Modulators of actin

The invention provides methods and compositions which find use, inter alia, for modulating the stabilization of actin filaments. The compositions may comprise one or more polypeptide moieties derived from a novel human diaphanous polypeptide and/or one or more nucleic acid moieties derived from a novel human diaphanous gene or gene transcript. The invention also provides agents which specifically modify the binding of a natural human diaphanous gene or gene product with a natural binding target thereof, isolated human diaphanous hybridization probes and primers capable of specifically hybridizing with the disclosed human diaphanous genes, human diaphanous-specific binding agents such as specific antibodies, and methods of making and using the subject compositions in diagnosis, therapy and in the biopharmaceutical industry.

INTRODUCTION
 1. Field of the Invention
 The invention relates to a class of polypeptides involved in actin
 stabilization.
 2. Background of the Invention
 The actin cytoskeleton plays a central role in defining cellular structure
 and effecting dynamic changes in morphology. By selectively stabilizing
 and destabilizing actin polymerization, the cell is able to effect a wide
 range of structural reorganization and effect phenomena such as cell
 motility, phagocytosis, cytokinesis, mitosis, etc. Because these
 phenomenon are involved in myriad medically significant physiologies and
 pathologies, e.g. the progress of many pathogenic infections, invasion and
 metastisis of neoplasia, fertilization, clotting and wound repair, etc.,
 the stability of actin polymerization is a choice target for therapuetic
 intervention. In fact, potent a drugs effecting actin filament
 destabilization and stabilization such as fungal-derived alkaloids
 including the cytochalasins and phalloidins are well known. Here we
 disclose a new family of modulators of actin polymer stabilization derived
 from a novel human diaphanous protein and gene.
 Relevant Literature
 Lynch ED, et al. (1997) Science 278(5341): 1315-1318 disclose nonsyndromic
 deafness DFNA1 associated with mutation of a human homolog of the
 Drosophila gene diaphanous.
 Watanabe N, et al. (1997) EMBO J 16:3044-3056, disclose a mouse gene with
 sequence similarity to the disclosed human gene. Bione S, et al. (1998) Am
 J Hum Genet 62(3): 533-541, report that a human homologue of the
 Drosophila melanogaster diaphanous gene is disrupted in premature ovarian
 failure. Vahava O, et al. (1998) Science 279(5358): 1950-1954. Mutation in
 transcription factor POU4F3 associated with inherited progressive hearing
 loss in humans.
 SUMMARY OF THE INVENTION
 The invention provides methods and compositions which find use, inter alia,
 for modulating the stabilization of actin filaments. The compositions may
 comprise one or more polypeptide moieties derived from a novel human
 diaphanous polypeptide and/or one or more nucleic acid moieties derived
 from a novel human diaphanous gene or gene transcript. The invention also
 provides agents which specifically modif the binding of a natural human
 diaphanous gene or gene product with a natural binding target thereof.
 Polypeptide components of subject compositions provide human
 diaphanous-specific structure and activity and may be produced
 recombinantly from transformed host cells from the subject human
 diaphanous polypeptide encoding nucleic acids. The invention provides
 isolated human diaphanous hybridization probes and primers capable of
 specifically hybridizing with the disclosed human diaphanous genes, human
 diaphanous-specific binding agents such as specific antibodies, and
 methods of making and using the subject compositions in diagnosis (e.g.
 genetic hybridization screens for human diaphanous transcripts), therapy
 (e.g. modulating a cellular function such as auditory signal transduction
 by introducing into the cell a subject modulator) and in the
 biopharmaceutical industry (e.g. as immunogens, reagents for isolating
 additional natural human diaphanous genes and alleles, reagents for
 screening bio/chemical libraries for ligands and lead and/or
 pharmacologically active agents, etc.).
 DESCRIPTION OF TICULAR EMBODIMENTS OF THE INVENTION
 In one embodiment, the modulators of the invention comprise a human
 diaphanous polypeptide (a plurality of amino acids linearly joined through
 peptide bonds) having a natural human diaphanous polypeptide-specific
 sequence and bioactivity (i.e. distinguished from natural murine and
 drosophila diaphanous sequences and bioactivities). SEQ ID NO: 1 depicts
 an exemplary natural cDNA encoding a human diaphanous polypeptide and SEQ
 ID NO: 2 depicts the corresponding encoded natural human diaphanous
 polypeptide. The subject polypeptides comprise at least a 6, preferably at
 least a 12, more preferably at least a 18, most preferably at least a 24
 residue domain of SEQ ID NO:2, not found in natural mouse or drosophila
 diaphanous polypeptides. Human specific sequences are readily identified
 by aligning the respectivel sequences. In a particular embodiment, the
 subject polypeptides comprise at least a 36, preferably at least a 72,
 more preferably at least a 144, most preferably at least a 288 residue
 domain of SEQ ID NO:2.
 The polypeptides provide natural human diaphanous polypeptide specific
 bioactivity or function, such as specific ligand binding or binding
 inhibition, antigenicity, immunogenicity, etc. Human diaphanous
 polypeptide-specific activity or function may be determined by convenient
 in vitro, cell-based, or in vivo assays: e.g. in vitro binding assays,
 cell culture assays, in animals (e.g. gene therapy, transgenics, etc.),
 etc. Binding assays encompass any assay where the molecular interaction of
 a human diaphanous polypeptide with a binding target is evaluated. The
 binding target may be a natural intracellular binding target such as a
 human diaphanous polypeptide regulating protein, effector or other
 regulator that directly modulates a human diaphanous polypeptide activity
 or its localization; or non-natural binding target such a specific immune
 protein such as an antibody, or an human diaphanous polypeptide specific
 agent such as those identified in bio/chemical screening assays. Exemplary
 binding targets include human prolifin and Rho polypeptides. Human
 diaphanous polypeptide-binding specificity may assayed by functional
 assays described below, binding equilibrium constants (usually at least
 about 10.sup.7 M.sup.-1, preferably at least about 10.sup.8 M.sup.-1, more
 preferably at least about 10.sup.9 M.sup.-1), by the ability of the
 subject polypeptides to function as negative mutants in a human diaphanous
 polypeptide-expressing cells, to elicit a human diaphanous polypeptide
 specific antibody in a heterologous host (e.g. a rodent or rabbit), etc.
 The human diaphanous polypeptide binding specificity of the human
 diaphanous polypeptides necessarily distinguishes that of natural murine
 and drosophila homologs. In a particular embodiment, the sequence and
 function also distinguishes those of the natural human diaphanous 2
 polypeptide.
 In particular embodiments, modulators comprising human diaphanous
 polypeptides are isolated, pure or recombinant: an "isolated" polypeptide
 is unaccompanied by at least some of the material with which it is
 associated in its natural state, preferably constituting at least about
 0.5%, and more preferably at least about 5% by weight of the total
 polypeptide in a given sample and a pure polypeptide constitutes at least
 about 90%, and preferably at least about 99% by weight of the total
 polypeptide in a given sample. A recombinant polypeptide comprises a
 non-natural terminus residue or is joined to other than an amino acid
 which it is joined to in a natural polypeptide. The polypeptides may be
 synthesized, produced by recombinant technology, or purified from cells. A
 wide variety of molecular and biochemical methods are available for
 biochemical synthesis, molecular expression and purification of the
 subject compositions, see e.g.
 Molecular Cloning, A Laboratory Manual (Sambrook, et al. Cold Spring Harbor
 Laboratory), Current Protocols in Molecular Biology (Eds. Ausubel, et al.,
 Greene Publ. Assoc., Wiley-Interscience, NY) or that are otherwise known
 in the art. Material and methods for the expression of heterologous
 recombinant polypeptides in bacterial cells (e.g. E. coli), yeast (e.g. S.
 Cerevisiae), animal cells (e.g. CHO, 3T3, BHK, baculovirus-compatible
 insect cells, etc.). The polypeptides may be provided uncomplexed with
 other moieties including other polypeptide moieties, complexed in a wide
 variety of covalent and/or non-covalent associations and binding
 complexes, etc., which may provide enhanced activity, stability,
 availability, targeting, etc.
 Exemplary active modulators comprising human diaphanous polypeptides
 moieties include (using N.fwdarw.C nomenclature convention):
 hDia1-del-1: MRG--residues 121-151 of SEQ ID NO:2 fusion polypeptide
 hDia1-del-2: GFP--residues 197-205 of SEQ ID NO:2 fusion polypeptide
 hDia1-del-3: FLAGG--residues 350-382 of SEQ ID NO:2 fusion polypeptide
 hDia1-del-4: CYCLIN A--residues 439454 of SEQ ID NO:2 fusion polypeptide
 hDia1-del-5: CYCLIN B1--residues 515-524 of SEQ ID NO:2 fusion polypeptide
 hDia1-del-6: CYCLIN B2--residues 551-569 of SEQ ID NO:2 fusion polypeptide
 hDia1-del-7: CYCLIN B3--residues 590-610 of SEQ ID NO:2 fusion polypeptide
 hDia1-del-8: SH2--residues 611-630 of SEQ ID NO: 2 fusion polypeptide
 hDia1-del-9: SH3--residues 651-670 of SEQ ID NO: 2 fusion polypeptide
 hDia1-del-10: MRG--residues 674-773 of SEQ ID NO:2 fusion polypeptide
 hDia1-del-11: GFP--residues 740-840 of SEQ ID NO:2 fusion polypeptide
 hDia1-del-12: FLAGG--residues 841-940 of SEQ ID NO:2 fusion polypeptide
 hDia1-del-13: CYCLIN A--residues 941-1040 of SEQ ID NO:2 fusion polypeptide
 hDia1-del-14: CYCLIN B1--residues 1041-1140 of SEQ ID NO:2 fusion
 polypeptide
 hDia1-del-15: CYCLIN B2--residues 1141-1171 of SEQ ID NO:2 fusion
 polypeptide
 The invention provides methods and compositions of selectively modulating
 cytoskeletal de/stabilization and/or the effective concentration of a
 human diaphanous protein within a target cell. The general methods involve
 introducing into the target cell an effective amount of a subject
 modulator, sufficient to selectively modulate actin cytoskeltal function
 of a cell. As demonstrated herein, the invention encompasses a wide
 variety of suitable methods of introduction, amounts, and modulator
 compositions, which are readily optimized empirically. In addition to the
 human diaphanous polypeptide moiety, the modulator may comprise a wide
 variety of additional moieties, including moieties which provide for
 detection, targeting, stability, proteolytic resistance, etc. Preferred
 modulators demonstrate cytoskelatal de/stabilization with several
 alternative methods of introduction, including direct medium uptake,
 uptake facilitated by chaotropic agents including detergents (e.g.
 TWEEN20, etc.), guanadine salts, etc., pulsed electric field, liposome
 fusion, etc.
 The compositions may be advantageously combined and/or used in combination
 with other therapeutic or prophylactic agents, different from the subject
 compounds. In many instances, administration in conjunction with the
 subject compositions enhances the efficacy of such agents, see e.g.
 Goodman & Gilman's The Pharmacological Basis of Therapeutics, 9.sup.th
 Ed., 1996, McGraw-Hill. In particular embodiments, such as where the
 modulators are polypeptides, the modulators may also be introduced
 indirectly by expression within the targeted cell. Such expression may be
 effected at least in part by transiently transfecting or by upregulation
 of a stably introduced polypeptide-encoding gene. A wide variety of
 well-established methods are known in the art for facilitating
 introduction, expression and/or stable integration of exogenous genes in
 targeted host cells (below).
 The invention provides binding agents specific to the claimed modulators,
 including substrates, agonists, antagonists, natural intracellular binding
 targets, etc., methods of identifying and making such agents, and their
 use in diagnosis, therapy and pharmaceutical development. For example,
 novel polypeptide-specific binding agents include human diaphanous
 polypeptide--specific receptors, such as somatically recombined
 polypeptide receptors like specific antibodies or T-cell antigen receptors
 (see, e.g Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold
 Spring Harbor Laboratory) and other natural intracellular binding agents
 identified with assays such as one-, two- and three-hybrid screens,
 non-natural intracellular binding agents identified in screens of chemical
 libraries, etc. For diagnostic uses, such binding agents are frequently
 labeled, such as with fluorescent, radioactive, chemiluminescent, or other
 easily detectable molecules, either conjugated directly to the binding
 agent or conjugated to a probe specific for the binding agent. Agents of
 particular interest modulate human diaphanous polypeptide function, e.g.
 human diaphanous polypeptide-dependent actin de/stabilization.
 The invention also provides efficient methods of identifying agents active
 at the level of a human diaphanous modulatable cellular function.
 Generally, these screening methods involve assaying for compounds which
 modulate a human diaphanous polypeptide interaction with a natural human
 diaphanous polypeptide binding target, etc. A wide variety of assays for
 binding agents are provided including labeled in vitro protein-protein
 binding assays, immunoassays, cell based assays, etc. The methods are
 amenable to automated, cost-effective high throughput screening of
 chemical libraries for lead compounds. Agents that modulate the
 interactions of a human diaphanous polypeptide with its ligands/natural
 binding targets can be used to modulate biological processes associated a
 human diaphanous polypeptide function, e.g. by contacting a cell
 comprising a human diaphanous polypeptide (e.g. administering to a subject
 comprising such a cell) with such an agent. Biological processes mediated
 by human diaphanous polypeptides include a wide variety of cellular events
 which are mediated when a human diaphanous polypeptide binds a ligand e.g.
 cytoskeletal modifications.
 The amino acid sequences of the subject polypeptides are used to
 back-translate polypeptide-encoding nucleic acids optimized for selected
 expression systems (Holler et al. (1993) Gene 136, 323-328; Martin et al.
 (1995) Gene 154, 150-166) or used to generate degenerate oligonucleotide
 primers and probes for use in the isolation of natural human diaphanous
 polypeptide-encoding nucleic acid sequences ("GCG" software, Genetics
 Computer Group, Inc, Madison Wis.). Modulator polypeptide-encoding nucleic
 acids are used in polypeptide-expression vectors and incorporated into
 recombinant host cells, e.g. for expression and screening, e.g. for
 functional studies such as the efficacy of candidate agents to manipulate
 modulator polypeptide-modulated cell function, etc.
 The invention also provides human diaphanous nucleic acids including
 hybridization probes and replication/amplification primers having a human
 diaphanous cDNA specific sequence comprising a fragment of a strand of SEQ
 ID NO: 1 sufficient to effect specific hybridization to the complementary
 strand of SEQ ID NO: 1 (i.e. specifically hybridize with a nucleic acid
 comprising the corresponding opposite strand of SEQ ID NO: 1, in the
 presence of a natural murine diaphanous gene and in a particular
 embodiment, in the presence of a natural human diaphanous 2 gene). Such
 primers or probes are at least 12, preferably at least 24, more preferably
 at least 36 and most preferably at least 96 bases in length. Demonstrating
 specific hybridization generally requires stringent conditions, i.e. those
 that (1) employ low ionic strength and high temperature for washing, for
 example, 0.015 M NaCl/0.0015 M sodium titrate/0.1% SDS at 50.degree. C.,
 or (2) employ during hybridization a denaturing agent such as formamide,
 for example, 50% (vol/vol) formamide with 0.1% bovine serum albumin/0.1%
 Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5
 with 750 mM NaCl, 75 mM sodium citrate at 42.degree. C. Another example is
 use of 50% formamide, 5.times.SSC (0.75 M NaCl, 0.075 M sodium citrate),
 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate,
 5.times.Denhardt's solution, sonicated salmon sperm DNA (50 (g/ml), 0.1%
 SDS, and 10% dextran sulfate at 42.degree. C., with washes at 42.degree.
 C. in 0.2.times.SSC and 0.1% SDS. human diaphanous nucleic acids can also
 be distinguished using alignment algorithms, such as BLASTX (Altschul et
 al. (1990) Basic Local Alignment Search Tool, J Mol Biol215, 403-410).
 The subject nucleic acids are of synthetic/non-natural sequences and/or are
 isolated, i.e. unaccompanied by at least some of the material with which
 it is associated in its natural state, preferably constituting at least
 about 0.5%, preferably at least about 5% by weight of total nucleic acid
 present in a given fraction, and usually recombinant, meaning they
 comprise a non-natural sequence or a natural sequence joined to
 nucleotide(s) other than that which it is joined to on a natural
 chromosome. Recombinant nucleic acids comprising the nucleotide sequence
 of SEQ ID NO: 1, or the subject fragments thereof, contain such sequence
 or fragment at a terminus, immediately flanked by (i.e. contiguous with) a
 sequence other than that which it is joined to on a natural chromosome, or
 flanked by a native flanking region fewer than 10 kb, preferably fewer
 than 2 kb, which is at a terminus or is immediately flanked by a sequence
 other than that which it is joined to on a natural chromosome. While the
 nucleic acids are usually RNA or DNA, it is often advantageous to use
 nucleic acids comprising other bases or nucleotide analogs to provide
 modified stability, etc.
 The subject nucleic acids find a wide variety of applications including use
 as translatable transcripts, knock-in/out vectors, hybridization probes,
 PCR primers, diagnostic nucleic acids, etc.; use in detecting the presence
 of human diaphanous genes and gene transcripts and in detecting or
 amplifying nucleic acids encoding additional human diaphanous homologs and
 structural analogs. Accordingly, the invention provides suitable nucleic
 acid vectors and tranformed host cells comprising the subject nucleic
 acids, especially wherein the nucleic acids are operably linked to a
 homologous or heterologous promoter and expressed in bacterial or insect
 cells. In diagnosis, human diaphanous hybridization probes find use in
 identifying wild-type and mutant human diaphanous alleles. Human
 diaphanous nucleic acids are used to effect and/or modulate cellular
 expression or intracellular concentration or availability of active human
 diaphanous.
 Methods for effecting the targeted expression of genes encoding the subject
 modulators are known in the art; see, e.g. Lalwani AK, et al. (1996) Gene
 Ther Jul;3(7):588-592; Tait, DL et al. (1997) A Phase I Trial of
 Retroviral BRCA1sv Gene Therapy in Ovarian Cancer, Clinical Cancer
 research, in press and excerpted below; Altenschmidt et al., 1997, J Mol
 Med 75:259-266; Perales et al. 1997, Proc Natl Acad Sci USA 94:6450-6455;
 Schmidt et al., 1997, Gene 190:211-216; Oldfield et al., 1993, human Gene
 Therapy 4: 39-46; Asgari et al., 1997, Int J. Cancer 71:377-382; He D, et
 al. 1997, Cancer Res 57:1868-1872. In a particular embodiment, the subject
 human diaphanous polypeptide is introduced by transfecting the cell with a
 nucleic acid encoding the polypeptide particularly, wherein the nucleic
 acid comprises SEQ ID NO: 1 or a fragment thereof. Therapeutic nucleic
 acid compositions may be advantageously combined and/or used in
 combination with other therapeutic or prophylactic agents, different from
 the subject compounds. In many instances, administration in conjunction
 with the subject compositions enhances the efficacy of such agents, see
 e.g. Goodman & Gilman's The Pharmacological Basis of Therapeutics,
 9.sup.th Ed., 1996, McGraw-Hill.
 Without further description, one of ordinary skill in the art can, using
 the preceding description and the following illustrative examples, make
 and utilize the compounds of the present invention and practice the
 claimed methods. The following working examples therefore, specifically
 point out preferred embodiments of the present invention, and are not to
 be construed as limiting in any way the remainder of the disclosure. Other
 generic configurations will be apparent to one skilled in the art. All
 publications and patent applications cited in this specification are
 herein incorporated by reference as if each individual publication or
 patent application were specifically and individually indicated to be
 incorporated by reference.

EXAMPLES
 Example 1
 We initially isolated the disclosed natural human diaphanous 1 gene through
 our studies of hereditary deafness (the actin cytoskeleton of hair cells
 of the inner ear is critical to hearing). Kindred M of Costa Rica defines
 the autosomal dominant, fully penetrant, progressive hearing loss DFNA1
 (OMIM 124900; 1, 2). Deafness in kindred M is a sensorineural
 cochleosaccular dysplasia specific to the membranous structures of the
 inner ear. DFNA1 in kindred M was mapped to a region of 1 cM on chromosome
 5q31 by linkage analysis, then a complete 800 kb BAC contig was
 constructed of the linked region (3). In order to identify all genes in
 the linked region, we sequenced BACs comprising the contig, after shotgun
 subdoning each into M13 (4, 5). We developed the computer program SeqHelp
 to organize sequences from the chromatograms, to call bases and align
 sequences using the computer programs PHRED and PHRAP, and to apply
 existing, publicly available software to evaluate the novel genomic
 sequences (6).
 A novel human gene homologous to Drosophila diaphanous (Genbank U11288) and
 to mouse p140mDia (Genbank U96963, SEQ ID NOS:3, 4) was revealed by
 genomic sequence of BACs 293C24, and 45M22, 249H5 (7). Given that the
 mouse and human predicted amino acid sequences were 97% identical for the
 regions identified from BACs, we estimated the sizes of gaps from the
 mouse sequence, constructed primers from the human coding sequence, and
 used these to amplify intervening exons from human cDNA and to carry out
 5' RACE on polyA+RNA from lymphoblastoid lines (8). Human diaphanous, or
 Dia1 (SEQ ID NOS: 1, 2), comprises at least 18 exons with approximately
 3800 bp coding sequence and 3=UTR of 918 bp or 1891 bp (9).
 In order to screen the Dia1 gene for mutation in the M family, primers were
 designed to amplify exons and flanking splice junctions from genomic DNA
 of affected and unaffected members of the M family and from controls. Each
 product was screened for single strand conformation polymorphisms (SSCP).
 Variant bands were gel-purified, reamplified, and sequenced (10).
 A guanine to thymine substitution in the splice donor of the penultimate
 exon of human Dia1 was observed in affected members of the M kindred. The
 guanine to thymine substitution at this site disrupts the canonical splice
 donor sequence AAGgtaagt. In order to determine the consequences of this
 mutation at the level of RNA message, polyA+cDNA was prepared from
 lymphoblast cell lines of three affected members of the M kindred and from
 unaffected family members and unrelated, unaffected controls. Insertion of
 TTAA was observed in cDNA of affected individuals. The mechanism for the
 insertion was splicing at a cryptic site four basepairs 3' of the wildtype
 site. The TTAA insertion leads to a frameshift, encoding 21 aberrant amino
 acids, followed by protein termination that truncates 32 amino acids
 (Table 1). All 78 affected members of the M kindred are heterozygous for
 the mutation. The site was wildtype in 330 hearing, control individuals
 (660 chromosomes) of the following ancestries: 12 Costa Ricans unrelated
 to the M family, 94 Latin Americans from other countries, 32 Spanish, 154
 Europeans (other than Spanish) and North Americans of European ancestry,
 and 38 African-Americans.
 Table 1. DFNA1 mutation in human diaphanous associated with deafness in the
 Monge family. The wildtype human diaphanous sequence of the splice
 junctions of the penultimate and ultimate exons and coding sequence of the
 ultimate exon are shown at top of the figure. Sequence present in the RNA
 message is capitalized; intronic sequence is in lower case; amino acid
 sequence is indicated. A guanine residue (g) at the donor splice junction
 is the site of the DFNA1 mutation. The DFNA1 mutant human diaphanous
 sequence of the same regions is shown at bottom of the figure. The mutant
 thymine (T) is indicated in bold. The G.fwdarw.T substitution abrogates
 the normal donor splice, so splicing occurs instead at the Ag four
 nucleotides 3' of the normal site. Consequently, TTAA is inserted in the
 mutant message, causing a frameshift and premature stops, as indicated.
 Wild Type (see SEQ ID NOS:1, 2)
 CCC CGT CAA Ggtaagtaa ... cagaatctctcgtcttctcttgcagCC AAC AGG AAG
 Pro Arg Gln Ala Asn Arg Lys
 GCC GGG TGT GCA GTC ACA TCT CTG CTA GCT TCG GAG CTG ACC AAG GAT
 Ala Gly Cys Ala Val Thr Ser Leu Leu Ala Ser Glu Leu Thr Lys Asp
 GAT GCC ATG GCT GCT GTT CCT GCC AAG GTG TCC AAG AAC AGT GAG ACA
 Asp Ala Met Ala Ala Val Pro Ala Lys Val Ser Lys Asn Ser Glu Thr
 TTC CCC ACA ATC CTT GAG GAA GCC AAG GAG TTG GTT GGC CGT GCA AGC TAA
 Phe Pro Thr Ile Leu Glu Glu Ala Lys Glu Leu Val Gly Arg Ala Ser *
 Mutant (see SEQ ID NOS:1, 2)
 CCC CGT CAA GTT Aagtaa ... cagaatctctcgtcttctcttgcagC CAA CAG GAA
 Pro Arg Gln Val Asn Gln Gln Glu
 GGC CGG GTG TGC AGT CAC ATC TCT GCT AGC TTC GGA GCT GAC CAA GGA
 Gly Arg Val Cys Ser His Ile Ser Ala Ser Phe Gly Ala Asp Gln Gly
 TGA TGC CAT GGC TGC TGT TCC TGC CAA GGT GTC CAA GAA CAG TGA GAC
 * Cys His Gly Cys Cys Ser Cys Gln Gly Val Gln Glu Gln * Asp
 ATT CCC CAC AAT CCT TGA GGA AGC CAA GGA GTT GGT TGG CCG TGC AAG CTA A
 Ile Pro His Arn Pro * Gly Ser Gln Gly Val Gly Trp Pro Cys Lys Lue
 Expression of human Dia1 message in brain, heart, placenta, lung, kidney,
 pancreas, liver and skeletal muscle was confirmed by Northern
 hybridization. A single transcript of 4.7 kb was observed in all tissues
 with highest expression in skeletal muscle. RNA from lymphoblastoid cell
 fines of affected and unaffected members of the M family similarly
 revealed a single transcript of 4.7 kb in all individuals, consistent with
 a 4 bp insertion in the mutant message. Expression of the human Dia1 gene
 in the cochlea was confirmed by RT-PCR of cochlear RNA using PCR primers
 that amplified the region of Dia1 that harbors the mutation in family M
 (10, 11). The sequence of the RT-PCR product from cochlear RNA was wild
 type. Hence, if alternate splice forms of Dia1 exist, normal cochlear
 transcripts include the region of Dia1 that is improperly spliced in
 affected members of kindred M.
 Human Dia1, mouse p140mDia, and Drosophila diaphanous proteins are homologs
 of Saccharomyces cervisiae gene Bnilp (12). The genes encoding these
 proteins are members of the formin gene family, which also includes the
 mouse limb deformity gene, Drosophila cappuccino, Aspergillus nidulins
 gene sepA, and S.pombe genes fus1 and cdc12 (13). These genes are involved
 in cytokinesis and establishment of cell polarity. All formins share
 Rho-binding domains formin-homology domains in the C-terminal region (12).
 Multiple mutants of mouse formin have been characterized (13). A truncated
 mouse formin allele Id.sup.In2 lacking the 42 C-terminal amino acids leads
 to mislocalization of the formin protein to the cytoplasm (14).
 We find that Dia1 affects hearing through the regulation of actin
 polymerization in hair cells. Actin polymerization involves proteins known
 to interact with diaphanous in Drosophila and mouse. The protein profilin
 binds actin monomers and is a regulator of actin polymerization (15).
 Mammalian and Drosophila diaphanous are effectors of Rho (12). Diaphanous
 acts in a Rho-dependent manner to recruit profilin to the membrane, where
 it promotes actin polymerization. As predicted by this model, transient
 expression of p140mDia induced homogeneous actin filament formation in COS
 cells (7). Rho-induced actin polymerization is conserved from yeast to
 mammals.
 The DFNA1 mutation observed in Dia1 in the M family is relatively subtle,
 in that it affects only the C-terminal 52 amino acids. Given that human
 Dia1 appears to be ubiquitously expressed, and the only observed phenotype
 in the M family is hearing loss, we conclude that the hair cells of the
 cochlea are particularly sensitive to proper maintenance of the actin
 cytoskeleton and that this mutation can effect a partial loss of function
 of the Dia1 protein. One process in the inner ear uniquely sensitive to
 disruption of actin polymerization is amplification of sound reception by
 the inner hair cells, which is due to the concerted action of outer hair
 cells and pillar cells. Relay of kinetic energy from outer hair cells to
 inner hair cells relies critically on the presence of a rigid structure of
 actin fibers. Additional structural support in hair cells is provided by
 the cuticular plate, a dense network of actin fibers at the apical ends of
 hair cells into which stereocilia are anchored. The DFNA1 mutation of Dia1
 can impair maintenance of the dynamic organization of the actin fibers of
 the cuticular plate.
 Hair cell stereocilia provides an additional site that can be affected by
 the aberrant protein. The structural support providing rigidity to the
 stereocilia is comprised largely of cross-linked actin filaments packed in
 a paracrystaline array (16). Upon acoustic overstimulation, the
 paracrystal is disordered (17) and Dia1 is involved in the reordering of
 the array. In the M family, mutant Dia1 can compete with the wild-type
 protein to repair damage from normal exposure to sound. Trangenic mice
 with the DFNA1 mutation in p140mDia are used to characterize the effects
 of acoustic exposures.
 A second human homolog of Drosophila diaphanous (SEQ ID NOS:5, 6) was
 revealed during the cloning of Dia1. This second human diaphanous, Dia2,
 maps to chromosome Xq22 (18). Non-syndromic X-linked deafness, DFN2, also
 maps to Xq22 (19), indicating the Dia2 gene as a candidate gene for DFN2
 hearing loss. In fact, we disclose that mutations in Dia1 and/or Dia2 can
 affect a wide range of pathologies in humans, including deafness,
 infertility, neuropathology, etc. Furthermore, Dia1 and/or Dia2 mutations
 can also manifest symptoms characterized as Perrault syndrome, Pallister P
 D, Opitz J M, Am J Med Genet 1979;4(3):239-246; Gottschalk M E, Coker S B,
 Fox L A, Am J Med Genet 1996 Nov 11;65(4):274-276. Accordingly, the
 disclosed modulators, nucleic acids and binding agents find a wide variety
 of diagnostic, biotechnological and clinical applications.
 REFERENCES
 1. Leon PE, et al., Amer J Hum Genet 33:209-214 (1981); Leon PE, Raventos
 H, Lynch E, Morrow J, King MC Proc Natl Acad Sci USA 89:181-184 (1992)
 2. This project has been approved by the Committee on Human Subjects in
 Research of the Ministry of Health of Costa Rica, and by the Human
 Subjects Division of the Institutional Review Board of the University of
 Washington. The criterion for deafness in the family is a hearing
 threshold greater than 50 dB at 250 Hz and 500 Hz. Of the participants, 78
 are deaf and 69 are older than 30 years with normal hearing. All deaf
 relatives are included in the analysis, as are all hearing relatives older
 than age 30 years and all persons marrying into the family. No relatives
 younger than age 30 with normal hearing are included in the analysis Cell
 lines were established from lymphocytes of 147 informative relatives using
 established techniques (1).
 3. Lynch ED, Lee M K, Lalwani A, Jackler R K, Sweetow R W, Raventos H,
 Kujawa S, Morrow J, King M C, Leon PE Localization, physical mapping, and
 description of the clinical phenotype of DFNA1, a gene for post-lingual
 non-syndromic deafness on chromosome 5q31. In review.
 4. Kim U J, et al., Genomics 34:213-218 (1996)
 5. Sequencing of BACs was performed as follows: 30 ug of BAC DNA was
 sonicated to 50 to 5000 bp, then treated with mung bean exonuclease. Blunt
 ended fragments were electrophoresed on agarose gels, DNA in the 1.5 kb to
 3 kb range was excised from the gel for DNA isolation with a Qiaex gel
 extraction kit. Recovered fragments were ligated into Smal digested,
 phosphatase-treated, M13mpl8 vector. Ligations were electroporated into E.
 coli strain DH12S. Transformations were plated in LB top agarose with
 DH12S lawn cells, X-gal, and IPTG, onto LB plates and incubated overnight
 at 37 C. The following day, clear plaques were picked and inoculated into
 1 mL of LB with DH12S host cells in 96 well 2mL plates. Phage cultures
 were incubated for 24 hours at 37 C, shaking at 250 rpm. Single stranded
 M13 DNA was prepared by standard methods using PEG precipitation of phage
 particles and NaI solution to remove proteins. A detailed copy of the DNA
 preparation method can be found on the Internet at &lt;hyper text transfer
 protocol://chroma.mbt.washington.edu/.about.kwseq/preps/amy_NaI_prep.html&gt;
 This preparation method yielded 1-2 ug of M13 DNA for sequencing. The
 resulting DNA pellets were diluted in 30 ul of water, and 6 microliters
 used in 10 microliter sequencing reactions with dichloroRhodamine Dye
 Terminator Chemistry from ABI. The remaining DNA was stored at -80 C for
 future use. Sequencing reactions were precipitated with 100 microliters of
 70% EtOH and 5mM MgCl2 at room temperature for 15 minutes. Precipitated
 reactions were pelleted by centrifugation for 15 minutes at 3500 rpm in
 Beckman SH-3000 rotor with 96 well plate adapters. Supernatants were
 removed by centrifugation of the inverted plate at 500 rpm for 1 minute
 then pellets dried at 37 C for 5 minutes. Pellets were resuspended in 3
 microliters of formamide loading dye, denatured at 95 C for two minutes,
 then placed on ice. One microliter of sequencing reaction was loaded onto
 a 36 cM Longranger gel (FMC) and electrophoresed on an ABI377 automated
 sequencer. ABI377 collection software Version 1.1 was used to support
 48-well combs and nine hour data collection in the 2.times. collection
 mode. The chromatograms generated by ABI Sequence Analysis software
 version 3.0 were transferred to a UNIX-based Sun workstation for contig
 assembly and blast analysis. The computer program PHRED (Green P and Ewing
 B. 1996. hyper text transfer protocol://world wide
 web.bozeman.mbt.washington.edu/ phrap.docs/ phred.html) was used to assign
 bases to the electropherograms. After eliminating vector sequences, the
 program PHRAP (Green P and Ewing B. 1996. hyper text transfer
 protocol://world wide web.bozeman.mbt.washington.edu/ phrap.docs/
 phrap.html) was used to analyze the sequences, identify overlapping
 individual sequences, and assemble them into contigs. To achieve
 approximately 6 fold coverage of a region, we sequenced an average of 600
 M13 subclones per BAC.
 6. The SeqHelp program incorporates several sequence analysis programs and
 creates output in HTML files for browsing with any WWW browser (Lee et al
 Genomics submitted). The core programs used by Seqhelp are PHRED to read
 the ABI sequence files and assign bases, PHRAP to generate contigs of
 overlapping sequences, Repeat Masker (Arian Smit) to identify and mask
 common repetative elements prior to database searching, and BLAST
 (Altschul S, Gish W, Miller W, Myers E, Lipman D J Mol Biol 215:403-410
 (1990)) comparison of project specific sequences to the NR and dbEST
 databases at the NCBI. An example of the SeqHelp output for analysis of
 the BRCA1 genomic region is available online at &lt;hyper text transfer
 protocol://polaris.mbt.washington.edu&gt;
 7. Castrillon D H, Wasserman S A. Development 120:3367-3377 (1994);
 Watanabe N, et al., EMBO J 16:3044-3056 (1997)
 8. Polyadenylated RNA [poly(A+)] RNA was purified from lymphoblastoid cell
 lines using oligo-dT cellulose (Sambrook J, Fritsch E F, Maniatis T
 Molecular Cloning. Cold Srping Harbor (1989)). 5' cDNA sequence was
 obtained using the 5' RACE (Rapid Amplification of cDNA Ends) System,
 Version 2.0 (Gibco BRL). 5' RACE was performed on 1 microgram of
 polyA+lymphoblast RNA according to the manufacturer's specifications.
 First strand cDNA synthesis was primed using the human diaphanous specific
 primer H2a (5'-AGTCATCCATCTCCATGCGAATG-3') (SEQ ID NO:7). Following cDNA
 synthesis and homopolymeric 3' tailing with Tdt (terminal deoxynucleotidyl
 transferase), first strand cDNA was amplified using the human diaphanous
 specific primer H2b (5'-ATGCGAATGTCATCCAGCCGTC-3') (SEQ ID NO:8), a nested
 primer which anneals 3' to H2a. 5' RACE products of approximately 1 kb
 were gel purified and TA cloned into the pGEM-T vector (Invitrogen)
 according to the manufactures directions. 5' RACE clones were amplified
 using M13-40F and M13-40R PCR products of 5' RACE clones were purified.
 Templates were sequenced using M13-40 R primers and the gene specific
 primers H6f (5'-TTGCGGGATATGCCTCTG-3') (SEQ ID NO:9) and H7a
 (5'-GGTTGTTGTTGAGAGACACAC-3') (SEQ ID NO: 10). Sequencing was done using
 dichloroRhodamine Dye Terminators (ABI) and an ABI 377 sequencer.
 9. IMAGE clones 51234, 52194, 124697, 261240, 262633, 612749, and 926002
 are cDNA clones of portions of human diaphanous (Lennon G, Auffray C,
 Polymeropoulos M, Soares MB. Genomics.33:151-152 (1996)). The ESTs for all
 clones are confined to the most 3' exon of human diaphanous.
 10. PCR primers used to amplify the the variant sequence which includes the
 involved splice donor region are Dia9F (5'-TGTGGGAGAGGGGAAATCAAG-3') (SEQ
 ID NO:11) and Dia9R (5'-TTGCTCTTTAGCCGCAGACTGG-3') (SEQ ID NO: 12). The
 278bp product was labeled by incorporation of a-p32 dCT? during PCR,
 diluted 1:10 in formamide loading buffer, denatured at 95 C for 2 minuted,
 then placed on ice for 10 minutes. Eight microliters of each sample was
 loaded onto an MDE (FMC Biochem) gel and electrophoresed at 6W for 18
 hours at room temperature to resolve single strand comformation
 polymorphisms. Gels were dried and exposed to X-ray film for 18 hours.
 Variant bands on SSCP gels were individually excised from dried gels,
 eluted with water, and used as a template for reamplification with the
 Dia9F and Dia9R primers. PCR products were purified by centrifugation
 through 300 microliters of Sephacryl-300 resin then sequenced using the
 Dia 9F and Di9R primers. Sequencing was done using dichloroRhodamine Dye
 Terminators (ABI) and an ABI 377 sequencer as described in footnote 2. PCR
 amplification for cDNA analysis of the variant region was done using
 primers Dia8-10F (5'-CGGCGGAAGACAGAAGAAAAG-3') (SEQ ID NO: 13) and
 Dia8-10R (5'-TAGCAGAGATGTGACTGCACACCC-3') (SEQ ID NO: 14) which are
 designed to amplify a 234 bp product that spans the second to last exon
 and ends in the last exon of human mDia. PCR products were labeled and
 analyzed by SSCP as describe above. Variant bands were sequenced using the
 Dia8-10F and Dia8-10R primers.
 11. Total cochlear RNA was extracted using the guanidine isothiocyanate
 method (Chirgwin J M, Przybyla A E, MacDonald R J, Rutter W J.
 Biochemistry 18:5294-5299 (1979). One microgram of total cochlear RNA was
 used in a 50 microliter random primed reverse transcription reaction with
 Superscript MMLV RTase (Gibco/BRL) according to manufacturers
 instructions. Five microliters of the resulting cDNA was used as template
 in a 50 microliter gene specific PCR reaction using the Dia8-10F and
 Dia8-10R primers (10). PCR products were resolved on a 2% agarose gel and
 visualized with ethidium bromide staining.
 12. Evangelista M, et al., Science 276:118-121 (1997); Narumiya S, Ishizaki
 T, Watanabe N FEBS Lett 410:68-72 (1997)
 13. Woychik R P, et al., Nature 346:850-853 (1990); Maas R L, et al.,
 Nature 346:853-855 (1990); Maas R L, et al., Am J Hum Genet 48:687-695
 (1991); Vogt T F, et al., Proc Natl Acad Sci USA 90:5554-5558 (1993); Wang
 C C, et al., Genomics 39:303-311 (1997); Wynshaw-Boris A, et al., Mol Med
 3:372-384 (1997); Frazier J A, et al., Curr Biol 7:414-417 (1997)
 14. Chan D C, Leder P J Biol Chem 271:23472-23477 (1996)
 15. Theriot J A, Mitchison T J. Cell 75:835-838 (1993).
 16. Flock A, et al., J Cell Biol 75:339-343 (1977); Itoh M Hearing Res
 6:227-289 (1982)
 17. Tilney L G, Saunders J C, Egelman E H, DeRosier D J Hear Res 7:181-197
 (1982)
 18. Dia2 is represented by several IMAGE clones including 626664, a 3.1 kb
 cDNA clone from a HeLa cDNA library. When searched against the Genbank
 database, a portion of this clone was identical to genomic DNA from 
 117P19, sequenced and mapped by the Sanger Center to Xq21.3. The
 Drosophila Related Expressed Sequences homepage &lt;hyper text transfer
 protocol://world wide web.tigem.it/LOCAL/drosophila/dros.html&gt;(Banfi S,
 Borsani G, Bulfome A, Ballbio A. Hum Mol Genet 6:1745-1753 (1997))
 indicates that a human homolog of Drosophila diaphanous maps to human
 chromosome Xq22.
 19. Tyson J, et al., Hum Mol Genet 5:2055-2060 (1996)
 Example 2
 Retroviral hDia1sv Gene Therapy
 LXSN-hDia1 vector is constructed by cloning a hDia1 cDNA into the
 well-characterized retroviral vector LXSN (Holt J T, et al. Nature
 Genetics 12:298-302,1996). Retroviral vector is manufactured under cGMP
 (current Good Manufacturing Practices) conditions employing a CellCube
 (Corning-Costar, Cambridge, Mass.) apparatus perfused with Aim V media
 (Life Technologies, Gaithersburg, Md.) under continuous monitoring of pH
 and O.sub.2. Once the oxygen and glucose consumption are consistent and
 appropriate, supernatant is collected as long as the oxygen and glucose
 levels assure optimal vector production. No post-production manufacturing
 is performed on the supernatants collected in Aim V media which are frozen
 and stored in aliquots at -70.degree. C. The titer of the vector
 preparations is determined by counting the number of particles present
 that confer G418 resistance to transduced MCF-7 cells, employing
 appropriate dilutions. Vector from this production lot is confirmed
 negative for bacterial, mycoplasm, viral contamination and endotoxin.
 Replication-competent retroviruses are confirmed absent using PG4
 indicator cells following amplification on Mus Dunni. In addition to the
 tests performed on the clinical grade vector described above, a number of
 tests are performed on the producer cells in the master cell bank:
 including tests for pathogenic viruses and replication-competent
 retroviruses. A toxicity study is done in mice: 92 Balb/C female mice were
 injected with either high-dose gene therapy (clinical grade) or low-dose
 (clinical grade diluted 1:10 in AimV) once daily for four days with and
 without oyster glycogen pre-treatment (48hrs prior) to simulate patient
 peritonitis. Mice are harvested at 4 hours, 24 hours, 48 hours, one week
 and two weeks post-injections, at which time blood and 14 tissues are
 removed for histological and molecular assays.
 Vector Administration.
 Aliquots of vector are thawed and 8 ug/ml of polybrene is added sterilely.
 Infusions of vector into patients are initiated within one hour of thawing
 the vector aliquot. The initial dose (between 3 mls to 300 mls depending
 on the dose escalation) is given with 1.5 liters of sterile saline ip and
 the three subsequent doses are given with sterile saline to a total volume
 of 100-300 ml.
 Study design.
 Patients undergo initial placement of a peritoneal port-a-cath for access
 to the peritoneal cavity and are subsequently treated for four consecutive
 days with intraperitoneal LXSN-hDia1 gene therapy. The starting dose level
 in patients is that dose which corresponds to the no effect dose in mice
 (10.sup.8), and a half-log dose escalation is performed up to the dose
 which corresponds to the LD10 dose in mice (10.sup.10). Five dose levels
 are studied: 10.sup.8, 3.3.times.10.sup.8, 10.sup.9, 3.3.times.10.sup.9,
 and 10.sup.10 viral particles. Objective endpoints to assess toxicity
 include: daily blood and peritoneal sample to evaluate peritoneal fluid
 cell counts, hematological cell counts, serum chemistries, bacterial
 cultures as needed, vector stability, viral uptake by cells, expression of
 hDia1 gene and presence of antibodies to vector envelope proteins. At four
 week intervals patients are evaluated for response to therapy; and if
 positive, retreatment allowed. The first three patients are treated at the
 first dose level. After the next higher dose level is tolerated by a new
 patient, any repeat patients are graduated to that dose. The dose is again
 elevated after three patients tolerate it without toxicity.
 Detection of vector stability and expression.
 DNA is prepared from cell samples by hypotonic lysis, digestion with
 proteinase K (Boehringer Mannheim, Indianapolis, Ind.) and SDS, followed
 by phenol/chloroform extraction and ethanol precipitation. DNA is prepared
 from tissue samples by freezing samples at -70.degree. C. and then finely
 mincing cold samples with a blade, prior to treatment with proteinase K as
 described above. RNA are purified from cells by lysis in guanidinium
 isothiocyanate.
 PCR primers specific for the neo sequences within the LXSN-hDia1sv vector
 are employed for determination of vector presence and stability within
 patient samples. RT-PCR is performed by our published methods (Thompson,
 M. E., et al. Nature Genetics 9, 444-450,1995.).
 Southern blotting of Ava I digested DNA is performed with a human hDia1
 probe. Percent transduction is estimated by quantitating hybridization
 with the phosphoimager and then comparing hybridization of the presumed
 haploid vector lower band to that of the diploid hDia1 upper band (percent
 transduction=2X vector signal/genomic signal.times.100). Nuclease
 protection assays are performed with MRNA isolated from patient samples
 and then probed with a T7 polymerase generated probe from a digested hDia1
 DNA template. Radiolabelled probe is hybridized with patient MRNA samples
 for 8 hours at 52.degree. C. in 80% formamide and then digested for 30
 minutes with RNAse A and RNAse T1 at 25.degree. C. and then products
 resolved on a 10% denaturing polyacrylamide gel (supra).
 Immunologic studies
 Patient plasmas and peritoneal fluids are frozen and then used for
 measurements of CH50 or western blotting for envelope antibodies. CH50 is
 performed following manufacturer's instructions on plasma and peritoneal
 samples, using antibody-sensitized sheep erythrocytes (Sigma, St. Louis,
 Mo.). Basically, patient peritoneal fluid or sera are incubated with
 antibody-sensitized sheep erythrocytes in sodium barbital buffer for 30
 minutes at 37.degree. C. The extent of antibody-dependent lysis is then
 determined by pelleting unlysed red cells and measuring hemolysis in the
 supernatant by spectrophometry against a standard curve. Standard
 complement serum (Sigma, St. Louis, Mo.) are employed as a control
 standard.