WO2000023084A1 - Procedes pour stimuler la regeneration des cellules de l'oreille interne - Google Patents

Procedes pour stimuler la regeneration des cellules de l'oreille interne Download PDF

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WO2000023084A1
WO2000023084A1 PCT/US1999/024829 US9924829W WO0023084A1 WO 2000023084 A1 WO2000023084 A1 WO 2000023084A1 US 9924829 W US9924829 W US 9924829W WO 0023084 A1 WO0023084 A1 WO 0023084A1
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inner ear
cell
cells
nucleic acid
pou4f3
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PCT/US1999/024829
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English (en)
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Jonathan Kil
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Jonathan Kil
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Priority to AU12235/00A priority Critical patent/AU1223500A/en
Publication of WO2000023084A1 publication Critical patent/WO2000023084A1/fr
Priority to US10/458,108 priority patent/US7132406B2/en
Priority to US11/588,910 priority patent/US7741303B2/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—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
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0618—Cells of the nervous system
    • C12N5/062—Sensory transducers, e.g. photoreceptors; Sensory neurons, e.g. for hearing, taste, smell, pH, touch, temperature, pain
    • 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
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/40—Regulators of development
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/60—Transcription factors

Definitions

  • the invention relates to the promotion of inner ear sensory cell regeneration by introducing transcription factor genes such as the transcription factor POU4F3.
  • SNHL Sensorineuronal hearing loss
  • hair cells Loss of the inner ear sensory cell (i.e., hair cells) is thought to be a major cause of this deficit.
  • SNHL can be caused by a multitude of events including age- related loss (presbycusis), noise exposure, drug exposure (e.g., antibiotics and anti- cancer therapeutics), infections, genetic mutations (syndromic and non-syndromic) and autoimmune disease.
  • age- related loss Presbycusis
  • drug exposure e.g., antibiotics and anti- cancer therapeutics
  • infections e.g., antibiotics and anti- cancer therapeutics
  • genetic mutations syndromic and non-syndromic
  • apical turn hair cells are highly resistant to aminoglycoside-induced death and therefore such cultures are not suitable to the study of hair cell regeneration.
  • P7-14 a method for lesioning the apical turn of the older neonatal organ of Corti, i.e., P7-14.
  • P7-14 mice apical turn hair cells are more susceptible to aminoglycoside ototoxicity than younger embryos, and can be effectively lesioned.
  • Reasons for this increased susceptibility remain unknown, but may be due to the onset of auditory function at this time in development.
  • POU4F3 a POU domain transcription factor called POU4F3.
  • Brn 3 c the mouse homolog of human POU4F3 is expressed in limited regions of the brain, retina, dorsal root and trigeminal ganglia, and in inner ear sensory hair cells (Xiang, M. et al., J. Ne roscience, 15(7):4762-4785 (1995)).
  • mice of the Brn 3 c gene results in the failure of inner ear hair cells to develop and differentiate in the postnatal period of mice (Erkman, L., et al., Nature, 381:603-606 (1996); Xiang, M et al., Proc. Natl. Acad. Sci. USA, 94:9445-9450 (1997)).
  • Mice with this deletion have permanent deficits in hearing and balance, while heterozygous mice have no obvious deficits as compared with wild-type mice.
  • a form of adult-onset SNHL was identified in an Israeli family with a mutation in one allele of POU4F3 that develops SNHL from the age of 18 to 30 (Vahava, O.
  • the present invention provides methods for stimulating the regeneration of inner ear cells comprising introducing a nucleic acid molecule (such as the nucleic acid molecule set forth in SEQ ID NO:l), that encodes an inner ear cell transcription factor capable of stimulating the regeneration of inner ear cells, into at least one inner ear cell under conditions that enable expression of the transcription factor in the inner ear cell.
  • inner ear cell transcription factors are capable of stimulating the regeneration of sensory hair cells from non-sensory cells and are at least 25% homologous, more preferably at least 50% homologous, most preferably at least 75% homologous to the POU4F3 transcription factor having the amino acid sequence set forth in SEQ ID NO:2.
  • nucleic acid molecules encoding an inner ear cell transcription factor encode a POU4F3 homologue and hybridize to the nucleic acid molecule set forth in SEQ ID NO: 1 (or to its complementary sequence) under stringent conditions.
  • Any type of inner ear cell can be treated in accordance with the present invention, but the presently preferred cells treated in accordance with the present invention are inner ear supporting cells.
  • inner ear sensory hair cells are regenerated from inner ear supporting cells.
  • any art-recognized gene delivery method can be used to introduce a nucleic acid molecule, encoding an inner ear cell transcription factor, into inner ear cells for expression therein.
  • the presently preferred gene delivery method is lipofection.
  • the present invention provides methods for ameliorating the symptoms of an inner ear disease.
  • the methods for ameliorating the symptoms of an inner ear disease comprise introducing a nucleic acid molecule that encodes an inner ear cell transcription factor, capable of stimulating the regeneration of inner ear cells, into a portion of the inner ear (preferably the cochlea, semicircular canals and/or otolithic organs) under conditions that enable the uptake of the nucleic acid molecule by inner ear cells and expression of the transcription factor within the inner ear cells.
  • Presently preferred inner ear cells suitable for treatment in accordance with this aspect of the present invention are inner ear supporting cells.
  • inner ear sensory hair cells are regenerated from inner ear supporting cells.
  • a nucleic acid molecule that encodes an inner ear cell transcription factor can be introduced into a portion of the inner ear by any art-recognized means, such as by injection.
  • a presently preferred method for introducing a nucleic acid molecule, that encodes an inner ear cell transcription factor, into the cochlea is by cochleostomy.
  • the present invention provides genetically-transformed inner ear cells, preferably supporting cells or sensory hair cells, including a transgene encoding an inner ear cell transcription factor.
  • FIGURE shows a cross section of the Organ of Corti.
  • amino acid and “amino acids” refer to all naturally occurring L- ⁇ -amino acids or their residues.
  • the amino acids are identified by either the single-letter or three-letter designations:
  • nucleotide means a monomeric unit of D ⁇ A or R ⁇ A containing a sugar moiety (pentose), a phosphate and a nitrogenous heterocyclic base.
  • the base is linked to the sugar moiety via the glycosidic carbon (1* carbon of pentose) and that combination of base and sugar is called a nucleoside.
  • the base characterizes the nucleotide with the four bases of D ⁇ A being adenine ("A"), guanine (“G”), cytosine ("C”) and thymine (“T”).
  • Inosine is a synthetic base that can be used to substitute for any of the four, naturally-occurring bases (A, C, G or T).
  • the four R ⁇ A bases are A,G,C and uracil ("U").
  • the nucleotide sequences described herein comprise a linear array of nucleotides connected by phosphodiester bonds between the 3' and 5' carbons of adjacent pentoses.
  • Oligonucleotide refers to short length single or double stranded sequences of deoxyribonucleotides linked via phosphodiester bonds.
  • the oligonucleotides are chemically synthesized by known methods and purified, for example, on polyacrylamide gels.
  • supporting cells means cells that physically support, or provide nutrients to, sensory inner ear cells, such as the sensory hair cells of the Organ of Corti.
  • supporting cells include border cells , inner pillar cells, outer pillar cells, inner phalangeal cells, Dieter's cells and Hensen's cells.
  • inner ear cell transcription factor refers to transcription factors that are normally expressed in at least one type of developing and/or mature inner ear cell, and have the ability to stimulate regeneration of inner ear cells, preferably stimulate the regeneration of inner ear sensory hair cells from supporting cells, when utilized in the practice of the methods of the present invention.
  • inner ear cell transcription factors useful in the practice of the present invention are required for the normal development, and or for the normal functioning, of the inner ear cell type(s) in which they are normally expressed.
  • Representative examples of inner ear cell transcription factors useful in the practice of the present invention include POU4F1 (Collum, R.G.
  • the term "homeodomain” means an amino acid sequence that is at least 50% homologous, more preferably at least 75% homologous, most preferably at least 90% homologous to the homeodomain amino acid sequence set forth in SEQ ID NO:3.
  • amino acid sequence homology also referred to as amino acid sequence identity
  • amino acid sequence identity is defined as the percentage of amino acid residues in the amino acid sequence set forth in SEQ ID NO: 3 that are identical with part or all of a candidate protein sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent homology (identity), and not considering any conservative substitutions as part of the sequence homology. Neither N- or C- terminal extensions nor insertions shall be construed as reducing homology.
  • POU-specific domain means an amino acid sequence that is at least 50% homologous, more preferably at least 75% homologous, most preferably at least 90% homologous to the POU-specific domain amino acid sequence set forth in SEQ ID NO:4.
  • amino acid sequence homology (also referred to as amino acid sequence identity) is defined as the percentage of amino acid residues in the amino acid sequence set forth in SEQ ID NO: 4 that are identical with part or all of a candidate protein sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent homology (identity), and not considering any conservative substitutions as part of the sequence homology. Neither N- or C- terminal extensions nor insertions shall be construed as reducing homology. No weight is given to the number or length of gaps introduced, if necessary, to achieve the maximum percent homology (identity).
  • POU4F3 transcription factor homologues are POU4F3 transcription factor homologues (hereinafter referred to as POU4F3 homologues).
  • POU4F3 homologues useful in the practice of the present invention are capable of stimulating the regeneration of inner ear cells (preferably capable of stimulating the regeneration of sensory hair cells from supporting cells) and are at least 25% homologous, preferably at least 50% homologous, more preferably at least 75% homologous, most preferably at least 90% homologous to the POU4F3 transcription factor having the amino acid sequence set forth in SEQ ID NO:2.
  • amino acid sequence homology is defined as the percentage of amino acid residues in the POU4F3 transcription factor having the amino acid sequence set forth in SEQ ID NO: 2 that are identical with part or all of a candidate protein sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent homology (identity), and not considering any conservative substitutions as part of the sequence homology. Neither N- or C- terminal extensions nor insertions shall be construed as reducing homology. No weight is given to the number or length of gaps introduced, if necessary, to achieve the maximum percent homology (identity).
  • POU4F3 homologues includes the POU4F3 protein having the amino acid sequence set forth in SEQ ID NO:2, which is the presently most preferred inner ear cell transcription factor useful in the practice of the present invention. Representative examples of other POU4F3 homologues useful in the practice of the present invention are set forth in Xiang, M. et al., J. Neuroscience, 15(7):4762-4785 (1995), incorporated herein by reference.
  • the term “stimulate regeneration of an inner ear cell”, and grammatical equivalents means to stimulate the complete or partial regeneration of an inner ear cell.
  • inner ear sensory hair cells are regenerated from inner ear supporting cells.
  • fully functional inner ear cells are regenerated.
  • alteration refers to protein molecules with some differences in their amino acid sequences as compared to the corresponding, native, i.e., naturally-occurring, proteins. Ordinarily, the variants will possess at least about 70% homology with the corresponding native protein, and preferably, they will be at least about 80% homologous with the corresponding, native protein.
  • the amino acid sequence variants of the transcription factors useful in the practice of the present invention possess substitutions, deletions, and/or insertions at certain positions. Sequence variants of inner ear cell transcription factors may, for example, be more effective at stimulating regeneration of inner ear cells when used in accordance with the present invention.
  • Substitutional protein variants are those that have at least one amino acid residue in the native protein sequence removed and a different amino acid inserted in its place at the same position.
  • the substitutions may be single, where only one amino acid in the molecule has been substituted, or they may be multiple, where two or more amino acids have been substituted in the same molecule.
  • Substantial changes in the activity of the proteins useful in the practice of the present invention may be obtained by substituting an amino acid with a side chain that is significantly different in charge and/or structure from that of the native amino acid. This type of substitution would be expected to affect the structure of the polypeptide backbone and/or the charge or hydrophobicity of the molecule in the area of the substitution.
  • Moderate changes in the activity of the transcription factor molecules useful in the present invention would be expected by substituting an amino acid with a side chain that is similar in charge and/or structure to that of the native molecule.
  • This type of substitution referred to as a conservative substitution, would not be expected to substantially alter either the structure of the polypeptide backbone or the charge or hydrophobicity of the molecule in the area of the substitution.
  • Insertional protein variants are those with one or more amino acids inserted immediately adjacent to an amino acid at a particular position in the native protein molecule. Immediately adjacent to an amino acid means connected to either the ⁇ -carboxy or ⁇ -amino functional group of the amino acid.
  • the insertion may be one or more amino acids. Ordinarily, the insertion will consist of one or two conservative amino acids. Amino acids similar in charge and/or structure to the amino acids adjacent to the site of insertion are defined as conservative. Insertional protein variants also include insertion of an amino acid with a charge and/or structure that is substantially different from the amino acids adjacent to the site of insertion.
  • Deletional variants are those where one or more amino acids in the native protein molecules have been removed. Ordinarily, deletional variants will have one or two amino acids deleted in a particular region of the protein molecule.
  • replicable vector refers to a piece of DNA, usually double-stranded, which may have inserted into it another piece of DNA (the insert DNA) such as, but not limited to, a cDNA molecule.
  • the vector is used to transport the insert DNA into a suitable host cell.
  • the insert DNA may be derived from the host cell, or may be derived from a different cell or organism. Once in the host cell, the vector can replicate independently of or coincidental with the host chromosomal DNA, and several copies of the vector and its inserted DNA may be generated.
  • the terms “replicable expression vector” and “expression vector” refer exclusively to vectors that contain the necessary elements that permit the expression of a polypeptide encoded by the insert DNA. Many molecules of the polypeptide encoded by the insert DNA can thus be rapidly synthesized.
  • transformed host cell refers to the introduction of DNA into a cell.
  • the cell is termed a "host cell”, and it may be a prokaryotic or a eukaryotic cell.
  • Typical prokaryotic host cells include various strains of E. coli.
  • Typical eukaryotic host cells are insect cells or animal cells, such as inner ear supporting cells.
  • the introduced DNA is usually in the form of a vector containing an inserted piece of DNA.
  • the introduced DNA sequence may be from the same species as the host cell or from a different species from the host cell, or it may be a hybrid DNA sequence, containing some foreign DNA and some DNA derived from the host species.
  • the present invention provides methods for stimulating the regeneration of inner ear cells comprising introducing a nucleic acid molecule that encodes an inner ear cell transcription factor, capable of stimulating the regeneration of inner ear cells (preferably capable of stimulating the regeneration of sensory hair cells from supporting cells), into at least one inner ear cell under conditions that enable expression of the transcription factor in the inner ear cell.
  • any type of inner ear cell can be treated in accordance with the present invention, but the presently preferred cells treated in accordance with the present invention are supporting cells. Supporting cells underlie and physically support sensory hair cells within the inner ear.
  • the anatomy of the inner ear is well known to those of ordinary skill in the art (see, e.g., Gray's Anatomy, Revised American Edition (1977), pages 859-867, incorporated herein by reference).
  • the inner ear includes three sensory portions: the cochlea, which senses sound; the semicircular canals, which sense angular acceleration; and the otolithic organs, which sense linear acceleration.
  • specialized sensory hair cells are arrayed upon one or more layers of inner ear supporting cells. In operation, the sensory hair cells are physically deflected in response to sound or motion, and their deflection is transmitted to nerves which send nerve impulses to the brain for processing and interpretation.
  • the cochlea includes the Organ of Corti which is primarily responsible for sensing sound.
  • the Organ of Corti 10 includes a basilar membrane 12 upon which are located a variety of supporting cells 14, including border cells 16, inner pillar cells 18, outer pillar cells 20, inner phalangeal cells 22, Dieter's cells 24 and Hensen's cells 26.
  • Supporting cells 14 support inner hair cells 28 and outer hair cells 30.
  • Tectorial membrane 32 is disposed above inner hair cells 28 and outer hair cells 30.
  • the present invention is adapted to stimulate regeneration of sensory hair cells 28 and 30 from underlying supporting cells 14.
  • the nucleic acid molecules that encode inner ear cell transcription factors useful in the practice of the present invention are cDNA molecules or genomic DNA molecules.
  • Presently preferred nucleic acid molecules useful in the practice of the present invention are cDNA molecules that encode an inner ear cell transcription factor, capable of stimulating the regeneration of inner ear cells (preferably capable of stimulating the regeneration of sensory hair cells from supporting cells), that possesses at least one homeodomain and/or at least one POU-specific domain, and has a molecular weight in the range of from about 33 kDa to about 37 kDa..
  • nucleic acid molecules useful in the practice of the present invention encode a POU4F3 homologue capable of stimulating the regeneration of inner ear cells (preferably capable of stimulating the regeneration of sensory hair cells from supporting cells).
  • a presently most preferred nucleic acid molecule that encodes a POU4F3 homologue is the nucleic acid molecule having the nucleic acid sequence set forth in SEQ ID NO: 1.
  • nucleic acid molecules encoding transcription factors useful in the practice of the present invention can be isolated by using a variety of cloning techniques known to those of ordinary skill in the art.
  • cloned POU4F3 homologues cDNAs or genes, or fragments thereof can be used as hybridization probes utilizing, for example, the technique of hybridizing radiolabelled nucleic acid probes to nucleic acids immobilized on nitrocellulose filters or nylon membranes as set forth at pages 9.52 to 9.55 of Molecular Cloning, A Laboratory Manual (2nd edition), J. Sambrook, E.F. Fritsch and T. Maniatis eds., the cited pages of which are incorporated herein by reference.
  • Presently preferred hybridization probes for identifying additional nucleic acid molecules encoding POU4F3 homologues are fragments, of at least 15 nucleotides in length, of the cDNA molecule (or its complementary sequence) having the nucleic acid sequence set forth in SEQ ID NO:l, although the complete cDNA molecule having the nucleic acid sequence set forth in SEQ ID NO:l is also useful as a hybridization probe for identifying additional nucleic acid molecules encoding POU4F3 homologue.
  • a presently most preferred hybridization probe for identifying additional nucleic acid molecules encoding POU4F3 homologues is the oligonucleotide having the nucleic acid sequence TAG AAG TGC AGG GCA CGC TGC TCA TGG TAT G (SEQ ID NO:5).
  • Exemplary high stringency hybridization and wash conditions useful for identifying (by Southern blotting) additional nucleic acid molecules encoding POU4F3 homologues are: hybridization at 68°C in 0.25 M Na 2 HPO 4 buffer (pH 7.2) containing 1 mM Na 2 EDTA, 20% sodium dodecyl sulfate; washing (three washes of twenty minutes each at 65°C) is conducted in 20 mM Na 2 HPO 4 buffer (pH 7.2) containing 1 mM Na 2 EDTA, 1% (w/v) sodium dodecyl sulfate.
  • Exemplary moderate stringency hybridization and wash conditions useful for identifying (by Southern blotting) additional nucleic acid molecules encoding POU4F3 homologues are: hybridization at 45°C in 0.25 M Na 2 HPO 4 buffer (pH 7.2) containing 1 mM Na 2 EDTA, 20% sodium dodecyl sulfate; washing is conducted in 5X SSC, containing 0.1% (w/v) sodium dodecyl sulfate, at 55°C to 65°C.
  • SSC refers to a buffer used in nucleic acid hybridization solutions.
  • One liter of the 20X (twenty times concentrate) stock SSC buffer solution (pH 7.0) contains 175.3 g sodium chloride and 88.2 g sodium citrate.
  • PCR primers will be designed against conserved amino acid sequence motifs found in most or all of the known, target protein sequences. Examples of conserved amino acid sequence motifs against which PCR primers can be designed for cloning additional POU4F3 homologues are the POU-specific domain having the amino acid sequence set forth in SEQ ID NO:4, and the homeodomain having the amino acid sequence set forth in SEQ ID NO:3.
  • nucleic acid molecules encoding transcription factors useful in the practice of the present invention can also be isolated, for example, by utilizing antibodies that recognize transcription factor proteins.
  • Methods for preparing monoclonal and polyclonal antibodies are well known to those of ordinary skill in the art and are set forth, for example, in chapters five and six of Antibodies A Laboratory Manual, E. Harlow and D. Lane, Cold Spring Harbor Laboratory (1988), the cited chapters of which are incorporated herein by reference.
  • antibodies were successfully raised against a fusion protein constructed from the C-terminal end of Brn3. Xiang M. et al., J. Neuroscience 15(7):4762-4785 (1995) and Xiang M. et al., P.N.AS.
  • a cDNA expression library can be screened using anti-POU4F3 homologue antibodies in order to identify one or more clones that encode a POU4F3 homologue protein.
  • DNA expression library technology is well known to those of ordinary skill in the art.
  • An exemplary protocol for synthesizing and screening a cDNA expression library is set forth in Chapter 12 of Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, the cited chapter of which is incorporated herein by reference.
  • Sequence variants, produced by deletions, substitutions, mutations and/or insertions, of the transcription factors useful in the practice of the present invention can also be used in the methods of the present invention.
  • the amino acid sequence variants of the transcription factors useful in the practice of the present invention may be constructed by mutating the DNA sequences that encode the wild-type transcription factor proteins, such as by using techniques commonly referred to as site-directed mutagenesis.
  • Nucleic acid molecules encoding the transcription factors useful in the practice of the present invention can be mutated by a variety of PCR techniques well known to one of ordinary skill in the art. (See, for example, the following publications, the cited portions of which are incorporated by reference herein: "PCR Strategies", M.A. Innis, D.H.
  • the two primer system utilized in the Transformer Site-Directed Mutagenesis kit from Clontech may be employed for introducing site-directed mutants into nucleic acid molecules encoding transcription factors useful in the practice of the present invention.
  • two primers are simultaneously annealed to the plasmid; one of these primers contains the desired site-directed mutation, the other contains a mutation at another point in the plasmid resulting in elimination of a restriction site.
  • Second strand synthesis is then carried out, tightly linking these two mutations, and the resulting plasmids are transformed into a mutS strain of E. coli.
  • Plasmid DNA is isolated from the transformed bacteria, restricted with the relevant restriction enzyme (thereby linearizing the unmutated plasmids), and then retransformed into E. coli.
  • This system allows for generation of mutations directly in an expression plasmid, without the necessity of subcloning or generation of single-stranded phagemids.
  • the tight linkage of the two mutations and the subsequent linearization of unmutated plasmids results in high mutation efficiency and allows minimal screening. Following synthesis of the initial restriction site primer, this method requires the use of only one new primer type per mutation site.
  • a set of "designed degenerate" oligonucleotide primers can be synthesized in order to introduce all of the desired mutations at a given site simultaneously.
  • Transformants can be screened by sequencing the plasmid DNA through the mutagenized region to identify and sort mutant clones. Each mutant DNA can then be fully sequenced or restricted and analyzed by electrophoresis on Mutation Detection Enhancement gel (J.T. Baker) to confirm that no other alterations in the sequence have occurred (by band shift comparison to the unmutagenized control).
  • the two primer system utilized in the QuikChangeTM Site-Directed Mutagenesis kit from Stratagene may be employed for introducing site-directed mutants into nucleic acid molecules encoding transcription factors useful in the practice of the present invention.
  • Double- stranded plasmid DNA containing the insert bearing the target mutation site, is denatured and mixed with two oligonucleotides complementary to each of the strands of the plasmid DNA at the target mutation site.
  • the annealed oligonucleotide primers are extended using Pfu DNA polymerase, thereby generating a mutated plasmid containing staggered nicks.
  • the unmutated, parental DNA template is digested with restriction enzyme Dpnl which cleaves methylated or hemimethylated DNA, but which does not cleave unmethylated DNA.
  • the parental, template DNA is almost always methylated or hemimethylated since most strains of E. coli, from which the template DNA is obtained, contain the required methylase activity.
  • the remaining, annealed vector DNA incorporating the desired mutation(s) is transformed into E. coli.
  • DNA sequence encoding transcription factors useful in the practice of the present invention may be used to generate deletion variants of transcription factors useful in the practice of the present invention, as described in Section 15.3 of Sambrook et al. Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, New York, NY (1989), incorporated herein by reference. A similar strategy may be used to construct insertion variants, as described in section 15.3 of Sambrook et al., supra.
  • Oligonucleotide-directed mutagenesis may also be employed for preparing substitution variants of transcription factors useful in the practice of the present invention. It may also be used to conveniently prepare the deletion and insertion variants of transcription factors useful in the practice of the present invention.
  • This technique is well known in the art as described by Adelman et al. (DNA 2:183 [1983]); Sambrook et al., supra; "Current Protocols in Molecular Biology", 1991, Wiley (NY), F.T. Ausubel, R. Brent, R.E. Scientific, D.D. Moore, J.D. Seidman, J.A. Smith and K. Struhl, eds., incorporated herein by reference.
  • oligonucleotides of at least 25 nucleotides in length are used to insert, delete or substitute two or more nucleotides in the nucleic acid molecules encoding transcription factors useful in the practice of the present invention.
  • An optimal oligonucleotide will have 12 to 15 perfectly matched nucleotides on either side of the nucleotides coding for the mutation.
  • the oligonucleotide is annealed to the single-stranded DNA template molecule under suitable hybridization conditions.
  • a DNA polymerizing enzyme usually the Klenow fragment of E. coli DNA polymerase I, is then added.
  • This enzyme uses the oligonucleotide as a primer to complete the synthesis of the mutation-bearing strand of DNA.
  • a heteroduplex molecule is formed such that one strand of DNA encodes the wild-type protein inserted in the vector, and the second strand of DNA encodes the mutated form of the protein inserted into the same vector.
  • This heteroduplex molecule is then transformed into a suitable host cell. Mutants with more than one amino acid substituted may be generated in one of several ways. If the amino acids are located close together in the polypeptide chain, they may be mutated simultaneously using one oligonucleotide that codes for all of the desired amino acid substitutions.
  • the amino acids are located some distance from each other (separated by more than ten amino acids, for example) it is more difficult to generate a single oligonucleotide that encodes all of the desired changes.
  • one of two alternative methods may be employed. In the first method, a separate oligonucleotide is generated for each amino acid to be substituted. The oligonucleotides are then annealed to the single-stranded template DNA simultaneously, and the second strand of DNA that is synthesized from the template will encode all of the desired amino acid substitutions.
  • An alternative method involves two or more rounds of mutagenesis to produce the desired mutant.
  • the first round is as described for the single mutants: wild-type protein DNA is used for the template, an oligonucleotide encoding the first desired amino acid substitutions) is annealed to this template, and the heteroduplex DNA molecule is then generated.
  • the second round of mutagenesis utilizes the mutated DNA produced in the first round of mutagenesis as the template.
  • this template already contains one or more mutations.
  • the oligonucleotide encoding the additional desired amino acid substitutions) is then annealed to this template, and the resulting strand of DNA now encodes mutations from both the first and second rounds of mutagenesis.
  • This resultant DNA can be used as a template in a third round of mutagenesis, and so on.
  • Prokaryotes may be used as host cells for the initial cloning steps of inner ear cell transcription factors useful in the practice of the present invention. They are particularly useful for rapid production of large amounts of DNA, for production of single-stranded DNA templates used for site-directed mutagenesis, for screening many mutants and/or putative inner ear cell transcription factors simultaneously, and for DNA sequencing of the mutants generated.
  • Suitable prokaryotic host cells include E. coli K12 strain 94 (ATCC No. 31,446), E. coli strain W3110 (ATCC No. 27,325) E. coli X1776 (ATCC No. 31,537), and E. coli B; however many other strains of E.
  • coli such as HB101, JM101, NM522, NM538, NM539, and many other species and genera of prokaryotes including bacilli such as Bacillus subtilis, other enterobacteriaceae such as Salmonella typhimurium or Serratia marcesans, and various Pseudomonas species may all be used as hosts.
  • Prokaryotic host cells or other host cells with rigid cell walls are preferably transformed using the calcium chloride method as described in section 1.82 of Sambrook et al., supra. Alternatively, electroporation may be used for transformation of these cells.
  • any plasmid vectors containing replicon and control sequences that are derived from species compatible with the host cell may also be used to clone, express and/or manipulate nucleic acid molecules encoding inner ear cell transcription factors useful in the practice of the present invention.
  • the vector usually has a replication site, marker genes that provide phenotypic selection in transformed cells, one or more promoters, and a polylinker region containing several restriction sites for insertion of foreign DNA. Plasmids typically used for transformation of E.
  • coli include pBR322, pUC18, pUC19, pUCI18, pUC119, and Bluescript M13, all of which are described in sections 1.12-1.20 of Sambrook et al., supra.
  • pBR322 pUC18, pUC19, pUCI18, pUC119, and Bluescript M13, all of which are described in sections 1.12-1.20 of Sambrook et al., supra.
  • suitable vectors are available as well. These vectors contain genes coding for ampicillin and/or tetracycline resistance which enables cells transformed with these vectors to grow in the presence of these antibiotics.
  • the promoters most commonly used in prokaryotic vectors include the ⁇ -lactamase (penicillinase) and lactose promoter systems (Chang et al. Nature, 375:615 [1978]; Itakura et al., Science, 198:1056 [1977]; Goeddel et al., Nature, 281:544 [1979]) and a tryptophan (trp) promoter system (Goeddel et al., Nucl. Acids Res., 8:4057 [1980]; ⁇ PO Appl. Publ. No. 36,776), and the alkaline phosphatase systems.
  • suitable vectors containing DNA encoding replication sequences, regulatory sequences, phenotypic selection genes and the DNA encoding a inner ear cell transcription factor useful in the practice of the present invention are prepared using standard recombinant DNA procedures. Isolated plasmids and DNA fragments are cleaved, tailored, and ligated together in a specific order to generate the desired vectors, as is well known in the art (see, for example, Sambrook et al., supra).
  • inner ear cells are genetically transformed with one or more nucleic acid molecules encoding one or more inner ear transcription factor proteins capable of stimulating the regeneration of inner ear cells (preferably capable of stimulating the regeneration of sensory hair cells from supporting cells).
  • Any art-recognized gene delivery method can be used to introduce a nucleic acid molecule encoding an inner ear cell transcription factor into inner ear cells for expression therein.
  • the following gene delivery methods can be used to introduce a nucleic acid molecule encoding an inner ear cell transcription factor into inner ear cells for expression therein: direct injection, electroporation, virus-mediated gene delivery, amino acid-mediated gene delivery, biolistic gene delivery and heat shock.
  • Non-viral methods of gene delivery into inner ear cells are disclosed in Huang, L., Hung, M-C, and Wagner, ⁇ ., Non-Viral Vectors for Gene Therapy, Academic Press, San Diego, California (1999), which is incorporated herein by reference.
  • genes can be introduced into cells in situ, or after removal of the cells from the body, by means of viral vectors.
  • retroviruses are RNA viruses that have the ability to insert their genes into host cell chromosomes after infection. Retroviral vectors have been developed that lack the genes encoding viral proteins, but retain the ability to infect cells and insert their genes into the chromosomes of the target cell (A.D. Miller, Hum. Gen. Ther. 1:5-14 (1990)).
  • Retroviruses will only efficiently infect dividing cells, thus when retroviruses are used to introduce genes into cells that have been removed from the body, cell division is stimulated with growth-promoting media or specific factors. In vivo application of retroviruses has been achieved by administration of virus-producing cells directly into tumors. Virus particle released by the infected cell will infect adjacent tumor cells, hence only a relatively small percentage of cells in a tumor need be initially infected in order to ultimately introduce the targeted gene into most or all of the tumor cells. (K.W. Culver et al., Science 256:1550-1552 (1992)). Adenoviral vectors are designed to be administered directly to patients.
  • adenoviral vectors do not integrate into the chromosome of the host cell. Instead, genes introduced into cells using adenoviral vectors are maintained in the nucleus as an extrachromosomal element (episome) that persists for a limited time period. Adenoviral vectors will infect dividing and non-dividing cells in many different tissues in vivo including airway epithelial cells, endothelial cells, hepatocytes and various tumors (B.C. Trapnell, Adv Drug Del Rev. 12:185-199 (1993)).
  • herpes simplex virus a large, double-stranded DNA virus that has been used in some initial applications to deliver therapeutic genes to neurons and could potentially be used to deliver therapeutic genes to some forms of brain cancer
  • Recombinant forms of the vaccinia virus can accommodate large inserts and are generated by homologous recombination.
  • this vector has been used to deliver interleukins (ILs), such as human IL-l ⁇ and the costimulatory molecules B7-1 and B7-2 (G.R. Peplinski et al., Ann. Surg. Oncol. 2:151-9 (1995); J.W. Hodge et al., Cancer Res. 54:5552-55 (1994)).
  • ILs interleukins
  • plasmid DNA is taken up by cells within the body and can direct expression of recombinant proteins.
  • plasmid DNA is delivered to cells in the form of liposomes in which the DNA is associated with one or more lipids, such as DOTMA (l,2,-diolcyloxypropyl-3-trimethyl ammonium bromide) and DOPE (dioleoylphosphatidylethanolamine).
  • DOTMA l,2,-diolcyloxypropyl-3-trimethyl ammonium bromide
  • DOPE dioleoylphosphatidylethanolamine
  • Intramuscular administration of plasmid DNA results in gene expression that lasts for many months (J.A. Wolff et al., Hum. Mol. Genet. 1:363-369 (1992); M. Manthorpe et al, Hum. Gene Ther. 4:419-431 (1993); G. Ascadi et al., New Biol. 3:71-81 (1991), D. Gal et al, Lab. Invest. 68:18-25 (1993)). Additionally, uptake and expression of DNA has also been observed after direct injection of plasmid into the thyroid (M. Sikes et al., Hum. Gene Ther. 5:837-844 (1994)) and synovium (J. Yovandich et al., Hum.
  • BiolisticTM a ballistic device that projects DNA-coated micro-particles directly into the nucleus of cells in vivo. Once within the nucleus, the DNA dissolves from the gold or tungsten microparticle and can be expressed by the target cell.
  • This method has been used effectively to transfer genes directly into the skin, liver and muscle (N.S. Yang et al., Proc. Natl. Acad. Sci. 87:9568-9572 (1990); L. Cheng et al., Proc. Natl. Acad. Sci. USA. 90:4455-4459 (1993); R.S. Williams et al, Proc. Natl. Acad. Sci. 88:2726-2730 (1991)).
  • molecular conjugates consist of protein or synthetic ligands to which a nucleic acid- or DNA-binding agent has been attached for the specific targeting of nucleic acids to cells
  • R.J. Cristiano et al Proc. Natl. Acad. Sci. USA 90:11548-52 (1993); B.A Bunnell et al., Somat. Call Mol. Genet. 18:559-69 (1992); M. Cotten et al., Proc. Natl. Acad. Sci. USA 89:6094-98 (1992)
  • This gene delivery system has been shown to be capable of targeted delivery to many cell types through the use of different ligands (R.J. Cristiano et al, Proc. Natl. Acad, Sci. USA 90:11548-52 (1993)).
  • the vitamin folate has been used as a ligand to promote delivery of plasmid DNA into cells that overexpress the folate receptor (e.g., ovarian carcinoma cells) (S. Gottschalk et al., Gene Ther. 1:185-91 (1994)).
  • the malaria circumsporozoite protein has been used for the liver-specific delivery of genes under conditions in which ASOR receptor expression on hepatocytes is low, such as in cirrhosis, diabetes, and hepatocellular carcinoma (Z.
  • an apparatus for culturing inner ears include a gas permeable bioreactor comprising a tubular vessel with walls that may be constructed at least partially of a gas permeable material, such as silicone rubber.
  • the vessel in one preferred embodiment is constructed such that half of it is comprised of gas permeable material and the remaining portion is made of nonpermeable material.
  • the gas permeable materials commonly available are opaque.
  • using nonpermeable material for at least part of the bioreactor may provide an advantage in allowing visual inspection of the tubular vessel chamber.
  • the tubular vessel has closed ends, a substantially horizontal longitudinal central axis, and one or more vessel access ports.
  • the vessel access ports provide access to the bioreactor for input of medium and cells, and for removal of old medium from the tubular vessel. This is easily done through the vessel access ports which are also referred to as valves or syringe ports.
  • the vessel access ports are constructed of valves with syringe ports.
  • the vessel is rotatable about its horizontal longitudinal central axis.
  • a preferred means for rotation is a motor assembly which sits on a mounting base and has means for attachment to the tubular vessel.
  • the speed of rotation can be adjusted so that the inner ear within the tubular vessel is constantly in motion, but rotation of the tubular vessel should not be fast enough to cause significant turbulence in the aqueous medium within the tubular vessel.
  • the use of gas permeable material in the construction of at least part of the tubular vessel wall permits O 2 to diffuse through the vessel walls and into the cell culture media in the vessel chamber.
  • CO 2 diffuses through the walls and out of the vessel
  • the use of gas permeable material in the construction of at least part of the tubular vessel wall typically overcomes the need for air injection into the bioreactor vessel.
  • Air injection into the aqueous medium within the bioreactor vessel may be utilized, however, if additional oxygen is required to culture an inner ear.
  • an air filter is also employed to protect the air pump valves from dirt.
  • An alternative embodiment of the bioreactor useful in the practice of the present invention is an annular vessel with walls that may be constructed at least partially of a gas permeable material.
  • Annular is defined herein to include annular, toroidal and other substantially symmetrical ring-like shaped tubular vessels.
  • the annular vessel has closed ends and a substantially horizontal longitudinal central axis.
  • the bioreactor useful in the practice of the present invention comprises a tubular vessel constructed at least partially of a gas permeable material.
  • the vessel has closed ends and a substantially horizontal longitudinal central axis around which it rotates.
  • the vessel furthermore has two slidably interconnected members wherein a first member fits slidably into a second member, forming a liquid tight seal therebetween and providing a variable volume tubular vessel.
  • the bioreactor has means for rotating the tubular vessel about its substantially horizontal longitudinal central axis.
  • One or more vessel access ports are provided for transferring materials into and out of the vessel.
  • bioreactor useful in the practice of the present invention is a particular advantage.
  • embodiment of the bioreactor with slidably interconnected members may be adjusted to provide the exact size bioreactor needed.
  • HARVTM High Aspect Ratio Vessel
  • CCCVTM Cylindrical Cell Culture Vessel
  • NeuralbasalTM media from Gibco BRL Gibco BRL media are produced by Life Technologies, Corporate Headquarters, Gaithersburg, MD
  • B27 or N2 media supplement is the presently preferred culture medium.
  • Other culture media can be successfully used, however, to culture fluid-filled sensory organs in the practice of the present invention.
  • Other suitable media include DME, BME and M-199 with fetal calf serum or horse serum. All of the foregoing media are sold by Gibco -BRL.
  • N2 or B27 supplements play a more significant role when extended periods of culture (>96 hr) are attempted.
  • the present invention provides methods for ameliorating the symptoms of an inner ear disease.
  • the methods for ameliorating the symptoms of an inner ear disease comprise introducing a nucleic acid molecule that encodes an inner ear cell transcription factor, capable of stimulating the regeneration of inner ear cells (preferably capable of stimulating the regeneration of sensory hair cells from supporting cells), into a portion of the inner ear (preferably the cochlea, semicircular canals and/or otolithic organs) under conditions that enable the uptake of the nucleic acid molecule by inner ear cells and expression of the transcription factor within the inner ear cells.
  • Presently preferred inner ear cells suitable for treatment in accordance with this aspect of the present invention are inner ear supporting cells.
  • a nucleic acid molecule that encodes an inner ear cell transcription factor can be introduced into a portion of the inner ear by any art- recognized means, such as by injection.
  • a presently preferred method for introducing a nucleic acid molecule, that encodes an inner ear cell transcription factor, into the cochlea is by cochleostomy, i.e., by puncturing the cochlea and inserting a catheter through which nucleic acid molecules encoding an inner ear cell transcription factor can be introduced into the cochlea.
  • cochleostomy method is disclosed, for example, in Lalwani, A.K. et al, Hearing Research 114:139-147 (1997), incorporated herein by reference.
  • Regeneration of sensory hair cells within the inner ear is desirable in treating subjects suffering from an inner ear disease caused, at least in part, by the degeneration and/or death of inner ear sensory hair cells.
  • diseases that affect inner ear sensory hair cells include: tinnitus, Meniere's Disease, vertigo, dysequilibrium, labyrinthitis and vestibulitis deafness.
  • EXAMPLE 1 Presently Preferred Protocol for Transfecting Cultured Mouse Organ of Corti Cells
  • the present Example describes presently preferred protocols for transfecting cultured mouse Organ of Corti cells, and for identifying transcription factors that are effective to promote regeneration of inner ear cells.
  • Previous attempts to transfect the Organ of Corti focused on unlesioned apical turn cochlear explants obtained from postnatal day 7-10 mice grown for a total of 5 days in vitro (DIN). For this procedure, mice prior to explant were sedated with halothone anesthesia before being sacrificed.
  • a presently preferred method involves lipofecting the cultures with a plasmid encoding green fluorescent protein (GFP; Clonetech). Detection of GFP only requires a standard FITC filter set and this gene has been successfully transfected into sensory cells and neurons within the cochlea using an AAV vector system (Lalwani, A.K. et al, Hear. Res., 114:139- 147 (1997)).
  • GFP green fluorescent protein
  • AAV vector system readily penetrated the central nervous system (CNS) and transfected the contralateral cochlea when injected unilaterally in vivo. Therefore, lipofection is a preferred method over viral mediated transfections.
  • CNS central nervous system
  • Organ of Corti cultures will be established from P7-P10 Swiss Webster mice derived from a breeding colony.
  • the cultured organs will be transfected using a variety of commercially available lipofection reagents (i.e., FuGENE Transfection Reagent; Boehringer-Mannheim). These efficiencies will be compared against the transfection efficiencies achieved by the InVitrogen Kit.
  • the optimal lipofection reagent and the optimal lipid to DNA ratio (3:1, 6:1 or 9:1) will be determined by counting the number of GFP-positive cells within the organ of Corti along a 250 ⁇ M length taken at the middle of the explant. Cells will be visualized using a Nikon epifluorescent microscope equipped with a CCD digital camera that will output images directly into Adobe Photoshop on a Macintosh computer where cell counts can be performed.
  • the inner ear of a mouse was excised in the following manner. Postnatal day 7-14 Swiss Webster mice were decapitated and their skulls immersed in 70% ethanol for 5 min to disinfect. Under sterile conditions, the skull was cut into halves along the mid-sagittal axis and placed into 3 ml of culture media (NeuralbasalTM Media at pH 7.4; Gibco) in a 35 mm plastic culture dish (Nalge Nunc International, 2000 North Aurora Road, Naperville, IL 60563). Using surgical forceps, the bony inner ear labyrinth was visualized and separated from the temporal bone. The overlying connective tissue, stapes bone, facial nerve and stapedial artery were removed.
  • an inner ear excised and prepared in the foregoing manner is transferred to the HARVTM or CCCVTM vessel which contains 50 or 55 ml of NeuralbasalTM Media supplemented with either N2 or B27 media supplement (both sold by Gibco-BRL, Catalogue number 17504-036), lO U/ml of penicillin and .25 ⁇ g/ ⁇ l of fungizone.
  • the B27 supplement is sold as a 50X concentrate which is used at a working concentration of 0.5X (e.g., 550 ⁇ l of 50X B27 stock solution is added to 55 ml of NeuralbasalTM Media).
  • the N2 supplement stock solution is 100X and is used at a working concentration of IX (e.g., 550 ⁇ l of 100X N2 stock solution is added to 55 ml of NeuralbasalTM Media).
  • the vessel is then placed in a tissue culture incubator at 37°C and in a 95% air/5% CO 2 environment.
  • the vessel is then rotated at 39 rpm for periods of 24-168 hr. 50% media changes are made every 48 hr. As few as 2 and as many as 12 inner ears have been successfully cultured in one vessel.
  • the inner ear is placed in NeuralbasalTM/N2 or B27 media that contain 1 mM neomycin sulfate (Sigma, P.O. Box 14508, St. Louis, MO 63178) for 24-48 hr. After this culture period, the media is completely replaced with media devoid of neomycin.
  • NeuralbasalTM/N2 or B27 media that contain 1 mM neomycin sulfate (Sigma, P.O. Box 14508, St. Louis, MO 63178) for 24-48 hr. After this culture period, the media is completely replaced with media devoid of neomycin.
  • NeuralbasalTM medium sold by Gibco. All concentrations are working concentrations, i.e., the concentrations of the components in the medium in which the fluid-filled sensory organ is incubated. Table I. NeuralbasalTM media com osition
  • NeuralbasalTM medium The following antibiotics may be added to NeuralbasalTM medium.
  • Fungizone reagent amphotericin B, 0.25 ⁇ g/ml, and sodium desoxycholate, 0.25 ⁇ g/ml
  • Penicillin G (10 units/ml) which is sold by Sigma, Catalog number P 3414.
  • NeuralbasalTM medium may also be supplemented with L-Glutamine (2mM).
  • Example 4 Assay for Sensory Epithelium Vitality During Long Term Culture
  • the microgravitational environment provided by the rotation of the culture vessel allows the sensory epithelium of the inner ear to be maintained for prolonged periods of culture (>168 hr.) without significant degradation or loss of the sensory hair-cells or non-sensory supporting- cells.
  • Data demonstrating the continued vitality of the sensory hair cells during prolonged culture were obtained by labeling the sensory epithelia with a probe against F-actin (phalloidin-FITC) that labels the surfaces of sensory and non-sensory cells, and with a hair-cell specific antibody against calbindin, a calcium binding protein. Both labels were detected and photographed under epifluorescence microscopy.
  • F-actin phalloidin-FITC
  • the Organ of Corti has several fluid- filled spaces called the tunnel of Corti and spaces of Nuel that are necessary for normal auditory function. These spaces occur between hair-cells and supporting-cells and are maintained after prolonged periods of culture.
  • the sensory epithelia In normal gravitational environments, (i.e., when the inner ear is floated without rotating the culture vessel) the sensory epithelia begin to degenerate. Without rotation, within 24 hr. the hair- cells are either completely missing or appear to be undergoing various endstages of cell death. After 48 hr., the supporting-cells are completely missing, or are present but with the total loss of the tunnel of Corti and spaces of Nuel. Rotating the vessel prevents this degradation and maintains normal cytoarchitecture.
  • POU4F3 is a DNA binding transcription factor that is specifically expressed in the hair cells in the inner ear. Mutations in POU4F3 are known to cause developmental failures in mice, and hearing loss in both mice and humans. A construct encoding POU4F3 will be spliced into a GFP encoding plasmid. This plasmid also contains an internal ribosome entry site (IRES) that permits the translation of two open reading frames (i.e., POU4F3 and GFP) from one mRNA.
  • IRES internal ribosome entry site
  • GFP production will coincide with the production of POU4F3.
  • Transfected GFP-positive cells will be tested directly to ascertain that they are expressing POU4F3.
  • Detection of exogenous POU4F3 will be determined using a polyclonal antibody (Santa Cruz Biotechnology or Babco).
  • double-labeled cells i.e., GFP and POU4F3 will be analyzed to determine whether they also express hair cell selective markers such as calbindin, a calcium binding protein, using an anti-calbindin monoclonal antibody (Sigma; Chemicon).
  • Cultures from P7-P10 mice will be established and lesioned with 1 mM neomycin for 2 DIV. The media will be removed and the cultures lipofected with pIRES-GFP-POU4F3 for four hours and permitted to recover in fresh media for 3-6 DI . Cultures will be aldehyde-fixed and processed for POU4F3 and calbindin immunocytochemistry. Cultures lipofected with only pIRES-GFP will serve as controls. The presence of a triple labeled cell (positive for GFP, POU4F3, and calbindin immunoreactivity) would indicate that POU4F3 is capable of promoting the adoption of a hair cell phenotype in the lesioned organ of Corti. Further determination of this phenotype will be corroborated with other immunologic hair cell markers, such as Myo7a.
  • RNA from cochlear explant cultures will be isolated and radiolabeled via incorporation of 32 P-dCTP using a reverse transcription.
  • One organ culture contains approximately 10,000 cells and yields up to 2 micrograms of total RNA. Two micrograms of total RNA is sufficient for expression analysis by hybridization to one set of cDNA grids.
  • the detection limit and dynamic range of cDNA hybridization arrays using nylon membranes to array cDNA clones and radioactively labeled cDNA probes are the two most critical parameters governing the types of genes which may be identified using this technology.
  • Transcript abundance in a typical eukaryotic cell can be categorized as follows: low (1-10 copies per cell), medium high (100-500 copies per cell), and high (several thousand copies per cell) (Bishop, J.O. et al., Nature, 250: 199-204 (1974)).
  • Several groups have now reported the detection of 1 transcript in 20,000 (Pietu, G. et al., Genome Res., 6:492-503 (1996); Nguyen, C.
  • the membrane-based high density cDNA array hybridization can detect between 5 and 50 mRNA molecules in a cell. While low level changes in the expression of low copy transcripts are not reliably detectable by this system, changes in transcript levels can be picked up within the medium-high or the high copy categories with reproducible results.
  • the two major factors governing the accuracy of the reading for one particular cDNA on a membrane are the amount of cDNA spotted on the membrane and the hybridization of the probe.
  • the remaining membrane will be hybridized with cDNA from aminoglycoside-lesioned organ of Corti cultures that have been transfected with the reporter GFP plasmid only, i.e., the control plasmid probe. Hybridization patterns and intensities will be compared between these two filters. Transfection efficiency will be monitored by determining expression of GFP from the construct containing the POU4F3 gene. Transfection efficiencies of the construct containing these two genes will be compared by fluorescence microscopy.
  • RNA/cDNA hybridization pairs will be repeated with four independent sets of RNA on four independent sets of filters. This repetition will allow for statistical evaluation of the standard deviation of the hybridization intensity between filter groups probed with 32 P-labeled RNA from the same transfected cell culture system. True positive up-regulated or down-regulated genes will provide a reproducible result over the set of four membrane pairs. Imaging of the hybridization pattern will be done using the Phosphorlmager storage phosphor system (Molecular Dynamics). Nylon membrane images stored on the phosphor screens are transferred to a Macintosh computer using an image splitting, spot finding, and spot integration software package (Crazy Quant VI.3) available freely through the laboratory of Dr. Leroy Hood.

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Abstract

L'invention porte, selon une réalisation, sur des procédés visant à stimuler la régénération des cellules de l'oreille interne et consistant à introduire une molécule d'acide nucléique qui code un facteur de transcription cellulaire de l'oreille interne, capable de stimuler la régénération d'une cellule de l'oreille interne, dans au moins une cellule, dans des conditions permettant l'expression du facteur de transcription dans la cellule de l'oreille interne. Tout type de cellule de l'oreille interne peut être traité selon cette invention, mais les cellules préférées, traitées, sont des cellules de soutien de l'oreille interne. Le procédé d'administration de gène actuellement préféré est la lipofection. Selon une autre réalisation, l'invention porte sur des procédés visant à réduire les symptômes d'une pathologie de l'oreille interne et consistant à introduire une molécule d'acide nucléique qui code un facteur de transcription cellulaire de l'oreille interne, capable de stimuler la régénération d'une cellule de l'oreille interne, dans une partie de l'oreille interne dans des conditions permettant la capture de la molécule d'acide nucléique par des cellules de l'oreille interne et l'expression du facteur de transcription dans les cellules de l'oreille interne. Selon encore une autre réalisation, cette invention porte sur des cellules de l'oreille interne régénérées, génétiquement transformées, de préférence des cellules de soutien ou des cellules ciliées sensorielles, comprenant un transgène codant un facteur de transcription des cellules de l'oreille interne.
PCT/US1999/024829 1998-02-23 1999-10-21 Procedes pour stimuler la regeneration des cellules de l'oreille interne WO2000023084A1 (fr)

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US7442688B2 (en) 1999-06-01 2008-10-28 Baylor College Of Medicine Composition and methods for the therapeutic use of an atonal-associated sequence for deafness, osteoarthritis and abnormal cell proliferation
US7470673B2 (en) 1999-06-01 2008-12-30 Baylor College Of Medicine Composition and methods for the therapeutic use of an atonal-associated sequence for deafness, osteoarthritis and abnormal cell proliferation
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