WO1998000014A1 - Transformation et therapie genique des cellules de l'oreille interne - Google Patents

Transformation et therapie genique des cellules de l'oreille interne Download PDF

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Publication number
WO1998000014A1
WO1998000014A1 PCT/US1997/011602 US9711602W WO9800014A1 WO 1998000014 A1 WO1998000014 A1 WO 1998000014A1 US 9711602 W US9711602 W US 9711602W WO 9800014 A1 WO9800014 A1 WO 9800014A1
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cell
inner ear
interest
cells
cochlear
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PCT/US1997/011602
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English (en)
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Anil Lalwani
Robert A. Schindler
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The Regents Of The University Of California
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Priority to AU35915/97A priority Critical patent/AU3591597A/en
Publication of WO1998000014A1 publication Critical patent/WO1998000014A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/185—Nerve growth factor [NGF]; Brain derived neurotrophic factor [BDNF]; Ciliary neurotrophic factor [CNTF]; Glial derived neurotrophic factor [GDNF]; Neurotrophins, e.g. NT-3

Definitions

  • This invention relates generally to the field of eukaryotic cell transformation, particularly transformation of cells of the inner ear, including cells of the auditory nerves, cochlea, and vestibular system, and application of transformation techniques of the invention for use in gene therapy.
  • the ear facilitates sound perception by first transforming the pressure changes of sound waves into physical movements of the tympanic membrane (Figs. 1 and 2). Movements of the tympanic membrane are then translated into movements of the bones of the middle ear, so that the footplate of the stapes induces movements of the oval window. These oval window movements are transmitted as waves in the endocochlear fluids (perilymph, endolymph, and cortilymph) . Pressure waves induced in the cochlear fluids then cause movements of the basilar membrane of the organ of Corti, located in the cochlear duct (Fig.
  • the hair cells transduce the mechanical deformation of the hairs into nerve impulses in the terminal fibers of the cochlear branch of the vestibulocochlear nerve (cranial nerve VIII) . Transmission of these signals to the appropriate cortical areas of the brain results in sound perception.
  • Hearing impairment which can range from a minor inability to perceive particular sound frequencies to complete deafness, is classified into three general categories: conductive losses, sensorineural losses, and mixed losses.
  • Conductive losses interfere with transmission of sound to the cochlea, and are usually caused by abnormalities of the outer and middle ear. For example, total occlusion of the external canal by cerumen is the most common cause of a conductive hearing loss in elderly patients, while fluid in the middle ear space (serus otitis media) is the most common cause of conductive hearing loss in children.
  • Sensorineural hearing loss is associated with damage to the cochlea, cranial nerve VIII, or its central connection (the brain stem auditory pathways and the auditory crest) . Damage can result from age-related changes, environmental influence, or pathological processes. For example, presbycusis, a sensorineural hearing loss that accompanies aging, is caused by a degenerative process of the inner ear and is characterized by a loss of hair cells, atrophy of the spinal ganglion, altered endolymph production, thickening of the basilar membrane, or neural degeneration. Other causes of sensorineural losses include noise trauma, ototoxic drugs, involvement of the acoustic nerve by benign or malignant tumors, cerebrovascular disease, and possible complications of arteriosclerosis.
  • Hearing loss that is caused by both conductive and sensorineural impairments is termed mixed or combined hearing loss.
  • Conventional treatment of hearing loss depends on the type and degree of hearing loss and the age at onset .
  • Surgical and medical intervention, corrective amplification (e.g., hearing aids), and education are most commonly used.
  • Medical management, surgical management, or both are generally appropriate for conductive hearing loss, depending on the degree of impairment and the status of bone conduction and speech discrimination.
  • Surgical removal of tumors of cranial nerve VIII may preserve hearing; however, the primary objective of such procedures is relief of vertigo.
  • Cochlear lesions usually are not correctable using conventional therapies.
  • Management of a disease or damaged cochlea using conventional therapy consists of hearing aid fitting, audiological rehabilitation, and for patients without aidable hearing, cochlear implants.
  • the conventional methods of treatment and study of hearing loss associated with the inner ear are limited in scope and success.
  • the structures of the inner ear are so delicate, attempts to study the mechanisms of hearing and hearing loss are severely hampered by the lack of adequate means to examine the function of specific genes and gene products within the cells of the inner ear.
  • gene therapy is currently used to treat a variety of genetic conditions and infections in other settings, application of gene therapy to genetic defects and other conditions of the inner ear is hampered by the availability of a method for delivery of transforming DNA to the inner ear and transformation of the cells.
  • the present invention addresses this problem.
  • the present invention features compositions and methods for transformation of cells of the inner ear and treatment of conditions of the inner ear using such methods .
  • cells of an inner ear of a subject are genetically altered to operatively incorporate a nucleotide sequence which expresses a gene product of interest (e.g., a therapeutic gene product) .
  • the inner ear cell into which the DNA of interest is introduced and expressed in a cell of the cochlea more preferably a cell of the spiral ligament, spiral limbus, stria vascularis, organ of Corti, spiral ganglion, and/or Reissner's membrane, and/or an auditory hair cell.
  • the DNA of interest preferably present within an adeno-associated viral vector, is introduced through a cannula inserted in the round or oval window and in communication with the peri lymph or endolymph.
  • introduction of the DNA of interest is accomplished with an osmotic minipump.
  • the invention also features recombinant cells of the inner ear, preferably a recombinant cochlear cell, more preferably a recombinant cell of the spiral ligament, spiral limbus, organ of Corti, spiral ganglion, Hansen's cell, and/or Reissner's mernbrane and/or an auditory hair cell, containing a DNA of interest operatively inserted in the genome of the cell and operatively linked to a promoter for expression of the DNA of interest.
  • the invention features an isolated cochlea (e.g, in an organotypic cochlear culture) which cochlea contains transformed cochlear cells.
  • a primary object of the invention is to provide a method for transformation of inner ear cells useful in the examination of the mechanisms associated with hearing and hearing impairment. Another object is to provide a method of gene therapy wherein cells of an inner ear of a subject are genetically modified to express a therapeutically useful gene product.
  • Another object is to produce genetically transformed inner ear cells which cells have incorporated into their genome genetic material which expresses a gene product of interest, e.g., a therapeutic gene product.
  • An advantage of the present invention is that gene therapy can be achieved in the ear to provide treatment of various conditions associated with defects (e.g., genetic defects, or defects associated with environmental damage or age-related deterioration) and/or infections of the peripheral auditory system, including the cochlea and its associated cells and structures.
  • defects e.g., genetic defects, or defects associated with environmental damage or age-related deterioration
  • infections of the peripheral auditory system including the cochlea and its associated cells and structures.
  • the method of transformation of the invention provides a means to access the inner ear and transform cells of the inner ear without substantially disrupting the delicate structures of the inner ear or eliciting a significant inflammatory response.
  • Fig. 1 is a schematic illustration of the human ear.
  • Fig. 2 is a schematic illustration of the human inner ear
  • Fig. 3 is a schematic illustration of a cross-section through a turn of a human cochlea.
  • Fig. 4 is a schematic illustration (enlarged relative to Fig. 3) of a cross -section through a turn of a human cochlea.
  • Fig. 5 is a schematic of a recombinant construct useful in producing recombinant inner ear cells according to the invention.
  • Fig. 6 is a schematic view of a recombinant adeno- associated viral construct containing DNA encoding the marker gene ⁇ -galactosidase (S-gal) .
  • TR represents the terminal repeat of adeno-associated virus (145 bp) ;
  • P mP represents the Ad 2 major late promoter (695 bp) ;
  • SD/SA SV40 represents the late gene 16S/19S splice donor/splice acceptor signal of SV40 (180 bp) ;
  • jS-gal represents that 0-gal gene (3530 bp) ;
  • pAl represents the polyadenylation signal of the SV40 late genes (196 bp) ; and
  • pAe represents the polyadenylation signal of SV40 early genes (50 bp) .
  • Fig. 7 is a schematic illustration of a recombinant adeno-associated viral vector for expression of neurotrophin-3 (NT-3) .
  • Fig. 8 is a schematic illustration of a recombinant adeno-associated viral vector for expression of brain-derived neurotrophin factor (BDNF) .
  • BDNF brain-derived neurotrophin factor
  • Fig. 9 is a schematic illustration of a recombinant adeno-associated viral vector for expression of nerve growth factor (NGF) .
  • NGF nerve growth factor
  • Fig. 10 is a schematic illustration of a recombinant adeno-associated viral vector expressing j ⁇ -galactoidase under the control of the CMV immediate/early gene promoter (Pcmv) .
  • IRES represents the internal ribosome entry site
  • SV40 SD/SA represents the late gene 16S/19S splice donor/splice acceptor signal of SV40
  • SV40poly(A) represents polyadenylation signal of SV40 late genes
  • bGHpoy(A) represents the polyadenylation signal .
  • FIG. 11 is a schematic illustration of a recombinant adeno-associated viral vector for expression of green fluorescent protein (GFP) from the CMV early/immediate gene promoter (Pcmv) .
  • IRES represents the internal ribosome entry site
  • SV40 SD/SA represents the late gene 16S/19S splice donor/splice acceptor signal of SV40
  • SV40poly(A) represents polyadenylation signal of SV40 late genes
  • bGHpoy(A) represents the polyadenylation signal.
  • Fig. 12 is a schematic illustrating use of the osmotic minipump for steady state delivery of AAV to the cochlea of a guinea pig via a cannula inserted through an opening created near the cochlear base .
  • Fig. 13 is a panel of photographs showing in vi tro transfection of rat cochlear cells either as dissociated cells (upper left panel) or as cochlear explants (bottom left and right panels) .
  • Fig. 14 is a panel of photographs showing organotypic rat cochlear explants stained with neurofilament antibody after exposure to amikacin either with or without transformation with BDNF-encoding DNA (AAV-BDNF) .
  • Control untreated cells, top left panel; cells treated with amikacin only, top right panel; cells infected with AAV-BDNF, bottom left and right panels.
  • inner ear is meant the portion of the ear positioned medial to the middle ear of a mammalian or nonma rnalian subject, and includes the cochlea, the auditory nerves, the semicircular canals and ducts, the ampules, the saccule, the endolymphatic sac, and the cochlear and vestibular aqueducts.
  • Fig. 1 is a schematic showing the position of the inner ear relative to the outer and middle ear.
  • Fig. 2 is a schematic showing a cross section of the inner ear and its relation to the middle ear.
  • inner ear cell or “cell of the inner ear” is meant a cell of any of the structures of the inner ear.
  • cochlea is meant the structure of the inner ear in the shape of a coiled tube of about 35 mm in length. In humans the cochlea makes about 2 3/4 turns.
  • the cochlea is divided into three chambers, or scala, throughout its length by the basilar membrane and Reissner's membrane.
  • the upper scala vestibuli and the lower scala tympani contain perilymph and communicate with each other at the apex of the cochlea through a small opening called the helicotrema.
  • the scala vestibuli ends at the oval window, which is closed by the foot plate of the stapes.
  • scala tympani ends at the round window, a foramen on the medial wall of the middle ear that is closed by the flexible secondary tympanic membrane.
  • the scala media, the middle cochlear chamber that contains endolymph, is continuous with the membranous labyrinth and does not communicate with the other two scalae.
  • Figs. 2 and 3 are schematics of a the human cochlea and structures within the human cochlea.
  • Fig. 4 is a schematic of a cross section of a turn of the cochlea of the guinea pig.
  • isolated cochlea By “isolated cochlea,” “cultured cochlea,” or “organotypic cochlear culture” is meant a cochlea that is removed and separate from an inner ear.
  • cochlear cell is meant a cell of any portion of or structure of the cochlea, including, but not limited to, any cell of the spiral ligament, spiral limbus, stria vascularis, organ of Corti, Reissner's membrane, basilar membrane, and spiral ganglion, as well as auditory hair cells. Structures and cells of the cochlea are shown in Figs. 3 and 4.
  • a substantially enriched transformed cochlear cell culture is an in vi tro culture of cochlear cells genetically transformed according to the method of the invention.
  • Window of the inner ear is meant to include the round window and oval window of the inner ear .
  • the round window is a foramen on the medial wall of the middle ear positioned at the at the base of the cochlea at the end of the scala tympani.
  • the round window is closed by the secondary tympanic membrane.
  • the oval window is a foramen that defines the end of the scala vestibuli and is closed by the foot plate of the stapes .
  • the position of the oval and round windows in the ear are shown in Fig. 2.
  • transformation is meant any permanent or transient genetic change induced in a cell following incorporation of new DNA (i.e., DNA exogenous to the cell) .
  • target cell is meant a cell(s) that is to be transformed using the methods and compositions of the invention. Transformation may be designed to nonselectively or selectively transform the target cell(s) .
  • target cell as used herein means a cell of the inner ear that is to be transformed using the method and compositions of the invention.
  • transformed cell is meant a cell into which (or into an ancestor of which) has been introduced, by means of recombinant DNA techniques, a DNA molecule encoding a molecule (e.g., RNA and/or protein) of interest (e.g., nucleic acid encoding a therapeutic cellular product) .
  • a DNA molecule encoding a molecule e.g., RNA and/or protein
  • a DNA molecule encoding a molecule (e.g., RNA and/or protein) of interest (e.g., nucleic acid encoding a therapeutic cellular product) .
  • nucleotide sequence of interest or “DNA of interest” is meant any nucleotide sequence (e.g., RNA or DNA sequence) or DNA sequence that encodes a protein or other molecule that is desirable for expression in a target cell (e.g., for production of the protein or other biological molecule (e.g., a therapeutic cellular product) in the target cell) .
  • the nucleotide sequence of interest is generally operatively linked to other sequences which are needed for its expression, e.g., a promoter.
  • Use of "DNA of interest” throughout the specification is not meant to limit the invention to deoxyribonucleic acid.
  • Gene product of interest is meant a polypeptide, RNA molecule, or other gene product that is desired for expression in a cell of the inner ear.
  • Gene products of interest can include, for example, polypeptides that serve as marker proteins to assess cell transformation and expression, fusion proteins, polypeptides having a desired biological activity, gene products that can complement a genetic defect, RNA molecules, transcription factors, and other gene products that are of interest in the regulation and/or expression of the cellular functions of inner ear cells.
  • Gene products of interest can also include nucleotide sequences that provide a desired effect or regulatory function, but do not necessarily encode an RNA molecule or polypeptide per se (e.g., transposons, introns, promoters, enhancers, splice signals, etc. ) .
  • RNA molecule a polypeptide, RNA molecule or other gene product that, when expressed in a cell of the inner ear, provides a desired therapeutic effect, e.g., repair of a genetic defect in the inner ear cell genome (e.g., by complement), expression of a polypeptide having a desired biological activity, and/or expression of an RNA molecule for antisense therapy (e.g., regulation of expression of a endogenous or heterologous gene in the inner ear cell genome) .
  • vector is meant any compound or formulation, biological or chemical, that facilitates transformation or transfection of a target cell (e.g., a cochlear or vestibular cell) with a DNA of interest.
  • exemplary biological vectors include viruses, particularly attenuated and/or replication- deficient viruses.
  • exemplary chemical vectors include lipid complexes and DNA constructs.
  • promoter is meant a minimal DNA sequence sufficient to direct transcription of a DNA sequence to which it is operably linked.
  • Promoter is also meant to encompass those promoter elements sufficient for promoter-dependent gene expression controllable for cell-type specific expression, tissue-specific expression, or inducible by external signals or agents; such elements may be located in the 5' or 3 ' regions of the native gene.
  • operably linked is meant that a DNA sequence and a regulatory sequence (s) are connected in such a way as to permit gene expression when the appropriate molecules (e.g., transcriptional activator proteins) are bound to the regulatory sequence (s) .
  • operatively inserted is meant that the DNA of interest is positioned adjacent a DNA sequence that directs transcription and translation of the introduced DNA (i.e., facilitates the production of, e.g., a polypeptide encoded by a DNA of interest) .
  • subject or “patient” is meant any mammalian or nonmammalian subject for which inner ear cell transformation and/or gene therapy is desired. Such subjects include, but are not limited to, humans, guinea pigs, primates, mice, cattle, goats, sheep, horses, dogs, cats, chicks, chick embryos, bullfrogs (as well as other reptiles), and fish.
  • Subjects include subjects that are to be treated for a hearing disorder or condition (e.g., humans) as well as mature and immature subjects that can serve as models for the study of hearing, cochlear and vestibular function, hearing loss, disorders, and conditions associated with hearing loss.
  • a hearing disorder or condition e.g., humans
  • mature and immature subjects that can serve as models for the study of hearing, cochlear and vestibular function, hearing loss, disorders, and conditions associated with hearing loss.
  • transgenic organism is meant a nonhuman organism (e.g., mammal or nonmammal) , having a nonendogenous (i.e., heterologous) nucleic acid sequence present as an extrachromosomal element in a portion of its cells or stably integrated into its germ line DNA.
  • transgenic animal is meant a nonhuman animal subject, usually a mammal, having a nonendogenous (i.e., heterologous) nucleic acid sequence present as an extrachromosomal element in a portion of its cells or stably integrated into its germ line DNA (i.e., in the genomic sequence of most or all of its cells) .
  • Heterologous nucleic acid is introduced into the germ line of such transgenic animals by genetic manipulation of, for example, embryos or embryonic stem cells of the host animal.
  • exemplary biological vectors include viruses, particularly attenuated and/or replication-deficient viruses.
  • exemplary chemical vectors include lipid complexes and various formulations comprising the nucleotide sequence of interest .
  • the vectors can contain or be derived from any of a variety of viral constructs, bacterial constructs, or constructs capable of replication in eukaryotic and prokaryotic hosts.
  • the construct is capable of replication in both eukaryotic and prokaryotic hosts in order to facilitate efficient production of the DNA of interest for use in the method of the invention.
  • Numerous constructs that can replicate in eukaryotic and prokaryotic hosts are known in the art and are commercially available.
  • the construct may be a stably integrating construct or a stable nonintegrating construct . Examples of such constructs include viral constructs and artificial chromosomes (e.g., human artificial chromosomes) .
  • a generic construct for use in the method of the invention is shown in Fig. 5.
  • the basic vector components include a promoter operably linked to a nucleotide sequence of interest. Additional components of a basic vector include a polyadenylation signal, a splice signal, and terminal repeat sequences (TR) , e.g., TR sequences corresponding to the viral sequence from which a viral vector is derived.
  • TR terminal repeat sequences
  • Transformation of inner ear cells may be accomplished by introduction of a DNA- or RNA-liposome complex formulations into the inner ear.
  • DNA- or RNA- complex formations comprise a mixture of lipids which bind to genetic material (DNA or RNA) , providing a hydrophobic coat which allows the genetic material to be delivered into cells.
  • Liposomes which can be used in accordance with the invention include DOPE (dioleyl phosphatidyl ethanol amine) , CUDMEDA (N- (5-cholestrum-3-3-ol 3-urethanyl) -N' ,N' -dimethylethylene diamine) .
  • the optimal values for the DNA lipid ratios and the absolute concentrations of DNA and lipid as a function of cell death and transformation efficiency. These values can then be used in or extrapolated for use in in vivo transformation.
  • the in vi tro determinations of these values can be readily carried out using techniques which are well known in the art.
  • nonviral vectors may also be used in accordance with the present invention.
  • chemical formulations include DNA or RNA coupled to a carrier molecule (e.g., an antibody or a receptor ligand) which facilitates delivery to host cells for the purpose of altering the biological properties of the host cells.
  • a carrier molecule e.g., an antibody or a receptor ligand
  • modifications of nucleic acids to allow coupling of the nucleic acid compounds to a carrier molecule such as a protein or lipid, or derivative thereof.
  • Exemplary protein carrier molecules include antibodies specific to the cells of a targeted inner ear cell or receptor ligands, i.e., molecules capable of interacting with receptors associated with a cell of a targeted inner ear cell.
  • the DNA of interest may be naked (i.e., not encapsulated) , or may be provided as a formulation of DNA and cationic compounds (e.g., dextran sulfate, DEAC-dextran, or poly-L-lysine) .
  • cationic compounds e.g., dextran sulfate, DEAC-dextran, or poly-L-lysine
  • a viral vector is used in the method of inner ear gene therapy of the invention.
  • viral vectors used in accordance with the invention are composed of a viral particle derived from a naturally-occurring virus which has been genetically altered to render the virus replication-defective and to express a recombinant gene of interest.
  • the virus delivers its genetic material to a cell, it does not generate additional infectious virus but does introduce exogenous recombinant genes into the cell, preferably into the genome of the cell.
  • the virus containing the DNA of interest is attenuated, i.e. does not cause significant pathology or morbidity in the infected host (i.e., the virus is nonpathogenic or causes only minor disease symptoms) .
  • viral vectors are well known in the art, including, for example, adeno-associated virus (AAV) , retrovirus, adenovirus, herpes simplex virus (HSV) , cytomegalovirus (CMV) , vaccinia and poliovirus vectors.
  • AAV adeno-associated virus
  • retrovirus retrovirus
  • adenovirus retrovirus
  • adenovirus herpes simplex virus
  • CMV cytomegalovirus
  • vaccinia vaccinia
  • poliovirus vectors lentivirus may be used to deliver a DNA of interest to inner ear cells.
  • Retroviral vectors are characterized by their ability to preferentially integrate into the genome of rapidly dividing cells, making them an ideal vector for introducing tumoricidal factors into proliferating neoplastic cells.
  • retroviral vectors are not necessarily as well-suited to the transformation of the neurosensory epithelia of the inner ear, which are post- mitotic and thus not rapidly dividing.
  • Adenoviral vectors infect both dividing and nondividing cells with high efficiency.
  • Adenoviral vectors do not integrate into the genome of the target cell (Berkner, 1992, Curr. Topics Microbiol . Immunol . , 158 : 39-66; Boviatsis et al . , 1994, Human Gene Therap . , 5.: 183-191) and thus provide temporal recombinant gene expression from an extra-chromosomal element for a period of several weeks to a month.
  • herpes virus vectors Replication-defective recombinant viruses and plasmid- derived amplicons derived from herpes virus vectors have been developed for gene delivery into cells and tissues (Leib et al., 1993, Bioessays, L5.: 547-54; Boviatsis et al . , 1994, Human Gene Therap. , 5_: 183-191). Both herpes -derived gene delivery vectors are relatively nonpathogenic to neural tissues and can mediate transgene expression in a substantial number of neurons and other cell types. The recombinant herpes vectors have the distinct advantage that they can enter a latent state in some neuronal cells and thus could potentially mediate stable transgene expression.
  • Adeno- associated virus has several desirable characteristics as a vector for gene therapy (Kotin, R.M., 1990, Proc . Natl . Acad . Sci . USA, 2 : 2211-5; Muzyczka, N., 1992, Curr. Topics Microbiol . Immunol . , 158 : 97-129).
  • AAV is nonpathogenic in both humans and animals and has a broad host range including human, primate, canine and murine . Its ability to infect and integrate into nondividing cells with high frequency makes it a desirable vector for transfecting post-mitotic epithelia of the inner ear (Kaplitt et al . , 1994, .Nature Genet, 8 .
  • AAV integration is stable; AAV remained stably integrated in the genome of transformed cells through 150 passages.
  • AAV and AAV-derived vectors are preferred for use in the present invention in the transformation of cells of the inner ear.
  • the viral vector is preferably a replication- deficient virus.
  • infective virus particles containing either DNA or RNA corresponding to the desired therapeutic gene product can be produced by introducing the viral construct into a recombinant cell line which provides the missing components essential for viral replication in trans.
  • transformation of the recombinant cell line with the recombinant viral vector will not result in production of replication-competent viruses (e.g., by homologous recombination of the viral sequences of the recombinant cell line into the introduced viral vector) .
  • the vector for transformation is composed of (in the case of a nonviral vector) or derived from (in the case of recombinant viral vectors) a DNA construct.
  • the DNA construct contains a promoter to facilitate expression of the DNA of interest within the inner ear cell.
  • the promoter is a strong, eukaryotic promoter.
  • Exemplary eukaryotic promoters include promoters from cytomegalovirus (CMV) , mouse mammary tumor virus (MMTV) , Rous sarcoma virus (RSV) , adenovirus, herpes simplex virus (HSV) (e.g., HSV thymidine kinase promoter), and SV40. More specifically, exemplary promoters include the Ad 2 major late promoter (Wong et al . 1986 J. Virol .
  • constructs suitable for use include a marker(s) (e.g., an antibiotic resistance gene (such as an ampicillin resistance gene) , 3-galactosidase or green fluorescent protein (GFP) ) to aid in selection of cells containing the construct, an origin of replication for stable replication of the construct in a bacterial cell (preferably, a high copy number origin of replication) , a nuclear localization signal, or other elements which facilitate production of the DNA construct, the protein encoded thereby, or both.
  • a marker(s) e.g., an antibiotic resistance gene (such as an ampicillin resistance gene) , 3-galactosidase or green fluorescent protein (GFP)
  • GFP green fluorescent protein
  • Such secretion signals can provide for delivery of the gene product of interest into an inner ear lumen (i.e., the cochlear duct) and/or into a fluid of the inner ear (e.g., the endolymph, perilymph, and or cortilymph).
  • the construct should contain at a minimum a eukaryotic promoter operably linked to the DNA of interest, which is in turn operably linked to a polyadenylation sequence.
  • the polyadenylation signal sequence may be selected from any of a variety of polyadenylation signal sequences known in the art.
  • the polyadenylation signal sequences are the polyadenylation signal sequences of the SV40 late and/or early genes.
  • the construct may also include one or more introns, which can increase levels of expression of the DNA of interest.
  • introns Any of a variety of introns known in the art may be used.
  • the human S-globin intron can be inserted in the construct at a position 5' to the DNA of interest to provide enhanced expression.
  • the DNA of interest can be inserted into a construct so that the therapeutic protein is expressed as a fusion protein.
  • the therapeutic protein can be a portion of a fusion protein having / S-galactosidase or a portion thereof at the N-terminus and the therapeutic protein at the C-terminal portion.
  • the therapeutic protein (or a portion thereof) can be fused to green fluorescent protein (or a portion thereof) . Methods for production of such fusion proteins are well known in the art (see, for example, Sambrook et al . Molecular Cloning: A Laboratory Manual , 2nd Ed., 1989, Cold Spring Harbor
  • Production of a fusion protein can facilitate monitoring of therapy, e.g., through detection of the fusion protein from a sample of perilymph fluid where the therapeutic protein is secreted into the perilymph of the cochlea.
  • therapeutic proteins that are, for example, protease resistant or have enhanced activity relative to the wild-type protein.
  • therapeutic protein is a hormone, it may be desirable to alter the protein's ability to form dimers or multimeric complexes.
  • the construct containing the DNA of interest can also be designed so as to provide for site-specific integration into the genome of the target inner ear cell. Methods and compositions for preparation of such site-specific constructs are described in, for example,
  • the DNA of interest can be any DNA encoding any gene product, e.g., protein, RNA molecule, or other gene product, for which therapy is desirable.
  • protein therapy i.e., through intracellular or secretory protein expression
  • Such protein-deficient states are amenable to treatment by replacement therapy, i.e., expression of a protein to restore at least normal protein expression levels.
  • the DNA of interest can encode proteins involved in the development of inner ear structures.
  • the DNA of interest can encode gene product (s) to facilitate the development of functional hair cells.
  • hearing loss is associated with dysfunctional hair cells or loss of hair cells
  • Such application of the methodology of the invention can be useful where hearing loss is due at least in part to stereocilia retraction and scarring of the tectorial plate associated with a region in the cochlea devoid of functional hair cells.
  • the subject may have or be susceptible to a condition that is amenable to treatment by expression or over-expression of a protein which is either normally present in a healthy subject or is foreign to the subject.
  • antibiotic or antiviral protein therapy may be desired for treatment of a mammalian subject having a viral, bacterial, fungal, and/or parasitic infection of the inner ear, particularly where the infection is chronic, i.e., persisting over a relatively long period of time.
  • Autoimmune disorders can be treated by expression of an anti-inflammatory agent, cytokine, anti-antibody or fragment thereof, or other gene product (s) that can be useful in treating such disorders.
  • bone formation in the cochlear following meningitis can be treated or prevented by expression of gene product that inhibit such bone formation processes.
  • Expression of nerve growth factor (NGF) may be desired for regeneration of the auditory nerve. Auditory nerve regeneration is desirable where, for example, hearing loss is associated with regression of neurites of the cochlear nerve from the spiral organ of Corti in a direction toward the cell body of the spiral ganglion (see Fig. 3) .
  • Expression of nerve growth factors can facilitate neurite growth toward the organ of Corti and the auditory nerve cells, or retard degeneration of auditory dendrites, to restore cochlear function, thereby improving hearing or stabilizing hearing loss .
  • Expression of the DNA of interest can result in production of a gene product that is either maintained within the transformed cell or secreted by the transformed cell to achieve the desired effect.
  • structural proteins will generally be maintained as intracellular components of the transformed cell, while gene products that achieve their desired effect through antibiotic activity are generally secreted into a space or fluid of the inner ear.
  • Gene products for which secretion may be desirable include gene products that affect inner ear fluid osmolarity, fluid viscosity, promote contractility, affect fluid pH, act as anti -inflammatory agents, and/or affect ion channels (e.g., sodium, potassium and/or calcium ion channels) .
  • ion channels e.g., sodium, potassium and/or calcium ion channels
  • the DNA of interest is preferably obtained from a source of the same species as the subject to be treated (e.g. human to human) , but this is not an absolute requirement .
  • DNA obtained from a species different from the subject can also be used, particularly where the amino acid sequences of the proteins are highly conserved and the xenogeneic protein is not highly immunogenic so as to elicit a significant, undesirable antibody response against the protein in the host.
  • Exemplary, preferred DNAs of interest include DNA encoding nerve growth factors, (e.g., NGF, BDNF, NT-3, NT4/5, p70) structural proteins (e.g., actin, myosin, dystrophin, dystrophin-related protein (DRP) , MERLIN, and neurofibronin) , electrolyte channels (e.g., for the potassium, calcium and sodium ions), mechanotransduction proteins water channels, and transcription factors (e.g., homeobox and paxeal transcription factors) .
  • the subject is a human subject and the DNA expressed encodes a human protein (e.g., human nerve growth factor) .
  • Table 1 provides a list of exemplary proteins and protein classes which can be delivered by the inner ear gene therapy of the invention. Table 1. Exemplary Gene Products, Proteins and Protein Classes for Delivery by Gene Therapy to the Inner Ear
  • Various disease conditions are amenable to treatment using the inner ear gene therapy of the invention.
  • One skilled in the art can recognize the appropriate gene product (e.g., polypeptide or RNA) that should be produced for the treatment of specific disease conditions.
  • a list of diseases and conditions associated with hearing loss (sensorineural or mixed) that are amenable to treatment according to the method of the invention are provided in Table 2. The genetics of hereditary hearing impairment is reviewed in Mhatre and Lalwani , 1996 , "Molecular Genetics of
  • ototoxic pharmaceuticals e.g., through the expression of nerve growth factors (e.g., NGF, NT-3, BDNF, NT 4/5, and p70) ) and to treat or prevent other conditions associated with auditory nerve degeneration or damage.
  • nerve growth factors e.g., NGF, NT-3, BDNF, NT 4/5, and p70
  • DNA encoding a protein of interest has not been isolated, this can be accomplished by various, standard protocols well known to those of skill in the art (see, for example, Sambrook et al . , ibid; Suggs et al . , Proc . Natl . Acad. Sci . USA 28 66 -6617, 1981; USPN 4,394,443; each of which are incorporated herein by reference with respect to identification and isolation of DNA encoding a protein of interest) .
  • genomic or cDNA clones encoding a specific protein can be isolated from genomic or cDNA libraries using hybridization probes designed on the basis of the nucleotide or amino acid sequences for the desired gene.
  • the probes can be constructed by chemical synthesis or by polymerase chain reaction (PCR) using primers based upon sequence data to amplify DNA fragments from pools or libraries (USPNs 4,683,195 and 4,683,202). Nucleotide substitutions, deletions, additions, and the like can also be incorporated into the polynucleotides, so long as the ability of the polynucleotide to hybridize is not substantially disrupted. (Sambrook et al . ibid) . The clones may be expressed or the DNA of interest can be excised or synthesized for use in other constructs. If desired, the DNA of interest can be sequenced using methods well known in the art .
  • transformation is accomplished by introducing the nucleotide sequence of interest (e.g., the DNA of interest contained in a construct that is formulated in a chemical vector solution or as a viral vector) directly into the inner ear, and may be inserted into or near the desired inner ear structure or cell (e.g., directly into the cochlear or vestibular structure) .
  • the DNA of interest can be accomplished by any means, generally by injection or infusion.
  • the DNA of interest is delivered to the inner ear in vivo by infusion, preferably using an osmotic minipump.
  • Implantation of an osmotic minipump and its use in the delivery of pharmacologic agents to the brain, kidney, and guinea pig cochlea have been described (Schindler et al . , 1977 Arch . Otolaryngol , 103 :691- 699; Nau, 1985 Toxical . Appl . Pharmacol . , 80:243-250;
  • introduction of the DNA of interest into the inner ear can be accomplished delivery through the round or oval window of the inner ear.
  • the cells to be transformed are cells of the cochlea or cochlear structures, it may be preferable to introduce the DNA of interest into the inner ear through the round window.
  • the cells to be transformed are vestibular cells, or where the subject is undergoing stapes- related surgery, it may be preferable to introduce the DNA into the inner ear via the oval window.
  • cochlear cells can be transformed in vi tro, and the transformed cochlear cells subsequently implanted into the subject's cochlea. Isolation, maintenance, and implantation of cochlear cells are well known in the art.
  • the form of the preparation for transformation of the inner ear cells will depend upon several factors such as the cochlear cell targeted for gene transfer and whether a biological or nonbiological vector is employed.
  • the vector solution can also contain therapeutic compounds (e.g, nerve growth factors, anti-inflammatory agents, antibiotic agents) in addition to the DNA of interest, as well as compounds to adjust, for example, the pH, osmolarity, and/or viscosity of the vector solution.
  • the preparation can additionally contain compounds that facilitate entry of the nucleic acid of interest into the inner ear cells such as lipofectin, permeability-enhancing agents (e.g., detergents), or other transformation-enhancing agents.
  • the vector is a viral vector
  • the preparation can also include a co-infecting virus to facilitate infection and transformation.
  • the DNA of interest is administered in a recombinant viral vector, e.g., an AAV vector
  • the vector solution is preferably normal saline .
  • the amount of DNA and/or number of viral particles administered will vary greatly according to a number of factors including the susceptibility of the inner ear cells to transformation, subject-dependent variables such as age, weight, sensitivity or responsiveness to therapy, susceptibility of targeted inner ear cells to transformation, the levels of protein expression desired, and the condition to be treated.
  • the total delivered viral dosage can be in the range of 1 virus per 5 cochlear cells, preferably 1 virus per 10 cochlear cells, more preferably 1 virus per 20 cochlear cells or less.
  • the amounts of DNA for transformation of human inner ear cells can be extrapolated from the amounts of DNA effective for gene therapy in an animal model .
  • successful transformation of the inner ear cells of a guinea pig was accomplished using 10 5 particles of AAV containing the DNA of interest at an moi (multiplicity of infection) of approximately 1:20 (1 viral particle per 20 cochlear cells) .
  • the amount of DNA and/or viral particles necessary to accomplish transformation of inner ear cells will decrease with an increase in the efficiency of the transformation method used.
  • the amount of DNA and/or the number of infectious viral particles is an amount effective to infect the targeted inner ear cells or structure, transform a sufficient number of inner ear cells, and provide for expression of desired or therapeutic levels of the protein or other gene product.
  • transformation is transient (e.g., the DNA of interest is maintained for some period as an extrachromosomal element)
  • the time period over which expression is desired may also be taken into consideration.
  • the desired number of copies (e.g., copy number) of the DNA of interest in the cell may additionally be taken into account in determining the amount of DNA and/or number of viral particles to be delivered to the subject, and such may be adjusted as desired to, for example, achieve varying levels of gene product expression. Transformation can be accomplished such that expression of the gene product of interest is either transient, inducible, or stable. For example, where the DNA of interest is present in the transformed cell as an extrachromosomal element (e.g., as with AAV vectors), expression of the gene product is generally transient.
  • Inducible expression can be achieved so that expression of the gene product of interest is induced only in the presence of some signal that is, for example, specific to a certain type of cell of the inner ear (e.g., is only expressed in cochlear cells or a specific type of cochlear cell due to the presence of a cell-specific or tissue-specific transcription factor in the transformed cell) .
  • gene product can be inducible by the presence of an extracellular factor that can be introduced at the same time as the transforming vector solution is introduced into the inner ear, and/or subsequent to inner ear cell transformation.
  • Stable expression of the gene product can be achieved by, for example, introduction of the DNA of interest in a vector to provide for stable genomic integration into the inner ear cell and expression of the gene product from the DNA of interest via a constitutive promoter.
  • expression of the gene product of interest is transient
  • expression can be maintained in the inner ear cell for a period ranging from several days to several months or years, e.g., for 6 months to 1 year, for 4 months to 6 months, for 2 weeks to 8 weeks, or for as little as one week or a few days (e.g., 3 to 5 days, or 1 to 3 days) .
  • Transient expression of the gene product of interest may be desirable where the subject is being exposed to the therapeutic regimen for the first time (e.g., where it is desirable to monitor the responsiveness and/or sensitivity of the subject) , or where expression is desired only over a specific period (e.g., for a period after cochlear implantation without permanent expression, or for a period during a specific stage of development) .
  • the period of transient expression can be adjusted by , for example, adjusting the transformation protocol to achieve a desired number of transformed cells or, where a viral vector is used, by adjusting aspects of the vector associated with maintenance in a cell (e.g., replication functions or other functions associated with vector stability and/or copy number) .
  • DNA and/or number of infectious viral particles required can be readily determined based upon such factors as the levels of protein expression achieved in cell lines in vi tro, and the susceptibility of the inner ear cells to transformation.
  • Any patient having a sensorineural or mixed hearing loss and/or vestibular dysfunction can be treated according to the method of the invention.
  • the type of hearing loss suffered by the candidate individuals can be determined using various methods well known in the art such as pure tone air- and bone-conduction audiometry, impedance (or immittance) batteries, and speech audiometry.
  • stimuli are presented to the ear through air conduction (i.e., by earphones) or through bone conduction, in which a vibrator is placed on portions of the skull.
  • a pure tone air- conduction test measures hearing impairment in the outer, middle, and inner ear, whereas bone conduction conducts sound directly to the inner ear.
  • Those individuals having a hearing loss associated with the inner ear are amenable to the method of treatment according to the invention.
  • a pure tone audiometer can be used to determining the degree and type of hearing loss and produces an audiogram that measures hearing sensitivity as a function of frequency.
  • Simple and reliable screening can be accomplished using a hand-held Welch Allyn audioscope, an otoscope with a special attachment that delivers a 40 dB tone at frequencies form 500 to 4,000 Hz. When compared with pure tone audiometry, this simple test has a 91 to 97 percent sensitivity and 69 to 85 percent specificity. The range from -10 dB to 25 dB on the audiogram is considered within normal limits.
  • a greater than 25 dB loss constitutes a hearing impairment.
  • a 25 dB loss is a mild impairment
  • a 40 dB loss is a moderate impairment
  • a 70 dB loss is a severe impairment.
  • Impedance (or immittance) measurements evaluate the integrity and performance of the peripheral auditory system.
  • the basic battery of immittance measurements include static compliance, measurement of the acoustic reflex, and tympanometry, the most sensitive indicator of middle-ear function available. Impedance evaluation is useful in distinguishing ossicular-chain discontinuity from otosclerosis .
  • Speech audiometry measure overall hearing performance for functional speech stimuli.
  • Two aspects of auditory performance are assessed: speech reaction threshold (SRT) and speech discrimination.
  • SRT is the level at which the patient recognizes two-syllable spondaic words, which have equal stress on both syllables, 50 percent of the time.
  • Speech discrimination measures the subject's ability to understand speech presented at 30 to 40 dB above the patient's SRT. For example, subjects with presbycusis lose the ability to differentiate consonants, in part because these sounds are usually of a higher frequency. Thus, speech discrimination deteriorates.
  • the patient is scored according to the percentage of speech understood (e.g., 50 percent speech discrimination indicates that the patient understands 50 percent of speech heard) . If difficulty in speech discrimination is suspected, audiological testing is recommended. Questioning the patient about he ability to understand speech may aid in identifying discrimination difficulty.
  • Tinnitus is a form of hearing impairment that occurs in 10 to 37 percent of the elderly population and can result from any disease or injury that affects the auditory system. All ototoxic effects are signaled by tinnitus, but this condition may also result from impairment of any part of the auditory pathway and have a vascular, muscular, or hormonal origin.
  • the method of the invention can be used to treat sensorineural and mixed hearing losses associated with exposure to ototoxic agents, genetic defects associated with defects in the structures and components of the inner ear
  • cochlear structures e.g., auditory hair cells, spiral ganglion, scala vestibuli, Hansen's cell, spiral organ of Corti, stria vascularis, and scala tympani
  • endolymph perilymph
  • semicircular ducts ampullae, utricle, and saccule
  • vestibular disorders inheritable hearing loss
  • infectious disease e.g, mumps
  • cancerous growths exposure to noise levels of greater than 85 to 90 dBA (decibels to the a scale, which approximates response of the human ear) for more than 8 hr intervals, exposure to hazardous materials associated with decrements of audiovisual performance, and/or hearing losses and vestibular disorders associated with age.
  • individuals who are at risk of developing a hearing loss may be treated according to the method of the invention prior to the actual onset of hearing loss.
  • the method of the invention can be used as a preventive or prophylactic measure to avoid, prevent, or reduce sensorineural and/or mixed hearing loss in individuals who are or will be receiving therapy with ototoxic agents, have a genetic defect associated with hearing loss or the development of hearing loss, have a family history of heritable hearing loss, have or are susceptible to a infectious disease (e.g., mumps) or tumorous growth associated with hearing loss, are exposed to or have been exposed to noise level of greater than 85 to 90 dBA (decibels to the a scale, which approximates response of the human ear) for more than 8 hr intervals, are exposed to hazardous materials associated with decrements of audiovisual performance; and/or exhibit early signs of hearing loss associated with age.
  • infectious disease e.g., mumps
  • noise level e.g., dBA (decibels to the a scale, which approximate
  • the methodology of the invention can also be used in conjunction with cochlear implantation.
  • Methods for cochlear implantation are known in the art (see, for example, Schindler et al . 1977 Arch . Otolaryngol . 103 :691-699; Schindler et al . 1993 A . J. O . JL4:263-272; Souliere et al . 1994 Otolaryngol . Clin . North . Am . 2 ⁇ 7:533-536; Parkin et al . 1994 E. N. T. J.
  • an osmotic minipump can be put in place at the time of cochlear implantation to provide for infusion of nerve growth factor and/or infusion of a transforming vector that encodes a nerve growth factor (e.g., NGF, NT-3, BDNF, NT4/5, p70, etc.) to facilitate the success of the cochlear implant through induction of nerve growth toward the electrode .
  • a nerve growth factor e.g., NGF, NT-3, BDNF, NT4/5, p70, etc.
  • the methodology of the invention can be used in conjunction with conventional methods of treatment of hearing loss .
  • the methods and compositions of the invention can be used to provide transgenic animal models useful as models of human hearing, development of inner ear structures (e.g., cochlear and/or vestibular development), and/or hearing conditions or disorders.
  • the methods of the invention can be used to transform inner ear cells of a nonhuman subject, e.g., a mouse, primate, chick, chick embryo, guinea pig, frog, bullfrog, or other animal useful as a model of hearing.
  • the methods of the invention can be applied to transform the inner ear cells of animals presently accepted as models for hearing.
  • Such conventional animal models for hearing are described in, for example, Davies et al . 1994 Am. J.
  • the ability of the transformed inner ear cells to express the DNA of interest can be assessed by various methods known in the art.
  • therapy can be assessed using the conventional basic audiological evaluation composed of pure tone air- and bone-conduction audiometry, the impedance battery, and speech audiometry as described above to identify subjects amenable to treatment using the method of the invention.
  • Prevention of hearing loss, or stabilization or an improvement in auditory acuity after treatment is indicative of a subject's positive responsiveness to therapy. Auditory acuity can be measured by use of an instrument called an audiometer, which consists of an earphone connected to an electronic oscillator capable of generating pure tones ranging from low to high frequencies.
  • Positive responsiveness to therapy can also be associated with, for example, prevention of osteoneogenesis (e.g., following meningitis), amelioration of vestibulopathy, tinnitus, and/or enhanced rehabilitation with cochlear implantation.
  • therapeutic gene product or other gene product of interest is secreted into the endolymph or perilymph of the inner ear
  • successful transformation of inner ear cells and expression of the DNA of interest can also be assessed by, for example, assaying for the presence of the gene product in the endocochlear fluid.
  • a sample of fluid can be obtained from the inner ear, and expression of a protein of interest detected by performing an ELISA on the sample using an antibody which specifically binds the protein encoded by the DNA of interest.
  • the ELISA can be performed either qualitatively or quantitatively.
  • the ELISA assay, as well as other immunological assays for detecting a protein in a sample, are described in Antibodies: a Laboratory Manual (1988, Harlow and Lane, ed.s Cold Spring Harbor Laboratory, Cold Spring Harbor, NY) .
  • the efficacy of the protein therapy can be assessed by testing a sample of fluid from the inner ear for an activity associated with the therapeutic protein (e.g., an enzymatic activity).
  • an activity associated with the therapeutic protein e.g., an enzymatic activity
  • the efficacy of therapy can be tested by examining the ability of the test sample to inhibit bacterial growth.
  • the efficacy of inner ear gene therapy can be assessed by monitoring the condition of the subject for improvement .
  • Constructs for production of recombinant AAV vectors were prepared according to methods well known in the art (see, e.g., Sambrook et al , 1989, Molecular Cloning: A Laboratory Manual, 2nd Ed.). Briefly, the commercially available plasmid pUC19 (Yanish-Perron et al . , 1985, Gene 11:103-19) was used as a "backbone" for construction. An x ⁇ mer NotI linker (New England Biolabs cat #1029) was cloned into the Smal site of pUC19. This destroys the Smal site, but generates in the process two SacII sites, one immediately on each side of the NotI linker. This vector was designated pN (Notl) .
  • a poly adenylylation signal a 196bp fragment from the SV40 genome (nucleotides 2533-2729) , as purified from an SV40 containing vector and, following the addition of BamHI linkers, was cloned into the unique BamHI site within pN.
  • the fragment was oriented such that RNA polymerase transcribing from a promoter upstream of the NotI site passes through the NotI site and into the SV40 fragment, encountering the SV40 late gene polyadenylyation signal (the early gene polyadenylation signals appear in the opposite orientation.
  • This vector was designated pNA (Adenylation signal) .
  • Two promoters containing DNA fragments were cloned into pNA.
  • a 180 bp region of the SV40 genome containing late viral protein gene 16S/19S splice donor and acceptor signals (obtained as a Xhol - PstI fragment from pLI (Okayama and Berg, 1983, Mol . Cell Biol . 1:280-9) was cloned into pNA to provide the appropriate RNA splicing signals as needed.
  • This vector was designated pNAss (splice site) .
  • the promoter-containing DNA fragments were cloned into pNA or pNAss . These fragments and vectors were all treated with T4 DNA polymerase prior to blunt end ligation.
  • the herpes simplex thymidine kinase gene promoter was obtained from pXII (Boulter and Wagner, 1987, Nucleic Acids Res . ljj: 7194) as a 925bp Sal - Xhol fragment.
  • the human cytomegalovirus immediate early gene promoter and enhancer was obtained as a 619bp Tha I fragment from pCM5029 (Boshart et al., 1985, Cell ⁇ :521-30).
  • the adenovirus 2 major late promoter with a fused tri -partite leader containing a 5' splice donor signal with a 3' splice acceptor signal derived from an IgG gene was obtained as a 695bp Xhol - EcoRI fragment from p91023 (B) (Wong et al . 1986 J. Virol . £0.(1) :149-56) .
  • These vectors were designated pNAss TK (Thymidine kinase promoter) , pNAssCMV (CMV promoter) , pNASV (SV40 promoter) and pNAAd (adenovirus promoter) .
  • the E-coli / .-gal gene from pC4AUG «3-gal was excised as a 3530 bp EcoRI - Xbal fragment and following the addition of NotI linkers, cloned into the unique NotI site of each of the four expression vectors. These constructions were designated pSV ⁇ , pCMV3, pAd/3 and pTK3. Restriction endonuclease sites in parenthesis refer to sites lost during cloning.
  • An AAV vector for expression of the marker ⁇ - galactosidase (pTR-MAP / 3) was constructed using procedures similar to those described above except the Ad 2 major late promoter was fused to the late gene 16s/19s splice donor/splice acceptor signal of SV40 (SD/SA SV40) .
  • the final plasmid construct contained E. coli ⁇ -galactosidase ( / 3-gal) gene, driven by a late promoter of Adenovirus 2, flanked at both sides with wild type AAV terminal repeats (Fig. 6) .
  • the construct additionally contains polyadenylation signals of the SV40 late (pAR) and early (pAe) genes.
  • a recombinant AAV vector for expression of neurotrophin-3 (NT-3) (Fig. 7) and a recombinant AAV vector for expression of brain-derived neurotrophin factor (BDNF) (Fig. 8) were also constructed using similar techniques and substituting the nucleotide sequence encoding NT- 3 and BDNF for the nucleotide sequence encoding /3-gal.
  • a recombinant AAV vector encoding nerve growth factor (NGF) can be similarly constructed (Fig. 9) .
  • constructs expressing / 3-gal (Fig. 10) or green fluorescent protein (GFP) (Fig. 11) from the CMV immediate/early gene promoter were constructed according to methods well known in the art.
  • Recombinant AAV for use in transformation of the cochlea was produced by co-transfecting 293 cells (human embryonic kidney cells, transformed with E1A-E1B region of Ad) with pTR-MAP3 (see Example 1) and a helper plasmid pIM29 (McCarty 92) carrying wild type AAV genome without terminal repeats. The same cells were also infected with Ad5 at a multiplicity of infection (moi) of 10. Recombinant AAV was harvested after 60 hr by freeze/thawing cells three times, centrifuging the lysed cells to remove cell debris, and heat inactivating adenovirus helper by incubating the lysate for 1 hr at 56°C.
  • the AAV titer was determined in an infectious center assay according to methods known in the art. Briefly, cells in a well of 96-well dish, plated at 75% of confluence, were in-infected with varying dilutions of recombinant AAV, wild type AAV (moi 2) , and Ad (moi 20) . Thirty-six hours after infection, the cells were resuspended in media, harvested in phosphate-buffered saline (PBS) , transferred onto a nylon filter 47 mm in diameter, and placed in to a microanalysis funnel with Frit support. The cells were then lysed in si tu following a colony lift protocol (Sambrook et al .
  • Example 3 Transformation of the Inner Ear in a Guinea Pig Model
  • Hartley guinea pigs wee used due to the relatively large size of their cochleae compared to the cochleae of mice and rats, and the ease of the surgical manipulation in this species. Guinea pig care was in accordance with the guidelines of the Committee on Animal Care, University of California, San Francisco.
  • Alzet osmotic minipumps (model 1007, Alza Corporation, Palo Alto, California) were prepared by connecting PESO polyethylene tubing (Intramedic, Becton Dickinson and Company, Parsippany, New Jersey) to the flow moderator on the pump as previously described (Davies et al . , 1994 Am. J " . Otology, 15 . : 757-761) .
  • the pump was filled with 100 ⁇ l PBS containing recombinant adeno-associated virus pTR- ML / 3 (10 6 viral particles/ml) which contained the bacterial ⁇ - gal sequence .
  • the catheter of the pump was then introduced into the basal turn of the cochlea into the perilymphatic space (scala tympani) via the cochleostomy (Fig. 7) .
  • the body of the pump was inserted in a subcutaneous pocket and the skin incision closed in layers.
  • AAV transfused cochleae were assayed for expression of the marker gene, 3-galactosidase, via in situ immunohistochemistry .
  • Two weeks post-cochleostomy animals were sacrificed by intraperitoneal injection of an overdose of sodium phenobarbital (250 mg/kg) and bilateral thoracotomy. Temporal bones were harvested from both sides of the head. Each bulla was opened using rongeurs to expose the cochlea, the stapes was removed, and the cochlea fixed by perfusion of 4.5% paraformaldehyde through the round window. The cochlea was then removed from the remaining temporal bone and immersed in 4.5% paraformaldehyde overnight at 4°C.
  • specimens were decalcified in 0.2 M EDTA/1X PBS/4.5% paraformaldehyde for 2-3 weeks with at least three solution changes. Following decalcification, the specimens were placed in 0.9% saline, dehydrated through a graded alcohol series and then xylenated. Specimens were embedded in paraffin and sectioned at 4-8 ⁇ m on a microtome (Leica RM2035) .
  • the paraffin-embedded cochlear sections were dewaxed, blocked with 10% NHS, 0.1% Tween 20 in PBS, and hybridized overnight with mouse anti-E. coli / 3-gal antibody.
  • the sections were washed to remove unbound antibody and then hybridized with a secondary antibody (biotin labeled anti- mouse IgG monoclonal antibody. Bound label was amplified using the ABC reagent (Vector) and developed with DAB. Sections were examined under low and high power magnification and the presence or absence of staining in different parts of the cochlea noted.
  • the jS-gal expression was the strongest in the basal turn of the cochlea and decreased in the subsequent turns toward the cochlear apex.
  • This tissue and regional selectivity within the cochlea may be consequence of differential susceptibility of inner ear cells/tissue to AAV transfection, and/or the viral titre gradient generated by the mode of vector delivery to the cochlea and its subsequent dissemination in the perilymphatic and endolymphatic space.
  • recombinant AAV viral vector particles were prepared using the / 3-gal-expressing construct shown in Fig. 10. Approximately 10 5 particles of AAV containing the CMV-driven / 3-gal expression construct were infused into the cochlea of guinea pigs with the aid of a an osmotic minipump as described above in Example 3. Animals were sacrificed after 1, 2, 4, 8, 12, and 24 weeks. Four Hartley guinea pigs infused with saline and four nonimplanted animals served as negative control. The infused cochleae was harvested from each animal, processed as paraffin sections, and assayed for /3-gal expression using immunohistochemistry .
  • Recombinant AAV viral vector particles were prepared using the GFP-expressing construct shown in Fig. 10. Approximately 10 5 particles of AAV containing the CMV-driven GFP expression construct were infused into the cochlea of guinea pigs over two days to one week with the aid of a an osmotic minipump as described above in Example 3. Animals infused with saline and noninfused animals were used as negative controls. Cochleae were fixed, decalcified, and embedded in paraffin. Sections of 8 ⁇ m were cut dewaxed, coverslipped, and viewed under fluorescence. Animals infused with AAV containing GFP showed intense fluorescent activity in spiral ganglia, Reissner's membrane, spiral ligament, and organ of Corti as compared to saline and noninfused animals.
  • Recombinant viral vectors and recombinant AAV particles are prepared as described above such that the viral vector expresses a nucleotide sequence encoding a therapeutic gene product of interest.
  • An osmotic minipump is filled with approximately 100 ⁇ l to 1000 ⁇ l PBS containing the recombinant adeno-associated virus.
  • Placement of the minipump in the patient can be achieved by a number of different incisions. After the incision is made, the periosteum is elevated off the mastoid.
  • the temporalis muscle In adults, the temporalis muscle is left in si tu at this stage, whereas in children it is elevated with the flap. After performing an intact canal wall mastoidectomy, the facial recess is opened. A cochleostomy is then created by drilling anteriorly from the round window into the basal turn of the cochlea. The catheter of the osmotic minipump is introduced into the basal turn of the cochlea into the perilymphatic space (scala tympani) via the cochleostomy.
  • the osmotic minipump is filled with a transforming vector solution composed of recombinant AAV viral particles encoding a nucleotide sequence of interest (e.g., a nerve growth factor (e.g., NGF (Fig. 9), NT-3 (Fig. 7), BDNF (Fig. 8) suspended in saline.
  • a nerve growth factor e.g., NGF (Fig. 9), NT-3 (Fig. 7), BDNF (Fig. 8) suspended in saline.
  • the solution in the mini-pump can additionally contain other agents desirable for introduction into the inner ear (e.g., antiinflammatory agents).
  • the body of the pump is inserted in a subcutaneous pocket, the round window and facial recess packed with gelfoam pledgets, and the skin incisions closed with sutures.
  • the osmotic minipump is left in place for several days to several weeks to allow infusion of the recombinant viral particles into the inner ear and transformation of inner
  • the method of the invention can be performed in conjunction with cochlear implantation.
  • Cochlear implantation can be achieved by a number of different incisions for the placement of the cochlear implant device. It is important to design a flap that is both well-vascularized and large enough to accommodate the receiver. In addition, it is desirable that both the device and its lead wires be located well away from the skin incision. After the incisions is made, the periosteum is elevated off the mastoid. In adults, the temporalis muscle is left in si tu at this stage, whereas in children it is elevated with the flap. A dummy device is used as a template to mark the location for the receiver, and a well is drilled into the calvarium to seat the receiver. A trough is drilled to accommodate the lead wire in its route to the mastoid cavity. Drill holes are placed on either side of the well to serve as anchors for retention sutures.
  • a cochleostomy is the created by drilling anteriorly from the round window into the basal turn of the cochlea.
  • the Clarion device Minimed, Inc., Sylmar, CA
  • an electrode inserter is utilized to facilitate placement of the active lead of the receiver into the scala tympani. This tool straightens the coiled electrode to ease its insertion.
  • the multichannel electrode is gently eased into the scala tympani .
  • the lead wire is secured by a suture through the mastoid cortex and then coiled in the mastoid cavity.
  • the catheter of the osmotic minipump is also introduced into the basal turn of the cochlea into the perilymphatic space (scala tympani) via the cochleostomy.
  • the osmotic minipump is filled with a transforming vector solution composed of recombinant AAV viral particles encoding a nucleotide sequence of interest (e.g., a nerve growth factor (e.g., NGF (Fig. 9), NT-3 (Fig. 7), BDNF (Fig. 8) suspended in saline.
  • the solution in the minipump can additionally containing other agents desirable for introduction into the inner ear (e.g., antiinflammatory agents).
  • the body of the pump is inserted in a subcutaneous pocket, the round window and facial recess packed with gelfoam pledgets, and the skin incisions closed with sutures.
  • the osmotic minipump is left in place for as long as desired (e.g., for several days to several weeks to several years) to allow infusion of the recombinant viral particles into the inner ear and transformation of inner ear cells.
  • Example 9 Transformation of Rat Cochlear Cells In Vi tro Recombinant viral vectors and recombinant AAV particles containing «3-gal-encoding DNA were prepared as described above using the CMV-driven /3-gal expression construct (Fig. 10). Dissection of cochlear explants and spiral ligament/stria vascularis explant followed the methods of Zheng et al . (1995, supra; 1996, supra) . The cochleae were dissected from postnatal day (PN) 3 Wistar rats. After spiral ligament and stria vascularis tissues were removed and kept, the remaining cochlear explant containing the spiral ganglion and organ of Corti was cut into three pieces consisting of basal, middle, and apical turns.
  • PN postnatal day
  • the culture media was changed every two days. Uninfected cochlear explants (mock infected) served as a control . After 72 hours of incubation, the cochlear explants were assayed for ⁇ -gal expression by histochemical detection using the methods described by Sanes et al . (1986 EMBO J. 5:3133-3142) with modifications. Briefly, the explants were fixed for 15 min at 4°C in 2% formaldehyde and 0.2% glutaraldehyde in PBS, and then washed three times with PBS +
  • ImM MgCl 2 - The explants were immersed in histochemical mixture of 1 mg/ml X-gal, 5 mM potassium ferricyanide, 5 mM potassium ferrocyanide, and 2 mM MgCl 2 , 0.02% NP-40, and 0.01% sodium deoxycholate in PBS. Incubation was done at 37°C for 6 to 10 hrs.
  • FIG. 13 bottom left panel shows an exemplary transformed organotypic rat cochlear explant after infection with AAV- ⁇ -gal and X-gal staining. Several transformed cells expressing ⁇ -gal are observable within the cochlear structure; a slight endogenous ⁇ -gal activity is detectable in the vicinity of the outer hair cells. At higher magnification (Fig. 13, right panel) shows a high magnification of the cochlear explant in the region of outer hair cells and Hansen's cells. Numerous Hansen's cells expressing ⁇ -gal following AAV- ⁇ -gal infection are noted.
  • SGN spiral ganglion neurons
  • SGN cell cultures were prepared according to methods well known in the art. Briefly, after dissection of the cochlea and removal of spiral ligament and stria vascularis tissues, the remaining spiral ganglion cells were incubated in a mixture of 0.125% trypsin and 0.125% collagenase for 25-30 min at 37°C. The enzyme was inactivated with a mixture of 0.005% soybean trypsin inhibitor and 0.005% DNASE before trituration with 0.05% DNase in BME.
  • the dissociated cells were counted on hemocytometer and plated at a density of -80,000/well on polylysine (100 ⁇ g/ml)/ laminin (10 ⁇ g/ml) coated 16-well Lab-Tek slides with 200 ⁇ l of serum free media as described above. Infection of dissociated SGN was accomplished by introducing approximately 10 6 AAV- -gal viral particles into the media. The infection was allowed to continue for 6 hrs at which time media was replaced with fresh media .
  • top left panel shows a culture of dissociated rat cochlear cells infected with AAV- ⁇ -gal. As shown, a substantial number of cochlear cells in the test samples were successfully transformed with the ⁇ -gal-encoding construct and expressed ⁇ -gal at readily detectable levels. Uninfected dissociated cochlear cultures demonstrated no blue cells.
  • Example 10 Prevention of Amikacin- Induced Auditory Nerve Dama ⁇ e in BDNF-Transgenic Cochlea
  • Recombinant viral vectors and recombinant AAV particles containing brain-derived neurotrophin factor- encoding DNA were prepared as described above.
  • Cochlear explants were prepared as described above and infected with approximately 10 6 particles/ml of AAV containing the BDNF expression construct according to the protocols described above .
  • Uninfected cochlear explants (mock infected) served as a control. The cochlear explants were then maintained in organotypic culture as described above.
  • the ototoxic drug amikacin was introduced into the culture media of the control cochlear (mock- infected) and AAV-BDNF transfected cochlear explants to a concnetration of 4.5 mM. After incubation for 2 days, the cochleae's neurofilaments were examined via in si tu fluorescent immunohistochemistry using AN anti-neurofilament antibody. The samples were first fixed in 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) for 30 min.
  • the fixed cultures were then washed with PBS, and then incubated with the primary anti-neurofilament antibody (N52, Boehringer) in 1% Triton X-100 containing 3% normal goat serum at 4°C for approximately 48 hrs. After washing in PBS, the samples were then incubated at room temperature for 1 hr with species- specific secondary antibody conjugated to Texas Red. The samples were then washed with PBS, mounted in Fluoromount-G (Southern Biotech. Associates) and visualized under fluorescent microscope. As shown in Fig. 14, top left panel, numerous radial neurite projections are observed in the control cochlea (mock infected, no amikacin) .

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Abstract

La présente invention concerne des compositions et des procédés permettant de transformer les cellules de l'oreille interne et, ce faisant, de traiter les affections de l'oreille interne. Plus particulièrement, on modifie génétiquement les cellules de l'oreille interne d'un patient afin d'incorporer de manière fonctionnelle une séquence nucléotidique exprimant un produit génique désiré (c'est-à-dire un produit génique thérapeutique). La cellule de l'oreille interne dans laquelle l'ADN désiré est introduit et exprimé, est de préférence une cellule de la cochlée, et de idéalement une cellule du ligament spiral, du limbe spiral, de la strie vasculaire, de l'organe de Corti, du ganglion spiral, et/ou de la membrane de Reissner, et/ou une cellule de cil auditif. On introduit l'ADN désiré, se trouvant de préférence à l'intérieur d'un vecteur viral adéno-associé, au travers d'une canule insérée dans la fenêtre ronde ou dans la fenêtre ovale et qui communique avec la périlymphe ou l'endolymphe. L'ADN désiré est de préférence introduit au moyen d'une minipompe osmotique.
PCT/US1997/011602 1996-06-28 1997-06-27 Transformation et therapie genique des cellules de l'oreille interne WO1998000014A1 (fr)

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WO1999061066A2 (fr) * 1998-05-27 1999-12-02 Avigen, Inc. Apport ameliore par la convection de vecteurs viraux adeno-associes (aav)
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EP1621625A2 (fr) * 1998-05-27 2006-02-01 Avigen Incorporated Vecteurs AAV pour la fabrication des médicaments pour l'administration amélioré par la convection
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
US7166433B2 (en) 2004-03-03 2007-01-23 The United States Of America As Represented By The Department Of Health And Human Services Transductin-1 and transductin-2 and applications to hereditary deafness
US7192705B2 (en) 2001-09-19 2007-03-20 United States Of America, Represented By The Secretary, Department Of Health And Human Services Transductin-1 and applications to hereditary deafness
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US9265933B2 (en) 2005-09-08 2016-02-23 Massachusetts Eye And Ear Infirmary Cochlear implants containing biological cells and uses thereof
US9951351B2 (en) 2014-10-09 2018-04-24 Genvec, Inc. Adenoviral vector encoding human atonal homolog-1 (HATH1)
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WO2022178419A1 (fr) * 2021-02-22 2022-08-25 Decibel Therapeutics, Inc. Méthodes et compositions pour réduire la toxicité induite par un vecteur d'acide nucléique dans l'oreille interne
CN116035800A (zh) * 2023-04-03 2023-05-02 首都医科大学附属北京友谊医院 一种皮下植入式经半规管内耳重复给药装置

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WO1999042088A2 (fr) * 1998-02-23 1999-08-26 Otogene Aktiengesellschaft Procede pour le traitement de maladies ou de troubles de l'oreille interne
WO1999042088A3 (fr) * 1998-02-23 2000-03-02 Otogene Ag Procede pour le traitement de maladies ou de troubles de l'oreille interne
EP1621626A1 (fr) * 1998-05-27 2006-02-01 Avigen, Inc. Vecteurs AAV pour la fabrication des médicaments pour l'administration amélioré par la convection
WO1999061066A2 (fr) * 1998-05-27 1999-12-02 Avigen, Inc. Apport ameliore par la convection de vecteurs viraux adeno-associes (aav)
US7534613B2 (en) 1998-05-27 2009-05-19 Genzyme Corporation Methods of treating parkinson's disease using viral vectors
US6953575B2 (en) 1998-05-27 2005-10-11 Avigen, Inc. Methods of treating central nervous system disorders using viral vectors
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US8309355B2 (en) 1998-05-27 2012-11-13 Genzyme Corporation Methods of treating Parkinson's disease using viral vectors
EP1621625A3 (fr) * 1998-05-27 2006-05-10 Avigen Incorporated Vecteurs AAV pour la fabrication des médicaments pour l'administration amélioré par la convection
US6309634B1 (en) 1998-05-27 2001-10-30 Avigen, Inc. Methods of treating Parkinson's disease using recombinant adeno-associated vector (rAAV)
WO1999061066A3 (fr) * 1998-05-27 2000-05-04 Avigen Inc Apport ameliore par la convection de vecteurs viraux adeno-associes (aav)
US9492415B2 (en) 1998-05-27 2016-11-15 Genzyme Corporation Methods of treating Parkinson's disease using viral vectors
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
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
US7192705B2 (en) 2001-09-19 2007-03-20 United States Of America, Represented By The Secretary, Department Of Health And Human Services Transductin-1 and applications to hereditary deafness
US7659115B2 (en) 2001-09-19 2010-02-09 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Nucleic acid encoding human transductin-1 polypeptide
US7390482B2 (en) 2002-10-02 2008-06-24 Anges Mg, Inc. Drug for auditory dysfunction
US7166433B2 (en) 2004-03-03 2007-01-23 The United States Of America As Represented By The Department Of Health And Human Services Transductin-1 and transductin-2 and applications to hereditary deafness
US9265933B2 (en) 2005-09-08 2016-02-23 Massachusetts Eye And Ear Infirmary Cochlear implants containing biological cells and uses thereof
WO2011006204A1 (fr) * 2009-07-15 2011-01-20 Newsouth Innovations Pty Limited Procédé permettant d'amener des principes actifs jusqu'à la cochlée
US9533138B2 (en) 2009-07-15 2017-01-03 Newsouth Innovations Pty Limited Method of providing agents to the cochlea
US11026842B2 (en) 2009-07-15 2021-06-08 Newsouth Innovations Pty Limited Method of providing agents to the cochlea
US11013917B2 (en) 2013-06-21 2021-05-25 Newsouth Innovations Pty Limited Method and apparatus for close-field electroporation
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)
WO2022178419A1 (fr) * 2021-02-22 2022-08-25 Decibel Therapeutics, Inc. Méthodes et compositions pour réduire la toxicité induite par un vecteur d'acide nucléique dans l'oreille interne
CN116035800A (zh) * 2023-04-03 2023-05-02 首都医科大学附属北京友谊医院 一种皮下植入式经半规管内耳重复给药装置
CN116035800B (zh) * 2023-04-03 2023-10-31 首都医科大学附属北京友谊医院 一种皮下植入式经半规管内耳重复给药装置

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