgene in CNS cells (eg, neurons or glial cells). Any powerful delivery device can be adapted for delivery of viral vectors. In a preferred embodiment, the device is an osmotic pump or an infusion pump. Osmotic pumps and infusion pumps are commercially available from a variety of suppliers (eg Alzet, Hamilton, Alza, Inc., Palo Alto, California). Viral vectors are typically delivered by the CED device below. A catheter, cannula or other injection device is inserted into the CNS tissue of the selected patient. One of ordinary skill in the art can readily determine a conventional region of the CNS suitable as a target. For example, when AAV-MANF is delivered to treat PD, the striatum is a suitable region of the brain's target. Stereotactic maps and positioning devices are available, for example, from ASI Instruments. Warren, MI. Localization can also be accomplished using an anatomical map obtained from CT and/or MRI imaging of the patient's brain to aid in manipulating the injection to a selected target site. Moreover, because the methods described herein can be practiced such that the associated larger brain regions receive viral vectors, fewer infusion cannulas are required. Therefore, surgical complications are related to the number of penetrations, and the delivery system of this model has fewer side effects than conventional delivery techniques. See U.S. Patent No. 6,309,6,340 for a detailed description of CED delivery.

In the case of PD, it is passed to patients who demonstrate the presence of nigrostriatal dopamine (DA) denervation, such as moderate to severe denervation. Moderate to severe denervation occurs when a patient exhibits visible PD symptoms, and at least about 60%-70% to over 90% of existing neurons have been lost. Thus, "moderate" denervation refers to the loss of at least about 60% to 70% of neurons, while "severe" denervation refers to the loss of at least about 90% of neurons.

One measure of neuronal loss is the level of dopamine activity in the CNS. Thus, "moderate" denervation refers to the loss of at least about 60% to 70% of dopamine content in one or both striatum hemispheres, while the "severe" denervation reaction is at least one or both of the striatum hemispheres. Lost about 90%. To measure the amount of dopamine, the patient is labeled with a tracer. The detection of the label indicates the activity of dopamine. The labeled tracer is preferably observed in vivo in the brain of the whole animal, for example, a positron emission tomography (PET) scan or other CNS imaging technique. Suitable labels for the selection of tracers include any component detectable by spectroscopic, photochemical, immunochemical, electrical, optical or chemical means. Markers useful in the present invention include radiolabels (e.g., ¹⁸ F, ³ H, ¹²⁵ I, ³⁵ S, ³² P, etc.), enzymes, colorimetric labels, fluorescent staining, and the like. In a preferred embodiment, the markers ¹⁸ F and DOPA are used for quantification of dopamine activity. Thus, in this embodiment, the degree of neuronal loss with ^[18 p] - fluoro -DOPA measured as tracer. Another measure of dopamine activity was the labeling tracer 6-[ ¹⁸ F]-fluoro-Lm-tyrosine ( ¹⁸ F-FMT). FMT binds to cells that use dopamine. See, for example, U.S. Patent No. 6,309,634 for the measurement of dopaquinone content in vivo.

The regeneration of neurons and the treatment of this disease can also be monitored by measuring the level of dopamine as described above. Treatment of a disease as used herein refers to alleviating or eliminating the symptoms of the disease, as well as the regeneration of neurons. Therefore, neuronal regeneration can be measured by comparing the levels of dopamine before and after treatment. Alternatively, use the symptoms of the disease that can be seen as a measure of treatment.

Neural tissue analysis and biochemical analysis

Tissue can be obtained from treated patients and conventional procedures are used to treat degeneration, regeneration and differentiation of Pingyuan neurons. In the present invention, it can be used to evaluate, for example, cells of various striatum and substantia nigra (SN), for example, to detect coronal sections of striatum and SN. Measurements over time may indicate an increase in cell correction away from the site of administration of the vector.

The levels of dopamine and its metabolites HVA and DOPAC can be determined using the high performance liquid chromatography HPLC previously described (Shen, Y., Hum. Gene Ther. (2000) 11:1509-1519). Especially if the vector encodes a secretable MANF, CSF can also be collected and used to assess protein levels or enzymatic activity.

The patient's periodic rotational behavior can also be tested by intraperitoneal injection of apomorphine-HCl.

The recombinant adeno-associated virus vector provided by the present invention and the materials and reagents used in the application thereof are commercially available.

The present invention is further illustrated below in conjunction with the embodiments:

Example 1 Design and Construction of Recombinant AAV Vector

The present invention relates to the expression of an exogenous MANF gene in vivo. To distinguish between an exogenously introduced gene and an endogenous MANF, it is necessary to distinguish between the transgene expression of MANF and the endogenous MANF for evaluating gene delivery. In the present example, HIS was labeled to the recombinant AAV vector target gene MANF, and the recombinant AAV vector was constructed as follows (Fig. 1).

The AAV vector plasmid pAAV2-MANF-HIS was derived from p AAV-LacZ particles. Between the inverted terminal repeat region (ITR) of the AAV-2 genome, transcription is initiated with the human cytomegalovirus (CMV) promoter, and the human MANF carboxyl terminal gene sequence will be tagged with the HIS tag (SEQ ID No. 3). Insert into this plasmid. The AAV expression vector sequence was obtained as shown in SEQ ID No. 4.

The AAV helper vector comprises two major AAV open reading frames (ORFs), rep and cap, plasmid pAAV/Package encoding Rep and Cap expression products. AAV helper constructs are typically used to provide transient expression of the AAV rep and/or cap genes to complement the AAV function necessary for lost cell lytic AAV replication; however, the helper construct lacks AAV ITR and is not self-replicating and self-packaging.

The AAV accessory function plasmid pAAV/Help includes an adenovirus VA RNA coding region, an adenovirus E4ORF6 coding region, an adenovirus E2A 72kD coding region, an adenovirus E1A coding region, and an adenoviral E1B coding region lacking the entire E1B 55k region.

pAAV2-MANF-HIS was co-transfected into 293 cells with the helper plasmid and the accessory plasmid, and after 72 hours of transfection, the cells and supernatant were harvested. The cells were lysed by three freezing and thawing cycles, and the supernatant was combined with the original supernatant after centrifugation. The virus was purified by affinity chromatography using a column of cross-linked AAV-8 capsid antibody, and viral titer was evaluated by quantitative DNA dot blot hybridization and real-time PCR analysis.

SEQ ID No. 1. Human Manf sequence:

SEQ ID No. 2. Human Manf amino acid sequence:

SEQ ID No. 3: His tag sequence

Example 2 Expression of AAV-MANFhis in vitro

To detect the MANFhis fusion protein expressed in vitro, HEK293 and Hep G2 cells were transduced with AAV MANFhis or AAV-EGFP (5000 vector genome copy number/cell). 36 hours after transfection, the cells were homogenized, and the transcription level of MANF mRNA produced in the cells was detected by real-time PCR. An increase in the transcription level of MANF mRNA was detected in the lysates of HEK293 and Hep G2 cells transduced by AAV MANFhis, whereas no change in the transcription level of MANF mRNA was observed in the cells without infection (Fig. 3, Table 1).

Table 1 Figure 3 Raw data

Note: ** indicates that the difference between the experimental group and the control group is extremely significant (P<0.01).

Example 3 Expression of MANFHIS in the nigrostriatal striatum

Expression of the midbrain MANFHIS transgene was detected after 2, 4, 8 and 20 weeks of AAV-MANFHIS injection. The rats were anesthetized by intraperitoneal injection of 2% sodium pentobarbital and fixed on a stereotaxic instrument. Insert the tip of the double-sided ear stem into the external auditory canal and place the sides of the head in a horizontal position. After routine disinfection, the epidermis was cut and the periosteum was removed. The stereotactic map of the rat brain was determined by Paxinos et al., and the coordinates of the substanianigta parscompacta (SNc) were determined to be 5.5 mm after the anterior iliac crest and the right side of the sagittal suture. 2.2mm, subdural 8.2mm. The skull drill carefully drilled through the skull, and the micro syringe was placed vertically into the skull according to the coordinates, and the needle was slowly inserted to reach the predetermined position. Four doses of AAV-MANFHIS (1 μl, 3 μl, 1 μl, 3 μl, respectively) of 1×10 ⁹ , 3×10 ⁹ , 1× ^{10 10} , 3×10 ¹⁰ were injected into the right substantia nigra pars compact of SD rats using a microinjection pump. The injection speed was 1 μl/min, and the needle was slowly withdrawn after the needle was left for 10 minutes. After routinely suturing the wound, place it on a heating pad until the rat wakes up. Two weeks after AAV-MANFHIS injection, the rats were perfused, fixed, and brain-extracted. After dehydration by 20% and 30% sucrose, the whole brain was frozen and sliced to a thickness of 10 μm. The brain tissue sections were subjected to serial scribing, dried at room temperature, and then added to the immunostaining blocking solution, and blocked at room temperature for 1 h. The blocking solution was discarded and the primary antibody against the HIS tag was added and incubated overnight at 4 °C. The next day, the primary antibody was discarded and washed 3 times with PBS for 10 min each time. After adding the fluorescent secondary antibody, it was incubated in a constant temperature water bath at 37 ° C for 1 h. The secondary antibody was discarded and washed 3 times with PBS for 5 min each time. The sealing liquid containing DAPI was added dropwise, and after sealing, the laser confocal microscope was used for observation and photographing. The results showed that the target gene was expressed in the injected nigrostriatal striatum, and no significant expression was observed in the other brain regions (Fig. 4). No expression was observed in the contralateral uninjected nigrostriatal region.

Example 4 Restoration of dopaminergic neuronal activity in the nigral striatum of the midbrain

3×10 ¹⁰ titers of AAV-MANFHIS were injected into the right substantia nigra parsing portion of the rat as described in Example 3, and the amount of the injected liquid was 3 μl. The control group was injected with 3 μl of PBS in the right substantia nigra pars compacta. Two weeks later, 20 μg of 6-OHDA was injected into the right striatum using brain stereotactic injection technique. The coordinates were 0 mm after anterior iliac crest, 3.5 mm to the right of sagittal suture, and 5.0 mm under subdural. The injection speed was 1 μl/min, and the needle was slowly withdrawn after the needle was left for 10 minutes. After 3 weeks, the rats were perfused, fixed, brained, and sectioned. The brain tissue sections were air-dried at room temperature, and then added to the blocking solution. After blocking at room temperature for 1 hour, the blocking solution was discarded, and the primary antibody dilution containing the anti-TH antibody was added and incubated at 4 ° C overnight. The next day, the tissue was discarded and washed three times with PBS for 10 min each time. Fluorescence was added and incubated for 1 h in a 37 ° C constant temperature water bath. The tissue was discarded and washed 3 times with PBS for 5 min each time. The sealing liquid containing DAPI was added dropwise, and after sealing, the laser confocal microscope was used for observation and photographing. The TH positive cells in the bilateral substantia nigra were subjected to morphological observation and counting. The results showed that the number of TH positive cells on the 6-OHDA injection side was significantly lower than that on the contralateral side in the 6-OHDA injury group and the injected AAV empty group. The number of TH positive cells on the injection side of the AAV-MANFHIS group was higher than that of the 6-OHDA injury group. And the AAV no-load group increased significantly, and the number of TH-positive cells on the uninjured side was similar. (Figure 5, Table 2)

Table 2 Figure 5 Raw data

Note: ΔΔ indicates that the difference between 6-OHDA and Control group is extremely significant (P<0.01), and ** indicates that 6-OHDA+AAV-MANF is significantly different from 6-OHDA group (P<0.01).

Example 5 Restoration of Behavior

3×10 ¹⁰ titers of AAV-MANFHIS were injected into the right substantia nigra parsing portion of the rat as described in Example 3, and the amount of the injected liquid was 3 μl. The control group was injected with 3 μl of PBS in the right substantia nigra pars compacta. Two weeks later, 20 μg of 6-OHDA was injected into the right striatum using brain stereotactic injection technique. Three weeks after the injection, the time of the rat drop was recorded using a rotary bar instrument, and the behavioral test was performed on the rat. The results showed that the rod time of the model group was significantly lower than that of the control group, suggesting that the injection of 6-OHDA in the striatum area significantly reduced the exercise capacity of rats; the rod time of the AAV empty group was compared with the model group. No significant increase was observed, and exercise capacity was not significantly improved compared with the model group. The AAV-MANFHIS injection group showed a significant increase in rod time compared with the AAV empty group, suggesting that AAV-MANFHIS injection can effectively improve the behavior of rats. Features. (Figure 6, Table 3)

Table 3 Raw data of Figure 6

Note: ## indicates that the difference between 6-OHDA and Control group is extremely significant (P<0.01), and ** indicates that 6-OHDA+AAV-MANF is significantly different from 6-OHDA group (P<0.01).

Example 6 Tissue-specific expression of the target gene

To study whether the target gene has non-specific expression and spread, dopamine neurons, astrocytes and oligodendrocytes were counterstained by anti-TH, GFAP and MBP antibodies, respectively. 3×10 ¹⁰ titers of AAV-MANFHIS were injected into the right substantia nigra parsing part of the rat as described in Example 3, and then perfused, fixed, taken, and sectioned after 2 weeks. The brain tissue sections were air-dried and added to the blocking solution. After blocking for 1 h at room temperature, the blocking solution was discarded, and the primary antibody dilution containing anti-HIS, TH, GFAP and MBP antibodies was added and incubated overnight at 4 °C. The next day, the tissue was discarded and washed three times with PBS for 10 min each time. Fluorescent secondary antibody was added and incubated in a constant temperature water bath at 37 ° C for 1 h. After discarding the tissue fluid, it was washed 3 times with PBS for 10 min each time. The sealing liquid containing DAPI was added dropwise, and after sealing, the laser confocal microscope was used for observation and photographing. The results showed that there was intracellular colocalization between HIS and TH, but no colocalization with GFAP and MBP. It is suggested that MANFHIS is only expressed in dopaminergic neurons in the substantia nigra, and no expression is found in astrocytes and oligodendrocytes (Fig. 7).

Example 7 Tissue Distribution of Capsid Protein

In order to study whether the AAV capsid protein has a tissue distribution spread, the present invention utilizes the AAV8 capsid protein antibody to immunohistochemically stain the main tissues such as heart, spleen, lung and kidney. The results showed no significant difference between the injected virus group and the injected PBS group (Fig. 8).

The recombinant adeno-associated virus vector provided by the present invention and its application are described in detail above. The principles and embodiments of the present invention have been described with reference to specific examples, and the description of the above embodiments is only to assist in understanding the method of the present invention and its core idea. It should be noted that those skilled in the art can make various modifications and changes to the present invention without departing from the spirit and scope of the invention.

Claims (13)

1. A recombinant adeno-associated virus vector, characterized in that it comprises:

   I. having a nucleotide sequence encoding an astrocyte-derived neurotrophic factor polypeptide;

   II. A nucleotide sequence obtained by modifying, substituting, deleting or adding one or more bases of a nucleotide sequence as shown in I;

   III. A sequence having at least 80% homology with a nucleotide sequence as shown in I or a polypeptide sequence obtained after translation is a nucleotide sequence corresponding to an amino acid sequence of an astrocyte-derived neurotrophic factor;

   IV, a complement of a sequence as shown by I, II or III.
2. The recombinant adeno-associated virus vector according to claim 1, wherein the adeno-associated virus vector comprises: AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV. -7, AAV-8 or AAV9.
3. The recombinant adeno-associated virus vector according to claim 1 or 2, wherein the nucleotide sequence further comprises a secretion sequence located at the 5' position and conforming to the reading frame encoding the MANF polypeptide sequence.
4. The recombinant adeno-associated virus vector according to claim 3, wherein the secretion sequence is a viral promoter, and the viral promoter comprises MLP, CMV or RSVLTR.
5. The recombinant adeno-associated virus vector according to any one of claims 1 to 4, which comprises an expression vector comprising, in order from the 5' to the 3' end:

   V, upstream ITR, CMV promoter, MANF gene and downstream ITR; or

   VI, upstream ITR, CMV promoter, MANF gene, HIS sequence, polyA and downstream ITR.
6. The recombinant adeno-associated virus vector according to claim 5, further comprising an auxiliary function carrier and/or an accessory function carrier;

   The helper function carrier comprises trans providing a protein Rep and/or Cap having AAV replication and/or packaging function;

   The accessory function vector includes an adenovirus VA RNA coding region, an adenovirus E4ORF6 coding region, an adenovirus E2A72kD coding region, an adenovirus E1A coding region, an adenoviral E1B region lacking a complete E1B 55k coding region, or a helper virus Ad5. Element E1a, E1b, E2a or E4.
7. The recombinant adeno-associated virus vector according to any one of claims 1 to 6, wherein the nucleotide sequence has any one of nucleotide sequences represented by VII, VIII, IX or X:

   VII, having the nucleotide sequence shown in SEQ ID NO: 1;

   VIII. A nucleotide sequence having the nucleotide sequence of SEQ ID NO: 1 modified, substituted, deleted or added with one or more bases;

   IX, a sequence having at least 80% homology with the nucleotide sequence shown in SEQ ID NO: 1 or a polypeptide sequence obtained after translation is a nucleotide sequence corresponding to the amino acid sequence shown in SEQ ID NO: 2;

   X. The complement of the sequence as shown in VII, VIII or IX.
8. The recombinant adeno-associated virus vector according to any one of claims 5 to 7, wherein the expression vector has any one of nucleotide sequences represented by XI, XII, XIII or XIV:

   XI, having the nucleotide sequence shown in SEQ ID NO: 4;

   XII, a nucleotide sequence obtained by modifying, substituting, deleting or adding one or more bases of the nucleotide sequence shown by SEQ ID NO: 4;

   XIII, a sequence having at least 80% homology to the nucleotide sequence set forth in SEQ ID NO: 4;

   XIV, the complement of the sequence as shown by XI, XII or XIII.
9. Use of the recombinant adeno-associated virus vector according to any one of claims 1 to 8 for the preparation of a medicament for preventing and/or treating a neurodegenerative disease.
10. The use according to claim 9, wherein the neurodegenerative disease is Parkinson's disease.
11. The use according to claim 9 or 10, wherein the Parkinson's disease comprises mild nigrostriatal dopamine damage and/or moderate nigrostriatal dopamine damage.
12. The use according to any one of claims 9 to 11, wherein the recombinant adeno-associated virus vector specifically acts on neurons of an animal.
13. The use according to any one of claims 9 to 12, wherein the recombinant adeno-associated virus vector specifically acts on the striatum and/or substantia nigra of the animal.

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