Note: Descriptions are shown in the official language in which they were submitted.
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COMPOSITIONS FOR THE THERAPEUTIC USE OF AN ATONAL-
ASSOCIATED SEQUENCE FOR DEAFNESS, OSTEOARTHRITIS,
AND ABNORMAL CELL PROLIFERATION
This application is a divisional of Canadian Patent Application No.
2,375,106, filed on June 1, 2000.
HELD OF THE INVENTION
The present invention relates in general to the field of genetic diagnosis and
therapy and, more particularly, to the characterization and use of an atonal-
associated
nucleic acid or amino acid sequence, or any of its homologs or orthologs, as a
therapeutic
agent for the treatment of deafness, partial hearing loss, vestibular defects
due to damage
or loss inner ear hair cells, osteoarthritis, and abnormal cell proliferation.
BACKGROUND OF THE INVENTION
An intricate pattern of interactions within and between cells directs the
sequential
development of neurons from dividing neuroepithelial progenitor cells.
Multiple
extracellular and intracellular signals moderate this process. Among the key
intracellular
signals are transcription factors, which induce the expression of a cascade of
genes. One
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subclass of transcription factors, belonging to the basic helix-loop-helix
(bHLH) family
of proteins, is expressed early on when the decision to proliferate or
differentiate is made.
This function is a particularly crucial one as mutations in these genes early
in
development can wipe out entire neural structures.
In Drosophila, the gene atonal (ato), which is homologous to Math], Math2,
Hathl and Hath2, encodes a bHLH protein essential for the development of
chordotonal
organs (sensory organs found in the body wall, joints and antenna that
function in
proprioception, balance and audition) (Eberl, 1999; McIver, 1985; van Staaden
and
Romer, 1998). CHOs populate the peripheral nervous system (PNS) in the body
wall and
joints (thorax, abdomen, sternum, wings, legs) and antennae (Moulins, 1976),
providing
the fly with sensory information much as touch and mechanoreceptors do in
vertebrates
(McIver, 1985; Moulins, 1976). Boyan (Boyan, 1993) proposed that, in the
course of
evolution, different CHOs became specialized for hearing in different insects.
This
hypothesis was recently confirmed by van Staaden and Romer (1998). In
Drosophila,
CHOs in the Johnston organ, located in the second antennal segment, function
in near
field hearing (Dreller and Kirschner, 1993; Eberl, 1999) and negative
geotaxis.
During development ato is expressed in a cluster of progenitor cells from
which
the CHO founder cells are selected (Jarman et al., 1993). It likely functions
by regulating
the expression of genes necessary for the specification and development of the
CHO
lineage; as it encodes a basic helix-loop-helix protein (bHLH) that dimerizes
with the
Daughterless protein and binds to E-box sequences, thereby activating genes
(Jarman et
al., 1993). CHO specificity is encoded by the ato basic domain, which is
required for
DNA binding in bHLH proteins (Chien et al., 1996; Davis et al., 1990; Jarman
and
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Ahmed, 1998; Vaessin et al., 1990). ato is both necessary and sufficient for
the
generation of CHOs in the fly: loss of ato function leads to the loss of CH0s,
while
ectopic ato expression causes ectopic CHO formation (Jarman et al., 1993).
Adult flies
that lack atonal function are uncoordinated, do not fly, and are deficient in
hearing.
Overexpression of the fly atonal gene can generate new chordotonal neurons,
indicating
that atonal is both essential and sufficient for the development of this
neuronal
population.
In vertebrates, during myogenesis and neurogenesis, cell fate specification
requires basis helix-loop-helix (bHLH) transcription factors. Math] (for mouse
atonal
homolog-1) is such a factor, and is expressed in the hindbrain, dorsal spinal
cord, external
germinal layer of the cerebellum, gut, joints, ear and Merkel cells of the
skin (which
function as mechanoreceptors) (Alcazawa et al.,1995; Ben-Arie et al., 1996;
Ben-Arie
et al., 1997). Mice heterozygous for a targeted deletion of Math] (Mathl") are
viable
and appear normal, but Math] null mice (Math-4) die shortly after birth and
lack
cerebellar granule neurons.
Math] is one of ato's closest known homologs, with 82% amino acid similarity
in the bHLH domain and 100% conservation of the basic domain that determines
target
recognition specificity (Ben-Arie et al., 1996; Chien et al., 1996). Math] is
transiently
expressed in the CNS starting at embryonic day 9 (E9) in the dorsal portion of
the neural
tube. Math] is also expressed in the rhombic lip of the fourth ventricle of
the brain,
where cerebellar granule cell precursors are born at E13-15 (Alder et al.,
1996). Upon
proliferation and differentiation, these progenitor cells migrate to form the
external
granule layer (EGL) of the cerebellar primordia (Hatten and Heintz, 1995).
Proliferating
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EGL cells continue to express Math I during the first three postnatal weeks,
until shortly
before they migrate to their final adult destination to generate the internal
granule layer
(IGL) of the cerebellum (Akazawa et al., 1995; Ben-Arie et al., 1996). Another
group
of cells, a small population of neuronal precursors in the dorsal spinal cord,
expresses
Math] during E10-E14 (Akazawa et al., 1995; Ben-Arie et al., 1996). These
precursor
cells also express the LIM homeodomain proteins (LH2A and LH2B), markers of
the DI
class of commissural interneurons (Lee et al., 1998). Helms and Johnson (1998)
reported
that lacZ expression under the control of Math] regulatory elements reproduced
Math/
expression patterns in the developing cerebellum and spinal cord, and
demonstrated that
Math] is expressed in precursors that give rise to a subpopulation of dorsal
commissural
interneurons.
To determine the in vivo function of Math] , the inventors generated mice
(Math]') lacking the MATH1 protein. This null mutation causes major cerebellar
abnormalities: lack of granule cell proliferation and migration from the
rhombic lip at
E14.5, and absence of the entire EGL at birth (Ben-Arie et al., 1997). It is
not clear
whether the agenesis of cerebellar granule neurons is due to failure of
progenitor
specification or the cells' inability to proliferate and/or differentiate.
Neonates cannot
breathe and die shortly after birth, but there are no gross defects in any
cranial nerves or
brain stem nuclei that could explain respiratory failure.
The fact that Math/ is expressed in the inner ear suggests that Math/
expression
is necessary for the development of auditory or balance organs. The inner ear
initially
forms as a thickening of the ectoderm, termed the otic placode, between
rhombomeres
5 and 6 in the hindbrain. The otic placode gives rise to neurons of the VIII'
cranial
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nerve and invaginates to become the otocyst, from which the inner ear will
develop. The
mature mammalian inner ear comprises one auditory organ, the cochlea, and five
vestibular organs: the utricle, the saccule, and three semicircular canals.
The sensory
epithelia of these organs consist of mechanoreceptive hair cells, supporting
cells and
nerve endings. Hair cells serve as mechanoreceptors for transducing sound
waves and
head motion into auditory and positional information. Hair cells and
supporting cells
both arise from a common progenitor cell and proliferate and differentiate
within the
sensory epithelia, with peak mitoses between embryonic day 13 and 18 (El 3-18)
in mice.
Although several genes have been implicated in the development of the inner
ear, such
as int2 (Mansour et al., 1993; pax2 (Torres et al., 1996; and Hmx3 (Wang et
al., 1998).
None have been shown to be required for the genesis of hair cell specifically.
Damage to hair cells is a common cause of deafness and vestibular dysfunction,
which are themselves prevalent diseases. Over 28 million Americans have
impaired
hearing; vestibular disorders affect about one-quarter of the general
population, and half
of our elderly. The delicate hair cells are vulnerable to disease, aging, and
environmental
trauma (i.e., antibiotics, toxins, persistent loud noise). Once these cells
are destroyed,
they cannot regenerate in mammals. Therefore, a need exists to address the
problems of
patients with congenital, chronic or acquired degenerative hearing impairment
and loss
or balance problems, and to provide compositions, methods and reagents for use
in
treating hearing loss and vestibular function.
In support of the teaching of the present invention, others have demonstrated
that
Math], upon overexpression, induces significant production of extra hair cells
in
postnatal rat inner ears (Zheng and Gao, 2000). Briefly, although fate
determination is
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usually completed by birth for mammalian cochlear hair cells, overexpression
of Math]
in postnatal rat cochlear explant cultures results in additional ear hair
cells which derive
from columnar epithelial cells located outside the sensory epithelium in the
greater
epithelial ridge. Furthermore, conversion ofpostnatal utricular supporting
cells into hair
cells is facilitated by Math] expression. The ability of Math] to permit
production of
hair cells in the ear is strong evidence in support of the claimed invention.
SUMMARY OF THE INVENTION
In one embodiment of the present invention there is an animal having a
heterologous nucleic acid sequence replacing an allele of an atonal-associated
nucleic
acid sequence under conditions wherein said heterologous sequence inactivates
said
allele. In a preferred embodiment said heterologous sequence is expressed
under control
of an atonal-associated regulatory sequence. In a specific embodiment both
atonal-
associated alleles are replaced. In an additional specific embodiment both
atonal-
associated alleles are replaced with nonidentical heterologous nucleic acid
sequences.
In an additional embodiment said animal has a detectable condition wherein
said
condition is selected from the group consisting of loss of hair cells,
cerebellar granule
neuron deficiencies, hearing impairment, imbalance, joint disease,
osteoarthritis,
abnormal proliferation of neoplastic neuroectodermal cells and formation of
medulloblastoma. In another embodiment of the present invention said
heterologous
nucleic acid sequence is a reporter sequence selected from the group
consisting of p-
galactosidase, green fluorescent protein (GFP), blue fluorescent protein
(BFP), neomycin,
kanamycin, luciferase, f3-glucuronidase and chloramphenicol transferase (CAT).
In
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another specific embodiment said reporter sequence regulatable or is expressed
in brain
tissue, neural tissue, skin tissue, non-ossified cartilage cells, joint
chondrocytes, Merkel
cells, inner ear sensory epithelia and brain stem nuclei. In additional
specific
embodiments said atonal-associated allele is replaced with an atonal-
associated nucleic
acid sequence under control of a regulatable promoter sequence or a tissue-
specific
promoter sequence wherein said tissue is selected from the group consisting of
brain
tissue, neural tissue, skin tissue, non-ossified cartilage cells, joint
chondrocytes, Merkel
cells, inner ear sensory epithelia and brain stem nuclei. In additional
embodiments said
animal is a mouse, Drosophila, zebrafish, frog, rat, hamster or guinea pig.
In another embodiment of the present invention is a method for screening for a
compound in an animal, wherein said compound affects expression of an atonal-
associated nucleic acid sequence comprising delivering said compound to said
animal
wherein said animal has at least one allele of an atonal-associated nucleic
acid sequence
inactivated by insertion of a heterologous nucleic acid sequence wherein said
heterologous nucleic acid sequence is under control of an atonal-associated
regulatory
sequence, and monitoring for a change in said expression of said atonal-
associated
nucleic acid sequence. In specific embodiments said compound upregulates or
downregulates said expression of an atonal-associated nucleic acid sequence.
In
additional embodiments said animal is a mouse or Drosophila. In a specific
embodiment
the heterologous nucleic acid sequence is a reporter sequence. In an
additional specific
embodiment the heterologous nucleic acid sequence is selected from the group
consisting
of 13-galactosidase, green fluorescent protein (GFP), blue fluorescent protein
(BFP),
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neomycin, kanamycin, luciferase, -glucuronidase and chloramphenicol
transferase
(CAT).
Another embodiment of the present invention is a compound which affects
expression of an atonal-associated nucleic acid sequence. In specific
embodiments said
compound upregulates or downregulates expression of an atonal-associated
nucleic acid
sequence. In a specific embodiment said compound affects a detectable
condition in an
animal wherein said condition is selected from the group consisting of loss of
hair cells,
cerebellar granule neuron deficiencies, hearing impairment, an imbalance
disorder, joint
disease, osteoarthritis, abnormal proliferation of neoplastic neuroectodermal
cells and
formation of medulloblastoma.
Another embodiment of the present invention is a method for screening for a
compound in an animal, wherein said compound affects a detectable condition in
said
animal, comprising delivering said compound to said animal wherein at least
one allele
of an atonal-associated nucleic acid sequence in said animal is inactivated by
insertion
of a heterologous nucleic acid sequence, wherein said heterologous nucleic
acid sequence
is under the control of an atonal-associated regulatory sequence, and
monitoring said
animal for a change in the detectable condition. In a specific embodiment said
detectable
condition is selected from the group consisting of loss of hair cells,
cerebellar granule
neuron deficiencies, hearing impairment, an imbalance disorder, joint disease,
osteoarthritis, abnormal proliferation of neoplastic neuroectodermal cells and
formation
of medulloblastoma. In another embodiment said delivery of said compound
affects
expression of said heterologous nucleic acid sequence. In specific embodiments
said
expression of said heterologous nucleic acid sequence is upregulated or
downregulated.
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In additional specific embodiments said animal is a mouse, Drosophila,
zebrafish, frog,
rat, hamster or guinea pig.
Another embodiment of the present invention is a compound wherein said
compound affects said detectable condition. In specific embodiments said
compound
affects expression of a heterologous nucleic acid sequence. In additional
specific
embodiments said compound upregulates or downregulates expression of a
heterologous
nucleic acid sequence.
In other embodiments of the present invention are methods of treating an
animal,
including a human, for cerebellar granule neuron deficiencies, for promoting
mechanoreceptive cell growth, for generating hair cells, for treating hearing
impairment
or an imbalance disorder, for treating a joint disease, for treating for an
abnormal
proliferation of cells, and for treating for a disease that is a result of
loss of functional
atonal-associated nucleic acid or amino acid sequence. Said methods include
administering a therapeutically effective amount of an atonal-associated
nucleic acid or
amino acid sequence. In specific embodiments said administration is by a
vector selected
from the group consisting o f an adenoviral vector, a retroviral vector, an
adeno-associated
vector, a plasmid, or any other nucleic acid based vector, a liposome, a
nucleic acid, a
peptide, a lipid, a carbohydrate and a combination thereof of said vectors. In
a specific
embodiment said vector is a non-viral vector or a viral vector. In another
specific
embodiment said vector is a cell. In a preferred embodiment said vector is an
adenovirus
vector comprising a cytomegalovirus IE promoter sequence and a SV40 early
polyadenylation signal sequence. In another specific embodiment said cell is a
human
cell. In an additional specific embodiment said joint disease is
osteoarthritis. In a
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specific embodiment said atonal-associated nucleic acid or amino acid sequence
is Hathl
or Math 1 . In another specific embodiment the cell contains an alteration in
an atonal-
associated nucleic acid or amino acid sequence. In an additional specific
embodiment
the amino acid sequence has at least about 80% identity to about 20 contiguous
amino
acid residues of SEQ ID NO:58. In an additional specific embodiment the
nucleic acid
sequence encodes a polypeptide which has at least about 80% identity to about
20
contiguous amino acid residues of SEQ ID NO:58.
In another embodiment of the present invention is a method for treating an
animal
for an abnormal proliferation of cells comprising altering atonal-associated
nucleic acid
or amino acid sequence levels in a cell. In a specific embodiment said
alteration is
reduction or said nucleic acid or amino acid sequence contains an alteration.
In another embodiment of the present invention is a composition comprising an
atonal-associated amino acid sequence or nucleic acid sequence in combination
with a
delivery vehicle wherein said vehicle delivers a therapeutically effective
amount of an
atonal-associated nucleic acid sequence or amino acid sequence into a cell. In
specific
embodiments said vehicle is the receptor-binding domain of a bacterial toxin
or any
fusion molecule or is a protein transduction domain. In a specific embodiment
said
protein transduction domain is from the HIV TAT peptide. In a specific
embodiment
said atonal-associated amino acid sequence or nucleic acid sequence is Hathl
or Mathl .
In another embodiment of the present invention there is a composition to treat
an
organism for loss of hair cells, wherein said organism comprises a defect in
an atonal-
associated nucleic acid sequence. In a specific embodiment the defect is a
mutation or
alteration of said atonal-associated nucleic acid sequence. In another
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embodiment the defect affects a regulatory sequence of said atonal-associated
nucleic
acid sequence. In an additional embodiment of the present invention there is a
composition to treat an organism for loss of hair cells, wherein said organism
comprises
defect in a nucleic acid sequence which is associated with regulation of an
atonal-
associated nucleic acid sequence. In an additional embodiment of the present
invention
there is a composition to treat an organism for loss of hair cells, wherein
said organism
comprises a defect in an amino acid sequence which is associated with
regulation of an
atonal-associated nucleic acid sequence.
In another embodiment of the present invention there is a composition to treat
an
organism for a cerebellar neuron deficiency, wherein said organism comprises a
defect
in an atonal-associated nucleic acid sequence. In a specific embodiment the
defect is a
mutation or alteration of said atonal-associated nucleic acid sequence. In
another specific
embodiment the defect affects a regulatory sequence of said atonal-associated
nucleic
acid sequence. In an additional embodiment of the present invention there is a
composition to treat an organism for a cerebellar neuron deficiency, wherein
said
organism comprises defect in a nucleic acid sequence which is associated with
regulation
of an atonal-associated nucleic acid sequence. In an additional embodiment of
the
present invention there is a composition to treat an organism for a cerebellar
neuron
deficiency, wherein said organism comprises a defect in an amino acid sequence
which
is associated with regulation of an atonal-associated nucleic acid sequence.
In another embodiment of the present invention there is a composition to treat
an
organism for hearing impairment, wherein said organism comprises a defect in
an atonal-
associated nucleic acid sequence. In a specific embodiment the defect is a
mutation or
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alteration of said atonal-associated nucleic acid sequence. In another
specific
embodiment the defect affects a regulatory sequence of said atonal-associated
nucleic
acid sequence. In an additional embodiment of the present invention there is a
composition to treat an organism for hearing impairment, wherein said organism
comprises defect in a nucleic acid sequence which is associated with
regulation of an
atonal-associated nucleic acid sequence. In an additional embodiment of the
present
invention there is a composition to treat an organism for hearing impairment,
wherein
said organism comprises a defect in an amino acid sequence which is associated
with
regulation of an atonal-associated nucleic acid sequence.
In another embodiment of the present invention there is a composition to treat
an
organism for imbalance, wherein said organism comprises a defect in an atonal-
associated nucleic acid sequence. In a specific embodiment the defect is a
mutation or
alteration of said atonal-associated nucleic acid sequence. In another
specific
embodiment the defect affects a regulatory sequence of said atonal-associated
nucleic
acid sequence. In an additional embodiment of the present invention there is a
composition to treat an organism for imbalance, wherein said organism
comprises defect
in a nucleic acid sequence which is associated with regulation of an atonal-
associated
nucleic acid sequence. In an additional embodiment of the present invention
there is a
composition to treat an organism for imbalance, wherein said organism
comprises a
defect in an amino acid sequence which is associated with regulation of an
atonal-
associated nucleic acid sequence.
In another embodiment of the present invention there is a composition to treat
an
organism for osteoarthritis, wherein said organism comprises a defect in an
atonal-
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associated nucleic acid sequence. In a specific embodiment the defect is a
mutation or
alteration of said atonal-associated nucleic acid sequence. In another
specific
embodiment the defect affects a regulatory sequence of said atonal-associated
nucleic
acid sequence. In an additional embodiment of the present invention there is a
composition to treat an organism for osteoarthritis, wherein said organism
comprises
defect in a nucleic acid sequence which is associated with regulation of an
atonal-
associated nucleic acid sequence. In an additional embodiment of the present
invention
there is a composition to treat an organism for osteoarthritis, wherein said
organism
comprises a defect in an amino acid sequence which is associated with
regulation of an
atonal-associated nucleic acid sequence.
In another embodiment of the present invention there is a composition to treat
an
organism for a joint disease, wherein said organism comprises a defect in an
atonal-
associated nucleic acid sequence. In a specific embodiment the defect is a
mutation or
alteration of said atonal-associated nucleic acid sequence. In another
specific
embodiment the defect affects a regulatory sequence of said atonal-associated
nucleic
acid sequence. In an additional embodiment of the present invention there is a
composition to treat an organism for a joint disease, wherein said organism
comprises
defect in a nucleic acid sequence which is associated with regulation of an
atonal-
associated nucleic acid sequence. In an additional embodiment of the present
invention
there is a composition to treat an organism for a joint disease, wherein said
organism
comprises a defect in an amino acid sequence which is associated with
regulation of an
atonal-associated nucleic acid sequence.
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In another embodiment of the present invention there is a composition to treat
an
organism for abnormal proliferation of cells, wherein said organism comprises
a defect
in an atonal-associated nucleic acid sequence. In a specific embodiment the
defect is a
mutation or alteration of said atonal-associated nucleic acid sequence. In
another specific
embodiment the defect affects a regulatory sequence of said atonal-associated
nucleic
acid sequence. In an additional embodiment of the present invention there is a
composition to treat an organism for abnormal proliferation of cells, wherein
said
organism comprises defect in a nucleic acid sequence which is associated with
regulation
of an atonal-associated nucleic acid sequence. In an additional embodiment of
the
present invention there is a composition to treat an organism for abnormal
proliferation
of cells, wherein said organism comprises a defect in an amino acid sequence
which is
associated with regulation of an atonal-associated nucleic acid sequence.
In another embodiment of the present invention there is a composition to treat
an
organism for cancer, wherein said organism comprises a defect in an atonal-
associated
nucleic acid sequence. In a specific embodiment the defect is a mutation or
alteration of
said atonal-associated nucleic acid sequence. In another specific embodiment
the defect
affects a regulatory sequence of said atonal-associated nucleic acid sequence.
In an
additional embodiment of the present invention there is a composition to treat
an
organism for cancer, wherein said organism comprises defect in a nucleic acid
sequence
which is associated with regulation of an atonal-associated nucleic acid
sequence. In an
additional embodiment of the present invention there is a composition to treat
an
organism for cancer, wherein said organism comprises a defect in an amino acid
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sequence which is associated with regulation of an atonal-associated nucleic
acid
sequence. In a specific embodiment said cancer is medulloblastoma.
Other and further objects, features and advantages would be apparent and
eventually more readily understood by reading the following specification and
by
reference to the company drawing forming a part thereof, or any examples of
the
presently preferred embodiments of the invention are given for the purpose of
the
disclosure.
DESCRIPTION OF THE DRAWINGS
Figure lA and 1B demonstrate that the inner ear 0-Gal staining (blue) of Mathl
heterozygous embryos as described hereinabove. Figure IA shows the otic
vesicle (OV)
at E12.5 and Figure 1B the inner ear at E14.5 of Mathl'i' embryos. Sensory
epithelia
stained positively in the cochlea (C), saccule (S), utricle (U), and
semicircular canal
ampullae (SCA). A schematic diagram of the inner ear is depicted alongside the
staining
for reference, blue indicates location of the sensory epithelia. The original
magnifications of the images taken under the microscope were x100 For Figure
lA and
x50 for Figure 1B.
Figures 2A through 2F are scanning electron micrographs of E18.5 inner ear
sensory epithelia in wild-type and Mathlfl'I'' mice. Wild-type mice epithelia
are
shown in Figures 2A, 2C, and 2E and null mouse epithelia in Figures 2B, 2D,
and 2F.
The organ of Corti of the cochlea are shown and indicated in Figures 2A and
2B. In the
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wild-type mouse there are three rows of outer hair cells (1, 2, 3), one row of
inner hair
cells (I), all with hair bundles (HB). The tectorial membrane (TM), an
accessory
structure of the cochlea, may be observed at the bottom. Above the sensory
epithelium
are squamous cells (SQ) with rudimentary kinocilia (RK). In null mice (Figure
2B), there
are only squamous cells. Crista ampullaries of a vertical semicircular canal
are depicted
in Figures 2C and 2D. The null mouse crista is similar to the wild-type in
overall shape,
including the septum (eminentia) cruciatum (EC), but is smaller. The macula of
the
uticle is the focus of Figure 2E and 2F. Again, the macula of the null mouse
is smaller
than the wild-type. Scale bars are as follows: 10 p.m in Figures 2A and 2B, 50
pin in
Figures 2C and 2D, and 100 j.rm in Figures 2E and 2F.
Figures 3A through 3F are light micrographs of semi-thin transverse sections
of
inner ear sensory epithelia in wild-type mice (Figures 3A, 3C, and 3E) and
Mathlfl-Galal
(Figures 3B, 3D, and 3F), all mice were observed at E18.5. As observed in the
cochlea
of wild-type mice, Figure 3A, three outer hair cells (1, 2, 3) and one inner
(I) hair cell are
present. Conversely, the null mouse cochlea (Figure 3B) has only squamous
cells (SQ)
in the same region. Hair cells (HC) and supporting cells (SC) are present in
the wild-type
crista ampullaris (Figure 3C) and utricular macula (Figure 3E), but only
supporting cells
are present in null mice (Figure 3D and 3F). The crista was cut obliquely,
accounting for
the multiple layers of hair cells in Figure 3C. The otolithic membrane (OM),
an
accessory structure of the utricle, is present in both wild-type mice (Figure
3E) and null
mice (Figure 3F). Scale bars equal 100 gm in (Figures 3A and 3B); 50 gm in
(Figures
3C and 3D); and 25 pm in (Figures 3E and 3F).
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Figures 4A and 4B are transmission electron micrographs of E18.5 utricular
macula in wild-type and Math Ifl'aufl-'1 mice. Figure 4A shows that the hair
cells (HC)
and supporting cells (SC) are present in wild-type utricular macula. By
contrast, only
supporting cells are present in the null mouse (Figure 4B). Hair cells have
hair bundles
(FIB) and supporting cells have miicrovilli (MV). Hair cells are less electron-
dense and
have more apical nuclei than supporting cells, but only the latter have
secretory granules
(SG). Some immature hair cells (JIM) are evident in the wild-type, but not in
the null
mouse. The scale bar in all the figures equals 1011m.
Figures 5A through 5F show the Calretinin staining pattern of inner ear
sensory
epithelia. Sections through the utricle of E16.5 wild-type (Figures AS and 5C)
and
Math] fl-Gal/fl-Gal (Figure 5B and 5D) littermates were counterstained with
propidium iodide
for confocal microscopy. Sections were cut through the crista ampullaris of
E18.5
wild-type (Figure 5E) and Math lfl'avfl-Gal (Figure 5F) were counterstained
with DAPI
for immunofluorescent microscopy. The crista is cut at an oblique angle, which
accounts for the multiple layers of hair cells in (Figure 5E). Immunostaining
of
Calretinin (arrows) is evident in hair cells of wild-type (Figures 5A, 5C, and
5E)
but not null mice (Figures 5B, 5D, and 5F). Boxed areas in Figures 5A and 5B
indicate
the regions magnified in Figures 5C and 5D. Scale bar equals 100 p.m in
(Figure 5A and
5B), 15 p.m in (Figures 5C and 5D) and an original magnification of x200 in
(Figures 5E
and 5F).
Figure 6A and 6B show the expression pattern of Math] in mouse articular
cartilage using the _Math/ heterozygote. Figure 6A shows the staining
pattern of a
P14 mouse forelimb and demonstrates expression in all joints. Figure 6B is a
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magnification (20X) of an elbow joint from the same mouse that demonstrates
that Math]
is expressed exclusively in the non-ossified articular chondrocytes.
Figure 7A through 7C show replacement of Math] coding region by lacZ gene.
Figure 7A, Top, has a map of the Mathl genomic locus. The coding region is
shown as
a black box. The sites of the probes used to detect the wild-type and mutant
alleles are
shown as black bars. The targeting vector is in the middle with the sites for
homologous
recombination indicated by larger Xs. In the targeted locus shown at the
bottom, lacZ
is translated under the control of Math] regulatory elements. Figure 7B
demonstrates
Southern blot analysis of embryonic stem cells using the 3' external probe.
The upper
band represents wt allele and the lower band the targeted mutant allele (mut)
in targeted
clones. Figure 7C demonstrates Southern blot analysis of DNA from the progeny
of
heterozygous mice demonstrating the presence of the targeted allele and
absence of the
wild-type allele in Mathl b-galitl-gal mice (asterisks). The abbreviations are
as follows: (A)
ApaI; (H) Hindi-II; (RI) EcoRI; (S) Sall; and (X) XbaI.
Figures 8A through 8H show Mathl/lacZ expression and cerebellar phenotype
in Math] 'gal and Mathl blaill"al mice. Figure 8A shows Mathl/lacZ expression
in the
dorsal neural tube at E9.5 and (Figure 8B) E10.5. Figure 8C indicates a
section through
the hind brain at E10.5 has Mathl/lacZ expression in the dorsal portion
(arrows). Figure
8D demonstrates that in a spinal cord section from E12.5 embryo, dorsal cells
migrate
ventrally (arrows). Figure 8E shows at E14.5 expression is observed in the EGL
progenitors at the rhombic lip and in migrating cells that will populate the
EGL. Figure
8F demonstrates in Math] b-gailbial mice, Math] /lacZ expression is limited to
a few cells
in the rhombic lip, which is significantly reduced in size. Figure 8G shows
that at PO
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Mathl/lacZ is expressed in the EGL. Figure 8H demonstrates that the EGL is
absent in
the null mice. Original magnification for Figures 8C through 8H was 1 00x.
Figure 9A through 9G shows expression ofMathl/lacZ in the inner ear and brain
stem and histological analysis of ventral pontine nucleus. X-gal staining of
El 8.5
Mathl'gal utricular crista (Figure 9A) and inner ear sensory epithelia of
MathI'gal
(Figure 9B) and Math/ boTho (Figure 9C). The Mathl/lacZ expression in the
upper hair
cell layer of the sensory epithelia of (Figures 9A and 9B) and the
characteristic calyx
appearance (arrowhead). In the null mice X-gal staining of epithelial cells is
non-specific
in the absence of hair cells (Figure 9C). Whole-brain X-gal staining of
Mathl'gal
(Figure 9D) and Math/bgaubgaT (Figure 9E) at E18.5 is demonstrated. There is
positive
staining of the pontine nucleus (arrowhead) and cerebellum (arrow) in Math)ib-
0 mice,
which is lacking or greatly reduced in null mutants in both the cerebellum,
and pontine
nucleus (inset). Figures 9F and 9G show haematoxylin and eosin staining of
sagittal
sections through the pons of a wild type and null mutant (Figures 9F and 9G,
respectively), showing the loss of the ventral pontine nucleus in null
mutants. The
original magnifications were as follows: (A) 400x (B & C) 1000x, (D & E) 8x,
inset in
D &E 100x, (F & G) 10x.
Figures 10A through 10E demonstrate Mathl /lacZ is expressed in joint
chondrocytes. X-gal staining of whole embryos at (Figure 10A) E12.5 and
(Figure 10B)
E16.5 illustrates that Mathl/lacZ is expressed in all joints (Figure 10C).
Horizontal
section through the elbow joint of El 8.5 Math) 'O mouse shows that it is
expressed in
resting chondrocytes (arrow). Figure 10D shows a horizontal section through a
humero-radial joint at P10 that has expression in the articular chondrocytes
(arrowhead)
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and resting chondrocytes (arrow). Figure 10E shows high magnification of a
section
through a wrist joint indicating Math 1 /lacZ is expressed in articular
chondrocytes. The
original magnification is was follows: (C) 10x; (D) 20x; and (E) 40x.
Figures 11A through 11L show Math I /lacZ expression in Merkel cells. To
identify the structures stained on the hairy and non-hairy skin, El 6.5
littermate embryos
were stained as whole mounts, sectioned, and microscopically examined. Shown
are
sections through the vibrissae (Figure 11A), foot pad at low (Figure 11B) and
high
magnification of the region marked by an arrow in B (Figure 10C), and hairy
skin (Figure
11D). In all sections the localization of the stained cells was as expected
from Merkel
cells. To look for macroscopic defects in null mice, close-up pictures were
taken through
a stereomicroscope of Mat111'0 (control, panels E-H) and Math 1 b-ga'gal
(null, panels
I-L) littermate mice. Staining in null mice appeared stronger because of a
dosage effect
in the vibrissae (E, I), limb joints (F, J), and foot pads (G, K). In
contrast, the staining
intensity of null (J, L) mice was markedly weaker than that of heterozygous
(F, H) mice
in the touch domes associated with the hairy skin. The original magnification
is was
follows: A x200; B x50; C x400; D x500; E-G-H-I-K-L x32; F-J x16.
Figures 12A through 12E show lack of /acZ-stained touch domes in Tabby mice.
Tabby/Tabby females were crossed with Math]'fb-0 males, and their progeny were
X-gal
stained and gender-determined at E16.5. Staining around primary vibrissae in
the snout
was detected in both female embryos heterozygous for the Tabby mutation
(Figure 12A)
and male embryos hemizygous for the mutation (Figure 12B). Secondary
vibrissae,
which are known to vary in number in the Tabby mutants (black arrows), were
also
stained. The staining of the touch domes was less intense in the Tabby/Xfemale
(Figure
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12D) than Mad/rib-gal (wt for Tabby) embryos (Figure 12C), since Tabby is a
semidominant mutation. However, patches of stained touch domes were detected
in a
female embryo that carried a wild-type allele at the Tabby locus (Figure 12A,
red arrow,
and 12D). In contrast, a hemizygous male completely lacked both staining and
touch
domes, due to the loss of hair follicles that abolishes the development of
Merkel cells
(Figures 12B and 12E).
Figures 13 A through 13F demonstrate marker analysis of Merkel cells in wild
type and Math I null mice. Skin sections from Math 1" and Math] b-galib-gal
reacted with
antibodies against MATH1 (Figures 13A and 13B), cytokeratin 18 (Figures 13C
and
13D), and ehromogranin A (Figures 13E and 13F). Polyclonal antibodies to MATH1
identify multiple basal nuclei in rare abdominal hair follicles of wild type
(Figure 13A)
but not mutant mice (Figure 13B). Monoclonal antibodies to cytokeratin 18 and
chromogranin A identify Merkel cells in both wild type (Figures 13C and 13E)
and
mutant (Figures 13D and 13F) mice. The original magnification was 100X.
Figures 14A through 14G show Math] rescues the lack of chordotonal neurons
in Drosophila ato mutant embryos. Figure 14A shows a dorsal view of the thorax
of a
wild-type fly. Note there are regular array of bristles or macrochaetae.
Figure 14B
shows a similar view of a transgenic fly in which Math] was overexpressed
using the
UAS/GAL4 system (Brand and Perrimon, 1993). This ectopic expression leads to
numerous extra bristles that are external sensory organs (another type of
mechano
receptor), not CHOs. Ectopic CHOs were produced in many other regions. Figure
14C
shows a lateral view of two abdominal clusters containing 6 CHOs in addition
to external
sensory organs, revealed by a neuronal-specific antibody (Mab 22C10). The 5
lateral
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CHOs form a cluster, and the sixth is dorsal to the cluster. Figure 14D shows
a similar view
of an ato mutant embryo showing lack of the CHOs. Figure I 4E demonstrates
ubiquitous
expression of Math] induces new CHO neurons in ato mutant embryos in the
proper
location. Figure 14F shows in situ hybridization of whole mount third instar
brain using the
ato cDNA as a probe. Note expression in the developing optic lobes ("horse
shoe"
expression patterns) and two punctate clusters of cells in the middle of the
brain lobes
(arrow heads). Figure 14G shows Math] expression in Drosophila induces CHO
formation
in normal and ectopic locations. The (+) indicates presence of CHOs and (-)
indicates their
absence. Number of (+) in the first column is used to quantify the relative
increase in the
number of CHOs observed when Math] is expressed.
DETAILED DESCRIPTION OF THE INVENTION
It is readily apparent to one skilled in the art that various embodiments and
modifications may be made to the invention disclosed in this Application
without
departing from the scope of the invention. The scope of the claims should not
be limited
by particular embodiments set forth herein, but should be construed in a
manner
consistent with the specification as a whole.
The term "abnormal proliferation" as used herein is defined as any
proliferation
of any type of cell, wherein said cell is not under the constraints of normal
cell cycle
progression and wherein said proliferation may result in a tumor or any
cancerous
development.
The term "alteration" as used herein is defined as any type of change or
modification to a nucleic acid or amino acid. Said change or modification
includes any
mutation, deletion, rearrangment, addition to a nucleic acid. This includes
posttranscriptional processing such as addition of a 5' cap, intron processing
and
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polyadenylation. Mutations can be nonsense, missense, frameshi ft, or could
lead to a
truncated amino acid or could alter the conformation of the amino acid. The
alteration
to a nucleic acid may be present in regulatory sequences or may affect trans-
acting
factors. Also, multiple alterations may be present. Said change or
modification also
includes any change to an amino acid including methylation, myristilation,
acetylation,
glycosylation, or a change to signals associated with processing of said amino
acid
including intracellular or intercellular localization signals and cleaving of
extraneous
amino acids. Said alteration may also affect degradation or folding of said
protein.
The term "atonal-associated" as used herein is defined as any nucleic acid
sequence or amino acid sequence which is the Drosophila atonal nucleic acid
sequence
or amino acid sequence, or is any sequence which is homologous to or has
significant
sequence similarity to said nucleic acid or amino acid sequence, respectively.
The
sequence can be present in any animal including mammals and insects. As used
herein,
significant sequence similarity means similarity is greater than 25% and can
occur in any
region of another sequence. Examples of atonal-associated include but are not
limited
to Math] (mouse atonal homolog D, Cathl (chicken atonal homolog 1), Hathl
(human
atonal homolog 1), and Xathl (Xenopus atonal homolog 1). Furthermore, multiple
homologous or similar sequences may exist in an animal.
The term "cerebellar granule neuron deficiencies" as used herein is defined as
any
deficiency associated with cerebellar granule neurons and can include loss of
cerebellar
granule neurons, cerebellar granule neuron precursor cells, lack of granule
cell
proliferation, lack of granule cell migration and lack of cerebellar external
granule layer
cells.
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The term "defect" as used herein is defined as an alteration, mutation, flaw
or loss
of expression of an atonal-associated sequence. A skilled artisan is aware
that loss of
expression concerns expression levels of an atonal-associated sequence which
are not
significant or detectable by standard means in the art. A skilled artisan is
also aware that
loss, or absence, of expression levels in an adult organism, such as a human,
occurs
naturally and leads to impairment of hearing over time. Thus, "defect" as used
herein
includes the natural reduction or loss of expression of an atonal-associated
sequence.
The term "delivering" as used herein is defined as bringing to a destination
and
includes administering, as for a therapeutic purpose.
The term "delivery vehicle" as used herein is defined as an entity which is
associated with transfer of another entity. Said delivery vehicle is selected
from the
group consisting of an adenoviral vector, a retroviral vector, an adeno-
associated vector,
a plasmid, a liposome, a nucleic acid, a peptide, a lipid, a carbohydrate and
a combination
thereof.
The term "detectable condition" as used herein is defined as any state of
health
or status of an animal, organ or tissue characterized by specific
developmental or
pathological symptoms. Examples include but are not limited to loss of hair
cells,
cerebellar granule neuron deficiencies, hearing impairment, imbalance, joint
disease,
osteoarthritis and abnormal proliferation of cells.
The term "heterologous" as used herein is defined as nucleic acid sequence
which
is of or relating to nucleic acid sequence not naturally occurring in a
particular locus. In
an alternative embodiment, the heterologous nucleic acid sequence naturally
occurs in
a particular locus, but contains a molecular alteration compared to the
naturally occurring
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locus. For instance, a wild-type locus of an atonal-associated sequence may be
used to
replace a defective copy of the same sequence.
The term "imbalance disorder" as used herein is defined as a medical condition
wherein an organism has impaired balance. In a specific embodiment the
impairment is
due to a defect of vestibular origin. In another specific embodiment the
disorder is a
vestibular disorder of balance perception including but not limited to Meniere
disease,
vertigo and layrinthitis.
The term "inactivated" as used herein is defined as a state in which
expression of
a nucleic acid sequence is reduced or completely eliminated. Said inactivation
can occur
by transfer or insertion of another nucleic acid sequence or by any means
standard in the
art to affect expression levels of a nucleic acid sequence.
The term "precursors" as used herein is defined as progenitor cells from which
other cells derive their origin and/or properties.
The term "regulatable reporter sequence" as used herein is defined as any
sequence which directs transcription of another sequence and which itself is
under
regulatory control by an extrinsic factor or state. Examples of extrinsic
factors or states
include but are not limited to exposure to chemicals, nucleic acids, proteins,
peptides,
lipids, carbohydrates, sugars, light, sound, hormones, touch, or tissue-
specific milieu.
Examples of regulatable reporter sequences include the GAL promoter sequence
and the
tetracycline promoter/transactivator sequence.
The term "regulatory sequence" as used herein is defined as any sequence which
controls either directly or indirectly the transcription of another sequence.
Said control
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can be either regarding the initiation or cessation of transcription or
regarding quantity
or tissue distribution of transcription.
The term "reporter sequence" as used herein is defined as any sequence which
demonstrates expression by a regulatory sequence. Said reporter sequence can
be used
as a marker in the form of an RNA or in a protein. Examples of reporter
sequences are
b-galactosidase, green fluorescent protein (GFP), blue fluorescent protein
(BFP),
neomycin, kanamycin, luciferase, b-glucuronidase and chloramphenicol
transferase
(CAT). In a specific aspect of the present invention, the presence and
quantity of the
reporter sequence product, whether it be a nucleic acid or amino acid,
reflects the level
of transcription by the promoter sequence which regulates it.
The term "therapeutically effective" as used herein is defined as the amount
of
a compound required to improve some symptom associated with a disease. For
example,
in the treatment of hearing impairment, a compound which improves hearing to
any
degree or arrests any symptom of hearing impairment would be therapeutically
effective.
In the treatment of a joint disease, a compound which improves the health or
movement
of a joint to any degree or arrests any symptom of a joint disease would be
therapeutically
effective. In the treatment of abnormal proliferation of cells, a compound
which reduces
the proliferation would be therapeutically effective. In the treatment of
cancer, a
compound which reduces proliferation of the cells, reduces tumor size, reduces
metastases, reduces proliferation of blood vessels to said cancer, facilitates
an immune
response against the cancer would be therapeutically effective, for example. A
therapeutically effective amount of a compound is not required to cure a
disease but will
provide a treatment for a disease.
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The term "vector" as used herein is defined as a biological vehicle for
delivery
of a specific entity. In a specific embodiment the entity is an atonal-
associated nucleic
acid.
In one aspect of the present invention there are methods and reagents which
include utilization of an atonal-associated nucleic acid or amino acid
sequence for the
therapeutic use of detectible conditions such as loss of hair cells,
cerebellar granule
neuron deficiencies, hearing impairment, an imbalance disorder, joint disease,
osteoarthritis and abnormal proliferation cells. Thus, any homolog or ortholog
of atonal
(from Drosophila) including but not limited to Cathl (from chicken), Hathl
(from
human), Mathl (from mice) or Xathl (from Xenopus) may be used in the present
invention. In a preferred embodiment these sequences are directed to treatment
of an
animal, specifically a human, for the detectable conditions stated above. It
is within the
scope of the invention to encompass any sequence which is homologous to or has
significant sequence similarity to said nucleic acid or amino acid sequence,
respectively.
The sequence can be present in any animal including mammals and insects. As
used
herein, significant sequence similarity means similarity (identity of amino
acid residues
or nucleic acid bases) is greater than 25% and can occur in any region of the
sequence.
In another embodiment an atonal-associated sequence as used herein has greater
than
. about 50% sequence similarity, greater than about 70% similarity, or greater
than about
80% similarity.
It is within the scope of the present invention that an atonal-associated
nucleic
acid sequence or amino acid sequence is utilized wherein domains important for
activity,
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such as the basic HLH region, are included in a molecule but further comprise
alterations,
mutations, deletions or substitutions in regions of the nucleic acid or amino
acid sequence
which are not part of a domain important for an activity and do not affect its
function.
Examples of atonal-associated include but are not limited to Math! (mouse
atonal
homolog 1), Cath I (chicken atonal homolog 1), Hath l (human atonal homolog
1), and
Xath I (Xenopus atonal homolog 1). Such examples are represented in SEQ ID
NO:!
through SEQ ID NO:66, although others very likely exist in related organisms.
A skilled
artisan is cognizant of means to identify such sequences which have
significant
similarity, such as searching database collections of nucleic and amino acid
sequence
located on the World Wide Web.
The sequences provided herein and the corresponding GenBank Accession
numbers are listed parenthetically as follows: SEQ ID NO:1 (NI\4_005172); SEQ
ID
NO:2 (NP_005163.1 ); SEQ 1D NO:3 (AW413228); SEQ ID NO: 4 (NM 009719); SEQ
1D NO:5 (NP 033849.1); SEQ ID NO:6 (NM 009718); SEQ ID NO: 7 (NP_033848.1)
SEQ ID NO:8 (NM 009717); SEQ ID NO: 9 (NP_033847.1); SEQ ID NO:10
(NM_007500); SEQ NO: 1 1 (NP_031526.1); SEQ ID NO:12 (NM_007501 ); SEQ ID
NO:13 (AW280518); SEQ ID NO:14(AW236965 ); SEQ ID NO:15(AW163683); SEQ
ID NO:16 (AF134869); SEQ ID NO: 17(AAD31451.1); SEQ ID NO:18 (AJ012660);
SEQ ID NO:19 (CAA10106.1); SEQ ID NO:20 (AJ012659); SEQ ID NO:21
(CAA10105.1); SEQ lD NO:22 (AF071223); SEQ ID NO:23 (AAC68868.1); SEQ ID
NO:24 (U76208); SEQ ID NO:25 (AAC53029.1); SEQ ID NO:26 (U76210); SEQ ID
NO:27 (AAC53033.1); SEQ ID NO:28 (U76209); SEQ ID NO:29 (AAC53032.1); SEQ
*Trade-mark
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ID NO:30 (U76207); SEQ ID NO:31 (AAC53028.1); SEQ ID NO:32 (AF036257); SEQ
ID NO:33 (AAC15969.1); SEQ ID NO:34 (AF034778); SEQ ID NO:35 (AJ001178);
SEQ ID NO:36 (CAA04572.1); SEQ ID NO:37 (Y07621); SEQ ID NO:38
(CAA68900.1); SEQ ID NO:39 (AF024536); SEQ ID NO:40 (AAB82272.1); SEQ ID
NO:41 (D85188); SEQ ID NO:42 (BAA12738.1); SEQ ID NO:43 (D44480); SEQ ID
NO:44(BAA07923.1); SEQ ID NO:45 (D43694); SEQ ID NO:46 (BAA07791.1); SEQ
ID NO:47 (D85845); SEQ ID NO:48 (BAA12880.1); SEQ ID NO:49 (U93171); SEQ ID
NO:50 (AAB58669.1); SEQ ID NO:51 (U93170); SEQ ID NO:52 (AAB58668.1); SEQ
ID NO:53 (U61152); SEQ ID NO:54 (AAB41307.1); SEQ ID NO:55 (U61151); SEQ ID
NO:56 (AA1341306.1); SEQ ID NO:57 (U61148); SEQ ID NO:58 (AAB41305.1); SEQ
ID NO:59 (U61149); SEQ ID NO:60 (AAB41304.1); SEQ ID NO:61 (U61150); SEQ ID
NO:62 (AAB41303.1); SEQ ID NO:63 (L36646); SEQ ID NO:64 (AAA21879.1); SEQ
ID NO:69 (AA625732).
In an aspect of the invention there is an animal having a heterologous nucleic
acid
sequence replacing an allele of an atonal-associated nucleic acid sequence
under
conditions wherein said heterologous sequence inactivates said allele. In an
alternative
embodiment a heterologous sequence is delivered to a cell for extrachromosomal
propagation. In another alternative embodiment a heterologous sequence is
integrated
into the chromosome of a cell in a locus other than the locus of an atonal-
associated
nucleic acid sequence. In a preferred embodiment said heterologous sequence is
expressed under control of an atonal-associated regulatory sequence. In a
specific
embodiment both atonal-associated alleles are replaced. In an additional
specific
embodiment both atonal-associated alleles are replaced with nonidentical
heterologous
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nucleic acid sequences. Methods to generate transgenic animals are well known
in the
art, and a skilled artisan would refer to such references as Transgenic
Animals by
Grosveld and Kollias (eds.) or Mouse Genetics and Transgenics : A Practical
Approach
by Jackson et al. (eds.).
In an additional embodiment a transgenic animal of the present invention has a
detectable condition wherein said condition is selected from the group
consisting of loss
ofhair cells, cerebellar granule neuron deficiencies, hearing impairment,
imbalance, joint
disease, osteoarthritis and abnormal proliferation of cells. In another
embodiment of the
present invention a heterologous nucleic acid sequence is a reporter sequence
selected
from the group consisting of P-galactosidase, green fluorescent protein (GFP),
blue
fluorescent protein (BFP), neomycin, kanamycin, luciferase, P-glucuronidase
and
chlorampheni col transferase (CAT). In another specific embodiment, a reporter
sequence
is regulatable or is expressed in brain tissue, neural tissue, skin tissue,
non-ossified
cartilage cells, joint chondrocytes, Merkel cells, inner ear epithelial cells
and brain stem
nuclei. In additional specific embodiments said atonal-associated allele is
replaced with
an atonal-associated nucleic acid sequence under control of a regulatable
promoter
sequence or a tissue-specific promoter sequence wherein said tissue is
selected from the
group consisting of brain tissue, neural tissue, skin tissue, non-ossified
cartilage cells,
joint chondrocytes, Merkel cells, inner ear epithelial cells and brain stem
nuclei. In
additional embodiments a transgenic animal is a mouse, Drosophila, frog,
zebrafish, rat,
guinea pig, or hamster.
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In another embodiment of the present invention is a method for screening for a
compound in an animal, wherein said compound affects expression of an atonal-
associated nucleic acid sequence comprising delivering said compound to said
animal
wherein said animal has at least one allele of an atonal-associated nucleic
acid sequence
inactivated by insertion of a heterologous nucleic acid sequence wherein said
heterologous nucleic acid sequence is under control of an atonal-associated
regulatory
sequence, and monitoring for a change in said expression of said atonal-
associated
nucleic acid sequence. Examples of regulatory sequences may include promoter
sequences, enhancers or silencers.
In specific embodiments there is a compound which upregulates or downregulates
said expression of an atonal-associated nucleic acid sequence. The
upregulation or
downregulation may be by increasing the rate of transcription or decreasing
the rate of
mRNA decay.
Another embodiment of the present invention is a compound which affects
expression of an atonal-associated nucleic acid sequence. In specific
embodiments said
compound upregulates or downregulates expression of an atonal-associated
nucleic acid
sequence. In a specific embodiment said compound affects a detectable
condition in an
animal wherein said condition is selected from the group consisting of loss of
hair cells,
cerebellar granule neuron deficiencies, hearing impairment, an imbalance
disorder, joint
disease, osteoarthritis, abnormal proliferation of cells and formation of
cancer.
Another embodiment of the present invention is a method for screening for a
compound in an animal, wherein the compound affects a detectable condition in
the
animal, comprising delivering the compound to the animal wherein at least one
allele of
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an atonal-associated nucleic acid sequence in said animal is inactivated by
insertion of
a heterologous nucleic acid sequence, wherein said heterologous nucleic acid
sequence
is under the control of an atonal-associated regulatory sequence, and
monitoring said
animal for a change in the detectable condition. In a specific embodiment said
detectable
condition is selected from the group consisting of loss of hair cells,
cerebellar granule
neuron deficiencies, hearing impairment, an imbalance disorder, joint disease,
osteoarthritis and abnormal proliferation of cells. In another embodiment said
delivery
of said compound affects expression of said heterologous nucleic acid
sequence. In
specific embodiments said expression of said heterologous nucleic acid
sequence is
upregulated or downregulated. In additional specific embodiments the animal is
a mouse,
Drosophila, frog, zebrafish, rat, hamster and guinea pig.
Another embodiment of the present invention is a compound wherein said
compound affects a detectable condition in a transgenic animal of the present
invention.
In specific embodiments said compound affects expression of a heterologous
nucleic acid
sequence. In additional specific embodiments said compound upregulates or
downregulates expression of a heterologous nucleic acid sequence.
In other embodiments of the present invention are methods of treating an
animal,
including a human, for cerebellar granule neuron deficiencies, for promoting
mechanoreceptive cell growth, for generating hair cells, for treating hearing
impairment
or imbalance, for treating a joint disease, for treating for an abnormal
proliferation of
cells, and for treating for a disease that is a result of loss of functional
atonal-associated
nucleic acid or amino acid sequence. Said methods include administering a
therapeutically effective amount of an atonal-associated nucleic acid or amino
acid
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sequence. In specific embodiments said administration is by a vector selected
from the
group consisting of a viral vector (including bacteriophage, animal and plant
viruses), a
plasmid, cosmid or any other nucleic acid based vector, a liposome, a nucleic
acid, a
peptide, a lipid, a carbohydrate and a combination thereof of said vectors. In
a specific
embodiment said viral vector is an adenovirus vector, a retrovirus vector, or
an adeno-
associated vector, including a lentivirus vector, Herpes virus vector, alpha
virus vector,
etc. Thus, the vector may be viral or non-viral. In a preferred embodiment
said vector
is an adenovirus vector comprising a cytomegalovirus IE promoter sequence and
a SV40
early polyadenylation signal sequence. In another specific embodiment said
cell is a
human cell. In an additional specific embodiment said joint disease is
osteoarthritis. In
an additional specific embodiment the cell contains an alteration in an atonal-
associated
amino acid sequence, wherein said amino acid sequence has at least about 80%
identity
to about 20 contiguous amino acid residues of SEQ ID NO:58.
In a specific embodiment, the present invention also provides a method of
treating
an animal in need of treatment for a deficiency in cerebellar granule neurons,
a hearing
impairment, an imbalance disorder, a joint disease, or in need of promoting
mechanoreceptive cell growth, or a disease that is a result of loss of
functional atonal-
associated nucleic acid or amino acid sequences. This method comprises
delivering a
transcription factor having an amino acid with at least about 70% identity,
preferably at
least about 80% identity, and more preferably at least about 90% identity to
the sequence
AANARERRRMHGLNHAFDQLR (SEQ ID NO: 70) to a cell in the animal.
In some embodiments, the cell in the animal is located in the inner ear of the
animal.
Preferably, the transcription
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factor competes with atonal for binding to Daughterless protein (Jarman et
al., 1993) or
competes for binding with Math-1 to E47 protein (Akazawa et al., 1995).
In an embodiment of the present invention there is provided a method for
treating
an organism for a disease that is a result of loss of functional atonal-
associated nucleic
acid or amino acid sequence. A skilled artisan is aware that this loss may be
due to
natural reduction or absence of significant (or to detectable levels)
expression which
occurs in an adult human.
In another embodiment of the present invention is a method for treating an
animal
for an abnormal proliferation of cells comprising altering atonal-associated
nucleic acid
or amino acid sequence levels in a cell. In a specific embodiment said
alteration is
reduction or said nucleic acid or amino acid sequence contains an alteration.
In a preferred embodiment of the present invention there are compositions to
treat
an organism for various medical conditions, discussed herein, comprising an
atonal-
associated nucleic acid sequence or amino acid sequence in combination with a
delivery
vehicle, wherein said organism comprises a defect in an atonal-associated
nucleic acid
sequence. A skilled artisan is aware that an adult organism, such as an adult
human,
naturally does not express atonal to significant or detectable levels, but
instead expresses
atonal in an embryonic stage of development (see the Examples). Thus, in a
preferred
embodiment, compositions to treat an organism as discussed herein, include
compositions to treat organisms who do not contain a mutation in an atonal
nucleic acid
or amino acid sequence but who naturally have atonal no longer expressed to
significant
or detectable levels.
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In another embodiment of the present invention is a composition comprising an
atonal-associated amino acid sequence or nucleic acid sequence in combination
with a
delivery vehicle wherein said vehicle delivers a therapeutically effective
amount of an
atonal-associated nucleic acid sequence or amino acid sequence into a cell. In
specific
embodiments said vehicle is the receptor-binding domain of a bacterial toxin
or any
fusion molecule or is a protein transduction domain. In a specific embodiment
said
protein transduction domain is from the HIV TAT peptide.
In another embodiment of the present invention there is a composition to treat
an
organism for loss of hair cells, wherein said organism comprises a defect in
an atonal-
associated nucleic acid sequence. In a specific embodiment the defect is a
mutation or
alteration of said atonal-associated nucleic acid sequence. In another
specific
embodiment the defect affects a regulatory sequence of said atonal-associated
nucleic
acid sequence. In an additional embodiment of the present invention there is a
composition to treat an organism for loss of hair cells, wherein said organism
comprises
defect in a nucleic acid sequence which is associated with regulation of an
atonal-
associated nucleic acid sequence. In an additional embodiment of the present
invention
there is a composition to treat an organism for loss of hair cells, wherein
said organism
comprises a defect in an amino acid sequence which is associated with
regulation of an
atonal-associated nucleic acid sequence.
In another embodiment of the present invention there is a composition to treat
an
organism for a cerebellar neuron deficiency, wherein said organism comprises a
defect
in an atonal-associated nucleic acid sequence. In a specific embodiment the
defect is a
mutation or alteration of said atonal-associated nucleic acid sequence. In
another specific
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embodiment the defect affects a regulatory sequence of said atonal-associated
nucleic
acid sequence. In an additional embodiment of the present invention there is a
composition to treat an organism for a cerebellar neuron deficiency, wherein
said
organism comprises defect in a nucleic acid sequence which is associated with
regulation
of an atonal-associated nucleic acid sequence. In an additional embodiment of
the
present invention there is a composition to treat an organism for a cerebellar
neuron
deficiency, wherein said organism comprises a defect in an amino acid sequence
which
is associated with regulation of an atonal-associated nucleic acid sequence.
In another embodiment of the present invention there is a composition to treat
an
organism for hearing impairment, wherein said organism comprises a defect in
an atonal-
associated nucleic acid sequence. In a specific embodiment the defect is a
mutation or
alteration of said atonal-associated nucleic acid sequence. In another
specific
embodiment the defect affects a regulatory sequence of said atonal-associated
nucleic
acid sequence. In an additional embodiment of the present invention there is a
composition to treat an organism for hearing impairment, wherein said organism
comprises defect in a nucleic acid sequence which is associated with
regulation of an
atonal-associated nucleic acid sequence. In an additional embodiment of the
present
invention there is a composition to treat an organism for hearing impairment,
wherein
said organism comprises a defect in an amino acid sequence which is associated
with
regulation of an atonal-associated nucleic acid sequence.
In another embodiment of the present invention there is a composition to treat
an
organism for an imbalance disorder, wherein said organism comprises a defect
in an
atonal-associated nucleic acid sequence. In a specific embodiment the defect
is a
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mutation or alteration of said atonal-associated nucleic acid sequence. In
another specific
embodiment the defect affects a regulatory sequence of said atonal-associated
nucleic
acid sequence. In an additional embodiment of the present invention there is a
composition to treat an organism for an imbalance disorder, wherein said
organism
comprises defect in a nucleic acid sequence which is associated with
regulation of an
atonal-associated nucleic acid sequence. In an additional embodiment of the
present
invention there is a composition to treat an organism for imbalance, wherein
said
organism comprises a defect in an amino acid sequence which is associated with
regulation of an atonal-associated nucleic acid sequence.
In another embodiment of the present invention there is a composition to treat
an
organism for osteoarthritis, wherein said organism comprises a defect in an
atonal-
associated nucleic acid sequence. In a specific embodiment the defect is a
mutation or
alteration of said atonal-associated nucleic acid sequence. In another
specific
embodiment the defect affects a regulatory sequence of said atonal-associated
nucleic
acid sequence. In an additional embodiment of the present invention there is a
composition to treat an organism for osteoarthritis, wherein said organism
comprises
defect in a nucleic acid sequence which is associated with regulation of an
atonal-
associated nucleic acid sequence. In an additional embodiment of the present
invention
there is a composition to treat an organism for osteoarthritis, wherein said
organism
comprises a defect in an amino acid sequence which is associated with
regulation of an
atonal-associated nucleic acid sequence.
In another embodiment of the present invention there is a composition to treat
an
organism for a joint disease, wherein said organism comprises a defect in an
atonal-
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associated nucleic acid sequence. In a specific embodiment the defect is a
mutation or
alteration of said atonal-associated nucleic acid sequence. In another
specific
embodiment the defect affects a regulatory sequence of said atonal-associated
nucleic
acid sequence. In an additional embodiment of the present invention there is a
composition to treat an organism for a joint disease, wherein said organism
comprises
defect in a nucleic acid sequence which is associated with regulation of an
atonal-
associated nucleic acid sequence. In an additional embodiment of the present
invention
there is a composition to treat an organism for a joint disease, wherein said
organism
comprises a defect in an amino acid sequence which is associated with
regulation of an
atonal-associated nucleic acid sequence.
In another embodiment of the present invention there is a composition to treat
an
organism for abnormal proliferation of cells, wherein said organism comprises
a defect
in an atonal-associated nucleic acid sequence. In a specific embodiment the
defect is a
mutation or alteration of said atonal-associated nucleic acid sequence. In
another specific
embodiment the defect affects a regulatory sequence of said atonal-associated
nucleic
acid sequence. In an additional embodiment of the present invention there is a
composition to treat an organism for abnormal proliferation of cells, wherein
said
organism comprises defect in a nucleic acid sequence which is associated with
regulation
of an atonal-associated nucleic acid sequence. In an additional embodiment of
the
present invention there is a composition to treat an organism for abnormal
proliferation
of cells, wherein said organism comprises a defect in an amino acid sequence
which is
associated with regulation of an atonal-associated nucleic acid sequence.
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In another embodiment of the present invention there is a composition to treat
an
organism for cancer, wherein said organism comprises a defect in an atonal-
associated
nucleic acid sequence. In a specific embodiment the defect is a mutation or
alteration of
said atonal-associated nucleic acid sequence. In another specific embodiment
the defect
affects a regulatory sequence of said atonal-associated nucleic acid sequence.
In an
additional embodiment of the present invention there is a composition to treat
an
organism for cancer, wherein said organism comprises defect in a nucleic acid
sequence
which is associated with regulation of an atonal-associated nucleic acid
sequence. In an
additional embodiment of the present invention there is a composition to treat
an
organism for cancer, wherein said organism comprises a defect in an amino acid
sequence which is associated with regulation of an atonal-associated nucleic
acid
sequence. In a specific embodiment said cancer is medulloblastorna.
NUCLEIC ACID-BASED EXPRESSION SYSTEMS
1. Vectors
One of skill in the art would be well equipped to construct a vector through
standard recombinant techniques, which are described in Sambrook et at., 1989
and
Ausubel et al., 1994,.
The term "expression vector" refers to a vector containing a nucleic acid
sequence coding
for at least part of a gene product capable of being transcribed. In some
cases, RNA
molecules are then translated into a protein, polypeptide, or peptide. In
other cases, these
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sequences are not translated, for example, in the production of antisense
molecules or
ribozymes. Expression vectors can contain a variety of "control sequences,"
which refer
to nucleic acid sequences necessary for the transcription and possibly
translation of an
operably linked coding sequence in a particular host organism. In addition to
control
sequences that govern transcription and translation, vectors and expression
vectors may
contain nucleic acid sequences that serve other functions as well and are
described infra.
a. Promoters and Enhancers
A "promoter" is a control sequence that is a region of a nucleic acid sequence
at
which initiation and rate of transcription are controlled. It may contain
genetic elements
at which regulatory proteins and molecules may bind such as RNA polymerase and
other
transcription factors. A promoter may or may not be used in conjunction with
an
"enhancer," which refers to a cis-acting regulatory sequence involved in the
transcriptional activation of a nucleic acid sequence.
Naturally, it will be important to employ a promoter and/or enhancer that
effectively directs the expression of the DNA segment in the cell type,
organelle, and
organism chosen for expression. Those of skill in the art of molecular biology
generally
know the use ofpromoters, enhancers, and cell type combinations for protein
expression,
for example, see Sambrook et al. (1989). The
promoters employed canbe constitutive, tissue-specific, inducible, and/or
useful under
the appropriate conditions to direct high level expression of the introduced
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segment, such as is advantageous in the large-scale production of recombinant
proteins
and/or peptides. The promoter canbe heterologous or endogenous.
The identity of tissue-specific promoters or elements, as well as assays to
characterize their activity, is well known to those of skill in the art.
Examples of such
regions include the human LIMK2 gene (Nomoto et al. 1999), the somatostatin
receptor
2 gene (Kraus et al., 1998), murine epididymal retinoic acid-binding gene
(Lareyre etal.,
1999), human CD4 (Zhao-Emonet et al., 1998), mouse alpha2 (XI) collagen
(Tsumaki,
etal., 1998), DIA dopamine receptor gene (Lee, et al., 1997), insulin-like
growth factor
II (Wu et al., 1997), human platelet endothelial cell adhesion molecule-1
(Almendro et
al., 1996).
b. Initiation Signals and Internal Ribosome Binding Sites
A specific initiation signal also canbe required for efficient translation of
coding
sequences. These signals include the ATG initiation codon or adjacent
sequences.
Exogenous translational control signals, including the ATG initiation codon,
canneed to
be provided. One of ordinary skill in the art would readily be capable of
determining this
and providing the necessary signals. It is well known that the initiation
codon must be
"in-frame" with the reading frame of the desired coding sequence to ensure
translation
of the entire insert. The exogenous translational control signals and
initiation codons can
be either natural or synthetic. The efficiency of expression canbe enhanced by
the
inclusion of appropriate transcription enhancer elements.
In certain embodiments of the invention, the use of internal ribosome entry
sites
(TRES) elements are used to create multigene, or polycistronic, messages. 1RES
elements
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can be linked to heterologous open reading frames. Multiple open reading
frames can
be transcribed together, each separated by an TRES, creating polycistronic
messages. By
virtue of the IRES element, each open reading frame is accessible to ribosomes
for
efficient translation. Multiple genes can be efficiently expressed using a
single
promoter/enhancer to transcribe a single message (see U.S. Patent 5,925,565
and
5,935,819).
c. Multiple Cloning Sites
Vectors can include a multiple cloning site (MCS), which is a nucleic acid
region
that contains multiple restriction enzyme sites, any of which can be used in
conjunction
with standard recombinant technology to digest the vector. (See Carbonelli et
al., 1999,
Levenson et al., 1998, and Cocea, 1997).
d. Splicing Sites
Most transcribed eukaryotic RNA molecules will undergo RNA splicing to
remove introns from the primary transcripts. Vectors containing genomic
eukaryotic
sequences canrequire donor and/or acceptor splicing sites to ensure proper
processing of
the transcript for protein expression. (See Chandler et al., 1997).
e. Polyadenylation Signals
In expression, one will typically include a polyadenylation signal to effect
proper
polyadenylation of the transcript. Specific
embodiments include the SV40
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polyadenylation signal and/or the bovine growth hormone polyadenylation
signal,
convenient and/or known to function well in various target cells.
1. Origins of Replication
In order to propagate a vector in a host cell, it cancontain one or more
origins of
replication sites (often termed "on'), which is a specific nucleic acid
sequence at which
replication is initiated.
g. Selectable and Screenable Markers
In certain embodiments of the invention, the cells contain nucleic acid
construct
of the present invention, a cell canbe identified in vitro or in vivo by
including a marker
in the expression vector. Such markers would confer an identifiable change to
the cell
permitting easy identification of cells containing the expression vector.
Generally, a
selectable marker is one that confers a property that allows for selection. A
positive
selectable marker is one in which the presence of the marker allows for its
selection,
while a negative selectable marker is one in which its presence prevents its
selection. An
example of a positive selectable marker is a drug resistance marker.
Usually the inclusion of a drug selection marker aids in the cloning and
identification of transformants, for example, genes that confer resistance to
neomycin,
puromycin, hygromycin, DHFR, GPT, zeocin and histidinol are useful selectable
markers. In addition to markers conferring a phenotype that allows for the
discrimination
of transformants based on the implementation of conditions, other types of
markers
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including screenable markers such as GFP or enhanced GFP, whose basis is
colorimetrie
analysis, are also contemplated. Alternatively, screenable enzymes such as
herpes
simplex virus thymidine kinase (tk) or chloramphenicol acetyltransferase (CAT)
canbe
utilized. One of skill in the art would also know how to employ immunologic
markers,
possibly in conjunction with FACS analysis. Further examples of selectable and
screenable markers are well known to one of skill in the art.
2. Expression Systems
Numerous expression systems exist that comprise at least a part or all of the
compositions discussed above. Prokaryote- and/or eukaryote-based systems can
be
employed for use with the present invention to produce nucleic acid sequences,
or their
cognate polypeptides, proteins and peptides. Many such systems are
commercially and
widely available.
The insect cell/baculovirus system can produce a high level of protein
expression
of a heterologous nucleic acid segment, such as described in U.S. Patent No.
5,871,986,
4,879,236 and which can be bought, for example,
under the name MAXBAC 2.0 from INVITROGENS and BACPACKTm
BACULOVIRUS EXPRESSION SYSTEM FROM CLONTECHO. Other examples of
expression systems are well known in the art.
Nucleic Acid Detection
In addition to their use in directing the expression of atonal-associated
proteins,
polypeptides and/or peptides, the nucleic acid sequences disclosed herein have
a variety
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of other uses. For example, they have utility as probes or primers or in any
of the
methods for embodiments involving nucleic acid hybridization, amplification of
nucleic
acid sequences, detection of nucleic acids, and other assays. A skilled
artisan is aware
of the following patents regarding details of these methods: U.S. Patent No.
5,840,873;
U.S. Patent No. 5,843, 640; U.S. Patent No. 5,843,650; U.S. Patent No.
5,843,651; U.S.
Patent No. 5,843,663; U.S. Patent No. 5,846,708; U.S. Patent No. 5,846,709;
U.S. Patent
No. 5,846,717; U.S. Patent No.5,846,726; U.S. Patent No. 5,846,729; U.S.
Patent No.
5,846,783; U.S. Patent No. 5,849,481; U.S. Patent No. 5,849,483; U.S. Patent
No.
5,849,486; U.S. Patent No. 5,849,487; U.S. Patent No. 5,849,497; U.S. Patent
No.
5,849,546; U.S. Patent No. 5,849,547; U.S. Patent No. 5,851,770; U.S. Patent
No.
5,851,772; U.S. Patent No.5,853,990; U.S. Patent No. 5,853, 993; U.S. Patent
No.
5,853,992; U.S. Patent No. 5,856,092; U.S. Patent No. 5,858,652; U.S. Patent
No.
5,861,244; U.S. Patent No.5,863,732; U.S. Patent No. 5,863,753; U.S. Patent
No.
5,866,331; U.S. Patent No. 5,866,336; U.S. Patent No. 5,866,337; U.S. Patent
No.
5,900,481; U.S. Patent No. 5,905,024; U.S. Patent No. 5,910,407; U.S. Patent
No.
5,912,124; U.S. Patent No. 5,912,145; U.S. Patent No. 5,912,148; U.S. Patent
No.
5,916,776; U.S. Patent No. 5,916,779; U.S. Patent No. 5,919,626; U.S. Patent
No.
5,919,630; U.S. Patent No. 5,922, 574; U.S. Patent No. 5,925,517; U.S. Patent
No.
5,925,525; U.S. Patent No. 5,928,862; U.S. Patent No. 5,928,869; U.S. Patent
No.
5,928,870; U.S. Patent No. 5,928,905; U.S. Patent No. 5,928,906; U.S. Patent
No.
5,929,227; U.S. Patent No. 5,932,413; U.S. Patent No. 5,932,451; U.S. Patent
No.
5,935,791; U.S. Patent No. 5,935,825; U.S. Patent No. 5,939,291; U.S. Patent
No.
5,942,391; European Application No. 320 308; European Application No. 329 822;
GB
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Application No. 2 202 328; PCT Application No. PCT/US87/00880; PCT Application
No. PCT/US89/01025; PCT Application WO 88/10315; PCT Application WO
89/06700; and PCT Application WO 90/07641.
Kits
All the essential materials and/or reagents required for detecting a sequence
selected from SEQ ID NO:1 through SEQ ID NO:66 in a sample canbe assembled
together in a kit. This generally will comprise a probe or primers designed to
hybridize
specifically to individual nucleic acids of interest in the practice of the
present invention,
such as the nucleic acid sequences in SEQ ID NO:1 through SEQ ID NO:66. Also
included canbe enzymes suitable for amplifying nucleic acids, including
various
polymerases (reverse transcriptase, Taq, etc.), deoxynucleotides and buffers
to provide
the necessary reaction mixture for amplification. Such kits canals() include
enzymes and
other reagents suitable for detection of specific nucleic acids or
amplification products.
Such kits generally will comprise, in suitable means, distinct containers for
each
individual reagent or enzyme as well as for each probe or primer pair.
Atonal-Associated Nucleic Acids
A. Nucleic Acids and Uses Thereof
The term "nucleic acid" will generally refer to at least one molecule or
strand of
DNA, RNA or a derivative or mimic thereof, comprising at least one nucleobase,
such
as, for example, a naturally occurring purine or pyrimidine base found in DNA
(e.g.
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adenine "A," guanine "G," thymine "T" and cytosine "C") or RNA (e.g. A, G,
uracil "U"
and C). The term "nucleic acid" encompass the terms "oligonucleotide" and
"polynucleotide." The term "oligonucleotide" refers to at least one molecule
of between
about 3 and about 100 nucleobases in length. The term "polynucleotide" refers
to at least
one molecule of greater than about 100 nucleobases in length. These
definitions
generally refer to at least one single-stranded molecule, but in specific
embodiments will
also encompass at least one additional strand that is partially, substantially
or fully
complementary to the at least one single-stranded molecule. Thus, a nucleic
acid
canencompass at least one double-stranded molecule or at least one triple-
stranded
molecule that comprises one or more complementary strand(s) or "complement(s)"
of a
particular sequence comprising a strand of the molecule. As used herein, a
single
stranded nucleic acid canbe denoted by the prefix "ss", a double stranded
nucleic acid by
the prefix "ds", and a triple stranded nucleic acid by the prefix "ts."
Thus, the present invention also encompasses at least one nucleic acid that is
complementary to a atonal-associated nucleic acid. In particular embodiments
the
invention encompasses at least one nucleic acid or nucleic acid segment
complementary
to the nucleic acid sequences set forth in SEQ ID NO:1 through SEQ II) NO:66,
of those
which are nucleic acid sequences. Nucleic acid(s) that are "complementary" or
"complement(s)" are those that are capable of base-pairing according to the
standard
Watson-Crick, Hoogsteen or reverse Hoogsteen binding complementarity rules. As
used
herein, the term "complementary" or "complement(s)" also refers to nucleic
acid(s) that
are substantially complementary, as canbe assessed by the same nucleotide
comparison
set forth above. The term "substantially complementary" refers to a nucleic
acid
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comprising at least one sequence of consecutive nucleobases, or
semiconsecutive
nucleobases if one or more nucleobase moieties are not present in the
molecule, are
capable of hybridizing to at least one nucleic acid strand or duplex even if
less than all
nucleobases do not base pair with a counterpart nucleobase.
Herein certain embodiments, a "gene" refers to a nucleic acid that is
transcribed.
As used herein, a "gene segment" is a nucleic acid segment of a gene. In
certain aspects,
the gene includes regulatory sequences involved in transcription, or message
production
or composition. In particular embodiments, the gene comprises transcribed
sequences
that encode for a protein, polypeptide or peptide. In other particular
aspects, the gene
comprises an atonal-associated nucleic acid, and/or encodes an atonal-
associated
polypeptide or peptide coding sequences. In keeping with the terminology
described
herein, an "isolated gene" cancomprise transcribed nucleic acid(s), regulatory
sequences,
coding sequences, or the like, isolated substantially away from other such
sequences,
such as other naturally occurring genes, regulatory sequences, polypeptide or
peptide
encoding sequences, etc. In this respect, the term "gene" is used for
simplicity to refer
to a nucleic acid comprising a nucleotide sequence that is transcribed, and
the
complement thereof. In particular aspects, the transcribed nucleotide sequence
comprises
at least one functional protein, polypeptide and/or peptide encoding unit. As
will be
understood by those in the art, this function term "gene" includes both
genomic
sequences, RNA or cDNA sequences or smaller engineered nucleic acid segments,
including nucleic acid segments of a non-transcribed part of a gene, including
but not
limited to the non-transcribed promoter or enhancer regions of a gene. Smaller
engineered gene nucleic acid segments canexpress, or canbe adapted to express
using
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nucleic acid manipulation technology, proteins, polypeptides, domains,
peptides, fusion
proteins, mutants and/or such like.
In certain embodiments, the nucleic acid sequence is a nucleic acid or nucleic
acid segment. As used herein, the term "nucleic acid segment", are smaller
fragments of
a nucleic acid, such as for non-limiting example, those that encode only part
of the
atonal-associated peptide or polypeptide sequence. Thus, a "nucleic acid
segment"
cancomprise any part of the atonal-associated gene sequence(s), of from about
2
nucleotides to the full length of the atonal-associated peptide or polypeptide
encoding
region. In certain embodiments, the "nucleic acid segment" encompasses the
full length
atonal-associated gene(s) sequence. In particular embodiments, the nucleic
acid
comprises any part of the SEQ ID NO:1 through SEQ ID NO:66, of from about 2
nucleotides to the full length of the sequence disclosed in SEQ ID NO:1
through SEQ ID
NO:66.
In certain embodiments, the nucleic acid segment canbe a probe or primer. As
used herein, a "probe" is a nucleic acid utilized for detection of another
nucleic acid and
is generally at least about 10 nucleotides in length. As used herein, a
"primer" is a
nucleic acid utilized for polymerization of another nucleic acid is generally
at least about
10 nucleotides in length. A non-limiting example of this would be the creation
of nucleic
acid segments of various lengths and sequence composition for probes and
primers based
on the sequences disclosed in SEQ ID NO:1 through SEQ ID NO:66, of those which
are
nucleic acid sequences.
The nucleic acid(s) of the present invention, regardless of the length of the
sequence itself, canbe combined with other nucleic acid sequences, including
but not
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limited to, promoters, enhancers, polyadenylation signals, restriction enzyme
sites,
multiple cloning sites, coding segments, and the like, to create one or more
nucleic acid
construct(s). As used herein, a "nucleic acid construct" is a recombinant
molecule
comprising at least two segments of different nucleic acid sequence. The
overall length
canvary considerably between nucleic acid constructs. Thus, a nucleic acid
segment of
almost any length canbe employed, with the total length preferably being
limited by the
ease of preparation or use in the intended recombinant nucleic acid protocol.
In certain embodiments, the nucleic acid construct is a recombinant vector. As
used herein, a "recombinant vector" is a nucleic acid comprising multiple
segments of
nucleic acids utilized as a vehicle for a nucleic acid sequence of interest.
In certain
aspects, the recombinant vector is an expression cassette. As used herein, an
expression
cassette is a segment of nucleic acid which comprises a gene of interest which
canbe
transfered between different recombinant vectors by means well known in the
art.
In particular embodiments, the invention concerns one or more recombinant
vector(s) comprising nucleic acid sequences that encode an atonal-associated
protein,
polypeptide or peptide that includes within its amino acid sequence a
contiguous amino
acid sequence in accordance with, or essentially as set forth in, SEQ ID NO:2
through
SEQ ID NO:66, of which sequences are amino acid sequences, corresponding to
Homo
sapiens or Mus muscu/us atonal-associated sequence.. In other embodiments, the
invention concerns recombinant vector(s) comprising nucleic acid sequences
from other
species that encode an atonal-associated protein, polypeptide or peptide that
includes
within its amino acid sequence a contiguous amino acid sequence in accordance
with, or
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essentially as set forth in SEQ ID NO:2 through SEQ ID NO:66, of which
sequences are
amino acid sequences. In particular aspects, the recombinant vectors are DNA
vectors.
It will also be understood that amino acid sequences or nucleic acid sequences
caninclude additional residues, such as additional N- or C-terminal amino
acids or 5' or
3' sequences, or various combinations thereof, and yet still be essentially as
set forth in
one of the sequences disclosed herein, so long as the sequence meets the
criteria set forth
above, including the maintenance of biological protein, polypeptide or peptide
activity
where expression of a proteinaceous composition is concerned. The addition of
terminal
sequences particularly applies to nucleic acid sequences that may, for
example, include
various non-coding sequences flanking either of the 5' and/or 3' portions of
the coding
region or can include various internal sequences, i.e., introns, which are
known to occur
within genes.
It will also be understood that this invention is not limited to the
particular nucleic
acid or amino acid sequences of SEQ ID NO: through SEQ ID NO:66, of which
sequences are amino acids. Recombinant vectors and isolated nucleic acid
segments can
therefore variously include these coding regions themselves, coding regions
bearing
selected alterations or modifications in the basic coding region, and they
canencode larger
polypeptides or peptides that nevertheless include such coding regions or
canencode
biologically functional equivalent proteins, polypeptide or peptides that have
variant
amino acids sequences.
The nucleic acids of the present invention encompass biologically functional
equivalent atonal-associated proteins, polypeptides, or peptides or atonal-
associated
proteins, polypeptides or polypeptides. Such sequences canarise as a
consequence of
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codon redundancy or functional equivalency that are known to occur naturally
within
nucleic acid sequences or the proteins, polypeptides or peptides thus encoded.
Alternatively, functionally equivalent proteins, polypeptides or peptides can
be created
via the application of recombinant DNA technology, in which changes in the
protein,
polypeptide or peptide structure can be engineered, based on considerations of
the
properties of the amino acids being exchanged. Changes designed by man can be
introduced, for example, through the application of site-directed mutagenesis
techniques
as discussed herein below, e.g., to introduce improvements or alterations to
the
antigenicity of the protein, polypeptide or peptide, or to test mutants in
order to examine
atonal-associated protein, polypeptide or peptide activity at the molecular
level.
Fusion proteins, polypeptides or peptides can be prepared, e.g., where the
atonal
associated coding regions are aligned within the same expression unit with
other proteins,
polypeptides or peptides having desired functions. Non-limiting examples of
such
desired functions of expression sequences include purification or
immunodetection
purposes for the added expression sequences, e.g., proteinaceous compositions
that canbe
purified by affinity chromatography or the enzyme labeling of coding regions,
respectively EP 266,032, or via deoxynucleoside H-phosphonate intermediates as
described by Froehler et al., Nucl. Acids Res., 14:5399-5407, 1986,
As used herein an "organism" can be a prokaryote, eukaryote, virus and the
like.
As used herein the term "sequence" encompasses both the terms "nucleic acid"
and
"proteancecous" or "proteanaceous composition." As used
herein, the term
"proteinaceous composition" encompasses the terms "protein", "polypeptide" and
"peptide." As used herein "artificial sequence" refers to a sequence of a
nucleic acid not
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derived from sequence naturally occurring at a genetic locus, as well as the
sequence of
any proteins, polypeptides or peptides encoded by such a nucleic acid. A
"synthetic
sequence", refers to a nucleic acid or proteinaceous composition produced by
chemical
synthesis in vitro, rather than enzymatic production in vitro (i.e. an
"enzymatically
produced" sequence) or biological production in vivo (i.e. a "biologically
produced"
sequence).
Cancer Therapies
Given the present invention is directed to methods and compositions for the
treatment of abnormal cell proliferation, a discussion of therapies of cancer,
which is the
state of abnormal cell proliferation, is warranted.
A wide variety of cancer therapies, such as radiotherapy, surgery,
chemotherapy
and gene therapy, are known to one of skill in the art, canbe used regarding
the methods
and compositions of the present invention.
Radiotherapeutic agents
Radiotherapeutic agents and factors include radiation and waves that induce
DNA
damage for example, g-irradiation, X-rays, UV-irradiation, microwaves,
electronic
emissions, radioisotopes, and the like. Therapy can be achieved by irradiating
the
localized tumor site with the above described forms of radiations.
Dosage ranges for X-rays range from daily doses of 50 to 200 roentgens for
prolonged periods of time (3 to 4 weeks), to single doses of 2000 to 6000
roentgens.
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Dosage ranges for radioisotopes vary widely, and depend on the half-life of
the isotope,
the strength and type of radiation emitted, and the uptake by the neoplastic
cells.
Surgery
Surgical treatment for removal of the cancerous growth is generally a standard
procedure for the treatment of tumors and cancers. This attempts to remove the
entire
cancerous growth. However, surgery is generally combined with chemotherapy
and/or
radiotherapy to ensure the destruction of any remaining neoplastic or
malignant cells.
Thus, surgery can be used in the context of the present invention.
Chemotherapeutic Agents
These can be, for example, agents that directly cross-link DNA, agents that
intercalate into DNA, or agents that lead to chromosomal and mitotic
aberrations by
affecting nucleic acid synthesis.
Agents that directly cross-link nucleic acids, specifically DNA, are envisaged
and
are shown herein, to eventuate DNA damage leading to a synergistic
antineoplastic
combination. Agents such as cisplatin, and other DNA alkylating agents can be
used.
Agents that damage DNA also include compounds that interfere with DNA
replication, mitosis, and chromosomal segregation. Examples of these compounds
include adriamycin (also known as doxorubicin), VP-16 (also known as
etoposide),
verapamil, podophyllotoxin, and the like. Widely used in clinical setting for
the
treatment of neoplasms these compounds are administered through bolus
injections
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intravenously at doses ranging from 25-75 mg/m2 at 21 day intervals for
adriamycin, to
35-100 mg/m2 for etoposide intravenously or orally.
Cancer therapies also include a variety of combination therapies with both
chemical and other types of treatments. Chemotherapeutics include, for
example,
cisplatin (CDDP), carboplatin, procarbazine, mechlorethamine,
cyclophosphamide,
camptothecin, ifosfamide, melphalan, chlorambucil, busulfan, nitrosurea,
dactinomycin,
daunorubicin, doxorubicin, bleomycin, plicomycin, mitomycin, etoposide (VP16),
tamoxifen, raloxifene, estrogen receptor binding agents, taxol, gemcitabien,
navelbine,
farnesyl-proteintansferase inhibitors, transplatinum, 5-fluorouracil,
vincristin, vinblastin
and methotrexate, or any analog or derivative variant of the foregoing.
Genes
Gene Therapy Administration
For gene therapy, a skilled artisan would be cognizant that the vector to be
utilized must contain the gene of interest or a suitable fragment thereof
operatively linked
to a promoter. For antisense gene therapy, the antisense sequence of the gene
of interest
or a suitable fragment thereof would be operatively linked to a promoter. One
skilled in
the art recognizes that in certain instances other sequences such as a 3' UTR
regulatory
sequences are useful in expressing the gene of interest. Where appropriate,
the gene
therapy vectors can be formulated into preparations in solid, semisolid,
liquid or gaseous
forms in the ways known in the art for their respective route of
administration. Means
known in the art can be utilized to prevent release and absorption of the
composition until
it reaches the target organ or to ensure timed-release of the composition. A
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pharmaceutically acceptable form should be employed which does not
ineffectuate the
compositions of the present invention. In pharmaceutical dosage forms, the
compositions
can be used alone or in appropriate association, as well as in combination,
with other
pharmaceutically active compounds. A sufficient amount of vector containing
the
therapeutic nucleic acid sequence must be administered to provide a
pharmacologically
effective dose of the gene product.
One skilled in the art recognizes that different methods of delivery can be
utilized
to administer a vector into a cell. Examples include: (1) methods utilizing
physical
means, such as electroporation (electricity), a gene gun (physical force) or
applying large
volumes of a liquid (pressure); and (2) methods wherein said vector is
complexed to
another entity, such as a liposome, viral vector or transporter molecule.
Accordingly, the present invention provides a method oftransferring a
therapeutic
gene to a host, which comprises administering the vector of the present
invention,
preferably as part of a composition, using any of the aforementioned routes of
administration or alternative routes known to those skilled in the art and
appropriate for
a particular application. Effective gene transfer of a vector to a host cell
in accordance
with the present invention to a host cell can be monitored in terms of a
therapeutic effect
(e.g. alleviation of some symptom associated with the particular disease being
treated)
or, further, by evidence of the transferred gene or expression of the gene
within the host
(e.g., using the polymerase chain reaction in conjunction with sequencing,
Northern or
Southern hybridizations, or transcription assays to detect the nucleic acid in
host cells,
or using immunoblot analysis, antibody-mediated detection, mRNA or protein
half-life
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studies, or particularized assays to detect protein or polypeptide encoded by
the
transferred nucleic acid, or impacted in level or function due to such
transfer).
These methods described herein are by no means all-inclusive, and further
methods to suit the specific application will be apparent to the ordinary
skilled artisan.
Moreover, the effective amount of the compositions can be further approximated
through
analogy to compounds known to exert the desired effect.
Furthermore, the actual dose and schedule can vary depending on whether the
compositions are administered in combination with other pharmaceutical
compositions,
or depending on interindividual differences in pharmacokinetics, drug
disposition, and
metabolism. Similarly, amounts can vary in in vitro applications depending on
the
particular cell line utilized (e.g., based on the number of vector receptors
present on the
cell surface, or the ability of the particular vector employed for gene
transfer to replicate
in that cell line). Furthermore, the amount of vector to be added per cell
will likely vary
with the length and stability of the therapeutic gene inserted in the vector,
as well as also
the nature of the sequence, and is particularly a parameter which needs to be
determined
empirically, and can be altered due to factors not inherent to the methods of
the present
invention (for instance, the cost associated with synthesis). One skilled in
the art can
easily make any necessary adjustments in accordance with the exigencies of the
particular
situation.
It is possible that cells containing the therapeutic gene canals() contain a
suicide
gene (i.e., a gene which encodes a product that can be used to destroy the
cell, such as
herpes simplex virus thymidine Idnase). In many gene therapy situations, it is
desirable
to be able to express a gene for therapeutic purposes in a host cell but also
to have the
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capacity to destroy the host cell once the therapy is completed, becomes
uncontrollable,
or does not lead to a predictable or desirable result. Thus, expression of the
therapeutic
gene in a host cell can be driven by a promoter although the product of said
suicide gene
remains harmless in the absence of a prodrug. Once the therapy is complete or
no longer
desired or needed, administration of a prodrug causes the suicide gene product
to become
lethal to the cell. Examples of suicide gene/prodrug combinations which can be
used are
Herpes Simplex Virus-thymidine kinase (HSV-tk) and ganciclovir, acyclovir or
FIAU;
oxidoreductase and cycloheximide; cytosine deaminase and 5-fluorocytosine;
thymidine
kinase thymidilate kinase (Tdk::Tmk) and AZT; and deoxycytidine kinase and
cytosine
arabinoside.
The method of cell therapy can be employed by methods known in the art wherein
a cultured cell containing a copy of a nucleic acid sequence or amino acid
sequence of
Math I is introduced.
In yet another embodiment, the secondary treatment is a secondary gene therapy
in which a second therapeutic polynucleotide is administered before, after, or
at the same
time a first therapeutic polynucleotide encoding all of part of an atonal-
associated
polypeptide. Delivery of a vector encoding either a full length or partial
atonal-
associated polypeptide in conjuction with a second vector encoding another
gene product
will have a combined anti-hyperproliferative effect on target tissues.
Alternatively, a
single vector encoding both genes can be used.
Immunotherapy
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Immunotherapeutics, generally, rely on the use of immune effector cells and
molecules to target and destroy cancer cells. The immune effector can be, for
example,
an antibody specific for some marker on the surface of a tumor cell. The
antibody alone
canserve as an effector of therapy or it canrecruit other cells to actually
effect cell killing.
The antibody also can be conjugated to a drug or toxin (chemotherapeutic,
radionuclide,
ricin A chain, cholera toxin, pertussis toxin, etc.) and serve merely as a
targeting agent.
Alternatively, the effector can be a lymphocyte carrying a surface molecule
that interacts,
either directly or indirectly, with a tumor cell target. Various effector
cells include
cytotoxic T cells and NK cells.
Immunotherapy, thus, could be used as part o f a combined therapy, in
conjunction
with Ad-mda7 gene therapy. The general approach for combined therapy is
discussed
below. Generally, the tumor cell must bear some marker that is amenable to
targeting,
i.e., is not present on the majority of other cells. Many tumor markers exist
and any of
these can be suitable for targeting in the context of the present invention.
Common
tumor markers include carcino embryonic antigen, prostate specific antigen,
urinary tumor
associated antigen, fetal antigen, tyrosinase (p97), gp68, TAG-72, HMFG,
Sialyl Lewis
Antigen, MucA, MucB, PLAP, estrogen receptor, laminin receptor, erb B and
p155.
Combination Treatments
It can be desirable in utilizing the present invention to combine the
compositions
with other agents effective in the treatment of hyperproliferative disease,
such as
anti-cancer agents. An "anti-cancer" agent is capable of negatively affecting
cancer in
a subject, for example, by killing cancer cells, inducing apoptosis in cancer
cells,
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reducing the growth rate of cancer cells, reducing the incidence or number of
metastases,
reducing tumor size, inhibiting tumor growth, reducing the blood supply to a
tumor or
cancer cells, promoting an immune response against cancer cells or a tumor,
preventing
or inhibiting the progression of cancer, or increasing the lifespan of a
subject with cancer.
More generally, these other compositions would be provided in a combined
amount
effective to kill or inhibit proliferation of the cell. This process
caninvolve contacting the
cells with the expression construct and the agent(s) or multiple factor(s) at
the same time.
This can be achieved by contacting the cell with a single composition or
pharmacological
formulation that includes both agents, or by contacting the cell with two
distinct
compositions or formulations, at the same time, wherein one composition
includes the
expression construct and the other includes the second agent(s).
Tumor cell resistance to chemotherapy and radiotherapy agents represents a
major
problem in clinical oncology. One goal of current cancer research is to find
ways to
improve the efficacy of chemo- and radiotherapy by combining it with gene
therapy. For
example, the herpes simplex-thymidine kinase (HS-tK) gene, when delivered to
brain
tumors by a retroviral vector system, successfully induced susceptibility to
the antiviral
agent ganciclovir (Culver, et al., 1992). In the context of the present
invention, it is
contemplated that mda-7 gene therapy could be used similarly in conjunction
with
chemotherapeutic, radiotherapeutic, or inmiunotherapeutic intervention, in
addition to
other pro-apoptotic or cell cycle regulating agents.
Alternatively, the gene therapy canprecede or follow the other agent treatment
by
intervals ranging from minutes to weeks. In embodiments where the other agent
and
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expression construct are applied separately to the cell, one would generally
ensure that
a significant period of time did not expire between the time of each delivery,
such that
the agent and expression construct would still be able to exert an
advantageously
combined effect on the cell. In such instances, it is contemplated that one
cancontact the
cell with both modalities within about 12-24 h of each other and, more
preferably, within
about 6-12 h of each other. In some situations, it can be desirable to extend
the time
period for treatment significantly, however, where several d (2, 3, 4, 5, 6 or
7) to several
wk (1, 2, 3, 4, 5, 6, 7 or 8) lapse between the respective administrations.
Various combinations can be employed, gene therapy is "A" and the secondary
agent,
such as radio- or chemotherapy, is "B":
A/B/A B/A/B B/B/A A/A/B A/B/B B/A/A A/B/B/B B/A/I3/B
B/B/B/A B/B/A/B AJAJ1313 AJB/A/B A/B/B/A B/B/A/A
B/A/B/A B/A/AJB A/A/A/B B/A/A/A A/B/A/A A/A/B/A
Administration of the therapeutic expression constructs of the present
invention to a
patient will follow general protocols for the administration of
chemotherapeutics, taking
into account the toxicity, if any, of the vector. It is expected that the
treatment cycles
would be repeated as necessary. It also is contemplated that various standard
therapies,
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as well as surgical intervention, can be applied in combination with the
described
hyperproliferative cell therapy.
Inhibitors of Cellular Proliferation
The tumor suppressor oncogenes function to inhibit excessive cellular
proliferation. The inactivation of these genes destroys their inhibitory
activity, resulting
in unregulated proliferation. The tumor suppressors p53, p16 and C-CAM are
specific
embodiments utilized in the present invention. Other genes that can be
employed
according to the present invention include Rb, APC, DCC, NF-1, NF-2, WT-1, MEN-
I,
MEN-II, zac 1 , p73, VHL, MMAC1 / PTEN, DBCCR-1, FCC, rsk-3, p27, p27/p16
fusions, p2 1/p27 fusions, anti-thrombotic genes (e.g., COX-1, TFPI), PGS, Dp,
E2F, ras,
myc, neu, raf, erb, fms, trk, ret, gsp, hst, abl, El A, p300, genes involved
in angiogenesis
(e.g., VEGF, FGF, thrombospondin, BAI-1, GDAIF, or their receptors) and MCC.
Regulators of Programmed Cell Death
Apoptosis, or programmed cell death, is an essential process for normal
embryonic development, maintaining homeostasis in adult tissues, and
suppressing
carcinogenesis (Kerr et al., 1972). The Bc1-2 family of proteins and ICE-like
proteases
have been demonstrated to be important regulators and effectors of apoptosis
in other
systems. The BcI-2 protein, discovered in association with follicular
lymphoma, plays
a prominent role in controlling apoptosis and enhancing cell survival in
response to
diverse apoptotic stimuli (Balchshi et al., 1985; Cleary and Sklar, 1985;
Cleary et al.,
1986; Tsujimoto et al., 1985; Tsujimoto and Croce, 1986). The evolutionarily
conserved
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Bc1-2 protein now is recognized to be a member of a family of related
proteins, which can
be categorized as death agonists or death antagonists. Different family
members have
been shown to either possess similar functions to Bc1-2 (e.g., Bc1XL, Bc1W,
Bc1S, Mc1-1,
Al, Bfl-1) or counteract Bc1-2 function and promote cell death (e.g., Bax,
Bak, Bik, Bim,
Bid, Bad, Harakiri).
Other agents
It is contemplated that other agents can be used in combination with the
present
invention to improve the therapeutic efficacy of treatment. These additional
agents
include immunomodulatory agents, agents that affect the upregulation of cell
surface
receptors and GAP junctions, cytostatic and differentiation agents, inhibitors
of cell
adehesion, or agents that increase the sensitivity of the hyperproliferative
cells to
apoptotic inducers.
Dosage and Formulation
The nucleic acid sequences and amino acid seqeunces (active ingredients) of
this
invention can be formulated and administered to treat a variety of disease
states by any
means that produces contact of the active ingredient with the agent's site of
action in the
body of an animal. They can be administered by any conventional means
available for
use in conjunction with pharmaceuticals, either as individual therapeutic
active
ingredients or in a combination of therapeutic active ingredients. They can be
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administered alone, or with a pharmaceutically acceptable carrier selected on
the basis
of the chosen route of administration and standard pharmaceutical practice.
The dosage administered will be a therapeutically effective amount of active
ingredient and will, of course, vary depending upon known factors such as the
pharmacodynamic characteristics of the particular active ingredient and its
mode and
route of administration; age, sex, health and weight of the recipient; nature
and extent of
symptoms; kind of concurrent treatment, frequency of treatment and the effect
desired.
The active ingredient can be administered orally in solid dosage forms such as
capsules, tablets and powders, or in liquid dosage forms such as elixirs,
syrups, emulsions
and suspensions. The active ingredient can also be formulated for
administration
parenterally by injection, rapid infusion, nasopharyngeal absorption or
dermoabsorption.
The agent can be administered intramuscularly, intravenously, or as a
suppository. In
addition, parenteral solutions can contain preservatives such as benzalkonium
chloride,
methyl- or propyl-paraben and chlorobutanol. Suitable pharmaceutical carriers
are
described in Remington 's Pharmaceutical Sciences, a standard reference text
in this field.
Additionally, standard pharmaceutical methods can be employed to control the
duration of action. These are well known in the art and include control
release
preparations and can include appropriate macromolecules, for example polymers,
polyesters, polyamino acids, polyvinyl, pyrolidone, ethylenevinylacetate,
methyl
cellulose, carboxymethyl cellulose or protamine sulfate. The concentration of
macromolecules as well as the methods of incorporation can be adjusted in
order to
control release. Additionally, the agent can be incorporated into particles of
polymeric
materials such as polyesters, polyamino acids, hydrogels, poly (lactic acid)
or
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ethylenevinylacetate copolymers. In addition to being incorporated, these
agents can also
be used to trap the compound in microcapsules.
Useful pharmaceutical dosage forms for administration of the compounds of this
invention can be illustrated as follows.
Capsules: Capsules are prepared by filling standard two-piece hard gelatin
capsulates each with a therapeutically effective amount of powdered active
ingredient,
175 milligrams of lactose, 24 milligrams of talc and 6 milligrams magnesium
stearate.
Soft Gelatin Capsules: A mixture of active ingredient in soybean oil is
prepared
and injected by means of a positive displacement pump into gelatin to form
soft gelatin
capsules containing a therapeutically effective amount of the active
ingredient. The
capsules are then washed and dried.
Tablets: Tablets are prepared by conventional procedures so that the dosage
unit
is a therapeutically effective amount of active ingredient. 0.2 milligrams of
colloidal
silicon dioxide, 5 milligrams of magnesium stearate, 275 milligrams of
microcrystalline
cellulose, 11 milligrams of cornstarch and 98.8 milligrams of lactose.
Appropriate
coatings can be applied to increase palatability or to delay absorption.
Injectable: A parenteral composition suitable for administration by injection
is
prepared by stirring 1.5% by weight of active ingredients in 10% by volume
propylene
glycol and water. The solution is made isotonic with sodium chloride and
sterilized.
Suspension: An aqueous suspension is prepared for oral administration so that
each 5 millimeters contain a therapeutically effective amount of finely
divided active
ingredient, 200 milligrams of sodium carboxymethyl cellulose, 5 milligrams of
sodium
benzoate, 1.0 grams of sorbitol solution U.S.P. and 0.025 millimeters of
vanillin.
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Accordingly, the pharmaceutical composition of the present invention can be
delivered via various routes and to various sites in an animal body to achieve
a particular
effect (see, e.g., Rosenfeld et al. (1991), supra; Rosenfeld et al., Clin.
Res., 39(2), 311A
(1991a); Jaffe et al., supra; Berkner, supra). One skilled in the art will
recognize that
although more than one route can be used for administration, a particular
route can
provide a more immediate and more effective reaction than another route. Local
or
systemic delivery can be accomplished by administration comprising application
or
instillation of the formulation into body cavities, inhalation or insufflation
of an aerosol,
or by parenteral introduction, comprising intramuscular, intravenous,
peritoneal,
subcutaneous, intradermal, as well as topical administration.
The composition of the present invention can be provided in unit dosage form
wherein each dosage unit, e.g., a teaspoonful, tablet, solution, or
suppository, contains
a predetermined amount of the composition, alone or in appropriate combination
with
other active agents. The term "unit dosage form" as used herein refers to
physically
discrete units suitable as unitary dosages for human and animal subjects, each
unit
containing a predetermined quantity of the compositions of the present
invention, alone
or in combination with other active agents, calculated in an amount sufficient
to produce
the desired effect, in association with a pharmaceutically acceptable diluent,
carrier, or
vehicle, where appropriate. The specifications for the unit dosage forms of
the present
invention depend on the particular effect to be achieved and the particular
pharmacodynamics associated with the pharmaceutical composition in the
particular host.
These methods described herein are by no means all-inclusive, and further
methods to suit the specific application will be apparent to the ordinary
skilled artisan.
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Moreover, the effective amount of the compositions can be further approximated
through
analogy to compounds known to exert the desired effect.
The following examples are offered by way of example, and are not intended to
limit the scope of the invention in any manner.
EXAMPLE 1
Mouse atonal homolog 1 (Math 1)
It has been found that the present methods for the treatment of the hearing
impaired have failed to address the problem directly, that is, the
regeneration of auditory
hair cell populations. The present invention in a preferred embodiment is
directed to a
member of the bHLH family, the Math] gene or an another atonal-associated
nucleic
acid sequence, and its requirement for generation of cerebellar granule
neurons and inner
ear hair cells. This discovery has wide ramifications not only for
understanding
neurodevelopment but also for therapies for a variety of prevalent disorders,
as described
below.
The mouse atonal homolog 1 (Math]) is expressed in the precursors of the
cerebellar granule neurons; a few cells in the dorsal portion of the
developing spinal cord;
the inner ear; Merkel cells (touch receptors on the skins); and joints.
Overexpressing
Math] in an otherwise differentiated cell can induce the formation or
differentiation into
a progenitor or mature inner ear hair cell-like cell.
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Math] expression in the precursors of the cerebellar granule neurons suggests
it
is required for function in the cerebellum and brain. The cerebellum is
essential for fine
motor coordination and posture, and its dysfunction disrupts balance, speech
and limb
movements. Cerebellar development typically begins at about embryonic day 9.5
(E9.5)
when a small group of cells in the hindbrain proliferates and migrates
rostrally to form
the external granule layer, brain stem, and pontine neurons. This population
of neuronal
progenitors, which continues to express Math], further proliferates and
migrates
internally to form the cerebellar granule neurons that are the predominant
neuronal
population in the cerebellum and brain. Mice that do not express Math I
completely lack
cerebellar granule neurons and their precursors. Math] is thus essential for
the
generation of these neurons and endows the very sparse population of neurons
at E9.5
with the ability to proliferate into billions and then differentiate (Ben-Arie
et al., 1997).
Both these functions are of great medical significance. To understand normal
proliferation provides necessary insight into abnormal proliferation, as
observed in
cancer. Cerebellar tumors of the primitive neuroectodermal type (e.g.,
medulloblastoma)
are the most common solid malignancy in children. Math] expressing cells
contribute
significantly to these tumors.
Math] is expressed in the non-ossified joint cartilage (see Figure 6) that
typically
degenerates in osteoarthritis. This is the most prevalent form of arthritis,
with 90% of
people over 40 showing some degree of osteoarthritis in one or more joints.
Given the
properties of Math] in cellular generation and proliferation, its artificial
expression in
affected joints can allow regeneration of the cells that constitute non-
ossified cartilage.
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Disclosed herein are compositions and methods for the use of the Math/ gene,
its
human homolog (Hath I) or any of its homologs, orthologs, chimeric fusion
proteins or
derivatives of any suitable atonal-associated nucleic acid sequence or any
another atonal-
associated nucleic acid sequence. To learn about the functions of Math/ in
mammals,
the Math] gene was deleted from a mouse using a strategy that permitted
detection of
cells that express Math]. Disclosed are the creation and characterization of
mice that
can be used to screen for compounds which could be utilized to decrease or
augment
Math/ expression in inner ear hair cells and other cells in which Math/
expression is
associated.
Methods are also disclosed for the study, characterization and treatment of
neoplastic proliferation of neuroectodermal origin since Math/ expression is
essential for
the generation and proliferation of cerebellar granule neurons. Also, it has
been
discovered that Math] plays a role in the development of cells that produce
non-ossified
joint cartilage, which are associated with the development of osteoarthitis.
These
discoveries have led to a method of screening for compounds that can be
helpful for the
treatment of inner ear hair cell loss and other diseases that occur due to the
functional loss
of Math I , such as osteoarthritis.
More particularly, the present invention provides an animal heterozygous for
Mathl gene inactivation or an another atonal-associated nucleic acid sequence,
wherein
at least one Math] allele or another atonal-associated nucleic acid sequence
has been
replaced by insertion of a heterologous nucleic acid sequence, wherein the
inactivation
of the Math] or atonal-associated sequence prevents expression of the Math] or
atonal-
associated allele. The mouse can be further used to generate mice homozygous
for
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Math] or another atonal-associated sequence gene inactivation and can further
include
a second heterologous nucleic acid sequence, wherein at least one of the
heterologous
genes is used to detect expression driven by the Math] or atona/-associated
sequence
regulatory elements. The complete or partial inactivation of the functional
Math] or
atonal-associated sequence can be detected in, e.g., proprioreceptory cells,
granule
neurons and their progenitor cells, or non-ossified cartilage cells.
Examples of heterologous nucleic acid sequences are reporter sequences such as
b-galactosidase, green fluorescent protein (GFP), blue fluorescent protein
(BFP),
neomycin, kanamycin, luciferase, b-glucuronidase and chloramphenicol
transferase
(CAT). The Math] or atona/-associated sequence can also be replaced under the
control
of regulatable promoter sequences or can be a tissue-specific promoter
sequences. Said
promoter sequences can be partial or can contain the entire promoter.
The present invention can also be used as, or as part of, a method for
screening
for a compound, wherein the administration of the compound affects a
developmental
and/or pathological condition wherein said condition is a result of reduction
in expression
of the Math] or atonal-associated sequence, the method including,
administering the
compound to a transgenic mouse that is homozygous for Math] or atonal-
associated
sequence inactivation, wherein at least one Math] or atonal-associated allele
is
inactivated by insertion of a heterologous nucleic acid sequence, wherein the
inactivation
of the Math I or atonal-associated sequence prevents expression of the Math 1
or atonal-
associated gene, and monitoring the mouse for a change in the developmental
and/or
pathological condition. The types of pathological conditions that can be
examined
include, but are not limited to loss of hair cells, loss of cerebellar granule
neurons or their
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precursors, lack of granule cell proliferation or migration, lack of
cerebellar external
granule layer cells, hearing impairment, an imbalance disorder, joint disease,
osteoarthritis, abnormal proliferation of neoplastic neuroectodermal cells and
formation
of medulloblastoma. As used herein, the screen provides for a compound that by
upregulating expression of a heterologous nucleic acid sequence is a positive
effector and
for a compound that by downregulating expression of a heterologous nucleic
acid
sequence is a negative effector.
Yet another embodiment of the present invention is a method of promoting
mechanoreceptive cell growth, that includes contacting a cell with a Math] or
atonal-
associated protein or gene in an amount effective to cause said cell to
express an inner
ear hair cell marker. An example of a hair cell marker for use with the method
is
calretinin. The cell can be contacted with a vector that expresses a Mathl or
atonal-
associated nucleic acid sequence or amino acid sequence. Math] or atonal-
associated
nucleic acid sequence-expressing recombinant vectors can include an adenoviral
vector,
a retroviral vector, an adeno-associated vector, a plasmid, a liposome, a
protein, a lipid,
a carbohydrate and a combination thereof of said vectors. Math] or atona/-
associated
sequence can be under the control of, e.g., a cytomegalovirus IE promoter
sequence or
the cytomegalovirus IE promoter sequence and a SV40 early polyadenylation
signal
sequence, or any other combination of appropriate promoter sequences, enhancer
sequence, and polyadenylation.
Furthermore, a method is disclosed for treating hearing impairment or an
imbalance disorder that includes administering to an animal, including a
human, with
hearing loss or an imbalance disorder a therapeutically effective amount of a
Mathl or
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atonal-associated amino acid sequence or nucleic acid sequence. The hearing or
balance
impairment can be complete or partial and can affect either one ear or both
ears. In a
preferred embodiment, there is a substantial impairment of hearing. Hearing
and an
imbalance disorder can be affected separately or concomitantly in an animal to
be
treated, and said hearing and/or an imbalance disorder could be as a result of
trauma,
disease, age-related condition, or could be due to loss of hair cells for any
reason.
The present invention is also directed to a composition that includes a Math I
or
atonal-associated protein or gene in combination with a delivery vehicle,
wherein the
delivery vehicle causes a therapeutically effective amount ofMath/ or atonal-
associated
sequence to be delivered into a cell. The delivery vehicle can be further
defined as a
vector that comprises a Math] or atonal-associated amino acid sequence or
nucleic acid
sequence in an animal cell. The vector can be a retroviral or an adenoviral
vector or any
other nucleic acid based vector which can even be dispersed in a
pharmacologically
acceptable formulation, and used for intralesional administration. The
composition can
even be a partially or fully purified protein that is delivered using a
liposome, a protein,
a lipid or a carbohydrate that promotes the entry of a Math/ or atonal-
associated protein
into a cell. Examples of proteins that can be used as delivery vehicles
include the
receptor-binding domains (the non-catalytic regions) of bacterial toxins, such
as, e.g.,
Exotoxin A, cholera toxin and Ricin toxin or protein transduction domains,
such as from
the }ITV TAT protein (Schwarze et al., 1999) (see Example 22). The composition
for
delivering Math] can be a fusion protein.
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A skilled artisan is aware that methods to treat animals as disclosed in the
invention can be either in utero or after birth. Treatment can be given to an
embryo and
can occur either ex vivo or in vivo.
EXAMPLE 2
Animal Model for Organogenesis
An effective animal model for deficiency in a gene that controls organogenesis
will most often have both alleles stably inactivated so that, throughout
embryogenesis,
one or more tissues cannot revert to a functional wild-type allele. One method
of
generating animals with an altered genotype is gene targeting (Mansour et al.,
1993), in
which homologous recombination of newly introduced DNA sequence (i.e., the
targeting
sequence or construct) and a specific targeted DNA sequence residing in the
chromosome
results in the insertion of a portion of the newly introduced DNA sequence
into the
targeted chromosomal DNA sequence. This method is capable of generating
animals of
any desired genotype, and is especially useful for gene disruption (i.e., to
"knock out")
at a specific chromosomal gene sequence by inserting a selectable marker into
the gene
or completely replacing the gene with another nucleotide sequence.
To knock out a genomic sequence, a cloned fragment must be available and
intron-exon boundaries within the fragment defined (Mansour et al., 1993).
Typically,
the targeting construct contains a selectable marker such as Neo (neomycin
resistance,
see Mansour et al., 1993) flanked by sequences homologous to the chromosomal
target
DNA, and beyond one of these flanking sequences the herpes simplex virus
thymidine
kinase gene (HSV-TK, see generally, McKnight et al., 1980). The targeting
construct is
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introduced, e.g., by electroporation, into embryo-derived stem (ES) cells
where
homologous recombination results in an insertion of the Neomycin resistance
marker
(Neo), but not the HSV-TK gene, into the targeted chromosomal DNA sequence.
The
altered ES cells are neomycin resistant and HSV-TK- and so are able to grow in
the
presence of both G418 and gancyclovir antibiotics. Random insertions contain
the
HSV-TK gene and are thus sensitive to gancyclovir (Mansour, et al.). Positive
ES clones
are then microinjected into blastocysts to generate germ-line chimeric mice,
which are
then bred to obtain progeny that are homozygous for the knock out gene. Such
general
methods of generating knock out animals have been demonstrated using mice.
Genes in
other animals such as rats, guinea pigs, gerbils, hamsters, and rabbits,
canalso be used as
long as sufficient DNA sequence data are available to make an appropriate
targeting
construct to knock out the gene of interest.
Although ato and Math] share a high degree of sequence conservation, there was
an apparent discrepancy between their expression patterns and the consequences
of their
loss of function. Whereas ato is expressed primarily in the PNS of the fly and
its absence
causes loss of almost all CHOs (Jarman et al., 1993), Math] is expressed in
the CNS and
its loss leads to absence of cerebellar granule neurons, the largest neuronal
population in
the CNS (Ben-Arie et al., 1997). To better understand the functional relations
between
ato and Mathl , the present invention describes generation of a second Math]
null allele
in mice (Mathlb-galfb- gal) by replacement of theMathl coding region with a b-
galactosidase
gene (lacZ) and performing a subsequent search for CNS expression of ato in
the fruit
fly. The Examples describe a functional link between ato and Math 1 : ato is
expressed
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in the fly brain, and lacZ expression under the control of Math] regulatory
elements
(Mathl/lacZ) not only replicated the known expression pattern in the CNS
(i.e., the
neural tube, spinal cord and cerebellum), but appeared in many other cells of
the murine
PNS. Overexpression of Math] inDrosophila caused ectopic CHO formation,
providing
further evidence that ato and Math] are functionally conserved.
The connections and consistency of the relationship between atonal in
Drosophila
and Math] in the mouse suggests that their use as model systems in the art is
justified.
A family of homologues have been cloned and analyzed in the mouse including
MATH1,2,3,4A, 4B, 4C and 5 (Azakawa et al., 1995; Bartholoma and Nave, 1994;
Ben-
Arie et al., 1997; Ben-Arie et al., 1996, Fode et al., 1998; Ma et al., 1998;
McCormick
et al., 1996; Shimizu et al., 1995; Takebayashi et al., 1997). A Xenopus
atonal homolog,
Xathl has been ectopically expressed in Drosophila and shown to behave
similarly to ato
(Kim et al., 1997). Furthermore, the ability of Mathl to induce ectopic CHO
formation
and to restore CHOs to ato mutant embryos (see Example 13) is strong evidence
that
Math 1 , and particularly its basic domain, encodes lineage identity
information not unlike
that encoded by ato and that mammalian cells expressing Math] are functionally
similar
and perhaps evolutionarily related to Drosophila cells that require ato. Thus,
the
similarities between atonal in Drosophila, Xathl in Xenopus and Math] in the
mouse
indicate that these animals are comparable animal model systems. Furthermore,
the
widespread use of mice in particular as a model system for humans also
suggests that it
similarly would allow utilization of the invention in humans.
With advances in molecular genetics now standard in the art, sequences from
humans and other species can be used interchangeably in a variety of
organisms. For
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example, the rat inducible hsp70 gene was used to produce transgenic mice that
overexpressed inducible hsp70, allowing organs from transgenic mice to be
protected
from ischemic injury (Marber et al. J. din. Invest. 95:1446-1456 (1995)) due
to the
increase in rat hsp70. Sequences in other animals have been interchanged
including
between humans and rodents to develop rodent models to study human disease,
i.e.
neurodegenerative diseases. One such example is the expression of the human
SCAI
gene, which encodes ataxin-1, in mice (Burright, E. N. et al. Cell 82:937-948
(1995)).
Transgenic mice were generated expressing the human SCA I gene with either a
normal
or an expanded CAG tract. The data illustrated that the expanded CAG repeats
were
expressed in sufficient amounts in the Purkinje cells to produce degeneration
and ataxia.
This example illustrates that a mouse model can be established to study
spinocerebellar
ataxia type 1, which is an autosomal dominant inherited neurologic disorder.
In addition
to developing mouse models, Drosophila is a hallmark model system in the
field.
Warrick et al. (1999) produced transgenic flies which co-expressed human hsp70
and a
human mutant polyglutamine (MJDtr-Q78). Expression of the human mutant
polyglutamineMJDtr-Q78 alone in the flies resulted in the formation of large
aggregates
in neurons. However, co-expression with human hsp 70 resulted in suppressed
aggregation. These examples illustrate that interchangeability of genes is
routine in the
field of molecular genetics and model systems provide powerful tools to
characterize
gene function.
EXAMPLE 3
Generation of Transgenic Mathl Mice
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To detect subtle Math] expression patterns not identified by RNA in situ
hybridization, and thus further illuminate this gene's role during embryonic
development,
Math] null alleles (Math] a9 Gal) were generated by replacing the Mathl coding
region
with 13-galactosidase (13-Gal).
The targeting construct, containing a lacZ cassette and a PGK-neo cassette
(Fig.
7A), was used to replace the Math] coding region. To delete the entire coding
region of
Math 1, a targeting construct was generated that contained the 5' and 3'
genomic flanking
fragments as described previously (Ben-Arie et al., 1997) flanking a
pSAbgal/PGK-neo
cassette (Friedrich and Soriano, 1991). The construct is designed so that lacZ
expression
is driven by endogenous Math] control elements, while an independent PGK
promoter
drives the expression of the selectable marker neo.
The construct was electroporated into ES cells and selection for neo was
achieved
with G418. Fourteen out of 76 (18%) clones underwent homologous recombination.
Genotyping of ES cells, yolk sac and tail DNA was performed using Southern
analysis
of EcoR I digested DNA and probes previously described (Ben-Arie et al.,
1997). The
targeting construct was electroporated into embryonic stem (ES) cells; 14/76
(18%)
clones exhibited correct homologous recombination at the Math] locus (Fig.
7B).
Three ES cell lines carrying the Mathrib-gal allele were injected into host
blastocysts to generate chimeric mice. Mathrib-gal mice were identified and
intercrossed
to generate homozygotes (Fig. 7C). The Math 1 deletion was confirmed by
Southern
analysis using both flanking and internal probes (Fig. 7A).
Mathlfi-Gal/fl-Gal mice show all the phenotypic features reported in the Math]
mice
(Ben-Arie et al., 1997; 2000).
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EXAMPLE 4
X-gal staining, histological and immunohistochemical analyses
Embryos were staged by vaginal plug, with the morning of the plug designated
E0.5. Embryos were dissected out of the uterus, separated from extraembryonic
membranes, and placed in cold phosphate buffered saline (PBS). The embryos
were then
fixed in 4% paraformaldehyde (PFA) in PBS for 30 minutes, and washed in cold
PBS.
Yolk sacs or tails were collected before fixation for DNA extraction and
genotyping.
Equilibration to improve the penetrability of the staining reagents was
performed in
0.02% NP40, 0.01% sodium deoxycholate in PBS for 10 minutes at room
temperature.
Whole mount staining with X-gal (Bonnerot and Nicolas, 1993) was performed for
16-24
hours at 30oC while shaking in the same equilibration buffer, which also
contained 5mM
potassium ferricyanide, 5mM potassium ferrocyanide, and 40 mg/ml X-gal
(dissolved in
DMS0). When the desired intensity of staining was achieved, usually within 18
hours,
embryos were washed in PBS, postfixed for 30 minutes in buffered formalin,
serially
dehydrated in 25, 50, and 70% ethanol, and stored at 4 C.
For histological analysis embryos were further dehydrated in 80, 90, and 100%
ethanol, treated in Histoclear (National Diagnostics), and embedded in
Paraplast (Oxford
Labware). Seven to 20 p.m sections were cut using in a microtome (Microme).
Counterstaining was performed using nuclear fast red (Vector Laboratories).
Lmmunohistochemistry was performed as detailed previously (Ben-Arie et al.,
1997).
Antibodies: Anti-cytokeratin 18 (DAKO) 1:20; Anti-human Chromogranin A (DAKO)
1:100; Anti-MATH1 (see below) 1:200.
*Trademark
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EXAMPLE 5
Expression Patterns in Transgenic Math] Mice
As expected, P-Gal expression in the cerebellum and dorsal spinal cord is
identical to that of Math I , and interestingly, 3-Gal is also expressed
throughout the otic
vesicle epithelia at E12.5 and in the sensory epithelia of the utricle,
saccule, semicircular
canals, and cochlea at El 4.5 and El 5.5 (Figures lA and 1B). Utricles were
obtained from
C57BL/129SVEV mice.
Gross morphological analysis of the inner ear o fMath 1 fi-Gailfl'Gal mice at
E18.5, one
day before full gestation, revealed no obvious defects in overall structure
and size
compared with wild type (wt) littermates. The branches of the VIII' cranial
nerve were
present and reached the epithelia, but degenerated due to absence of the hair
cells.
The sensory epithelia were examined in detail. The utricles and cochleas of
wild-
type, Math and Mathlfi-Gal/fl-Gal mice were excised to allow viewing of
the sensory
epithelia with Nomarski optics. Hair bundles were present in both organs of
wild-type
and heterozygotes, but were completely absent in Math] null litter-mates.
Scanning
electron microscopy (SEM) of the cochlea and vestibular organs confirmed the
absence
of hair bundles in null mice (Figures 2A through 2F). To determine whether
lack of hair
bundles reflects the absence of hair cells, cross-sections of the sensory
epithelia of all
inner ear organs using both light and transmission electron microscopy (LM and
TEM,
respectively) were examined (Figures 3A through 3F). LM and TEM were earned
out
as described previously (Lysakowski and Goldberg, 1997). Tissue preparation
for SEM
consisted of osmication (1% 0504 in cacodylate buffer), dehydration, critical-
point
drying, sputter-coating with gold, and examination in a JEOL 35S electron
microscope.
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Light microscopy revealed that sensory epithelia in null mice are considerably
thinner, lack the normal stratification of cell nuclei and stain uniformly,
all of which are
consistent with the absence of hair cells. TEM clearly distinguishes between
hair cells
and supporting cells in normal utricles: hair cells have hair bundles, less
electron-dense
cytoplasm, more apical nuclei, and no secretory granules (Figures 4A and 4B).
The
sensory epithelia of the null mutants lack hair cells entirely but do have
supporting cells
with normal appearance (Riisch, et al.,1998), including electron-dense
cytoplasm, basal
nuclei, and secretory granules. However heterozygous Math] '' mice retain hair
cells.
EXAMPLE 6
Expression of a Hair Cell Specific Marker in Transgenic Mathl Mice
Lack of hair cells at E18.5 can be due to (1) lack of sensory cell
progenitors, (2)
the inability of progenitors to differentiate into hair cells, or (3) the
inability of hair cells
to maintain the differential states, as has been observed in the absence of
the POU
domain transcription factor Brn3c. The first possibility is unlikely because
progenitors
give rise to both hair cells and supporting cells. To evaluate the remaining
possibilities,
the expression of the hair cell specific marker, calretinin and myosin VI were
examined.
Calretinin is a member of the calcium binding family of proteins and is
expressed in
differentiating hair cells (prior to hair bundle formation) and mature inner
ear and
fl-Gal/fi-Gl
auditory hair cells, but not in supporting cells. Calretinin expression in
Mathl a
and wild-type mice was studied by immunofluorescense on coronal sections of
E15.5,
E16.5 and El 8.5 embryos (Figures 5A through 5F).
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For immunofluorescence, embryos were fixed for 1.5 hours in 4%
paraformaldehyde/PBS at 4 C, sunk through 15% sucrose/PBS for 5 hours then 30%
sucrose/PBS overnight, and snap frozen in a 2-methylbutane dry ice bath. 14 um
sections were cut on a cryostat and mounted onto gelatin-coated slides.
Sections were
fixed onto slides by dipping for 10 minutes in Streck tissue fixative (Streck
laboratories)
and air drying. Sections were blocked in 30% normal goat serum and 0.3% triton
X-100 *
in PBS for 1 hr at room temperature (RT). Rabbit anti-calretinin polyclonal
antibody
(Chemicon laboratories) was diluted 1:200 in blocking solution and incubated
overnight
on sections at 4 C. Sections were washed 3 times (20 minutes each) in
Phosphate-
Buffered Saline (PBS) at RT. The secondary antibody anti-rabbit antibody,
Alexa 488%
(Molecular Probes), was diluted 1:400 in blocking solution and used to detect
calretinin.
Sections were covered and incubated at RT for 2 hours before washing and
mounting in
Vectashield containing DAPI (Vector). For confocal microscopy, sections were
treated
with 25 ptg/m1 RNAse before counterstaining with 50 fig/m1 of propidium iodide
and
mounted in Vectashield without DAPI. Stained sections were viewed under a Bio-
Rad
1024 confocal microscope.
Caketinin-positive cells are clearly visible in the sensory epithelia of the
semicircular canals and utricles of wild-type mice, but Mat/all-Gal/II-Gal
embryos lack
calretinin expression at all three states. Using the mouse model disclosed
herein the
present inventors demonstrate that hair cells never develop within the sensory
epithelia
of Math I fl-Ga1715-Gal mice. The presence of the tectorial and otolithic
membranes (secreted
in part by the supporting cells), together with the TEM results, suggests that
the
*Trade-mark
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remaining cells in the sensory epithelia of the MathP-Galifl-Gal mice are
functional
supporting cells.
EXAMPLE 7
MathillacZ expression mimics Math] expression in the developing CNS
The developing cerebellum at E14.5 and postnatal day 0 (PO) in Mathl'gal and
Math] b-gamb-gal mice were analyzed by RNA in situ hybridization analysis.
The analysis showed that the expression pattern of the lacZ gene faithfully
reproduced the Math/ expression pattern observed by RNA in situ hybridization
analysis
shown previously (Akazawa et al., 1995; Ben-Arie et al., 1996) (Fig. 2A, B, E,
G).
Moreover, the cerebellar phenotype in Math] b-gallb-gal mice (Fig. 8F and 8H)
was identical
to that observed in Math] null mice (Ben-Arie et al., 1997). At E14.5, the
precursors of
the EGL are present in the rhombic lip from which they migrate over the
cerebellar
anlage to populate the EGL (Fig. 8E). Mutant mice displayed far fewer of these
cells
than heterozygous mice (Fig. 8F). At PO, the neurons of the external granule
layer (EGL)
were completely lacking (Fig. 8H).
Mathl/lacZ expression in the developing hind brain and spinal cord similarly
reproduced the expression pattern of Math] (Fig. 8 C, 8D). The only notable
difference
between the expression patterns established by in situ hybridization and lacZ
staining is
that ligalactosidase expression persists in differentiating or migrating cells
of the spinal
cord because of the stability of the b-GAL protein (Fig. 8D). In summary, the
neural
tissue expression pattern and cerebellar phenotype associated with the
replacement of the
Math] coding region by lacZ is consistent with previously published data on
Math]
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expression (Akazawa et al., 1995; Ben-Arie et al., 1997; Ben-Arie etal., 1996;
Helms and
Johnson, 1998), demonstrating that the endogenous control elements were not
disrupted
by insertion of the lacZ gene. Moreover, many previously undetected clusters
of
lacZ-expressing cells became apparent upon X-gal staining of whole embryos and
sections in Math 1" mice (see below). It is likely that limitations in the
spatial
resolution of RNA in situ hybridization techniques used to detect the
transcript in earlier
studies prevented these sites of expression from being discerned (Akazawa et
al., 1995;
Ben-Arie et al., 1996). Alternatively, the stability of the lacZ gene product
and the
increased sensitivity due to signal amplification allowed us to identify sites
of relatively
low expression levels.
EXAMPLE 8
Math.14acZ is expressed in inner ear sensory epithelia
The sensory organs of the inner ear were among the newly identified sites of
Mathi /lacZ expression, demonstrated utilizing the methods described in
Example 2.
Expression in the otic vesicle was first detected at E12.5 and continued until
El 8.5
throughout much of the sensory epithelia (Bermingham et al., 1999) (Figure 9A,
9B).
Null mutants displayed Mathl/lacZ expression in the inner ear throughout
embryogenesis
(Fig. 9C). Math] null mutants lack hair cells in all of the sensory organs
(Bermingham
et al., 1999), but maintain supporting cells, the other sensory epithelia-
derived cells (Fig.
9C). These supporting cells seem to be functional, based on their morphology
and the
presence of overlying membranes secreted in part by these cells. Although the
expression of Math] in inner ear sensory epithelia was not demonstrated by RNA
in situ
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hybridization analysis, the complete lack of inner ear hair cells in the null
mutants leaves
little doubt about the authenticity of the Mathl/lacZ expression pattern.
Math] is clearly essential for hair cell development in the inner ear. Its
expression pattern and in vivo function are akin to those of Math] 's
proneural homolog,
atonal (ato) (A. P. Jarman, Y. Grau, L. Y. Jan, Y. N. Jan, Cell 73, 1307-21
(1994)). ato
is expressed in a ring of epithelial cells within the antennal disc of
Drosophila. Some of
these epithelial cells will subsequently develop into mechanoreceptors in the
Johnston
organ, which is necessary for hearing and negative geotaxis. It is interesting
to note that
mechanoreceptor progenitor cells are absent in ato mutants, whereas only the
mechanoreceptors, and not their progenitors, are absent in Math] null mice.
Based on the observations made herein, the present inventors have recognized
that
Math] is required for the specification of inner ear hair cells. In a sense,
Math] acts as
a "pro-hair cell gene" in the developing sensory epithelia. In conjunction
with two recent
studies, the present inventors have recognized that the results provided
herein provide
evidence supporting a lateral inhibition model for the determination of hair
cells and
supporting cells (Haddon et al., 1998; Adam, et al., 1998), in which the
interplay of
Delta, Notch, and Serrate] results in the selection of individual hair cells
from clusters
of competent cells. Such a model entails that the sensory epithelia express a
"pro-hair
cell gene" whose function is essential for hair cell fate specification.
The ectopic expression of ato in the fruitfly and its homolog Xathl in Xenopus
(Kim et al.,1997) can recruit epithelial cells into specific neuronal fates,
and the
expression of Math] in inner ear epithelia strongly suggests loss of a
functional Math]
gene is likely to be a common cause of deafness and vestibular dysfunction.
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EXAMPLE 9
MathlfiacZ is expressed in brain stem nuclei
In the brainstem Mathl/lacZ staining appeared from El 8.5 to P7 in the ventral
pons in the regions corresponding to the pontine nuclei (Fig. 9D and inset).
This finding
is consistent with the hypothesis of Akazawa and colleagues that Math /-
positive cells in
the developing hind brain are precursors to the bulbopontine neurons (Akazawa
et al.,
1995). No such staining appeared in the null mutants (Fig. 9E and inset).
These data
raise the possibility that the absence of lacZ staining in pontine nuclei can
be due to
failure of their precursors to migrate, proliferate, and/or differentiate.
Ventral pontine
nuclei were examined upon haematoxylin and eosin staining of sections and were
found
to be missing in the brain stem of null mice (Fig. 9F, G). Furthermore, the
failure of null
mouse newborns to breathe can be due to absence of these brainstem neurons.
EXAMPLE 10
Mathl/lacZ is expressed in chondrocytes
Math]
heterozygotes displayed expression of Math/ in articular cartilage
(Figures 6A and 6B). Figure 6A demonstrates expression in all joints of a
forelimb.
Upon closer examination of an elbow joint, Math] is noted to be expressed
exclusively
in the non-ossified articular chondrocytes.
Expression of Mathl/lacZ was detected in the developing proximal joints, such
as those of the hip and shoulder, as early as E 12.5 (Fig 10A). X-gal positive
staining was
detected at subsequent developmental stages in a progressive proximal-distal
pattern that
paralleled the normal development of joints (Figure 10B). In the joints,
Mathl/lacZ
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expression immediately follows mesenchymal condensation, which begins at
E11.5.
Condensed mesenchyme cells differentiate into chondrocytes (Bi et al., 1999;
Horton et
al., 1993; Karsenty, 1998).
Chondrocytes differentiate in three major phases during bone formation:
resting,
proliferating and hypertrophic. The resting chondrocytes that populate the
articular
cartilage are referred to as articular chondrocytes (Bucicwalter and Mankin,
1998; Poole,
1997). Prior to birth, resting chondrocytes constitute the entire chondrocyte
population
in joints. To establish which cells expressed Mathl/lacZ, sections from El 8.5
and P7
Mathre'ga) mice were stained with X-gal. Math 1 /lacZ is expressed in the
resting
chondrocytes of all joints analyzed at E18.5; resting chondrocytes in the
elbow joint are
shown in Figure 10C, and Fig. 10D shows the resting, proliferating, and
articular
chondrocytes of a P7 mouse.
The joints ofE18.5 embryos were examined with anti-MATH1 antibody prepared
by the following methods. An EcoRI-Hind III fragment encoding the N-terminal
156
amino acids of the Math] open reading frame (Math1D) was cloned into the pET
28a+
expression vector (Novagen). MathlD fragment was expressed as a His tag fusion
protein. Soluble MATH1D protein was purified according to His-tag kit
specifications
(Novagen) and 2mg of protein were used to immunize Chickens (Cocalico
Biologicals
Inc.).
Expression was found in resting chondrocytes, whereas no expression was
observed in null embryos. It should be noted that not all articular cartilage
cells express
MathMacZ (Fig. 10E). Mathl/lacZ expression in Math] null mutants is similar to
that
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in heterozygous mice at El 8.5, suggesting that Math] is not required for
resting
chondrocyte development.
EXAMPLE 11
Mathl/lacZ is expressed in Merkel cells
By El 4.5 Math///acZ-positive cells were apparent around the vibrissae and in
the
skin of much of the body (Fig. 10B). In the trunk, the stained cells were
arranged in a
striped pattern defined by the epidermal ridges. This staining was apparent
only in the
hairy, not the glabrous, skin. All the primary (mystical) vibrissae, including
the lateral
nasal, maxillary and four large hairs, were positive for Math] /lacZ. Staining
was also
detected in the secondary vibrissae, including the labial, submental, rhinal,
and isolated
orbital vibrissae (supra-, infra- and post-orbital) (Yamakado and Yohro,
1979). By E 15.5
staining appeared in clusters of cells in the foot pads (Fig. 10B).
To identify theMathMacZ-positive cells in the vibrissae, footpad, and hairy
skin,
we examined histological sections from Math ] mice (Fig. 11A-D). Sections
through
the vibrissae showed that the stained cells are localized to the more apical
half of the hair
shaft, but are not in the hair itself. Cross sections through the foot pad
illustrated staining
of cluster of cells in the epidermal layer (Fig. 11 B,C). As shown in Fig.
11D, sections
through the truncal skin identified clusters of Math MacZ-stained cells. The
stained cells
were arranged in a horseshoe-shaped pattern centered within an elevated button-
like
structure in the hairy skin. These button-like structures were identified as
touch domes
or Haarscheiben (Pinkus, 1905), which are characterized by a thickened
epidermis and
an elevated dermal papilla with a capillary network. Touch domes are
associated with
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large guard hairs dispersed between other hair types in the coat. The spatial
distribution
of Mathl/lacZ-stained cells, the timing of their appearance at E14.5, and
their
localization within the mystical pads of the vibrissae and the touch domes in
the hairy
skin suggest that these cells correspond to Merkel cells, specialized cells in
the epidermis
that form slow-adapting type I mechanoreceptor complexes with neurites
(Munger,
1991).
The results of comparative analysis of the Mathl/lacZ expression pattern in
heterozygous and homozygous E16.5 animals are shown in Figure 11E-L. Mathlb-
ga'gal
embryos displayed a staining pattern similar to that of MathrTh-gal
littermates in the
vibrissae and footpads (Fig.11E-G, I-K). In contrast, staining in the touch
domes of the
hairy skin was barely detectable in Math] b-gailb-gal embryos (Fig. 11H,L).
The reduction
of staining in null animals was also obvious at E18.5.
To further define Mathl/lacZ-positive cells in the skin, Mathrib-o mice were
mated to Tabby mice. Tabby (Ta) is a spontaneous X-linked mutation displaying
a
similar phenotype in hemizygous males and homozygous females (Ferguson et at.,
1997).
Tabby mutants lack hair follicles (tylotrich), a subset of Merkel cells that
are associated
with touch domes in the hairy skin of the trunk (Vielkind et al., 1995), and
some of the
five secondary vibrissae on the head (Gruneberg, 1971). Hence, in a cross of
Ta/Ta
females with a heterozygous Math 1'' male, 50% of the male progeny are Ta/Y:
Math1'0, allowing us to assess whether the Mathl/lacZ-positive cells
correspond to
Merkel cells.
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Ta/Ta females were time-mated with Math rib-gal males, and embryos were
harvested at E16.5. Each pup's gender was determined by PCR on tail DNA, using
primers (forward 5'-TGAAGCTTTTGGCTTTGAG-3% SEQ ID NO:67, and reverse
CCGCTGCCAAATTCTTTGG-3'; SEQ ID NO:68) that yielded a 320 bp product
from chromosome X, and a 300 bp product from chromosome Y (Liu et al., 1999).
Amplification conditions were: 92 C/1 min, 55 C/1 min, 72 C/1 min for 32
cycles, with
an initial denaturation step of 94 C/7 min and last extension step of 72 C/7
min.
Amplification products were separated on 2% agarose gels. X-gal-stained
embryos were
scored independently by 2 individuals, and only then were results matched with
the
determined gender.
Both Tabby females and males carrying the Math rib-gal allele displayed X-gal
staining in the vibrissae and foot pads (Fig. 12A,B). The effect of the Tabby
mutation
on the number of secondary vibrissae was quite clear; hemizygous males
completely
lacked Math MacZ-positive cells in the secondary vibrissae (typically lacking
in Ta
mutants) and on the trunk (Fig. 12E). Females that are heterozygous for Tabby
showed
patchy staining in the touch domes (although less than wt), as should be
anticipated in
female carriers of a mutation in a gene that undergoes random X chromosome
inactivation (Fig. 12C,D). The localization and distribution of the positive
cells, as well
as their absence in selected vibrissae and the trunk of Tabby males, strongly
indicate that
Math] is expressed in the Merkel cells associated with guard follicles in the
touch domes
of the hairy skin.
To ascertain whether Mathl/lacZ staining pattern reflects normal Mathl
expression pattern, immunohistochemical analysis of MATH I was performed on
sections
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from abdominal skin (see Example 2). As seen in Fig. 13A and B, MATH1-positive
cells
were detected around the hair follicles of Math 1 but not Math I b-galib-gal
mice. Antibodies
against two Merkel cells markers were chosen for further analysis: anti-
cytokeratin18,
expressed in simple epithelia, and chromogranin, localized to secretory
granules of
neuroendocrine, endocrine, and neuronal tissues. Both cytokeratin 18 (Fig.
13C,D) and
chromogranin A (Fig. 13E,F) confirmed the identity of the Math /fiacZ-positive
cells as
Merkel cells, but did not reveal staining abnormalities in Math/b-gal'o mice.
Thus,
Math] does not seem to be essential for the genesis of the neuroendocrine
Merkel cells,
in contrast to pure neuronal cell types like cerebellar EGL and pontine
nuclei. Because
Math] null mutants die at birth, we can not assess whether the entire cluster
of Merkel
cells is formed or the functional integrity of Merkel cells in these mutants
is affected.
EXAMPLE 12
Mathl partially rescues Chinese Hamster
Ovary Cells in flies deleted for ato
This Example demonstrates that atonal-associated genes can induce the
development of CNS cells in animals deficient in a native atonal-associated
gene or gene
product. This Example also demonstrates that atonal-associated genes can
therapeutically function in species in which they are not natively expressed.
Given the remarkable similarity in expression patterns of ato and Math] , and
their
identical basic domains, Math] was tested to see if it would mimic the effects
of ato
overexpression by producing ectopic chordotonal organs as described by the
following
methods. Wild-type, also known as yw flies, were transformed with a UAS-Mathl
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construct as described (Brand and Perrimon, 1993). To overexpress Math I in
wild type
flies, yw; UAS-Mathl flies were mated to IIS-Ga14 flies. The progeny were heat
shocked
as previously described (Jarman et al., 1993). To rescue the loss of
chordotonal organs
in ato mutant flies, w; UAS-Mathl/UAS-Mathl ; atol /TM6 flies were crossed to
w;
HS-Ga14/Cy0; atol/TM6 flies. Embryos were collected for 3 hr., aged for 3 hr.,
heat
shocked for 30 min. at 37 C and allowed to develop for the next 12-15 hr.
Embryos were
fixed in 4% formaldehyde in PBS with 50% heptane. Embryos were washed with
100%
ethanol, transferred to PBT and stained with mAb 22C10 as previously described
(Kania
et al., 1995) to detect PNS neurons. Chordotonal neurons were identified by
their distinct
morphology and position.
Expressing Math) during pupal development by heat shock using the UAS-Ga14
system (Brand and Perrimon, 1993) resulted in supernumerary external sense
organs on
the notum (Fig. 14A,B) and the wing blade, as reported for ato (Jarman et al.,
1993) and
the Achaete-Scute complex (AS-C) genes (Brand and Perrimon, 1993; Rodriguez et
al.,
1990). Math] expression in flies, like ato, produced ectopic chordotonal
organs (Fig.
80), although with less efficiency. Overexpression of the AS-C genes does not,
however,
result in ectopic chordotonal organs (Jarman et al., 1993). Mathl thus has a
similar
functional specificity to ato.
Since several ato enhancers are ato-dependent (Sun et al., 1998), they can be
activated by Math), which would then lead to ectopic CHO specification. To
determine
whether Math] can substitute for ato function in the fly, and to rule out the
possibility
that production of CHOs by Math) is due to ato activation, Math) was expressed
in ato
mutant embryos. The mutants lack all chordotonal neurons (Fig. 14C), but
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overexpressing Mathl partially rescues the loss of these neurons (Fig. 14D) in
a manner
similar to ato (Chien et al., 1996).
EXAMPLE 13
Significance of atonal and Mathl in the CNS and PNS
Over the past few years significant progress has been made towards unraveling
the roles o fbHLH proteins in vertebrate neurogenesis. Neural vertebrate bHLH-
encoding
genes were isolated and characterized because Drosophila homologues such as
ato or the
AS-C genes had been previously shown to be required for neurogenesis
(Anderson, 1995;
Guillemot, 1046 1995; Lee, 1997; Takebayashi et al., 1997). Indeed, several
genes were
shown to be proneural because their absence caused a failure of neuroblast or
sensory
organ precursor (SOP) specification, whereas their overexpression lead to the
recruitment
of supernumerary neuronal precursors (Ghysen and Dambly-Chaudiere, 1989). With
the
exception of neurogenin (Ngn) 1 and 2 (Fode et al., 1998; Ma et al., 1998), it
remains
uncertain which of the vertebrate homologues play roles similar to their
Drosophila
counterparts, and what precise role different bHLH proteins play in neural
development.
In Drosophila, ato is required for the development of a specific subset of
sense organs,
the chordotonal organs (Jarman et al., 1993). CHOs are internal mechanosensors
of the
PNS (McIver, 1985). Thus, ato and the CHOs provide an excellent system in
which to
ascertain not only the molecular and developmental relationship between
invertebrate and
vertebrate neurogenesis vis-à-vis the function of the proneural genes, but
also the
evolutionary conservation of sensory organ function and specification. Seven
ato
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homologues have been cloned and analyzed in the mouse: Mouse Atonal Homologues
(MATH) 1, 2, 3, 4A (also known as Ngn2), 4B (Ngn3), 4C (Ngnl), and 5 (Akazawa
et al.,
1995; Bartholorna and Nave, 1994; Ben-Arie et al., 1997, 1996; Fode et al.,
1998; Ma et
al., 1998; McCormick et al., 1996; Shimizu et al., 1995; Takebayashi et al.,
1997). Most
are expressed during neurogenesis in both the CNS and PNS. These homologues
vary in
the degree of their sequence conservation, and can be divided into three
groups. The most
distantly related group, the neurogenins, includes Ngn 1, 2 and 3. These gene
products
share, on average, 53% identity in the bHLH domain with ATO. They are
expressed
largely in mitotic CNS and sensory ganglia progenitor cells. Recent work
suggests that
these genes canplay a role in neurob last determination, and cantherefore be
true proneural
genes (Fode et at., 1998; Ma et al., 1998). The second group includes MATH2
and
MATH3, which share 57% identity in the bHLH domain with ATO. These proteins
have
been postulated to function in postmitotic neural cells (Bartholoma and Nave,
1994;
Shimizu et al., 1995). Math2 expression is confined to the CNS, while Math3 is
expressed in both the CNS and the trigeminal and dorsal root ganglia. The
third group
includes MATH1 and MATHS, which share 67% and 71% identity with the bHLH
domain of ATO, respectively. It is noteworthy that both genes encode a basic
domain
identical to that of ATO. Interestingly, the basic domain of ATO was shown to
be
sufficient, in the context of another proneural protein (SCUTE), to substitute
for the loss
of ato function (Chien et al., 1996). Mathl was initially shown to be
expressed in the
precursors of the cerebellar EGL and in the dorsal spinal cord (Ben-Arie et
al., 1997,
1996). Math5 is expressed in the dividing progenitors in the developing retina
and in the
vagal ganglion (Brown et al., 1998). With the exception of Math5 expression in
the
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neural retina, these observations pose a paradox: none of the vertebrate
homologs
appeared to be expressed in peripheral organs or tissues similar to those
where ato is
expressed. Jarman et al. (1993) reported that ato is expressed in the CNS. In
the examples
described herein it is shown that, in addition to the inner proliferation
center of the optic
lobe, ato is expressed in a small anteriomedial patch of cells in each brain
lobe (Fig. 8F).
Because it remains unclear, however, precisely what role ato plays in
Drosophila CNS
development, it has been difficult to argue that ato and its vertebrate
homologues display
functional conservation. Our experiments reveal sites of previously
uncharacterized
Math I expression. As expected, we found that Math 1 /lacZ expression in the
CNS
corresponds to that of Math] , but we also found that Math] is expressed in
the skin, the
joints, and the inner ear, in striking parallel to ato expression in the fly.
Moreover, the
expression in the ear (sensory epithelium) and the skin (Merkel cells) is
restricted to
sensory structures whose function is to convert mechanical stimuli into
neuronal
electrochemical signals. It is important to point out that in Drosophila, ato
appears to play
two roles simultaneously. It is required not only to select the precursors of
the CHOs
(proneural role), but also to specify these precursors as CHO precursors
(lineage identity
role) (Jarman and Ahmed, 1998; Jarman et al., 1993). The specificity of Math]
expression in the periphery makes it tempting to speculate that it, too,
canendow specific
cells with very specific lineage identities to distinguish them functionally
from other
sensory structures. The ability of Math] to induce ectopic CHO formation and
to restore
CHOs to ato mutant embryos supports the notion that Math], and particularly
its basic
domain, encodes lineage identity information not unlike that encoded by ato.
This
suggests that the mammalian cells expressing Math1, at least in the ear and
the skin, are
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functionally similar and perhaps evolutionarily related to Drosophila cells
that require
ato. Furthermore, Math5 expression in the neural retina suggests that the
functions of
atonal in the fly are carried out by two genes in the mouse: the development
of some
mechanoreceptors is under the control of Math/ and retinal development is
possibly
under the control of Math5. It is interesting to note that in the fully
sequenced nematode
C.elegans, only one homolog of atonal, lin-32, was identified (Zhao and
Emmons, 1995).
Mutants with the u282 allele of lin-32 are touch-insensitive, which
strengthens the
argument for evolutionary conservation of atonal function in mechanoreeeption.
The
pattern of Mathl/lacZ expression in the pontine nuclei suggested this region
should be
carefully evaluated in null mutants. Although no defects in the pons of Math/
null mice
(Ben-Arie et al., 1997) were originally detected, closer analysis revealed the
lack of
pontine nuclei at this site. These neurons derive from the rhombic lip (Altman
and Bayer,
1996) as do the EGL neurons, which are also lacking in Math] null mice. While
it is
possible to draw parallels between Math] and ato expression in the skin and
ear, it is not
clear that such is the case for the joints. ato expression in the fly joints
is required for the
formation of leg CHOs. In contrast, Math] is expressed in resting and
articular
chondrocytes that do not have any described neural function, and for which no
parallels
exist in the fly. It can be that Math] expression in cartilage indicates a
novel role for a
mechanosensory gene, or it cansimply reflect similarities in the molecular
events
underlying the development of the various Math]-expressing cell types.
Alternatively,
CHOs canalso function as joint structural elements in the fly, or articular
cartilage
canhave a mechanoreceptive or transducive capacity yet to be described. There
is no
evidence at this point to support one or another of these possibilities.
Analyzing the
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functions of ato and Mathl will enhance our understanding of neural
development and
the evolutionary conservation of sensory function. The sites and specificity
of Math]
expression canmake it suitable as a tool of gene therapy or gene activation
approaches
to illnesses such as hearing loss and osteoarthritis that are due to age-
related or
environmental damage.
EXAMPLE 14
Atonal-Associated Nucleic Acid Delivery using Adenovirus
Human adenoviruses are double-stranded DNA tumor viruses with genome sizes
of approximate 36 kb. As a model system for eukaryotic gene expression,
adenoviruses
have been widely studied and well characterized, which makes them an
attractive system
for development of adenovirus as a gene transfer system. This group of viruses
is easy
to grow and manipulate and they exhibit a broad host range in vitro and in
vivo. In
lytically infected cells, adenoviruses are capable of shutting off host
protein synthesis,
directing cellular machineries to synthesize large quantities of viral
proteins, and
producing copious amounts of virus.
The El region of the genome includes E1A and El B, which encode proteins
responsible for transcription regulation of the viral genome, as well as a few
cellular
genes. E2 expression, including E2A and E2B, allows synthesis of viral
replicative
functions, e.g. DNA-binding protein, DNA polymerase, and a terminal protein
that
primes replication. E3 gene products prevent cytolysis by cytotoxic T cells
and tumor
necrosis factor and appear to be important for viral propagation. Functions
associated
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with the E4 proteins include DNA replication, late gene expression, and host
cell shutoff.
The late gene products include most of the virion capsid proteins, and these
are expressed
only after most of the processing of a single primary transcript from the
major late
promoter has occurred. The major late promoter (MLP) exhibits high efficiency
during
the late phase of the infection.
As only a small portion of the viral genome appears to be required in cis,
adenovirus-derived vectors offer excellent potential for the substitution of
large DNA
fragments when used in connection with cell lines such as 293 cells. Ad5-
transformed
human embryonic kidney cell lines have been developed to provide the essential
viral
proteins in trans. The inventors thus reasoned that the characteristics of
adenoviruses
rendered them good candidates for use in targeting Mathl deficient cells in
vivo. In
another embodiment these constructs include a Hathl or any atonal-associated
nucleic
acid sequence.
Particular advantages of an adenovirus system for delivering foreign proteins
to
a cell include: (i) the ability to substitute relatively large pieces of viral
DNA by foreign
DNA; (ii) the structural stability of recombinant adenoviruses; (iii) the
safety of
adenoviral administration to humans; (iv) lack of any known association of
adenoviral
infection with cancer or malignancies; (v) the ability to obtain high titers
of the
recombinant virus; and (vi) the high infectivity of Adenovinis.
One advantage of adenovirus vectors over retroviruses is a higher level of
gene
expression. Additionally, adenovirus replication is independent of host gene
replication,
unlike retroviral sequences. Because adenovirus transforming genes in the El
region can
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be readily deleted and still provide efficient expression vectors, oncogenic
risk from
adenovirus vectors is thought to be negligible.
In general, adenovirus gene transfer systems are based upon recombinant,
engineered adenovirus that is rendered replication-incompetent by deletion of
a portion
of its genome, such as El, and yet still retains its competency for infection.
Relatively
large foreign proteins can be expressed when additional deletions are made in
the
adenovirus genome. For example, adenoviruses deleted in both El and E3 regions
are
capable of carrying up to 10 Kb of foreign DNA and can be grown to high titers
in 293.
Surprisingly, persistent expression of transgenes following adenoviral
infection is
possible. Use of the adenovirus gene transfer system can be more useful for
the delivery
of Math] to cells in nascent or damaged cartilage in joints. In particular,
the Mathl
adenovirus can be used to deliver Math], and confer Mathl gene expression in,
non-
ossified joint cartilage that has been damaged as a consequence of
osteoarthritis.
EXAMPLE 15
Math/-Adenovirus Constructs
Recombinant virions for the controlled expression of Mathl can be constructed
to exploit the advantages of adenoviral vectors, such as high titer, broad
target range,
efficient transduction, and non-integration in target cells for the
transformation of cells
into hair cells. In one embodiment these constructs include a Hathl or any
atonal-
associated nucleic acid sequence. In one embodiment of the invention, a
replication-
defective, helper-independent adenovirus is created that expresses wild type
Math]
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sequences under the control of the human cytomegalovirus promoter or the
metallothionine promoter.
Control functions on expression vectors are often provided from viruses when
expression is desired in mammalian cells. For example, commonly used promoters
are
derived from polyoma, adenovirus 2 and simian virus 40 (SV40). The early and
late
promoters of SV40 virus are particularly useful because both are obtained
easily from the
virus as a fragment which also contains the SV40 viral origin of replication.
Smaller or
larger SV40 fragments canalso be used provided there is included the
approximately 250
bp sequence extending from the Hind111 site toward the Bgll site located in
the viral
origin of replication. Further, it is also possible, and often desirable, to
use promoter or
control sequences normally associated with the Math 1 gene sequence, namely
the Math]
promoter, provided such control sequences are compatible with the host cell
systems or
the target cell. One such target cell is located in the inner ear of a human
patient in need
of inner ear hair cells.
An origin of replication can be provided by construction of the vector to
include
an exogenous origin, such as can be derived from SV40 or other viral (e.g.,
polyoma,
adeno, VSV, BPV) source, or can be provided by the host cell chromosomal
replication
mechanism. If the vector is integrated into the host cell chromosome, the
latter is often
sufficient.
EXAMPLE 16
Atonal-Associated Nucleic Acid Delivery using Retrovirus
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Another approach for gene delivery capitalizes on the natural ability of
viruses
to enter cells, bringing their own genetic material with them. Retroviruses
have promise
as gene delivery vectors due to their ability to integrate their genes into
the host genome,
transferring a large amount of foreign genetic material, infecting a broad
spectrum of
species and cell types and because they are easily packaged in special cell-
lines.
Retroviruses can be particularly useful for the delivery of Math 1 into inner
ear hair cells
that have reduced expression of Math], or that are in need of over-expression
of Math].
EXAMPLE 17
Mathl Retroviral Constructs
The Math! open reading frame (ORF) was excised from pBluescript by an EcoR
I-XbaI digest. The fragment was gel purified, and blunt ended using Klenow DNA
polymerase. The retroviral vector pLNCX (purchased from CLONTECH) was
linearized
with HpaI, and ligated with the Math] ORF fragment. The ligation was
transformed into
transformation competent E. coli cells. The resulting antibiotic resistant
colonies were
assayed for the presence of the correct construct.
The cloning, reproduction and propagation retroviral expression vectors is
well
known to those of skill in the art. One example of a retroviral gene transfer
and
expression system that has been used to express Mathl is the CLONTECH pLNCX,
pLXSN and LAPSN expression vectors. For propagation of these vectors PT67 and
EcoPack packaging cell lines can be used. For more information on mammalian
cell
culture, the following general references can be used: Culture of Animal
Cells, Third
*Trade mark
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Edition, edition by R. I. Freshney (Wiley-Liss, 1993); and Current Protocols
in
Molecular Biology, ed. By F. M. Ausubel, et al., (Greene Publishing Associates
and
Wiley & Sons, 1994)
In another embodiment these constructs could be constructed with al-lath] or
any
atonal-associated nucleic acid sequence.
EXAMPLE 18
Maintenance of Packaging Cell Lines
The maintenance of packaging cell lines, such as the 293 and P167 packaging
cell
lines, is described briefly. A vial of frozen cells is transferred from liquid
N2 to a 37 C
water bath until just thawed. In order avoid osmotic shock to the cells, and
to maximize
cell survival, 1 ml of (Dulbecco's Modified Eagle Medium) DMEM is added to the
tube
and the mixture is transferred to a 15-ml tube. Another 5 ml of DMEM is added
and the
cells are mixed. After repeating these steps the final volume in the tube
should be about
12 ml. Next, the cells are centrifuged at 500 x g for 10 min. Finally, the
supernatant is
removed and the cells are resuspended in maintenance media as described in the
next
step. Generally, the cells are maintained in DMEM (high glucose: 4.5 g/L)
containing
10% Fetal Bovine Serum (FBS), and 4 mM L-glutamine. If desired or necessary,
100
U/ml penicillin/100 g/m1 streptomycin can be added. It is recommended that are
plated
at 3-5 x 105 per 100-mm plate and split every 2 to 3 days, when they reach 70-
80%
confluency (confluence is 3-4 x 106 per 100-mm plate). The PT67 cell line, for
example,
has a very short doubling time (<16h) and should be split before they become
confluent.
The doubling time for EcoPack-293 cells is 24-36 h.
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Cells are split be removing the medium and washing the cells once with PBS.
After treatment with 1-2 ml of trypsin-EDTA solution for 0.5-1 min, 5 to 10 ml
of media
and serum is added to stop trypsinization. The cells are dispersed gently, but
thoroughly,
by pipetting and are resuspended. Alternatively, a predetermined portion of
the cells is
replated in a 100-mm plate in 10 ml of medium, followed by rotation or shaking
of the
plate to distribute the cells evenly. A ratio of up to 1:20 for the PT67 or
EcoPack-293
cells is common.
Generally, the percentage of PT67 or EcoPack-293 cells capable of packaging
retroviral vectors decreases slowly with continued passage of the cell line.
Therefore,
packaging cells should be reselected after 2 months of growth in culture.
Alternatively,
new high-titer cells can be purchased from, e.g., CLONTECH, or low passage
number
stocks can be frozen, stored and thawed to increase the viral yield.
EXAMPLE 19
Methods Utilizing a Retroviral Vector
The following protocol is used to transfect the retroviral vector for virus
production, infection of target cells, and selection of stable clones. Other
methods and
vectors canals be used with the present invention to express Math I, such as
those
described in Retroviruses, ed. by J. M. Coffin & H. E. Varmus (1996, Cold
Spring
Harbor Laboratory Press, NY) and Current Protocols in Molecular Biology, ed.
by F. M.
Ausubel et al. (1994, Greene Publishing Associates and Wiley & Sons).
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Briefly, the transfection of the retroviral vector into PT67 cells was as
follows.
Math I was cloned into pLNX as described hereinabove. The packaging cells were
plated
to a density of 5-7 x 105 cells per 100-mm plate 12-24 hours before
transfection. 1-2
hours before transfection, the medium replace with fresh medium. 25 uM
chloroquine
can be added just prior to transfection. Chloroquine increases transfection
efficiency 2-3
fold. A 25 mM stock solution of chloroquine can be made in distilled water and
filter
sterilized.
To each 100-mm plate 10-15 jig of plasmid DNA using the desired method is
transfected using, e.g., standard calcium-phosphate procedures (CalPhos
Mammalian
Transfection Kit, #K2050-1). The final volume of transfection mixture should
not exceed
1 ml. The transfection solution is added to the medium and the plate is
rotated to ensure
even distribution. About 8 hours after transfection, a glycerol shock
treatment can be
performed to increase the uptake of DNA. After 10 to 24 hours post-
transfection the
medium was removed and the cells were washed twice with PBS, before adding 5
ml
DMEM containing 10% FBS. The culture was incubated for an additional 12-48
hours
to allow increase in virus titer. The virus titer reaches a maximum - 48 hours
post-
transfection and is generally at least 30% of maximum between 24 and 72 hours
post-
transfection.
Alternatively, a stable virus-producing cell lines canalso be selected. To
obtain
stable virus-producing cell lines, the transfected packaging cells are plated
in a selection
medium 2-3 days post-transfection. For G418 selection of neomycin resistance,
the cells
are selected in the presence of G418 (0.5 mg/ml "active") for one week.
Vectors carrying
other selectable markers such as Puro, Bleo, or Hyg, can be used to obtain
stable virus
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producing cell populations as well. Cell populations producing virions that
produce titers
of 105-106 recombinant virus particles per ml are common. Generally, 105-106
recombinant virus particles per ml is suitable for most purposes. For some
studies,
higher titer clones can be required. In this case, after antibiotic selection,
individual
clones are selected using, e.g., clone cylinders or limiting dilution, prior
to propagation.
Viral titer can be determined in a variety or ways, one such method is
described
hereinbelow. The viral titer produced by transiently transfected or stable
virus-producing
packaging cell lines is determined as follows, NTH/3T3 cells are plated one
day prior to
beginning the titer procedure. Cells are plated in 6-well plates at a density
of 5 x 104-1
x 105 cells per well and 4 ml of media are added per well. Virus-containing
medium is
collected from packaging cells, and polybrene is added to a final
concentration of 4
kig/ml. The medium is filter-sterilized through a 0.45- m filter. Polybrene is
a
polycation that reduces the charge repulsion between the virus and the
cellular
membrane. The filter should be cellulose acetate or polysulfonic (low protein
binding)
but not nitrocellulose. Nitrocellulose binds proteins in the retroviral
membrane, and
consequently destroys the virus. Serial dilutions are prepared as follows: six
10-fold
serial dilutions are usually sufficient. To dilute the virus use fresh medium
containing
4 kighnl ofpolybrene. Next, NIH/3T3 target cells are infected by adding virus-
containing
medium to the wells. After 48 hours, the NIH/3T3 cells are stained. The titer
of virus
corresponds to the number of colonies present at the highest dilution that
contains
colonies, multiplied by the dilution factor. For example, the presence of four
colonies
in the 105 dilution would represent a viral titer of 4 x 105.
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For the infection of cells, the following procedure was followed. The target
cells
were plated 12-18 hours before infection at a cell density of 3-5 x 10 per 100-
mm plate.
For the infection of cells that can be used for a biological assay, control
cells can be
treated with an insert-free virus produced under identical conditions. Half-
maximal
infection generally occurs after 5-6 hours of exposure of cells to virus, with
maximal
infection occurring after approximately 24 hours of exposure. The actual
reverse
transcription and integration o f the retrovirus takes place within 24-36
hours of infection,
depending on cell growth kinetics. Expression can be observed at 24 hours, and
reaches
a maximum at approximately 48 hours. Alternatively, infections can be
conducted
sequentially, about 12 hours apart. Sequential infection generally increases
the efficiency
of infection and also increases viral copy number. A minimum of 12 hours
between each
infection is recommended in order to ensure that cellular receptors will be
unoccupied
by viral envelope.
EXAMPLE 20
Screening Assays
Finally, the present invention also provides candidate substance screening
methods that are based upon whole cell assays, in vivo analysis and
transformed or
immortal cell lines in which a reporter gene is employed to confer on its
recombinant
hosts a readily detectable phenotype that emerges only under conditions where
Mathl
would be expressed, is under-expressed or is over-expressed. Generally,
reporter genes
encode a polypeptide not otherwise produced by the host cell that is
detectable by
analysis, e.g., by chromogenic, fluorometric, radioisotopic or
spectrophotometric
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analysis. In the present invention the Math] gene has been replaced with I3-
galactosidase
in a mouse.
An example of a screening assay of the present invention is presented herein.
Math] expressing cells are grown in microtiter wells, followed by addition of
serial molar
proportions of the small molecule candidate to a series of wells, and
determination of the
signal level after an incubation period that is sufficient to demonstrate,
e.g., calretinin
expression in controls incubated solely with the vehicle used to resuspend or
dissolve the
compound. The wells containing varying proportions of candidate are then
evaluated for
signal activation. Candidates that demonstrate dose related enhancement of
reporter gene
transcription or expression are then selected for further evaluation as
clinical therapeutic
agents. The stimulation of transcription can be observed in the absence of
expressed
Math), in which case the candidate compound might be a positive stimulator of
hair cell
differentiation. Alternatively, the candidate compound might only give a
stimulation in
the presence of low levels of Math 1 , which would suggest that it functions
to stabilize
the formation of Math) dimers or the interaction of Math I with one or more
transcriptional factors. Candidate compounds of either class might be useful
therapeutic
agents that would stimulate production of inner ear hair cells and thereby
address the
need of patients with hearing loss or balance control impairments.
EXAMPLE 21
Transfection of Cells with Mathl Retroviral Vectors
The present invention provides recombinant host cells transformed or
transfected
with a polynucleotide that encodes Math], as well as transgenic cells derived
from those
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transformed or transfected cells. In another embodiment these constructs could
be
constructed with a Hath1 or any atonal-associated nucleic acid sequence. A
recombinant
host cell of the present invention is transfected with a polynucleotide
containing a
functional Math I nucleic acid sequence or a chimeric Math] gene. Methods of
transforming or transfecting cells with exogenous polynucleotides, such as DNA
molecules, are well known in the art and include techniques such as calcium-
phosphate-
*
or DEAE-dextran-mediated transfection, protoplast fusion, electroporation,
liposome
mediated transfection, direct microinjection and adenovirus infection.
Math] expression using recombinant constructs can be used to target the
delivery
of Math Ito cells in need thereof. Different promoter-vector combinations can
be chosen
by a person skilled in these arts to drive Math I expression in different cell
types. In some
cases, the desired outcome cannot be protein, but RNA, and recombinant vectors
would
include those with inserts present in either forward or reverse orientations.
In addition,
some vectors, for instance retroviruses or artificial recombination systems,
can be
designed to incorporate sequences within a cellular or viral genome in order
to achieve
constitutive or inducible expression of protein or RNA.
Many of the vectors and hosts are available commercially and have specific
features that facilitate expression or subsequent purification. For instance
DNA
sequences to be expressed as proteins often appear as fusion with unrelated
sequences
that encode polyhistidine tags, or HA, FLAG, myc and other epitope tags for
immunochemical purification and detection, or phosphorylation sites, or
protease
recognition sites, or additional protein domains such as glutathione S-
transferase (GST),
maltose binding protein (MBP) (New England Biolabs), and so forth that
facilitate
*Trade-mark
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purification. Vectors canalso be designed that contain elements for
polyadenylation,
splicing, and termination, such that incorporation of naturally occurring
genomic DNA
sequences that contain introns and exons can be produced and processed, or
such that
unrelated introns and other regulatory signals require RNA processing prior to
production
of mature, translatable RNAs. Proteins produced in the systems described above
are
subject to a variety of post-translational modifications, such as
glycosylation,
phosphorylation, nonspecific or specific proteolysis or processing.
EXAMPLE 22
Delivery of Mathl as an Amino Acid Sequence
A peptide (11 amino acids) derived from HIV has been recently described that
when fused to full length proteins and injected into mice allow a rapid
dispersal to the
nucleus of all cells of the body (Schwarze et al., 1999). Schwarze et al. made
fusion
proteins to Tat ranging in size from 15 to 120 kDa. They documented a rapid
uptake of
the fusion proteins to the nuclei of cells throughout the animal, and the
functional activity
of said proteins was retained.
In an embodiment of the present invention there are constructs containing the
Tat
or Tat-HA nucleic acid sequence operatively linked to a Math] nucleic acid
sequence.
In another embodiment these constructs include a Hathl or any atonal-
associated nucleic
acid sequence. The vectors are expressed in bacterial cultures and the fusion
protein is
purified. This purified Tat-Mathl protein or Tat-Hathl protein is injected
into animal to
determine the efficiency of the Tat delivery system into the inner ear, skin,
cerebellum,
brain stem, spinal cord and joints. Analysis is carried out to determine the
potential of
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the Tat-Mathl /Tat-Hathl protein in hair cell and neuronal regeneration. This
is a viable
therapeutic approach either in its own right or in association with other
methods or genes.
It should be understood that the methods to screen for compounds which affect
Math/ expression disclosed herein are useful notwithstanding that effective
candidates
cannot be found, since it is of practical utility to know what upstream
effector is
necessary for Math/ transcription.
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,
PCT Application No. PCT/US89/01025
PCT Application WO 88/10315
PCT Application WO 89/06700
PCT Application WO 90/07641
One skilled in the art readily appreciates that the present invention is well
adapted to carry out the objectives and obtain the ends and advantages
mentioned as well
as those inherent therein. Sequences, mutations, complexes, methods,
treatments,
pharmaceutical compositions, procedures and techniques described herein are
presently
representative of the preferred embodiments and are intended to be exemplary
and are not
intended as limitations of the scope. Changes therein and other uses will
occur to those
skilled in the art, which are defined by the scope of the pending claims. The
scope of the
claims should not be limited by particular embodiments set forth herein, but
should be
construed in a manner consistent with the specification as a whole.
121
