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WO2025041032A1 - Promoteur pour l'expression spécifique de gènes dans les cellules de la couche 5 du cortex de la souris - Google Patents

Promoteur pour l'expression spécifique de gènes dans les cellules de la couche 5 du cortex de la souris Download PDF

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Publication number
WO2025041032A1
WO2025041032A1 PCT/IB2024/058069 IB2024058069W WO2025041032A1 WO 2025041032 A1 WO2025041032 A1 WO 2025041032A1 IB 2024058069 W IB2024058069 W IB 2024058069W WO 2025041032 A1 WO2025041032 A1 WO 2025041032A1
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nucleic acid
cells
cell
sequence
gene
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Georg Keller
Hassana K. OYIBO
Beatriz Lucia SAENZ DE BURUAGA CORROCHANO
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Novartis Forschungsstiftung Zweigniederlassung Friedrich Miescher Institute for Biomedical Research
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Novartis Forschungsstiftung Zweigniederlassung Friedrich Miescher Institute for Biomedical Research
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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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • A61K48/0058—Nucleic acids adapted for tissue specific expression, e.g. having tissue specific promoters as part of a contruct
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/0075—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the delivery route, e.g. oral, subcutaneous
    • 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
    • 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 relates to a nucleic acid sequence leading to the expression of genes specifically in layer 5 cells in mouse cortex.
  • recombinant genes are usually transfected into the target cells, cell populations or tissues, as cDNA constructs in the context of an active expression cassette to allow transcription of the heterologous gene.
  • the DNA construct is recognized by the cellular transcription machinery in a process that involves the activity of many trans-acting transcription factors (TF) at cis-regulatory elements, including enhancers, silencers, insulators and promoters (herein globally referred to as “promoters”).
  • TF trans-acting transcription factors
  • Gene promoters are involved in all these levels of regulation, serving as the determinant in gene transcription by integrating the influences of the DNA sequence, transcription factor binding and epigenetic features. They determine the strength of e.g., transgene expression which is encoded by a plasmid vector as well as in which cell type or types said transgene will be expressed.
  • CMV human and mouse cytomegalovirus
  • RSV Rous Sarcoma Virus
  • LTR long-terminal-repeat
  • cellular promoters can also be used.
  • known promoters are those from house-keeping genes that encode abundantly transcribed cellular transcripts, such as beta-actin, elongation factor 1-alpha (EF-1alpha), or ubiquitin.
  • EF-1alpha elongation factor 1-alpha
  • ubiquitin elongation factor 1-alpha
  • One of the aspects concerning the use of endogenous regulatory elements for transgene expression is the generation of stable mRNA and that expression can take place in the native environment of the host cell where trans-acting transcription factors are provided accordingly. Since expression of eukaryotic genes is controlled by a complex machinery of cis- and transacting regulatory elements, most cellular promoters suffer from a lack of extensive functional characterization. Parts of the eukaryotic promoter are usually located immediately upstream of its transcribed sequence and serve as the point of transcriptional initiation. The core promoter immediately surrounds the transcription start site (TSS) which is sufficient to be recognized by the transcription machinery.
  • TSS transcription start site
  • the proximal promoter comprises the region upstream of the core promoter and contains the TSS and other sequence features required for transcriptional regulation.
  • Transcription factors act sequence-specific by binding to regulatory motifs in the promoter and enhancer sequence thereby activating chromatin and histone modifying enzymes that alter nucleosome structure and its position which finally allows initiation of transcription.
  • the identification of a functional promoter is mainly dependent on the presence of associated upstream or downstream enhancer elements.
  • Another crucial aspect concerning the use of endogenous regulatory elements for transgene expression is that some promoters can act in a cell specific manner and will lead to the expression of the transgene on in cells of a specific type or, depending on the promoter, in cells of a particular subset.
  • the present inventors have serendipitously found that a fragment of the CHODL gene is able to drive the expression of heterologous genes specifically in layer 5 cells in mouse cortex.
  • the nucleic acid sequence of the sequence of the invention is: GTTTTACAGAAGCTGATCCTAAGAACATTTTCAGGTTTCCTATTATGGACTATGTGTAAAC TGTCATTATGAGAACTCTCACGAACATCTCTGACTCCATAAAAATGGTAAGCCAGCCATT GGACTGTTACAGAAGAGTGGGTCAGGTTTGGAAAGGCACATACATATTCCTTAAAGTGA GACTTACTTTTGTTTGTTTATTTGTTTGTTTGTTTGGTTGGTTGGTTGGTTGGTTGGTTTTTTGAGAC AGGGTTTCTCTGTATAGCCCTGGCTGTCCTGGAACTCACTTTGTAGACCAGGCTGGCTT ACTTTTAATGGAATCCATAGACCACTGATGGATACGTTCAGAGAAATACACTCATTTTGCA TTCGTGAAGTTCTCAACAATTCAGATTATTTATTTTGCCTGGACTTTCCCCTTGGAGTT GCAAACACTGAGACTGAAATTAGTCGGGGGGGGGGCTTGTTTCCAAGATTCTTTTCAAAT TACTTC
  • the present invention hence provides an isolated nucleic acid molecule comprising, or consisting of, the nucleic acid sequence of SEQ ID NO:1 or a nucleic acid sequence of at least 1400 bp having at least 70% identity to said nucleic acid sequence of SEQ ID NO:1 , wherein said isolated nucleic acid molecule specifically leads to the expression in layer 5 cells in mouse cortex of a gene operatively linked to said nucleic acid sequence coding for said gene.
  • the nucleic acid sequence is at least 1400 bp, has at least 80 % identity to said nucleic acid sequence of SEQ ID NO:1 .
  • the nucleic acid sequence is at least 1400 bp, and has at least 85 % identity to said nucleic acid sequence of SEQ ID NO:1. In some embodiments, the nucleic acid sequence is at least 1400 bp, and has at least 90 % identity to said nucleic acid sequence of SEQ ID NO:1 . In some embodiments, the nucleic acid sequence is at least 1400 bp, and has at least 95 % identity to said nucleic acid sequence of SEQ ID NO:1 . In some embodiments, the nucleic acid sequence is at least 1400 bp, and has at least 96 % identity to said nucleic acid sequence of SEQ ID NO:1 .
  • the nucleic acid sequence is at least 1000 bp, and has at least 97 % identity to said nucleic acid sequence of SEQ ID NO:1 . In some embodiments, the nucleic acid sequence is at least 1400 bp, and has at least 98 % identity to said nucleic acid sequence of SEQ ID NO:1 . In some embodiments, the nucleic acid sequence is at least 1400 bp, and has at least 99 % identity to said nucleic acid sequence of SEQ ID NO:1. In some embodiments, the nucleic acid sequence is at least 1400 bp, and has 100 % identity to said nucleic acid sequence of SEQ ID NO:1 .
  • Said identity is the identity of the sequence of the molecule over the overlapping segment(s).
  • the nucleic acid molecule of the invention having the identities described herein above, can have a length of at least 1400 bp, at least 1450 bp, at least 1500 bp, at least 1550 bp. at least 1600 bp, at least 1650 bp, at least 1700 bp, at least 1750 bp, at least 1800 bp, at least 1850 bp, at least 1900 bp, at least 1950 bp, at least 1980 bp, at least 1990 bp, at least 2000 bp.
  • the isolated nucleic acid molecule of the invention can additionally comprise a minimal promoter, for instance a SV40 minimal promoter, e.g. the SV40 minimal promoter or ATCCTCACATGGTCCTGCTGGAGTTAGTAGAGGGTATATAATGGAAGCTCGACTTCCAG CTATCACATCCACTGTGTTGTTGTGAACTGGAATCCACTATAGGCCA (SEQ ID NO:2).
  • a SV40 minimal promoter e.g. the SV40 minimal promoter or ATCCTCACATGGTCCTGCTGGAGTTAGTAGAGGGTATATAATGGAAGCTCGACTTCCAG CTATCACATCCACTGTGTTGTTGTGAACTGGAATCCACTATAGGCCA (SEQ ID NO:2).
  • the present invention also provides an expression cassette comprising an isolated nucleic acid of the invention as described above, wherein said promoter is operatively linked to at least a nucleic acid sequence encoding for a gene to be expressed specifically in layer 5 cells in mouse cortex.
  • the present invention further provides a vector comprising the expression cassette of the invention.
  • said vector is a viral vector.
  • the present invention also encompasses the use of a nucleic acid of the invention, of an expression cassette of the invention or of a vector of the invention for the expression of a gene in layer 5 cells in mouse cortex.
  • the present invention further provides a method of expressing gene in layer 5 cells in mouse cortex comprising the steps of transfecting an isolated cell, a cell line or a cell population (e.g. a tissue) with an expression cassette of the invention, wherein the gene to be expressed will be expressed by the isolated cell, the cell line or the cell population if said cell is, or said cells comprise, layer 5 cells of mouse cortex.
  • the isolated cell, cell line or cell population or tissue is human.
  • the present invention also provides an isolated cell comprising the expression cassette of the invention.
  • the expression cassette or vector is stably integrated into the genome of said cell.
  • a typical gene which can be operatively linked to the promoter of the invention is a gene encoding for a halorhodopsin or a channelrhodosin.
  • Therapeutic genes i.e. genes encoding for a therapeutic protein useful for the treatment of a pathological condition, can also be used.
  • the present invention also provides a kit for expressing a gene in layer 5 cells in mouse cortex, which kit comprises an isolated nucleic acid molecule of the invention.
  • Figure 1 Plasmid Map of AAV vector
  • Figure 2 Coronal sections, of a brain injected with an AAV construct corresponding to figure 1 and having SEQ ID NO:1 as promoter.
  • Figure 3 Injections of ubiquitous EF1 a promoter AAV compared to promoter of SEQ ID NO:1.
  • the present inventors have serendipitously found that a fragment of the CHODL gene is able to drive the expression of heterologous genes specifically in layer 5 cells in mouse cortex.
  • nucleic acid sequence of the sequence of the invention is:
  • the present invention hence provides an isolated nucleic acid molecule comprising, or consisting of, the nucleic acid sequence of SEQ ID NO:1 or a nucleic acid sequence of at least 1400 bp having at least 70% identity to said nucleic acid sequence of SEQ ID NO:1 , wherein said isolated nucleic acid molecule specifically leads to the expression in layer 5 cells in mouse cortex of a gene operatively linked to said nucleic acid sequence coding for said gene.
  • the cells are in the cortex.
  • the cells are from a specific animal, e.g. mouse cells.
  • the nucleic acid sequence is at least 1400 bp, has at least 80 % identity to said nucleic acid sequence of SEQ ID NO:1 .
  • the nucleic acid sequence is at least 1400 bp, and has at least 85 % identity to said nucleic acid sequence of SEQ ID NO:1 . In some embodiments, the nucleic acid sequence is at least 1400 bp, and has at least 90 % identity to said nucleic acid sequence of SEQ ID NO:1. In some embodiments, the nucleic acid sequence is at least 1400 bp, and has at least 95 % identity to said nucleic acid sequence of SEQ ID NO:1 . In some embodiments, the nucleic acid sequence is at least 1400 bp, and has at least 96 % identity to said nucleic acid sequence of SEQ ID NO:1 .
  • the nucleic acid sequence is at least 1000 bp, and has at least 97 % identity to said nucleic acid sequence of SEQ ID NO:1. In some embodiments, the nucleic acid sequence is at least 1400 bp, and has at least 98 % identity to said nucleic acid sequence of SEQ ID NO:1 . In some embodiments, the nucleic acid sequence is at least 1400 bp, and has at least 99 % identity to said nucleic acid sequence of SEQ ID NO:1 . In some embodiments, the nucleic acid sequence is at least 1400 bp, and has 100 % identity to said nucleic acid sequence of SEQ ID NO:1. Said identity is the identity of the sequence of the molecule over the overlapping segment(s).
  • the nucleic acid molecule of the invention having the identities described herein above, can have a length of at least 1400 bp, at least 1450 bp, at least 1500 bp, at least 1550 bp. at least 1600 bp, at least 1650 bp, at least 1700 bp, at least 1750 bp, at least 1800 bp, at least 1850 bp, at least 1900 bp, at least 1950 bp, at least 1980 bp, at least 1990 bp, at least 2000 bp.
  • the isolated nucleic acid molecule of the invention can additionally comprise a minimal promoter, for instance a SV40 minimal promoter, e.g. the SV40 minimal promoter or ATCCTCACATGGTCCTGCTGGAGTTAGTAGAGGGTATATAATGGAAGCTCGACTTCCAG CTATCACATCCACTGTGTTGTTGTGAACTGGAATCCACTATAGGCCA (SEQ ID NO:2).
  • a SV40 minimal promoter e.g. the SV40 minimal promoter or ATCCTCACATGGTCCTGCTGGAGTTAGTAGAGGGTATATAATGGAAGCTCGACTTCCAG CTATCACATCCACTGTGTTGTTGTGAACTGGAATCCACTATAGGCCA (SEQ ID NO:2).
  • the present invention also provides an expression cassette comprising an isolated nucleic acid of the invention as described above, wherein said promoter is operatively linked to at least a nucleic acid sequence encoding for a gene to be expressed specifically in layer 5 cells in mouse cortex.
  • the present invention further provides a vector comprising the expression cassette of the invention.
  • said vector is a viral vector.
  • the present invention also encompasses the use of a nucleic acid of the invention, of an expression cassette of the invention or of a vector of the invention for the expression of a gene in layer 5 cells in mouse cortex.
  • the present invention further provides a method of expressing gene in layer 5 cells in mouse cortex comprising the steps of transfecting an isolated cell, a cell line or a cell population (e.g. a tissue) with an expression cassette of the invention, wherein the gene to be expressed will be expressed by the isolated cell, the cell line or the cell population if said cell is, or said cells comprise, layer 5 cells of mouse cortex.
  • the isolated cell, cell line or cell population or tissue is human.
  • the present invention also provides an isolated cell comprising the expression cassette of the invention.
  • the expression cassette or vector is stably integrated into the genome of said cell.
  • a typical gene which can be operatively linked to the promoter of the invention is a gene encoding for a halorhodopsin or a channelrhodosin.
  • Therapeutic genes i.e. genes encoding for a therapeutic protein useful for the treatment of a pathological condition, can also be used.
  • the present invention also provides a kit for expressing a gene in layer 5 cells in mouse cortex, which kit comprises an isolated nucleic acid molecule of the invention.
  • promoter refers to any cis-regulatory elements, including enhancers, silencers, insulators and promoters.
  • a promoter is a region of DNA that is generally located upstream (towards the 5' region) of the gene that is needed to be transcribed. The promoter permits the proper activation or repression of the gene which it controls. In the context of the present invention, the promoters lead to the specific expression of genes operably linked to them in the layer 5 cells of mouse cortex.
  • Specific expression of an exogenous gene also referred to as “expression only in a certain type of cell” means that at least more than 75%, preferably more than 85%, more that 90% or more than 95%, of the cells expressing the exogenous gene of interest are of the type specified, i.e. in layer 5 cells of mouse cortex in the present case.
  • Expression cassettes are typically introduced into a vector that facilitates entry of the expression cassette into a host cell and maintenance of the expression cassette in the host cell.
  • vectors are commonly used and are well known to those of skill in the art. Numerous such vectors are commercially available, e. g., from Invitrogen, Stratagene, Clontech, etc., and are described in numerous guides, such as Ausubel, Guthrie, Strathem, or Berger, all supra.
  • Such vectors typically include promoters, polyadenylation signals, etc. in conjunction with multiple cloning sites, as well as additional elements such as origins of replication, selectable marker genes (e. g., LEU2, URA3, TRP 1 , HIS3, GFP), centromeric sequences, etc.
  • Viral vectors for instance an AAV, a PRV or a lentivirus, are suitable to target and deliver genes to in layer 5 cells of mouse cortex using a promoter of the invention.
  • the output of cells can be measured using an electrical method, such as a multi-electrode array or a patch-clamp, or using a visual method, such as the detection of fluorescence.
  • an electrical method such as a multi-electrode array or a patch-clamp
  • a visual method such as the detection of fluorescence.
  • the methods using nucleic acid sequence of the invention can be used for identifying therapeutic agents for the treatment of a neurological disorder or of a disorder involving layer 5 cells of mouse cortex, said method comprising the steps of contacting a test compound with layer 5 cells of mouse cortex expressing one or more transgene under a promoter of the invention, and comparing at least one output of layer 5 cells of mouse cortex obtained in the presence of said test compound with the same output obtained in the absence of said test compound.
  • the methods using promoters of the invention can also be used for /n vitro testing of vision restoration, said method comprising the steps of contacting layer 5 cells of mouse cortex expressing one or more transgene under the control of a promoter of the invention with an agent, and comparing at least one output obtained after the contact with said agent with the same output obtained before said contact with said agent.
  • Channelrhodopsins are a subfamily of opsin proteins that function as light-gated ion channels. They serve as sensory photoreceptors in unicellular green algae, controlling phototaxis, i.e. movement in response to light. Expressed in cells of other organisms, they enable the use of light to control intracellular acidity, calcium influx, electrical excitability, and other cellular processes. At least three “natural” channelrhodopsins are currently known: Channelrhodopsin-1 (ChR1), Channelrhodopsin-2 (ChR2), and Volvox Channelrhodopsin (VChR1). Moreover, some modified/improved versions of these proteins also exist.
  • ChR1 Channelrhodopsin-1
  • ChR2 Channelrhodopsin-2
  • VhR1 Volvox Channelrhodopsin
  • Halorhodopsin is a light-driven ion pump, specific for chloride ions, and found in phylogenetically ancient “bacteria” (archaea), known as halobacteria. It is a seven- transmembrane protein of the retinylidene protein family, homologous to the light-driven proton pump bacteriorhodopsin, and similar in tertiary structure (but not primary sequence structure) to vertebrate rhodopsins, the pigments that sense light in the retina. Halorhodopsin also shares sequence similarity to channelrhodopsin, a light-driven ion channel.
  • Halorhodopsin contains the essential light-isomerizable vitamin A derivative all-trans-retinal.
  • Halorhodopsin is one of the few membrane proteins whose crystal structure is known. Halorhodopsin isoforms can be found in multiple species of halobacteria, including H. salinarum, and N. pharaonis. Much ongoing research is exploring these differences, and using them to parse apart the photocycle and pump properties. After bacteriorhodopsin, halorhodopsin may be the best type I (microbial) opsin studied. Peak absorbance of the halorhodopsin retinal complex is about 570 nm. Recently, halorhodopsin has become a tool in optogenetics.
  • halorhodopsin opens up the ability to silence excitable cells with brief pulses of yellow light.
  • halorhodopsin and channelrhodopsin together enable multiple-color optical activation, silencing, and desynchronization of neural activity, creating a powerful neuroengineering toolbox.
  • the promoter is part of a vector targeted to the cortex, said vector expressing at least one reporter gene which is detectable in living layer 5 cells of mouse cortex.
  • Suitable viral vectors for the invention are well-known in the art.
  • an AAV, a PRV or a lentivirus are suitable to target and deliver genes to layer 5 cells of mouse cortex.
  • the output of transfected cells can be measured using well-known methods, for instance using an electrical method, such as a multi-electrode array or a patch-clamp, or using a visual method, such as the detection of fluorescence.
  • an electrical method such as a multi-electrode array or a patch-clamp
  • a visual method such as the detection of fluorescence.
  • the inner limiting membrane is removed by micro-surgery the inner limiting membrane.
  • recording is achieved through slices performed to the inner limiting membrane.
  • the term "animal” is used herein to include all animals.
  • the non-human animal is a vertebrate. Examples of animals are human, mice, rats, cows, pigs, horses, chickens, ducks, geese, cats, dogs, etc.
  • the term “animal” also includes an individual animal in all stages of development, including embryonic and fetal stages.
  • a "genetically-modified animal” is any animal containing one or more cells bearing genetic information altered or received, directly or indirectly, by deliberate genetic manipulation at a sub-cellular level, such as by targeted recombination, microinjection or infection with recombinant virus.
  • genetically-modified animal is not intended to encompass classical crossbreeding or in vitro fertilization, but rather is meant to encompass animals in which one or more cells are altered by, or receive, a recombinant DNA molecule.
  • This recombinant DNA molecule may be specifically targeted to a defined genetic locus, may be randomly integrated within a chromosome, or it may be extrachromosomally replicating DNA.
  • germ-line genetically-modified animal refers to a genetically-modified animal in which the genetic alteration or genetic information was introduced into germline cells, thereby conferring the ability to transfer the genetic information to its offspring. If such offspring in fact possess some or all of that alteration or genetic information, they are genetically-modified animals as well.
  • the alteration or genetic information may be foreign to the species of animal to which the recipient belongs, or foreign only to the particular individual recipient, or may be genetic information already possessed by the recipient.
  • the altered or introduced gene may be expressed differently than the native gene, or not expressed at all.
  • genes used for altering a target gene may be obtained by a wide variety of techniques that include, but are not limited to, isolation from genomic sources, preparation of cDNAs from isolated mRNA templates, direct synthesis, or a combination thereof.
  • ES cells A type of target cells for transgene introduction is the ES cells.
  • ES cells may be obtained from pre-implantation embryos cultured in vitro and fused with embryos (Evans et al. (1981), Nature 292:154-156; Bradley et al. (1984), Nature 309:255-258; Gossler et al. (1986), Proc. Natl. Acad. Sci. USA 83:9065-9069; Robertson et al. (1986), Nature 322:445-448; Wood et al. (1993), Proc. Natl. Acad. Sci. USA 90:4582- 4584).
  • Transgenes can be efficiently introduced into the ES cells by standard techniques such as DNA transfection using electroporation or by retrovirus-mediated transduction.
  • the resultant transformed ES cells can thereafter be combined with morulas by aggregation or injected into blastocysts from a non-human animal.
  • the introduced ES cells thereafter colonize the embryo and contribute to the germline of the resulting chimeric animal (Jaenisch (1988), Science 240:1468-1474).
  • the use of gene- targeted ES cells in the generation of gene-targeted genetically-modified mice was described 1987 (Thomas et al. (1987), Cell 51 :503-512) and is reviewed elsewhere (Frohman et al.
  • a "targeted gene” is a DNA sequence introduced into the germline of a non- human animal by way of human intervention, including but not limited to, the methods described herein.
  • the targeted genes of the invention include DNA sequences which are designed to specifically alter cognate endogenous alleles.
  • isolated refers to material removed from its original environment (e.g., the natural environment if it is naturally occurring), and thus is altered “by the hand of man” from its natural state.
  • an isolated polynucleotide could be part of a vector or a composition of matter, or could be contained within a cell, and still be “isolated” because that vector, composition of matter, or particular cell is not the original environment of the polynucleotide.
  • isolated does not refer to genomic or cDNA libraries, whole cell total or mRNA preparations, genomic DNA preparations (including those separated by electrophoresis and transferred onto blots), sheared whole cell genomic DNA preparations or other compositions where the art demonstrates no distinguishing features of the polynucleotide/sequences of the present invention.
  • isolated DNA molecules include recombinant DNA molecules maintained in heterologous host cells or purified (partially or substantially) DNA molecules in solution.
  • Isolated RNA molecules include in vivo or in vitro RNA transcripts of the DNA molecules of the present invention.
  • a nucleic acid contained in a clone that is a member of a library e.g., a genomic or cDNA library
  • a chromosome removed from a cell or a cell lysate e.g. , a "chromosome spread", as in a karyotype
  • isolated nucleic acid molecules according to the present invention may be produced naturally, recombinantly, or synthetically.
  • Polynucleotides can be composed of single-and double-stranded DNA, DNA that is a mixture of single-and double-stranded regions, single-and double-stranded RNA, and RNA that is mixture of single-and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions.
  • polynucleotides can be composed of triple-stranded regions comprising RNA or DNA or both RNA and DNA. Polynucleotides may also contain one or more modified bases or DNA or RNA backbones modified for stability or for other reasons.
  • Modified bases include, for example, tritylated bases and unusual bases such as inosine.
  • polynucleotide embraces chemically, enzymatically, or metabolically modified forms.
  • polynucleotide encoding a polypeptide encompasses a polynucleotide which includes only coding sequence for the polypeptide as well as a polynucleotide which includes additional coding and/or non-coding sequence.
  • Stringent hybridization conditions refers to an overnight incubation at 42 degree C in a solution comprising 50% formamide, 5x SSC (750 mM NaCI, 75 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5x Denhardt's solution, 10% dextran sulfate, and 20 pg/ml denatured, sheared salmon sperm DNA, followed by washing the filters in 0.1x SSC at about 50 degree C. Changes in the stringency of hybridization and signal detection are primarily accomplished through the manipulation of formamide concentration (lower percentages of formamide result in lowered stringency); salt conditions, or temperature.
  • washes performed following stringent hybridization can be done at higher salt concentrations (e.g. 5X SSC). Variations in the above conditions may be accomplished through the inclusion and/or substitution of alternate blocking reagents used to suppress background in hybridization experiments.
  • Typical blocking reagents include Denhardt's reagent, BLOTTO, heparin, denatured salmon sperm DNA, and commercially available proprietary formulations.
  • the inclusion of specific blocking reagents may require modification of the hybridization conditions described above, due to problems with compatibility.
  • fragment when referring to polypeptides means polypeptides which either retain substantially the same biological function or activity as such polypeptides.
  • An analog includes a pro-protein which can be activated by cleavage of the proprotein portion to produce an active mature polypeptide.
  • gene means the segment of DNA involved in producing a polypeptide chain; it includes regions preceding and following the coding region “leader and trailer” as well as intervening sequences (introns) between individual coding segments (exons).
  • Polypeptides can be composed of amino acids joined to each other by peptide bonds or modified peptide bonds, i.e., peptide isosteres, and may contain amino acids other than the 20 gene-encoded amino acids.
  • the polypeptides may be modified by either natural processes, such as posttranslational processing, or by chemical modification techniques which are well known in the art. Such modifications are well described in basic texts and in more detailed monographs, as well as in a voluminous research literature. Modifications can occur anywhere in the polypeptide, including the peptide backbone, the amino acid sidechains and the amino or carboxyl termini. It will be appreciated that the same type of modification may be present in the same or varying degrees at several sites in a given polypeptide.
  • polypeptides may contain many types of modifications.
  • Polypeptides may be branched, for example, as a result of ubiquitination, and they may be cyclic, with or without branching. Cyclic, branched, and branched cyclic polypeptides may result from posttranslation natural processes or may be made by synthetic methods.
  • Modifications include, but are not limited to, acetylation, acylation, biotinylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, denivatization by known protecting/blocking groups, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, linkage to an antibody molecule or other cellular ligand, methylation, myristoylation, oxidation, pegylation, proteolytic processing (e.g., cleavage), phosphorylation, prenylation
  • a polypeptide fragment "having biological activity” refers to polypeptides exhibiting activity similar, but not necessarily identical to, an activity of the original polypeptide, including mature forms, as measured in a particular biological assay, with or without dose dependency. In the case where dose dependency does exist, it need not be identical to that of the polypeptide, but rather substantially similar to the dose-dependence in a given activity as compared to the original polypeptide (i.e., the candidate polypeptide will exhibit greater activity or not more than about 25-fold less and, in some embodiments,, not more than about tenfold less activity, or not more than about three-fold less activity relative to the original polypeptide.)
  • Species homologs may be isolated and identified by making suitable probes or primers from the sequences provided herein and screening a suitable nucleic acid source for the desired homologue.
  • Variant refers to a polynucleotide or polypeptide differing from the original polynucleotide or polypeptide, but retaining essential properties thereof. Generally, variants are overall closely similar, and, in many regions, identical to the original polynucleotide or polypeptide.
  • nucleic acid molecule or polypeptide is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, 99%, or 100%identical to a nucleotide sequence of the present invention can be determined conventionally using known computer programs.
  • a preferred method for determining the best overall match between a query sequence (a sequence of the present invention) and a subject sequence, also referred to as a global sequence aligmnent, can be determined using the FASTDB computer program based on the algorithm of Brutlag et al. (Comp. App. Blosci. (1990) 6:237-245). In a sequence alignment the query and subject sequences are both DNA sequences.
  • RNA sequence can be compared by converting U's to T's.
  • the result of said global sequence alignment is in percent identity.
  • the FASTDB program does not account for 5' and 3' truncations of the subject sequence when calculating percent identity.
  • the percent identity is corrected by calculating the number of bases of the query sequence that are 5' and 3' of the subject sequence, which are not matched/aligned, as a percent of the total bases of the query sequence. Whether a nucleotide is matched/aligned is determined by results of the FASTDB sequence alignment. This percentage is then subtracted from the percent identity, calculated by the above FASTDB program using the specified parameters, to arrive at a final percent identity score. This corrected score is what is used for the purposes of the present invention.
  • a 90 base subject sequence is compared with a 100 base query sequence. This time the deletions are internal deletions so that there are no bases on the 5' or 3' of the subject sequence which are not matched/aligned with the query. In this case the percent identity calculated by FASTDB is not manually corrected. Once again, only bases 5' and 3' of the subject sequence which are not matched/aligned with the query sequence are manually corrected for.
  • a polypeptide having an amino acid sequence at least, for example, 95% "identical" to a query amino acid sequence of the present invention it is intended that the amino acid sequence of the subject polypeptide is identical to the query sequence except that the subject polypeptide sequence may include up to five amino acid alterations per each 100 amino acids of the query amino acid sequence.
  • the subject polypeptide sequence may include up to five amino acid alterations per each 100 amino acids of the query amino acid sequence.
  • up to 5% of the amino acid residues in the subject sequence may be inserted, deleted, or substituted with another amino acid.
  • These alterations of the reference sequence may occur at the amino or carboxy terminal positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence or in one or more contiguous groups within the reference sequence.
  • any particular polypeptide is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, 99%, or 100% identical to, for instance, the amino acid sequences shown in a sequence or to the amino acid sequence encoded by deposited DNA clone can be determined conventionally using known computer programs.
  • a preferred method for determining, the best overall match between a query sequence (a sequence of the present invention) and a subject sequence, also referred to as a global sequence alignment, can be determined using the FASTDB computer program based on the algorithm of Brutlag et al. (Comp. App. Biosci. (1990) 6:237-245).
  • the query and subject sequences are either both nucleotide sequences or both amino acid sequences.
  • the result of said global sequence alignment is in percent identity.
  • the FASTDB program does not account for N-and C-terminal truncations of the subject sequence when calculating global percent identity.
  • the percent identity is corrected by calculating the number of residues of the query sequence that are N-and C-terminal of the subject sequence, which are not matched/aligned with a corresponding subject residue, as a percent of the total bases of the query sequence. Whether a residue is matched/aligned is determined by results of the FASTDB sequence alignment. This percentage is then subtracted from the percent identity, calculated by the above FASTDB program using the specified parameters, to arrive at a final percent identity score.
  • This final percent identity score is what is used for the purposes of the present invention. Only residues to the N-and C-termini of the subject sequence, which are not matched/aligned with the query sequence, are considered for the purposes of manually adjusting the percent identity score. That is, only query residue positions outside the farthest N-and C-terminal residues of the subject sequence. Only residue positions outside the N-and C-terminal ends of the subject sequence, as displayed in the FASTDB alignment, which are not matched/aligned with the query sequence are manually corrected for. No other manual corrections are to be made for the purposes of the present invention.
  • Naturally occurring protein variants are called "allelic variants," and refer to one of several alternate forms of a gene occupying a given locus on a chromosome of an organism. (Genes 11 , Lewin, B., ed., John Wiley & Sons, New York (1985).) These allelic variants can vary at either the polynucleotide and/or polypeptide level. Alternatively, non-naturally occurring variants may be produced by mutagenesis techniques or by direct synthesis.
  • Label refers to agents that are capable of providing a detectable signal, either directly or through interaction with one or more additional members of a signal producing system. Labels that are directly detectable and may find use in the invention include fluorescent labels. Specific fluorophores include fluorescein, rhodamine, BODIPY, cyanine dyes and the like.
  • fluorescent label refers to any label with the ability to emit light of a certain wavelength when activated by light of another wavelength.
  • Fluorescence refers to any detectable characteristic of a fluorescent signal, including intensity, spectrum, wavelength, intracellular distribution, etc.
  • Detecting fluorescence refers to assessing the fluorescence of a cell using qualitative or quantitative methods. In some of the embodiments of the present invention, fluorescence will be detected in a qualitative manner. In other words, either the fluorescent marker is present, indicating that the recombinant fusion protein is expressed, or not.
  • the fluorescence can be determined using quantitative means, e. g., measuring the fluorescence intensity, spectrum, or intracellular distribution, allowing the statistical comparison of values obtained under different conditions. The level can also be determined using qualitative methods, such as the visual analysis and comparison by a human of multiple samples, e. g., samples detected using a fluorescent microscope or other optical detector (e. g., image analysis system, etc.).
  • an “alteration” or “modulation” in fluorescence refers to any detectable difference in the intensity, intracellular distribution, spectrum, wavelength, or other aspect of fluorescence under a particular condition as compared to another condition. For example, an “alteration” or “modulation” is detected quantitatively, and the difference is a statistically significant difference. Any “alterations” or “modulations” in fluorescence can be detected using standard instrumentation, such as a fluorescent microscope, CCD, or any other fluorescent detector, and can be detected using an automated system, such as the integrated systems, or can reflect a subjective detection of an alteration by a human observer.
  • the “green fluorescent protein” is a protein, composed of 238 amino acids (26.9 kDa), originally isolated from the jellyfish Aequorea victoria/ Aequorea aequorea/Aequorea forskalea that fluoresces green when exposed to blue light.
  • the GFP from A. victoria has a major excitation peak at a wavelength of 395 nm and a minor one at 475 nm. Its emission peak is at 509 nm which is in the lower green portion of the visible spectrum.
  • the GFP from the sea pansy (Renilla reniformis) has a single major excitation peak at 498 nm.
  • GFP GFP has an extinction coefficient (denoted e), also known as its optical cross section of 9.13x10-21 m 2 /molecule, also quoted as 55,000 L/(mol «cm).
  • YFP yellow fluorescent protein
  • a “virus” is a sub-microscopic infectious agent that is unable to grow or reproduce outside a host cell.
  • Each viral particle, or virion consists of genetic material, DNA or RNA, within a protective protein coat called a capsid.
  • the capsid shape varies from simple helical and icosahedral (polyhedral or near-spherical) forms, to more complex structures with tails or an envelope.
  • Viruses infect cellular life forms and are grouped into animal, plant and bacterial types, according to the type of host infected.
  • transsynaptic virus refers to viruses able to migrate from one neurone to another connecting neurone through a synapse.
  • transsynaptic virus examples include rhabodiviruses, e.g. rabies virus, and alphaherpesviruses, e.g. pseudorabies or herpes simplex virus.
  • transsynaptic virus as used herein also encompasses viral sub-units having by themselves the capacity to migrate from one neurone to another connecting neurone through a synapse and biological vectors, such as modified viruses, incorporating such a sub-unit and demonstrating a capability of migrating from one neurone to another connecting neurone through a synapse.
  • Transsynaptic migration can be either anterograde or retrograde.
  • a virus will travel from a postsynaptic neuron to a presynaptic one. Accordingly, during anterograde migration, a virus will travel from a presynaptic neuron to a postsynaptic one.
  • Homologs refer to proteins that share a common ancestor. Analogs do not share a common ancestor, but have some functional (rather than structural) similarity that causes them to be included in a class (e.g. trypsin like serine proteinases and subtilisin's are clearly not related - their structures outside the active site are completely different, but they have virtually geometrically identical active sites and thus are considered an example of convergent evolution to analogs).
  • trypsin like serine proteinases and subtilisin's are clearly not related - their structures outside the active site are completely different, but they have virtually geometrically identical active sites and thus are considered an example of convergent evolution to analogs).
  • Orthologs are the same gene (e.g. cytochome 'c'), in different species. Two genes in the same organism cannot be orthologs. Paralogs are the results of gene duplication (e.g. hemoglobin beta and delta). If two genes/proteins are homologous and in the same organism, they are paralogs.
  • layer 5 cells refers to neurons that are located in the layer 5 of the brain cortex.
  • the cerebral cortex also known as the cerebral mantle, is the outer layer of neural tissue of the cerebrum of the brain in humans and other mammals.
  • the cerebral cortex mostly consists of the six-layered neocortex, with just 10% consisting of allocortex.
  • the neocortex is formed of six layers, numbered I to VI (1-6), from the outermost layer I - near to the pia mater, to the innermost layer VI - near to the underlying white matter.
  • Each cortical layer has a characteristic distribution of different neurons and their connections with other cortical and subcortical regions. There are direct connections between different cortical areas and indirect connections via the thalamus.
  • Layer V or layer 5, the internal pyramidal layer, contains, among other cells, large pyramidal neurons. Axons from these leave the cortex and connect with subcortical structures including the basal ganglia. In the primary motor cortex of the frontal lobe, layer V contains giant pyramidal cells called Betz cells, whose axons travel through the internal capsule, the brain stem, and the spinal cord forming the corticospinal tract, which is the main pathway for voluntary motor control.
  • Betz cells giant pyramidal cells
  • disorder refers to an ailment, disease, illness, clinical condition, or pathological condition.
  • the term "pharmaceutically acceptable carrier” refers to a carrier medium that does not interfere with the effectiveness of the biological activity of the active ingredient, is chemically inert, and is not toxic to the patient to whom it is administered.
  • pharmaceutically acceptable derivative refers to any homolog, analog, or fragment of an agent, e.g. identified using a method of screening of the invention, that is relatively non-toxic to the subject.
  • therapeutic agent refers to any molecule, compound, or treatment, that assists in the prevention or treatment of disorders, or complications of disorders.
  • compositions comprising such an agent formulated in a compatible pharmaceutical carrier may be prepared, packaged, and labeled for treatment.
  • the complex is water-soluble, then it may be formulated in an appropriate buffer, for example, phosphate buffered saline or other physiologically compatible solutions.
  • an appropriate buffer for example, phosphate buffered saline or other physiologically compatible solutions.
  • the resulting complex may be formulated with a non-ionic surfactant such as Tween, or polyethylene glycol.
  • a non-ionic surfactant such as Tween, or polyethylene glycol.
  • the compounds and their physiologically acceptable solvates may be formulated for administration by inhalation or insufflation (either through the mouth or the nose) or oral, buccal, parenteral, rectal administration or, in the case of tumors, directly injected into a solid tumor.
  • the pharmaceutical preparation may be in liquid form, for example, solutions, syrups or suspensions, or may be presented as a drug product for reconstitution with water or other suitable vehicle before use.
  • Such liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e. g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e. g., lecithin or acacia); non-aqueous vehicles (e. g., almond oil, oily esters, or fractionated vegetable oils); and preservatives (e. g., methyl or propyl-p- hydroxybenzoates or sorbic acid).
  • suspending agents e. g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats
  • emulsifying agents e. g., lecithin or acacia
  • non-aqueous vehicles e. g., almond oil, oily esters, or fractionated vegetable oils
  • the pharmaceutical compositions may take the form of, for example, tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e. g., pregelatinized maize starch, polyvinyl pyrrolidone or hydroxypropyl methylcellulose); fillers (e. g., lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e. g., magnesium stearate, talc or silica); disintegrants (e. g., potato starch or sodium starch glycolate); or wetting agents (e. g., sodium lauryl sulphate).
  • binding agents e. g., pregelatinized maize starch, polyvinyl pyrrolidone or hydroxypropyl methylcellulose
  • fillers e. g., lactose, microcrystalline cellulose or calcium hydrogen phosphate
  • lubricants e. g., magnesium stearate, talc or silica
  • Preparations for oral administration may be suitably formulated to give controlled release of the active compound.
  • the compounds may be formulated for parenteral administration by injection, e. g., by bolus injection or continuous infusion.
  • Formulations for injection may be presented in unit dosage form, e. g., in ampoules or in multi-dose containers, with an added preservative.
  • compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • the active ingredient may be in powder form for constitution with a suitable vehicle, e. g., sterile pyrogen-free water, before use.
  • the compounds may also be formulated as a topical application, such as a cream or lotion.
  • the compounds may also be formulated as a depot preparation.
  • Such long acting formulations may be administered by implantation (for example, intraocular, subcutaneous or intramuscular) or by intraocular injection.
  • the compounds may be formulated with suitable polymeric or hydrophobic materials (for example, as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.
  • suitable polymeric or hydrophobic materials for example, as an emulsion in an acceptable oil
  • ion exchange resins for example, as an emulsion in an acceptable oil
  • sparingly soluble derivatives for example, as a sparingly soluble salt.
  • Liposomes and emulsions are well known examples of delivery vehicles or carriers for hydrophilic drugs.
  • the compositions may, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the active ingredient.
  • 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.
  • kits for carrying out the therapeutic regimens of the invention comprise in one or more containers therapeutically or prophylactically effective amounts of the compositions in pharmaceutically acceptable form.
  • composition in a vial of a kit may be in the form of a pharmaceutically acceptable solution, e. g., in combination with sterile saline, dextrose solution, or buffered solution, or other pharmaceutically acceptable sterile fluid.
  • the complex may be lyophilized or desiccated; in this instance, the kit optionally further comprises in a container a pharmaceutically acceptable solution (e. g., saline, dextrose solution, etc.), preferably sterile, to reconstitute the complex to form a solution for injection purposes.
  • a pharmaceutically acceptable solution e. g., saline, dextrose solution, etc.
  • kits further comprises a needle or syringe, preferably packaged in sterile form, for injecting the complex, and/or a packaged alcohol pad. Instructions are optionally included for administration of compositions by a clinician or by the patient.
  • the present inventors generated an adeno associated virus (AAV) vector with the promoter of SEQ ID NO:1 followed by the sequence for EGFP. It was made into AAV (serotype AAV2/1) using the standard protocol. A titer of 4.2x10 A 11 was generated and used for injections.
  • AAV adeno associated virus
  • mice were anesthetized using a mix of fentanyl (0.05 mg/kg), medetomidine (0.5 mg/kg) and midazolam (5 mg/kg), as well as analgesics.
  • a hole was drilled through cortex above V1 , bilaterally (2.5 mm lateral of lambda),, and virus was injected at a depth of 500 micrometers (3-4 injections per mouse, approx. 100-150 nL per injection). Mice were returned to their home cage after injection.
  • tissues were collected after 14 days and fixed overnight in 4% PFA. Approximately 14 days after viral injections, mice were euthanized and tissues were isolated and fixed in paraformaldehyde (PFA) using standard protocol.
  • PFA fixed tissue was washed with PBS and sectioned via Vibratome (Leica VT1000 S) into 60 micrometer slices. Sections were slide mounted with mounting medium and visualized by confocal microscopy.

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Abstract

La présente invention présente une molécule d'acide nucléique isolée comportant, ou consistant en, la séquence d'acide nucléique de SEQ ID NO : 1 ou une séquence d'acide nucléique d'au moins 1400 pb ayant au moins 80 % d'identité avec ladite séquence de SEQ ID NO : 1, dans laquelle ladite molécule d'acide nucléique isolée conduit à l'expression spécifique d'un gène exogène dans les cellules de la couche 5 du cortex de souris lorsqu'une séquence d'acide nucléique codant pour ledit gène exogène est liée de manière fonctionnelle à ladite molécule d'acide nucléique isolée.
PCT/IB2024/058069 2023-08-22 2024-08-20 Promoteur pour l'expression spécifique de gènes dans les cellules de la couche 5 du cortex de la souris Pending WO2025041032A1 (fr)

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WO2012061741A2 (fr) * 2010-11-05 2012-05-10 The Board Of Trustees Of The Leland Stanford Junior University. Contrôle et caractérisation d'états psychotiques

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