U.S. patent application number 14/426520 was filed with the patent office on 2015-07-30 for methods and compositions for regenerating hair cells and/or supporting cells.
The applicant listed for this patent is MASSACHUSETTS EYE AND EAR INFIRMARY. Invention is credited to Zheng-Yi Chen.
Application Number | 20150209406 14/426520 |
Document ID | / |
Family ID | 50237658 |
Filed Date | 2015-07-30 |
United States Patent
Application |
20150209406 |
Kind Code |
A1 |
Chen; Zheng-Yi |
July 30, 2015 |
METHODS AND COMPOSITIONS FOR REGENERATING HAIR CELLS AND/OR
SUPPORTING CELLS
Abstract
Provided are methods and compositions for inducing cells of the
inner ear (for example, cochlear and utricular hair cells) to
reenter to cell cycle and to proliferate. More particularly, the
invention relates to the use of agents that increase c-myc activity
and/or Notch activity for inducing cell cycle reentry and
proliferation of cochlear or utricular hair cells and/or cochlear
or utricular supporting cells. The methods and compositions can be
used to promote the proliferation of hair cells and/or supporting
cells to treat a subject at risk of or affected with, hearing loss
or a subject at risk of or affected with vestibular
dysfunction.
Inventors: |
Chen; Zheng-Yi; (Somerville,
MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
MASSACHUSETTS EYE AND EAR INFIRMARY |
Boston |
MA |
US |
|
|
Family ID: |
50237658 |
Appl. No.: |
14/426520 |
Filed: |
September 6, 2013 |
PCT Filed: |
September 6, 2013 |
PCT NO: |
PCT/US13/58626 |
371 Date: |
March 6, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61698246 |
Sep 7, 2012 |
|
|
|
Current U.S.
Class: |
514/16.5 ;
435/377 |
Current CPC
Class: |
A61P 25/28 20180101;
C12N 2710/10341 20130101; A61P 27/16 20180101; A61K 38/177
20130101; A61K 48/005 20130101; A61K 31/65 20130101; C12N 2501/606
20130101; C12N 5/062 20130101; C12N 2501/42 20130101; A61K 38/1709
20130101; A61K 48/00 20130101; A61K 35/30 20130101; A61P 43/00
20180101; A61K 38/16 20130101 |
International
Class: |
A61K 38/17 20060101
A61K038/17; C12N 5/0793 20060101 C12N005/0793 |
Claims
1. A method of inducing proliferation or cell cycle reentry of a
differentiated cochlear cell or a utricular cell, the method
comprising increasing both c-myc activity and Notch activity within
the cell sufficient to induce proliferation or cell cycle reentry
of the cochlear cell or utricular cell.
2. The method of claim 1, wherein the cell dedifferentiates upon
reentry into the cell cycle.
3. The method of claim 1, wherein the cochlear cell or the
utricular cell is a hair cell or a supporting cell.
4. The method of claim 1, wherein c-myc activity is increased by
administering an effective amount of a c-myc protein or a c-myc
activator.
5. The method of claim lany one of claim 1, wherein Notch activity
is increased by administering an effective amount of a Notch
protein, a Notch Intracellular Domain (NICD) protein or a Notch
activator.
6. The method of claim 1, wherein, after c-myc activity is
increased, c-myc activity is inhibited to limit proliferation of
the cochlear cell or utricular cell and/or to promote survival of
the cochlear cell or utricular cell.
7. The method of claim 3, wherein when the cochlear cell or the
utricular cell is a supporting cell, the method further comprises
the step of inhibiting Notch activity after proliferation of the
supporting cell thereby to induce differentiation of the supporting
cell and/or at least one of its daughter cells into a hair
cell.
8. The method of claim 7, wherein the Notch activity is inhibited
by administering an effective amount of a Notch inhibitor.
9. The method of claim 3, wherein when the cochlear cell or the
utricular cell is a supporting cell, the method further comprises
the step of increasing Atoh1 activity after proliferation of the
supporting cell thereby to induce differentiation of the supporting
cell and/or at least one of its daughter cells into a hair
cell.
10. A method for regenerating a cochlear or utricular hair cell,
the method comprising: (a) increasing both c-myc activity and Notch
activity within the hair cell thereby to induce cell proliferation
to produce a daughter cell; and (b) after cell proliferation,
decreasing Notch activity thereby to induce differentiation of at
least one of the cell and the daughter cell to produce a
differentiated cochlear or utricular hair cell.
11. The method of claim 10, wherein the cochlear cell or the
utricular cell is a hair cell or a supporting cell.
12. The method of claim 10, wherein, in step (a), c-myc is
increased by contacting the cell with an effective amount of a
c-myc protein or a c-myc activator.
13. The method of claim 10, wherein, in step (b), Notch is
increased by contacting the cell with an effective amount of a
Notch protein, a Notch Intracellular Domain (NICD) protein or a
Notch activator.
14. The method of claim 10, wherein, in step (b), Notch activity is
decreased by contacting the cell with an effective amount of a
Notch inhibitor.
15. The method of claim 12, wherein the c-myc protein or the c-myc
activator is administered to an inner ear of a subject.
16. The method of claim 13, wherein the Notch protein, NICD
protein, or the Notch activator is administered to an inner ear of
a subject.
17. The method of claim 14, wherein the Notch inhibitor is
administered to an inner ear of a subject.
Description
CROSS REFERENCE TO RELATED APPLICATION
[0001] This application claims priority to and the benefit of U.S.
Provisional Patent Application No. 61/698,246, which was filed on
Sep. 7, 2012, the entire contents of which are incorporated by
reference herein.
FIELD OF THE INVENTION
[0002] The field of the invention relates generally to methods and
compositions for inducing inner ear cells to reenter the cell cycle
and to proliferate. More particularly, the invention relates to
increasing c-myc and/or Notch activity within cells to induce cell
cycle reentry and proliferation of hair cells and/or supporting
cells of the inner ear.
BACKGROUND OF THE INVENTION
[0003] One of the most common types of hearing loss is
sensorineural deafness that is caused by the loss of hair cells or
hair cell function. Hair cells are sensory cells in the cochlea
responsible for transduction of sound into an electrical signal.
The human inner ear contains only about 15,000 hair cells per
cochlea at birth, and, although these cells can be lost as a result
of various genetic or environmental factors (e.g., noise exposure,
ototoxic drug toxicity, viral infection, aging, and genetic
defects), the lost or damaged cells cannot be replaced. Hair cells
also are found in the utricle of the vestibule, an organ which
regulates balance. Therefore, hair cell regeneration is an
important approach to restoring hearing and vestibular
function.
[0004] Studies of regeneration of hair cells in mature mammalian
inner ear to date have focused on transdifferentiation of existing
supporting cells. Supporting cells underlie, at least partially
surround, and physically support sensory hair cells within the
inner ear. Examples of supporting cells include inner rod (pillar
cells), outer rod (pillar cells), inner phalangeal cells, outer
phalangeal cells (of Deiters), cells of Held, cells of Hensen,
cells of Claudius, cells of Boettcher, interdental cells and
auditory teeth (of Huschke). Transdifferentiation of supporting
cells to hair cells by overexpression or activation of Protein
Atonal Homolog 1 (Atoh1) in supporting cells or by exposure of
supporting cells to Atoh1 agonists is one such approach to
generating new hair cells. One limitation to this approach,
however, is that transdifferentiation of supporting cells to hair
cells diminishes the existing population of supporting cells, which
can impair inner ear function. In addition, overexpression of Atoh1
in aged inner ear or flat epithelium, which lacks supporting cells,
is not sufficient to induce hair cells. Furthermore, it is not
clear if all types of supporting cells can be transdifferentiated
into hair cells upon Atoh1 overexpression.
[0005] Other studies of hair cell regeneration have examined cell
cycle reentry for hair cells in embryonic or neonatal mice by, for
example, blocking Rb1 and p27kip1. However similar manipulations in
the adult inner ear have not induced cell cycle reentry. In
addition, the hair cells in embryonic and neonatal mice that
reenter the cell cycle in general subsequently die.
[0006] Over 150 types of genetic deafness are due to mutations in
genes that affect both hair cells and supporting cells. For
example, mutations in Myosin VIIa (Myo7a) cause hair cell
stereocilia abnormalities that lead to permanent deafness.
Mutations in GJB2 (connexin 26) cause damage to supporting cells
that lead to the most common form of genetic deafness. Approaches
(e.g., gene therapy and anti-sense oligonucleotide therapy) have
been developed as potential treatments for hereditary deafness.
However most of these defects occur during embryonic development.
By birth, affected hair cells and supporting cells already have
died or are severely degenerated, making intervention difficult.
Therefore, to treat genetic deafness, there is an ongoing need to
regenerate hair cells and/or supporting cells in utero and after
birth, which can be combined with other approaches to correct the
genetic defects underlying the disease.
[0007] In addition, inner ear non-sensory cells (e.g., fibrocytes
in the ligament) play essential roles in hearing. Inner ear
non-sensory cells can be damaged by factors such as noise and
aging, which contribute to hearing loss. These cell types, like
many of those in the inner ear, lack the capacity to regenerate
spontaneously after damage.
[0008] Because spontaneous regeneration does not occur in the
mammalian inner ear, recovery from hearing loss requires
intervention to replace any inner ear cell types that are lost or
degenerated. Therefore, there is an ongoing need to regenerate hair
and/or supporting cells within the mammalian ear, in particular in
the inner ear, to replace those lost, for example, by genetic or
environmental factors. The regenerated hair and supporting cells
may be used to slow the loss of hearing and/or vestibular function
and/or partially or fully to restore loss of hearing and/or
vestibular function.
SUMMARY OF THE INVENTION
[0009] The invention is based, in part, upon the discovery that
increasing c-myc activity, Notch activity, or both c-myc and Notch
activity in an ear cell, for example, a cell of an inner ear,
promotes cell cycle reentry and proliferation of the cell. When the
cell is, for example, a hair cell or a supporting cell, it is
contemplated that proliferation and subsequent differentiation of
the cell into hair and/or supporting cells can restore or improve
hearing and/or vestibular function.
[0010] In one aspect, the invention relates to a method of inducing
proliferation or cell cycle reentry of a differentiated cochlear
cell or a utricular cell. The method comprises increasing both
c-myc activity and Notch activity within the cell sufficient to
induce proliferation or cell cycle reentry of the cochlear cell or
utricular cell. Upon entry into the cell cycle, the cell may
dedifferentiate but retain aspects of its differentiated state. In
certain embodiments, the cochlear or utricular cell can be, for
example, a hair cell or a supporting cell. The method may also
include the step of inhibiting c-myc and/or Notch activity after
proliferation of the cochlear or the utricular hair or supporting
cell to induce differentiation or transdifferentiation of the cell
and/or at least one of its daughter cells into a hair cell
Inhibition of c-myc and/or Notch activity after proliferation can
be important in promoting cell survival.
[0011] In another aspect, the invention relates to a method for
regenerating a cochlear or utricular hair cell. The method includes
increasing both c-myc activity and Notch activity within the hair
cell thereby to induce cell proliferation to produce one, two or
more daughter hair cells, and, after cell proliferation, decreasing
c-myc and/or Notch activity to induce and/or maintain
differentiation of the daughter hair cells. In certain embodiments,
the cochlear or utricular cell can be, for example, a hair cell or
a supporting cell. These steps can be performed in vivo (for
example, in the inner ear of a mammal, in particular the cochlea or
utricle), or ex vivo, wherein the resulting cells are cultured
and/or introduced into the inner ear of a recipient.
[0012] In another aspect, the invention relates to a method for
reducing the loss of, maintaining, or promoting hearing in a
subject. The method comprises increasing both c-myc activity and
Notch activity within a hair cell and/or a supporting cell of the
inner ear thereby to induce cell proliferation to produce daughter
cells, and, after cell proliferation, decreasing c-myc and/or Notch
activity, and permitting daughter cells of hair cell origin to
differentiate into hair cells or permitting daughter cells of
supporting cell origin to transdifferentiate into hair cells
thereby to reduce the loss of, maintain or promote hearing in the
subject. The daughter cells of supporting cell origin can be
induced to transdifferentiate into hair cells by activating Atoh1
activity, for example, by gene expression, by administration of an
effective amount of Atoh1 or an Atoh1 agonist. The steps can be
performed in vivo (for example, in the inner ear of a mammal, in
particular in the cochlea), or ex vivo, wherein the resulting cells
are cultured and/or introduced into the inner ear of the
subject.
[0013] In another aspect, the invention relates to a method for
reducing the loss of, maintaining, or promoting vestibular function
in a subject. The method comprises increasing both c-myc activity
and Notch activity within a hair cell and/or a supporting cell of
the inner ear thereby to induce cell proliferation to produce
daughter cells, and, after cell proliferation, decreasing c-myc
and/or Notch activity, and permitting daughter cells of hair cell
origin to differentiate into hair cells or permitting daughter
cells of supporting cell origin to transdifferentiate into hair
cells thereby to reduce the loss of, maintain or promote vestibular
function in the subject. The daughter cells of supporting cell
origin can be induced to transdifferentiate into hair cells by
activating Atoh1 activity, for example, by gene expression, by
administration of an effective amount of Atoh1 or an Atoh1 agonist.
The steps can be performed in vivo (for example, in the inner ear
of a mammal, in particular in the utricle), or ex vivo, wherein the
resulting cells are cultured and/or introduced into the inner ear
of the subject.
[0014] In each of the foregoing aspects of the invention, c-myc
activity may be increased by contacting the cell with an effective
amount of a c-myc protein or a c-myc activator. After c-myc
activity is increased, c-myc activity can be inhibited to limit
proliferation of the cochlear cell or utricular cell and/or to
promote survival of the cochlear cell or utricular cell. Similarly,
in each of the foregoing aspects of the invention, Notch activity
may be increased by contacting the cell with an effective amount of
a Notch protein, a Notch Intracellular Domain (NICD) protein or a
Notch activator. Notch activity can be inhibited by contacting the
cell with an effective amount of a Notch inhibitor.
[0015] In certain embodiments, the c-myc protein or c-myc activator
may be administered to the inner ear of a subject. In certain
embodiments, the Notch protein, NICD protein, Notch activator,
and/or Notch inhibitor may be administered to the inner ear of a
subject. In other embodiments, the c-myc protein or c-myc activator
may be co-administered together with the Notch protein, the NICD
protein, the Notch activator, and/or the Notch inhibitor to the
inner ear of the subject.
[0016] The foregoing aspects and embodiments of the invention may
be more fully understood by reference to the following figures,
detailed description and claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0017] The objects and features of the invention may be more fully
understood by reference to the drawings described herein.
[0018] FIG. 1(A) shows the full-length protein sequence of human
c-myc (NP.sub.--002458.2; SEQ ID NO: 1) and (B) shows the c-myc
protein consensus protein sequence (SEQ ID NO: 9).
[0019] FIG. 2(A) shows the full-length protein sequence of human
Notch (NP.sub.--060087.3; SEQ ID NO: 2), (B) shows the protein
sequence of human Notch intracellular domain (NP.sub.--060087.3
residues 1754-2555; SEQ ID NO: 7), and (C) shows a consensus
protein sequence of the Notch Intracellular domain (SEQ ID NO:
10).
[0020] FIG. 3(A) shows the full-length protein sequence of human
Atoh1 (NP.sub.--005163.1; SEQ ID NO: 3) and (B) shows an Atoh1
consensus protein sequence (SEQ ID NO: 11).
[0021] FIG. 4 shows the nucleic acid sequence of human c-myc mRNA
(NM.sub.--002467.4; SEQ ID NO: 4).
[0022] FIG. 5(A) shows the nucleic acid sequence of human Notch
mRNA (NM.sub.--017617.3; SEQ ID NO: 5) and (B) shows the nucleotide
sequence of human Notch intracellular domain (NM.sub.--017617.3
nucleotide positions 5260 to 7665; SEQ ID NO: 8).
[0023] FIG. 6 shows the nucleic acid sequence of human Atoh1 mRNA
(NM.sub.--005172.1; SEQ ID NO: 6).
[0024] FIG. 7 shows cochlear hair and supporting cells
double-labeled with cell-type specific markers and BrdU 4 days
(A-E), 8 days (K-O), or 12 days (P-T) post-injection of Ad-Cre-GFP
virus and Ad-Myc virus into cochleas of 45-day-old
NICD.sup.flox/flox mice. Solid arrows indicate BrdU labeled hair
cells and open arrows indicate BrdU labeled supporting cells. FIG.
7(F-J) shows an uninjected control cochlea in which no hair and
supporting cells double-labeled with cell-type specific markers and
BrdU could be found. FIGS. 7(A, F, K, and P) show BrdU labeling.
FIGS. 7(B, G, L, and Q) show Myo7a labeling of hair cells. FIGS.
7(C, H, M, and R) show Sox2 labeling of supporting cells. FIGS.
7(D, I, N, and S) show DAPI labeling of cell nuclei. FIGS. 7(E, J,
O, and T) show merged images.
[0025] FIG. 8 shows cochlear hair and supporting cells
double-labeled with cell-type specific markers and BrdU in the
cochlear epithelium of NICD.sup.flox/flox mice 35 days
post-injection of an Ad-Cre-GFP/Ad-Myc mixture followed by 5 days
of daily BrdU administration. FIGS. 8(A, F, and K) show BrdU
labeling. FIGS. 8(B, G, and L) show Myo7a labeling of hair cells.
FIGS. 8(C, H, and M) show Sox2 labeling of supporting cells. FIGS.
8(D, I, and N) show DAPI labeling of cell nuclei. FIGS. 8(E, J, and
O) show merged images. FIG. 8(A-E) shows labeling with BrdU and
Myo7a, demonstrating that proliferating hair cells survive 35 days
post-injection (solid arrows, FIGS. 8A, B, C, and E). FIG. 8(F-J)
shows an enlarged image of two hair cells displaying stereocilia
(solid arrowhead, FIG. 8J) derived from division of one mother hair
cell. FIG. 8(K-O) shows cells labeled with BrdU and Sox2 (open
arrows, FIGS. 8K, M, and O), demonstrating that proliferating
supporting cells survive 35 days post-injection. Closed arrows in
FIGS. 8 (K, L, M, and O) show Myo7a+/BrdU+ hair cells. Arrowhead in
FIGS. 8(K,L,M, and O) show Myo7a+/Sox2+/BrdU+ hair cell.
[0026] FIG. 9 shows cochlear hair and supporting cells
double-labeled with cell-type specific markers and BrdU in the
cochlear epithelium of aged NICD.sup.flox/flox mice injected with
an Ad-Cre-GFP/Ad-Myc mixture over the course of 15 days. FIGS. 9(A,
F, and K) show Myo7a labeling of hair cells. FIGS. 9(B, G, and L)
show BrdU labeling of dividing cells. FIGS. 9(C, H, and M) show
Sox2 labeling of supporting cells. FIGS. 9(D, I, and N) show DAPI
labeling of cell nuclei. FIGS. 9(E, J, and O) show merged images.
FIG. 9(A-J) shows Myo7a+/BrdU+ hair cells (A, B, and E; arrows) and
Sox2+/BrdU+ supporting cells (B, C, E, G, H, and J; arrowheads)
following injection with Ad-Myc and Ad-Cre-GFP adenovirus. FIG.
9(K-O) shows the same staining in 17-month old NICD.sup.flox/flox
mice injected with Ad-Cre-GFP virus alone. No BrdU labeled hair
cells or supporting cells were found in the latter group. Scale
bars: 10 .mu.M.
[0027] FIG. 10 shows BrdU (FIGS. 10A and F), Myo7a (FIGS. 10B and
G) and Sox2 (FIGS. 10C and H) labeled hair and supporting cells in
cultured adult human cochlear (FIG. 10A-E) and utricular (FIG.
10F-J) tissue transduced with Ad-Myc/Ad-NICD for 10 days. Open
arrows (FIGS. 10A, C, D, E, F, H, I, and J) indicate proliferating
supporting cells (Sox2+/BrdU+) and solid arrow (F-J) indicates a
proliferating hair cell (Myo7a+/BrdU+). Nuclear staining is shown
by DAPI (D and I).
[0028] FIG. 11 shows Myo7+ hair (A and F) and Sox2+ supporting (C
and H) cells in adult monkey cochlear cultures. Dividing cells were
labeled with EdU (B and G). FIG. 11(A-E) shows Ad-GFP infected
control monkey cochlea, in which no EdU+ cells were identified.
FIG. 11(G, H, J) shows EdU+/Sox2+ supporting cells (arrowheads) in
monkey cochlea cultures exposed to Ad-Myc/Ad-NICD virus. In both
control and Ad-Myc/Ad-NICD virus infected cultures, no hair cells
were observed to re-enter the cell cycle (A, E, F, and J; arrows).
Scale bars: 20 .mu.M.
[0029] FIG. 12 shows selective induction of proliferation in
supporting cells (arrows; B, C, and E), but not inner hair cells
(arrowheads; A, C, and E), of rtTa/tet-on-Myc/tet-on-NICD mice
exposed to doxycycline administered by an implanted osmotic pump
for 9 days to induce expression of NICD and Myc. Cells that
reentered the cell cycle were labeled via daily EdU (FIG. 12B)
administration during the same period. Cell nuclei were stained for
DAPI (FIG. 12D). Inner hair cells were stained for Parvalbumin
(Parv; FIG. 12A). Supporting cells were stained for Sox2 (FIG.
12C). A single Parv+ hair cell is shown that also expressed Sox2
due to Notch activation (rightmost arrowhead in FIGS. 12A, C, and
E). Outer hair cells are not shown as they were lost during
surgical implantation of the osmotic pump. Scale bar: 20 .mu.M.
[0030] FIG. 13 shows outer hair cells are selectively induced to
undergo cell cycle reentry following exposure to elevated c-Myc and
Notch activity in vivo. rtTa/tet-on-Myc/tet-on-NICD mice were
exposed to doxycycline administered by an implanted osmotic pump
for 12 days to induce expression of NICD and Myc, after which
tissue was harvested for staining. Cells that reentered the cell
cycle were labeled via daily EdU (FIG. 13B) administration during
the period of doxycycline exposure. Cell nuclei were stained for
DAPI (FIG. 13D). Inner and outer hair cells were stained for Espin
(Esp; FIG. 13A). Supporting cells were stained for Sox2 (FIG. 13C).
Note that outer hair cells were spared during implantation of the
osmotic pump in this experiment, as opposed to the experiment shown
in FIG. 12. A dividing Esp+/EdU+ outer hair cell is shown in FIGS.
13(B and E; arrows), demonstrating selective induction of outer
hair cell proliferation at this level of exposure to elevated c-Myc
and Notch activity.
[0031] FIG. 14 shows Espin-positive (Esp+) hair cells labeled with
FM-143FX (FM1) to reveal cells with functional membrane channels.
Cochlea of 45-day-old NICD.sup.flox/flox mice were exposed to
Ad-Myc/Ad-Cre-GFP virus and EdU was injected once daily for 5 days
following virus injection to label dividing cells. 35 days
post-virus injection, cochlea were harvested, briefly exposed to
FM1, fixed, and stained. FIG. 14(A-E) shows an Esp+/FM1+/EdU-
control hair cell that has not undergone cell cycle reentry, but
which expresses Esp and takes up FM1. FIG. 14(F-J) shows an
Esp+/FM1+/EdU+ hair cell in a cochlea exposed to Ad-Myc/Ad-NICD
virus, indicating the presence of functional membrane channels in a
cell that has undergone cell cycle reentry. Arrowhead (FIG. 14H)
indicates EdU labeling; arrow (FIG. 14F) indicates the presence of
Esp+ hair bundles. Scale bars: 10 .mu.M.
[0032] FIG. 15 shows that production of Myo7a+ hair cells induced
to undergo cell proliferation following exposure to elevated levels
of c-Myc and Notch activity is accompanied by production of
neurofilament-positive (NF+; FIG. 15B) neurofibers. Cochlea of
45-day-old NICD.sup.flox/flox mice were exposed to
Ad-Myc/Ad-Cre-GFP virus and BrdU was injected once daily for 15
days following virus injection to label dividing cells (FIG. 15C).
Tissue was harvested and stained 20 days post-virus injection. FIG.
15(A) shows Myo7+ hair cells. Cell nuclei were stained using DAPI
(FIG. 15D). FIG. 15(E) shows a merge of all stains and an enlarged
view of the boxed area indicated by the rightmost arrow in the
panel. Arrows (FIGS. 15A, C, and E) indicate Myo7a+/BrdU+ hair
cells in contact with NF+ ganglion neuron neurofibers. Scale bar:
10 .mu.M.
[0033] FIG. 16(A-E) shows an example of an inner hair cell induced
to proliferate via exposure to elevated levels of c-Myc and Notch
activity and expressing an inner hair cell-specific marker (Vglut3;
FIGS. 16B and G) and a marker of functional synapses (CtBP2; FIGS.
16A and F; brackets). Cochlea of 45-day-old NICD.sup.flox/flox mice
were exposed to Ad-Myc/Ad-Cre-GFP (FIG. 16A-E) or Ad-GFP (FIG.
16F-J) virus via a single injection of virus, and BrdU was injected
once daily for 15 days following virus injection to label dividing
cells (FIGS. 16C and H). Tissue was then harvested and stained.
Cell nuclei were stained with DAPI (FIGS. 16 D and I). FIG. 16(A-E)
show a CtBP2+/VGlut3+/BrdU+ inner hair cell (FIG. 16B; arrow)
induced to proliferate following exposure to elevated c-Myc and
Notch activity, and a CtBP2+/Vglut3+/BrdU- inner hair cell (FIG.
16B; arrowhead) that did not undergo cell cycle reentry. (FIG.
16F-J) shows inner hair cells exposed to Ad-GFP that did not stain
positive for BrdU but expressed the inner hair cell-specific marker
Vglut3 and the presynaptic marker CtBP2. IHC=inner hair cell
layer.
[0034] FIG. 17 shows cultured cochlear support cells from
doxycycline-inducible rtTa/tet-on-Myc/tet-on-Notch mice induced to
transdifferentiate or proliferate and transdifferentiate to
functional hair cells following exposure to either Atoh1-expressing
adenovirus alone (FIG. 17F-J) or doxycycline and Atoh1-expressing
adenovirus (Ad-Atoh1; FIGS. 17A-E and K-O). Cochlea from adult
rtTa/tet-on-Myc/tet-on-Notch mice were dissected and cultured for 5
days in the presence (FIGS. 17A-E and K-O) or absence (FIG. 17F-J)
of doxycycline, followed by Ad-Atoh1 infection and an additional 14
days of culture. EdU was added daily to label dividing cells (FIGS.
17A, F, and M). Cell nuclei were stained with DAPI (FIGS. 17D, I,
and N). FIG. 17(A-E) shows supporting cells exposed to doxycycline
followed by Ad-Atoh1, and labeled with EdU, reenter the cell cycle
and/or transdifferentiate into Myo7a+/Parv+ hair cells (closed
arrows in FIGS. 17A, B, C, and E). Open arrow in FIGS. 17(B, C, and
E) indicates the presence of a Myo7a+/Parv+ supporting cell that
has transdifferentiated into a hair cell, but has not undergone
cell cycle reentry. Arrowhead in FIGS. 17(A and E) indicates an
EdU+ supporting cell. FIG. 17(F-J) shows supporting cells exposed
to Ad-Atoh1, but not doxycycline, transdifferentiate to
Myo7a+/Parv+ hair cells. Arrow in FIGS. 17(G, H, and J) indicates a
supporting cell that has transdifferentiated into a Myo7a+/Parv+
hair cell, but which has not undergone cell cycle reentry. FIG.
17(K-O) shows supporting cells exposed to doxycycline followed by
Ad-Atoh1 and labeled with FM1 (FIG. 17L) and Edu (FIG. 17M) have
Esp+ hair bundles (FIG. 17K) and take up FM1 dye. Arrow in FIGS.
17(K and O) indicates an Esp+/FM1+/EdU+ hair cell displaying
stereocilia derived from a transdifferentiated supporting cell that
has undergone cell cycle reentry. Arrowhead in FIGS. 17 (K and O)
indicates an Esp+/FM1+/EdU- hair cell derived from a
transdifferentiated supporting cell that has not undergone cell
cycle reentry. Scale bar: 10 .mu.M.
[0035] FIG. 18 shows the results of semi-quantitative RT-PCR
analysis of sets of mRNA transcripts produced in control cochlear
cells and in cochlear cells following exposure to elevated c-Myc
and NICD levels. Adult NICD.sup.flox/flox mouse cochleas were
exposed to Ad-Myc/Ad-Cre-GFP (Myc+Nicd) or Ad-GFP (Ctr) and
cultured for 4 days, mRNA was extracted, and semi-quantitative
RT-PCT was performed. Changes in expression of stem cell genes
(Nanog, ALPL, and SSEA) and ear progenitor cell genes/Notch genes
(Eya1, DLX5, Six1, Pax2, p27kip1, Islet-1, Sox2, Math1, NICD,
Prox1, and HesS) was examined GAPDH expression was used as an
internal control.
[0036] FIG. 19(A) shows the full-length protein sequence of human
N-myc (NP.sub.--005369.2; SEQ ID NO: 12) and (B) shows the nucleic
acid sequence of human N-myc (NM.sub.--005378.4; SEQ ID NO:
13).
[0037] FIG. 20(A) shows the full-length protein sequence of human
Notch2 (NP.sub.--077719.2; SEQ ID NO: 14) and (B) shows the nucleic
acid sequence of human Notch2 (NM.sub.--024408.3; SEQ ID NO:
15).
[0038] FIG. 21(A) shows the full-length protein sequence of human
Notch3 (NP.sub.--000426.2; SEQ ID NO: 16) and (B) shows the nucleic
acid sequence of human Notch3 (NM.sub.--000435.2; SEQ ID NO:
17).
[0039] FIG. 22(A) shows the full-length protein sequence of human
Notch4 (NP.sub.--004548.3; SEQ ID NO: 18) and (B) shows the nucleic
acid sequence of human Notch4 (NM.sub.--004557.3; SEQ ID NO:
19).
[0040] FIG. 23 (A) shows the full-length protein sequence of human
Atoh7 (NP.sub.--660161.1; SEQ ID NO: 20) and (B) shows the nucleic
acid sequence of human Atoh7 (NM.sub.--145178.3; SEQ ID NO:
21).
[0041] FIG. 24 shows the nucleic acid sequence for an Atoh1
enhancer (SEQ ID NO: 22), which controls expression in hair
cells.
[0042] FIG. 25 shows the nucleic acid sequence for a Pou4f3
promoter (SEQ ID NO: 23), which controls expression in hair
cells.
[0043] FIG. 26 shows the nucleic acid sequence for a Myo7a promoter
(SEQ ID NO: 24), which controls expression in hair cells.
[0044] FIG. 27 shows the nucleic acid sequence for a HesS promoter
(SEQ ID NO: 25), which controls expression in vestibular supporting
cells and cochlear inner phalangeal cells, Deiters cells and Pillar
cells.
[0045] FIG. 28 shows the nucleic acid sequence for a GFAP promoter
(SEQ ID NO: 26), which controls expression in vestibular supporting
cells and cochlear inner phalangeal cells, Deiters cells and Pillar
cells.
DETAILED DESCRIPTION
[0046] The invention relates to methods and compositions for
inducing cell cycle reentry and proliferation of hair and/or
supporting cells in the ear, in particular, the inner ear. The
methods and compositions can be used to increase a population of
hair cells and/or supporting cells diminished by environmental or
genetic factors. Using the methods and compositions described
herein, it may be possible to preserve or improve hearing and/or
vestibular function in the inner ear.
[0047] As demonstrated herein, simultaneously increasing c-myc and
Notch activity appears to be an important step in inducing cell
cycle reentry and proliferation in cells of the inner ear. As shown
in the Examples below, overexpression of c-myc and Notch in the
inner ear of a mammal results in the reentry of hair and supporting
cells into the cell cycle and the proliferation of those cells. The
proliferation of hair cells (or the proliferation of supporting
cells followed by transdifferentiation of those cells into hair
cells) may lead to improved hearing and/or vestibular function in a
subject.
Definitions
[0048] For convenience, certain terms in the specification,
examples, and appended claims are collected in this section.
[0049] As used herein, the term "effective amount" is understood to
mean the amount of an active agent, for example, a c-myc or Notch
activator, that is sufficient to induce cell cycle reentry and/or
proliferation of the cells of the inner ear (e.g., a hair cell or a
supporting cell). The cells are contacted with amounts of the
active agent effective to induce cell cycle reentry and/or
proliferation.
[0050] As used herein, "pharmaceutically acceptable" or
"pharmacologically acceptable" mean molecular entities and
compositions that do not produce an adverse, allergic or other
untoward reaction when administered to an animal, or to a human, as
appropriate. The term, "pharmaceutically acceptable carrier"
includes any and all solvents, dispersion media, coatings,
antibacterial and antifungal agents, isotonic and absorption
delaying agents and the like. The use of such media and agents for
pharmaceutical active substances is well known in the art. Except
insofar as any conventional media or agent is incompatible with the
active ingredient, its use in the therapeutic compositions is
contemplated. Supplementary active ingredients can also be
incorporated into the compositions.
[0051] The term "subject" is used throughout the specification to
describe an animal, human or non-human, to whom treatment according
to the methods of the present invention is provided. Veterinary and
non-veterinary applications are contemplated. The term includes,
but is not limited to, birds and mammals, e.g., humans, other
primates, pigs, rodents such as mice and rats, rabbits, guinea
pigs, hamsters, cows, horses, cats, dogs, sheep and goats. Typical
subjects include humans, farm animals, and domestic pets such as
cats and dogs.
[0052] As used herein "target cell" and "target cells" refers to a
cell or cells that are capable of reentering the cell cycle and/or
proliferating and/or transdifferentiating to or towards a cell or
cells that have or result in having characteristics of auditory or
vestibular hair cells. Target cells include, but are not limited
to, e.g., hair cells, e.g., inner ear hair cells, which includes
auditory hair cells (inner and outer hair cells) and vestibular
hair cells (located in the utricle, saccule and three semi-circular
canals, for example), progenitor cells (e.g., inner ear progenitor
cells), supporting cells (e.g., Deiters' cells, pillar cells, inner
phalangeal cells, tectal cells and Hensen's cells), supporting
cells expressing one or more of p27.sub.kip, p75, S100A, Jagged-1,
Proxl, and/or germ cells. "Inner hair cell" refers to a sensory
cell of the inner ear that is anatomically situated in the organ of
Corti above the basilar membrane. "Outer hair cell" refers to a
sensory cell of the inner ear that is anatomically situated in the
organ of Corti below the tectorial membrane near the center of the
basilar membrane. Examples of target cells also include fibrocytes,
marginal cells or interdental cells expressing one or more of Gjb2,
Slc26a4 and Gjb6. As described herein, prior to treatment with the
methods, compounds, and compositions described herein, each of
these target cells can be identified using a defined set of one or
more markers (e.g., cell surface markers) that is unique to the
target cell. A different set of one or more markers (e.g., cell
surface markers) can also be used to identify target cells have
characteristics of an auditory hair cell or supporting cell.
[0053] As used herein, the term "host cell" refers to cells
transfected, infected, or transduced in vivo, ex vivo, or in vitro
with a recombinant vector or a polynucleotide. Host cells may
include packaging cells, producer cells, and cells infected with
viral vectors. In particular embodiments, host cells infected with
viral vector of the invention are administered to a subject in need
of therapy. In certain embodiments, the term "target cell" is used
interchangeably with host cell and refers to transfected, infected,
or transduced cells of a desired cell type.
[0054] The term "vector" is used herein to refer to a nucleic acid
molecule capable transferring or transporting another nucleic acid
molecule. The transferred nucleic acid is generally linked to, for
example , inserted into, the vector nucleic acid molecule. A vector
may include sequences that direct autonomous replication in a cell,
or may include sequences sufficient to allow integration into host
cell DNA. Useful vectors include, for example, plasmids (e.g., DNA
plasmids or RNA plasmids), transposons, cosmids, bacterial or yeast
artificial chromosomes, and viral vectors. Useful viral vectors
include, for example, adenoviruses, replication defective
retroviruses, and lentiviruses.
[0055] As used herein, the term "viral vector" refers either to a
nucleic acid molecule that includes virus-derived nucleic acid
elements that typically facilitate transfer of the nucleic acid
molecule or integration into the genome of a cell or to a viral
particle that mediates nucleic acid transfer. Viral particles will
typically include various viral components and sometimes also host
cell components in addition to nucleic acid(s). The term "viral
vector" may also refer either to a virus or viral particle capable
of transferring a nucleic acid into a cell or to the transferred
nucleic acid itself Viral vectors and transfer plasmids contain
structural and/or functional genetic elements that are primarily
derived from a virus.
[0056] The term "retroviral vector" refers to a viral vector or
plasmid containing structural and functional genetic elements, or
portions thereof, that are primarily derived from a retrovirus.
[0057] The term "lentiviral vector" refers to a viral vector or
plasmid containing structural and functional genetic elements, or
portions thereof, that are primarily derived from a lentivirus.
[0058] The terms "lentiviral vector" or "lentiviral expression
vector" may be used to refer to lentiviral transfer plasmids and/or
infectious lentiviral particles. It is understood that nucleic acid
sequence elements such as cloning sites, promoters, regulatory
elements, heterologous nucleic acids, etc., are present in RNA form
in the lentiviral particles of the invention and are present in DNA
form in the DNA plasmids of the invention.
[0059] The term "hybrid" refers to a vector, LTR or other nucleic
acid containing both retroviral (e.g., lentiviral) sequences and
non-lentiviral viral sequences. A hybrid vector may refer to a
vector or transfer plasmid comprising retroviral (e.g., lentiviral)
sequences for reverse transcription, replication, integration
and/or packaging. In some embodiments of the invention, a hybrid
vector may be used to practice the invention described herein.
[0060] The term "transduction" refers to the delivery of a gene(s)
or other polynucleotide sequence using a retroviral or lentiviral
vector by means of viral infection rather than by transfection. In
certain embodiments, a cell (e.g., a target cell) is "transduced"
if it comprises a gene or other polynucleotide sequence delivered
to the cell by infection using a viral (e.g., adenoviral) or
retroviral vector. In particular embodiments, a transduced cell
comprises one or more genes or other polynucleotide sequences
delivered by a retroviral or lentiviral vector in its cellular
genome.
[0061] As used herein, the term "c-myc" refers to a
multifunctional, nuclear phosphoprotein that plays a role in cell
cycle progression, apoptosis and cellular transformation and/or has
an amino sequence or consensus amino acid sequence set forth in
Section 1(i) below. The full length sequence of human c-myc
appears, for example, in the NCBI protein database under accession
no. NP.sub.--002458.2 (see ncbi.nlm.nih.gov and SEQ ID NO: 1). A
consensus sequence for c-myc built from an alignment of human, rat,
mouse and chimpanzee using ClustalW is set forth in SEQ ID NO: 9.
C-myc functions as a transcription factor that regulates
transcription of specific target genes. Mutations, overexpression,
rearrangement and translocation of this gene have been associated
with a variety of hematopoietic tumors, leukemias and lymphomas,
including Burkitt lymphoma. C-myc is also known in the art as MYC,
v-myc myelocytomatosis viral oncogene homolog (avian),
transcription factor p64, bHLHe39, MRTL, avian myelocytomatosis
viral oncogene homolog, v-myc avian myelocytomatosis viral oncogene
homolog, myc proto-oncogene protein, class E basic helix-loop-helix
protein 39, myc-related translation/localization regulatory factor,
and proto-oncogene c-Myc, and BHLHE39.
[0062] As used herein, the term, "Notch" refers to the Notch family
of signaling proteins, which includes Notch1, Notch2, Notch3 and
Notch4, a NICD, and/or a protein having an amino acid sequence or
consensus amino acid sequence set forth in Section (1)(i) below.
The full length sequence of human Notchl appears, for example, in
the NCBI protein database under accession no. NP.sub.--060087.3
(see ncbi.nlm.nih.gov and SEQ ID NO: 2). Members of this Type 1
transmembrane protein family share structural characteristics
including an extracellular domain consisting of multiple epidermal
growth factor-like (EGF) repeats, and an intracellular domain
consisting of multiple, different domain types. Notch family
members play a role in a variety of developmental processes by
controlling cell fate decisions.
[0063] Notch1 is cleaved in the trans-Golgi network, and presented
on the cell surface as a heterodimer. Notchl functions as a
receptor for membrane bound ligands Jaggedl, Jagged2 and Deltal to
regulate cell-fate determination. Upon ligand activation through
the released notch intracellular domain (NICD) it forms a
transcriptional activator complex with RBPJ/RBPSUH and activates
genes of the enhancer of split locus. Notch 1 affects the
implementation of differentiation, proliferation and apoptotic
programs.
[0064] Disclosed herein is a method of inducing proliferation or
cell cycle reentry of a differentiated cochlear cell or a utricular
cell. The method comprises increasing c-myc, Notch or both c-myc
activity and Notch activity within the cell sufficient to induce
proliferation or cell cycle reentry of the cochlear cell or
utricular cell.
[0065] In certain embodiments, the method includes increasing c-myc
activity within a cell when Notch activity is already increased,
for example, when Notchl has been upregulated in response to damage
to the inner ear. In certain embodiments, the invention relates to
a method of inducing proliferation or cell cycle reentry of a
differentiated cochlear cell or a utricular cell in which Notch
activity is increased in response to damage to the cochlear cell or
utricular cell, as compared to the level of Notch activity in
undamaged cochlear cells or utricular cells, respectively. The
method comprises increasing c-myc activity within the cochlear cell
or utricular cell sufficient to induce proliferation or cell cycle
reentry of the cochlear cell or utricular cell.
[0066] In other embodiments, the method includes increasing Notch
activity within a cell, when c-myc activity is already increased.
(See, for example, Lee et al. (2008) Assoc. RES. OTOLARYNGOL. ABS.:
762.) In particular, the invention relates to a method of inducing
proliferation or cell cycle reentry of a differentiated cochlear
cell or a utricular cell in which c-myc activity is increased in
response to damage to the cochlear cell or utricular cell, as
compared to the level of c-myc activity in undamaged cochlear cells
or utricular cells, respectively. The method comprises increasing
Notch activity within the cochlear cell or utricular cell
sufficient to induce proliferation or cell cycle reentry of the
cochlear cell or utricular cell.
[0067] After c-myc activity, Notch activity, or both c-myc and
Notch activities, as appropriate, is or are increased, Notch may be
inhibited according to methods known in the art and/or described
herein to cause proliferating supporting cells to
transdifferentiate into hair cells. Alternatively, or in addition,
after c-myc activity, Notch activity, or both c-myc and Notch
activity is or are increased, as appropriate, Atoh1 activity can be
increased to cause proliferating supporting cells to
transdifferentiate into hair cells. Methods of increasing Atohl
activity (including use of Atoh1 agonists) are known in the art
(see, for example, U.S. Pat. No. 8,188,131; U.S. Patent Publication
No. 20110305674; U.S. Patent Publication No. 20090232780; Kwan et
al. (2009) INT'L SYMPOSIUM ON OLFACTION AND TASTE: ANN. N.Y. ACAD.
SCI. 1170:28-33; Daudet et al. (2009) DEV. BIO. 326:86-100;
Takebayashi et al. (2007) DEV. BIO. 307:165-178; and Ahmed et al.
(2012) DEV. CELL 22(2):377-390.)
[0068] Also disclosed is a method of regenerating a cochlear or
utricular hair cell. The method includes (a) increasing c-myc,
Notch, or both c-myc activity and Notch activity, as appropriate,
\within the hair cell thereby to induce cell proliferation to
produce one, two or more daughter cells, and (b) after cell
proliferation, decreasing Notch activity to induce differentiation
of at least one of the cell and the daughter cells to produce a
differentiated cochlear or utricular hair cell. The process can
occur in vivo or ex vivo. In one embodiment, Notch activity is
decreased in a cell that originated from a supporting cell to cause
the supporting cell to transdifferentiate into a hair cell. In
another embodiment, Atoh1 activity is increased in a cell that
originated from a supporting cell to cause the supporting cell to
transdifferentiate into a hair cell.
[0069] In certain embodiments, after c-myc and Notch induce
proliferation within a hair cell or supporting cell, c-myc activity
is decreased to induce differentiation of at least one of the cell
and the daughter cell to produce a differentiated cochlear or
utricular hair cell. Decreasing c-myc activity after proliferation
can promote survival of the proliferating cell.
[0070] Also disclosed is a method for reducing the loss of,
maintaining, or promoting hearing in a subject. The method
comprises increasing c-myc activity, Notch activity, or both c-myc
activity and Notch activity, as appropriate, within a hair cell
and/or a supporting cell of the inner ear thereby to induce cell
proliferation to produce daughter cells, and, after cell
proliferation, decreasing c-myc and/or Notch activity, and
permitting daughter cells of hair cell origin to differentiate into
hair cells or permitting daughter cells of supporting cell origin
to transdifferentiate into hair cells thereby to reduce the loss
of, maintain or promote hearing in the subject. The daughter cells
of supporting cell origin can be induced to transdifferentiate into
hair cells by activating Atoh1 activity, for example, by gene
expression, by administration of an effective amount of Atoh1 or an
Atoh1 agonist. The steps can be performed in vivo (for example, in
the inner ear of a mammal, in particular in the cochlea), or ex
vivo, wherein the resulting cells are cultured and/or introduced
into the inner ear of the subject.
[0071] Also disclosed is a method for reducing the loss of,
maintaining, or promoting vestibular function in a subject. The
method comprises increasing c-myc activity, Notch activity, or both
c-myc activity and Notch activity, as appropriate, within a hair
cell and/or a supporting cell of the inner ear thereby to induce
cell proliferation to produce daughter cells, and, after cell
proliferation, decreasing c-myc and/or Notch activity, and
permitting daughter cells of hair cell origin to differentiate into
hair cells or permitting daughter cells of supporting cell origin
to transdifferentiate into hair cells thereby to reduce the loss
of, maintain or promote vestibular function in the subject. The
daughter cells of supporting cell origin can be induced to
transdifferentiate into hair cells by activating Atoh1 activity,
for example, by gene expression, by administration of an effective
amount of Atoh1 or an Atoh1 agonist. The steps can be performed in
vivo (for example, in the inner ear of a mammal, in particular in
the utricle), or ex vivo, wherein the resulting cells are cultured
and/or introduced into the inner ear of the subject.
[0072] The methods and compositions described herein can be used
for treating subjects who have, or who are at risk for developing,
an auditory disorder resulting from a loss of auditory hair cells,
e.g., sensorineural hair cell loss. Patients having an auditory
disorder can be identified using standard hearing tests known in
the art. The method can comprise (a) increasing c-myc activity,
Notch activity, or both c-myc activity and Notch activity, as
appropriate, within the hair cell of the subject thereby to induce
cell proliferation to produce a daughter cell, and (b) after cell
proliferation, decreasing Notch activity to induce differentiation
of at least one of the cell and the daughter cell to produce a
differentiated cochlear or utricular hair cell. This can be
accomplished by administering an agent or agents to the subject to
modulate c-myc and Notch activity. Alternatively, the process can
occur in cells (e.g., cochlear and/or utricular cells) ex vivo,
after which the resulting cells are transplanted into the inner ear
of the subject. In certain embodiments, the methods and
compositions described herein can be used to promote growth of
neurites from the ganglion neurons of the inner ear. For example,
the regeneration of hair cells may promote the growth of new
neurites from ganglion neurons and formation of new synapses with
the regenerated hair cells to transmit sound and balance signals
from the hair cells to the brain.
[0073] In certain embodiments, the methods and compositions
described herein can be used to promote growth of neurites from the
ganglion neurons of the inner ear. For example, the regeneration of
hair cells may promote the growth of new neurites from ganglion
neurons and formation of new synapses with the regenerated hair
cells to transmit sound and balance signals from the hair cells to
the brain. In some embodiments, the methods and compositions
described herein can be used to reestablish proper synaptic
connections between hair cells and auditory neurons to treat, for
example, auditory neuropathy.
[0074] Subjects with sensorineural hair cell loss experience the
degeneration of cochlea hair cells, which frequently results in the
loss of spiral ganglion neurons in regions of hair cell loss. Such
subjects may also experience loss of supporting cells in the organ
of Corti, and degeneration of the limbus, spiral ligament, and
stria vascularis in the temporal bone material.
[0075] In certain embodiments, the present invention can be used to
treat hair cell loss and any disorder that arises as a consequence
of cell loss in the ear, such as hearing impairments, deafness,
vestibular disorders, tinnitus (see, Kaltenbach et al. (2002) J
NEUROPHYSIOL. 88(2):699-714s), and hyperacusis (Kujawa et al.
(2009) J. NEUROSCI. 29(45):14077-14085), for example, by promoting
differentiation (e.g., complete or partial differentiation) of one
or more cells into one or more cells capable of functioning as
sensory cells of the ear, e.g., hair cells.
[0076] In certain embodiments, the subject can have sensorineural
hearing loss, which results from damage or malfunction of the
sensory part (the cochlea) or non-sensory part (the limbus, spiral
ligament and stria vascularis) or the neural part (the auditory
nerve) of the ear, or conductive hearing loss, which is caused by
blockage or damage in the outer and/or middle ear. Alternatively or
in addition, the subject can have mixed hearing loss caused by a
problem in both the conductive pathway (in the outer or middle ear)
and in the nerve pathway (the inner ear). An example of a mixed
hearing loss is a conductive loss due to a middle-ear infection
combined with a sensorineural loss due to damage associated with
aging.
[0077] In certain embodiments, the subject may be deaf or have a
hearing loss for any reason, or as a result of any type of event.
For example, a subject may be deaf because of a genetic or
congenital defect; for example, a human subject can have been deaf
since birth, or can be deaf or hard-of-hearing as a result of a
gradual loss of hearing due to a genetic or congenital defect. In
another example, a human subject can be deaf or hard-of-hearing as
a result of a traumatic event, such as a physical trauma to a
structure of the ear, or a sudden loud noise, or a prolonged
exposure to loud noises. For example, prolonged exposures to
concerts, airport runways, and construction areas can cause inner
ear damage and subsequent hearing loss.
[0078] In certain embodiments, a subject can experience
chemical-induced ototoxicity, wherein ototoxins include therapeutic
drugs including antineoplastic agents, salicylates, quinines, and
aminoglycoside antibiotics, contaminants in foods or medicinals,
and environmental or industrial pollutants.
[0079] In certain embodiments, a subject can have a hearing
disorder that results from aging. Alternatively or in addition, the
subject can have tinnitus (characterized by ringing in the ears) or
hyperacusis (heightened sensitivity to sound).
[0080] In addition, the methods and compositions described herein
can be used to treat a subject having a vestibular dysfunction,
including bilateral and unilateral vestibular dysfunction.
Vestibular dysfunction is an inner ear dysfunction characterized by
symptoms that include dizziness, imbalance, vertigo, nausea, and
fuzzy vision and may be accompanied by hearing problems, fatigue
and changes in cognitive functioning. Vestibular dysfunction can be
the result of a genetic or congenital defect; an infection, such as
a viral or bacterial infection; or an injury, such as a traumatic
or nontraumatic injury. Vestibular dysfunction is most commonly
tested by measuring individual symptoms of the disorder (e.g.,
vertigo, nausea, and fuzzy vision).
[0081] Alternatively or in addition, the methods and compositions
described herein can be used prophylactically, such as to prevent,
reduce or delay progression of hearing loss, deafness, or other
auditory disorders associated with loss of inner ear function. For
example, a composition containing one or more of the agents can be
administered with (e.g., before, after or concurrently with) a
second composition, such as an active agent that may affect hearing
loss. Such ototoxic drugs include the antibiotics neomycin,
kanamycin, amikacin, viomycin, gentamycin, tobramycin,
erythromycin, vancomycin, and streptomycin; chemotherapeutics such
as cisplatin; nonsteroidal anti-inflammatory drugs (NSAIDs) such as
choline magnesium trisalicylate, diclofenac, diflunisal,
fenoprofen, flurbiprofen, ibuprofen, indomethacin, ketoprofen,
meclofenamate, nabumetone, naproxen, oxaprozin, phenylbutazone,
piroxicam, salsalate, sulindac, and tolmetin; diuretics;
salicylates such as aspirin; and certain malaria treatments such as
quinine and chloroquine. For example, a human undergoing
chemotherapy can be treated using the compounds and methods
described herein. The chemotherapeutic agent cisplatin, for
example, is known to cause hearing loss. Therefore, a composition
containing one or more agents that increase the activity of c-myc
and Notch can be administered with cisplatin therapy (e.g., before,
after or concurrently with) to prevent or lessen the severity of
the cisplatin side effect. Such a composition can be administered
before, after and/or simultaneously with the second therapeutic
agent. The two agents may be administered by different routes of
administration.
[0082] In certain embodiments, the methods and compositions
described herein can be used to increase the levels (e.g., protein
levels) and/or activity (e.g., biological activity) of c-myc
and
[0083] Notch in cells (e.g., inner ear cells). Exemplary methods
and compositions include, but are not limited to methods and
compositions for increasing c-myc or Notch expression (e.g.,
transcription and/or translation) or levels (e.g., concentration)
in cells. It is contemplated that such modulation can be achieved
in hair cells and/or supporting cells in vivo and ex vivo.
1. Methods and Compositions for Increasing C-myc and Notch and
Atoh1 Activity
[0084] (i) C-myc, Notch, or Atoh1 Polypeptides
[0085] It is contemplated that c-myc, Notch, and Atoh1 proteins,
including full length proteins, biologically active fragments, and
homologs of c-myc and Notch can be introduced into target cells
using techniques known in the art.
[0086] Exemplary c-myc polypeptides include, for example,
NP.sub.--002458.2 (SEQ ID NO: 1), as referenced in the NCBI protein
database. Exemplary Notch polypeptides include, for example,
NP.sub.--060087.3 (SEQ ID NO: 2), as referenced in the NCBI protein
database. Exemplary Atoh1 polypeptides include, for example,
NP.sub.--005163.1 (SEQ ID NO: 3), as referenced in the NCBI protein
database.
[0087] In certain embodiments, nucleic acid sequences encoding
c-myc, Notch, and Atoh1 family members may be used in accordance
with the methods described herein. Exemplary c-myc family members
include N-myc, referenced in the NCBI protein database as
NP.sub.--005369.2 (SEQ ID NO: 12). Exemplary Notch family members
include Notch2, referenced in the NCBI protein database as
NP.sub.--077719.2 (SEQ ID NO: 14); Notch3, referenced in the NCBI
protein database as NP.sub.--000426.2 (SEQ ID NO: 16); and Notch4,
referenced in the NCBI protein database as NP.sub.--004548.3 (SEQ
ID NO: 18). Exemplary Atoh1 family members include Atoh7,
referenced in the NCBI protein database as NP.sub.--660161.1 (SEQ
ID NO: 20).
[0088] In certain embodiments, a protein sequence of the invention
may comprise a consensus protein sequence or a nucleotide sequence
encoding a consensus protein sequence. Consensus protein sequences
of c-myc, Notch intracellular domain, and Atoh1 of the invention
are set forth below.
[0089] A consensus protein sequence of c-myc built from human,
mouse, rat and chimpanzee sequences using ClustalW is as
follows:
[0090]
MPLNVX.sub.1FX.sub.2NRNYDLDYDSVQPYFX.sub.3CDEEENFYX.sub.4QQQQSELQPP-
APSEDI
WKKFELLPTPPLSPSRRSGLCSPSYVAVX.sub.5X.sub.6X.sub.7FSX.sub.8RX.sub.9D-
X.sub.10DGGGGX.sub.11FSTADQLEM
X.sub.12TELLGGDMVNQSFICDPDDETFIKNIIIQDCMWSGFSAAAKLVSEKLASYQAARKDS
X.sub.13
SX.sub.14X.sub.15PARGHSVCSTSSLYLQDLX.sub.16AAASECIDPSVVFPYPLNDSS
SPKSCX.sub.17SX.sub.18D
SX.sub.19AFSX.sub.20SSDSLLSSX.sub.21ESSPX.sub.22X.sub.23X.sub.24PEPLVLHEE-
TPPTTSSDSEEEQX.sub.25DEEEIDVVS
VEKRQX26PX.sub.27KRSESGSX.sub.28X.sub.29X.sub.30GGHSKPPHSPLVLKRCHVSTHQHNY-
AAPPSTRKD
YPAAKRX.sub.31KLDSX.sub.32RVLX.sub.33QISNNRKCX.sub.34SPRSSDTEENX-
.sub.35KRRTHNVLERQRRNELK
RSFFALRDQIPELENNEKAPKVVILKKATAYILSX.sub.36QAX.sub.37EX.sub.38KLX.sub.39SE-
X.sub.40DLLRKRRE QLKHKLEQLRNSX.sub.41A (SEQ ID NO: 9), wherein
X.sub.1 is S or N; X.sub.2 is T or A; X.sub.3 is Y or I; X.sub.4 is
Q or H; X.sub.5 is T or A; X.sub.6 is P or T; X.sub.7 is S or a
bond; X.sub.8 is L or P; X.sub.9 is G or E; X.sub.10 is N or D;
X.sub.11 is S or N; X.sub.12 is V or M; X.sub.13 is G or T;
X.sub.14 is P or L; X.sub.15 is N or S; X.sub.16 is S or T;
X.sub.17 is P or A or T; X.sub.18 is Q or S; X.sub.19 is S or T;
X.sub.20 is P or S; X.sub.21 is T or a bond; X.sub.22 is Q or R;
X.sub.23 is A or G; X.sub.24 is S or T; X.sub.25 is E or D;
X.sub.26 is A or T or P; X.sub.27 is G or A; X.sub.28 is P or S;
X.sub.29 is P or S; X.sub.30 is A or S; X.sub.31 is V or A;
X.sub.32 V or G; X.sub.33 is K or R; X.sub.34 is T or S; X.sub.35
is D or V; X.sub.36 is V or I; X.sub.37 is E or D; X.sub.38 is Q or
H; X.sub.39 is T or I; X.sub.40 is E or K; and X.sub.41 is C or
G.
[0091] A consensus protein sequence of the Notch intracellular
domain build from human, rat and mouse sequences using ClustalW is
as follows:
[0092] VLLSRKRRRQHGQLWFPEGFKVSEASKKKRREPLGEDSVGLKPLKNASDG
ALMDDNQNEWGDEDLETKKFRFEEPVVLPDLX.sub.1DQTDHRQWTQQHLDAADLRX.sub.2SA
MAPTPPQGEVDADCMDVNVRGPDGFTPLMIASCSGGGLETGNSEEEEDAPAVISDFIY
QGASLHNQTDRTGETALHLAARYSRSDAAKRLLEASADANIQDNMGRTPLHAAVSAD
AQGVFQILX.sub.3RNRATDLDARMHDGTTPLILAARLAVEGMLEDLINSHADVNAVDDLG
KSALHWAAAVNNVDAAVVLLKNGANKDMQNNX.sub.4EETPLFLAAREGSYETAKVLLDH
FANRDITDHMDRLPRDIAQERMHHDIVRLLDEYNLVRSPQLHGX.sub.5X.sub.6LGGTPTLSPX.sub.7-
LC
SPNGYLGX.sub.8LKX.sub.9X.sub.10X.sub.11QGKKX.sub.12RKPSX.sub.13KGLACX.s-
ub.14SKEAKDLKARRKKSQDGKGCL
LDSSX.sub.15MLSPVDSLESPHGYLSDVASPPLLPSPFQQSPSX.sub.16PLX.sub.17HLPGMPDTHL-
GIX.sub.18H
LNVAAKPEMAALX.sub.19GGX.sub.20RLAFEX.sub.21X.sub.22PPRLSHLPVASX.sub.23X.s-
ub.24STVLX.sub.25X.sub.26X.sub.27X.sub.28X.sub.29G
AX.sub.30NFTVGX.sub.31X.sub.32X.sub.33SLNGQCEWLX.sub.34RLQX.sub.35GMVPX.s-
ub.36QYNPLRX.sub.37X.sub.38VX.sub.39PGX.sub.40LST
QAX.sub.41X.sub.42LQHX.sub.43MX.sub.44GPX.sub.45HS
SLX.sub.46X.sub.47X.sub.48X.sub.49LSX.sub.50X.sub.51X.sub.52X.sub.53YQGLP-
X.sub.54TRLATQPHL
VQTQQVQPQNLQX.sub.55QX.sub.56QNLQX.sub.57X.sub.58X.sub.59X.sub.60X.sub.61-
X.sub.62X.sub.63X.sub.64X.sub.65X.sub.66X.sub.67X.sub.68X.sub.69X.sub.70PP-
X.sub.71QP
HLX.sub.72VSSAAX.sub.73GHLGRSFLSGEPSQADVQPLGPSSLX.sub.74VHTILPQ-
ESX.sub.75ALPTSLPSSX.sub.76
VPPX.sub.77TX.sub.78X.sub.79QFLTPPSQHSYSSX.sub.80PVDNTPSHQLQVPEHPFLTPSPES-
PDQWSSS SX.sub.81H
SNX.sub.82SDWSEGX.sub.83SSPPTX.sub.84MX.sub.85SQIX.sub.86X.sub.87IPEAFK
(SEQ ID NO: 10), wherein X.sub.1 is D or S; X.sub.2 is M or V;
X.sub.3 is L or I; X.sub.4 is K or R; X.sub.5 is T or A; X.sub.6 is
A or P; X.sub.2 is T or P; X.sub.8 is S or N; X.sub.9 is S or P;
X.sub.10 is A or G; X.sub.1 1 is T or V; X.sub.12 is A or V;
X.sub.13 is T or S; X.sub.14 is G or S; X.sub.15 is G or S;
X.sub.16 is M or V; X.sub.17 is S or N; X.sub.18 is S or G;
X.sub.19 is A or G; X.sub.20 is S or G; X.sub.21 is P or T;
X.sub.22 is P or G; X.sub.23 is S or G; X.sub.24 is A or T;
X.sub.25 is S or G; X.sub.26 is T or S; X.sub.27 is N or S;
X.sub.28 is G or S; X.sub.29 is T or G; X.sub.30 is M or L;
X.sub.31 is A or G; X.sub.32 is P or S; X.sub.33 is A or T;
X.sub.34 is P or S; X.sub.35 is N or S; X.sub.36 is S or N;
X.sub.37 is P or G; X.sub.38 is G or S; X.sub.39 is T or A;
X.sub.40 is T or P; X.sub.41 is A or P; X.sub.42 is G or S;
X.sub.43 is G or S; X.sub.44 is M or V; X.sub.45 is L or I;
X.sub.46 is S or A; X.sub.47 is T or A; X.sub.48 is N or S;
X.sub.49 is T or A; X.sub.50 is P or Q; X.sub.51 is M or I;
X.sub.52 is M or I; X.sub.53 is S or a bond; X.sub.54 is S or N;
X.sub.55 is L or I or M; X.sub.56 is Q or P; X.sub.57 is a bond or
P; X.sub.58 is a bond or A; X.sub.59 is a bond or N; X.sub.60 is a
bond or I; X.sub.61 is a bond or Q; X.sub.62 is a bond or Q;
X.sub.63 is a bond or Q; X.sub.64 is a bond or Q; X.sub.65 is a
bond or S; X.sub.66 is a bond or L; X.sub.67 is a bond or Q;
X.sub.68 is a bond or P; X.sub.69 is a bond or P; X.sub.70 is a
bond or P; X.sub.71 is P or S; X.sub.72 S or G; X.sub.73 is N or S;
X.sub.74 is P or A; X.sub.75 is Q or P; X.sub.76 is M or L;
X.sub.77 is M or V; X.sub.78 is T or A; X.sub.79 is T or A;
X.sub.80 is S or a bond; X.sub.81 is P or R; X.sub.82 is I or V;
X.sub.83 is I or V; X.sub.84 is T or S; X.sub.85 is P or Q;
X.sub.86 is T or A; X.sub.87 is H or R.
[0093] A consensus protein sequence of Atoh1 built from human,
mouse and chimpanzee sequences using ClustalW is as follows:
[0094]
MSRLLHAEEWAEVKELGDHHRX.sub.1PQPHHX.sub.2PX.sub.3X.sub.4PPX.sub.5X.s-
ub.6QPPATLQARX.sub.7X.sub.8
PVYPX.sub.9ELSLLDSTDPRAWLX.sub.10PTLQGX.sub.11CTARAAQYLLHSPELX.sub.12ASEA-
AAPRDEX.sub.13
DX.sub.14X.sub.15GELVRRSX.sub.16X.sub.17GX.sub.18X.sub.19X.sub.20SKSPGPVK-
VREQLCKLKGGVVVDELGCSRQRAPS
SKQVNGVQKQRRLAANARERRRMHGLNHAFDQLRNVIPSFNNDKKLSKYETLQMAQ
IYINALSELLQTPX.sub.21X.sub.22GEQPPPPX.sub.23ASCKX.sub.24DHHHLRTAX.sub.25S-
YEGGAGX.sub.26X.sub.27X.sub.28X.sub.29A GAQX.sub.30AX.sub.31
GGX.sub.32X.sub.33RPTPPGX.sub.34CRTRF
SX.sub.35PASX.sub.36GGYSVQLDALHFX.sub.37X.sub.38FEDX.sub.39A
LTAMMAQKX.sub.40LSPSLPGX.sub.41ILQPVQEX.sub.42NSKTSPRSHRSDGEFSPHSHYSDSDEA-
S (SEQ ID NO: 11), wherein X.sub.1 is Q or H; X.sub.2 is L or V;
X.sub.3 is Q or a bond; X.sub.4 is P or a bond; X.sub.5 is P or a
bond; X.sub.6 is P or a bond; X.sub.7 is E or D; X.sub.8 is H or L;
X.sub.9 is P or A; X.sub.10 is A or T; X.sub.11 is I or L; X.sub.12
is S or G; X.sub.13 is V or A; X.sub.14 is G or S; X.sub.15 is R or
Q; X.sub.16 is S or G; X.sub.17 is G or C; X.sub.18 is A or G;
X.sub.19 is S or a bond; X.sub.20 is S or L; X.sub.21 is S or N;
X.sub.22 is G or V; X.sub.23 is P or T; X.sub.24 is S or N;
X.sub.25 is A or S; X.sub.26 is A or N; X.sub.27 is A or S;
X.sub.28 is T or A; X.sub.29 is A or V; X.sub.30 is Q or P;
X.sub.31 is S or P; X.sub.32 is S or G; X.sub.33 is Q or P;
X.sub.34 is S or P; X.sub.35 is A or G; X.sub.36 is A or S;
X.sub.37 is S or P; X.sub.38 is T or A; X.sub.39 is S or R;
X.sub.40 is N or D; X.sub.41 is S or G; and X.sub.42 is E or D.
[0095] As used herein, the term "Atoh1" refers to a protein
belonging to the basic helix-loop-helix (BHLH) family of
transcription factors that is involved in the formation of hair
cells in an inner ear of a mammal, and/or is a protein having an
amino sequence or consensus sequence as set forth herein.
[0096] The c-myc, Notch, or Atoh1 polypeptides can be used in
combination with compositions to enhance uptake of the polypeptides
into biological cells. In certain embodiments, the Atoh1, c-myc, or
Notch polypeptides can be mutated to include amino acid sequences
that enhance uptake of the polypeptides into a biological cell. In
certain embodiments, Atoh1, c-myc, or Notch polypeptides can be
altered or mutated to increase the stability and/or activity of the
polypeptide (e.g., c-myc, Notch or Atoh-1 point mutants). In
certain embodiments, c-myc, Notch or Atoh1 polypeptides can be
altered to increase nuclear translocation of the polypeptide. In
certain embodiments, altered c-myc, Notch or Atohl polypeptides or
biologically active fragments of c-myc, Notch, or Atoh1 retain at
least 50%, 60%, 70%, 80%, 90%, or 95% of the biological activity of
full length, wild type respective c-myc, Notch or Atoh1 protein in
the species that is the same species as the subject that is or will
be treated with the methods and compositions described herein.
[0097] In certain embodiments, c-myc polypeptides sequences can be
50%, 60%, 70%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to
NP.sub.--002458.2 (SEQ ID NO.: 1). In certain embodiments, Notch
polypeptides sequences are 50%, 60%, 70%, 80%, 85%, 90%, 95%, 98%,
99%, or 100% identical to NP.sub.--060087.3 (SEQ ID NO.: 2). In
certain embodiments, Atoh1 polypeptides sequences can be 50%, 60%,
70%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to
NP.sub.--005163.1 (SEQ ID NO.: 3). In certain embodiments, agents
encoded by modified Atoh1, c-myc, or Notch nucleic acid sequences
and Atoh1, c-myc, or Notch polypeptide sequences possess at least a
portion of the activity (e.g., biological activity) of the
molecules encoded by the corresponding, e g., unmodified,
full-length Atoh1, c-myc, or Notch nucleic acid sequences and
Atoh1, c-myc, or Notch polypeptide sequences. For example,
molecules encoded by modified Atoh1, c-myc, or Notch nucleic acid
sequences and modified Atoh1, c-myc, or Notch polypeptides retain
50%, 60%, 70%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% of the
activity (e.g., biological activity) of the molecules encoded by
the corresponding, e g., unmodified, respective Atoh1, c-myc, or
Notch nucleic acid sequences and/or full length Atoh1, c-myc, or
Notch polypeptide sequences.
[0098] In certain embodiments, the c-myc protein of the invention
comprises functional domains at least 50%, 60%, 70%, 80%, 85%, 90%,
95%, 98%, 99%, or 100% identical to a Myc-N domain comprising amino
acid residues 16-360 of SEQ ID NO: 1, a helix-loop-helix domain
comprising amino acid residues 370-426 of SEQ ID NO: 1, a Myc
leucine zipper domain comprising amino acid residues 423-454 of SEQ
ID NO: 1, and/or surrounding and/or intervening sequences of SEQ ID
NO: 1. In certain embodiments, the Notch protein of the invention
comprises functional domains at least 50%, 60%, 70%, 80%, 85%, 90%,
95%, 98%, 99%, or 100% identical to a Notch intracellular domain
comprising amino acid residues 1754-2555 of SEQ ID NO: 2. In
certain embodiments, the Atoh1 protein of the invention comprises
functional domains at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 98%,
99%, or 100% identical to a basic helix-loop-helix domain
comprising amino acids 158-214 of SEQ ID NO: 3, a helix-loop-helix
domain comprising amino acids 172-216 of SEQ ID NO: 3, and/or
surrounding and/or intervening sequences of SEQ ID NO: 3.
[0099] In certain embodiments, the c-myc and Notch proteins of the
invention can be administered to cells as a single protein
containing both c-myc and Notch (or active domains thereof),
preferably separated by a cleavable linker. Examples of cleavable
linkers are known in the art (see, e.g., U.S. Pat. No. 5,258,498
and U.S. Pat. No. 6,083,486.)
[0100] C-myc, Notch or Atoh1 levels (e.g., protein levels) and/or
activity (e.g., biological activity) in target cells and/or in the
nucleus of target cells can be assessed using standard methods such
as Western Blotting, in situ hybridization, reverse transcriptase
polymerase chain reaction, immunocytochemistry, viral titer
detection, and genetic reporter assays. Increases in c-myc, Notch
or Atoh1 levels (e.g., protein levels) and/or activity (e.g.,
biological activity) in target cells and/or in the nucleus of
target cells can be assessed by comparing c-myc, Notch or Atoh1
levels and/or activity in a first cell sample or a standard with
c-myc, Notch or Atoh1 levels and/or activity in a second cell
sample, e.g., contacting the cell sample with an agent contemplated
to increase c-myc, Notch or Atoh1 levels and/or activity.
[0101] Sequence identity may be determined in various ways that are
within the skill in the art, e.g., using publicly available
computer software such as BLAST, BLAST-2, ALIGN or Megalign
(DNASTAR) software, which are used to perform sequence alignments
and then calculate sequence identity. Exemplary software programs
available from the National Center for Biotechnology Information
(NCBI) on the website ncbi.nlm.nih.gov include blastp, blastn,
blastx, tblastn and tblastx. Those skilled in the art can determine
appropriate parameters for measuring alignment, including any
algorithms needed to achieve maximal alignment over the full length
of the sequences being compared. The search parameters for
histogram, descriptions, alignments, expect (i.e., the statistical
significance threshold for reporting matches against database
sequences), cutoff, matrix and filter are used at the default
settings. The default scoring matrix used by blastp, blastx,
tblastn, and tblastx is the BLOSUM62 matrix (Henikoff et al.,
(1992) PROC. NATL. ACAD. SCI. USA 89:10915-10919). In one approach,
the percent identity can be determined using the default parameters
of blastp, version 2.2.26 available from the NCBI.
(ii) DNA Encoding Atoh1, C-myc, or Notch
[0102] Atoh1, c-myc, or Notch can be expressed in target cells
using one or more expression constructs known in the art. Such
expression constructs include, but are not limited to, naked DNA,
viral, and non-viral expression vectors. Exemplary c-myc nucleic
acid sequences that may be expressed in target cells include, for
example, NM.sub.--002467.4 (SEQ ID NO: 4), as referenced in the
NCBI gene database. Exemplary Notch nucleic acid sequences that may
be expressed include, for example, NM.sub.--017617.3 (SEQ ID NO:
5), as referenced in the NCBI gene database. Exemplary Atoh1
nucleic acid sequences that may be expressed in target cells
include, for example, NM.sub.--005172.1 (SEQ ID NO: 6), as
referenced in the NCBI gene database.
[0103] In certain embodiments, c-myc, Notch, and Atoh1 family
members may be used. Exemplary c-myc family members include N-myc,
referenced in the NCBI gene database as NM.sub.--005378.4 (SEQ ID
NO: 13). Exemplary Notch family members include Notch2, referenced
in the NCBI gene database as NM.sub.--024408.3 (SEQ ID NO: 15);
Notch3, referenced in the NCBI gene database as NM.sub.--000435.2
(SEQ ID NO: 17); and Notch4, referenced in the NCBI gene database
as NM.sub.--004557.3 (SEQ ID NO: 19). Exemplary Atoh1 family
members include Atoh7, referenced in the NCBI gene database as
NM.sub.--145178.3 (SEQ ID NO: 21).
[0104] In certain embodiments, DNA encoding c-myc, Notch or Atoh1
can be an unmodified wild type sequence. Alternatively, DNA
encoding c-myc, Notch or Atoh1 can be modified using standard
techniques. For example, DNA encoding c-myc, Notch or Atoh1 can be
modified or mutated, e.g., to increase the stability of the DNA or
resulting polypeptide. Polypeptides resulting from such altered
DNAs should retain the biological activity of wild type c-myc,
Notch or Atoh1. In certain embodiments, DNA encoding Atoh1, c-myc,
or Notch can be altered to increase nuclear translocation of the
resulting polypeptide. In certain embodiments, DNA encoding c-myc,
Notch or Atoh1 can be modified using standard molecular biological
techniques to include an additional DNA sequence that can encode
one or more of, e.g., detectable polypeptides, signal peptides, and
protease cleavage sites.
[0105] In certain embodiments, c-myc nucleic acid sequences can be
50%, 60%, 70%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to
NM.sub.--002467.4 (SEQ ID NO: 4). In certain embodiments, Notch
nucleic acid sequences are 50%, 60%, 70%, 80%, 85%, 90%, 95%, 98%,
99%, or 100% identical to NM.sub.--017617.3 (SEQ ID NO: 5). In
certain embodiments, Atoh1 nucleic acid sequences are 50%, 60%,
70%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to
NM.sub.--005172.1 (SEQ ID NO: 6).
[0106] In certain embodiments, the c-myc nucleic acid sequence of
the invention comprises functional domains at least 50%, 60%, 70%,
80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to DNA encoding a
Myc-N domain comprising amino acid residues 16-360 of SEQ ID NO: 1,
a helix-loop-helix domain comprising amino acid residues 370-426 of
SEQ ID NO: 1, DNA encoding a Myc leucine zipper domain comprising
amino acid residues 423-454 of SEQ ID NO: 1, and/or DNA encoding
the surrounding and/or intervening sequences of SEQ ID NO: 1. In
certain embodiments, the Notch nucleic acid sequence of the
invention comprises functional domains at least 50%, 60%, 70%, 80%,
85%, 90%, 95%, 98%, 99%, or 100% identical to DNA encoding a Notch
intracellular domain comprising amino acid residues 1754-2555 of
SEQ ID NO: 2. In certain embodiments, the Atoh1 nucleic acid
sequence of the invention comprises functional domains at least
50%, 60%, 70%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to
DNA encoding a basic helix-loop-helix domain comprising amino acids
158-214 of SEQ ID NO: 3, DNA encoding a helix-loop-helix domain
comprising amino acids 172-216 of SEQ ID NO: 3, and/or DNA encoding
surrounding and/or intervening sequences of SEQ ID NO: 3.
(iii) C-myc, Notch or Atoh1 Pathway Modulators
[0107] In certain embodiments, c-myc or Notch levels (e.g., protein
levels) and/or activity (e.g., biological activity) can be
increased or decreased using compounds or compositions that target
c-myc or Notch, or one or more components of the c-myc or Notch
pathway. Similarly, Atoh1 levels (e.g., protein levels) and/or
activity (e.g., biological activity) can be increased using
compounds that target Atoh1 or one or more components of the Atoh1
pathway.
[0108] Exemplary c-myc activators include microRNAs that target
FBXW-7 (Ishikawa Y et al., Oncogene 2012 Jun. 4;
doi:10.1038/onc.2012.213) and activators that increase c-myc
expression levels or activity such as nordihydroguaiaretic acid
(NDGA) (Park S et al. (2004) J. CELL BIOCHEM. 91(5):973-86), CD19
(Chung et a.l, (2012) J. CLIN. INVEST. 122(6):2257-2266, cohesin
(McEwan et al, (2012) PLoS ONE 7(11): e49160), bryostatin 1 (Hu et
al. (1993) LEUK. LYMPHOMA 10(1-2):135-42),
2'-3-dimethyl-4-aminoazobenzene (ortho-aminoazotoluene, OAT)
(Smetanina et al. (2011) TOXICOL. APPL. PHARMACOL. 255(1):76-85),
2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) (Lauber et
al. (2004) CARCINOGENESIS 25(12):2509-17), .beta.-estradiol (U.S.
Pat. No. 7,544,511 B2), RU38486 (U.S. Pat. No. 7,544,511 B2),
dexamethasone (U.S. Pat. No. 7,544,511 B2), thyroid hormones (U.S.
Pat. No. 7,544,511 B2), retinoids (U.S. Pat. No. 7,544,511 B2), and
ecdysone (U.S. Pat. No. 7,544,511 B2).
[0109] Exemplary c-myc inhibitors include
7-nitro-N-(2-phenylphenyl)-2,1,3-benzoxadiazol-4-amine (10074-G5)
(Clausen D M et al., (2010) J. PHARMACOL. EXP. THER.
335(3):715-27), thioxothiazolidinone
[Z-E]-5-[4-ethylbenzylidene]-2-thioxo-1,3-thiazolidin-4-one
(10058-F4) (Clausen et al. (2010) J. PHARMACOL. EXP. THER.
335(3):715-27; Lin C P et al. (2007) ANTICANCER DRUGS.
18(2):161-70; Huang et al. (2006) EXP. HEMATOL. 34(11):1480-9),
4-phenylbutyrate (phenylbutyrate) (Engelhard et al. (1998) J.
NEUROONCOL. 37(2):97-108), Compound 0012 (Hurley et al. (2010) J.
VASC. RES. 47(1): 80-90), curcumin (Aggarwal et al. (2005) CLIN.
CANCER RES. 11(20):7490-8), magnesium hydroxide (Mori et al. (1997)
J. CELL BIOCHEM. SUPPL. 27:35-41), BP-1-102 (Zhang et al. (2012)
PROC. NATL. ACAD. SCI. U.S.A. 109(24):9623-8), WP1193 (Sai et al.
(2012) J. NEUROONCOL. 107(3):487-501), BP-1-107 (Page et al. (2012)
J. MED. CHEM. 55(3):1047-55), BP-1-108 (Page et al. (2012) J. MED.
CHEM. 55(3):1047-55), SF-1-087 (Page et al. (2012) J. MED. CHEM.
55(3):1047-55), SF-1-088 (Page et al. (2012) J. MED. CHEM.
55(3):1047-55), STX-0119 (Ashizawa et al. (2011) INT. J. ONCOL.
38(5):1245-52), substituted thiazol-4-one compounds (U.S. Pat. No.
7,872,027), (Z,E)-5-(4-ethylbenzylidene)-2-thioxothiazolidin-4-one
(10058-F4) (U.S. Pat. No. 7,026,343), S2T1-6OTD (U.S. Publication
No. 20120107317A1), Quarfloxin (CX-3543) (U.S. Publication No.
20120107317A1), benzoylanthranilic acid (U.S. Publication No.
20120107317A1), cationic porphyrin TMPyP4 (U.S. Publication No.
20120107317A1), tyrphostin and tryphostin-like compounds (European
Patent No. EP2487156A1), AG490 (European Patent No. EP2487156A1),
FBXW-7 expression vectors (Ishikawa Y et al., supra), and siRNAs
targeting c-Myc transcript (Id.).
[0110] Exemplary Notch activators include microRNAs that target
FBXW-7 (Ishikawa Y et al. supra), AG-370, 5 (U.S. Pat. No.
8,114,422), AG-1296 (6,7-dimethoxy-3-phenylquinoxaline) (Id.),
nigericin.Na (Id.), cytochalasin D (Id.), FCCP
(carbonylcyanide-4-(trifluoromethoxy)-phenylhydrazone) (Id.),
SP60012 (Id.), and vectors that produce protein of or isolated
protein of Jagged-1, Jagged-2, Jagged-3, Serrate, any member of the
Jagged/Serrate protein family, Delta, Delta-like-1, Delta-like-3,
Delta-like-4, Delta-like homolog-1 (DLK1), any member of the Delta
protein family, and any portion of any of these proteins (PCT
Publication WO2004090110A3). Exemplary Notch activators may also
include chemical activators such as valproic acid (VPA, see, U.S.
Pat. No. 8,338,482), resveratrol and phenethyl isothiocyanate.
[0111] Exemplary Notch inhibitors include gamma-secretase
inhibitors such as an arylsulfonamide, a benzodiazepine, L-685,458
(U.S. Patent Publication No. 2001/0305674), MK-0752 (Purow B.
(2012) ADV. EXP. MED. BIOL. 727:305-19; Imbimbo BP (2008) CURR.
TOP. MED. CHEM. 8(1):54-61), DAPT
([N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl
ester) (Id.; Ishikawa Y et al. supra; PCT Publication
WO2011149762A3), LY-374973
(N-[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl
ester) (PCT Publication WO2011149762A3),
N-[(3,5-Difluorophenyl)acetyl]-L-alanyl-2-phenyl]glycine-1,1-dimethylethy-
l ester (Id.); Lilly GSI L685,458 (Purow B, supra), compound E
((2S)-2-{[(3,5-Difluorophenyl)acetyl]amino}-N-[(3S)-1-methyl-2-oxo-5-phen-
yl-2,3-dihydro-1H-1,4-benzodiazepin-3-yl]propanamide) (Purow B,
supra), DBZ (dibenzazepine) (Purow B, supra), isocoumarin (Purow B,
supra), JLK6 (7-amino-4-chloro-3-methoxyisocoumarin) (Purow B
(2012) ADV. EXP. MED. BIOL. 727:305-19), Compound 18
([11-endo]-N-(5,6,7,8,9,10-hexahydro-6,9-methano
benzo[9][8]annulen-11-yl)-thiophene-2-sulfonamide) (Purow B,
supra), E2012 (Imbimbo BP, supra; PCT Publication WO2009005688A3),
MRK560 (Imbimbo BP, supra), LY-411575 (Imbimbo BP, supra),
LY-450139 (Imbimbo BP, supra; PCT Publication WO2009005688A3),
y-secretase inhibitor XII (PCT Publication WO2011149762A3; PCT
Publication WO2009005688A3),
2,2-dimethyl-N--((S)-6-oxo-6,7-dihydro-5H-dibenzo(b,
d)azepin-7-yl)-N'-(2,2,3,3,3-pentafluoro-propyl)-malonamide (U.S.
Patent Publication No. 20090181944A1), GSI-IX (EP1949916B1), GSI-X
(EP1949916B1), tocopherol derivatives (PCT Publication
WO2009040423A1),
[(2S)-2-{[(3,5-Difluorophenyl)acetyl]amino}-N-[(3S)1-methyl-2-oxo-5-pheny-
l-2,3-dihydro-1H-1,4-benzodiazepin-3-yl]propanamide] (PCT
Publication WO2009005688A3),
N-[N-(3,5-difluorophenacetyl)-L-alanyl]-Sphenylglycine-t-butylester
(Id.), [1,1'-Biphenyl]-4-acetic acid (Id.), 2-fluoro-alpha-methyl
(Id.), NGX-555 (Id.), LY-411575 (Id.), Cellzome (Id.),
2-Thiophenesulfonamide (Id.),
5-chloro-N-[(1S)-3,3,3-trifluoro-1-(hydroxymethyl)-2-(trifluoromet-
hyl)propyl] (Id.), NIC5-15 (Id.), BMS (Id.), CHF-5074 (Id.),
BMS-299897 (Imbimbo BP, supra), RO4929097; L-685458
((5S)-(t-Butoxycarbonylamino)-6-phenyl-(4R)hydroxy-(2R)benzylhexanoyl)-L--
leu-L-phe-amide); BMS-708163 (Avagacestat); BMS-299897
(2-[(1R)-1-[[(4-Chlorophenyl)sulfonyl](2,5-difluorophenyl)amino]ethyl-5-f-
luorobenzenebutanoic acid); MK-0752; YO-01027; MDL28170 (Sigma);
LY411575
(N-2((2S)-2-(3,5-difluorophenyl)-2-hydroxyethanoyl)-N1-((7S)-5-methyl-6-o-
xo-6,7-dihydro-5H-dibenzo[b,d]azepin-7-yl)-1-alaninamide, see U.S.
Pat. No. 6,541,466); ELN-46719 (2-hydroxy-valeric acid amide analog
of LY411575 (where LY411575 is the 3,5-difluoro-mandelic acid
amide) (U.S. Pat. No. 6,541,466)); PF-03084014
((S)-2-((S)-5,7-difluoro-1,2,3,4-tetrahydronaphthalen-3-ylamino)-N-(1-(2--
methyl-1-(neopentylamino)propan-2-yl)-1H-imidazol-4-yl)pentanamide,
Samon et al., MOL CANCER THER 2012; 11:1565-1575); and Compound E
((2S)-2-{[(3,5-Diflurophenyl)acetyl]amino}-N-[(3S)-1-methyl-2-oxo-5-pheny-
l-2,3-dihydro-1H-1,4-benzodiazepin-3-yl]propanamide; see WO
98/28268 and Samon et al., MOL CANCER THER 2012; 11:1565-1575;
available from Alexis Biochemicals)), or pharmaceutically
acceptable salts thereof. In some embodiments, suitable gamma
secretase inhibitors include: semagacestat (also known as LY450139,
(2S)-2-hydroxy-3-methyl-N-[(1S)-1-methyl-2-oxo-2-[[(1S)-2,3,4,5-tetrahydr-
o-3-methyl-2-oxo-1H-3-benzazepin-1-yl]amino]ethyl]butanamide,
available from Eli Lilly; WO 02/47671 and U.S. Pat. No. 7,468,365);
LY411575
(N-2((2S)-2-(3,5-difluorophenyl)-2-hydroxyethanoyl)-N1-((7S)-5-methyl-6-o-
xo-6,7-dihydro-5H-dibenzo[b,d]azepin-7-yl)-L-alaninamide, available
from Eli Lilly, Fauq et al., (2007) BIOORG MED CHEM LETT 17:
6392-5);begacestat (also known as GSI-953, U.S. Pat. No.
7,300,951); arylsulfonamides (A S, Fuwa et al., (2006) BIOORG MED
CHEM LETT. 16(16):4184-4189);
N-[N-(3,5-difluorophenacetyl)-L-alanyl]-(S)-phenylglycine t-butyl
ester (DAPT, Shih et al., (2007) CANCER RES. 67: 1879-1882);
N-[N-3,5-Difluorophenacetyl]-L-alanyl-S-phenylglycine Methyl Ester
(also known as DAPM, gamma-Secretase Inhibitor XVI, available from
EMD Millipore); Compound W (3,5-bis(4-Nitrophenoxy)benzoic acid,
available from Tocris Bioscience); L-685,458
((5S)-(tert-Butoxycarbonylamino)-6-phenyl-(4R)-hydroxy-(2R)-benzylhexanoy-
l)-L-leucy-L-phenylalaninamide, available from Sigma-Aldrich,
Shearmen et al., (2000) BIOCHEMISTRY 39, 8698-8704); BMS-289948
(4-chloro-N-(2,5-difluorophenyl)-N-((1R)-{4-fluoro-2-[3-(1H-imidazol-1-yl-
)propyl]phenyl}ethyl)benzenesulfonamide hydrochloride, available
from Bristol Myers Squibb); BMS-299897
(4-[2-((1R)-1-{[(4-chlorophenyl)sulfonyl]-2,5-difluoroanilino}ethyl)-5-fl-
uorophenyl]butanoic acid, available from Bristol Myers Squibb, see
Zheng et al., (2009) XENOBIOTICA 39(7):544-55); avagacestat (also
known as BMS-708163,
(R)-2-(4-chloro-N-(2-fluoro-4-(1,2,4-oxadiazol-3-yl)benzyl)phenylsulfonam-
ido)-5,5,5-trifluoropentanamide, available from Bristol Myers
Squibb, Albright et al., (2013) J PHARMACOL. EXP. THER.
344(3):686-695); MK-0752
(3-(4-((4-chlorophenyl)sulfonyl)-4-(2,5-difluorophenyl)cyclohexyl)propano-
ic acid, available from Merck); MRK-003
((3'R,6R,9R)-5'-(2,2,2-trifluoroethyl)-2-((E)-3-(4-(trifluoromethyl)piper-
idin-1-yl)prop-1-en-1-yl)-5,6,7,8,9,10-hexahydrospiro[6,9-methanobenzo[8]a-
nnulene-11,3'-[1,2,5]thiadiazolidine]1',1'-dioxide, available from
Merck, Mizuma et al., (2012) MOL CANCER THER. 11(9):1999-2009);
MRK-560
(N-[cis-4-[(4-Chlorophenyl)sulfonyl]-4-(2,5-difluorophenyl)cyclohexyl]-1,-
1,1-trifluoro-methanesulfonamide, Best et. al., (2006) J PHARMACOL
EXP Ther. 317(2):786-90);RO-4929097 (also known as R4733,
(S)-2,2-dimethyl-N1-(6-oxo-6,7-dihydro-5H-dibenzo[b,d]azepin-7-yl)-N3-(2,-
2,3,3,3-pentafluoropropyl)malonamide, available from Hoffman-La
Roche Inc., Tolcher et al., (2012) J CLIN. ONCOL.
30(19):2348-2353); JLK6 (also known as
7-Amino-4-chloro-3-methoxyisocoumarin, available from Santa Cruz
Biotechnology, Inc., Petit et al., (2001) NAT. CELL. BIOL. 3:
507-511); Tarenflurbil (also known as (R)-Flurbiprofen,
(2R)-2-(3-fluoro-4-phenylphenyl)propanoic acid); ALX-260-127 (also
known as Compound 11, described by Wolfe et al., (1998) J. MED.
CHEM. 41: 6);Sulindac sulfide (SSide, et al., (2003) J BIOL CHEM.
278(20): 18664-70);
1,1,1-trifluoro-N-(4-[5-fluoro-2-(trifluoromethyl)phenyl]-4-{[4
(trifluoromethyl)phenyl]sulfonyl}cyclohexyl)methanesulfonamide
(U.S. Patent Publication No. 20110275719);
N-[trans-3-[(4-chlorophenyl)sulfonyl]-3
-(2,5-difluorophenyl)cyclobutyl]-1,1,1-trifluoromethanesulfonamide
(U.S. Patent Publication No. 20110263580);
N-[cis-3-[(4-chlorophenyl)sulfonyl]-3-(2,5-difluorophenyl)cyclobutyl]-1,1-
,1-trifluoromethanesulfonamide (U.S. Patent Publication No.
20110263580);
N-[cis-3-[(4-chlorophenyl)sulfonyl]-3-(2-cyano-5-fluorophenyl)cyclobutyl]-
-1,1,1-trifluoromethanesulfonamide (U.S. Patent Publication No.
20110263580);
N-[cis-3-[(4-chlorophenyl)sulfonyl]-3-(2,5-dichlorophenyl)cyclobutyl]-1,1-
,1-trifluoromethanesulfonamide (U.S. Patent Publication No.
20110263580);
N-(cis-3-(2,5-difluorophenyl)-3-{[4-(trifluoromethyl)phenyl]sulfonyl}cycl-
obutyl)-1,1,1-trifluoromethanesulfonamide (U.S. Patent Publication
No. 20110263580);
N-{cis-3-(5-chloro-2-fluorophenyl)-3-[(4-chlorophenyl)sulfonyl]cyclobutyl-
}-1,1,1-trifluoromethanesulfonamide (U.S. Patent Publication No.
20110263580);
N-{cis-3-(2,5-difluorophenyl)-3-[(4-fluorophenyl)sulfonyl]cyclobutyl}-1,1-
,1-trifluoromethanesulfonamide (U.S. Patent Publication No.
201102635 80);
N-{cis-3-(2,5-difluorophenyl)-3-[(3,4-difluorophenyl)sulfonyl]cyclobutyl}-
-1,1,1-trifluoromethanesulfonamide (U.S. Patent Publication No.
20110263580);
N-[cis-3-[(4-cyanophenyl)sulfonyl]-3-(2,5-difluorophenyl)cyclobutyl]-1,1,-
1-trifluoromethanesulfonamide (U.S. Patent Publication No.
20110263580);
4-{[cis-3-[(4-chlorophenyl)sulfonyl]-3-(2,5-difluorophenyl)cyclobutyl][tr-
ifluoromethyl) sulfonyl]amino}butanoic acid (U.S. Patent
Publication No. 20110263580);
N-[cis-3-[(4-chlorophenyl)sulfonyl]-3-(2,5-difluorophenyl)cyclobutyl]-1,1-
,1-trifluoro-N-[2-(tetrahydro-2-pyran-2-yloxy)ethyl]methanesulfonamide
(U.S. Patent Publication No. 20110263580); Methyl
{[cis-3-[(4-chlorophenyl)sulfonyl]-3-(2,5-difluorophenyl)cyclobutyl][(tri-
fluoromethyl)sulfonyl]amino}acetate (U.S. Patent Publication No.
20110263580);
N-[3-[(4-chlorophenyl)sulfonyl]-3-(2,5-difluorophenyl)cyclobutyl]-1,1,1-t-
rifluoro-N-methylmethanesulfonamide (U.S. Patent Publication No.
20110263580);
N-[3-[(4-chlorophenyl)sulfonyl]-3-(2,5-difluorophenyl)cyclobutyl]-1,1,1-t-
rifluoro-N-methylmethanesulfonamide (U.S. Patent Publication No.
20110263580); Methyl
4-{[cis-3-[(4-chlorophenyl)sulfonyl]-3-(2,5-difluorophenyl)cyclobutyl][(t-
rifluoro-methyl)sulfonyl]amino}butanoate (U.S. Patent Publication
No.
20110263580);N-[cis-3-[(4-chlorophenyl)sulfonyl]-3-(2,5-difluorophenyl)cy-
clobutyl]-N-[(trifluoromethyl)sulfonyl]glycine (U.S. Patent
Publication No. 20110263580);
N-[cis-3-[(4-chlorophenyl)sulfonyl]-3-(2,5-difluorophenyl)-1-methylcyclob-
utyl]-1,1,1-trifluoromethanesulfonamide (U.S. Patent Publication
No. 20110263580);
N-(cis-3-(2,5-difluorophenyl)-1-methyl-3-{[4-(trifluoromethyl)phenyl]sulf-
onyl}cyclobutyl)-1,1,1-trifluoromethanesulfonamide (U.S. Patent
Publication No. 20110263580);
N-[cis-3-[(4-chlorophenyl)sulfonyl]-3-(2,5-difluorophenyl)cyclobutyl]-1,1-
,1-trifluoro-N-[(trifluoromethyl)sulfonyl]methanesulfonamide (U.S.
Patent Publication No. 20110263580); Sodium[cis-3
-[(4-chlorophenyl)sulfonyl]-3-(2,5-difluorophenyl)cyclobutyl][(trifluorom-
ethyl)sulfonyl]azanide (U.S. Patent Publication No. 20110263580);
Potassium[cis-3-[(4-chlorophenyl)sulfonyl]-3-(2,5-difluorophenyl)cyclo
butyl][(trifluoromethyl)sulfonyl]azanide (U.S. Patent Publication
No. 20110263580);
W[cis-3-[(4-trifluoromethoxyphenyl)sulfonyl]-3-(2,5-difluorophenyl)cyclob-
utyl]-1,1,1-trifluoromethanesulfonamide (U.S. Patent Publication
No. 20110263580);
1,1,1-trifluoro-N-(4-[5-fluoro-2-(trifluoromethyl)phenyl]-4-{[4-(trifluor-
omethyl)phenyl]sulfonyl}cyclohexyl)methanesulfonamide (U.S. Patent
Publication No. 20110263580); gamma-Secretase Inhibitor I (also
known as Z-Leu-Leu-Nle-CHO,
benzyloxycarbonyl-leucyl-leucyl-norleucinal, available from
Calbiochem); gamma-secretase inhibitor II:
##STR00001##
(MOL)(CDX) (available from Calbiochem);gamma secretase inhibitor
III, (N-Benzyloxycarbonyl-Leu-leucinal, available from
Calbiochem);gamma secretase inhibitor IV,
(N-(2-Naphthoyl)-Val-phenylalaninal, available from Calbiochem);
gamma-secretase inhibitor V (also known as Z-LF-CHO,
N-Benzyloxycarbonyl-Leu-phenylalaninal, available from EMD
Millipore);gamma-secretase inhibitor VI
(1-(S)-endo-N-(1,3,3)-Trimethylbicyclo[2.2.1]hept-2-yl)-4-fluorophenyl
Sulfonamide, available from EMD Millipore);gamma secretase
inhibitor VII, (also known as Compound A, MOC-LL-CHO,
Menthyloxycarbonyl-LL-CHO, available from Calbiochem);gamma
secretase inhibitor X,
({1S-Benzyl-4R-[1-(1S-carbamoyl-2-phenethylcarbamoyl)-1S-3-methylbutylcar-
bamoyl]-2R-hydroxy-5-phenylpentyl}carbamic acid tert-butyl ester,
available from Calbiochem); gamma secretase inhibitor XI,
(7-Amino-4-chloro-3-methoxyisocoumarin, available from
Calbiochem);gamma secretase inhibitor XII, (also known as
Z-Ile-Leu-CHO, Shih and Wang, (2007) CANCER RES. 67: 1879-1882);
gamma secretase inhibitor XIII, (Z-Tyr-Ile-Leu-CHO, available from
Calbiochem); gamma secretase inhibitor XIV,
(Z-Cys(t-Bu)-Ile-Leu-CHO, available from Calbiochem); gamma
secretase inhibitor XVII, (also known as WPE-III-31C),
##STR00002##
(MOL)(CDX) (available from Calbiochem);gamma secretase inhibitor
XIX, (also known as benzodiazepine,
(2S,3R)-3-(3,4-Difluorophenyl)-2-(4-fluorophenyl)-4-hydroxy-N-((3S)-2-oxo-
-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]diazepin-3-yl)-butyramide,
Churcher et al., (2003) J MED CHEM. 46(12):2275-8); gamma secretase
inhibitor XX, (also known as dibenzazepine,
(S,S)-2-[2-(3,5-Difluorophenyl)acetylamino]-N-(5-methyl-6-oxo-6,7-dihydro-
-5H-dibenzo[b,d]azepin-7-yl)propionamide.
##STR00003##
(MOL)(CDX) (Weihofen et al., Science 296: 2215-2218, 2002,
available from Calbiochem);gamma secretase inhibitor XXI,
((S,S)-2-[2-(3,5-Difluorophenyl)-acetylamino]-N-(1-methyl-2-oxo-5-phenyl--
2,3-dihydro-1H-benzo[e][1,4]diazepin-3-yl)-propionamide, available
from Calbiochem);
5-methyl-2-propan-2-ylcyclohexyl)N-[4-methyl-1-[(4-methyl-1-oxopentan-2-y-
l)amino]-1-oxopentan-2-yl]carbamate (available from HDH Pharma
Inc.); N-trans-3,5-Dimethoxycinnamoyl-Ile-leucinal (available from
Calbiochem); N-tert-Butyloxycarbonyl-Gly-Val-Valinal; isovaleryl-V
V-Sta-A-Sta-OCH3 (available from
Calbiochem);diethyl-(5-phenyl-3H-azepin-2-yl)-amine (U.S. Pat. No.
8,188,069);diethyl-(5-isopropyl-3H-azepin-2-yl)-amine (U.S. Pat.
No. 8,188,069); diethyl-(4-phenyl-3H-azepin-2-yl)-amine (U.S. Pat.
No. 8,188,069); diethyl-(6-phenyl-3H-azepin-2-yl)-amine (U.S. Pat.
No. 8,188,069); 5-phenyl-1,3-dihydro-azepin-2-one (U.S. Pat. No.
8,188,069); 5-Isopropyl-1,3-dihydro-azepin-2-one (U.S. Pat. No.
8,188,069); 4-phenyl-1,3-dihydro-azepin-2-one (U.S. Pat. No.
8,188,069); 6-phenyl-1,3-dihydro-azepin-2-one (U.S. Pat. No.
8,188,069); 2-butoxy-5-phenyl-3H-azepine (U.S. Pat. No. 8,188,069);
1-methyl-5-phenyl-1,3-dihydro-azepin-2-one (U.S. Pat. No.
8,188,069); 5-isopropyl-1-methyl-1,3-dihydro-azepin-2-one (U.S.
Pat. No. 8,188,069); 1-methyl-4-phenyl-1,3-dihydro-azepin-2-one
(U.S. Pat. No. 8,188,069);
1-methyl-6-phenyl-1,3-dihydro-azepin-2-one (U.S. Pat. No.
8,188,069); 1-methyl-5-phenyl-1H-azepine-2,3-dione-3-oxime (U.S.
Pat. No. 8,188,069);
5-isopropyl-1-methyl-1H-azepine-2,3-dione-3-oxime (U.S. Pat. No.
8,188,069); 1-methyl-6-phenyl-1H-azepine-2,3-dione-3-oxime (U.S.
Pat. No. 8,188,069); 1-methyl-4-phenyl-1H-azepine-2,3-dione-3-oxime
(U.S. Pat. No. 8,188,069);
3-amino-1-methyl-5-phenyl-1,3-dihydro-azepin-2-one (U.S. Pat. No.
8,188,069); 3-amino-5-isopropyl-1-methyl-1,3-dihydro-azepin-2-one
(U.S. Pat. No. 8,188,069);
3-amino-1-methyl-4-phenyl-1,3-dihydro-azepin-2-one (U.S. Pat. No.
8,188,069); 3-amino-1-methyl-6-phenyl-1,3-dihydro-azepin-2-one
(U.S. Pat. No. 8,188,069);
(S)-[1-(1-methyl-2-oxo-5-phenyl-2,3-dihydro-1H-azepin-3-ylcarbamoyl)-ethy-
l]-carbamic acid tertbutyl ester (U.S. Pat. No. 8,188,069);
[(S)-1-(5-isopropyl-1-methyl-2-oxo-2,3-dihydro-1H-azepin-3-ylcarbamoyl)-e-
thyl]carbamic acid tert-butyl ester (U.S. Pat. No. 8,188,069);
[(S)-1-(1-methyl-2-oxo-4-phenyl-2,3-dihydro-1H-azepin-3-ylcarbamoyl)-ethy-
l]carbamic acid tert-butyl ester (U.S. Pat. No. 8,188,069);
[(S)-1-(1-methyl-2-oxo-6-phenyl-2,3-dihydro-1H-azepin-3-ylcarbamoyl)-ethy-
l]-carbamic acid tert-butyl ester (U.S. Pat. No. 8,188,069);
(S)-2-amino-N-(1-methyl-2-oxo-5-phenyl-2,3-dihydro-1H-azepin-3-yl)-propio-
namide (U.S. Pat. No. 8,188,069);
(S)-2-amino-N-(5-isopropyl-1-methyl-2-oxo-2,3-dihydro-1H-azepin-3-yl)prop-
ionarnide (U.S. Pat. No. 8,188,069);
(S)-2-Amino-N-(1-methyl-2-oxo-6-phenyl-2,3-dihydro-1H-azepin-3-yl)propion-
amide hydrochloride (U.S. Pat. No. 8,188,069);
(S)-2-Amino-N-(1-methyl-2-oxo-4-phenyl-2,3-dihydro-1 H
-azepin-3-yl)propionamide hydrochloride (U.S. Pat. No. 8,188,069);
(S)-2-fluoro-3-methyl-butyric acid (U.S. Pat. No. 8,188,069);
(S)-2-hydroxy-3-methyl-N-[(S)-1-((S)-1-methyl-2-oxo-5
-phenyl-2,3-dihydro-1H-azepin-3-ylcarbamoyl)-ethyl]-butyramide
(U.S. Pat. No. 8,188,069);
(S)-2-fluoro-3-methyl-N-[(S)-1-(1-methyl-2-oxo-5-phenyl-2,3-dihydro-1H-az-
epin-3-ylcarbamoyl)-ethyl]-butyramide (U.S. Pat. No. 8,188,069);
(S)-2-hydroxy-N-[(S)-1-(5-isopropyl-1-methyl-2-oxo-2,3-dihydro-1H-azepin--
3-ylcarbamoyl)ethyl]-3-methyl-butyramide (U.S. Pat. No. 8,188,069);
(S)-2-hydroxy-3-methyl-N-[(S)-1-(1-methyl-2-oxo-4-phenyl-2,3-dihydro-1H-a-
zepin-3-ylcarbamoyl)-ethyl]-butyramide (U.S. Pat. No. 8,188,069);
(S)-2-hydroxy-3-methyl-N-[(S)-1-(1-methyl-2-oxo-6-phenyl-2,3-dihydro-1H-a-
zepin-3-ylcarbamoyl)-ethyl]-butyramide (U.S. Pat. No. 8,188,069);
and
(S)-2-fluoro-3-methyl-N-[(S)-1-(1-methyl-2-oxo-6-phenyl-2,3-dihydro-1H-az-
epin-3-ylcarbamoyl)-ethyl]-butyramide (U.S. Pat. No. 8,188,069),
and pharmaceutically acceptable salts thereof.
[0112] Additional examples of gamma-secretase inhibitors are
disclosed in U.S. Patent Application Publication Nos. 2004/0029862,
2004/0049038, 2004/0186147, 2005/0215602, 2005/0182111,
2005/0182109, 2005/0143369, 2005/0119293, 2007/0190046,
2008/008316, 2010/0197660 and 2011/0020232; U.S. Pat. Nos.
6,756,511; 6,890,956; 6,984,626; 7,049,296; 7,101,895; 7,138,400;
7,144,910; 7,183,303; 8,188,069; and International Publication Nos.
WO 1998/28268; WO 2001/70677, WO 2002/049038, WO 2004/186147, WO
2003/093253, WO 2003/093251, WO 2003/093252, WO 2003/093264, WO
2005/030731, WO 2005/014553, WO 2004/039800, WO 2004/039370, WO
2009/023453, EP 1720909, EP 2178844, EP 2244713.
[0113] Additional exemplary Notch inhibitors include nonsteroidal
anti-inflammatory drugs (NSAIDs) such as flurbiprofen (Purow B,
supra), MPC-7869 (Imbimbo BP, supra), ibuprofen (Id.), sulindac
sulphide, indomethacin, alpha-secretase inhibitors (ASIs) (Purow B,
supra), the Na+/H+ antiporter Monensin (Id.); small molecules that
block Notch binding to interacting proteins such as Jagged, Numb,
Numb-like, CBF1 transcription factor, and mastermind-like (MAML)
(Id.; Ishikawa Y et al, supra.); antibodies that bind Notch
proteins or Notch ligands such as Delta-Like-4 (Purow B, supra);
stapled peptides that bind Notch such as SAHM1 (Id.);
dominant-negative forms of genes such as MAML (Id; Ishikawa Y et
al., supra), Numb/Numb-Like (Purow B, supra), and FBXW-7 (Id.);
expression vectors that increase levels of Notch regulators such as
FBXW-7 (Id.; Ishikawa Y et al., supra); siRNAs that target Notch
transcripts (Purow B, supra); microRNAs such as miR-326, miR-34a,
microRNA-206, and miR-124 (Id.); and Notch antibodies (U.S. Pat.
No. 8,226,943, U.S. Publication No. 20090258026A2, PCT Publication
WO2012080926A2).
[0114] Exemplary Atoh1 activators include, for example,
.beta.-Catenin or .beta.-catenin pathway agonists, e.g., Wnt
ligands, DSH/DVL1, 2, 3, LRP6.delta.N, WNT3A, WNT5A, and WNT3A, 5A.
Additional Wnt/.beta.-catenin pathway activators and inhibitors are
reviewed in the art (Moon et al., Nature Reviews Genetics,
5:689-699, 2004). In some embodiments, suitable Wnt/.beta.-catenin
pathway agonists can include antibodies and antigen binding
fragments thereof, and peptides that bind specifically to frizzled
(Fzd) family of receptors.
[0115] Kinase inhibitors, e.g., casein kinase 1 (CK1) and glycogen
synthase kinase 3.beta. (GSK3.beta.) inhibitors can also act as
.beta.-Catenin or .beta.-catenin pathway agonists to activate
Atoh1. GSK3.beta. inhibitors include, but are not limited to,
lithium chloride (LiCl), Purvalanol A, olomoucine, alsterpaullone,
kenpaullone, benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione
(TDZD-8), 2-thio(3-iodobenzyl)-5-(1-pyridyl)[1,3,4]-oxadiazole
(GSK3 inhibitor II), 2,4-dibenzyl-5-oxothiadiazolidine-3-thione
(OTDZT), (2'Z,3'E)-6-Bromoindirubin-3'-oxime (BIO),
.alpha.-4-Dibromoacetophenone (i.e., Tau Protein Kinase I (TPK I)
Inhibitor), 2-Chloro-1-(4,5-dibromo-thiophen-2-yl)-ethanone,
N-(4-Methoxybenzyl)-N'-(5-nitro-1,3-thiazol-2-yl)urea (AR-A014418),
and indirubins (e.g., indirubin-5-sulfonamide; indirubin-5-sulfonic
acid (2-hydroxyethyl)-amide indirubin-3'-monoxime;
5-iodo-indirubin-3'-monoxime; 5-fluoroindirubin;
5,5'-dibromoindirubin; 5-nitroindirubin; 5-chloroindirubin;
5-methylindirubin, 5-bromoindirubin),
4-Benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione (TDZD-8),
2-thio(3-iodobenzyl)-5-(1-pyridyl)-[1,3,4]-oxadiazole (GSK3
inhibitor II), 2,4-Dibenzyl-5-oxothiadiazolidine-3-thione (OTDZT),
(2'Z,3'E)-6-Bromoindirubin-3'-oxime (BIO),
.alpha.-4-Dibromoacetophenone (i.e., Tau Protein Kinase I (TPK I)
Inhibitor), 2-Chloro-1-(4,5-dibromo-thiophen-2-yl)-ethanone, (vi)
N-(4-Methoxybenzyl)-N'-(5-nitro-1,3-thiazol-2-yl)urea (AR-A014418),
and H-KEAPPAPPQSpP-NH2 (L803) or its cell-permeable derivative
Myr-N-GKEAPPAPPQSpP-NH2 (L803-mts). Other GSK3.beta. inhibitors are
disclosed in U.S. Pat. Nos. 6,417,185; 6,489,344; and 6,608,063. In
some embodiments, suitable kinase inhibitors can include RNAi and
siRNA designed to decrease GSK3.beta. and/or CK1 protein levels. In
some embodiments, useful kinase inhibitors include FGF pathway
inhibitors. In some embodiments, FGF pathway inhibitors include,
for example, SU5402.
[0116] Additional Atoh1 activators include gamma secretase
inhibitors (e.g., arylsulfonamides, dibenzazepines,
benzodiazepines,
N-[N-(3,5-difluorophenacetyl)-L-alanyl]-(S)-phenylglycine t-butyl
ester (DAPT; EMD Biosciences, San Diego, Calif., USA), L-685,458,
or MK0752ho, in addition to those listed above under Notch
inhibitors), gentamycin, and the combination of transcription
factors Eya1 and Six1 (and optionally Sox2), as described in Ahmed
et al. (2012) DEV. CELL 22(2):377-390.
[0117] Additional Atoh1 activators are described in U.S. Pat. No.
8,188,131, including a compound represented by Formula I:
##STR00004##
wherein:
[0118] each of R.sub.118, R.sub.119, R.sub.120, and R.sub.121 is,
independently selected from H, halo, OH, CN, NO.sub.2,
C.sub.1-C.sub.3 alkyl, C.sub.1-C.sub.3 haloalkyl, C.sub.1-C.sub.3
alkoxy, and C.sub.1-C.sub.3 haloalkoxy;
[0119] R.sub.122 is hydrogen or --Z--R.sup.a; wherein:
[0120] Z is O or a bond; and
[0121] R.sup.a is: [0122] (i) C.sub.1-C.sub.6 alkyl or
C.sub.1-C.sub.6 haloalkyl, each of which is optionally substituted
with from 1-3 R.sup.b; or [0123] (ii) C.sub.3-C.sub.10 cycloalkyl,
C.sub.3-C.sub.10 cycloalkenyl, each of which is optionally
substituted with from 1-5 R.sup.c; or [0124] (iii) C.sub.7-C.sub.11
aralkyl, or heteroaralkyl including 6-11 atoms, each of which is
optionally substituted with from 1-5 R.sup.c; [0125] (iv)
C.sub.6-C.sub.10 aryl or heteroaryl including 5-10 atoms, each of
which is optionally substituted with from 1-5 R.sup.d;
[0126] R.sub.123 is: [0127] (i) hydrogen; or [0128] (ii)
C.sub.1-C.sub.6 alkyl or C.sub.1-C.sub.6haloalkyl, each of which is
optionally substituted with from 1-3 R.sup.b; or [0129] (iii)
C.sub.6-C.sub.10 aryl or heteroaryl including 5-10 atoms, each of
which is optionally substituted with from 1-5 R.sup.d; or [0130]
(iv) C.sub.7-C.sub.11 aralkyl, or heteroaralkyl including 6-11
atoms, each of which is optionally substituted with from 1-5
R.sup.c; or [0131]
(v)--(C.sub.1-C.sub.6alkyl)-Z.sup.1--(C.sub.6-C.sub.10aryl),
wherein Z.sup.1 is O, S, NH, or N(CH.sub.3); the alkyl portion is
optionally substituted with from 1-3 R.sup.b; and the aryl portion
is optionally substituted with from 1-5 R.sup.d; or [0132]
(vi)--(C.sub.1-C.sub.6 alkyl)-Z.sup.2-(heteroaryl including 5-10
atoms), wherein Z.sup.2 is O, S, NH, or N(CH.sub.3); the alkyl
portion is optionally substituted with from 1-3 R.sup.b; and the
heteroaryl portion is optionally substituted with from 1-5 R.sup.d;
or [0133] (vii)--(C.sub.1-C.sub.6
alkyl)-Z.sup.3--(C.sub.3-C.sub.10cycloalkyl), wherein Z.sup.3 is O,
S, NH, or N(CH.sub.3); the alkyl portion is optionally substituted
with from 1-3 R.sup.b; and the cycloalkyl portion is optionally
substituted with from 1-5 R.sup.c;
[0134] R.sup.b at each occurrence is, independently: [0135] (i)
NH.sub.2; NH(C.sub.1-C.sub.3 alkyl); N(C.sub.1-C.sub.3
alkyl).sub.2; hydroxy; C.sub.1-C.sub.6 alkoxy or C.sub.1-C.sub.6
haloalkoxy; or [0136] (ii) C.sub.3-C.sub.7 cycloalkyl optionally
substituted with from 1-3 substituents independently selected from
C.sub.1-C.sub.6 alkyl, NH.sub.2; NH(C.sub.1-C.sub.3 alkyl);
N(C.sub.1-C.sub.3 alkyl).sub.2; hydroxy; C.sub.1-C.sub.6alkoxy or
C.sub.1-C.sub.6haloalkoxy;
[0137] R.sup.c at each occurrence is, independently: [0138] (i)
halo; NH.sub.2; NH(C.sub.1-C.sub.3 alkyl); N(C.sub.1-C.sub.3
alkyl).sub.2; hydroxy; C.sub.1-C.sub.6 alkoxy; C.sub.1-C.sub.6
haloalkoxy; or oxo; or [0139] (ii) C.sub.1-C.sub.6 alkyl or
C.sub.1-C.sub.6haloalkyl; and
[0140] R.sup.d at each occurrence is, independently: [0141] (i)
halo; NH.sub.2; NH(C.sub.1-C.sub.3 alkyl); N(C.sub.1-C.sub.3
alkyl).sub.2; hydroxy; C.sub.1-C.sub.6 alkoxy or C.sub.1-C.sub.6
haloalkoxy; nitro; --NHC(O)(C.sub.1-C.sub.3 alkyl); or cyano; or
[0142] (ii) C.sub.1-C.sub.6 alkyl or C.sub.1-C.sub.6haloalkyl; or a
pharmaceutically acceptable salt thereof.
[0143] Other exemplary Atoh1 activators described in U.S. Pat. No.
8,188,131 include
4-(4-chlorophenyl)-1-(5H-pyrimido[5,4-b]indo1-4-yl)-1H-pyrazol-3-amine;
6-chloro-1-(2-chlorobenzyloxy)-2-phenyl-1H-benzo[d]imidazole;
6-chloro-1-(2-chlorobenzyloxy)-2-(4-methoxyphenyl)-1H-benzo[d]imidazole;
6-chloro-2-(4-methoxyphenyl)-1-(4-methylbenzyloxy)-1H-benzo[d]imidazole;
6-chloro-1-(3,5-dimethylbenzyloxy)-2-(4-methoxyphenyl)-1H-benzo[d]imidazo-
le;
6-chloro-1-(4-methoxybenzyloxy)-2-(4-methoxyphenyl)-1H-benzo[d]imidazo-
le; 1-(4-methylbenzyloxy)-6-nitro-2-phenyl-1H-benzo[d]imidazole;
4-(1H-benzo[d]imidazol-2-yl)phenol;
2,5-dichloro-N-((1-methyl-1H-benzo[d]imidazol-2-yl)methyl)aniline;
4-(2-(1-methyl-1H-benzo [d]imidazol-2-yl)ethyl)aniline;
2-((2-methoxyphenoxy)methyl)-1H-benzo[d]imidazole;
2-((4-fluorophenoxy)methyl)-1-methyl-1H-benzo[d]imidazole;
2-(phenylthiomethyl)-1H-benzo[d]imidazole;
3-(6-methyl-1H-benzo[d]imidazole-2-yl)-2H-chromen-2-imine;
N-(2-(1H-benzo[d]imidazole-2-yl)phenyl)isobutyramide;
2-(o-tolyloxymethyl)-1H-benzo[d]imidazole;
2-(4-methoxyphenyl)-1-phenethyl-1H-benzo[d]imidazole;
N-(6-bromobenzo[d]thiazole-2-yl)thiophene-2-carboxamide;
N-(benzo[d]thiazole-2-yl)-1-methyl-1H-pyrazole-5-carboxamide;
2-(4-fluorobenzylthio)benzo[d]thiazole;
5-chloro-N-methylbenzo[d]thiazole-2-amine;
N-(6-acetamidobenzo[d]thiazol-2-yl)furan-2-carboxamide;
N-(6-fluorobenzo[d]thiazole-2-yl)-3-methoxybenzamide;
2-(benzo[d]oxazol-2-ylthio)-N-(2-chlorophenyl)acetamide;
5-chloro-2-phenylbenzo[d]oxazole;
5-methyl-2-m-tolylbenzo[d]oxazole;
2-(4-isobutoxyphenyl)-3-(naphthalen-2-yl)-2,3-dihydroquinazolin-4(1H)-one-
;
N-(2-(2-(4-fluorophenyl)-2-oxoethylthio)-4-oxoquinazolin-3(4H)-yl)benzam-
ide;
2-(4-chlorophenyl)-4-(4-methoxyphenyl)-1,4-dihydrobenzo[4,5]imidazo
[1,2-a]pyrimidine;
2-(3-pyridyl)-4-(4-bromophenyl)-1,4-dihydrobenzo[4,5]imidazo
[1,2-a]pyrimidine;
N-sec-butyl-1,7,7-trimethyl-9-oxo-8,9-dihydro-7H-furo[3,2-f]chromene-2-ca-
rboxamide;
N-(3-carbamoyl-5,6-dihydro-4H-cyclopenta[b]thiophen-2-yl)benzof-
uran-2-carboxamide;
3-chloro-N-(5-chloropyridin-2-yl)benzo[b]thiophene-2-carboxamide;
3-chloro-N-((tetrahydrofuran-2-yl)methyl)benzo[b]thiophene-2-carboxamide;
N-(3-(5-chloro-3-methylbenzo[b]thiopen-2-yl)-1H-pyrazol-5-yl)acetamide;
2-(naphthalen-2-yl)-1H-indole; 2-(pyridin-2-yl)-1H-indole;
N-(2-chlorophenyl)-2-(1H-indole-3-yl)-2-oxoacetamide;
2-m-tolylquinoline; 2-(4-(2-methoxyphenyl)piperazin-1-yl)quinolone;
2-(1H-
benzo[d][1,2,3]triazol-1-yl)-N-(2,3-dihydro-1H-inden-2-yl)acetamide;
1-phenethyl-1H-benzo[d][1,2,3]triazole;
7-(4-fluorobenzyloxy)-2H-chromen-2-one;
N-(2,4-dichlorophenyl)-8-methoxy-2H-chromene-3-carboxamide;
N-(3-chlorophenyl)-8-methyl-3,4-dihydroquinoline-1(2H)-carbothioamide;
7-methoxy-5-methyl-2-phenyl-4H-chromen-4-one;
2-(3,4-dimethylphenyl)quinoxaline;
4-bromo-N-(5-chloropyridin-2-yl)benzamide;
3-amino-6,7,8,9-tetrahydro-5H-cyclohepta[e]thieno[2,3-b]pyridine-2-carbox-
amide; (Z)-3-methyl-N'-(nicotinoyloxy)benzimidamide;
N,N-diethyl-6-methoxythieno[2,3-b]quinoline-2-carboxamide;
6-(4-methoxyphenyl)-1,2,3,4-tetrahydro-1,5-naphthyridine;
5-bromo-N-(2-(phenylthio)ethyl) nicotinamide;
N-(6-methylpyridin-2-yl)-2,3-dihydrobenzo[b][1,4]dioxine-6-carboxamide;
2-(4-methylbenzylthio)oxazolo [4,5-b]pyridine;
N-(2-methoxyethyl)-5-p-tolylpyrimidin-2-amine;
4-(5-(benzo[b]thiophen-2-yl)pyrimidin-2-yl)morpholino;
4-(5-(4-fluorophenyl)pyrimidin-2-yl)morpholino;
N-(4-bromo-3-methylphenyl)quinazoline-4-amine;
N-(4-methoxyphenyl)quinazolin-4-amine;
N-(3-methoxyphenyl)-9H-purin-6-amine;
N,N-diethyl-1-m-tolyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine;
(5-(4-bromophenyl)furan-2-yl)(morpholino)methanone;
(Z)-4-bromo-N'-(furan-2-carbonyloxy)benzimidamide;
N-(4-iodophenyl)furan-2-carboxamide;
5-(5-(2,4-difluorophenyl)furan-2-yl)-1-(methylsulfonyl)-1H-pyrazole;
1-(3-amino-5-(4-tert-butylphenyl)thiophen-2-yl)ethanone;
N-(3-cayano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)-2-fluorobenzamide;
N-(5-chloropyridin-2-yl)thiophene-2-carboxamide;
N-(2-(4-fluorophenoxy)ethyl)thiophene-2-carboxamide;
2,5-dimethyl-N-phenyl-1-(thiophen-2-ylmethyl)-1H-pyrrole-3-carboxamide;
N-(3-cyanothiophen-2-yl)-4-isopropoxybenzamide;
2-(4-methoxyphenoxy)-N-(thiazol-2-yl)acetamide;
4-(4-methoxyphenyl)-N-(3-methylpyridin-2-yl)thiazol-2-amine;
4-(biphenyl-4-yl)thiazol-2-amine;
4-(4-(4-methoxyphenyl)thiazol-2-yl)-3-methylisoxazol-5-amine;
N-(2-methoxyphenyl)-4-phenylthiazol-2-amine;
1-(4-amino-2-(m-tolylamino)thiazol-5-yl)-2-methylpropan-1-one;
4-(4-chlorophenyl)-1-(5H-pyrimido[5,4-b]indol-4-yl)-1H-pyrazol-3-amine;
2-(4-chlorophenyl)-6-ethyl-5-methylpyrazolo[1,5-a]pyrimidin-7(4H)-one;
5-methoxy-2-(5-phenyl-1H-pyrazol-3-yl)phenol;
(3-(4-bromophenyl)-1-phenyl-1H-pyrazol-4-yl)methanol;
N-(2,5-dichlorophenyl)-1-ethyl-1H-pyrazole-3-carboxamide;
4-chloro-1-methyl-N-(2-oxo-2-phenylethyl)-1H-pyrazole-3-carboxamide;
N-(3-(5-tert-butyl-2-methylfuran-3-yl)-1H- pyrazole-5-yl)benzamide;
N-(5-methylisoxazol-3-yl)benzo[d][1,3]dioxole-5-carboxamide; (5-(4-
bromophenyl)isoxazole-3-yl)(morpholino)methanone;
N-(4-bromophenyl)-5-isopropylisoxazole-3-carboxamide;
5-((4-chloro-2-methylphenoxy)methyl)-3-(pyridin-4-yl)-1,2,4-oxadiazole;
5-(2-methoxyphenyl)-3-p-tolyl-1,2,4-oxadiazole;
5-(phenoxymethyl)-3-(pyridin-3-yl)-1,2,4-oxadiazole;
5-(2-chloro-4-methylphenyl)-3-(pyridin-3-yl)-1,2,4-oxadiazole;
3-(2-chlorophenyl)-5-p-tolyl-1,2,4-oxadiazole;
5-(piperidin-1-ylmethyl)-3-p-toyl-1,2,4-oxadiazole;
5-(4-bromophenyl)-3-(pyridin-3-yl)-1,2,4-oxadiazole;
5-(2-bromophenyl)-3-(4-bromophenyl)-1,2,4-oxadiazole;
5-(2-bromo-5-methoxyphenyl)-3-(thiophenyl-2-yl)-1,2,4-oxadiazole;
3-(2-fluorophenyl)-N-(3-(piperidin-1-yl)propyl)-1,2,4-oxadiazol-5-amine;
2-(2-chlorobenzoyl)-N-(4-fluorophenyl)hydrazinecarbothioamide;
2-(methylamino)-N-phenethylbenzamide;
4-tert-butyl-N-((tetrahydrofuran-2-yl)methyl)benzamide;
2-phenyl-5-o-tolyl-1,3,4-oxadiazole;
4-(3-(4-chlorophenyl)-4,5-dihydro-1H-1,2,4-triazole-5-yl)-N,N-dimethylani-
line;
7-methoxy-2-(4-methoxyphenyl)-1,10b-dihydrospiro[benzo[e]pyrazolo[1,-
5-c][1,3]oxazine-5,1'-cyclohexane];
6-oxo-2-(4-(3-(trifluoromethyl)phenoxy)phenyl)-1,4,5,6-tetrahydropyridine-
-3- carbonitrile; 6-(4-methoxyphenyl)imidazo[2,1-b]thiazole;
2-(2-bromophenoxy)-N-(4H-1,2,4-triazol-3-yl)acetamide;
1-(indolin-1-yl)-2-phenoxyethanone;
2-(4-chlorophenyl)-6,7,8,9-tetrahydrobenzo[e]imidazo
[1,2-b][1,2,4]triazine; and pharmaceutically acceptable salts
thereof.
2. Delivery of Agents for Modulating c-myc, Notch and Atoh1
Delivery of Proteins, Activators and Inhibitors
[0144] The method of delivery of modulators of c-myc, Notch or
Atoh1 activity will depend, in part, upon whether the hair cells or
supporting cells are being contacted with the agents of interest in
vivo or ex vivo. In the in vivo approach, the agents are delivered
into the inner ear of a mammal In the ex vivo approach, cells are
contacted with the agents ex vivo. The resulting hair cells can
then be transplanted into the inner ear of a recipient using
techniques known and used in the art.
[0145] In certain embodiments, c-myc activity is increased by
administering c-myc protein or a c-myc activator in the inner ear
of a recipient to give, for example, a final concentration of
greater than about 30 .mu.M, for example, in the range of about 30
.mu.M to about 1000 .mu.M. In certain embodiments, the c-myc
protein or c-myc activator can be administered in an amount
sufficient to give a final concentration of greater than about 30
.mu.M. For example, the c-myc protein or c-myc activator may be
administered in an amount sufficient to give a final concentration
in the range from about 30 .mu.M to about 1000 .mu.M, to about 1000
.mu.M, 80 .mu.M, to about 1000 .mu.M, about 100 .mu.M to about 1000
.mu.M, about 150 .mu.M, to about 1000 .mu.M, from about 200 .mu.M,
to about 800 .mu.M, or from about 200 .mu.M, to about 600
.mu.M.
[0146] In other embodiments, c-myc protein or a c-myc activator is
administered at a dose from about 0.025 mg to about 4 mg, from
about 0.035 mg to about 2 mg, from about 0.05 mg to about 2 mg,
from about 0.1 mg to about 2 mg, from about 0.2 mg to about 1 mg,
or from about 0.2 mg to about 0.8 mg of the c-myc protein or c-myc
activator can be administered locally to the inner ear of a mammal
In one embodiment, 0.5 mg of c-myc protein or c-myc activator is
administered locally to the inner ear. In certain other
embodiments, from about 0.05 mg to about 2 mg, from about 0.2 mg to
about 2 mg, from about 0.05 mg to about 1.5 mg, from about 0.15 mg
to about 1.5 mg, from about 0.4 mg to about 1 mg, or from about 0.5
mg to about 0.8 mg of c-myc protein or c-myc activator can be
administered locally to the inner ear of a mammal.
[0147] In certain embodiments, Notch activity is increased by
administering a Notch protein, a NICD protein or a Notch activator
to an inner ear of a recipient to give a final concentration of
greater than about 30 .mu.M, for example, in the range of about 30
.mu.M to about 1000 .mu.M. In certain embodiments, a Notch protein,
NICD protein or Notch activator can be administered in an amount
sufficient to give a final concentration of greater than about 30
.mu.M. For example, the Notch protein, NICD protein or Notch
activator may be administered in an amount sufficient to give a
final concentration in the range from about 30 .mu.M to about 1000
.mu.M, 50 .mu.M to about 1000 .mu.M, 80 .mu.M to about 1000 .mu.M,
about 100 .mu.M to about 1000 .mu.M, about 150 .mu.M to about 1000
.mu.M, from about 200 .mu.M to about 800 .mu.M, or from about 200
.mu.M to about 600 .mu.M.
[0148] In other embodiments, Notch protein, NICD protein or Notch
activator is administered at a dose from about 0.025 mg to about 4
mg, from about 0.035 mg to about 2 mg, from about 0.05 mg to about
2 mg, from about 0.1 mg to about 2 mg, from about 0.2 mg to about 1
mg, or from about 0.2 mg to about 0.8 mg of the Notch protein, NICD
protein or Notch activator can be administered locally to the inner
ear of a mammal In one embodiment, 0.5 mg of Notch protein, NICD
protein or Notch activator is administered locally to the inner ear
of a mammal In certain other embodiments, from about 0.05 mg to
about 2 mg, from about 0.2 mg to about 2 mg, from about 0.05 mg to
about 1.5 mg, from about 0.15 mg to about 1.5 mg, from about 0.4 mg
to about 1 mg, or from about 0.5 mg to about 0.8 mg of Notch
protein, NICD protein or Notch activator can be administered
locally to the inner ear of a mammal.
[0149] In certain embodiments, after cell proliferation has
occurred, Notch activity is inhibited by administering a Notch
inhibitor. A Notch inhibitor can be administered to give a final
concentration of greater than about 30 .mu.M, for example, in the
range of about 30 .mu.M to about 1000 .mu.M. In certain
embodiments, a Notch inhibitor can be administered in an amount
sufficient to give a final concentration of greater than about 30
.mu.M. For example, the Notch inhibitor may be administered in an
amount sufficient to give a final concentration in the range from
about 30 .mu.M to about 1000 .mu.M, 50 .mu.M to about 1000 .mu.M,
80 .mu.M to about 1000 .mu.M, about 100 .mu.M to about 1000 .mu.M,
about 150 .mu.M to about 1000 .mu.M, from about 200 .mu.M to about
800 .mu.M, or from about 200 .mu.M to about 600 .mu.M. In certain
embodiments, the Notch inhibitor is administered in an amount
sufficient to give a final concentration of about 400 .mu.M.
[0150] In other embodiments, a Notch inhibitor is administered at a
dose from about 0.025 mg to about 4 mg, from about 0.035 mg to
about 2 mg, from about 0.05 mg to about 2 mg, from about 0.1 mg to
about 2 mg, from about 0.2 mg to about 1 mg, or from about 0.2 mg
to about 0.8 mg of the Notch inhibitor can be administered locally
to the inner ear of a mammal In one embodiment, 0.5 mg of Notch
inhibitor is administered locally to the inner ear of a mammal In
certain other embodiments, from about 0.05 mg to about 2 mg, from
about 0.2 mg to about 2 mg, from about 0.05 mg to about 1.5 mg,
from about 0.15 mg to about 1.5 mg, from about 0.4 mg to about 1
mg, or from about 0.5 mg to about 0.8 mg of Notch inhibitor can be
administered locally to the inner ear of a mammal In certain
embodiments, about 0.7 mg Notch inhibitor is administered locally
to the inner ear of a mammal
[0151] In certain embodiments, Atoh1 activity is increased by
administering Atoh1 protein or an Atoh1 activator in the inner ear
of a recipient to give, for example, a final concentration of
greater than about 30 .mu.M, for example, in the range of about 30
.mu.M to about 1000 .mu.M. In certain embodiments, the Atohlprotein
or Atoh1 activator can be administered in an amount sufficient to
give a final concentration of greater than about 30 .mu.M. For
example, the Atoh1 protein or Atoh1 activator may be administered
in an amount sufficient to give a final concentration in the range
from about 30 .mu.M to about 1000 .mu.M, 50 .mu.M to about 1000
.mu.M, 80 .mu.M to about 1000 .mu.M, about 100 .mu.M to about 1000
.mu.M, about 150 .mu.M to about 1000 .mu.M, from about 200 .mu.M to
about 800 .mu.M, or from about 200 .mu.M to about 600 .mu.M.
[0152] In other embodiments, Atoh1 protein or a Atoh1 activator is
administered at a dose from about 0.025 mg to about 4 mg, from
about 0.035 mg to about 2 mg, from about 0.05 mg to about 2 mg,
from about 0.1 mg to about 2 mg, from about 0.2 mg to about 1 mg,
or from about 0.2 mg to about 0.8 mg of the Atoh1 protein or Atoh1
activator can be administered locally to the inner ear of a mammal
In one embodiment, 0.5 mg of Atoh1 protein or Atoh1 activator is
administered locally to the inner ear. In certain other
embodiments, from about 0.05 mg to about 2 mg, from about 0.2 mg to
about 2 mg, from about 0.05 mg to about 1.5 mg, from about 0.15 mg
to about 1.5 mg, from about 0.4 mg to about 1 mg, or from about 0.5
mg to about 0.8 mg of Atoh1 protein or Atoh1 activator can be
administered locally to the inner ear of a mammal.
Delivery of DNA
[0153] In some aspects, the activity of c-myc, Notch or Atoh1 can
be increased in a target cell using expression constructs known in
the art, e.g., naked DNA constructs, DNA vector based constructs,
and/or viral vector and/or viral based constructs to express
nucleic acids encoding a desired c-myc, Notch or Atoh1 protein. In
certain embodiments, a single DNA construct expressing c-myc and
Notch or NICD as two separate genes can be delivered into the inner
ear of a subject. In certain embodiments, a single DNA construct
expressing c-myc and Notch or NICD and Atoh1 as three separate
genes can be delivered into the inner ear of a subject.
[0154] Exemplary expression constructs can be formulated as a
pharmaceutical composition, e.g., for administration to a
subject.
[0155] DNA constructs and the therapeutic use of such constructs
are well known to those of skill in the art (see, e.g., Chiarella
et al. (2008) RECENT PATENTS ANTI-INFECT. DRUG DISC. 3:93-101; Gray
et al. (2008) EXPERT OPIN. BIOL. THER. 8:911-922; Melman et al.
(2008) HUM. GENE THER. 17:1165-1176). Naked DNA constructs
typically include one or more therapeutic nucleic acids (e.g., DNA
encoding c-myc and/or Notch) and a promoter sequence. A naked DNA
construct can be a DNA vector, commonly referred to as pDNA. Naked
DNA typically do not integrate into chromosomal DNA. Generally,
naked DNA constructs do not require, or are not used in conjunction
with, the presence of lipids, polymers, or viral proteins. Such
constructs may also include one or more of the non-therapeutic
components described herein.
[0156] DNA vectors are known in the art and typically are circular
double stranded DNA molecules. DNA vectors usually range in size
from three to five kilo-base pairs (e.g., including inserted
therapeutic nucleic acids). Like naked DNA, DNA vectors can be used
to deliver and express one or more therapeutic proteins in target
cells. DNA vectors do not integrate into chromosomal DNA.
[0157] Generally, DNA vectors include at least one promoter
sequence that allows for replication in a target cell. Uptake of a
DNA vector may be facilitated by combining the DNA vector with, for
example, a cationic lipid, and forming a DNA complex. Typically,
viral vectors are double stranded circular DNA molecules that are
derived from a virus. Viral vectors typically are larger in size
than naked DNA and DNA vector constructs and have a greater
capacity for the introduction of foreign (i.e., not virally
encoded) genes. Like naked DNA and DNA vectors, viral vectors can
be used to deliver and express one or more therapeutic nucleic
acids in target cells. Unlike naked DNA and DNA vectors, certain
viral vectors stably incorporate themselves into chromosomal DNA.
Typically, viral vectors include at least one promoter sequence
that allows for replication of one or more vector encoded nucleic
acids, e.g., a therapeutic nucleic acid, in a host cell. Viral
vectors may optionally include one or more non-therapeutic
components described herein. Advantageously, uptake of a viral
vector into a target cell does not require additional components,
e.g., cationic lipids. Rather, viral vectors transfect or infect
cells directly upon contact with a target cell.
[0158] The approaches described herein include the use of
retroviral vectors, adenovirus-derived vectors, and/or
adeno-associated viral vectors as recombinant gene delivery systems
for the transfer of exogenous genes in vivo, particularly into
humans. Protocols for producing recombinant retroviruses and for
infecting cells in vitro or in vivo with such viruses can be found
in Current Protocols in Molecular Biology, Ausubel, F. M. et al.
(eds.) Greene Publishing Associates, (1989), Sections 9.10-9.14,
and other standard laboratory manuals.
[0159] Viruses that are used as transduction agents of DNA vectors
and viral vectors such as adenoviruses, retroviruses, and
lentiviruses may be used in practicing the present invention.
Illustrative retroviruses include, but are not limited to: Moloney
murine leukemia virus (M-MuLV), Moloney murine sarcoma virus
(MoMSV), Harvey murine sarcoma virus (HaMuSV), murine mammary tumor
virus (MuMTV), gibbon ape leukemia virus (GaLV), feline leukemia
virus (FLV), spumavirus, Friend murine leukemia virus, Murine Stem
Cell Virus (MSCV) and Rous Sarcoma Virus (RSV)) and lentivirus. As
used herein, the term "lentivirus" refers to a group (or genus) of
complex retroviruses. Illustrative lentiviruses include, but are
not limited to: HIV (human immunodeficiency virus; including HIV
type 1, and HIV type 2); visna-maedi virus (VMV) virus; the caprine
arthritis-encephalitis virus (CAEV); equine infectious anemia virus
(EIAV); feline immunodeficiency virus (FIV); bovine immune
deficiency virus (BIV); and simian immunodeficiency virus
(SIV).
[0160] In certain embodiments, an adenovirus can be used in
accordance with the methods described herein. The genome of an
adenovirus can be manipulated such that it encodes and expresses a
gene product of interest but is inactivated in terms of its ability
to replicate in a normal lytic viral life cycle. Suitable
adenoviral vectors derived from the adenovirus strain Ad type 5
d1324 or other strains of adenovirus (e.g., Ad2, Ad3, Ad7 etc.) are
known to those skilled in the art. Recombinant adenoviruses can be
advantageous in certain circumstances in that they are not capable
of infecting nondividing cells and can be used to infect a wide
variety of cell types, including epithelial cells Furthermore, the
virus particle is relatively stable and amenable to purification
and concentration, and as above, can be modified so as to affect
the spectrum of infectivity. Additionally, introduced adenoviral
DNA (and foreign DNA contained therein) is not integrated into the
genome of a host cell but remains episomal, thereby avoiding
potential problems that can occur as a result of insertional
mutagenesis in situ where introduced DNA becomes integrated into
the host genome (e.g., retroviral DNA). Moreover, the carrying
capacity of the adenoviral genome for foreign DNA is large (up to 8
kilobases) relative to other gene delivery vectors.
[0161] Adeno-associated virus is a naturally occurring defective
virus that requires another virus, such as an adenovirus or a
herpes virus, as a helper virus for efficient replication and a
productive life cycle. It is also one of the few viruses that may
integrate its DNA into non-dividing cells, and exhibits a high
frequency of stable integration.
[0162] In various embodiments, one or more viral vectors that
expresses a therapeutic transgene or transgenes encoding a
polypeptide or polypeptides of the invention (e.g., Atoh1, Notch,
c-myc) is administered by direct injection to a cell, tissue, or
organ of a subject, in vivo.
[0163] In various other embodiments, cells are transduced in vitro
or ex vivo with such a vector encapsulated in a virus, and
optionally expanded ex vivo. The transduced cells are then
administered to the inner ear of a subject. Cells suitable for
transduction include, but are not limited to stem cells, progenitor
cells, and differentiated cells. In certain embodiments, the
transduced cells are embryonic stem cells, bone marrow stem cells,
umbilical cord stem cells, placental stem cells, mesenchymal stem
cells, neural stem cells, liver stem cells, pancreatic stem cells,
cardiac stem cells, kidney stem cells, hematopoietic stem cells,
inner ear hair cells, iPS cells, inner ear supporting cells,
cochlear cells, or utricular cells.
[0164] In particular embodiments, host cells transduced with viral
vector of the invention that expresses one or more polypeptides,
are administered to a subject to treat and/or prevent an auditory
disease, disorder, or condition. Other methods relating to the use
of viral vectors, which may be utilized according to certain
embodiments of the present invention, can be found in, e.g., Kay
(1997) CHEST 111(6 Supp.):138S-142S; Ferry et al. (1998) HUM. GENE
THER. 9:1975-81; Shiratory et al. (1999) LIVER 19:265-74; Oka et
al. (2000) CURR. OPIN. LIPIDOL. 11:179-86; Thule et al. (2000) Gene
Ther. 7: 1744-52; Yang (1992) CRIT. REV. BIOTECHNOL. 12:335-56; Alt
(1995) J. HEPATOL. 23:746-58; Brody et al. (1994) ANN. N.Y. ACAD.
SCI. 716:90-101; Strayer. (1999) EXPERT OPIN. INVESTIG. DRUGS
8:2159-2172; Smith-Arica et al. (2001) CURR. CARDIOL. REP. 3:43-49;
and Lee et al. (2000) NATURE 408:483-8.
[0165] In some embodiments of the invention, it may be desirable to
use a cell, cell type, cell lineage or tissue specific expression
control sequence to achieve cell type specific, lineage specific,
or tissue specific expression of a desired polynucleotide sequence,
for example, to express a particular nucleic acid encoding a
polypeptide in only a subset of cell types, cell lineages, or
tissues, or during specific stages of development. Illustrative
examples of cell, cell type, cell lineage or tissue specific
expression control sequences include, but are not limited to: an
Atoh1 enhancer for all hair cells (see, e.g., FIG. 24); a Pou4f3
promoter for all hair cells (see, e.g., FIG. 25); a Myo7a promoter
for all hair cells (see, e.g., FIG. 26); a HesS promoter for
vestibular supporting cells and cochlear inner phalangeal cells,
Deiters cells and Pillar cells (see, e.g., FIG. 27); and GFAP
promoter for vestibular supporting cells and cochlear inner
phalangeal cells, Deiters cells and Pillar cells (see, e.g., FIG.
28).
[0166] Certain embodiments of the invention provide conditional
expression of a polynucleotide of interest. For example, expression
is controlled by subjecting a cell, tissue, organism, etc., to a
treatment or condition that causes the polynucleotide to be
expressed or that causes an increase or decrease in expression of
the polynucleotide encoded by the polynucleotide of interest.
Illustrative examples of inducible promoters/systems include, but
are not limited to, steroid-inducible promoters such as promoters
for genes encoding glucocorticoid or estrogen receptors (inducible
by treatment with the corresponding hormone), metallothionine
promoter (inducible by treatment with various heavy metals), MX-1
promoter (inducible by interferon), the "GeneSwitch"
mifepristone-regulatable system (Sirin et al., 2003, GENE, 323:67),
the cumate inducible gene switch (WO 2002/088346),
tetracycline-dependent regulatory systems, etc.
[0167] Conditional expression can also be achieved by using a site
specific DNA recombinase. According to certain embodiments of the
invention the vector comprises at least one (typically two) site(s)
for recombination mediated by a site specific recombinase. As used
herein, the terms "recombinase" or "site specific recombinase"
include excisive or integrative proteins, enzymes, co-factors or
associated proteins that are involved in recombination reactions
involving one or more recombination sites (e.g., two, three, four,
five, seven, ten, twelve, fifteen, twenty, thirty, fifty, etc.),
which may be wild-type proteins (see Landy (1993) CURRENT OPINION
IN BIOTECHNOLOGY 3:699-707), or mutants, derivatives (e.g., fusion
proteins containing the recombination protein sequences or
fragments thereof), fragments, and variants thereof Illustrative
examples of recombinases suitable for use in particular embodiments
of the present invention include, but are not limited to: Cre, Int,
IHF, Xis, Flp, Fis, Hin, Gin, OC31 , Cin, Tn3 resolvase, TndX,
XerC, XerD, TnpX, Hjc, Gin, SpCCE1. and ParA.
[0168] The vectors may comprise one or more recombination sites for
any of a wide variety of site specific recombinases. It is to be
understood that the target site for a site specific recombinase is
in addition to any site(s) required for integration of a vector
(e.g., a retroviral vector or lentiviral vector).
[0169] In certain embodiments, vectors comprise a selection gene,
also termed a selectable marker. Typical selection genes encode
proteins that (a) confer resistance to antibiotics or other toxins,
e.g., ampicillin, neomycin, hygromycin, methotrexate, Zeocin,
Blastocidin, or tetracycline, (b) complement auxotrophic
deficiencies, or (c) supply critical nutrients not available from
complex media, e.g., the gene encoding D-alanine racemase for
Bacilli. Any number of selection systems may be used to recover
transformed cell lines. These include, but are not limited to, the
herpes simplex virus thymidine kinase (Wigler et al., (1977) CELL
11:223-232) and adenine phosphoribosyltransferase (Lowy et al.,
(1990) CELL 22:817-823) genes which can be employed in tk- or
aprt-cells, respectively.
[0170] All the molecular biological techniques required to generate
an expression construct described herein are standard techniques
that will be appreciated by one of skill in the art.
[0171] In certain embodiments, DNA delivery may occur auricularly,
parenterally, intravenously, intramuscularly, or even
intraperitoneally as described, for example, in U.S. Pat. Nos.
5,543,158; 5,641,515; and 5,399,363 (each specifically incorporated
herein by reference in its entirety). Solutions of the active
compounds as free base or pharmacologically acceptable salts may be
prepared in water suitably mixed with a surfactant, such as
hydroxypropylcellulose. Dispersions may also be prepared in
glycerol, liquid polyethylene glycols, and mixtures thereof and in
oils. Under ordinary conditions of storage and use, these
preparations contain a preservative to prevent the growth of
microorganisms.
[0172] In certain embodiments, DNA delivery may occur by use of
liposomes, nanocapsules, microparticles, microspheres, lipid
particles, vesicles, optionally mixing with cell penetrating
polypeptides, and the like, for the introduction of the
compositions of the present invention into suitable host cells. In
particular, the compositions of the present invention may be
formulated for delivery either encapsulated in a lipid particle, a
liposome, a vesicle, a nanosphere, a nanoparticle or the like. The
formulation and use of such delivery vehicles can be carried out
using known and conventional techniques.
[0173] Exemplary formulations for ex vivo DNA delivery may also
include the use of various transfection agents known in the art,
such as calcium phosphate, electroporation, heat shock and various
liposome formulations (i.e., lipid-mediated transfection).
Particular embodiments of the invention may comprise other
formulations, such as those that are well known in the
pharmaceutical art, and are described, for example, in Remington:
The Science and Practice of Pharmacy, 20th Edition. Baltimore, Md.:
Lippincott Williams & Wilkins, 2000.
Duration of Delivery
[0174] The duration of c-myc, Notch and Atoh1 activation can be
varied to achieve a desired result. For example, it may be
beneficial to expose a target cell to a c-myc protein or c-myc
activator and a Notch protein, NICD protein, or a Notch activator
for one to six days, one week, two weeks, three weeks, one month,
three months, six months, nine months, one year, two years or more.
Alternatively, when c-myc is increased by constitutive activation
(e.g., using an adenovirus to overexpress c-myc), the duration of
increased c-myc activity can be controlled by administering a c-myc
inhibitor following administration of a myc protein or a myc
activator. Inhibiting c-myc activity after a period of increased
c-myc activity can be used to control proliferation, promote cell
survival, and avoid tumorigenesis.
[0175] Similarly, the duration of increased Notch activity can be
controlled by administering a Notch inhibitor, as discussed above,
following administration of a Notch protein, NICD protein, or a
Notch activator.
Route of Administration and Formulation
[0176] The route of administration will vary depending on the
disease being treated. Hair cell loss, sensorineural hearing loss,
and vestibular disorders can be treated using direct therapy using
systemic administration and/or local administration. In certain
embodiments, the route of administration can be determined by a
subject's health care provider or clinician, for example following
an evaluation of the subject.
[0177] The invention provides (i) a composition for use in
proliferating or regenerating a cochlear or a utricular hair cell,
(ii) a composition for use in proliferating or regenerating a
cochlear or a utricular supporting cell, (iii) a composition for
use in reducing the loss of, maintaining, or promoting hearing in a
subject, and (iv) a composition for use in reducing the loss of,
maintaining, or promoting vestibular function in a subject.
Accordingly, the invention provides a first composition comprising
an agent, for example, each of the agents discussed hereinabove,
for example, an agent that increases c-myc activity and/or an agent
that increases Notch activity within a hair or supporting cell,
either alone or in combination with a pharmaceutically acceptable
carrier for use in each of the foregoing approaches. In addition,
the invention provides a second composition comprising an agent,
for each of the agents discussed hereinabove, for example, an agent
that reduces or inhibits c-myc activity and/or an agent that
reduces or inhibits Notch activity within a hair or supporting
cell, either alone or in combination with in a pharmaceutically
acceptable carrier for use in each of the foregoing approaches.
When supporting cells are regenerated, the invention provides a
third composition comprising an agent, for example, an agent for
increasing Atoh1 activity, to induce transdifferentiation of a
proliferated supporting cell into a hair cell.
[0178] In certain embodiments, a c-myc protein or c-myc activator
and a Notch protein, NICD protein or Notch activator can be
formulated as a pharmaceutical composition containing the
appropriate carriers and/or excipients.
[0179] The c-myc protein or activator and/or the Notch protein,
NICD protein, or Notch activator, and/or the Atoh1 protein or
activator can be solubilized in a carrier, for example, a
viscoelastic carrier, that is introduced locally into the inner
ear. In other embodiments, the c-myc protein or activator and/or
the Notch protein, NICD protein, or Notch activator, and/or Atoh1
protein or activator can be solubilized in a liposome or
microsphere. Methods for delivery of a drug or combination of drugs
in liposomes and/or microspheres are well-known in the art.
[0180] In addition, it is contemplated that the c-myc protein or
activator and/or the Notch protein, NICD protein, or Notch
activator, and/or Atoh1 protein or activator can be formulated so
as to permit release of one or more proteins and/or activators over
a prolonged period of time. A release system can include a matrix
of a biodegradable material or a material, which releases the
incorporated active agents. The active agents can be homogeneously
or heterogeneously distributed within a release system. A variety
of release systems may be useful in the practice of the invention,
however, the choice of the appropriate system will depend upon the
rate of release required by a particular drug regime. Both
non-degradable and degradable release systems can be used. Suitable
release systems include polymers and polymeric matrices,
non-polymeric matrices, or inorganic and organic excipients and
diluents such as, but not limited to, calcium carbonate and sugar
(for example, trehalose). Release systems may be natural or
synthetic.
[0181] In certain embodiments, the agents can be administered to a
subject, e.g., a subject identified as being in need of treatment
for hair cell loss, using a systemic route of administration.
Systemic routes of administration can include, but are not limited
to, parenteral routes of administration, e.g., intravenous
injection, intramuscular injection, and intraperitoneal injection;
enteral routes of administration, e.g., administration by the oral
route, lozenges, compressed tablets, pills, tablets, capsules,
drops (e.g., ear drops), syrups, suspensions and emulsions; rectal
administration, e.g., a rectal suppository or enema; a vaginal
suppository; a urethral suppository; transdermal routes of
administration; and inhalation (e.g., nasal sprays).
[0182] Alternatively or in addition, the agents can be administered
to a subject, e.g., a subject identified as being in need of
treatment for hair cell loss, using a local route of
administration. Such local routes of administration include
administering one or more compounds into the ear of a subject
and/or the inner ear of a subject, for example, by injection and/or
using a pump.
[0183] In certain embodiments, the agents may be injected into the
ear (e.g., auricular administration), such as into the luminae of
the cochlea (e.g., the Scala media, Sc vestibulae, and Sc tympani).
For example, the agents can be administered by intratympanic
injection (e.g., into the middle ear), and/or injections into the
outer, middle, and/or inner ear. Such methods are routinely used in
the art, for example, for the administration of steroids and
antibiotics into human ears. Injection can be, for example, through
the round window of the ear or through the cochlea capsule.
[0184] In other embodiments, the agents can be delivered via
nanoparticles, for example, protein-coated nanoparticles.
Nanoparticles can be targeted to cells of interest based on
cell-type specific receptor affinity for ligands coating the
nanoparticles. The dosage of the agent can be modulated by
regulating the number of nanoparticles administered per dose.
[0185] Alternatively, the agent may be administered to the inner
ear using a catheter or pump. A catheter or pump can, for example,
direct the agent into the cochlea luminae or the round window of
the ear. Exemplary drug delivery systems suitable for administering
one or more compounds into an ear, e.g., a human ear, are described
in U.S. Patent Publication No. 2006/0030837 and U.S. Pat. No.
7,206,639. In certain embodiments, a catheter or pump can be
positioned, e.g., in the ear (e.g., the outer, middle, and/or inner
ear) of a subject during a surgical procedure.
[0186] Alternatively or in addition, the agents can be delivered in
combination with a mechanical device such as a cochlea implant or a
hearing aid, which is worn in the outer ear. An exemplary cochlea
implant that is suitable for use with the present invention is
described in U.S. Patent Publication No. 2007/0093878.
[0187] In certain embodiments, the modes of administration
described above may be combined in any order and can be
simultaneous or interspersed. For example, the agents may be
administered to a subject simultaneously or sequentially. It will
be appreciated that when administered simultaneously, the agents
may be in the same pharmaceutically acceptable carrier (e.g.,
solubilized in the same viscoelastic carrier that is introduced
into the inner ear) or the two agents may be dissolved or dispersed
in separate pharmaceutical carriers, which are administered at the
same time. Alternatively, the agents may be provided in separate
dosage forms and administered sequentially.
[0188] Alternatively or in addition, the agents may be administered
according to any of the Food and Drug Administration approved
methods, for example, as described in CDER Data Standards Manual,
version number 004 (which is available at
fda.give/cder/dsm/DRG/drg00301.htm).
3. Delivery of Agents to Hair Cells and Supporting Cells Ex
Vivo
[0189] It is understood that the concepts for delivering agents of
interest to hair cells and supporting cells in vivo can also apply
to the delivery of the agents of interest to hair cells and
supporting cells ex vivo. The hair cells and supporting cells can
be harvested and cultured using techniques known and used in the
art. The agents (protein expression vectors, activators and
inhibitors (for example, as discussed above)) can then be contacted
with the cultured hair cells or supporting cells to induce the
cells to reenter the cell cycle, and proliferate. Thereafter, once
the cells have proliferated, the c-myc and Notch activities can be
inhibited using appropriate inhibitors, for example, those
discussed above. The resulting hair cells can then be maintained in
culture for any number of uses, including, for example, to study
the biological, biophysical, physiological and pharmacological
characteristics of hair cells and/or supporting cells.
Alternatively, the resulting hair cells can then be implanted in to
the inner ear of a recipient using standard surgical
procedures.
[0190] In certain embodiments, suitable cells can be derived from a
mammal, such as a human, mouse, rat, pig, sheep, goat, or non-human
primate. In certain embodiments, the cells can be harvested from
the inner ear of a subject, and cells can be obtained from the
cochlea organ of Corti, the modiolus (center) of the cochlea, the
spiral ganglion of the cochlea, the vestibular sensory epithelia of
the saccular macula, the utricular macula, or the cristae of the
semicircular canals. Alternatively or in addition, methods include
obtaining tissue from the inner ear of the animal, where the tissue
includes at least a portion of the utricular maculae.
[0191] Tissue isolated from a subject can be suspended in a neutral
buffer, such as phosphate buffered saline (PBS), and subsequently
exposed to a tissue-digesting enzyme (e.g., trypsin, leupeptin,
chymotrypsin, and the like) or a combination of enzymes, or a
mechanical (e.g., physical) force, such as trituration, to break
the tissue into smaller pieces. Alternatively, or in addition, both
mechanisms of tissue disruption can be used. For example, the
tissue can be incubated in about 0.05% enzyme (e.g., about 0.001%,
0.01%, 0.03%, 0.07%, or 1.0% of enzyme) for about 5, 10, 15, 20, or
30 minutes, and following incubation, the cells can be mechanically
disrupted. The disrupted tissue can be passed through a device,
such as a filter or bore pipette, that separates a stem cell or
progenitor cell from a differentiated cell or cellular debris. The
separation of the cells can include the passage of cells through a
series of filters having progressively smaller pore size. For
example, the filter pore size can range from about 80 .mu.m or
less, about 70 .mu.m or less, about 60 .mu.m or less, about 50
.mu.m or less, about 40 .mu.m or less, about 30 .mu.m or less,
about 35 .mu.m or less, or about 20 .mu.m or less.
[0192] Partially and/or fully differentiated cells, e.g., generated
by the methods described above, can be maintained in culture for a
variety of uses, including, for example, to study the biological,
biophysical, physiological and pharmacological characteristics of
hair cells and/or supporting cells. Cell cultures can be
established using inner ear cells from subjects with hearing loss
and/or loss in vestibular function to develop potential treatments
(e.g., to screen for drugs effective in treating the hearing loss
and/or loss in vestibular function). Further, the methods of the
present invention can be used in combination with induced
pluripotent stem (iPS) cell technology to establish cell lines
(e.g., hair cell lines and/or supporting cell lines). For example,
fibroblasts from a subject with hearing loss can be induced to form
iPS cells using known techniques (see, for example, Oshima et al.
(2010) CELL 141(4):704-716). However, because the numbers of cells
generated using iPS cell technology is limited, the methods
provided herein can be used in combination with iPS cell technology
to produce sufficient numbers of cells to establish cell lines
(e.g., hair cell lines and/or supporting cell lines).
[0193] Partially and/or fully differentiated cells, e.g., generated
by the methods described above, can be transplanted or implanted,
such as in the form of a cell suspension, into the ear by
injection, such as into the luminae of the cochlea. Injection can
be, for example, through the round window of the ear or through the
bony capsule surrounding the cochlea. The cells can be injected
through the round window into the auditory nerve trunk in the
internal auditory meatus or into the scala tympani. In certain
embodiments, the cells described herein can be used in a cochlea
implant, for example, as described in U.S. Patent Publication No.
2007/0093878.
[0194] To improve the ability of transplanted or implanted cells to
engraft, cells can be modified prior to differentiation. For
example, the cells can be engineered to overexpress one or more
anti-apoptotic genes. The Fak tyrosine kinase or Akt genes are
candidate anti-apoptotic genes that can be used for this purpose;
overexpression of FAK or Akt can prevent cell death in spiral
ganglion cells and encourage engraftment when transplanted into
another tissue, such as an explanted organ of Corti (see, for
example, Mangi et al., (2003) NAT. MED. 9:1195-201). Neural
progenitor cells overexpressing .alpha..sub.v.beta..sub.3 integrin
may have an enhanced ability to extend neurites into a tissue
explant, as the integrin has been shown to mediate neurite
extension from spiral ganglion neurons on laminin substrates
(Aletsee et al., (2001) AUDIOL. NEUROOTOL. 6:57-65). In another
example, ephrinB2 and ephrinB3 expression can be altered, such as
by silencing with RNAi or overexpression with an exogenously
expressed cDNA, to modify EphA4 signaling events. Spiral ganglion
neurons have been shown to be guided by signals from EphA4 that are
mediated by cell surface expression of ephrin-B2 and -B3 (Brors et
al., (2003) J. COMP. NEUROL. 462:90-100). Inactivation of this
guidance signal may enhance the number of neurons that reach their
target in an adult inner ear. Exogenous factors such as the
neurotrophins BDNF and NT3, and LIF can be added to tissue
transplants to enhance the extension of neurites and their growth
towards a target tissue in vivo and in ex vivo tissue cultures.
Neurite extension of sensory neurons can be enhanced by the
addition of neurotrophins (BDNF, NT3) and LIF (Gillespie et al.
(2010) NEUROREPORT 12:275-279).
4. Measurement of c-myc, Notch or Atoh1 Activity in Target
Cells
[0195] The methods and compositions described herein can be used to
induce cells, e.g., adult mammalian inner ear cells, to reenter the
cell cycle and proliferate. For example, the number of hair cells
can be increased about 2-, 3-, 4-, 6-, 8-, or 10-fold, or more, as
compared to the number of hair cells before treatment. The hair
cell can be induced to reenter the cell cycle in vivo or ex vivo.
It is contemplated that using these approaches it may be possible
to improve the hearing of a recipient. For example, using the
methods and compositions described herein, it may be possible to
improve the hearing of a recipient by at least about 5, 10, 15, 20,
40, 60, 80, or 90% relative to the hearing prior to the treatment.
Tests of auditory or vestibular function also can be performed to
measure hearing improvement.
[0196] Cells that have been contacted with (i) a c-myc protein or
c-myc activator and/or (ii) a Notch protein, NICD protein or Notch
activator, can be assayed for markers indicative of cell cycle
reentry and proliferation. In one example, a cell can be assayed
for incorporation of EdU (5-ethynyl-2'-deoxyuridine) followed
sequentially by BrdU (5-bromo-2'-deoxyuridine) by using, for
example, an anti-EdU antibody and an anti-BrdU antibody. Labelling
by EdU and/or BrdU is indicative of cell proliferation. In
addition, double labeling of EdU and BrdU can be used to
demonstrate that a cell has undergone division at least two times.
Alternatively or in addition, a cell can be assayed for the
presence of phosphorylated histone H3 (Ph3) or aurora B, which are
indicative of a cell that has reentered the cell cycle and is
undergoing metaphase and cytokinesis.
[0197] Cell markers can also be used to determine whether a target
cell, e.g., a hair cell or a supporting cell, has entered the cell
cycle. Exemplary markers indicative of hair cells include Myo7a,
Myo6, Prestin, Lhx3, Dner, espin, parvalbumin, and calretinin.
Exemplary markers indicative of supporting cells include Sox2,
S100a1, Prox1, Rps6, and Jag1. Double labeling of a cell cycle
and/or proliferation marker and a cell-type molecule can be used to
determine which cells have reentered the cell cycle and are
proliferating.
[0198] In addition, neuronal markers, e.g., acetylated tubulin,
neurofilament and CtBP2, can be used to detect neuronal structure,
to determine whether proliferating hair cells are in contact with
neurons. The presence of neuronal markers adjacent to or in contact
with hair cells suggests that newly-generated hair cells have
formed synapses with neurons (e.g., ganglion neurons) and that the
hair cells are differentiated.
[0199] Where appropriate, following treatment, the subject, for
example, a human subject, can be tested for an improvement in
hearing or in other symptoms related to inner ear disorders.
[0200] Methods for measuring hearing are well-known and include
pure tone audiometry, air conduction, auditory brainstem response
(ABR) and bone conduction tests. These exams measure the limits of
loudness (intensity) and pitch (frequency) that a human can hear.
Hearing tests in humans include behavioral observation audiometry
(for infants to seven months), visual reinforcement orientation
audiometry (for children 7 months to 3 years) and play audiometry
for children older than 3 years. Oto-acoustic emission testing can
be used to test the functioning of the cochlea hair cells, and
electro-cochleography provides information about the functioning of
the cochlea and the first part of the nerve pathway to the brain.
In certain embodiments, treatment can be continued with or without
modification or can be stopped.
[0201] Throughout the description, where compositions are described
as having, including, or comprising specific components, or where
processes are described as having, including, or comprising
specific process steps, it is contemplated that compositions of the
present invention also consist essentially of, or consist of, the
recited components, and that the processes of the present invention
also consist essentially of, or consist of, the recited processing
steps. Further, it should be understood that the order of steps or
order for performing certain actions are immaterial so long as the
invention remains operable. Moreover, two or more steps or actions
may be conducted simultaneously.
EXAMPLES
[0202] The invention is further illustrated by the following
examples, which are provided for illustrative purposes only, and
should not be construed as limiting the scope or content of the
invention in any way.
Example 1
In Vivo Induction of Cell Cycle Reentry in Adult Cochlear Cells Via
C-Myc and Notch
[0203] This example demonstrates that providing c-myc and Notch to
cells of the inner ear of an adult animal can induce cell cycle
reentry and cell proliferation among differentiated cochlear hair
and supporting cells.
[0204] Adult mice aged between 1 and 15 months were used to
investigate the potential for c-myc and Notch to induce cell cycle
reentry, proliferation, differentiation, and survival among
cochlear hair and supporting cells. In separate experiments, the
mice used were either wild type (WT) background mice or mice
harboring a LoxP-flanked NICD cassette (NICD.sup.flox/flox)
susceptible to Cre-mediated recombination resulting in activation
of NICD expression. The NICD cassette encoded (from 5' to 3') an
intracellular fragment of mouse Notch1 (amino acids 1749-2293,
lacking the C-terminal PEST domain, see Murthaugh et al. (2003)
PROC. NATL. ACAD. SCI. U.S.A. 100(25):14920-14925.) Mice were
anaesthetized and cochleostomy was performed to allow injection of
adenovirus. Virus was injected via the scala media, facilitating
infection of hair and supporting cells within the cochlear sensory
epithelium. A mixture of adenovirus carrying a combination of
either human c-myc (Ad-Myc) and CRE-GFP (Ad-Cre-GFP) expression
cassettes or c-myc and NICD (Ad-NICD) expression cassettes was
injected into the cochlea of either NICD.sup.flox/flox or WT mice,
respectively. One ear per mouse was injected, while the other ear
served as an uninjected control. An additional control was used in
which cochlea were injected with Ad-Cre-GFP alone. Ad-Myc induced
myc overexpression, Ad-NICD induced NICD overexpression, and
Ad-CRE-GFP induced overexpression of CRE-GFP, recombination at loci
flanked by LoxP sequences, and--in the case of NICD.sup.flox/flox
mice--NICD overexpression. Virus titered at 2.times.10.sup.12
plaque-forming units (pfu) was mixed in equal parts, and a total of
0.6 .mu.L virus was injected per animal. Following viral injection,
5-bromo-2-deoxyuridine (BrdU) was injected daily between 1 and 5
days.
[0205] Mice were sacrificed and cochlea were harvested at either 4,
8, 12, 35, or 60 days post-viral injection. Cochlea were dissected,
fixed, and decalcified prior to whole mount immunostaining. Hair
cells were identified via labeling with antibodies directed against
Myo7a and espin. Supporting cells were identified via labeling with
antibodies directed against Sox2. Cell cycle reentry and
proliferation were assessed via labeling antibodies directed
against BrdU. Nuclear labeling was achieved via DAPI exposure.
[0206] Cells of the cochlear epithelium exposed to c-myc and NICD
via viral injection were analyzed to determine whether cell cycle
reentry and proliferation occurred. Cochlea from NICD.sup.flox/flox
mice injected with Ad-Cre-GFP and Ad-Myc followed by BrdU
administration were harvested at 4, 8, or 12 days post-virus
injection and immunostained (FIG. 7). At all time points analyzed,
immunostained sections revealed the presence of cycling hair cells
as determined by BrdU+/Myo7a+ (FIG. 7A, B, E, K, L, O, P, Q, T,
closed arrows) staining. At 4 days post-injection, BrdU+/Sox2+
(FIG. 7A, B, E, open arrows) staining showed that supporting cells
also reentered the cell cycle in this population. These findings
demonstrate that cochlear hair cells and supporting cells can be
induced to reenter the cell cycle following exposure to c-myc and
NICD. BrdU-labeled hair cell doublets (assumed to be daughter cells
derived from the same cell division) at 12 days post-virus
injection were observed, demonstrating that cells induced to
reenter the cell cycle following c-Myc and NICD exposure can
subsequently proliferate (FIG. 7, P-T, arrows). Furthermore, BrdU
staining in cochlear cells was not observed in uninjected control
ears at any time point (FIG. 7, F-J, showing 4 day time point).
These observations suggest that exposing differentiated cochlear
hair and supporting cells to increased c-myc and Notch activity
induces cell cycle reentry within these populations.
[0207] The in vivo cell survival of hair and supporting cells
induced to reenter the cell cycle at more distant time points after
viral injection was assessed. Cochlear tissue from
NICD.sup.flox/flox mice infected with Ad-Cre-GFP and Ad-Myc virus
and subsequently subjected to BrdU injection was harvested 35 days
post-virus injection and immunostained to assess cell cycle reentry
and survival of cycling hair and supporting cells. Analysis of
stained cochlea at this time point again revealed the presence of
proliferating hair and supporting cells (FIG. 8). Myo7a-positive
hair cells stained positive for BrdU in cochlear epithelia
subjected to BrdU labeling and harvested 35 days post-virus
injection were observed (FIG. 8, A-E, arrows). In the same animals,
BrdU-labeled Sox2-positive supporting cells were observed (FIG. 8,
K-O, open arrows). A dividing hair cell in which Sox2 is activated
by Notch is also shown (FIG. 8M, arrowhead). These observations
demonstrate that supporting cells and hair cells induced to reenter
the cell cycle following exposure to increased c-myc and Notch
activity can survive for at least 35 days in vivo. BrdU-labeled
hair cells displaying stereocilia following c-Myc and NICD virus
exposure at this time point were also observed (FIG. 8, F-J,
arrowhead in panel J). This finding demonstrates that hair cells
induced to reenter the cell cycle or their progeny retain physical
characteristics of differentiated hair cells.
[0208] In a similar set of experiments, a mixture of Ad-Myc and
Ad-NICD was injected into the scala media of WT mice followed by
daily administration of BrdU from one to five days. Cochlea were
harvested at time points between 2 and 35 days post-virus injection
and immunostained. Immunostaining with antibodies directed against
BrdU, Myo7a, and Sox2 antigens revealed the presence of
double-labeled hair (BrdU+/Myo7a+) and supporting (BrdU+/Sox2+)
cells in harvested cochlea. (Data not shown.) Accordingly, exposure
to increased c-myc and Notch activity in differentiated hair and
supporting cells of WT background also induces cell cycle reentry
and proliferation.
Example 2
In Vivo Induction of Cell Cycle Reentry in Cochlear Cells of Aced
Mice via C-Myc and Notch
[0209] The following example demonstrates that providing c-myc and
Notch to cells of the inner ear can also induce cell cycle reentry
and cell proliferation among differentiated cochlear hair and
supporting cells in aged animal subjects.
[0210] Ad-Myc and Ad-Cre-GFP were injected once into 17-month old
NICD.sup.flox/flox mouse cochlear scala media via cochleostomy and
the animals were harvested 15 days later. 0.3 .mu.l of a mixture of
an equal amount of Ad-Cre-GFP and Ad-Myc with a titer of
2.times.10.sup.12 was injected. BrdU (50 .mu.g/g body weight) was
also injected once per day for 15 days to label cycling cells. The
same protocol was used as a control, in which only Ad-Cre was
injected into the cochlea. Cochlear tissue harvested following BrdU
and virus injection demonstrated that cells of the aged mouse
cochlea underwent cell re-entry, as evidenced by the presence of
double-labeled hair (BrdU+/Myo7a+) and supporting (BrdU+/Sox2+;
FIG. 9, A-J; arrows identify double-labeled hair cells; arrowheads
identify double-labeled support cells). By contrast, no BrdU
labeling was observed in Sox2+ support or Myo7a+ hair cells in
17-month old NICD.sup.flox/flox control animals injected with
Ad-Cre alone and subjected to the same BrdU labeling time course
(FIG. 9K-O).
[0211] These results demonstrate that inner ear hair and support
cell proliferation can be achieved in aged mice, which suggest that
similar effects can be achieved in the aged human inner ear.
Example 3
Induction of Cell Cycle Reentry in Cultured Adult Cells Harvested
from Inner Ear Tissue of Various Mammals
[0212] The following example demonstrates that exposure to
increased c-myc and Notch activity supports cell cycle reentry and
proliferation of adult mouse, monkey and human hair and supporting
cells of the inner ear.
[0213] In order to investigate whether increased c-myc and Notch
activity induce cell cycle reentry and proliferation in human
cells, adult human cochlear and utricular tissue was collected.
Samples were derived from surgeries during which such tissue was
discarded. Cells were cultured in high glucose Dulbecco's modified
Eagle's medium and F 12 medium supplemented with N2 and B27 (Media
and supplements were from Invitrogen/GIBCO/BRL, Carlsbad, Calif.),
and 1% FBS was added.
[0214] A working viral titer of 10.sup.8 was used for 5 mL of
culture. Cultures of harvested tissue and transduced cultured cells
were contacted with a mixture of Ad-Myc and Ad-NICD, to elevate
cellular levels of c-myc and NICD. Following virus exposure, the
cycling cells were labeled via 3 .mu.g/ml BrdU administration to
the culture. As in the in vivo studies of transduced mouse tissue,
BrdU-labeled supporting (Sox2+) cells and at least one BrdU-labeled
hair (Myo7a+) cell in cultured human tissue (FIG. 10) were
identified.
[0215] BrdU+/Sox2+ supporting cells were identified in the cochlear
cultures (FIG. 10A, C, D, E) and utricular cultures (FIG. 10F, H,
I, J; all panels, open arrows). The cochlear cell cultures
contained virtually no hair cells, so no BrdU-labeled cochlear hair
cells were detected.
[0216] Exposure to virus resulted in few labeled hair cells in
utricular cultures, which may be the result of low infection rate
of hair cells by adenovirus. However, at least one BrdU+/Myo7a+
hair cell was identified in the human utricular cultures (FIG. 10F,
G, I, J; closed arrow).
[0217] Similar culture-based experiments were performed utilizing
harvested mouse utricle as the culture tissue. In the latter
experiments, tissue was derived from either NICD.sup.flox/flox or
WT mice and infected with a mixture of Ad-Myc/Ad-Cre-GFP or
Ad-Myc/Ad-NICD, respectively. Following viral transduction, the
cells were exposed to BrdU to label the cycling cells. BrdU was
added to a final concentration of 3 .mu.g/ml. As in the human
utricle culture-based experiments, BrdU-labeled hair and supporting
cells in the murine cultures were observed, demonstrating that
these cells can reenter the cell cycle upon exposure to increased
levels of Notch and c-myc activity. Examples of BrdU-labeled hair
and supporting cells were observed in these cultures, although the
majority of BrdU-labeled cells were supporting cells. Based on
these findings, it appears that increased c-myc and Notch activity
induces cell cycle reentry and proliferation in cultured hair and
supporting cells of the inner ear.
[0218] Additionally, experiments were performed in cultured cochlea
harvested from adult monkeys. The culture medium contained DMEM/F12
supplied with N2 and B27 without serum. Cultured cochlea were
exposed to an Ad-Myc/Ad-NICD mixture (final titer of 10.sup.9) for
16 hours, and the medium was replaced with fresh medium for 4 days.
EdU was added at the final concentration of 10 .mu.M. Cycling cells
were additionally labeled via EdU administration. Cultured cochlea
were fixed and stained for hair and supporting cell markers, as
well as EdU. Cycling Sox2+/EdU+ supporting cells were observed
following exposure to elevated levels of c-Myc and NICD (FIG. 11G,
H, J; arrowheads). Thus, this example demonstrates that cells of
the monkey inner ear can also be induced to proliferate following
exposure to elevated levels of c-Myc and Notch activity, suggesting
that the disclosed method can be applied to mammals other than
mice, e.g., primates. In cultured control monkey cochlea infected
with Ad-Cre in the presence of EdU, no EdU labeled cells were seen
(FIG. 11A-E), a demonstration that no cells underwent
proliferation. It is generally observed, both in cultured mouse and
monkey cochlea that surviving inner hair cells rarely re-entered
cell cycle, in contrast to mouse cochlea in vivo, in which inner
hair cells could readily be induced to proliferation by the
combination of c-Myc and NICD. It is likely that inner hair cells
require a higher concentration of Myc and NICD and more time to
proliferate, as the titer used in culture was not as high as in
vivo (10.sup.9 vs. 10.sup.12) and the tissues were harvested within
a short period of time after infection (4 days).
Example 4
Dose-Dependent Induction of Cell Proliferation in Cochlear Cell
Subpopulations
[0219] The following example illustrates that different populations
of cochlear hair cells are induced to proliferate upon varying
degrees of exposure to c-myc and Notch activity.
[0220] An osmotic pump (Alzet) was implanted in the back of adult
(45-day-old) doxycycline-inducible mice
(rtTa/tet-on-Myc/tet-on-NICD) with tubing inserted to the round
window niche to continuously dispense doxycycline (150 mg/ml in
DMSO) at a rate of 1 .mu.l per hour for 9 days, with concurrent EdU
administration (200 .mu.g/g body weight) by ip injection once daily
to label proliferating cells. Using this procedure, c-Myc and NICD
were activated in all cochlear cell types including supporting
cells and hair cells (data not shown). Due to the surgical
procedure, the cochlea in this sample lost all outer hair cells
with only supporting cells and some inner hair cells remaining.
Exposure of cochlear cells to this level of c-myc and NICD resulted
in proliferation of Sox2+ supporting cells (FIG. 12B, C, E;
arrows). By contrast Parv+ inner hair cells did not appear to
divide upon exposure to these levels of c-myc and NICD (FIG. 12A,
E; arrowheads).
[0221] Additionally, the rTta/Tet-on-myc/Tet-on-NICD mouse model
was used to examine induction of proliferation in outer hair cells.
rTta/Tet-on-myc/Tet-on-NICD mice were exposed to doxycycline
exposure for 12 days, accompanied by EdU administration once daily
during the 12 day period to label cycling cells, following the same
procedure described for FIG. 12. Tissue was then harvested and
stained for markers of hair cells (Esp) and supporting cells
(Sox2). In this case, EdU+/Esp+ proliferating outer hair cells were
observed following tissue harvest and staining (FIG. 13A, B, E;
arrows). No cell proliferation was observed in inner hair cells. As
this method activates c-Myc and NICD in all cochlear cell types,
this example demonstrates that exposure of outer hair cells to
elevated c-Myc and Notch activity can selectively induce outer hair
cell cycle reentry and proliferation. In the same cochlea, fewer
supporting cells (compared to outer hair cells) labeled with EdU
were also seen (data not shown), which is consistent with the
observation that outer hair cells have a greater capacity for cell
cycle re-entry following c-Myc and NICD activation. This sample
(FIG. 13) contrasts with the sample shown in FIG. 12 in that most
of the outer hair cells survived and showed heightened
proliferation capacity. It further indicates that after loss of
outer hair cells, supporting cells can be induced to proliferate
upon c-Myc and NICD activation (FIG. 12).
[0222] Taken together, these results indicate that while all
populations of cochlear hair and supporting cells can be induced to
differentiate upon exposure to elevated levels of c-myc and Notch
activity, different subpopulations within the cochlea respond to
different levels of c-myc and Notch exposure. For example, outer
hair cells respond to lower levels of c-myc and Notch stimulation
than supporting cells and inner hair cells. Supporting cells
respond to lower levels of c-myc and Notch stimulation than inner
hair cells, but require higher levels of c-myc and Notch
stimulation than outer hair cells. Inner hair cells appear to
require higher levels of c-myc and Notch stimulation than
supporting cells and outer hair cells to promote cell
proliferation.
Example 5
Functional Characteristics of Hair Cells Produced by Myc and Notch
Exposure
[0223] The following examples demonstrate that hair cells produced
by applying the methods described herein possess characteristics of
functional hair cells.
[0224] The presence of signal transduction channels necessary for
hair cell function was assessed in hair cells produced by elevated
Myc and Notch exposure. 45-day-old NICD.sup.flox/flox mice were
injected with Ad-Cre-GFP and Ad-Myc mixture in the scala media
using cochleostomy. EdU was injected for 5 days daily following
adenovirus injection to label proliferating hair cells. 35 days
post-virus injection, mouse cochleas were dissected and incubated
with fluorescence dye FM1-43FX for 30 seconds before cochleas were
washed and fixed. Fixed tissues were decalcified and stained with
Espin (Esp) for hair cells. Cells that underwent proliferation were
labeled by EdU. FIG. 14 shows that control Esp+ hair cells that did
not undergo cell cycle reentry following EdU exposure (EdU-) took
up FM1-43FX (FIG. 14, A-E). Significantly, Esp+ hair cells that
reenter the cell cycle following Ad-Myc/Ad-NICD virus injection and
EdU exposure (EdU+) also took up FM1-43FX (FIG. 14, F-J). As
FM1-43FX rapidly enters hair cells through functional transduction
channels, labeling by FM1-43FX demonstrates the presence of
functional transduction channels in proliferating hair cells
similar to non-proliferating hair cells. This result demonstrates
that hair cells produced by exposure to elevated Myc and Notch
activity possess functional membrane channels that are essential
for hair cell function.
[0225] Synapse formation was also assessed in cells exposed to
elevated levels of c-Myc and Notch activity in vivo. Adult
(45-day-old) NICD.sup.flox/flox mice were transduced with an
Ad-Myc/Ad-Cre virus mixture, exposed to BrdU administration, and
analyzed for evidence of functional synapse formation as described
for FIG. 9. Tissue was harvested 20 days post-injection of virus
and stained for neurofilament (NF) to identify neurofibers of
ganglion neurons. Analysis of stained sections revealed the
presence of proliferating hair cells (Myo7a+/BrdU+) that were in
contact with NF+ neurofibers (FIG. 15A, C, E; arrows). This result
suggests that production of hair cells via the methods disclosed
herein is accompanied by regrowth of neurofibers and formation of
functional synapses crucial for hair cell function.
Example 6
Hair Cells Induced to Proliferate In Vivo Maintain Specific Hair
Cell Identity
[0226] The following example illustrates that inner hair cells
produced in vivo via induced proliferation of existing inner hair
cells maintain characteristics specific to inner hair cells.
[0227] Cochlea of adult NICD.sup.flox/flox mice were transduced in
vivo with an Ad-Myc/Ad-Cre virus mixture for 15 days with BrdU
injected daily for the first 5 days. The methods used are the same
as those described for FIG. 9. Cochlear tissue was harvested and
analyzed for inner hair cell-specific markers. Both inner hair
cells that underwent cell cycle reentry (FIG. 16A-E; arrow) and
those that did not undergo cell cycle reentry (FIG. 16A-E;
arrowhead) stained positive for Vesicular Glutamate Transporter-3
(Vglut3), an inner hair cell-specific marker. Furthermore, the same
cells also stained positive for C-Terminal Binding Protein 2
(CtBP2) (brackets), a presynaptic marker, indicating the presence
of functional synapses. By contrast, in control animals exposed to
Ad-GFP, no BrdU labeling was observed, although Vglut3+/CtBP2+
inner hair cells were detected (FIG. 16F-J, bracket). The results
show that induced proliferation of inner hair cells via exposure to
elevated c-myc and Notch activity produce inner hair cells with
markers of functional synapses.
Example 7
Transdifferentiation of Proliferating Supporting Cells in
Culture
[0228] The following example demonstrates that application of the
methods described herein can be used to induce proliferation and
transdifferentiation of inner ear support cells to a hair cell
fate.
[0229] Experiments were performed using a mouse model capable of
expressing elevated levels of myc and Notch following doxycycline
induction (rTta/Tet-on-Myc/Tet-on-NICD). Adult mouse
(rTta/Tet-on-Myc/Tet-on-NICD) cochlea was dissected, with three
holes drilled to the bone for efficient media exposure and cultured
in the DMEM/F12 supplied with N2 and B27 without serum. Doxycycline
(1 mg/ml) was added to the culture for 5 days to activate
c-Myc/NICD, followed by Ad-Atoh1 (2.times.10.sup.12, 1:100
dilution) infection for 16 hours. The culture was exchanged with
fresh medium for additional 14 days, with medium changed every 3
days. EdU (final concentration 10 .mu.M) was added to the culture
throughout the entire period. Support cells induced to express
elevated NICD and myc levels via doxycycline exposure were observed
to undergo cell proliferation as evidenced by EdU labeling (FIG.
17A-E, arrowheads and closed arrows). Furthermore, exposure to
Ad-Atoh1 resulted in transdifferentiation of both cycling (FIG.
17A, C, E, closed arrows) and non-cycling (FIG. 17B, C, E, open
arrow) support cells to a hair cell fate as evidenced by Myo7a and
Parvalbumin (Parv) staining. Control, cultured
rTta/Tet-on-Myc/Tet-on-NICD support cells exposed to Ad-Atoh1, but
not doxycycline, underwent transdifferentiation but failed to
undergo cell cycle reentry (FIG. 17F-J, arrow), as evidenced by the
presence of Myo7a+/Parv+/EdU- cells. In a similar experiment,
cultured cochlear supporting cells harvested from
rTta/Tet-on-Myc/Tet-on-NICD mice were exposed to doxycycline and
Ad-Atoh1 virus, and then exposed to FM1-43FX (3 .mu.M) for 30
seconds to investigate whether hair cells produced by this process
possess characteristics of functional hair cells. Esp staining of
cells subjected to this protocol revealed the presence of hair
bundles in transdifferentiated supporting cells that also stained
positive for FM1 uptake, revealing the presence of functional
membrane channels (FIG. 17K, O; arrow). Other transdifferentiated
cells were labeled with FM1, but did not show signs of cell cycle
reentry as they are EdU negative (FIG. 17K, O; arrowhead). Thus,
exposure of cultured cochlear support cells to elevated levels of
myc and Notch, followed by Atoh1 induced proliferation of
supporting cells and transdifferentiation to a hair cell fate,
where the cells generated possessed characteristics of functional
hair cells.
Example 8
Induction of Inner Ear Progenitor Gene Expression
[0230] In order to understand how cell fate is affected by elevated
c-myc and Notch activity, a study of mRNA transcripts expressed
following exposure to c-Myc and NICD was performed.
[0231] Adult NICD.sup.flox/flox mouse cochleas were cultured and
infected with Ad-Myc/Ad-Cre-GFP overnight (2.times.10.sup.12 in
1:100 dilution). Beginning the next day, the media was changed
daily for the next 4 days. Ad-Cre-GFP infected NICD.sup.flox/flox
mouse cochleas were used as controls. The infected cochleas were
harvested for mRNA isolation using QIAGEN mRNA isolation kit. cDNAs
were synthesized using Life Science Technology SuperScript III
reverse transcriptase kit. Semi-quantitative RT-PCR was performed
using standard protocol. Analysis of different sets of transcripts
revealed that stem cell gene transcripts (e.g., Nanog, ALPL, SSEA)
were not noticeably upregulated following c-myc and NICD exposure.
By contrast, most of the analyzed transcripts specific to ear
progenitor cells (e.g., Eya1, DLX5 , Six2, Pax2, p27kip1, NICD,
Prox1, HesS) were upregulated following exposure to c-myc and NICD
(FIG. 18). GAPDH served as an internal control for normalization of
signal intensity. These results suggest a decisive advantage
inherent in using the method disclosed herein, as opposed to using
embryonic stem cells. Specifically, these results demonstrate that
exposure to elevated c-Myc and Notch activity results in elevated
levels of progenitor, rather than stem cell gene expression, which
likely allows the inner ear cells to both re-enter the cell cycle
and maintain the desired cell fate.
INCORPORATION BY REFERENCE
[0232] The entire disclosure of each of the patent documents and
scientific articles cited herein are incorporated by reference in
their entirety for all purposes.
EQUIVALENTS
[0233] The invention can be embodied in other specific forms with
departing from the essential characteristics thereof The foregoing
embodiments therefore are to be considered illustrative rather than
limiting on the invention described herein. The scope of the
invention is indicated by the appended claims rather than by the
foregoing description, and all changes that come within the meaning
and range of equivalency of the claims are intended to be embraced
therein.
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