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Title:  Stimulation of cellular regeneration and differentiation in the inner ear
United States Patent: 
7,132,406
Issued: 
November 7, 2006

Inventors: 
Kil; Jonathan (Seattle, WA), Lowenheim; Hubert (Tubingen, DE), Gu; Rende (Seattle, WA), Grigeur; Corinne (Seattle, WA)
Assignee: 
Sound Pharmaceuticals Incorporated (Seattle, WA)
Appl. No.:  
10/458,108
Filed: 
June 9, 2003


 

Executive MBA in Pharmaceutical Management, U. Colorado


Abstract

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

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

As used herein, the abbreviation "SSC" refers to a buffer used in nucleic acid hybridization solutions. One liter of the 20.times. (twenty times concentrate) stock SSC buffer solution (pH 7.0) contains 175.3 g sodium chloride and 88.2 g sodium citrate.

As used herein, the phrase "damaging one or more inner ear sensory hair cells", or "damaging a first inner ear sensory hair cell", or grammatical equivalents thereof, means causing a deleterious change in the structure, biochemistry and/or physiology of the damaged, sensory hair cell (including killing the damaged cell) compared to an inner ear sensory hair cell that is cultured under substantially the same conditions as the damaged cell, but which is not damaged.

As used herein, the phrase "improving auditory function" or "improvement in auditory function", or grammatical equivalents thereof, means improving, by at least 10%, the sensitivity to sound of an inner ear by treating the inner ear in accordance with the methods of the present invention, or effecting any measurable improvement in the sensitivity to sound of an inner ear that is completely unresponsive to sound prior to treatment in accordance with the present invention. The sensitivity to sound of the treated inner ear is measured by any art-recognized means (such as the auditory brainstem response) and compared to the sensitivity to sound of a control inner ear that is not treated in accordance with the present invention and which is cultured under substantially the same conditions as the treated inner ear.

As applied to nucleic acid sequence comparisons or amino acid sequence comparisons herein, the term "sequence homology" (also referred to as "sequence identity") is defined as the percentage of amino acid residues or nucleic acid residues in a subject amino acid sequence or nucleic acid sequence that are identical with part or all of a candidate amino acid sequence or nucleic acid sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent homology (identity), and not considering any conservative substitutions as part of the sequence homology. Neither N- or C-terminal extensions nor insertions shall be construed as reducing homology. No weight is given to the number or length of gaps introduced, if necessary, to achieve the maximum percent homology (identity).

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

The anatomy of the inner ear is well known to those of ordinary skill in the art (see, e.g., Gray's Anatomy, Revised American Edition (1977), pages 859 867, incorporated herein by reference). In particular, the cochlea includes the Organ of Corti which is primarily responsible for sensing sound. As shown in FIG. 1, the Organ of Corti 10 includes a basilar membrane 12 upon which are located a variety of supporting cells 14, including border cells 16, inner pillar cells 18, outer pillar cells 20, inner phalangeal cells 22, Dieter's cells 24 and Hensen's cells 26. Supporting cells 14 support inner hair cells 28 and outer hair cells 30. Tectorial membrane 32 is disposed above inner hair cells 28 and outer hair cells 30. The present invention is adapted, in one aspect, to stimulate regeneration of sensory hair cells 28 and 30 from underlying supporting cells 14. In another aspect, the present invention is adapted to stimulate the formation of supporting cells 14.

The present inventors have observed that destruction of existing inner ear sensory hair cells promotes the re-entry of normally quiescent inner ear supporting cells into the cell cycle to yield progeny cells that can be induced to form inner ear sensory hair cells as disclosed herein. In some instances, destruction of existing inner ear sensory hair cells is sufficient to stimulate underlying and/or surrounding inner ear support cells to develop into sensory hair cells. In other instances, efficient regeneration of sensory hair cells from support cells requires destruction of existing inner ear sensory hair cells in combination with another stimulus, as described herein.

In the practice of one aspect of the present invention, inner ear sensory hair cells are damaged, for example by contact with an amount of an ototoxic agent that is effective to damage inner ear sensory hair cells. Representative examples of ototoxic agents useful for damaging inner ear sensory hair cells include aminoglycoside antibiotics (such as, neomycin, gentamycin, streptomycin, kanamycin, amikacin and tobramycin). In the practice of the present invention, the foregoing aminoglycoside antibiotics are typically used in vitro at an effective concentration in the range of from about 0.01 mM 10 mM, and in vivo at an effective concentration in the range of from about 100 to about 1,000 milligrams per kilogram body weight per day (mg/kg/d). Additional, representative examples of chemical agents useful for damaging inner ear sensory hair cells include the following anti-cancer agents: cisplatin, carboplatin and methotrexate which are typically used in vitro at an effective concentration in the range of from about 0.01 0.1 mM, and in vivo at an effective concentration in the range of from about 5 to about 10 mg/kg/d. Other useful chemical agents include poly-L-lysine at an effective concentration in the range of from about 0.1 1.0 mM in vitro, and magnesium chloride at an effective concentration in vitro in the range of from about 5 100 mM.

The ototoxic agent, or agents, can be introduced into the inner ear by any art-recognized means, for example by injection using a needle and syringe, or by cochleostomy. Cochleostomy involves puncturing the cochlea and inserting a catheter through which a chemical agent can be introduced into the cochlea. A cochleostomy method is disclosed, for example, in Lalwani, A. K. et al., Hearing Research 114:139 147 (1997), which publication is incorporated herein by reference.

In one embodiment of the methods of the present invention, the formation of inner ear sensory hair cells from inner ear support cells is stimulated by damaging inner ear sensory hair cells and expressing (before, during, and/or after damaging the inner ear sensory hair cells) within at least some of the inner ear support cells a transcription factor capable of stimulating the formation of an inner ear sensory hair cell from an inner ear support cell. For example, in one embodiment, a nucleic acid molecule encoding a transcription factor capable of stimulating the formation of an inner ear sensory hair cell is introduced into inner ear support cells under conditions that enable expression of the transcription factor.

Transcription factors useful in this aspect of the present invention have the ability to stimulate regeneration of inner ear sensory hair cells from inner ear supporting cells when utilized in the practice of the methods of the present invention. Some transcription factors useful in this aspect of the present invention are required for the normal development, and/or for the normal functioning, of inner ear sensory hair cells.

Representative examples of transcription factors useful in this aspect of the present invention include POU4F1 (Collum, R. G. et al., Nucleic Acids Research 20(18):4919 4925 (1992)), POU4F2 (Xiang et al., Neuron 11:689 701 (1993)), POU4F3 (Vahava, O., Science 279(5358):1950 1954 (1998), Brn3a (also known as Brn3.0), Brn3b (also known as Brn3.2) and Brn3c (also known as Brn3.1) as disclosed in Gerrero et al., Proc. Nat'l. Acad. Sci. (U.S.A.) 90(22):10841 10845 (1993), Xiang, M. et al., Proc. Nat'l. Acad. Sci. (U.S.A.) 93(21):11950 11955 (1996), Xiang, M. et al., J. Neurosci. 15(7Part 1):4762 4785 (1995), Erkman, L. et al., Nature 381(6583):603 606 (1996), Xiang, M. et al., Proc. Nat'l. Acad. Sci. (U.S.A.) 94(17): 9445 9450 (1997), each of which publications is incorporated herein by reference. Some transcription factors useful in this aspect of the present invention possess at least one homeodomain and/or at least one POU-specific domain, and have a molecular weight in the range of from about 33 kDa to about 37 kDa.

As used herein, the term "homeodomain" means an amino acid sequence that is at least 50% homologous (such as at least 75% homologous, or at least 90% homologous) to the homeodomain amino acid sequence set forth in SEQ ID NO:1.

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

An example of an algorithm that can be used to determine the percentage homology between two protein sequences, or between two nucleic acid sequences, is the algorithm of Karlin and Altschul (Proc. Natl. Acad. Sci. USA 87:2264 2268 (1990)), modified as in Karlin and Altschul (Proc. Natl. Acad. Sci. USA 90:5873 5877 (1993)). Such an algorithm is incorporated into the NBLEST and XBLEST programs of Altschul et al. (J. Mol. Biol. 215:403 410 (1990)).

Presently more preferred inner ear cell transcription factors useful in the practice of the present invention are POU4F3 transcription factor homologues (hereinafter referred to as POU4F3 homologues). POU4F3 homologues useful in the practice of the present invention are capable of stimulating the regeneration of inner ear sensory hair cells from supporting cells and are at least 25% homologous (such as at least 50% homologous or at least 75% homologous, or at least 90% homologous) to the POU4F3 transcription factor having the amino acid sequence set forth in SEQ ID NO:4 and which is encoded by the nucleic acid molecule of SEQ ID NO:3. As used herein, the term "POU4F3 homologues" includes the POU4F3 protein having the amino acid sequence set forth in SEQ ID NO:4, which is the presently most preferred inner ear cell transcription factor useful in the practice of the present invention. Representative examples of other POU4F3 homologues useful in the practice of the present invention are set forth in Xiang, M. et al., J. Neuroscience 15(7):4762 4785 (1995), which publication is incorporated herein by reference.

Additional nucleic acid molecules encoding transcription factors useful in the practice of the present invention can be isolated by using a variety of cloning techniques known to those of ordinary skill in the art. For example, cloned POU4F3 homologues cDNAs or genes, or fragments thereof, can be used as hybridization probes utilizing, for example, the technique of hybridizing radiolabeled nucleic acid probes to nucleic acids immobilized on nitrocellulose filters or nylon membranes as set forth at pages 9.52 to 9.55 of Molecular Cloning, A Laboratory Manual (2nd edition), J. Sambrook, E. F. Fritsch and T. Maniatis eds., the cited pages of which are incorporated herein by reference. Presently preferred hybridization probes for identifying additional nucleic acid molecules encoding POU4F3 homologues are fragments, of at least 15 nucleotides in length, of the cDNA molecule (or its complementary sequence) having the nucleic acid sequence set forth in SEQ ID NO:3, although the complete cDNA molecule having the nucleic acid sequence set forth in SEQ ID NO:3 is also useful as a hybridization probe for identifying additional nucleic acid molecules encoding POU4F3 homologue. A presently most preferred hybridization probe for identifying additional nucleic acid molecules encoding POU4F3 homologues is the oligonucleotide having the nucleic acid sequence 5'-TAG AAG TGC AGG GCA CGC TGC TCA TGG TAT G-3' (SEQ ID NO:5).

Exemplary high stringency hybridization and wash conditions useful for identifying (by Southern blotting) additional nucleic acid molecules encoding POU4F3 homologues are: hybridization at 68.degree. C. in 0.25 M Na.sub.2HPO.sub.4 buffer (pH 7.2) containing 1 mM Na.sub.2EDTA, 20% sodium dodecyl sulfate; washing (three washes of twenty minutes each at 65.degree. C.) is conducted in 20 mM Na.sub.2HPO.sub.4 buffer (pH 7.2) containing 1 mM Na.sub.2EDTA, 1% (w/v) sodium dodecyl sulfate.

Exemplary moderate stringency hybridization and wash conditions useful for identifying (by Southern blotting) additional nucleic acid molecules encoding POU4F3 homologues are: hybridization at 45.degree. C. in 0.25 M Na.sub.2HPO.sub.4 buffer (pH 7.2) containing 1 mM Na.sub.2EDTA, 20% sodium dodecyl sulfate; washing is conducted in 5.times.SSC, containing 0.1% (w/v) sodium dodecyl sulfate, at 55.degree. C. to 65.degree. C.

Again, by way of example, nucleic acid molecules encoding transcription factors useful in the present invention can be isolated by the polymerase chain reaction (PCR) described in The Polymerase Chain Reaction (K. B. Mullis, F. Ferre, R. A. Gibbs, eds), Birkhauser Boston (1994), incorporated herein by reference. Thus, for example, first strand DNA synthesis can be primed using an oligo (dT) primer, and second strand cDNA synthesis can be primed using an oligonucleotide primer that corresponds to a portion of the 5'-untranslated region of a cDNA molecule that is homologous to the target DNA molecule. Subsequent rounds of PCR can be primed using the second strand cDNA synthesis primer and a primer that corresponds to a portion of the 3'-untranslated region of a cDNA molecule that is homologous to the target DNA molecule.

By way of non-limiting example, representative PCR reaction conditions for amplifying nucleic acid molecules encoding transcription factors useful in the present invention are as follows. The following reagents are mixed in a tube (on ice) to form the PCR reaction mixture: DNA template (e.g., up to 1 .mu.g genomic DNA, or up to 0.1 .mu.g cDNA), 0.1 0.3 mM dNTPs, 10 .mu.l 10.times.PCR buffer (10.times.PCR buffer contains 500 mM KCl, 15 mM MgCl.sub.2, 100 mM Tris-HCl, pH 8.3), 50 pmol of each PCR primer (PCR primers should preferably be greater than 20 bp in length and have a degeneracy of 10.sup.2 to 10.sup.3 ), 2.5 units of Taq DNA polymerase (Perkin Elmer, Norwalk, Conn.) and deionized water to a final volume of 50 .mu.l. The tube containing the reaction mixture is placed in a thermocycler and a thermocycler program is run as follows. Denaturation at 94.degree. C. for 2 minutes, then 30 cycles of: 94.degree. C. for 30 seconds, 47.degree. C. to 55.degree. C. for 30 seconds, and 72.degree. C. for 30 seconds to two and a half minutes.

Preferably, PCR primers will be designed against conserved amino acid sequence motifs found in some or all of the known target protein sequences. Examples of conserved amino acid sequence motifs against which PCR primers can be designed for cloning additional POU4F3 homologues are the POU-specific domain having the amino acid sequence set forth in SEQ ID NO:2, and the homeodomain having the amino acid sequence set forth in SEQ ID NO:1.

Further, additional nucleic acid molecules encoding transcription factors useful in the practice of the present invention can also be isolated, for example, by utilizing antibodies that recognize transcription factor proteins. Methods for preparing monoclonal and polyclonal antibodies are well known to those of ordinary skill in the art and are set forth, for example, in chapters five and six of Antibodies A Laboratory Manual, E. Harlow and D. Lane, Cold Spring Harbor Laboratory (1988), the cited chapters of which are incorporated herein by reference. By way of non-limiting example, antibodies were successfully raised against a fusion protein constructed from the C-terminal end of Brn3 as described in Xiang, M. et al., J. Neuroscience 15(7):4762 4785 (1995) and Xiang, M. et al., P.N.A.S. (U.S.A.) 94:9445 9450 (1997), which publication is incorporated herein by reference.

Nucleic acid molecules that encode transcription factors useful in the practice of the present invention can be isolated, for example, by screening expression libraries. By way of non-limiting example, a cDNA expression library can be screened using anti-POU4F3 homologue antibodies in order to identify one or more clones that encode a POU4F3 homologue protein. DNA expression library technology is well known to those of ordinary skill in the art. Screening cDNA expression libraries is fully discussed in Chapter 12 of Sambrook, J., Fritsch, E. F. and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., the cited chapter of which is incorporated herein by reference.

By way of representative example, antigen useful for raising antibodies for screening expression libraries can be prepared in the following manner. A full-length cDNA molecule encoding a transcription factor, such as a POU4F3 homologue, (or a cDNA molecule that is not full-length, but which includes all of the coding region) can be cloned into a plasmid vector, such as a Bluescript plasmid (available from Stratagene, Inc., La Jolla, Calif.). The recombinant vector is then introduced into an E. coli strain (such as E. coli XL1-Blue, also available from Stratagene, Inc.) and the protein encoded by the cDNA is expressed in E. coli and then purified. For example, E. coli XL 1-Blue harboring a Bluescript vector including a cDNA molecule of interest can be grown overnight at 37.degree. C. in LB medium containing 100 .mu.g ampicillin/ml. A 50 .mu.l aliquot of the overnight culture can be used to inoculate 5 mg of fresh LB medium containing ampicillin, and the culture grown at 37.degree. C. with vigorous agitation to A.sub.600=0.5 before induction with 1 mM IPTG. After an additional two hours of growth, the suspension is centrifuged (1000.times.g, 15 min, 4.degree. C.), the media removed, and the pelleted cells resuspended in 1 ml of cold buffer that preferably contains 1 mM EDTA and one or more proteinase inhibitors. The cells can be disrupted by sonication with a microprobe. The chilled sonicate is cleared by centrifugation and the expressed, recombinant protein purified from the supernatant by art-recognized protein purification techniques, such as those disclosed in Methods in Enzymology, Vol. 182, Guide to Protein Purification, Murray P. Deutscher, ed (1990), which publication is incorporated herein by reference.

Methods for preparing monoclonal and polyclonal antibodies are well known to those of ordinary skill in the art and are set forth, for example, in chapters five and six of Antibodies A Laboratory Manual, E. Harlow and D. Lane, Cold Spring Harbor Laboratory (1988), the cited chapters of which are incorporated herein by reference. In one representative example, polyclonal antibodies specific for a purified protein can be raised in a New Zealand rabbit implanted with a whiffle ball. One .mu.g of protein is injected at intervals directly into the whiffle ball granuloma. A representative injection regime is injections (each of 1 .mu.g protein) at day 1, day 14 and day 35. Granuloma fluid is withdrawn one week prior to the first injection (preimmune serum), and forty days after the final injection (postimmune serum).

Sequence variants, produced by deletions, substitutions, mutations and/or insertions, of the transcription factors useful in the practice of the present invention can also be used in the methods of the present invention. The amino acid sequence variants of the transcription factors useful in the practice of the present invention may be constructed by mutating the DNA sequences that encode the wild-type transcription factor proteins, such as by using techniques commonly referred to as site-directed mutagenesis. Nucleic acid molecules encoding the transcription factors useful in the practice of the present invention can be mutated by a variety of PCR techniques well known to one of ordinary skill in the art. (See, for example, the following publications, the cited portions of which are incorporated by reference herein: "PCR Strategies", M. A. Innis, D. H. Gelfand and J. J. Sninsky, eds., 1995, Academic Press, San Diego, Calif. (Chapter 14); "PCR Protocols: A Guide to Methods and Applications", M. A. Innis, D. H. Gelfand, J. J. Sninsky and T. J. White, eds., Academic Press, N.Y. (1990)).

By way of non-limiting example, the two primer system utilized in the Transformer Site-Directed Mutagenesis kit from Clontech, may be employed for introducing site-directed mutants into nucleic acid molecules encoding transcription factors useful in the practice of the present invention. Following denaturation of the target plasmid in this system, two primers are simultaneously annealed to the plasmid; one of these primers contains the desired site-directed mutation, the other contains a mutation at another point in the plasmid resulting in elimination of a restriction site. Second strand synthesis is then carried out, tightly linking these two mutations, and the resulting plasmids are transformed into a mutS strain of E. coli. Plasmid DNA is isolated from the transformed bacteria, restricted with the relevant restriction enzyme (thereby linearizing the unmutated plasmids), and then retransformed into E. coli. This system allows for generation of mutations directly in an expression plasmid, without the necessity of subcloning or generation of single-stranded phagemids. The tight linkage of the two mutations and the subsequent linearization of unmutated plasmids results in high mutation efficiency and allows minimal screening. Following synthesis of the initial restriction site primer, this method requires the use of only one new primer type per mutation site. Rather than prepare each positional mutant separately, a set of "designed degenerate" oligonucleotide primers can be synthesized in order to introduce all of the desired mutations at a given site simultaneously. Transformants can be screened by sequencing the plasmid DNA through the mutagenized region to identify and sort mutant clones. Each mutant DNA can then be fully sequenced or restricted and analyzed by electrophoresis on Mutation Detection Enhancement gel (J. T. Baker) to confirm that no other alterations in the sequence have occurred (by band shift comparison to the unmutagenized control).

Again, by way of non-limiting example, the two primer system utilized in the QuikChange.TM. Site-Directed Mutagenesis kit from Stratagene (La Jolla, Calif.), may be employed for introducing site-directed mutants into nucleic acid molecules encoding transcription factors useful in the practice of the present invention. Double-stranded plasmid DNA, containing the insert bearing the target mutation site, is denatured and mixed with two oligonucleotides complementary to each of the strands of the plasmid DNA at the target mutation site. The annealed oligonucleotide primers are extended using Pfu DNA polymerase, thereby generating a mutated plasmid containing staggered nicks. After temperature cycling, the unmutated, parental DNA template is digested with restriction enzyme DpnI which cleaves methylated or hemimethylated DNA, but which does not cleave unmethylated DNA. The parental, template DNA is almost always methylated or hemimethylated since most strains of E. coli, from which the template DNA is obtained, contain the required methylase activity. The remaining, annealed vector DNA incorporating the desired mutation(s) is transformed into E. coli.

In the design of a particular site directed mutagenesis experiment, it is generally desirable to first make a non-conservative substitution (e.g., Ala for Cys, His or Glu) and determine if activity is greatly impaired as a consequence. If the residue is by this means demonstrated to be important by activity impairment, or knockout, then conservative substitutions can be made, such as Asp for Glu to alter side chain length, Ser for Cys, or Arg for His. For hydrophobic segments, it is largely size that is usefully altered, although aromatics can also be substituted for alkyl side chains.

Other site directed mutagenesis techniques may also be employed with nucleic acid molecules encoding transcription factors useful in the practice of the present invention. For example, restriction endonuclease digestion of DNA followed by ligation may be used to generate deletion variants of transcription factors useful in the practice of the present invention, as described in Section 15.3 of Sambrook et al. Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, New York, N.Y. (1989), incorporated herein by reference. A similar strategy may be used to construct insertion variants, as described in section 15.3 of Sambrook et al., supra.

Oligonucleotide-directed mutagenesis may also be employed for preparing substitution variants of transcription factors useful in the practice of the present invention. It may also be used to conveniently prepare the deletion and insertion variants of transcription factors useful in the practice of the present invention. This technique is well known in the art as described by Adelman et al. (DNA 2:183 [1983]); Sambrook et al., supra; "Current Protocols in Molecular Biology", 1991, Wiley (NY), F. T. Ausubel, R. Brent, R. E. Kingston, D. D. Moore, J. D. Seidman, J. A. Smith and K. Struhl, eds., incorporated herein by reference.

Generally, oligonucleotides of at least 25 nucleotides in length are used to insert, delete or substitute two or more nucleotides in the nucleic acid molecules encoding transcription factors useful in the practice of the present invention. An optimal oligonucleotide will have 12 to 15 perfectly matched nucleotides on either side of the nucleotides coding for the mutation. To mutagenize wild-type transcription factor proteins useful in the practice of the present invention, the oligonucleotide is annealed to the single-stranded DNA template molecule under suitable hybridization conditions. A DNA polymerizing enzyme, usually the Klenow fragment of E. coli. DNA polymerase I, is then added. This enzyme uses the oligonucleotide as a primer to complete the synthesis of the mutation-bearing strand of DNA. Thus, a heteroduplex molecule is formed such that one strand of DNA encodes the wild-type protein inserted in the vector, and the second strand of DNA encodes the mutated form of the protein inserted into the same vector. This heteroduplex molecule is then transformed into a suitable host cell.

Mutants with more than one amino acid substituted may be generated in one of several ways. If the amino acids are located close together in the polypeptide chain, they may be mutated simultaneously using one oligonucleotide that codes for all of the desired amino acid substitutions. If, however, the amino acids are located some distance from each other (separated by more than ten amino acids, for example) it is more difficult to generate a single oligonucleotide that encodes all of the desired changes. Instead, one of two alternative methods may be employed. In the first method, a separate oligonucleotide is generated for each amino acid to be substituted. The oligonucleotides are then annealed to the single-stranded template DNA simultaneously, and the second strand of DNA that is synthesized from the template will encode all of the desired amino acid substitutions. An alternative method involves two or more rounds of mutagenesis to produce the desired mutant. The first round is as described for the single mutants: wild-type protein DNA is used for the template, an oligonucleotide encoding the first desired amino acid substitution(s) is annealed to this template, and the heteroduplex DNA molecule is then generated. The second round of mutagenesis utilizes the mutated DNA produced in the first round of mutagenesis as the template. Thus, this template already contains one or more mutations. The oligonucleotide encoding the additional desired amino acid substitution(s) is then annealed to this template, and the resulting strand of DNA now encodes mutations from both the first and second rounds of mutagenesis. This resultant DNA can be used as a template in a third round of mutagenesis, and so on.

Prokaryotes may be used as host cells for the initial cloning steps of transcription factors useful in the practice of the present invention. They are particularly useful for rapid production of large amounts of DNA, for production of single-stranded DNA templates used for site-directed mutagenesis, for screening many mutants and/or putative inner ear cell transcription factors simultaneously, and for DNA sequencing of the mutants generated. Suitable prokaryotic host cells include E. coli K12 strain 94 (ATCC No. 31,446), E. coli strain W3110 (ATCC No. 27,325) E. coli X1776 (ATCC No. 31,537), and E. coli B; however many other strains of E. coli, such as HB101, JM101, NM522, NM538, NM539, and many other species and genera of prokaryotes including bacilli such as Bacillus subtilis, other enterobacteriaceae such as Salmonella typhimurium or Serratia marcesans, and various Pseudomonas species may all be used as hosts. Prokaryotic host cells or other host cells with rigid cell walls are preferably transformed using the calcium chloride method as described in section 1.82 of Sambrook et al., supra. Alternatively, electroporation may be used for transformation of these cells. Prokaryote transformation techniques are set forth in Dower, W. J., in Genetic Engineering, Principles and Methods, 12:275 296, Plenum Publishing Corp., 1990; Hanahan et al., Meth. EnzyMol. 204:63 (1991).

As will be apparent to those skilled in the art, any plasmid vectors containing replicon and control sequences that are derived from species compatible with the host cell may also be used to clone, express and/or manipulate nucleic acid molecules encoding transcription factors useful in the practice of the present invention. The vector usually has a replication site, marker genes that provide phenotypic selection in transformed cells, one or more promoters, and a polylinker region containing several restriction sites for insertion of foreign DNA. Plasmids typically used for transformation of E. coli include pBR322, pUC18, pUC19, pUCI118, pUC119, and Bluescript M13, all of which are described in sections 1.12 1.20 of Sambrook et al., supra. However, many other suitable vectors are available as well. These vectors contain genes coding for ampicillin and/or tetracycline resistance which enables cells transformed with these vectors to grow in the presence of these antibiotics.

The promoters most commonly used in prokaryotic vectors include the .beta.-lactamase (penicillinase) and lactose promoter systems (Chang et al. Nature 375:615 [1978]; Itakura et al., Science 198:1056 [1977]; Goeddel et al., Nature 281:544 [1979]) and a tryptophan (trp) promoter system (Goeddel et al., Nucl. Acids Res. 8:4057 [1980]; EPO Appl. Publ. No. 36,776), and the alkaline phosphatase systems. While these are the most commonly used, other microbial promoters have been utilized, and details concerning their nucleotide sequences have been published, enabling a skilled worker to ligate them functionally into plasmid vectors (see Siebenlist et al., Cell 20:269 [1980]).

The construction of suitable vectors containing DNA encoding replication sequences, regulatory sequences, phenotypic selection genes and the DNA encoding a transcription factor useful in the practice of the present invention are prepared using standard recombinant DNA procedures. Isolated plasmids and DNA fragments are cleaved, tailored, and ligated together in a specific order to generate the desired vectors, as is well known in the art (see, for example, Sambrook et al., supra).

In another embodiment of the methods of the present invention, the formation of inner ear sensory hair cells from inner ear support cells is stimulated by damaging inner ear sensory hair cells and inhibiting the expression (before, during and/or after damaging the inner ear sensory hair cells) of one or more cell cycle inhibitors active in inner ear support cells. In this way, inner ear support cells that are in contact with damaged sensory hair cells can be stimulated to divide and at least some of the resulting progeny form inner ear sensory hair cells. By way of representative example, cell cycle inhibitors active in inner ear support cells include cyclin-dependent kinase inhibitors, such as cyclin-dependent kinase inhibitors of the so-called CIP/KIP family including p21.sup.Cip1, p27.sup.Kip1 and p57.sup.Kip2.

Specific examples of cell cycle inhibitors active within inner ear support cells include: p57.sup.Kip2 (Lee et al., Genes Dev. 9(6):639 649 (1995)(SEQ ID NO:6)); p27.sup.Kip1 (Cell 78(1):59 66 (1994)(SEQ ID NOS:8 and 9)); p21.sup.Cip1 (El-Diery et al., Cell 75(4):817 825 (1993)(SEQ ID NOS:10 and 11)); p19 Ink 4d (Chan et al., Mol. Cell. Biol. 15(5):2682 2688 (1995)(SEQ ID NOS:12 and 13)); p18 Ink 4c (Guan et al., Genes Dev. 8(24):2939 2952 (1994)(SEQ ID NOS:14 and 15)); p15 Ink 4b (Hannon and Beach, 371(6494):257 261 (1994)(SEQ ID NOS:16 and 17)); and p16 Ink 4a (Serrano, M. et al., Nature 366(6456):704 707 (1993)(SEQ ID NOS:18 and 19)). Nucleic acid molecules that encode cell cycle inhibitors useful in the practice of the present invention hybridize to the antisense strands of any one of the nucleic acid molecules set forth in SEQ ID NOS: 6, 8, 10, 12, 14, 16 and 18 under at least one hybridization stringency greater than 2.times.SSC at 55.degree. C., such as 1.times.SSC at 60.degree. C., or 0.2.times.SSC at 60.degree. C.

Inhibitors of cell cycle inhibitors can be substances, such as proteins, that act on the cell cycle inhibitor in an intracellular, direct or indirect manner. Additionally, inhibitors of cell cycle inhibitors can be antisense nucleic acid molecules that are complementary to all or a portion of a nucleic acid molecule (such as an mRNA molecule) that encodes a cell cycle inhibitor protein, and that hybridize to the nucleic acid molecule encoding a cell cycle inhibitor protein under stringent conditions (such as a stringency greater than 2.times.SSC at 55.degree. C., e.g., 1.times.SSC at 60.degree. C. or 0.2.times.SSC at 60.degree. C.).

Any art-recognized method can be used to inhibit cell cycle inhibitor gene expression in inner ear support cells. For example, the expression of a cell cycle inhibitor active in inner ear support cells can be inhibited by introducing into inner ear support cells a vector that includes a portion (or all) of a nucleic acid molecule, in antisense orientation relative to a promoter sequence, that encodes a cell cycle inhibitor active in inner ear support cells.

In general, antisense technology utilizes a DNA sequence that is inverted relative to its normal orientation for transcription and so expresses an RNA transcript that is complementary to a target mRNA molecule expressed within the host cell (i.e., the RNA transcript of the anti-sense gene can hybridize to the target mRNA molecule through Watson-Crick base pairing). An anti-sense gene may be constructed in a number of different ways provided that it is capable of interfering with the expression of a target gene, such as a gene encoding a cell cycle inhibitor. The anti-sense gene can be constructed by inverting the coding region (or a portion thereof) of the target gene relative to its normal orientation for transcription to allow the transcription of its complement, hence the RNAs encoded by the anti-sense and sense gene are complementary.

The anti-sense gene generally will be substantially identical to at least a portion of the target gene or genes. The sequence, however, need not be perfectly identical to inhibit expression. Generally, higher homology can be used to compensate for the use of a shorter anti-sense gene. The minimal identity will typically be greater than about 65%, but a higher identity might exert a more effective repression of expression of the endogenous sequences. Substantially greater identity of more than about 80% is preferred, though about 95% to absolute identity would be most preferred.

Furthermore, the anti-sense gene need not have the same intron or exon pattern as the target gene, and non-coding segments of the target gene may be equally effective in achieving anti-sense suppression of target gene expression as coding segments. Normally, a DNA sequence of at least about 30 or 40 nucleotides should be used as the anti-sense gene, although a longer sequence is preferable. The construct is then introduced into one or more inner ear support cells and the antisense strand of RNA is produced.

Catalytic RNA molecules or ribozymes can also be used to inhibit expression of target genes. It is possible to design ribozyme transgenes that encode RNA ribozymes that specifically pair with a target RNA and cleave the phosphodiester backbone at a specific location, thereby functionally inactivating the target RNA. In carrying out this cleavage, the ribozyme is not itself altered, and is thus capable of recycling and cleaving other molecules. The inclusion of ribozyme sequences within antisense RNAs confers RNA-cleaving activity upon them, thereby increasing the activity of the antisense constructs. Tabler et al. (1991, Gene 108:175) have greatly simplified the construction of catalytic RNAs by combining the advantages of the anti-sense RNA and the ribozyme technologies in a single construct. Smaller regions of homology are required for ribozyme catalysis, therefore this can promote the repression of different members of a large gene family if the cleavage sites are conserved.

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

Any art-recognized gene delivery method can be used to introduce a nucleic acid molecule encoding a transcription factor (or a vector including an antisense DNA molecule) into inner ear cells for expression therein, including: direct injection, electroporation, virus-mediated gene delivery, amino acid-mediated gene delivery, biolistic gene delivery, lipofection and heat shock. Non-viral methods of gene delivery into inner ear cells are disclosed in Huang, L., Hung, M-C, and Wagner, E., Non-Viral Vectors for Gene Therapy, Academic Press, San Diego, Calif. (1999), which is incorporated herein by reference.

For example, genes can be introduced into cells in situ, or after removal of the cells from the body, by means of viral vectors. For example, retroviruses are RNA viruses that have the ability to insert their genes into host cell chromosomes after infection. Retroviral vectors have been developed that lack the genes encoding viral proteins, but retain the ability to infect cells and insert their genes into the chromosomes of the target cell (A. D. Miller, Hum. Gen. Ther. 1:5 14 (1990)).

Adenoviral vectors are designed to be administered directly to patients. Unlike retroviral vectors, adenoviral vectors do not integrate into the chromosome of the host cell. Instead, genes introduced into cells using adenoviral vectors are maintained in the nucleus as an extrachromosomal element (episome) that persists for a limited time period. Adenoviral vectors will infect dividing and non-dividing cells in many different tissues in vivo including airway epithelial cells, endothelial cells, hepatocytes and various tumors (B. C. Trapnell, Adv Drug Del Rev. 12:185 199 (1993)).

Another viral vector is the herpes simplex virus, a large, double-stranded DNA virus that has been used in some initial applications to deliver therapeutic genes to neurons and could potentially be used to deliver therapeutic genes to some forms of brain cancer (D. S. Latchman, Mol. Biotechnol. 2:179 95 (1994)). Recombinant forms of the vaccinia virus can accommodate large inserts and are generated by homologous recombination. To date, this vector has been used to deliver interleukins (ILs), such as human IL-1.beta. and the costimulatory molecules B7-1 and B7-2 (G. R. Peplinski et al., Ann. Surg. Oncol. 2:151 9 (1995); J. W. Hodge et al., Cancer Res. 54:5552 55 (1994)).

Another approach to gene therapy involves the direct introduction of DNA plasmids into patients. (F. D. Ledley, Hum. Gene Ther. 6:1129 1144 (1995)). The plasmid DNA is taken up by cells within the body and can direct expression of recombinant proteins. Typically plasmid DNA is delivered to cells in the form of liposomes in which the DNA is associated with one or more lipids, such as DOTMA (1,2,-diolcyloxypropyl-3-trimethyl ammonium bromide) and DOPE (dioleoylphosphatidylethanolamine). Formulations with DOTMA have been shown to provide expression in pulmonary epithelial cells in animal models (K. L. Brigham et al., Am. J. Med. Sci. 298:278 281 (1989); A. B. Canonico et al., Am. J. Respir. Cell. Mol. Biol. 10:24 29 (1994)). Additionally, studies have demonstrated that intramuscular injection of plasmid DNA formulated with 5% PVP (50,000 kDa) increases the level of reporter gene expression in muscle as much as 200-fold over the levels found with injection of DNA in saline alone (R. J. Mumper et al., Pharm. Res. 13:701 709 (1996); R. J. Mumper et al., Proc. Intern. Symp. Cont. Rol. Bioac. Mater. 22:325 326 (1995)). Intramuscular administration of plasmid DNA results in gene expression that lasts for many months (J. A. Wolff et al., Hum. Mol. Genet. 1:363 369 (1992); M. Manthorpe et al., Hum. Gene Ther. 4:419 431 (1993); G. Ascadi et al., New Biol. 3:71 81 (1991), D. Gal et al., Lab. Invest. 68:18 25 (1993)).

Additionally, uptake and expression of DNA has also been observed after direct injection of plasmid into the thyroid (M. Sikes et al., Hum. Gene Ther. 5:837 844 (1994)) and synovium (J. Yovandich et al., Hum. Gene Ther. 6:603 610 (1995)). Lower levels of gene expression have been observed after interstitial injection into liver (M. A. Hickman et al., Hum. Gene Ther. 5:1477 1483 (1994)), skin (E. Raz et al., Proc. Natl. Acad. Sci. 91:9519 9523 (1994)), instillation into the airways (K. B. Meyer et al., Gene Therapy 2:450 460 (1995)), application to the endothelium (G. D. Chapman et al., Circulation Res. 71:27 33 (1992); R. Riessen et al., Human Gene Therapy 4:749 758 (1993)), and after intravenous administration (R. M. Conry et al., Cancer Res. 54:1164 1168 (1994)).

Various devices have been developed for enhancing the availability of DNA to the target cell. A simple approach is to contact the target cell physically with catheters or implantable materials containing DNA (G. D. Chapman et al., Circulation Res. 71:27 33 (1992)). Another approach is to utilize needle-free, jet injection devices which project a column of liquid directly into the target tissue under high pressure. (P. A. Furth et al., Anal Biochem. 20:365 368 (1992); (H. L. Vahlsing et al., J. Immunol. Meth. 175:11 22 (1994); (F. D. Ledley et al., Cell Biochem. 18A:226 (1994)).

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

Cochleostomy involves puncturing the cochlea and inserting a catheter through which a chemical agent, such as a nucleic acid molecule, can be introduced into the cochlea. A cochleostomy method is disclosed, for example, in Lalwani, A. K. et al., Hearing Research 114:139 147 (1997), which publication is incorporated herein by reference.

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

When the methods of the present invention are utilized in vitro, the whole inner ear, including the Organ of Corti, is preferably excised and cultured and manipulated in a culture vessel. Presently preferred embodiments of an apparatus that is useful for culturing inner ears in vitro are disclosed in U.S. patent No. 5,437,998; U.S. Pat. No. 5,702,941 and U.S. Pat. Ser. No. 5,763,279, each of which is incorporated herein by reference.

In general, presently preferred embodiments of an apparatus for culturing inner ears include a gas permeable bioreactor comprising a tubular vessel with walls that may be constructed at least partially of a gas permeable material, such as silicone rubber. The vessel in one preferred embodiment is constructed such that half of it is comprised of gas permeable material and the remaining portion is made of nonpermeable material. The gas permeable materials commonly available are opaque. Thus, using nonpermeable material for at least part of the bioreactor may provide an advantage in allowing visual inspection of the tubular vessel chamber.

The tubular vessel has closed ends, a substantially horizontal longitudinal central axis, and one or more vessel access ports. The vessel access ports provide access to the bioreactor for input of medium and cells, and for removal of old medium from the tubular vessel. This is easily done through the vessel access ports which are also referred to as valves or syringe ports. In the preferred embodiment, the vessel access ports are constructed of valves with syringe ports.

Preferably the vessel is rotatable about its horizontal longitudinal central axis. A preferred means for rotation is a motor assembly which sits on a mounting base and has means for attachment to the tubular vessel. The speed of rotation can be adjusted so that the inner ear within the tubular vessel is constantly in motion, but rotation of the tubular vessel should not be fast enough to cause significant turbulence in the aqueous medium within the tubular vessel.

If so desired, the use of gas permeable material in the construction of at least part of the tubular vessel wall permits O.sub.2 to diffuse through the vessel walls and into the cell culture media in the vessel chamber. Correspondingly, CO.sub.2 diffuses through the walls and out of the vessel. Thus, the use of gas permeable material in the construction of at least part of the tubular vessel wall typically overcomes the need for air injection into the bioreactor vessel. Air injection into the aqueous medium within the bioreactor vessel may be utilized, however, if additional oxygen is required to culture an inner ear. When an air pump is utilized to inject air into the aqueous medium, an air filter is also employed to protect the air pump valves from dirt.

An alternative embodiment of the bioreactor useful in the practice of the present invention is an annular vessel with walls that may be constructed at least partially of a gas permeable material. Annular is defined herein to include annular, toroidal and other substantially symmetrical ring-like shaped tubular vessels. The annular vessel has closed ends and a substantially horizontal longitudinal central axis.

In another embodiment, the bioreactor useful in the practice of the present invention comprises a tubular vessel constructed at least partially of a gas permeable material. The vessel has closed ends and a substantially horizontal longitudinal central axis around which it rotates. The vessel furthermore has two slidably interconnected members wherein a first member fits slidably into a second member, forming a liquid tight seal therebetween and providing a variable volume tubular vessel. The bioreactor has means for rotating the tubular vessel about its substantially horizontal longitudinal central axis. One or more vessel access ports are provided for transferring materials into and out of the vessel.

In situations where minimization of contamination is necessary (e.g., AIDS or human tissue research), disposability of the bioreactor useful in the practice of the present invention is a particular advantage. Moreover, the embodiment of the bioreactor with slidably interconnected members may be adjusted to provide the exact size bioreactor needed.

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

Neuralbasal.TM. media from Gibco BRL (Gibco BRL media are produced by Life Technologies, Corporate Headquarters, Gaithersburg, Md.), which requires the addition of B27 or N2 media supplement, is the presently preferred culture medium for culturing inner ears in vitro. Other culture media can be successfully used, however, to culture fluid-filled sensory organs in the practice of the present invention. Other suitable media include DME, BME and M-199 with fetal calf serum or horse serum. All of the foregoing media are sold by Gibco -BRL. When using Neuralbasal.TM. medium, N2 or B27 supplements play a more significant role when extended periods of culture (>96 hr) are attempted.

In another aspect, the present invention provides methods for stimulating the formation of inner ear support cells. The methods of this aspect of the invention include the steps of damaging inner ear support cells under conditions that promote the formation of new inner ear support cells (for example by cell division of inner ear support cells that are in contact with damaged inner ear support cells). In this aspect of the invention, the inner ear support cell is damaged, and the formation of new inner ear support cells is stimulated, using the same techniques described herein for the methods of the present invention that stimulate the formation of inner ear sensory hair cells from inner ear support cells. Thus, for example, inner ear support cells can be damaged by contact with an amount of an ototoxic agent, such as an aminoglycoside antibiotic, that is effective to damage inner ear support cells. Again by way of example, new inner ear support cell formation can be further stimulated by damaging inner ear support cells and expressing (before, during and/or after damaging inner ear support cells) within inner ear support cells a transcription factor (such as POU4F1, POU4F2, POU4F3, Brn3a, Brn3b and Brn3c) capable of stimulating inner ear support cells to divide and form new inner ear support cells. In preferred embodiments of this aspect of the invention, the proliferation of inner ear support cells results in improvement in the auditory function of the treated inner ear.

 

Claim 1 of 8 Claims

1. A method for stimulating the formation of an inner ear sensory hair cell from an inner ear support cell in vivo, the method comprising the steps of: (a) damaging a first inner ear sensory hair cell by contacting the sensory hair cell, in vivo, with an effective amount of an antibiotic; and (b) locally introducing into an inner ear support cell, that is in contact with the damaged sensory hair cell, a nucleic acid molecule that is complementary to a portion of an mRNA molecule that encodes p27Kip1 and inhibits mammalian p27Kip1.

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