U.S. patent application number 11/226555 was filed with the patent office on 2006-02-02 for morphogen-induced dendritic growth.
This patent application is currently assigned to Curis, Inc.. Invention is credited to Charles M. Cohen, Dennis Higgins, Hermann Oppermann, Engin Ozkaynak, David C. Rueger, Kuber T. Sampath, John E. Smart.
Application Number | 20060025571 11/226555 |
Document ID | / |
Family ID | 35094083 |
Filed Date | 2006-02-02 |
United States Patent
Application |
20060025571 |
Kind Code |
A1 |
Rueger; David C. ; et
al. |
February 2, 2006 |
Morphogen-induced dendritic growth
Abstract
Disclosed are methods and compositions for maintaining neural
pathways in a mammal including: enhancing survival of neurons at
risk of dying; inducing cellular repair of damaged neurons and
neural pathways; stimulating neurons to maintain their
differentiated phenotype; and promoting dendritic outgrowth,
including maintaining dendritic arbors and regenerating dendritic
architecture. In one embodiment, the invention provides means for
stimulating CAM expression in neurons. The invention also provides
means for evaluating the status of nerve tissue, including means
for detecting and monitoring neuropathies in a mammal. The methods,
devices and compositions include a morphogen or
morphogen-stimulating agent provided to the mammal in a
therapeutically effective concentration. In another embodiment, the
invention provides methods and compositions which include a
morphogen or morphogen-stimulating agent, and a nerve trophic
factor or nerve trophic factor-stimulating agent at concentrations
effective for stimulating dendrite outgrowth. The morphogen and the
nerve trophic factor can be admixed in combination.
Inventors: |
Rueger; David C.;
(Hopkinton, MA) ; Sampath; Kuber T.; (Holliston,
MA) ; Smart; John E.; (Weston, MA) ;
Oppermann; Hermann; (Medway, MA) ; Ozkaynak;
Engin; (Milford, MA) ; Cohen; Charles M.;
(Weston, MA) ; Higgins; Dennis; (Amherst,
NY) |
Correspondence
Address: |
Ropes & Gray LLP
1251 Avenue of the Americas
New York
NY
10020-1104
US
|
Assignee: |
Curis, Inc.
Cambridge
MA
|
Family ID: |
35094083 |
Appl. No.: |
11/226555 |
Filed: |
September 13, 2005 |
Related U.S. Patent Documents
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Application
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Filing Date |
Patent Number |
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08292782 |
Aug 18, 1994 |
6949505 |
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11226555 |
Sep 13, 2005 |
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08260675 |
Jun 16, 1994 |
6800603 |
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08292782 |
Aug 18, 1994 |
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08126100 |
Sep 23, 1993 |
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08260675 |
Jun 16, 1994 |
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07922813 |
Jul 31, 1992 |
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08126100 |
Sep 23, 1993 |
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07752764 |
Aug 30, 1991 |
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07922813 |
Jul 31, 1992 |
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07667274 |
Mar 11, 1991 |
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07752764 |
Aug 30, 1991 |
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07753059 |
Aug 30, 1991 |
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07922813 |
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07971091 |
Nov 3, 1992 |
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08292782 |
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07752764 |
Aug 30, 1991 |
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07971091 |
Nov 3, 1992 |
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07667274 |
Mar 11, 1991 |
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07752764 |
Aug 30, 1991 |
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07938336 |
Aug 28, 1992 |
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08292782 |
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07938337 |
Aug 28, 1992 |
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08292782 |
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07667274 |
Mar 11, 1991 |
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07938337 |
Aug 28, 1992 |
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07938021 |
Aug 28, 1992 |
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08292782 |
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07752861 |
Aug 30, 1991 |
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07938021 |
Aug 28, 1992 |
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07667274 |
Mar 11, 1991 |
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07752861 |
Aug 30, 1991 |
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07752764 |
Aug 30, 1991 |
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08292782 |
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Current U.S.
Class: |
530/350 |
Current CPC
Class: |
C07K 14/51 20130101;
A61K 38/00 20130101; A61K 38/185 20130101; A01N 1/021 20130101;
A61K 38/1841 20130101 |
Class at
Publication: |
530/350 ;
514/012 |
International
Class: |
A61K 38/18 20060101
A61K038/18; C07K 14/475 20060101 C07K014/475 |
Claims
1. A method for regenerating a neural pathway, the method
comprising contacting cells forming said pathway with a morphogen
and a nerve trophic factor at concentrations and for times
effective for maintaining said pathway.
2-11. (canceled)
12. A method for preserving a neural pathway comprising: contacting
neurons forming said pathway with a morphogen and a nerve trophic
factor at concentrations and for times effective for preserving
said pathway.
13-31. (canceled)
32. A method for promoting dendrite outgrowth of neural cells, the
method comprising administering to a mammal an agent effective for
stimulating production of an endogenous morphogen by cells or
tissue of said mammal competent to produce said endogenous
morphogen; and a nerve trophic factor.
33-47. (canceled)
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] The following is a continuation-in-part of copending U.S.
Ser. No. 08/260,675, filed Jun. 16, 1994 as a file wrapper
continuation of U.S. Ser. No. 08/126,100, filed Sep. 23, 1993 as a
file wrapper continuation of U.S. Ser. No. 07/922,813, filed Jul.
31, 1992 as a continuation-in-part of U.S. Ser. No. 07/752,764,
filed Aug. 30, 1991 as a continuation-in-part of U.S. Ser. No.
07/667,274, filed Mar. 11, 1991. Prior related application U.S.
Ser. No. 07/922,813 was also a continuation-in-part of U.S. Ser.
No. 07/753,059, filed Aug. 30, 1991. The following is also a
continuation-in-part of copending U.S. Ser. No. 07/971,091, filed
Nov. 3, 1992 as a continuation-in-part of U.S. Ser. No. 07/752,764,
filed Aug. 30, 1991 as a continuation-in-part of U.S. Ser. No.
07/667,274, filed Mar. 11, 1991, now abandoned. The following is
also a continuation-in-part of U.S. Ser. No. 07/938,336, filed Aug.
28, 1992 and of U.S. Ser. No. 07/938,337, also filed Aug. 28, 1992;
both continuations-in-part are a continuation-in-part of U.S. Ser.
No. 07/667,274, filed Mar. 11, 1991, now abandoned. The following
is also a continuation-in-part of U.S. Ser. No. 07/938,021, filed
Aug. 28, 1992, which itself is a continuation-in-part of U.S. Ser.
No. 07/752,861, filed Aug. 30, 1991, now abandoned, as a
continuation-in-part of U.S. Ser. No. 07/667,274, filed Mar. 11,
1991, now abandoned. The following is also a continuation-in-part
of U.S. Ser. No. 07/752,764, filed Aug. 30, 1991. The teachings of
each of the above-mentioned applications are incorporated herein by
reference.
BACKGROUND OF THE INVENTION
[0002] The present invention relates to methods for enhancing the
survival of neuronal cells in vivo and to methods, compositions and
devices for maintaining neural pathways in vivo. More particularly,
the invention provides methods for enhancing survival of neuronal
cells at risk of dying, including methods for redifferentiating
transformed cells of neural origin and methods for maintaining
phenotypic expression of differentiated neuronal cells. The
invention also provides means for repairing damaged neural
pathways, including methods for stimulating axonal growth over
extended distances, and methods for alleviating
immunologically-related nerve tissue damage. In a particular
embodiment, this invention provides a method for stimulating cell
adhesion molecule expression in cells, and particularly nerve cell
adhesion molecule expression in neurons. The invention further
provides means for evaluating nerve tissue stasis and identifying
neural dysfunction in a mammal. In other specific embodiments, the
invention provides methods and compositions for promoting dendrite
outgrowth of neural cells from both the central and peripheral
nervous system.
[0003] The mammalian nervous system comprises a peripheral nervous
system (PNS) and a central nervous system (CNS, comprising the
brain and spinal cord), and is composed of two principal classes of
cells: neurons and glial cells. The glial cells fill the spaces
between neurons, nourishing them and modulating their function.
Certain glial cells, such as Schwann cells in the PNS and
oligodendrocytes in the CNS, also provide a protective myelin
sheath that surrounds and protects neuronal axons, which are the
processes that extend from the neuron cell body and through which
the electric impulses of the neuron are transported. In the
peripheral nervous system, the long axons of multiple neurons are
bundled together to form a nerve or nerve fiber. These, in turn,
may be combined into fascicles, wherein the nerve fibers form
bundles embedded, together with the intraneural vascular supply, in
a loose collagenous matrix bounded by a protective multilamellar
sheath. In the central nervous system, the neuron cell bodies are
visually distinguishable from their myelin-ensheathed processes,
and are referenced in the art as grey and white matter,
respectively.
[0004] During development, differentiating neurons from the central
and peripheral nervous systems send out axons that must grow and
make contact with specific target cells. In some cases, growing
axons must cover enormous distances; some grow into the periphery,
whereas others stay confined within the central nervous system. In
mammals, this stage of neurogenesis is completed during the
embryonic phase of life and neuronal cells do not multiply once
they have fully differentiated.
[0005] In the vertebrate nervous system, dendrites are the primary
site of synapse formation and neurons that lack dendrites typically
receive fewer synaptic inputs than cells with complex dendritic
arbors. Dendritic growth can be considered to occur in two phases:
initial extension followed by elongation and ramification (Purves
et al. (1988), 336 Nature 123-128). Some molecules, including
neurotransmitters and hormones, have been shown to regulate
expansion of an existing dendritic arbor. Much less is known,
however, about the factors that influence earlier events and cause
a neuron to initially form dendrites. In certain classes of
neurons, initial dendritic sprouting occurs as part of an intrinsic
development program which is relatively independent of trophic
interactions (Dotti et al. (1988), 8 J. Neurosci. 1454-1468). In
other classes of neurons, however, the regulation of the initial
stages of dendritic growth appears to be quite different. For
example, rat sympathetic neurons fail to form dendrites and extend
only axons when they are cultured in the absence of nonneuronal
cells. In contrast, co-culture with Schwann cells or astrocytes
causes these neurons to form dendritic processes and to eventually
generate a dendritic arbor which is comparable in size to that
observed in situ (Tropea et al. (1988), 1 Glia 380-392). This
change in cell shape is not observed when either fibroblasts or
heart cells are co-cultured with Schwann cells. Thus, it would
appear that specific trophic interactions are required to allow
sympathetic neurons to form dendrites.
[0006] The foregoing observations have been taken to support a
theory that the in situ environment specifies formation of a
dendritic arbor. The environment in the vicinity of neural cells or
developing neural processes is thus thought to include
electromagnetic, electrochemical and/or biochemical fields or
gradients which positively and negatively influence the extent and
specificity of dendritic outgrowth as well as the formation of
contacts (synapses) between dendrites and nerve cell bodies and
axons. This theory, however, suffers from a paucity of identified
mediators which have the capacity to cause neurons to sprout
dendrites. Currently, the only molecule for which a role in the
regulation of the initial stages of dendritic growth has been
established is the nerve trophic factor, nerve growth factor (NGF).
This growth factor can cause a subpopulation of nodose neurons to
form dendrites in culture (de Koninck et al. (1993), 13 J.
Neurosci. 577-585) and can enhance the growth of sympathetic
dendrites when injected in situ (Snider, W. D. (1988), 8 J.
Neurosci. 2628-2634, the teachings of which are herein incorporated
by reference). NGF alone, however, does not support dendritic
growth in cultures of sympathetic neurons (Bruckenstein and Higgins
(1988), 128 Dev. Biol. 324-336, the teachings of which are herein
incorporated by reference). It therefore appears that there are
other molecules which can regulate the morphological development of
neurons.
[0007] The neural pathways of a mammal are particularly at risk if
neurons are subjected to mechanical or chemical trauma or to
neuropathic degeneration sufficient to put the neurons that define
the pathway at risk of dying. A host of neuropathies, some of which
affect only a subpopulation or a system of neurons in the
peripheral or central nervous systems (PNS or CNS) have been
identified to date. The neuropathies, which may affect the neurons
themselves or the associated glial cells, may result from cellular
metabolic dysfunction, infection, exposure to toxic agents,
autoimmunity dysfunction, malnutrition or ischemia. In some cases
the cellular dysfunction is thought to induce cell death directly.
In other cases, the neuropathy may induce sufficient tissue
necrosis to stimulate the body's immune/inflammatory system and the
mechanisms of the body's immune response to the initial neural
injury then destroys the neurons and the pathway defined by these
neurons.
[0008] Currently no satisfactory method exists to repair the damage
caused by these neuropathies, which include multiple sclerosis,
amyotrophic lateral sclerosis (ALS), Huntington's chorea,
Alzheimer's disease, Parkinson's disease (parkinsonism), and
metabolically derived disorders, such as hepatic encephalopathy.
Current attempts to counteract the effects of severe traumatic or
neural degenerative lesions of the brain and/or spinal cord have to
date primarily involved implantation of embryonic neurons in an
effort to replace functionally, or otherwise compensate for, lost
or deficient neurons. Currently, however, human fetal cell
transplantation research is severely restricted. Administration of
nerve trophic factors such as nerve growth factor and insulin-like
growth factor also have been suggested to stimulate neuronal growth
within the CNS. (See, for example, Lundborg, (1987) Acta Orthop.
Scand. 58:145-169 and U.S. Pat. No. 5,093,317.) Administration of
nerve trophic factors to the CNS requires bypassing the blood-brain
barrier. The barrier may be overcome by direct infusion, or by
modifying the molecule to enhance its transport across the barrier,
as by chemical modification or conjugation, or by molecule
truncation. Schwann cells also have been grafted to a site of a CNS
lesion in an attempt to stimulate and maintain growth of damaged
neuronal processes (Paino et al. (1991) Exp. Neurology
114(2):254-257).
[0009] Where the damaged neural pathway results from CNS axonal
damage, autologous peripheral nerve grafts have been used to bridge
lesions in the central nervous system and to allow axons to make it
back to their normal target area. In contrast to CNS neurons,
neurons of the peripheral nervous system can extend new peripheral
processes in response to axonal damage. This regenerative property
of peripheral nervous system axons is thought to be sufficient to
allow grafting of these segments to CNS axons. Successful grafting
appears to be limited, however, by a number of factors, including
the length of the CNS axonal lesion to be bypassed, and the
distance of the graft sites from the CNS neuronal cell bodies, with
successful grafts occurring near the cell body.
[0010] Within the peripheral nervous system, this cellular
regenerative property of neurons has limited ability to repair
function to a damaged neural pathway. Specifically, the new axons
and dendrites extend randomly, and are often misdirected, making
contact with inappropriate targets that can cause abnormal
function. For example, if a motor nerve is damaged, regrowing axons
may contact the wrong muscles, resulting in paralysis. In addition,
where severed nerve processes result in a gap of longer than a few
millimeters, e.g., greater than 10 millimeters (mm), appropriate
nerve regeneration does not occur, either because the processes
fail to grow the necessary distance, or because of misdirected
axonal growth. Efforts to repair peripheral nerve damage by
surgical means has met with mixed results, particularly where
damage extends over a significant distance. In some cases, the
suturing steps used to obtain proper alignment of severed nerve
ends stimulates the formulation of scar tissue which is thought to
inhibit axon regeneration. Even where scar tissue formation has
been reduced, as with the use of nerve guidance channels or other
tubular prostheses, successful regeneration generally still is
limited to nerve damage of less than 10 millimeters in distance. In
addition, the reparative ability of peripheral neurons is
significantly inhibited where an injury or neuropathy affects the
cell body itself or results in extensive degeneration of a distal
axon.
[0011] Mammalian neural pathways also are at risk due to damage
caused by neoplastic lesions. Neoplasias of both the neurons and
glial cells have been identified. Transformed cells of neural
origin generally lose their ability to behave as normal
differentiated cells and can destroy neural pathways by loss of
function. In addition, the proliferating tumors may induce lesions
by distorting normal nerve tissue structure, inhibiting pathways by
compressing nerves, inhibiting cerebrospinal fluid or blood supply
flow, and/or by stimulating the body's immune response. Metastatic
tumors, which are a significant cause of neoplastic lesions in the
brain and spinal cord, also similarly may damage neural pathways
and induce neuronal cell death.
[0012] One type of morphoregulatory molecule associated with
neuronal cell growth, differentiation and development and the
proper formation of cell-cell contacts (synapses) between neurons
is the cell adhesion molecule ("CAM"), most notably the nerve cell
adhesion molecule (N-CAM). CAMs belong to the immunoglobulin
super-family and mediate cell-cell interactions in developing and
adult tissues through homophilic binding, i.e., CAM-CAM binding on
apposing cells. A number of different CAMs currently have been
identified. Of these, the most thoroughly studied to date are N-CAM
and L-CAM (liver cell adhesion molecules), both of which have been
identified on all cells at early stages of development, as well as
in different adult tissues. In neural tissue development, N-CAM
expression is believed to be important in tissue organization,
neuronal migration, nerve-muscle tissue adhesion, retinal
formation, synaptogenesis, and neural degeneration. Reduced N-CAM
expression also is thought to be associated with nerve dysfunction.
For example, expression of at least one form of N-CAM, N-CAM-180,
is reduced in a mouse dysmyelinating mutant (Bhat (1988) Brain Res.
452:373-377). Reduced levels of N-CAM also have been associated
with normal pressure hydrocephalus (Werdelin (1989) Acta Neurol.
Scand. 79:177-181), and with type II schizophrenia (Lyons et al.,
(1988) Biol. Psychiatry 23:769-775.) In addition, antibodies to
N-CAM have been shown to disrupt functional recovery in injured
nerves (Remsen (1990) Exp. Neurobiol. 110:268-273).
[0013] It is an object of this invention to provide methods for
enhancing survival of neurons at risk of dying in a mammal. Another
object is to provide methods for maintaining neural pathways in
vivo at risk of injury, or following damage to nerve tissue due to
mechanical or chemical trauma, a neuropathy, or a neoplastic
lesion. Another object is to provide compositions and devices for
repairing gaps in a neural pathway of the peripheral nervous
system. Yet another object is to provide a means for
redifferentiating transformed cells defining neural pathways,
particularly transformed cells of neural origin. Another object is
to provide a means for stimulating CAM expression, particularly
N-CAM expression in a cell. Yet another object is to provide
methods for monitoring the status of nerve tissue by monitoring
fluctuations in protein levels present in nerve tissue, serum
and/or cerebrospinal fluid. Still other objects are to provide
methods and compositions for promoting dendrite outgrowth of neural
cells from both the central and peripheral nervous system; to
provide methods and compositions for preserving a dendritic arbor
which may be at risk of deterioration or may be damaged by
mechanical or chemical trauma, neuropathy, or a neoplastic lesion;
and to provide methods and compositions for amelioration and
clinical management of neurodegenerative disorders including
memory, motor, associative, and metabolic disorders. Yet another
object of this invention is to provide a composition for repairing
a dendritic arbor and promoting regeneration of dendritic
architecture at a site of injury, damage, or impairment.
[0014] These and other objects and features of the invention will
be apparent from the description, drawings, and claims which
follow.
SUMMARY OF THE INVENTION
[0015] Embodiments of the present invention provide methods and
compositions for maintaining neural pathways in a mammal in vivo,
including methods for enhancing the survival of neural cells.
[0016] As used herein, a "neural pathway" describes a nerve circuit
for the passage of electric signals from a source to a target cell
site. The pathway includes the neurons through which the electric
impulse is transported, including groups of interconnecting
neurons, the nerve fibers formed by bundled neuronal axons, and the
glial cells surrounding and associated with the neurons. At the
level of cell-cell interaction, the pathway includes neural
processes such as axons an dendrites, which provide and participate
in specific sites of electrical contact and/or signal transmission
between the cells forming the pathway. Sites of contact and signal
transmission between nerve cells or between a nerve cell and
another type of cell are referred to herein as synapses.
[0017] In one aspect, the present invention features compositions
and therapeutic treatment methods that comprise the step of
administering to a mammal a therapeutically effective amount of a
morphogenic protein ("morphogen"), as defined herein and set forth
in detail below, upon injury to a neural pathway, or in
anticipation of such injury, for a time and at a concentration
sufficient to maintain or preserve the neural pathway, including
repairing damaged pathways, or inhibiting additional damage
thereto. Thus, in certain preferred embodiments, the present
invention provides methods for promoting dendrite outgrowth,
including methods for preserving dendritic arbors and promoting
regeneration of dendritic architecture.
[0018] In another aspect, the invention features compositions and
therapeutic treatment methods for maintaining neural pathways in a
mammal in vivo which include administering to the mammal, upon
injury to a neural pathway or in anticipation of such injury, a
compound that stimulates in vivo a therapeutically effective
concentration of an endogenous morphogen within the body of the
mammal sufficient to maintain or preserve the neural pathway,
including repairing damaged pathways or inhibiting additional
damage thereto. These compounds are referred to herein as
morphogen-stimulating agents, and are understood to include
substances which, when administered to a mammal, act on tissue(s)
or organ(s) that normally are responsible for, or competent for,
producing or secreting a morphogen and which cause the endogenous
level of the morphogen to be altered. The agent may act, for
example, by stimulating expression, secretion, or both, of an
endogenous morphogen.
[0019] In particular, the invention provides methods for enhancing
the survival of neurons at risk of dying, including protecting
neurons from the tissue destructive effects associated with the
body's immune/inflammatory response to a nerve injury. The
invention also provides methods for stimulating neurons to maintain
their differentiated phenotype or to preserve expression of this
phenotype, including inducing the redifferentiation of transformed
cells of neuronal origin to a morphology characteristic of
untransformed neurons. In one embodiment, the invention provides
means for stimulating production of cell adhesion molecules in
cells, particularly nerve cell adhesion molecules (N-CAM) in
neurons. The invention also provides methods, compositions and
devices for stimulating cellular repair of damaged neurons and
neural pathways, including regenerating damaged axons of the
peripheral and central nervous systems. In addition, the invention
also provides means for evaluating the status of nerve tissue, and
for detecting and monitoring neuropathies in a mammal by monitoring
fluctuations in the morphogen levels or endogenous morphogen
antibody levels present in a mammal's serum or cerebrospinal
fluid.
[0020] As used herein, "dendrites" or "dendritic outgrowth" refers
to nerve cell processes distinguishable from axons by virtue of
their morphological and immunological characteristics (Lein and
Higgins (1989), 136 Dev. Biol. 330-345, the teachings of which are
herein incorporated by reference). For example, dendrites are
morphologically distinguishable in that dendrites are broad-based
(up to 5 .mu.m in diameter), exhibit a distinct taper, and branch
in a "Y"-shaped pattern with daughter processes being distinctly
smaller than the parent process. Additionally, dendrites are
thicker than axons, and unlike axons, they terminate locally
usually extending less than 300 .mu.m from the soma. With respect
to immunological characteristics, dendrites and axons are also
readily distinguishable using certain antibody-based probes. For
example, antibodies to MAP2, non-phosphorylated forms of M and H
neurofilaments, and the transferrin receptor are considered by the
skilled artisan to be dendrite-specific surface markers.
Axon-specific markers include antibodies to synaptophysin, Tau1,
and phosphorylated forms of M and H neurofilament subunits. The
appearance of these surface antigens can be differentially detected
using, e.g., indirect immunofluoresence, thereby permitting
identification of nerve cell processes as dendrites or axons.
[0021] In one aspect, the invention features compositions and
methods that comprise the step of contacting neural cells with a
morphogenic protein ("morphogen") as defined herein, for a time and
at a concentration effective for promoting dendrite outgrowth,
including preserving a dendritic arbor or regenerating dendritic
architecture in both the central and peripheral nervous
systems.
[0022] In another aspect of the invention, the morphogens described
herein are useful in repairing damaged neural pathways of the
peripheral nervous system. In particular, the morphogens are useful
for repairing damaged pathways, including transected or otherwise
damaged nerve fibers (nerves) requiring regeneration of neuronal
processes, particularly axons, over extended distances to bridge a
gap in the nerve itself, or between the nerve and a post-synaptic
cell. Specifically, the morphogens described herein are capable of
stimulating complete axonal nerve regeneration, including
vascularization and reformation of the protective myelin sheath.
The morphogen preferably is provided to the site of injury
dispersed in a biocompatible, bioresorbable carrier material
capable of maintaining the morphogen at the site and, where
necessary, means for directing axonal growth from the proximal to
the distal ends of a severed neuron or nerve. For example, means
for directing axonal growth may be required where nerve
regeneration is to be induced over an extended distance, such as
greater than 10 mm. Many carriers capable of providing these
functions are envisioned. For example, useful carriers include
substantially insoluble materials or viscous solutions prepared as
disclosed herein comprising laminin, hyaluronic acid or collagen,
or other suitable synthetic, biocompatible polymeric materials such
as polylactic, polyglycolic or polybutyric acids and/or copolymers
thereof. The currently preferred carrier comprises an extracellular
matrix composition, such as one described herein derived, for
example, from mouse sarcoma cells. Also envisioned as especially
useful are brain tissue-derived extracellular matrices.
[0023] In a particularly preferred embodiment, the morphogen is
provided to the site as part of a device wherein the morphogen is
disposed in a nerve guidance channel which spans the distance of
the damaged pathway. The channel acts both as a protective covering
and a physical means for guiding growth of a neuronal process such
as an axon. Useful channels comprise a biocompatible membrane or
casing, which may be tubular in structure, having a dimension
sufficient to span the gap or break in the nerve to be repaired,
and having openings adapted to receive severed nerve ends. The
casing or membrane may be made of any biocompatible, nonirritating
material, such as silicone or a biocompatible polymer such as
polyethylene or polyethylene vinyl acetate. The casing also may be
composed of biocompatible, bioresorbable polymers, including, for
example, collagen, hyaluronic acid, polylactic, polybutyric and
polyglycolic acids. In a currently preferred embodiment, the outer
surface of the channel is substantially impermeable.
[0024] The morphogen may be disposed in the channel in association
with a biocompatible carrier material, or it may be adsorbed to or
otherwise associated with the inner surface of the casing, such as
is described in U.S. Pat. No. 5,011,486, provided that the
morphogen is accessible to the severed nerve ends. Additionally,
although the nerve guidance channels described herein generally are
tubular in shape, it should be evident to those skilled in the art
that various alternative shapes may be employed. The lumen of the
guidance channels may, for example, be oval or even square in cross
section. Moreover the guidance channels may be constructed of two
or more parts which may be clamped together to secure the nerve
stumps. Nerve endings may be secured to the nerve guidance channels
by means of sutures, biocompatible adhesives such as fibrin glue,
or other means known in the medical art.
[0025] The morphogens described herein also are envisioned to be
useful in autologous peripheral nerve segment implants to bypass
damaged neural pathways in the central nervous system, such as in
the repair of damaged or detached retinas, or other damage to the
optic nerve. Here the morphogen is provided to the site of
attachment to stimulate axonal growth at the graft site,
particularly where the damaged axonal segment to be bypassed occurs
far from the neuronal cell body.
[0026] The morphogens described herein also are useful for
enhancing survival of neuronal cells at risk of dying, thereby
preventing, limiting or otherwise inhibiting damage to neural
pathways. Non-mitotic neurons are at risk of dying as a result of a
neuropathy or other cellular dysfunction of a neuron or glial cell
inducing cell death, or following a chemical or mechanical lesion
to the cell or its surrounding tissue. The chemical lesions may
result from known toxic agents, including lead, ethanol, ammonia,
formaldehyde and many other organic solvents, as well as the toxins
in cigarette smoke and opiates. Excitatory amino acids, such as
glutamate also may play a role in the pathogenesis of neuronal cell
death (see Freese et al. (1990) Brain Res. 521:254-264). Neuronal
cell death also is thought to be a significant contributing factor
in a number of progressive neuropathies currently classified as
neurodegenerative diseases, including Alzheimer's disease,
Huntington's chorea, and Parkinson's disease, amyotrophic lateral
sclerosis and multiple sclerosis. The etiology of these diseases
may be metabolic, as results in hepatic encephalopathy, infectious,
toxic, autoimmune, nutritional or ischemic. In addition, ethanol
and a number of other toxins also have been identified as
significant contributing factors in neurodegenerative diseases. The
morphogens described herein may be provided to cells at risk of
dying to enhance their survival and thereby protect the integrity
of the neural pathway. The morphogens may be provided directly to
the site, or they may be provided systemically. Alternatively, as
described above, an agent capable of stimulating endogenous
morphogen expression and/or secretion, preferably in cells
associated with the nerve tissue of interest, may be administered
to the mammal.
[0027] In another aspect of the invention, the method disclosed is
useful for redifferentiating transformed cells, particularly
transformed cells of neuronal or glial origin, such that the
morphogen-treated cells are induced to display a morphology
characteristic of untransformed cells. Where the transformed cells
are cells of neuronal origin, morphogen treatment preferably
induces cell rounding and cell aggregation (clumping), cell-cell
adhesion, neurite outgrowth formation and elongation, and N-CAM
production. The methods described herein are anticipated to
substantially inhibit or reduce neural cell tumor formation and/or
proliferation in nerve tissue. It is anticipated that the methods
of this invention will be useful in substantially reducing the
effects of various carcinomas of nerve tissue origin such as
retinoblastomas, neuroblastomas, and gliomas or glioblastomas. In
addition, the method also is anticipated to aid in inhibiting
neoplastic lesions caused by metastatic tissue. Metastatic tumors
are one of the most common neoplasms of the CNS, as they can reach
the intracranial compartment through the bloodstream. Metastatic
tumors may damage neural pathways for example, by distorting normal
nerve tissue structure, compressing nerves, blocking flow of
cerebrospinal fluid or the blood supply nourishing brain tissue,
and/or by stimulating the body's immune response.
[0028] In another aspect of the invention, the morphogens described
herein are useful for providing neuroprotective effects to
alleviate neural pathway damage associated with the body's
immune/inflammatory response to an initial injury to nerve tissue.
Such a response may follow trauma to nerve tissue, caused, for
example, by an autoimmune dysfunction, neoplastic lesion,
infection, chemical or mechanical trauma, disease, by interruption
of blood flow to the neurons or glial cells, for example following
ischemia or hypoxia, or by other trauma to the nerve or surrounding
material. For example, the primary damage resulting from hypoxia or
ischemia-reperfusion following occlusion of a neural blood supply,
as in an embolic stroke, is believed to be immunologically
associated. In addition, at least part of the damage associated
with a number of primary brain tumors also appears to be
immunologically related. Application of the morphogen directly to
the cells to be treated, or providing the morphogen to the mammal
systemically, for example, intravenously or indirectly by oral
administration, may be used to alleviate and/or inhibit the
immunologically related response to a neural injury. Alternatively,
administration of an agent capable of stimulating morphogen
expression and/or secretion in vivo, preferably at the site of
injury, also may be used. Where the injury is to be induced, as
during surgery or other aggressive clinical treatment, the
morphogen or agent may be provided prior to induction of the injury
to provide a neuroprotective effect to the nerve tissue at
risk.
[0029] In still another aspect, the invention described herein
provides methods for supporting the growth and maintenance of
differentiated neurons, including inducing neurons to continue
expressing their active differentiated phenotype. It is anticipated
that this activity will be particularly useful in the treatment of
nerve tissue disorders where loss of function is caused by reduced
or lost cellular metabolic function and cells become senesent or
quiescent, such as is thought to occur in aging cells and to be
manifested in Alzheimer's disease. Application of the morphogen
directly to cells to be treated, or providing it systemically by
parenteral or oral administration stimulates these cells to
continue expressing their active differentiated phenotype,
significantly inhibiting and/or reversing the effects of the
cellular metabolic dysfunction, thereby maintaining the neural
pathway at risk. Alternatively, administration of an agent capable
of stimulating endogenous morphogen expression and/or secretion in
vivo may be used.
[0030] In still another aspect, the invention provides methods for
stimulating CAM expression levels in a cell, particularly N-CAM
expression in neurons. CAMs are molecules defined as carrying out
cell-cell interactions necessary for tissue formation. CAMs are
believed to play a fundamental regulatory role in tissue
development, including tissue boundary formation, embryonic
induction and migration, and tissue stabilization and regeneration.
Thus, patterns of expression of individual CAMs may guide, in a
positive (attractant) or negative (deterrent) sense, the formation
of specific cell-cell contacts, including synapses, within nerve
tissue and among nerve and innervated tissues. Alternatively, CAM
expression may respond to fluctuations in electromagnetic and/or
biochemical signals within and between cells and tissues. Gradients
of biochemical signals (e.g., chemoattractants) long have been
thought to exist in developing tissues and to provide cells of
these tissues with positional information necessary for the proper
course of morphogenesis of defined tissues and organs in the
embryo. Without being limited to any particular theory, it is
believed that morphogens released by particular cells of an organ
or tissue can diffuse within developing tissues and organs to
provide a biochemical gradient from which cells can extract
positional information needed for normal pattern formation or
morphogenesis. Thus, morphogens can be viewed as chemotrophic or
chemotactic factors that guide and promote differentiation of
particular cell or tissue types and/or dissuade differentiation of
inappropriate cell or tissue types. This concept is presented in
Basler et al. (1993), 73 Cell 687-702, the teachings of which are
incorporated herein by reference.
[0031] Altered CAM levels have been implicated in a number of
tissue disorders, including congenital defects, neoplasias, and
degenerative diseases. In particular, N-CAM expression is
associated with normal neuronal cell development and
differentiation, including retinal formation, synaptogenesis, and
nerve-muscle tissue adhesion. Inhibition of one or more of the
N-CAM isoforms is known to prevent proper tissue development.
Altered N-CAM expression levels also are associated with
neoplasias, including neuroblastomas (see infra), as well as with a
number of neuropathies, including normal pressure hydrocephalous
and type II schizophrenia. Application of the morphogen directly to
the cells to be treated, or providing the morphogen to the mammal
systemically, for example, parenterally, or indirectly by oral
administration, may be used to induce cellular expression of one or
more CAMs, particularly N-CAMs. Alternatively, administration of an
agent capable of stimulating morphogen expression and/or secretion
in vivo, preferably at the site of injury, also may be used to
induce CAM production.
[0032] CAMs also have been postulated as part of a morphoregulatory
pathway whose activity is induced by a to date unidentified
molecule (See, for example, Edelman, G. M. (1986) Ann. Rev. Cell
Biol. 2:81-116). Without being limited to any given theory, the
morphogens described herein may act as the inducer of this
pathway.
[0033] Still further, modulations of endogenous morphogen levels
may be monitored as part of a method of detecting nerve tissue
dysfunction. Specifically, modulations in endogenous morphogen
levels are anticipated to reflect changes in nerve tissue status.
Morphogen expression may be monitored directly in biopsied cell
samples, in cerebrospinal fluid, or serum. Alternatively, morphogen
levels may be assessed by detecting changes in the levels of
endogenous antibodies to the morphogen. For example, one may obtain
serum samples from a mammal, and then detect the concentration of
morphogen or antibody present in the fluid by standard protein
detection means known to those skilled in the art. As an example,
binding protein capable of interacting specifically with the
morphogen of interest such as an anti-morphogen antibody may be
used to detect a morphogen in a standard immunoassay. The morphogen
levels detected then may be compared to a previously determined
standard or reference level, with changes in the detected levels
being indicative of the status of the tissue.
[0034] In certain embodiments of the invention, the morphogen or
morphogen-stimulating agent is administered systemically to the
individual, e.g., orally or parenterally. In other embodiments of
the invention, the morphogen can be provided directly to the nerve
tissue, e.g., by injection to the cerebral spinal fluid or to a
nerve tissue locus.
[0035] In any treatment method of the invention, "administration of
morphogen" refers to the administration of the morphogen, either
alone or in combination with other molecules. For example, the
mature form of the morphogen may be provided in association with
its precursor "pro" domain, which is known to enhance the
solubility of the protein. Other useful molecules known to enhance
protein solubility include casein and other milk components, as
well as various serum proteins. Additional useful molecules which
may be associated with the morphogen or morphogen-stimulating agent
include tissue targeting molecules capable of directing the
morphogen or morphogen-stimulating agent to nerve tissue. Tissue
targeting molecules envisioned to be useful in the treatment
protocols of this invention include antibodies, antibody fragments
or other binding proteins which interact specifically with surface
molecules on nerve tissue cells.
[0036] Still another useful tissue targeting molecule is part or
all of the morphogen precursor "pro" domain, particularly that of
OP-1 or GDF-1. These proteins are found naturally associated with
nerve tissue but also may be synthesized in other tissues and
targeted to nerve tissue after secretion from the synthesizing
tissue. For example, while the protein has been shown to be active
in bone tissue, the primary source of OP-1 synthesis appears to be
the tissues of the urogenic system (e.g., renal and bladder
tissue), with secondary expression levels occurring in the brain,
heart and lungs (see below.) Moreover, the protein has been
identified in serum, saliva and various milk forms. In addition,
the secreted form of the protein comprises the mature dimer in
association with the pro domain of the intact morphogen sequence.
Accordingly, the associated morphogen pro domains may act to target
specific morphogens to different tissues in vivo.
[0037] Associated tissue targeting or solubility-enhancing
molecules also may be covalently linked to the morphogen using
standard chemical means, including acid-labile linkages, which
likely will be preferentially cleaved in acidic environments.
[0038] Finally, the morphogens or morphogen-stimulating agents
provided herein also may be administered in combination with other
molecules known to be beneficial in maintaining neural pathways,
including, for example, anti-inflammatory agents and nerve trophic
(growth) factors.
[0039] In this regard, the present invention also features
compositions and methods that comprise the step of contacting
neural cells with a morphogenic protein and a nerve trophic factor
as defined below, for a time and at a concentration effective for
promoting dendrite outgrowth, including preserving a dendritic
arbor or regenerating dendritic architecture in both the central
and peripheral nervous systems.
[0040] "Nerve trophic factors" as defined herein refer to proteins
able to stimulate survival and growth of nerve cells (Hefti and
Lapchak (1993), 24 Adv. in Pharmacol. 239-273). Most of the
characterized actions of nerve trophic actors relate to
developmental events and suggest that the temporal and local
regulation of expression of these proteins plays a role during
maturation of the nervous system. Nerve trophic factors are also
important in the function of the adult nervous system for the
maintenance of structural integrity and regulation of plasticity.
Such processes are altered by diseases and neurodegenerative events
following acute injury to the nervous system. This has prompted
speculation that nerve trophic factors are involved in the
structural alterations which occur in response to injury and
disease.
[0041] Nerve trophic factors are found among several protein
families, including neurotrophins, fibroblast growth factors, the
epidermal growth factor protein family, and lymphokines to name a
few. Nerve growth factor (NGF) is the best characterized member of
the nerve trophic factor protein families. NGF belongs to the
protein family called neurotrophins, the other known members of
which are brain-derived neurotrophic factor (BDNF), neurotrophin-3
(NT-3), neurotrophin-4 (NT-4), and neurotrophin-5 (NT-5).
Individual neurotrophins are highly conserved among mammalian
species and share at least about 50% amino acid sequence homology
with each other known member of this group. Preferred for use
herein are nerve trophic factors having amino acid sequences that
comprise a sequence sharing at least about 50%, preferably at least
about 60%, homology with each of the sequences of human NGF, human
BDNF, human NT-3, human NT-4, and human NT-5. Alternatively,
mammalian nerve trophic factors can be used herein that have amino
acid sequences comprising six cysteine residues in relative
positions that are strictly conserved and in common with the
sequences of the currently known nerve trophic factors of rats,
humans, chickens, and frogs (Xenopus). See, e.g., Bradshaw et al.
(1993), Trends in Biotechology Sciences (TIBS); Ebendal (1992), 32
J. of Neurosci. Res. 461; Meakin and Shooter (1992), 15 Trends in
Neurosciences (TINS) 323; Swindells (1992), 258 Science 1160;
Daopin et al. (1992), 258 Science 1161.
[0042] In a related aspect, the present invention features
compositions and methods for promoting dendritic outgrowth which
include administering to a mammal a first agent that stimulates
production of an endogenous morphogen within the mammal at a
concentration effective for promoting dendritic outgrowth. As
described above, these agents are referred to herein as
morphogen-stimulating agents, and are understood to include
substances which, when administered to a mammal, act on tissues or
organs that normally are responsible for, or capable of, producing
a morphogen and/or secreting a morphogen, and which cause the
endogenous level of the morphogen to be altered. The agent can act,
for example, by stimulating expression and/or secretion of an
endogenous morphogen. Another feature of the present invention
includes administering this first agent and a nerve trophic factor
at concentrations and for times effective for promoting dendritic
outgrowth. The first agent and the nerve trophic factor can be
co-administered in a composition.
[0043] In yet another related aspect, the invention also features
compositions and methods for promoting dendritic outgrowth which
include administering to a mammal the above-described
morphogen-stimulating factor and a second agent that stimulates
production of an endogenous nerve trophic factor at concentrations
effective for promoting dendritic outgrowth. It is understood that
production of the endogenous nerve trophic factor includes cells or
tissue normally responsible for, or capable of, producing the
endogenous level of factor. Both agents can be co-administered in a
composition. Preferably, both agents can cross the blood-brain
barrier, either passively or actively.
[0044] In a particularly preferred embodiment, a morphogen is
provided to the site of nerve impairment or damage as a composition
comprising a biocompatible, in vivo bioresorbable carrier suitable
for maintaining a protein at a site in vivo, wherein the morphogen
is dispersed in the carrier to a concentration effective for
stimulating dendritic outgrowth at the site of impairment or
damage. The carrier can be composed of a polymeric material such as
laminin or collagen, or comprise brain-tissue-derived extracellular
matrix. In another preferred embodiment, a morphogen and a nerve
trophic factor are disposed at the site of impairment or damage by
means of a biocompatible, in vivo resorbable carrier suitable for
maintaining both at the site in vivo. Both can be admixed with the
carrier and co-provided to the site of impairment or damage.
[0045] Another preferred embodiment of the present invention
comprises a composition for ameliorating a neuropathy, comprising a
morphogen in combination with a nerve trophic factor, the
concentrations of which in combination are competent to promote
dendrite outgrowth. "Neuropathy" in this context refers to a
memory, motor, associative, or metabolic disorder, and includes
Alzheimer's disease, Parkinson's disease, Lewy body dementia,
progressive supranuclear palsy, dementia pugilistica,
olivopontocerebellar atrophy, Wernicke-Korsakoff's syndrome and
diabetic neuropathy. Amelioration refers to clinical management of
the disorder, including attenuation, alleviation, remission or cure
of the disorder. This composition also comprises a biocompatible
acellular support matrix suitable for maintaining said morphogen
and said nerve trophic factor at a site in vivo. The support matrix
can be composed of a malleable gel or a solid polymeric material
that permits dendrite growth around and within the matrix.
Exemplary matrices include laminin or collagen.
[0046] Where the morphogen is intended for use as a therapeutic for
disorders of the CNS, an additional problem must be addressed:
overcoming the so-called "blood-brain barrier", the brain capillary
wall structure that effectively screens out all but selected
categories of molecules present in the blood, preventing their
passage into the brain. The blood-brain barrier can be bypassed
effectively by direct infusion of the morphogen or
morphogen-stimulating agent into the brain. Alternatively, the
morphogen or morphogen-stimulating agent can be modified to enhance
its transport across the blood-brain barrier. For example,
truncated forms of the morphogen or a morphogen-stimulating agent
may be most successful. Alternatively, the morphogen or
morphogen-stimulating agent may be modified to render it more
lipophilic, or it may be conjugated to another molecule which is
naturally transported across the barrier, using standard means
known to those skilled in the art, as, for example, described in
Pardridge, Endocrine Reviews 7:314-330 (1986) and U.S. Pat. No.
4,801,575.
[0047] Accordingly, as used herein, a functional "analog" of a
morphogen refers to a protein having morphogenic biological
activity but possessing additional structural differences compared
to a morphogen as defined herein, e.g., having additional chemical
moieties not normally a part of a morphogen. Such moieties
(introduced, for example, by acylation, alkylation, cationization,
or glycosylation reactions, or other means for conjugating the
moiety to the morphogen) can improve the molecule's solubility,
absorption, biological half-life, or transport, e.g., across the
blood-brain barrier.
[0048] Among the morphogens useful in this invention are proteins
originally identified as osteogenic proteins, such as the OP-1,
OP-2 and CBMP2 proteins, as well as amino acid sequence-related
proteins such as DPP (from Drosophila), Vgl (from Xenopus), Vgr-1
(from mouse, see U.S. Pat. No. 5,011,691 to Oppermann et al.),
GDF-1 (from mouse, see Lee (1991) PNAS 88:4250-4254), all of which
are presented in Table II and Seq. ID Nos.5-14), and the recently
identified 60A protein (from Drosophila, Seq. ID No. 24, see
Wharton et al. (1991) PNAS 88:9214-9218.) The morphogens
collectively comprise a family or genus, which includes members of
a structurally distinct sub-family of the TGF-.beta. super-family
of proteins. That is, the morphogens share substantial amino acid
sequence homology in their C-terminal regions, including a
conserved arrangement or motif of cysteine residues shared by
members of the TGF.beta. super-family. The proteins are translated
as a precursor, having an N-terminal signal peptide sequence,
typically less than about 30 residues, followed by a "pro" domain
that is cleaved to yield the mature C-terminal domain. The signal
peptide is cleaved rapidly upon translation, at a cleavage site
that can be predicted in a given sequence using the method of Von
Heijne ((1986) Nucleic Acids Research 14:4683-4691.) Table I,
below, describes the various morphogens identified to date,
including their nomenclature as used herein, their Seq. ID
references, and publication sources for the amino acid sequences
for the full length proteins not included in the Seq. Listing. The
disclosure of these publications is incorporated herein by
reference. TABLE-US-00001 TABLE I "OP-1" Refers generally to a
morphogenically active protein expressed from a DNA sequence
encoding at least a portion of OP-1 protein bearing the
above-mentioned conserved cysteine residues. Thus, for example, a
human OP-1 protein has an amino acid sequence comprising at least
the conserved seven cysteine skeleton defined by residues 38-139 of
Seq. ID No. 5. In a broader sense, however, "OP1" refers to
naturally occuring or biosynthetic conservative variants of the
human OP1 disclosed in Seq. ID No. 5. Naturally occurring variants
include OP1 variants isolated from individuals of a single
phylogenetic species (e.g., humans). Such variants are referred to
herein as "allelic variants." Other naturally occurring variants
include counterparts of human OP1 isolated from phylogenetically
distinct species (e.g., mouse, Drosophila). These variants are
referred to herein as "species variants" and can also be referred
to as phylogenetic counterparts or homologs. The mature protein
amino acid sequence of human OP-1 ("hOP-1") is set forth in Seq. ID
No. 5 and that of mouse OP-1 ("mOP-1") is set forth in Seq. ID No.
6. The conserved seven cysteine skeleton in each protein is defined
by residues 38 to 139 of Seq. ID Nos. 5 and 6. The cDNA sequences
and the amino acids encoding the full length proteins are provided
in Seq. Id Nos. 16 and 17 (hOP1) and Seq. ID Nos. 18 and 19 (mOP1.)
The mature proteins are defined by residues 293-431 (hOP1) and
292-430 (mOP1). The "pro" regions of the proteins, cleaved to yield
the mature, morphogenically active proteins are defined essentially
by residues 30-292 (hOP1) and residues 30-291 (mOP1). "OP-2" refers
generally to a morphogenically active protein expressed from a DNA
sequence encoding at least a portion of OP-2 protein bearing the
above-mentioned conservative arrangement of cysteine residues.
Thus, for example, a human OP2 protein has an amino acid sequence
comprising at least the conserved seven cysteine skeleton defined
by residues 38- 139 of Seq. ID No. 7. As explained above, "OP2"
refers in a broader sense to naturally occurring or biosynthetic
conservative variants of the human OP2 disclosed in Seq. ID No. 7,
including allelic and species variants thereof, e.g., human OP-2
("hOP-2", Seq. ID No. 7, mature protein amino acid sequence) or
mouse OP-2 ("mOP-2", Seq. ID No. 8, mature protein amino acid
sequence). The conserved seven cysteine skeleton is defined by
residues 38 to 139 of Seq. ID Nos. 7 and 8. The cDNA sequences and
the amino acids encoding the full length proteins are provided in
Seq. ID Nos. 20 and 21 (hOP2) and Seq. ID Nos. 22 and 23 (mOP2.)
The mature proteins are defined essentially by residues 264-402
(hOP2) and 261-399 (mOP2). The "pro" regions of the proteins,
cleaved to yield the mature, morphogenically active proteins likely
are defined essentially by residues 18-263 (hOP2) and residues
18-260 (mOP2). (Another cleavage site also occurs 21 residues
upstream for both OP-2 proteins.) "CBMP2" refers generally to a
morphogenically active protein expressed from a DNA sequence
encoding at least a portion of the CBMP2 proteins bearing the
above- mentioned conserved arrangement of cysteine residues,
including allelic and species variants thereof, e.g., human CBMP2A
("CBMP2A(fx)", Seq ID No. 9) or human CBMP2B DNA ("CBMP2B(fx)",
Seq. ID No. 10). The amino acid sequence for the full length
proteins, referred to in the literature as BMP2A and BMP2B, or BMP2
and BMP4, appear in Wozney, et al. (1988) Science 242: 1528-1534.
The pro domain for BMP2 (BMP2A) likely includes residues 25- 248 or
25-282; the mature protein, residues 249-396 or 283-396. The pro
domain for BMP4 (BMP2B) likely includes residues 25-256 or 25-292;
the mature protein, residues 257-408 or 293-408. "DPP(fx)" refers
to protein sequences encoded by the Drosophila DPP gene and
defining the conserved seven cysteine skeleton (Seq. ID No. 11).
The amino acid sequence for the full length protein appears in
Padgett, et al (1987) Nature 325: 81-84. The pro domain likely
extends from the signal peptide cleavage site to residue 456; the
mature protein likely is defined by residues 457-588. "Vg1(fx)"
refers to protein sequences encoded by the Xenopus Vg1 gene and
defining the conserved seven cysteine skeleton (Seq. ID No. 12).
The amino acid sequence for the full length protein appears in
Weeks (1987) Cell 51: 861-867. The prodomain likely extends from
the signal peptide cleavage site to residue 246; the mature protein
likely is defined by residues 247-360. "Vgr-1(fx)" refers to
protein sequences encoded by the murine Vgr-1 gene and defining the
conserved seven cysteine skeleton (Seq. ID No. 13). The amino acid
sequence for the full length protein appears in Lyons, et al,
(1989) PNAS 86: 4554-4558. The prodomain likely extends from the
signal peptide cleavage site to residue 299; the mature protein
likely is defined by residues 300-438. "GDF-1(fx)" refers to
protein sequences encoded by the human GDF-1 gene and defining the
conserved seven cysteine skeleton (Seq. ID No. 14). The cDNA and
encoded amino sequence for the full length protein is provided in
Seq. ID. No. 32. The prodomain likely extends from the signal
peptide clavage site to residue 214; the mature protein likely is
defined by residues 215-372. "60A" refers generally to
morphogenically active proteins expressed from a DNA sequence (from
the Drosophila 60A gene) encoding at least a portion of the 60A
protein that bears the conserved arrangement of cysteine residues
(see Seq. ID No. 24 wherein the cDNA and encoded amino acid
sequence for the full length protein is provided). Drosphila 60A
protein is thought to be a species variant of human OP1. "60A(fx)"
refers to the protein sequences defining the conserved seven
cysteine skeleton (residues 354 to 455 of Seq. ID No. 24.) The
prodomain likely extends from the signal peptide cleavage site to
residue 324; the mature protein likely is defined by residues
325-455. "BMP3(fx)" refers to protein sequences encoded by the
human BMP3 gene and defining the conserved seven cysteine skeleton
(Seq. ID No. 26). The amino acid sequence for the full length
protein appears in Wozney et al. (1988) Science 242: 1528-1534. The
pro domain likely extends from the signal peptide cleavage site to
residue 290; the mature protein likely is defined by residues
291-472. "BMP5(fx)" refers to protein sequences encoded by the
human BMP5 gene and defining the conserved seven cysteine skeleton
(Seq. ID No. 27). The amino acid sequence for the full length
protein appears in Celeste, et al. (1991) PNAS 87: 9843-9847. The
pro domain likely extends from the signal peptide cleavage site to
residue 316; the mature protein likely is defined by residues
317-454. "BMP6(fx)" refers to protein sequences encoded by the
human BMP6 gene and defining the conserved seven cysteine skeleton
(Seq. ID No. 28). The amino acid sequence for the full length
protein appears in Celeste, et al. (1990) PNAS 87: 9843-5847. The
pro domain likely includes extends from the signal peptide cleavage
site to residue 374; the mature sequence likely includes residues
375-513.
[0049] The OP-2 proteins have an additional cysteine residue in
this region (e.g., see residue 41 of Seq. ID Nos. 7 and 8), in
addition to the conserved cysteine skeleton in common with the
other proteins in this family. The GDF-1 protein has a four amino
acid insert within the conserved skeleton (residues 44-47 of Seq.
ID No. 14) but this insert likely does not interfere with the
relationship of the cysteines in the folded structure. In addition,
the CBMP2 proteins are missing one amino acid residue within the
cysteine skeleton.
[0050] The morphogens are inactive when reduced, but are active as
oxidized homodimers and when oxidized in combination with other
morphogens of this invention. Thus, as defined herein, a morphogen
is a dimeric protein comprising a pair of polypeptide chains,
wherein the sequence of each polypeptide chain comprises at least
the C-terminal seven cysteine skeleton defined by the positionally
conserved seven cysteine residues included within residues 38-139
of Seq. ID No. 5, including functionally equivalent arrangements of
these cysteines (e.g., amino acid insertions or deletions which
alter the linear arrangement of the cysteines in the sequence but
not their relationship in the folded structure), such that, when
the polypeptide chains are folded, the dimeric protein species
comprising the pair of polypeptide chains has the appropriate
three-dimensional structure, including the appropriate intra- or
inter-chain disulfide bonds such that the protein has morphogenic
activity as defined herein. Specifically, the morphogens generally
are competent to induce all of the following biological functions
in a morphogenically permissive environment: stimulating
proliferation of progenitor cells; stimulating the differentiation
of progenitor cells; stimulating the proliferation of
differentiated cells; and supporting the growth and maintenance of
differentiated cells. In addition, it is also anticipated that
these morphogens are capable of inducing redifferentiation of
committed cells under appropriate environmental conditions.
[0051] The positional locations of cysteine residues conserved
among the morphogens of this invention are set forth in Generic
Sequence 1 (Seq. ID No. 1) and Generic Sequence 2 (Seq. ID No. 2);
where each Xaa indicates one of the 20 naturally-occurring
L-isomer, .alpha.-amino acids or a derivative thereof. Generic
Sequence 1 comprises the conserved six cysteine skeleton and
Generic Sequence 2 comprises the conserved six cysteine skeleton
plus the additional cysteine identified in OP-2 (see residue 36,
Seq. ID No. 2). The conserved seven cysteine skeleton of the
morphogens is produced when the following additional sequence is
present in the morphogen polypeptide chain N-terminal to and
adjoining the region bearing the positionally conserved cysteines
of Generic Sequences 1 and 2: TABLE-US-00002 Cys Xaa Xaa Xaa Xaa
(Seq. ID No. 15) 1 5
[0052] Preferred amino acid sequences within the foregoing generic
sequences include: Generic Sequence 3 (Seq. ID No. 3), Generic
Sequence 4 (Seq. ID No. 4), Generic Sequence 5 (Seq. ID No. 30) and
Generic Sequence 6 (Seq. ID No. 31), listed below. These Generic
Sequences accommodate homologies shared among various preferred
members of the morphogen family or genus identified in Table II, as
well as the amino acid sequence variation among them. Specifically,
Generic Sequences 3 and 4 are composite amino acid sequences of the
following proteins presented in Table II and identified in Seq. ID
Nos. 5-14: human OP-1 (hOP-1, Seq. ID Nos. 5 and 16-17), mouse OP-1
(mOP-1, Seq. ID Nos. 6 and 18-19), human and mouse OP-2 (Seq. ID
Nos. 7, 8, and 20-22), CBMP2A (Seq. ID No. 9), CBMP2B (Seq. ID No.
10), DPP (from Drosophila, Seq. ID No. 11), Vgl, (from Xenopus,
Seq. ID No. 12), Vgr-1 (from mouse, Seq. ID No. 13), and GDF-1
(from mouse, Seq. ID No. 14.) The generic sequences include both
the amino acid identity shared by the sequences in Table II, as
well as alternative residues for the variable positions within the
sequence. Note that these generic sequences allow for an additional
cysteine at position 41 or 46 in Generic Sequences 3 or 4,
respectively, providing an appropriate cysteine skeleton where
inter- or intramolecular disulfide bonds can form, and contain
certain critical amino acids which influence the tertiary structure
of the proteins. TABLE-US-00003 Generic Sequence 3 Leu Tyr Val Xaa
Phe 1 5 Xaa Xaa Xaa Gly Trp Xaa Xaa Trp Xaa 10 Xaa Ala Pro Xaa Gly
Xaa Xaa Ala 15 20 Xaa Tyr Cys Xaa Gly Xaa Cys Xaa 25 30 Xaa Pro Xaa
Xaa Xaa Xaa Xaa 35 Xaa Xaa Xaa Asn His Ala Xaa Xaa 40 45 Xaa Xaa
Leu Xaa Xaa Xaa Xaa Xaa 50 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys 55 60
Cys Xaa Pro Xaa Xaa Xaa Xaa Xaa 65 Xaa Xaa Xaa Leu Xaa Xaa Xaa 70
75 Xaa Xaa Xaa Xaa Val Xaa Leu Xaa 80 Xaa Xaa Xaa Xaa Met Xaa Val
Xaa 85 90 Xaa Cys Gly Cys Xaa 95
[0053] wherein each Xaa is independently selected from a group of
one or more specified amino acids defined as follows: "Res." means
"residue" and Xaa at res.4=(Ser, Asp or Glu); Xaa at res.6=(Arg,
Gln, Ser or Lys); Xaa at res.7=(Asp or Glu); Xaa at res.8=(Leu or
Val); Xaa at res.11=(Gln, Leu, Asp, His or Asn); Xaa at
res.12=(Asp, Arg or Asn); Xaa at res.14=(Ile or Val); Xaa at
res.15=(Ile or Val); Xaa at res.18=(Glu, Gln, Leu, Lys, Pro or
Arg); Xaa at res.20=(Tyr or Phe); Xaa at res.21=(Ala, Ser, Asp,
Met, His, Leu or Gln); Xaa at res.23=(Tyr, Asn or Phe); Xaa at
res.26=(Glu, His, Tyr, Asp or Gln); Xaa at res.28=(Glu, Lys, Asp or
Gln); Xaa at res.30=(Ala, Ser, Pro or Gln); Xaa at res.31=(Phe, Leu
or Tyr); Xaa at res.33=(Leu or Val); Xaa at res.34=(Asn, Asp, Ala
or Thr); Xaa at res.35=(Ser, Asp, Glu, Leu or Ala); Xaa at
res.36=(Tyr, Cys, His, Ser or Ile); Xaa at res.37=(Met, Phe, Gly or
Leu); Xaa at res.38=(Asn or Ser); Xaa at res.39=(Ala, Ser or Gly);
Xaa at res.40=(Thr, Leu or Ser); Xaa at res.44=(Ile or Val); Xaa at
res.45=(Val or Leu); Xaa at res.46=(Gln or Arg); Xaa at
res.47=(Thr, Ala or Ser); Xaa at res.49=(Val or Met); Xaa at
res.50=(His or Asn); Xaa at res.51=(Phe, Leu, Asn, Ser, Ala or
Val); Xaa at res.52=(Ile, Met, Asn, Ala or Val); Xaa at
res.53=(Asn, Lys, Ala or Glu); Xaa at res.54=(Pro or Ser); Xaa at
res.55=(Glu, Asp, Asn, or Gly); Xaa at res.56=(Thr, Ala, Val, Lys,
Asp, Tyr, Ser or Ala); Xaa at res.57=(Val, Ala or Ile); Xaa at
res.58=(Pro or Asp); Xaa at res.59=(Lys or Leu); Xaa at res.60=(Pro
or Ala); Xaa at res.63=(Ala or Val); Xaa at res.65=(Thr or Ala);
Xaa at res.66=(Gln, Lys, Arg or Glu); Xaa at res.67=(Leu, Met or
Val); Xaa at res.68=(Asn, Ser or Asp); Xaa at res.69=(Ala, Pro or
Ser); Xaa at res.70=(Ile, Thr or Val); Xaa at res.71=(Ser or Ala);
Xaa at res.72=(Val or Met); Xaa at res.74=(Tyr or Phe); Xaa at
res.75=(Phe, Tyr or Leu); Xaa at res.76=(Asp or Asn); Xaa at
res.77=(Asp, Glu, Asn or Ser); Xaa at res.78=(Ser, Gln, Asn or
Tyr); Xaa at res.79=(Ser, Asn, Asp or Glu); Xaa at res.80=(Asn, Thr
or Lys); Xaa at res.82=(Ile or Val); Xaa at res.84=(Lys or Arg);
Xaa at res.85=(Lys, Asn, Gln or His); Xaa at res.86=(Tyr or His);
Xaa at res.87=(Arg, Gln or Glu); Xaa at res.88=(Asn, Glu or Asp);
Xaa at res.90=(Val, Thr or Ala); Xaa at res.92=(Arg, Lys, Val, Asp
or Glu); Xaa at res.93=(Ala, Gly or Glu); and Xaa at res.97=(His or
Arg); TABLE-US-00004 Generic Sequence 4 Cys Xaa Xaa Xaa Xaa Leu Tyr
Val Xaa Phe 1 5 10 Xaa Xaa Xaa Gly Trp Xaa Xaa Trp Xaa 15 Xaa Ala
Pro Xaa Gly Xaa Xaa Ala 20 25 Xaa Tyr Cys Xaa Gly Xaa Cys Xaa 30 35
Xaa Pro Xaa Xaa Xaa Xaa Xaa 40 Xaa Xaa Xaa Asn His Ala Xaa Xaa 45
50 Xaa Xaa Leu Xaa Xaa Xaa Xaa Xaa 55 Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Cys 60 65 Cys Xaa Pro Xaa Xaa Xaa Xaa Xaa 70 Xaa Xaa Xaa Leu Xaa
Xaa Xaa 75 80 Xaa Xaa Xaa Xaa Val Xaa Leu Xaa 85 Xaa Xaa Xaa Xaa
Met Xaa Val Xaa 90 95 Xaa Cys Gly Cys Xaa 100
wherein each Xaa is independently selected from a group of one or
more specified amino acids as defined by the following: "Res."
means "residue" and Xaa at res.2=(Lys or Arg); Xaa at res.3=(Lys or
Arg); Xaa at res.4=(His or Arg); Xaa at res.5=(Glu, Ser, His, Gly,
Arg or Pro); Xaa at res.9=(Ser, Asp or Glu); Xaa at res.11=(Arg,
Gln, Ser or Lys); Xaa at res.12=(Asp or Glu); Xaa at res.13=(Leu or
Val); Xaa at res.16=(Gln, Leu, Asp, His or Asn); Xaa at
res.17=(Asp, Arg, or Asn); Xaa at res.19=(Ile or Val); Xaa at
res.20=(Ile or Val); Xaa at res.23=(Glu, Gln, Leu, Lys, Pro or
Arg); Xaa at res.25=(Tyr or Phe); Xaa at res.26=(Ala, Ser, Asp,
Met, His, Leu, or Gln); Xaa at res.28=(Tyr, Asn or Phe); Xaa at
res.31=(Glu, His, Tyr, Asp or Gln); Xaa at res.33=Glu, Lys, Asp or
Gln); Xaa at res.35=(Ala, Ser or Pro); Xaa at res.36=(Phe, Leu or
Tyr); Xaa at res.38=(Leu or Val); Xaa at res.39=(Asn, Asp, Ala or
Thr); Xaa at res.40=(Ser, Asp, Glu, Leu or Ala); Xaa at
res.41=(Tyr, Cys, His, Ser or Ile); Xaa at res.42=(Met, Phe, Gly or
Leu); Xaa at res.44=(Ala, Ser or Gly); Xaa at res.45=(Thr, Leu or
Ser); Xaa at res.49=(Ile or Val); Xaa at res.50=(Val or Leu); Xaa
at res.51=(Gln or Arg); Xaa at res.52=(Thr, Ala or Ser); Xaa at
res.54=(Val or Met); Xaa at res.55=(His or Asn); Xaa at
res.56=(Phe, Leu, Asn, Ser, Ala or Val); Xaa at res.57=(Ile, Met,
Asn, Ala or Val); Xaa at res.58=(Asn, Lys, Ala or Glu); Xaa at
res.59=(Pro or Ser); Xaa at res.60=(Glu, Asp, or Gly); Xaa at
res.61=(Thr, Ala, Val, Lys, Asp, Tyr, Ser or Ala); Xaa at
res.62=(Val, Ala or Ile); Xaa at res.63=(Pro or Asp); Xaa at
res.64=(Lys or Leu); Xaa at res.65=(Pro or Ala); Xaa at res.68=(Ala
or Val); Xaa at res.70=(Thr or Ala); Xaa at res.71=(Gln, Lys, Arg
or Glu); Xaa at res.72=(Leu, Met or Val); Xaa at res.73=(Asn, Ser
or Asp); Xaa at res.74=(Ala, Pro or Ser); Xaa at res.75=(Ile, Thr
or Val); Xaa at res.76=(Ser or Ala); Xaa at res.77=(Val or Met);
Xaa at res.79=(Tyr or Phe); Xaa at res.80=(Phe, Tyr or Leu); Xaa at
res.81=(Asp or Asn); Xaa at res.82=(Asp, Glu, Asn or Ser); Xaa at
res.83=(Ser, Gln, Asn or Tyr); Xaa at res.84=(Ser, Asn, Asp or
Glu); Xaa at res.85=(Asn, Thr or Lys); Xaa at res.87=(Ile or Val);
Xaa at res.89=(Lys or Arg); Xaa at res.90=(Lys, Asn, Gln or His);
Xaa at res.91=(Tyr or His); Xaa at res.92=(Arg, Gln or Glu); Xaa at
res.93=(Asn, Glu or Asp); Xaa at res.95=(Val, Thr or Ala); Xaa at
res.97=(Arg, Lys, Val, Asp or Glu); Xaa at res.98=(Ala, Gly or
Glu); and Xaa at res.102=(His or Arg).
[0054] Similarly, Generic Sequence 5 (Seq. ID No. 30) and Generic
Sequence 6 (Seq. ID No. 31) accommodate the homologies shared among
all the morphogen protein family members identified in Table II.
Specifically, Generic Sequences 5 and 6 are composite amino acid
sequences of human OP-1 (hOP-1, Seq. ID Nos. 5 and 16-17), mouse
OP-1 (mOP-1, Seq. ID Nos. 6 and 18-19), human and mouse OP-2 (Seq.
ID Nos. 7, 8, and 20-22), CBMP2A (Seq. ID No. 9), CBMP2B (Seq. ID
No. 10), DPP (from Drosophila, Seq. ID No. 11), Vgl, (from Xenopus,
Seq. ID No. 12), Vgr-1 (from mouse, Seq. ID No. 13), and GDF-1
(from mouse, Seq. ID No. 14), human BMP3 (Seq. ID No. 26), human
BMP5 (Seq. ID No. 27), human BMP6 (Seq. ID No. 28) and 60(A) (from
Drosophila, Seq. ID Nos. 24-25). The generic sequences include both
the amino acid identity shared by these sequences in the C-terminal
domain, defined by the six and seven cysteine skeltons (Generic
Sequences 5 and 6, respectively), as well as alternative residues
for the variable positions within the sequence. As for Generic
Sequences 3 and 4, Generic Sequences 5 and 6 allow for an
additional cysteine at position 41 (Generic Sequence 5) or position
46 (Generic Sequence 6), providing an appropriate cysteine skeleton
where inter- or intramolecular disulfide bonds can form, and
containing certain critical amino acids which influence the
tertiary structure of the proteins. TABLE-US-00005 Generic Sequence
5 Leu Xaa Xaa Xaa Phe 1 5 Xaa Xaa Xaa Gly Trp Xaa Xaa Trp Xaa 10
Xaa Xaa Pro Xaa Xaa Xaa Xaa Ala 15 20 Xaa Tyr Cys Xaa Gly Xaa Cys
Xaa 25 30 Xaa Pro Xaa Xaa Xaa Xaa Xaa 35 Xaa Xaa Xaa Asn His Ala
Xaa Xaa 40 45 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 50 Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Cys 55 60 Cys Xaa Pro Xaa Xaa Xaa Xaa Xaa 65 Xaa Xaa
Xaa Leu Xaa Xaa Xaa 70 75 Xaa Xaa Xaa Xaa Val Xaa Leu Xaa 80 Xaa
Xaa Xaa Xaa Met Xaa Val Xaa 85 90 Xaa Cys Xaa Cys Xaa 95
[0055] wherein each Xaa is independently selected from a group of
one or more specified amino acids defined as follows: "Res." means
"residue" and Xaa at res.2=(Tyr or Lys); Xaa at res.3=Val or Ile);
Xaa at res.4=(Ser, Asp or Glu); Xaa at res.6=(Arg, Gln, Ser, Lys or
Ala); Xaa at res.7=(Asp, Glu or Lys); Xaa at res.8=(Leu, Val or
Ile); Xaa at res.11=(Gln, Leu, Asp, His, Asn or Ser); Xaa at
res.12=(Asp, Arg, Asn or Glu); Xaa at res.14=(Ile or Val); Xaa at
res.15=(Ile or Val); Xaa at res.16 (Ala or Ser); Xaa at
res.18=(Glu, Gln, Leu, Lys, Pro or Arg); Xaa at res.19=(Gly or
Ser); Xaa at res.20=(Tyr or Phe); Xaa at res.21=(Ala, Ser, Asp,
Met, His, Gln, Leu or Gly); Xaa at res.23=(Tyr, Asn or Phe); Xaa at
res.26=(Glu, His, Tyr, Asp, Gln or Ser); Xaa at res.28=(Glu, Lys,
Asp, Gln or Ala); Xaa at res.30=(Ala, Ser, Pro, Gln or Asn); Xaa at
res.31=(Phe, Leu or Tyr); Xaa at res.33=(Leu, Val or Met); Xaa at
res.34=(Asn, Asp, Ala, Thr or Pro); Xaa at res.35=(Ser, Asp, Glu,
Leu, Ala or Lys); Xaa at res.36=(Tyr, Cys, His, Ser or Ile); Xaa at
res.37=(Met, Phe, Gly or Leu); Xaa at res.38=(Asn, Ser or Lys); Xaa
at res.39=(Ala, Ser, Gly or Pro); Xaa at res.40=(Thr, Leu or Ser);
Xaa at res.44=(Ile, Val or Thr); Xaa at res.45=(Val, Leu or Ile);
Xaa at res.46=(Gln or Arg); Xaa at res.47=(Thr, Ala or Ser); Xaa at
res.48=(Leu or Ile); Xaa at res.49=(Val or Met); Xaa at
res.50=(His, Asn or Arg); Xaa at res.51=(Phe, Leu, Asn, Ser, Ala or
Val); Xaa at res.52=(Ile, Met, Asn, Ala, Val or Leu); Xaa at
res.53=(Asn, Lys, Ala, Glu, Gly or Phe); Xaa at res.54=(Pro, Ser or
Val); Xaa at res.55=(Glu, Asp, Asn, Gly, Val or Lys); Xaa at
res.56=(Thr, Ala, Val, Lys, Asp, Tyr, Ser, Ala, Pro or His); Xaa at
res.57=(Val, Ala or Ile); Xaa at res.58=(Pro or Asp); Xaa at
res.59=(Lys, Leu or Glu); Xaa at res.60=(Pro or Ala); Xaa at
res.63=(Ala or Val); Xaa at res.65=(Thr, Ala or Glu); Xaa at
res.66=(Gln, Lys, Arg or Glu); Xaa at res.67=(Leu, Met or Val); Xaa
at res.68=(Asn, Ser, Asp or Gly); Xaa at res.69=(Ala, Pro or Ser);
Xaa at res.70=(Ile, Thr, Val or Leu); Xaa at res.71=(Ser, Ala or
Pro); Xaa at res.72=(Val, Met or Ile); Xaa at res.74=(Tyr or Phe);
Xaa at res.75=(Phe, Tyr, Leu or His); Xaa at res.76=(Asp, Asn or
Leu); Xaa at res.77=(Asp, Glu, Asn or Ser); Xaa at res.78=(Ser,
Gln, Asn, Tyr or Asp); Xaa at res.79=(Ser, Asn, Asp, Glu or Lys);
Xaa at res.80=(Asn, Thr or Lys); Xaa at res.82=(Ile, Val or Asn);
Xaa at res.84=(Lys or Arg); Xaa at res.85=(Lys, Asn, Gln, His or
Val); Xaa at res.86=(Tyr or His); Xaa at res.87=(Arg, Gln, Glu or
Pro); Xaa at res.88=(Asn, Glu or Asp); Xaa at res.90=(Val, Thr, Ala
or Ile); Xaa at res.92=(Arg, Lys, Val, Asp or Glu); Xaa at
res.93=(Ala, Gly, Glu or Ser); Xaa at res.95=(Gly or Ala) and Xaa
at res.97=(His or Arg). TABLE-US-00006 Generic Sequence 6 Cys Xaa
Xaa Xaa Xaa Leu Xaa Xaa Xaa Phe 1 5 10 Xaa Xaa Xaa Gly Trp Xaa Xaa
Trp Xaa 15 Xaa Xaa Pro Xaa Xaa Xaa Xaa Ala 20 25 Xaa Tyr Cys Xaa
Gly Xaa Cys Xaa 30 35 Xaa Pro Xaa Xaa Xaa Xaa Xaa 40 Xaa Xaa Xaa
Asn His Ala Xaa Xaa 45 50 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 55 Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Cys 60 65 Cys Xaa Pro Xaa Xaa Xaa Xaa Xaa
70 Xaa Xaa Xaa Leu Xaa Xaa Xaa 75 80 Xaa Xaa Xaa Xaa Val Xaa Leu
Xaa 85 Xaa Xaa Xaa Xaa Met Xaa Val Xaa 90 95 Xaa Cys Xaa Cys Xaa
100
wherein each Xaa is independently selected from a group of one or
more specified amino acids as defined by the following: "Res."
means "residue" and Xaa at res.2=(Lys, Arg, Ala or Gln); Xaa at
res.3=(Lys, Arg or Met); Xaa at res.4=(His, Arg or Gln); Xaa at
res.5=(Glu, Ser, His, Gly, Arg, Pro, Thr, or Tyr); Xaa at
res.7=(Tyr or Lys); Xaa at res.8=(Val or Ile); Xaa at res.9=(Ser,
Asp or Glu); Xaa at res.11=(Arg, Gln, Ser, Lys or Ala); Xaa at
res.12=(Asp, Glu, or Lys); Xaa at res.13=(Leu, Val or Ile); Xaa at
res.16=(Gln, Leu, Asp, His, Asn or Ser); Xaa at res.17=(Asp, Arg,
Asn or Glu); Xaa at res.19=(Ile or Val); Xaa at res.20=(Ile or
Val); Xaa at res.21=(Ala or Ser); Xaa at res.23=(Glu, Gln, Leu,
Lys, Pro or Arg); Xaa at res.24=(Gly or Ser); Xaa at res.25=(Tyr or
Phe); Xaa at res.26=(Ala, Ser, Asp, Met, His, Gln, Leu, or Gly);
Xaa at res.28=(Tyr, Asn or Phe); Xaa at res.31=(Glu, His, Tyr, Asp,
Gln or Ser); Xaa at res.33=Glu, Lys, Asp, Gln or Ala); Xaa at
res.35=(Ala, Ser, Pro, Gln or Asn); Xaa at res.36=(Phe, Leu or
Tyr); Xaa at res.38=(Leu, Val or Met); Xaa at res.39=(Asn, Asp,
Ala, Thr or Pro); Xaa at res.40=(Ser, Asp, Glu, Leu, Ala or Lys);
Xaa at res.41=(Tyr, Cys, His, Ser or Ile); Xaa at res.42=(Met, Phe,
Gly or Leu); Xaa at res.43=(Asn, Ser or Lys); Xaa at res.44=(Ala,
Ser, Gly or Pro); Xaa at res.45=(Thr, Leu or Ser); Xaa at
res.49=(Ile, Val or Thr); Xaa at res.50=(Val, Leu or Ile); Xaa at
res.51=(Gln or Arg); Xaa at res.52=(Thr, Ala or Ser); Xaa at
res.53=(Leu or Ile); Xaa at res.54=(Val or Met); Xaa at
res.55=(His, Asn or Arg); Xaa at res.56=(Phe, Leu, Asn, Ser, Ala or
Val); Xaa at res.57=(Ile, Met, Asn, Ala, Val or Leu); Xaa at
res.58=(Asn, Lys, Ala, Glu, Gly or Phe); Xaa at res.59=(Pro, Ser or
Val); Xaa at res.60=(Glu, Asp, Gly, Val or Lys); Xaa at
res.61=(Thr, Ala, Val, Lys, Asp, Tyr, Ser, Ala, Pro or His); Xaa at
res.62=(Val, Ala or Ile); Xaa at res.63=(Pro or Asp); Xaa at
res.64=(Lys, Leu or Glu); Xaa at res.65=(Pro or Ala); Xaa at
res.68=(Ala or Val); Xaa at res.70=(Thr, Ala or Glu); Xaa at
res.71=(Gln, Lys, Arg or Glu); Xaa at res.72=(Leu, Met or Val); Xaa
at res.73=(Asn, Ser, Asp or Gly); Xaa at res.74=(Ala, Pro or Ser);
Xaa at res.75=(Ile, Thr, Val or Leu); Xaa at res.76=(Ser, Ala or
Pro); Xaa at res.77=(Val, Met or Ile); Xaa at res.79=(Tyr or Phe);
Xaa at res.80=(Phe, Tyr, Leu or His); Xaa at res.81=(Asp, Asn or
Leu); Xaa at res.82=(Asp, Glu, Asn or Ser); Xaa at res.83=(Ser,
Gln, Asn, Tyr or Asp); Xaa at res.84=(Ser, Asn, Asp, Glu or Lys);
Xaa at res.85=(Asn, Thr or Lys); Xaa at res.87=(Ile, Val or Asn);
Xaa at res.89=(Lys or Arg); Xaa at res.90=(Lys, Asn, Gln, His or
Val); Xaa at res.91=(Tyr or His); Xaa at res.92=(Arg, Gln, Glu or
Pro); Xaa at res.93=(Asn, Glu or Asp); Xaa at res.95=(Val, Thr, Ala
or Ile); Xaa at res.97=(Arg, Lys, Val, Asp or Glu); Xaa at
res.98=(Ala, Gly, Glu or Ser); Xaa at res.100=(Gly or Ala); and Xaa
at res.102=(His or Arg).
[0056] Particularly useful sequences for use as morphogens in this
invention include the C-terminal domains, e.g., the C-terminal
96-102 amino acid residues of Vgl, Vgr-1, DPP, OP-1, OP-2, CBMP-2A,
CBMP-2B, GDF-1 (see Table II, below, and Seq. ID Nos. 5-14), as
well as proteins comprising the C-terminal domains of 60A, BMP3,
BMP5 and BMP6 (see Seq. ID Nos. 24-28), all of which include at
least the conserved seven cysteine skeleton. In addition,
biosynthetic constructs designed from the generic sequences, such
as COP-1,3-5, 7, 16, disclosed in U.S. Pat. No. 5,011,691, also are
useful. Accordingly, other useful proteins are those exhibiting
morphogenic activity and having amino acid sequences sharing at
least 70% amino acid sequence homology or "similarity", and
preferably 80% homology or similarity with any of the sequences
above. These are anticipated to include allelic variants, species
variants and other sequence variants (e.g., "muteins" or "mutant
proteins"), whether naturally occurring or biosynthetically
produced, as well as novel members of this morphogenic family of
proteins.
[0057] As used herein, "amino acid sequence homology" is understood
to mean amino acid sequence similarity, and homologous sequences
share identical or similar amino acids, where similar amino acids
are conserved amino acids as defined by Dayoff et al., Atlas of
Protein Sequence and Structure; vol. 5, Suppl. 3, pp. 345-362 (M.
O. Dayoff, ed., Nat'l BioMed. Research Fdn., Washington D.C. 1978.)
Thus, a candidate sequence sharing 70% amino acid homology with a
reference sequence requires that, following alignment of the
candidate sequence with the reference sequence, 70% of the amino
acids in the candidate sequence are identical to the corresponding
amino acid in the reference sequence, or constitute a conserved
amino acid change thereto. "Amino acid sequence identity" is
understood to require identical amino acids between two aligned
sequences. Thus, a candidate sequence sharing 60% amino acid
identity with a reference sequence requires that, following
alignment of the candidate sequence with the reference sequence,
60% of the amino acids in the candidate sequence are identical to
the corresponding amino acid in the reference sequence.
[0058] As used herein, all homologies and identities calculated use
OP-1 as the reference sequence. Also as used herein, sequences are
aligned for homology and identity calculations using the method of
Needleman et al. (1970) J. Mol. Biol. 48:443-453 and identities
calculated by the Align program (DNAstar, Inc.) In all cases,
internal gaps and amino acid insertions in the candidate sequence
as aligned are ignored when making the homology/identity
calculation.
[0059] The currently most preferred protein sequences useful as
morphogens in this invention include those having greater than 60%
identity, preferably greater than 65% identity, with the amino acid
sequence defining the conserved seven cysteine skeleton of hOP1
(e.g., residues 38-139 of Seq. ID No. 5). These most preferred
sequences include both allelic and species variants of the OP-1 and
OP-2 proteins, including the Drosophila 60A protein. Accordingly,
in another preferred aspect of the invention, useful morphogens
include active proteins comprising species of polypeptide chains
having the generic amino acid sequence herein referred to as "OPX",
which accommodates the homologies between the various identified
species of OP1 and OP2 (Seq. ID No. 29).
[0060] In still another preferred aspect of the invention, useful
morphogens include active proteins comprising polypeptide chains
encoded by nucleic acids which hybridize to DNA or RNA sequences
encoding the C-terminal sequence defining the conserved cysteine
domain, e.g., nucleotides 1036-1341 and nucleotides 1390-1695 of
Seq. Id. Nos. 16 and 20, respectively, of OP1 or OP2 under
stringent hybridization conditions. As used herein, stringent
hybridization conditions are defined as hybridization in 40%
formamide, 5.times.SSPE, 5.times. Denhardt's Solution, and 0.1% SDS
at 37.degree. C. overnight, and washing in 0.1.times.SSPE, 0.1% SDS
at 50.degree. C.
[0061] The morphogens useful in the methods, composition and
devices of this invention include proteins comprising any of the
polypeptide chains described above, whether isolated from
naturally-occurring sources, or produced by recombinant DNA or
other synthetic techniques, and includes allelic and species
variants of these proteins, naturally-occurring or biosynthetic
mutants thereof, as well as various truncated and fusion
constructs. Deletion or addition mutants also are envisioned to be
active, including those which may alter the conserved C-terminal
cysteine skeleton, provided that the alteration does not
functionally disrupt the relationship of these cysteines in the
folded structure. Accordingly, such active forms are considered the
equivalent of the specifically described constructs disclosed
herein. The proteins may include forms having varying glycosylation
patterns, varying N-termini, a family of related proteins having
regions of amino acid sequence homology, and active truncated or
mutated forms of native or biosynthetic proteins, produced by
expression of recombinant DNA in host cells.
[0062] The morphogenic proteins can be expressed from intact or
truncated cDNA or from synthetic DNAs in procaryotic or eucaryotic
host cells, and purified, cleaved, refolded, and dimerized to form
morphogenically active compositions. Currently preferred host cells
include E. coli or mammalian cells, such as CHO, COS or BSC cells.
A detailed description of the morphogens useful in the methods,
compositions and devices of this invention is disclosed in
copending U.S. patent application Ser. No. 752,764, filed Aug. 30,
1991, and Ser. No. 667,274, filed Mar. 11, 1991, the disclosure of
which are incorporated herein by reference.
[0063] Thus, in view of this disclosure, skilled genetic engineers
can isolate genes from cDNA or genomic libraries of various
different species which encode appropriate amino acid sequences, or
construct DNAs from oligonucleotides, and then can express them in
various types of host cells, including both procaryotes and
eucaryotes, to produce large quantities of active proteins capable
of maintaining neural pathways in a mammal, including enhancing the
survival of neurons at risk of dying and stimulating nerve
regeneration and repair in a variety of mammals, including
humans.
[0064] The foregoing and other objects, features and advantages of
the present invention will be made more apparent from the following
detailed description of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0065] The foregoing and other objects and features of this
invention, as well as the invention itself, may be more fully
understood from the following description, when read together with
the accompanying drawings, in which:
[0066] FIGS. 1(A and B) are photomicrographs illustrating the
ability of morphogen (OP-1) to induce transformed
neuroblastoma.times.glioma cells (1A) to redifferentiate to a
morphology characteristic of untransformed neurons (1B);
[0067] FIG. 2A is a dose response curve for the induction of the
180 kDa and 140 kDa N-CAM isoforms in morphogen-treated NG108-15
cells;
[0068] FIG. 2B is a photomicrograph of a Western blot of whole cell
extracts from morphogen-treated NG108-15 cells with an
N-CAM-specific antibody; and
[0069] FIG. 3 is a plot of the mean number of cell aggregates
counted in 20 randomly selected fields as a function of morphogen
concentration.
[0070] FIG. 4 is a photomicrograph of an immunoblot demonstrating
the presence of OP-1 in human serum.
[0071] FIG. 5 is a time course of the response of cultured
sympathetic neurons to OP-1. Intracellular dye injections (N>30
for each point) were performed at various times to determine: a)
the percentage of cells with dendrites (open symbols, control;
closed symbols, cells supplemented with 100 ng/ml OP-1 during the
time course study); b) the mean number of dendrites/cell; and c)
the number of axons/cell (circles). The bars shown in b) represent
the SEM; where bars are not shown, the SEM was smaller than the
size of the symbol.
[0072] FIG. 6 is a plot of the effects of varying concentrations of
OP-1 on dendritic growth. Sympathetic neurons were exposed to OP-1
in culture for 3 days and then immunostained with a
dendrite-specific mAb (SMI 32). Percentage of cells with dendrites,
open circles; mean number of dendrites per cell, filled
circles.
[0073] FIG. 7 is a plot of the effects of varying concentrations of
NGF on OP-1 induced dendritic growth in cultured sympathetic
neurons. Percentage of cells with dendrites (97%, open triangles);
mean number of dendrites/cell (4.7.+-.0.3, solid circles); neurons
surviving/culture (2808.+-.267, open circles).
DETAILED DESCRIPTION OF THE INVENTION
[0074] It now has been discovered that the proteins described
herein are effective agents for enhancing the survival of neurons,
particularly neurons at risk of dying, and for maintaining neural
pathways in a mammal. As described herein, these proteins
("morphogens") can enhance survival of non-mitotic neurons,
stimulate neuronal CAM expression, maintain the phenotypic
expression of differentiated neurons, induce the redifferentiation
of transformed cells of neural origin, and stimulating axonal
growth over breaks in neural processes, particularly large gaps in
distal axons. The proteins also can provide a neuroprotective
effect to alleviate the tissue destructive effects associated with
immunologically-related nerve tissue damage. Finally, the proteins
can be used in a method for monitoring the viability of nerve
tissue in a mammal.
[0075] Provided below are detailed descriptions of suitable
morphogens useful in the methods, compositions and devices of this
invention, as well as methods for their administration and
application, and numerous, nonlimiting examples which 1) illustrate
the suitability of the morphogens and morphogen-stimulating agents
described herein as therapeutic agents for maintaining nerual
pathways in a mammal and enhancing survival of neuronal cells at
risk of dying; and 2) provide assays with which to test candidate
morphogens and morphogen-stimulating agents for their efficacy.
I. Useful Morphogens
[0076] As defined herein a protein is morphogenic if it can induce
the developmental cascade of cellular and molecular events that
culminates in the formation of new, organ-specific tissue and
comprises at least the conserved C-terminal seven cysteine skeleton
of human OP1 or its functional equivalent (see supra).
Specifically, the morphogens generally can elicit all of the
following biological responses in an orderly fashion, when disposed
in a morphogenically permissive environment: stimulating
proliferation of progenitor cells; stimulating the differentiation
of progenitor cells; stimulating the proliferation of
differentiated cells; and supporting the growth and maintenance of
differentiated cells. Each of these biological responses is
elicited upon productive interaction (binding) between the
morphogen and a morphogen-specific receptor displayed by the
stimulated cells. Further, each of the foregoing biological
responses, culminating with the morphogenesis of new or replacement
functional tissue of a desired, specific organ or tissue type, is
elicited in a morphogenically permissive environment. This
permissive environment is free of or contains reduced levels of
signals that inhibit morphogenesis. Further, the permissive
environment contains signals informing uncommitted progenitor cells
of a desired morphogenic pathway. Such informative signals can
induce progenitor cells to commit, for example, to morphogenetic
pathways leading to bone, liver, nerve, periodontal tissue,
gastrointestinal mucosa or other specific tissue types. "Signals"
thus include diffusible molecules (e.g., cytokines, lymphokines,
differentiative factors, chemoattractants, chemotrophic factors,
and other cell products) and substantially indiffusible molecules
(e.g., extracellular matrix components such as collagen, laminin,
fibronectin; cell attached or cell surface molecules, such as cell
adhesion molecules, cadherins and the like).
[0077] Details of how the morphogens useful in the method of this
invention first were identified, as well as a description on how to
make, use and test them for morphogenic activity are disclosed in
international application US92/01968 (WO92/15323), the disclosure
of which is hereby incorporated by reference. US92/01968 describes
in particular a preferred test for morphogenic activity which tests
whether a suspected morphogen can elicit the full developmental
cascade of endochondral bone morphogenesis when sorbed on a matrix
and implanted in a mammal at a nonbony site. It should be
recognized and appreciated that the endochondral bone assay is a
representative example of morphogenesis of a variety of specific
tissues and organs, particularly tissues and organs of mesenchymal
origin. Caplan (1991), 9 J. Orthopedic Res. 641-650. Thus,
analogous systems can be devised through no more than routine
experimentation in light of the guidance provided in US92/01968 to
assess morphogenesis of liver (U.S. Ser. No. 07/946,238, the
teachings of which are incorporated herein by reference) or other
tissues including nerve tissue as described herein. As disclosed in
US92/01968, the morphogens can be purified from naturally-sourced
material or recombinantly produced from procaryotic or eucaryotic
host cells, using the genetic sequences disclosed therein.
Alternatively, novel morphogenic sequences can be identified
following the procedures disclosed therein.
[0078] Particularly useful morphogens include the naturally-sourced
proteins listed in Table II. Other useful morphogens include
biosynthetic constructs such as those disclosed in U.S. Pat. No.
5,011,691, the disclosure of which is incorporated herein by
reference (e.g., COP-1, COP-3, COP-4, COP-5, COP-7, and COP-16).
Still other useful morphogens include OP3, disclosed in U.S. Ser.
No. 07/971,091, dorsalin-1, disclosed in Basler et al. (1993), 73
Cell 687-702 and GDF5, disclosed in Storm et al. (1994), 368 Nature
639-643, the teachings of each of which are incorporated herein by
reference.
[0079] Accordingly, the morphogens useful in the methods and
compositions of this invention also may be described by
morphogenically active proteins having amino acid sequences sharing
70% or, preferably, 80% homology (similarity) with any of the
sequences described above, where "homology" is as defined herein
above.
[0080] Structural features of the morphogens useful in the method
of this invention also can be described by any of the six Generic
Sequences described herein (Generic Sequences 1, 2, 3, 4, 5 and 6).
Generic Sequence 6 provides a preferred definition of the
structural features of a morphogen useful in the present
invention.
[0081] Table II, set forth below, compares the amino acid sequences
of the active regions of native proteins that have been identified
as morphogens, including human OP-1 (hOP-1, Seq. ID Nos. 5 and
16-17), mouse OP-1 (mOP-1, Seq. ID Nos. 6 and 18-19), human and
mouse OP-2 (Seq. ID Nos. 7, 8, and 20-23), CBMP2A (Seq. ID No. 9),
CBMP2B (Seq. ID No. 10), BMP3 (Seq. ID No. 26), DPP (from
Drosophila, Seq. ID No. 11), Vgl, (from Xenopus, Seq. ID No. 12),
Vgr-1 (from mouse, Seq. ID No. 13), GDF-1 (from mouse, Seq. ID Nos.
14, 32 and 33), 60A protein (from Drosophila, Seq. ID Nos. 24 and
25), BMP5 (Seq. ID No. 27) and BMP6 (Seq. ID No. 28). The sequences
are aligned essentially following the method of Needleman et al.
(1970) J. Mol. Biol., 48:443-453, calculated using the Align
Program (DNAstar, Inc.) In the table, three dots indicates that the
amino acid in that position is the same as the amino acid in hOP-1.
Three dashes indicates that no amino acid is present in that
position, and are included for purposes of illustrating homologies.
For example, amino acid residue 60 of CBMP-2A and CBMP-2B is
"missing". Of course, both these amino acid sequences in this
region comprise Asn-Ser (residues 58, 59), with CBMP-2A then
comprising Lys and Ile, whereas CBMP-2B comprises Ser and Ile.
TABLE-US-00007 TABLE II hOP-1 Cys Lys Lys His Glu Leu Tyr Val mOP-1
... ... ... ... ... ... ... ... hOP-2 ... Arg Arg ... ... ... ...
... mOP-2 ... Arg Arg ... ... ... ... ... DPP ... Arg Arg ... Ser
... ... ... Vg1 ... ... Lys Arg His ... ... ... Vgr-1 ... ... ...
... Gly ... ... ... CBMP-2A ... ... Arg ... Pro ... ... ... CBMP-2B
... Arg Arg ... Ser ... ... ... BMP3 ... Ala Arg Arg Tyr ... Lys
... GDF-1 ... Arg Ala Arg Arg ... ... ... 60A ... Gln Met Glu Thr
... ... ... BMP5 ... ... ... ... ... ... ... ... BMP6 ... Arg ...
... ... ... ... ... 1 5 hOP-1 Ser Phe Arg Asp Leu Gly Trp Gln Asp
mOP-1 ... ... ... ... ... ... ... ... ... hOP-2 ... ... Gln ... ...
... ... Leu ... mOP-2 Ser ... ... ... ... ... ... Leu ... DPP Asp
... Ser ... Val ... ... Asp ... Vg1 Glu ... Lys ... Val ... ... ...
Asn Vgr-1 ... ... Gln ... Val ... ... ... ... CBMP-2A Asp ... Ser
... Val ... ... Asn ... CBMP-2B Asp ... Ser ... Val ... ... Asn ...
BMP3 Asp ... Ala ... Ile ... ... Ser Glu GDF-1 ... ... ... Glu Val
... ... His Arg 60A Asp ... Lys ... ... ... ... His ... BMP5 ...
... ... ... ... ... ... ... ... BMP6 ... ... Gln ... ... ... ...
... ... 10 15 hOP-1 Trp Ile Ile Ala Pro Glu Gly Tyr Ala mOP-1 ...
... ... ... ... ... ... ... ... hOP-2 ... Val ... ... ... Gln ...
... Ser mOP-2 ... Val ... ... ... Gln ... ... Ser DPP ... ... Val
... ... Leu ... ... Asp Vg1 ... Val ... ... ... Gln ... ... Met
Vgr-1 ... ... ... ... ... Lys ... ... ... CBMP-2A ... ... Val ...
... Pro ... ... His CBMP-2B ... ... Val ... ... Pro ... ... Gln
BMP3 ... ... ... Ser ... Lys Ser Phe Asp GDF-1 ... Val ... ... ...
Arg ... Phe Leu 60A ... ... ... ... ... ... ... ... Gly BMP5 ...
... ... ... ... ... ... ... ... BMP6 ... ... ... ... ... Lys ...
... ... 20 25 hOP-1 Ala Tyr Tyr Cys Glu Gly Glu Cys Ala mOP-1 ...
... ... ... ... ... ... ... ... hOP-2 ... ... ... ... ... ... ...
... Ser mOP-2 ... ... ... ... ... ... ... ... ... DPP ... ... ...
... His ... Lys ... Pro Vg1 ... Asn ... ... Tyr ... ... ... Pro
Vgr-1 ... Asn ... ... Asp ... ... ... Ser CBMP-2A ... Phe ... ...
His ... Glu ... Pro CBMP-2B ... Phe ... ... His ... Asp ... Pro
BMP3 ... ... ... ... Ser ... Ala ... Gln GDF-1 ... Asn ... ... Gln
... Gln ... ... 60A ... Phe ... ... Ser ... ... ... Asn BMP5 ...
Phe ... ... Asp ... ... ... Ser BMP6 ... Asn ... ... Asp ... ...
... Ser 30 35 hOP-1 Phe Pro Leu Asn Ser Tyr Met Asn Ala mOP-1 ...
... ... ... ... ... ... ... ... hOP-2 ... ... ... Asp ... Cys ...
... ... mOP-2 ... ... ... Asp ... Cys ... ... ... DPP ... ... ...
Ala Asp His Phe ... Ser Vg1 Tyr ... ... Thr Glu Ile Leu ... Gly
Vgr-1 ... ... ... ... Ala His ... ... ... CBMP-2A ... ... ... Ala
Asp His Leu ... Ser CBMP-2B ... ... ... Ala Asp His Leu ... Ser
GDF-1 Leu ... Val Ala Leu Ser Gly Ser** ... BMP3 ... ... Met Pro
Lys Ser Leu Lys Pro 60A ... ... ... ... Ala His ... ... ... BMP5
... ... ... ... Ala His Met ... ... BMP6 ... ... ... ... Ala His
Met ... ... 40 hOP-1 Thr Asn His Ala Ile Val Gln Thr Leu mOP-1 ...
... ... ... ... ... ... ... ... hOP-2 ... ... ... ... ... Leu ...
Ser ... mOP-2 ... ... ... ... ... Leu ... Ser ... DPP ... ... ...
... Val ... ... ... ... Vg1 Ser ... ... ... ... Leu ... ... ...
Vgr-1 ... ... ... ... ... ... ... ... ... CBMP-2A ... ... ... ...
... ... ... ... ... CBMP-2B ... ... ... ... ... ... ... ... ...
BMP3 Ser ... ... ... Thr Ile ... Ser Ile GDF-1 Leu ... ... ... Val
Leu Arg Ala ... 60A ... ... ... ... ... ... ... ... ... BMP5 ...
... ... ... ... ... ... ... ... BMP6 ... ... ... ... ... ... ...
... ... 45 50 hOP-1 Val His Phe Ile Asn Pro Glu Thr Val mOP-1 ...
... ... ... ... ... Asp ... ... hOP-2 ... His Leu Met Lys ... Asn
Ala ... mOP-2 ... His Leu Met Lys ... Asp Val ... DPP ... Asn Asn
Asn ... ... Gly Lys ... Vg1 ... ... Ser ... Glu ... ... Asp Ile
Vgr-1 ... ... Val Met ... ... ... Tyr ... CBMP-2A ... Asn Ser Val
... Ser --- Lys Ile CBMP-2B ... Asn Ser Val ... Ser --- Ser Ile
BMP3 ... Arg Ala** Gly Val Val Pro Gly Ile GDF-1 Met ... Ala Ala
Ala ... Gly Ala Ala 60A ... ... Leu Leu Glu ... Lys Lys ... BMP5
... ... Leu Met Phe ... Asp His ... BMP6 ... ... Leu Met ... ...
... Tyr ... 55 60 hOP-1 Pro Lys Pro Cys Cys Ala Pro Thr Gln mOP-1
... ... ... ... ... ... ... ... ... hOP-2 ... ... Ala ... ... ...
... ... Lys mOP-2 ... ... Ala ... ... ... ... ... Lys DPP ... ...
Ala ... ... Val ... ... ... Vg1 ... Leu ... ... ... Val ... ... Lys
Vgr-1 ... ... ... ... ... ... ... ... Lys CBMP-2A ... ... Ala ...
... Val ... ... Glu CBMP-2B ... ... Ala ... ... Val ... ... Glu
BMP3 ... Glu ... ... ... Val ... Glu Lys GDF-1 Asp Leu ... ... ...
Val ... Ala Arg 60A ... ... ... ... ... ... ... ... Arg BMP5 ...
... ... ... ... ... ... ... Lys BMP6 ... ... ... ... ... ... ...
... Lys 65 70 hOP-1 Leu Asn Ala Ile Ser Val Leu Tyr Phe mOP-1 ...
... ... ... ... ... ... ... ... hOP-2 ... Ser ... Thr ... ... ...
... Tyr mOP-2 ... Ser ... Thr ... ... ... ... Tyr Vg1 Met Ser Pro
... ... Met ... Phe Tyr Vgr-1 Val ... ... ... ... ... ... ... ...
DPP ... Asp Ser Val Ala Met ... ... Leu CBMP-2A ... Ser ... ... ...
Met ... ... Leu CBMP-2B ... Ser ... ... ... Met ... ... Leu BMP3
Met Ser Ser Leu ... Ile ... Phe Tyr GDF-1 ... Ser Pro ... ... ...
... Phe ... 60A ... Gly ... Leu Pro ... ... ... His BMP5 ... ...
... ... ... ... ... ... ... BMP6 ... ... ... ... ... ... ... ...
... 75 80 hOP-1 Asp Asp Ser Ser Asn Val Ile Leu Lys mOP-1 ... ...
... ... ... ... ... ... ... hOP-2 ... Ser ... Asn ... ... ... ...
Arg mOP-2 ... Ser ... Asn ... ... ... ... Arg DPP Asn ... Gln ...
Thr ... Val ... ... Vg1 ... Asn Asn Asp ... ... Val ... Arg Vgr-1
... ... Asn ... ... ... ... ... ... CBMP-2A ... Glu Asn Glu Lys ...
Val ... ... CBMP-2B ... Glu Tyr Asp Lys ... Val ... ... BMP3 ...
Glu Asn Lys ... ... Val ... ... GDF-1 ... Asn ... Asp ... ... Val
... Arg 60A Leu Asn Asp Glu ... ... Asn ... ... BMP5 ... ... ...
... ... ... ... ... ... BMP6 ... ... Asn ... ... ... ... ... ... 85
hOP-1 Lys Tyr Arg Asn Met Val Val Arg mOP-1 ... ... ... ... ... ...
... ... hOP-2 ... His ... ... ... ... ... Lys mOP-2 ... His ... ...
... ... ... Lys DPP Asn ... Gln Glu ... Thr ... Val Vg1 His ... Glu
... ... Ala ... Asp Vgr-1 ... ... ... ... ... ... ... ... CBMP-2A
Asn ... Gln Asp ... ... ... Glu CBMP-2B Asn ... Gln Glu ... ... ...
Glu BMP3 Val ... Pro ... ... Thr ... Glu GDF-1 Gln ... Glu Asp ...
... ... Asp 60A ... ... ... ... ... Ile ... Lys BMP5 ... ... ...
... ... ... ... ... BMP6 ... ... ... Trp ... ... ... ... 90 95
hOP-1 Ala Cys Gly Cys His mOP-1 ... ... ... ... ... hOP-2 ... ...
... ... ... mOP-2 ... ... ... ... ... DPP Gly ... ... ... Arg Vg1
Glu ... ... ... Arg Vgr-1 ... ... ... ... ... CBMP-2A Gly ... ...
... Arg CBMP-2B Gly ... ... ... Arg BMP3 Ser ... Ala ... Arg GDF-1
Glu ... ... ... Arg 60A Ser ... ... ... ... BMP5 Ser ... ... ...
... BMP6 ... ... ... ... ... 100 **Between residues 56 and 57 of
BMP3 is a Val residue; between residues 43 and 44 of GDF-1 lies the
amino acid sequence Gly-Gly-Pro-Pro.
[0082] As is apparent from the foregoing amino acid sequence
comparisons, significant amino acid changes can be made within the
generic sequences while retaining the morphogenic activity. For
example, while the GDF-1 protein sequence depicted in Table II
shares only about 50% amino acid identity with the hOP1 sequence
described therein, the GDF-1 sequence shares greater than 70% amino
acid sequence homology (or "similarity") with the hOP1 sequence,
where "homology" or "similarity" includes allowed conservative
amino acid changes within the sequence as defined by Dayoff, et
al., Atlas of Protein Sequence and Structure vol. 5, supp. 3, pp.
345-362, (M. O. Dayoff, ed., Nat'l BioMed. Res. Fd'n, Washington
D.C. 1979.)
[0083] The currently most preferred protein sequences useful as
morphogens in this invention include those having greater than 60%
identity, preferably greater than 65% identity, with the amino acid
sequence defining the conserved seven cysteine skeleton of hOP1
(e.g., residues 38-139 of Seq. ID No. 5). These most preferred
sequences include both allelic and species variants of the OP-1 and
OP-2 proteins, including the Drosophila 60A protein. Accordingly,
in still another preferred aspect, the invention includes
morphogens comprising species of polypeptide chains having the
generic amino acid sequence referred to herein as "OPX", which
defines the seven cysteine skeleton and accommodates the identities
between the various identified mouse and human OP1 and OP2
proteins. OPX is presented in Seq. ID No. 29. As described therein,
each Xaa at a given position independently is selected from the
residues occurring at the corresponding position in the C-terminal
sequence of mouse or human OP1 or OP2 (see Seq. ID Nos. 5-8 and/or
Seq. ID Nos. 16-23).
II. Formulations and Methods for Administering Therapeutic
Agents
[0084] The morphogens can be administered to an individual by any
suitable means, preferably directly (e.g., locally, as by injection
to a nerve tissue locus) or systemically (e.g., parenterally or
orally). Where the morphogen is to be administered parenterally,
such as by intravenous, subcutaneous, intramuscular, intraorbital,
ophthalmic, intraventricular, intracranial, intracapsular,
intraspinal, intracisternal, intraperitoneal, buccal, rectal,
vaginal, intranasal or by aerosol administration, the morphogen
preferably comprises part of an aqueous solution. The solution is
physiologically acceptable so that in addition to delivery of the
desired morphogen to the patient, the solution does not otherwise
adversely affect the patient's electrolyte and volume balance. The
aqueous medium for the morphogen thus may comprise normal
physiologic saline (9.85% NaCl, 0.15M), pH 7-7.4. The aqueous
solution containing the morphogen can be made, for example, by
dissolving the protein in 50% ethanol containing acetonitrile in
0.1% trifluoroacetic acid (TFA) or 0.1% HCl, or equivalent
solvents. One volume of the resultant solution then is added, for
example, to ten volumes of phosphate buffered saline (PBS), which
further may include 0.1-0.2% human serum albumin (HSA). The
resultant solution preferably is vortexed extensively. If desired,
a given morphogen can be made more soluble by association with a
suitable molecule. For example, association of the mature dimer
with the pro domain of the morphogen increases solubility of the
protein significantly (see Section II.1, below). In fact, the
endogenous protein is thought to be transported in this form.
Another molecule effective for enhancing solubility and
particularly useful for oral administrations, is casein. For
example, addition of 0.2% casein increases solubility of the mature
active form of OP-1 by 80%. Other components found in milk and/or
various serum proteins also may be useful.
[0085] Useful solutions for parenteral administration can be
prepared by a variety of methods well known in the pharmaceutical
art, described, for example, in Remington's Pharmaceutical Sciences
(Gennaro, A., ed.), Mack Pub., 1990. Formulations can include, for
example, polyalkylene glycols such as polyethylene glycol, oils of
vegetable origin, hydrogenated naphthalenes, and the like.
Formulations for direct administration, in particular, can include
glycerol and other compositions of high viscosity. Biocompatible,
preferably bioresorbable, polymers, including, for example,
hyaluronic acid, collagen, polybutyrate, tricalcium phosphate,
lactide and lactide/glycolide copolymers, can be useful excipients
to control the release of the morphogen in vivo. Other potentially
useful parenteral delivery systems for these morphogens include
ethylene-vinyl acetate copolymer particles, osmotic pumps,
implantable infusion systems, and liposomes. Formulations for
inhalation administration contain as excipients, for example,
lactose, or can be aqueous solutions containing, for example,
polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or
oily solutions for administration in the form of nasal drops, or as
a gel to be applied intranasally. Formulations for parenteral
administration can also include glycocholate for buccal
administration, methoxysalicylate for rectal administration, or
cutric acid for vaginal administration.
[0086] Alternatively, the morphogens described herein can be
administered orally. Oral administration of proteins as
therapeutics generally is not practiced as most proteins are
readily degraded by digestive enzymes and acids in the mammalian
digestive system before they can be absorbed into the bloodstream.
However, the morphogens described herein typically are acid stable
and protease-resistant (see, for example, U.S. Pat. No. 4,968,590.)
In addition, at least one morphogen, OP-1, has been identified in
mammary gland extract, colostrum and 57-day milk. Moreover, the
OP-1 purified from mammary gland extract is morphogenically active.
Specifically, this protein induces endochondral bone formation in
mammals when implanted subcutaneously in association with a
suitable matrix material, using a standard in vivo bone assay, such
as is disclosed in U.S. Pat. No. 4,968,590. Moreover, the morphogen
also is detected in the bloodstream (see Example 9, below).
Finally, soluble form morphogen, e.g., mature morphogen associated
with the pro domain, is capable of maintaining neural pathways in a
mammal (See Examples 4 and 6 below). These findings indicate that
oral and parenteral administration are viable means for
administering morphogens to an individual. In addition, while the
mature forms of certain morphogens described herein typically are
sparingly soluble, the morphogen form found in milk (and mammary
gland extract and colostrum) is readily soluble, probably by
association of the mature, morphogenically active form with part or
all of the pro domain of the intact sequence and/or by association
with one or more milk components. Accordingly, the compounds
provided herein also can be associated with molecules capable of
enhancing their solubility in vitro or in vivo.
[0087] The compounds provided herein also can be associated with
molecules capable of targeting the morphogen or
morphogen-stimulating agent to nerve tissue. For example, an
antibody, antibody fragment, or other binding protein that
interacts specifically with a surface molecule on nerve tissue
cells, including neuronal or glial cells, can be used. Useful
targeting molecules can be designed, for example, using the single
chain binding site technology disclosed, for example, in U.S. Pat.
No. 5,091,513.
[0088] As described above, the morphogens provided herein share
significant sequence homology in the C-terminal active domains. By
contrast, the sequences typically diverge significantly in the
sequences which define the pro domain. Accordingly, the pro domain
is thought to be morphogen-specific. As described above, it is also
known that the various morphogens identified to date are
differentially expressed in the different tissues. Accordingly,
without being limited to any given theory, it is likely that, under
natural conditions in the body, selected morphogens typically act
on a given tissue. Accordingly, part or all of the pro domains
which have been identified associated with the active form of the
morphogen in solution, can serve as targeting molecules for the
morphogens described herein. For example, the pro domains can
interact specifically with one or more molecules at the target
tissue to direct the morphogen associated with the pro domain to
that tissue. Accordingly, another useful targeting molecule for
targeting morphogen to nerve tissue is part or all of a morphogen
pro domain, particularly part or all of the pro domains of OP-1 or
GDF-1, both of which proteins are found naturally associated with
nerve tissue.
[0089] Finally, the morphogens or morphogen-stimulating agents
provided herein can be administered alone or in combination with
other molecules known to be beneficial in maintaining neural
pathways, including anti-inflammatory agents and nerve trophic
(growth) factors.
[0090] Nerve trophic factors as defined herein refer to proteins
able to stimulate survival and growth of nerve cells. Most of the
characterized actions of nerve trophic actors relate to
developmental events and suggest that the temporal and local
regulation of expression of these proteins plays a role during
maturation of the nervous system. Nerve trophic factors are also
important in the function of the adult nervous system for the
maintenance of structural integrity and regulation of plasticity.
Such processes are altered in neurodegenerative diseases and
neurodegenerative events following acute injury to the nervous
system. This has prompted speculation that nerve trophic factors
are involved in the structural alterations which occur in response
to injury and disease.
[0091] Nerve trophic factors are found among several protein
families, including neurotrophins, fibroblast growth factors, the
epidermal growth factor protein family, and lymphokines to name a
few. Nerve growth factor (NGF) is the best characterized member of
the nerve trophic factor protein families. NGF belongs to the
protein family called neurotrophins, the other known members of
which are brain-derived neurotrophic factor (BDNF), neurotrophin-3
(NT-3), neurotrophin-4 (NT-4), and neurotrophin-5 (NT-S).
Individual neurotrophins are highly conserved among mammalian
species and share at least about 50%, preferably at least 60%,
amino acid sequence homology with other members of this group. In
particular, nerve trophic factors that are suitable for use herein
have amino acid sequences comprising a sequence sharing at least
60% homology with each of the sequences of human NGF, human BDNF,
human NT-3, human NT-4, and human NT-5. Alternatively, nerve
trophic factors can include mammalian proteins comprising amino
acid sequences having six cysteine residues at relative positions
that are strictly conserved and in common with the sequences of
nerve trophic factors from rats, humans, chickens, and frogs.
[0092] The compounds provided herein can be formulated into
pharmaceutical compositions by admixture with pharmaceutically
acceptable nontoxic excipients and carriers. As noted above, such
compositions can be prepared for parenteral administration,
particularly in the form of liquid solutions or suspensions; for
oral administration, particularly in the form of tablets or
capsules; or intranasally, particularly in the form of powders,
nasal drops, or aerosols.
[0093] The compositions can be formulated for parenteral or oral
administration to humans or other mammals in therapeutically
effective amounts, e.g., amounts which provide appropriate
concentrations for a time sufficient to eliminate or reduce the
patient's pathological condition, to provide therapy for the
neurological diseases and disorders described above, and amounts
effective to enhance neural cell survival an/or to protect neurons
and neural pathways in anticipation of injury to nerve tissue.
[0094] As will be appreciated by those skilled in the art, the
concentration of the compounds described in a therapeutic
composition will vary depending upon a number of factors, including
the dosage of the drug to be administered, the chemical
characteristics (e.g., hydrophobicity) of the compounds employed,
and the route of administration. The preferred dosage of drug to be
administered also is likely to depend on such variables as the type
and extent of progression of the neurological disease, the overall
health status of the particular patient, the relative biological
efficacy of the compound selected, the formulation of the compound,
excipients, and its route of administration. In general terms, the
compounds of this invention can be provided in an aqueous
physiological buffer solution containing about 0.1 to 10% w/v
compound for parenteral administration. Typical dose ranges are
from about 10 ng/kg to about 1 g/kg of body weight per day; a
preferred dose range is from about 0.1 .mu.g/kg to 100 mg/kg of
body weight per day. Optimally, the morphogen dosage given in all
cases is between 2-20 .mu.g of protein per kilogram weight of the
patient per day. No obvious OP-1 induced pathological lesions are
induced when mature morphogen (e.g., OP-1, 20 .mu.g) is
administered daily to normal growing rats for 21 consecutive days.
Moreover, 10 .mu.g systemic injections of morphogen (e.g., OP-1)
injected daily for 10 days into normal newborn mice does not
produce any gross abnormalties.
[0095] Since the ability of proteins and protein fragments to
penetrate the blood-brain barrier can be related to their size,
lipophilicity or their net ionic charge, suitable modifications of
the morphogens can be formulated (e.g., by substituting
pentafluorophenylalanine for phenylalanine, or by conjugation to a
cationized protein such as albumin) to increase their
transportability across the barrier, using standard methodologies
known in the art. See, for example, Kastin et al., Pharmac.
Biochem. Behav. (1979) 11:713-716; Rapoport et al., Science (1980)
207:84-86; Pardridge et al., (1987) Biochem. Biophys. Res. Commun.
146:307-313; Riekkinen et al., (1987) Peptides 8:261-265. The
efficacy of these functional analogs can be assessed for example,
by evaluating the ability of these compounds to induce
redifferentiation of transformed cells, or enhance survival of
neurons at risk of dying, as described in the Examples provided
herein.
[0096] In administering morphogens systemically in the methods of
the present invention, preferably a large volume loading dose is
used at the start of the treatment. The treatment then is continued
with a maintenance dose. Further administration then can be
determined by monitoring at intervals the levels of the morphogen
in the blood.
[0097] Where injury to neurons of a neural pathway is induced
deliberately as part of, for example, a surgical procedure, the
morphogen preferably is provided just prior to, or concomitant with
induction of the trauma. Preferably, the morphogen is administered
prophylactically in a surgical setting. Optimally, the morphogen
dosage given in all cases is between 2-20 .mu.g of protein per
kilogram weight of the patient.
[0098] Alternatively, an effective amount of an agent that
stimulates endogenous morphogen levels can be administered by any
of the routes described above. For example, an agent effective for
stimulating morphogen production and/or secretion from nerve tissue
cells can be provided to a mammal, e.g., by direct administration
of the morphogen stimulating agent to glial cells associated with
the nerve tissue to be treated. A method for identifying and
testing agents capable of modulating the levels of endogenous
morphogens in a given tissue is described generally herein in
Example 13, and in detail in internatinal application US92/07359
(WO93/015172), the disclosure of which is f incorporated herein by
reference. Briefly, candidate compounds can be identified and
tested by incubating the compound in vitro with a test tissue or
cells thereof, for a time sufficient to allow the compound to
affect the production, i.e., the expression and/or secretion, of a
morphogen that said cells are competent to produce. Here, suitable
tissue or cultured cells of a tissue preferably would comprise
neurons and/or glial cells. For example, suitable tissue for
testing can includes cultured cells isolated from the substantia
nigra, adendema glial cells, and the like.
[0099] A currently preferred detection means for evaluating the
level of the morphogen in culture upon exposure to the candidate
compound comprises an immunoassay utilizing an antibody or other
suitable binding protein capable of reacting specifically with a
morphogen and being detected as part of a complex with the
morphogen. Immunoassays can be performed using standard techniques
known in the art and antibodies raised against a morphogen and
specific for that morphogen. As described herein, morphogen
stimulating agents can be isolated from natural-sourced material or
they can be recombinantly produced or chemically synthesized by
those skilled in the art of medicinal chemistry. Agents effective
for stimulating endogenous morphogens then can formulated into
pharmaceutical preparations and administered as described
herein.
[0100] Where the morphogen is to be provided to a site to stimulate
axon regeneration, the morphogen preferably is provided to the site
in association with a biocompatible, preferably bioresorbable
carrier suitable for maintaining a protein at a site in vivo, and
through which a neural process can regenerate. A currently
preferred carrier also comprises sufficient structure to assist
direction of axonal growth. Currently preferred carriers include
structural molecules such as collagen, hyaluronic acid or laminin,
and/or synthetic polymers or copolymers of, for example, polylactic
acid, polyglycolic acid or polybutyric acid. Currently most
preferred are carriers comprising tissue extracellular matrix.
These can be obtained commercially. In addition, a brain
tissue-derived extracellular matrix can be prepared as described in
international application US92/01968 (WO92/15323), incorporated
hereinabove by reference, and/or by other means known in the
art.
[0101] The currently preferred means for repairing breaks in neural
pathways, particularly pathways of the peripheral nervous system,
include providing the morphogen to the site as part of a device
that includes a biocompatible membrane or casing of a dimension
sufficient to span the break and having openings adapted to receive
severed nerve ends. The morphogen is disposed within the casing,
preferably dispersed throughout a suitable carrier, and is
accessible to the severed nerve ends. Alternatively, the morphogen
can be adsorbed onto the interior surface of the casing, or
otherwise associated therewith. In addition, currently preferred
casings have an impermeable exterior surface. The casing acts as a
nerve guidance channel, aiding in directing axonal growth. In
addition, the casing also protects the damaged nerve from
immunologically-related agents which can assist in scar tissue
formation. Suitable channel or casing materials include silicone or
bioresorbable materials such as collagen, hyaluronic acid, laminin,
polylactic acid, polyglycolic acid, polybutyric acid and the like.
Additionally, although the nerve guidance channels described herein
generally are tubular in shape, it should be evident to those
skilled in the art that various alternative shapes can be employed.
The lumen of the guidance channels can, for example, be oval or
even square in cross section. Moreover the guidance channels can be
constructed of two or more parts which can be clamped together to
secure the nerve stumps. Nerve endings can be secured to the nerve
guidance channels by means of sutures, biocompatible adhesives such
as fibrin glue, or other means known in the medical art.
II.1 Soluble Morphogen Complexes
[0102] A currently preferred form of the morphogen useful in
therapeutic formulations, having improved solubility in aqueous
solutions and consisting essentially of amino acids, is a dimeric
morphogenic protein comprising at least the 100 amino acid peptide
sequence having the pattern of seven or more cysteine residues
characteristic of the morphogen family complexed with a peptide
comprising part or all of a pro region of a member of the morphogen
family, or an allelic, species or other sequence variant thereof.
Preferably, the dimeric morphogenic protein is complexed with two
peptides. Also, the dimeric morphogenic protein preferably is
noncovalently complexed with the pro region peptide or peptides.
The pro region peptides also preferably comprise at least the
N-terminal eighteen amino acids that define a given morphogen pro
region. In a most preferred embodiment, peptides defining
substantially the full length pro region are used.
[0103] Other soluble forms of morphogens include dimers of the
uncleaved pro forms of these proteins, as well as "hemi-dimers"
wherein one subunit of the dimer is an uncleaved pro form of the
protein, and the other subunit comprises the mature form of the
protein, including truncated forms thereof, preferably
noncovalently associated with a cleaved pro domain peptide.
[0104] As described above, useful pro domains include the full
length pro regions, as well as various truncated forms hereof,
particularly truncated forms cleaved at proteolytic Arg-Xaa-Xaa-Arg
cleavage sites. For example, in OP-1, possible pro sequences
include sequences defined by residues 30-292 (full length form);
48-292; and 158-292. Soluble OP-1 complex stability is enhanced
when the pro region comprises the full length form rather than a
truncated form, such as the 48-292 truncated form, in that residues
30-47 show sequence homology to the N-terminal portions of other
morphogens, and are believed to have particular utility in
enhancing complex stability for all morphogens. Accordingly,
currently preferred pro sequences are those encoding the full
length form of the pro region for a given morphogen. Other pro
sequences contemplated to have utility include biosynthetic pro
sequences, particularly those that incorporate a sequence derived
from the N-terminal portion of one or more morphogen pro
sequences.
[0105] As will be appreciated by those having ordinary skill in the
art, useful sequences encoding the pro region can be obtained from
genetic sequences encoding known morphogens. Alternatively,
chimeric pro regions can be constructed from the sequences of one
or more known morphogens. Still another option is to create a
synthetic sequence variant of one or more known pro region
sequences.
[0106] In another preferred aspect, useful pro region peptides
include polypeptide chains comprising an amino acid sequence
encoded by a nucleic acid that hybridizes under stringent
conditions with a DNA or RNA sequence encoding at least the
N-terminal eighteen amino acids of the pro region sequence for OP1
or OP2, e.g., nucleotides 136-192 and 152-211 of Seq. ID No. 16 and
20, respectively.
[0107] A. Isolation of Soluble Morphogen Complex from Conditioned
Media or Body Fluid
[0108] Morphogens are expressed from mammalian cells as soluble
complexes. Typically, however the complex is most often
disassociated during purification, generally by exposure to
denaturants often added to the purification solutions, such as
detergents, alcohols, organic solvents, chaotropic agents and
compounds added to reduce the pH of the solution. Provided below is
a currently preferred protocol for purifying the soluble proteins
from conditioned media (or, optionally, a body fluid such as serum,
cerebro-spinal or peritoneal fluid), under non-denaturing
conditions. The method is rapid, reproducible and yields isolated
soluble morphogen complexes in substantially pure form.
[0109] Soluble morphogen complexes can be isolated from conditioned
media using a simple, three step chromatographic protocol performed
in the absence of denaturants. The protocol involves running the
media (or body fluid) over an affinity column, followed by ion
exchange and gel filtration chromatographies. The affinity column
described below is a Zn-IMAC column. The present protocol has
general applicability to the purification of a variety of
morphogens, all of which are anticipated to be isolatable using
only minor modifications of the protocol described below. An
alternative protocol also envisioned to have utility an
immunoaffinity column, created using standard procedures and, for
example, using antibody specific for a given morphogen pro domain
(complexed, for example, to a protein A-conjugated Sepharose
column.) Protocols for developing immunoaffinity columns are well
described in the art, (see, for example, Guide to Protein
Purification, M. Deutscher, ed., Academic Press, San Diego, 1990,
particularly sections VII and XI.)
[0110] In this experiment human OP-1 was expressed in mammalian CHO
(chinese hamster ovary) cells as described in the art (see, for
example, international application US90/05903 (WO91/05802).) The
CHO cell conditioned media containing 0.5% FBS was initially
purified using Immobilized Metal-Ion Affinity Chromatography
(IMAC). The soluble OP-1 complex from conditioned media binds very
selectively to the Zn-IMAC resin and a high concentration of
imidazole (50 mM imidazole, pH 8.0) is required for the effective
elution of the bound complex. The Zn-IMAC step separates the
soluble OP-1 from the bulk of the contaminating serum proteins that
elute in the flow through and 35 mM imidazole wash fractions. The
Zn-IMAC purified soluble OP-1 is next applied to an S-Sepharose
cation-exchange column equilibrated in 20 mM NaPO.sub.4 (pH 7.0)
with 50 mM NaCl. This S-Sepharose step serves to further purify and
concentrate the soluble OP-1 complex in preparation for the
following gel filtration step. The protein was applied to a
Sephacryl S-200HR column equilibrated in TBS. Using substantially
the same protocol, soluble morphogens also can be isolated from one
or more body fluids, including serum, cerebro-spinal fluid or
peritoneal fluid.
[0111] IMAC was performed using Chelating-Sepharose (Pharmacia)
that had been charged with three column volumes of 0.2 M
ZnSO.sub.4. The conditioned media was titrated to pH 7.0 and
applied directly to the ZN-IMAC resin equilibrated in 20 mM HEPES
(pH 7.0) with 500 mM NaCl. The Zn-IMAC resin was loaded with 80 mL
of starting conditioned media per mL of resin. After loading, the
column was washed with equilibration buffer and most of the
contaminating proteins were eluted with 35 mM imidazole (pH 7.0) in
equilibration buffer. The soluble OP-1 complex then is eluted with
50 mM imidazole (pH 8.0) in 20 mM HEPES and 500 mM NaCl.
[0112] The 50 mM imidazole eluate containing the soluble OP-1
complex was diluted with nine volumes of 20 mM NaPO.sub.4 (pH 7.0)
and applied to an S-Sepharose (Pharmacia) column equilibrated in 20
mM NaPO.sub.4 (pH 7.0) with 50 mM NaCl. The S-Sepharose resin was
loaded with an equivalent of 800 mL of starting conditioned media
per mL of resin. After loading the S-Sepharose column was washed
with equilibration buffer and eluted with 100 mM NaCl followed by
300 mM and 500 mM NaCl in 20 mM NaPO.sub.4 (pH 7.0). The 300 mM
NaCl pool was further purified using gel filtration chromatography.
Fifty mls of the 300 mm NaCl eluate was applied to a 5.0.times.90
cm Sephacryl S-200HR (Pharmacia) equilibrated in Tris buffered
saline (TBS), 50 mM Tris, 150 mM NaCl (pH 7.4). The column was
eluted at a flow rate of 5 mL/minute collecting 10 mL fractions.
The apparent molecular of the soluble OP-1 was determined by
comparison to protein molecular weight standards (alcohol
dehydrogenase (ADH, 150 kDa), bovine serum albumin (BSA, 68 kDa),
carbonic anhydrase (CA, 30 kDa) and cytochrome C (cyt C, 12.5 kDa).
The purity of the S-200 column fractions was determined by
separation on standard 15% polyacrylamide SDS gels stained with
coomassie blue. The identity of the mature OP-1 and the pro-domain
was determined by N-terminal sequence analysis after separation of
the mature OP-1 from the pro-domain using standard reverse phase
C18 HPLC.
[0113] The soluble OP-1 complex elutes with an apparent molecular
weight of 110 kDa. This agrees well with the predicted composition
of the soluble OP-1 complex with one mature OP-1 dimer (35-36 kDa)
associated with two pro-domains (39 kDa each). Purity of the final
complex can be verified by running the appropriate fraction in a
reduced 15% polyacrylamide gel.
[0114] The complex components can be verified by running the
complex-containing fraction from the S-200 or S-200HR columns over
a reverse phase C18 HPLC column and eluting in an acetonitrile
gradient (in 0.1% TFA), using standard procedures. The complex is
dissociated by this step, and the pro domain and mature species
elute as separate species. These separate species then can be
subjected to N-terminal sequencing using standard procedures (see,
for example, Guide to Protein Purification, M. Deutscher, ed.,
Academic Press, San Diego, 1990, particularly pp. 602-613), and the
identity of the isolated 36 kD, 39 kDa proteins confirmed as mature
morphogen and isolated, cleaved pro domain, respectively.
N-terminal sequencing of the isolated pro domain from mammalian
cell produced OP-1 revealed 2 forms of the pro region, the intact
form (beginning at residue 30 of Seq. ID No. 16) and a truncated
form, (beginning at residue 48 of Seq. ID No. 16.) N-terminal
sequencing of the polypeptide subunit of the isolated mature
species reveals a range of N-termini for the mature sequence,
beginning at residues 293, 300, 313, 315, 316, and 318, of Seq. ID
No. 16, all of which are active as demonstrated by the standard
bone induction assay.
[0115] B. In Vitro Soluble Morphogen Complex Formation
[0116] As an alternative to purifying soluble complexes from
culture media or a body fluid, soluble complexes can be formulated
from purified pro domains and mature dimeric species. Successful
complex formation apparently requires association of the components
under denaturing conditions sufficient to relax the folded
structure of these molecules, without affecting disulfide bonds.
Preferably, the denaturing conditions mimic the environment of an
intracellular vesicle sufficiently such that the cleaved pro domain
has an opportunity to associate with the mature dimeric species
under relaxed folding conditions. The concentration of denaturant
in the solution then is decreased in a controlled, preferably
step-wise manner, so as to allow proper refolding of the dimer and
pro regions while maintaining the association of the pro domain
with the dimer. Useful denaturants include 4-6M urea or guanidine
hydrochloride (GuHCl), in buffered solutions of pH 4-10, preferably
pH 6-8. The soluble complex then is formed by controlled dialysis
or dilution into a solution having a final denaturant concentration
of less than 0.1-2M urea or GuHCl, preferably 1-2 M urea of GuHCl,
which then preferably can be diluted into a physiological buffer.
Protein purification/renaturing procedures and considerations are
well described in the art, and details for developing a suitable
renaturing protocol readily can be determined by one having
ordinary skill in the art. One useful text one the subject is Guide
to Protein Purification, M. Deutscher, ed., Academic Press, San
Diego, 1990, particularly section V. Complex formation also can be
aided by addition of one or more chaperone proteins.
[0117] C. Stability of Soluble Morphogen Complexes
[0118] The stability of the highly purified soluble morphogen
complex in a physiological buffer, e.g., tris-buffered saline (TBS)
and phosphate-buffered saline (PBS), can be enhanced by any of a
number of means. Currently preferred is by means of a pro region
that comprises at least the first 18 amino acids of the pro
sequence (e.g., residues 30-47 of Seq. ID NO. 16 for OP-1), and
preferably is the full length pro region. Residues 30-47 show
sequence homology to the N-terminal portion of other morphogens and
are believed to have particular utility in enhancing complex
stability for all morphogens. Other useful means for enhancing the
stability of soluble morphogen complexes include three classes of
additives. These additives include basic amino acids (e.g.,
L-arginine, lysine and betaine); nonionic detergents (e.g., Tween
80 or NonIdet P-120); and carrier proteins (e.g., serum albumin and
casein). Useful concentrations of these additives include 1-100 mM,
preferably 10-70 mM, including 50 mM, basic amino acid, 0.01-1.0%,
preferably 0.05-0.2%, including 0.1% (v/v) nonionic detergent, and
0.01-1.0%, preferably 0.05-0.2%, including 0.1% (w/v) carrier
protein.
III. EXAMPLES
Example 1
Identification of Morphogen-Expressing Tissue
[0119] Determining the tissue distribution of morphogens can be
used to identify different morphogens expressed in a given tissue,
as well as to identify new, related morphogens. Tissue distribution
also can be used to identify useful morphogen-producing tissue for
use in screening and identifying candidate morphogen-stimulating
agents. The morphogens (or their mRNA transcripts) readily are
identified in different tissues using standard methodologies and
minor modifications thereof in tissues where expression can be low.
For example, protein distribution can be determined using standard
Western blot analysis or immunofluorescent techniques, and
antibodies specific to the morphogen or morphogens of interest.
Similarly, the distribution of morphogen transcripts can be
determined using standard Northern hybridization or in situ
hybridization protocols and transcript-specific probes.
[0120] Any probe capable of hybridizing specifically to a
transcript, and distinguishing the transcript of interest from
other, related transcripts can be used. Because the morphogens
described herein share such high sequence homology in their active,
C-terminal domains, the tissue distribution of a specific morphogen
transcript can best be determined using a probe specific for the
pro region of the protein and/or the N-terminal region of the
mature protein. Another useful sequence is the 3' non-coding region
flanking and immediately following the stop codon. These portions
of the sequence vary substantially among the morphogens of this
invention, and accordingly, are specific for each protein. For
example, a particularly useful Vgr-1-specific probe sequence is the
PvuII-SacI fragment, a 265 bp fragment encoding both a portion of
the pro region and the N-terminus of the mature sequence (see Lyons
et al. (1989) PNAS 86:4554-4558 for a description of the cDNA
sequence). Similarly, particularly useful mOP-1-specific probe
sequences are the BstX1-BglI fragment, a 0.68 Kb sequence that
covers approximately two-thirds of the mOP-1 pro region; a
StuI-StuI fragment, a 0.2 Kb sequence immediately upstream of the
7-cysteine domain; and the Ear1-Pst1 fragment, an 0.3 Kb fragment
containing a portion of the 3'untranslated sequence (See Seq. ID
No. 18, where the pro region is defined essentially by residues
30-291.) Similar approaches can be used, for example, with hOP-1
(Seq. ID No. 16) or human or mouse OP-2 (Seq. ID Nos. 20 and
22.)
[0121] Using these morphogen-specific probes, which can be
synthetically engineered or obtained from cloned sequences,
morphogen transcripts can be identified in mammalian tissue, using
standard methodologies well known to those having ordinary skill in
the art. Briefly, total RNA is prepared from various adult murine
tissues (e.g., liver, kidney, testis, heart, brain, thymus and
stomach) by a standard methodology such as by the method of
Chomczyaski et al. ((1987) Anal. Biochem 162:156-159) and described
below. Poly (A)+ RNA is prepared by using oligo (dT)-cellulose
chromatography (e.g., Type 7, from Pharmacia LKB Biotechnology,
Inc.). Poly (A)+ RNA (generally 15 .mu.g) from each tissue is
fractionated on a 1% agarose/formaldehyde gel and transferred onto
a Nytran membrane (Schleicher & Schuell). Following the
transfer, the membrane is baked at 80.degree. C. and the RNA is
cross-linked under UV light (generally 30 seconds at 1
mW/cm.sup.2). Prior to hybridization, the appropriate probe is
denatured by heating. The hybridization is carried out in a lucite
cylinder rotating in a roller bottle apparatus at approximately 1
rev/min for approximately 15 hours at 37.degree. C. using a
hybridization mix of 40% formamide, 5.times. Denhardts,
5.times.SSPE, and 0.1% SDS. Following hybridization, the
non-specific counts are washed off the filters in 0.1.times.SSPE,
0.1% SDS at 50.degree. C.
[0122] Examples demonstrating the tissue distribution of various
morphogens, including Vgr-1, OP-1, BMP2, BMP3, BMP4, BMP5, GDF-1,
and OP-2 in developing and adult tissue are disclosed in
international application US92/01968 (WO92/15323), and in Ozkaynak,
et al., (1991) Biochem. Biophys. Res. Commn. 179:116-123, and
Ozkaynak, et al. (1992) J. Biol. Chem. 267: 25220-25227. Using the
general probing methodology described herein, northern blot
hybridizations using probes specific for these morphogens to probe
brain, spleen, lung, heart, liver and kidney tissue indicate that
kidney-related tissue appears to be the primary expression source
for OP-1, with brain, heart and lung tissues being secondary
sources. Lung tissue appears to be the primary tissue expression
source for Vgr-1, BMP5, BMP4 and BMP3. Lower levels of Vgr-1 also
are seen in kidney and heart tissue, while the liver appears to be
a secondary expression source for BMP5, and the spleen appears to
be a secondary expression source for BMP4. GDF-1 appears to be
expressed primarily in brain tissue. To date, OP-2 appears to be
expressed primarily in early embryonic tissue. Specifically,
northern blots of murine embryos and 6-day post-natal animals shows
abundant OP2 expression in 8-day embryos. Expression is reduced
significantly in 17-day embryos and is not detected in post-natal
animals.
Example 2
Morphogen Localization in the Nervous System
[0123] Morphogens have been identified in developing and adult rat
brain and spinal cord tissue, as determined both by northern blot
hybridization of morphogen-specific probes to mRNA extracts from
developing and adult nerve tissue (see Example 1, above) and by
immunolocalization studies. For example, northern blot analysis of
developing rat tissue has identified significant OP-1 mRNA
transcript expression in the CNS international application
US92/01968 (WO92/15323), and Ozkaynak et al. (1991) Biochem.
Biophys. Res. Comm., 179:11623 and Ozkaynak, et al. (1992) J. Biol.
Chem. 267:25220-25227. GDF-1 mRNA appears to be expressed primarily
in developing and adult nerve tissue, specifically in the brain,
including the cerebellum and brain stem, spinal cord and peripheral
nerves (Lee, S. (1991) PNAS 88: 4250-4254). BMP2B (also referred in
the art as BMP4) and Vgr-1 transcripts also have been reported to
be expressed in nerve tissue (Jones et al. (1991) Development
111:531-542), although the nerve tissue does not appear to be the
primary expression tissue for these genes (Ozkaynak, et al., (1992)
J. Biol. Chem. 267:25220-25227. Specifically, CBMP2 transcripts are
reported in the region of the diencephalon associated with
pituitary development, and Vgr-1 transcripts are reported in the
anteroposterior axis of the CNS, including in the roof plate of the
developing neural tube, as well as in the cells immediately
adjacent the floor plate of the developing neural tube. In older
rats, Vgr-1 transcripts are reported in developing hippocampus
tissue. In addition, the genes encoding OP-1 and BMP2 originally
were identified by probing human hippocampus cDNA libraries.
[0124] Immunolocalization studies, performed using standard
methodologies known in the art and disclosed in international
application US92/01968 (WO92/15323), the disclosure of which is
incorporated herein, localized OP-1 expression to particular areas
of developing and adult rat brain and spinal cord tissue.
Specifically, OP-1 protein expression was assessed in adult (2-3
months old) and five or six-day old mouse embryonic nerve tissue,
using standard morphogen-specific antisera, specifically, rabbit
anti-OP1 antisera, made using standard antibody protocols known in
the art and preferably purified on an OP-1 affinity column. The
antibody itself was labelled using standard fluorescent labelling
techniques, or a labelled anti-rabbit IgG molecule was used to
visualize bound OP-1 antibody.
[0125] As can be seen in FIGS. 1A and 1B, immunofluorescence
staining demonstrates the presence of OP-1 in adult rat central
nervous system (CNS.) Similar and extensive staining is seen in
both the brain (1A) and spinal cord (1B). OP-1 appears to be
localized predominantly to the extracellular matrix of the grey
matter (neuronal cell bodies), distinctly present in all areas
except the cell bodies themselves. In white matter, formed mainly
of myelinated nerve fibers, staining appears to be confined to
astrocytes (glial cells). A similar staining pattern also was seen
in newborn rat (10 day old) brain sections.
[0126] In addition, OP-1 has been specifically localized in the
substantia nigra, which is composed primarily of striatal basal
ganglia, a system of accessory motor neurons that function is
association with the cerebral cortex and corticospinal system.
Dysfunctions in this subpopulation or system of neurons are
associated with a number of neuropathies, including Huntington's
chorea and Parkinson's disease.
[0127] OP1 also has been localized at adendema glial cells, known
to secrete factors into the cerebrospinal fluid, and which occur
around the intraventricular valve, coroid fissure, and central
canal of the brain in both developing and adult rat.
[0128] Finally, morphogen inhibition in developing embryos inhibits
nerve tissue development. Specifically, 9-day mouse embryo cells,
cultured in vitro under standard culturing conditions, were
incubated in the presence and absence of an OP-1-specific
monoclonal antibody prepared using recombinantly produced, purified
mature OP-1 and the immunogen. The antibody was prepared using
standard antibody production means well known in the art and as
described generally in Example 13. After two days, the effect of
the antibody on the developing embryo was evaluated by histology.
As determined by histological examination, the OP-1-specific
antibody specifically inhibits eye lobe formation in the developing
embryo. In particular, the diencephalon outgrowth does not develop.
In addition, the heart is malformed and enlarged. Moreover, in
separate immunolocalization studies on embryo sections with
labelled OP-1 specific antibody, the OP-1-specific antibody
localizes to neural epithelia.
[0129] The endogenous morphogens which act on neuronal cells can be
expressed and secreted by nerve tissue cells, e.g., by neurons
and/or glial cells associated with the neurons, and/or they can be
transported to the neurons by the cerebrospinal fluid and/or
bloodstream. Recently, OP-1 has been identified in the human blood
(See Example 9, below). In addition, transplanted Schwann cells
recently have been shown to stimulate nerve fiber formation in rat
spinal cord, including inducing vascularization and myelin sheath
formation around at least some of the new neuronal processes (Bunge
(1991) Exp. Neurology 114:254-257.) The regenerative property of
these cells can be mediated by the secretion of a morphogen by the
Schwann cells.
Example 3
Morphogen Enhancement of Neuronal Cell Survival
[0130] The morphogens described herein enhance cell survival,
particularly of neuronal cells at risk of dying. For example, fully
differentiated neurons are non-mitotic and die in vitro when
cultured under standard mammalian cell culture conditions, using a
chemically defined or low serum medium known in the art, (see, for
example, Charness (1986) J. Biol. Chem. 26:3164-3169 and Freese et
al. (1990) Brain Res. 521:254-264.) However, if a primary culture
of non-mitotic neuronal cells is treated with a morphogen, the
survival of these cells is enhanced significantly. For example, a
primary culture of striatal basal ganglia isolated from the
substantia nigra of adult rat brain was prepared using standard
procedures, e.g., by dissociation by trituration with pasteur
pipette of substania nigra tissue, using standard tissue culturing
protocols, and grown in a low serum medium, e.g., containing 50%
DMEM (Dulbecco's modified Eagle's medium), 50% F-12 medium, heat
inactivated horse serum supplemented with penicillin/streptomycin
and 4 g/l glucose. Under standard culture conditions, these cells
are undergoing significant cell death by three weeks when cultured
in a serum-free medium. Cell death is evidenced morphologically by
the inability of cells to remain adherent and by changes in their
ultrastructural characteristics, e.g., by chromatin clumping and
organelle disintegration.
[0131] In this example, the cultured basal ganglia were were
treated with chemically defined medium conditioned with 0.1-100
ng/ml OP-1. Fresh, morphogen-conditioned medium was provided to the
cells every 3-4 days. Cell survival was enhanced significantly and
was dose dependent upon the level of OP-1 added: cell death
decreased significantly as the concentration of OP-1 was increased
in cell cultures. Specifically, cells remained adherent and
continued to maintain the morphology of viable differentiated
neurons. In the absence of morphogen treatment, the majority of the
cultured cells dissociated and underwent cell necrosis.
[0132] Dysfunctions in the basal ganglia of the sustantia nigra are
associated with Huntington's chorea and parkinsonism in vivo. The
ability of the morphogens defined herein to enhance neuron survival
indicates that these morphogens will be useful as part of a therapy
to enhance survival of neuronal cells at risk of dying in vivo due,
for example, to a neuropathy or chemical or mechanical trauma. It
is particularly anticipated that these morphogens will provide a
useful therapeutic agent to treat neuropathies which affect the
striatal basal ganglia, including Huntington's chorea and
Parkinson's disease. For clinical applications, the morphogen may
be administered or, alternatively, a morphogen-stimulating agent
can be administered.
Example 4
Morphogen-Induced Redifferentiation of Transformed Cells
[0133] The morphogens described herein also induce
redifferentiation of transformed cells to a morphology
characteristic of untransformed cells. In particular, the
morphogens are capable of inducing redifferentiation of transformed
cells of neuronal origin to a morphology characteristic of
untransformed neurons. The example provided below details morphogen
induced redifferentiation of a transformed human cell line of
neuronal origin, NG108-15. Morphogen-induced redifferentiation of
transformed cells also has been shown in mouse neuroblastoma cells
(N1E-115) and in human embryo carcimona cells (see international
application US92/01968 (WO92/15323).
[0134] NG108-15 is a transformed hybrid cell line produced by
fusing neuroblastoma.times.glioma cells (obtained from America Type
Tissue Culture, Rockville, Md.), and exhibiting a morphology
characteristic of transformed embryonic neurons, e.g., having a
fibroblastic morphology. Specifically, the cells have polygonal
cell bodies, short, spike-like processes and make few contacts with
neighboring cells (see FIG. 1A). Incubation of NG108-15 cells,
cultured in a chemically defined, serum-free medium, with 0.1 to
300 ng/ml of OP-1 for four hours induces an orderly, dose-dependent
change in cell morphology.
[0135] In the experiment NG108-15 cells were subcultured on
poly-L-lysine coated 6-well plates. Each well contained 40-50,000
cells in 2.5 ml of chemically defined medium. On the third day 2.5
.mu.l of OP-1 in 60% ethanol containing 0.025% trifluoroacetic was
added to each well. OP-1 concentrations of 0-300 ng/ml were tested.
Typically, the media was changed daily with new aliquots of OP-1,
although morphogenesis can be induced by a single four hour
incubation with OP-1. In addition, OP-1 concentrations of 10 ng/ml
were sufficient to induce redifferentiation. After one day,
hOP-1-treated cells undergo a significant change in their cellular
ultrastructure, including rounding of the soma, increase in phase
brightness and extension of the short neurite processes. After two
days, cells treated with OP-1 begin to form epithelioid sheets,
which provide the basis for the growth of mutilayered aggregates at
three day's post-treatment. By four days, the great majority of
OP-1-treated cells are associated in tightly-packed, mutilayered
aggregates (FIG. 1B). FIG. 2 plots the mean number of multi-layered
aggregates or cell clumps identified in twenty randomly selected
fields from six independent experiments, as a function of morphogen
concentration. Forty ng/ml of OP-1 is sufficient for half maximum
induction of cell aggregation.
[0136] The morphogen-induced redifferentiation occurred without any
associated changes in DNA synthesis, cell division, or cell
viability, making it unlikely that the morphologic changes were
secondary to cell differentiation or a toxic effect of hOP-1.
Moreover, the OP-1-induced morphogenesis does not inhibit cell
division, as determined by .sup.3H-thymidine uptake, unlike other
molecules which have been shown to stimulate differentiation of
transformed cells, such as butyrate, DMSO, retanoic acid or
Forskolin. The data indicate that OP-1 can maintain cell stability
and viability after inducing redifferentiation. In addition, the
effects are morphogen specific, and redifferentiation is not
induced when NG108-15 cells are incubated with 0.1-40 ng/ml
TGF-.beta..
[0137] The experiments also have been performed with highly
purified soluble morphogen (e.g., mature OP1 associated with its
pro domain) which also specifically induced redifferentiation of
NG108-15 cells.
[0138] The morphogens described herein accordingly provide useful
therapeutic agents for the treatment of neoplasias and neoplastic
lesions of the nervous system, particularly in the treatment of
neuroblastomas, including retinoblastomas, and gliomas. The
morphogens themselves can be administered or, alternatively, a
morphogen-stimulating agent can be administered.
Example 5
Nerve Tissue Protection from Chemical Trauma
[0139] The ability of the morphogens described herein to enhance
survival of neuronal cells and to induce cell aggregation and
cell-cell adhesion in redifferentiated cells, indicates that the
morphogens will be useful as therapeutic agents to maintain neural
pathways by protecting the cells defining the pathway from the
damage caused by chemical trauma. In particular, the morphogens can
protect neurons, including developing neurons, from the effects of
toxins known to inhibit the proliferation and migration of neurons
and to interfere with cell-cell adhesion. Examples of such toxins
include ethanol, one or more of the toxins present in cigarette
smoke, and a variety of opiates. The toxic effects of ethanol on
developing neurons induces the neurological damage manifested in
fetal alcohol syndrome. The morphogens also can protect neurons
from the cytoxic effects associated with excitatory amino acids
such as glutamate.
[0140] For example, ethanol inhibits the cell-cell adhesion effects
induced in morphogen-treated NG108-15 cells when provided to these
cells at a concentration of 25-50 mM. Half maximal inhibition can
be achieved with 5-10 mM ethanol, the concentration of blood
alcohol in an adult following ingestion of a single alcoholic
beverage. Ethanol likely interferes with the homophilic binding of
CAMs between cells, rather than their induction, as
morphogen-induced N-CAM levels are unaffected by ethanol. Moreover,
the inhibitory effect is inversely proportional to morphogen
concentration. Accordingly, it is envisioned that administration of
a morphogen or morphogen-stimulating agent to neurons, particularly
developing neurons, at risk of damage from exposure to toxins such
as ethanol, can protect these cells from nerve tissue damage by
overcoming the toxin's inhibitory effects. The morphogens described
herein also are useful in therapies to treat damaged neural
pathways resulting from a neuropathy induced by exposure to these
toxins.
Example 6
Morphogen-Induced CAM Expression
[0141] The morphogens described herein induce CAM expression,
particularly N-CAM expression, as part of their induction of
morphogenesis. CAMs are morphoregulatory molecules identified in
all tissues as an essential step in tissue development. N-CAMS,
which comprise at least 3 isoforms (N-CAM-180, N-CAM-140 and
N-CAM-120, where "180", "140" and "120" indicate the apparent
molecular weights of the isoforms as measured by polyacrylamide gel
electrophoresis) are expressed at least transiently in developing
tissues, and permanently in nerve tissue. Both the N-CAM-180 and
N-CAM-140 isoforms are expressed in both developing and adult
tissue. The N-CAM-120 isoform is found only in adult tissue.
Another neural CAM is L1.
[0142] N-CAMs are implicated in appropriate neural development,
including appropriate nuerulation, neuronal migration,
fasciculation, and synaptogenesis. Inhibition of N-CAM production,
as by complexing the molecule with an N-CAM-specific antibody,
inhibits retina organization, including retinal axon migration, and
axon regeneration in the peripheral nervous system, as well as axon
synapsis with target muscle cells. In addition, significant
evidence indicates that physical or chemical trauma to neurons,
oncogenic transformation and some genetic neurological disorders
are accompanied by changes in CAM expression, which alter the
adhesive or migratory behavior of these cells. Specifically,
increased N-CAM levels are reported in Huntington's disease
striatum (e.g., striatal basal ganglia), and decreased adhesion is
noted in Alzheimer's disease.
[0143] The morphogens described herein can stimulate CAM
production, particularly L1 and N-CAM production, including all
three isoforms of the N-CAM molecule. For example, N-CAM expression
is stimulated significantly in morphogen-treated NG108-15 cells.
Untreated NG108-15 cells exhibit a fibroblastic, or minimally
differentiated morphology and express only the 180 and 140 isoforms
of N-CAM normally associated with a developing cell. Following
morphogen treatment these cells exhibit a morphology characteristic
of adult neurons and express enhanced levels of all three N-CAM
isoforms. Using a similar protocol as described in the example
below, morphogen treatment of NG108-15 cells also induced L1
expression.
[0144] In this example NG108-15 cells were cultured for 4 days in
the presence of increasing concentrations of OP-1 and standard
Western blots performed on whole cells extracts. N-CAM isoforms
were detected with an antibody which crossreacts with all three
isoforms, mAb H28.123, obtained from Sigma Chemical Co., St. Louis,
the different isoforms being distinguishable by their different
mobilities on an electrophoresis gel. Control NG108-15 cells
(untreated) express both the 140 kDa and the 180 kDa isoforms, but
not the 120 kDa, as determined by western blot analyses using up to
100 .mu.g of protein. Treatment of NG108-15 cells with OP-1
resulted in a dose-dependent increase in the expression of the 180
kDa and 140 kDa isoforms, as well as the induction of the 120 kDa
isoform. See FIGS. 2A and 2B. FIG. 2B is a Western blot of
OP1-treated NG108-15 cell extracts, probed with mAb H28.123,
showing the induction of all three isoforms. FIG. 2A is a dose
response curve of N-CAM-180 and N-CAM-140 induction as a function
of morphogen concentration. N-CAM-120 is not shown in the graph as
it could not be quantitated in control cells. However, as is
clearly evident from the Western blot in FIG. 2A, N-CAM-120 is
induced in response to morphogen treatment. The differential
induction of N-CAM 180 and 140 isoforms seen can be because
constitutive expression of the 140 isoform is close to maximum.
[0145] The increase in N-CAM expression corresponded in a
dose-dependent manner with the morphogen induction of multicellular
aggregates. Compare FIG. 2A and FIG. 3. FIG. 3 graphs the mean
number of multilayered aggregates (clumps) counted per 20 randomly
selected fields in 6 independent experiments, versus the
concentration of morphogen. The induction of the 120 isoform also
indicates that morphogen-induced redifferentiation of transformed
cells stimulates not only redifferentiation of these cells from a
transformed phenotype, but also differentiation to a phenotype
corresponding to a developed cell. Standard immunolocalization
studies performed with the mAb H28.123 on morphogen-treated cells
show N-CAM cluster formation associated with the periphery and
processes of treated cells and no reactivity with untreated cells.
Moreover, morphogen treatment does not appear to inhibit cell
division as determined by cell counting or .sup.3H-thymidine
uptake. Finally, known chemical differentiating agents, such as
Forskolin and dimethylsulfoxide do not induce N-CAM production.
[0146] In addition, the cell aggregation effects of OP-1 on
NG108-15 cells can be inhibited with anti-N-CAM antibodies or
antisense N-CAM oligonucleotides. Antisense oligonucleotides can be
made synthetically on a nucleotide synthesizer, using standard
means known in the art. Preferably, phosphorothioate
oligonucleotides ("S-oligos") are prepared, to enhance transport of
the nucleotides across cell membranes. Concentrations of both N-CAM
antibodies and N-CAM antisense oliognucleotides sufficient to
inhibit N-CAM induction also inhibited formation of multilayered
cell aggregates. Specifically, incubation of morphogen-treated
NG108-115 cells with 0.3-3 .mu.M N-CAM antisense S-oligos, 5-500
.mu.M unmodified N-CAM antisense oligos, or 10 .mu.g/ml mAb H28.123
significantly inhibits cell aggregation. It is likely that
morphogen treatment also stimulates other CAMs, as inhibition is
not complete.
[0147] The experiments also have been performed with soluble
morphogen (e.g., mature OP-1 associated with its pro domain) which
also specifically induced CAM expression.
[0148] The morphogens described herein are useful as therapeutic
agents to treat neurological disorders associated with altered CAM
levels, particularly N-CAM levels, such as Huntington's chorea and
Alzheimers' disease, and the like. In clinical applications, the
morphogens themselves can be administered or, alternatively, a
morphogen-stimulating agent can be administered.
[0149] The efficacy of the morphogens described herein to affect
N-CAM expression can be assessed in vitro using a suitable cell
line and the methods described herein. In addition to a transformed
cell line, N-CAM expression can be assayed in a primary cell
culture of neural or glial cells, following the procedures
described herein. The efficacy of morphogen treatment on N-CAM
expression in vivo can be evaluated by tissue biopsy as described
in Example 9, below, and detecting N-CAM molecules with an
N-CAM-specific antibody, such as mAb H28.123, or using the animal
model described in Example 11.
[0150] Alternatively, the level of N-CAM proteins or protein
fragments present in cerebrospinal fluid or serum also can be
detected to evaluate the effect of morphogen treatment. N-CAM
molecules are known to slough off cell surfaces and have been
detected in both serum and cerebrospinal fluid. In addition,
altered levels of the soluble form of N-CAM are associated with
normal pressure hydrocephalus and type II schizophrenia. N-CAM
fluid levels can be detected following the procedure described in
Example 9 and using an N-CAM specific antibody, such as mAb
H28.123.
Example 7
Morphogen-Induced Nerve Gap Repair (PNS)
[0151] The morphogens described herein also stimulate peripheral
nervous system axonal growth over extended distances allowing
repair and regeneration of damaged neural pathways. While neurons
of the peripheral nervous system can sprout new processes following
injury, without guidance these sproutings typically fail to connect
appropriately and die. Where the break is extensive, e.g., greater
than 5 or 10 mm, regeneration is poor or nonexistent.
[0152] In this example morphogen stimulation of nerve regeneration
was assessed using the rat sciatic nerve model. The rat sciatic
nerve can regenerate spontaneously across a 5 mm gap, and
occasionally across a 10 mm gap, provided that the severed ends are
inserted in a saline-filled nerve guidance channel. In this
experiment, nerve regeneration across a 12 mm gap was tested.
[0153] Adult female Sprague-Dawley rats (Charles River
Laboratories, Inc.) weighing 230-250 g were anesthetized with
intraperitoneal injections of sodium pentobarbital 35 mg/kg body
weight). A skin incision was made parallel and just posterior to
the femur. The avascular intermuscular plane between vastus
lateralis and hamstring muscles were entered and followed to the
loose fibroareolar tissue surrounding the sciatic nerve. The loose
tissue was divided longitudinally thereby freeing the sciatic nerve
over its full extent without devascularizing any portion. Under a
surgical microscope the sciatic nerves were transected with
microscissors at mid-thigh and grafted with an OP-1 gel graft that
separated the nerve stumps by 12 mm. The graft region was encased
in a silicone tube 20 mm in length with a 1.5 mm inner diameter,
the interior of which was filled a morphogen solution.
Specifically, The central 12 mm of the tube consisted of an OP-1
gel prepared by mixing 1 to 5 .mu.g of substantially pure
CHO-produced recombinant OP-1 with approximately 100 .mu.l of
MATRIGEL.TM. (from Collaborative Research, Inc., Bedford, Mass.),
an extracellular matrix extract derived from mouse sarcoma tissue,
and containing solubilized tissue basement membrane, including
laminin, type IV collagen, heparin sulfate, proteoglycan and
entactin, in phosphate-buffered saline. The OP-1-filled tube was
implanted directly into the defect site, allowing 4 mm on each end
to insert the nerve stumps. Each stump was abutted against the OP-1
gel and was secured in the silicone tube by three stitches of
commercially available surgical 10-0 nylon through the epineurium,
the fascicle protective sheath.
[0154] In addition to OP-1 gel grafts, empty silicone tubes,
silicone tubes filled with gel only and "reverse" autografts,
wherein 12 mm transected segments of the animal's sciatic nerve
were rotated 180.degree. prior to suturing, were grafted as
controls. All experiments were performed in quadruplicate. All
wounds were closed by wound clips that were removed after 10 days.
All rats were grafted on both legs. At 3 weeks the animals were
sacrificed, and the grafted segments removed and frozen on dry ice
immediately. Frozen sections then were cut throughout the graft
site, and examined for axonal regeneration by immunofluorescent
staining using anti-neurofilament antibodies labeled with flurocein
(obtained from Sigma Chemical Co., St. Louis).
[0155] Regeneration of the sciatic nerve occurred across the entire
12 mm distance in all graft sites wherein the gap was filled with
the OP-1 gel. By contrast, empty silicone tubes and reverse
autografts did not show nerve regeneration, and only one graft site
containing the gel alone showed axon regeneration.
Example 8
Morphogen-Induced Nerve Gap Repair (CNS)
[0156] Following axonal damage in vivo the CNS neurons are unable
to resprout processes. Accordingly, trauma to CNS nerve tissue,
including the spinal cord, optic nerve and retina, severely damages
or destroys the neural pathways defined by these cells. Peripheral
nerve grafts have been used in an effort to bypass CNS axonal
damage. Successful autologous graft repair to date apparently
requires that the graft site occur near the CNS neuronal cell body,
and a primary result of CNS axotomy is neuronal cell death. The
efficacy of morphogens described herein on CNS nerve repair, can be
evaluated using a rat crushed optic nerve model such as the one
described by Bignami et al., (1979) Exp. Eye Res. 28: 63-69, the
disclosure of which is incorporated herein by reference. Briefly,
and as described therein, laboratory rats (e.g., from Charles River
Laboratories, Wilmington, Mass.) are anesthesized using standard
surgical procedures, and the optic nerve crushed by pulling the eye
gently out of the orbit, inserting a watchmaker forceps behind the
eyeball and squeezing the optic nerve with the forceps for 15
seconds, followed by a 30 second interval and second 15 second
squeeze. Rats are sacrificed at different time intervals, e.g., at
48 hours, and at 3, 4, 11, 15 and 18 days after operation. The
effect of morphogen on optic nerve repair can be assessed by
performing the experiment in duplicate and providing morphogen or
PBS (e.g., 25 .mu.l solution, and 25 .mu.g morphogen) to the optic
nerve, e.g., just prior to the operation, concommitant with the
operation, or at specific times after the operation.
[0157] In the absence of therapy, the surgery induces glial
scarring of the crushed nerve, as determined by immunofluoresence
staining for glial fibrillary acidic protein (GFA), a marker
protein for glial scarring, and by histology. Indirect
immunofluoresence on air-dried cryostat sections as described in
Bignami et al. (1974) J. Comp. Neur. 153: 27-38, using commercially
available antibodies to GFA (e.g., Sigma Chemical Co., St. Louis).
Reduced levels of GFA are anticipated in animals treated with the
morphogen, evidencing the ability of morphogens to inhibit glial
scar formation and to stimulate optic nerve regeneration.
Example 9
Nerve Tissue Diagnostics
[0158] Morphogen localization in nerve tissue can be used as part
of a method for diagnosing a neurological disorder or neuropathy.
The method can be particularly advantageous for diagnosing
neuropathies of brain tissue. Specifically, a biopsy of brain
tissue is performed on a patient at risk, using standard procedures
known in the medical art. Morphogen expression associated with the
biopsied tissue then is assessed using standard methodologies, as
by immunolocalization, using standard immunofluorescence techniques
in concert with morphogen-specific antisera or monoclonal
antibodies. Specifically, the biopsied tissue is thin sectioned
using standard methodologies known in the art, and fluorescently
labelled (or otherwise detectable) antibodies incubated with the
tissue under conditions sufficient to allow specific
antigen-antibody complex formation. The presence and quantity of
complex formed then is detected and compared with a predetermined
standard or reference value. Detection of altered levels of
morphogen present in the tissue then can be used as an indicator of
tissue dysfunction. Alternatively, fluctuation (increase or
decrease) in morphogen levels can be assessed by monitoring
morphogen transcription levels, either by standard northern blot
analysis or in situ hybridization, using a labelled probe capable
of hybridizing specifically to morphogen RNA and standard RNA
hybridization protocols well described in the art.
[0159] Monitoring fluctuations (increases or decreases) over time
in morphogen levels present in the cerebrospinal fluid or
bloodstream of an individual also can be used to evaluate nerve
tissue viability. For example, morphogens are detected in
association with adendema cells which are known to secrete factors
into the cerebrospinal fluid, and are localized generally
associated with glial cells, and in the extracellular matrix, but
not with neuronal cell bodies. Accordingly, the cerebrospinal fluid
may be a natural means of morphogen transport. Alternatively,
morphogens can be released from dying cells into cerebrospinal
fluid. In addition, OP-1 recently has been identified in human
blood, which also can be a means of morphogen transport, and/or a
repository for the contents of dying cells.
[0160] Spinal fluid can be obtained from an individual by a
standard lumbar puncture, using standard methodologies known in the
medical art. Similarly, serum samples can be obtained by standard
venipuncture and serum prepared by centrifugation at 3,000 RPM for
ten minutes. The presence of morphogen in the serum or cerebral
spinal fluid then can be assessed by standard Western blot
(immunoblot), ELISA or RIA procedures. Briefly, for example, with
the ELISA, samples can be diluted in an appropriate buffer, such as
phosphate-buffered saline, and 50 .mu.l aliquots allowed to absorb
to flat bottomed wells in microtitre plates pre-coated with
morphogen-specific antibody, and allowed to incubate for 18 hours
at 4.degree. C. Plates then can be washed with a standard buffer
and incubated with 50 .mu.l aliquots of a second morphogen-specific
antibody conjugated with a detecting agent, e.g., biotin, in an
appropriate buffer, for 90 minutes at room temperature.
Morphogen-antibody complexes then can be detected using standard
procedures.
[0161] Alternatively, a morphogen-specific affinity column can be
created using, for example, morphogen-specific antibodies adsorbed
to a column matrix, and passing the fluid sample through the matrix
to selectively extract the morphogen of interest. The morphogen
then is eluted. A suitable elution buffer can be determined
empirically by determining appropriate binding and elution
conditions first with a control (e.g., purified,
recombinantly-produced morphogen.) Fractions then are tested for
the presence of the morphogen by standard immunoblot, and confirmed
by N-terminal sequencing. Morphogen concentrations in serum or
other fluid samples then can be determined using standard portein
quantification techniques, including by spectrophotometric
absorbance or by quantitation by ELISA or RIA antibody assays.
Using this procedure, OP-1 has been identified in serum.
[0162] OP-1 was detected in human serum using the following assay.
A monoclonal antibody raised against mammalian, recombinantly
produced OP-1 using standard immunology techniques well described
in the art and described generally in Example 13, was immobilized
by passing the antibody over an activated agarose gel (e.g.,
Affi-Gel.TM., from Bio-Rad Laboratories, Richmond, Calif., prepared
following manufacturer's instructions), and used to purify OP-1
from serum. Human serum then was passed over the column and eluted
with 3M K-thiocyanate. K-thiocyanante fractions then were dialyzed
in 6M urea, 20 mM PO.sub.4, pH 7.0, applied to a C8 HPLC column,
and eluted with a 20 minute, 25-50% acetonitrile/0.1% TFA gradient.
Mature, recombinantly produced OP-1 homodimers elute between 20-22
minutes. Fractions then were collected and tested for the presence
of OP-1 by standard immunoblot. FIG. 4 is an immunoblot showing
OP-1 in human sera under reducing and oxidized conditions. In the
figure, lanes 1 and 4 are OP-1 standards, run under oxidized (lane
1) and reduced (lane 4) conditions. Lane 5 shows molecular weight
markers at 17, 27 and 39 kDa. Lanes 2 and 3 are human sera OP-1,
run under oxidized (lane 2) and reduced (lane 3) conditions.
[0163] Morphogens can be used in diagnostic applications by
comparing the quantity of morphogen present in a body fluid sample
with a predetermined reference value, with fluctuations in fluid
morphogen levels indicating a change in the status of nerve tissue.
Alternatively, fluctuations in the level of endogenous morphogen
antibodies can be detected by this method, most likely in serum,
using an antibody or other binding protein capable of interacting
specifically with the endogenous morphogen antibody. Detected
fluctuations in the levels of the endogenous antibody can be used
as indicators of a change in tissue status.
Example 10
Alleviation of Immune Response-Mediated Nerve Tissue Damage
[0164] The morphogens described herein can be used to alleviate
immunologically-related damage to nerve tissue. Details of this
damage and the use of morphogens to alleviate this injury are
disclosed in international application US92/07358 (WO93/04692). A
primary source of such damage to nerve tissue follows hypoxia or
ischemia-reperfusion of a blood supply to a neural pathway, such as
can result from an embolic stroke, or can be induced during a
surgical procedure. As described in international application
US92/07358 (WO93/04692), morphogens have been shown to alleviate
damage to myocardial tissue following ischemia-reperfusion of the
blood supply to the tissue. The effect of morphogens on alleviating
immunologically-related damage to nerve tissue can be assessed
using methodologies and models known to those skilled in the art
and described below.
[0165] For example, the rabbit embolic stroke model provides a
useful method for assessing the effect of morphogens on tissue
injury following cerebral ischemia-reperfusion. The protocol
disclosed below is essentially that of Phillips et al. (1989)
Annals of Neurology 25:281-285, the disclosure of which is herein
incorporated by reference. Briefly, white New England rabbits (2-3
kg) are anesthetized and placed on a respirator. The intracranial
circulation then is selectively catheterized by the Seldinger
technique. Baseline cerebral angiography then is performed,
employing a digital substration unit. The distal internal carotid
artery or its branches then is selectively embolized with 0.035 ml
of 18-hour-aged autologous thrombus. Arterial occlusion is
documented by repeat angiography immediately after embolization.
After a time sufficient to induce cerebral infarcts (15 minutes or
90 minutes), reperfusion is induced by administering a bolus of a
reperfusion agent such as the TPA analogue FB-FB-CF (e.g., 0.8
mg/kg over 2 minutes).
[0166] The effect of morphogen on cerebral infarcts can be assessed
by administering varying concentrations of morphogens, e.g., OP-1,
at different times before or following embolization and/or
reperfusion. The rabbits are sacrificed 3-14 days post embolization
and their brains prepared for neuropathological examination by
fixing by immersion in 10% neutral buffered formation for at least
2 weeks. The brains then are sectioned in a coronal plane at 2-3 mm
intervals, numbered and submitted for standard histological
processing in paraffin, and the degree of nerve tissue necrosis
determined visually. Morphogen-treated animals are anticipated to
reduce or significantly inhibit nerve tissue necrosis following
cerebral ischemia-reperfusion in the test animals as determined by
histology comparison with nontreated animals.
Example 11
Animal Model for Assessing Morphogen Efficacy In Vivo
[0167] The in vivo activities of the morphogens described herein
also are assessed readily in an animal model as described herein. A
suitable animal, preferably exhibiting nerve tissue damage, for
example, genetically or environmentally induced, is injected
intracerebrally with an effective amount of a morphogen in a
suitable therapeutic formulation, such as phosphate-buffered
saline, pH 7. The morphogen preferably is injected within the area
of the affected neurons. The affected tissue is excised at a
subsequent time point and the tissue evaluated morphologically
and/or by evaluation of an appropriate biochemical marker (e.g., by
morphogen or N-CAM localization; or by measuring the dose-dependent
effect on a biochemical marker for CNS neurotrophic activity or for
CNS tissue damage, using for example, glial fibrillary acidic
protein as the marker. The dosage and incubation time will vary
with the animal to be tested. Suitable dosage ranges for different
species can be determined by comparison with established animal
models. Presented below is an exemplary protocol for a rat brain
stab model.
[0168] Briefly, male Long Evans rats, obtained from standard
commercial sources, are anesthesized and the head area prepared for
surgery. The calvariae is exposed using standard surgical
procedures and a hole drilled toward the center of each lobe using
a 0.035K wire, just piercing the calvariae. 25 .mu.l solutions
containing either morphogen (e.g., OP-1, 25 .mu.g) or PBS then is
provided to each of the holes by Hamilton syringe. Solutions are
delivered to a depth approximately 3 mm below the surface, into the
underlying cortex, corpus callosum and hippocampus. The skin then
is sutured and the animal allowed to recover.
[0169] Three days post surgery, rats are sacrificed by decapitation
and their brains processed for sectioning. Scar tissue formation is
evaluated by immunofluoresence staining for glial fibrillary acidic
protein, a marker protein for glial scarring, to qualitatively
determine the degree of scar formation. Glial fibrillary acidic
protein antibodies are available commercially, e.g., from Sigma
Chemical Co., St. Louis, Mo. Sections also are probed with
anti-OP-1 antibodies to determine the presence of OP-1. Reduced
levels of glial fibrillary acidic protein are anticipated in the
tissue sections of animals treated with the morphogen, evidencing
the ability of morphogens to inhibit glial scar formation and
stimulate nerve regeneration.
Example 12
In Vitro Model for Evaluating Morphogen Transport Across the
Blood-Brain Barrier
[0170] Described below is an in vitro method for evaluating the
facility with which a selected morphogen likely will pass across
the blood-brain barrier. A detailed description of the model and
protocol are provided by Audus et al. (1987) Ann. N.Y. Acad. Sci.
507:9-18, the disclosure of which is incorporated herein by
reference.
[0171] Briefly, microvessel endothelial cells are isolated from the
cerebral gray matter of fresh bovine brains. Brains are obtained
from a local slaughter house and transported to the laboratory in
ice cold minimum essential medium (MEM) with antibiotics. Under
sterile conditions the large surface blood vessels and meninges are
removed using standard dissection procedures. The cortical gray
matter is removed by aspiration, then minced into cubes of about 1
mm. The minced gray matter then is incubated with 0.5% dispase
(BMB, Indianapolis, Ind.) for 3 hours at 37.degree. C. in a shaking
water bath. Following the 3 hour digestion, the mixture is
concentrated by centrifugation (1000.times.g for 10 min.), then
resuspended in 13% dextran and centrifuged for 10 min. at
5800.times.g. Supernatant fat, cell debris and myelin are discarded
and the crude microvessel pellet resuspended in 1 mg/ml
collagenase/dispase and incubated in a shaking water bath for 5
hours at 37.degree. C. After the 5-hour digestion, the microvessel
suspension is applied to a pre-established 50% Percoll gradient and
centrifuged for 10 min at 1000.times.g. The band containing
purified endothelial cells (second band from the top of the
gradient) is removed and washed two times with culture medium
(e.g., 50% MEM/50% F-12 nutrient mix). The cells are frozen
(-80.degree. C.) in medium containing 20% DMSO and 10% horse serum
for later use.
[0172] After isolation, approximately 5.times.10.sup.5
cells/cm.sup.2 are plated on culture dishes or 5-12 m.mu. pore size
polycarbonate filters that are coated with rat collagen and
fibronectin. 10-12 days after seeding the cells, cell monolayers
are inspected for confluency by microscopy.
[0173] Characterization of the morphological, histochemical and
biochemical properties of these cells has shown that these cells
possess many of the salient features of the blood-brain barrier.
These features include: tight intercellular junctions, lack of
membrane fenestrations, low levels of pinocytotic activity, and the
presence of gamma-glutamyl transpeptidase, alkaline phosphatase,
and Factor VIII antigen activities.
[0174] The cultured cells can be used in a wide variety of
experiments where a model for polarized binding or transport is
required. By plating the cells in multi-well plates, receptor and
non-receptor binding of both large and small molecules can be
conducted. In order to conduct transendothelial cell flux
measurements, the cells are grown on porous polycarbonate membrane
filters (e.g., from Nucleopore, Pleasanton, Calif.). Large pore
size filters (5-12 mp) are used to avoid the possibility of the
filter becoming the rate-limiting barrier to molecular flux. The
use of these large-pore filters does not permit cell growth under
the filter and allows visual inspection of the cell monolayer.
[0175] Once the cells reach confluency, they are placed in a
side-by-side diffusion cell apparatus (e.g., from Crown Glass,
Sommerville, N.J.). For flux measurements, the donor chamber of the
diffusion cell is pulsed with a test substance, then at various
times following the pulse, an aliquot is removed from the receiver
chamber for analysis. Radioactive or fluorescently-labelled
substances permit reliable quantitation of molecular flux.
Monolayer integrity is simultaneously measured by the addition of a
non-transportable test substance such as sucrose or inulin and
replicates of at least 4 determinations are measured in order to
ensure statistical significance.
Example 13
OP-1 Induces Dendritic Growth in Sympathetic Neurons
[0176] Suspensions of neurons dissociated from the superior
cervical ganglia of Sprague-Dawley rat fetuses (19-21 day) or rat
pups (1-3 day postnatal) were prepared according to the method of
(Higgins et al. (1991), Culturing Nerve Cells Banker and Goslin,
eds., MIT Press, pp. 177-205, the teachings of which herein
incorporated by reference). Equivalent results were obtained with
pre- and postnatal animals. Neurons were plated at low density
(about 10 cells/mm.sup.2) onto poly-D-lysine coated (100 .mu.g/ml)
coverslips and maintained in a serum-free medium (Higgins et al.
(1991), Culturing Nerve Cells) containing NGF (100 ng/ml).
Cytosine-.beta.-D-furanoside (1 .mu.M) was added to the medium of
all cultures for 48 hrs on the second day. This exposure was
sufficient to render them virtually free of nonneuronal cells for
30 days. To label sympathetic neuroblasts, ganglia from 15-day
embryos were grown in explant culture for 18 hrs in the presence of
.sup.3H-(methyl)-thymidine (0.3 .mu.Ci/ml, ICN) before being
dissociated. Because NT3 (50 ng/ml) enhances the survival of
immature sympathetic neurons (Birren et al. (1993), 119 Develop
597-610), it was added to the NGF-containing medium during the
period of explant culture and the next 4 days in vitro. As in
cultures of sympathetic neurons, exposure to NGF, OP-1 or both was
initiated after the elimination of nonneuronal cells.
[0177] Cellular morphology was routinely assessed by intracellular
injection of fluorescent dyes (4% Lucifer Yellow or 5% 5,6
dicarboxyfluorescein) (Bruckenstein and Higgins (1988), 128 Dev.
Biol. 924-936). Only neurons whose cell bodies were at least 150
.mu.m from their nearest neighbor were injected because
density-dependent changes in morphology occur when somata of
sympathetic neurons are separated by lesser distances. Mature human
recombinant OP-1 was isolated from medium conditioned by
transfected Chinese hamster ovary cells using S-Sepharose and
phenyl-Sepharose chromatography followed by reverse phase high
performance liquid chromatography as described previously.
[0178] Under control conditions, sympathetic neurons typically
extended a single process during the first 24-48 hrs in vitro. This
process has the cytoskeletal and ultrastructural characteristics of
an axon. The axon continued to elongate during the next few weeks
and generate an elaborate plexus. The basic morphology of the
cells, however, remained essentially unchanged, with 80% of the
neurons still being unipolar after 1 month in vitro. Most of the
remainder had either 2 axons (13% of the cells) or an axon and a
short dendrite (7%). Thus, the mean number of processes at this
time was 1.13.+-.0.06 axons/cells and 0.07.+-.0.04
dendrites/cell.
[0179] Exposure to OP-1 caused sympathetic neurons to form
additional processes. This response was relatively slow with only
42% of the cells forming a second process within 24 hrs. However,
virtually all cells (94%) had begun to respond to maximally
effective concentrations of OP-1 within 3 days. The processes that
formed in the presence of OP-1 had the appearance of dendrites in
that they were broad-based (up to 5 .mu.m diameter), exhibited a
distinct taper, and branched in a "Y"-shaped pattern, with daughter
processes being distinctly smaller than the parent process.
Dendrites were much thicker than sympathetic axons and, unlike
axons, they ended locally, usually extending less than 300 .mu.m
from the soma. The mean number of dendrites/cell continued to
increase during a 4 week exposure to OP-1 with most of the change
occurring during the first 10 days of treatment. After 4 weeks,
OP-1-treated neurons had a mean of 7.3.+-.0.3 dendrites/cell,
representing a 100-fold increase over control cells. During this
time, the size of the dendritic arbor also increased with cells
progressing from simple cells to a more complicated morphology.
These observations are summarized in panels A, B and C of FIG. 5.
TABLE-US-00008 TABLE III COMPARISON OF THE DENDRITIC ARBORS
GENERATED BY SYMPATHETIC NEURONS IN SITU AND AFTER A 2 WEEK
EXPOSURE TO OP-1. PARAMETER IN SITU* IN VITRO Numbier of
dendrites/cells 6.9 .+-. 2.1 7.6 .+-. 0.4 Total linear length of
779 .+-. 288 1078 .+-. 77 dendritic arbor (.mu.m) Maximum extent of
arbor (.mu.m) 99 .+-. 31 171 .+-. 6 Number of branches crossing 8.0
.+-. 2.8 9.3 .+-. 0.7 50% circle Soma diameter (.mu.m) 29.2 .+-.
6.0 32.9 .+-. 0.5 *in situ data are from Snider (1988) 8 J.
Neurosci. 2628-2634. Mean .+-. SD. After a 2 week exposure to OP-1,
cultures of sympathetic neurons from 2 different dissections were
immunostained with dendrite-specific antibody (SMI-32 or AP14)(Lein
and Higgins (1989), 136 Dev. # Biol. 330-345). Dendritic growth was
quantitated using the Image 1 Software image analysis system (Lein
and Higgins (1989), 136 Dev. Biol. 330-345). # The dendritic extent
is the radius of a circle encompassing the entire arbor. The number
of branches crossing a circle of half that diameter was used as an
index of branching (Scholl (1953), 87 J. Anat. # (London) 387, the
teachings of which are herein incorporated by reference). Data are
expressed as the mean .+-. SEM (N = 40).
[0180] The effects of OP-1 appeared to be dendrite-specific since
there was no increase in axon number when tested in the delayed
introduction paradigm used in the experiment shown in FIG. 5. The
effects of OP-1 on initial axon growth during the first 48 hrs. in
culture were also examined. It did not affect either the rate at
which axons were initially extended or the number of axons extended
per cell. Cell number also remained constant during the exposure to
OP-1, indicating that it was not acting by enhancing the survival
of a subpopulation of neurons as shown in FIG. 5.
Example 14
Properties of Dendrites Formed in the Presence of OP-1
[0181] Other techniques were used to confirm the light microscopic
identification of processes and to asses the state of
differentiation of the dendrites formed in the presence of OP-1.
For example, cultures were immunostained with antibodies previously
shown to react selectively with either axons or dendrites (Lein and
Higgins (1989), 136 Dev. Biol. 330-345). Monoclonal (mAb)
antibodies to MAP2 (AP14, a gift of L.I. Binder), to
nonphosphorylated forms of the M and H neurofilaments (SMI32,
Sternbery-Meyer Immunocytochemicals), and to the transferrin
receptor (MRC Ox-26, Serotech) were used as dendritic markers. The
teachings of each of the above-mentioned publications are
incorporated herein by reference. Axonal probes included mAb's to
synaptophysin (SY-38, Boehringer Mannheim), tau (Tau l, L.I.
Binder), and phosphorylated forms of the H (NE14, Boehringer
Mannheim) and the M and H (SMI31, Sternbery-Meyer
Immunocytochemicals) neurofilament subunits. All antigens were
localized by indirect immunofluoresence using previously described
procedures (Lein and Higgins (1989), 136 Dev. Biol. 330-345). Image
1 Software (Universal Imaging) was used for the morphometric
analyses of dendritic growth in immunostained cultures.
[0182] When cultures exposed to OP-1 were immunostained with a mAb
to MAP2, immunoreactivity was observed in the somata and the
dendritic processes, but not in the thinner axons. Moreover, the
mean number and average length of the MAP2-positive processes
corresponded closely (within 10%) to the values obtained from dye
injections suggesting that all dendrites were stained in their
entirety. A similar staining pattern was observed with mAbs to
non-phosphorylated neurofilaments and to the transferrin receptor.
In contrast, monoclonal antibodies to tau, synaptophysin, or
phosphorylated forms of the H or the M and H neurofilament subunits
selectively stained the thin axons, with little or no
immunoreactivity being observed in the dendrites. Additional
features ascertained by immunostaining are summarized in Table
III.
Example 15
Concentration Effect of OP-1
[0183] The effects of OP-1 were concentration-dependent (FIG. 6).
Significant changes in dendritic growth could be detected with
concentrations as low as 300 .mu.g/ml and half-maximal effects were
observed at about 2 ng/ml. Maximal dendritic growth was obtained
with medial concentrations between 30 and 100 ng/ml. Although
typically added to the medium on day 5, earlier initiation of
dendritic growth (by the third day of culture) could be obtained by
adding OP-1 to the medium at the time of plating. No dendritic
growth was detected in cultures in which the OP-1 (1 .mu.g/ml) had
been allowed to absorb to coverslips before plating cells.
[0184] It appeared that several parameters of dendritic growth,
including the percentage of cells with dendrites, the mean number
of dendrites/cell, dendritic length (not shown), changed over the
same concentration range (FIG. 6). In addition, three other changes
in cellular morphology in this concentration range were observed.
As had been observed while dendritic growth is occurring in vivo,
the somata became larger. In addition, the nuclei became less
eccentric and the axons formed small fascicles.
Example 16
Comparison of OP-1 to Other Growth Factors
[0185] Mature human recombinant Op-1 was isolated from medium
conditioned by transfected Chinese hamster ovary cells using
S-Sepharose and phenyl-Sepharose chromatography followed by reverse
phase high performance liquid chromatography as described above.
Ciliary neurotrophic factor (CNTF) was purified from rat sciatic
nerve (Manthorpe et al. (1986) 367 Brain Research 282-286, the
teachings of which are herein incorporated by reference) and
activin A was generously provided by Ralph Schwall (Genentech).
Other growth factors were obtained from commercial sources:
GIBCOBRL (IL-1.beta., 3, 4, 6, 7; LIF, EGF, GM-CSF, RANTES, MCAF,
TGF.alpha., TGF.beta.1 and 3, rat gamma interferon); Collaborative
Research (HGF, PDGF); Boehringer (IL 2); and Promega (IL 8).
[0186] Since OP-1 is a member of the TGF.beta. superfamily, its
actions were compared to those of other growth factors (Table IV).
Dendritic growth was not observed in the presence of TGF.beta.1,
TGF.beta.3, activin A or inhibin, all of which are members of the
TGF.beta. family but are not members of the structurally distinct
sub-family thereof to which the morphogens belong. In addition,
negative results were obtained with most neurotrophins and nine
other growth factors known to affect neuronal survival or
differentiation. In other experiments, negative results were also
obtained with: TGF.beta.2, interleukins 1.beta., 2, 3, 4, 6, 7, 8,
PDGF, HGF, GM-CSF, MCAF, RANTES, TGF.alpha. and gamma interferon.
It would thus appear that the dendrite-promoting effect of OP-1 is
a highly specific response that is observed with a very limited
subset of growth factors. It is believed that the OP-1 effect will
be reproduced or approximated by other morphogens. TABLE-US-00009
TABLE IV COMPARISON OF THE EFFECTS OF OP-1 AND OTHER GROWTH FACTORS
ON DENDRITIC GROWTH MEAN NUMBER OF GROWTH FACTOR DENDRITES/CELL
NONE 0.8 .+-. 0.04 OP1 3.08 .+-. 0.20 TGF.beta.1 0.17 .+-. 0.09
TGF.beta.3 0.00 .+-. 0.00 INHIBIN 0.20 .+-. 0.10 ACTIVIN A 0.08
.+-. 0.05 BDNF 0.11 .+-. 0.05 NT3 0.11 .+-. 0.07 NT4 0.32 .+-. 0.11
CNTF 0.10 .+-. 0.05 LIF 0.13 .+-. 0.07 EGF 0.07 .+-. 0.07 bFGF 0.03
.+-. 0.03 Beginning on day 5, cultures of sympathetic neurons were
exposed to varying concentrations of growth factors. Seven to eight
days later, the mean number of dendrites/cell was assessed by
intracellular # dye injection (N > 30 cells for each condition).
Only the results obtained with the highest concentration tested
(100 ng/ml) are shown in this table, but lower concentrations
yielded similar results.
Example 17
NGF and the Response to OP-1
[0187] Since NGF regulates the growth of sympathetic dendrites in
situ, the possibility that it might exert a similar action in vitro
was investigated. For this study, murine NGF was purified from
mouse salivary glands according to standard protocols. Recombinant
NGF also could have been used. Since dendritic growth was not
observed in the control medial which contained high levels of NGF,
it seemed reasonable that NGF might function as a modulator rather
than an inducer of dendritic growth. Neurons were, therefore,
exposed to a maximally effective concentration of OP-1 while the
concentration of NGF was varied. As the concentration of NGF was
decreased from 100 ng/ml to 0.3 ng/ml (FIG. 7), there was a dose
dependent decrease in the mean number of dendrites/cell, the
percentage of cells with dendrites, and the length of the longest
dendrite. Cell number decreased over this same concentration range
and the half-maximal concentration for cell survival differed less
than 2-fold from that for either the percentage of cells with
dendrites or the mean number of dendrites. These data suggest NGF
is a necessary cofactor for OP-1 induced dendritic growth and that
the amount of dendritic growth that occurs is highly dependent on
the trophic state of the cell.
Example 18
OP-1 and Neuronal Survival
[0188] A separate study investigated the effects of OP-1 on
neuronal survival in the primary culture system described in
Example 13 above. After 48 hrs, the number of neurons in OP-1
treated cultures was less than 3% of that in NGF-treated cultures
and by 72 hrs all neurons in the OP-1 cultures had died. Thus, the
effects of OP-1 are distinct from those NGF. In particular, NGF was
significantly more effective in promoting survival of primary
symphathetic neurons in culture.
Example 19
Comparison of Dendritic Growth In Vitro and In Situ
[0189] To assess the potential importance of OP-1 as a regulatory
molecule, the extent of dendritic growth that occurs in vitro was
compared to that which normally occurs during a comparable period
in situ (Table III). For the former, cultures were used that had
been exposed to optimal concentrations of OP-1 and NGF for 2 weeks
(and then immunostained with a dendrite-specific mAb). For the
latter, published in situ data (Snider (1988), 8 J. Neurosci.
2628-2634) was used that relates particularly to the dendrites of
superior cervical ganglion neurons of 2-week postnatal rats. Cells
exposed to OP-1 in vitro were at least as complex as their
counterparts in situ. They had about the same number of dendrites
as cells in situ but the length of their dendritic arbor was 38%
greater. In addition, the maximum extent of the arbor and
complexity of the branching pattern were increased in vitro. Since
these experiments were performed in a chemically defined medium, it
appears that the combination OP-1 and NGF presents a sufficient
condition for establishment of a normal dendritic arbor.
Example 20
Screening Assay for Candidate Compounds which Alter Endogenous
Morphogen and Nerve Trophic Factor Levels
[0190] The following screening assay can be used to establish
whether candidate compound(s) can affect the production (gene
expression and/or protein secretion) of a given morphogen by cells
competent to produce said morphogen. Cells that are competent to
produce a desired morphogen include, within their genetic material,
a gene encoding the desired morphogen. This morphogen gene is
preferably a cellular gene naturally present in the cells, but can
also be a foreign gene introduced by standard genetic engineering
techniques. The morphogen gene is in operative association with
nucleic acid expression regulatory elements thereof, including but
not necessarily limited to a promoter element. Thus, such cells,
although not necessarily actively expressing the morphogen gene,
are competent to express this gene when appropriately stimulated by
a candidate morphogen stimulating agent. The desired morphogen can
be a known morphogen (e.g., OP1, OP2 or other protein listed in
Table II or otherwise described herein) or a new, previously
unidentified morphogen. In the following screening assay, the level
of morphogen production by competent cells which can produce
measurable levels of the morphogen is determined with and without
incubating the cell in culture with the compound, in order to
assess the effects of the compound on the cell. This can be
accomplished by detection of the morphogen either at the protein or
RNA level. Classes of candidate compounds that can be tested for
morphogen stimulating activity include but are not limited to
biological response modifiers (lymphokines, cytokines, hormones,
vitamins, neurotransmitters, chemotrophic factors, chemoattractants
and other cell products), plant extracts and/or phytochemicals,
microbial products, including fungal and bacterial products, body
fluids, tissue or organ extracts, and broth or medium conditioned
by living cells of prokaryotic or eukaryotic origin. Other classes
of compounds that can be assessed for morphgen-stimulating activity
include naturally sourced or synthetic compounds with known or
suspected pharmacologic activity. Thus, for example, Ishibashi et
al. (1993), 193 Biochem. Biophys. Res. Comm. 235-239 (the
disclosure of which is hereby incorporated by reference), teaches
that naturally sourced and synthetic modulators of protein kinase A
and protein kinase C expression and/or activity can affect the
expression of OP1 (referred to in the reference as BMP-7). The
specific modulators tested in Ishibashi et al. included Forskolin,
8-bromo-cyclic AMP and phorbolmyristyl acetate (PMA). A more
detailed description of the screening assay also can be found in
international application US92/07359 (WO92/05172), the teachings of
which are incorporated herein by reference.
20.1 Growth of Cells in Culture
[0191] Primary or immortalized cultures of cells of kidney, adrenal
glands, bladder, brain, or other tissue specific origin can be
prepared as described widely in the literature. For example,
kidneys can be explanted from neonatal or new born or young or
adult rodents (mouse or rat) and used in organ culture as whole or
sliced (1-4 mm) tissues. Primary tissue cultures and established
cell lines, also derived from kidney, adrenals, urinary, bladder,
brain, mammary, or other tissues can be established in multiwell
plates (6 well or 24 well) according to conventional cell culture
techniques, and are cultured in the absence or presence of serum
for a period of time (1-7 days). Cells can be cultured, for
example, in Dulbecco's Modified Eagle medium (Gibco, Long Island,
N.Y.) containing serum (e.g., fetal calf serum at 1%-10%, Gibco) or
in serum-deprived medium, as desired, or in defined medium (e.g.,
containing insulin, transferrin, glucose, albumin, or other growth
factors).
[0192] Samples for testing the level of morphogen production
include culture supernatants or cell lysates, collected
periodically prior to and following exposure to one or more
concentrations of a known or candidate morphogen-stimulating agent,
and evaluated for morphogen production by immunoblot analysis with
an appropriate specific antibody or fragment thereof. (Sambrook et
al., eds., 1989, Molecular Cloning, Cold Spring Harbor Press, Cold
Spring Harbor, N.Y.). Alternatively, samples can be used to prepare
polyA+ RNA for RNA analysis with an appropriate morphogen specific
probe. To monitor de novo morphogen synthesis, some cultures are
labeled according to conventional procedures with an
.sup.35S-methionine/.sup.35S-cysteine mixture for 6-24 hours and
then evaluated to OP-1 synthesis by conventional
immunoprecipitation methods. Optionally, samples can be assayed for
morphogenic activity using a suitable bioassay therefor, (e.g., an
in vitro bioassay for indicia of tissue-specific morphogenesis.
Such as bioassay system can be particularly suitable for
investigating expression of novel or previously uncharacterized
morphogens.
20.2 Determination of Level of Morphogenic Protein
[0193] In order to quantitate the production of a morphogenic
protein by a cell type, an immunoassay can be performed to detect
the morphogen using a polyclonal or monoclonal antibody specific
for that protein. For example, OP-1 can be detected using a
polyclonal antibody specific for OP-1 in an ELISA, as follows.
[0194] 1 .mu.g/100 .mu.l of affinity-purified polyclonal rabbit IgG
specific for OP-1 is added to each well of a 96-well plate and
incubated at 37.degree. C. for an hour. The wells are washed four
times with 0.167M sodium borate buffer with 0.15 M NaCl (BSB), pH
8.2, containing 0.1% Tween 20. To minimize non-specific binding,
the wells are blocked by filling completely with 1% bovine serum
albumin (BSA) in BSB and incubating for 1 hour at 37.degree. C. The
wells are then washed four times with BSB containing 0.1% Tween 20.
A 100 .mu.l aliquot of an appropriate dilution of each of the test
samples of cell culture supernatant is added to each well in
triplicate and incubated at 37.degree. C. for 30 min. After
incubation, 100 .mu.l biotinylated rabbit anti-OP-1 serum (stock
solution is about 1 mg/ml and diluted 1:400 in BSB containing 1%
BSA before use) is added to each well and incubated at 37.degree.
C. for 30 min. The wells are then washed four times with BSB
containing 0.1% Tween 20. 100 .mu.l strepavidin-alkaline (Southern
Biotechnology Associates, Inc. Birmingham, Ala., diluted 1:2000 in
BSB containing 0.1% Tween 20 before use) is added to each well and
incubated at 37.degree. C. for 30 min. The plates are washed four
times with 0.5M Tris buffered Saline (TBS), pH 7.2. 50 .mu.l
substrate (ELISA Amplification System Kit, Life Technologies, Inc.,
Bethesda, Md.) is added to each well incubated at room temperature
for 15 min. Then, 50 .mu.l amplifier (from the same amplification
system kit) is added and incubated for another 15 min at room
temperature. The reaction is stopped by the addition of 50 .mu.l
0.3 M sulphuric acid. The OD at 490 nm of the solution in each well
is recorded. To quantitate OP-1 in culture media, a OP-1 standard
curve is performed in parallel with the test samples.
[0195] Polyclonal antibody can be prepared as follows. Each rabbit
is given a primary immunization of 100 .mu.g/500 .mu.l E. coli
produced OP-1 monomer (amino acids 328-431 in SEQ ID NO:5) in 0.1%
SDS mixed with 500 .mu.l Complete Freund's Adjuvant. The antigen is
injected subcutaneously at multiple sites on the back and flanks of
the animal. The rabbit is boosted after a month in the same manner
using incomplete Freund's Adjuvant. Test bleeds are taken from the
ear vein seven days later. Two additional boosts and test bleeds
are performed at monthly intervals until antibody against OP-1 is
detected in the serum using an ELISA assay. Then, the rabbit is
boosted monthly with 100 .mu.g of antigen and bled (15 ml per
bleed) at days seven and ten after boosting.
[0196] Monoclonal antibody specific for a given morphogen can be
prepared as follows. A mouse is given two injections of E. coli
produced OP-1 monomer. The first injection contains 100 .mu.g of
OP-1 in complete Freund's adjuvant and is given subcutaneously. The
second injection contains 50 .mu.g of OP-1 in incomplete adjuvant
and is given intraperitoneally. The mouse then receives a total of
230 .mu.g of OP-1 (amino acids 307-431 in SEQ ID NO:5) in four
intraperitoneal injections at various times over an eight month
period. One week prior to fusion, both mice are boosted
intraperitoneally with 100 .mu.g of OP-1 (307-431) and 30 .mu.g of
the N-terminal peptide (Ser.sub.293-Asn.sub.309-Cys) conjugated
through the added cysteine to bovine serum albumin with SMCC
crosslinking agent. This boost was repeated five days (IP), four
days (IP), three days (IP) and one day (IV) prior to fusion. The
mouse spleen cells are then fused to myeloma (e.g., 653) cells at a
ratio of 1:1 using PEG 1500 (Boeringer Mannheim), and the cell
fusion is plated and screened for OP-1-specific antibodies using
OP-1 (307-431) as antigen. The cell fusion and monoclonal screening
then are according to standard procedures well described in
standard texts widely available in the art.
20.3 Determination of Level of Nerve Trophic Factor
[0197] In a manner analogous to that described above, substances
which stimulate production of nerve trophic factors can also be
identified by the skilled artisan. See, e.g., Ebendal et al.
(1991), Plasticity and Regeneral of the Nervous System, pp. 207-225
(Timeras et al., eds.; Plenum Press, N.Y.; Caswell (1993), 124 Exp.
Neurol. 36, the teachings of both references being herein
incorporated by reference). Cells that are competent to produce a
desired nerve trophic factor include, within their genetic
material, a gene encoding the desired factor. This nerve trophic
factor gene is preferably a cellular gene naturally present in the
cells, but can also be a foreign gene introduced by standard
genetic engineering techniques. The nerve trophic factor gene is in
operative association with nucleic acid expression regulatory
elements thereof, including but not necessarily limited to a
promoter element. Thus, such cells, although not necessarily
actively expressing the nerve trophic factor gene, are competent to
express this gene when appropriately stimulated by a candidate
nerve trophic factor stimulating agent. In the exemplary assay, the
level of nerve trophic factor production is determined with and
without incubating the cell in culture with the putative
stimulating agent in order to assess the effects of the agent on
the cell. This can be accomplished by detection of the nerve
trophic factor either at the protein or RNA level. Classes of
candidate compounds that can be tested for stimulating activity
include but are not limited to those discussed above.
[0198] The invention can be embodied in other specific forms
without departing from the spirit or essential characteristics
thereof. The present embodiments are therefore to be considered in
all respects as illustrative and not restrictive, the scope of the
invention being indicated by the appended claims rather than by the
foregoing description, and all changes which come within the
meaning and range of equivalency of the claims are therefore
intended to be embraced therein.
Sequence CWU 1
1
* * * * *