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WO1998013048A1 - Nouveaux procedes induisant une proliferation dans l'epithelium des recepteurs auditifs - Google Patents

Nouveaux procedes induisant une proliferation dans l'epithelium des recepteurs auditifs Download PDF

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Publication number
WO1998013048A1
WO1998013048A1 PCT/US1997/017428 US9717428W WO9813048A1 WO 1998013048 A1 WO1998013048 A1 WO 1998013048A1 US 9717428 W US9717428 W US 9717428W WO 9813048 A1 WO9813048 A1 WO 9813048A1
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receptor
camp
patient
cells
hair
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PCT/US1997/017428
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J. Carl Oberholtzer
Dhasakumar S. Navaratnan
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Trustees Of The University Of Pennsylvania
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Priority to AU46002/97A priority Critical patent/AU4600297A/en
Priority to US09/077,264 priority patent/US6268351B1/en
Publication of WO1998013048A1 publication Critical patent/WO1998013048A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
    • A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/24—Heavy metals; Compounds thereof
    • A61K33/243—Platinum; Compounds thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 

Definitions

  • This invention relates to the field of auditory research. Specifically, the invention pertains to methods which promote the regeneration of auditory hair cells following loss due to trauma or disease.
  • Hair cells are sensory cells that transduce motion into neural signals. In the cochlea, they are used to detect sound waves in the environment and convert them into auditory signals that can be processed in the brain. Loss of receptor hair cells in the cochlea accounts for a significant proportion of hearing impairment in the population (1) . This loss can occur as a consequence of viral or bacterial insult, aging, and damage from intense sound or a inoglycoside antibiotics. The generation of replacement hair cells following damage by sound or drugs has been clearly demonstrated in birds (2-4) , the chick being the best-studied model for auditory hair cell regeneration (5) . New hair cells arise as progeny from an otherwise non-dividing supporting cell population induced to proliferate by the damage (6-12) .
  • the present invention provides a novel system for the regeneration of auditory receptor hair cells.
  • the generation of replacement hair cells following damage by sound or drugs has been clearly demonstrated in birds where the hair cells arise from a normally non- dividing supporting cell population induced to proliferate by the damage.
  • the present invention demonstrates that agents which increase cAMP levels induce cell proliferation in explanted but otherwise undamaged receptor epithelium of cochleas, and that protein kinase A (PKA) inhibitors block this proliferative response.
  • PKA protein kinase A
  • a research tool is provided for the elucidation of the molecular events involved in the restoration of auditory receptor hair cells.
  • stimulation of proliferation is achieved via the administration of agents which activate the cAMP pathway, such as stimulators of adenylate cyclase (i.e., forskolin).
  • forskolin may be used in a range of about 1- 200 ⁇ M.
  • Narrower ranges of forskolin concentration are also contemplated in practicing the present invention, i.e, between about 10 ⁇ M- 100 ⁇ M, or between about 75 ⁇ M- 150 ⁇ M.
  • cAMP levels may be augmented by direct delivery of cAMP or analogues thereof (i.e., 8-Br-cAMP) .
  • Proliferation of auditory receptor cells may be assessed by co-administration and detection of agents that are incorporated into replicating DNA (i.e., Bromo-deoxyuridine) .
  • the explant cochlea cultures are exposed to ototoxic agents. Damage to these cultures results in a proliferative response. Following damage, this regenerative response may be abrogated by agents that inhibit the cAMP mediated signal transduction pathway, such as PKA inhibitors.
  • PKA inhibitors agents that inhibit the cAMP mediated signal transduction pathway
  • a method for treating a patient suffering from hearing loss comprises delivering to a patient's auditory receptor epithelium, a cAMP activating agent in a suitable pharmaceutical carrier in an effective amount to activate the signal transduction pathway responsible for regeneration of hair cells in the cochlea epithelium.
  • the method is used to assess the regeneration of hair receptor cells in the bird.
  • an agent for detecting DNA replication is co-administered to the damaged auditory epithelium with the cAMP pathway activating agent.
  • the animal Following stimulation of a proliferative response, the animal is sacrificed and the level of proliferation assessed.
  • Receptor hair cells are cells which lie in the upper part of the epithelium with their stereocilia embedded in the tectorial membrane. Hair cells are surrounded by supporting cells. The transduction of the mechanical energy of sound to a coded neural signal is effected by hair cells. Receptor hair cells are located in the sensory epithelia of both the auditory and vestibular portions of the inner ear.
  • the vestibular system is composed of the utricle, the saccule and the three ampullae of the semicircular canals. Impairments in the vestibular sensory epithelia often cause impaired balance.
  • the auditory receptor epithelia is located within the cochlea and is referred to the organ of corti in mammals and basilar papilla in avian species. Damage to the sensory epithelium in the cochlea can lead to impaired hearing.
  • the methods of the invention provide a novel approach for stimulating the regeneration of auditory receptor epithelium.
  • the methods of the present invention may be beneficially used to elucidate the molecular mechanisms relevant in the restoration of hearing in subjects whose hearing has been damaged due to trauma or disease.
  • Figures 1A-1I are a series of micrographs depicting cochlear explants cultured in the presence of BrdU and exposed to forskolin with/without PKA inhibitors, or damaged with gentamicin with/without PKA inhibitors.
  • Fig. 1A - 1C Control whole-mounts fluorescently stained for filamentous actin with rhodamine-labeled phalloidin.
  • the cochlea shown in (Fig. 1A) and (Fig. IB) was fixed and stained immediately following removal; the explant shown in (Fig. 1C) was maintained in culture with no other treatment for 3 days prior to staining.
  • p and d identify the proximal (basal) and distal (apical) ends of the basilar papilla; n and a indicate the neural (superior) and abneural (inferior) aspects.
  • Se indicates the sensory epithelium in which the stereocilia bundles of the receptor hair cells can be identified.
  • Fig. ID - 1G Whole-mounts im unochemically stained for BrdU (purple-black) . In these panels the position of the exposed sensory epithelium is outlined with dashed white lines.
  • Fig. IH, Fig. II Cross-sections immunostained for both BrdU (purple-black) and for calbindin (brown) , to highlight the sensory epithelium.
  • the arrowhead indicates a labeled nucleus at the supporting cell level while the arrow indicates labeled nucleus at receptor hair cell level; arrow in inset indicates vertical cluster of labeled nuclei.
  • the arrows indicate a fold or bend in the sensory epithelium which was introduced in the explant during the period of culture. The lower arrow points to the basilar membrane, while the upper arrow points to the tectorial membrane. Note that cells outside the sensory epithelium were also labeled with BrdU, however their labeling is independent of forskolin treatment. DNA synthesis in these cell types, many of which are likely to be fibroblasts, is either constitutive or was induced by explantation alone. Calibration bars: 0.2 mm in (Figs. 1A - 1G) ; 50 ⁇ m in (Fig. IH, Fig. II) .
  • Figures 2A and 2B are graphs summarizing the data showing the involvement of the cAMP pathway in signaling proliferation in the undamaged and gentamicin-damaged auditory receptor epithelium. Bars show the mean number of labeled nuclei per sensory epithelium for each of the indicated groups. Error bars are the standard error of the mean (S.E.M.); n is the number of cochleas per group. Fig.
  • 2A shows results from undamaged cochlear explants receiving no treatment (CTRL) ; or treatment with 8-bromo-cAMP and IBMX (8BcAMP) , forskolin (FSK) , forskolin plus PKA inhibitor H89 (FSK+H89) , or forskolin plus PKA inhibitor KT5720 (FSK+KT5720) .
  • CTRL vs. FSK
  • FSK vs. FSK+H89
  • FSK vs. FSK+KT5720 .
  • Fig 2B shows results from explanted cochleas receiving no treatment (CTRL) , or damaged with gentamicin (GENTA) , damaged with gentamicin and treated with PKA inhibitor H89 (GENTA+H89) , and damaged with gentamicin and treated with PKA inhibitor KT5720 (GENTA+KT5720) .
  • CTRL cochleas receiving no treatment
  • GENTA damaged with gentamicin
  • GENTA+H89 damaged with gentamicin and treated with PKA inhibitor KT5720
  • the lack of a complete block of DNA synthesis by PKA inhibitors in these settings may be due to partial cellular accessibility of the inhibitors, or may indicate the involvement of additional mechanisms.
  • Figures 3A-3C are a series of autoradiographs showing 3 H-thymidine labeling of sensory epithelial cells in cochlear explants stimulated with forskolin.
  • Fig. 3A shows a portion of sensory epithelium of one cochlear explant with the tectorial membrane (t) , basilar membrane (b) , and innervating fibers (n) identified.
  • Fig. 3B and Fig. 3C show segments of the sensory epithelia of two additional cochlear explants.
  • 3 H-thymidine-labeled sensory hair cells are marked with arrows. These are identified as hair cells on the basis of their shapes, their characteristic dark-staining cytoplasm, and their flat apical surfaces forming part of the reticular lamina. Magnification 500x.
  • Figure 4 is a schematic diagram of the mammalian inner ear.
  • 1 ear canal; 2: middle ear; 3: oval window; 4: semicircular canals; 5: utricle; 6: pinna; 7: eardrum; 8: round window; 9: eustachian tube; 10: saccule; 11: cochlea.
  • cyclic AMP serves as a second messenger which is produced as a result of the activation of cell-surface receptors.
  • the present invention demonstrates that proliferation may be induced in undamaged receptor epithelium by agents which increase cAMP levels, and that following this stimulation, hair cells become labeled with proliferation markers. This remarkable proliferative response is blocked by inhibitors of the cAMP-regulated protein kinase (PKA) .
  • PKA protein kinase
  • the results presented illustrate that the proliferative response induced by in vitro gentamicin damage is also significantly blocked by PKA inhibitors.
  • the present invention provides a valuable research tool for the elucidation of the molecular events relevant to the restoration of hearing in patients who have lost receptor epithelium hair cells.
  • the cAMP pathway has been identified as playing a key role in regeneration of the receptor epithelium in the cochlea, it is possible to biochemically intervene at several points in the pathway and characterize the key steps leading to proliferation.
  • a cell line will be developed to provide a continuous culture system to study the signal transduction events which result in the regeneration of hair cells.
  • Gentamicin treatment of acute gram negative infections leads to significant loss of receptor hair cells in humans. Restoration of hearing in these patients by topical administration of agents that stimulate the cAMP pathway may now be possible. Similarly, chemotherapies used in cancer treatment also damage the hair cells in the auditory receptor epithelium. Acoustic trauma and aging also result in hearing loss. In situ administration of agents that affect the cAMP pathway may be effective in restoring hearing in these patients.
  • DMSO dimethyl sulfoxide
  • Forskolin (lOO ⁇ M) Forskolin
  • H89 and KT5720 each at 0.5 ⁇ M; from Calbiochem
  • IBMX isobutyl methylxanthine
  • 8-bromo-cAMP was added to a final concentration of 5mM from an aqueous stock solution.
  • Control cultures included carrier DMSO at a final concentration (1%) similar to that present in the experimental cultures.
  • Negative controls for forskolin were performed using the inactive analog 1,9-dideoxy-forskolin (lOO ⁇ M; Calbioche ) ; this compound did not stimulate DNA synthesis (data not shown) .
  • Those explants treated with 8-bromo-cAMP were pretreated for 24 hours with IBMX, and IBMX was maintained in the medium throughout the three days 8-bromo-cAMP was present.
  • PKA inhibitors H89 and KT5720
  • Gentamicin sulfate was added to explant cultures at a final concentration of 0.1% (20,21).
  • Cochleas used for autoradiography were maintained for five days in culture in the presence of forskolin and 3 H-thymidine (l ⁇ Ci/ml; 20Ci/mmol) .
  • forskolin and 3 H-thymidine l ⁇ Ci/ml; 20Ci/mmol
  • Nuclear BrdU incorporation was determined in whole-mounts essentially as described (11) . Briefly, cochleas were immobilized on a matrix (Silguard) and the tegmentum vasculosum was dissected away. The tectorial membrane was peeled away after a 5 minute treatment with 0.005% subtilisin (Sigma type 27). Cochleas were then immersed in 10% normal buffered formalin for 30 minutes followed by a 20 minute incubation in 2N HCl, 0.1% Tween 20.
  • BrdU was detected immunohistochemically by incubating the cochleas in the presence of anti-BrdU antibody (Becton Dickinson) for one hour (1:40 in PBS, 0.1% Tween 20) followed by secondary antibody (1:1000 alkaline phosphatase-conjugated goat anti-mouse, Boehringer Mannheim) for an additional hour. Secondary antibody was detected with 5-bromo-4-chloro-3-indolyl phosphate (0.34 mg/ml) and nitro-blue tetrazolium (0.175 mg/ml) in lOOmM NaCl, 50mM MgCl 2 , lOOmM Tris-HCl pH 9.5.
  • Cochleas were washed three times in PBS, 0.1% Tween 20 between each step. Fluorescent staining for filamentous actin in cochlear whole-mounts was performed using rhoda ine-labeled phalloidin as previously described (22) .
  • Tissue cross-sections were prepared from cochleas embedded in paraffin for immunohistochemistry, and in plastic for autoradiography. Paraffin sections (5 ⁇ m-thick) were cut from cochleas that were fixed for 24 hours in Bouin's fixative, dehydrated, and embedded. These were stained with anti-BrdU antibodies (see above) after deparaffination. In order to highlight the sensory epithelium in the paraffin sections they were also stained immuno-histochemically for calbindin D28K23 using a procedure similar to that for BrdU.
  • the primary antibody was a rabbit anti-calbindin antiserum (1:500 in PBS, 0.1% Tween 20) which was detected with a goat anti-rabbit antibody (1:1000) coupled to horseradish peroxidase using 3, 3'-diaminobenzidine and H 2 0 2 as substrates.
  • a goat anti-rabbit antibody (1:1000) coupled to horseradish peroxidase using 3, 3'-diaminobenzidine and H 2 0 2 as substrates.
  • One ⁇ m-thick plastic sections for autoradiography were cut from cochleas fixed in 2% glutaraldehyde overnight at 4°C and then for one hour in fixative with 1% tannic acid, followed by dehydration and embedding in Epon. Plastic sections were coated with undiluted NTB-2 emulsion (Eastman-Kodak, Rochester) . Slides were developed (Kodak D19) and fixed (Kodak Rapid Fixer) after 4 days exposure at 4°C. The tissue
  • Utricles may be prepared as described in (26) . Briefly, utricular epithelial sheets are separated from postnatal day 4-5 Wistar rats with 0.5 mg/ml thermolysin (Sigma, St. Louis, MO) in calcium- and magnesium-free Hanks buffered saline solution for 30 minutes at 37 °C. The epithelial sheets are then incubated in a mixture of 0.125% trypsin and 0.125% collagenase for 8 minutes at 37°C. The enzyme activity was inactivated with a mixture of 0.005% soybean trypsin inhibitor (Sigma) and 0.005% DNase
  • the cell suspension was then plated in poly-D- lysine-coated (500 ⁇ g/ml) 96 well plates or 16 well labtek slides in 200 ⁇ l of serum containing medium (DMEM + 5% fetal bovine serum, 4.5 mg/ml glucose, 2 mM glutamine, 25 ng/ l fungizone, and 10 U/ml penicillin) at a density of approximately 70 cells/mm 2 .
  • serum containing medium DMEM + 5% fetal bovine serum, 4.5 mg/ml glucose, 2 mM glutamine, 25 ng/ l fungizone, and 10 U/ml penicillin
  • the third group was treated with the membrane-permeable cAMP analog 8-bromo-cAMP and the phosphodiesterase inhibitor iso-butyl methylxanthine (IBMX) as an independent method of activating the cAMP pathway.
  • the fourth group was treated with both forskolin and the specific PKA inhibitor H89.
  • the fifth group was treated with both forskolin and the specific PKA inhibitor KT5720. Groups four and five were included to confirm that any proliferative response was mediated by PKA, the major cAMP pathway effector. After incubation for three days, cochleas were fixed and whole mounts examined for BrdU incorporation using immunohistochemistry.
  • Figure 1A Shown for orientation purposes in Figure 1A is a freshly-dissected chick cochlea from which the tegmentum vasculosum and tectorial membrane have been removed revealing the surface of the sensory epithelium (labeled "se"); Figure IB shows a portion of this sensory epithelium (basilar papilla) at higher power. The stereocilia bundles on the apical surfaces of the receptor hair cells can be seen studding the epithelium in this preparation stained to reveal filamentous actin using fluorescence-labeled phalloidin. A similar preparation of a cochlea maintained in culture for three days is shown in Figure 1C.
  • a measure of the integrity of the sensory epithelium maintained under the culture conditions used for these experiments can be determined by examining the preserved precise array of receptor hair cells across the papilla.
  • the similar whole-mount preparations shown in panels D-G, stained immunohistochemically for BrdU, are presented at a comparable magnification.
  • Figure ID shows a portion of the basilar papilla of a representative cochlear explant treated with forskolin. Numerous BrdU-labeled nuclei are seen across and along the sensory epithelium; some of these are paired suggesting that cell division has already occurred. Specificity controls using the inactive forskolin analog 1, 9-dideoxy-forskolin did not show proliferation (data not shown) .
  • Figure IE shows the sensory epithelium of a cochlea treated with both forskolin and the PKA inhibitor KT5720. In this representative case, the number of BrdU-labeled nuclei is greatly diminished.
  • FIG. 1H shows two examples of cochleas treated with forskolin. Multiple labeled nuclei are seen at the level of both supporting cells and hair cells. This is in contrast to the example of the untreated control cochlea shown in Figure II, in which no labeled nuclei can be identified within the sensory epithelium.
  • Figure 2A Treatment with either forskolin or 8-bromo-cAMP was highly effective in stimulating DNA synthesis in the normally quiescent basilar papilla.
  • Control papillas contained only one to three BrdU-labeled nuclei, while those treated with forskolin and 8-bromo-cAMP contained an average of 180 and 50, respectively.
  • Forskolin-induced BrdU incorporation was reduced 87% and 73% by H89 and KT5729, respectively.
  • Receptor Hair Cells are Stimulated by Agents that Activate the cAMP Pathway
  • a third set of experiments was performed to determine if the population of cells which become labeled with DNA synthesis markers after activation of the cAMP pathway includes receptor hair cells.
  • Cochlear explants were treated with forskolin in the presence of 3 H-thymidine, and autoradiography was performed on plastic-embedded cross-sections.
  • the explants were maintained in culture for five days rather than three to allow a longer time period for the presumptive differentiation of supporting cell progeny into hair cells (11) .
  • Figure 3 shows representative portions of the sensory epithelium from three cochlear explants. Several examples of hair cells with clearly-labeled nuclei are seen; other labeled cells within the epithelium resembled supporting cells. Essentially every section examined contained multiple labeled sensory epithelial cells; most sections contained at least one labeled hair cell. These results suggest that the cAMP-induced proliferation leads to the production of new hair cells.
  • EXAMPLE IV Treatment of Auditory Receptor Epithelium in situ
  • the results presented implicate the involvement of the cAMP pathway in the regeneration of auditory receptor epithelium. Accordingly, hearing compromised patients would be treated with agents that stimulate cAMP production.
  • forskolin 25 -100 ⁇ M dissolved in di ethylsulfoxide (DMSO)
  • DMSO di ethylsulfoxide
  • the drug delivery system may comprise an osmotic pump or other timed-release gel delivery systems known in the art. Restoration of hearing would then be assessed using standard methods.
  • the methods performed in explant culture may also be performed in situ.
  • ototoxic agents would be co-administered with agents for detecting DNA replication. After a predetermined time period the animal would be sacrificed and proliferation would be assessed.
  • Agents that stimulate (i.e., cAMP activators) or abrogate (i.e., PKA inhibitors) the proliferative response may also be simultaneously administered to the animal and their effects assessed. These procedures would result in the further elucidation of the molecular mechanisms involved in the regeneration of auditory epithelium. DISCUSSION These studies are the first to show induction of proliferation in the normally quiescent auditory sensory epithelium by any means other than damage.
  • Hashino, E. Tanaka, Y., Salvi, R. J. , et al. Hair cell regeneration in the adult budgerigar after kana ycin ototoxicity. Hear. Res. 59, 46-58 (1992).

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Abstract

La présente invention concerne de nouveaux procédés pour stimuler la prolifération dans l'épithélium des récepteurs auditifs. L'administration d'agents qui activent la production d'AMP cyclique entraîne une régénération manifeste des cellules sensorielles dans la cochlée. Ces agents peuvent être administrés in vivo afin de guérir la perte auditive ou la perte d'équilibre chez les patients ayant besoin de ce traitement.
PCT/US1997/017428 1996-09-27 1997-09-26 Nouveaux procedes induisant une proliferation dans l'epithelium des recepteurs auditifs WO1998013048A1 (fr)

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AU46002/97A AU4600297A (en) 1996-09-27 1997-09-26 Novel methods for inducing proliferation in auditory receptor epithelium
US09/077,264 US6268351B1 (en) 1997-09-26 1997-09-26 Methods for inducing proliferation in auditory receptor epithelium

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US60/026,800 1996-09-27

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999042088A3 (fr) * 1998-02-23 2000-03-02 Otogene Ag Procede pour le traitement de maladies ou de troubles de l'oreille interne
US6838444B1 (en) 1999-06-01 2005-01-04 Baylor College Of Medicine Compositions and methods for the therapeutic use of an atonal-associated sequence for deafness, osteoarthritis, and abnormal cell proliferation
US7053200B1 (en) 1999-06-01 2006-05-30 Baylor College Of Medicine Compositions and methods for the therapeutic use of an atonal-associated sequence for deafness, osteoarthritis, and abnormal cell proliferation
US9951351B2 (en) 2014-10-09 2018-04-24 Genvec, Inc. Adenoviral vector encoding human atonal homolog-1 (HATH1)

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5500902A (en) * 1994-07-08 1996-03-19 Stockham, Jr.; Thomas G. Hearing aid device incorporating signal processing techniques
US5554361A (en) * 1994-01-21 1996-09-10 Dixon; Gary W. Processed product for skin and hair treatment

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5554361A (en) * 1994-01-21 1996-09-10 Dixon; Gary W. Processed product for skin and hair treatment
US5500902A (en) * 1994-07-08 1996-03-19 Stockham, Jr.; Thomas G. Hearing aid device incorporating signal processing techniques

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999042088A3 (fr) * 1998-02-23 2000-03-02 Otogene Ag Procede pour le traitement de maladies ou de troubles de l'oreille interne
US7087581B1 (en) 1998-02-23 2006-08-08 Sound Pharmaceuticals Incorporated Method for the treatment of diseases or disorders of the inner ear
US6838444B1 (en) 1999-06-01 2005-01-04 Baylor College Of Medicine Compositions and methods for the therapeutic use of an atonal-associated sequence for deafness, osteoarthritis, and abnormal cell proliferation
US7053200B1 (en) 1999-06-01 2006-05-30 Baylor College Of Medicine Compositions and methods for the therapeutic use of an atonal-associated sequence for deafness, osteoarthritis, and abnormal cell proliferation
US7442688B2 (en) 1999-06-01 2008-10-28 Baylor College Of Medicine Composition and methods for the therapeutic use of an atonal-associated sequence for deafness, osteoarthritis and abnormal cell proliferation
US7470673B2 (en) 1999-06-01 2008-12-30 Baylor College Of Medicine Composition and methods for the therapeutic use of an atonal-associated sequence for deafness, osteoarthritis and abnormal cell proliferation
US9951351B2 (en) 2014-10-09 2018-04-24 Genvec, Inc. Adenoviral vector encoding human atonal homolog-1 (HATH1)
US11279951B2 (en) 2014-10-09 2022-03-22 Genvec, Inc. Adenoviral vector encoding human atonal homolog-1 (HATH1)

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