EP2271763A2 - Verfahren zur expression eines polypeptids im gehirn und dazu fähige nukleinsäurekonstrukte - Google Patents

Verfahren zur expression eines polypeptids im gehirn und dazu fähige nukleinsäurekonstrukte

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Publication number
EP2271763A2
EP2271763A2 EP09728297A EP09728297A EP2271763A2 EP 2271763 A2 EP2271763 A2 EP 2271763A2 EP 09728297 A EP09728297 A EP 09728297A EP 09728297 A EP09728297 A EP 09728297A EP 2271763 A2 EP2271763 A2 EP 2271763A2
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EP
European Patent Office
Prior art keywords
nucleic acid
acid construct
polypeptide
brain
inducer
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EP09728297A
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English (en)
French (fr)
Inventor
Alon Chen
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Yeda Research and Development Co Ltd
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Yeda Research and Development Co Ltd
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Publication of EP2271763A2 publication Critical patent/EP2271763A2/de
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    • 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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • 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
    • A01K2207/00—Modified animals
    • A01K2207/05—Animals modified by non-integrating nucleic acids, e.g. antisense, RNAi, morpholino, episomal vector, for non-therapeutic purpose
    • 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
    • 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
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
    • C12N2740/15041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/15043—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
    • 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
    • C12N2830/001—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
    • 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
    • C12N2840/00—Vectors comprising a special translation-regulating system
    • C12N2840/20—Vectors comprising a special translation-regulating system translation of more than one cistron
    • C12N2840/203—Vectors comprising a special translation-regulating system translation of more than one cistron having an IRES

Definitions

  • the present invention in some embodiments thereof, relates to methods of expressing polypeptides in the brain and nucleic acid constructs for same.
  • Pharmacological administration of synthetic peptides or recombinant proteins into the brain ventricles is a common method employed for exploring physiological and behavioral functions of novel or known gene products.
  • the need for direct administration into the ventricles rises from the presence of the Blood-Brain-Barrier (BBB), which prevents a non-selective transport of peptides and proteins from the periphery into the CNS.
  • BBB Blood-Brain-Barrier
  • the Receptor Mediated Transport uses monoclonal peptide-mimetic antibodies (MAb's) to help large molecules to cross the BBB [Pardridge WM. Pharm Res 2007; 24(9): 1733-44]. These MAb's are either conjugated or fused to a peptide of interest and then use endogenous receptors to gain entry across the BBB. While this systems offers easy non-invasive administration (intravenous) it offers only a transient effect, it lacks specificity to the brain (i.e. it will act on all tissues that present the receptor) and may elicit an immune response. Spencer and Verma [Proc Natl Acad Sci U S A.
  • the Choroid plexus is a secretory epithelial tissue suspended at multiple loci in the cerebroventricular system. In addition to manufacturing the CSF, it performs a diversity of homeostatic functions to stabilize the interstitial environment of neurons. Kidney, liver, and immune-type functions have been ascribed to the CP.
  • the CP-CSF nexus furnishes micronutrients, growth factors, and neurotrophins to neuronal networks, making it a valuable target for the development of pharmacological agents that will distribute along similar CSF pathways to targets in the brain.
  • the choroidal epithelium has structural and functional properties that distinguish it from the cerebral endothelium of the BBB. Tight junctions which contain the protein occludin are "spot welds" at the apical zone of neighboring cells block the passage from blood to CSF. CP cells are highly vascularized with fenestrated capillaries that provide a perfusion rate 5-10 times that of the mean Cerebral Blood Flow (CBF). This fact coupled with a microvili structure of the apical lamina and abundant ion transporters and mitichondria enable the CP to play a role in CSF secretion and reabsorption.
  • CBF Cerebral Blood Flow
  • the CSF effectively distributes native and foreign compounds, thus substances presented ICV have a larger volume of distribution in CNS than those injected intrathecally above the brain and spinal cord.
  • the CP strongly and specifically (in the brain) expresses the ⁇ splice variant of the type 2 CRF receptor [Chen A., MoI Endocrinol 2005; 19: 441-458].
  • Other genes which are specifically expressed in the CP include GPRl 25 [Pickering et al. BMC Neurosci. 2008; 9: 97] and transthyretin [Costa et al., Molecular and Cellular Biology, Jan, 1988, p.81-90; Chen et al., The EMBO Journal, Vol.9, No. 3, p869-878, 1990].
  • nucleic acid construct comprising a regulatory sequence which regulates inducible expression of a polypeptide of interest, said regulatory sequence comprising a choroid plexus specific promoter, with the proviso that said choroid plexus specific promoter is not a transthyretin promoter.
  • the nucleic acid construct comprises a polynucleotide sequence encoding said polypeptide of interest.
  • the regulatory sequence comprises a tetracycline response element.
  • the nucleic acid construct further comprises an additional polynucleotide sequence encoding a transactivator positioned under a control of said choroid plexus specific promoter, said transactivator in combination with an inducer are for regulating transcription of said polypeptide of interest.
  • a nucleic acid construct system comprising a first nucleic acid construct comprising a first regulatory sequence and a second expression construct including a second polynucleotide sequence encoding a transactivator positioned under the transcriptional control of a choroid plexus specific promoter, wherein said transactivator activates said first regulatory sequence to direct transcription of a polypeptide of interest operatively linked to said first regulatory sequence, and wherein a transactivating activity of said transactivator is controlled by an inducer.
  • the nucleic acid construct system further comprises the polynucleotide sequence encoding the polypeptide of interest.
  • a pharmaceutical composition comprising a polynucleotide comprising a nucleic acid sequence encoding a therapeutic polypeptide operatively linked to a choroid plexus specific promoter.
  • the choroid plexus specific promoter is a ⁇ splice variant of the type 2 corticotrophin releasing factor receptor (CRFR2 ⁇ ) promoter or a G protein-coupled receptor 125 (GPRl 25) promoter.
  • the choroid plexus specific promoter is selected from the group consisting of a ⁇ splice variant of the type 2 corticotrophin releasing factor receptor (CRFR2 ⁇ ) promoter, a G protein-coupled receptor 125 (GPRl 25) promoter and a transthyretin promoter.
  • CRFR2 ⁇ corticotrophin releasing factor receptor
  • GPRl 25 G protein-coupled receptor 125
  • the pharmaceutical composition comprises a nucleic acid construct system comprising a first nucleic acid construct comprising a first regulatory sequence and a second expression construct including a second polynucleotide sequence encoding a transactivator positioned under the transcriptional control of a choroid plexus specific promoter, wherein said transactivator activates said first regulatory sequence to direct transcription of a polypeptide of interest operatively linked to said first regulatory sequence, and wherein a transactivating activity of said transactivator is controlled by an inducer.
  • the pharmaceutical composition comprises a nucleic acid construct comprising a regulatory sequence which regulates inducible expression of a polypeptide of interest, said regulatory sequence comprising a choroid plexus specific promoter, with the proviso that said choroid plexus specific promoter is not a transthyretin promoter.
  • the nucleic acid construct further comprises an additional polynucleotide sequence encoding a transactivator positioned under a control of said choroid plexus specific promoter, said transactivator in combination with an inducer are for regulating transcription of said polypeptide of interest.
  • the nucleic acid construct system or pharmaceutical composition further comprises a third nucleic acid construct including a third polynucleotide sequence encoding a polypeptide processing enzyme positioned under the transcriptional control of said first regulatory sequence.
  • the inducer upregulates said transactivating activity of said transactivator.
  • the inducer downregulates said transactivating activity of said transactivator.
  • the inducer is a tetracycline.
  • the inducer is capable of crossing a blood brain barrier.
  • the inducer is doxycycline.
  • a method of expressing a polypeptide of interest in a brain of a subject comprising administering to the subject a polynucleotide comprising a nucleic acid sequence encoding the polypeptide, said polynucleotide being operatively linked to a choroid plexus specific promoter, thereby delivering the polypeptide to the brain of the subject.
  • the polypeptide of interest is a therapeutic agent.
  • a kit for expressing a polynucleotide in a brain of a subject comprising the nucleic acid construct of the present invention or the nucleic acid construct system of the present invention and in a separate container said inducer.
  • a method of treating a brain disorder or disease in a subject in need thereof comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition of the present invention, thereby treating the brain disorder or disease in the subject.
  • the therapeutic agent is a polypeptide selected from the group consisting of an enzyme, a growth factor, and an antibody.
  • the therapeutic agent is a neuropeptide.
  • the neuropeptide is selected from the group consisting of Oxytocin, Vasopressin, Corticotropin releasing hormone
  • CSH Control hormone releasing hormone
  • LHRH Luteinizing hormone releasing hormone
  • Somatostatin growth hormone release inhibiting hormone Thyrotropin releasing hormone
  • TRH Neurokinin ⁇
  • Substance K Neurokinin ⁇
  • Neuropeptide K Neuropeptide K, Substance P, ⁇ -endorphin, Dynorphin, Met- and leu-enkephalin, Neuropeptide tyrosine (NPY), Pancreatic polypeptide, Peptide tyrosine-tyrosine (PYY), Glucogen-like peptide- 1 (GLP-I), Peptide histidine isoleucine (PHI), Pituitary adenylate cyclase activating peptide (PACAP), Vasoactive intestinal polypeptide (VIP), Brain natriuretic peptide, Calcitonin gene-related peptide (CGRP) ( ⁇ - and ⁇ -form), Cholecystokinin (CCK), Galanin, Islet amyloid polypeptide (IAPP), Melanin concentrating hormone (MCH), ACTH, ⁇ -MSH, Neuropeptide FF, Neurotensin, Parathyroid hormone related protein, Agouti gene-related protein (AGRP), Cocaine and amphe
  • a cell comprising the nucleic acid construct system of the present invention. According to an aspect of some embodiments of the present invention there is provided a cell comprising the nucleic acid construct of the present invention.
  • the polynucleotide is comprised in a nucleic acid construct system comprising a first nucleic acid construct comprising a first regulatory sequence and a second expression construct including a second polynucleotide sequence encoding a transactivator positioned under the transcriptional control of a choroid plexus specific promoter, wherein said transactivator activates said first regulatory sequence to direct transcription of a polypeptide of interest operatively linked to said first regulatory sequence, and wherein a transactivating activity of said transactivator is controlled by an inducer.
  • the polynucleotide is comprised in a nucleic acid construct comprising a regulatory sequence which regulates inducible expression of a polypeptide of interest, said regulatory sequence comprising a choroid plexus specific promoter, with the proviso that said choroid plexus specific promoter is not a transthyretin promoter.
  • the method further comprises administering an inducer to the subject.
  • the method further comprises administering a polynucleotide sequence encoding a polypeptide processing enzyme positioned under a transcriptional control of a regulatory sequence.
  • the polynucleotide is administered to a brain ventricle of the subject.
  • the polynucleotide is administered to a spinal cord of the subject.
  • the inducer is administered orally.
  • the inducer is doxycycline.
  • the brain disease or disorder is selected from the group consisting of brain tumor, neuropathy, Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, motor neuron disease, traumatic nerve injury, multiple sclerosis, acute disseminated encephalomyelitis, acute necrotizing hemorrhagic leukoencephalitis, dysmyelination disease, mitochondrial disease, migrainous disorder, bacterial infection, fungal infection, stroke, aging, dementia, schizophrenia, depression, manic depression, anxiety, panic disorder, social phobia, sleep disorder, attention deficit, conduct disorder, hyperactivity, personality disorder, drug abuse, infertility and head injury.
  • FIGs. IA-B are schematic representations of the lenti viral based system for inducible over-expression of peptides/proteins of interest in the cerebro-spinal-fluid
  • CSF cerebro-spinal-fluid
  • FIGs. IC-E are schematic representation of the constructs used in the system according to an embodiment of the present invention.
  • FIGs. 2A-I are photographs illustrating choroid plexus specific expression of CRFR2 ⁇ .
  • Figure 2A RT-PCR showing CRFR2 ⁇ expressed by CP but not by hypothalamus (HT).
  • Figures 2B-C Dark-field photomicrographs showing in-situ hybridization for CRFR2B at the choroid plexus.
  • Figures 2D-I In-vivo demonstration of GFP expression by infected CP cells. Brain slices of mice ICV injected with "effector" virus were DAPI stained ( Figures 2D, G), and GFP visualized ( Figures 2E, H). A merge of DAPI and GFP images show GFP expression at CP cells, but not at cells surrounding the ventricle ( Figures 2F, I). CP- choroid plexus, LV- lateral ventricle.
  • FIGs. 3A-F are photographs illustrating the RT-PCR results for various processing enzymes in the choroid plexus.
  • Furin - Figure 3A
  • CPE CPE
  • Figure 3D peptidyl-glycine-alpha-amidating-mono-oxygenase 1 (PAMl)-
  • FIG. 3G is a schematic diagram of an inducible lentiviral construct for expression of additional processing enzymes.
  • FIGs. 3H-K are photomicrographs of HEK-293T cells infected with the three different viruses showing expression of RFP (Figure 3H), GFP ( Figure 31), and BFP (Figue 3J), merged in Figure 3K, proving the feasibility of a triple co-infection.
  • FIGs. 4A-N are photomicrographs illustrating inducible over-expression of mCRF in the cerebro-spinal-fluid (CSF) using a choroid plexus (CP) specific lentiviral based system. Brain sections were stained for the presence of GFP (Cy2) and mCRF (Cy3) with ( Figures 4 A-G) or without ( Figures 4H-N) administration of the inducer doxycycline.
  • FIGs. 5A-D are graphs illustrating an increase in anxiety like behavior in
  • C57B/6 mice conditionally over-expressing mCRF in the choroid plexus measured by the dark/light transfer test with or without the inducer doxycycline (Dox). Values are expressed as mean ⁇ SEM ** p ⁇ 0.005, * p ⁇ 0.05.
  • FIGs. 6A-D are graphs illustrating an increase in anxiety like behavior in
  • C57B/6 mice conditionally over-expressing mCRF in the choroid plexus measured by open field test with or without the inducer doxycycline (Dox). Values are expressed as mean ⁇ SEM ** p ⁇ 0.005, * p ⁇ 0.05.
  • FIGs. 7A-B are graphs illustrating an increase in anxiety like behavior in
  • FIG. 7C is a graph illustrating no change in anxiety in C57B/6 mice conditionally over-expressing mCRF in the choroid plexus as measured by the home cage locomotion with or without the inducer doxycycline (Dox). Values are expressed as mean ⁇ SEM ** p ⁇ 0.005, * p ⁇ 0.05.
  • FIGs. 8A-N are photomicrographs illustrating inducible over-expression of GnRH in the cerebro-spinal-fluid (CSF) using a choroid plexus (CP) specific lentiviral based system. Brain sections were stained for the presence of GFP (Cy2) and mGnRH
  • FIG. 9A is a graph illustrating the percent of animals showing an intact estrous cycle at pre-induced, induced and post-induced conditions. Values are expressed as mean ⁇ SEM ** p ⁇ 0.0001.
  • FIG. 9B is a graph illustrating the estrous cycle stage of a representative mouse as determined by vaginal smears throughout the experiment. Values are expressed as mean ⁇ SEM ** p ⁇ 0.0001. DESCRIPTION QF SPECIFIC EMBODIMENTS QF THE INVENTION
  • the present invention in some embodiments thereof, relates to methods of expressing polypeptides in the brain and nucleic acid constructs for same.
  • BBB blood-brain-barrier
  • the Choroid plexus is a secretory epithelial tissue constantly secreting cerebrospinal fluid.
  • the current inventor(s) have conceived of using choroid plexus specific promoters in constructs to mediate transcription of secreted peptides for CNS delivery via the cerebrospinal fluid.
  • secreted polypeptides may reach extensive areas of the brain since the cerebrospinal fluid has a large volume of distribution.
  • this approach allows for the chronic expression of polypeptides, whilst alleviating the need for expensive and complex micro-injection machinery and micropumps.
  • the present inventors Whilst reducing the present invention to practice, the present inventors have generated lentiviral constructs comprising the type 2 CRF receptor (CRFR2 ⁇ ) promoter to mediate specific (and inducible) transcription of two secreted peptides in the choroid plexus for CNS delivery via the CSF: The corticotrophin releasing factor (CRF) and the gonadotrophin releasing hormone (GnRH), which are accountable for mediating the stress-induced behavioral changes and the intact estrous cycle, respectively.
  • CRF corticotrophin releasing factor
  • GnRH gonadotrophin releasing hormone
  • mice under induced conditions demonstrated a significant increase in anxiety and depression-like behaviors, which corresponds to the established effects of central CRF over-expression.
  • the present inventors injected GnRH over-expressing lenti viruses into female mice injected and detected an inducible and reversible disruption of their estrous cycle Figures 9A-B.
  • a method of expressing a polypeptide of interest in a brain of a subject comprises administering to the subject a polynucleotide comprising a nucleic acid sequence encoding the polypeptide, the polynucleotide being operatively linked to a choroid plexus specific promoter.
  • polypeptide refers to a polymer of any length made up of natural amino acids only. Accordingly, the term polypeptide includes short peptides and full length proteins.
  • the polypeptide may be useful for research, diagnostic or therapeutic purposes, as further described below.
  • the term "subject” refers to a mammal (e.g. human, mouse, rat, rabbit, bovine, porcine, ovine, canine and feline).
  • the phrase "choroid plexus specific promoter" refers to a polynucleotide sequence capable of directing expression of a polynucleotide sequence to which it is operably linked, to the cells of the choroid plexus and not to other cells (such as neuronal cells or ependymal cells).
  • choroid plexus specific promoters include, but are not limited to a ⁇ splice variant of the type 2 corticotropin releasing factor receptor (CRFR2 ⁇ ) promoter
  • GPR125 G protein-coupled receptor 125
  • transthyretin promoter e.g. as set forth in SEQ ID NO: 21
  • the choroid plexus specific promoter is not a transthyretin promoter.
  • the Choroid plexus is a secretory epithelial tissue responsible for manufacturing the cerebrospinal fluid (CSF) suspended at multiple loci in the cerebroventricular system.
  • the choroidal epithelium has structural and functional properties that distinguish it from the cerebral endothelium of the blood brain barrier.
  • CP cells are highly vascularized with fenestrated capillaries that provide a perfusion rate 5-10 times that of the mean Cerebral Blood Flow (CBF). This fact coupled with a microvili structure of the apical lamina and the presence of abundant ion transporters and mitichondria enables the CP to play a role in CSF secretion and reabsorption.
  • the choroid plexus specific promoter sequence is placed 3' to the polynucleotide sequence encoding the polypeptide of interest on a nucleic acid construct such that expression thereof is constitutive, but tissue specific.
  • the choroid plexus specific promoter sequence is situated relative to the polynucleotide sequence encoding the polypeptide of interest on a nucleic acid construct such that expression thereof is tissue specific , but also may be controlled in an exogenously regulatable fashion.
  • a nucleic acid construct may be designed such that it comprises a polynucleotide encoding a transactivator under control of the choroid plexus specific promoter.
  • the polynucleotide encoding the polypeptide of interest may be inserted in the same nucleic acid construct or in an additional construct under control of an inducible promoter.
  • the transactivator in combination with an inducer act to regulate expression from the inducible promoter.
  • Inducible promoters suitable for use with the present invention are preferably response elements capable for directing transcription of the polynucleotide sequence so as to confer regulatable synthesis of the polypeptide of interest.
  • a suitable response element can be, for example, a tetracycline response element (such as described by Gossen and Bujard (Proc. Natl. Acad. Sci. USA 89:5547-551, 1992); an ectysone- inducible response element (No D et al., Proc Natl Acad Sci U S A. 93:3346-3351, 1996) a metal-ion response element such as described by Mayo et al. (Cell. 29:99-108, 1982); Brinster et al.
  • the response element is an ectysone-inducible response element, more preferably the response element is a tetracycline response element.
  • An exemplary system that may be used according to this embodiment of the present invention is a Tet-on expression system.
  • the reverse tetracycline transactivator rtTA
  • TRE Tet Response Element
  • the chimeric reverse transactivator (under control of the CP-specific promoter on a first construct), activates (in the presence of the inducer) transcription of the polypeptide of interest from a silent, inducible promoter (Tet response element; TRE) present on the second construct.
  • Tet response element Tet response element
  • the chimeric transactivator under control of the CP-specific promoter activates transcription of the polypeptide of interest from the silent inducible promoter all on the same construct (see for example Chtarto et al., Gene Therapy (2003) 10, 84-94).
  • Another exemplary construct that may be used according to this embodiment of the present invention is a Tet-off expression system.
  • the tetracycline transactivator under control of the CP-specific promoter, activates (in the absence of the inducer), transcription of the polypeptide of interest. In the presence of the inducer, the tTA loses its ability to bind the TRE, and expression of the polypeptide of interests is shut off.
  • the regimen for administration of the inducer is selected according to the construct (for example, tet-on or tet-off) and the type of expression required for the polypeptide of interest (continuous, intermittent etc.).
  • the inducer is administered directly into the brain or through the spine. If the inducer is capable of crossing the blood brain barrier (e.g. doxycycline) it may be administered centrally (e.g. orally). Typically, the inducer is administered following administration of the constructs of the present invention.
  • the nucleic acid constructs of the present invention may also include one or more enhancers.
  • Enhancer elements can stimulate transcription up to 1,000 fold from linked homologous or heterologous promoters. Enhancers are active when placed downstream or upstream from the transcription initiation site. Many enhancer elements derived from viruses have a broad host range and are active in a variety of tissues. For example, the SV40 early gene enhancer is suitable for many cell types.
  • Other enhancer/promoter combinations that are suitable for the present invention include those derived from polyoma virus, human or murine cytomegalovirus (CMV), the long term repeat from various retroviruses such as murine leukemia virus, murine or Rous sarcoma virus and HIV. See, Enhancers and Eukaryotic Expression, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. 1983, which is incorporated herein by reference.
  • CMV cytomegalovirus
  • Polyadenylation sequences can also be added to the nucleic acid construct in order to increase the translation efficiency of the polypeptide of interest expressed from the expression construct of the present invention.
  • Two distinct sequence elements are required for accurate and efficient polyadenylation: GU or U rich sequences located downstream from the polyadenylation site and a highly conserved sequence of six nucleotides, AAUAAA, located 11-30 nucleotides upstream.
  • Termination and polyadenylation signals that are suitable for the present invention include those derived from SV40.
  • the nucleic acid construct of the present invention may typically contain other specialized elements intended to increase the level of expression of cloned polynucleotides or to facilitate the identification of cells that carry the recombinant DNA.
  • a number of animal viruses contain DNA sequences that promote the extra chromosomal replication of the viral genome in permissive cell types. Plasmids bearing these viral replicons are replicated episomally as long as the appropriate factors are provided by genes either carried on the plasmid or with the genome of the host cell.
  • the nucleic acid constructs may or may not include a eukaryotic replicon. If a eukaryotic replicon is present, then the vector is amplifiable in eukaryotic cells using the appropriate selectable marker. If the construct does not comprise a eukaryotic replicon, no episomal amplification is possible. Instead, the recombinant DNA integrates into the genome of the engineered cell, where the promoter directs expression of the desired polynucleotide.
  • the nucleic acid constructs of the present invention can further include additional polynucleotide sequences that allow, for example, the translation of several proteins from a single mRNA such as an internal ribosome entry site (IRES) and sequences for genomic integration of the promoter-chimeric polypeptide.
  • IRS internal ribosome entry site
  • a single expression construct can be designed and co-express two distinct polypeptides one the polypeptide of interest, and one a processing enzyme as further described herein below.
  • mammalian expression constructs include, but are not limited to, pcDNA3, pcDNA3.1 (+/-), pGL3, pZeoSV2(+/-), pSecTag2, pDisplay, pEF/myc/cyto, pCMV/myc/cyto, pCR3.1, pSinRep5, DH26S, DHBB, pNMTl, pNMT41, pNMT81, which are available from Invitrogen, pCI which is available from Promega, pMbac, pPbac, pBK-RSV and pBK-CMV which are available from Strategene, pTRES which is available from Clontech, and their derivatives.
  • SV40 vectors include pSVT7 and pMT2.
  • Vectors derived from bovine papilloma virus include pBV- IMTHA, and vectors derived from Epstein Bar virus include pHEBO, and p2O5.
  • exemplary vectors include pMSG, pAV009/A + , pMTO10/A + , pMAMneo-5, baculovirus pDSVE, and any other vector allowing expression of proteins under the direction of the SV-40 early promoter, SV-40 later promoter, metallothionein promoter, murine mammary tumor virus promoter, Rous sarcoma virus promoter, polyhedrin promoter, or other promoters shown effective for expression in eukaryotic cells.
  • Viruses are specialized infectious agents that have evolved, in many cases, to elude host defense mechanisms. Typically, viruses infect and propagate in specific cell types.
  • the targeting specificity of viral vectors utilizes its natural specificity to specifically target predetermined cell types and thereby introduce a recombinant gene into the infected cell.
  • Recombinant viral vectors are useful for in vivo expression of transgenic polynucleotides since they offer advantages such as lateral infection and targeting specificity.
  • Lateral infection is inherent in the life cycle of, for example, retrovirus and is the process by which a single infected cell produces many progeny virions that bud off and infect neighboring cells. The result is that a large area becomes rapidly infected, most of which was not initially infected by the original viral particles. This is in contrast to vertical-type of infection in which the infectious agent spreads only through daughter progeny.
  • Viral vectors can also be produced that are unable to spread laterally. This characteristic can be useful if the desired purpose is to introduce a specified gene into only a localized number of targeted cells.
  • the constructs of the present invention are incorporated into lentiviruses. These viruses are advantageous because of their ability to integrate their DNA into the genome of mammalian non-dividing cells in-vivo.
  • nucleic acid construct can be designed as a gene knock-in construct in which case it will lead to genomic integration of construct sequences, or it can be designed as an episomal expression vector.
  • the nucleic acid construct can be generated using standard ligation and restriction techniques, which are well known in the art (see Maniatis et al., in: Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York, 1982). Isolated plasmids, DNA sequences, or synthesized oligonucleotides are cleaved, tailored, and religated in the form desired.
  • the constructs of the present invention may be administered directly into the brain (via the ventricle) or via the spinal cord (e.g. by an epidural catheter).
  • CP cells express a battery of processing enzymes necessary for precursor cleavage and amidation ( Figures 3A-F), the present invention also contemplates expression of additional processing enzymes.
  • polypeptide processing enzyme refers to an enzyme capable of cleaving and/or modifying a polypeptide.
  • the polypeptide processing enzyme is a neuropeptide processing enzyme.
  • processing enzymes contemplated for expression by the present invention include, but are not limited to furin (EC 3.4.21.75), Prohormone convertase 1
  • PCl Prohormone convertase 2
  • CPE carboxypeptidase E
  • the method of the present invention is effected by administering a polynucleotide sequence encoding a polypeptide processing enzyme positioned under a transcriptional control of a regulatory sequence in conjunction with the construct(s) encoding the polypeptide of interest.
  • the polypeptide processing enzyme sequence may be present on the constructs described herein above or may be administered on a separate construct.
  • the regulatory sequence for the polypeptide processing enzyme may be a constitutive promoter or an inducible promoter such as the ones described herein above.
  • the regulatory sequence is the identical regulatory sequence that is linked to the polypeptide of interest (e.g. the TRE).
  • the tetracycline inducer controls expression of both the processing enzyme and the polypeptide of interest.
  • the polypeptide processing enzyme construct may be administered to the subject prior to, concomitant with or following administration of the construct which encodes the polypeptide of interest, but prior to administration of the inducer.
  • the constructs of the present invention are used to express polypeptides of interest in the brain.
  • Exemplary polypeptides contemplated by the present invention include neuropeptides, enzymes, structural polypeptides, growth factors and antibodies.
  • the constructs may be used to express secreted polypeptides in the cells of the CP. This would enable the polypeptides to be secreted into the cerebrospinal fluid and reach large areas of the brain.
  • neuropeptides includes peptide hormones, peptide growth factors and other peptides.
  • Examples of neuropeptides which can be used in accordance with the present invention include, but are not limited to Oxytocin, Vasopressin, Corticotropin releasing hormone (CRH), Growth hormone releasing hormone (GHRH), Luteinizing hormone releasing hormone (LHRH), Somatostatin growth hormone release inhibiting hormone, Thyrotropin releasing hormone (TRH), Neurokinin a (substance K), Neurokinin ⁇ , Neuropeptide K, Substance P, ⁇ -endorphin, Dynorphin, Met- and leu-enkephalin, Neuropeptide tyrosine (NPY), Pancreatic polypeptide, Peptide tyrosine-tyrosine (PYY), Glucogen-like peptide- 1 (GLP-I), Peptide histidine iso leucine (PHI), Pituitary adenylate cyclas
  • the polypeptide is one that is expressed in the choroid plexus (CP) - for example a structural protein or an enzyme.
  • the polypeptide is one which comprises a detectable moiety. This may be useful for diagnostic imaging of choroid plexus disease.
  • Polypeptides comprising detectable moieties are well known to those of skill in the art. They include, but are not limited to, bacterial chloramphenicol acetyl transferase (CAT), beta-galactosidase, green fluorescent protein (GFP) and other fluorescent protein, various bacterial luciferase genes, e.g., the luciferase genes encoded by Vibrio harveyi, Vibrio fischeri, and Xenorhabdus luminescens, the firefly luciferase gene FFlux, and the like.
  • CAT bacterial chloramphenicol acetyl transferase
  • GFP green fluorescent protein
  • bacterial luciferase genes e.g., the luciferase genes encoded by Vibrio harveyi, Vibrio fischeri, and
  • polypeptide of interest is a therapeutic polypeptide it may be useful for treating a disease.
  • a method of treating a brain disorder or disease in a subject in need thereof comprises administering to the subject a therapeutically effective amount of a therapeutic polypeptide using the constructs of the present invention.
  • brain disorders which may be treated or diagnosed by the agents of the present invention include, but are not limited to brain tumor, neuropathy, Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotropic lateral sclerosis, motor neuron disease, traumatic nerve injury, multiple sclerosis, acute disseminated encephalomyelitis, acute necrotizing hemorrhagic leukoencephalitis, dysmyelination disease, mitochondrial disease, migrainous disorder, bacterial infection, fungal infection, stroke, aging, dementia, schizophrenia, depression, manic depression, anxiety, panic disorder, social phobia, sleep disorder, attention deficit, conduct disorder, hyperactivity, personality disorder, drug abuse, infertility and head injury.
  • the disease is one that is particular to the choroid plexus.
  • diseases include (1) neoplasms (papilloma, leukaemia, meningioma, lymphoma and metastases) of the choroid plexus; (2) infections (bacterial, fungal and viral) of the choroid plexus; (3) cysts in the choroid plexus; (4) haemorrhage in the choroid plexus; (5) congenital abnormalities (Sturge-Weber syndrome, Klippel- Trenaunay- Weber syndrome and vascular malformations); and (6) non-infectious inflammatory disorders (xanthogranulomas, inflammatory pseudotumour, neurosarcoidosis, rheumatoid nodule and villous hypertrophy).
  • neoplasms papilloma, leukaemia, meningioma, lymphoma and metastases
  • infections bacterial, fungal and viral
  • constructs of the present invention can be provided to the individual per se, or as part of a pharmaceutical composition where it is mixed with a pharmaceutically acceptable carrier.
  • a "pharmaceutical composition” refers to a preparation of one or more of the active ingredients described herein with other chemical components such as physiologically suitable carriers and excipients.
  • the purpose of a pharmaceutical composition is to facilitate administration of a compound to an organism.
  • active ingredient refers to the constructs which are accountable for the biological effect.
  • physiologically acceptable carrier refers to the constructs which are accountable for the biological effect.
  • pharmaceutically acceptable carrier refers to a carrier or a diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound.
  • An adjuvant is included under these phrases.
  • One of the ingredients included in the pharmaceutically acceptable carrier can be for example polyethylene glycol (PEG), a biocompatible polymer with a wide range of solubility in both organic and aqueous media (Mutter et al. (1979).
  • excipient refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient.
  • excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.
  • suitable routes of administration include direct injection into the brain or spinal cord (intrathecal).
  • compositions of the present invention may be manufactured by processes well known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.
  • compositions for use in accordance with the present invention may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active ingredients into preparations which, can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.
  • the active ingredients of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological salt buffer.
  • Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multidose containers with optionally, an added preservative.
  • the compositions may be suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • compositions for parenteral administration include aqueous solutions of the active preparation in water-soluble form. Additionally, suspensions of the active ingredients may be prepared as appropriate oily or water based injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acids esters such as ethyl oleate, triglycerides or liposomes. Aqueous injection suspensions may contain substances, which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the active ingredients to allow for the preparation of highly concentrated solutions.
  • compositions suitable for use in context of the present invention include compositions wherein the active ingredients are contained in an amount effective to achieve the intended purpose. More specifically, a therapeutically effective amount means an amount of active ingredients effective to prevent, alleviate or ameliorate symptoms of disease or prolong the survival of the subject being treated.
  • the therapeutically effective amount or dose can be estimated initially from in vitro assays.
  • a dose can be formulated in animal models and such information can be used to more accurately determine useful doses in humans.
  • Toxicity and therapeutic efficacy of the active ingredients described herein can be determined by standard pharmaceutical procedures in vitro, in cell cultures or experimental animals. The data obtained from these in vitro and cell culture assays and animal studies can be used in formulating a range of dosage for use in human. The dosage may vary depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. [See e.g., Fingl, et al., (1975) "The Pharmacological Basis of Therapeutics", Ch. 1 p. I].
  • dosing can be of a single or a plurality of administrations, with course of treatment lasting from several days to several weeks or until cure is effected or diminution of the disease state is achieved.
  • the amount of a composition to be administered will, of course, be dependent on the subject being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, etc.
  • constructs of the present invention can be provided to the individual with additional active agents to achieve an improved therapeutic effect as compared to treatment with each agent by itself.
  • measures e.g., dosing and selection of the complementary agent
  • adverse side effects are taken to adverse side effects which may be associated with combination therapies.
  • compositions including the preparation of the present invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition.
  • compositions of the present invention may, if desired, be presented in a pack or dispenser device, such as an FDA approved kit, which may contain one or more unit dosage forms containing the active ingredient.
  • the kit may also contain an inducer, such as the ones described herein above.
  • the pack may, for example, comprise metal or plastic foil, such as a blister pack.
  • the pack or dispenser device may be accompanied by instructions for administration.
  • the pack or dispenser may also be accommodated by a notice associated with the container in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the compositions or human or veterinary administration. Such notice, for example, may be of labeling approved by the U.S. Food and Drug Administration for prescription drugs or of an approved product insert.
  • choroid specific promoter is intended to include all such promoters a priori.
  • other inducible construct systems will be developed and the present invention contemplates the use of all.
  • compositions, methods or structure may include additional ingredients, steps and/or parts, but only if the additional ingredients, steps and/or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.
  • a compound or “at least one compound” may include a plurality of compounds, including mixtures thereof.
  • method refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.
  • treating includes abrogating, substantially inhibiting, slowing or reversing the progression of a condition, substantially ameliorating clinical or aesthetical symptoms of a condition or substantially preventing the appearance of clinical or aesthetical symptoms of a condition.
  • Tet-On gene expression system enables regulation of gene expression in a precise, reversible and quantitative manner.
  • the Tet system uses the chimeric transactivator (tTA) to activate transcription of the gene of interest from a silent promoter ( Figure IA).
  • tTA chimeric transactivator
  • Figure IA Gene expression is controlled through the interaction of two components, the product of an 'effector' transgene carried by one virus, acting on a 'target' transgene carried by a second virus ( Figure IA).
  • rtTA reverse tTA
  • TRE Tet Response Element
  • Dox Doxycycline
  • choroid plexus cells were infected with a mixture of both 'effector' and 'target' viruses.
  • tissue specific transgene expression the 5'-flanking region of the CRFR2 ⁇ gene, which was previously demonstrated to be expressed specifically in choroid plexus cells [Chen et al., MoI Endocrinol 2005; 19: 441-458], was selected to drive the transcription of the reverse transactivator (rtTA).
  • rtTA reverse transactivator
  • rtTA reverse tetracycline transactivator
  • the amplified sequence was then inserted into the pCSC-SP-PW-rtTA-IRES- GFP construct using CIaI and Agel sites, replacing the CMV promoter and resulting in the pCSC-SP-PW-CRFR2 ⁇ pro-rtTA-IRES-GFP lentiviral construct, as illustrated in Figure 1C.
  • Target plasmid a coding sequence for monomelic RFP was subcloned into a pIRES plasmid (Clonetech) using Xbal and Sail sites.
  • the IRES-RFP sequence was PCR amplified and transferred into the pCSC-SP-PW-IRES-GFP using Agel and Bspl 4071 sites, replacing the IRES-GFP sequence.
  • MCS multiple cloning site
  • the MCS was ligated into the pCSC-SP-PW-IRES-RFP plasmid, using the Agel site, downstream to the CMV promoter.
  • the resulting plasmid was digested with CIaI to excise the CMV promoter sequence.
  • a Tetracycline responsive element (TRE) was PCR amplified from a TRE- containing plasmid inserted using a HindIII site upstream to a mouse CRF (mCRF) cDNA sequence (GenBank accession no. NM 205769 (SEQ ID NO: 22)) in a pCDNA3-mCRF plasmid.
  • the TRE-mCRF sequence was subcloned into the pCSC-SP-PW-IRES-RFP plasmid using the Nhel and Xhol sites in the added MCS resulting in the pCSC-SP-PW-TRE-mCRF-IRES-RFP lentiviral construct - see Figure ID.
  • Mouse GnRH (mGnRH) cDNA was PCR amplified from mouse hypothalamic cDNA using the following specific oligonucleotide primers: 5'- GCTGGCCTCTTTGCTAA-3' (SEQ ID NO: 17) and 5'- AGTGCATCTACATCTTCTTCTG-3' (SEQ ID NO: 18) corresponding to nucleotides 20-36 and 335-356 respectively (GenBank accession no. BCl 16897 (SEQ ID NO: 23)), and TA-cloned into a pCR-TOPO2.1 plasmid (Invitrogen).
  • the mGnRH sequence (GenBank accession no.
  • BCl 16897 was then inserted into the pCSC-SP-P W-TRE- mCRF-IRES-RFP plasmid, using Pad and Xhol sites replacing the CRF sequence and resulting in the pCSC-SP-PW-TRE-mGnRH-IRES-RFP plasmid - see Figure IE.
  • lentiviral vectors' Production of lentiviral vectors'.
  • Recombinant lentiviruses were produced by transient transfection in HEK293T cells, as described previously [Naldini L, et al., Science 1996; 272: 263-267;Naldini L, et al., Proc Natl Acad Sci U S A., 1996; 93: 11382-11388; Pfeifer A, et al., Proc Natl Acad Sci U S A. 2000; 97: 12227-12232; Chen A, J Neurosci. 2006; 26: 5500-5510].
  • infectious lentiviruses were harvested at 48 and 72 hours post-transfection, filtered through 0.45 ⁇ m-pore cellulose acetate filters and concentrated by ultracentrifugation. Vector concentrations were analyzed using eGFP or RFP fluorescence in HEK293T cells infected with serial dilutions of the recombinant lentivirus.
  • mice 7 weeks old wild-type C57BL/6J male mice and ICR female mice (Harlan, Jerusalem, Israel) were used for the experimental procedures. Mice were housed in a temperature-controlled room (22 0 C ⁇ 1) on a reverse 12 hourts light/dark cycle. Food and water were given ad libitum. All experimental protocols were approved by the Institutional Animal Care and Use Committee of The Weizmann Institute of Science.
  • Surgical procedure Animals were anesthetized with lO ⁇ g ketamine, 0.8 ⁇ g xylazine, 4 ⁇ g acepromazine per gr. body weight, intraperitoneally, and placed in a Angle Two stereotaxic instrument (myNeuroLab, St. Louis, MO, USA). 2 ⁇ l of concentrated lentiviral vector preperation was injected into the left lateral ventricle of mice, using a 26s-gauge blunt-tip needle Hamilton microsyringe (Hamilton, Reno, NV, USA), at a rate of 0.5 ⁇ l/min. Injection coordinates relative to bregma were as follows: AP -0.22 mm; ML -1.15 mm; DV -2.06 mm.
  • RT-PCR analysis primers are listed in Table 1, herein below
  • Brain slices were analyzed 14 days following infection at 4 0 C. RESULTS
  • the cDNA products were used as templates for semiquantitative RT- PCR analysis, using specific primers for mouse CRFR2 ⁇ ( Figure 2A).
  • the RT-PCR data demonstrate expression of CRFR2 ⁇ that is abundant and restricted to the CP tissue ( Figure 2A).
  • a specific in situ probe was used and a positive hybridization signal for mouse CRFR2 ⁇ was found only in the CP ( Figures 2B-C).
  • Intracerebroventricular injection of the CP-specific lentiviruses show GFP expression specifically by the CP cells ( Figures 2D-I).
  • Co-staining with the nuclear marker, DAPI clearly demonstrates that only the CP-cells and not the ependymal cells (cells which line the brain ventricles) express GFP ( Figures 2D-F).
  • Higher magnification images demonstrate the ability of these lentiviruses to infect the CP-cells with high efficiency ( Figures 2G-I).
  • EXAMPLE 2 Verification of neuropeptide processing enzymes expression by choroid plexus cells.
  • neuropeptides are derived from larger biologically inactive polypeptide precursors and require post-translational processing to become biologically active.
  • Specific proteases/endopeptidases are thought to process precursors during transit through the ER/golgi secretory pathway. Following cleavage, the residual basic amino acids are removed by an exopeptidase (carboxypeptidase B/H/E), which is often followed by other post-translational modifications such as glycosylation, sulphation and amidation to obtain full bioactivity.
  • RNA isolated from the mouse choroid plexus was screened for a set of key processing enzymes known to expressed by neuroendocrine neurons and to be necessary for the proper processing of most neuropeptides.
  • Semi-quantitative RT-PCR analysis of the paired basic amino acid cleaving enzyme Furin ( Figure 3A) and the exopeptidase E (CPE), which responsible for the removal of an amino acid from the end of a polypeptide chain ( Figure 3D) showed similar expression levels in the CP cells, compared with the positive control tissue obtained from the mouse hypothalamus.
  • PC2 prohormone convertase 2
  • PAM1 peptidylglycine alpha-amidating monooxygenase 1
  • a third lentiviral DNA construct was designed to express a blue florescent protein (BFP) reporter, in an inducible manner (Figure 3G).
  • BFP blue florescent protein
  • mice 7 week old wild-type C57BL/6J male mice and ICR female mice (Harlan, Jerusalem, Israel) were used for the experimental procedures. Mice were housed in a temperature-controlled room (22 0 C ⁇ 1) on a reverse 12 hours light/dark cycle. Food and water were given ad libitum. All experimental protocols were approved by the Institutional Animal Care and Use Committee of The Weizmann Institute of Science.
  • Surgical procedure Animals were anesthetized with 10 ⁇ g ketamine, 0.8 ⁇ g xylazine, 4 ⁇ g acepromazine per gr. body weight, intraperitoneally, and placed in a Angle Two stereotaxic instrument (myNeuroLab, St. Louis, MO, USA). 2 ⁇ l of concentrated lentiviral vector preperation was injected into the left lateral ventricle of mice, using a 26s-gauge blunt-tip needle Hamilton microsyringe (Hamilton, Reno, NV, USA), at a rate of 0.5 ⁇ l/min. Injection coordinates relative to bregma were as follows:
  • mice were subjected to behavioral and physiological studies as described below. At the end of the tests, mice were anesthetized and perfused with phosphate buffered 4 % paraformaldehyde.
  • mice were subjected to the following tests: open field, dark/light transfer box and elevated plus maze (EPM) followed by a home cage locomotion analysis. Animals were then given a 0.5 mg/ml doxycycline, 0.2 % sucrose solution as drinking water for 4 days before repeating the same set of tests. To avoid experimental artifacts due to repetition, the EPM test was performed on animals only once (half group under each condition). Prior to sacrifice, half the group was kept on tap water and half on dox-containing water.
  • EPM elevated plus maze
  • mice were tested for anxiety related behaviors, under both induced and non-induced conditions using the following tests: Light/dark transfer test: The light/dark transfer test apparatus and experimental conditions were as previously described [Chen et al., J Neurosci. 2006; 26: 5500-5510].
  • the light/dark transfer test takes advantage of the natural conflict of a rodent between the exploration of a novel environment and the aversive properties of a large, brightly lit open field. A greater amount of time spent in the light compartment and a greater number of transitions are indicative of decreased anxiety-like behavior.
  • the activity and transitions were quantified with a video tracking system (VideoMot2; TSE Systems,
  • Open field test The open field test apparatus and experimental conditions was as previously described [Chen et al., J Neurosci. 2006; 26: 5500-551O]. Time (sec) spent in the center, number of visits to center, ambulation (total distance traveled), latency to enter center (sec) and rearings were quantified with a video tracking system
  • Elevated plus maze (EPM) test The EPM apparatus and experimental conditions are as previously described [Chen et al., J Neurosci. 2006; 26: 5500-5510; Lister R.G. Psychopharmacology 1987; 92: 180-185]. Anxiolytic and anxiogenic drugs respectively increase or decrease relative exploration of the open arms [Lister, supra]. Therefore, the number of entries into and the time spent on the open arms were expressed as a percentage of the total number of arm entries and test duration, respectively.
  • Locomotor activity To control for the possibility of behavioral effects originating from differences in ambulatory movement, locomotor activity of mice was examined over a 48 hr period, which proceeded a few days of habituation. Mice were single housed in specialized home cages and locomotion was measured using the InfraMot system (TSE Systems, Bad Hamburg, Germany).
  • mice were anesthetized with chloral hydrate (350 mg/kg, ip) and perfused with 4 % paraformaldehyde fixative. Coronal 30 ⁇ m thick sections throughout the brain were prepared for immunofluorecence localization of GFP (rtTA virus) or CRP immunoreactivity.
  • Sections were washed (5 minutes x3) with PBS and incubated over night at 4 °C with PBS containing 0.2 % triton, monoclonal ⁇ -GFP antibody (Chemicon) (1:1000) and rabbit ⁇ -CRF (1: 1000) serum. Following PBS washes (5 minutes x3) sections were incubated for 2 hours at room temperature with Cy2 ⁇ -mouse (Jackson ImmunoResearch) (1:2000) and Cy3 ⁇ -rabbit (Jackson ImmunoResearch) (1:2000) secondary antibodies. Sections were washed with PBS (5 minutes x3), mounted on slides and visualized using an Olympus 1X81 fluorescence microscope.
  • mice conditionally over- expressing CRF were calculated as mean values ⁇ SEM, and were analyzed using JMP 7 software (SAS, NC, USA). Student Mest was used to compare doxycycline induced and control groups for the elevated plus maze test. Paired Mest was used to compare between doxycycline induced and control conditions for the dark/light transfer test and the open field test.
  • CRF corticotrophin-releasing factor
  • mice conditionally over-expressing CRF at the choroid plexus were evaluated, with or without the presence of the inducer Dox.
  • Three days following doxycycline induction mice showed increase in anxiety-like behavior measured by the light/dark transfer test, with significantly less time spent in the light compartment, reduced exploration of light area, fewer transitions to the light compartment and increase in the latency to first enter the light compartment ( Figures 5A-D).
  • Results from the open field test were consistent with the results obtained from the light/dark transfer test.
  • Mice over-expressing CRF showed a reduced number of entries to the center, a greater latency entering the center, decrease in rearing events and lower exploratory behavior as measured by the shorter distance traveled during the test ( Figures 6 A-D).
  • mice treated with Dox and over express CRF acutely show significant differences between the control and the induced group of mice.
  • CRF over-expressing mice show a decrease in the percent of time spent in the open arm and the percent of entries into open arms ( Figures 7 A-B).
  • No significant differences were found between the experimental groups in their home cage locomotor activity (Figure 7C), suggesting that the observed phenotype is not a consequence of locomotion deficit but rather a genuine change in the mice anxiety-like behavior.
  • GnRH is the central hypothalamic hormone regulating reproduction.
  • the pulse- timing and concentration levels of GnRH are critical for the maintenance of gonadal steroidogenesis, normal reproductive function and estrous cycle.
  • Chronic, high concentrations of GnRH induce regulatory changes that lead to gonadal hypoactivity and cessation or abnormalities of the estrous cycle.
  • Estrous cycle determination The murine estrous cycle is 4-5 days long and its four stages are termed proestrus, estrus, metestrus, and diestrus.
  • the stage of estrous was determined by cytological evaluation of vaginal smears as follows: a smear consisting almost exclusively of leukocytes depicted diestrus; a thin smear of equal numbers of leukocytes and elongated nucleated epithelium indicated proestrus; large cornified epithelial cells were exclusively found in estrus; and metestrus was marked by a thick smear composed of equal numbers of nucleated epithelial cells and leukocytes.
  • Vaginal smears from mature virgin female ICR mice (12-14 weeks old) were obtained once in two days at 10:30 am. Only those mice undergoing normal estrous cycle changes for at least three consecutive 4-day cycles were included in the study. Mice were sacrificed at the stage of estrus / diestrus. To validate the accuracy of stage determination, vaginal smears were again obtained prior to sacrificing the animals.
  • mice were anesthetized with chloral hydrate (350 mg/kg, ip) and perfused with 4 % paraformaldehyde fixative. Coronal 30 ⁇ m thick sections throughout the brain were prepared for immunofluorecence localization of GFP (rtTA virus) or
  • Sections were washed (5 minutes x3) with PBS and incubated over night at 4 °C with PBS containing 0.2 % triton, monoclonal ⁇ -GFP antibody (Chemicon) (1:1000) and rabbit ⁇ -GnRH (1:1000) serum. Following PBS washes (5 minutes x3) sections were incubated for 2 hours at room temperature with Cy2 ⁇ -mouse (Jackson
  • GnRH gonadotropin-releasing factor

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