U.S. patent application number 10/419026 was filed with the patent office on 2004-03-25 for manipulation of non-terminally differentiated cells using the notch pathway.
This patent application is currently assigned to Yale University. Invention is credited to Artavanis-Tsakonas, Spyridon, Fortini, Mark Edward, Matsuno, Kenji.
Application Number | 20040058443 10/419026 |
Document ID | / |
Family ID | 24141687 |
Filed Date | 2004-03-25 |
United States Patent
Application |
20040058443 |
Kind Code |
A1 |
Artavanis-Tsakonas, Spyridon ;
et al. |
March 25, 2004 |
Manipulation of non-terminally differentiated cells using the Notch
pathway
Abstract
The present invention is directed to methods for the expansion
of non-terminally differentiated cells ("precursor cells") using
agonists of Notch function, by inhibiting the differentiation of
the cells without inhibiting proliferation (mitotic activity) such
that an expanded population of non-terminally differentiated cells
is obtained. The cells are preferably stem or progenitor cells.
These expanded cells can be used in cell replacement therapy to
provide desired cell populations and help in the regeneration of
diseased and/or injured tissues. The expanded cell populations can
also be made recombinant and used for gene therapy, or can be used
to supply functions associated with a particular precursor cell or
its progeny cell.
Inventors: |
Artavanis-Tsakonas, Spyridon;
(Hamden, CT) ; Fortini, Mark Edward; (New Haven,
CT) ; Matsuno, Kenji; (New Haven, CT) |
Correspondence
Address: |
JONES DAY
222 EAST 41ST STREET
NEW YORK
NY
10017
US
|
Assignee: |
Yale University
|
Family ID: |
24141687 |
Appl. No.: |
10/419026 |
Filed: |
April 18, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10419026 |
Apr 18, 2003 |
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09113824 |
Jul 10, 1998 |
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09113824 |
Jul 10, 1998 |
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08537210 |
Sep 29, 1995 |
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5780300 |
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Current U.S.
Class: |
435/366 ;
435/368; 435/369; 435/370; 435/371; 435/372 |
Current CPC
Class: |
C12N 5/0623 20130101;
C12N 5/0601 20130101; C12N 5/0018 20130101; A61K 35/12 20130101;
C07K 14/47 20130101; A61K 38/00 20130101; C12N 2501/42 20130101;
A61P 43/00 20180101 |
Class at
Publication: |
435/366 ;
435/368; 435/369; 435/370; 435/371; 435/372 |
International
Class: |
C12N 005/08 |
Goverment Interests
[0001] This invention was made with government support under grant
number NS 26084 awarded by the National Institutes of Health. The
government has certain rights in the invention.
Claims
What is claimed is:
1. A method for the expansion of a human precursor cell comprising
contacting the cell with an amount of an agonist of Notch function
effective to inhibit differentiation of the cell, and exposing the
cell to cell growth conditions such that the cell proliferates.
2. The method according to claim 1 wherein the precursor cell is of
ectodermal origin.
3. The method according to claim 1 wherein the precursor cell is of
endodermal origin.
4. The method according to claim 1 wherein the precursor cell is of
mesodermal origin.
5. The method according to claim 1 wherein the precursor cell is
selected from the group consisting of hematopoietic
precursor-cells, epithelial precursor cells, kidney precursor
cells, neural precursor cells, skin precursor cells, osteoblast
precursor cells, chondrocyte precursor cells, and liver precursor
cells.
6. The method according to claim 1 wherein the agonist is a Delta
protein or a derivative thereof which binds to Notch.
7. The method according to claim 1 wherein the agonist is a Serrate
protein or a derivative thereof which binds to Notch.
8. The method according to claim 1 wherein the agonist is an
antibody to a Notch protein or a fragment of the antibody
containing the binding region.
9. The method according to claim 1 wherein the precursor cell is an
hematopoietic stem cell.
10. The method according to claim 1 wherein the precursor cell
contains a recombinant nucleic acid encoding a protein of value in
the treatment of a human disease or disorder.
11. The method according to claim 1 wherein the agonist is a Delta
or Serrate protein and said contacting is carried out by a method
comprising exposing the precursor cells to cells recombinantly
expressing the agonist.
12. The method according to claim 1 wherein said contacting is
carried out by culturing said precursor cells in medium containing
a purified agonist in soluble form.
13. The method according to claim 1 wherein substantially no
differentiation of the cells occurs.
14. A method for the expansion of a precursor cell comprising
contacting the cell with an amount of a soluble agonist of Notch
function effective to inhibit differentiation of the cell, and
exposing the cell to cell growth conditions such that the cell
proliferates.
15. The method according to claim 14 wherein the precursor cell is
selected from the group consisting of hematopoietic precursor
cells, epithelial precursor cells, kidney precursor cells, neural
precursor cells, skin precursor cells, osteoblast precursor cells,
chondrocyte precursor cells, liver precursor cells, and muscle
cells.
16. The method according to claim 14 wherein the precursor cell is
an hematopoietic stem cell.
17. The method according to claim 14 wherein substantially no
differentiation of the cells occurs.
18. The method according to claim 14 wherein the soluble agonist is
a derivative of a Delta protein which binds to a Notch protein.
19. The method according to claim 14 wherein the soluble agonist is
a derivative of a Serrate protein which binds to a Notch
protein.
20. The method according to claim 18 wherein the derivative of
Delta consists essentially of the extracellular domain of a Delta
protein.
21. The method according to claim 19 wherein the derivative of
Serrate consists essentially of the extracellular domain of a
Serrate protein.
22. The method according to claim 14 wherein the soluble agonist is
an antibody to a Notch protein or a fragment of the antibody
containing the binding region.
23. A method for the expansion of a precursor cell comprising
recombinantly expressing within the cell an amount of a Deltex
protein or fragment thereof which binds to a Notch protein in the
precursor cell effective to inhibit differentiation of the cells;
and exposing the cell to cell growth conditions such that the cell
proliferates.
24. A method for the expansion of a hematopoietic precursor cell
comprising recombinantly expressing within the cell an amount of a
Notch protein consisting essentially of the intracellular domain of
a Notch protein in the precursor cell effective to inhibit
differentiation; and exposing the cell to cell growth conditions
such that the cell proliferates.
25. A method for the expansion of an epithelial precursor cell
comprising recombinantly expressing within the cell an amount of a
Notch protein consisting essentially of the intracellular domain of
a Notch protein in the precursor cell effective to inhibit
differentiation; and exposing the cell to cell growth conditions
such that the cell proliferates.
26. A method for the expansion of a liver precursor cell comprising
recombinantly expressing within the cell an amount of a Notch
protein consisting essentially of the intracellular domain of a
Notch protein in the precursor cell effective to inhibit
differentiation; and exposing the cell to cell growth conditions
such that the cell proliferates.
27. A method for the expansion of a human precursor cell comprising
contacting the precursor cell with a second cell wherein the second
cell recombinantly expresses on its surface a molecule consisting
of at least the extracellular domain of a Notch ligand; and
exposing the precursor cell to cell growth conditions such that the
precursor cell proliferates.
28. The method of claim 27 wherein the second cell recombinantly
expresses on its surface at least the extracellular domain of a
Delta protein.
29. The method of claim 27 wherein the second cell recombinantly
expresses on its surface at least the extracellular domain of a
Serrate protein.
30. A method for the expansion of an hematopoietic precursor cell
comprising contacting the precursor cell with a second cell wherein
the second cell recombinantly expresses on its surface a molecule
consisting of at least the extracellular domain of a Notch ligand;
and exposing the precursor cell to cell growth conditions such that
the precursor cell proliferates.
31. The method according to claim 14 wherein the precursor cell
contains a recombinant nucleic acid encoding a protein of value in
the treatment of a disease or disorder.
32. The method according to claim 14 which further comprises after
said contacting step the step of introducing into the cell a
recombinant nucleic acid encoding a protein of value in the
treatment of a disease or disorder.
33. A method for the expansion of a human precursor cell comprising
contacting the precursor cell with an amount of a second cell
expressing a Notch ligand effective to inhibit differentiation of
the cell; and exposing the precursor cell to cell growth conditions
such that the precursor cell proliferates.
34. A method for therapy comprising contacting a precursor cell
with an effective amount of an agonist of Notch function effective
to inhibit differentiation of the cell; exposing the cell to cell
growth conditions to form an expanded precursor cell population;
and administering a therapeutically effective amount of the
expanded precursor cell population or progeny cells produced
therefrom to a patient.
35. The method according to claim 1 or 14 which further comprises
removing the agonist of Notch function and inducing at least some
of the resulting expanded cells to differentiate.
36. A method for the inhibition of a function of a signaling
pathway that regulates cell growth or differentiation comprising
contacting a cell with an amount of an agonist of Notch function,
effective to inhibit a function of a signaling pathway in the cell
that regulates cell growth or differentiation.
37. The method according to claim 36 wherein the pathway is a
ras-mediated pathway.
38. The method according to claim 36 wherein the pathway is a wnt-1
or homologous locus-mediated pathway.
39. The method according to claim 1 in which said contacting and
exposing steps are carried out concurrently.
40. A method for promoting mammalian neuronal cell growth
comprising contacting a mammalian neuron with an antagonist of
Notch function and exposing the neuron to neuronal cell growth
conditions.
41. The method according to claim 1, 14 and 16 in which said
contacting and exposing steps are carried out in vitro.
Description
1. FIELD OF THE INVENTION
[0002] The present invention is directed to methods for the
expansion of non-terminally differentiated cells ("precursor
cells") using Notch reagents, by maintaining the differentiation
state of the cells without inhibiting proliferation ("mitotic
activity") such that an expanded population of non-terminally
differentiated cells is obtained. The cells are preferably stem or
progenitor cells. These expanded cells can be used in cell
replacement therapy to repopulate lost cell populations and help in
the regeneration of diseased and/or injured tissues. The expanded
cell populations can also be made recombinant and used for gene
therapy, or can be used to supply functions (e.g., expressed
protein products) associated with of a particular precursor cell or
its progeny cells.
2. BACKGROUND OF THE INVENTION
[0003] The developmental processes that govern the ontogeny of
multicellular organisms, including humans, depends on the interplay
between signaling pathways, which gradually narrow the
developmental potential of cells from the original totipotent stem
cell to the terminally differentiated mature cell, which performs a
specialized function, such as a heart cell or a nerve cell.
[0004] The fertilized egg is the cell from which all other cell
lineages derive, i.e., the ultimate stem cell. As development
proceeds, early embryonic cells respond to growth and
differentiation signals which gradually narrow the cells'
developmental potential, until the cells reach developmental
maturity, i.e., are terminally differentiated. These terminally
differentiated cells have specialized functions and
characteristics, and represent the last step in a multi-step
process of precursor cell differentiation into a particular
cell.
[0005] The transition from one step to the next in cell
differentiation is governed by specific biochemical mechanisms
which gradually control the progression until maturity is reached.
It is clear that the differentiation of tissues and cells is a
gradual process which follows specific steps until a terminally
differentiated state is reached.
[0006] Gastrulation, the morphogenic movement of the early
embryonic cell mass, results in the formation of three distinct
germ cell layers, the ectoderm, the mesoderm, and the endoderm. As
cells in each germ cell layer respond to various developmental
signals, specific organs are generated which are composed of
specific differentiated cells. For example, the epidermis and the
nervous system develop from ectoderm-derived cells, the respiratory
system and the digestive tract are developed from endoderm-derived
cells, and mesoderm-derived cells develop into the connective
tissues, the hematopoietic system, the urogenital system, muscle,
and parts of most internal organs.
[0007] The following is a brief outline of how ectoderm, endoderm
and mesoderm are developed and further, how these three dermal
layers give rise to the different tissues of the body. For a
general review of development see Scott F. Gilbert, 1991,
Developmental Biology, 3rd Edition, Sinauer Associates, Inc.,
Sunderland Mass.
[0008] The interaction between the dorsal mesoderm and the
overlaying ectoderm initiates organogenesis. In this interaction
the chordamesoderm directs the ectoderm above it to form the neural
tube which will eventually give rise to the brain and the spinal
cord. The differentiation of the neural tube into the various
regions of the central nervous system is clear at the gross
anatomical level where morphogenetic changes shape specific
constrictions and bulges to form the chambers of the brain and the
spinal cord. At the cellular level, cell migratory events rearrange
various groups of cells. The neuroepithelial cells respond to
growth and differentiation signals and eventually differentiate
into the numerous types of neurons and supportive (glial) cells.
Both neural tube and brain are highly regionalized with each
specific region serving distinct functional purposes (see FIG. 1).
Each cell in this tissue has specific morphological and biochemical
characteristics. Differentiated cells are the last step in a
lineage where precursor cells responding to developmental cues
progress to a more differentiated state until they reach their
terminal differentiation state. For example, ependymal cells which
are the integral components of the neural tube lining can give rise
to precursors which may differentiate into neurons or glia
depending on the developmental cues they will receive (Rakic et
al., 1982, Neurosci. Rev. 20:429-611).
[0009] The neural crest derives from the ectoderm and is the cell
mass from which an extraordinary large and complex number of
differentiated cell types are produced. (see Table I), including
the peripheral nervous system, pigment cells, adrenal medulla and
certain areas of the head cartilage.
1TABLE I Major Neural Crest Derivatives* Pigment Sensory Autonomic
Skeletal and Skeletal and cells nervous system nervous system
connective tissue connective tissue TRUNK CREST (INCLUDING CERVICAL
CREST) Melanocytes Spinal ganglia Symphathetic Mesenchyme of dorsal
Adrenal Xanthophores Some contributions to Superior cervical fin in
amphibia medulla (erythrophores) vagal (X) root ganglia ganglion
Walls of aortic Type I cells Iridophores Prevertebral ganglia
arches of carotid (guanophores) Paravertebral ganglia Connective
tissue of body in dermis Adrenal medulla parathyroid Parafollicle
epidermis Parasympahtetic (calcitonin- and epidermal Remark's
ganglion producing) derivates Pelvic plexus cells of Visceral and
enteric thyroid ganglia Some supportive cells Glia
(oligodendrocytes) Schwann sheath cells Some contribution to
meninges CRANIAL CREST Small, belated Trigeminal (V)
Parasympahtetic ganglia Most visceral contribution Facial (VII)
root Chary cartilages Glossopharyngeal (IX) Ethmoid Trabeculae
carneae (ant.) root (superior Sphenopalatine Contributes cells to
ganglia) Submandibular posterior trabeculae, Vagal (X) root
(jugular basal plate, para- ganglia) chordal cartilages Supportive
cells Odontoblasts Head mesenchyme (membrane bones) *Derived from
Gilbert, 1991, Developmental Biology, 3rd Edition, Sinauer
Associates, Inc., Sunderland MA, p. 182.
[0010] The fate of neural crest cells will depend on where they
migrate and settle during development since the cells will
encounter different differentiation and growth signals that govern
their ultimate differentiation. The pluripotentiality of neural
crest cells is well established (LeDouarin et al., 1975, Proc.
Natl. Acad. Sci USA 72:728-732). A single neural crest cell can
differentiate into several different cell types. Transplantation
experiments of cell populations or single neural crest cells point
to the remarkably plastic differentiation potential of these cells.
Even though the cell lineages of the various differentiation
pathways have not been established to the degree they have in the
hematopoietic development, the existence of multi-potential cell
precursors, reminiscent to those seen in the hematopoietic system,
is well founded.
[0011] The cells covering the embryo after neurulation form the
presumptive epidermis. The epidermis consists of several cellular
layers which define a differentiation lineage starting from the
undifferentiated, mitotically active basal cells to the terminally
differentiated non-dividing keratinocytes. The latter cells are
eventually shed and constantly replenished by the underlying less
differentiated precursors. Psoriasis, a pathogenic condition of the
skin results from the exfoliation of abnormally high levels of
epidermal cells.
[0012] Skin is not only the derivative of epidermis. Interactions
between mesenchymal dermis, a tissue of mesodermal origin and the
epidermis at specific sites, result in the formation of cutaneous
appendages, hair follicles, sweat glands and apocrine glands. The
cell ensemble that produces hairs is rather dynamic in that the
first embryonic hairs are shed before birth and replaced by new
follicles (vellus). Vellus, a short and silky hair, remains on many
parts of the body which are considered hairless, e.g., forehead and
eye lids. In other areas vellus can give way to "terminal" hair.
Terminal hair can revert into the production of unpigmented vellus,
a situation found normally in male baldness.
[0013] The endoderm is the source of the tissues that line two
tubes within the adult body. The digestive tube extends throughout
the length of the body. The digestive tube gives rise not only to
the digestive tract but also to, for example, the liver, the
gallbladder and the pancreas. The second tube, the respiratory
tube, forms the lungs and part of the pharynx. The pharynx gives
rise to the tonsils, thyroid, thymus, and parathyroid glands.
[0014] The genesis of the mesoderm which has also been referred to
as the mesengenic process gives rise to a very large number of
internal tissues which cover all the organs between the ectodermal
wall and the digestive and respiratory tubes. As is the case with
all other organs it is the intricate interplay between various
intercellular signaling events and the response of non-terminally
differentiated precursor cells that will eventually dictate
specific cellular identities. To a large degree organ formation
depends on the interactions between mesenchymal cells with the
adjacent epithelium. The interaction between dermis and epidermis
to form, e.g., hairs, has been described above. The formation of
the limbs, the gut organs, e.g., liver or pancreas, kidney, teeth,
etc., all depend on interactions between specific mesenchymal and
epithelial components. In fact, the differentiation of a given
epithelium depends on the nature of the adjacent mesenchyme. For
example, when lung bud epithelium is cultured alone, no
differentiation occurs. However, when lung bud epithelium is
cultured with stomach mesenchyme or intestinal mesenchyme, the lung
bud epithelium differentiates into gastric glands or villi,
respectively. Further, if lung bud epithelium is cultured with
liver mesenchyme or bronchial mesenchyme, the epithelium
differentiates into hepatic cords or branching bronchial buds,
respectively.
[0015] 2.1. Adult Tissues and Precursor Cells
[0016] Embryonic development produces the fully formed organism.
The morphologic, i.e., cellular boundaries of each organ are
defined and in the juvenile or adult individual the maintenance of
tissues whether during normal life or in response to injury and
disease, depends on the replenishing of the organs from precursor
cells that are capable of responding to specific developmental
signals.
[0017] The best known example of adult cell renewal via the
differentiation of immature cells is the hematopoietic system.
Here, developmentally immature precursors (hematopoietic stem and
progenitor cells) respond to molecular signals to gradually form
the varied blood and lymphoid cell types.
[0018] While the hematopoietic system is the best understood self
renewing adult cellular system it is believed that most, perhaps
all, adult organs harbor precursor cells that under the right
circumstances, can be triggered to replenish the adult tissue. For
example, the pluripotentiality of neural crest cells has been
described above. The adult gut contains immature precursors which
replenish the differentiated tissue. Liver has the capacity to
regenerate because it contains hepatic immature precursors; skin
renews itself, etc. Through the mesengenic process, most mesodermal
derivatives are continuously replenished by the differentiation of
precursors. Such repair recapitulates the embryonic lineages and
entails differentiation paths which involve pluripotent progenitor
cells.
[0019] Mesenchymal progenitor cells are pluripotent cells that
respond to specific signals and adopt specific lineages. For
example, in response to bone morphogenic factors, mesenchymal
progenitor cells adopt a bone forming lineage. For example, in
response to injury, mesodermal progenitor cells can migrate to the
appropriate site, multiply and react to local differentiation
factors, consequently adopting a distinct differentiation path. It
has been suggested that the reason that only a limited tissue
repair is observed in adults is because there are too few
progenitor cells which can adopt specific differentiation lineages.
It is clear that if such progenitor cells could be expanded, then
the tissue repair could be much more efficient. An expanded pool of
stem and progenitor cells, as well as non-terminally differentiated
cells supplying a desired differentiation phenotype, would be of
great value in gene therapy and myriad therapeutic regimens.
[0020] 2.2. The Notch Pathway
[0021] Genetic and molecular studies have led to the identification
of a group of genes which define distinct elements of the Notch
signaling pathway. While the identification of these various
elements has come exclusively from Drosophila using genetic tools
as the initial guide, subsequent analyses have lead to the
identification of homologous proteins in vertebrate species
including humans. FIG. 2 depicts the molecular relationships
between the known Notch pathway elements as well as their
subcellular localization (Artavanis-Tsakonas et al., 1995, Science
268:225-232).
[0022] The extracellular domain of Notch carries 36 EGF-like
repeats, two of which have been implicated in interactions with the
Notch ligands Serrate and Delta. Delta and Serrate are membrane
bound ligands with EGF homologous extracellular domains, which
interact physically with Notch on adjacent cells to trigger
signaling.
[0023] Functional analyses involving the expression of truncated
forms of the Notch receptor have indicated that receptor activation
depends on the six cdc10/ankyrin repeats in the intracellular
domain. Deltex and Suppressor of Hairless, whose over-expression
results in an apparent activation of the pathway, associate with
those repeats.
[0024] Deltex is a cytoplasmic protein which contains a ring zinc
finger. Suppressor of Hairless on the other hand, is the Drosophila
homologue of CBF1, a mammalian DNA binding protein involved in the
Epstein-Barr virus-induced immortalization of B cells. It has been
demonstrated that, at least in cultured cells, Suppressor of
Hairless associates with the cdc10/ankyrin repeats in the cytoplasm
and translocates into the nucleus upon the interaction of the Notch
receptor with its ligand Delta on adjacent cells (Fortini and
Artavanis, 1994, Cell 79:273-282). The association of Hairless, a
novel nuclear protein, with Suppressor of Hairless has been
documented using the yeast two hybrid system therefore, it is
believed that the involvement of Suppressor of Hairless in
transcription is modulated by Hairless (Brou et al., 1994, Genes
Dev. 8:2491; Knust et al. 1992, Genetics 129:803).
[0025] Finally, it is known that Notch signaling results in the
activation of at least certain bHLH genes within the Enhancer of
split complex (Delidakis et al., 1991, Genetics 129:803).
Mastermind encodes a novel ubiquitous nuclear protein whose
relationship to Notch signaling remains unclear but is involved in
the Notch pathway as shown by genetic analysis (Smoller et al.,
1990, Genes Dev. 4:1688).
[0026] The generality of the Notch pathway manifests itself at
different levels. At the genetic level, many mutations exist which
affect the development of a very broad spectrum of cell types in
Drosophila. Knockout mutations in mice are embryonic lethals
consistent with a fundamental role for Notch function (Swiatek et
al., 1994, Genes Dev. 8:707). Mutations in the Notch pathway in the
hematopoietic system in humans are associated with lymphoblastic
leukemia (Ellison et al., 1991, Cell 66:649-661). Finally the
expression of mutant forms of Notch in developing Xenopus embryos
interferes profoundly with normal development (Coffman et al.,
1993, Cell 73:659).
[0027] The expression patterns of Notch in the Drosophila embryo
are complex and dynamic. The Notch protein is broadly expressed in
the early embryo, and subsequently becomes restricted to
uncommitted or proliferative groups of cells as development
proceeds. In the adult, expression persists in the regenerating
tissues of the ovaries and testes (reviewed in Fortini et al.,
1993, Cell 75:1245-1247; Jan et al., 1993, Proc. Natl. Acad. Sci.
USA 90:8305-8307; Sternberg, 1993, Curr. Biol. 3:763-765;
Greenwald, 1994, Curr. Opin. Genet. Dev. 4:556-562;
Artavanis-Tsakonas et al., 1995, Science 268:225-232). Studies of
the expression of Notch1, one of three known vertebrate homologues
of Notch, in zebrafish and Xenopus, have shown that the general
patterns are similar; with Notch expression associated in general
with non-terminally differentiated, proliferative cell populations.
Tissues with high expression levels include the developing brain,
eye and neural tube (Coffman et al., 1990, Science 249:1438-1441;
Bierkamp et al., 1993, Mech. Dev. 43:87-100). While studies in
mammals have shown the expression of the corresponding Notch
homologues to begin later in development, the proteins are
expressed in dynamic patterns in tissues undergoing cell fate
determination or rapid proliferation (Weinmaster et al., 1991,
Development 113:199-205; Reaume et al., 1992, Dev. Biol.
154:377-387; Stifani et al., 1992, Nature Genet. 2:119-127;
Weinmaster et al., 1992, Development 116:931-941; Kopan et al.,
1993, J. Cell Biol, 121:631-641; Lardelli et al., 1993, Exp. Cell
Res. 204:364-372; Lardelli et al., 1994, Mech. Dev. 46:123-136;
Henrique et al., 1995, Nature 375:787-790; Horvitz et al., 1991,
Nature 351:535-541; Franco del Amo et al., 1992, Development
115:737-744). Among the tissues in which mammalian Notch homologues
are first expressed are the pre-somitic mesoderm and the developing
neuroepithelium of the embryo. In the pre-somitic mesoderm,
expression of Notch1 is seen in all of the migrated mesoderm, and a
particularly dense band is seen at the anterior edge of pre-somitic
mesoderm. This expression has been shown to decrease once the
somites have formed, indicating a role for Notch in the
differentiation of somatic precursor cells (Reaume et al., 1992,
Dev. Biol. 154:377-387; Horvitz et al., 1991, Nature 351:535-541).
Similar expression patterns are seen for mouse Delta (Simske et
al., 1995, Nature 375:142-145).
[0028] Within the developing mammalian nervous system, expression
patterns of Notch homologue have been shown to be prominent in
particular regions of the ventricular zone of the spinal cord, as
well as in components of the peripheral nervous system, in an
overlapping but non-identical pattern. Notch expression in the
nervous system appears to be limited to regions of cellular
proliferation, and is absent from nearby populations of recently
differentiated cells (Weinmster et al., 1991, Development
113:199-205; Reaume et al., 1992, Dev. Biol. 154:377-387;
Weinmaster et al., 1992, Development 116:931-941; Kopan et al.,
1993, J. Cell Biol. 121:631-641; Lardelli et al., 1993, Exp. Cell
Res. 204:364-372; Lardelli et al., 1994, Mech. Dev. 46:123-136;
Henrique et al., 1995, Nature 375:787-790; Horvitz et al., 1991,
Nature 351:535-541). A rat Notch ligand is also expressed within
the developing spinal cord, in distinct bands of the ventricular
zone that overlap with the expression domains of the Notch genes.
The spatio-temporal expression pattern of this ligand correlates
well with the patterns of cells committing to spinal cord neuronal
fates, which demonstrates the usefulness of Notch as a marker of
populations of cells for neuronal fates (Henrique et al., 1995,
Nature 375:787-790). This has also been suggested for vertebrate
Delta homologues, whose expression domains also overlap with those
of Notch1 (Larsson et al., 1994, Genomics 24:253-258; Fortini et
al., 1993, Nature 365:555-557; Simske et al., 1995, Nature
375:142-145). In the cases of the Xenopus and chicken homologues,
Delta is actually expressed only in scattered cells within the
Notch1 expression domain, as would be expected from the lateral
specification model, and these patterns "foreshadow" future
patterns of neuronal differentiation (Larsson et al., 1994,
Genomics 24:253-258; Fortini et al., 1993, Nature 365:555-557).
[0029] Other vertebrate studies of particular interest have focused
on the expression of Notch homologues in developing sensory
structures, including the retina, hair follicles and tooth buds. In
the case of the Xenopus retina, Notch1 is expressed in the
undifferentiated cells of the central marginal zone and central
retina (Coffman et al., 1990, Science 249:1439-1441; Mango et al.,
1991, Nature 352:811-815). Studies in the rat have also
demonstrated an association of Notch1 with differentiating cells in
the developing retina have been interpreted to suggest that Notch1
plays a role in successive cell fate choices in this tissue (Lyman
et al., 1993, Proc. Natl. Acad. Sci. USA 90:10395-10399).
[0030] A detailed analysis of mouse Notch1 expression in the
regenerating matrix cells of hair follicles was undertaken to
examine the potential participation of Notch proteins in
epithelial/mesenchymal inductive interactions (Franco del Amo et
al., 1992, Development 115:737-744). Such a role had originally
been suggested for Notch1 based on the its expression in rat
whiskers and tooth buds (Weinmaster et al., 1991, Development
113:199-205). Notch1 expression was instead found to be limited to
subsets of non-mitotic, differentiating cells that are not subject
to epithelial/mesenchymal interactions, a finding that is
consistent with Notch expression elsewhere.
[0031] Expression studies of Notch proteins in human tissue and
cell lines have also been reported. The aberrant expression of a
truncated Notch1 RNA in human T-cell leukemia results from a
translocation with a breakpoint in Notch1 (Ellisen et al., 1991,
Cell 66:649-661). A study of human Notch1 expression during
hematopoiesis has suggested a role for Notch1 in the early
differentiation of T-cell precursors (Mango et al., 1994,
Development 120:2305-2315). Additional studies of human Notch1 and
Notch2 expression have been performed on adult tissue sections
including both normal and neoplastic cervical and colon tissue.
Notch1 and Notch2 appear to be expressed in overlapping patterns in
differentiating populations of cells within squamous epithelia of
normal tissues that have been examined and are clearly not
expressed in normal columnar epithelia, except in some of the
precursor cells. Both proteins are expressed in neoplasias, in
cases ranging from relatively benign squamous metaplasias to
cancerous invasive adenocarcinomas in which columnar epithelia are
replaced by these tumors (Mello et al., 1994, Cell 77:95-106).
[0032] Insight into the developmental role and the general nature
of Notch signaling has emerged from studies with truncated,
constitutively activated forms of Notch in several species. These
recombinantly engineered Notch forms, which lack extracellular
ligand-binding domains, resemble the naturally occurring oncogenic
variants of mammalian Notch proteins and are constitutively
activated using phenotypic criteria (Greenwald, 1994, Curr. Opin.
Genet. Dev. 4:556; Fortini et al., 1993, Nature 365:555-557;
Coffman et al., 1993, Cell 73:659-671; Struhl et al., 1993, Cell
69:1073; Rebay et al., 1993, Genes Dev. 7:1949; Kopan et al., 1994,
Development 120:2385; Roehl et al., 1993, Nature 364:632).
[0033] Ubiquitous expression of activated Notch in the Drosophila
embryo suppresses neuroblast segregation without impairing
epidermal differentiation (Struhl et al., 1993, Cell 69:331; Rebay
et al., 1993, Genes Dev. 7:1949).
[0034] Persistent expression of activated Notch in developing
imaginal epithelia likewise results in an overproduction of
epidermis at the expense of neural structures (Struhl et al., 1993,
Cell 69:331).
[0035] Neuroblast segregation occurs in temporal waves that are
delayed but not prevented by transient expression of activated
Notch in the embryo (Struhl et al., 1993, Cell 69:331).
[0036] Transient expression in well-defined cells of the Drosophila
eye imaginal disc causes the cells to ignore their normal inductive
cues and to adopt alternative cell fates (Fortini et al., 1993,
Nature 365:555-557).
[0037] Studies utilizing transient expression of activated Notch in
either the Drosophila embryo or the eye disc indicate that once
Notch signaling activity has subsided, cells may recover and
differentiate properly or respond to later developmental cues
(Fortini et al., 1993, Nature 365:555-557; Struhl et al., 1993,
Cell 69:331).
[0038] For a general review on the Notch pathway and Notch
signaling, see Artavanis-Tsakonas et al., 1995, Science
268:225-232.
[0039] Citation or identification of any reference in Section 2 or
any other section of this application shall not be construed as an
admission that such reference is available as prior art to the
present invention.
3. SUMMARY OF THE INVENTION
[0040] The present invention is directed to methods for the
expansion of non-terminally differentiated cells ("precursor
cells") by activating the Notch pathway in a precursor cell such
that differentiation of the precursor cell is inhibited without
destroying the ability of the cell to proliferate. The precursor
cell is preferably a stem or progenitor cell. The present invention
is also directed to methods for the expansion of precursor cells in
precursor cell containing-populations by activating the Notch
pathway in the cells such that the differentiation of the stem cell
is inhibited without affecting the mitotic activity of the stem
cells. Further, the precursor cells can be isolated from a cell
population, if desired, before or after Notch pathway activation.
Activation of the Notch pathway is preferably achieved by
contacting the cell with a Notch ligand, e.g., in soluble form or
recombinantly expressed on a cell surface or immobilized on a solid
surface, or by introducing into the cell a recombinant nucleic acid
expressing a dominant active Notch mutant or an activating Notch
ligand, or other molecule that activates the Notch pathway.
[0041] Activating Notch in the precursor cell renders the precursor
cell refractory to differentiation signals, thus substantially
inhibiting differentiation and allowing maintenance of the cell in
its differentiation stage, and, optionally, expansion of the cell
upon exposure to cell growth conditions. Thus, the methods of the
invention provide precursor cells of a specific differentiation
state. Thus, in one embodiment, such a cell which expresses a
desired differentiation phenotype (e.g., production of a desired
hormone or growth factor) can be administered to a patient wherein
the differentiation phenotype is therapeutically useful (e.g.,
hormone or growth factor deficiency). Alternatively, an expanded
stem or progenitor cell population produced by activation of Notch
and cell growth can be used to replace or supplement the stem or
progenitor cell lineage in a patient by administration of such cell
population. If desired, members of the expanded cell population can
be induced to differentiate in vitro prior to in vivo
administration, so as to supply to the patient the function of a
more differentiated cell population. Preferably, the Notch
activation is carried out in vitro and is reversible so that upon
in vivo administration of the cells differentiation can occur.
Thus, for example, in a preferred embodiment, a Notch ligand is
used to activate Notch on the cells, e.g., by being added in
soluble form to the cell media, or contacting the cells with a
layer of cells in culture expressing the Notch ligand (e.g., Delta,
Serrate) on its surface.
[0042] The precursor cells to be expanded in the present invention
can be isolated from a variety of sources using methods known to
one skilled in the art (see Section 5.5, infra). The precursor
cells can be of any animal, preferably mammalian, most preferably
human, and can be of primary tissue, cell lines, etc. The precursor
cells can be of ectodermal, mesodermal or endodermal origin. Any
precursor cells which can be obtained and maintained in vitro can
potentially be used in accordance with the present invention. In a
preferred embodiment, the precursor cell is a stem cell. Such stem
cells include but are not limited to hematopoietic stem cells
(HSC), stem cells of epithelial tissues such as the skin and the
lining of the gut, embryonic heart muscle cells, and neural stem
cells (Stemple and Anderson, 1992, Cell 71:973-985). The stem cells
can be expanded under cell growth conditions, i.e., conditions that
promote proliferation ("mitotic activity") of the cells.
[0043] The least differentiated cell in a cell lineage is termed a
stem cell. However, stem cell is an operational term. The classic
definition of the stem cell is a cell which can divide to produce
another stem cell (self-renewal capacity), as well as a cell which
can differentiate along multiple specific differentiation paths. It
is often the case that a particular cell within a differentiation
lineage, has derived from a "less" differentiated parent and can
still divide and give rise to a "more" differentiated cellular
progeny. FIG. 3 describes diagrammatically hematopoietic
development. Totipotent, pluripotent and progenitor stem cells are
referred to in the figure.
[0044] A "precursor cell" may or may not divide and can be
triggered to adopt a different differentiation state but not
necessarily a fully differentiated state, by responding to specific
developmental signals.
[0045] The present invention is also directed to methods for use of
the expanded precursor cells for use in gene therapy as well as for
use in providing desired cell populations, e.g., for regenerating
injured and/or diseased tissues. The expanded precursor cell
populations can be administered to a patient using methods commonly
known to those skilled in the art (see Section 5.8, infra). In
other specific embodiments, after Notch activation and expansion,
the precursor can be induced to differentiate in vivo, or
alternatively in vitro, followed by administration to an
individual, to provide a differentiated phenotype to a patient.
Additionally, Notch activation and expansion can be carried out in
vitro subsequent to in vitro production of a precursor cell of a
desired phenotype from a stem or progenitor cell.
[0046] The present invention is also directed to precursor cells
containing recombinant genes, such that the gene is inheritable and
expressible by the precursor cell or its progeny. These recombinant
precursor cells can be transplanted into a patient such that the
desired gene is expressed in the patient to alleviate a disease
state caused by the lack of or deficient expression of the
recombinant gene. The precursor cells can be made recombinant
either before or after precursor cell expansion. Methods of
tranfecting the nucleic acid encoding the desired gene product such
that the precursor cell or its progeny stably expresses the gene
product are known to those of skill in the art and are described
infra.
4. BRIEF DESCRIPTION OF THE FIGURES
[0047] FIG. 1 is a diagram showing regional specialization during
human brain development. (Gilbert, 1991, Developmental Biology, 3rd
Edition, Sinauer Associates, Inc., Sunderland Mass., p. 166.)
[0048] FIG. 2 is a schematic diagram of the Notch signaling
pathway. The Notch receptor can bind to either Delta or Serrate
through its extracellular domain. Ligand binding can result in
receptor multimerization that is stabilized by interactions between
the intracellular ankyrin repeats of Notch and the cytoplasmic
protein Deltex. These events can control the nuclear translocation
of the DNA-binding protein Suppressor of Hairless and its known
association with the Hairless protein. The transcriptional
induction of the Enhancer of Split bHLH genes appears to depend on
Notch signaling.
[0049] FIG. 3 is a schematic diagram of the origin of mammalian
blood and lymphoid cells. (Gilbert, 1991, Developmental Biology,
3rd Edition, Sinauer Associates, Inc., Sunderland Mass., p.
232).
[0050] FIG. 4 shows the highly conserved ankyrin repeat region of
Notch.
[0051] FIGS. 5A-F. Expression of activated Notch and neural
differentiation in cone cell precursors of transgenic flies bearing
both sev-Notch.sup.nucl and the activated Raf construct
sE-raf.sup.torY9. Third-instar larval eye imaginal discs were
reacted with mouse monoclonal antibody C17.9C6 directed against the
intracellular domain of Notch and rat monoclonal antibody 7E8A10
directed against the neural antigen ELAV and visualized with
immunofluorescent secondary antibodies using confocal microscopy.
Low (5A-C) and high (5E-F) magnification images of posterior eye
disc regions showing nuclear Notch staining (green) in
sevenless-expressing cells (5A,D), nuclei expressing ELAV (red)
undergoing neural differentiation (5B,E) and corresponding image
overlays of both staining patterns (5C,F). The field shown in
(5A-C) spans ommatidial rows 10-23, with the posterior margin of
the disc visible at the left; nuclei that express Notch protein do
not express ELAV. Individual cone cell precursor nuclei of similar
developmental ages are labeled `N` in (5D) if they stain for Notch
but not ELAV, and are labelled `E` in (5E) if they stain for ELAV
but not Notch. Faint ELAV staining (red) was often observed beneath
strongly Notch-positive (green) cone cell precursor nuclei;
examples are indicated by asterisks in (5E). Optical sectioning
revealed that this ELAV staining corresponds to R1,3, 4, 6 and 7
photoreceptor cell precursor nuclei that are located immediately
below and partially intercalated with the cone cell precursor
nuclei. Identical staining patterns were observed for
sev-Notch.sup.nucl flies bearing the activated Sevenless tyrosine
kinase construct sev-S11 or the activated Ras1 construct
sevRas1.sup.Val12 instead of sE-raf.sup.torY9 (data not shown).
[0052] FIGS. 6A-B. Co-expression of activated Notch and activated
Sevenless proteins in cone cell precursors of sevenless.sup.d2
flies bearing sev-Notch.sup.nucl and sev-S11. Third-instar larval
eye imaginal discs were reacted with rat polyclonal antibody Rat5
directed against the intracellular domain of Notch and mouse
monoclonal antibody sev150C3 directed against the 60 kD subunit of
Sevenless and visualized with immunofluorescent secondary
antibodies using confocal microscopy. The sevenless.sup.d2 allele
produces no protein recognized by mAb sev150C3. (6A) Image overlay
of two horizontal optical sections collected at slightly different
apical levels within the same posterior eye disc quadrant, showing
expression of activated Notch (green) in most of the cone cell
precursor nuclei and expression of activated Sevenless (purple) in
most of the corresponding apical membranes of the cone cell
precursor population. The ring-shaped distribution of Sevenless
protein in each assembling ommatidium represents the apical
microvillar tufts of up to four cone cell precursors and the R7
precursor cell. (6B) Higher magnification image overlay similar to
that in (6A), showing a developing ommatidium in which all four
cone cell precursor nuclei express Notch (labelled `N`) and all or
most cone cell precursor apical membrane tufts exhibit strong
Sevenless expression (labelled `Sev`).
[0053] FIG. 7. Schematic representation of the epistatic
relationship between Notch activation and the signalling pathway
involving the sevenless receptor tyrosine kinase, Ras1 and Raf
during neural induction of the R7 cell precursor in Drosophila.
Sevenless protein (Sev) in the R7 cell precursor is activated by
binding to its ligand Bride of sevenless (Boss), presented by the
adjacent R8 cell, resulting in Ras1 activation presumably via
regulation of the activities of its guanine nucleotide exchange
factor Son-of-sevenless and its GTPase-activating protein Gap1.
Ras1 activation leads to the activation of Raf. This signalling
pathway is inhibited by Notch activation at some point downstream
of Raf.
5. DETAILED DESCRIPTION OF THE INVENTION
[0054] The present invention is directed to methods for the
expansion of non-terminally differentiated cells ("precursor
cells") by activating the Notch pathway in a precursor cell such
that the differentiation of the precursor cell is inhibited without
destroying the ability of the cell to proliferate. As used herein,
"precursor cells" shall mean any non-terminally differentiated
cells. The precursor cell is preferably a stem or progenitor cell.
The present invention is also directed to methods for the expansion
of precursor cells in precursor cell containing-populations by
activating the Notch pathway in the cells such that the
differentiation of the stem cell is inhibited without affecting the
mitotic activity of the cells. Further, the precursor cells can be
isolated from a cell population, if desired, before or after Notch
pathway activation. Activation of Notch pathway is preferably
achieved by contacting the cell with a Notch ligand, e.g., in
soluble form or recombinantly expressed on a cell surface or
immobilized on a solid surface, or by introducing into the cell a
recombinant nucleic acid expressing a dominant active Notch mutant
or an activating Notch ligand, or other molecule that activates the
Notch pathway.
[0055] Agonists of the Notch pathway are able to activate the Notch
pathway at the level of protein-protein interaction or protein-DNA
interaction. Agonists of Notch include but are not limited to
proteins and derivatives comprising the portions of toporythmic
proteins such as Delta or Serrate or Jagged (Lindsell et al., 1995,
Cell 80:909-917) that mediate binding to Notch, and nucleic acids
encoding the foregoing (which can be administered to express their
encoded products in vivo). In a preferred embodiment, the agonist
is a protein or derivative or fragment thereof comprising a
functionally active fragment such as a fragment of a Notch ligand
that mediates binding to a Notch protein. In another preferred
embodiment, the agonist is a human protein or portion thereof
(e.g., human Delta). In another preferred embodiment the agonist is
Deltex or Suppressor of Hairless or a nucleic acid encoding the
foregoing (which can be administered to express its encoded product
in vivo).
[0056] The Notch pathway is a signal transducing pathway comprising
elements which interact, genetically and/or molecularly, with the
Notch receptor protein. For example, elements which interact with
the Notch protein on both a molecular and genetic basis are, for
example, and not by way of limitation, Delta, Serrate and Deltex.
Elements which interact with the Notch protein genetically are, for
example, and not by way of limitation, Mastermind, Hairless and
Suppressor of Hairless.
[0057] Activating Notch function in the precursor cell renders the
precursor cell refractory to differentiation signals, thus
substantially inhibiting differentiation and allowing maintenance
of the cell in its differentiation stage, and, optionally,
expansion of the cell upon exposure to cell growth conditions.
Thus, the methods of the invention provide precursor cells of a
specific differentiation state. Thus, in one embodiment, such a
cell which expresses a desired differentiation phenotype (e.g.,
production of a desired hormone or growth factor) can be
administered to a patient wherein the differentiation phenotype is
therapeutically useful (e.g., hormone or growth factor deficiency).
Alternatively, an expanded stem or progenitor cell population
produced by activation of Notch and cell growth can be used to
replace or supplement the stem or progenitor cell lineage in a
patient by administration of such cell population. If desired,
members of the expanded cell population can be induced to
differentiate in vitro prior to in vivo administration, so as to
supply to the patient the function of a more differentiated cell
population. Preferably, the Notch activation is carried out in
vitro and is reversible so that upon in vivo administration of the
cells differentiation can occur. Thus, for example, in a preferred
embodiment, a Notch ligand is used to activate Notch on the dells,
e.g., by being added in soluble form to the cell media, or
contacting the cells with a layer of cells in culture expressing
the Notch ligand (e.g., Delta, Serrate) on its surface.
[0058] The precursor cells to be expanded in the present invention
can be isolated from a variety of sources using methods known to
one skilled in the art (see Section 5.5, infra). The precursor
cells can be of ectodermal, mesodermal or endodermal origin. Any
precursor cells which can be obtained and maintained in vitro can
potentially be used in accordance with the present invention. In a
preferred embodiment, the precursor cell is a stem cell. Such stem
cells include but are not limited to hematopoietic stem cells
(HSC), stem cells of epithelial tissues such as the skin and the
lining of the gut, embryonic heart muscle cells, and neural stem
cells (Stemple and Anderson, 1992, Cell 71:973-985). The stem cells
can be expanded under cell growth conditions, i.e., conditions that
promote proliferation ("mitotic activity") of the cells.
[0059] The least differentiated cell in a cell lineage is termed a
stem cell. However, stem cell is an operational term. The classic
definition of the stem cell is a cell which can divide to produce
another stem cell (self-renewal capacity), as well as a cell which
can differentiate along multiple specific differentiation paths. It
is often the case that a particular cell within a differentiation
lineage, has derived from a "less" differentiated parent and can
still divide and give rise to a "more" differentiated cellular
progeny. FIG. 3 describes diagrammatically hematopoietic
development. Totipotent, pluripotent and progenitor stem cells are
referred to in the figure.
[0060] A "precursor cell" has specific biochemical properties, may
or may not divide and can be triggered to adopt a different
differentiation state but not necessarily a fully differentiated
state, by responding to specific developmental signals.
[0061] The present invention is also directed to methods for use of
the expanded precursor cells for use in gene therapy as well as for
use in providing desired cell populations and for use in
regenerating injured and/or diseased tissues. The expanded
precursor cell populations can be administered to a patient using
methods commonly known to those skilled in the art (see Section
5.8, infra). In other specific embodiments, after Notch activation
and expansion, the precursor cell can be induced to differentiate
in vivo, or alternatively in vitro, followed by administration to
an individual, to provide a differentiated phenotype to a patient.
Additionally, Notch activation and expansion can be carried out in
vitro subsequent to in vitro production of a precursor cell of a
desired phenotype from a stem or progenitor cell.
[0062] The present invention is also directed to precursor cells
expressing recombinant genes, such that the precursor cells express
a desired gene. These recombinant precursor cells can be
transplanted into a patient such that the desired gene is expressed
in the patient to alleviate a disease state caused by the lack of
expression of the recombinant gene. The precursor cells can be made
recombinant either before or after precursor cell expansion.
Methods of tranfecting the nucleic acid encoding the desired gene
product such that the precursor cell or its progeny stably
expresses the gene product are known to those of skill in the art
and are described infra.
[0063] The subject into which the expanded cells or their progeny
are introduced, or from which precursor cells can be derived, is
preferably an animal, including but not limited to animals such as
cows, pigs, horses, chickens, cats, dogs, etc., and is preferably a
mammal, and most preferably human.
[0064] In an embodiment of the present invention, the subjects to
which the cells are administered are immunocompromised or
immunosuppressed or have an immune deficiency. For example, the
subject has Acquired Immune Deficiency Syndrome or has been exposed
to radiation or chemotherapy regimens for the treatment of cancer,
and the subjects are administered hematopoietic or immune precursor
cells such that the administered cells perform a needed immune or
hematopoietic function.
[0065] Preferably, the expanded precursor cell is originally
derived from the subject to which it is administered, i.e., the
transplant is autologous.
[0066] For clarity of disclosure, and by way of limitation, the
detailed description of the invention is divided into the following
sub-sections:
[0067] (i) Notch signaling and stem cell differentiation,
[0068] (ii) Notch activation inhibits the differentiation of stem
cells,
[0069] (iii) Activation of the Notch pathway,
[0070] (iv) Notch and terminal differentiation,
[0071] (v) Obtaining precursor cells,
[0072] (vi) Gene therapy,
[0073] (vii) Pharmaceutical compositions,
[0074] (viii) Transplantation.
[0075] 5.1. Notch Signaling and Stem Cell Differentiation
[0076] The progression of a precursor cell to a more mature or
differentiated state depends on a combination of signals that
ultimately govern the differentiation steps. Specific factors, for
example, bone morphogenic factors or the various factors known to
be important in hematopoiesis, for example, interleukin-5 or
thrombopoietin, together with intercellular and cell-extracellular
matrix interactions contribute to the differentiation of a
precursor cell along a specific differentiation path.
[0077] The effectors of such contributions are the various
signaling pathways which transmit the extracellular signal to the
nucleus, ultimately changing transcriptional expression patterns,
i.e., genes expressed only in the tissue that is the cells'
ultimate fate are switched on and conversely others are switched
off, such that, e.g., kidney cells express kidney-specific genes
and do not express liver cell-specific proteins. In order for a
precursor cell to respond to the various extracellular signals, it
must be competent to do so, for example, in order to respond to a
soluble factor the cell must express a receptor which can recognize
the factor. Tissue competence has been articulated in the classic
studies of Waddington, 1940, organisers and Genes, Cambridge
University Press, Cambridge, England.
[0078] The present invention is based, at least in part, on the
discovery that the Notch signaling pathway is not a pathway that
transmits specific developmental signals such that cell
differentiation is effected, but rather it controls the competence
of a precursor cell to interpret and respond to differentiation
signals. The Notch pathway is a general and evolutionarily
conserved developmental "switch." Specifically, when the Notch
pathway is activated in precursor cells, the precursor cells are
unable to respond to particular differentiation signals but
generally the mitotic ability of the precursor cells remains (i.e.,
the cells can proliferate). The existence of the Notch pathway
allows for the manipulation of the differentiation state of
precursor cells without knowing all of the differentiation signals,
e.g., growth factors, which are required for the maintenance of a
particular differentiation state or for advancing the cell to a
more differentiated state. In a preferred aspect, the inhibitory
effect on differentiation by activating the Notch pathway with a
Notch function agonist can be reversed by adding an antagonist of
the Notch pathway or diluting out the Notch pathway agonist.
[0079] 5.2. Notch Activation Inhibits the Differentiation of
Precursor Cells
[0080] Notch regulates the competence of many different cell types
to respond to more specific signals, with the particular cell fates
chosen depending upon the developmental history of each cell type
and the specific signaling pathways operating within it. When Notch
function is activated in a precursor cell (e.g., progenitor or stem
cell), the precursor cell can be prevented from differentiating
even in the presence of the correct differentiation signals. Once,
however, Notch function activation subsides, the cells can respond
again to developmental cues. We have shown, using human
keratinocytes which have been transfected with activated forms of
Notch, that while cells stably expressing activated Notch forms are
prevented from differentiating, their proliferation potential is
not affected.
[0081] The modulation of Notch pathway activity offers a novel and
unique tool to manipulate the fate of precursor cells. A precursor
at a given developmental state can be "frozen" into that state by
virtue of activating the Notch pathway. Importantly, these cells
may be expanded, since Notch signaling activity may not destroy or,
preferably, does not substantially impair, their ability to divide.
Thus, precursor cells may be expanded, ex vivo in order to provide
a source of precursors which are useful in gene therapy as well as
tissue repair. Notch agonists are also useful in cases where it is
important to maintain a cell in a particular differentiation state
in order to provide indefinitely, or for a given period of time, a
chemical produced by a cell of that differentiated state, to a
particular tissue. In this latter embodiment, for example, it may
be desired to activate Notch in the cells administered in vivo for
a long period of time (e.g., hours or days) or substantially
irreversibly, e.g., by excapsulating the cells with a soluble Notch
agonist, or having them recombinantly express a Notch dominant
active mutant from a constitutive promoter, respectively.
[0082] An embodiment of the present invention is to treat the
desired cell population with agonists of the Notch pathway and then
either allow these cells to proliferate in culture before
transplanting them back into the appropriate region, or directly
transplanting them without necessarily allowing them to proliferate
in vitro. Antagonists can be used to reverse or neutralize the
action of the Notch-function agonist. For example, and not by way
of limitation, a Notch ligand or a molecule that mimics the ligand
can be used to keep Notch receptor expressing cells in an
"activated" state while withdrawal of the ligand will reverse that
effect.
[0083] It is possible in many cases that the simple activation of
Notch may not suffice to expand the stem cells ex vivo. Subjecting
the cell to growth conditions, e.g., culturing it in the presence
of specific growth factors or combinations of growth factors may be
necessary, nevertheless, the importance of Notch pathway activation
in these events will be essential since the presence of only those
factors will generally not be sufficient to maintain those cells in
culture without differentiation occurring.
[0084] 5.3. Activation of Notch Function
[0085] An agonist of Notch function is an agent that promotes
activation of Notch function. As used herein, "Notch function"
shall mean a function mediated by the Notch signaling pathway.
[0086] Notch function activation is preferably carried out by
contacting a precursor cell with a Notch function agonist. The
agonist of Notch function can be a soluble molecule, recombinantly
expressed as a cell-surface molecule, on a cell monolayer with
which the precursor cells are contacted, a molecule immobilized on
a solid phase. In another embodiment, the Notch agonist can be
recombinantly expressed from a nucleic acid introduced into the
precursor cells. Notch function agonists of the present invention
include Notch proteins and analogs and derivatives (including
fragments) thereof; proteins that are other elements of the Notch
pathway and analogs and derivatives (including fragments) thereof;
antibodies thereto and fragments or other derivatives of such
antibodies containing the binding region thereof; nucleic acids
encoding the proteins and derivatives or analogs; as well as
toporythmic proteins and derivatives and analogs thereof which bind
to or otherwise interact with Notch proteins or other proteins in
the Notch pathway such that Notch function is promoted. Such
agonists include but are not limited to Notch proteins and
derivatives thereof comprising the intracellular domain, Notch
nucleic acids encoding the foregoing, and proteins comprising
toporythmic protein domains that interact with Notch (e.g., the
extracellular domain of Delta, Serrate or Jagged). Other agonists
include Deltex and Suppressor of Hairless. These proteins,
fragments and derivatives thereof can be recombinantly expressed
and isolated or can be chemically synthesized.
[0087] In a preferred embodiment the agonist is a protein
consisting of at least a fragment (termed herein "adhesive
fragment") of the proteins encoded by toporythmic genes which
mediate binding to Notch proteins or adhesive fragments thereof.
Toporythmic genes, as used herein, shall mean the genes Notch,
Delta, Serrate, Jagged, Suppressor of Hairless and Deltex, as well
as other members of the Delta/Serrate/Jagged family or Deltex
family which may be identified by virtue of sequence homology or
genetic interaction and more generally, members of the "Notch
cascade" or the "Notch group" of genes, which are identified by
molecular interactions (e.g., binding in vitro, or genetic
interactions (as depicted phenotypically, e.g., in Drosophila).
[0088] Vertebrate homologs of Notch pathway elements have been
cloned and sequenced. For example, these include Serrate (Lindsell
et al., 1995, Cell 80:909-917); Delta (Chitnis et al., 1995, Nature
375:761; Henrique et al., 1995, Nature 375:787-790; Bettenhausen et
al., 1995, Development 121:2407); and Notch (Coffman et al., 1990,
Science 249:1438-1441; Bierkamp et al., 1993, Mech. Dev. 43:87-100;
Stifani et al., 1992, Nature Genet. 2:119-127; Lardelli et al.,
1993, Exp. Cell Res. 204:364-372; Lardelli et al., 1994, Mech. Dev.
46:123-136; Larsson et al., 1994, Genomics 24:253-258; Ellisen et
al., 1991, Cell 66:649-661; Weinmaster et al., 1991, Development
113:199-205; Reaume et al., 1992, Dev. Biol. 154:377-387; Weinmster
et al., 1992, Development 116:931-941; Franco del Amo et al., 1993,
Genomics 15:259-264; and Kopan et al., 1993, J. Cell. Biol.
121:631-641).
[0089] In one embodiment, the Notch agonist is expressed from a
recombinant nucleic acid. For example, in vivo expression of
truncated, "activated" forms of the Notch receptor lacking the
extra cellular, ligand binding domain results in gain of function
mutant phenotypes. When analyzed at the single cell level, these
phenotypes demonstrate that expression of such molecules in
progenitor or stem cells, prevents the cells from responding to
differentiation signals, thus inhibiting differentiation. It has
also been mentioned that this process may be desired to be
reversible, since when the activated Notch receptor is no longer
expressed the undifferentiated stem cells can respond to
differentiation signals and differentiate. Thus, preferably the
Notch dominant active mutant is expressed inside the precursor
cells from an inducible promoter, such that expression can be
induced in vitro for expansion, with the inducer lacking in vivo so
that differentiation occurs after administration of the
transplanted cells.
[0090] Alternatively, in another embodiment the agonist of Notch
function is not a recombinant dominant Notch active mutant.
[0091] Alternatively, in another embodiment, contacting of the
precursor cells with a Notch agonist is not done by incubation with
other cells recombinantly expressing a Notch ligand on the cell
surface (although in other embodiments, this method can be
used).
[0092] In another embodiment, the recombinantly expressed Notch
agonist is a chimeric Notch protein which comprises the
intracellular domain of Notch and the extracellular domain of
another ligand-binding surface receptor. For example, a chimeric
Notch protein comprising the EGF receptor extracellular domain and
the Notch intracellular domain is expressed in a precursor cell.
However, the Notch pathway will not be active unless the EGF
receptor ligand EGF is contacted with the precursor cell-expressing
the chimera. As with the inducible promoter controlling the
expression of the truncated form of Notch, the activity of the
chimeric Notch protein is reversible; when EGF is removed from the
cells, Notch activity will cease and the cell can then
differentiate. Notch activity can again be turned on with the
addition of the ligand.
[0093] A systematic deletion analysis of the intracellular domain
of Notch demonstrates that the Notch sequences that are both
necessary and sufficient for the downstream signaling of the Notch
receptor are confined to the ankyrin repeats of the intracellular
region (Matsuno et al., 1995, Development 121:2633-2644 and
unpublished results). Using the yeast two hybrid system it was
discovered that the ankyrin repeats interact homotypically.
[0094] Expression of appropriate deletion constructs in the defined
cellular environment of the developing Drosophila eye demonstrates
that expression of a polypeptide fragment comprising just the
ankyrin repeats resulted in an activated phenotype. Not
surprisingly this is the part of the Notch protein which is most
highly conserved among various species. FIG. 4 shows the high
sequence homology of ankyrin repeats across evolution.
[0095] These findings suggest that any small molecules, for
example, but not by way of limitation, polypeptides or antibodies
which bind to the Notch ankyrin repeats, can block its function,
and hence behave as antagonists of the pathway. Conversely,
molecules that mimic the Notch ankyrin repeat activity can behave
as agonists of the Notch pathway. Since the expression of truncated
forms of Notch give mutant phenotypes in the developing Drosophila
eye, genetic screens for modifiers of these phenotypes can be used
for identifying and isolating additional gene products that can act
as agonists or antagonists of the pathway.
[0096] Genes that act as enhancers of the activated phenotypes are
potential agonists and those that act as suppressors are potential
antagonists.
[0097] Deltex and Suppressor of Hairless are also agonists of Notch
function that can be used. It has been shown that the activation of
the Notch pathway, as judged by the induction of activated
phenotypes similar to those induced by the expression of activated
forms of Notch, can be achieved by manipulating the expression of
Deltex (Schweisguth and Posakony, 1994, Development 120:1477), as
well as Suppressor of Hairless (Matsuno et al., 1995, Development
121:2633) both of which can interact with the ankyrin repeats of
Notch.
[0098] Using the yeast `interaction trap` assay (Zervos et al.,
1993, Cell 72:223-232), as well as cell culture co-localization
studies, the protein regions responsible for heterotypic
interactions between Deltex and the intracellular domain of Notch,
as well as homotypic interaction among Deltex molecules were
defined. The function of the Deltex-Notch interaction domains was
examined by in vivo expression studies. Taken together, data from
over-expression of Deltex fragments and from studies of physical
interactions between Deltex and Notch demonstrate that Deltex
positively regulates the Notch pathway through interactions with
the Notch ankyrin repeats.
[0099] Experiments involving cell cultures indicate that the
Deltex-Notch interaction prevents the cytoplasmic retention of
Suppressor of Hairless protein, which is normally sequestered in
the cytoplasm via association with the Notch ankyrin repeats and
translocates to the nucleus when Notch binds to its ligand, Delta.
On the basis of these findings Deltex appears to regulate Notch
activity by antagonizing the interaction between Notch and
Suppressor of Hairless. The translocation of the normally
cytoplasmic Suppressor of Hairless protein to the nucleus when
Notch binds to a ligand (Fortini and Artavanis-Tsakonas, 1994, Cell
79:273-282) is a convenient assay to monitor for Notch function as
well as for the ability of Notch agonists of the present invention
to activate Notch function.
[0100] Suppressor of Hairless has been shown to be a DNA binding
protein. Genetic and molecular data indicate that the activity of
Suppressor of Hairless can be influenced by its binding to the
nuclear protein Hairless. Moreover it appears that the
transcription of at least some of the bHLH genes of the Enhancer of
split complex depends directly on Notch signaling and the ability
of Suppressor of Hairless to recognize the appropriate binding
sites upstream of these genes. Manipulation of these various
interactions (e.g., disrupting the interaction between Notch and
Suppressor of Hairless with an antibody directed against the
ankyrin repeats) will result in modulating the activity of the
Notch pathway.
[0101] Finally, the Notch pathway can be manipulated by the binding
of Notch ligands (e.g., Delta, Serrate) to the extracellular
portion of the Notch receptor. Notch signaling appears to be
triggered by the physical interaction between the extracellular
domains of Notch and its membrane-bound ligands on adjacent cells.
The expression of full length ligands on one cell triggers the
activation of the pathway in the neighboring cell which expresses
the Notch receptor. Not surprisingly, the ligands act as agonists
of the pathway. On the other hand, the expression of truncated
Delta or Serrate molecules which lack intracellular domains
expressed in neighboring cells results in non-autonomous, dominant
negative phenotypes. This demonstrates that these mutant forms of
the receptor act as antagonists of the pathway.
[0102] The definition of the various molecular interactions among
the Notch pathway elements provides additional specific
pharmacological targets and assays which can be used to screen for
Notch function agonists and antagonists. Having evaluated the
consequences of a particular molecular manipulation in vivo, this
information can be used to design biochemical in vitro screening
assays for biological or pharmaceuticals that interfere or enhance
Notch function.
[0103] Screening for molecules that will trigger the dissociation
of the Notch ankyrin repeats with Suppressor of Hairless and the
subsequent translocation of Suppressor of Hairless from the
cytoplasm to the nucleus results in the identification of agonists.
The activation of transcription of a reporter gene which has been
engineered to carry several Suppressor of Hairless binding sites at
its 5' end in a cell that expresses Notch also results in the
identification of agonists of the pathway.
[0104] Reversing the underlying logic of these assays leads to the
identification of antagonists. For example, cell lines expressing
the aforementioned reporter gene can be treated with chemicals and
biologicals and those which have the capacity to stop the
expression of the reporter gene can be identified.
[0105] The precursor cell in which Notch function has been
activated is subjected to cell growth conditions to induce
proliferation. Such cell growth conditions (e.g., cell culture
medium, temperature, if growth is done in vitro) can be any of
those commonly known in the art. Preferably, both Notch activation
and exposure to cell growth conditions is carried out in vitro.
Contacting the cell with a Notch function agonist and exposing the
cell to cell growth conditions can be carried out concurrently or,
if the agonist acts over a sufficient period of time, sequentially
(as long as Notch function activation to inhibit differentiation is
present while cell growth occurs).
[0106] 5.3.1. Modulating Other Signaling Pathways with Notch
[0107] Notch defines a general cell interaction mechanism whose
biological function is to permit or block the action of
developmental signals that are essential for the progression of
undifferentiated progenitor cells to a more differentiated state.
Consistent with that is the discovery that one can modulate the
activity of other signaling pathways by modulating Notch. Thus, in
another embodiment, the invention provides methods of modulating
other cell signal transduction pathway, e.g., those that mediate
cell growth and differentiation.
[0108] A dramatic example of how Notch signaling regulates specific
differentiation pathways involves the Ras pathway in the developing
Drosophila eye, which is used to transmit an inductive signal
generated by ligand-induced activation of the Sevenless receptor
tyrosine kinase, and is blocked by appropriately timed activation
of the Notch pathway. We have demonstrated that in the cone cell
precursors of the developing Drosophila eye, Notch activation and
Ras1-mediated signalling separately cause opposite cell-fate
alterations. Co-expression studies in these cells demonstrate that
Notch activation inhibits the neural differentiation produced by
constitutively activated components of a well-defined inductive
signalling cascade, including the Sevenless receptor tyrosine
kinase, Ras1 and Raf. Therefore, the activation of Notch in a cell
blocks the action of activated ras (see Section 6, infra).
[0109] Consistent with the notion that Notch activation initiates a
distinct signalling pathway that modulates the cellular response to
signals transduced by diverse pathways is the finding that the
modulation of Notch activity controls the action of Drosophila
wingless (Hing et al., 1994, Mech. Dev. 47:261-268), a homologue of
the mouse wnt-1 locus, which encodes a secreted protein involved in
cell-signaling during various stages in development
(Nusslein-Volhard and Wieschaus, 1980, Nature 287:795-801; Martinez
Arias et al., 1988, Development 103:157-170; Nusse and Varmus,
1992, Cell 69:1073-1087; Struhl and Basler, 1993, Cell 72:527-540).
Therefore, agonists and antagonists of the Notch pathway provide a
novel and unique tool in manipulating the activity of specific
signals which control the differentiation of cells using pathways
unrelated to Notch. In a particular embodiment of the invention,
cells are contacted with an agonist of Notch function to inhibit
the function of a signaling pathway that regulates cell growth or
differentiation.
[0110] 5.4. Notch and Terminal Differentiation
[0111] The present invention is also directed to using agents to
inhibit the Notch pathway such that cells, which are maintained in
one differentiation state by Notch pathway activity, can be allowed
to change their differentiation state. Notch expression is
generally associated with non-terminally differentiated cells. One
exception to this general rule is that Notch is expressed in
post-mitotic neurons of rat and human adult retina (Ahmad et al.,
unpublished results). Immunocytochemical staining data indicates
that the Notch polypeptides recognized by the antibodies are
nuclear. The expression of engineered Notch fragments that are
localized in the nuclear has been documented (reviewed in
Artavanis-Tsakonas et al., 1995, Science 268:225-232), and these
fragments were shown to be associated with activated mutant
phenotypes. The presence of an activated form of Notch in the
nucleus may lock these cells into a particular state of
differentiation by restricting or completely blocking their
capacity to respond to differentiation and/or proliferation
stimuli. Therefore, it is conceivable that these post-mitotic
neurons maintain their differentiated state by virtue of an
activated Notch-1 form that is independent of Notch ligands. This
state may perhaps afford such cell populations a certain
plasticity. For example, an eventual cessation of nuclear Notch-1
activity might allow these cells to re-enter a mitotic state and/or
respond to specific differentiation signals. In this context, it is
interesting to note that retinal neurons in lower vertebrates such
as Goldfish and Xenopus have regenerative capacity. Chemical
ablation of specific neurons, such as degeneration of dopaminergic
amacrine cells by 6-OH dopamine result in their replacement by
regeneration (Reh and Tully, 1986, Dev. Biol. 114(2):463-469).
However, such plasticity for regenerative purposes have not been
observed in higher vertebrates. The observed Notch-1 activity in
mature retinal neurons in the rat may represent the recapitulation
of the functional significance of Notch-1 in-retinal regeneration
in lower vertebrates. The invention thus provides a method
comprising antagonizing Notch function to confer regenerative
properties on the mammalian neurons (e.g., of the central nervous
system), thus leading to regeneration. Such a method comprises
contacting a mammalian neuron with an antagonist of Notch function
and exposing the neuron to neuronal cell growth conditions.
[0112] 5.5. Obtaining Precursor Cells
[0113] Precursor cells can be obtained by any method known in the
art. The cells can be obtained directly from tissues of an
individual or from cell lines or by production in vitro from less
differentiated precursor cells, e.g., stem or progenitor cells. An
example of obtaining precursor cells from less differentiated cells
is described in Gilbert, 1991, Developmental Biology, 3rd Edition,
Sinauer Associates, Inc., Sunderland Mass. Briefly, progenitor
cells can be incubated in the presence of other tissues or growth
and differentiation factors which cause the cell to differentiate.
For example, when lung bud epithelium is cultured alone, no
differentiation occurs. However, when lung bud epithelium is
cultured with stomach mesenchyme or intestinal mesenchyme, the lung
bud epithelium differentiates into gastric glands or villi,
respectively. Further, if lung bud epithelium is cultured with
liver mesenchyme or bronchial mesenchyme, the epithelium
differentiates into hepatic cords or branching bronchial buds,
respectively. Once a progenitor cell has reached a desired
differentiation state, a Notch function agonist can be used to stop
differentiation.
[0114] 5.5.1. Isolation of Stem or Progenitor Cells
[0115] The following describes approaches which allow for the
isolation of precursor cells and precursor cell-containing tissues,
which are to be treated with agonists and, if subsequently desired,
antagonists of the Notch pathway according to the present
invention. As already alluded to, isolated cell types or even
mixtures of cell populations can be treated with Notch function
agonists. The isolated precursor cell or precursor cell population
can be cultured ex vivo for proliferation which under the influence
of the Notch function agonists and cell growth conditions can
continue to divide, i.e., expand, in order to reach the desired
numbers before transplantation. Optionally, a recombinant gene can
be introduced into the cell so that it or its progeny expresses a
desired gene product before transplantation. Introduction of a
recombinant gene can be accomplished either before or after
precursor cell expansion.
[0116] In a preferred embodiment, the precursor cell populations
are purified or at least highly enriched. However, in order to
treat precursor cells with Notch reagents it is not necessary that
the precursor cells are a pure population. Once a mixture is
treated, only Notch pathway-expressing non-differentiated
precursors will be refractory to differentiation signals but will
respond to growth signals while their differentiated partners will
eventually terminally differentiate and cease growing, such that
the precursor cells will outgrow the differentiated cells and can
be purified from the original mixed population. Consequently, the
precursor population can still be expanded selectively.
Furthermore, purification may not be necessary or desirable prior
to therapeutic administration in vivo.
[0117] The isolation of precursor cells for use in the present
invention can be carried out by any of numerous methods commonly
known to those skilled in the art. For example, one common method
for isolating precursor cells is to collect a population of cells
from a patient and using differential antibody binding, wherein
cells of one or more certain differentiation stages are bound by
antibodies to differentiation antigens, fluorescence activated cell
sorting is used to separate the desired precursor cells expressing
selected differentiation antigens from the population of isolated
cells. The following section describes exemplary methods for the
isolation of various types of stem cells.
[0118] 5.5.1.1. Mesenchymal Stem Cells
[0119] One of the most important type of progenitor cells vis a vis
for therapeutic applications are those derived from the mesenchyme.
Mesenchymal progenitors give rise to a very large number of
distinct tissues (Caplan, 1991, J. Orth. Res 641-650). Most work to
date involves the isolation and culture of cells which can
differentiate into chondrocytes and osteoblasts. The systems
developed to isolate the relevant progenitor cell populations were
worked out first in chick embryos (Caplan, 1970, Exp. Cell. Res.
62:341-355; Caplan, 1981, 39th Annual Symposium of the Society for
Developmental Biology, pp. 37-68; Caplan et al., 1980, Dilatation
of the Uterine Cervix 79-98; DeLuca et al., 1977, J. Biol. Chem.
252:6600-6608; Osdoby et al., 1979, Dev. Biol. 73:84-102; Syftestad
et al., 1985, Dev. Biol. 110:275-283). Conditions were defined
under which chick mesenchymal cells differentiated into
chondrocytes and bone. Id. With regard to cartilage and bone, the
properties of mouse or human mesenchymal limb appear to be quite
similar if not identical (Caplan, 1991, J. Orth. Res. 641-650).
Mesenchymal cells capable of differentiating into bone and
cartilage have also been isolated from marrow (Caplan, 1991, J.
Orth. Res. 641-650).
[0120] Caplan et al., 1993, U.S. Pat. No. 5,226,914 describes an
exemplary method for isolating mesenchymal stem cells from bone
marrow. These isolated marrow stem cells can be used in conjunction
with Notch reagents to expand the stem cell population. These
expanded cells may then be transplanted into a host where they can
differentiate into osteocytes, cartilage, chondocytes, adipocytes,
etc., depending on the surrounding microenvironment of the
transplant site.
[0121] Animal models involving mice, rats as well as avian
preparations, have suggested that the source for mesenchymal stem
cells is bone marrow. It has been possible to purify marrow
mesenchymal cells by their differential adhesion to culture dishes
and demonstrate that they can differentiate, e.g., into
osteoblasts. Expansion of such isolated stem cells using Notch
reagents can provide a source of cells which when transplanted to
the appropriate sites will be induces by the microenvironment to
differentiate into the appropriate lineage and help repair damaged
and/or diseased tissue. It is expected that the animal models
described to date will be applicable to humans. Indeed, as far as
cartilage and bone are concerned, the properties of mouse and human
limb mesenchymal cells in culture are quite similar, if not
identical (Hauska, 1974, Dev. Biol. 37:345-368; Owens and Solursh,
1981, Dev. Biol. 88:297-311). The isolation of human marrow and the
demonstration that cells deriving from it can sustain osteogenesis
has been described, e.g., by Bab et al., 1988, Bone Mineral
4:373-386.
[0122] Several bone marrow isolation protocols have been reported
and can be used to obtain progenitor or precursor cells. Single
cell suspensions from rat bone marrow can be prepared according to
Goshima et al., 1991, Clin. Orth. and Rel. Res. 262:298-311. Human
stem cell cultures from marrow can be prepared as described by Bab
et al., 1988, Bone Mineral 4:373-386 as follows: Whole marrow cells
are obtained from five patients. The marrow samples are separated
from either the iliac crest or femoral midshaft. Marrow samples, 3
ml in volume, are transferred to 6 ml of serum-free Minimal
Essential Medium (MEM) containing 50 U/ml penicillin and 0.05 mg/ml
streptomycin-sulfate. A suspension of predominantly single cells is
prepared as described previously (Bab et al., 1984, Calcif. Tissue
Int. 36:77-82; Ashton et al., 1984, Calcif. Tissue Int. 36:83-86)
by drawing the preparation into a syringe and expelling it several
times sequentially through 19, 21, 23 and 25 gauge needles. The
cells are counted using a fixed volume hemocytometer and the
concentration adjusted to 1-5.times.10.sup.8 total marrow cells per
ml suspension. Positive and negative control cell suspensions can
be set as described before (Shteyer et al., 1986, Calcif. Tissue
Int. 39:49-54), using rabbit whole marrow and spleen cells,
respectively.
[0123] 5.5.1.2. Neural Stem Cells
[0124] It is generally assumed that neurogenesis in the central
nervous system ceases before or soon after birth. In recent years,
several studies have presented evidence indicating that at least to
some degree new neurons continue to be added to the brain of adult
vertebrates (Alvarez-Buylla and Lois, 1995, Stem Cells (Dayt)
13:263-272). The precursors are generally located in the wall of
the brain ventricles. It is thought that from these proliferative
regions, neuronal precursors migrate towards target positions where
the microenvironment induces them to differentiate. Studies have
been reported where cells from the sub-ventricular zone can
generate neurons both in vivo as well as in vitro, reviewed in
Alvarez-Buylla and Lois, 1995, Stem Cells (Dayt) 13:263-272.
[0125] The neuronal precursors from the adult brain can be used as
a source of cells for neuronal transplantation (Alvarez-Buylla,
1993, Proc. Natl. Acad. Sci. USA 90:2074-2077). Neural crest cells
have also been long recognized to be pluripotent neuronal cells
which can migrate and differentiate into different cell neuronal
cell types according to the instructions they receive from the
microenvironment they find themselves in (LeDouarin and Ziller,
1993, Curr. Opin. Cell Biol. 5:1036-1043).
[0126] 5.5.1.3. Fetal Cells
[0127] The fact that fetal brain tissue has been shown to have
clear behavioral effects when transplanted into adult lesioned
brains, has focused attention on human fetal tissue as a potential
cell source in transplantation protocols designed to improve
neurodegenerative disorders (Bjorklund, 1993, Nature 362:414-415;
McKay, 1991, Trends Neurosci. 14:338-340). Nevertheless both
ethical, as well as practical considerations make fetal tissue a
difficult source to deal with. Expansion of neuronal stem cells
whether fetal or otherwise using Notch function agonists provides
an alternative source for obtaining the desired quantities of
precursor cells for transplantation purposes. Fetal tissues or
adult tissues containing precursors can be treated with Notch
function agonists as described earlier in order to expand the
undifferentiated progenitor cell populations. Fetal cells can
placed into primary culture using, for example, protocols developed
by Sabate et al., 1995, Nature Gen. 9:256-260, before being treated
with Notch function agonists. By way of example but not limitation,
the procedure is as follows: Primary cultures of human fetal brain
cells can be isolated from human fetuses, obtained from legal
abortions after 5 to 12 weeks of gestation. Expulsion can be done
by syringe-driven gentle aspiration under echographic control.
Fetuses collected in sterile hibernation medium are dissected in a
sterile hood under a stereomicroscope. Brains are first removed in
toto in hibernation medium containing penicillin G 500 U/ml,
streptomycin 100 .mu.g/ml, and fungizon 5 pg/ml. For fetuses of six
to eight weeks of age the brain is separated into an anterior
(telencephalic vesicles and diencephalon) and a posterior fraction
(mesencephalon, pons and cerebellar enlage) and a posterior in toto
after careful removal of meninges. For older fetuses, striatal
hippocampal, cortical and cerebellar zones expected to contain
proliferative precursor cells are visualized under the
stereomicroscope and dissected separately. Cells are transferred to
either Opti-MEM (Gibco BRL) containing 15% heat-inactivated fetal
bovine serum (FBS) (Seromed), or to a defined, serum-free medium
(DS-FM) with human recombinant bFGF (10 ng/ml, Boehringer), which
is a minor modification of the Bottenstein-Sato medium 39 with
glucose, 6 g/l, glutamine 2 mM (Gibco BRL), insulin 25 ug/ml
(Sigma) transferrin 100 .mu.g/ml (Sigma), sodium selenite 30 nM
(Gibco BRL), progesterone 20 nM (Sigma), putrescine 60 nM (Sigma),
penicillin G (500 U/ml), streptomycin 100 .mu.g/ml, and fungizon 5
.mu.g/ml. Cells, approximately 40,000 per cm.sup.2, are grown at
37.degree. C. in an atmosphere containing 10% CO.sub.2 in tissue
culture dishes (Falcon or Nunc) coated with gelatin (0.25% wt/vol)
followed by Matrigel (Gibco BRL, a basement membrane extract
enriched in laminin and containing trace amounts of growth factors
diluted one in 20). Cells in culture can be treated with Notch
function agonists in order to expand the population of the
appropriate cells until the desired cell mass is reached for
transplantation.
[0128] 5.5.1.4. Hematopoietic Stem Cells
[0129] Any technique which provides for the isolation, propagation,
and maintenance in vitro of hematopoietic stem cells (HSC) can be
used in this embodiment of the invention. Techniques by which this
can be accomplished include (a) the isolation and establishment of
HSC cultures from bone marrow cells isolated from the future host,
or a donor, or (b) the use of previously established long-term HSC
cultures, which may be allogeneic or xenogeneic. Non-autologous HSC
are used preferably in conjunction with a method of suppressing
transplantation immune reactions of the future host/patient. In a
particular embodiment of the present invention, human bone marrow
cells can be obtained from the posterior iliac crest by needle
aspiration (see, e.g., Kodo et al., 1984, J. Clin. Invest.
73:1377-1384). In a preferred embodiment of the present invention,
the HSCs can be made highly enriched or in substantially pure form.
This enrichment can be accomplished before, during, or after
long-term culturing, and can be done by any techniques known in the
art. Long-term cultures of bone marrow cells can be established and
maintained by using, for example, modified Dexter cell culture
techniques (Dexter et al., 1977, J. Cell Physiol. 91:335) or
Witlock-Witte culture techniques (Witlock and Witte, 1982, Proc.
Natl. Acad. Sci. USA 79:3608-3612).
[0130] Another technique for the isolation of HSC is described by
Milner et al., 1994, Blood 83:2057-2062. Bone marrow samples are
obtained and are separated by Ficoll-Hypaque density gradient
centrifugation, are washed, and stained using two-color indirect
immunofluorescent antibody binding and then separated by
fluorescence-activated cell sorting (FACS). The cells are labelled
simultaneously with IgG antibodies such that CD34.sup.+
hematopoietic stem cells, including the immature subset that lacks
expression of individual lineage associated antigens,
CD34.sup.+lin.sup.-, are isolated from the cells collected from
marrow.
[0131] Where hematopoietic progenitor cells are desired, the
presence of hematopoietic progenitor cells and/or their progeny can
be detected by commonly known in vitro colony forming assays (e.g.,
those that detect CFU-GM, BFU-E). As another example, assays for
hematopoietic stem cells are also known in the art (e.g., spleen
focus forming assays, assays that detect the ability to form
progenitors after replating).
[0132] 5.5.1.5. Epithelial Stem Cells
[0133] Epithelial stem cells (ESCs) or keratinocytes can be
obtained from tissues such as the skin and the lining of the gut by
known procedures (Rheinwald, 1980, Meth. Cell Bio. 21A:229). In
stratified epithelial tissue such as the skin, renewal occurs by
mitosis of precursor cells within the germinal layer, the layer
closest to the basal lamina. Precursor cells within the lining of
the gut provide for a rapid renewal rate of this tissue. ESCs or
keratinocytes obtained from the skin or lining of the gut of a
patient or donor can be grown in tissue culture (Rheinwald, 1980,
Meth. Cell Bio. 21A:229; Pittelkow and Scott, 1986, Mayo Clinic
Proc. 61:771). If the ESCs are provided by a donor, a method for
suppression of host versus graft reactivity (e.g., irradiation,
drug or antibody administration to promote moderate
immunosuppression) can also be used.
[0134] 5.5.1.6. Liver Stem Cells
[0135] Liver stem cells can be isolated by methods described in PCT
Publication WO 94/08598, dated Apr. 28, 1994.
[0136] 5.5.1.7. Kidney Stem Cells
[0137] Mammalian kidney emerges from the metanephric mesenchyme
which induces the uteric bud to undergo a series of morphogenetic
movements ultimately forming the mature urinary collecting system
(Nigam and Brenner, 1992, Curr. Opin. Nephrol. Huper 1:187-191. The
uteric bud, an epithelial outgrowth of the Wolfian duct, contracts
and induces condensing adjacent mesenchyme along differentiation
pathways of epithelial divergence in early embryonic life. Attempts
to study this process in vitro have been reported; metanephros in
organ culture can be induced to form tubules using embryonic spinal
cord as the inducer. While the specific transducing agents that
lead to the induction of metanephric mesenchyme by the uteric bud
in vivo or by spinal cord in vitro are not known, it is clear that
differentiation program is induced in progenitor cells (Karp et
al., 1994, Dev. Biol. 91:5286-5290).
[0138] 5.5.2. Expansion and Differentiation
[0139] After the precursors cells have been isolated according to
the methods described above or other methods known in the art, the
precursor cells can be contacted with an amount of an agonist of
Notch function effective to inhibit differentiation, and are
exposed to cell growth conditions (e.g., promoting mitosis) such
that the cell proliferates to obtain an expanded precursor
population according to the present invention.
[0140] In one embodiment, substantially no differentiation of the
precursor cells occurs during expansion. The amount of
differentiation that occurs can be assayed for by known assays,
e.g., those that detect the presence of more differentiated cells
by detecting functions associated with a particular stage of
differentiation, e.g., expression of differentiation antigens on
the cell surface or secretion of proteins associated with a
particular state, or ability to generate various cell types, or
detecting morphology associated with particular stages of
differentiation.
[0141] Once the population has reached a desired titer, the Notch
function agonist can be removed (e.g., by separation, dilution), or
a Notch function antagonist can be added, such that Notch function
is absent or inhibited allowing at least some of the cells in the
expanded population to differentiate in the presence of the desired
differentiation signals to a desired differentiation state or to a
differentiation state of the cell such that the cell expresses a
desired phenotype. Optionally, once the cells reach the desired
differentiation state the Notch pathway can again be activated with
a Notch agonist to freeze the cell in that differentiation state.
The cells can be differentiated to a terminally differentiated
state if the function of that terminally differentiated cell is
desired.
[0142] 5.6. Gene Therapy
[0143] The cells produced by manipulation of the Notch pathway can
be made recombinant and used in gene therapy. In its broadest
sense, gene therapy refers to therapy performed by the
administration of a nucleic acid to a subject. The nucleic acid,
either directly or indirectly via its encoded protein, mediates a
therapeutic effect in the subject. The present invention provides
methods of gene therapy wherein a nucleic acid encoding a protein
of therapeutic value (preferably to humans) is introduced into the
precursor cells expanded according to the invention, before or
after expansion, such that the nucleic acid is expressible by the
precursor cells and/or their progeny, followed by administration of
the recombinant cells to a subject.
[0144] The recombinant precursor cells of the present invention can
be used in any of the methods for gene therapy available in the
art. Thus, the nucleic acid introduced into the cells may encode
any desired protein, e.g., a protein missing or dysfunctional in a
disease or disorder. The descriptions below are meant to be
illustrative of such methods. It will be readily understood by
those of skill in the art that the methods illustrated represent
only a sample of all available methods of gene therapy.
[0145] For general reviews of the methods of gene therapy, see
Goldspiel et al., 1993, Clinical Pharmacy 12:488-505; Wu and Wu,
1991, Biotherapy 3:87-95; Tolstoshev, 1993, Ann. Rev. Pharmacol.
Toxicol. 32:573-596; Mulligan, 1993, Science 260:926-932; and
Morgan and Anderson, 1993, Ann. Rev. Biochem. 62:191-217; May,
1993, TIBTECH 11(5):155-215). Methods commonly known in the art of
recombinant DNA technology which can be used are described in
Ausubel et al. (eds.), 1993, Current Protocols in Molecular
Biology, John Wiley & Sons, NY; and Kriegler, 1990, Gene
Transfer and Expression, A Laboratory Manual, Stockton Press,
NY.
[0146] In an embodiment in which recombinant precursor cells are
used in gene therapy, a gene whose expression is desired in a
patient is introduced into the precursor cells such that it is
expressible by the cells and/or their progeny, and the recombinant
cells are then administered in vivo for therapeutic effect.
[0147] Precursor cells or expanded precursor cells can be used in
any appropriate method of gene therapy, as would be recognized by
those in the art upon considering this disclosure. The resulting
action of a recombinant precursor cell or its progeny cells
administered to a patient can, for example, lead to the activation
or inhibition of a pre-selected gene in the patient, thus leading
to improvement of the diseased condition afflicting the
patient.
[0148] The desired gene is transferred to precursor cells in tissue
culture by such methods as electroporation, lipofection, calcium
phosphate mediated transfection, or viral infection. Usually, the
method of transfer includes the transfer of a selectable marker to
the cells. The cells are then placed under selection to isolate
those cells that have taken up and are expressing the transferred
gene. Those precursor cells are then delivered to a patient.
[0149] In this embodiment, the desired gene is introduced into a
precursor cell prior to administration in vivo of the resulting
recombinant cell. Such introduction can be carried out by any
method known in the art, including but not limited to transfection,
electroporation, microinjection, infection with a viral or
bacteriophage vector containing the gene sequences, cell fusion,
chromosome-mediated gene transfer, microcell-mediated gene
transfer, spheroplast fusion, etc. Numerous techniques are known in
the art for the introduction of foreign genes into cells (see e.g.,
Loeffler and Behr, 1993, Meth. Enzymol. 217:599-618; Cohen et al.,
1993, Meth. Enzymol. 217:618-644; Cline, 1985, Pharmac. Ther.
29:69-92) and may be used in accordance with the present invention,
provided that the necessary developmental and physiological
functions of the recipient cells are not disrupted. The technique
should provide for the stable transfer of the gene to the cell, so
that the gene is expressible by the cell and preferably heritable
and expressible by its cell progeny.
[0150] One common method of practicing gene therapy is by making
use of retroviral vectors (see Miller et al., 1993, Meth. Enzymol.
217:581-599). A retroviral vector is a retrovirus that has been
modified to incorporate a preselected gene in order to effect the
expression of that gene. It has been found that many of the
naturally occurring DNA sequences of retroviruses are dispensable
in retroviral vectors. Only a small subset of the naturally
occurring DNA sequences of retroviruses is necessary. In general, a
retroviral vector must contain all of the cis-acting sequences
necessary for the packaging and integration of the viral genome.
These cis-acting sequences are:
[0151] a) a long terminal repeat (LTR), or portions thereof, at
each end of the vector;
[0152] b) primer binding sites for negative and positive strand DNA
synthesis; and
[0153] c) a packaging signal, necessary for the incorporation of
genomic RNA into virions.
[0154] The gene to be used in gene therapy is cloned into the
vector, which facilitates delivery of the gene into a precursor
cell by infection or delivery of the vector into the cell.
[0155] More detail about retroviral vectors can be found in Boesen
et al., 1994, Biotherapy 6:291-302, which describes the use of a
retroviral vector to deliver the mdr1 gene to hematopoietic stem
cells in order to make the stem cells more resistant to
chemotherapy. Other references illustrating the use of retroviral
vectors in gene therapy are: Clowes et al., 1994, J. Clin. Invest.
93:644-651; Kiem et al., 1994, Blood 83:1467-1473; Salmons and
Gunzberg, 1993, Human Gene Therapy 4:129-141; and Grossman and
Wilson, 1993, Curr. Opin. in Genetics and Devel. 3:110-114.
[0156] Adenoviruses are also of use in gene therapy. Adenoviruses
are especially attractive vehicles for delivering genes to
respiratory precursor cells. Adenoviruses can also be used to
deliver genes to precursor cells from the liver, the central
nervous system, endothelium, and muscle. Adenoviruses have the
advantage of being capable of infecting non-dividing cells.
Kozarsky and Wilson, 1993, Current Opinion in Genetics and
Development 3:499-503 present a review of adenovirus-based gene
therapy. Other instances of the use of adenoviruses in gene therapy
can be found in Rosenfeld et al., 1991, Science 252:431-434;
Rosenfeld et al., 1992, Cell 68:143-155; and Mastrangeli et al.,
1993, J. Clin. Invest. 91:225-234.
[0157] It has been proposed that adeno-associated virus (AAV) be
used in gene therapy (Walsh et al., 1993, Proc. Soc. Exp. Biol.
Med. 204:289-300).
[0158] A desired gene can be introduced intracellularly and
incorporated within host precursor cell DNA for expression, by
homologous recombination (Koller and Smithies, 1989, Proc. Natl.
Acad. Sci. USA 86:8932-8935; Zijlstra et al., 1989, Nature
342:435-438).
[0159] In a specific embodiment, the desired gene recombinantly
expressed in the precursor cell to be introduced for purposes of
gene therapy comprises an inducible promoter operably linked to the
coding region, such that expression of the recombinant gene is
controllable by controlling the presence or absence of the
appropriate inducer of transcription.
[0160] In another embodiment, if a greater number of differentiated
cells is desired before administering to a patient then the
precursor cells can be differentiated prior to expansion. In
another embodiment, one can expand and differentiate the precursor
cells simultaneously such that greater numbers of differentiated
cells are obtained.
[0161] 5.7. Pharmaceutical Compositions
[0162] The invention provides methods of treatment by
administration to a subject of a pharmaceutical (therapeutic)
composition comprising a therapeutically effective amount of a
recombinant or non-recombinant cell, preferably a stem or
progenitor cell. Such a stem cell or recombinant stem cell
envisioned for therapeutic use is referred to hereinafter as a
"Therapeutic" or "Therapeutic of the invention." In a preferred
aspect, the Therapeutic is substantially purified. The subject is
preferably an animal, including but not limited to animals such as
cows, pigs, horses, chickens, cats, dogs, etc., and is preferably a
mammal, and most preferably human.
[0163] The present invention provides pharmaceutical compositions.
Such compositions comprise a therapeutically effective amount of a
Therapeutic, and a pharmaceutically acceptable carrier or
excipient. Such a carrier includes but is not limited to saline,
buffered saline, dextrose, water, glycerol, ethanol, and
combinations thereof. The carrier and composition can be sterile.
The formulation should suit the mode of administration.
[0164] The composition, if desired, can also contain minor amounts
of wetting or emulsifying agents, or pH buffering agents. The
composition can be a liquid solution, suspension, or emulsion.
[0165] In a preferred embodiment, the composition is formulated in
accordance with routine procedures as a pharmaceutical composition
adapted for intravenous administration to human beings. Typically,
compositions for intravenous administration are solutions in
sterile isotonic aqueous buffer. Where necessary, the composition
may also include a solubilizing agent and a local anesthetic such
as lignocaine to ease pain at the site of the injection.
[0166] 5.8. Transplantation
[0167] The expanded stem cell populations of the present invention
whether recombinantly expressing a desired gene or not can be
transplanted into a patient for the treatment of disease or injury
or for gene therapy by any method known in the art which is
appropriate for the type of stem cells being transplanted and the
transplant site. Hematopoietic stem cells can be transplanted
intravenously, as can liver stem cells which will locate to the
liver. Neural stem cells can be transplanted directly into the
brain at the site of injury or disease.
[0168] Methods of introduction include but are not limited to
intradermal, intramuscular, intraperitoneal, intravenous,
subcutaneous, intranasal, and epidural routes. The compounds may be
administered by any convenient route, for example by infusion or
bolus injection, by absorption through epithelial or mucocutaneous
linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and
may be administered together with other biologically active agents.
Administration can be systemic or local. In addition, it may be
desirable to introduce the pharmaceutical compositions of the
invention into the central nervous system by any suitable route,
including intraventricular and intrathecal injection;
intraventricular injection may be facilitated by an
intraventricular catheter, for example, attached to a reservoir,
such as an Ommaya reservoir.
[0169] In a specific embodiment, it may be desirable to administer
the Therapeutics of the invention locally to the area in need of
treatment; this may be achieved by, for example, and not by way of
limitation, local infusion during surgery, topical application,
e.g., in conjunction with a wound dressing after surgery, by
injection, by means of a catheter, or by means of an implant, said
implant being of a porous, non-porous, or gelatinous material,
including membranes, such as sialastic membranes, or fibers.
[0170] The following describes exemplary methods which can be
modified for the transplantation of precursor cells: Protocols for
the isolation and transplantation of fetal tissues in humans have
been reported and clinical trials involving these studies having
been carried out. For example, Lindvall et al., 1990, Science
247:574-577, have described results regarding grafts and survival
of fetal dopamine neurons after transplantation into brain. Rinsing
and partial dissociation of precursor cells, if necessary, can be
carried out by a modification of that described in Lindvall et al.,
1989, Arch. Neurol. 46:615.
[0171] By way of example, implantation of cells into the brain can
be performed as follows. Implantation is done at three sites in the
left putamen with a stereotactic technique (Lindvall et al., 1989,
Arch. Neurol. 46:615). For each site, 20 .mu.l of the dissociated
cells is drawn into the instrument (outer diameter, 1.0 mm). The
cells are injected along a 10, 12 and 14 mm linear tract,
respectively, in either 2.5 .mu.l portions for 15 to 20 seconds
each. Between each injection there is a 2 minute delay, and the
cannula is then retracted 1.5 to 1.7 mm. After the final injection,
the cannula is left in situ for 8 minutes before being slowly
withdrawn from the brain. After surgery the cell viability is
assessed following the procedure of Brundin et al., 1985, Brain.
Res. 331:251.
[0172] Another example is outlined by Caplan et al., 1993, U.S.
Pat. No. 5,226,914. Briefly, after marrow cells are harvested from
bone marrow plugs and the marrow mesenchymal, stem cells are
separated by centrifugation. The stem cells are isolated further by
selective adherence to the plastic or glass surface of a tissue
culture dish. The stem cells are allowed to proliferate but not
differentiate. Porous ceramic cubes composed of 60% hydroxyapatite
and 40% .beta.-tricalcium phosphate are added to the cells under a
slight vacuum. The cubes with adhered cells are implanted into
incisional pockets along the backs of nude mice. The mesenchymal
stem cells differentiate into bone.
[0173] The titer of stem cells transplanted or the amount of the
Therapeutic of the invention which will be effective in the
treatment of a particular disorder or condition will depend on the
nature of the disorder or condition, and can be determined by
standard clinical techniques. In addition, in vitro assays may
optionally be employed to help identify optimal dosage ranges. The
precise dose to be employed in the formulation will also depend on
the route of administration, and the seriousness of the disease or
disorder, and should be decided according to the judgment of the
practitioner and each patient's circumstances.
[0174] 6. Inhibition of Ras-1-Mediated Signaling by Activated Notch
in Drosophila Eye
[0175] In the cone cell precursors of the developing Drosophila
eye, Notch activation and Ras1-mediated signaling separately cause
opposite cell-fate alterations. Co-expression studies in these
cells demonstrate that Notch activation inhibits the neural
differentiation produced by constitutively activated components of
a well-defined inductive signaling cascade, including the Sevenless
receptor tyrosine kinase, Ras1 and Raf.
[0176] The sevenless signaling pathway is required only for the
induction of the R7 photoreceptor cell by the previously determined
R8 cell of each ommatidium. The interaction of the sevenless gene
product and its ligand, encoded by the bride of sevenless (boss)
gene, normally occurs only between two particular cell types during
a narrow developmental time window (Basler and Hafen, 1989,
Development 107:723-731; Mullins and Rubin, 1991, Proc. Natl. Acad.
Sci. USA 88:9387-9391; Kramer et al., 1991, Nature 352:207-212).
Plies mutant for either or both genes display a very specific
phenotype: misrouting of the R7 cell precursor into the cone-cell
fate (Tomlinson and Ready, 1986, Science 231:400-402, 1987, Dev.
Biol. 123:264-275; Reinke and Zipursky, 1988, Cell 55:321-330).
Within each ommatidium, sevenless is expressed in a small set of
cells separated by no more than a few cell diameters, consisting of
the R3, R4, and R7 precursor cells, the four cone cell precursor
cells, and up to two so-called `mystery cells` (Tomlinson et al.,
1987, Cell 51:143-150; Bowtell et al., 1989, Proc. Natl. Acad. Sci.
USA 86:6245-6249; Basler et al., 1989, EMBO J. 8:2381-2386). In
wild-type flies, only the R7 precursor cell ever comes into contact
with the R8 cell, which is the only eye disc cell type that
expresses bride of sevenless, resulting in the recruitment of one
R7 cell per ommatidium (Kramer et al., 1991, Nature 352:207-212).
Experiments in which sev and boss were expressed ubiquitously under
heat-shock gene control have demonstrated that the spatially
restricted presentation of ligand by the R8 cell is a crucial
feature of this inductive signalling mechanism (Basler and Hafen,
1989, Science 243:931-934; Bowtell et al., 1989, Cell 56:931-936;
Van Vactor et al., 1991, Cell 67:1145-1155). Recent studies have
shown that activation of the Sevenless receptor tyrosine kinase
initiates a signaling cascade involving the activation of Ras1 and
the subsequent activation of Raf (Simon et al., 1991, Cell
67:701-716; Bonfini et al., 1992, Science 255:603-606; Dickson et
al., 1992, Genes Dev. 6:2327-2339; Dickson et al., Nature
360:600-603). Ras1 and Raf are also downstream targets of other
receptor tyrosine kinases in Drosophila, including the torso kinase
and the Drosophila EGF receptor homolog (Ambrosio et al., 1989,
Nature 342:288-291; Simon et al., 1991, Cell 67:701-716; Doyle and
Bishop, 1993, Genes Dev. 7:633-646; Melnick et al., 1993,
Development 118:127-138; Diaz-Benjumea and Hafen, 1994, Development
120:569-578).
[0177] In contrast to sevenless-mediated signalling, the signalling
mechanism involving Notch appears to regulate a common step in
cell-fate commitment throughout development. The Drosophila Notch
gene encodes a large transmembrane receptor protein with an
extracellular domain consisting of 36 tandem EGF-like repeats and 3
Notch/lin-12 repeats as well as an intracellular domain containing
6 tandem cdc10/ankyrin repeats (Wharton et al., 1985, Cell
43:567-581; Kidd et al., 1986, Mol. Cell. Biol. 6:3094-3108).
Unlike the sev and boss gene products, the Drosophila Notch protein
is widely expressed in developing tissues, including all or most
cells of the imaginal eye disc (Johansen et al., 1989, J. Cell
Biol. 109:2427-2440; Kidd et al., 1989, Genes Dev. 3:1113-1129;
Fehon et al., 1991, J. Cell. Biol. 113:657-669). Analysis of Notch
gene mutant phenotypes has revealed that Notch function is required
for numerous developmental processes, including embryonic
neurogenesis (Poulson, 1937, Proc. Natl. Acad. Sci. USA 23:133-137,
1940, J. Exp. Zool. 83:271-325), mesoderm differentiation (Corbin
et al., 1991, Cell 67:311-323), axonal pathfinding (Giniger et al.,
1993, Development 117:431-440), oogenesis (Ruohola et al., 1991,
Cell 66:433-449; Xu et al., 1992, Development 115:913-922; Cummings
and Cronmiller, 1994, Development 120:381-394), and differentiation
of adult peripheral nervous system structures (Cagan and Ready,
1989, Genes Dev. 3:1099-1112; Palka et al., 1990, Development
109:167-175; Hartenstein and Posakony, 1990, Dev. Biol. 142:13-30;
Hartenstein et al., 1992, Development 116:1203-1220). A detailed
study of the phenotypic effects of the conditional loss-of-function
allele Notch.sup.tsl has shown that every cell type of the adult
eye, including the R7 cell, requires Notch activity at some stage
for its proper cell-fate specification (Cagan and Ready, 1989,
Genes Dev. 3:1099-1112).
[0178] In the absence of boss gene function, the
sevenless-expressing cells of each ommatidium may be induced to
differentiate as neurons by ectopic activation of the Sevenless
protein, Ras1, or Raf (Basler et al., 1991, Cell 64:1069-1081;
Fortini et al., 1992, Nature 355:559-561; Dickson et al., 1992,
Genes Dev. 6:2327-2339; Dickson et al., Nature 360:600-603).
Evidence is presented below that the neural induction of these
cells by activated sevenless pathway components is blocked by
constitutive Notch activation in the developing eye imaginal disc.
These results indicate that the signal mediated by Notch and its
ligands are integrated with the cell type-specific inductive signal
mediated by Sevenless at a point downstream of Raf during R7
photoreceptor cell fate specification. Since both Ras1 and Raf are
utilized by other tissue-specific inductive signalling pathways,
our data implies that Notch may exert regulatory effects on these
pathways as well.
[0179] 6.1. Materials and Methods
[0180] Drosophila Culture:
[0181] Flies were grown on standard medium at 18.degree. C. for
optimal imaginal disc growth.
[0182] Immunohistochemistry:
[0183] Antibody staining of eye imaginal discs was performed as
described in Gaul et al., 1992, Cell 68:1007-1019. For the
Notch/ELAV stainings, mouse mAb C17.9C6 (Fehon et al., 1990, Cell
61:523-534) and rat mAb 7E8A10 (Robinow and White, 1991, J.
Neurobiol. 22:443-461) were used at 1:2000 and 1:1 dilutions,
respectively. For the Notch/Sevenless double stainings, rat
polyclonal Ab Rat5 (R. G. Fehon, I. Rebay, and S.
Artavanis-Tsakonas, unpublished) and mouse mAb sev150C3 (Banerjee
et al., 1987, Cell 51:151-158) were used at 1:500 and 1:1000
dilutions, respectively. In both cases, goat anti-mouse
FITC-conjugated and goat anti-rat Texas Red-conjugated double-label
grade secondary antibodies (Jackson ImmunoResearch Laboratories,
Inc.) were used at 1:250 and 1:500 dilutions, respectively.
[0184] Confocal Microscopy:
[0185] Confocal microscopy and image processing were performed as
described by Xu et al., 1992, Development 115:913-922.
6.2. Results
[0186] Previous studies on R7 photoreceptor cell determination in
Drosophila have shown that specification of neural fate in the R7
precursor cell is initiated by ligand-induced activation of the
receptor tyrosine kinase encoded by sevenless (reviewed in
Greenwald and Rubin, 1992, Cell 68:271-281), which is expressed
strongly in a subset of uncommitted cells in each developing
ommatidium, namely the R3, R4, and R7 precursor cells, the four
cone cell precursors, and up to two so-called `mystery cells`
(Tomlinson et al., 1987, Cell 51:143-150; Bowtell et al., 1989,
Proc. Natl. Acad. Sci. USA 86:6245-6249; Basler et al., 1989, EMBO
J. 8:2381-2386). Activation of the Sevenless tyrosine kinase
results in the subsequent activation of Ras1 (Simon et al., 1991,
Cell 67:701-716; Bonfini et al., 1992, Science 255:603-606), which
in turn activates Raf (Dickson et al., 1992, Nature 360:600-603).
These studies have also led to the production of transgenic fly
lines bearing constitutively activated Sevenless, Ras1, and Raf
proteins, all expressed under sevenless gene control in the above
mentioned cells (Basler et al., 1991, Cell 64:1069-1081; Fortini et
al., 1992, Nature 355:559-561; Dickson et al., 1992, Genes Dev.
6:2327-2339; Dickson et al., Nature 360:600-603). In each case,
expression of the activated sevenless pathway component drives
sevenless-expressing cells into neural fates, as judged by the
expression of neural-specific antigens such as BP-104 (Hortsch et
al., 1990, Neuron 4:697-709) or ELAV (Bier et al., 1988, Science
240:913-916; Robinow and White, 1991, J. Neurobiol. 22:443-461) in
the eye disc. While the wild-type Notch gene is expressed in and
required for normal development of all or most eye disc cells
(Cagan and Ready, 1989, Genes Dev. 3:1099-1112; Fehon et al., 1991,
J. Cell. Biol. 113:657-669), a constitutively activated Notch
receptor lacking the extracellular and transmembrane domains
expressed under sevenless gene control blocks cell-fate commitment,
preventing ELAV expression in neural precursors and causing
cell-fate misspecifications among the sevenless-expressing cells
(Fortini et al., 1993, Nature 365:555-557).
[0187] To determine whether the block imposed by activated Notch
upon neural differentiation can be circumvented by constitutive
activation of any of the sevenless signalling pathway components,
transgenic flies were produced co-expressing activated Notch and
activated sevenless pathway factors in the sevenless-expressing
cells. Eye discs of these flies were double-stained with antibodies
directed against Notch and against the ELAV protein to determine
whether cells expressing activated Notch are capable of neural
induction by activated Sevenless, activated Ras, or activated Raf.
Since ELAV is a nuclear antigen, we chose to use an activated Notch
construct, termed sev-Notch.sup.nucl, that produces nuclear Notch
protein localization (Fortini et al., 1993, Nature 365:555-557).
This nuclear Notch expression is easily distinguished from the
apical membrane distribution of the endogenous wild-type Notch
protein (Fehon et al., 1991, J. Cell. Biol. 113:657-669; Fortini et
al., 1993, Nature 365:555-557). Nuclear translocation of the Notch
protein apparently is not required for its activated behavior,
since the same phenotypic effects are caused by a truncated Notch
protein lacking extracellular but not transmembrane sequences that
is apically localized, as judged by antibody staining experiments
(Fortini et al., 1993, Nature 365:555-557).
[0188] The analysis was restricted to the four cone cell precursors
of each developing ommatidium for the following reasons. First, the
cone cell precursor nuclei are easily identified by their
distinctive sausage-shaped morphology. Second, the cone cell
precursors are normally non-neural and thus should only be
ELAV-positive as a result of the transgene-driven activated
sevenless pathway components. Third, Notch expression in
sev-Notch.sup.nucl cone cell precursor nuclei persists throughout
those ommatidial rows of the posterior eye disc in which cone cell
precursors exhibit strong ELAV expression if they are transformed
into neurons (Fortini et al., 1992, Nature 355:559-561, 1993,
Nature 365:555-557; Gaul et al., 1992, Cell. 68:1007-1019; Dickson
et al., 1992, Nature 360:600-603). By contrast, the nuclear Notch
expression in R3 and R4 precursor cells and mystery cells is more
transient, subsiding prior to the onset of ELAV expression (Fortini
et al., 1993, Nature 365:555-557).
[0189] Notch-positive cone cell precursor nuclei were scored for
ELAV expression in sev-Notchnucl flies also carrying either the
activated Sevenless tyrosine kinase construct sev-S11 (Basler et
al., 1991, Cell 64:1069-1081), the activated Ras1 construct
sevRas1.sup.Val12 (Fortini et al., 1992, Nature 355:559-561), or
the activated Raf construct sE-raf.sup.torY9 (Dickson et al., 1992,
Genes Dev. 6:2327-2339; Dickson et al., Nature 360:600-603). For
each genotype, 500 Notch-positive cone cell precursor nuclei in
ommatidial rows 15-25 were examined, representing at least six
separate pairs of eye discs. In no case did we observe any cone
cell precursor nuclei positive for both Notch and ELAV antigens
(FIG. 5). We frequently found that not all four cone cell precursor
nuclei in an ommatidium express Notch, and that those which do not
are often ELAV-positive (FIG. 5). These nuclei presumably
correspond to cone cell precursors that do not express
sev-Notch.sup.nucl efficiently but do express sufficient amounts of
an activated sevenless pathway molecule to induce neural
differentiation. Identical results were obtained with different
sev-Notch.sup.nucl, sev-S11, and sevRas1.sup.Val12 transgenic lines
as well as with an alternative activated Raf construct
sE-raf.sup.tor4021 (Dickson et al., 1992, Genes Dev. 6:2327-2339;
Dickson et al., Nature 360:600-603).
[0190] To rule out the possibility that our failure to detect
co-expression of activated Notch and ELAV antigens in cone cell
precursor nuclei is due to some mechanism that prevents two
different sevenless promoter constructs from being expressed in the
same cell, we double-stained eye discs of transgenic flies bearing
both sev-Notchnucl and sev-S11 in a sevenless.sup.d2 genetic
background with antibodies against Notch and against the
intracellular 60-kD subunit of Sevenless (Banerjee et al., 1987,
Cell 51:151-158). Since sevenless.sup.d2 flies do not express this
subunit (Banerjee et al, 1987), the only Sevenless immunoreactivity
detected corresponds to the extracellularly truncated protein
produced by the sev-S11 transgene (Basler et al., 1991, Cell
64:1069-1081). It was found that most cone cell precursors showing
strong nuclear expression of activated Notch also display strong
apical membrane expression of activated Sevenless, demonstrating
that both transgenes are coexpressed in these cells (FIG. 6).
6.3. Discussion
[0191] The class of transmembrane receptor proteins encoded by the
Notch locus and related genes appears to regulate a common step in
cell-fate selection in organisms ranging from nematodes to humans
(reviewed in Greenwald and Rubin, 1992, Cell 68:271-281; Fortini
and Artavanis-Tsakonas, 1993, Cell 75:1245-1247). In many different
cell types, the signal generated by Notch activation renders cells
temporarily unable to respond to developmental cues from
neighboring cells (Coffman et al., 1993, Cell 73:659-671; Rebay et
al., 1993, Cell 74:319-329; Struhl et al., 1993, Cell 74:331-345;
Fortini et al., 1993, Nature 365:555-557; Lieber et al., 1993,
Genes Dev. 7:1949-1965). The Notch protein and its ligands Delta
and Serrate may thus be part of a general mechanism that limits the
competence of undifferentiated cells to undergo cell-fate
commitment (Coffman et al., 1993, Cell 73:659-671; Fortini and
Artavanis-Tsakonas, 1993, Cell 75:1245-1247). This mechanism may
play a crucial role in the timing of inductive events by allowing
an uncommitted cell to ignore irrelevant signals from adjacent
cells until it is presented with the appropriate inductive signal,
presumably preceded or accompanied by a signal that inactivates
Notch in the recipient cell. Consistent with this notion, genetic
analyses in Caenorhabditis and Drosophila have revealed an
interdependence between Notch-mediated signaling and several
distinct cell type-specific inductive signaling events (reviewed in
Horvitz and Sternberg, 1991, Nature 351:535-541; Artavanis-Tsakonas
and Simpson, 1991, Trends Genet. 7:403-408; Greenwald and Rubin,
1992, Cell 68:271-281), although little is known about how the
different signals are integrated at the molecular level. We have
sought to address this question by performing epistasis tests
between a constitutively activated Notch receptor and various
activated components of the inductive signalling pathway involving
the Sevenless receptor tyrosine kinase, Ras1 and Raf in the
developing Drosophila eye.
[0192] The results presented here indicate that the Notch receptor
protein, in its active state, interferes with the intracellular
signal generated by constitutively activated versions of Sevenless,
Ras1, and Raf (FIG. 7). Our epistasis data are difficult to
reconcile with models in which the Notch protein mediates cell
signaling primarily by promoting cell adhesion (Hoppe and
Greenspan, 1986, Cell 46:773-783; Greenspan, 1990, New Biologist
2:595-600) or by recruiting cell type-specific receptors and their
ligands to specialized membrane regions of polarized epithelia
(Singer, 1992, Science 255:1671-1677). Instead, Notch apparently
mediates a separate signalling pathway whose input is integrated
with that of the Ras1 pathway at some point downstream of Raf, at
least in this case. Ras1 and Raf, unlike the sevenless receptor
tyrosine kinase, act in many different tissues throughout
Drosophila development, as does Notch. For example, genetic studies
have identified both Ras1 and Raf as essential components of the
signaling pathways initiated by the torso and Drosophila EGF
receptor (DER) tyrosine kinases (Ambrosio et al., 1989, Nature
342:288-291; Simon et al., 1991, Cell 67:701-716; Doyle and Bishop,
1993, Genes Dev. 7:633-646; Melnick et al., 1993, Development
118:127-138; Diaz-Benjumea and Hafen, 1994, Development
120:569-578). Moreover, cell-fate specifications involving other
types of signalling molecules, such as the Drosophila scabrous,
wingless and daughterless gene products, also depend upon Notch
gene function (Baker et al., 1990, Science 250:1370-1377; Hing et
al., 1994, Mech. Dev., in press; Cummings and Cronmiller, 1994,
Development 120:381-394). Thus, the activity state of Notch is
likely to play an important regulatory role in modulating
signalling by Ras1, Raf, and other signalling molecules in a
variety of developmental processes.
[0193] The present invention is not to be limited in scope by the
specific embodiments described herein. Indeed, various
modifications of the invention in addition to those described
herein will become apparent to those skilled in the art from the
foregoing description and accompanying figures. Such modifications
are intended to fall within the scope of the appended claims.
[0194] Various publications are cited herein, the disclosures of
which are incorporated by reference in their entireties.
Sequence CWU 1
1
4 1 1015 PRT Homo sapiens 1 Ser Asn Pro Cys Gln His Gly Ala Thr Cys
Ser Asp Phe Ile Gly Gly 1 5 10 15 Tyr Arg Cys Glu Cys Val Pro Gly
Tyr Gln Gly Val Asn Cys Glu Tyr 20 25 30 Glu Val Asp Glu Cys Gln
Asn Gln Pro Cys Gln Asn Gly Gly Thr Cys 35 40 45 Ile Asp Leu Val
Asn His Phe Lys Cys Ser Cys Pro Pro Gly Thr Arg 50 55 60 Gly Leu
Leu Cys Glu Glu Asn Ile Asp Asp Cys Ala Arg Gly Pro His 65 70 75 80
Cys Leu Asn Gly Gly Gln Cys Met Asp Arg Ile Gly Gly Tyr Ser Cys 85
90 95 Arg Cys Leu Pro Gly Phe Ala Gly Glu Arg Cys Glu Gly Asp Ile
Asn 100 105 110 Glu Cys Leu Ser Asn Pro Cys Ser Ser Glu Gly Ser Leu
Asp Cys Ile 115 120 125 Gln Leu Thr Asn Asp Tyr Leu Cys Val Cys Arg
Ser Ala Phe Thr Gly 130 135 140 Arg His Cys Glu Thr Phe Val Asp Val
Cys Pro Gln Met Pro Cys Leu 145 150 155 160 Asn Gly Gly Thr Cys Ala
Val Ala Ser Asn Met Pro Asp Gly Phe Ile 165 170 175 Cys Arg Cys Pro
Pro Gly Phe Ser Gly Ala Arg Cys Gln Ser Ser Cys 180 185 190 Gly Gln
Val Lys Cys Arg Lys Gly Glu Gln Cys Val His Thr Ala Ser 195 200 205
Gly Pro Arg Cys Phe Cys Pro Ser Pro Arg Asp Cys Glu Ser Gly Cys 210
215 220 Ala Ser Ser Pro Cys Gln His Gly Gly Ser Cys His Pro Gln Arg
Gln 225 230 235 240 Pro Pro Tyr Tyr Ser Cys Gln Cys Ala Pro Pro Phe
Ser Gly Ser Arg 245 250 255 Cys Glu Leu Tyr Thr Ala Pro Pro Ser Thr
Pro Pro Ala Thr Cys Leu 260 265 270 Ser Gln Tyr Cys Ala Asp Lys Ala
Arg Asp Gly Val Cys Asp Glu Ala 275 280 285 Cys Asn Ser His Ala Cys
Gln Trp Asp Gly Gly Asp Cys Ser Leu Thr 290 295 300 Met Glu Asn Pro
Trp Ala Asn Cys Ser Ser Pro Leu Pro Cys Trp Asp 305 310 315 320 Tyr
Ile Asn Asn Gln Cys Asp Glu Leu Cys Asn Thr Val Glu Cys Leu 325 330
335 Phe Asp Asn Phe Glu Cys Gln Gly Asn Ser Lys Thr Cys Lys Tyr Asp
340 345 350 Lys Tyr Cys Ala Asp His Phe Lys Asp Asn His Cys Asn Gln
Gly Cys 355 360 365 Asn Ser Glu Glu Cys Gly Trp Asp Gly Leu Asp Cys
Ala Ala Asp Gln 370 375 380 Pro Glu Asn Leu Ala Glu Gly Thr Leu Val
Ile Val Val Leu Met Pro 385 390 395 400 Pro Glu Gln Leu Leu Gln Asp
Ala Arg Ser Phe Leu Arg Ala Leu Gly 405 410 415 Thr Leu Leu His Thr
Asn Leu Arg Ile Lys Arg Asp Ser Gln Gly Glu 420 425 430 Leu Met Val
Tyr Pro Tyr Tyr Gly Glu Lys Ser Ala Ala Met Lys Lys 435 440 445 Gln
Arg Met Thr Arg Arg Ser Leu Pro Gly Glu Gln Glu Gln Glu Val 450 455
460 Ala Gly Ser Lys Val Phe Leu Glu Ile Asp Asn Arg Gln Cys Val Gln
465 470 475 480 Asp Ser Asp His Cys Phe Lys Asn Thr Asp Ala Ala Ala
Ala Leu Leu 485 490 495 Ala Ser His Ala Ile Gln Gly Thr Leu Ser Tyr
Pro Leu Val Ser Val 500 505 510 Val Ser Glu Ser Leu Thr Pro Glu Arg
Thr Gln Leu Leu Tyr Leu Leu 515 520 525 Ala Val Ala Val Val Ile Ile
Leu Phe Ile Ile Leu Leu Gly Val Ile 530 535 540 Met Ala Lys Arg Lys
Arg Lys His Gly Ser Leu Trp Leu Pro Glu Gly 545 550 555 560 Phe Thr
Leu Arg Arg Asp Ala Ser Asn His Lys Arg Arg Glu Pro Val 565 570 575
Gly Gln Asp Ala Val Gly Leu Lys Asn Leu Ser Val Gln Val Ser Glu 580
585 590 Ala Asn Leu Ile Gly Thr Gly Thr Ser Glu His Trp Val Asp Asp
Glu 595 600 605 Gly Pro Gln Pro Lys Lys Val Lys Ala Glu Asp Glu Ala
Leu Leu Ser 610 615 620 Glu Glu Asp Asp Pro Ile Asp Arg Arg Pro Trp
Thr Gln Gln His Leu 625 630 635 640 Glu Ala Ala Asp Ile Arg Arg Thr
Pro Ser Leu Ala Leu Thr Pro Pro 645 650 655 Gln Ala Glu Gln Glu Val
Asp Val Leu Asp Val Asn Val Arg Gly Pro 660 665 670 Asp Gly Cys Thr
Pro Leu Met Leu Ala Ser Leu Arg Gly Gly Ser Ser 675 680 685 Asp Leu
Ser Asp Glu Asp Glu Asp Ala Glu Asp Ser Ser Ala Asn Ile 690 695 700
Ile Thr Asp Leu Val Tyr Gln Gly Ala Ser Leu Gln Ala Gln Thr Asp 705
710 715 720 Arg Thr Gly Glu Met Ala Leu His Leu Ala Ala Arg Tyr Ser
Arg Ala 725 730 735 Asp Ala Ala Lys Arg Leu Leu Asp Ala Gly Ala Asp
Ala Asn Ala Gln 740 745 750 Asp Asn Met Gly Arg Cys Pro Leu His Ala
Ala Val Ala Ala Asp Ala 755 760 765 Gln Gly Val Phe Gln Ile Leu Ile
Arg Asn Arg Val Thr Asp Leu Asp 770 775 780 Ala Arg Met Asn Asp Gly
Thr Thr Pro Leu Ile Leu Ala Ala Arg Leu 785 790 795 800 Ala Val Glu
Gly Met Val Ala Glu Leu Ile Asn Cys Gln Ala Asp Val 805 810 815 Asn
Ala Val Asp Asp His Gly Lys Ser Ala Leu His Trp Ala Ala Ala 820 825
830 Val Asn Asn Val Glu Ala Thr Leu Leu Leu Leu Lys Asn Gly Ala Asn
835 840 845 Arg Asp Met Gln Asp Asn Lys Glu Glu Thr Pro Leu Phe Leu
Ala Ala 850 855 860 Arg Glu Gly Ser Tyr Glu Ala Ala Lys Ile Leu Leu
Asp His Phe Ala 865 870 875 880 Asn Arg Asp Ile Thr Asp His Met Asp
Arg Leu Pro Arg Asp Val Ala 885 890 895 Arg Asp Arg Met His His Asp
Ile Val Arg Leu Leu Asp Glu Tyr Asn 900 905 910 Val Thr Pro Ser Pro
Pro Gly Thr Val Leu Thr Ser Ala Leu Ser Pro 915 920 925 Val Ile Cys
Gly Pro Asn Arg Ser Phe Leu Ser Leu Lys His Thr Pro 930 935 940 Met
Gly Lys Lys Ser Arg Arg Pro Ser Ala Lys Ser Thr Met Pro Thr 945 950
955 960 Ser Leu Pro Asn Leu Ala Lys Glu Ala Lys Asp Ala Lys Gly Ser
Arg 965 970 975 Arg Lys Lys Ser Leu Ser Glu Lys Val Gln Leu Ser Glu
Ser Ser Val 980 985 990 Thr Leu Ser Pro Val Asp Ser Leu Glu Ser Pro
His Thr Tyr Val Ser 995 1000 1005 Asp Thr Thr Ser Ser Pro Met 1010
1015 2 1068 PRT Homo sapiens misc_feature (612)..(612) Xaa can be
any naturally occurring amino acid 2 Pro Ser Pro Cys Gln Asn Gly
Ala Thr Cys Thr Asp Tyr Leu Gly Gly 1 5 10 15 Tyr Ser Cys Lys Cys
Val Ala Gly Tyr His Gly Val Asn Cys Ser Glu 20 25 30 Glu Ile Asp
Glu Cys Leu Ser His Pro Cys Gln Asn Gly Gly Thr Cys 35 40 45 Leu
Asp Leu Pro Asn Thr Tyr Lys Cys Ser Cys Pro Trp Gly Thr Gln 50 55
60 Gly Val His Cys Glu Ile Asn Val Asp Asp Cys Asn Pro Pro Val Asp
65 70 75 80 Pro Val Ser Trp Ser Pro Lys Cys Phe Asn Asn Gly Thr Cys
Val Asp 85 90 95 Gln Val Gly Gly Tyr Ser Cys Thr Cys Pro Pro Gly
Phe Val Gly Glu 100 105 110 Arg Cys Glu Gly Asp Val Asn Glu Cys Leu
Ser Asn Pro Cys Asp Ala 115 120 125 Arg Gly Thr Gln Asn Cys Val Gln
Arg Val Asn Asp Phe His Cys Glu 130 135 140 Cys Arg Ala Gly His Thr
Gly Arg Arg Cys Glu Ser Val Ile Asn Gly 145 150 155 160 Cys Lys Gly
Lys Pro Cys Lys Asn Gly Gly Thr Cys Ala Val Ala Ser 165 170 175 Asn
Thr Ala Arg Gly Phe Ile Cys Lys Cys Pro Ala Gly Phe Glu Gly 180 185
190 Ala Thr Cys Glu Asn Asp Ala Arg Thr Cys Gly Ser Leu Arg Cys Leu
195 200 205 Asn Gly Gly Thr Cys Ile Ser Gly Pro Arg Ser Pro Thr Cys
Leu Cys 210 215 220 Leu Gly Pro Phe Thr Gly Pro Glu Cys Gln Phe Pro
Ala Ser Ser Pro 225 230 235 240 Cys Leu Gly Gly Asn Pro Cys Tyr Asn
Gln Gly Thr Cys Glu Pro Thr 245 250 255 Ser Glu Ser Pro Phe Tyr Arg
Cys Leu Cys Pro Ala Lys Phe Asn Gly 260 265 270 Leu Leu Cys His Ile
Leu Asp Tyr Ser Phe Gly Gly Gly Ala Gly Arg 275 280 285 Asp Ile Pro
Pro Pro Leu Ile Glu Glu Ala Cys Glu Leu Pro Glu Cys 290 295 300 Gln
Glu Asp Ala Gly Asn Lys Val Cys Ser Leu Gln Cys Asn Asn His 305 310
315 320 Ala Cys Gly Trp Asp Gly Gly Asp Cys Ser Leu Asn Phe Asn Asp
Pro 325 330 335 Trp Lys Asn Cys Thr Gln Ser Leu Gln Cys Trp Lys Tyr
Phe Ser Asp 340 345 350 Gly His Cys Asp Ser Gln Cys Asn Ser Ala Gly
Cys Leu Phe Asp Gly 355 360 365 Phe Asp Cys Gln Arg Ala Glu Gly Gln
Cys Asn Pro Leu Tyr Asp Gln 370 375 380 Tyr Cys Lys Asp His Phe Ser
Asp Gly His Cys Asp Gln Gly Cys Asn 385 390 395 400 Ser Ala Glu Cys
Glu Trp Asp Gly Leu Asp Cys Ala Glu His Val Pro 405 410 415 Glu Arg
Leu Ala Ala Gly Thr Leu Val Val Val Val Leu Met Pro Pro 420 425 430
Glu Gln Leu Arg Asn Ser Ser Phe His Phe Leu Trp Glu Leu Ser Arg 435
440 445 Val Leu His Thr Asn Val Val Phe Lys Arg Asp Ala His Gly Gln
Gln 450 455 460 Met Ile Phe Pro Tyr Tyr Gly Arg Glu Glu Glu Leu Arg
Lys His Pro 465 470 475 480 Ile Lys Arg Ala Ala Glu Gly Trp Ala Ala
Pro Asp Ala Leu Leu Gly 485 490 495 Gln Val Lys Ala Ser Leu Leu Pro
Gly Gly Ser Glu Gly Gly Trp Trp 500 505 510 Trp Arg Glu Leu Asp Pro
Met Asp Val Arg Gly Ser Ile Val Tyr Leu 515 520 525 Glu Ile Asp Asn
Trp Gln Cys Val Gln Ala Ser Ser Gln Cys Phe Gln 530 535 540 Ser Ala
Thr Asp Val Ala Ala Phe Leu Gly Ala Leu Ala Ser Leu Gly 545 550 555
560 Ser Leu Asn Ile Pro Tyr Lys Ile Glu Ala Val Gln Ser Glu Thr Val
565 570 575 Glu Pro Pro Pro Pro Ala Gln Leu His Phe Met Tyr Val Ala
Ala Ala 580 585 590 Ala Phe Val Leu Leu Phe Phe Val Gly Cys Gly Val
Leu Leu Ser Arg 595 600 605 Lys Arg Trp Xaa Gln His Gly Gln Leu Trp
Phe Pro Glu Gly Phe Lys 610 615 620 Val Ser Glu Ala Ser Lys Lys Lys
Trp Trp Glu Xaa Leu Gly Glu Asp 625 630 635 640 Ser Val Gly Leu Lys
Pro Leu Lys Asn Ala Ser Asp Gly Ala Leu Met 645 650 655 Asp Asp Asn
Gln Asn Glu Trp Gly Asp Glu Asp Leu Glu Thr Lys Lys 660 665 670 Phe
Trp Phe Glu Glu Pro Val Val Leu Pro Asp Leu Asp Asp Gln Thr 675 680
685 Asp His Trp Gln Trp Thr Gln Gln His Leu Asp Ala Ala Asp Leu Arg
690 695 700 Met Ser Ala Met Ala Pro Thr Pro Pro Gln Gly Glu Val Asp
Ala Asp 705 710 715 720 Cys Met Asp Val Asn Val Arg Gly Pro Asp Gly
Phe Thr Pro Leu Met 725 730 735 Ile Ala Ser Cys Ser Gly Gly Gly Leu
Glu Thr Gly Asn Ser Glu Glu 740 745 750 Glu Glu Asp Ala Pro Ala Val
Ile Ser Asp Phe Ile Tyr Gln Gly Ala 755 760 765 Ser Leu His Asn Gln
Thr Asp Arg Thr Gly Glu Thr Ala Leu His Leu 770 775 780 Ala Ala Arg
Tyr Ser Arg Ser Asp Ala Ala Lys Arg Leu Leu Glu Ala 785 790 795 800
Ser Ala Asp Ala Asn Ile Gln Asp Asn Met Gly Arg Thr Pro Leu His 805
810 815 Ala Ala Val Ser Ala Asp Ala Gln Gly Val Phe Gln Ile Leu Ile
Trp 820 825 830 Asn Arg Ala Thr Asp Leu Asp Ala Arg Met His Asp Gly
Thr Thr Pro 835 840 845 Leu Ile Leu Ala Ala Arg Leu Ala Val Glu Gly
Met Leu Glu Asp Leu 850 855 860 Ile Asn Ser His Ala Asp Val Asn Ala
Val Asp Asp Leu Gly Lys Ser 865 870 875 880 Ala Leu His Trp Ala Ala
Ala Val Asn Asn Val Asp Ala Ala Val Val 885 890 895 Leu Leu Lys Asn
Gly Ala Asn Lys Asp Met Gln Asn Asn Arg Glu Glu 900 905 910 Thr Pro
Leu Phe Leu Ala Ala Trp Glu Gly Ser Tyr Glu Thr Ala Lys 915 920 925
Val Leu Leu Asp His Phe Ala Asn Trp Asp Ile Thr Asp His Met Asp 930
935 940 Arg Leu Pro Arg Asp Ile Ala Gln Glu Arg Met His His Asp Ile
Val 945 950 955 960 Arg Leu Leu Asp Glu Tyr Asn Leu Val Arg Ser Pro
Gln Leu His Gly 965 970 975 Ala Pro Leu Gly Gly Thr Pro Thr Leu Ser
Pro Pro Leu Cys Ser Pro 980 985 990 Asn Gly Tyr Leu Gly Ser Leu Lys
Pro Gly Val Gln Gly Lys Lys Val 995 1000 1005 Arg Lys Pro Ser Ser
Lys Gly Leu Ala Cys Gly Ser Lys Glu Ala 1010 1015 1020 Lys Asp Leu
Lys Ala Trp Arg Lys Lys Ser Gln Asp Gly Lys Gly 1025 1030 1035 Cys
Leu Leu Asp Ser Ser Gly Met Leu Ser Pro Val Asp Ser Leu 1040 1045
1050 Glu Ser Pro His Gly Tyr Leu Ser Asp Val Ala Ser Pro Pro Leu
1055 1060 1065 3 1064 PRT Homo sapiens 3 Pro Asn Pro Cys Gln Asn
Gly Ala Thr Cys Thr Asp Tyr Leu Gly Gly 1 5 10 15 Tyr Ser Cys Glu
Cys Val Ala Gly Tyr His Gly Val Asn Cys Ser Glu 20 25 30 Glu Ile
Asn Glu Cys Leu Ser His Pro Cys Gln Asn Gly Gly Thr Cys 35 40 45
Ile Asp Leu Ile Asn Thr Tyr Lys Cys Ser Cys Pro Arg Gly Thr Gln 50
55 60 Gly Val His Cys Glu Ile Asn Val Asp Asp Cys Thr Pro Phe Tyr
Asp 65 70 75 80 Ser Phe Thr Leu Glu Pro Lys Cys Phe Asn Asn Gly Lys
Cys Ile Asp 85 90 95 Arg Val Gly Gly Tyr Asn Cys Ile Cys Pro Pro
Gly Phe Val Gly Glu 100 105 110 Arg Cys Glu Gly Asp Val Asn Glu Cys
Leu Ser Asn Pro Cys Asp Ser 115 120 125 Arg Gly Thr Gln Asn Cys Ile
Gln Leu Val Asn Asp Tyr Arg Cys Glu 130 135 140 Cys Arg Gln Gly Phe
Thr Gly Arg Arg Cys Glu Ser Val Val Asp Gly 145 150 155 160 Cys Lys
Gly Met Pro Cys Arg Asn Gly Gly Thr Cys Ala Val Ala Ser 165 170 175
Asn Thr Glu Arg Gly Phe Ile Cys Lys Cys Pro Pro Gly Phe Asp Gly 180
185 190 Ala Thr Cys Glu Tyr Asp Ser Arg Thr Cys Ser Asn Leu Arg Cys
Gln 195 200 205 Asn Gly Gly Thr Cys Ile Ser Val Leu Thr Ser Ser Lys
Cys Val Cys 210 215 220 Ser Glu Gly Tyr Thr Gly Ala Thr Cys Gln Tyr
Pro Val Ile Ser Pro 225 230 235 240 Cys Ala Ser His Pro Cys Tyr Asn
Gly Gly Thr Cys Gln Phe Phe Ala 245 250 255 Glu Glu Pro Phe Phe Gln
Cys Phe Cys Pro Lys Asn Phe Asn Gly Leu 260 265 270 Phe Cys His Ile
Leu Asp Tyr Glu Phe Pro Gly Gly Leu Gly Lys Asn 275 280 285 Ile Thr
Pro Pro Asp Asn Asp Asp Ile Cys Glu Asn Glu Gln Cys Ser 290 295 300
Glu Leu Ala Asp Asn Lys Val Cys Asn Ala Asn Cys Asn Asn His Ala 305
310 315 320 Cys Gly Trp Asp Gly Gly Asp Cys Ser Leu Asn Phe Asn Asp
Pro Trp 325 330 335 Lys Asn Cys Thr Gln Ser Leu Gln Cys Trp Lys Tyr
Phe Asn Asp Gly
340 345 350 Lys Cys Asp Ser Gln Cys Asn Asn Thr Gly Cys Leu Tyr Asp
Gly Phe 355 360 365 Asp Cys Gln Lys Val Glu Val Gln Cys Asn Pro Leu
Tyr Asp Gln Tyr 370 375 380 Cys Lys Asp His Phe Gln Asp Gly His Cys
Asp Gln Gly Cys Asn Asn 385 390 395 400 Ala Glu Cys Glu Trp Asp Gly
Leu Asp Cys Ala Asn Met Pro Glu Asn 405 410 415 Leu Ala Glu Gly Thr
Leu Val Leu Val Val Leu Met Pro Pro Glu Arg 420 425 430 Leu Lys Asn
Asn Ser Val Asn Phe Leu Arg Glu Leu Ser Arg Val Leu 435 440 445 His
Thr Asn Val Val Phe Lys Lys Asp Ser Lys Gly Glu Tyr Lys Ile 450 455
460 Tyr Pro Tyr Tyr Gly Asn Glu Glu Glu Leu Lys Lys His His Ile Lys
465 470 475 480 Arg Ser Thr Asp Tyr Trp Ser Asp Ala Pro Ser Ala Ile
Phe Ser Thr 485 490 495 Met Lys Glu Ser Ile Leu Leu Gly Arg His Arg
Arg Glu Leu Asp Glu 500 505 510 Met Glu Val Arg Gly Ser Ile Val Tyr
Leu Glu Ile Asp Asn Arg Gln 515 520 525 Cys Tyr Lys Ser Ser Ser Gln
Cys Phe Asn Ser Ala Thr Asp Val Ala 530 535 540 Ala Phe Leu Gly Ala
Leu Ala Ser Leu Gly Ser Leu Asp Thr Leu Ser 545 550 555 560 Tyr Lys
Ile Glu Ala Val Lys Ser Glu Asn Met Glu Thr Pro Lys Pro 565 570 575
Ser Thr Leu Tyr Pro Met Leu Ser Met Leu Val Ile Pro Leu Leu Ile 580
585 590 Ile Phe Val Phe Met Met Val Ile Val Asn Lys Lys Arg Arg Arg
Glu 595 600 605 His Asp Ser Phe Gly Ser Pro Thr Ala Leu Phe Gln Lys
Asn Pro Ala 610 615 620 Lys Arg Asn Gly Glu Thr Pro Trp Glu Asp Ser
Val Gly Leu Lys Pro 625 630 635 640 Ile Lys Asn Met Thr Asp Gly Ser
Phe Met Asp Asp Asn Gln Asn Glu 645 650 655 Trp Gly Asp Glu Glu Thr
Leu Glu Asn Lys Arg Phe Arg Phe Glu Glu 660 665 670 Gln Val Ile Leu
Pro Glu Leu Val Asp Asp Lys Thr Asp Pro Arg Gln 675 680 685 Trp Thr
Arg Gln His Leu Asp Ala Ala Asp Leu Arg Ile Ser Ser Met 690 695 700
Ala Pro Thr Pro Pro Gln Gly Glu Ile Glu Ala Asp Cys Met Asp Val 705
710 715 720 Asn Val Arg Gly Pro Asp Gly Phe Thr Pro Leu Met Ile Ala
Ser Cys 725 730 735 Ser Gly Gly Gly Leu Glu Thr Gly Asn Ser Glu Glu
Glu Glu Asp Ala 740 745 750 Ser Ala Asn Met Ile Ser Asp Phe Ile Gly
Gln Gly Ala Gln Leu His 755 760 765 Asn Gln Thr Asp Arg Thr Gly Glu
Thr Ala Leu His Leu Ala Ala Arg 770 775 780 Tyr Ala Arg Ala Asp Ala
Ala Lys Arg Leu Leu Glu Ser Ser Ala Asp 785 790 795 800 Ala Asn Val
Gln Asp Asn Met Gly Arg Thr Pro Leu His Ala Ala Val 805 810 815 Ala
Ala Asp Ala Gln Gly Val Phe Gln Ile Leu Ile Arg Asn Arg Ala 820 825
830 Thr Asp Leu Asp Ala Arg Met Phe Asp Gly Thr Thr Pro Leu Ile Leu
835 840 845 Ala Ala Arg Leu Ala Val Glu Gly Met Val Glu Glu Leu Ile
Asn Ala 850 855 860 His Ala Asp Val Asn Ala Val Asp Glu Phe Gly Lys
Ser Ala Leu His 865 870 875 880 Trp Ala Ala Ala Val Asn Asn Val Asp
Ala Ala Ala Val Leu Leu Lys 885 890 895 Asn Ser Ala Asn Lys Asp Met
Gln Asn Asn Lys Glu Glu Thr Ser Leu 900 905 910 Phe Leu Ala Ala Arg
Glu Gly Ser Tyr Glu Thr Ala Lys Val Leu Leu 915 920 925 Asp His Tyr
Ala Asn Arg Asp Ile Thr Asp His Met Asp Arg Leu Pro 930 935 940 Arg
Asp Ile Ala Gln Glu Arg Met His His Asp Ile Val His Leu Leu 945 950
955 960 Asp Glu Tyr Asn Leu Val Lys Ser Pro Thr Leu His Asn Gly Pro
Leu 965 970 975 Gly Ala Thr Thr Leu Ser Pro Pro Ile Cys Ser Pro Asn
Gly Tyr Met 980 985 990 Gly Asn Met Lys Pro Ser Val Gln Ser Lys Lys
Ala Arg Lys Pro Ser 995 1000 1005 Ile Lys Gly Asn Gly Cys Lys Glu
Ala Lys Glu Leu Lys Ala Arg 1010 1015 1020 Arg Lys Lys Ser Gln Asp
Gly Lys Thr Thr Leu Leu Asp Ser Gly 1025 1030 1035 Ser Ser Gly Val
Leu Ser Pro Val Asp Ser Leu Glu Ser Thr His 1040 1045 1050 Gly Tyr
Leu Ser Asp Val Ser Ser Pro Pro Leu 1055 1060 4 1139 PRT Homo
sapiens 4 Ser Gln Pro Cys Gln Asn Gly Gly Thr Cys Arg Asp Leu Ile
Gly Ala 1 5 10 15 Tyr Glu Cys Gln Cys Arg Gln Gly Phe Gln Gly Gln
Asn Cys Glu Leu 20 25 30 Asn Ile Asp Asp Cys Ala Pro Asn Pro Cys
Gln Asn Gly Gly Thr Cys 35 40 45 His Asp Arg Val Met Asn Phe Ser
Cys Ser Cys Pro Pro Gly Thr Met 50 55 60 Gly Ile Ile Cys Glu Ile
Asn Lys Asp Asp Cys Lys Pro Gly Ala Cys 65 70 75 80 His Asn Asn Gly
Ser Cys Ile Asp Arg Val Gly Gly Phe Glu Cys Val 85 90 95 Cys Gln
Pro Gly Phe Val Gly Ala Arg Cys Glu Gly Asp Ile Asn Glu 100 105 110
Cys Leu Ser Asn Pro Cys Ser Asn Ala Gly Thr Leu Asp Cys Val Gln 115
120 125 Leu Val Asn Asn Tyr His Cys Asn Cys Arg Pro Gly His Met Gly
Arg 130 135 140 His Cys Glu His Lys Val Asp Phe Cys Ala Gln Ser Pro
Cys Gln Asn 145 150 155 160 Gly Gly Asn Cys Asn Ile Arg Gln Ser Gly
His His Cys Ile Cys Asn 165 170 175 Asn Gly Phe Tyr Gly Lys Asn Cys
Glu Leu Ser Gly Gln Asp Cys Asp 180 185 190 Ser Asn Pro Cys Arg Val
Gly Asn Cys Val Val Ala Asp Glu Gly Phe 195 200 205 Gly Tyr Arg Cys
Glu Cys Pro Arg Gly Thr Leu Gly Glu His Cys Glu 210 215 220 Ile Asp
Thr Leu Asp Glu Cys Ser Pro Asn Pro Cys Ala Gln Gly Ala 225 230 235
240 Ala Cys Glu Asp Leu Leu Gly Asp Tyr Glu Cys Leu Cys Pro Ser Lys
245 250 255 Trp Lys Gly Lys Arg Cys Asp Ile Tyr Asp Ala Asn Tyr Pro
Gly Trp 260 265 270 Asn Gly Gly Ser Gly Ser Gly Asn Asp Arg Tyr Ala
Ala Asp Leu Glu 275 280 285 Gln Gln Arg Ala Met Cys Asp Lys Arg Gly
Cys Thr Glu Lys Gln Gly 290 295 300 Asn Gly Ile Cys Asp Ser Asp Cys
Asn Thr Tyr Ala Cys Asn Phe Asp 305 310 315 320 Gly Asn Asp Cys Ser
Leu Gly Ile Asn Pro Trp Ala Asn Cys Thr Ala 325 330 335 Asn Glu Cys
Trp Asn Lys Phe Lys Asn Gly Lys Cys Asn Glu Glu Cys 340 345 350 Asn
Asn Ala Ala Cys His Tyr Asp Gly His Asp Cys Glu Arg Lys Leu 355 360
365 Lys Ser Cys Asp Thr Leu Phe Asp Ala Tyr Cys Gln Lys His Tyr Gly
370 375 380 Asp Gly Phe Cys Asp Tyr Gly Cys Asn Asn Ala Glu Cys Ser
Trp Asp 385 390 395 400 Gly Leu Asp Cys Glu Asn Lys Thr Gln Ser Pro
Val Leu Ala Glu Gly 405 410 415 Ala Met Ser Val Val Met Leu Met Asn
Val Glu Ala Phe Arg Glu Ile 420 425 430 Gln Ala Gln Phe Leu Arg Asn
Met Ser His Met Leu Arg Thr Thr Val 435 440 445 Arg Leu Lys Lys Asp
Ala Leu Gly His Asp Ile Ile Ile Asn Trp Lys 450 455 460 Asp Asn Val
Arg Val Pro Glu Ile Glu Asp Thr Asp Phe Ala Arg Lys 465 470 475 480
Asn Lys Ile Leu Tyr Thr Gln Gln Val His Gln Thr Gly Ile Gln Ile 485
490 495 Tyr Leu Glu Ile Asp Asn Arg Lys Cys Thr Glu Cys Phe Thr His
Ala 500 505 510 Val Glu Ala Ala Glu Phe Leu Ala Ala Thr Ala Ala Lys
His Gln Leu 515 520 525 Arg Asn Asp Phe Gln Ile His Ser Val Arg Gly
Ile Lys Asn Pro Gly 530 535 540 Asp Glu Asp Asn Gly Glu Pro Pro Ala
Asn Val Lys Tyr Val Ile Thr 545 550 555 560 Gly Ile Ile Leu Val Ile
Ile Ala Leu Ala Phe Phe Gly Met Val Leu 565 570 575 Ser Thr Gln Arg
Lys Arg Ala His Gly Val Thr Trp Phe Pro Glu Gly 580 585 590 Phe Arg
Ala Pro Ala Ala Val Met Ser Arg Arg Arg Arg Asp Pro His 595 600 605
Gly Gln Glu Met Arg Asn Leu Asn Lys Gln Val Ala Met Gln Ser Gln 610
615 620 Gly Val Gly Gln Pro Gly Ala His Trp Ser Asp Asp Glu Ser Asp
Met 625 630 635 640 Pro Leu Pro Lys Arg Gln Arg Ser Asp Pro Val Ser
Gly Val Gly Leu 645 650 655 Gly Asn Asn Gly Gly Tyr Ala Ser Asp His
Thr Met Val Ser Glu Tyr 660 665 670 Glu Glu Ala Asp Gln Arg Val Trp
Ser Gln Ala His Leu Asp Val Val 675 680 685 Asp Val Arg Ala Ile Met
Thr Pro Pro Ala His Gln Asp Gly Gly Lys 690 695 700 His Asp Val Asp
Ala Arg Gly Pro Cys Gly Leu Thr Pro Leu Met Ile 705 710 715 720 Ala
Ala Val Arg Gly Gly Gly Leu Asp Thr Gly Glu Asp Ile Glu Asn 725 730
735 Asn Glu Asp Ser Thr Ala Gln Val Ile Ser Asp Leu Leu Ala Gln Gly
740 745 750 Ala Glu Leu Asn Ala Thr Met Asp Lys Thr Gly Glu Thr Ser
Leu His 755 760 765 Leu Ala Ala Arg Phe Ala Arg Ala Asp Ala Ala Lys
Arg Leu Phe His 770 775 780 Ala Gly Ala Asp Ala Asn Cys Gln Asp Asn
Thr Gly Arg Thr Pro Leu 785 790 795 800 His Ala Ala Val Ala Ala Asp
Ala Met Gly Val Phe Gln Ile Leu Leu 805 810 815 Arg Asn Arg Ala Thr
Asn Leu Asn Ala Arg Met His Asp Gly Thr Thr 820 825 830 Pro Leu Ile
Leu Ala Ala Arg Leu Ala Ile Glu Gly Met Val Glu Asp 835 840 845 Leu
Ile Thr Ala Asp Ala Asp Ile Asn Ala Ala Asp Asn Ser Gly Lys 850 855
860 Thr Ala Leu His Trp Ala Ala Ala Val Asn Asn Thr Glu Ala Val Asn
865 870 875 880 Ile Leu Leu Met His His Ala Asn Arg Asp Ala Gln Asp
Asp Lys Asp 885 890 895 Glu Thr Pro Leu Phe Leu Ala Ala Arg Glu Gly
Ser Tyr Glu Ala Cys 900 905 910 Lys Ala Leu Leu Asp Asn Phe Ala Asn
Arg Glu Ile Thr Asp His Met 915 920 925 Asp Arg Leu Pro Arg Asp Val
Ala Ser Glu Arg Leu His His Asp Ile 930 935 940 Val Arg Leu Leu Asp
Glu His Val Pro Arg Ser Pro Gln Met Leu Ser 945 950 955 960 Met Thr
Pro Gln Ala Met Ile Gly Ser Pro Pro Pro Gly Gln Gln Gln 965 970 975
Pro Gln Leu Ile Thr Gln Pro Thr Val Ile Ser Ala Gly Asn Gly Gly 980
985 990 Asn Asn Gly Asn Gly Asn Ala Ser Gly Lys Gln Ser Asn Gln Thr
Ala 995 1000 1005 Lys Gln Lys Ala Ala Lys Lys Ala Lys Leu Ile Glu
Gly Ser Pro 1010 1015 1020 Asp Asn Gly Leu Asp Ala Thr Gly Ser Leu
Arg Arg Lys Ala Ser 1025 1030 1035 Ser Lys Lys Thr Ser Ala Ala Ser
Lys Lys Ala Ala Asn Leu Asn 1040 1045 1050 Gly Leu Asn Pro Gly Gln
Leu Thr Gly Gly Val Ser Gly Val Pro 1055 1060 1065 Gly Val Pro Pro
Thr Asn Ser Ala Val Gln Ala Ala Ala Ala Ala 1070 1075 1080 Ala Ala
Ala Val Ala Ala Met Ser His Glu Leu Glu Gly Ser Pro 1085 1090 1095
Val Gly Val Gly Met Gly Gly Asn Leu Pro Ser Pro Tyr Asp Thr 1100
1105 1110 Ser Ser Met Tyr Ser Asn Ala Met Ala Ala Pro Leu Ala Asn
Gly 1115 1120 1125 Asn Pro Asn Thr Gly Ala Lys Gln Pro Pro Ser 1130
1135
* * * * *