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WO2016059166A1 - Rongeur contenant de la neuromélanine humanisée - Google Patents

Rongeur contenant de la neuromélanine humanisée Download PDF

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Publication number
WO2016059166A1
WO2016059166A1 PCT/EP2015/073909 EP2015073909W WO2016059166A1 WO 2016059166 A1 WO2016059166 A1 WO 2016059166A1 EP 2015073909 W EP2015073909 W EP 2015073909W WO 2016059166 A1 WO2016059166 A1 WO 2016059166A1
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rodent
gene
human tyrosinase
human
transgenic
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Inventor
Miquel Vila Bover
Iria CARBALLO CARBAJAL
Jordi BOVÉ BADELL
Thais CUADROS ARASA
Ariadna LAGUNA TUSET
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Institucio Catalana de Recerca i Estudis Avancats ICREA
Centro de Investigacion Biomedica en Red de Enfermedades Neurodegenerativas CIBERNED
Fundacio Institut de Recerca Hospital Universitari Vall dHebron
Original Assignee
Institucio Catalana de Recerca i Estudis Avancats ICREA
Centro de Investigacion Biomedica en Red de Enfermedades Neurodegenerativas CIBERNED
Fundacio Institut de Recerca Hospital Universitari Vall dHebron
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/0004—Screening or testing of compounds for diagnosis of disorders, assessment of conditions, e.g. renal clearance, gastric emptying, testing for diabetes, allergy, rheuma, pancreas functions
    • A61K49/0008—Screening agents using (non-human) animal models or transgenic animal models or chimeric hosts, e.g. Alzheimer disease animal model, transgenic model for heart failure
    • 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
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • A01K67/0278—Knock-in vertebrates, e.g. humanised vertebrates
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0055—Oxidoreductases (1.) acting on diphenols and related substances as donors (1.10)
    • C12N9/0057—Oxidoreductases (1.) acting on diphenols and related substances as donors (1.10) with oxygen as acceptor (1.10.3)
    • C12N9/0059—Catechol oxidase (1.10.3.1), i.e. tyrosinase
    • 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
    • A01K2267/0306—Animal model for genetic diseases
    • A01K2267/0318—Animal model for neurodegenerative disease, e.g. non- Alzheimer's
    • 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
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/001—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination
    • C12N2830/002—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination inducible enhancer/promoter combination, e.g. hypoxia, iron, transcription factor
    • C12N2830/003—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination inducible enhancer/promoter combination, e.g. hypoxia, iron, transcription factor tet inducible
    • 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
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/008—Vector systems having a special element relevant for transcription cell type or tissue specific enhancer/promoter combination

Definitions

  • the present invention provides a rodent that expresses the human tyrosinase gene in the brain, an enzyme which might be involved in the production of neuromelanin, and accumulates the latter pigment in dopaminergic neurons.
  • This rodent has potential applications in the pre-clinical research of
  • Parkinson's disease enabling: (i) the search of potential neuromelanin- related biomarkers for early detection, diagnosis and/or progression of PD, and (ii) the screening for treatments aimed at modulating neuromelanin accumulation to eventually slow down or halt the progression of PD.
  • Parkinson's disease is one of the main degenerative diseases of the central nervous system, second only to Alzheimer's. It is estimated to affect around 7 million people worldwide, and its prevalence is estimated to be around 0.3% in the industrialized nations. It is characterized by a progressive and sustained neurodegeneration in different brain regions, mostly of dopaminergic neurons in the substantia nigra pars compacta (SNPC), located in the midbrain. This neuronal death causes a variety of symptoms which become more severe with time, such as motor impairment, dementia and depression. PD is treated with Levodopa, dopamine agonists, anticholinergics and MAO inhibitors, among others. However, there is currently no cure for the disease. Therefore, new disease-modifying treatments are badly needed.
  • NM pigment neuromelanin
  • LC locus coeruleus
  • DMNV dorsal motor nucleus of the vagus
  • Aging is one of the main risk factors associated to the development of PD.
  • NM progressively accumulates with age since it cannot be degraded or eliminated by neurons.
  • NM to PD accumulation of NM to PD is not known mainly due to the fact that, unlike humans, common experimental animal species used in pre-clinical studies (mainly rodents) lack NM. In fact, the great abundance of NM in the SNPC seems to be characteristic of humans, as a macroscopic dark pigmentation of the midbrain is not observed in other species or NM is only present at moderate levels in some species of monkeys, horses or sheep.
  • a rodent that expresses the human tyrosinase gene in the brain not only accumulates NM in its neurons, but also reveals physiological changes at the cellular level that correspond to those seen in the neurons of PD patients, and what is even more remarkable, it additionally displays motor dysfunctions and neurodegeneration typical of PD.
  • the rodent of the invention does accumulate NM progressively with age as is the case of human subjects, that the accumulation takes place in the dopaminergic neurons (especially those of the SNPC) and further, that it is significant enough to surpass a certain pathologic threshold as it ultimately is linked with the appearance of neurodegeneration and motor dysfunction typically experienced by PD patients.
  • the rodent that is part of the invention can thus be used as a faithful preclinical model of PD, and be exploited as a screening tool for the search of potential neuromelanin-related biomarkers for the early detection, diagnosis and/or progression of PD, and for the testing of new therapies with disease- modifying potential that combat NM accumulation as a potential mechanism of action with which to treat PD.
  • a first aspect of the invention is a rodent that comprises the human tyrosinase gene, the gene being expressed in at least the dopaminergic neurons of the substantia nigra pars compacta.
  • a second aspect of the invention is a method of screening for therapeutic agents that prevent, delay or halt the development of neurodegenerative disease in the rodent of the first aspect of the invention, comprising: 1 ) exposing said rodent to at least one candidate therapeutic agent; and 2) assessing the effect of the at least one therapeutic agent on the progression of neurodegenerative disease in said rodent.
  • a third aspect of the invention is a method of screening for a biomarker for the early detection, diagnosis and/or prognosis of neurodegenerative disease in the rodent of the first aspect of the invention, comprising: 1 ) isolating from said rodent and a second reference rodent at least one sample; 2) determining the differential expression and/or level of a potential biomarker between said rodent and the second reference rodent, wherein if there is a difference in expression and/or level of the potential biomarker, then the potential biomarker is taken as a biomarker for the early detection, diagnosis and/or prognosis of neurodegenerative disease.
  • nucleotide sequence or gene construct comprising the human tyrosinase gene under the control of or operably linked to a promoter of a tyrosine hydroxylase gene.
  • vectors comprising the above mentioned nucleotide sequence, as well as host cells in the rodent comprising said vectors or said nucleotide sequence
  • nucleotide sequences comprising: (a) a sequence that comprises the human tyrosinase gene operably linked to an inducible promoter, and (b) a nucleotide sequence comprising a neuron specific expression promoter (in particular a rodent tyrosine hydroxylase promoter) linked to a sequence the expression of which promotes expression of the adjacent gene, in this case human tyrosinase (a) by interaction with the inducible promoter.
  • a neuron specific expression promoter in particular a rodent tyrosine hydroxylase promoter
  • All these gene constructs comprise sequences with the human tyrosinase gene sequence and also sequences promoting the expression of the human tyrosinase gene in neurons. By promoting it is to be understood that the expession is neuron specific or only achieved in some brain tissue types.
  • FIG.1 Human-like NM production in the SNpc of AAV-hTyr-injected rats, (a) Left panel, adult Sprague-Dawley rats received a single unilateral stereotaxic injection with 2 ⁇ of AAV2/1 -CMV-hTyr into the region immediately above the right SNpc. Right panel, by 4 weeks post-injection, 80% of ipsilateral DA SNpc neurons (TH, top right) were transduced with the tyrosinase gene (Tyr, middle right).
  • the image in the bottom right panel is a composite of the top and middle images
  • Ipsilateral SNpc pigmentation in AAV-hTyr-injected rats corresponds to the presence of intracellular NM within SNpcA/TA neurons, as shown microscopically in a 30 ⁇ -thick unstained section from the same brain.
  • FIG.2 PD-like neuropathological features in AAV-hTyr-injected rats.
  • ipsilateral rat SNpc bottom panels exhibit several neuropathological features typical of PD brains (top panels), including extracellular (E NM) and perivascular NM (P NM), neuronophagia (NP), Marinesco bodies (MB, arrows) and pale bodies (PB, LB precursors, arrowheads).
  • E NM extracellular
  • P NM perivascular NM
  • NP neuronophagia
  • MB Marinesco bodies
  • PB pale bodies
  • LB precursors arrowheads
  • FIG.3 SNpc dopaminergic neuronal dysfunction/degeneration in AAV-hTyr- injected rats.
  • the x-axis represents weeks after the AW-hTyr injection and the y-axis represents the Contralateral forepaw contacts - CFC (% of total contacts).
  • FIG. 1 Bottom panel - Number of SNpc Tyrosine Hydroxilase (TH)-positive neurons
  • the x-axis represents weeks after the AW-hTyr injection and the y-axis represents the number of SNpc TH-positive neurons (ipsilateral to the injected side) - SNpc TH+ .
  • A/ 6-8 animals per group. * p ⁇ 0.05, compared to control animals (basal); #p ⁇ 0.05, compared to 8 weeks and 12 months post-AAV- hTyr injection (one way ANOVA, Student-Newmann-Keuls post-hoc test).
  • FIG.4 NM accumulation in catecholaminergic brain structures of 12 month-old Tg-TH-hTyr mice.
  • Nissl-stained cryosections showing intracellular accumulation of NM (dark brown) in the cytoplasm of neurons from the substantia nigra pars compacta (SNpc), ventral tegmental area (VTA) and locus coeruleus (LC) regions.
  • SNpc substantia nigra pars compacta
  • VTA ventral tegmental area
  • LC locus coeruleus
  • the human tyrosinase gene is a copper-containing enzyme that catalyzes the production of melanin and other pigments from tyrosine oxydation. In humans, it is encoded by the TYR gene. Its protein product is found in the Uniprot database with the entry P14679 (last update on 1 1 .06.2014).
  • the sequence of the human tyrosinase gene used in the present invention is (SEQ ID NO 1 ):
  • the term “expresses” when referring to a gene is to be understood here as the process by which the information stored in the DNA of the gene is used for the synthesis of a functional gene product (usually a protein, but can also sometimes be an RNA).
  • a functional gene product usually a protein, but can also sometimes be an RNA.
  • the expression of the human tyrosinase gene is to be understood as the production of the human tyrosinase enzyme.
  • the term “progressively accumulates” as used herein refers to the
  • the rodent of young age features a certain amount of NM in its dopaminergic neurons
  • the adults feature higher amounts than the young rodents
  • the older animals are the ones featuring the highest amounts of NM, mimcking the progression of NM accumulation in humans, that is, the older the animal is, the higher the amount of intracellular NM.
  • transduction refers to the process whereby heterologous (foreign) DNA is introduced into a host cell via a viral vector.
  • the aim of this process is for the piece of DNA to be integrated in the genome of the host cell so that the protein or RNA that the foreign DNA codes for is stably expressed in the host cell.
  • the transduction of the human tyrosinase gene is carried out with the aim of having the host cell (and especially the neurons of the SNPC of a rodent) stably expressing the h-tyrosinase gene.
  • viral vector refers to a virus that is used as a delivery device to insert heterologous genetic material into a host cell
  • AAV adeno-associated virus
  • A2/1 -CMV-TYR is SEQ ID NO 2 (the coding sequence for the human tyrosinase gene is highlighted in bold):
  • a second sequence corresponds to a promoter
  • a second sequence which in the invention at hand can be the hTyr gene (the DNA coding for human tyrosinase) or the gene coding for the tetracycline transactivator (tTA).
  • the promoter sequence regulates transcription of the DNA corresponding to the second sequence.
  • operably linked means that the two nucleic acid sequences being linked are contiguous and, when necessary, in the same reading frame.
  • screening for therapeutic agents refers to the use of the rodent that forms part of the invention for testing organic molecules, peptides, proteins, DNA, antisense oligonucleotide, small interfering RNA, therapeutic vaccines or any cell-based or radiation-based therapy for the treatment of any pathology related to the accumulation of NM, and especially PD.
  • the expression "screening for a biomarker” relates to the use of the rodent that forms part of the invention for determining whether a potential biomarker between said rodent and a second reference rodent, is differentially expressed and/or has different levels. If this is the case, then the potential biomarker is taken as a biomarker for the early detection, diagnosis and/or prognosis of neurodegenerative disease.
  • the reference rodent can be a wild type rodent (that does not express the human tyrosinase gene) or a rodent of the invention which expresses human tyrosinase, but which is taken at a different developmental stage to the first rodent.
  • the comparison allows defining which biomarkers (metabolites, proteins, etc.) express differentially between both rodents, and can therefore be used in the detection, diagnosis and/or prognosis of neurodegenerative disease.
  • constitutive refers to a gene that is transcribed (and optionally also translated) continually and does not depend on the presence of an external stimulus in order to give its protein product.
  • inducible refers to a gene that is transcribed (and optionally also translated) only when there is a certain stimulus present in the environment. Thus, an inducible gene is not transcribed continually, as opposed to a “constitutive” gene.
  • the stimulus can be, for instance, the presence of an effector molecule.
  • Tet-Off refers to a gene control system where the controlled genes can be activated (expressed) in the absence of tetracycline or one of its analogues (such as doxycycline - dox).
  • tTA tetracycline transactivator
  • TetO Tet operators
  • Tet-Off systems several repeats of such TetO sequences are placed upstream of a promoter.
  • the coupling of several TetO sequences to the promoter is called a tetracycline response element (TRE), because it responds to binding of the tetracycline transactivator protein tTA by increased expression of the gene or genes downstream of its promoter.
  • TRE tetracycline response element
  • tTA refers to a tetracycline transactivator protein of a Tet-Off system, able to bind TetO sequences.
  • expression of TRE-controlled genes can be repressed by tetracycline and its derivatives, i.e., they bind tTA and render it incapable of binding to TRE sequences, thereby preventing transactivation of TRE-controlled genes.
  • therapeutic agent refers to any organic small molecule, peptide, protein, DNA, antisense oligonucleotide, small interfering RNA,vaccine, radiation or combinations thereof that could be used to cure, slow down the progression of, or alleviate a neurodegenerative disease. In this particular case, it would modulate the accumulation of NM in order to prevent the initiation of PD or to slow down or halt its progression.
  • transgenic rodent refers to a rodent whose genome has been altered by in vitro manipulation, with the aim of inserting or deleting certain genetic material with the aim of driving a phenotypic change.
  • the rodent genome is altered at the embryonic stage so that the animal expresses the human tyrosinase gene. This is typically achieved by pronuclear injection of an expression cassette into the male pronucleus of purified zygotes and reimplantation of the injected zygotes into pseudo- pregnant mouse females. The transgene randomly integrates into the mouse genome and its expression will therefore depend on the insertion position.
  • the DNA fragment for microinjection in mice to generate the constitutive transgenic mice is (SEQ ID NO 3):
  • a first aspect of the invention is a rodent that comprises the human tyrosinase gene, the gene being expressed in at least the dopaminergic neurons of the substantia nigra pars compacta.
  • the rodent progressively accumulates NM in said neurons along its lifetime.
  • the human tyrosinase gene is the human tyrosinase gene of SEQ ID NO 1 .
  • the human tyrosinase gene has been transduced with a viral vector.
  • the rodent is a rodent transduced with a viral vector comprising the human tyrosinase gene.
  • the former embodiment can be reformulated into a method of obtaining the rodent of the first aspect of the invention, comprising transducing the human tyrosinase gene via a viral vector which is injected into the brain of the rodent.
  • the viral vector is an adeno-associated viral vector.
  • the adeno-associated viral vector is the adeno-associated viral vector of SEQ ID NO 2.
  • the rodent is a transgenic rodent.
  • the expression of the human tyrosinase gene is
  • the human tyrosinase gene in the transgenic rodent, is operably linked to the promoter of a rodent tyrosine hydroxylase.
  • the transgenic rodent departs from an embryo that has been injected with the construct comprising SEQ ID NO 3.
  • the construct is the construct consisting of SEQ ID NO 3.
  • the former embodiment can be reformulated into a method of obtaining the rodent of the first aspect of the invention, by using the following protocol: a) designing a gene construct comprising SEQ ID NO 3; b) microinjecting the gene construct in the masculine pronucleus of a rodent fertilized ovus; c) culturing the obtained ovus; d) transplanting the latter into the oviduct of a female pseudopregnant rodent; c) selecting, among the offspring, those animals which contain the cDNA coding for the transgene.
  • the former embodiment can be reformulated into a method of obtaining the rodent of the first aspect of the invention, by using the following protocol: a) designing a gene construct consisting of SEQ ID NO 3; b) microinjecting the gene construct in the masculine pronucleus of a rodent fertilized ovus; c) culturing the obtained ovus; d) transplanting the latter into the oviduct of a female pseudopregnant rodent; c) selecting, among the offspring, those animals which contain the cDNA coding for the transgene.
  • the expression of the human tyrosinase gene is inducible.
  • the transgenic rodent is an inducible double transgenic rodent derived from mating a first parent rodent comprising the gene coding for the tTA transactivator operably linked to the promoter of a rodent tyrosine hydroxylase, and a second parent comprising the gene coding for the human tyrosinase operably linked to a promoter under the control of the target sequence for the tTA transactivator.
  • the former embodiment can be reformulated into a method of obtaining the rodent of the first aspect of the invention, by using the following protocol: a) generating a first parent rodent comprising the gene coding for the tTA transactivator operably linked to the promoter of a rodent tyrosine
  • the rodent is selected from the group consisting of rat and mouse.
  • a second aspect of the invention is a method of screening for therapeutic agents that prevent, delay or halt the development of neurodegenerative disease in the rodent of the first aspect of the invention, comprising: 1 ) exposing said rodent to at least one candidate therapeutic agent; and 2) assessing the effect of the at least one therapeutic agent on the progression of neurodegenerative disease in said rodent.
  • the neurodegenerative disease is associated with neuromelanin accumulation.
  • the neurodegenerative disease is associated with a-synuclein accumulation.
  • the neurodegenerative disease is PD.
  • the pcDNA4_TYR construct containing the coding sequence of the human tyrosinase gene (TYR, 2.082 bp) (Hasegawa et al. "Increased dopamine and its metabolites in SH-SY5Y neuroblastoma cells that express tyrosinase" J. Neurochem. 2003, vol. 87, pp. 470-475) was used as a parent construct to generate the adeno-associated viral (AAV) vector and the transgenic mice.
  • AAV adeno-associated viral
  • the human TYR was subcloned from the pcDNA4_TYR construct into an AAV of serotype 2/1 , commonly used to transfect distinct areas of the brain.
  • the AAV encodes an internal ribosome entry site (IRES) under the control of cytomegalovirus immediate-early (CMV-IE) promoter and contains a SV40 polyadenylation signal, flanked by two inverted terminal repeats (ITRs).
  • SEQ ID NO 4 were used as control (its sequence found below).
  • AAVs were generated and purified by the Viral Vector Production Unit (UPV) of the Center of Animal Biotechnology and Gene Therapy (CBATEG) at the Universitat Autonoma de Barcelona (Barcelona, Spain). Titers obtained were in the range of 1 .2x10 12 to 2.5x10 13 vector genomes per ml (vg/ml).
  • the 9 Kb upstream rat tyrosine hydroxylase (TH) promoter was cloned into the pcDNA4_TYR construct using the restriction sites Hindi 11 and EcoRI.
  • the TH_pcDNA4_TYR construct was then amplified in XL1 -Blue bacteria and isolated using the NucleoBond Extra Midi Kit (Macherey-Nagel). 10 ⁇ g of construct DNA were sequentially digested with the enzymes Hindi 11 and BciVI and run overnight in a 1 % agarose gel electrophoresis to isolate the 1 1 Kb DNA fragment to be injected.
  • This expression cassette a lineal DNA fragment containing the rat tyrosine hydroxylase (TH) promoter, the human TYR gene and a polyadenylation site, was purified by electroelution.
  • Control rats/mice were injected with an empty viral vector (AAV2/1 -CMV-null, 2,48E+13 vg/ml) or with vehicle (sham). At different times post-injection (2, 4, 8, 16 weeks and 6, 12, 18 y 24 months) animals were subjected to behavioral tests and processed for further characterization.
  • AAV2/1 -CMV-null 2,48E+13 vg/ml
  • vehicle sham
  • Transgenic embryos were generated by microinjecting the expression cassette into zygote pronuclei that were afterwards transferred into
  • Transgenic lines were generated using mice of a mixed genetic background (C57BL6/J and SJLF2), or rats of the Sprague Dawley strain following the same protocol. Founders were identified by Southern blotting of tail DNA using transgene specific probes. Each individual line was then backcrossed with mice or rats from the same strain and the expression level of the litters assessed by real time PCR or
  • the expression cassettes corresponding to the tTa and the tet-O elements are microinjected in the pronuclei of Sprague Dawley rats or hybrid mice to produce two different lines of transgenic animals: (i) one transactivator line expressing the activator (tTA) under the control of the tissue-specific TH promoter and (ii) a responder line containing the tetracycline responder element (tet-O) that controls the expression of the downstream human tyrosinase gene.
  • tTA activator
  • tet-O tetracycline responder element
  • the double-transgenic progeny express tyrosinase with a tissue specific pattern determined by the TH promoter (including dopaminergic neurons from the substantia nigra pars compacta and noradrenergic neurons from the locus coeruleus).
  • the system is activated resulting in the suppression of the transgene over-expression.
  • AAV-hTyr-injected rats corresponds to the presence of intracellular NM within SNpc/VTA neurons (FIG. 1 c).
  • intracellular NM reached levels similar to that of elder human brains, with NM occupying most of the cellular cytoplasm of SNpc neurons (FIG. 1 d).
  • pigmented SNpc from AAV-hTyr-injected rodents can be detected by NM-sensitive high-resolution T1 -weighted magnetic resonance imaging (FIG. 1 e). Ultrastructurally, NM from AAV-hTyr-injected rats is indistinguishable from human NM (FIG. 1f).
  • NM-containing SNpc neurons from AAV-hTyr-injected rats exhibit several neuropathological features typical of PD brains, such as extracellular and perivascular NM, neuronophagia, Marinesco bodies and pale bodies (FIG. 2)
  • AAV-Tyr-injected rats were subjected to motor behavioral tests, such as the cylinder test, and neuropathological analyses, by stereological cell counts of dopaminergic TH-positive SNpc neurons.
  • the cylinder test is designed to evaluate forelimb use asymmetry by quantifying the number of forelimb contacts against the wall within an open-top, clear plastic cylinder while rearing. The number of contacts with the forelimb ipsilateral or contralateral to the injection site is expressed as a percentage of total contacts.
  • the rodent of the present invention can be employed as a faithful model of the progression of PD in humans, and therefore as a promising in vivo screening tool for the search of novel biomarkers and therapies to control and combat the disease.

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  • Pathology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention concerne un rongeur humanisé faisant preuve d'une production et d'une accumulation intracellulaire de neuromélanine de type humain en fonction de l'âge, jusqu'à des niveaux atteints chez les êtres humains âgés, au sein de groupes neuronaux vulnérables vis-à-vis de la maladie de Parkinson. Le rongeur contenant de la neuromélanine humanisée exprime de façon stable le gène de la tyrosinase humaine dans au moins les neurones dopaminergiques du substantia nigra pars compacta et peut être un rongeur de type sauvage auquel le gène de la tyrosinase humaine a été transduit par l'intermédiaire d'un vecteur viral, ou un rongeur transgénique qui exprime le gène humain sous une forme constitutive ou inductible. Dans l'un quelconque de ces cas, le rongeur développe des caractéristiques de la maladie de Parkinson telles que la neurodégénérescence dopaminergique nigro-striée et un handicap moteur, ce qui fait de lui un outil de valeur pour (i) la recherche de biomarqueurs pour la détection précoce, le diagnostic ou l'évolution de la maladie, et (ii) le criblage de nouvelles thérapies avec potentiel modificateur de la maladie pour combattre la maladie de Parkinson.
PCT/EP2015/073909 2014-10-15 2015-10-15 Rongeur contenant de la neuromélanine humanisée Ceased WO2016059166A1 (fr)

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