WO2008130949A2 - Modulation de l'expression de nurr1 dans une cellule - Google Patents

Modulation de l'expression de nurr1 dans une cellule Download PDF

Info

Publication number
WO2008130949A2
WO2008130949A2 PCT/US2008/060392 US2008060392W WO2008130949A2 WO 2008130949 A2 WO2008130949 A2 WO 2008130949A2 US 2008060392 W US2008060392 W US 2008060392W WO 2008130949 A2 WO2008130949 A2 WO 2008130949A2
Authority
WO
WIPO (PCT)
Prior art keywords
nurrl
seq
nucleic acid
vector
expression
Prior art date
Application number
PCT/US2008/060392
Other languages
English (en)
Other versions
WO2008130949A3 (fr
Inventor
Corinna Burger
Maria Estela Andres-Coke
Jose Antonio Fuentealba-Evans
Original Assignee
University Of Florida Research Foundation, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by University Of Florida Research Foundation, Inc. filed Critical University Of Florida Research Foundation, Inc.
Publication of WO2008130949A2 publication Critical patent/WO2008130949A2/fr
Publication of WO2008130949A3 publication Critical patent/WO2008130949A3/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70567—Nuclear receptors, e.g. retinoic acid receptor [RAR], RXR, nuclear orphan receptors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1138—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against receptors or cell surface proteins
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A01K2217/054—Animals comprising random inserted nucleic acids (transgenic) inducing loss of function
    • A01K2217/058—Animals comprising random inserted nucleic acids (transgenic) inducing loss of function due to expression of inhibitory nucleic acid, e.g. siRNA, antisense
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A01K2267/035—Animal model for multifactorial diseases
    • A01K2267/0356—Animal model for processes and diseases of the central nervous system, e.g. stress, learning, schizophrenia, pain, epilepsy
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/11—Antisense
    • C12N2310/111—Antisense spanning the whole gene, or a large part of it
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/12—Type of nucleic acid catalytic nucleic acids, e.g. ribozymes
    • C12N2310/121—Hammerhead
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2799/00—Uses of viruses
    • C12N2799/02—Uses of viruses as vector
    • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
    • C12N2799/025—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from a parvovirus

Definitions

  • the invention relates generally to the fields of molecular biology, gene therapy, and medicine. More particularly, the invention relates to methods and compositions for modulating Nurrl expression in a cell. BACKGROUND
  • Nurrl is a transcription factor that belongs to the nuclear receptor superfamily. It has been demonstrated that Nurrl is essential for the development of ventral dopaminergic neurons. The role of Nurrl in adult animals is unknown, however, because Nurrl knock-out mice die after birth. Nurrl is suspected of playing a role in Parkinson's disease because a mutation in a familial form of Parkinson's disease was found in the Nurrl gene. Nurrl is also suspected of playing a role in learning and memory. Because to dopaminergic system is involved in drug addiction, it is possible that Nurrl plays a role in this disorder as well. A means for studying the role of Nurrl in the adult brain (e.g. maintenance of the adult dopaminergic phenotype), and in several neurological diseases, is thus highly desirable.
  • the invention relates to compositions and methods for modulating Nurrl expression in a cell, including a cell in a subject. Somatic knock-outs of Nurrl in the adult rat midbrain were performed using viral-delivered ribozymes, and overexpression of Nurrl in the adult rat brain was achieved using a recombinant Adeno- Associated Virus (AAV) vector encoding Nurrl.
  • AAV Adeno- Associated Virus
  • the invention features an isolated nucleic acid including a nucleotide sequence having at least 80% sequence identity with one of : SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3.
  • a vector having at least one isolated nucleic acid including a nucleotide sequence having at least 80% sequence identity with one of : SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO: 3.
  • the vector can be a recombinant AAV vector, e.g., a serotype 5 recombinant AAV vector.
  • the invention also features a recombinant AAV vector including a nucleotide sequence encoding Nurrl .
  • the invention in another aspect, relates to a method for modulating Nurrl expression in a eukaryotic cell.
  • the method includes the steps of: a) providing at least one isolated nucleic acid including a nucleotide sequence having at least 80% sequence identity with one of: SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3; and b) contacting the cell with the at least one isolated nucleic acid under conditions in which the nucleotide sequence having at least 80% sequence identity with one of: SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3 can be expressed.
  • Modulating Nurrl expression in a eukaryotic cell can include decreasing Nurrl expression in the eukaryotic cell.
  • the invention features a method of expressing Nurrl in the hippocampus of a subject.
  • the method includes the steps of: a) providing the subject; and cO administering to the subject at least one isolated nucleic acid that encodes Nurrl protein.
  • the at least one isolated nucleic acid can be within a vector, e.g., within a recombinant AAV vector.
  • the recombinant AAV vector can be serotype 5, for example.
  • Another method within the invention includes a step of administering to a cell a recombinant AAV virion including: (a) a nucleotide sequence that when expressed modulates Nurrl expression in the cell, the nucleotide sequence interposed between a first AAV inverted terminal repeat and second AAV inverted terminal repeat: and (b) at least one AAV serotype 5 capsid protein.
  • the nucleotide sequence can be one of: SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, and a nucleic acid that encodes Nurrl protein.
  • nucleic acid means a chain of two or more nucleotides such as RNA (ribonucleic acid) and DNA (deoxyribonucleic acid).
  • a “purified” nucleic acid molecule is one that has been substantially separated or isolated away from other nucleic acid sequences in a cell or organism in which the nucleic acid naturally occurs (e.g., 30, 40, 50, 60, 70, 80, 90, 95, 96, 97, 98, 99, 100% free of contaminants).
  • the term includes, e.g., a recombinant nucleic acid molecule incorporated into a vector, a plasmid, a virus, or a genome of a prokaryote or eukaryote.
  • a recombinant nucleic acid molecule incorporated into a vector, a plasmid, a virus, or a genome of a prokaryote or eukaryote.
  • “Nurrl gene” or “Nurrl nucleic acid” is meant a native Nurrl - encoding nucleic acid sequence, genomic sequences from which Nurrl cDNA can be transcribed, and/or allelic variants and homologues of the foregoing.
  • the terms encompass double-stranded DNA, single-stranded DNA, and RNA.
  • protein or “polypeptide” are used synonymously to mean any peptide-linked chain of amino acids, regardless of length or post-translational modification, e.g., glycosylation or phosphorylation.
  • Neuronal protein an expression product of a Nurrl gene: or a protein that shares at least 65% (but preferably 75, 80, 85, 90, 95, 96, 97, 98, or 99%) amino acid sequence identity with Nurrl (e.g., accession number NM 019328 in rat) and displays a functional activity of Nurrl .
  • a "functional activity" of a protein is any activity associated with the physiological function of the protein.
  • Nurrl can mean a native Nurrl -encoding nucleic acid sequence, genomic sequences from which Nurrl cDNA can be transcribed, and/or allelic variants and homologues of the foregoing, as well as an expression product of a Nurrl gene, or a protein that shares at least 65% (but preferably 75, 80, 85, 90, 95, 96, 97, 98, or 99%) amino acid sequence identity with Nurrl )e.g., accession number NM Ol 9328 in rat) and displays a functional activity of Nurrl.
  • nucleic acid molecule or polypeptide when referring to a nucleic acid molecule or polypeptide, term “native” refers to a naturally-occurring (e.g., a wild-type; "WT”) nucleic acid or polypeptide.
  • WT wild-type
  • hybridization or “hybridizing” is meant the pairing of complementary RNA and DNA strands, or the pairing of complementary DNA single strands.
  • sequence identity means the percentage of identical subunits at corresponding positions in two sequences when the two sequences are aligned to maximize subunit matching, i.e., taking into account gaps and insertions. Sequence identity can be measured using sequence analysis software (e.g., Sequence Analysis Software Package from Accelrys CGC, San Diego, CA).
  • vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • An example of a type of vector is an episome, i.e., a nucleic acid capable of extra-chromosomal replication.
  • Another example of a vector is a viral vector (e.g., recombinant AAV vector).
  • Vectors capable of directing the expression of genes to which they are operatively linked are referred to herein as "expression vectors.”
  • a first nucleic acid sequence is "operably" linked with a second nucleic acid sequence when the first nucleic acid sequence is place in a functional relationship with the second nucleic acid sequence.
  • a promoter is operable linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence.
  • operably linked nucleic acid sequences are contiguous and, where necessary to join two protein coding regions, in reading frame.
  • expression control sequence refers to a nucleic acid that regulates the replication, transcription and translation of a coding sequence in a recipient cell.
  • expression control sequences include promoter sequences, polyadenylation (pA) signals, introns, transcription termination sequences, enhancers, upstream regulatory domains, origins of replication, and internal ribosome entry sites ("IRES").
  • promoter is used herein to refer to a DNA regulatory sequence to which RNA polymerase binds, initiating transcription of a downstream (3' direction) coding sequence.
  • pseudotyped is meant a nucleic acid or genome derived from a first AAV serotype that is encapsidated or packaged by an AAV capsid containing at least one AAV capsid protein of a second serotype.
  • AAV inverted terminal repeats AAV terminal repeats, “ITRs” and “TRs” are meant those sequences required in cis for replication and packaging of the AAV virion including any fragments or derivatives of an ITR which retain activity of a full-length or WT ITR.
  • rAAV vector and “recombinant AAV vector” refer to a recombinant nucleic acid derived from an AAV serotype, including without limitation, AAVl, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, etc.
  • rAAV vectors can have one or more of the AAV WT genes deleted in Whole or in part, preferably the rep and/or cap genes, but retain functional flanking ITR sequences.
  • a “recombinant AAV virion” or “rAAV virion” is defined herein as an infectious, replication-defective virus composed of and AAV protein shell encapsulating a heterologous nucleotide sequence that is flanked on both sides by AAV ITRs.
  • FIG. 1 is a graph showing relative abundance of the Nurrl mRNA in four rats infected with the AAV5-Rzl844 virus. Rats were infected with the virus in the ipsilateral side, the contralateral side being the uninfected side.
  • FIG. 2 is a graph showing relative abundance of the Nurrl mRNA in eight rats infected with the AAV5-Rzl844 virus. Rats were infected with the virus in the ipsilateral side, the contralateral side being the uninfected side.
  • FIG. 3 shows AAVl, AAV2 and AAV5 transduction in the substantia nigra of adult rats.
  • Panels A, D, G show a montage of rostral to caudal coronal sections to illustrate the extent of expression of GFP in the substantia nigra with AAVl , AA V2 or AAV5.
  • Panels C-O high magnification of the transduced areas on B, G, L. J.
  • the dotted line indicates that the number of cells and the mean estimated volume counted for AAVl were done for the entire midbrain, whereas for AA V2 and AAV5, cells in the substantia nigra were counted. Number of cells transduced with each virus pseudotype estimated from stereological counts. The dots individual animals.
  • AAVl shows a higher transduction rate through the entire midbrain area (A, B, P, Q).
  • AA V2 number of cells and volume
  • SN pars compacta of the substantia nigra
  • AAV5 transduces the entire SN (K, L, P, Q).
  • FIG. 4 is a photograph of an electrophoretic gel showing cleavage of Nurrl mRNA by ten ribozymes. Of these, three were found to cleave the in vitro transcribed full- length Nurl mRNA. The three ribozymes that cut are indicated on the top: Nurrl 1363 hh, Nurrl 1884 hh, andNurrl 1869hh. In the control lane the labeled IVR mRNA was incubated with no ribozymes under the same conditions. Arrow on the far left indicates the size of the full-length Nurrl mRNA. The arrows after the third lane show the size of the cleaved fragments. The larger cleaved fragments for 1844 and 1869 barely resolved in this gel (they can be appreciated just underneath the full length band). Only the smaller fragments of the mRNA are marked by arrows (arrows 1844 and 1869).
  • FIG. 5 is a graph showing the effects of Nurrl ribozymes in the transcriptional activity of endogenous Nurrl.
  • Transient transfection in PC 12 cells of the reporter 3x NBRE tk luciferase plus control expression vector (endo) or ribozyme (Rz) (1363, 1844 or 1869).
  • the vectors were transfected at a 1 :2 molar ratio of reporter DNA to ribozyme, as indicated.
  • Two of the ribozymes caused an increase in the transcriptional activation of the reporter gene. Only Rz 1844hh decreases Nurrl transactivation by almost half- fold.
  • the invention encompasses compositions and methods relating to the use of rAAV-based vectors encoding Nurrl and rAAV-based vectors encoding ribozymes against Nurrl for modulating Nurrl expression in a eukaryotic cell.
  • the below described preferred embodiments illustrate adaptations of these compositions and methods. Nonetheless, from the description of these embodiments, other aspects of the invention can be made and/or practiced based on the description provided below.
  • the invention provides nucleic acids for modulating Nurrl expression in a cell.
  • a typical nucleic acid as described herein for modulating Nurrl in a cell is an isolated nucleic acid including a nucleotide sequence having at least 80% sequence identity with one of : SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3.
  • the sequences of SEQ ID :1, SEQ ID NO:2, and SEQ ID NO:3 are ribozyme sequences that decreased or prevented Nurrl expression in rodents as described in the Examples section below.
  • Additional nucleic acids of the invention are variants of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3 that differ from SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3 in one or more bases and that substantially maintain a functional activity (e.g., decreasing or preventing Nurrl expression in a cell) of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3.
  • Nucleic acids for modulating Nurrl expression in a cell can be delivered to a cell or a subject by any suitable route, but are typically delivered by a viral vector (e.g., recombinant AAV vector). Modulating Nurrl Expression In A Cell
  • a typical method includes the steps of: a) providing at least one isolated nucleic acid including a nucleotide sequence having at least 80% sequence identity with one of: SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3 and b) contacting the cell with the at least one isolated nucleic acid under conditions in which the nucleotide sequence having at least 80% sequence identity with one of: SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3 can be expressed.
  • modulating Nurrl expression involves decreasing or preventing Nurrl expression in the eukaryotic cell, hi the Examples described herein, ribozyme molecules designed to catalytically cleave Nurrl mRNA transcripts (e.g. SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3) were used to decrease translation of Nurrl mRNA and expression of Nurrl protein (see, e.g. Lewin AS, and Hauswirth WW, Trends MoI Med 7:221-228, 2001; and Lewin et al, Nat. Med 6:967-971, 1998).
  • SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3 were used to decrease translation of Nurrl mRNA and expression of Nurrl protein (see, e.g. Lewin AS, and Hauswirth WW, Trends MoI Med 7:221-228, 2001; and Lewin et al, Nat. Med 6:967-971, 1998).
  • ribozymes that cleave mRNA at site-specific recognition sequences can be used to destroy Nurrl mRNAs
  • the use of hammerhead ribozymes may be preferred some embodiments.
  • Hammerhead ribozymes cleave mRNAs at locations dictated flanking regions that form complementary base pairs with the target mRNA.
  • the construction and production of hammerhead ribozymes is well known in the art and are described more fully in Vazquez- Tello et al., Nucleic Acid Res. 30(7): 1606-1612, 2002; Canny et al., J. Am Chem Soc.
  • Ribozymes as described herein can be delivered to a cell by any suitable means, such as a vector (e.g., recombinant AAV vector).
  • a vector e.g., recombinant AAV vector.
  • Methods of increasing Nurrl expression in a cell are also encompassed by the invention. Such methods typically include delivering to the cell a nucleic acid that encodes Nurrl protein.
  • a method of expressing Nurrl in the hippocampus of a subject includes the steps of providing the subject and administering to the subject at least one isolated nucleic acid that encodes Nurrl protein. rAAV Vectors And Virions
  • the nucleic acids of the invention may be incorporated into vectors and/or virions in order to facilitate their introduction into a cell.
  • rAAV vectors useful in the invention are recombinant nucleic acid constructs that include (1) a heterologous sequence to be expressed (e.g., Nurrl, ribozymes against Nurrl, etc.) and (2) viral sequences that facilitate expression of the heterologous sequence.
  • the viral sequences may include those sequences of AAV that are required in cis for replication and packaging (e.g., functional TRs) of the DNA into a virion, hi addition to containing a heterologous sequence (e.g., Nurrl .
  • rAAV vectors may also contain marker or reporter genes.
  • Useful rAAV vectors have one or more of the AAV WT genes deleted in whole or in par, but retain functional flanking TR sequences.
  • the AAV TRs may be of any serotype (e.g., derived from serotype 2, serotype 5, etc.) suitable for a particular application.
  • the nucleic acids and vectors of the invention may be incorporated into a rAAV virion in order to facilitate introduction of the nucleic acid or vector into a cell.
  • the capsid proteins of AAV compose the exterior, non-nucleic acid portion of the virion and are encoded by the AAV cap gene.
  • the cap gene encodes three viral coat proteins, VPl, VP2 and VP3, which are required for virion assembly.
  • the construction and use of rAAV virions has been described. See, e.g., U.S. Pat. Nos. 5,173,414, 5,139,941, 5,863,541, and 5,869,305, 6,057,152, 6,376,237; Rabinowitz et al., J. Virol.
  • rAAV virions useful in the invention include those derived from a number of AAV serotypes including 1, 2, 3, 4, 5, 6, and 7.
  • rAAV virions that have serotype 2 capsid proteins may be particularly useful as the experiments reported herein show they induce significantly higher cellular expression of ribozymes against Nurrl as well as higher cellular expression of nurrl than do rAAV virions having only serotype 2 capsids. Construction and use of AAV vectors and AAV proteins of different serotypes, including mosaic rAAV, are discussed in Chao et al., MoI. Ther.
  • Pseudotyped vectors of the invention include AAV vectors of a given serotype (e.g., AA V2) pseudotyped with a capsid gene derived from a serotype other than the given serotype (e.g., AAVl, AAV3, AAV4, AA V5, AAV6 or AAV 7 capsids).
  • AAV vectors of a given serotype e.g., AA V2
  • a capsid gene derived from a serotype other than the given serotype e.g., AAVl, AAV3, AAV4, AA V5, AAV6 or AAV 7 capsids.
  • An example of a pseudotyped vector that can be used in methods and compositions as described herein is an AA V2 vector encoding Nurrl or ribozymes against Nurrl pseudotyped with a capsid gene derived from AAV serotype 5.
  • AA V2 vector encoding Nurrl or ribozymes against nurrl is pseudotyped with a capsid gene derived from AAV serotype 9 or serotype 10.
  • the serotype of the vector and of the capsid protein(s) typically depends on the heterologous sequence to be delivered, as well as the type of cell or tissue into which the heterologous sequence is to be delivered. For example, when delivering a heterologous sequence to cells of the substantia nigra, AA V2 is preferred. As another example, when delivering a heterologous sequence to cells of the ventral tegmantal area, AAV5 is preferred.
  • AAV virions that have mutations within the virion capsid may be used to infect particular cell types more effectively than non-mutated capsid virions.
  • suitable AAV mutants may have ligand insertion mutations for the facilitation of targeting AAV to specific cell types.
  • the construction and characterization of AAV capsid mutants including insertion mutants, alanine screening mutants, and epitope tag mutants is described in Wu et al., J. Virol. 74:8635-45, 2000.
  • Other rAAV virions that can be used in methods of the invention include those capsid hybrids that are mosaic capsids, as well as those that are generated by molecular breeding of viruses as well as by exon shuffling.
  • compositions described above may be administered to subjects including mammals (e.g., rodents, human beings) in any suitable formulation by any suitable method.
  • mammals e.g., rodents, human beings
  • rAAV virions i.e., particles
  • target tissue e.g., substantia nigra
  • IV intravenous
  • IP intraperitoneal
  • a conventional syringe and needle can be used to inject an rAAV virion suspension into a subject.
  • injection can be in situ (i.e., to as particular tissue or location on a tissue), IM, IV, IP, or by another parenteral route.
  • Parenteral administration of virions by injection can be performed, for example, by bolus injection of continuous infusion.
  • Formulations for injection may be presented in unit dosage form, for example, in ampoules or in multi-dose containers, with an added preservative.
  • the compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • the rAAV virions may be in powder form (e.g., lyophilized) for constitution with a suitable vehicle, for example, sterile pyrogen-free water, before use.
  • the virions of the invention can be mixed with a carrier or excipient.
  • Carriers and excipients that might be used include saline (especially sterilized, pyrogen-free saline) saline buffers (for example, citrate buffer, phosphate buffer, acetate buffer, and bicarbonate buffer), amino acids, urea, alcohols, ascorbic acid, phospholipids, proteins (for example, serum albumin), EDTA, sodium chloride, liposomes, mannitol, sorbitol, glycerol, and mannitol (which increases the spread of viral transduction).
  • USP grade carriers and excipients are particularly preferred for delivery of virions to human subjects. Methods for making such formulations are well known and can be found in, for example, Remington's Pharmaceutical Sciences.
  • the virions can also be formulated as a depot preparation. Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by IM injection.
  • the virions may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives.
  • rAAV vectors may be administered to a subject using a variety of methods.
  • rAAV vectors may be directly introduced into a subject by peritoneal administration (e.g., IP injection, oral administration), as well as parenteral administration (e.g., IV injection, IM injection, and in situ injection into target tissue).
  • parenteral administration e.g., IV injection, IM injection, and in situ injection into target tissue.
  • Methods and formulations for parenteral administration described above for rAAV virions may be used to administer rAAV vectors.
  • Ex vivo delivery of cells transduced with rAAV vectors or virions is also provided _for within the invention.
  • Ex vivo gene delivery may be used to transplant rAAV- transduced host cells back into the host.
  • ex vivo stem cell e.g., mesenchymal stem cell
  • a suitable ex vivo protocol may include several steps.
  • a segment of target_tissue e.g., substantia nigra
  • rAAV virions may be used to transduce a Nurrl -encoding nucleic acid into the host's cells.
  • These genetically modified cells may then be transplanted back into the host.
  • Several approaches may be used for the reintroduction of cells into the host, including intravenous injection, intraperitoneal injection, or in situ injection into target tissue.
  • Microencapsulation of cells transduced or infected with rAAV modified ex vivo is another technique that may be used within the invention.
  • Autologous and allogeneic cell transplantation may be used according to the invention. Effective Doses
  • compositions described above are preferably administered to a subject (e.g., a mammal) in an effective amount, that is, and amount capable of producing a desirable result in a treated subject (e.g., decreasing or increasing Nurrl expression in a target tissue in the subject).
  • a therapeutically effective amount can be determined as described below.
  • Toxicity and therapeutic efficacy of the compositions utilized in methods of the invention can be determined by standard pharmaceutical procedures, using either cells in culture or experimental animals to determine the LD so (dose lethal to 50% of the population).
  • the dose ration between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LDso/EDso.
  • Those compositions that exhibit large therapeutic indices are preferred. While those that exhibit toxic side effects may be used, care should be taken to design a delivery system that minimizes the potential damage of such side effects.
  • the dosage of preferred compositions lies preferably within a range that includes an EDso with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.
  • dosage for any one subject depends on may factors, including the subject's size, body surface area, age, the particular composition to be administered, time and route of administration, general health, and other drugs being administered concurrently.
  • Example 1 - Somatic knockdown of Nurrl an approach to evaluate its function in the dopaminergic system in the adult rat brain.
  • Ribozymes against the mRNA of Nurrl were designed and delivered via rAAV, resulting in a somatic knock-out of Nurrl.
  • rAAV ribozymes against the mRNA of Nurrl
  • a decrease in Nurrl expression in the rats infected with rAAV expressing the ribozymes was detected.
  • the ribozymes described herein can be used to investigate the physiological role of Nurrl in the mesencephalic dopaminergic system, for example.
  • RNA is stored at -80 0 C.
  • Example 2 Knock-down of Nurrl expression in the normal adult substantia nigra to determine its role in the maintenance of the mature midbrain dopagminergic phenotype.
  • Example 3 Determination of the impact of Nurrl knock-down on the mature nigrostriatal dopagminergic system.
  • a full-length rat Nurrl gene was cloned into a vector containing AA V5 TRs and this vector was encapsidated into virions with AAV5 capsid proteins. This virus was expressed in adult rat brain.
  • a vector containing AA V2 TRs and Nurrl gene is constructed for encapsidation by capsid proteins of other AAV seroptypes (e.g., AAVl, AAV5, AA V9, AAVlO, or combinations thereof).
  • AAV seroptypes e.g., AAVl, AAV5, AA V9, AAVlO, or combinations thereof.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Zoology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • General Health & Medical Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Physics & Mathematics (AREA)
  • Biotechnology (AREA)
  • Cell Biology (AREA)
  • Immunology (AREA)
  • High Energy & Nuclear Physics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Toxicology (AREA)
  • Plant Pathology (AREA)
  • Medicinal Chemistry (AREA)
  • Microbiology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

La présente invention concerne des compositions et des procédés permettant de moduler l'expression de Nurr1 dans une cellule, y compris une cellule d'un sujet. Des inactivations somatiques de Nurr1 dans le mésencéphale de rats adultes ont été réalisées à l'aide de ribozymes délivrés par voie virale et la sur-expression de Nurr1 dans le cerveau de rats adultes a été obtenue à l'aide d'un virus adéno-associé (Adeno-Associated Virus : AAV) recombinant codant pour Nurr1.
PCT/US2008/060392 2007-04-16 2008-04-16 Modulation de l'expression de nurr1 dans une cellule WO2008130949A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US91200207P 2007-04-16 2007-04-16
US60/912,002 2007-04-16

Publications (2)

Publication Number Publication Date
WO2008130949A2 true WO2008130949A2 (fr) 2008-10-30
WO2008130949A3 WO2008130949A3 (fr) 2009-04-16

Family

ID=39876148

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2008/060392 WO2008130949A2 (fr) 2007-04-16 2008-04-16 Modulation de l'expression de nurr1 dans une cellule

Country Status (1)

Country Link
WO (1) WO2008130949A2 (fr)

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000066780A2 (fr) * 1999-04-30 2000-11-09 University Of Florida Ribozymes fournis par virus adeno-associes pour la decouverte de fonctions geniques
US20020049151A1 (en) * 2000-05-12 2002-04-25 Evelyn Murphy Therapeutic approaches to diseases by suppression of the NURR subfamily of nuclear transcription factors
US20040052764A1 (en) * 2000-04-28 2004-03-18 Markus Hildinger Recombinant aav vectors with aav5 capsids and aav5 vectors pseudotyped in heterologous capsids

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000066780A2 (fr) * 1999-04-30 2000-11-09 University Of Florida Ribozymes fournis par virus adeno-associes pour la decouverte de fonctions geniques
US20040052764A1 (en) * 2000-04-28 2004-03-18 Markus Hildinger Recombinant aav vectors with aav5 capsids and aav5 vectors pseudotyped in heterologous capsids
US20020049151A1 (en) * 2000-05-12 2002-04-25 Evelyn Murphy Therapeutic approaches to diseases by suppression of the NURR subfamily of nuclear transcription factors

Also Published As

Publication number Publication date
WO2008130949A3 (fr) 2009-04-16

Similar Documents

Publication Publication Date Title
Klein et al. Dose and promoter effects of adeno-associated viral vector for green fluorescent protein expression in the rat brain
US7238674B2 (en) Methods for delivering DNA to muscle cells using recombinant adeno-associated virus vectors
JP7504967B2 (ja) ＭｅＣＰ２発現カセット
KR102629418B1 (ko) 최적화된 cln1 유전자 및 발현 카세트 및 그의 용도
EP2121914B1 (fr) Systemes de fourniture d'acides nucleiques aux mitochondries
US20180042991A1 (en) Recombinant glut1 adeno-associated viral vector constructs and related methods for restoring glut1 expression
US20220177539A1 (en) Gene therapies for stargardt disease (abca4)
EP3737423B1 (fr) Compositions et méthodes de traitement de troubles de la rétine
US20230270884A1 (en) Compositions useful for treatment of charcot-marie-tooth disease
US20230250422A1 (en) Cpg-free itrs for aav gene therapy
KR20220140537A (ko) Cdkl5 결핍 장애를 치료하기 위한 유전자 요법
CN115029360A (zh) 用于治疗粘多糖贮积症iiia型的转基因表达盒
WO2022226183A2 (fr) Polynucléotides ap4m1 optimisés et cassettes d'expression, utilisations de ceux-ci
WO2022232575A1 (fr) Compositions comprenant des protéines de capside chimériques de virus adéno-associé et leurs méthodes d'utilisation
WO2022020697A1 (fr) Gènes slc13a5 optimisés, cassettes d'expression et leur utilisation
WO2021207077A1 (fr) Compositions de virus adéno-associés pour le transfert de gènes ids et leurs procédés d'utilisation
WO2008130949A2 (fr) Modulation de l'expression de nurr1 dans une cellule
EP3963073A1 (fr) Gènes sumf1 optimisés et cassettes d'expression et leur utilisation
WO2024079661A1 (fr) Thérapie génique atp7b
TW202434729A (zh) 含有星狀細胞導向啟動子構築體之合成核酸及其使用方法
BR102020023425A2 (pt) Vetor biológico para tratamento de doenças neurodegenerativas com base em terapia gênica
EP4405469A1 (fr) Compositions utiles pour le traitement de la maladie de charcot-marie-tooth
CN116670159A (zh) 组合物及其用于治疗安格尔曼综合征的用途
WO2024079667A1 (fr) Éléments régulateurs d'acide nucléique pour l'expression génique dans le système nerveux central et méthodes d'utilisation
JP2022506066A (ja) 最適化されたｆｉｇ４遺伝子および発現カセットならびにそれらの使用

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 08745900

Country of ref document: EP

Kind code of ref document: A2

NENP Non-entry into the national phase in:

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 08745900

Country of ref document: EP

Kind code of ref document: A2