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WO2018112815A1 - Modèle animal pour syndrome de perte auditive et sa méthode t de traitement - Google Patents

Modèle animal pour syndrome de perte auditive et sa méthode t de traitement Download PDF

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Publication number
WO2018112815A1
WO2018112815A1 PCT/CN2016/111403 CN2016111403W WO2018112815A1 WO 2018112815 A1 WO2018112815 A1 WO 2018112815A1 CN 2016111403 W CN2016111403 W CN 2016111403W WO 2018112815 A1 WO2018112815 A1 WO 2018112815A1
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Prior art keywords
pou4f3
transgenic mouse
gene
dfna15
disease
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PCT/CN2016/111403
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English (en)
Inventor
Minsheng Zhu
Guangjie ZHU
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Nanjing University
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Priority to PCT/CN2016/111403 priority Critical patent/WO2018112815A1/fr
Priority to CN201680091790.6A priority patent/CN110099999B/zh
Publication of WO2018112815A1 publication Critical patent/WO2018112815A1/fr

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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • A01K67/0278—Knock-in vertebrates, e.g. humanised vertebrates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/0004—Screening or testing of compounds for diagnosis of disorders, assessment of conditions, e.g. renal clearance, gastric emptying, testing for diabetes, allergy, rheuma, pancreas functions
    • A61K49/0008—Screening agents using (non-human) animal models or transgenic animal models or chimeric hosts, e.g. Alzheimer disease animal model, transgenic model for heart failure
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • 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
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A01K2267/0306—Animal model for genetic diseases

Definitions

  • the present disclosure generally relates to animal models for progressive hearing loss, and particularly to animal models for DFNA15 disease and treatment method thereof.
  • ADNSHL autosomal dominant inheritance of nonsyndromic hearing loss
  • DFNA15 Autosomal dominant deafness-15
  • DFNA15 is a form of progressive non-syndromic sensorineural hearing loss with post lingual onset between the second and sixth decades of life. While it has been suggested that mutation of one or more genes might be associated with DFNA15, the direct gene-to-phenotype evidence has been yet determined.
  • Embodiments of the present disclosure relate to a transgenic mouse whose genome includes a disruption of Pou4f3 gene.
  • the transgenic mouse exhibits reduced hearing ability as compared to hearing ability of a wild-type mouse and/or exhibits a symptom of DFNA15 disease.
  • Some embodiments further relate to a method of testing for therapeutic efficacy of an agent on one or more symptoms of DFNA15 disease.
  • the method may include applying one or more agents to be tested to a transgenic mouse whose genome may include a disruption of Pou4f3 gene.
  • the transgenic mouse exhibits reduced hearing ability as compared to hearing ability of a wild-type mouse and/or exhibits a symptom of DFNA15 disease. Further, whether one or more symptoms of DFNA15 disease have changed as a result of application of the one or more agents may be determined.
  • the method may include disrupting Pou4f3 gene in an embryonic stem cell of a mouse to generate an embryonic stem cell or a somatic cell isolated from a mouse to generate an embryo.
  • the method may further include transferring the embryonic stem cell or the embryo into a recipient female mouse such that the genome of the transgenic mouse may include a disruption of Pou4f3 gene.
  • the transgenic mouse exhibits reduced hearing ability as compared to hearing ability of a wild-type mouse and/or exhibits a symptom of DFNA15 disease
  • the disruption of Pou4f3 gene may include a heterozygous disruption of Pou4f3 gene, and the transgenic mouse expresses wild-type Pou4f3 gene.
  • the disruption of Pou4f3 gene may include a homozygous disruption of Pou4f3 gene, and the transgenic mouse does not express wild-type Pou4f3 gene.
  • the disruption of Pou4f3 gene may include a homozygous or heterozygous deletion of one or more nucleotides of Pou4f3 gene and a C/T transition of Pou4f3 gene.
  • the disruption of Pou4f3 gene is a deletion of a portion of exon 2 of Pou4f3 gene.
  • exon 1 of Pou4f3 gene of the transgenic mouse is not disrupted.
  • the symptom of the DFNA15 disease may include at least one of progressive hearing loss and impaired balance ability.
  • the one or more agents may include an agent regulating Espin expression.
  • the agent regulating Espin expression is associated with a retinoid acid signaling pathway.
  • one or more tests may include at least one of auditory brainstem responses (ABRs) , distortion product optoacoustic emissions (DPOAEs) and rotarod tests on the transgenic mouse before and after the application of the one or more agents may be performed. Further, whether results of the one or more tests have changed as the result of application of the one or more agents may determine.
  • ABRs auditory brainstem responses
  • DPOAEs distortion product optoacoustic emissions
  • rotarod tests on the transgenic mouse before and after the application of the one or more agents may be performed. Further, whether results of the one or more tests have changed as the result of application of the one or more agents may determine.
  • the improvement of the results of the one or more tests indicates the therapeutic efficacy of an agent on one or more symptoms of DFNA15 disease
  • Some embodiments further relate to an isolated tissue of the transgenic mouse as described above.
  • Some embodiments further relate to a method of treating (e.g., reducing) one or more symptoms of the DFNA15 disease of a subject.
  • the method may include administering to the subject an amount of an agent that inhibits a retinoid acid signaling pathway of the subject.
  • FIG. 1 illustrates ablation of the 8 bp and a C-T reversion in Pou4f3 gene.
  • A Schematic strategy for introducing Pou4f3 gene with 8 bp and a C-T reversion.
  • B Southern blot analysis for homologous recombinant allele. The wild-type and mutant alleles yield 6.7-kilobase (Kb) and 8.6-Kb fragments, respectively.
  • C Gross appearance of CTR and Pou4f3 ⁇ /+ mice.
  • D PCR genotyping for knock-in mice. A band in size of 542-bp can be detected in the genomic DNA from knock-in mice.
  • E Western blot analysis for POU4F3 protein from cochlea. The small band indicated the truncated protein of Pou4f3 ⁇ /+ mice.
  • FIG. 2 shows examinations for the hearing function of Pou4f3 ⁇ /+ mice.
  • A A typical recording of ABR traces. Left panel shows the ABR trace of control mice (6-7 months) in response to 16kHz stimuli with indicated intensities; Right panel shows for Pou4f3 ⁇ /+ mice (6-7 months) . *indicates the ABR threshold.
  • Error bars indicate standard error at each time point.
  • FIG. 3. illustrates measurement of vestibular function of Pou4f3 ⁇ /+ mice. Both control and Pou4f3 ⁇ /+ mice were allowed a maximum retention time of 120 seconds per trial. The results showed the ratio of animals remaining on drum of speed setting at 12 rpm (A) and 20 rpm (B) respectively.
  • Pou4f3 ⁇ /+ mice (filled circles) show shorter latencies to fall compared with the control mice (filled squares) .
  • FIG. 4 shows histological morphology of the organ of Corti of Pou4f3 ⁇ /+ mice .
  • the plastic sections of the organ of Corti were stained with Hematoxylin & eosin (H&E) .
  • H&E Hematoxylin & eosin
  • A The organ of Corti from control mice (4 months) .
  • B, C, D The organ of Corti from Pou4f3 ⁇ /+ mice (4 months) .
  • Scale bars 50 mm.
  • FIG. 5 shows SEM ultrastructure of the organ of Corti of Pou4f3 ⁇ /+ mice.
  • the organs of Corti at the middle turn from control (A) and Pou4f3 ⁇ /+ mice (B) were sampled and examined under a scan electric microscope.
  • the mutant IHCs from Pou4f3 ⁇ /+ mice (B, C, D, E) show stereocilia fusion ( ⁇ ) and overgrowth (arrowheads) . Some mutant OHCs disappeared (asterisks) .
  • the number of stereocilia of the existing OHCs (G, J) were less in contrast to control (F, I) .
  • FIG. 6 shows TEM ultrastructure of the hair cells of Pou4f3 ⁇ /+ mice.
  • the cochlea at middle turn was isolated from control and Pou4f3 ⁇ /+ mice (6-7 months) and sampled for TEM examination.
  • Two upper panels of the TEM images represent structures of IHCs, and two lower panels represent the structure of OHCs.
  • the cells delineated with white dash lines are IHC and OHC cells.
  • Asterisks indicate the vacuoles of the hair cells. Note that the number of mitochondria in the mutant hair cells is less than that of control. Quantitation for the mitochondria of hair cells is shown at left panels. Scale bars: 1 um. ***, p ⁇ 0.001.
  • FIG. 7 shows overexpression of Espin in Pou4f3 ⁇ /+ cochlea
  • B Western blot assay for Espin protein.
  • C Quantitation for Espin protein. The bars represent the means of IODESPIN/IODGAPDH ⁇ SD (integral optical density, IOD) .
  • D Regulation of Espin expression by POU4F protein. HEK293 cells were co-transfected with reporter containing Espin regulatory region and Pou4f3-or Pou4f3 ⁇ /+ -expressible vectors. 48 hours after transfection, luciferase activities were then measured. *, p ⁇ 0.05.
  • FIG. 8 shows administration ALDH inhibitor affect the hearing function of DFNA15 animals.
  • the present disclosure provides animal models of human diseases and conditions (e.g., DFNA15 deafness) .
  • the animal models can be used in methods such as identification and characterization of approaches for treatment of the diseases and conditions.
  • Embodiments of the present disclosure relate to genetic modifications that result in animals having one or more symptoms characteristic of the disease or condition. Animals exhibiting such symptoms are particularly advantageous in the development of therapeutic approaches, as candidate drugs and other approaches to treatment can be evaluated for effects on the symptoms in such animals. Thus, in addition to the animal models themselves, the present disclosure further provides methods of using the animals for identifying and characterizing treatments. In some embodiments, the present disclosure includes methods of making transgenic non-human animal models and cells that can be used in these methods. In some embodiments, the present disclosure further relates to methods for treating the diseases and conditions.
  • an element means one element or more than one element.
  • binding means that one molecule recognizes and adheres to a particular second molecule in a sample or organism, but does not substantially recognize or adhere to other structurally unrelated molecules in the sample.
  • coding sequence is meant any nucleic acid sequence that contributes to the code for the polypeptide product of a gene.
  • non-coding sequence refers to any nucleic acid sequence that does not contribute to the code for the polypeptide product of a gene.
  • complementarity refers to polynucleotides (i.e., a sequence of nucleotides) related by the base-pairing rules.
  • sequence “A-G-T, ” is complementary to the sequence “T-C-A. ”
  • Complementarity may be “partial, ” in which only some of the nucleic acids’bases are matched according to the base pairing rules. Or, there may be “complete” or “total” complementarity between the nucleic acids. The degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of hybridization between nucleic acid strands.
  • a “decreased” or “reduced” or “lesser” amount is typically a “statistically significant” or a physiologically significant amount, and may include a decrease that is about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6 1.7, 1.8, 1.9, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, or 50 or more times (e.g., 100, 500, 1000 times) (including all integers and decimal points in between and above 1, e.g., 1.5, 1.6, 1.7. 1.8, etc. ) an amount or level described herein.
  • targeted gene may also be accomplished by targeting the mRNA of that gene, such as by using various antisense technologies (e.g., antisense oligonucleotides and siRNA) known in the art. Accordingly, targeted genes may be considered “non-functional” when the polypeptide or enzyme encoded by that gene is not expressed by the modified cell, or is expressed in negligible amounts, such that the modified cell produces or accumulates less of the polypeptide or enzyme product (e.g., albumin) than an unmodified or differently modified cell.
  • the polypeptide or enzyme product e.g., albumin
  • exogenous refers to a polynucleotide sequence that does not naturally-occur in a wild-type cell or organism, but is typically introduced into the cell by molecular biological techniques.
  • exogenous polynucleotides include vectors, plasmids, and/or man-made nucleic acid constructs encoding a desired protein.
  • endogenous or “native” refers to naturally-occurring polynucleotide sequences that may be found in a given wild-type cell or organism.
  • polynucleotide sequences that is isolated from a first organism and transferred to second organism by molecular biological techniques is typically considered an “exogenous” polynucleotide with respect to the second organism.
  • polynucleotide sequences can be “introduced” by molecular biological techniques into a microorganism that already contains such a polynucleotide sequence, for instance, to create one or more additional copies of an otherwise naturally-occurring polynucleotide sequence, and thereby facilitate overexpression of the encoded polypeptide.
  • the terms “function” and “functional” and the like refer to a biological, enzymatic, or therapeutic function.
  • gene is meant a unit of inheritance that occupies a specific locus on a chromosome and consists of transcriptional and/or translational regulatory sequences and/or a coding region and/or non-translated sequences (i.e., introns, 5’and 3’untranslated sequences) .
  • Homology refers to the percentage number of amino acids that are identical or constitute conservative substitutions. Homology may be determined using sequence comparison programs such as GAP (Deveraux et al., 1984, Nucleic Acids Research 12, 387-395) which is incorporated herein by reference. In this way sequences of a similar or substantially different length to those cited herein could be compared by insertion of gaps into the alignment, such gaps being determined, for example, by the comparison algorithm used by GAP.
  • host cell includes an individual cell or cell culture which can be or has been a recipient of any recombinant vector (s) or isolated polynucleotide of the invention.
  • Host cells include progeny of a single host cell, and the progeny may not necessarily be completely identical (in morphology or in total DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation and/or change.
  • a host cell includes cells transfected or infected in vivo or in vitro with a recombinant vector or a polynucleotide of the invention.
  • a host cell which includes a recombinant vector of the invention is a recombinant host cell.
  • isolated is meant material that is substantially or essentially free from components that normally accompany it in its native state.
  • an “isolated polynucleotide” refers to a polynucleotide, which has been purified from the sequences which flank it in a naturally-occurring state, e.g., a DNA fragment which has been removed from the sequences that are normally adjacent to the fragment.
  • an “isolated peptide” or an “isolated polypeptide” and the like, as used herein refer to in vitro isolation and/or purification of a peptide or polypeptide molecule from its natural cellular environment, and from association with other components of the cell.
  • labeled with regard to a probe or antibody, is intended to encompass direct labeling of the probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody.
  • locus is the specific physical location of a DNA sequence (e.g. of a gene) on a chromosome.
  • locus usually refers to the specific physical location of a target sequence on a chromosome.
  • a sample such as, for example, a polynucleotide or polypeptide is isolated from, or derived from, a particular source, such as a desired organism or a specific tissue within a desired organism.
  • Obtained from can also refer to the situation in which a polynucleotide or polypeptide sequence is isolated from, or derived from, a particular organism or tissue within an organism.
  • a polynucleotide sequence encoding a reference polypeptide described herein may be isolated from a variety of prokaryotic or eukaryotic organisms, or from particular tissues or cells within certain eukaryotic organism.
  • polynucleotide or “nucleic acid” as used herein designates mRNA, RNA, cRNA, rRNA, cDNA or DNA.
  • the term typically refers to polymeric form of nucleotides of at least 10 bases in length, either ribonucleotides or deoxynucleotides or a modified form of either type of nucleotide.
  • the term includes single and double stranded forms of DNA and RNA.
  • polynucleotide variant and “variant” and the like refer to polynucleotides displaying substantial sequence identity with a reference polynucleotide sequence or polynucleotides that hybridize with a reference sequence under stringent conditions that are defined hereinafter. These terms also encompass polynucleotides that are distinguished from a reference polynucleotide by the addition, deletion or substitution of at least one nucleotide. Accordingly, the terms “polynucleotide variant” and “variant” include polynucleotides in which one or more nucleotides have been added or deleted, or replaced with different nucleotides.
  • polynucleotide variants include, for example, polynucleotides having at least 50% (and at least 51%to at least 99%and all integer percentages in between, e.g., 90%, 95%, or 98%) sequence identity with a reference polynucleotide sequence described herein.
  • polynucleotide variant and variant also include naturally-occurring allelic variants and orthologs that encode these enzymes.
  • a targeted gene may be rendered “non-functional” by changes or mutations at the nucleotide level that alter the amino acid sequence of the encoded polypeptide, such that a modified polypeptide is expressed, but which has reduced function or activity with respect to its activity (e.g., introducing transportation of albumin) , whether by modifying that polypeptide’s active site, its cellular localization, its stability, or other functional features apparent to a person skilled in the art.
  • modifications to the coding sequence of a polypeptide involved in albumin expression may be accomplished according to known techniques in the art, such as site directed mutagenesis at the genomic level and/or natural selection (i.e., directed evolution) of a given cell.
  • Polypeptide, ” “polypeptide fragment, ” “peptide” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues and to variants and synthetic analogues of the same. Thus, these terms apply to amino acid polymers in which one or more amino acid residues are synthetic non-naturally occurring amino acids, such as a chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally-occurring amino acid polymers.
  • polypeptide variant refers to polypeptides that are distinguished from a reference polypeptide sequence by the addition, deletion or substitution of at least one amino acid residue.
  • a polypeptide variant is distinguished from a reference polypeptide by one or more substitutions, which may be conservative or non-conservative.
  • the polypeptide variant includes conservative substitutions and, in this regard, it is well understood in the art that some amino acids may be changed to others with broadly similar properties without changing the nature of the activity of the polypeptide.
  • Polypeptide variants also encompass polypeptides in which one or more amino acids have been added or deleted, or replaced with different amino acid residues.
  • reference sequence refers generally to a nucleic acid coding sequence, or amino acid sequence, to which another sequence is being compared. All polypeptide and polynucleotide sequences described herein are included as references sequences, including those described by name (e.g., ⁇ 2 microglobulin proteins) and those described in the Sequence Listing.
  • sample is used herein in its broadest sense.
  • a sample including polynucleotides, peptides, antibodies and the like may include a bodily fluid, a soluble fraction of a cell preparation or media in which cells were grown, genomic DNA, RNA or cDNA, a cell, a tissue, skin, hair and the like.
  • samples include saliva, serum, biopsy specimens, blood, urine, and plasma.
  • sequence identity or, for example, including a “sequence 50%identical to, ” as used herein, refer to the extent that sequences are identical on a nucleotide-by-nucleotide basis or an amino acid-by-amino acid basis over a window of comparison.
  • a “percentage of sequence identity” may be calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, I) or the identical amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys and Met) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size) , and multiplying the result by 100 to yield the percentage of sequence identity.
  • the identical nucleic acid base e.g., A, T, C, G, I
  • the identical amino acid residue e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp,
  • nucleotides and polypeptides having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%or 100%sequence identity to any of the reference sequences described herein (see, e.g., Sequence Listing) , typically where the polypeptide variant maintains at least one biological activity of the reference polypeptide.
  • references to describe sequence relationships between two or more polynucleotides or polypeptides include “reference sequence” , “comparison window” , “sequence identity” , “percentage of sequence identity” and “substantial identity” .
  • a “reference sequence” is at least 12 but frequently 15 to 18 and often at least 25 monomer units, inclusive of nucleotides and amino acid residues, in length.
  • two polynucleotides may each include (1) a sequence (i.e., only a portion of the complete polynucleotide sequence) that is similar between the two polynucleotides, and (2) a sequence that is divergent between the two polynucleotides
  • sequence comparisons between two (or more) polynucleotides are typically performed by comparing sequences of the two polynucleotides over a “comparison window” to identify and compare local regions of sequence similarity.
  • a “comparison window” refers to a conceptual segment of at least 6 contiguous positions, usually about 50 to about 100, more usually about 100 to about 150 in which a sequence is compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned.
  • the comparison window may include additions or deletions (i.e., gaps) of about 20%or less as compared to the reference sequence (which does not include additions or deletions) for optimal alignment of the two sequences.
  • Optimal alignment of sequences for aligning a comparison window may be conducted by computerized implementations of algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive Madison, WI, USA) or by inspection and the best alignment (i.e., resulting in the highest percentage homology over the comparison window) generated by any of the various methods selected.
  • GAP Garnier et al.
  • BESTFIT Pearson FASTA
  • FASTA Pearson's Alignment of sequences
  • TFASTA Pearson's Alignin
  • Statistical significance By “statistically significant, ” it is meant that the result was unlikely to have occurred by chance. Statistical significance can be determined by any method known in the art. Commonly used measures of significance include the p-value, which is the frequency or probability with which the observed event would occur, if the null hypothesis were true. If the obtained p-value is smaller than the significance level, then the null hypothesis is rejected. In simple cases, the significance level is defined at a p-value of 0.05 or less.
  • substantially or “essentially” means nearly totally or completely, for instance, 95%, 96%, 97%, 98%, 99%or greater of some given quantity.
  • Transformation refers to the permanent, heritable alteration in a cell resulting from the uptake and incorporation of foreign DNA into the host-cell genome; also, the transfer of an exogenous gene from one organism into the genome of another organism.
  • vector is meant a polynucleotide molecule, preferably a DNA molecule derived, for example, from a plasmid, bacteriophage, yeast or virus, into which a polynucleotide can be inserted or cloned.
  • a vector preferably contains one or more unique restriction sites and can be capable of autonomous replication in a defined host cell including a target cell or tissue or a progenitor cell or tissue thereof, or be integrable with the genome of the defined host such that the cloned sequence is reproducible.
  • the vector can be an autonomously replicating vector, i.e., a vector that exists as an extra-chromosomal entity, the replication of which is independent of chromosomal replication, e.g., a linear or closed circular plasmid, an extra-chromosomal element, a mini-chromosome, or an artificial chromosome.
  • the vector can contain any means for assuring self-replication.
  • the vector can be one which, when introduced into the host cell, is integrated into the genome and replicated together with the chromosome (s) into which it has been integrated.
  • Such a vector may include specific sequences that allow recombination into a particular, desired site of the host chromosome.
  • a vector system can include a single vector or plasmid, two or more vectors or plasmids, which together contain the total DNA to be introduced into the genome of the host cell, or a transposon.
  • the choice of the vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced.
  • the vector is preferably one which is operably functional in a host cell, such as a plasmid.
  • the vector can include a reporter gene, such as a green fluorescent protein (GFP) , which can be either fused in frame to one or more of the encoded polypeptides, or expressed separately.
  • the vector can also include a selection marker such as an antibiotic resistance gene that can be used for selection of suitable transformants.
  • wild-type refers to a gene or gene product that has the characteristics of that gene or gene product when isolated from a naturally-occurring source.
  • a wild-type gene or gene product e.g., a polypeptide
  • a wild-type gene or gene product is that which is most frequently observed in a population and is thus arbitrarily designed the “normal” or “wild-type” form of the gene.
  • heterologous refers to a combination of elements not naturally occurring.
  • heterologous DNA refers to DNA not naturally located in the cell, or in a chromosomal site of the cell. It is contemplated that the heterologous DNA includes a gene foreign to the cell.
  • a heterologous expression regulatory element is such an element operatively associated with a different gene than the one it is operatively associated with in nature.
  • homologous refers to the relationship between proteins that possess a “common evolutionary origin, ” including proteins from super-families (e.g., the immunoglobulin superfamily) and homologous proteins from different species (e.g., myosin light chain, etc. ) (Reeck et al., Cell 50: 667, 1987) .
  • proteins and their encoding genes
  • sequence homology as reflected by their sequence similarity, whether in terms of percent similarity or the presence of specific residues or motifs at conserved positions.
  • the terms “express” and “expression” refer to allowing or causing the information in a gene or DNA sequence to become manifest, for example producing a protein by activating the cellular functions involved in transcription and translation of a corresponding gene or DNA sequence.
  • a DNA sequence is expressed in or by a cell to form an “expression product” such as a protein.
  • the expression product itself e.g. the resulting protein, may also be said to be “expressed. ”
  • An expression product is, in various aspects, characterized as intracellular, extracellular or secreted.
  • intracellular means inside a cell.
  • extracellular means outside a cell, such as a transmembrane protein.
  • a substance is “secreted” by a cell if it appears in significant measure outside the cell, from somewhere on or inside the cell.
  • transfection refers to the introduction of a foreign nucleic acid into a cell.
  • transformation refers to the introduction of a “foreign” (i.e. exogenous, heterologous, extrinsic or extracellular) gene, DNA or RNA sequence to an embryonic stem (ES) cell or pronucleus, so that the cell will express the introduced gene or sequence to produce a desired substance in a transgenic animal.
  • ES embryonic stem
  • a coding sequence is “under the control of, ” “operably linked to” or “operatively associated with” transcriptional and translational control sequences in a cell when RNA polymerase transcribes the coding sequence into RNA, which is then trans-RNA spliced (if it contains introns) and translated, in the case of mRNA, into the protein encoded by the coding sequence.
  • a “transgenic animal” is a non-human animal in which one or more, and preferably essentially all, of the cells of the animal contain a transgene introduced by way of human intervention, such as by transgenic techniques known in the art.
  • the transgene can be introduced into the cell, directly or indirectly by introduction into a precursor of the cell, by way of deliberate genetic manipulation, such as by microinjection or by infection with a recombinant virus.
  • the term “genome” as used herein, can refer to sequences, either DNA, RNA or cDNA derived from a patient, a tissue, an organ, a single cell, a tumor, a specimen of an organic fluid taken from a patient, freely circulating nucleic acid, a fungus, a prokaryotic organism and a virus.
  • Chimeric as used herein (e.g., “chimeric animal” or “chimeric liver” ) is meant to describe an organ or animal including xenogeneic tissues or cells.
  • a “knock-out” of a gene means an alteration in the sequence of the gene that results in a decrease of function of the target gene, preferably such that target gene expression is undetectable or insignificant.
  • Transgenic knock-out animals can include a heterozygous knock-out of a target gene, or a homozygous knock-out of a target gene.
  • “Knock-outs” as used herein also include conditional knock-outs, where alteration of the target gene can occur upon, for example, exposure of the animal to a substance that promotes target gene alteration, introduction of an enzyme that promotes recombination at the target gene site (e.g., Cre in the Cre-lox system) , or other method for directing the target gene alteration postnatally.
  • a “knock-in” of a target gene means an alteration in a host cell genome that results in altered expression (e.g., increased (including ectopic) or decreased expression) of a target gene, e.g., by introduction of an additional copy of the target gene, or by operatively inserting a regulatory sequence that provides for enhanced expression of an endogenous copy of the target gene.
  • “Knock-in” transgenics can include a heterozygous knock-in of the target gene or a homozygous knock-in of a target gene. “Knock-ins” also encompass conditional knock-ins.
  • treatment generally mean obtaining a desired pharmacologic and/or physiologic effect.
  • the effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of a partial or complete cure for a disease and/or adverse effect attributable to the disease.
  • Treatment covers any treatment of a disease in a mammal, particularly a human, and includes: (a) preventing the disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development; or (c) relieving the disease, i.e., causing regression of the disease.
  • Embodiments of the present disclosure relate to a transgenic mouse whose genome includes a disruption of Pou4f3 gene.
  • the transgenic mouse exhibits reduced hearing ability as compared to hearing ability of a wild-type mouse and/or exhibits a symptom of DFNA15 disease.
  • Some embodiments further relate to a method of testing for therapeutic efficacy of an agent on one or more symptoms of DFNA15 disease.
  • the method may include applying one or more agents to be tested to a transgenic mouse whose genome may include a disruption of Pou4f3 gene.
  • the transgenic mouse exhibits reduced hearing ability as compared to hearing ability of a wild-type mouse and/or exhibits a symptom of DFNA15 disease. Further, whether one or more symptoms of DFNA15 disease have changed as a result of application of the one or more agents may be determined.
  • the method may include disrupting Pou4f3 gene in an embryonic stem cell of a mouse to generate an embryonic stem cell or a somatic cell isolated from a mouse to generate an embryo.
  • the method may further include transferring the embryonic stem cell or the embryo into a recipient female mouse such that the genome of the transgenic mouse may include a disruption of Pou4f3 gene.
  • the transgenic mouse exhibits reduced hearing ability as compared to hearing ability of a wild-type mouse and/or exhibits a symptom of DFNA15 disease.
  • DFNA15 Autosomal dominant deafness-15
  • DFNA15 is a form of progressive non-syndromic sensorineural hearing loss with post-lingual onset between the second and sixth decades of life. Examples of symptoms of DFNA15 includes congenital hearing loss, progressive hearing loss and impaired balance ability. While DFNA15 is believed to be associated with mutation of Pou4f3 gene, its pathology has long been unknown due to the impossible collection of human biopsy. The embodiments of the present disclosure provide a DFNA15 disease model in mice with a mutation of Pou4f3 gene of the family H.
  • the mutant mice (Pou4f3 ⁇ /+ ) has a heterozygous disruption of Pou4f3 gene of mice and exhibits a progressive hearing loss and vestibular dysfunction in a similar manner of DFNA15 patients.
  • Histology of the mutant Corti’s organ shows progressive spare inner hair cells with long and fused stereocilia.
  • the out hair cells of the mutant mice show slight longer but less stereocilia, along with disorganized cell bodies. Both inner and out hair cells have degenerative alterations as evidenced by less mitochondrion.
  • the mutant cochlea shows overexpression of Espin, a gene for hearing development and stereocilia maintenance. Biochemical analysis indicates that Pou4f3 may transcriptionally inhibit Espin expression and Pou4f3 mutation eliminated this effect.
  • Pou4f3 is a member of the POU family of transcription factors that regulate a wide array of neuroendocrine developmental pathways. This family is characterized by the presence of a biparitite DNA binding domain known as the POU domain which may include a POU-homeo domain and a POU-specific domain separated by a linker. All of these components are required for sequence-specific DNA binding.
  • Mutant mice provided by the present disclosure shows symptoms consisting with the appearance of progressive hearing loss. Further, IHC and OHC cells of cochlea from the mutant mice show multiple pathologies of hearing cells, such as less, fused and lengthen stereolcilia in IHC and lengthen stereoliclia in OHC. Hearing cells displayed degenerative pathology as evidenced by less mitochondria. In light of the high similarity of the functional phenotypes as well as their phenotypic occurrences between the mutant mice and human patients, the histological pathology in Pou4f3 ⁇ /+ mice may be used as animal models for pathology studies of DFNA15 cochlea.
  • the disruption of Pou4f3 gene may include a heterozygous disruption of Pou4f3 gene, and the transgenic mouse expresses wild-type Pou4f3 gene.
  • the disruption of Pou4f3 gene may include a homozygous disruption of Pou4f3 gene, and the transgenic mouse does not express wild-type Pou4f3 gene.
  • the disruption of Pou4f3 gene may include a homozygous or heterozygous deletion of one or more nucleotides of Pou4f3 gene and a C/T transition of Pou4f3 gene.
  • the disruption of Pou4f3 gene is a deletion of a portion of exon 2 of Pou4f3 gene.
  • exon 1 of Pou4f3 gene of the transgenic mouse is not disrupted.
  • the present disclosure further relates to a surprising discovery that the phenotype of Pou4f3 knockout (Pou4f3 +/- and Pou4f3 -/- ) is different from that of Pou4f3 ⁇ /+ and Pou4f3 ⁇ / ⁇ mice.
  • Pou4f3 -/- mice show deaf and lack of both IHC and OHC cells, while Pou4f3 +/- mice appear normal at embryonic and postnatal stage.
  • both Pou4f3 ⁇ /+ and Pou4f3 ⁇ / ⁇ mice showed normal cochlea at young stage, although the extent of hearing impairment varies at adulthood.
  • the one or more agents may include an agent regulating Espin expression.
  • the agent regulating Espin expression is associated with a retinoid acid signaling pathway.
  • Pou4f3 may regulate a number of target genes (such as Bdnf, NT-3, Gfi1 and Lhx3) possibly through a complex manner. Mutation of Pou4f3 in DFNA15 expectedly impair this regulatory apparatus and affect the expression of target genes. Surprisingly, the expression of the target genes including Bdnf, NT-3, Gfi1 and Lhx3 is not altered in the mutant cochlea, which is inconsistent with in vitro assay. Surprising, it has been found that Espin gene expression is elevated in the mutant cochlea.
  • the symptom of the DFNA15 disease may include at least one of progressive hearing loss and impaired balance ability.
  • one or more tests including at least one of auditory brainstem responses (ABRs) , distortion product optoacoustic emissions (DPOAEs) and rotarod tests on the transgenic mouse before and after the application of the one or more agents may be performed. Further, whether results of the one or more tests have changed as the result of application of the one or more agents may determine.
  • ABRs auditory brainstem responses
  • DPOAEs distortion product optoacoustic emissions
  • Symptoms of progressive hearing loss may include progressive loss of hearing after exposure to loud noises or with age, difficulty hearing high-frequency sounds, difficulty distinguishing words during conversations, especially when in noisy or crowded circumstances, and/or difficulty hearing over the telephone.
  • Symptom examples of impaired balance ability include an impairment in hearing, whether permanent or fluctuating, that adversely affects a subject’performance.
  • Symptoms of progressive hearing loss may include feel unsteady or dizzy, and cannot stand still.
  • the auditory brainstem response (ABR) or auditory evoked potential (AEP) test gives information about the inner ear (cochlea) and brain pathways for hearing.
  • the test can be used with a subject who have a difficult time with conventional behavioral methods of hearing screening.
  • the ABR is also indicated for the subject with signs, symptoms, or complaints suggesting a type of hearing loss in the brain or a brain pathway.
  • the ABR may be performed by pasting electrodes on the head—similar to electrodes placed around the heart when an electrocardiogram is run—and recording brain wave activity in response to sound.
  • the subject being tested rests quietly or sleeps while the test is performed. No response is necessary.
  • ABR can also be used as a screening test in newborn hearing screening programs. When used as a screening test, only one intensity or loudness level is checked, and the baby either passes or fails the screen.
  • Distortion product otoacoustic emissions are sounds generated within a listener's ears upon physical and physiological interactions between spectral components in a given auditory input.
  • the relationship between sounds generated by loudspeakers and sounds generated in the listener's ears offers fertile ground for the exploration of spatial depth in sounds.
  • the rotarod performance test is a performance test based on a rotating rod with forced motor activity being applied, usually by a rodent.
  • the test measures parameters such as riding time (seconds) or endurance.
  • the improvement of the results of the one or more tests indicates the therapeutic efficacy of an agent on one or more symptoms of DFNA15 disease
  • Some embodiments further relate to an isolated tissue of the transgenic mouse as described above.
  • Some embodiments further relate to a method of treating (e.g., reducing) one or more symptoms of the DFNA15 disease of a subject.
  • the method may include administering to the subject an amount of an agent that reduces expression or activity of Epsin sufficient to reduce one or more symptoms of the DFNA15 disease.
  • the agent may include an inhibitor that inhibits a retinoid acid signaling pathway of the subject.
  • retinoid acid may promote Espin expression in cochlea.
  • DEAB which is a ALDH inhibitor causing less production of endogenous retinoid acid
  • the ABR value of the subject may be reduced upon treating the subject with DFNA15 disease with DEAB, which is a ALDH inhibitor causing less production of endogenous retinoid acid, the ABR value of the subject may be reduced. In some embodiments, the ABR value of the subject may be reduced significantly.
  • mice were obtained by injecting the positive recombinant ES cells into embryos.
  • the germ line transmission of the offspring was identified by genomic PCR and Southern blot. PCR primer pairs shown in SEQ ID NO: 2 and 3. All the mice used here were specific pathogen-free (SPF) animals that maintained in standard animal rooms of the National Resource Center for Mutant Mice (NRCMM) of China. All experiments were approved by the Animal Care and Use Committee and carried out in accordance with the animal protocol of Model Animal Research Center of Nanjing University (permit number AP#MZ15) .
  • the intact basilar membrane of cocholea was freshly isolated from mice and lyzed with a lysis buffer containing 2%SDS, 10 mM dithiothreitol, 10%glycerol, a trace amount of Bromophenol Blue and 50 mM Tris HCl, pH 7.4 in 4°C. After homogenization and centrifugation, the protein samples were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transfer to PVDF membrane.
  • SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis
  • the membrane was respectively probed with anti-Pou4f3 (Abcam, UK) , anti- ⁇ -actin (Sigma, USA) anti-Espin (abcam, UK) and anti-GAPDH (Sigma, USA) antibodies followed by incubation with a corresponding secondary antibody.
  • the signals were visualized by incubation with the ECL substrate (Mucyte Co., Ltd, Nanjing)
  • ABR auditory brainstem response
  • At least four animals were tested in each group, and the number of tested mouse was 28, 29, 17, 17, 7 for control mice and 26, 27, 15, 20, 5 for Pou4f3 ⁇ /+ mice at 5weeks, 2-3, 4-5, 6-7, 8-9 moths respectively.
  • Quantification of waveIamplitude at supra-threshold levels of intensities at 8 kHz, 16 kHz and 32 kHz at 5 weeks and 2-3 months was here employed to detect the mutation in hearing defection at early stage.
  • Distortion product optoacoustic emissions (DPOAEs) were measured with an evoked generation workstation system III (Tucker Davis Technologies Incorporated, Gainesville, FL, USA) powered by SigGen32 software.
  • a custom plastic ear tip (diameter of 3 mm) was inserted into the ear canal after intraperitoneal anesthesia.
  • DPOAE amplitude was measured at f2 frequencies of 4, 8, 12, 16, 24 and 32 kHz and plotted after subtraction of noise floor amplitude.
  • mice motor coordination and balance were tested in the afternoon with an accelerating rotarod (UGO Basile Accelerating Rotarod, Italy) . All animals were placed on textured drums to avoid slipping. When an animal drops onto the individual sensing platforms, test results are recorded. The speed of the rotarod was set at 12 and 20 rpm for each 2-min period. Mice were given three trials with a 30-60 min interatrial rest interval. Rotarod data were analyzed with a two-way ANOVA with repeated measures.
  • mice were sacrificed with an overdose of anesthesia and then infused with phosphate-buffered solution (PBS) .
  • the isolated cochlea was fixed with 4%paraformaldehyde (PFA) .
  • Decalcification was performed with 10% (W/V) ethylenedi-amine tetra-acetic acid (EDTA) for 3 days on a shaker followed by gradient dehydration using ethanol.
  • the dehydrated specimens were penetrated and embedded with MC-Plastic I Kit (MuCyte, Nanjing) in 4°C overnight.
  • the embedding blocks were cut and stained by hematoxylin/eosin (H&E) .
  • mice were fixed by perfusion with PBS containing 2.5%glutaraldehyde following an overdose of anesthesia.
  • the inner ear tissues were isolated and decalcificated with 10% (W/V) EDTA for 2 days.
  • the epithelia of Corti’s organ were exposed and then fixed with 1%OsO 4 in H 2 O for 2 h.
  • SEM scanning electron microscopy
  • the tissues were dehydrated in an ethanol series and point-dried.
  • the dried samples were mounted on stubs, sputter-coated with gold and examined on S-3000 N scanning electron microscope (Hitachi, Tokyo, Japan) at 15 kV.
  • For TEM (transmission electron microscope) examination samples were dehydrated, infiltrated, and polymerized in araldite. Ultrathin sections (70 nm) were post-stained and examined under a Hitachi-7650 transmission electron microscope at 70kV.
  • Reverse transcription reactions were performed by using the HiScriptTM Q RT Super Mix (Vazyme, China) and q–PCR was performed with a Rremix Ex TaqTM kit (TaKaRa, Japan) and a Step One Plus TM Real-time PCR System.
  • NT-3 SEQ ID NO: 4, (R) SEQ ID NO: 5; Bdnf (F) SEQ ID NO: 6, (R) SEQ ID NO: 7; Gfi1 (F) SEQ ID NO: 8, (R) SEQ ID NO: 9; Lhx3 (F) SEQ ID NO: 10, (R) SEQ ID NO: 11; Gapdh (F) SEQ ID NO: 12, (R) SEQ ID NO: 13.
  • a 3.5Kb fragment of the regulatory region of mouse Espin gene (gene ID: 56226) was amplified from C57/BL6 mice tail DNA with a high-fidelity polymerase ExTaq.
  • the primers are shown in SEQ ID NO: 14 and SEQ ID NO: 15, where a nucleotide sequence of SEQ ID NO: 16 and a nucleotide sequence of SEQ ID NO: 17 are restriction sites for Kpn I and Mlu I, respectively.
  • PCR products were confirmed by sequencing and then ligated into a pGL3-Basic vectors containing a luciferase reporter gene (Takara, Japan) .
  • the resultant reporter pGL-Espin was transfected in HEK293 together with an internal control pRL-TK. Firefly and Renilla luciferase activities were measured 24 h after transfection with the Dual-Luciferase Reporter Assay System in a GloMax96 luminescence reader (both from Promega, Madison, WI, USA) according to the manufacturer's instructions. Relative luciferase activity was normalized by Renilla luciferase activity. All experiments were repeated independently at least for three times.
  • FIG. 1A A mouse line with an 8bp deletion and a C/T reversion of mouse Pou4f3 gene was established (Fig 1A) . Mutation of Pou4f3 allele in the target ES cells was confirmed by Southern blots analysis and sequencing (Fig. 1B) . The Ki mice with 129: B6 genetic background had been backcrossed to C57BL/6 mice for 9 generations, and the resultant heterozygotes (Pou4f3 ⁇ /+ ) were used as DFNA15 disease model. Genotyping analysis for Pou4f3 ⁇ /+ mice with tail genomic DNA showed a specific PCR product in size of 542 bp, suggesting a successful germ line transmission of the mutation (Fig 1D) .
  • Human DFNA15 deafness presents as postlingual progressive hearing loss and vestibular dysfunction.
  • Cochlear and vestibular function were measured in Pou4f3 ⁇ /+ mice using auditory brainstem responses (ABRs) , distortion product optoacoustic emissions (DPOAEs) and rotarod tests.
  • ABRs auditory brainstem responses
  • DPOAEs distortion product optoacoustic emissions
  • the Pou4f3 ⁇ /+ mutant mice showed an increase in ABR thresholds for pure tones as early as 5 weeks after birth.
  • the typical ABR trace recording was shown in Fig. 2A.
  • the threshold of 16 kHz significantly elevated from 18.45 ⁇ 1.00 dB of control to 25.74 ⁇ 1.76 dB (p ⁇ 0.01) , while the thresholds at frequency of 8 kHz and 32 kHz slightly increased without no statistical significance (p>0.05) (Fig. 2. B, C, D, E) .
  • the ABR threshold of the mutant mice elevated further at more frequencies of tone, even at click.
  • the ABR thresholds of Pou4f3 ⁇ /+ mice (4-5 month-old) to click (22.33 ⁇ 2.12 dB vs.
  • the ABR waveforms in response to clicks or tonepips usually contain five peaks (waves I-V) in mouse (Fig. 2F) , in which wave I amplitude may reflect gross activity of mouse cochlear auditory nerve. Quantification of wave I amplitude at suprathreshold levels of intensities at 8 kHz, 16 kHz and 32 kHz at 5 weeks and 2-3 months were performed [Fig. 2F, 2G] . At 60dB intensity, Pou4f3 ⁇ /+ mice showed an apparent reduction (12.56 ⁇ v in average) of wave I amplitude at their respective tonepip frequency at 5 weeks (p ⁇ 0.05, 2-way ANOVA) (Fig.
  • DPOAEs reflect functional out hair cell-derived cochlear amplifier in sensitivity of hearing and selectivity of tone frequency.
  • DPOAE of Pou4f3 ⁇ /+ mice at 4-6 months was measured when the ABR threshold elevated apparently. The result showed that DPOAE of the mutant mice was reduced significantly (p ⁇ 0.001, 2-way ANOVA, with significant pairwise post-hoc comparison at all frequency) (Fig. 2I) , suggesting the out hair cells was also affected by Pou4f3 mutation.
  • mice were placed on a rotating drum and counted the mice felling down the drum at different time point. 77.78%of Pou4f3 ⁇ /+ mice fell down the rod at a speed of 12 rpm within 120 seconds, while 27.27%for the control mice (Fig. 3A) . As the speed of rod increased to 20 rpm, 100%of the mutant mice fell off within 54 seconds, while 35.36%of control mice were still able to stay on the rod as long as 120 seconds (Fig. 3B) .
  • mice In average, both 2-3 month-old mice and 20-30 years old patients show no apparent hearing loss, while both 8-9 month-old mice and 41-50 years old patients show an increase in ABR threshold at higher frequency tone. Interestingly, almost all 11-12 month-old mice show high ABR threshold (>70 dB) , indicating a consistent deafness occurring in the mice. However, some patients at age of 51-60 still have a relative small ABR threshold although the average threshold values elevated. Such a phenotypic variation may reflect the diverse gene interaction affected by POU4F3. It is suggested that Pou4f3 ⁇ /+ mice display a comparable phenotypic onset and progression to family H patients, but the phenotypes of patients may be more variable.
  • mutant hair cells To examine the structure of the mutant hair cells, TEM (transmission electron microscope) analyses was performed. The result showed that the mutant IHCs and OHCs had normal nuclei, cuticular plate, cellular organelles and tight junctions. Interestingly, the mutant IHCs and OHCs showed reduced mitochondria by 50%and serval vacuoles were observed in the mitochondria (Fig. 6) . This observation implied a degenerative alteration in both hearing cells.
  • Espin Over expression of Espin may underlie the phenotypic alteration of DFNA15A.
  • POU4F3 presumably regulate the expression of several target genes.
  • the mRNA from control and Pou4f3 ⁇ /+ cochlear tissues was prepared and then subjected to micro-array and real time PCR analysis.
  • the genes with 2-folds reduction include ion channels (e.g. chloride channel and non-selective sodium leak channel) , matrix proteins (e.g. fibrinogen alpha chain) , olfactory receptors; the genes with significant elevation include protein tyrosine phosphatase, caspase 7 etc. Functional prediction implies these genes unlikely involved in the processes of hearing loss of these mice.
  • mice at age of two months in C57/BL6j background were i. p administrated daily with N, N-diethylaminobenzaldehyde (DEAB) a dose of 100mg/kg/day for 38 days. DEAB was dissolved in 100%dimethylsulfoxide DMSO and was diluted to the final 10%DMSO concentration with corn oil. The mice were anesthetized by intraperitoneal injection with Avertin at an initial dose of 500 mg/kg body weight, and the anesthesia was maintained with a half-dose delivered every 20 min.
  • DEAB N, N-diethylaminobenzaldehyde

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Abstract

Une souris transgénique dont le génome comprend une interruption du gène Pou4f3 présente une capacité auditive réduite par rapport à la capacité auditive d'une souris de type sauvage et/ou présente un symptôme de maladie DFNA15. L'invention porte sur une méthode de préparation d'une souris transgénique. Une méthode de réduction d'un ou plusieurs symptômes de la maladie DFNA15 d'un sujet comprend l'administration au sujet d'une quantité d'un agent qui inhibe une voie de signalisation de l'acide rétinoïque du sujet.
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