KR100896489B1 - Stimulation of cellular regeneration and differentiation in the inner ear - Google Patents

Abstract

The present invention provides a method for stimulating the formation of inner ear cells, including inner ear sensory hair cells and inner ear support cells. The methods of the invention damage and / or kill inner ear cells and stimulate the formation of new inner ear cells.

Description

Stimulation of cell regeneration and differentiation in the inner ear {STIMULATION OF CELLULAR REGENERATION AND DIFFERENTIATION IN THE INNER EAR}

The present invention relates to methods and compositions for stimulating the formation of inner ear cells, including inner ear sensory hair cells and inner ear support cells.

Sensory Neuron Hearing Loss (SNHL), the so-called "neural hearing loss", is a serious communication disorder affecting tens of millions of Americans. Loss of inner ear sensory hair cells that detect sound is thought to be a major cause of this disorder. Inner ear anatomy is well known to those skilled in the art (see Gray's Anatomy , Revised American Edition (1977), pages 859-867, which is incorporated herein by reference). In short, the inner ear includes three sensory parts: a cochlear angle to detect sound; a semi-circular tube to detect angular acceleration; and an equilibrium organ to detect linear velocity. In each of these sensory parts, specialized sensory hair cells align over one or more layers of inner ear support cells. Supporting cells are under, or at least partially surround, and physically support sensory hair cells within the inner ear. In operation, sensory hair cells are physically deflected in response to sound or motion, which deflects to nerves that send nerve impulses to the brain for processing and translation.

In mammals, the inner ear typically cannot regenerate damaged or dead inner ear sensory hair cells. Therefore, hearing abnormalities due to the death or weakening of sensory hair cells typically cause permanent hearing impairment. Sensory neuronal deafness includes age-related loss (senile deafness), noise exposure, drug exposure (eg antibiotics and anti-cancer treatment), infections, genetic mutations (syndromes and non-syndromes) and autoimmune diseases It can be caused by a number of events, including.

Currently, treatments for acquired sensorineural hearing loss include the use of external hearing aids and cochlear implants. Both devices have somewhat limited therapeutic potential and, more importantly, do not pose a problem of restoring the structure or function to hearing sensory epithelium.

A more recent approach to the problem of regenerating sensory inner hair cells is disclosed in published international application PCT / US99 / 24829 which discloses a method for stimulating regeneration of inner ear cells (including sensory hair cells). Introducing into an inner cell nucleic acid molecule encoding a transcription factor capable of stimulating regeneration.

As disclosed herein, the inventors have found that the destruction of the inner ear sensory hair cells present normally stops the inner ear support cells (which express reduced levels of one or more cell cycle inhibitor proteins, or where cell cycle protein activity is reduced). Was found to produce progeny cells that could induce reintroduction into the cell cycle, leading to formation of inner ear sensory hair cells. In some instances, destruction of the inner ear sensory hair cells present is sufficient to stimulate the underlying and / or surrounding inner ear support cells to develop into sensory hair cells. In another example, effective regeneration of sensory hair cells from support cells requires the destruction of the inner ear sensory hair cells present in combination with at least one other stimulus as disclosed herein. In addition, the inventors have found that stimulating the differentiation of inner ear support cells (with or without stimulating the regeneration of inner ear sensory hair cells) improves the hearing function of the inner ear.

Summary of the Invention

The present invention provides a method for stimulating the formation of inner ear cells, including inner ear sensory hair cells and inner ear support cells. The method of the present invention relies on the unexpected observation that damaged and / or killed inner ear cells stimulate the formation of new, inner ear cells.

In one aspect, the present invention provides a method for stimulating the formation of inner ear sensory hair cells from inner ear support cells. The method of this aspect of the invention comprises the step of (a) damaging one or more inner ear sensory hair cells under conditions that promote the formation of one or more new sensory hair cells from one or more support cells in contact with the damaged sensory hair cells. do. Preferably, the plurality of inner ear sensory hair cells are formed from the plurality of inner ear support cells. The method of this aspect of the invention further comprises the step (b) of further stimulating the formation of one or more inner ear sensory hair cells from the inner ear support cells adjoining the optionally damaged inner ear sensory hair cells. Step (b) may occur before, during, after or simultaneously with step (a). In one embodiment, stimulating the formation of one or more inner ear sensory hair cells from one or more inner ear support cells in contact with an damaged inner ear sensory hair cell, stimulating the inner ear support cells to enter the cell cycle, and then the inner ear support cells. Stimulating at least some progeny cells of the differentiation to form inner ear sensory hair cells.

For example, the inner ear sensory hair cells may be damaged by contact with a large amount of inner ear neurotoxic agents, such as antibiotics, preferably aminoglycoside antibiotics, which are effective in damaging the inner ear sensory hair cells. Inner ear neurotoxic agents may be introduced into the inner ear by any industry-certified means, for example by injection (with needles and syringes, etc.) or through cannula. In one embodiment of this aspect of the invention, the inner ear sensory hair cells are sufficiently damaged to cause cell death.

In some embodiments of the invention, the damage applied to the inner ear sensory hair cells stimulates the formation of one or more new inner ear sensory hair cells from the inner ear support cells that are in contact with the damaged inner ear sensory hair cells. However, in other embodiments, the damage itself on the inner ear sensory hair cells is insufficient to effectively stimulate the formation of one or more new inner ear sensory hair cells from the inner ear support cells that are in contact with the damaged inner ear sensory hair cells. Therefore, in one embodiment of the method of this aspect of the invention, the formation of the inner ear sensory hair cells from the inner ear support cells is a transcription that can stimulate the inner ear sensory hair cells and form the inner ear sensory hair cells from the inner ear support cells. The factor is stimulated by expressing in the inner ear support cells (before, during and / or after damaging the sensory hair cells). For example, in one embodiment of the invention, a nucleic acid molecule encoding a transcription factor capable of stimulating the formation of an inner ear sensory hair cell from an inner ear support cell is introduced into the inner ear support cell under conditions in which the transcription factor can be expressed. . Representative examples of transcription factors that can stimulate the formation of inner ear sensory hair cells from inner ear support cells include POU4F1, POU4F2, POU4F3, Brn3a, Brn3b, and Brn3c.

In another embodiment of the method of this aspect of the invention, the formation of the inner ear sensory hair cells from the inner ear support cells impairs the expression of the one or more cell cycle inhibitors that are active in the inner ear support cells (sense). Is inhibited before, during and / or after damaging the hair cells. Inhibitors of cell cycle inhibitors may be substances that act on the cell cycle directly or indirectly in cells, such as proteins. As a specific example, cell cycle inhibitors active in inner ear support cells include cyclin-dependent kinase inhibitors such as cyclin-dependent kinase inhibitors of the CIP / KIP family, including p21 ^Cip1 , p27 ^Kip1 and p57 ^Kip2 . For example, expression of cell cycle inhibitors that are active in inner ear support cells can be inhibited by introducing an expression vector into the inner ear support cells, wherein the vector is a nucleic acid molecule encoding a cell cycle inhibitor that is active in the inner ear support cells (55 ° C.). Express nucleic acid molecules that hybridize under constriction conditions (such as constriction greater than 2 × SSC).

In addition, various recombinant growth factors such as TGF-alpha, insulin and IGF-1 can be used to stimulate the formation of inner ear sensory hair cells from inner ear support cells. Representative effective concentration ranges for recombinant growth factors used in vitro in the practice of the present invention are 1 to 1000 ng / ml. More specifically, TGF-alpha is preferably used at an effective concentration of 1 to 100 ng / ml; Insulin is preferably used at an effective concentration of 100 to 1000 ng / ml; IGF-1 is preferably used at an effective concentration of 10 to 1000 ng / ml. For in vivo applications, a sufficient amount of recombinant growth factor can be administered in vivo to produce the preceding concentrations.

In a preferred embodiment of this aspect of the invention, the formation of inner ear sensory hair cells from inner ear support cells results in an improvement in auditory function of the treated inner ear. Therefore, in one aspect, the present invention provides a method for improving auditory function in the inner ear, comprising the steps of: (a) one or more new inner ear sensory hairs from supporting cells abutting damaged, original inner ear sensory hair cells; Damaging the first inner ear sensory hair cells under conditions that promote the formation of cells; And (b) measuring an improvement in auditory function in the inner ear treated according to step (a).

In another aspect, the present invention provides a method for stimulating the formation of inner ear support cells. The method of this aspect of the invention includes damaging the inner ear support cells under conditions that promote the formation of new inner ear support cells (eg, by cell division of the inner ear support cells in contact with the damaged inner ear support cells). In this aspect of the invention, the formation of new inner ear support cells is stimulated using the same techniques as the methods of the invention disclosed herein, which damage the inner ear support cells and stimulate the formation of inner ear sensory hair cells from the inner ear support cells. Thus, for example, inner ear support cells may be damaged by contact with large amounts of inner ear neurotoxic agents, such as aminoglycoside antibiotics, which are effective at damaging the inner ear support cells. As another example, the formation of new inner ear support cells may be caused by a transcription factor (eg, POU4F1, POU4F2, POU4F3, Brn3a, which may damage inner ear support cells and stimulate the inner ear support cells to divide to form new inner support cells). Can be further stimulated by expressing Brn3b and Brn3c) in inner ear support cells. In a preferred embodiment of this aspect of the invention, proliferation of the inner ear support cells results in an improvement in the auditory function of the treated inner ear.

The methods of the invention are useful for stimulating the formation of inner ear cells such as sensory hair cells and support cells. Moreover, the methods of the present invention are useful for ameliorating symptoms of hearing loss caused by the death or damage of inner ear cells in mammals such as humans. In addition, the methods of the present invention can be used to identify genes and / or proteins that can stimulate the formation of inner ear support cells and the formation of inner ear sensory hair cells from the inner ear support cells.

1 shows a cross section of a Corti organ.

Figure 2 shows the number of BrdU-labeled, JH4 cells in guinea pigs after serum deprivation for 24 hours and BrdU pulses for the last 4 hours of the 24 hour period. Cells were counted under fluorescence microscopy. Combination of lipofection and p27 ^Kip1 AS ^reverses growth arrest by 40% of what appears as 10% FBS stimulation (p <.0001). (+) FBS; (-) FBS free; (AS) antisense oligonucleotides; (lipid) Refection.

Figure 3 shows the ABR threshold of the right ear of mice two weeks after treatment of mice's inner ear with amikacin sulfate. Abbreviations are: ABR, hearing brainstem response; dB, decibels; SPL, acoustic pressure level; Wt, wild type; Het, p27 heterozygote; Ko, p27 knockout; kHZ, kilohertz.

4 shows the ABR threshold of the left ear of mice two weeks after treatment of mice's inner ear with amikacin sulfate. Abbreviations are the same as those shown in the description of FIG. 3.

5 shows the ABR threshold of the right ear of mice 4 weeks after treatment of mice's inner ear with amikacin sulfate. Abbreviations are the same as those shown in the description of FIG. 3.

Figure 6 shows the ABR threshold of the left ear of mice 4 weeks after treatment of mice's inner ear with amikacin sulfate. Abbreviations are the same as those shown in the description of FIG. 3.

As used herein, the abbreviation “SSC” refers to the buffer used in the nucleic acid hybrid forming solution. One liter of 20 × (20-fold concentrated) stock SSC buffer solution (pH 7.0) contains 175.3 g sodium chloride and 88.2 g sodium citrate.

As used herein, the phrase "damaging one or more inner ear sensory hair cells", or "damaging the first inner ear sensory hair cells", or a grammatical equivalent thereof, has been incubated under substantially the same conditions as the damaged cells, but not damaged. Compared to non-inner ear sensory hair cells, this refers to structural, biochemical and / or physiologically toxic changes (including death of damaged cells) in damaged sensory hair cells.

As used herein, the phrase "improving hearing function" or "improvement of hearing function", or its grammatical equivalents, refers to the sensitivity of the inner ear to sound by treating the inner ear according to the method of the present invention, at least 10%. Improvement to, or resulted in some measurable improvement in sensitivity to the sound of the inner ear that did not respond to sound at all prior to treatment in accordance with the present invention. Sensitivity to the sound of the treated inner ear is measured by any industry-certified means (such as an auditory brainstem response) and is compared to the sound of a control inner ear that has not been treated in accordance with the invention but incubated under substantially the same conditions as the treated inner ear. Sensitivity is compared.

When applied to nucleic acid sequence comparison or amino acid sequence comparison herein, the term “sequence homology” (also referred to as “sequence identity”), refers to a gap in order to align sequences and achieve maximum percent homology (identity), if necessary. It is defined as the percentage of amino acid residues or nucleic acid residues in a subject amino acid sequence or nucleic acid sequence that is introduced but excludes any conservative substitutions as part of sequence homology and that matches part or all of the candidate amino acid sequence or nucleic acid sequence. N- or C-terminal extension or insertions are not to be interpreted as reducing homology. The weight is not given the number or length of gaps introduced to achieve maximum percent homology (identity), if necessary.

In one aspect, the present invention provides a method for stimulating the formation of inner ear sensory hair cells from inner ear support cells. The method of this aspect of the present invention comprises the step (a) of damaging one or more inner ear sensory hair cells under conditions that promote the formation of one or more new sensory hair cells from one or more support cells in contact with the damaged sensory hair cell (s). Include. Preferably, the plurality of inner ear sensory hair cells are formed from the plurality of inner ear support cells. The method of this aspect of the invention further comprises the step (b) of further stimulating the formation of one or more inner ear sensory hair cells from the inner ear support cells in contact with the damaged inner ear sensory hair cells. Step (b) may occur before, during, after or simultaneously with step (a). The method of this aspect of the invention can be used in vivo and in vitro.

Inner ear anatomy is well known to those skilled in the art (see Gray's Anatomy , Revised American Edion (1977), pages 859-867, which is incorporated herein by reference). In particular, the cochlear angle includes corti organs that can primarily respond to sound sensing. As shown in FIG. 1, the corti organ 10 comprises a basement membrane 12 located on various support cells 14, which are border cells 16, internal strut cells 18, external support cells ( 20), internal phalanx cells 22, Dieter cells 24 and Hensen cells 26. Support cells 14 support inner hair cells 28 and outer hair cells 30. The overcoat 32 is disposed over the inner hair cells 28 and the outer hair cells 30. The invention is, in one aspect, adapted to stimulate the regeneration of sensory hair cells 28 and 30 from underlying support cells 14. In another embodiment, the invention is adapted to stimulate the formation of support cells 14.

The inventors have observed that the destruction of the inner ear sensory hair cells present promotes the re-entry of normally resting inner ear support cells into the cell cycle to produce progeny cells that can lead to the formation of inner ear sensory hair cells as disclosed herein. did. Representative examples of inner ear neurotoxic agents useful for damaging inner ear sensory hair cells include aminoglycoside antibiotics (eg, neomycin, gentamycin, streptomycin, kanamycin, amikacin, and tobramycin). In the practice of the present invention, the aforementioned aminoglycoside antibiotics are typically in vitro in effective concentrations ranging from about 0.01 mM to 10 mM, and in vivo about 100 to about 1,000 milligrams per kilogram body weight (mg / kg / d) Used at effective concentrations in the range. In addition, representative examples of chemicals useful for damaging inner ear sensory hair cells include the following anti-cancer agents: cisplatin, carboplatin and methotrexate (these are typically effective concentrations ranging from about 0.01 mM to 0.1 mM in vitro). As an effective concentration in the range of about 5 to about 10 mg / kg / d in vivo). Other useful chemistries include poly-L-lysine, which ranges from about 0.1 to 1.0 mM in effective concentrations in vitro, and magnesium chloride, in a range of about 5 to 100 mM in effective concentrations in vitro.

Inner ear neurotoxic agents or drugs can be introduced into the inner ear by any industry-certified means, for example by injection with a needle and syringe, or by cochleostomy. Cochlear opening involves drilling a cochlear angle and inserting a catheter, through which a chemical can be introduced into the cochlear angle. Cochlear aperture surgery methods are disclosed, for example, in Lalwani, AK et al., Hearing Research 114 : 139-147 (1997), which publications are incorporated herein by reference.

In one embodiment of the method of the invention, the formation of inner ear sensory hair cells from inner ear support cells is characterized by damaging the inner ear sensory hair cells and at least some inner ear (before, during, and / or after damaging the inner ear sensory hair cells). In support cells, they are stimulated by expressing transcription factors that can stimulate the formation of inner ear sensory hair cells from the inner ear support cells. For example, in one embodiment, nucleic acids encoding transcription factors capable of stimulating the formation of inner ear sensory hair cells are introduced into the inner ear support cells under conditions that allow expression of the transcription factors.

Transcription factors useful in this aspect of the invention have the ability to stimulate regeneration of inner ear sensory hair cells therefrom when inner ear support cells are used in the practice of the invention. Several transcription factors useful in this aspect of the invention are required for normal development and / or normal function of inner ear sensory hair cells.

Representative examples of transcription factors useful in this embodiment of the invention include POU4F1 (Collum, RG et al., Nucleic Acids Research 20 (18) : 4919-4925 (1992)), POU4F2 (Xiang et al., Neuron 11 : 689- 701 (1993)), POU4F3 (Vahava, O., Science 279 (5358) : 1950-1954 (1998), Brn3a (also known as Brn3.0), Brn3b (also known as Brn3.2) and Brn3c (Also known as Brn3.1), which includes Gerrero et al., Proc. Nat'l. Acad. Sci. (USA) 90 (22) : 10841-10845 (1993), the publications of which are incorporated herein by reference . , Xiang, M. et al., Proc. Nat'l.Acad . Sci. (USA) 93 (21) : 11950-11955 (1996), Xiang, M. et al., J. Neurosci. 15 (7-1) ) : 4762-4785 (1995), Erkman, L. et al., Nature 381 (6583) : 603-606 (1996), Xiang, M. et al., Proc. Nat'l. Acad. Sci. (USA) 94 17: the same as that disclosed in 9445-9450 (1997). some transcription factors useful in the present invention has at least one call Meo domain (homeodomain) and at least one POU- specific domain, about 33kDa Stand has a molecular weight in the range of about 37kDa.

As used herein, the term “homeodomain” refers to an amino acid sequence that is at least 50% homologous (such as at least 75% homologous, or at least 90% homologous) with the homeodomain amino acid sequence shown in SEQ ID NO: 1.

As used herein, the term “POU-specific domain” refers to an amino acid sequence that is at least 50% homologous (such as at least 75% homologous, or at least 90% homologous) to the POU-specific domain amino acid sequence shown in SEQ ID NO: 2. Means.

One example of a computational approach that can be used to determine percent homology between two protein sequences, or between two nucleic acid sequences, is described in Karlin and Altschul ( Proc. Natl. Acad. Sci. USA 90: 5873-5877 (1993)). Karlin and Altschul ( Proc. Natl. Acad. Sci. USA 87: 2264-2268 (1990)), modified as follows. This algorithm is described in Altschul et al. ( J. Mol. Biol. 215: 403-410 (1990)) in the NBLEST and XBLEST programs.

A more preferred inner ear cell transcription factor in the practice of the present invention is the POU4F3 transcription factor homologue (hereinafter POU4F3 homolog). POU4F3 homologues useful in the practice of the present invention are capable of stimulating regeneration of inner ear sensory hair cells from support cells and (at least 50% homologous or at least 75% homologous, with a POU4F3 transcription factor having the amino acid sequence shown in SEQ ID NO: 4, Or at least 25% homology (as is at least 90% homology), which homolog is encoded by the nucleic acid molecule of SEQ ID NO: 3. As used herein, the term “POU4F3 homologue” includes the POU4F3 protein having the amino acid sequence shown in SEQ ID NO: 4, which is the present 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 shown in Xiang, M. et al., J. Neuroscience , 15 (7) : 4762-4785 (1995), incorporated herein by reference.

Additional nucleic acid molecules encoding transcription factors useful in the practice of the present invention can be isolated using various cloning techniques well known to those skilled in the art. For example, cloned POU4F3 homologous cDNAs or genes, or fragments thereof, can be used as hybrid forming probes, for example, Molecular Cloning, A Laboratory Manual (2nd edition), J. Sambrook, As shown on pages 9.52 to 9.55 of EF Fritsch and T. Maniatis eds., Hybridization techniques of nucleic acid immobilized on nitrocellulose filters or nylon membranes and radiolabeled nucleic acid probes can be used, see here. It is recorded as a literature. Currently most preferred hybridization probes for identifying additional nucleic acid molecules encoding POU4F3 homologues are fragments of cDNA molecules (or their complementary sequences) having the nucleic acid sequence shown in SEQ ID NO: 3, which is at least 15 nucleotides in length. However, complete cDNA molecules with the nucleic acid sequence shown in SEQ ID NO: 3 are also useful as hybridization probes for identifying additional nucleic acids encoding POU4F3 homologs. Currently most preferred hybridization probes for identifying additional nucleic acid molecules encoding POU4F3 homologues are oligonucleotides having the nucleic acid sequence 5'-TAG AAG TGC AGG GCA CGC TGC TCA TGG TAT G-3 '(SEQ ID NO: 5). to be.

Typical highly constricted hybrid formation and wash conditions useful for identifying additional nucleic acid molecules encoding POU4F3 homologs (by Southern blotting) are: 1 mM Na ₂ EDTA, 0.25 containing 20% sodium dodecyl sulfate. Hybrid formation at 68 ° C. in M Na ₂ HPO ₄ buffer, pH 7.2; Wash in 20 mM Na ₂ HPO ₄ buffer (pH 7.2) containing 1 mM Na ₂ EDTA, 1% (weight / volume) sodium dodecyl sulfate (3 washes at 65 ° C. for 20 minutes each).

Typical intermediate contractile hybrid formation and wash conditions useful for identifying (by Southern blotting) additional nucleic acid molecules encoding POU4F3 homologues are: 0.25 M with 1 mM Na ₂ EDTA, 20% sodium dodecyl sulfate. Hybrid formation at 45 ° C. in Na ₂ HPO ₄ buffer, pH 7.2; Wash at 55 ° C.-65 ° C. in 5 × SSC containing 1% (weight / volume) sodium dodecyl sulfate.

Again, by way of example, nucleic acid molecules encoding transcription factors useful in the present invention are polymerases disclosed in The Polymerase chain Reaction (KB Mullis, F. Ferre, RA Gibbs, eds), Birkhauser Boston (1994), incorporated herein by reference. Can be separated by chain reaction (PCR). Thus, for example, primary strand DNA synthesis can be initiated using oligo (dT) primers and secondary strand cDNA synthesis is consistent with the portion of the 5'-untranslated region of the cDNA molecule homologous to the target DNA molecule. Can be started using oligonucleotide primers. The next round of PCR can be initiated using a second strand cDNA synthesis primer and a primer that matches the portion of the 3'-untranslated region of cDNA homologous to the target DNA molecule.

As a non-limiting example, representative PCR reaction conditions for amplifying a nucleic acid molecule encoding a transcription factor useful in the present invention are as follows. The following reagents are mixed in (ice) tubes to form a PCR reaction mixture: DNA template (eg, genomic DNA up to 1 μg, or cDNA up to 0.1 μg), 0,1 to 0.3 mM dNTPs, 10 10 μl 10X PCR buffer (10X PCR buffer contains 500 mM KCl, 15 mM MgCl ₂ , 100 mM Tris-HCl at pH 8.3), 50 pmol of PCR primer each (PCR primer should preferably be greater than 20 bp in length) And have a degeneracy of 10 ² to 10 ³ ), 2.5 units of Taq DNA polymerase (Perkin Elmer, Norwalk, CT) and deionized water to a total volume of 50 μl. The tube containing the reaction mixture is placed in a thermocycler and the thermocycler program is operated as follows. Denaturation at 94 ° C. for 2 minutes, then 30 cycles at 94 ° C., 30 seconds at 47 ° C. to 55 ° C., and 30 cycles at 72 ° C. for 30 seconds to 2 minutes 30 seconds.

Preferably, PCR primers can be designed for conservative amino acid sequence motifs found in some or all known target protein sequences. Examples of conservative amino acid sequence motifs against PCR primers that can be designed to clone additional POU4F3 homologues are POU-specific domains having the amino acid sequence shown in SEQ ID NO: 2, and shown in SEQ ID NO: 1. Homeodomain with amino acid sequence.

In addition, additional nucleic acid molecules encoding transcription factors useful in the practice of the present invention may also be isolated, for example, using antibodies that recognize transcription factor proteins. Methods for preparing monoclonal and polyclonal antibodies are well known to those skilled in the art and are described, for example, in Chapters 5 and 6 of the Antibodies A Laboratory Manual , E. Harlow and D. Lane, Cold Spring Harbor Laboratory (1988). This chapter is incorporated herein by reference. As a non-limiting example, Xiang, M. et al., J. Neuroscience 15 (7): 4762-4782 (1995) and Xiang, M. et al., PNAS (USA) 94: As described in 9445-9450 (1997), antibodies to fusion proteins made from the C-terminus of Brn3 were successfully created.

Nucleic acid molecules encoding transcription factors useful in the practice of the present invention can be isolated, for example, by screening expression libraries. As a non-limiting example, the cDNA expression library can be screened using anti-POU4F3 homologous antibodies to identify one or more clones encoding POU4F3 homolog proteins. DNA expression library techniques are well known to those skilled in the art. Screening of cDNA expression libraries is fully reviewed in Chapter 12 of Sambrook, J., Fritsch, EF and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. The chapter is incorporated herein by reference.

As a representative example, antigens useful for making antibodies for screening expression libraries can be prepared by the following means. Full-length cDNA molecules (or cDNA molecules, including but not full-length, including all of the coding region) encoding transcription factors, such as the POU4F3 homologues, were obtained from the Bluescript plasmid (Stratagene, Inc., La Jolla, California). Can be cloned into a plasmid vector such as commercially available. Recombinant plasmids are then introduced into E. coli strains (such as E. coli XL1-Blue, also available from Stratagene, Inc.) and the proteins encoded by cDNA are expressed and purified in E. coli . For example, E. coli XL1-Blue with a BlueScript vector containing cDNA molecules of interest can be incubated overnight at 37 ° C. in LB medium containing 100 μg ampicillin / ml. 50 μl aliquots of overnight cultures can be used to inoculate 5 ml of fresh LB medium containing ampicillin and incubate the culture at 37 ° C. with vigorous stirring until A ₆₀₀ = 0.5 before induction with 1 mM IPTG. After an additional 2 hours of incubation, the supernatant is centrifuged (1000 xg, 15 minutes, 4 ° C), the medium is removed, preferably 1 ml cold buffer containing 1 mM EDTA and one or more protease inhibitors. Resuspend the pelleted cells in. Cells can be milled by sonication with microprobes. Cooled sonicates are made transparent by centrifugation, and the technique is disclosed in the industry, such as those disclosed in Methods in Enzymology, Vol. 182, Guide to Protein Purification, Murray P. Deutscher, ed (199), which is incorporated by reference. Purify the expressed recombinant protein from the supernatant by certified protein purification techniques.

Methods for preparing monoclonal and polyclonal antibodies are well known to those skilled in the art and are described, for example, in Chapters 5 and 6 of the Antibodies A Laboratory Manual , E. Harlow and D. Lane, Cold Spring Harbor Laboratory (1988). The above chapters are incorporated herein by reference. In one representative example, polyclonal antibodies specific for the purified protein may arise from New Zealand rabbits implanted with whiffballs. At intervals, 1 μg of protein is injected directly into the whiffball granuloma. Exemplary injection dosage regimens (each of 1 μg protein) are injected on days 1, 14 and 35. Granulomatous fluid is collected one week before the first injection (preimmune serum) and 5 days after the last injection (serum after immunization).

Sequence variants of transcription factors useful in the practice of the invention, resulting from deletions, substitutions, mutations and / or insertions, can also be used in the methods of the invention. Amino acid sequence variants of transcription factors useful in the practice of the present invention can be made by mutating DNA sequences encoding wild type transcription factor proteins, for example by using techniques commonly referred to as site specific mutagenesis. Nucleic acid molecules encoding transcription factors useful in the practice of the present invention can be mutated by various PCR techniques well known to those skilled in the art (e.g., see the following publications, which are incorporated herein by reference: " PCR Strategies ", MA Innis, DH Gelfand and JJ Sninsky, eds., 1995, Academic Press, San Diego, CA (Chapter 14);" PCR Protocols: A Guide to Methods and Applications ", MA Innis, DH Gelfand, JJ Sninsky and TJ White, eds., Academic Press, NY (1990)).

As a non-limiting example, a two primer system used in Clontech's Transformer Site-Directed Mutagenesis kit can be employed to introduce site-specific mutants into nucleic acid molecules encoding transcription factors useful in the practice of the present invention. After denaturation of the target plasmid in this system, both primers are annealed to the plasmid at the same time; One of these primers contains the desired site-specific mutation, and the other contains mutations at different points in the plasmid, resulting in the removal of restriction sites. Synthesis of the second strand is then performed and these two mutations are tightly linked and the resulting plasmid is transformed into the mutS strain of E. coli . Plasmid DNA is isolated from the transformed bacteria, enzymatically treated with appropriate restriction enzymes (thus straightening unmutated plasmids) and retransformed into E. coli . This system allows for the generation of mutations directly in expression plasmids without the need for subcloning or single stranded phagemid production. Tight binding of both mutations and straight chaining of unmutated plasmids results in high mutation efficiency and allows for minimal screening. After synthesis of the start restriction enzyme site primer, this method requires the use of only one new primer type per mutation site. Rather than making each point-specific mutant separately, it is better to synthesize a set of "designed denatured" oligonucleotide primers to simultaneously introduce all the desired mutations at a given site. Transformants can be screened by sequencing plasmid DNA through the mutated site to identify and classify mutant clones. Each mutant DNA was then sequenced or restriction enzyme treated entirely and analyzed by electrophoresis (JTBaker) on a Mutation Detection Enhancement gel (by band shift comparison with non-mutated controls) to ensure no other changes in the sequence. can confirm.

By way of non-limiting example, the two primer system used in the Quick-Change ™ Site-Directed Mutagenesis kit from Stratagene (La Joola, California) is a nucleic acid encoding a site-specific mutant that is a transcription factor useful for the practice of the present invention. It is adopted to introduce into the molecule. Double stranded plasmid DNA containing the insert with the target mutation site was denatured and mixed with two oligonucleotides complementary to each strand of plasmid DNA at the target mutation site. By extending the annealed oligonucleotides with Pfu DNA polymerase, a mutated plasmid containing staggered nicks is produced. After the temperature cycle test, the original, unmutated DNA template is enzymatically treated with DpnI , a restriction enzyme that cleaves methylated or semimethylated DNA but does not cleave unmethylated DNA. Since most E. coli strains from which template DNA has been obtained have essential methylase activity, the original, template DNA is almost always methylated or half-methylated. The remaining annealed vector DNA belonging to the desired mutation (group) is transformed into E. coli .

In the design of particular site-specific mutagenesis experiments, it is generally desirable to first make non-conservative substitutions (eg, Ala instead of Cys, His or Glu) and determine whether the activity significantly decreases as a result. If residues are described as important for reduced activity or knockout by this means, conservative substitutions can be made with Arg instead of Ser or His instead of Cys, for example Asp instead of Glu, to alter the length of the side chain. have. For hydrophobic sections, this is the major size that is usefully converted, while the aromatics can be substituted for alkyl side chains.

Other site specific mutagenesis techniques can also be employed in nucleic acid molecules encoding transcription factors useful in the practice of the present invention. For example, Sambrook et al. As described in Section 15.3 of Molecular Cloning: Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, New York, NY (1989), the restriction reactions following DNA restriction endonuclease treatment are performed in the practice of the present invention. Can be used to generate missing variants of transcription factors.

Oligonucleotide-specific mutagenesis may also be employed to prepare substitutional variants of transcription factors useful in the practice of the present invention. It can also be used to conveniently make deletions or insertion variants of transcription factors useful in the practice of the present invention. Adelman et al., Incorporated herein by reference. (DNA 2 : 183 [1983]); Sambrook et al, above;.. "Current Protocols in Molecular Biology", 1991, Wiley (NY), FT Ausubel, R. Brent, RE Kingston, DD Moore, JD Seidman, JA Smith and K. Struhl, eds, described in As noted, this technique is well known in the industry.

In general, oligonucleotides of at least 25 nucleotides in length are used to insert, delete or substitute two or more nucleotides in a nucleic acid molecule encoding a transcription factor useful in the practice of the present invention. The optimal oligonucleotide may have 12 to 15 nucleotides that perfectly match both sides of the nucleotide encoding the mutation. For mutagenesis of wild-type transcription factor proteins useful in the practice of the present invention, oligonucleotides are annealed to single-stranded DNA template molecules under appropriate hybridization conditions. Klenow fragments of DNA polymerase, usually E. coli DNA polymerase I, are then added. This enzyme uses oligonucleotides as primers to complete the synthesis of mutation-producing strands of DNA. Thus, heteroduplex molecules are formed such that one strand of DNA encodes a wild type protein inserted into a vector and the second strand of DNA encodes a mutant form of the protein inserted into the same vector. This heteroduplex molecule is then transformed into a suitable host cell.

Mutants with one or more substituted amino acids can be generated by one of several pathways. If amino acids are located in close proximity to the polypeptide chain, they can be mutated simultaneously using one oligonucleotide encoding all desired amino acid substituents. However, if the amino acids are located slightly apart from each other (eg more than 10 amino acids), it is more difficult to produce a single oligonucleotide encoding all the desired changes. Instead, either alternative method may be adopted. In a first method, an isolated oligonucleotide is generated for each amino acid to be substituted. The oligonucleotides are then annealed simultaneously to the single-stranded template DNA, and the second strand of DNA synthesized from the template will encode all desired amino acid substituents. Alternative methods include two or more rounds of mutagenesis to produce the desired mutant. The first round is as described for the single mutant: the DNA of the wild type protein is used as a template, the oligonucleotides encoding the first desired amino acid substituent (group) are annealed to this template, and then a heteroduplex DNA molecule is generated. do. The second round of mutagenesis uses the mutant DNA produced in the first round of mutagenesis as a template. Therefore, this template already contains one or more mutations. Oligonucleotides encoding additional desired amino acid substituents (groups) are then annealed to this template and the resulting DNA strands now encode mutations from both the first and second rounds of mutagenesis. This resulting DNA can be used as a template in the third round of mutagenesis and the like.

Prokaryotes can be used as host cells during the initial cloning step of transcription factors useful in the practice of the present invention. This is particularly true for the rapid production of large amounts of DNA, the production of single-stranded DNA templates used for site-specific mutagenesis, the simultaneous screening of multiple mutants and / or putative inner cell transcription factors, and the DNA sequence of the resulting mutants. Useful for decision 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 other enterobacteria such as Bacillus, Bacillus subtilis , Salmonella typhimurium or Serratia marcesans , and many other prokaryotes, including various Pseudomonas species. The species and genus of can be used as host cells. Other host cells with rigid cell walls or a prokaryotic host cell may be preferably transformation using the calcium chloride method as described in Sambrook et al., The section of the 1.82. Alternatively, electroporation can be used for transformation of these cells. Prokaryotic transformation techniques are described in Dower, WJ, in Genetic Engineering, Principles and Methods, 12 : 275-296, Plenum Publishing Corp., 1990; Hanahan et al., Meth. EnzyMol., 204 : 63 (1991).

As will be apparent to those skilled in the art, any plasmid vector containing replicon and control sequences derived from species compatible with the host cell can also be used to clone nucleic acid molecules encoding transcription factors useful in the practice of the present invention, It can be used to express and / or manipulate. Vectors usually have a polylinker region that contains a replication site, a marker gene that provides phenotypic selection in transformed cells, one or more promoters, and several restriction enzyme sites for insertion of foreign DNA. To plasmids that are typically used for transformation of E.coli include pBR322, pUC18, pUC19, pUCI18, pUC119, and blue script M13, those of which are described in Sambrook et al., The 1.12 to 1.20 of the section. However, many other suitable vectors are also available. Such vectors contain genes encoding ampicillin and / or tetracycline resistance, allowing cells transformed with these vectors to grow in the presence of such antibiotics.

The most commonly used promoters for prokaryotic vectors are β-lactamase (penicillinase) and lactose promoter system (Chang et al. Nature, 375: 615 [1978]; Itakura et al., Science, 198: 1056 [1977) Goeddel et al., Nature, 281: 544 [1979]) and tryptophan (trp) promoter system (Goeddel et al., Nucl, Acids Res., 8: 4057 [1980]; European Patent Application Publication No. 36,776) , And alkaline phosphatase systems. Although these are the most commonly used, other microbial promoters have also been used, and details regarding their nucleotide sequences have been published, allowing the skilled person to functionally bind this promoter into plasmid vectors (Siebenlist et al. , Cell, 20: 269 [1980]).

Construction of suitable vectors containing DNA encoding the replication sequences, regulatory sequences, phenotype selection genes and transcription factors useful in the practice of the present invention are made using standard recombinant DNA methods. Separate plasmids and DNA fragments are, as is known in the art, is cut in a specific order to generate the desired vectors, and cutting, are joined together (e. G., Sambrook et al., See above).

In another embodiment of the method of the invention, the formation of the inner ear sensory hair cells from the inner ear support cells is characterized by damage of the inner ear sensory hair cells and one or more cell cycle inhibitors that are active in the inner ear support cells (before, during the damage of the inner ear sensory hair cells). And / or after) inhibition of expression. In this way, the inner ear support cells in contact with the damaged sensory hair cells can be stimulated to divide and at least some of the resulting offspring form the inner ear sensory hair cells. As a representative example, cell cycle inhibitors that are active in inner ear support cells include cyclin-dependent kinase inhibitors, such as cyclin-dependent kinase inhibitors of the CIP / KIP family, including p21 ^Cip1 , p27 ^Kip1 and p57 ^Kip2 .

Specific examples of cell cycle inhibitors active in inner ear support cells are: p57 ^Kip2 (Lee et al., Genes Dev. 9 (6): 639-649 (1995) (SEQ ID NO: 6)); p27 ^Kip1 ( Cell 78 (l): 59-66 (1994) (SEQ ID NOS: 8 and 9)); p21 ^CiP1 (El-Diery et al., Cell 75 (4): 817-825 (1993) (SEQ ID NOS: 10 and 11)); pl9 Inlc 4d (Chan et al., Mol. Cell. Biol. 15 (5): 2682-2688 (1995) (SEQ ID NOS: 12 and 13)); pl8 Ink 4c (Guan et al., Genes Dev. 8 (24): 2939-2952 (1994) (SEQ ID NOS: 14 and 15)); pl5 Ink 4b (Hannon and Beach, 371 (6494): 257-261 (1994) (SEQ ID NOS: 16 and 17)); And p16 Ink 4a (Serrano, M. et al., Nature 366 (6456): 704-707 (1993) (SEQ ID NOS: 18 and 19)). Nucleic acid molecules encoding cell cycle inhibitors useful in the practice of the present invention are subjected to at least one hybrid formation constriction condition that is more constricted than 2 X SSCs at 55 ° C, such as 1 X SSC at 60 ° C or 0.2 X SSC at 60 ° C. Hybridization is made to the antisense strand of either nucleic acid molecule shown in SEQ ID NOS: 6, 8, 10, 12, 14, 16 and 18.

Inhibitors of cell cycle inhibitors can be substances, such as proteins, that act as cell cycle inhibitors in cells in a direct or indirect manner. Moreover, the inhibitor of cell cycle inhibitor can be an antisense nucleic acid molecule complementary to all or part of a nucleic acid molecule (such as an mRNA molecule), which encodes a cell cycle inhibitor protein and (2 × SSC at 55 ° C.). Hybridization to nucleic acid molecules encoding cell cycle inhibitor proteins under constriction conditions (such as greater constriction, for example, 1 X SSC at 60 ° C. or 0.2 X SSC at 60 ° C.).

Any industry-certified method can be used to inhibit cell cycle inhibitor gene expression in inner ear support cells. For example, expression of an active cell cycle inhibitor in an inner ear support cell may comprise a vector comprising a portion (or all) of the nucleic acid molecule encoding the cell cycle inhibitor active in the inner ear support cell in antisense orientation with respect to the promoter sequence. May be inhibited by introduction into support cells.

In general, antisense technology involves RNA transcripts that are inverted relative to the normal orientation for transcription and complementary to target mRNA molecules expressed in the host cell (ie, RNA transcripts of anti-sense genes exhibit Watson-Crick base pairing). Can be hybridized to the target mRNA). Anti-sense genes can be constructed in a number of different ways that can interfere with the expression of target genes, such as genes encoding cell cycle inhibitors. Anti-sense genes can be constructed by inverting the coding region (or a portion thereof) of the target gene against normal transcriptional orientations that allow transcription of its complement, so that the RNA encoded by the anti-sense and sense genes is complementary. to be.

Anti-sense genes are generally substantially identical to at least a portion of a target gene or group of genes. However, the sequences need not be identical to inhibit expression. In general, higher homology can be used to compensate for the use of shorter anti-sense genes. Minimal identity is typically at least about 65%, but higher identity can exert more effective inhibition of expression of endogenous sequences. Substantially greater identity of at least about 80% is preferred, while from about 95% to complete identity is most preferred.                 

Furthermore, the anti-sense gene need not have the same intron or exon pattern as the target gene, and the non-coding compartment of the target gene can be equally effective in the coding compartment in achieving anti-sense inhibition of target gene expression. . Typically, DNA sequences of at least about 30 or 40 nucleotides are used as anti-sense genes, but longer sequences are preferred. The construct is then introduced into one or more inner ear support cells and an antisense strand of RNA is produced.

Catalytic RNA molecules or ribozymes can also be used to inhibit the expression of a target gene. It is possible to design ribozyme transgenes that encode RNA ribozymes, which specifically pair with the target RNA and cleave the phosphodiester backbone at specific points, thereby functionally inactivating the target RNA. In carrying out this cleavage, the ribozyme does not change on its own, so it can be recycled to cleave other molecules. Inclusion of ribozyme sequences into antisense RNA confers RNA-cleaving activity on them, increasing the activity of the antisense constructs. Tabler et al. (1991, Gene 108: 175) greatly simplifies the fabrication of catalytic RNA by combining the advantages of anti-sense RNA and ribozyme technology into a single construct. Since smaller homology regions are required for ribozyme catalysis, this can promote the inhibition of different members of a large gene family if the cleavage site is conserved.

An additional strategy suitable for the inhibition of target gene activity involves the expression of a sense in a mutated or partially deleted form of a protein encoded by the target gene according to the general standard for the production of dominant negative mutations (Herskowitz I, Nature 329). : 219-222 (1987)).

Any industry-certified gene delivery method can be used to introduce nucleic acid molecules (or vectors comprising antisense DNA molecules) encoding transcription factors into these cells for expression in the inner ear cells, which are directly injected, electrophoretic. Migration, virus-mediated gene delivery, amino acid-neutral gene delivery, biolistic gene delivery, lipofection and heat shock. Non-viral gene delivery methods to inner ear cells are described in Huang, L., Hung, MC, and Wagner, E., Non-Viral Vectors for Gene Therapy, Academic Press, San Diego, California (1999). Is initiated.

For example, the gene may be introduced in vivo by viral vectors in situ or after separating cells from the body. For example, retroviruses are RNA viruses that have the ability to insert their genes into the chromosome of a host cell after infection. Retroviral vectors have been developed that lack the genes encoding viral proteins, but retain the ability to infect cells and insert the genes into the chromosome of target cells (AD Miller, Hum. Gen. Ther. 1: 5-14 (1990)).

Adenoviral vectors are designed to be administered directly to a patient. Unlike retroviral vectors, adenovirus vectors do not integrate into the chromosome of a host cell. Instead, genes introduced in vivo using adenovirus vectors remain in the nucleus as episomes that last for a limited time. Adeno viral vectors can infect in vivo dividing and non-dividing cells in many different tissues including airway epithelial cells, endothelial cells, hepatocytes and various tumors (BC Trapnell, Adv Drug Del Rev. 12: 185 -199 (1993)).

Another viral cell is the herpes single virus, a huge, double-stranded DNA virus that has been used in some early applications of delivering therapeutic genes to neurons and potentially can be used to deliver therapeutic genes to some forms of brain cancer. (DS Latchman, Mol. Biotechnol. 2: 179-95 (1994)). Recombinant forms of vaccinia virus can accommodate large inserts and are produced by homologous recombination. To date, this vector has been used to deliver interleukin (ILs) such as human IL-1β and costimulatory molecules B7-1 and B7-2 (GR Peplinski et al., Ann. Surg. Oncol. 2: 151-9 (1995) JW Hodge et al., Cancer Res. 54: 5552-55 (1994)).

Another approach to gene therapy involves the direct introduction of DNA plasmids into patients (FD Ledley, Hum. Gene Ther. 6: 1129-1144 (1995)). Plasmid DNA can be taken up by cells in the body to direct the expression of recombinant proteins. Typically plasmid DNA is delivered to cells in the form of liposomes, wherein the DNA is one or more, such as DOTMA (1,2-diolsiloxypropyl-3-trimethyl ammonium bromide) and DOPE (dioleoylphosphatidylethanolamine) Combined with lipids. Formulations with DOTMA have been shown to provide expression in lung epithelial cells in animal models (KL Brigham et al., Am. J Med. Sci, 298: 278-281 (1989); AB Canonico et al., Am. J) Respir.Cell.Mol. Biol. 10: 24-29 (1994)). In addition, it has been demonstrated that intramuscular injection of plasmid DNA formulated with 5% PVP (50,000 kDa) increases the level of reporter gene expression in muscle by more than 200 times the level found in injection of DNA formulated with saline only. (JJ Mumper et al., Pharm. Res. 13: 701-709 (1996); RJ Mumper et al., Proc. Intern. Symp. Cont. Rol. Bioac. Mater. 22: 325-326 (1995)). Intramuscular administration of plasmid DNA results in gene expression that lasts for months (JA 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).

In addition, the uptake and expression of DNA was found in the thyroid gland (M. Sikes et al., Hum. Gene Ther. 5: 837-844 (1994)) and the synovial membrane (J. Yovandich et al., Hum. Gene Ther. 6: 603-610 (1995)) was also observed after direct injection of the plasmid. Lower levels of gene expression include liver (MA Hickman et al., Hum. Gene Ther. 5: 1477-1483 (1994)), skin (E. Raz et al., Proc. Natl. Acad. Sci. 91: 9519- 9523 (1994)), interstitial injections into the airways (KB Meyer et al., Gene Therapy 2: 450-460 (1995)), applied to the epithelium (GD Chapman et al., Circulafion Res. 71: 27-33 ( 1992); R. Riessen et al., Human Gene Therapy, 4: 749-758 (1993)), and after vascular administration (RM Conry et al., Cancer Res. 54: 1164-1168 (1994)) .

Various devices have been developed to improve the availability of DNA to target cells. A simple approach is to physically contact target cells with an implantable material containing a catheter or DNA (GD Chapman et al., Circulation Res. 71: 27-33 (1992)). Another approach is to use a needleless jet injection device that launches a column of liquid directly into the target cell under high pressure (PA Furth et al., Anal Biochem. 20: 365-368 (1992); (HL Vahlsing et al) J. Immunol. Meth. 175: 11-22 (1994); (FD Ledley et al., Cell Biochem. 18A: 226 (1994)).

Another device for gene delivery is a "gene gun" or Biolistic ™, which is a ballistic device that launches DNA-coated micro-particles in vivo directly into the nucleus of a cell. Once in the nucleus, DNA can be isolated from gold or tungsten fine particles and expressed by target cells. This method has been used to directly and efficiently transport genes into skin, liver and muscle (NS Yang et al., Proc. Natl. Acad. Sci. 87: 9568-9572 (1990); L. Cheng et al., Proc. Natl.Acad . Sci. USA. 90: 4455-4459 (1993); RS Williams et al., Proc. Natl. Acad. Sci. 88: 2726-2730 (1991).

Cochlear opening involves perforation of the cochlear angle and insertion of the catheter through where chemicals, such as nucleic acid molecules, can be introduced into the cochlear angle. Cochlear aperture surgery methods are disclosed, for example, in Lalwani, A. K. et al., Hearing Research 114: 139-147 (1997), the publications of which are incorporated herein by reference.

Another approach to targeted gene delivery is the use of molecular conjugates, which consist of proteins or synthetic ligands where nucleic acid- or DNA-binding agents are attached to cells for specific targeting of nucleic acids (RJ Cristiano et al., Proc Natl.Acad . Sci. USA 90: 11548-52 (1993); BA Bunnell et al., Somat.Call Mol.Genet . 18: 559-69 (1992); M. Cotten et al., Proc. Natl. Acad. Sci. USA 89: 6094-98 (1992). Once the DNA is coupled with the molecular conjugate, a protein-DNA complex is created. This gene delivery system appears to be capable of targeted delivery to many cell types through the use of different ligands (RJ Cristiano et al., Proc. NatZ. Acad. Sci. USA 90: 11548-52 (1993)). For example, vitamin folate is used as a ligand to facilitate delivery of plasmid DNA into cells that overexpress folate receptors (eg ovarian cancer cells) (S. Gottschalk et al., Gene Ther. 1: 185-91 (1994)). Malaria sporeosomal proteins have been used for liver-specific delivery of genes under conditions of low ASOR receptor expression in hepatocytes, such as cirrhosis, diabetes, and hepatocellular carcinoma (Z. Ding et al., J. Biol. Chem. 270: 3667-76 (1995)). Overexpression of epidermal growth factor (EGF) receptors in cancer cells allowed specific uptake of the EGF / DNA complex by lung cancer cells (R. Cristiano et al., Cancer Gene Ther. 3: 4-10 (1996)). Currently preferred method of gene delivery is lipofection.

When the method of the present invention is used in vitro, the entire inner ear, including the Corti organ, is preferably cut, cultured and manipulated in a culture vessel. Current preferred embodiments of the apparatus useful for culturing the inner ear in vitro are described in US Pat. No. 5,437,998; US Pat. No. 5,702,941 and US Pat. No. 5,763,279, which are incorporated herein by reference.

Generally presently preferred embodiments of the apparatus for culturing the inner ear include a gas permeable bioreactor, which includes a tubular vessel with walls that can be made, at least in part, of a gas permeable material such as silicone synthetic rubber. In one preferred embodiment the container is made of half of it gas permeable material and the remaining part is made of non-permeable material. Commonly available gas permeable materials are opaque. Therefore, the use of a non-permeable material in at least a portion of the bioreactor can provide the advantage of allowing visual inspection of the tubular container chamber.

The tubular vessel has a closed end, a substantially horizontal longitudinal center axis, and one or more vessel access ports. The vessel proximity provides proximity to the bioreactor for the introduction of media and cells, and removal of old media from the tubular vessel. This is easily accomplished through a vessel proximity port, also referred to as a valve or syringe port. In a preferred embodiment, the container proximity port is made of a valve with a syringe.

Preferably the container is rotatable about its horizontal length centerworm. Preferred means for rotation are motor assemblies seated on the mount and have means for attachment to the tubular container. The rate of rotation causes the inner ear to constantly move in the tubular vessel, but the rotation of the tubular vessel should not be fast enough to cause significant turbulence in the aqueous medium in the tubular vessel.

If so, the use of a gas permeable material in the fabrication of at least a portion of the tubular vessel wall allows O ₂ to diffuse through the vessel wall into the cell culture medium in the vessel chamber. Correspondingly, CO 2 diffuses out of the vessel through the wall. Therefore, the use of gas permeable material in at least a portion of the tubular vessel wall typically overcomes the need for air injection into the bioreactor vessel. However, if additional oxygen is needed to culture the inner ear, air injection into the aqueous medium in the bioreactor vessel may be used. When an air pump is used to inject air into the aqueous medium, an air filter is also employed to protect the air pump valve from dust.

An alternative embodiment of a bioreactor useful for the practice of the present invention is an annular vessel having a wall made of at least partly gas permeable material. The annulus includes the annular, toroidal and other substantially symmetrical, ring-like tubular containers. The annular container has a closed end and a substantially horizontal longitudinal center axis.

In another embodiment, a bioreactor useful in the practice of the present invention comprises a tubular vessel made at least in part of a gas permeable material. The container has a closed end and a substantially horizontal longitudinal center axis and rotates around it. Moreover, the vessel has two slidably interconnected components, one component slidably fixed within the other component, forming a liquid resistant seal therebetween to provide various volumes to the tubular vessel. The bioreactor has means for rotating the tubular vessel about its substantially horizontal longitudinal center axis. One or more vessel proximity ports provide for the movement of mass into and out of the vessel.

In situations where minimization of contamination is required (eg AIDS or human tissue studies), the one-off nature of bioreactors useful in the practice of the present invention is a particular advantage. Moreover, embodiments of bioreactors having slidably interconnected components can be adjusted to provide the correct size of bioreactor required.

Presently preferred commercially available bioreactors useful in the practice of the present invention for culturing fluid-filled sensory organs are described in Synthecon, Inc. High Aspect Ratio Vessel (HARV ™) and Cylindrical Cell Culture Vessel (CCCV ™) produced by (8054 El Rio, Houston, Texas).

Neuralbasal ™ of Gibco BRL (Gibco BRL medium is produced by Life Technologies (Corporate Headquarters, Gaithersburg, MD)), which requires the addition of B27 or N2 medium supplementation, is currently the preferred culture medium for culturing the inner ear in vitro. . However, other culture media can also be used successfully to culture fluid-filled sensory organs in the practice of the present invention. Other suitable media include DME, BME and M-199 with fetal bovine serum or equine serum. All the above badges are sold by Gibco-BRL. When using Neuralbasal ™ media, N2 and B27 play a more important role when attempting extended periods of culture (> 96 hours).

In another aspect, the invention provides a method for stimulating the formation of inner ear support cells. The method of this aspect of the invention comprises damaging the inner ear support cells under conditions that promote the formation of new inner ear support cells (eg, by cell division of the inner ear support cells in contact with the damaged inner ear support cells). In this aspect of the invention, the inner ear support cells are damaged and the formation of new inner ear support cells is stimulated using the same technique as the method of the present invention that stimulates the formation of inner ear sensory hair cells from the inner ear support cells described herein. Thus, for example, inner ear support cells can be damaged by contacting a significant amount of inner ear neurotoxic agent, such as an aminoglycoside antibiotic effective to damage the inner ear support cells. As another example, new inner ear support cell formation may be caused by damage to inner ear support cells and within inner ear support cells (prior to, during and / or after damage to inner ear support cells) such as POU4F1, POU4F2, POU4F3, Brn3a, Brn3b, and Brn3c. It can be further stimulated by the expression of transcription factors, which can stimulate the inner ear support cells to divide to form new inner ear support cells. In a preferred embodiment of this aspect of the invention, proliferation of the inner ear support cells indicates an improvement in the auditory function of the treated inner ear.

The following examples merely illustrate the best mode currently planned for carrying out the invention and should not be construed as limiting the invention.

Example 1

In Vitro Immortal Support-overexpression of POU4F3 in Cell Lines

POU4F3 is a DNA binding transcription factor that shows pronounced specificity for hair-cells in the inner ear. Mutations in POU4F3 are known to cause developmental failure in mice and to cause hearing loss in both mice and humans. The role of POU4F3 in directing the development of hair-cell precursors is studied by transfecting inner ear support-cell lines with POU4F3.

To detect the expression of POU4F3 in living cultures, Enhanced Green Fluorescent Protein (EGFP), which is translated from bicistronic mRNA containing both POU4F3 and GFP coding regions, is monitored. In particular, a 1250 bp cDNA encoding POU4F3, including 70 bp of 5'UTR and 73bp of 3'UTR, into the only EcoRV restriction enzyme site of the pIRES2-EGFP vector (Clonetech) immediately downstream from the human CMV main mediated promoter / enhancer. Cloning directionally. The intervening synthetic introns are cloned downstream from POU4F3 to enhance the stability of the mRNA. An internal ribosome entry point (IRES) derived from Encepelomyocarditis virus is cloned between POU4F3 and GFP to allow translation of GFP and POU4F3 proteins from the same mRNA. Immediately following the GFP coding region is the polyadenylation signal from the bovine growth hormone gene. This expression cassette is designed to take advantage of the two-cystronic promoter that allows tracking of transfection and expression of POU4F3 and GFP by visualization of GFP expressed under fluorescence microscopy. This secondary production GFP vector has a red shifted variant (stimulation maximum = 488 nm; emission minimum = 507 nm) of wild-type GFP optimized for brighter fluorescence. To optimize the identification of cells expressing high levels of POU4F3, the pIRES2 vector uses partially neutralized IRES sequences (Rees, S. et al., BioTechniques 20: 102110 (1996)). This declined IRES reduces the rate of translation start in the GFP start codon compared to that of POU4F3.

Support-cell lines (UCLs) are obtained from the inner ear anterior sensory epithelium of 112k ^b - ts A 58 transgenic mice (Immortamouse). The inner ear oocytes were excised from 1 day old mice to isolate sensory epithelium (hair-cell population and support-cell population) after simple thermolysin treatment. Resulting support-cell lines were induced after multiple passages in acceptable conditions (33 ° C. and γINF) to stimulate renal cell proliferation. Concentration cultures were made on days 3-4. This process caused the death of all hair-cell populations as measured by ICC and electron microscopy (EM). Further characterize the UCL group with an antibody called ZO-1 (labels a tight bond that appears in the support-cell population) and EM (shows a tight binding complex, secretory vesicles and luminescent surface villi, all of which are characteristic of the support-cell population) It was.

Culture medium for the UCL cell line consists of DMEM / F12 (Gibco), fetal bovine serum (10%) and γINF (20 u / ml). Medium exchange was performed 2-3 times a week depending on the growth rate of the cells. Single-cell clones were developed using special seeding methods that could allow a single cell population to become confluent and passable at 3-4 weeks. Cell growth was stopped at non-acceptable conditions (37 ° C. or 39 ° C., no γINF and low or no FBS).

Since high transfection efficacy was already observed in the UCL group, this cell group was grown and passaged in specific medium which was serum free. Once this was established, this cell population was refected with IRES-GFP-POU4F3 encoding the plasmid. Cultures were monitored for GFP expression over 1-6 DIV. If a period of high GFP fluorescence was observed in living cultures, the cultures were fixed and prepared for POU4F3 and Calvindine ICC.

Example 2

Overexpression of POU4F3 in Injured Corti Organ Cultures

Cultures were obtained from P7-P14 mice and damaged with 1 mM neomycin for 2 DIV. After removing the medium the cultures were refected with pIRES2-GFP-POU4F3 for 6 hours and recovered in fresh medium for 1-6 DIV. Cultures were fixed with aldehyde and processed for POU4F3 and Calvindine immunocytochemistry. Cultures lipofected only with pIRES2-GFP serve as controls. The presence of triple labeled cells (positive for GFP, POU4F3 and Calvindine immunoreactivity) indicates that POU4F3 can promote uptake of the hair-cell phenotype in the damaged Corti organs. Further measurements of this phenotype are confirmed as antibodies against other hair-cell selectable markers using induced polyclonal antibodies against proteins such as myosin 6 and myosin 7a.

Expression of hair-cell specific markers such as myosin 6 and 7a is observed in the Corti organs of mouse embryos at E16, where expression of the Brn 3.1 transcription factor begins 2-3 days after E13.5.

Example 3

p27 ^Kip1  Regeneration of Inner Ear Hair Cells in Mice

Previous reports in p27 ^Kip1 -/-and +/- mice show qualitative evidence of excess hair cell populations (HCs) on both the inner hair cell (IHC) and outer hair cell (OHC) zones (Chen, P., & Segil, N. Development 126: 1581-1590 (1999); Lowenheim, H., et al., Proc. Natl. Acad. Sci. USA. 96: 4084-4088 (1999). Unfortunately, significant dysplasia of peripheral support cells could explain some very well, if not all of these observations. Therefore, the number of IHC and OHC at the cochlear ^{angle of} p27 ^Kip1 − / −, +/− and + / + mice was measured. In order to more accurately assess whether there was a true increase in HC number, several zones from the same cochlear angle were analyzed. Using the HC specific marker myosin-VIIa antibody, a 20% increase in the number of IHCs in the p27 ^Kip1 − / − cochlea was observed when compared to the number of IHCs in the p27 ^Kip1 +/− and + / + angles of view. However, except for a 10% increase in the number of OHCs in one analyzed zone, there was no statistically significant difference in the overall OHC numbers between p27 ^Kip1 − / −, +/− and + / + cochlea (Table 1). Table 1 shows the number of hair cells (n) in p27 ^Kip1 + / +, +/−, − / − mice at 4 weeks of age. Counts were made on sensory epithelium 100 μm long from three different locations along the length axis of the Corti organ. The distance corresponds to the angle (± sd) from the peak of the cochlear angle. Comparison was made to the same hair cell zones over + / +, +/- and-/-. Statistical significance was measured using ANOVA.

To determine whether auditory hair cells are produced after initiation of hearing on day 10, ^bromode , a nucleotide analogue that is incorporated into proliferating cells during S phase in p27 ^Kip1 + / +, +/-,-/-mice at 2 weeks of age Oxyuridine (BrdU; 30 mg / kg / sc) was injected systemically for 3 days. Mice were allowed to recover without further injection for 2 days or 2 weeks. BrdU positive HC was identified by immunocytochemistry using optical and fluorescence microscopy. Cochlear angles were also labeled with antibodies against myosin-VI and myosin-VIIa. At 2 weeks of age p27 ^Kip1 − / − cochlea, restored for 2 days after the last injection, no BrdU / myosin-VIIa positive cells were observed in the BrdU positive cells. However, BrdU / myosin-VIIa positive cells were observed at 4 weeks of age p27 ^Kip1 − / − cochlea, which recovered for 14 days after the last injection. Most of the double labeled was IHC. This qualitative finding is similar to our quantitative assessment of IHC and OHC. p27 ^Kip1 + / +, +/− mice had no BrdU positive cells on days 2 or 14 of recovery. This data is summarized in Table 2. Table 2 shows BrdU labeled cells in the Corti organs of p27 ^Kip1 + / +, +/-,-/-mice at 2 and 4 weeks of age with a recovery of 2 or 4 days after injection with BrdU BrdU / ^amicasin The number (n) of is shown. Counting was performed on the 1000 μm long sensory epithelium obtained in the upper half of the cochlear angle (± sd). Proliferation in the BrdU / amicasin group was compared with that in the BrdU group of the same genotype. Statistical significance was measured using ANOVA.

To confirm that auditory HC can be regenerated, systemic injection of amikacin sulfate was used to damage HC (P7-P12) and then injected with BrdU (P10-12). Mice were sacrificed 2 or 14 days after the last injection. First, in both p27 ^Kip1 − / − and +/− mice, the number of BrdU positive cells increased after treatment with Amikacin / BrdU vs BrdU alone. In the p27 ^Kip1 +/- cochlear angle, the number of BrdU labeled cells increased in most of the samples tested, but not all p27 ^Kip1 +/- cochlear angles showed BrdU positive cells. Second, a greater number of BrdU positive HCs were observed after amikacin damage at p27 ^Kip1 − / − cochlea. Most of the BrdU positive HC appeared in the region of the cochlea (lower half of the cochlea) where amikacin sulfate damaged or killed HC. No BrdU positive cells were observed in wild-type cochlea after treatment with Amikacin / BrdU or BrdU alone. This data is summarized in Table 2.

To determine specific protein levels, single cochlear cell lysates were serially diluted and run on polyacrylamide gels. Western blotting showed that myosin-VI and VIIa levels were approximately equal across p27 ^Kip1 − / −, +/−, and + / +, although stronger myosin-VIIa bands were observed at the p27 ^Kip1 − / − cochlear angle It appears. The p27 ^Kip1 +/- cochlear ^angle contains approximately 50% of the p27 ^Kip1 protein levels found in wild-type cochlear angle, where a decrease in p27 ^Kip1 from normal to 50% can stimulate support cell proliferation and occurs after treatment with amikacin sulfate Indicates that it may allow for some hair cell regeneration.

The protocol used to measure protein levels in the cochlea is as follows. The cochlear angle is transferred to a tube containing 10 μl of extraction buffer, which comprises: HEPES (25 mM), NP-40 (0.7%), Aprotinin (1 mM), Leupeptin (1 g / ml), Pepstatin (10 μM), phenylmethylsulfonylfluoride (PMSF) (1 mM), dithiothritol (DTT) (1 mM), and ethylenediaminetetraacetic acid (EDTA) (2 mM). Immediately break the cochlea and place the tube on ice for about 30 minutes. 5 μl of 4 × sample buffer is added and the salt concentration is adjusted to 0.5 M. The supernatant is collected by heating the sample to 90-100 ° C. for 5 minutes and then centrifuging at 13,000 rpm for 10 minutes. Protein aliquots from the supernatant are run at 200V for 50 minutes on a 15% SDS-PAGE gel and the protein is transferred onto PVDF membrane at 100V for 1 hour. The membrane is blocked for 1 hour or overnight with 10% Amersham blocking buffer solution. Primary antibody in blocking buffer solution is added to the membrane for 1 hour and then washed 5 times with PBS / Tween 5 minutes per wash. Probe membranes for 1 hour with anti-biotin-AP + chlorine anti-mouse or goat anti-rabbit-alkaline phosphatase and wash 5 times per wash with PBS / Tween for 5 minutes.

p27 ^Kip1 is to determine whether a role similar to the peripheral vestibular system, examined the proliferative capacity of p27 ^Kip1 +/- and oval sac, sac sphere and Krista of + / + mice. Mice (P7-P12) were injected systemically with amikacin sulfate sulfate for 6 consecutive days (500 mg / kg / d / sc) and a combination of injection of bromodeoxyuridine (BrdU), a replication marker, between P10-P12 (30 mg / kg / d / sc). BrdU alone was also injected into mice in a similar manner. After 14 days mice were sacrificed and vestibular sensory organs were fixed, incised and processed for BrdU immunocytochemistry. BrdU positive nuclei were counted from the entire slide using an optical microscope and Nomarski optical lens. Selected organs were further processed for translaminar analysis.

In p27 ^Kip1 mice administered with BrdU alone, very low levels of BrdU-labeled cells were observed in the globular and oval cysts. However, after combining Amikacin / BrdU treatment, a 40-fold increase in BrdU positive cells was observed in both organs. Approximately half of labeled cells appeared as doublets presenting recent or ongoing cell division. The plastic lamella showed that most of the BrdU labeled cells were in the basal layer of the sensory epithelium along the basement membrane. BrdU positive HC was also observed in both ear organs. Most of these regenerated HCs appeared as Type I HCs bordering the cup-shaped organs. In p27 ^Kip1 +/− mice injected with BrdU alone, no proliferation was observed in the globules or oocytes. After a combination of amikacin / BrdU treatment, very low levels of proliferation were induced. In p27 ^Kip1 + / + mice, no BrdU positive nuclei were observed in the globules or oocytes after treatment with amikacin / BrdU or BrdU alone. Interestingly, no BrdU positive cells were observed in any genotyped crista after treatment with Amikacin / BrdU or BrdU alone. This data indicates a significant discussed effect on p27 ^Kip1 deficiency between various vestibular sensory organs and between vestibular sensory organs and Corti organs. This data is summarized in Table 3. Table 3 shows the number of BrdU labeled cells in the vestibular organs of 4 week-old p27 ^Kip1 + / +, +/- and − / − mice with 14 days of recovery after injection with BrdU or BrdU / ^amicasin . Count (± sd) from total oocyte, globular sac and crista sensory epithelium. In the BrdU group, statistical significance was measured by comparing proliferation levels in the same sensory organs across + / +, +/- and-/-. In the BrdU / amicasin group, statistical significance was determined by comparing proliferation levels in the same sensory organs of the same genotype. Statistical significance was measured using ANOVA.

Selected mid-thickness flakes were re-embedded in plastic, thinly cut and tested under electron microscope. BrdU positive HC showed neural distribution of steric ciliary bundles, keratinous plaques and goblet organs, evidence of synapse formation.

Example 4

p27 ^Kip1  Antisense inhibition of expression

To test whether inhibition of p27 ^Kip1 gene product in p27 ^Kip1 + / + cochlea proliferates non-mitotic support cells, explants of wild-type Corti organs (P7-P10, at hearing initiation age) were ^examined for p27 ^Kip1 antisense oligonucleotides. (ONs). Explant cultures were prepared by dissecting the corti organs from the cochlea, removing the overcoat and attaching the corti organs to the slide glass coated with Cell-tak and maintained at 37 ° C. in a 5% CO ₂ atmosphere. The explants were then incubated for 48 hours with an inner ear neurotoxic antagonist (1 mM neomycin sulfate) (Kil, J., et al., ARO abs., 21: 672 (1998)) that kills> 95% of HC. Exposed. The neomycin containing medium was removed and p27 ^Kip1 antisense oligonucleotides (Ons) (40 nM) were administered (lipofection) using cationic lipids for 24 to 48 hours. Some of these live cultures were tested under fluorescence to detect the presence of FITC bound antisense ON.

FITC-positive support cells were detected between 18 and 24 hours and increased in number and fluorescence intensity between 24 and 48 hours. Cultures were fixed with aldehyde and processed for BrdU immunocytochemistry. BrdU-positive support cells appeared in most cochlear cultures after 24 hours of antisense oligonucleotide (ON) treatment. BrdU-positive doublets appeared in antisense ON treated cultures recovered for an additional 24 hours without antisense ONs. This indicates that successful completion of the M phase and sequential cell division can occur. Lipofection-only treated cultures have very low levels of BrdU-labeled support cells.

We have observed that administration of p27 ^Kip1 antisense oligonucleotides can induce proliferation of support cells in wild type cochlea. This observation is unique and of great importance. Previous studies exemplifying p27 ^Kip1 antisense ON induced proliferation have been shown in active dividing cells that have been transiently or reversibly grown (Coats, S., et al, Science, 272: 877-880 (1996); Dao, MA, et al, Proc. Natl. Acad. Sci. USA, 95: 13006-13011 (1998)). Our results are the first illustration that the final mitotic cells in the finally differentiated organs can ^reenter the cell-cycle after inhibition of the p27 ^Kip1 gene product.

In other words, support cell proliferation typically stops between E12 and 14 in the mouse corti organ. After this point, support cell differentiation continues over the second week of life. When explants were transplanted, the treated cultures as described herein had already developed some adult-like morphological properties. These data further support the role for p27 ^Kip1 antisense ON as a potential means to induce hair cell regeneration. In p27 ^Kip1 +/- mice, a 50% reduction in normal protein levels allows any finally differentiated support cell to overcome p27 ^Kip1 blockade and re-enter and proliferate into the cell cycle.

The addition of growth factors that induce cell proliferation in other epidermal organs does not promote cell proliferation or hair cell regeneration in postnatal corti organs, either in vitro or in vivo. The experiments reported here have identified a rather ubiquitous and potent cell cycle inhibitor that, when defective, allows the Corti organ to regenerate its hair cells to some degree spontaneously. Corti organs in mice containing one copy of the gene and 50% of normal protein can regenerate auditory hair cells.

In addition, organotyping can be established on slide glass (Nunc) coated with CellTak (Collaborative Research) in 100 μl of NeuroBasal medium containing 1 mM neomycin. This treatment kills 95% of hair cells and also facilitates the level of transfection. Cultures are lipofected with antisense molecules using commercially available lipofection reagents (eg, Perfect Lipofection Kit; InVitrogen, Inc.). The medium also contains BrdU (10 μM) to identify proliferating cells. In addition, various recombinant growth factors such as TGF-alpha (1-100 nM), insulin (10-100 μM) and IGF-1 (1-100 μM) can be used to increase or control this proliferative effect. .

Example 5

P27 to stimulate cell proliferation in guinea pig fibroblasts in vitro ^Kip1  Use of antisense oligonucleotides

Cultures of cell lines that respond to p27 ^Kip1 antisense oligonucleotides (ON) during the period of serum recruitment and growth arrest were established. This culture contains guinea pig fibroblast line (JH4). Lipofection of hexaelement antisense ON (with nucleic acid sequence described in SEQ ID NO: 20) reverses growth arrest by at least 40% of normal in JH4 cell line.

Example 6

Guinea pig wow from p27 ^Kip1  Stimulation of support cell proliferation using antisense molecules

Two recent, independent studies indicate that antisense ONs can be successfully delivered through the peripheral lymphatic space, leading to changes in the corti organs of mature guinea pigs (D'Aldin, C., et al., Mol Brain Res., 55: 151-164 (1998); Leblanc, CR, et al. Hear. Res., 135: 105-112 (1999). The presence of FITC-antisense ON against the sequence specific for mRNA of GluR2 is seen in spiral ganglion, support cells, and inner and outer spiral brain fissure cells within 24 hours after osmotic pump installation. Sequential decreases in GluR2 / 3 protein were also observed in situ (D'Aldin et al., 1998, supra).

The expression pattern of p27 ^Kip1 in mature guinea pig corti organs was found to be similar to that observed in developing and adult mice. Supporting cell proliferation in guinea pig cochlea is stimulated as follows:

Induces general anesthesia by inhalation of isoflurane (5% for induction and 2-3% for maintenance) or intramuscular injection of catamine (50 mg / kg) and xylazine (9 mg / kg) . 1% lidocaine is injected topically behind the earlobe. The animal is placed on a warming pad to maintain basal body temperature during the surgical procedure. Respiration and circulation are carefully monitored. The drug is delivered into the guinea pig's inner ear by surgically implanting an injection unit consisting of a coiled catheter and an osmotic small pump (Alzet, catalog no. 2002, flow rate 0.5 μl / hour by day 14). The drug is loaded into the coil and the osmotic small pump carries the dye. The total volume of drug ejected into the inner ear can be monitored by the total amount of dye ejected into the coil. The drug is dissolved in a solution of artificial peripheral lymph, which is: 137 mM NaCl, 5 mM KC1, 2 mM CaCl ₂ , 1 mM MgCl ₂ , 10 mM Hepes, 11 mM glucose, pH 7.4 and osmolarity 300mOsm / L It is composed.

All surgical procedures are performed under a dissecting microscope and under sterile conditions. The incision behind the auricle exposes the papillary bleb (medium space) and is open to view the base protrusion of the cochlea using a 1 mm cutting burr. Using a 0.5 mm diamond paste burr, cochlear opening is performed about 1 mm below the insole. Observe the correct inner positioning of the cochlear opening with the observed inner ear peripheral lymph bleeding from this site. The tip of the injection unit is inserted into the cochlear opening and the tube secured with dental cement on the wall of the middle ear. The infusion unit is stored in a hypodermic bag created behind the neck. Skin incisions were closed with 2-0 silk sutures. This procedure is performed to avoid any concomitant imbalance or behavioral changes in both ears and to reduce the number of animals needed to complete the experiment. The procedure takes approximately 30 minutes per side.

Repeat surgery to replace the infusion unit is performed under general anesthesia one week after implantation. The procedure is done only through the skin incision behind the initial auricle and does not include re-entry into the middle ear space. This procedure takes 5 to 10 minutes per injection unit. The postoperative condition of the animals is monitored daily by checking activity, appetite, watering, feces and body weight. Animals are sacrificed if their body weight is reduced by more than 20% after surgery, or if they exhibit severe behavioral changes or head hindrance. No surgical discomfort should occur and this procedure causes only minor postoperative discomfort.

As shown in Table 4 below, the normal control is given a solution of artificial peripheral lymph for 1 or 2 weeks. The hair cell loss group is given gentamicin sulfate for 1 week to kill hair cells with immediate sacrifice and 2 weeks recovery. Three concentrations are used that should provide the total loss of HCs as well as the gradient loss of HCs. Cationic liposomes are delivered in 5% (weight / volume) dextrose solution for 1-2 weeks. The purpose of this group is to see what damage is caused by lipofection. Lipofection after hair cell loss involves replacing the gentamicin containing pump with a lipid containing pump. Lipofection + antisense after hair cell loss involves lipofection of FITC-antisense for another week. Post-injury lipofection + antisense + growth factor is delivered for 1 week followed by lipofection and growth factor of antisense for another week. Several groups include combinations of growth factors with antisense, including TGF-alpha, insulin, and IGF-1. In all animals, a single osmotic pump loaded with BrdU is implanted subcutaneously to identify mitotically active cells.                 

Hair cell damage caused by gentamicin is checked as myosin-VIIa immunocytochemistry (hair cell specific markers) and paloidine histochemistry (F-actin markers) using whole slide staining techniques. Transfection efficiency of liposomes and p27 ^Kip1 antisense oligonucleotides is assessed by observing the presence of FITC-labeled nuclei under fluorescence. BrdU immunohistochemistry is used to determine if proliferation was induced by p27 ^Kip1 antisense ON treatment. Selected samples are analyzed under an electron microscope.

Some experiments were conducted using double labeling of BrdU and myosin-VIIa to assess the number of new support cells versus the total number of hair cells.                 

Another experiment is done using double labeling of BrdU and non-mentin to assay the number of new support cells. The double label of BrdU, vimentin and BrdU, myosin distinguishes the number of new hair cells compared to the number of new support cells. The baseline for corti organ support cell proliferation and auditory hair cell regeneration in mammals is zero, making statistical significance easier to reach a small number of observed events with unidirectional ANOVAs.

Example 7

Inner ear neurotoxic damage in guinea pigs and / or p27 ^Kip1  Evaluation of hearing function after antisense treatment

Auditory brainstem response (ABR) is tested in guinea pigs after inner ear neurotoxic damage and / or p27 ^Kip1 antisense treatment. ABR thresholds are compared within the same animal over time and across animals of the same or different groups (before and after surgery). Significance is measured using analysis of unidirectional variation for each stimulus frequency and intensity (ANOVA). The difference is considered to be statistically significant that the p-value <0.05. Previous studies have shown that this surgery does not attenuate the ABR response after surgery.

To record induced ABRs, guinea pigs are anesthetized with Avatin (0.2 ml / 10 g body weight / im 1.2% stock). The active electrode is placed subcutaneously near the ear canal (0.1-mm silver wire; Narishige). Place the dura mater electrode in the beak-shaped hole perforated in the crown (or insert the earphone into the ear canal and acoustic delivery tube with surgical tape on the ear canal). The base electrode (Ag / AgCl ₂ pellets) is fixed to the back. The acoustic stimulus is a 100 kHz wideband click or 10 ms tone burst (1 ms rise / fall time). Guinea pigs are placed in an acoustic damping chamber (TDT model AC-1). The response is measured and recorded via the Auditory Evoked Response Workstation (SmartEP; Intelligent Hearing System). The guinea pig receives a stimulus intensity scale that is increased in 5 dB increments from 20 to 85 dB for both the click and tone burst stimuli. Stimulus frequency tones (1, 2, 4, 8, 16, 32 Hz) are also used for 50 dB of continuous intensity in tone burst. The stimulus is repeated 5 times / second and averaged over 512 trials in total. The threshold is defined as the minimum intensity that can lead to replicable and apparently detectable ABR.

Example 8

Improving Hearing Function in Sulfate Amikacin-treated p27 Heterozygous Mice

In this sample, the experimental animals were treated the same as the mice described in the experiments reported in Table 2, except that there was an additional recovery time four weeks after the amicacin treatment. Hearing function was measured using an auditory brainstem response (ABR) using a subcutaneous recording electrode located at the crown of mice anesthetized with isoflurane. Sound intensity thresholds were measured by representing single frequencies with different sound intensities (intensities measured in decibels). The higher the pitch intensity required to induce ABR, the higher the hearing threshold (i.e., deterioration of hearing function). Data shown in FIGS. 3-6 show hearing improvement in 5 of 8 p27 heterozygotes.                 

In Table 2 herein, 5 of 10 p27 heterozygotes show evidence of internal cell proliferative regeneration as assessed by BrdU-label and morphological standards. This data indicates that the majority of BrdU-labeled cells are support cells, not hair cells. Therefore, the improved hearing function in the p27 heterozygote may be due only to the regeneration of support cells or due to combination with the regeneration of hair cells.

Example 9

Lipofection Methods of Gene Delivery and Gene Expression in a Mouse Corti Organ Culture System

Lipofection of the Corti organs utilizes cochlear explants obtained from mice aged 7 to 14 days of age in vitro (DIV) developed for a total of 8 days. Cultures are cultured in a specific culture medium (Gibco) consisting of Neurobasal medium containing B27 additive. The culture is exposed (for 48 hours) to an aminoglycoside antibiotic (1 mM neomycin sulfate) that selectively kills inner ear sensory hair cells. Then eight different lipid combinations were examined from the Perfect Lipofection Kit (Invitrogen). The bacterial plasmid (InVitrogen), which encodes the beta galactosidase reporter gene, operated by the CMV mediated / added gene promoter, was delivered over 7 hours. Cultures were fixed with aldehyde and processed for beta galactosidase expression using x-gal histochemistry. X-gal labeling appeared in support cells (54.3 +/- 15.3 labeled cells per 1000 μm in the area of sensory epithelium that once contained hair cells vs. 5 to 10 labeled cells per 1000 μm in intact tissue ).                 

Given the activity involved in the detection of beta-galactosidase expression, the size of the beta galactosidase coding construct (ie 4.1 kbp) and the reduced concordance of this technique with other desired ICC methods, Green Fluorescent Protein Repopulate the culture with a plasmid encoding (GFP; Clonetech). Detection of GFP requires a standard FITC filter set (stimulation max = 488 nm; emission min = 509 nm) and is successfully transfected into cochlear hair cells, support cells and neurons using an AAV vector system.

Corti organ cultures established from P7-P14 Swiss Webster mice from our breeding grounds were re-fected using a variety of commercially available lipofection reagents (ie, FuGENE Transfection Reagent; Boehringer-Mannheim). do. This efficiency is compared to the transfection efficiency achieved by the InVitrogen Kit. GFP-positive cells within 1000 μm length of the Corti organs obtained in the middle of explants are counted to determine good lipofection reagent and good lipid to DNA ratio (3: 1, 6: 1, 9: 1). Cells are visualized using a NiKon fluorescence microscope equipped with a CCD digital camera that outputs images directly into an image software program where cell counting is performed.

Aminoglycoside antibiotic damage of hair cells is combined with subsequent lipofection. Hair cells are killed by administering medium (Sigma) containing 1 mM neomycin sulfate over 48 hours in culture. Unlike cultures from neonatal mice, two-week-old mice with developed hearing functions are more easily affected by this concentration of neomycin, 95% as measured by calvindine immunoreactivity and plastic cross-wafer analysis. Causes abnormal hair cell defects. Remaining support cells are lipofected for 6 hours with GFP encoding plasmid. The cultures are washed and incubated in fresh medium for a total of 3-6 DIV by adding 1-4 DIV. Cultures are fixed with aldehyde and GFP is visualized directly under fluorescence. Several reports illustrate the loss of GFP fluorescence after aldehyde fixation. This may require the use of commercially available GFP antibodies (Clonetech) to enhance fluorescence of lipofected cells.

Example 10

Incision and In Vitro Culture of Mouse Inner Ear

The inner ear of the mouse was dissected by the following means. 7-14 days old Swiss Webster mice were cut out of the neck and sterilized by soaking the skull in 70% ethanol for 5 minutes. Under sterile conditions, the skull is cut in half along the medial sagittal axis and placed in 3 ml culture medium (Neuralbasal ™ Media at pH 7.4; Gibco) in a 35 mm plastic petri dish (Nalge Nunc International, 2000 North Aurora Road, Naperville, IL 60563). Located. Using a surgical perceptor, the inner labyrinth of the bone was visible and separated from the temporal bone. Covered connective tissue, middle ear, facial nerve and spinal artery were removed. Using a micro perceptor, a small hole about 2 mm in diameter was made at the vertex edge of the lateral cochlear wall. Along the open oval and insole of the cochlea, this surgically constructed conduit allows for easy diffusion of the culture medium into the fluid-filled ear canal.

Typically, the inner ear that is cut and prepared by the aforementioned means is transferred to a HARV ™ and CCCV ™ vessel, which has one of N2 or B27 medium additives (both sold on Gibco-BRL, catalog No. 17504-036). The 50 degrees added contains 55 ml of Neuralbasal ™ medium, 10 U / ml penicillin and 25 μg / μl fungizone. The B27 additive is sold at a concentration of 50 × and used at a 0.5 × working concentration (eg, 550 μl of 50 × B27 stock solution is added to 55 ml Neuralbasal ™ medium). The stock of N2 additive is 100X and used at a working concentration of 1X (for example 550 μl of 100X N2 stock is added to 55 ml of Neuralbasal ™ medium). The vessel is then placed in a tissue culture incubator at 37 ° C. and 95% air / 5% CO ₂ environment. The vessel is then rotated at 39 rpm for 24 to 168 hours. 50% medium change is performed every 48 hours. A minimum of 2 to 12 inner ear were successfully cultured in one vessel.

To damage the inner ear sensory hair cells, the inner ear is placed in Neuralbasal ™ / N2 or B27 medium containing 1 mM neomycin sulfate (Sigma, P.O.Box 14508, St. Louis, MO 63178) for 24 to 48 hours. After this incubation period, complete replacement with neomycin-free medium.

Example 11

Culture medium

Table 5 shows the composition (1 ×) of Neuralbasal ™ medium sold by Gibco. All concentrations are working concentrations, ie the concentrations of the constituents in the medium incubating the fluid-filled sensory organs.                 

The following antibiotics can be added to the Neuralbasal ™ medium. Fungizone reagent (amphotericin B, 0.25 μg / ml, and sodium desoxycholate, 0.25 μg / ml); Sold by Gibco-BRL, catalog No.17504-036. Penicillin G (10 units / ml); Sold by Sigma, catalog No.P 3414. Neomycin sulfate (1 mM); Sigma, catalog No.N 6386. L-glutamine (2 mM) can also be added to the Neuralbasal ™ medium.

Example 12

Test for sensory epithelial vitality during long term culture

In the practice of one aspect of the present invention, the microgravity environment provided by the rotation of the culture vessel allows the sensory epithelium of the inner ear to prolong the incubation period without significant degradation or loss of sensory hair cells or non-sensory support cells. 168 hours). Data illustrating the continued viability of sensory hair cells in prolonged culture include probes against F-actin (palloydin-FITC) that labels the surface of sensory and non-sensory cells, and the calcium binding protein, calvindine. Obtained by labeling the sensory epithelium with a counterpart hair cell specific antibody. Both labels were detected and photographed under fluorescence microscopy.

Cross- lamella data indicate that normal cell construction of the inner ear sensory epithelium is maintained. For example, Corti organs have several fluid-filled spaces called corti tubes and noel spaces required for normal hearing function. These spaces arise between the hair cells and the support cells and are maintained after an extended incubation period. In a normal gravity environment, the sensory epithelium begins to collapse (ie, when supporting the inner ear without rotating the culture vessel). If not rotated, within 24 hours hair cells appear to be completely lost or undergo various late stages of cell death. After 48 hours, the support cells are completely lost or present but the corti ducts and noel space are completely lost. Rotation of the container prevents this disruption and maintains normal cell building.

While the preferred embodiments of the invention have been illustrated and described, it will be understood that various changes may occur without departing from the spirit and scope of the invention.

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(1014) <400> 3 atg atg gcc atg aac tcc aag cag cct ttc ggc atg cac ccg gtg ctg 48 Met Met Ala Met Asn Ser Lys Gln Pro Phe Gly Met His Pro Val Leu   1 5 10 15 caa gaa ccc aaa ttc tcc agt ctg cac tct ggc tcc gag gcc atg cgc 96 Gln Glu Pro Lys Phe Ser Ser Leu His Ser Gly Ser Glu Ala Met Arg              20 25 30 cga gtc tgt ctc cca gcc ccg cag ctg cag ggt aat ata ttt gga agc 144 Arg Val Cys Leu Pro Ala Pro Gln Leu Gln Gly Asn Ile Phe Gly Ser          35 40 45 ttt gat gag agc ctg ctg gca cgc gcc gaa gct ctg gcg gcg gtg gat 192 Phe Asp Glu Ser Leu Leu Ala Arg Ala Glu Ala Leu Ala Ala Val Asp      50 55 60 atc gtc ttc cac ggg aag aac cat ccg ttt aag ccc gac gcc acc tac 240 Ile Val Phe His Gly Lys Asn His Pro Phe Lys Pro Asp Ala Thr Tyr  65 70 75 80 cat acc atg agc agc gtg ccc tgc acg tcc act tcg tcc acc gtg ccc 288 His Thr Met Ser Ser Val Pro Cys Thr Ser Thr Ser Ser Thr Val Pro                  85 90 95 atc tcc cac cca gct gcg ctc acc tca cac cct cac cac gcc gtg cac 336 Ile Ser His Pro Ala Ala Leu Thr Ser His Pro His His Ala Val His             100 105 110 cag ggc ctc gaa ggc gac ctg ctg gag cac atc tcg ccc acg ctg agt 384 Gln Gly Leu Glu Gly Asp Leu Leu Glu His Ile Ser Pro Thr Leu Ser         115 120 125 gtg agc ggc ctg ggc gct ccg gaa cac tcg gtg atg ccc gca cag atc 432 Val Ser Gly Leu Gly Ala Pro Glu His Ser Val Met Pro Ala Gln Ile     130 135 140 cat cca cac cac ctg ggc gcc atg ggc cac ctg cac cag gcc atg ggc 480 His Pro His His Leu Gly Ala Met Gly His Leu His Gln Ala Met Gly 145 150 155 160 atg agt cac ccg cac acc gtg gcc cct cat agc gcc atg cct gca tgc 528 Met Ser His Pro His Thr Val Ala Pro His Ser Ala Met Pro Ala Cys                 165 170 175 ctc agc gac gtg gag tca gac ccg cgc gag ctg gaa gcc ttc gcc gag 576 Leu Ser Asp Val Glu Ser Asp Pro Arg Glu Leu Glu Ala Phe Ala Glu             180 185 190 cgc ttc aag cag cgg cgc atc aag ctg ggg gtg acc cag gcg gac gtg 624 Arg Phe Lys Gln Arg Arg Ile Lys Leu Gly Val Thr Gln Ala Asp Val         195 200 205 ggc gcg gct ctg gct aat ctc aag atc ccc ggc gtg ggc tcg ctg agc 672 Gly Ala Ala Leu Ala Asn Leu Lys Ile Pro Gly Val Gly Ser Leu Ser     210 215 220 caa agc acc atc tgc agg ttc gag tct ctc act ctc tcg cac aac aac 720 Gln Ser Thr Ile Cys Arg Phe Glu Ser Leu Thr Leu Ser His Asn Asn 225 230 235 240 atg atc gct ctc aag ccg gtg ctc cag gcc tgg ttg gag gag gcc gag 768 Met Ile Ala Leu Lys Pro Val Leu Gln Ala Trp Leu Glu Glu Ala Glu                 245 250 255 gcc gcc tac cga gag aag aac agc aag cca gag ctc ttc aac ggc agc 816 Ala Ala Tyr Arg Glu Lys Asn Ser Lys Pro Glu Leu Phe Asn Gly Ser             260 265 270 gaa cgg aag cgc aaa cgc acg tcc atc gcg gcg ccg gag aag cgt tca 864 Glu Arg Lys Arg Lys Arg Thr Ser Ile Ala Ala Pro Glu Lys Arg Ser         275 280 285 ctc gag gcc tat ttc gct atc cag cca cgt cct tca tct gag aag atc 912 Leu Glu Ala Tyr Phe Ala Ile Gln Pro Arg Pro Ser Ser Glu Lys Ile     290 295 300 gcg gcc atc gct gag aaa ctg gac ctt aaa aag aac gtg gtg aga gtc 960 Ala Ala Ile Ala Glu Lys Leu Asp Leu Lys Lys Asn Val Val Arg Val 305 310 315 320 tgg ttc tgc aac cag aga cag aaa cag aaa cga atg aag tat tcg gct 1008 Trp Phe Cys Asn Gln Arg Gln Lys Gln Lys Arg Met Lys Tyr Ser Ala                 325 330 335 gtc cac 1014 Val his <210> 4 <211> 338 <212> PRT <213> Homo sapiens <400> 4 Met Met Ala Met Asn Ser Lys Gln Pro Phe Gly Met His Pro Val Leu   1 5 10 15 Gln Glu Pro Lys Phe Ser Ser Leu His Ser Gly Ser Glu Ala Met Arg              20 25 30 Arg Val Cys Leu Pro Ala Pro Gln Leu Gln Gly Asn Ile Phe Gly Ser          35 40 45 Phe Asp Glu Ser Leu Leu Ala Arg Ala Glu Ala Leu Ala Ala Val Asp      50 55 60 Ile Val Phe His Gly Lys Asn His Pro Phe Lys Pro Asp Ala Thr Tyr  65 70 75 80 His Thr Met Ser Ser Val Pro Cys Thr Ser Thr Ser Ser Thr Val Pro                  85 90 95 Ile Ser His Pro Ala Ala Leu Thr Ser His Pro His His Ala Val His             100 105 110 Gln Gly Leu Glu Gly Asp Leu Leu Glu His Ile Ser Pro Thr Leu Ser         115 120 125 Val Ser Gly Leu Gly Ala Pro Glu His Ser Val Met Pro Ala Gln Ile     130 135 140 His Pro His His Leu Gly Ala Met Gly His Leu His Gln Ala Met Gly 145 150 155 160 Met Ser His Pro His Thr Val Ala Pro His Ser Ala Met Pro Ala Cys                 165 170 175 Leu Ser Asp Val Glu Ser Asp Pro Arg Glu Leu Glu Ala Phe Ala Glu             180 185 190 Arg Phe Lys Gln Arg Arg Ile Lys Leu Gly Val Thr Gln Ala Asp Val         195 200 205 Gly Ala Ala Leu Ala Asn Leu Lys Ile Pro Gly Val Gly Ser Leu Ser     210 215 220 Gln Ser Thr Ile Cys Arg Phe Glu Ser Leu Thr Leu Ser His Asn Asn 225 230 235 240 Met Ile Ala Leu Lys Pro Val Leu Gln Ala Trp Leu Glu Glu Ala Glu                 245 250 255 Ala Ala Tyr Arg Glu Lys Asn Ser Lys Pro Glu Leu Phe Asn Gly Ser             260 265 270 Glu Arg Lys Arg Lys Arg Thr Ser Ile Ala Ala Pro Glu Lys Arg Ser         275 280 285 Leu Glu Ala Tyr Phe Ala Ile Gln Pro Arg Pro Ser Ser Glu Lys Ile     290 295 300 Ala Ala Ile Ala Glu Lys Leu Asp Leu Lys Lys Asn Val Val Arg Val 305 310 315 320 Trp Phe Cys Asn Gln Arg Gln Lys Gln Lys Arg Met Lys Tyr Ser Ala                 325 330 335 Val his <210> 5 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: oligonucleotide <220> <221> misc_feature (222) (1) .. (31) Oligonucleotide for identifying POU4F3 homologs <400> 5 tagaagtgca gggcacgctg ctcatggtat g 31 <210> 6 <211> 1355 <212> DNA <213> Mus musculus <220> <221> CDS (222) (41) .. (1084) <400> 6 ccgcaggagc cgtccatcac caatcagcca gccttcgacc atg ggc atg tcc gac 55                                             Met Gly Met Ser Asp                                               1 5 gtg tac ctc cgc agc aga aca gcg atg gaa cgc ttg gcc tcc agc gat 103 Val Tyr Leu Arg Ser Arg Thr Ala Met Glu Arg Leu Ala Ser Ser Asp                  10 15 20 acc ttc cca gtg ata gcg cgt agc agc gcc tgc cgc agc ctc ttc ggg 151 Thr Phe Pro Val Ile Ala Arg Ser Ser Ala Cys Arg Ser Leu Phe Gly              25 30 35 cct gta gac cac gag gag ctg ggc cgc gag ctg cgg atg cgc ctg gcc 199 Pro Val Asp His Glu Glu Leu Gly Arg Glu Leu Arg Met Arg Leu Ala          40 45 50 gag ctg aac gcc gag gac cag aac cgc tgg gac ttc aac ttc cag cag 247 Glu Leu Asn Ala Glu Asp Gln Asn Arg Trp Asp Phe Asn Phe Gln Gln      55 60 65 gat gtg cct ctt cga ggc cct ggt cgt ctg cag tgg atg gag gtg gac 295 Asp Val Pro Leu Arg Gly Pro Gly Arg Leu Gln Trp Met Glu Val Asp  70 75 80 85 agc gag tct gtg ccc gcc ttc tac cgc gag acg gtg cag gtg ggg cgc 343 Ser Glu Ser Val Pro Ala Phe Tyr Arg Glu Thr Val Gln Val Gly Arg                  90 95 100 tgt cgc ctg cag ctg ggg ccc cgg cca ccc ccg gtg gcc gtg gct gtc 391 Cys Arg Leu Gln Leu Gly Pro Arg Pro Pro Pro Val Ala Val Ala Val             105 110 115 atc ccg cgt tct ggg ccg ccg gct ggc gag gcc ccc gac ggc cta gag 439 Ile Pro Arg Ser Gly Pro Pro Ala Gly Glu Ala Pro Asp Gly Leu Glu         120 125 130 gag gcg cct gag cag ccg ccc agc gcc cca gcc tcg gcc gtg gtc gcg 487 Glu Ala Pro Glu Gln Pro Pro Ser Ala Pro Ala Ser Ala Val Val Ala     135 140 145 gac gcc acc cca ccc gcg acc ccg gcc ccg gct tca gat ctg acc tca 535 Asp Ala Thr Pro Pro Ala Thr Pro Ala Pro Ala Ser Asp Leu Thr Ser 150 155 160 165 gac cca att ccg gag gtg acc ctg gtc gcg acc tcc gac ccg act ccg 583 Asp Pro Ile Pro Glu Val Thr Leu Val Ala Thr Ser Asp Pro Thr Pro                 170 175 180 gac ccg atc ccg gac gcg aac ccg gac gtg gcg act cgg gac ggc gag 631 Asp Pro Ile Pro Asp Ala Asn Pro Asp Val Ala Thr Arg Asp Gly Glu             185 190 195 gaa cag gtc cct gag cag gtc tct gag cag ggc gag gag tcg ggt gct 679 Glu Gln Val Pro Glu Gln Val Ser Glu Gln Gly Glu Glu Ser Gly Ala         200 205 210 gag ccg ggt gat gag ctg gga act gag ccg gtc tct gag cag ggc gag 727 Glu Pro Gly Asp Glu Leu Gly Thr Glu Pro Val Ser Glu Gln Gly Glu     215 220 225 gag cag ggc gca gag ccg gtc gag gag aag gac gag gag ccg gag gag 775 Glu Gln Gly Ala Glu Pro Val Glu Glu Lys Asp Glu Glu Pro Glu Glu 230 235 240 245 gag cag ggc gcg gag ccg gtc gag gag cag ggt gcg gag ccg gtc gag 823 Glu Gln Gly Ala Glu Pro Val Glu Glu Gln Gly Ala Glu Pro Val Glu                 250 255 260 gag cag aat ggg gag ccg gtc gag gag cag gac gag aat caa gag cag 871 Glu Gln Asn Gly Glu Pro Val Glu Glu Gln Asp Glu Asn Gln Glu Gln             265 270 275 cgc ggc cag gag ctg aag gac cag cct ctc tcg ggg att cca gga cgt 919 Arg Gly Gln Glu Leu Lys Asp Gln Pro Leu Ser Gly Ile Pro Gly Arg         280 285 290 cct gca ccc ggg act gct gcg gcc aat gcg aac gac ttc ttc gcc aag 967 Pro Ala Pro Gly Thr Ala Ala Ala Asn Ala Asn Asp Phe Phe Ala Lys     295 300 305 cgc aag aga act gcg cag gag aac aag gcg tcg aac gac gtc cct cca 1015 Arg Lys Arg Thr Ala Gln Glu Asn Lys Ala Ser Asn Asp Val Pro Pro 310 315 320 325 ggg tgt ccc tct cca aac gtg gct cct ggg gtg ggc gcg gtg gag cag 1063 Gly Cys Pro Ser Pro Asn Val Ala Pro Gly Val Gly Ala Val Glu Gln                 330 335 340 acc ccg cgc aaa cgt ctg aga tgagtt agtttagagg ctaacggcca 1110 Thr Pro Arg Lys Arg Leu Arg             345 gagagaactt gctgggcatc tgggcagcgg acgatggaag aactctgggc ttcggctggg 1170 acctttcgtt catgtagcag gaaccggaga tggttgcgta gagcagccca cggttttgtg 1230 gaaatctgaa aactgtgcaa tgtattgaga acactctgta ccatgtgcaa ggagtacgct 1290 ggtcccaagg tgtaaagctt taaatcattt atgtaaaatg tttaatctct actcgctctc 1350 agtgc 1355 <210> 7 <211> 348 <212> PRT <213> Mus musculus <400> 7 Met Gly Met Ser Asp Val Tyr Leu Arg Ser Arg Thr Ala Met Glu Arg   1 5 10 15 Leu Ala Ser Ser Asp Thr Phe Pro Val Ile Ala Arg Ser Ser Ala Cys              20 25 30 Arg Ser Leu Phe Gly Pro Val Asp His Glu Glu Leu Gly Arg Glu Leu          35 40 45 Arg Met Arg Leu Ala Glu Leu Asn Ala Glu Asp Gln Asn Arg Trp Asp      50 55 60 Phe Asn Phe Gln Gln Asp Val Pro Leu Arg Gly Pro Gly Arg Leu Gln  65 70 75 80 Trp Met Glu Val Asp Ser Glu Ser Val Pro Ala Phe Tyr Arg Glu Thr                  85 90 95 Val Gln Val Gly Arg Cys Arg Leu Gln Leu Gly Pro Arg Pro Pro Pro             100 105 110 Val Ala Val Ala Val Ile Pro Arg Ser Gly Pro Pro Ala Gly Glu Ala         115 120 125 Pro Asp Gly Leu Glu Glu Ala Pro Glu Gln Pro Pro Ser Ala Pro Ala     130 135 140 Ser Ala Val Val Ala Asp Ala Thr Pro Pro Ala Thr Pro Ala Pro Ala 145 150 155 160 Ser Asp Leu Thr Ser Asp Pro Ile Pro Glu Val Thr Leu Val Ala Thr                 165 170 175 Ser Asp Pro Thr Pro Asp Pro Ile Pro Asp Ala Asn Pro Asp Val Ala             180 185 190 Thr Arg Asp Gly Glu Glu Gln Val Pro Glu Gln Val Ser Glu Gln Gly         195 200 205 Glu Glu Ser Gly Ala Glu Pro Gly Asp Glu Leu Gly Thr Glu Pro Val     210 215 220 Ser Glu Gln Gly Glu Glu Gln Gly Ala Glu Pro Val Glu Glu Lys Asp 225 230 235 240 Glu Glu Pro Glu Glu Glu Gln Gly Ala Glu Pro Val Glu Glu Gln Gly                 245 250 255 Ala Glu Pro Val Glu Glu Gln Asn Gly Glu Pro Val Glu Glu Gln Asp             260 265 270 Glu Asn Gln Glu Gln Arg Gly Gln Glu Leu Lys Asp Gln Pro Leu Ser         275 280 285 Gly Ile Pro Gly Arg Pro Ala Pro Gly Thr Ala Ala Ala Asn Ala Asn     290 295 300 Asp Phe Phe Ala Lys Arg Lys Arg Thr Ala Gln Glu Asn Lys Ala Ser 305 310 315 320 Asn Asp Val Pro Pro Gly Cys Pro Ser Pro Asn Val Ala Pro Gly Val                 325 330 335 Gly Ala Val Glu Gln Thr Pro Arg Lys Arg Leu Arg             340 345 <210> 8 <211> 597 <212> DNA <213> Homo sapiens <220> <221> CDS (222) (1) .. (594) <400> 8 atg tca aac gtg cga gtg tct aac ggg agc cct agc ctg gag cgg atg 48 Met Ser Asn Val Arg Val Ser Asn Gly Ser Pro Ser Leu Glu Arg Met   1 5 10 15 gac gcc agg cag gcg gag cac ccc aag ccc tcg gcc tgc agg aac ctc 96 Asp Ala Arg Gln Ala Glu His Pro Lys Pro Ser Ala Cys Arg Asn Leu              20 25 30 ttc ggc ccg gtg gac cac gaa gag tta acc cgg gac ttg gag aag cac 144 Phe Gly Pro Val Asp His Glu Glu Leu Thr Arg Asp Leu Glu Lys His          35 40 45 tgc aga gac atg gaa gag gcg agc cag cgc aag tgg aat ttc gat ttt 192 Cys Arg Asp Met Glu Glu Ala Ser Gln Arg Lys Trp Asn Phe Asp Phe      50 55 60 cag aat cac aaa ccc cta gag ggc aag tac gag tgg caa gag gtg gag 240 Gln Asn His Lys Pro Leu Glu Gly Lys Tyr Glu Trp Gln Glu Val Glu  65 70 75 80 aag ggc agc ttg ccc gag ttc tac tac aga ccc ccg cgg ccc ccc aaa 288 Lys Gly Ser Leu Pro Glu Phe Tyr Tyr Arg Pro Pro Arg Pro Pro Lys                  85 90 95 ggt gcc tgc aag gtg ccg gcg cag gag agc cag gat gtc agc ggg agc 336 Gly Ala Cys Lys Val Pro Ala Gln Glu Ser Gln Asp Val Ser Gly Ser             100 105 110 cgc ccg gcg gcg cct tta att ggg gct ccg gct aac tct gag gac acg 384 Arg Pro Ala Ala Pro Leu Ile Gly Ala Pro Ala Asn Ser Glu Asp Thr         115 120 125 cat ttg gtg gac cca aag act gat ccg tcg gac agc cag acg ggg tta 432 His Leu Val Asp Pro Lys Thr Asp Pro Ser Asp Ser Gln Thr Gly Leu     130 135 140 gcg gag caa tgc gca gga ata agg aag cga cct gca acc gac gat tct 480 Ala Glu Gln Cys Ala Gly Ile Arg Lys Arg Pro Ala Thr Asp Asp Ser 145 150 155 160 tct act caa aac aaa aga gcc aac aga aca gaa gaa aat gtt tca gac 528 Ser Thr Gln Asn Lys Arg Ala Asn Arg Thr Glu Glu Asn Val Ser Asp                 165 170 175 ggt tcc cca aat gcc ggt tct gtg gag cag acg ccc aag aag cct ggc 576 Gly Ser Pro Asn Ala Gly Ser Val Glu Gln Thr Pro Lys Lys Pro Gly             180 185 190 ctc aga aga cgt caa acg taa 597 Leu Arg Arg Arg Gln Thr         195 <210> 9 <211> 198 <212> PRT <213> Homo sapiens <400> 9 Met Ser Asn Val Arg Val Ser Asn Gly Ser Pro Ser Leu Glu Arg Met   1 5 10 15 Asp Ala Arg Gln Ala Glu His Pro Lys Pro Ser Ala Cys Arg Asn Leu              20 25 30 Phe Gly Pro Val Asp His Glu Glu Leu Thr Arg Asp Leu Glu Lys His          35 40 45 Cys Arg Asp Met Glu Glu Ala Ser Gln Arg Lys Trp Asn Phe Asp Phe      50 55 60 Gln Asn His Lys Pro Leu Glu Gly Lys Tyr Glu Trp Gln Glu Val Glu  65 70 75 80 Lys Gly Ser Leu Pro Glu Phe Tyr Tyr Arg Pro Pro Arg Pro Pro Lys                  85 90 95 Gly Ala Cys Lys Val Pro Ala Gln Glu Ser Gln Asp Val Ser Gly Ser             100 105 110 Arg Pro Ala Ala Pro Leu Ile Gly Ala Pro Ala Asn Ser Glu Asp Thr         115 120 125 His Leu Val Asp Pro Lys Thr Asp Pro Ser Asp Ser Gln Thr Gly Leu     130 135 140 Ala Glu Gln Cys Ala Gly Ile Arg Lys Arg Pro Ala Thr Asp Asp Ser 145 150 155 160 Ser Thr Gln Asn Lys Arg Ala Asn Arg Thr Glu Glu Asn Val Ser Asp                 165 170 175 Gly Ser Pro Asn Ala Gly Ser Val Glu Gln Thr Pro Lys Lys Pro Gly             180 185 190 Leu Arg Arg Arg Gln Thr         195 <210> 10 <211> 2121 <212> DNA <213> Homo sapiens <220> <221> CDS <222> (76) .. (567) <400> 10 gccgaagtca gttccttgtg gagccggagc tgggcgcgga ttcgccgagg caccgaggca 60 ctcagaggag gcgcc atg tca gaa ccg gct ggg gat gtc cgt cag aac 108                       Met Ser Glu Pro Ala Gly Asp Val Arg Gln Asn                         1 5 10 cca tgc ggc agc aag gcc tgc cgc cgc ctc ttc ggc cca gtg gac agc 156 Pro Cys Gly Ser Lys Ala Cys Arg Arg Leu Phe Gly Pro Val Asp Ser              15 20 25 gag cag ctg agc cgc gac tgt gat gcg cta atg gcg ggc tgc atc cag 204 Glu Gln Leu Ser Arg Asp Cys Asp Ala Leu Met Ala Gly Cys Ile Gln          30 35 40 gag gcc cgt gag cga tgg aac ttc gac ttt gtc acc gag aca cca ctg 252 Glu Ala Arg Glu Arg Trp Asn Phe Asp Phe Val Thr Glu Thr Pro Leu      45 50 55 gag ggt gac ttc gcc tgg gag cgt gtg cgg ggc ctt ggc ctg ccc aag 300 Glu Gly Asp Phe Ala Trp Glu Arg Val Arg Gly Leu Gly Leu Pro Lys  60 65 70 75 ctc tac ctt ccc acg ggg ccc cgg cga ggc cgg gat gag ttg gga gga 348 Leu Tyr Leu Pro Thr Gly Pro Arg Arg Gly Arg Asp Glu Leu Gly Gly                  80 85 90 ggc agg cgg cct ggc acc tca cct gct ctg ctg cag ggg aca gca gag 396 Gly Arg Arg Pro Gly Thr Ser Pro Ala Leu Leu Gln Gly Thr Ala Glu              95 100 105 gaa gac cat gtg gac ctg tca ctg tct tgt acc ctt gtg cct cgc tca 444 Glu Asp His Val Asp Leu Ser Leu Ser Cys Thr Leu Val Pro Arg Ser         110 115 120 ggg gag cag gct gaa ggg tcc cca ggt gga cct gga gac tct cag ggt 492 Gly Glu Gln Ala Glu Gly Ser Pro Gly Gly Pro Gly Asp Ser Gln Gly     125 130 135 cga aaa cgg cgg cag acc agc atg aca gat ttc tac cac tcc aaa cgc 540 Arg Lys Arg Arg Gln Thr Ser Met Thr Asp Phe Tyr His Ser Lys Arg 140 145 150 155 cgg ctg atc ttc tcc aag agg aag ccc taa tccgcccaca ggaagcctgc 590 Arg Leu Ile Phe Ser Lys Arg Lys Pro                 160 agtcctggaa gcgcgagggc ctcaaaggcc cgctctacat cttctgcctt agtctcagtt 650 tgtgtgtctt aattattatt tgtgttttaa tttaaacacc tcctcatgta cataccctgg 710 ccgccccctg ccccccagcc tctggcatta gaattattta aacaaaaact aggcggttga 770 atgagaggtt cctaagagtg ctgggcattt ttattttatg aaatactatt taaagcctcc 830 tcatcccgtg ttctcctttt cctctctccc ggaggttggg tgggccggct tcatgccagc 890 tacttcctcc tccccacttg tccgctgggt ggtaccctct ggaggggtgt ggctccttcc 950 catcgctgtc acaggcggtt atgaaattca ccccctttcc tggacactca gacctgaatt 1010 ctttttcatt tgagaagtaa acagatggca ctttgaaggg gcctcaccga gtgggggcat 1070 catcaaaaac tttggagtcc cctcacctcc tctaaggttg ggcagggtga ccctgaagtg 1130 agcacagcct agggctgagc tggggacctg gtaccctcct ggctcttgat acccccctct 1190 gtcttgtgaa ggcaggggga aggtggggta ctggagcaga ccaccccgcc tgccctcatg 1250 gcccctctga cctgcactgg ggagcccgtc tcagtgttga gccttttccc tctttggctc 1310 ccctgtacct tttgaggagc cccagcttac ccttcttctc cagctgggct ctgcaattcc 1370 cctctgctgc tgtccctccc ccttgtcttt cccttcagta ccctctcatg ctccaggtgg 1430 ctctgaggtg cctgtcccac ccccaccccc agctcaatgg actggaaggg gaagggacac 1490 acaagaagaa gggcacccta gttctacctc aggcagctca agcagcgacc gccccctcct 1550 ctagctgtgg gggtgagggt cccatgtggt ggcacaggcc cccttgagtg gggttatctc 1610 tgtgttaggg gtatatgatg ggggagtaga tctttctagg agggagacac tggcccctca 1670 aatcgtccag cgaccttcct catccacccc atccctcccc agttcattgc actttgatta 1730 gcagcggaac aaggagtcag acattttaag atggtggcag tagaggctat ggacagggca 1790 tgccacgtgg gctcatatgg ggctgggagt agttgtcttt cctggcacta acgttgagcc 1850 cctggaggca ctgaagtgct tagtgtactt ggagtattgg ggtctgaccc caaacacctt 1910 ccagctcctg taacatactg gcctggactg ttttctctcg gctccccatg tgtcctggtt 1970 cccgtttctc cacctagact gtaaacctct cgagggcagg gaccacaccc tgtactgttc 2030 tgtgtctttc acagctcctc ccacaatgct gaatatacag caggtgctca ataaatgatt 2090 cttagtgact ttaaaaaaaa aaaaaaaaaa a 2121 <210> 11 <211> 164 <212> PRT <213> Homo sapiens <400> 11 Met Ser Glu Pro Ala Gly Asp Val Arg Gln Asn Pro Cys Gly Ser Lys   1 5 10 15 Ala Cys Arg Arg Leu Phe Gly Pro Val Asp Ser Glu Gln Leu Ser Arg              20 25 30 Asp Cys Asp Ala Leu Met Ala Gly Cys Ile Gln Glu Ala Arg Glu Arg          35 40 45 Trp Asn Phe Asp Phe Val Thr Glu Thr Pro Leu Glu Gly Asp Phe Ala      50 55 60 Trp Glu Arg Val Arg Gly Leu Gly Leu Pro Lys Leu Tyr Leu Pro Thr  65 70 75 80 Gly Pro Arg Arg Gly Arg Asp Glu Leu Gly Gly Gly Arg Arg Pro Gly                  85 90 95 Thr Ser Pro Ala Leu Leu Gln Gly Thr Ala Glu Glu Asp His Val Asp             100 105 110 Leu Ser Leu Ser Cys Thr Leu Val Pro Arg Ser Gly Glu Gln Ala Glu         115 120 125 Gly Ser Pro Gly Gly Pro Gly Asp Ser Gln Gly Arg Lys Arg Arg Gln     130 135 140 Thr Ser Met Thr Asp Phe Tyr His Ser Lys Arg Arg Leu Ile Phe Ser 145 150 155 160 Lys Arg Lys Pro <210> 12 <211> 706 <212> DNA <213> Homo sapiens <220> <221> CDS (222) (1) .. (498) <400> 12 atg ctg ctg gag gag gtt cgc gcc ggc gac cgg ctg agt ggg gcg gcg 48 Met Leu Leu Glu Glu Val Arg Ala Gly Asp Arg Leu Ser Gly Ala Ala   1 5 10 15 gcc cgg ggc gac gtg cag gag gtg cgc cgc ctt ctg cac cgc gag ctg 96 Ala Arg Gly Asp Val Gln Glu Val Arg Arg Leu Leu His Arg Glu Leu              20 25 30 gtg cat ccc gac gcc ctc aac cgc ttc ggc aag acg gcg ctg cag gtc 144 Val His Pro Asp Ala Leu Asn Arg Phe Gly Lys Thr Ala Leu Gln Val          35 40 45 atg atg ttt ggc agc acc gcc atc gcc ctg gag ctg ctg aag caa ggt 192 Met Met Phe Gly Ser Thr Ala Ile Ala Leu Glu Leu Leu Lys Gln Gly      50 55 60 gcc agc ccc aat gtc cag gac acc tcc ggt acc agt cca gtc cat gac 240 Ala Ser Pro Asn Val Gln Asp Thr Ser Gly Thr Ser Pro Val His Asp  65 70 75 80 gca gcc cgc act gga ttc ctg gac acc ctg aag gtc cta gtg gag cac 288 Ala Ala Arg Thr Gly Phe Leu Asp Thr Leu Lys Val Leu Val Glu His                  85 90 95 ggg gct gat gtc aac gtg cct gat ggc acc ggg gca ctt cca atc cat 336 Gly Ala Asp Val Asn Val Pro Asp Gly Thr Gly Ala Leu Pro Ile His             100 105 110 ctg gca gtt caa gag ggt cac act gct gtg gtc agc ttt ctg gca gct 384 Leu Ala Val Gln Glu Gly His Thr Ala Val Val Ser Phe Leu Ala Ala         115 120 125 gaa tct gat ctc cat cgc agg gac gcc agg ggt ctc aca ccc ttg gag 432 Glu Ser Asp Leu His Arg Arg Asp Ala Arg Gly Leu Thr Pro Leu Glu     130 135 140 ctg gca ctg cag aga ggg gct cag gac ctc gtg gac atc ctg cca ggc 480 Leu Ala Leu Gln Arg Gly Ala Gln Asp Leu Val Asp Ile Leu Pro Gly 145 150 155 160 cac atg gtg gcc ccg ctg tg atctggggtc accctctcca gcaagagaac 530 His Met Val Ala Pro Leu                 165 ccccccgtgg ttatgtatca gaagagaggg gaagaaacac tttctcttct tgtttctcct 590 gcccactgct gcagtagggg aggagcacag tttgtggctt ataggtgttg gttttggggg 650 tgtgagtgtt tgggggacgt tctcatttgt ttttctcact ccttttggtg tgttgg 706 <210> 13 <211> 166 <212> PRT <213> Homo sapiens <400> 13 Met Leu Leu Glu Glu Val Arg Ala Gly Asp Arg Leu Ser Gly Ala Ala   1 5 10 15 Ala Arg Gly Asp Val Gln Glu Val Arg Arg Leu Leu His Arg Glu Leu              20 25 30 Val His Pro Asp Ala Leu Asn Arg Phe Gly Lys Thr Ala Leu Gln Val          35 40 45 Met Met Phe Gly Ser Thr Ala Ile Ala Leu Glu Leu Leu Lys Gln Gly      50 55 60 Ala Ser Pro Asn Val Gln Asp Thr Ser Gly Thr Ser Pro Val His Asp  65 70 75 80 Ala Ala Arg Thr Gly Phe Leu Asp Thr Leu Lys Val Leu Val Glu His                  85 90 95 Gly Ala Asp Val Asn Val Pro Asp Gly Thr Gly Ala Leu Pro Ile His             100 105 110 Leu Ala Val Gln Glu Gly His Thr Ala Val Val Ser Phe Leu Ala Ala         115 120 125 Glu Ser Asp Leu His Arg Arg Asp Ala Arg Gly Leu Thr Pro Leu Glu     130 135 140 Leu Ala Leu Gln Arg Gly Ala Gln Asp Leu Val Asp Ile Leu Pro Gly 145 150 155 160 His Met Val Ala Pro Leu                 165 <210> 14 <211> 600 <212> DNA <213> Homo sapiens <220> <221> CDS (222) (94) .. (597) <400> 14 ccgatgccat catgcagcct ggttaggagc aaaggaaagg ggaaaaagaa aaacgactaa 60 ttcatctttt cctgatcgtc aggaccctaa aga atg gcc gag cct tgg 108                                             Met Ala Glu Pro Trp                                               1 5 ggg aac gag ttg gcg tcc gca gct gcc agg ggg gac cta gag caa ctt 156 Gly Asn Glu Leu Ala Ser Ala Ala Ala Arg Gly Asp Leu Glu Gln Leu                  10 15 20 act agt ttg ttg caa aat aat gta aac gtc aat gca caa aat gga ttt 204 Thr Ser Leu Leu Gln Asn Asn Val Asn Val Asn Ala Gln Asn Gly Phe              25 30 35 gga agg act gcg ctg cag gtt atg aaa ctt gga aat ccc gag att gcc 252 Gly Arg Thr Ala Leu Gln Val Met Lys Leu Gly Asn Pro Glu Ile Ala          40 45 50 agg aga ctg cta ctt aga ggt gct aat ccc gat ttg aaa gac cga act 300 Arg Arg Leu Leu Leu Arg Gly Ala Asn Pro Asp Leu Lys Asp Arg Thr      55 60 65 ggt ttc gct gtc att cat gat gcg gcc aga gca ggt ttc ctg gac act 348 Gly Phe Ala Val Ile His Asp Ala Ala Arg Ala Gly Phe Leu Asp Thr  70 75 80 85 tta cag act ttg ctg gag ttt caa gct gat gtt aac atc gag gat aat 396 Leu Gln Thr Leu Leu Glu Phe Gln Ala Asp Val Asn Ile Glu Asp Asn                  90 95 100 gaa ggg aac ctg ccc ttg cac ttg gct gcc aaa gaa ggc cac ctc cgg 444 Glu Gly Asn Leu Pro Leu His Leu Ala Ala Lys Glu Gly His Leu Arg             105 110 115 gtg gtg gag ttc ctg gtg aag cac acg gcc agc aat gtg ggg cat cgg 492 Val Val Glu Phe Leu Val Lys His Thr Ala Ser Asn Val Gly His Arg         120 125 130 aac cat aag ggg gac acc gcc tgt gat ttg gcc agg ctc tat ggg agg 540 Asn His Lys Gly Asp Thr Ala Cys Asp Leu Ala Arg Leu Tyr Gly Arg     135 140 145 aat gag gtt gtt agc ctg atg cag gca aac ggg gct ggg gga gcc aca 588 Asn Glu Val Val Ser Leu Met Gln Ala Asn Gly Ala Gly Gly Ala Thr 150 155 160 165 aat ctt caa taa 600 Asn Leu Gln <210> 15 <211> 168 <212> PRT <213> Homo sapiens <400> 15 Met Ala Glu Pro Trp Gly Asn Glu Leu Ala Ser Ala Ala Ala Arg Gly   1 5 10 15 Asp Leu Glu Gln Leu Thr Ser Leu Leu Gln Asn Asn Val Asn Val Asn              20 25 30 Ala Gln Asn Gly Phe Gly Arg Thr Ala Leu Gln Val Met Lys Leu Gly          35 40 45 Asn Pro Glu Ile Ala Arg Arg Leu Leu Leu Arg Gly Ala Asn Pro Asp      50 55 60 Leu Lys Asp Arg Thr Gly Phe Ala Val Ile His Asp Ala Ala Arg Ala  65 70 75 80 Gly Phe Leu Asp Thr Leu Gln Thr Leu Leu Glu Phe Gln Ala Asp Val                  85 90 95 Asn Ile Glu Asp Asn Glu Gly Asn Leu Pro Leu His Leu Ala Ala Lys             100 105 110 Glu Gly His Leu Arg Val Val Glu Phe Leu Val Lys His Thr Ala Ser         115 120 125 Asn Val Gly His Arg Asn His Lys Gly Asp Thr Ala Cys Asp Leu Ala     130 135 140 Arg Leu Tyr Gly Arg Asn Glu Val Val Ser Leu Met Gln Ala Asn Gly 145 150 155 160 Ala Gly Gly Ala Thr Asn Leu Gln                 165 <210> 16 <211> 837 <212> DNA <213> Homo sapiens <220> <221> CDS <222> (328) .. (738) <400> 16 gaggactccg cgacggtccg caccctgcgg ccagagcggc tttgagctcg gctgcttccg 60 cgctaggcgc tttttcccag aagcaatcca ggcgcgcccg ctggttcttg agcgccagga 120 aaagcccgga gctaacgacc ggccgctcgg cactgcacgg ggccccaagc cgcagaagaa 180 ggacgacggg agggtaatga agctgagccc aggtctccta ggaaggagag agtgcgccgg 240 agcagcgtgg gaaagaaggg aagagtgtcg ttaagtttac ggccaacggt ggattatccg 300 ggccgctgcg cgtctggggg ctgcgga atg cgc gag gag aac aag ggc atg 351                                  Met Arg Glu Glu Asn Lys Gly Met                                    1 5 ccc agt ggg ggc ggc agc gat gag ggt ctg gcc acg ccg gcg cgg gga 399 Pro Ser Gly Gly Gly Ser Asp Glu Gly Leu Ala Thr Pro Ala Arg Gly      10 15 20 cta gtg gag aag gtg cga cac tcc tgg gaa gcc ggc gcg gat ccc aac 447 Leu Val Glu Lys Val Arg His Ser Trp Glu Ala Gly Ala Asp Pro Asn  25 30 35 40 gga gtc aac cgt ttc ggg agg cgc gcg atc cag gtc atg atg atg ggc 495 Gly Val Asn Arg Phe Gly Arg Arg Ala Ile Gln Val Met Met Met Gly                  45 50 55 agc gcc cgc gtg gcg gag ctg ctg ctg ctc cac ggc gcg gag ccc aac 543 Ser Ala Arg Val Ala Glu Leu Leu Leu Leu His Gly Ala Glu Pro Asn              60 65 70 tgc gca gac cct gcc act ctc acc cga ccg gtg cat gat gct gcc cgg 591 Cys Ala Asp Pro Ala Thr Leu Thr Arg Pro Val His Asp Ala Ala Arg          75 80 85 gag ggc ttc ctg gac acg ctg gtg gtg ctg cac cgg gcc ggg gcg cgg 639 Glu Gly Phe Leu Asp Thr Leu Val Val Leu His Arg Ala Gly Ala Arg      90 95 100 ctg gac gtg cgc gat gcc tgg ggt cgt ctg ccc gtg gac ttg gcc gag 687 Leu Asp Val Arg Asp Ala Trp Gly Arg Leu Pro Val Asp Leu Ala Glu 105 110 115 120 gag cgg ggc cac cgc gac gtt gca ggg tac ctg cgc aca gcc acg ggg 735 Glu Arg Gly His Arg Asp Val Ala Gly Tyr Leu Arg Thr Ala Thr Gly                 125 130 135 gac tg acgccaggtt ccccagccgc ccacaacgac tttattttct tacccaattt 790 Asp cccaccccca cccacctaat tcgatgaagg ctgccaacgg ggagcgg 837 <210> 17 <211> 137 <212> PRT <213> Homo sapiens <400> 17 Met Arg Glu Glu Asn Lys Gly Met Pro Ser Gly Gly Gly Ser Asp Glu   1 5 10 15 Gly Leu Ala Thr Pro Ala Arg Gly Leu Val Glu Lys Val Arg His Ser              20 25 30 Trp Glu Ala Gly Ala Asp Pro Asn Gly Val Asn Arg Phe Gly Arg Arg          35 40 45 Ala Ile Gln Val Met Met Met Gly Ser Ala Arg Val Ala Glu Leu Leu      50 55 60 Leu Leu His Gly Ala Glu Pro Asn Cys Ala Asp Pro Ala Thr Leu Thr  65 70 75 80 Arg Pro Val His Asp Ala Ala Arg Glu Gly Phe Leu Asp Thr Leu Val                  85 90 95 Val Leu His Arg Ala Gly Ala Arg Leu Asp Val Arg Asp Ala Trp Gly             100 105 110 Arg Leu Pro Val Asp Leu Ala Glu Glu Arg Gly His Arg Asp Val Ala         115 120 125 Gly Tyr Leu Arg Thr Ala Thr Gly Asp     130 135 <210> 18 <211> 987 <212> DNA <213> Homo sapiens <220> <221> CDS (222) (41) .. (508) <400> 18 cggagagggg gagaacagac aacgggcggc ggggagcagc atg gag ccg gcg gcg 55                                             Met Glu Pro Ala Ala                                               1 5 ggg agc agc atg gag cct tcg gct gac tgg ctg gcc acg gcc gcg gcc 103 Gly Ser Ser Met Glu Pro Ser Ala Asp Trp Leu Ala Thr Ala Ala Ala                  10 15 20 cgg ggt cgg gta gag gag gtg cgg gcg ctg ctg gag gcg ggg gcg ctg 151 Arg Gly Arg Val Glu Glu Val Arg Ala Leu Leu Glu Ala Gly Ala Leu              25 30 35 ccc aac gca ccg aat agt tac ggt cgg agg ccg atc cag gtc atg atg 199 Pro Asn Ala Pro Asn Ser Tyr Gly Arg Arg Pro Ile Gln Val Met Met          40 45 50 atg ggc agc gcc cga gtg gcg gag ctg ctg ctg ctc cac ggc gcg gag 247 Met Gly Ser Ala Arg Val Ala Glu Leu Leu Leu Leu His Gly Ala Glu      55 60 65 ccc aac tgc gcc gac ccc gcc act ctc acc cga ccc gtg cac gac gct 295 Pro Asn Cys Ala Asp Pro Ala Thr Leu Thr Arg Pro Val His Asp Ala  70 75 80 85 gcc cgg gag ggc ttc ctg gac acg ctg gtg gtg ctg cac cgg gcc ggg 343 Ala Arg Glu Gly Phe Leu Asp Thr Leu Val Val Leu His Arg Ala Gly                  90 95 100 gcg cgg ctg gac gtg cgc gat gcc tgg ggc cgt ctg ccc gtg gac ctg 391 Ala Arg Leu Asp Val Arg Asp Ala Trp Gly Arg Leu Pro Val Asp Leu             105 110 115 gct gag gag ctg ggc cat cgc gat gtc gca cgg tac ctg cgc gcg gct 439 Ala Glu Glu Leu Gly His Arg Asp Val Ala Arg Tyr Leu Arg Ala Ala         120 125 130 gcg ggg ggc acc aga ggc agt aac cat gcc cgc ata gat gcc gcg gaa 487 Ala Gly Gly Thr Arg Gly Ser Asn His Ala Arg Ile Asp Ala Ala Glu     135 140 145 ggt ccc tca gac atc ccc gat tg aaagaaccag agaggctctg agaaacctcg 540 Gly Pro Ser Asp Ile Pro Asp 150 155 ggaaacttag atcatcagtc accgaaggtc ctacagggcc acaactgccc ccgccacaac 600 ccaccccgct ttcgtagttt tcatttagaa aatagagctt ttaaaaatgt cctgcctttt 660 aacgtagata taagccttcc cccactaccg taaatgtcca tttatatcat tttttatata 720 ttcttataaa aatgtaaaaa agaaaaacac cgcttctgcc ttttcactgt gttggagttt 780 tctggagtga gcactcacgc cctaagcgca cattcatgtg ggcatttctt gcgagcctcg 840 cagcctccgg aagctgtcga cttcatgaca agcattttgt gaactaggga agctcagggg 900 ggttactggc ttctcttgag tcacactgct agcaaatggc agaaccaaag ctcaaataaa 960 aataaaataa ttttcattca ttcactc 987 <210> 19 <211> 156 <212> PRT <213> Homo sapiens <400> 19 Met Glu Pro Ala Ala Gly Ser Ser Met Glu Pro Ser Ala Asp Trp Leu   1 5 10 15 Ala Thr Ala Ala Ala Arg Gly Arg Val Glu Glu Val Arg Ala Leu Leu              20 25 30 Glu Ala Gly Ala Leu Pro Asn Ala Pro Asn Ser Tyr Gly Arg Arg Pro          35 40 45 Ile Gln Val Met Met Met Gly Ser Ala Arg Val Ala Glu Leu Leu Leu      50 55 60 Leu His Gly Ala Glu Pro Asn Cys Ala Asp Pro Ala Thr Leu Thr Arg  65 70 75 80 Pro Val His Asp Ala Ala Arg Glu Gly Phe Leu Asp Thr Leu Val Val                  85 90 95 Leu His Arg Ala Gly Ala Arg Leu Asp Val Arg Asp Ala Trp Gly Arg             100 105 110 Leu Pro Val Asp Leu Ala Glu Glu Leu Gly His Arg Asp Val Ala Arg         115 120 125 Tyr Leu Arg Ala Ala Ala Gly Gly Thr Arg Gly Ser Asn His Ala Arg     130 135 140 Ile Asp Ala Ala Glu Gly Pro Ser Asp Ile Pro Asp 145 150 155 <210> 20 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: oligonucleotide <220> <221> misc_feature (222) (1) .. (15) P223 antisense oligonucleotide (5 'to 3' orientation) <400> 20 tggctctcct gcgcc 15

Claims (48)

(i) a first agent for damaging the first inner ear sensory hair cells to promote the formation of one or more new inner ear sensory hair cells from support cells bordering the damaged first inner ear sensory hair cells, which contain an antibiotic as a chemical; Pharmaceutical composition; And (ii) a second pharmaceutical composition for inhibiting cell cycle inhibitors active in inner ear support cells, containing one or more antisense nucleic acid molecules complementary to a portion of SEQ ID NO: 8 A combination of pharmaceutical compositions for treating hearing disorders by stimulating the formation of inner ear sensory hair cells from inner ear support cells. The combination of claim 1, wherein the support cells are selected from the group consisting of Henssen cells, dieter cells, inner strut cells, border cells and outer strut cells. The combination of claim 1, wherein the first pharmaceutical composition contains an amount of a chemical effective to damage sensory hair cells. delete The combination of claim 1, wherein the antibiotic is an aminoglycoside. The combination of claim 1, wherein the concentration of antibiotic is from 0.01 mM to 10 mM when the first pharmaceutical composition is used in vitro. The combination of claim 1, wherein the concentration of antibiotic is from 100 mg / kg / d to 1000 mg / kg / d when the first pharmaceutical composition is used in vivo. delete delete The combination of claim 1, wherein the first pharmaceutical composition is suitable for delivery to the inner ear by injection. The combination of claim 1, wherein the first pharmaceutical composition is suitable for delivery to the inner ear via a cannula. delete delete delete delete The combination of claim 1, wherein said at least one antisense nucleic acid molecule is SEQ ID NO: 20. delete delete (i) antibiotics as chemicals; And (ii) one or more antisense nucleic acid molecules complementary to a portion of SEQ ID NO: 8 A pharmaceutical composition for treating hearing disorders by improving hearing function in the inner ear, containing. delete (i) a first pharmaceutical composition for damaging the first inner ear support cell to promote the formation of one or more new inner ear support cells, containing an antibiotic as a chemical; And (ii) a second pharmaceutical composition for inhibiting cell cycle inhibitors active in inner ear support cells, containing one or more antisense nucleic acid molecules complementary to a portion of SEQ ID NO: 8 A combination of pharmaceutical compositions for treating hearing disorders by stimulating the formation of inner ear support cells. The combination of claim 21, wherein the first inner ear support cell is selected from the group consisting of Hensen cells, dieter cells, inner strut cells, border cells and outer strut cells. 22. The combination according to claim 21, wherein the first pharmaceutical composition contains an amount of a chemical effective to damage the inner ear support cells. delete The combination of claim 21, wherein the antibiotic is an aminoglycoside. The combination of claim 21, wherein the concentration of antibiotic is from 0.01 mM to 10 mM when the first pharmaceutical composition is used in vitro. 22. The combination according to claim 21, wherein when the first pharmaceutical composition is used in vivo, the concentration of antibiotic is from 100 mg / kg / d to 1000 mg / kg / d. delete delete The combination of claim 21, wherein the first pharmaceutical composition is suitable for delivery to the inner ear by injection. The combination of claim 21, wherein the first pharmaceutical composition is suitable for delivery to the inner ear via a cannula. 22. The combination of claim 21, wherein said at least one antisense nucleic acid molecule is SEQ ID NO: 20. delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete

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