U.S. patent application number 11/953797 was filed with the patent office on 2008-10-30 for use of stem cells to generate inner ear cells.
Invention is credited to Albert Edge, Stefan Heller, Huawei Li.
Application Number | 20080267929 11/953797 |
Document ID | / |
Family ID | 36000669 |
Filed Date | 2008-10-30 |
United States Patent
Application |
20080267929 |
Kind Code |
A1 |
Li; Huawei ; et al. |
October 30, 2008 |
Use of stem cells to generate inner ear cells
Abstract
This invention relates generally to methods and compositions for
inducing stem cell or progenitor cell differentiation, and more
particularly to methods and compositions for inducing
differentiation of stem cells and/or progenitor cells into cells
that function within the inner ear.
Inventors: |
Li; Huawei; (Shanghai,
CN) ; Edge; Albert; (Newton, MA) ; Heller;
Stefan; (Rockland, MA) |
Correspondence
Address: |
FISH & RICHARDSON PC
P.O. BOX 1022
MINNEAPOLIS
MN
55440-1022
US
|
Family ID: |
36000669 |
Appl. No.: |
11/953797 |
Filed: |
December 10, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10989649 |
Nov 15, 2004 |
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11953797 |
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60519712 |
Nov 13, 2003 |
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60605746 |
Aug 31, 2004 |
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Current U.S.
Class: |
424/93.21 ;
435/377; 435/6.1; 435/6.12; 435/7.8 |
Current CPC
Class: |
C12N 5/0619 20130101;
A61P 27/16 20180101; C12N 2506/02 20130101; A61K 35/12 20130101;
A61K 38/1709 20130101; C12N 2501/11 20130101; G01N 33/5073
20130101; A61K 35/55 20130101; C12N 5/062 20130101; A61K 35/30
20130101; C12N 2501/115 20130101; A61K 9/0046 20130101; C12N
2501/105 20130101; C12N 2501/41 20130101 |
Class at
Publication: |
424/93.21 ;
435/377; 435/7.8; 435/6 |
International
Class: |
A61K 35/30 20060101
A61K035/30; C12N 5/02 20060101 C12N005/02; C12Q 1/68 20060101
C12Q001/68; A61P 27/16 20060101 A61P027/16; G01N 33/53 20060101
G01N033/53 |
Claims
1. A method of producing a population of neural progenitor cells,
the method comprising: providing a first population of cells
comprising stem cells that have undetectable levels of Sox1
expression; culturing the first population of cells in suspension
in the absence of serum and feeder cells, under conditions and for
a time period sufficient to induce differentiation of said first
population of cells into neural progenitor cells, thereby obtaining
a second population of cells comprising neural progenitor
cells.
2. The method of claim 1, further comprising assaying the levels of
Sox1 in the first and second population of cells, wherein a higher
level of Sox1 expression in the second population of cells as
compared to the first population of cells indicates that the second
population of cells comprises neural progenitor cells.
3. The method of claim 1, further comprising assaying expression of
one or more of nestin, Pax2, Math1, NeuroD, and GFAP in the second
population of cells, wherein expression one or more of nestin,
Pax2, Math1, NeuroD, and GFAP indicates that the second population
of cells comprises neural progenitor cells.
4. The method of claim 1, wherein the second population of cells
express one or more of nestin, Pax2, Math1, NeuroD, GFAP, BMP4,
BMP7, Notch1, Jag1, and Jag2.
5. The method of claim 1, wherein the stem cells comprise embryonic
stem cells.
6. The method of claim 5, wherein the embryonic stem cells comprise
murine embryonic stem cells.
7. The method of claim 5, wherein the embryonic stem cells comprise
human embryonic stem cells.
8. The method of claim 1, wherein the first and second populations
of cells comprise one or more of a reporter under the control of a
beta-actin promoter or a reporter under the control of a Sox1
promoter.
9. The method of claim 8, wherein the reporter is a fluorescent
protein.
10. The method of claim 1, wherein following differentiation of the
first population of cells, the method further comprises: disrupting
clusters of two or more neural progenitor cells to yield individual
neural progenitor cells; culturing the individual neural progenitor
cells for a time and under conditions sufficient to promote
attachment of the cells to a surface; and maintaining the attached
cells in the presence of one or more growth factors for a time and
under conditions sufficient to promote cell proliferation, thereby
increasing the number of neural progenitor cells.
11. The method of claim 10, wherein the attached cells are cultured
in the presence of 10% serum.
12. The method of claim 10, wherein the second and third population
of cells express one or more of Sox1, nestin, Pax2, and Math1.
13. The method of claim 10, wherein the growth factor is basic
fibroblast growth factor.
14. The method of claim 10, further comprising culturing the neural
progenitor cells in the absence of growth factor for a time
sufficient to promote differentiation of the neural progenitor
cells into sensory neural cells.
15. The method of claim 1, further comprising culturing the neural
progenitor cells in the absence of growth factor for a time
sufficient to promote differentiation of the neural progenitor
cells into sensory neural cells.
16. The method of claim 14, wherein the sensory neural cells
comprise one or more of neurons, glial cells, and
oligodendrocytes.
17. The method of claim 14, wherein the sensory neural cells
express one or both of (i) less Sox1 than the neural progenitor
cells and (ii) higher levels of one or more of TrkC and Map2 than
the neural progenitor cells.
18. The method of claim 14, wherein one or more of .beta.-III
tubulin and GFAP is detectable in the sensory neural cells.
19. The method of claim 1, further comprising culturing the neural
progenitor cells in the presence of an effective amount of bone
morphogenetic protein 4 (BMP4) for a time period sufficient to
promote differentiation of the cells into sensory neural cells.
20. The method of claim 19, wherein the sensory neural cells
express elevated levels of GATA3, TrkB, and TrkC when compared to
the neural progenitor cells.
21. A method of producing a population of sensory neural cells, the
method comprising: providing a population of stem cells having
undetectable levels of Sox1 expression; culturing the stem cells in
the absence of serum for a time period sufficient to promote
differentiation of the stem cells into a population of cells
comprising neural progenitor cells; contacting the neural
progenitor cells with an effective amount of one or both of (i)
bone morphogenetic protein 4 (BMP4) or (ii) retinoic acid for a
time period sufficient to promote differentiation of the neural
progenitor cells into sensory neural cells, thereby producing a
population of sensory neural cells.
22. The method of claim 21, wherein the neural progenitor cells
express detectable levels of Sox1.
23. The method of claim 21, wherein the stem cells comprise
embryonic stem cells.
24. The method of claim 21, wherein the stem cells are human stem
cells or murine stem cells.
25. The method of claim 21, wherein the stem cells are cultured in
the presence of human leukemia inhibitory factor (LIF).
26. The method of claim 21, wherein one or both of the stem cells
and neural progenitor cells are cultured in the presence of one or
more growth factors.
27. The method of claim 21, wherein the sensory neural cells have
elevated levels of GATA3, TrkB, and TrkC as compared to the stem
cells.
28. The method of claim 1, further comprising culturing the neural
progenitor cells in the presence of an effective amount of retinoic
acid for a time period sufficient to promote differentiation of the
cells into sensory neural cells.
29. A method of producing a population of sensory neural cells, the
method comprising: providing a population of cells comprising inner
ear stem cells; culturing the inner ear stem cells in the absence
of serum and in the presence of one or more of (i) an effective
amount of retinoic acid or (ii) bone morphogenetic protein 4 (BMP4)
for a time sufficient to promote differentiation of the inner ear
stem cells into sensory neural cells.
30. The method of claim 29, wherein the inner ear stem cells are
cultured in the presence of one or more growth factors.
31. The method of claim 29, wherein the inner ear stem cells
express detectable levels of Sox1.
32. The method of claim 29, wherein the inner ear stem cells
express undetectable levels of .beta.-III tubulin, GFAP, and myosin
VIIa.
33. The method of claim 29, wherein the sensory neural cells
express higher levels of Pax2 as compared to the inner ear stem
cells.
34. The method of claim 33, further comprising, prior to culturing
the inner ear cells in the presence of retinoic acid or BMP4: (a)
culturing the inner ear stem cells in suspension in the absence of
serum for a time period sufficient for the cells to form clusters
of cells comprising two or more cells; (b) disrupting the cluster
of cells to yield a population of individual cells; (c) culturing
the individual cells in suspension in the absence of serum
suspension for a time period sufficient for the cells to form
clusters of cells comprising two or more cells; and optionally
repeating steps (a) to (c) until a desired number of inner ear stem
cells is obtained, and then culturing the inner ear stem cells in
the presence of serum for a time sufficient to promote attachment
of the cells to a surface.
35. A method of treating, or preventing the progression of,
sensorineural hearing loss in a subject, the method comprising:
providing a population of neural progenitor cells obtained using
the method of claim 1, and administering said population of cells
into the inner ear of a subject, thereby treating or preventing the
development or progression of sensorineural hearing loss in the
subject.
36. The method of claim 35, wherein the cells are administered by
injection into the luminae of the cochlea, into the auditory nerve
trunk in the internal auditory meatus, or into the scala
tympani.
37. A method of treating, or preventing the development or
progression of, sensorineural hearing loss in a subject, the method
comprising: providing a population of sensory neural cells obtained
using the method of claim 14, and administering said population of
sensory neural cells into the inner ear of a subject, thereby
treating or preventing the progression of sensorineural hearing
loss in the subject.
38. The method of claim 37, wherein the cells are administered by
injection into the luminae of the cochlea, into the auditory nerve
trunk in the internal auditory meatus, or into the scala
tympani.
39. A method of treating, or preventing the progression of,
sensorineural hearing loss in a subject, the method comprising:
providing a population of sensory neural cells obtained using the
method of claim 15, and administering said population of sensory
neural cells into the inner ear of a subject, thereby treating or
preventing the progression of sensorineural hearing loss in the
subject.
40. The method of claim 39, wherein the cells are administered by
injection into the luminae of the cochlea, into the auditory nerve
trunk in the internal auditory meatus, or into the scala
tympani.
41. A method of treating, or preventing the development or
progression of, sensorineural hearing loss in a subject, the method
comprising: providing a population of cells obtained using the
method of claim 21, and administering said population of cells into
the inner ear of a subject, thereby treating or preventing the
progression of sensorineural hearing loss in the subject.
42. The method of claim 41, wherein the cells are administered by
injection into the luminae of the cochlea, into the auditory nerve
trunk in the internal auditory meatus, or into the scala
tympani.
43. A method of treating, or preventing the development or
progression of, sensorineural hearing loss in a subject, the method
comprising: providing a population of cells obtained using the
method of claim 29, and administering said population of cells into
the inner ear of a subject, thereby treating or preventing the
progression of sensorineural hearing loss in the subject.
44. The method of claim 43, wherein the cells are administered by
injection into the luminae of the cochlea, into the auditory nerve
trunk in the internal auditory meatus, or into the scala
tympani.
45. A method of identifying a candidate compound that promotes
differentiation of cells into mature cells of the inner ear, the
methods comprising: providing a cell expressing a reporter
construct comprising a Math-1 regulatory region operably linked to
a reporter gene; contacting the cell with a test compound; and
detecting expression of the reporter gene; wherein an increase in
expression of the reporter gene in that presence of the test
compound as compared to expression of the reporter gene in the
absence of the test compound indicates that the test compound is a
candidate compound that promotes differentiation of cells into
mature cells of the inner ear.
46. The method of claim 45, further comprising contacting a stem
cell, inner ear stem cell, or neural progenitor cell with the
candidate compound and evaluating the ability of the candidate
compound to promote differentiation into a mature cell of the inner
ear, and selecting a candidate compound that promotes
differentiation into a mature cell of the inner ear.
47. The method of claim 45, wherein the reporter gene is selected
from the group consisting of a fluorescent protein, an
enzymatically active protein, or a protein detectable in an
antibody-based assay.
48. The method of claim 45, wherein the mature cell of the inner
ear is a hair cell or spiral ganglion neuron.
49. The method of claim 46, further comprising administering the
candidate compound to the inner ear of an animal model of
sensorineural hearing loss, and evaluating the ability of the
candidate compound to promote differentiation of inner ear stem
cells into mature cells of the inner ear.
Description
[0001] This application is a continuation of U.S. patent
application Ser. No. 10/989,649, filed Nov. 15, 2004, which claims
the benefit of U.S. Provisional Patent Application Ser. No.
60/519,712, filed on Nov. 13, 2003, and U.S. Provisional Patent
Application Ser. No. 60/605,746, filed on Aug. 31, 2004. The
contents of the prior applications are hereby incorporated in the
present application in their entirety.
TECHNICAL FIELD
[0002] This invention generally relates to compositions and methods
for inducing cellular differentiation (e.g., complete or partial
differentiation of stem cells into cells capable of functioning as
sensory cells of the ear) and to assays and methods of treatment
that employ the stem cells or the more fully differentiated cells
into which they develop.
BACKGROUND
[0003] More than 5% of the people in industrialized nations have
significant hearing problems that range in severity from modest
difficulty with speech comprehension to profound deafness. Hearing
loss is age-related, as about 4% of people under 45 years old and
about 34% of those over 65 years old have debilitating hearing
loss. In most cases, the cause is related to degeneration and death
of hair cells and their associated spiral ganglion neurons.
[0004] The ear is composed of four main sections: the external ear,
middle ear, inner ear, and the transmission pathway to the hearing
center in the brain. The inner ear is a capsule of very dense bone
containing a fluid that communicates with the middle ear. Small
bones within the middle ear (the malleus, incus, and stapes)
transmit sound energy from the tympanic membrane to the oval window
at the entrance to the cochlea of the inner ear. The action of the
stapes at the oval window exerts pressure on the fluid within the
cochlea. The pressure is transmitted through the cochlea,
ultimately causing a second window, the round window to oscillate.
A basilar membrane that defines the fluid-filled chambers of the
cochlea then transmits the oscillations to the organ of Corti,
which contains about 13,000 mechanosensory cells called hair cells.
Hair cells are located in the epithelial lining of the inner ear
(in the cochlear organ of Corti, as mentioned), as well as in the
vestibular sensory epithelia of the saccular macula, the utricular
macula, and the cristae of the three semicircular canals of the
labyrinth. The cochlear hair cells send signals to the cochlear
spiral ganglion, and the clustered neuronal cell bodies convey
those signals to the cochlear nucleus of the brain stem (see FIGS.
5A, 5B, and 5C).
SUMMARY
[0005] The present invention features compositions and methods
related to stem cells and cells of the inner ear. The methods
include those for producing (e.g., isolating or obtaining) stem
cells or progenitor cells from a tissue (e.g., a tissue within the
inner ear) and for identifying agents that mediate complete or
partial differentiation of those cells to or toward a mature cell
type of the inner ear (e.g., a hair cell or spiral ganglion
neuron). We may refer to these agents as "differentiation" agents
or compounds. Other methods provide treatment for patients who
have, or who are at risk for developing, an auditory disorder. The
methods of treatment include steps whereby one administers a
differentiation agent (e.g., an agent identified by a screening
method described herein), a stem cell or progenitor cell (e.g., a
cell isolated by the methods described herein), or both (i.e., both
a differentiation agent and a stem cell and/or progenitor cell) to
the inner ear of the patient. The compositions include stem cells
and progenitor cells isolated by the methods described herein as
well as pharmaceutical compositions and kits containing them. The
methods of the invention can be practiced using either stem cells
or cells that are partially differentiated (progenitor cells).
[0006] In one aspect, the invention features screening methods for
identifying agents that can increase or decrease the expression of
one or more auditory proteins within a cell (regardless of the
extent to which that cell has differentiated). The change in
expression can be, but is not necessarily, a robust change. For
example, a candidate agent may increase the expression of an
auditory protein from an essentially undetectable level to a
readily detectable level. It may also increase expression to a
certain degree (e.g., there may be about a 1-, 2-, or 5-fold
increase in expression). The protein analyzed (i.e., the auditory
protein) can be any protein that is ordinarily expressed in a
mature cell of the inner ear (e.g., a hair cell or spiral ganglion
cell of an adult who has normal hearing), but expression is not
necessarily specific for an inner ear cell. For example, the
protein can be one that is expressed in other cell types, and it
may be expressed at varying levels as a stem cell differentiates
into a progenitor cell and finally into a completely differentiated
cell. Proteins that are expressed in inner ear cells (e.g., in hair
cells and spiral ganglion cells) are well known in the art.
[0007] The screening methods include providing a cell or a
population of cells, which may contain a single cell type or a
variety of cell types, including cells that may be undifferentiated
(i.e., pluripotent stem cells) less than fully differentiated
(i.e., progenitor cells) or fully differentiated (e.g.,
recognizable as hair cells or spiral ganglion cells). Where a
population of test cells is used, the proportion of stem cells
within the test population can vary. For example, the population
can contain few stem cells (e.g., about 1-10%) a moderate
proportion of stem cells (e.g., about 10-90% (e.g., about 20, 25,
30, 40, 50, 60, 70, 75, 80, or 85% stem cells)) or many stem cells
(e.g., at least 90% of the population (e.g., 92, 94, 96, 97, 98, or
99%) can be stem cells). The cells will have the potential to
differentiate into a completely or partially differentiated cell of
the inner ear (e.g., the cell can be a pluripotent stem cell that
differentiates into a cell that expresses one or more auditory
proteins). Partially differentiated cells are useful in the
treatment methods (whether therapeutic or prophylactic) so long as
they express a sufficient number and type of auditory-specific
proteins to confer a benefit on the patient (e.g., improved
hearing).
[0008] With respect to their source, the cells employed in the
screening or treatment methods can be obtained from a mammal, such
as a human, from any developmental stage. For example, the cells
can be derived from an embryo, fetus or post-natal mammal (e.g., an
infant, child, adolescent, or adult (e.g., an adult human)). More
specifically, the stem cell or the progenitor cell can be obtained
from the cochlear 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 (see FIGS. 5A, 5B, and 5C). The
stem cell or progenitor cell can also be obtained, however, from
other tissues such as bone marrow, blood, skin, or an eye. The
cells employed can be obtained from a single source (e.g., the ear
or a structure or tissue within the ear) or a combination of
sources (e.g., the ear and one or more peripheral tissues (e.g.,
bone marrow, blood, skin, or an eye)). The cells can also be
obtained from a patient to whom they will subsequently be
readministered.
[0009] Where the methods are carried out in cell culture, one can
use an essentially pure population of cells (e.g., an essentially
pure population of stem cells (e.g., a population in which about
90% or more of the cells are stem cells). Individual cells (e.g., a
single cell placed within the well of a tissue culture plate) can
also be analyzed (by, for example, an amplification technique such
as "single-cell" PCR). Once the cell or cell population is
selected, the cell(s) can be contacted with a candidate agent or
exposed to certain environmental conditions (e.g., conditions that
vary from physiologic conditions (e.g., increased or decreased
temperature, abnormal levels of CO.sub.2 or other gases (e.g.,
oxygen), or non-physiological pH)). Following exposure to the
candidate agent or environmental change, one can determine whether
the level of expression of an auditory protein is more (or less)
than the level prior to exposure to the agent (or relative to a
reference standard). More than one auditory protein can be
assessed, at the same time or sequentially. To assess expression,
one can examine protein levels per se or the level of RNA
transcription. Numerous methods are known in the art that can be
suitably employed to assess either protein or RNA expression. An
increase in expression of the auditory protein indicates that the
agent can promote the expression of the auditory protein within the
cell, thereby promoting at least partial differentiation of a cell
(e.g., a stem cell) into a more mature cell of the inner ear. The
ultimate goal of the screening methods is to identify an agent or
group of agents or conditions that increase the expression of
auditory proteins that mediate the sense of hearing and can,
therefore, be used to generate cells that improve a patient's
ability to hear or maintain their balance. No particular mechanism
of action is required or implied. The agent(s) and/or condition(s)
may act directly or indirectly on the transcriptional machinery for
the auditory protein in question.
[0010] The candidate agents can be essentially any nucleic acid
(e.g., a gene or gene fragment that encodes a polypeptide (e.g., a
functional protein) such as a growth factor or other cytokine
(e.g., an interleukin)), any polypeptide per se (which may be a
full-length protein or a biologically active fragment or other
mutant thereof), or any small molecule. The small molecules can
include those contained within commercially available compound
libraries (suppliers include ChemBridge Corp (San Diego, Calif.)
and ChemDiv (San Diego, Calif.)). The screening assays can be
configured as "high throughput" assays to screen many such agents
at once. For example, the agents and/or cells to be assessed can be
presented in an array. More specifically, the candidate agent can
be, for example, a nucleic acid that encodes, or a polypeptide that
is, a polypeptide active in the cellular biochemical pathway of
which Notch, WNT, or Sonic hedgehog are a part (e.g., WNT1, WNT10B,
WNT11, WNT13, WNT14, WNT15, WNT2, WNT2B, WNT5a, WNT7a, or WNT8B); a
homolog of Notch, WNT, or Sonic hedgehog; or a biologically active
fragment or other variant of Notch, WNT, or Sonic hedgehog. For
example, the nucleic acid can encode a fragment of Sonic hedgehog,
such as SHH-N or a variant thereof (e.g., an SHH-N fragment that
contains a limited number (e.g., 1-10) of conservative amino acid
substitutions), or a homolog of Sonic hedgehog, such as Indian
hedgehog or Desert hedgehog or fragments or other mutants thereof
(e.g., a fragment of Indian hedgehog or Desert hedgehog that
corresponds to SHH-N). A homolog is a nucleic acid or polypeptide
that is substantially identical to, for example, a Notch, WNT, or
Sonic hedgehog nucleic acid or polypeptide and, preferably,
functions in the pathways in which Notch, WNT, and Sonic hedgehog
are active. Notch, WNT, or Sonic hedgehog from different species
may also be described as homologs (e.g., a human sequence may be
described as the homolog of a Notch protein from Drosophila or
mouse). A first nucleic acid (whether genomic DNA, cDNA, RNA or a
nucleic acid containing non-naturally occurring nucleotides) or
polypeptide is substantially identical to a second nucleic acid or
polypeptide, respectively, when the two are exhibit sequence
similarity and at least one shared activity. Nucleic acids and
polypeptides useful in the screening and therapeutic methods of the
present invention can be substantially identical to a human Sonic
hedgehog cDNA (SEQ ID NO:2; FIG. 2) or amino acid sequence (SEQ ID
NO:8; FIG. 1). For example, a nucleic acid sequence substantially
identical to human Sonic hedgehog cDNA is at least 80% identical
(e.g., 85%, 90%, 95%, 98%, or 99%) to SEQ ID NO:2, and a
substantially identical amino acid sequence is at least 80%
identical (e.g., 85%, 90%, 95%, 98%, or 99%) to SEQ ID NO:1.
[0011] In particular embodiments, the nucleic acid can encode, or
the polypeptide can be: Math1, parvalbumin 3, Brn3.1, Brn3.2, Hes1,
Hes5, neurogenin-1, NeuroD, Jagged1, Jagged2, Delta1, Notch1,
Lunatic fringe, Numb, Wnt7a, p27Kip1, Shh, Bmp4, Fgfr3, Fgfr1,
Fgfr2, Fgf10, Fgf2, Fgf3, GATA3, Pax2, neurotrophin-3, BDNF, or a
fragment or other mutant thereof (e.g., a fragment or other mutant
that retains sufficient biological activity to function in a
screening method or therapeutic method described herein).
[0012] Rather than, or in addition to, assessing the expression of
one or more auditory proteins, the screening methods can be carried
out by assessing a reporter gene that has been placed under the
control of a sequence that regulates the expression of an auditory
protein (e.g., a promoter and/or enhancer that directs expression
of an auditory protein in vivo). Accordingly, in another aspect,
the invention features methods of identifying differentiation
agents that promote the expression of an auditory protein within a
cell by providing a cell (any of the cells or populations of cells
described above would be appropriate) containing a reporter gene
operably linked to a promoter or promoter element (e.g., an
enhancer region) of an auditory protein gene. As with the screening
method described above, the cell(s) can be contacted with the
candidate agent in vivo or in cell culture, and the level of
expression of the reporter gene within the cell can be assessed. An
increase in expression following exposure to the candidate agent
indicates that the agent promotes the expression of the auditory
protein within the cell. A decrease in reporter gene expression
identifies the agent as a candidate inhibitor of auditory protein
expression (proteins that inhibit the expression of an auditory
protein are potential targets for inhibition; by inhibiting a
protein that inhibits the expression of an auditory protein, one
can promote expression of the auditory protein). Cells (e.g., stem
cells, progenitor cells, or differentiated cells from the inner ear
or another tissue) that contain the reporter constructs described
herein (e.g., a plasmid bearing an auditory protein regulatory
region operably linked to a reporter gene) are also within the
scope of the present invention, as are the reporter constructs per
se (e.g., the invention features nucleic acids, which may be
further contained within a vector such as a plasmid, in which a
regulatory region of an auditory protein (e.g., a Math1 regulatory
region of a sonic hedgehog regulatory region) is operably linked to
a reporter gene). The reporter gene can encode any detectable
polypeptide. For example, the reporter gene can be a gene that
encodes a fluorescent protein, an enzymatically active protein
(e.g., .beta.-galactosidase and chloramphenicol acetyltransferase),
or a protein detectable in an antibody-based assay. Other markers
are known in the art and additional exemplary markers are described
further below.
[0013] The screening methods described herein can be performed on a
cell in cell culture under ex vivo conditions of pH and temperature
suitable to maintain viability (such conditions are generally known
in the art and exemplary conditions are provided below). Cells can
also be treated in cell culture prior to administration to a
patient.
[0014] The invention also features methods of isolating a stem cell
or progenitor cell from the inner ear of an animal (e.g., a mammal
such as a human, non-human primate, or other mammal such as a pig,
cow, sheep, goat, horse, dog, cat, or rodent). These methods
include providing tissue from the inner ear (e.g., a piece of
tissue that includes hair cells or the membrane with which they are
associated, or spiral ganglion cells). For example, the tissue can
include at least a portion of the utricular maculae. The tissue can
be disrupted by exposure to a chemical or mechanical force (or
both). For example, the tissue can be exposed to a tissue-digesting
enzyme, such as trypsin, and/or to a mechanical (e.g., physical)
force such as trituration to break the tissue into smaller pieces.
The treated tissue (e.g., enzyme-treated tissue (e.g., the
enzyme-treated utricular maculae)) can optionally be soaked in
fetal calf serum or other protein solution to neutralize or exhaust
the enzyme (fully or partially); washed; and the disrupted tissue
can be passed through a device such as a cell strainer that
separates the stem cells or progenitor cells within the disrupted
tissue from differentiated cells or cellular debris. The cells
obtained may constitute an enriched population of stem cells and/or
progenitor cells; isolation from all (or essentially all)
differentiated cells or other cellular material within the tissue
may be achieved but is not required to meet the definition of
"isolated." Absolute purity is not required. The invention
encompasses cells obtained by the isolation procedures described
herein. The cells may be mixed with a cryoprotectant and stored or
packaged into kits. Once obtained, the stem cells and/or progenitor
cells can be expanded in culture.
[0015] Methods for treating patients (e.g., humans) who have, or
who are at risk for developing, an auditory disorder, are also
described and are within the scope of the present invention. These
methods include administering a cell or population of cells (as
described above; e.g. a stem cell and/or progenitor cell obtained
from a tissue such as the ear) to the ear of the patient. The
administered cells may be obtained by the methods described herein,
and the starting material may be tissue obtained from the patient
to be treated. In other embodiments, the methods include the step
of administering a therapeutic agent that promotes the expression
of an auditory protein within a cell within the inner ear (e.g., a
differentiation agent as described herein or as identified by the
screening methods described herein). When used, the differentiation
agent can be administered to cells in culture or can be
administered to the patient either alone (to stimulate the
differentiation of stem cells or progenitor cells within the
patient's inner ear) or together with undifferentiated cells (e.g.,
undifferentiated cells isolated by the methods described herein).
The differentiation agent can be, for example, an agonist of the
hedgehog pathway, such as an agonist of Sonic hedgehog (e.g.,
Hh-Ag1.3).
[0016] As noted, the invention also features a stem cell or
progenitor cell (either of which may cluster into cellular spheres)
isolated by the methods described herein, compositions containing
them, and kits that include them (with, for example, instructions
for inducing differentiation; for expanding the cells in culture;
and/or for administering the cells to a patient or to a cell (e.g.,
a cell in culture) to promote its differentiation). The
instructions can be printed or in another form (e.g., provided on
audio- or videotape).
[0017] There may be certain advantages to the use of stem cells
and/or progenitor cells for the treatment of hearing disorders. For
example, stem cells are readily expandable and can be expanded to
generate a desired tissue or cell type (e.g., hair cells or spiral
ganglion cells) for application to a patient. The stem cells can be
obtained from humans for clinical applications. Because the stem
cells can be harvested from a human, and in particular can be
harvested from the human in need of treatment, the immunological
hurdles common in xeno- and allotransplantation experiments can be
largely avoided.
[0018] Other features and advantages of the invention will be
apparent from the accompanying description and the claims. The
contents of all references, pending patent applications and
published patents, cited throughout this application are hereby
expressly incorporated by reference. In case of conflict, the
present specification, including definitions, will control.
BRIEF DESCRIPTION OF THE DRAWINGS
[0019] FIG. 1 is the amino acid sequence of an SHH polypeptide from
human (GenBank Accession No. AY422195; SEQ ID NO:1). The amino
acids of the SHH-N polypeptide are underlined.
[0020] FIG. 2 is a protein-coding nucleic acid sequence of SHH from
human (GenBank Accession No. AY422195; SEQ ID NO:2).
[0021] FIG. 3 is the amino acid sequence of an Indian hedgehog
(Ihh) polypeptide from human (GenBank Accession No.
XM.sub.--050846; SEQ ID NO:3).
[0022] FIG. 4 is the amino acid sequence of a Desert hedgehog (Dhh)
polypeptide from human (GenBank Accession No. NM.sub.--021044; SEQ
ID NO:4).
[0023] FIG. 5A is a diagram of the inner ear (from Clinical
Neuroanatomy and Related Neuroscience, Fourth ed., Fitzgerald and
Folan, eds., Saunders publishing, 2001).
[0024] FIG. 5B is a diagram of the semicircular canals and the
saccular macula of the inner ear (from Clinical Neuroanatomy and
Related Neuroscience, Fourth ed., Fitzgerald and Folan, eds.,
Saunders publishing, 2001).
[0025] FIG. 5C is a diagram of the cochlea, in section, of the
inner ear.
[0026] FIG. 6 is a gel indicating the expression of marker genes in
embryonic stem (ES) cells, progenitor cells, and differentiated
cells. Expression was detected by reverse transcription followed by
polymerase chain reaction (RT-PCR), and examination of the
amplified products by gel electrophoresis.
[0027] FIG. 7A is a graph illustrating the compound action
potential (CAP) threshold elevation in de-afferented and control
cat ears. The auditory nerve was cut 10 weeks prior to taking the
measurements.
[0028] FIG. 7B is a graph illustrating the distortion product
otoacoustic emissions (DPOAEs) in the de-afferented and control cat
ears. The auditory nerve was cut 10 weeks prior to taking these
measurements.
[0029] FIG. 8A is a graph illustrating a quantitative analysis of
the promoting effect of SHH on the number of hair cells generated
in otic vesicles after 3 days in culture. The basic serum-free
culture conditions ("no GF") include serum-free knockout DMEM
medium with N2 supplement.
[0030] FIG. 8B is a graph illustrating a quantitative analysis of
the promoting effect of SHH on the number of hair cells generated
in otic vesicles after seven days in culture. Serum conditions are
as described in FIG. 8A.
DETAILED DESCRIPTION
[0031] We have developed, inter alia, methods for identifying
agents that cause stem cells or progenitor cells to differentiate
(fully or partially) into cells of the inner ear. The methods are
amenable for use in identifying genes that, when expressed or
silenced, can promote or inhibit the differentiation of stem cells
into inner ear cells. The methods and agents are useful for
treating any disorder that arises as a consequence of cell loss in
the ear, such as hearing impairments, deafness, and vestibular
disorders.
[0032] Stem cells are unspecialized cells capable of extensive
proliferation. Stem cells are pluripotent and are believed to have
the capacity to differentiate into most cell types in the body
(Pedersen, Scientif. Am. 280:68, 1999), including neural cells,
muscle cells, blood cells, epithelial cells, skin cells, and cells
of the inner ear (e.g., hair cells and cells of the spiral
ganglion). Stem cells are capable of ongoing proliferation in vitro
without differentiating. As they divide, they retain a normal
karyotype, and they retain the capacity to differentiate to produce
adult cell types. Stem cells can differentiate to varying degrees.
For example, stem cells can form cell aggregates called embryoid
bodies in hanging drop cultures. The embryoid bodies contain neural
progenitor cells that can be selected by their expression of an
early marker gene such as Sox1 and the nestin gene, which encodes
an intermediate filament protein (Lee et al., Nat. Biotech.
18:675-9, 2000).
[0033] Stem cells useful for generating cells of the inner ear can
be derived from a mammal, such as a human, mouse, rat, pig, sheep,
goat, or non-human primate. Furthermore, stem cells can be derived
from any number of tissues including, but not limited to, an ear,
eye, bone marrow, blood, or skin. For example, stem cells have been
identified and isolated from the mouse utricular macula (Li et al.,
Nature Medicine 9:1293-1299, 2003). Stem cells useful for
generating cells of the inner ear can be adult stem cells, and
therefore derived from differentiated tissue, or the cells can be
from embryonic tissue.
[0034] The changes that induce a cell to differentiate, such as
into a hair cell or a spiral ganglion neuron, involve altered
biochemical pathways that lead to a specific phenotype. These
alterations are a result of the expression of specific genes, and
this expression pattern is influenced by signals from the
environment of the cell including cell-cell contact, oxygen
content, nutrient availability, ligands that bind to receptors on
the cells, temperature, and other factors. Stem cells are adaptive
in nature, and their response to changes in these signals triggers
the differentiation process.
[0035] Proteins that influence (e.g., promote or inhibit
differentiation) the phenotype of inner ear cells include
developmental regulators, cell cycle inhibitors, transcription
factors and other regulatory proteins that act on stem cells. The
phenotype of the cell includes the characteristics that distinguish
it from other cell types. For example, the phenotype of a hair cell
is distinct from the phenotype of a spiral ganglion cell.
[0036] Agents capable of causing stem cells to differentiate are
referred to as differentiation agents. Differentiation agents can
be, for example, small molecules, antibodies, peptides (e.g.,
peptide aptamers), antisense RNAs, small inhibitory RNAs (siRNA),
or ribozymes. Differentiation agents, such as small molecules, can
modulate the activity of one or more of the proteins that influence
cell phenotype by altering the activity of a growth factor or
receptor, an enzyme, a transcription factor, or a cell-specific
inhibitor. These molecules can change the binding affinity of a
protein for another protein, or can bind in an active site of an
enzyme or act as an agonist or antagonist of a ligand binding to a
receptor. Some types of differentiation agents, such as small
inhibitory RNAs (siRNAs), antisense RNAs, or ribozymes, can modify
the expression pattern of genes that encode these proteins.
Furthermore, the agents can be useful as therapeutic agents for
treating hearing disorders or vestibular dysfunction.
[0037] Many different genes are required for the development of the
structure and different cell types of the ear. The methods featured
in the invention are useful for identifying these genes. The
identified genes and gene products can be targets for therapeutic
agents and methods for treating hearing disorders and vestibular
dysfunction. Indications suited for the methods and therapeutic
agents featured in the invention are discussed in greater detail
below.
[0038] Screening Methods. Screening methods are provided. For
example, methods of identifying a differentiation agent that can
cause a stem cell to differentiate, at least partially, into a cell
of the inner ear or a precursor of the inner ear are features of
the invention. A differentiation agent can be a polypeptide, such
as an aptamer or antibody; a nucleic acid, such as DNA or RNA; or a
compound, such as a small molecule. According to one exemplary
method, an agent is contacted with a stem cell, and the stem cell
is determined to differentiate, at least partially, into a cell of
the inner ear, such as a hair cell or cell of the spiral ganglion.
The agent can be naturally occurring or synthetic. The agent can be
obtained from a library, or the agent can be a candidate molecule
identified by other methods. The candidate agent can have been
previously identified as a modulator of a gene or protein known to
be active in cells of the inner ear.
[0039] A variety of methods can be utilized to determine that a
stem cell has differentiated at least partially into a cell of the
inner ear. For example, the cell can be examined for the expression
of a cell marker gene. Hair cell marker genes include myosin VIIa
(myoVIIa), Math1, .alpha.9 acetylcholine receptor, espin,
parvalbumin 3, and Brn3.1. A pluripotent stem cell does not express
these genes. A stem cell that propagates and produces a cell
expressing one or more of these genes, has produced a hair cell,
i.e., the stem cell has differentiated at least partially into a
hair cell. A stem cell that has differentiated into a progenitor
cell (a precursor of hair cells) expresses early ear marker genes
such as Sox1, Nestin, Pax2, Bmp7, Jagged1, or p27.sup.Kip1. A
progenitor cell can express one or more of these genes. The
progenitor cells can be propagated in serum-free medium in the
presence of growth factors. Removal of growth factors will induce
the cells to differentiate further, such as into hair cells.
[0040] Identification of a hair cell or hair cell progenitor (e.g.,
a hair cell or progenitor cell that differentiated from a stem
cell) can be facilitated by the detection of expression of
tissue-specific genes. Detection of gene expression can be by
immunocytochemistry. Immunocytochemistry techniques involve the
staining of cells or tissues using antibodies against the
appropriate antigen. In this case, the appropriate antigen is the
protein product of the tissue-specific gene expression. Although,
in principle, a first antibody (i.e., the antibody that binds the
antigen) can be labeled, it is more common (and improves the
visualization) to use a second antibody directed against the first
(e.g., an anti-IgG). This second antibody is conjugated either with
fluorochromes, or appropriate enzymes for calorimetric reactions,
or gold beads (for electron microscopy), or with the biotin-avidin
system, so that the location of the primary antibody, and thus the
antigen, can be recognized. The protein marker can also be detected
by flow cytometry using antibodies against these antigens, or by
Western blot analysis of cell extracts.
[0041] Tissue-specific gene expression can also be assayed by
detection of RNA transcribed from the gene. RNA detection methods
include reverse transcription coupled to polymerase chain reaction
(RT-PCR), Northern blot analysis, and RNAse protection assays.
[0042] Identification of a differentiated hair cell or spiral
ganglion cell can also be assayed by physiological testing to
determine if the cells generate conductance channels characteristic
of mature hair or spiral ganglion cells.
[0043] In some embodiments, a candidate differentiation agent can
be tested against stem cells that have been engineered to express a
reporter gene that facilitates detection of cells converted into
inner ear cells. These engineered stem cells make up a reporter
cell line. A reporter gene is any gene whose expression may be
assayed; such genes include, without limitation, green fluorescent
protein (GFP), .alpha.-glucuronidase (GUS), luciferase,
chloramphenicol transacetylase (CAT), horseradish peroxidase (HRP),
alkaline phosphatase, acetylcholinesterase and
.beta.-galactosidase. Other optional fluorescent reporter genes
include but are not limited to red fluorescent protein (RFP), cyan
fluorescent protein (CFP) and blue fluorescent protein (BFP), or
any paired combination thereof, provided the paired proteins
fluoresce at distinguishable wavelengths.
[0044] A reporter gene can be under control of a promoter that is
active in cells of the inner ear, including progenitor cells and
cells at varying degrees of differentiation, but not in stem cells.
Ideally, the promoter is stably upregulated in the differentiated
cells or progenitors cells to allow assessment of the partially or
fully differentiated phenotype (e.g., expression of the reporter
gene and further identification of genes known to be expressed in
the inner ear). In one exemplary embodiment, the luciferase gene is
the reporter gene, which is under control of a promoter active in
hair cells, such as a myoVIIa promoter. Since myoVIIa is primarily
expressed in hair cells and in only a few other cell types, the
partial or full conversion of the stem cells to hair cells will
result in increased luminescent signal, whereas conversion of stem
cells to most other cell types will not increase luciferase
expression. Other promoters appropriate for use with a reporter
gene for identifying differentiated hair cells include myoVIIa,
Math1, .alpha.9 acetylcholine receptor, espin, parvalbumin 3, and
Brn3.1. In some cases it may be necessary to optimize the
expression system by performing initial control experiments with
various promoters to determine which will work best in the given
culture conditions with the particular stem cells (e.g., origin of
stem cells) and reporter gene used.
[0045] Different types of stem cells can be used for the screening
assays, including mouse and human adult stem cells from the ear,
bone marrow, or other tissue sources, and embryonic stem cells from
mouse or human. Stem cells isolated from other mammalian species
are also acceptable for the screening methods described herein.
[0046] To determine whether a differentiation agent can induce stem
cells to differentiate at least partially into a cell of the spiral
ganglion, rather than a hair cell, methods are provided for
determining the expression of genes known to be expressed in such
cells in vivo. Genes expressed in the spiral ganglion, and useful
as cell marker genes, include ephrinB2, ephrinB3, trkB, trkC,
GATA3, BF1, FGF10, FGF3, CSP, GFAP, and Islet1.
[0047] Secondary assays can be used to confirm, or provide more
definitive evidence, that a cell has differentiated into a cell of
the inner ear. For example, a gene useful as a marker for
identifying a cell of the inner ear can be expressed exclusively in
a particular cell type (e.g., exclusively in a hair cell or
exclusively in cells of the spiral ganglion), or the cell may also
be expressed in a few other cell types (preferably not more than
one, two, three, four, or five other cell types). For example,
ephrinB1 and ephrinB2 are expressed in spiral ganglion cells, and
also in retinal cells. Thus detection of ephrinB1 or ephrinB2
expression is not definitive proof that a stem cell has
differentiated into a cell of the spiral ganglion. Secondary assays
can be used to confirm that a cell has developed into a cell of the
spiral ganglion. Such assays include detection of multiple genes
known to be expressed in the suspected cell type. For example, a
cell that expresses ephrinB1 and/or ephrinB2, can also be assayed
for expression of one or more of GATA3, trkB, trkC, BF1, FGF10,
FGF3, CSP, GFAP, and Islet1. A determination that these additional
genes are expressed is additional evidence that a stem cell has
differentiated into a spiral ganglion cell.
[0048] In embodiments where a primary assay includes the use of a
reporter gene under control of a tissue-specific promoter, a
secondary assay can include detection of the endogenous protein
expressed from the endogenous promoter. For example, in a primary
screen that assays for expression of luciferase fused to an
ephrinB1 promoter in a plasmid, the secondary screen can include an
immunocytochemistry assay to detect endogenous ephrinB1 protein,
which is expressed from the endogenous ephrinB1 promoter.
[0049] Secondary assays also include detection of the absence of
gene expression or the absence of proteins that are not typically
expressed in hair cells. Such negative markers include the
pan-cytokeratin gene, which is not expressed in mature hair cells
but is expressed in supporting cells of the inner ear (Li et al.,
Nature Medicine 9:1293-1299, 2003).
[0050] The agents identified as being capable of causing stem cells
to differentiate into cells of the inner ear can function by
activating a gene or protein necessary for differentiation of a
stem cell. For example, a differentiation agent can activate or
increase expression or activity of a gene of the hedgehog pathway,
such as Sonic hedgehog (Shh). Alternatively, an identified agent
can function by inhibiting activity of a gene or protein that
prevents differentiation of a stem cell into a cell of the inner
ear. For example, the agent can inhibit the gene expression or
protein activity of hes1, hes5, p19.sup.Ink4d, or proteins of the
Notch family. Many different proteins have been identified as being
important for establishing and maintaining the phenotype of the
inner ear. These include developmental regulators, cell cycle
inhibitors, transcription factors, and other regulatory proteins
known to influence the activity of stem cells. It is not necessary
that the effect of an agent on a cell be the complete
differentiation of the stem cell. A stem cell that is partially
differentiated may continue to express some genes that typically
inhibit stem cell differentiation (although expression may be
weaker). If the agent triggers the cell to differentiate at least
partially into a cell of the inner ear, the agent may be useful as
a therapeutic agent or as an agent for generating cells having
therapeutic value for treatment of hearing disorders by the methods
described herein.
[0051] Small molecule libraries can be screened against proteins
known to be required for preventing the conversion of stem cells to
hair cells or spiral ganglion cells. Transcription factors, for
example, are required for proper timing of the differentiation of
an embryo, and they can prevent the formation of inner ear cells,
such as by preventing mitosis. Inhibition of these factors in a
stem cell can increase the number of cells that will eventually be
converted to the inner ear phenotype. Screening for molecules that
can interact with such factors will lead to the discovery of agents
that have high affinity for the polypeptide factors.
Protein/protein interaction assays are known in the art and include
co-immunoprecipitation-based assays; binding assays, such as
bead-based binding assays; or cell-based assays such as the yeast
two-hybrid assay, or a related method.
[0052] The ability of the differentiation agents to inhibit or
enhance the biological activity of the proteins can be assessed
using assays that measure the conversion of the stem cells to inner
ear cells. Such assays are described herein and include the
detection of inner ear cell-specific markers, or reporter gene
assays, wherein expression of a reporter gene indicates conversion
of a stem cell to an inner ear cell.
[0053] The screens featured in the invention can also be used to
identify agents that increase the yield or rate of differentiation
of stem cells. Retinoic acid, for example, can induce stem cells to
differentiate into a variety of cell types including, but not
specific for, hair cells. Agents can be identified that are more
specific for inducing differentiation of cells to hair or spiral
ganglion cells.
[0054] Stem cells that are grown in the presence of supplemental
growth factors, and then transferred to growth medium lacking
supplemental growth factors will be induced to differentiate into
hair cells. Supplemental growth factors are added to the culture
medium. They are not required for cell survival, but the type and
concentration of the supplementary growth factors can be adjusted
to modulate the growth characteristics of the cells (e.g., to
stimulate or sensitize the cells to differentiate). Thus a
candidate differentiation agent (e.g., a polypeptide, nucleic acid,
or small molecule) can be tested for an effect on the
differentiation of the stem cell when the cell is transferred to a
medium lacking growth factors and contacted with the agent, as
compared to the differentiation of a stem cell that is not
contacted with a test agent. Alternatively, or additionally, an
effect of the agent can be examined in the presence of growth
factors, and the concentration of growth factors can be lowered to
increase the likelihood of triggering the cells to differentiate.
Concentrations of growth factors can range from about 100 ng/mL to
about 0.5 ng/mL (e.g., from about 80 ng/mL to about 3 ng/mL, such
as about 60 ng/mL, about 50 ng/mL, about 40 ng/mL, about 30 ng/mL,
about 20 ng/mL, about 10 ng/mL, or about 5 ng/mL).
[0055] Exemplary supplementary growth factors are discussed in
detail below, and include, but are not limited to basic fibroblast
growth factor (bFGF), insulin-like growth factor (IGF), and
epidermal growth factor (EGF).
[0056] Screens provided herein include screens to identify genes
that can influence development of cells of the inner ear. The
identified genes can be targets of the agents discovered by the
screens described above. Genes that can influence development of
cells of the inner ear can promote differentiation or inhibit
differentiation.
[0057] To identify genes that promote differentiation, the reporter
stem cells described above can be utilized. These cells express a
reporter gene, such as luciferase, under control of a cell specific
promoter, or promoter fragment. The promoter can be specific for
hair cells (e.g., a myoVIIa, Math1, .alpha.9 acetylcholine
receptor, espin, parvalbumin 3, or Brn3.1 promoter) or auditory
neural cells, such as spiral ganglion cells (e.g., an ephrinB2,
ephrinB3, trkB, trkC, GATA3, BF1, FGF10, FGF3, CSP, GFAP, or Islet1
promoter), for example.
[0058] According to one exemplary screen, such as a library (e.g.,
a cDNA library) screen, the candidate genes of the library are
cloned into plasmids (standard library screening protocols such as
those described in Brent et al. (Current Protocols in Molecular
Biology, New York: John Wiley & Sons Inc, 2003) can be
followed). The plasmid used in the library can contain a
constitutive promoter, such as a CMV promoter, that drives
expression of the candidate gene. The plasmids of the library are
introduced into a reporter stem cell line that is cultured in
medium containing supplemental growth factors. The transfection of
the plasmids into the reporter cell line is performed such that
only one plasmid is introduced into any one cell. The cell is
examined for an increase in luminescence, by comparison to a
reporter cell that has been transfected with a plasmid lacking the
candidate gene. An increase in luminescence indicates that the gene
promotes the differentiation of the stem cell into a cell of the
inner ear. The specific promoter driving expression of the
luciferase gene dictates the cell type for which the reporter assay
is useful for monitoring differentiation. For example, if the
luciferase gene is under control of a hair cell specific promoter,
an increase in luminescence indicates that the candidate gene
promotes differentiation of hair cells. If the luciferase gene is
under control of a spiral ganglion-specific promoter, an increase
in luminescence indicates that the candidate gene promotes
differentiation of spiral ganglion cells.
[0059] The increase in luminescence can be observed while the cells
remain cultured in the presence of growth factors, or the cells can
be transferred to lower concentrations of growth factors, or to
other modified conditions that may sensitize the cells for
differentiation. In yet another alternative, the cells can be
completely removed from the supplemental growth factors, to compare
the luminescence in the presence and absence of the candidate
gene.
[0060] The screening method can be modified to identify genes that
inhibit differentiation. According to one such modified screen, an
inhibitory agent, such as a small interfering RNA (siRNA),
antisense RNA, ribozyme, antibody, or small molecule, is contacted
with a reporter stem cell. The inhibitory agent targets a candidate
gene (e.g., an endogenous candidate gene) for down regulation. For
example, an siRNA or antisense RNA can block translation of a
target RNA, or an antibody or small molecule compound can block the
activity of a target protein.
[0061] The reporter stem cells are cultured in the presence of
growth factors, and they can remain in the presence of growth
factors, when the cell is contacted with the inhibitory agent.
Alternatively, the cells can be transferred to a lower
concentration of growth factors to sensitize the cells for
differentiation. In yet another alternative, the cells can be
completely removed from the supplemental growth factors, to compare
the luminescence in the presence and absence of the candidate
gene.
[0062] Following contact with the inhibitory agent, the cell is
examined for an increase in luminescence, and the signal intensity
is compared to a control cell. The control cell can be contacted
with an agent that does not target any gene in the cell, or an
agent that targets a gene known not to influence (promote or
inhibit) differentiation of stem cells into cells of the inner ear,
or the control cell may not be contacted with any agent. An
increase in luminescence indicates that the gene can inhibit the
differentiation of the stem cell into a cell of the inner ear. As
described above, the specific promoter driving expression of the
luciferase gene dictates the cell type for which the reporter assay
is useful for monitoring differentiation. For example, if the
luciferase gene is under control of a hair cell specific promoter,
an increase in luminescence indicates that the candidate gene
inhibits differentiation of hair cells. If the luciferase gene is
under control of a spiral ganglion-specific promoter, an increase
in luminescence indicates that the candidate gene inhibits
differentiation of spiral ganglion cells. The agent can be tested
against different reporter cell lines (e.g., lines for testing
differentiation of hair cells, and lines for testing
differentiation of spiral ganglion cells). Some candidate genes may
be found to inhibit differentiation of stem cells to multiple
different tissue cell types.
[0063] The screens are useful for determining whether a candidate
gene can influence stem cell differentiation. Known candidate genes
have previously been implicated in ear development or in disorders
related to the ear, and many of these genes are listed in Table 1.
The screens are also useful for identifying genes not previously
recognized as being involved in ear cell differentiation or
function. To identify such genes, libraries can be assayed with the
described screens. Libraries can be commercially obtained or can be
constructed from nucleic acids isolated from specific desired
tissues. The libraries can be cDNA libraries constructed from RNA
isolated from a mammal, such as a mouse or a human. The RNA can be
isolated from a specific tissue of a mammal, such as the brain
(e.g., mouse brain or human striatum). The described screens can be
modified for high throughput, such as for use in 96-well plates. An
agent identified in a screen as being capable of influencing the
differentiation of a stem cell into an ear cell can be used to
generate ear cells in the laboratory for further research or for
treatment of a hearing disorder or other ear-related disorders.
[0064] A plasmid can drive overexpression or low-level expression
of a candidate gene or inhibitory agent in a reporter stem cell
line. In one embodiment, the plasmid can be an adenoviral vector.
For example, an adenoviral vector can drive expression of a
candidate gene or an inhibitory agent, such as an siRNA, antisense
RNA, or ribozyme. The adenoviral vector can drive expression of the
candidate gene or inhibitory agent from a promoter, such as a
constitutive promoter (e.g., a CMV or human U6 promoter).
Libraries, including overexpression or knockdown libraries, are
also suitable for use in the methods described herein.
[0065] Treatment methods. The agents (e.g., polypeptides, nucleic
acids, small molecules, and the like) identified by the screening
methods described above can be used to generate cells for
therapeutic use. Treatment methods include generating cells of the
inner ear (e.g., hair cells or cells of the spiral ganglion) from
stem cells for transplantation into an ear of a human in need
thereof. Methods of culturing cells of the inner ear include
culturing stem cells under conditions that cause the stem cell to
differentiate into a cell of the inner ear. Transplantation of the
cells into the inner ear of a subject can be useful for restoring
or improving the ability of the subject to hear, or for decreasing
the symptoms of vestibular dysfunction. Inner ear cells derived
from stem cells according to the methods described herein need not
be fully differentiated to be therapeutically useful. A partially
differentiated cell that improves any symptom of a hearing disorder
in a subject is useful for the therapeutic compositions and methods
described herein.
[0066] Methods of generating cells of the inner ear are provided.
Ear cells or ear cell progenitors can be generated from stem cells
isolated from a mammal, such as a mouse or human, and the cells can
be embryonic stem cells or stem cells derived from mature (e.g.,
adult) tissue, such as the inner ear, central nervous system,
blood, skin, eye or bone marrow. Any of the methods described above
for culturing stem cells and inducing differentiation into ear
cells (e.g., hair cells or cells of the spiral ganglion) can be
used.
[0067] Methods of isolating a stem cell or progenitor cell from the
inner ear of an animal are also featured in the invention. These
methods include providing tissue from the inner ear of the animal,
where the tissue includes at least a portion of the utricular
maculae. The animal can be a mammal, such as a mouse, rat, pig,
rabbit, goat, horse, cow, dog, cat, primate, or human. The isolated
tissue 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. In one alternative, both mechanisms of tissue
disruption are 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. The cells can be frozen for future use or
placed in culture for differentiation.
[0068] The separated cells can be placed in individual wells of a
culture dish at a low dilution, and cultured to differentiate and
into cells of the inner ear, or to differentiate into inner-ear
like cells to various stages of the differentiation process. Thus,
partially or fully differentiated cells are useful for the methods
described herein. The cells can be separated into one cell per
well. Formation of spheres (clonal floating colonies) from the
isolated cells can be monitored, and the spheres can be amplified
by disrupting them (e.g., by physically means) to separate the
cells, and the cells can be cultured again to form additional
spheres. Further culturing of the cells in the absence of or in
lower amounts of growth factors will induce the spheres (and the
cells of the spheres) to differentiate further into more highly
developed cells of the inner ear.
[0069] Appropriate culture medium is described in the art, such as
in Li et al (supra). For example, stem cells can be cultured in
serum free DMEM/high-glucose and F12 media (mixed 1:1), and
supplemented with N2 and B27 solutions and growth factors. Growth
factors such as EGF, IGF-1, and bFGF have been demonstrated to
augment sphere formation in culture. In vitro, stem cells often
show a distinct proliferation potential for forming spheres. Thus,
the identification and isolation of spheres can aid in the process
of isolating stem cells from mature tissue for use in making
differentiated cells of the inner ear. The growth medium for
cultured stem cells can contain one or more or any combination of
growth factors, provided that the stem cells do not differentiate.
To induce the cells (and the cells of the spheres) to
differentiate, the medium can be exchanged for medium lacking
growth factors. For example, the medium can be serum-free DMEM/high
glucose and F12 media (mixed 1:1) supplemented with N2 and B27
solutions. Equivalent alternative media and nutrients can also be
used. Culture conditions can be optimized using methods known in
the art.
[0070] The cells can be monitored for expression of cell-specific
markers. For example, hair cells can be identified by the
expression of myoVIIa, Math1, .alpha.9 acetylcholine receptor,
espin, parvalbumin 3, or Brn3.1. Cells of the spiral ganglion can
be identified by the expression of ephrinB2, ephrinB3, trkB, trkC,
GATA3, BF1, FGF10, FGF3, CSP, GFAP, and Islet1.
[0071] An agent capable of causing differentiation of a stem cell
into a cell of the inner ear can be administered directly to the
ear of a human requiring such treatment, and the administration of
the agent can generate hair cell growth in the ear (e.g., in the
inner, middle, and/or outer ear). The number of hair cells in the
ear can be increased about 2-, 3-, 4-, 6-, 8-, or 10-fold or more
as compared to the number of hair cells before treatment with the
agent. This new hair cell growth can effectively restore or
establish at least a partial improvement in the subject's ability
to hear. For example, administration of an agent can improve
hearing loss by about 5, 10, 15, 20, 40, 60, 80, 100% or more.
[0072] Pharmaceutical compositions can include one or more ear cell
differentiation agents identified as being capable of causing a
pluripotent stem cell to differentiate into a cell of the inner
ear. The pharmaceutical compositions provided herein can generate
hair cell growth in any region of the ear, such as in the inner,
middle, and/or outer regions of the ear. For example, a
differentiation agent can generate hair cell growth in the cochlea
or the vestibular system of the inner ear. Pharmaceutical
compositions can also include any of the secondary factors
discussed above, including factors to enhance cell engraftment or
neurite extension. Exemplary formulations are described in greater
detail below. A composition as described herein can be packaged and
labeled for use as a treatment for a hearing disorder.
[0073] A human having a disorder of the inner ear, or at risk for
developing such a disorder, can be treated with inner ear cells
(hair cells or spiral ganglion cells) generated from stem cells. In
a successful engraftment, at least some transplanted spiral
ganglion neurons, for example, will form synaptic contacts with
hair cells and with targets in the cochlear nucleus. To improve the
ability of the cells to engraft, the stem cells can be modified
prior to differentiation. For example, the cells can be engineered
to overexpress one or more anti-apoptotic genes in the progenitor
or differentiated cells. The Fak tyrosine kinase or Akt genes are
candidate anti-apoptotic genes that can be useful 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., Nat. Med. 9:1195-201, 2003). 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., Audiol. Neurootol. 6:57-65, 2001). 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., J. Comp. Neurol. 462:90-100, 2003). 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.,
NeuroReport 12:275-279, 2001). A Sonic hedgehog (Shh) polypeptide
or polypeptide fragment (e.g., SHH-N), can also be useful as an
endogenous factor to enhance neurite extension. Shh is a
developmental modulator for the inner ear and a chemoattractant for
axons (Charron et al., Cell 113:11 23, 2003).
[0074] Any human experiencing or at risk for developing a hearing
loss is a candidate for the treatment methods described herein. For
example, the human can receive a transplant of inner ear hair cells
or spiral ganglion cells generated by exposure to a differentiation
agent, or the human can be administered an agent identified as
being capable of causing a stem cell to differentiate into a cell
of the inner ear. A human having or at risk for developing a
hearing loss can hear less well than the average human being, or
less well than a human before experiencing the hearing loss. For
example, hearing can be diminished by at least 5, 10, 30, 50% or
more. The human can have sensorineural hearing loss, which results
from damage or malfunction of the sensory part (the cochlea) 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, or the human can have mixed hearing loss, which is
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.
[0075] The subject can be deaf or have a hearing loss for any
reason or as a result of any type of event. For example, a human
can be deaf because of a genetic or congenital defect; for example,
a human 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 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 concert venues, airport runways, and construction
areas can cause inner ear damage and subsequent hearing loss. A
human 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. A human can have a hearing disorder that
results from aging, or the human can have tinnitus (characterized
by ringing in the ears).
[0076] A human suitable for the therapeutic compositions and
methods featured in the invention can include a human 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).
[0077] Following treatment with an agent or inner ear cell or inner
ear cell progenitor described herein, the human can be tested for
an improvement in hearing or in other symptoms related to inner ear
disorders. Methods for measuring hearing are well-known and include
pure tone audiometry, air conduction, 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 cochlear hair cells, and electro-cochleography provides
information about the functioning of the cochlea and the first part
of the nerve pathway to the brain.
[0078] The therapeutic compositions and methods featured in the
invention can be used prophylactically, such as to prevent hearing
loss, deafness, or other auditory disorder associated with loss of
inner ear function. For example, a composition containing a
differentiation agent can be administered with a second
therapeutic, such as a therapeutic that may effect a hearing
disorder. 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.
[0079] For example, a human undergoing chemotherapy can also be
administered a differentiation agent described herein or an agent
identified by a method described herein. The chemotherapeutic agent
cisplatin, for example, is known to cause hearing loss. Therefore,
a composition containing a differentiation agent can be
administered with cisplatin therapy to prevent or lessen the
severity of the cisplatin side effect. A composition containing a
differentiation agent can be administered before, after and/or
simultaneously with the second therapeutic agent. The two agents
may be administered by different routes of administration.
[0080] The compositions and methods featured in the invention are
appropriate for the treatment of hearing disorders resulting from
sensorineural hair cell loss or auditory neuropathy. Patients
suffering from auditory neuropathy experience a loss of cochlear
sensory neurons while the hair cells of the inner ear remain
intact. Such patients will benefit particularly from treatment that
causes cells (stem cells or progenitor cells) to differentiate into
spiral ganglion cells, or from administration of spiral ganglion
cells into the inner ear. Patients with sensorineural hair cell
loss experience the degeneration of cochlear hair cells, which
frequently results in the loss of spiral ganglion neurons in
regions of hair cell loss. Such patients 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. These patients can receive treatment with an agent that
causes cells to differentiate into hair cells, or a tissue
transplant containing hair cells grafted or injected into the inner
ear. The patients may additionally benefit from treatment that
causes cells to differentiate into spiral ganglion cells, or from
administration of spiral ganglion cells into the inner ear.
[0081] Formulations and Routes of Administration. Differentiation
agents identified by the methods described above can be formulated
for administration to a subject diagnosed as having or at risk for
developing a hearing loss or vestibular disorder. Pharmaceutical
compositions containing a differentiation agent can be formulated
in a conventional manner using one or more physiologically
acceptable carriers or excipients. For example, a differentiation
agent can be formulated for administration by drops into the ear,
insufflation (such as into the ear), topical, or oral
administration.
[0082] In another mode of administration, the differentiation agent
can be directly administered in situ to the cochlea of the inner
ear, such as via a catheter or pump. A catheter or pump can, for
example, direct a differentiation agent into the cochlear luminae
or the round window of the ear.
[0083] In another route of administration, a differentiation agent
can be injected into the ear, such as into the luminae of the
cochlea (e.g., the Scala media, Sc vestibulae, and Sc tympani).
Injection can be, for example, through the round window of the ear
or through the cochlear capsule.
[0084] Ear cells generated by the methods described above can be
transplanted, 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.
[0085] The nature of the pharmaceutical compositions for
administration is dependent on the mode of administration and can
readily be determined by one of ordinary skill in the art. The
therapeutic compositions feature in the invention can contain
carriers or excipients, many of which are known to skilled
artisans. Excipients that can be used include buffers (for example,
citrate buffer, phosphate buffer, acetate buffer, and bicarbonate
buffer), amino acids, urea, alcohols, ascorbic acid, phospholipids,
polypeptides (for example, serum albumin), EDTA, sodium chloride,
liposomes, mannitol, sorbitol, and glycerol. The nucleic acids,
polypeptides, small molecules, and other modulatory compounds
featured in the invention can be administered by any standard route
of administration. For example, administration can be parenteral,
intravenous, subcutaneous, or oral. A modulatory compound can be
formulated in various ways, according to the corresponding route of
administration. For example, liquid solutions can be made for
administration by drops into the ear, for injection, or for
ingestion; gels or powders can be made for ingestion or topical
application. Methods for making such formulations are well known
and can be found in, for example, "Remington's Pharmaceutical
Sciences."
[0086] The differentiation agents described herein or identified by
a method described herein, can be administered directly to the
inner ear (e.g., by injection or through surgical placement). Other
compositions (e.g., pharmaceutically acceptable compositions
containing stem cells, progenitor cells, or auditory cells
differentiated by a method described herein) can also be
administered directly to the inner ear. The amount of the
differentiation agent or the amount of a cell-based composition may
be described as a therapeutically effective amount. Where
application over a period of time is advisable or desirable, the
compositions of the invention can be placed in sustained released
formulations or implantable devices (e.g., a pump).
[0087] The pharmaceutical compositions can be formulated for
parenteral administration by injection, for example, by bolus
injection or continuous infusion. Formulations for injection may be
presented in unit dosage form, for example, in ampoules or in
multi-dose containers, with an added preservative. The compositions
may take such forms as suspensions, solutions or emulsions in oily
or aqueous vehicles, and may contain formulatory agents such as
suspending, stabilizing and/or dispersing agents. Alternatively,
the active ingredient may be in powder form for constitution with a
suitable vehicle, for example, sterile pyrogen-free water, before
use.
[0088] In addition to the formulations described previously, the
compositions can also be formulated as a depot preparation. Such
long acting formulations can be administered by implantation (e.g.,
subcutaneously). Thus, for example, the compositions can be
formulated with suitable polymeric or hydrophobic materials (for
example as an emulsion in an acceptable oil) or ion exchange
resins, or as sparingly soluble derivatives, for example, as a
sparingly soluble salt.
[0089] Pharmaceutical compositions formulated for oral
administration can take the form of tablets or capsules prepared by
conventional means with pharmaceutically acceptable excipients such
as binding agents (for example, pregelatinised maize starch,
polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers
(for example, lactose, microcrystalline cellulose or calcium
hydrogen phosphate); lubricants (for example, magnesium stearate,
talc or silica); disintegrants (for example, potato starch or
sodium starch glycolate); or wetting agents (for example, sodium
lauryl sulphate). The tablets can be coated by methods well known
in the art. Liquid preparations for oral administration may take
the form of, for example, solutions, syrups or suspensions, or they
may be presented as a dry product for constitution with water or
other suitable vehicle before use. Such liquid preparations may be
prepared by conventional means with pharmaceutically acceptable
additives such as suspending agents (for example, sorbitol syrup,
cellulose derivatives or hydrogenated edible fats); emulsifying
agents (for example, lecithin or acacia); non-aqueous vehicles (for
example, almond oil, oily esters, ethyl alcohol or fractionated
vegetable oils); and preservatives (for example, methyl or
propyl-p-hydroxybenzoates or sorbic acid). The preparations may
also contain buffer salts, flavoring, coloring and sweetening
agents as appropriate. Preparations for oral administration may be
suitably formulated to give controlled release of the active
compound.
[0090] The compositions may, if desired, be presented in a pack or
dispenser device that may contain one or more unit dosage forms
containing the active ingredient. The pack may for example comprise
metal or plastic foil, such as a blister pack. The dispenser device
may include a liquid dropper for administration of a therapeutic
agent dropwise into the ear. The pack or dispenser device can be
accompanied by instructions for administration.
[0091] The efficacy of the treatment methods described herein can
be assayed by determining an improvement in the subject's ability
to hear, or by an improvement in other symptoms such as balance.
Alternatively, efficacy can be assayed by measuring distortion
product otoacoustic emissions (DPOAEs) or compound action potential
(CAP).
[0092] The pharmaceutical compositions and methods described herein
can be used independently or in combination with one another. That
is, subjects can be administered one or more of the pharmaceutical
compositions, for example, pharmaceutical compositions containing a
differentiation agent subjected to one or more of the therapeutic
methods described herein, or both, in temporally overlapping or
non-overlapping regimens. The subject can also be administered a
solution or tissue containing the differentiated cells generated
from stem cells as described above. One or both of these therapies
can be administered in addition to a mechanical device such as a
cochlear implant or a hearing aid, which is worn in the outer ear.
When therapies overlap temporally, the therapies may generally
occur in any order and can be simultaneous or interspersed.
[0093] The differentiation agents for use in the methods featured
in the invention can be packaged as pharmaceutical compositions and
labeled for any use as described herein. For example, the package
can be labeled for use to treat a hearing disorder.
[0094] Effective Dose. Toxicity and therapeutic efficacy of the
compositions disclosed in the invention (e.g., pharmaceutical
compositions including the differentiation agents), can be
determined by standard pharmaceutical procedures, using either
cells in culture or experimental animals to determine the LD.sub.50
(the dose lethal to 50% of the population) and the ED.sub.50 (the
dose therapeutically effective in 50% of the population). The dose
ratio between toxic and therapeutic effects is the therapeutic
index and can be expressed as the ratio LD.sub.50/ED.sub.50.
Polypeptides or other compounds that exhibit large therapeutic
indices are preferred.
[0095] Data obtained from cell culture assays and further animal
studies can be used in formulating a range of dosage for use in
humans. The dosage of such compounds lies preferably within a range
of circulating concentrations that include the ED.sub.50 with
little or no toxicity, and with little or no adverse effect on a
human's ability to hear. The dosage may vary within this range
depending upon the dosage form employed and the route of
administration utilized. For any compound used in the methods
described herein, the therapeutically effective dose can be
estimated initially from cell culture assays. A dose can be
formulated in animal models to achieve a circulating plasma
concentration range that includes the IC.sub.50 (that is, the
concentration of the test compound which achieves a half-maximal
inhibition of symptoms) as determined in cell culture. Such
information can be used to more accurately determine useful doses
in humans. Exemplary dosage amounts of a differentiation agent are
at least from about 0.01 to 3000 mg per day, e.g., at least about
0.00001, 0.0001, 0.001, 0.01, 0.1, 1, 2, 5, 10, 25, 50, 100, 200,
500, 1000, 2000, or 3000 mg per kg per day, or more.
[0096] The formulations and routes of administration can be
tailored to the specific hearing disorder being treated, and for
the specific human being treated. For example, the human can have
been deaf from birth due to a genetic or environmental event, or a
child or adult human can be losing hearing due to environmental
factors such as prolonged exposure to loud noises, or a human can
be experiencing a hearing loss due to aging. Therefore the human
can be any age (e.g., an infant or an elderly person), and
formulation and route of administration can be adjusted
accordingly. A subject can receive a dose of the agent once or
twice or more daily for one week, one month, six months, one year,
or more. The treatment can continue indefinitely, such as
throughout the lifetime of the human. Treatment can be administered
at regular or irregular intervals (once every other day or twice
per week), and the dosage and timing of the administration can be
adjusted throughout the course of the treatment. The dosage can
remain constant over the course of the treatment regimen, or it can
be decreased or increased over the course of the treatment.
[0097] Generally the dosage facilitates an intended purpose for
both prophylaxis and treatment without undesirable side effects,
such as toxicity, irritation or allergic response. Although
individual needs may vary, the determination of optimal ranges for
effective amounts of formulations is within the skill of the art.
Human doses can readily be extrapolated from animal studies (Katocs
et al., Chapter 27 In: "Remington's Pharmaceutical Sciences", 18th
Ed., Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990).
Generally, the dosage required to provide an effective amount of a
formulation, which can be adjusted by one skilled in the art, will
vary depending on several factors, including the age, health,
physical condition, weight, type and extent of the disease or
disorder of the recipient, frequency of treatment, the nature of
concurrent therapy, if required, and the nature and scope of the
desired effect(s) (Nies et al., Chapter 3, In: Goodman &
Gilman's "The Pharmacological Basis of Therapeutics", 9th Ed.,
Hardman et al., eds., McGraw-Hill, New York, N.Y, 1996).
[0098] Kits. A differentiation agent described herein or identified
by a method described herein can be provided in a kit, as can cells
that have been induced to differentiate (e.g., stem cells or
progenitor cells that have differentiated into, for example, hair
cells or hair-like cells). The kit can include (a) the agent, such
as in a composition that includes the agent, and (b) informational
material. The informational material can be descriptive,
instructional, marketing or other material that relates to the
methods described herein and/or to the use of the agent for the
methods described herein. For example, the informational material
relates to the use of a differentiation agent to treat a subject
who has, or who is at risk for developing, a hearing disorder. The
kits can also include paraphernalia for administering a
differentiation agent to a cell (in culture or in vivo) and/or for
administering a cell to a patient.
[0099] In one embodiment, the informational material can include
instructions for administering the differentiation agent and/or
cell(s) in a suitable manner to treat a human, e.g., in a suitable
dose, dosage form, or mode of administration (e.g., a dose, dosage
form, or mode of administration described herein). For example,
doses, dosage forms, or modes of administration can be by liquid
drops into the ear, such as from a dropper bottle, or the
composition can be administered directly to the ear such as through
a catheter or pump. In another embodiment, the informational
material can include instructions to administer the differentiation
agent to a suitable subject, e.g., a human, e.g., a human having,
or at risk for developing, a hearing disorder. For example, the
material can include instructions to administer the agonist to a
subject who has experienced a hearing loss due to a traumatic
event, or to a subject who has received a separate therapeutic
agent that causes hearing loss, such as the antibiotics and
chemotherapeutic agents discussed herein.
[0100] The informational material of the kits is not limited in its
form. In many cases, the informational material (e.g.,
instructions) is provided in printed matter, such as in a printed
text, drawing, and/or photograph, such as a label or printed sheet.
However, the informational material can also be provided in other
formats, such as Braille, computer readable material, video
recording, or audio recording. In another embodiment, the
informational material of the kit is contact information, such as a
physical address, email address, website, or telephone number,
where a user of the kit can obtain substantive information about
the hedgehog pathway agonist and/or its use in the methods
described herein. Of course, the informational material can also be
provided in any combination of formats.
[0101] In addition to the differentiation agent, the composition of
the kit can include other ingredients, such as a solvent or buffer,
a stabilizer, a preservative, a fragrance or other cosmetic
ingredient, and/or a second agent for treating a condition or
disorder described herein (e.g., a hearing disorder).
Alternatively, the other ingredients can be included in the kit,
but in different compositions or containers than the agent. In such
embodiments, the kit can include instructions for admixing the
agent and the other ingredients, or for using the agent together
with the other ingredients.
[0102] The differentiation agent (e.g., a hedgehog agonist) can be
provided in any form, including a liquid, dried or lyophilized
form. The agent is preferably substantially pure and/or sterile.
When the agent is provided in a liquid solution, the liquid
solution preferably is an aqueous solution, with a sterile aqueous
solution being preferred. When the agent is provided as a dried
form, reconstitution generally is by the addition of a suitable
solvent. The solvent, e.g., sterile water or buffer, can optionally
be provided in the kit.
[0103] The kit can include one or more containers for the
composition containing the differentiation agent. In some
embodiments, the kit contains separate containers, dividers or
compartments for the composition and informational material. For
example, the composition can be contained in a bottle (e.g., a
dropper bottle, such as for administering drops into the ear),
vial, or syringe, and the informational material can be contained
in a plastic sleeve or packet. In other embodiments, the separate
elements of the kit are contained within a single, undivided
container. For example, the composition is contained in a bottle,
vial or syringe that has attached thereto the informational
material in the form of a label. In some embodiments, the kit
includes a plurality (e.g., a pack) of individual containers, each
containing one or more unit dosage forms (e.g., a dosage form
described herein) of the hedgehog pathway agonist. For example, the
kit can include a plurality of syringes, ampoules, foil packets, or
blister packs, each containing a single unit dose of the hedgehog
pathway agonist. The containers of the kits can be air tight and/or
waterproof, and the containers can be labeled for a particular use.
For example, a container can be labeled for use to treat a hearing
disorder.
[0104] As noted above, the kits optionally include a device
suitable for administration of the composition (e.g., a syringe,
pipette, forceps, dropper (e.g., ear dropper), swab (e.g., a cotton
swab or wooden swab), or any such delivery device). The device can
be a dropper for administration to the ear.
[0105] Hedgehog Pathway Agonists as Differentiation Agents.
Exemplary candidates for use in the treatment methods and
pharmaceutical compositions featured in the invention include
hedgehog pathway agonists. A hedgehog pathway agonist is a
molecule, such as a polypeptide, drug, or nucleic acid that
stimulates a hedgehog signaling pathway.
[0106] One exemplary hedgehog pathway agonist is a Sonic hedgehog
(SHH) polypeptide (SEQ ID NO:1), or fragment of an SHH polypeptide,
particularly an N-terminal fragment (FIG. 1). In vivo, SHH
undergoes an autoproteolysis event to generate two biochemically
distinct products, an 18K amino-terminal fragment, "N," and a 25K
carboxy-terminal fragment, "C" (Lee et al., Science 266:1528-1537,
1994). In Drosophila, cleavage occurs between residues Gly257 and
Cys258 (of a conserved Gly-Cys-Phe tripeptide), and the cleavage at
this site is conserved in other organisms, including at the site of
the corresponding conserved Gly-Cys-Phe tripeptide in mouse and
human SHH proteins Cys197 (Porter et al., Nature 374:363-366). For
example, the cleavage of human SHH occurs between Gly196 and
Cys197. The N-terminal cleavage product is referred to as SHH-N.
SHH-N polypeptides can perform the signaling functions of SHH, and
are suitable for use in the compositions and treatment methods
described herein. An SHH-N polypeptide from any species, preferably
a mammal, more preferably a human, can be used for the compositions
and treatment methods.
[0107] A SHH polypeptide (FIG. 1; SEQ ID NO:1) can be any spliced
isoform of SHH, or fragment or modified polypeptide thereof. For
example, a "modified" polypeptide can be ubiquitinated,
phosphorylated, methylated, or conjugated to any natural or
synthetic molecule, such as a fluorescent tag or heterologous
polypeptide tag. A hedgehog pathway agonist can be a known homolog
of Sonic hedgehog, such as an Indian or Desert hedgehog
polypeptide, or any spliced isoform, or fragment or modified
polypeptide thereof.
[0108] A hedgehog pathway agonist can also be a polypeptide having
a sequence that is substantially identical to the amino acid
sequence of SHH (SEQ ID NO:1). A "substantially identical" gene or
polypeptide is similar in sequence to the human Shh cDNA (SEQ ID
NO:2; FIG. 2) or amino acid sequence (SEQ ID NO:8; FIG. 1),
respectively. A substantially identical nucleic acid sequence is at
least 80% identical to SEQ ID NO:2, and a substantially identical
amino acid sequence is at least 80% identical to SEQ ID NO:1. For
example, a target DNA or RNA sequence can be 80%, 85%, 95%, or 100%
identical. A fragment of a target nucleic acid sequence, e.g., a
sequence that encodes an exon, can be at least 80% identical to a
fragment of SEQ ID NO:2. To determine the percent identity of two
amino acid sequences, or of two nucleic acid sequences, the
sequences are aligned for optimal comparison purposes (e.g., gaps
can be introduced in one or both of a first and a second amino acid
or nucleic acid sequence for optimal alignment and non-homologous
sequences can be disregarded for comparison purposes). In a
preferred embodiment, the length of a reference sequence aligned
for comparison purposes is at least 30%, preferably at least 40%,
more preferably at least 50%, even more preferably at least 60%,
and even more preferably at least 70%, 80%, 90%, or 100% of the
length of the reference sequence (e.g., when aligning a second
sequence to the SHH amino acid sequence of SEQ ID NO:1, having 462
amino acid residues, at least 139, preferably at least 185, more
preferably at least 231, even more preferably at least 277, and
even more preferably at least 323, 370, or 416 amino acid residues
are aligned). The amino acid residues or nucleotides at
corresponding amino acid positions or nucleotide positions are then
compared. When a position in the first sequence is occupied by the
same amino acid residue or nucleotide as the corresponding position
in the second sequence, then the molecules are identical at that
position. The determination of percent identity between two amino
acid sequences is accomplished using the BLAST 2.0 program.
Sequence comparison is performed using an ungapped alignment and
using the default parameters (Blossom 62 matrix, gap existence cost
of 11, per residue gapped cost of 1, and a lambda ratio of 0.85).
The mathematical algorithm used in BLAST programs is described in
Altschul et al. (Nucleic Acids Res. 25:3389-3402, 1997). An SHH
polypeptide or polypeptide fragment, such as SHH-N, or
substantially identical polypeptide, such as Dhh or Ihh, can have
up to about 20 (e.g., up to about 10, 5, or 3) amino acid
deletions, additions, or substitutions, such as conservative
substitutions, to be useful for the compositions and methods
described herein.
[0109] In another aspect, a hedgehog agonist can be a polypeptide
that is substantially identical to the amino acid sequence of
Indian hedgehog (Ihh; see FIG. 3), or Desert hedgehog (Dhh; see
FIG. 4). A hedgehog pathway agonist can also be a polypeptide
fragment (e.g., an N-terminal peptide fragment) of Ihh or Dhh.
[0110] A hedgehog pathway agonist can act on a nucleic acid of a
second (or the same) hedgehog pathway agonist. For example, an
agonist can increase gene expression of a hedgehog polypeptide,
such as by acting as a transcription factor or an enhancer of
transcription (e.g., of a Sonic hedgehog gene), or the agonist can
stabilize (e.g., protect from degradation) a RNA transcript of a
hedgehog pathway agonist. The hedgehog pathway agonist can also (or
alternatively) act on a nucleic acid of a gene that is not a
hedgehog pathway agonist, but which otherwise influences
differentiation of a stem cell or progenitor cell into a cell of
the inner ear.
[0111] Nucleic acids, such as DNA plasmids, can be used in the
methods and compositions described herein, such as for gene
therapy. For example, nucleic acids (and nucleic acid vectors) can
encode polypeptides that act as hedgehog pathway agonists, such as
by any method described herein.
[0112] A hedgehog pathway agonist can be a small molecule, such as
Hh-Ag1.3. A small molecule is a chemical compound that affects the
phenotype of a cell or organism by, for example, modulating the
activity of a specific polypeptide or nucleic acid, such as a
hedgehog polypeptide or nucleic acid, within a cell. A small
molecule can, for example, affect a cell by directly interacting
with a polypeptide or by interacting with a molecule that acts
upstream or downstream of the biochemical cascade that results in
polypeptide expression or activity.
[0113] Other members of the hedgehog signaling pathway, besides
hedgehog polypeptides themselves (e.g., SHH, Ihh, Dhh), can be used
for the treatment methods and compositions described herein. For
example, overexpression or modification of a transcription factor
that regulates expression of a hedgehog pathway agonist can
stimulate hair cell growth. For example, a Gli transcription factor
polypeptide (or a nucleic acid expressing a Gli polypeptide) can be
administered. Alternatively, a polypeptide, a small molecule, drug,
or other modulatory compound that stimulates Gli activity can
function as a hedgehog pathway agonist. The Gli family of
transcription factors is known to stimulate transcription of Sonic
hedgehog in vivo.
[0114] In one alternative, the methods and compositions can include
an activator of a hedgehog pathway agonist receptor. For example, a
polypeptide, small molecule, or other modulatory compound can
activate a Patched and/or Smoothened receptor, both of which are
recognized by Sonic hedgehog in vivo. In another alternative, cells
can be induced to overexpress one or more of the hedgehog pathway
agonist receptors, or nucleic acids can be administered (e.g., by
gene therapy) and induced to express exogenous receptors. For
example, the receptors are expressed on a cell surface to
facilitate interaction with a hedgehog polypeptide and activation
of a hedgehog signaling pathway that ultimately leads to the
development of a hair cell.
[0115] In some embodiments, a hedgehog pathway agonist can
stimulate endogenous hedgehog proteins. For example, the methods
and compositions can include morphogens, growth factors, hormones,
and the like, that stimulate hedgehog protein activity (e.g.,
upregulate gene expression, stimulate protein modification, or
otherwise activate protein activity).
[0116] In some embodiments, a hedgehog pathway agonist can inhibit
an inhibitor of a hedgehog signaling pathway. An inhibitor can be,
for example, a polypeptide (such as an antibody), small molecule,
or other modulatory compound that binds, sequesters, or otherwise
downregulates a component of the hedgehog signaling pathway or
inhibits an inhibitor of a hedgehog signaling pathway.
[0117] A hedgehog pathway agonist can also be applied to a tissue
ex vivo to induce and/or expand the number of hair cells (or
hair-like cells) or the hair cell density of the tissue, such as in
culture conditions. The resulting tissue can be administered, such
as by grafting to the ear (e.g., to the inner ear) of a subject,
thereby treating the subject for a hearing disorder.
[0118] While not being bound by theory, a hedgehog pathway agonist
(e.g., a polypeptide or small molecule agonist) can stimulate hair
cell growth by acting on non-hair cells of the ear and instructing
these cells to differentiate into hair cells. The hair cells of the
mammalian inner ear, for example, are located in the cochlear organ
of Corti, as well as in the vestibular sensory epithelia of the
saccular macula, the utricular macula, and the cristae of the three
semicircular canals. A hedgehog pathway agonist (e.g., a
polypeptide or small molecule agonist) can therefore stimulate hair
cell growth, for example, by acting on non-hair cells of the
cochlear organ of Corti (supporting cells and other cells) and
instructing these cells to differentiate into hair cells.
[0119] The invention is further illustrated by the following
examples, which should not be construed as further limiting.
EXAMPLES
Example 1
Neurons were Isolated from the Inner Ear of a Pig Fetus for Use in
Transplantation Studies
[0120] We isolated pig fetal spiral ganglion cells from the inner
ear after timed pregnancies and placed the cells in culture for
periods up to two weeks. Gestational ages of E36, E41, E49, E60 and
E63 were compared. Following isolation of whole cochlea, the
tissues containing spiral ganglion cells were separated from other
tissues and incubated with trypsin-EDTA at 37.degree. C. for 10
minutes. After three washes with PBS plus DNAse, tissues were
triturated with three pre-calibrated flame polished Pasteur
pipettes with progressively smaller apertures. Cells were
resuspended in PBS plus glucose solution at approximately
100.times.106/ml. The viability of the cells was determined by
trypan blue exclusion assay prior to transplantation. Some cells
were plated on poly-D lysine coated 12-well culture plate in
complete neurobasal medium.
[0121] Immunohistochemical staining revealed that the E36 neurons
did not express neurofilament but did express neuron specific
enolase. At days E49 and later, the neurons expressed neuron
specific enolase and neurofilament as well as galactocerebrosidase.
The later time points yielded an increased ratio of connective
tissue components relative to neurons. The best yield of cells was
at E41 and these cells could be stained with all of these markers.
This time point was therefore selected for the isolation of cells
for transplantation.
Example 2
Embryonic Stem Cell Cultures were Established and Controlled
Differentiation of Different Cell Types was Observed
[0122] We established cultures of the murine ES cell lines
YC5/EYFP, a derivative of the totipotent cell line R1 (Nagy et al.,
Proc Natl Acad Sci USA 90:8424-8, 1993); R1; ROSA26-6; and Sox1-GFP
(Aubert et al., Nat. Biotechnol. 20:1240-5, 2002). YC5/EYFP cells
carry the gene for enhanced yellow fluorescent protein (EYFP) under
control of a promoter composed of a cytomegalovirus immediate early
enhancer coupled to the .beta.-actin promoter (Hadjantonakis et
al., Mech. Dev. 76:79-90, 1998). ROSA26-6 cells and their
derivatives express the lacZ gene encoding the bacterial
beta-galactosidase enzyme (Pirity et al., Methods Cell Biol.
57:279-93, 1998). The Sox1-GFP cells express GFP controlled by the
promoter for the early neural marker Sox1.
[0123] Low passage ES cells are maintained on a feeder layer of
mitotically inactivated primary mouse embryonic fibroblasts (Pirity
et al., Methods Cell Biol. 57:279-93, 1998). Undifferentiated ES
cells proliferate actively and form compact clusters of small
cells. We initiated in vitro differentiation of ES cells in hanging
drop cultures in the absence of embryonic fibroblast feeder cells
and of leukemia inhibitory factor, a cytokine that promotes the
pluripotency of ES cells. Within two days, cell aggregates of
uniform size termed embryoid bodies form in the hanging drops.
[0124] Using a published protocol (Lee et al., Nat. Biotechnol.
18:675-9, 2000), we were able to select neuronal progenitor cell
populations that express the defining marker protein nestin.
Nestin-positive progenitors were subjected to in vitro
differentiation conditions (see Lee et al., Nat. Biotechnol.
18:675-9, 2000) that led to differentiation of astrocytes and
neurons.
[0125] Using protocols for the selection of progenitor cells, we
were able to select inner ear progenitor cells that express a
variety of marker genes indicative of the developing inner ear. In
particular, we found after selection from embryoid body-derived
cells, cell populations that expressed genes indicative of the otic
placode, such as Pax2, BMP4, and BMP7 (Morsli et al., J. Neurosci.
18:3327-35, 1998; Groves and Bronner-Fraser, Development
127:3489-99, 2000). In addition, we found expression of marker
genes for the developing sensory epithelia--for example Math1
(Bermingham et al., Science 284:1837-41, 1999), delta1, jagged1 and
jagged2 (Lanford et al., Nat. Genet. 21:289-92, 1999; Morrison et
al., Mech. Dev. 84:169-72, 1999). Gene expression was detected by
reverse transcription followed by polymerase chain reaction
(RT-PCR). The differentiated cells were analyzed 14 days after the
removal of bFGF from the culture. The expression of the marker
genes correlated with the developmental stage of the progenitor or
mature cells as nestin and Pax2 and BMP7 expression decreased upon
differentiation of the cells and appearance of hair cell markers
(FIG. 6).
[0126] Hair cell markers in differentiated cells were also detected
by immunohistochemistry. The hair cells produced in this system
co-expressed markers important for hair cell differentiation
(Math1) and survival (Brn3.1) and markers present in the more fully
differentiated cells (myosin VIIa).
[0127] In preliminary experiments we explored whether it was
feasible to isolate from embryoid bodies clonal lines that
represent hair cell and neural progenitors. We were able to
generate spheres that contained progenitors, which we identified by
expression of the early neural marker Sox1 and the intermediate
filament protein nestin. We were able to propagate these progenitor
cells in serum-free conditions for more than three months either in
form of spheres or as adherent cultures in the presence of
mitogenic growth factors. We routinely observed differentiation of
the progenitor cells after removal of growth factors in adherent
cultures.
Example 3
Different Neuronal Progenitor Cells were Generated from ES
Cells
[0128] We explored whether it was feasible to use embryoid bodies
to isolate clonal lines that represent neural progenitors. One goal
of the project was to generate neurons with different features that
could be used to generate neural populations that are very similar
to spiral ganglion neurons. The principal idea of this technique
was to use the sphere-forming capacity of neural stem cells to
clone different cell lines. Our initial results indicated that we
were able, for example, to generate spheres that contain neural
progenitors, which we identified by expression of the early neural
marker Sox1 and the intermediate filament protein nestin.
[0129] We were able to propagate these neural progenitor cells in
serum-free conditions for more than three months either as spheres
or as adherent cultures in the presence of mitogenic growth
factors. We routinely observed neural differentiation of the
progenitor cells either in aging spheres or after removal of growth
factors in adherent cultures. In experiments done with Sox1-GFP ES
cells, we were able to generate proliferating neural progenitor
lines that expressed nestin and Sox1, visible in real-time by green
fluorescence. These cells readily differentiated into
morphologically and immunologically distinct neurons after removal
of mitogenic growth factors.
[0130] We examined the electrophysiological properties of neurons
generated from embryonic stem cells and from stem cells harvested
from adult ears. Using the strategy outlined above we examined
embryonic stem cells differentiated to become presumptive auditory
sensory neurons. The cells adopted neuronal morphology and acquired
negative resting potentials and the ability to fire action
potentials.
Example 4
Development of an Assay for Differentiation of ES Cells
[0131] In order to more systematically test the effects of
different genes or compounds on the conversion of ES cells to
spiral ganglion neurons, we developed a luciferase assay system in
which the conversion of the progenitors to the desired cell types
is readily detected by a reporter construct. The aim was to have
the reporter construct under the control of a promoter that is
activated in the differentiated cell but is inactive in the
progenitor cells, so that a luciferase signal is generated by
differentiation of the cells. The assay can be performed using
conditions known to be useful for generating neurons from ES cells.
Cells that are grown in the presence of growth factors are cultured
in medium without growth factors, and this induces their
differentiation to neurons based on the expression of markers.
Under these conditions, the reporter cells will differentiate and
generate a signal. We used mouse ES cells (ROSA 26) to generate
neural progenitors in the presence of EGF, IGF-1 and bFGF. The
neural progenitors were used for construction of the reporter cell
lines. The progenitor cells were positive for nestin expression and
were kept in culture in the presence of bFGF.
[0132] To determine whether a cell specific promoter could be
measured in this assay, neural progenitors were co-transfected with
the firefly luciferase gene controlled by a GFAP promoter and a
vector that contains the Renilla luciferase gene under control of a
CMV promoter. The firefly luciferase construct was made in the pGL3
basic vector (Promega, Madison, Wis.) that contains the firefly
luciferase gene and a multiple cloning site for the promoter. The
GFAP promoter inserted into this site allowed us to measure the
activity of this promoter relative to the constitutively active
control promoter in a separate vector driving the Renilla
luciferase. Co-transfection of the vectors into the neural
progenitors followed by lysis of the cells and measurement of
luciferase activity (using two substrates for measurement of
firefly and Renilla luciferase) allowed us to demonstrate that the
neural progenitors were initially negative for GFAP expression but
after removing bFGF from the culture medium, had increasing amounts
of luciferase activity (at 24, 48 and 72 hours). Furthermore, the
neural progenitor cells expressed the Renilla and firefly
luciferases at levels that were proportional to the amount of
vector used for transfection. These results indicate that the assay
is useful for determining quantitative effects relating to the
differentiation of the cells in response to individual genes or
factors.
Example 5
ES Cell-Derived Progenitors were Grafted into a Developing Chicken
Inner Ear
[0133] We established microsurgical techniques to manipulate
developing chicken ears. For injection of ES cell-derived
progenitors, we used beveled glass-capillary micropipettes for
injections into the otic pits or vesicles of stage 10-16 chicken
embryos (1.5-2.5 days of embryonic development, (Hamburger and
Hamilton, J. Morphol. 88:49-92, 1951)). Genetically labeled ES
cell-derived inner ear progenitors were implanted into the inner
ear of chicken embryos and their fate was followed through early
otic development. The cells were observed to be engrafted into a
preexisting epithelium and certain criteria were identified as
being necessary for the cells to engraft. Progenitor cells only
survived when implanted as cell aggregates. Progenitor cells that
were injected into the otic vesicle in the form of suspensions were
not traceable. Integration of cells from the progenitor cell
aggregates into the epithelial layers that form the otic vesicle
occurred preferentially at sites of epithelial damage. The
progenitor-derived cells were incorporated throughout the inner
ear, but in our study, we only focused on hair cell development.
Murine cells only upregulated hair cell markers when situated in a
developing sensory epithelium and only when they were located on
the luminal site of the epithelium--in the correct orientation for
hair cells. Progenitor-derived cells that we found elsewhere in the
inner ear did not display expression of hair cell markers.
[0134] In addition to the repopulation of the sensory epithelium
(Li et al., Proc. Natl. Acad. Sci. USA 100: 13495-500, 2003), we
also found progenitor cell derivatives outside of the sensory
epithelia in the auditory ganglion. In fact, we initially observed
more efficient integration of cells into the auditory ganglion than
into the cochlear sensory epithelium.
Example 6
An Explant of the Organ of Corti was Established
[0135] The organ of Corti from C57BL6 mice at P0-P3 was removed
from the cochlea and placed in culture in a collagen matrix or on a
collagen coated plate. The morphology of the explants remained
intact for up to two weeks. The progenitor cells and differentiated
neurons can be tested for their ability to engraft into an explant
of the organ of Corti.
Example 7
Transplantation-Repair Studies in the De-Afferented Cat
[0136] A unilaterally de-afferented cat is a useful animal model
for the study of sensorineural hearing loss with either primary
neuronal degeneration or primary hair cell damage followed by
secondary neuronal degeneration. We cut the auditory nerve in cats
and allowed them to survive for up to 1 yr post surgery. Such
surgery can result in near complete loss of the auditory nerve, yet
all other structures of the cochlea remain normal. Months after
nerve section, there appeared to be a reinnervation of the organ of
Corti by branches of the facial nerve, which can be seen, in serial
sections, streaming through the ganglion without a soma. Within the
organ of Corti, this reinnervation appeared as spiraling fibers
lining either side of the inner hair cell. These results suggested
1) that hair cells can survive in the adult ear without their
afferent innervation, and 2) that the surviving hair cells are
likely expressing signals that remain capable of attracting new
neuronal contacts.
[0137] This animal model was used as a platform for neuronal
transplantation studies. As shown in FIGS. 7A and 7B, the
distortion product otoacoustic emissions (DPOAEs) remained normal
in the de-afferented ear, while there was a dramatic elevation of
compound action potential (CAP) thresholds in the de-afferented
ear. These results indicated that all the processes underlying
transduction and amplification in the cochlea were normal in the
de-afferented ear. Therefore, this model system is ideally suited
to a neuronal transplantation experiment.
[0138] We have performed a number of xenotransplantation
experiments in the unilaterally de-afferented cat and assessed the
extent of incorporation and differentiation of progenitor cells up
to 8 weeks post transplantation. The basic approach in the eight
animals studied to date has been to 1) cut the auditory nerve
bundle near the Schwann glial border, 2) put the animals on
cyclosporin immunosuppression therapy, 3) inject neural progenitor
cells after a variable recovery interval from 0 to 12 weeks, 4)
allow a post-implantation survival of 1-8 weeks, 5) assess
functional recovery via a terminal electrophysiological session,
and 6) harvest the cochlea and the brain for histological
verification of the extent of the primary neural degeneration and
the survival and differentiation of transplanted cells.
[0139] The progenitor cells injected have included 1) immature
spiral ganglion neurons isolated from fetal pigs and 2) mouse ES
cells, expressing .beta.-galactosidase reporters. In some cases,
the exogenous cells were transplanted into the round window and in
other cases into the auditory nerve, just peripheral to the site of
the surgical section.
[0140] In one study, ES cells were transplanted into the auditory
nerve 4 weeks after surgery. When the animal was sacrificed 6 weeks
after transplantation, .beta.-galactosidase positive cells were
seen only in the vicinity of the electrode track (none were seen
anywhere else in the nerve or cochlear nucleus). Some of these
cells had neuronal morphology. In one case, a total of 150
.beta.-gal positive cells were seen near the electrode track.
Example 8
An Amino-Terminal Polypeptide of Sonic Hedgehog (SHH-N) Stimulates
Growth of Hair Cells in Murine Cochlear Explants
[0141] Explants of the organ of Corti from postnatal day 1 mice
were cultured in basic serum-free medium (no growth factor),
consisting of serum-free knockout DMEM medium with N2 supplement.
Experimental explants were treated with the soluble reagent SHH-N.
After 7 days in culture, in situ analysis was performed to examine
hair cells through the detection of the hair cell markers myosin
VIIA (Myo7a) and Math1. In situ staining revealed that more inner
and outer hair cells were present in cultures supplemented with 25
nM SHH-N than in control cultures (N2).
[0142] In another experiment, inner ear progenitor cells derived
from adult murine inner ear stem cells were cultured in serum-free
medium (see Li et al., Nature Medicine 9:1293-1299, 2003), and
explants were treated with the soluble reagent SHH-N as described
above. The number of cells expressing hair cell markers was greater
in cultures supplemented with 25 nM SHH-N as compared to control
(N2+b27) cultures, and typically the number of cells expressing
hair cell markers was about 3-fold greater. Hair cells were
identified by in situ staining with an antibody against Myo7a.
Example 9
An Amino-Terminal Polypeptide of Sonic Hedgehog (SHH-N) Stimulates
Growth of Hair Cells in Murine Cochlear Explants
[0143] Chicken otic vesicles were cultured in basic serum-free
medium (no growth factor), consisting of serum-free knockout DMEM
medium with N2 supplement. Explants were treated with the soluble
reagent SHH-N as described above. After 7 days in culture, more
hair cells were present in cultures supplemented with 50 nM SHH-N
than in control cultures (N2). Hair cell markers were identified by
in situ staining with antibodies against myosin VIIA (Myo7a) and
hair cell antigen (HCA).
[0144] Dosage studies examined the effect of varying concentrations
of SHH on hair cell growth in the otic vesicles after three and
seven days in culture. After three days in culture, the greatest
number of hair cells was observed in cultures containing 12.5 nM
SHH-N (FIG. 8A). After seven days in culture, the greatest number
of hair cells was observed in cultures containing 50 nM SHH-N (FIG.
8B).
Example 10
Assays to Monitor Differentiation of a Stem Cell into a Hair Cell
of the Inner Ear
[0145] We have developed assays to monitor the differentiation of a
pluripotent stem cell into a hair cell of the inner ear. According
to one assay, a luciferase gene can be cloned downstream of a
myoVIIa promoter. This promoter will activate expression of a
reporter gene in any cells that have been converted to hair cells.
As an alternative, the Math1 promoter is well characterized and can
be used to drive expression of reporter genes in hair cells. Other
alternative promoters include the .alpha.9 acetylcholine receptor
promoter and the espin promoter.
[0146] The myoVIIa (or Math1) promoter can be obtained by PCR of
mouse genomic DNA. PCR can be performed using primers with specific
restriction sites for cloning the DNA into the pGL3-Basic Vector
(Promega, Madison, Wis.). The PCR product can be purified by
agarose gel electrophoresis, gel purified, and cleaved with
restriction enzymes. The pGL3-Basic Vector contains the firefly
luciferase gene and a multiple cloning site upstream of the open
reading frame. The purified and cleaved PCR product can be cloned
into the multiple cloning site in the proper orientation for
directing expression of the luciferase gene.
[0147] The myoVIIa promoter-luciferase construct can be transformed
into bacteria for plasmid amplification, and plasmids purified from
the resulting clones can be transfected into derivatives of the
mouse stem cell lines ROSA26 or R1. ROSA26 and R1 cell lines are
maintained and propagated in medium containing the growth factors
EGF (20 ng/mL), IGF-1 (50 ng/mL), and bFGF (10 ng/mL) (Li et al.,
Proc. Natl. Acad. Sci. USA 100:13495-13500). These cells have the
characteristics of progenitor cells and have the ability to
differentiate into hair cells, but the myoVIIa gene is not
activated. A baseline level of luciferase expression can be
measured while the cells are in this progenitor state. Removal of
the cells to medium lacking growth factors will induce the cells to
differentiate into hair cells, which can be detected by an increase
in luciferase expression. To detect luminescence, the cells can be
lysed and incubated with substrate and the luminescence measured
with a device, such as luminescence spectrometer.
[0148] Following the initial assay in mouse embryonic stem cells
for transfection and conversion of stem cells to hair cells, the
assay can be performed in other cell types, such as neural stem
cells and bone marrow derived stem cells.
[0149] The assay can be performed using a clonal population of
cells. To obtain a clonal cell line, cells transfected with
myoVIIa-luciferase are selected by growth on G418. The
myoVIIa-luciferase reporter cells can be grown in a 10 cm dish
until colonies are apparent. The individual colonies can be then be
ring-cloned. Alternatively, if the cells are capable of growth at
low density, the cells can be grown in 96 well plates at dilutions
of up to 1 cell per well, and wells that have apparent cell growth
will be harvested. The cells can be propagated to obtain large
numbers and can then be subjected to the luciferase assay to
determine the effect of candidate genes on the conversion to the
hair cell phenotype.
Example 11
Assays to Identify Genes Involved in the Differentiation of Cells
of the Inner Ear
[0150] We have developed assays to identify genes that influence
the differentiation of pluripotent stem cells or progenitor cells
into hair cells or spiral ganglion cells (or cells that have
differentiated to a point sufficient to act as hair cells or spiral
ganglion cells; we may refer to these cells herein as "hair-like"
cells or "ganglion-like" cells). These genes can have a positive
influence, in which case expression of the gene promotes cell
differentiation (whether through a positive action or by inhibiting
an inhibitor), or a negative influence, in which case expression of
the gene inhibits cell differentiation. According to one assay, the
myoVIIa-luciferase reporter cells described in Example 10 can be
grown in medium containing EGF, IGF-1, and bFGF. The cells can be
transfected with a candidate gene (or a biologically active
fragment thereof) expressed from a vector such as a plasmid.
Expression can be regulated by an inducible promoter or by a
constitutively active promoter such as a CMV promoter. Exemplary
candidate genes are described in Table 1, and any of the genes or
types of genes described in Table 1 can be used in the screening
methods of the invention, regardless of the exact manner in which
the screen is configured (e.g., regardless of whether the screen is
conducted with a single cell; a population of cells; a stem cell or
progenitor cell; a pure or impure population of stem cells or
progenitor cells; or in culture or in vivo).
TABLE-US-00001 TABLE 1 Genes that may influence differentiation of
pluripotent stem cells to hair cells or spiral ganglion cells. Gene
family Exemplary Candidate Genes Basic helix-loop-helix Math1,
Brn3.1, Brn3.2, Hes1, Hes5, transcription factors neurogenin-1,
NeuroD Notch Pathway factors Jagged1, Jagged2, Delta1, Notch1,
Lunatic fringe, Numb WNT pathway genes Wnt7a Cell Cycle regulators
p27.sup.Kip1 Sonic hedgehog pathway genes Shh, Bmp4 Growth factors
and growth Fgfr3, Fgfr1, Fgfr2, Fgf10, Fgf2, Fgf3 factor receptors
Zinc finger and homeobox GATA3, Pax2 transcription factors
Neurotrophins Neurotrophin-3, BDNF
[0151] The cDNAs for the candidate genes can be obtained from RNA
prepared from various sources include mouse brain or human striatum
(Stratagene, La Jolla, Calif.). The RNA can be reverse-transcribed
using the Superscript First Strand Synthesis System (Invitrogen,
Carlsbad, Calif.) with an oligo(dT) primer and Superscript II
reverse transcriptase. The cDNA for each can be cloned into an
expression vector containing a hygromycin resistance gene, and
transformed into bacterial cells for amplification
(pcDNA3.1/hygromycin). The purified vector can be transfected into
luciferase expressing cells, such as those described in Example 10,
and the cells cultured in medium containing hygromycin. Clonal cell
lines can be obtained and expanded in medium containing hygromycin
and the growth factor bFGF, EGF, and IGF, as described in Example
10. The expanded cell culture can then be diluted into 96 well
plates. The cells can be cultured overnight for initial growth and
spreading to take place, and then the cells can be grown in medium
without growth factors to induce differentiation. The individual
wells can be subjected to the luciferase assay at various times. A
method to measure luciferase activity is described above.
[0152] As a control experiment, a plasmid expressing a gene that is
known to bias the cells to development of a phenotype other than
hair cells or spiral ganglion cells can be transfected into
luciferase gene-expressing cells. For example, a myoD gene can be
transfected into the luciferase-reporter cells to induce
differentiation of the cells into muscle cells, or a GFAP gene can
be transfected to induce differentiation into ganglion cells.
[0153] An alternative assay can be appropriate for identifying
genes that inhibit differentiation of stem cells to hair cells.
According to this assay, the luciferase reporter stem cells are
cultured in low concentrations of growth factors, which will
maintain the cells in the precursor state. Small interfering RNAs
(siRNAs) that target candidate genes will be introduced into the
cells to inhibit the expression of genes whose expression is
necessary to maintain the cells in a progenitor state, and thus
inhibit the differentiation of the cells into hair cells.
Down-regulation of these inhibitory genes will be detected by an
increase in luminescence.
[0154] To construct the siRNA molecules, short sequences of
nucleotides (e.g., sequences of about 21-23 nucleotides) will be
selected from the coding sequence of the same group of candidate
genes listed above. Synthetic RNAs can be incubated at 90.degree.
C. for 1 minute followed by 37.degree. C. for 1 hour to allow the
two 21-23 nucleotide strands to anneal. Cationic liposomes can be
formed by mixing the siRNAs with oligofectamine (Invitrogen,
Carlsbad, Calif.), and this mixture can be used to introduce the
siRNA into the luciferase reporter stem cells. Following
transfection, the expression of the target gene can be assessed by
flow cytometry (for the proteins for which antibodies are
available) and by RT-PCR. If a reduction in expression of the
targeted protein (or RNA) correlates with an increase in
luminescence, it can be concluded that the target gene is an
inhibitor of hair cell development. If a decrease in luminescence
is detected upon introduction of the siRNA into the cell culture,
it can be concluded that the target gene may promote
differentiation of stem cells to hair cells. The results of this
assay can be compared to the results of the gene overexpression
assays described above.
Example 12
Library Screens to Identify Genes Involved in the Differentiation
of Stem Cells to Cells of the Inner Ear
[0155] The inner ear cell development assays described above are
amenable to screening large numbers of genes that can be introduced
from an expression library.
[0156] According to one screening approach, a library can be
constructed that includes the luciferase reporter cells described
above, transformed with expression vectors containing the test
cDNAs. The library can be constructed from inner ear mRNA. The
screening assay can be performed in 96-well plates as described
above. Detection of luminescence can be performed after various
time periods, following the course of differentiation in response
to cDNA expression.
[0157] In another screening approach, a commercial library of genes
cloned into adenoviral vectors can be used to express human genes
in the luciferase reporter cell line described above. These assays
take advantage of the efficient transduction and long-term
expression of the adenoviral delivery system.
Example 13
Purification of Inner Ear Cells Obtained from Stem Cells
[0158] The genes identified by the methods described above can be
used to induce the differentiation of embryonic stem cells into
hair cells or neural cells of the spiral ganglion. These newly
generated cells are suitable for transplantation.
[0159] Before the cells can be used for transplantation, the
differentiated ear cells must be separated from remaining
pluripotent cells, and from cells that are otherwise not hair cells
or neural cells. To purify the cells, the promoters described above
for tissue specific expression in hair cells or spiral ganglion
cells can be cloned upstream of a selectable marker, such as the
hygromycin resistance gene. Other selectable markers, such as a GFP
gene, are appropriate. The methods can be applied to human or mouse
embryonic stem cells.
[0160] Thus, according to the purification method, a myoVIIa
promoter can be cloned upstream of a hygromycin resistance gene.
The fusion product can be constructed in a plasmid containing a
second selectable marker, such as neomycin, under control of a
constitutive promoter, such as CMV. Another plasmid can be
constructed, wherein a gene identified by the methods of Examples
10-12 is placed under control of a constitutive promoter, such as
CMV. The two plasmids can be transfected into a pluripotent human
embryonic stem cell. Cells containing the plasmids are selected by
growth in medium containing neomycin. Cells that grow in neomycin
are expressing the gene of interest, and the neomycin resistance
gene. The stem cells are cultured in growth factors such as EGF,
IGF-1, and bFGF to maintain the cell in a progenitor state.
[0161] To induce differentiation of the cells to form hair cells,
the cells containing the engineered plasmid are cultured on
hygromycin. The hygromycin media can also include supplemental
growth factors. The concentration of growth factors can be reduced
to sensitize the cells for differentiation. Some cells may be
induced to differentiate by plating on hygromycin in the absence of
supplemental growth factors. Cells cultured in hygromycin are newly
formed hair cells and can be isolated for use in
transplantation.
OTHER EMBODIMENTS
[0162] A number of embodiments featured in the invention have been
described. Nevertheless, it will be understood that various
modifications may be made without departing from the spirit and
scope of the invention. Accordingly, other embodiments are within
the scope of the following claims.
Sequence CWU 1
1
41461PRTHomo sapiens 1Met Leu Leu Leu Ala Arg Cys Leu Leu Leu Val
Leu Val Ser Ser Leu1 5 10 15Leu Val Cys Ser Gly Leu Ala Cys Gly Pro
Gly Arg Gly Phe Gly Lys 20 25 30Arg Arg His Pro Lys Lys Leu Thr Pro
Leu Ala Tyr Lys Gln Phe Ile 35 40 45Pro Asn Val Ala Glu Lys Thr Leu
Gly Ala Gly Arg Tyr Glu Gly Lys 50 55 60Ile Ser Arg Asn Ser Glu Arg
Phe Lys Glu Leu Thr Pro Asn Tyr Asn65 70 75 80Pro Asp Ile Ile Phe
Lys Asp Glu Glu Asn Thr Gly Ala Asp Arg Leu 85 90 95Met Thr Gln Arg
Cys Lys Asp Lys Leu Asn Ala Leu Ala Ile Ser Val 100 105 110Met Asn
Gln Trp Pro Gly Val Lys Leu Arg Val Thr Glu Gly Trp Asp 115 120
125Glu Asp Gly His His Ser Glu Glu Ser Leu His Tyr Glu Gly Arg Ala
130 135 140Val Asp Ile Thr Thr Ser Asp Arg Asp Arg Ser Lys Tyr Gly
Met Leu145 150 155 160Ala Arg Leu Ala Val Glu Ala Gly Phe Asp Trp
Val Tyr Tyr Glu Ser 165 170 175Lys Ala His Ile His Cys Ser Val Lys
Ala Glu Asn Ser Val Ala Ala 180 185 190Lys Ser Gly Gly Cys Phe Pro
Gly Ser Ala Thr Val His Leu Glu Gln 195 200 205Gly Gly Thr Lys Leu
Val Lys Asp Leu Ser Pro Gly Asp Arg Val Leu 210 215 220Ala Ala Asp
Asp Gln Gly Arg Leu Leu Tyr Ser Asp Phe Leu Thr Phe225 230 235
240Leu Asp Arg Asp Asp Gly Ala Lys Lys Val Phe Tyr Val Ile Glu Thr
245 250 255Arg Glu Pro Arg Glu Arg Leu Leu Leu Thr Ala Ala His Leu
Leu Phe 260 265 270Val Ala Pro His Asn Asp Ser Ala Thr Gly Glu Pro
Glu Ala Ser Ser 275 280 285Gly Ser Gly Pro Pro Ser Gly Gly Ala Leu
Gly Pro Arg Ala Leu Phe 290 295 300Ala Ser Arg Val Arg Pro Gly Gln
Arg Val Tyr Val Val Ala Glu Arg305 310 315 320Asp Gly Asp Arg Arg
Leu Leu Pro Ala Ala Val His Ser Val Thr Leu 325 330 335Ser Glu Glu
Ala Ala Gly Ala Tyr Ala Pro Leu Thr Ala Gln Gly Thr 340 345 350Ile
Leu Ile Asn Arg Val Leu Ala Ser Cys Tyr Ala Val Ile Glu Glu 355 360
365His Ser Trp Ala His Arg Ala Phe Ala Pro Phe Arg Leu Ala His Ala
370 375 380Leu Leu Ala Ala Leu Ala Pro Ala Arg Thr Asp Arg Gly Gly
Asp Ser385 390 395 400Gly Gly Gly Asp Arg Gly Gly Gly Gly Gly Arg
Val Ala Leu Thr Ala 405 410 415Pro Gly Ala Ala Asp Ala Pro Gly Ala
Gly Ala Thr Ala Gly Ile His 420 425 430Trp Tyr Ser Gln Leu Leu Tyr
Gln Ile Gly Thr Trp Leu Leu Asp Ser 435 440 445Glu Ala Leu His Pro
Leu Gly Met Ala Val Lys Ser Ser 450 455 46021389DNAHomo sapiens
2atgctgctgc tggcgagatg tctgctgcta gtcctcgtct cctcgctgct ggtatgctcg
60ggactggcgt gcggaccggg cagggggttc gggaagagga ggcaccccaa aaagctgacc
120cctttagcct acaagcagtt tatccccaat gtggccgaga agaccctagg
cgccagcgga 180aggtatgaag ggaagatctc cagaaactcc gagcgattta
aggaactcac ccccaattac 240aaccccgaca tcatatttaa ggatgaagaa
aacaccggag cggacaggct gatgactcag 300aggtgtaagg acaagttgaa
cgctttggcc atctcggtga tgaaccagtg gccaggagtg 360aaactgcggg
tgaccgaggg ctgggacgaa gatggccacc actcagagga gtctctgcac
420tacgagggcc gcgcagtgga catcaccacg tctgaccgcg accgcagcaa
gtacggcatg 480ctggcccgcc tggcggtgga ggccggcttc gactgggtgt
actacgagtc caaggcacat 540atccactgct cggtgaaagc agagaactcg
gtggcggcca aatcgggagg ctgcttcccg 600ggctcggcca cggtgcacct
ggagcagggc ggcaccaagc tggtgaagga cctgagcccc 660ggggaccgcg
tgctggcggc ggacgaccag ggccggctgc tctacagcga cttcctcact
720ttcctggacc gcgacgacgg cgccaagaag gtcttctacg tgatcgagac
gcgggagccg 780cgcgagcgcc tgctgctcac cgccgcgcac ctgctctttg
tggcgccgca caacgactcg 840gccaccgggg agcccgaggc gtcctcgggc
tcggggccgc cttccggggg cgcactgggg 900cctcgggcgc tgttcgccag
ccgcgtgcgc ccgggccagc gcgtgtacgt ggtggccgag 960cgtgacgggg
accgccggct cctgcccgcc gctgtgcaca gcgtgaccct aagcgaggag
1020gccgcgggcg cctacgcgcc gctcacggcc cagggcacca ttctcatcaa
ccgggtgctg 1080gcctcgtgct acgcggtcat cgaggagcac agctgggcgc
accgggcctt cgcgcccttc 1140cgcctggcgc acgcgctcct ggctgcactg
gcgcccgcgc gcacggaccg cggcggggac 1200agcggcggcg gggaccgcgg
gggcggcggc ggcagagtag ccctaaccgc tccaggtgct 1260gccgacgctc
cgggtgcggg ggccaccgcg ggcatccact ggtactcgca gctgctctac
1320caaataggca cctggctcct ggacagcgag gccctgcacc cgctgggcat
ggcggtcaag 1380tccagctga 13893411PRTHomo sapiens 3Met Ser Pro Ala
Arg Leu Arg Pro Arg Leu His Phe Cys Leu Val Leu1 5 10 15Leu Leu Leu
Leu Val Val Pro Ala Ala Trp Gly Cys Gly Pro Gly Arg 20 25 30Val Val
Gly Ser Arg Arg Arg Pro Pro Arg Lys Leu Val Pro Leu Ala 35 40 45Tyr
Lys Gln Phe Ser Pro Asn Val Pro Glu Lys Thr Leu Gly Ala Ser 50 55
60Gly Arg Tyr Glu Gly Lys Ile Ala Arg Ser Ser Glu Arg Phe Lys Glu65
70 75 80Leu Thr Pro Asn Tyr Asn Pro Asp Ile Ile Phe Lys Asp Glu Glu
Asn 85 90 95Thr Gly Ala Asp Arg Leu Met Thr Gln Arg Cys Lys Asp Arg
Leu Asn 100 105 110Ser Leu Ala Ile Ser Val Met Asn Gln Trp Pro Gly
Val Lys Leu Arg 115 120 125Val Thr Glu Gly Trp Asp Glu Asp Gly His
His Ser Glu Glu Ser Leu 130 135 140His Tyr Glu Gly Arg Ala Val Asp
Ile Thr Thr Ser Asp Arg Asp Arg145 150 155 160Asn Lys Tyr Gly Leu
Leu Ala Arg Leu Ala Val Glu Ala Gly Phe Asp 165 170 175Trp Val Tyr
Tyr Glu Ser Lys Ala His Val His Cys Ser Val Lys Ser 180 185 190Glu
His Ser Ala Ala Ala Lys Thr Gly Gly Cys Phe Pro Ala Gly Ala 195 200
205Gln Val Arg Leu Glu Ser Gly Ala Arg Val Ala Leu Ser Ala Val Arg
210 215 220Pro Gly Asp Arg Val Leu Ala Met Gly Glu Asp Gly Ser Pro
Thr Phe225 230 235 240Ser Asp Val Leu Ile Phe Leu Asp Arg Glu Pro
His Arg Leu Arg Ala 245 250 255Phe Gln Val Ile Glu Thr Gln Asp Pro
Pro Arg Arg Leu Ala Leu Thr 260 265 270Pro Ala His Leu Leu Phe Thr
Ala Asp Asn His Thr Glu Pro Ala Ala 275 280 285Arg Phe Arg Ala Thr
Phe Ala Ser His Val Gln Pro Gly Gln Tyr Val 290 295 300Leu Val Ala
Gly Val Pro Gly Leu Gln Pro Ala Arg Val Ala Ala Val305 310 315
320Ser Thr His Val Ala Leu Gly Ala Tyr Ala Pro Leu Thr Lys His Gly
325 330 335Thr Leu Val Val Glu Asp Val Val Ala Ser Cys Phe Ala Ala
Val Ala 340 345 350Asp His His Leu Ala Gln Leu Ala Phe Trp Pro Leu
Arg Leu Phe His 355 360 365Ser Leu Ala Trp Gly Ser Trp Thr Pro Gly
Glu Gly Val His Trp Tyr 370 375 380Pro Gln Leu Leu Tyr Arg Leu Gly
Arg Leu Leu Leu Glu Glu Gly Ser385 390 395 400Phe His Pro Leu Gly
Met Ser Gly Ala Gly Ser 405 4104396PRTHomo sapiens 4Met Ala Leu Leu
Thr Asn Leu Leu Pro Leu Cys Cys Leu Ala Leu Leu1 5 10 15Ala Leu Pro
Ala Gln Ser Cys Gly Pro Gly Arg Gly Pro Val Gly Arg 20 25 30Arg Arg
Tyr Ala Arg Lys Gln Leu Val Pro Leu Leu Tyr Lys Gln Phe 35 40 45Val
Pro Gly Val Pro Glu Arg Thr Leu Gly Ala Ser Gly Pro Ala Glu 50 55
60Gly Arg Val Ala Arg Gly Ser Glu Arg Phe Arg Asp Leu Val Pro Asn65
70 75 80Tyr Asn Pro Asp Ile Ile Phe Lys Asp Glu Glu Asn Ser Gly Ala
Asp 85 90 95Arg Leu Met Thr Glu Arg Cys Lys Glu Arg Val Asn Ala Leu
Ala Ile 100 105 110Ala Val Met Asn Met Trp Pro Gly Val Arg Leu Arg
Val Thr Glu Gly 115 120 125Trp Asp Glu Asp Gly His His Ala Gln Asp
Ser Leu His Tyr Glu Gly 130 135 140Arg Ala Leu Asp Ile Thr Thr Ser
Asp Arg Asp Arg Asn Lys Tyr Gly145 150 155 160Leu Leu Ala Arg Leu
Ala Val Glu Ala Gly Phe Asp Trp Val Tyr Tyr 165 170 175Glu Ser Arg
Asn His Val His Val Ser Val Lys Ala Asp Asn Ser Leu 180 185 190Ala
Val Arg Ala Gly Gly Cys Phe Pro Gly Asn Ala Thr Val Arg Leu 195 200
205Trp Ser Gly Glu Arg Lys Gly Leu Arg Glu Leu His Arg Gly Asp Trp
210 215 220Val Leu Ala Ala Asp Ala Ser Gly Arg Val Val Pro Thr Pro
Val Leu225 230 235 240Leu Phe Leu Asp Arg Asp Leu Gln Arg Arg Ala
Ser Phe Val Ala Val 245 250 255Glu Thr Glu Trp Pro Pro Arg Lys Leu
Leu Leu Thr Pro Trp His Leu 260 265 270Val Phe Ala Ala Arg Gly Pro
Ala Pro Ala Pro Gly Asp Phe Ala Pro 275 280 285Val Phe Ala Arg Arg
Leu Arg Ala Gly Asp Ser Val Leu Ala Pro Gly 290 295 300Gly Asp Ala
Leu Arg Pro Ala Arg Val Ala Arg Val Ala Arg Glu Glu305 310 315
320Ala Val Gly Val Phe Ala Pro Leu Thr Ala His Gly Thr Leu Leu Val
325 330 335Asn Asp Val Leu Ala Ser Cys Tyr Ala Val Leu Glu Ser His
Gln Trp 340 345 350Ala His Arg Ala Phe Ala Pro Leu Arg Leu Leu His
Ala Leu Gly Ala 355 360 365Leu Leu Pro Gly Gly Ala Val Gln Pro Thr
Gly Met His Trp Tyr Ser 370 375 380Arg Leu Leu Tyr Arg Leu Ala Glu
Glu Leu Leu Gly385 390 395
* * * * *