U.S. patent application number 10/806852 was filed with the patent office on 2005-03-24 for methods and compositions for the treatment of motor neuron injury and neuropathy.
Invention is credited to Cohen, Charles M., Oppermann, Hermann, Pang, Roy H.L., Rueger, David C., Sampath, Kuber T..
Application Number | 20050065083 10/806852 |
Document ID | / |
Family ID | 46276184 |
Filed Date | 2005-03-24 |
United States Patent
Application |
20050065083 |
Kind Code |
A1 |
Rueger, David C. ; et
al. |
March 24, 2005 |
Methods and compositions for the treatment of motor neuron injury
and neuropathy
Abstract
Disclosed are therapeutic treatment methods, compositions and
devices 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, and stimulating
neurons to maintain their differentiated phenotype. 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.
Inventors: |
Rueger, David C.;
(Southborough, MA) ; Sampath, Kuber T.;
(Holliston, MA) ; Oppermann, Hermann; (Medway,
MA) ; Pang, Roy H.L.; (Etna, NH) ; Cohen,
Charles M.; (Weston, MA) |
Correspondence
Address: |
ROPES & GRAY LLP
ONE INTERNATIONAL PLACE
BOSTON
MA
02110-2624
US
|
Family ID: |
46276184 |
Appl. No.: |
10/806852 |
Filed: |
March 23, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10806852 |
Mar 23, 2004 |
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08937755 |
Sep 25, 1997 |
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6723698 |
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08937755 |
Sep 25, 1997 |
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08260675 |
Jun 16, 1994 |
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6800603 |
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08260675 |
Jun 16, 1994 |
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08126100 |
Sep 23, 1993 |
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08126100 |
Sep 23, 1993 |
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07922813 |
Jul 31, 1992 |
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07922813 |
Jul 31, 1992 |
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07752764 |
Aug 30, 1991 |
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07922813 |
Jul 31, 1992 |
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07753059 |
Aug 30, 1991 |
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07753059 |
Aug 30, 1991 |
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07667274 |
Mar 11, 1991 |
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Current U.S.
Class: |
514/8.8 ;
514/17.7; 514/19.1; 514/8.3 |
Current CPC
Class: |
A61K 38/1875 20130101;
G01N 2500/10 20130101; A61K 38/17 20130101; A61L 27/227 20130101;
A01N 1/0226 20130101; A61F 2310/00365 20130101; C07K 14/495
20130101; C07K 16/22 20130101; A01N 1/0205 20130101; C07K 14/51
20130101; A01N 1/02 20130101; A61K 38/1703 20130101; A61L 27/24
20130101; A61K 38/1875 20130101; A61K 2300/00 20130101 |
Class at
Publication: |
514/012 |
International
Class: |
A61K 038/17 |
Claims
1-9. (Canceled)
10. A method of preserving motor function in a mammal with symptoms
of or at risk of amyotrophic lateral sclerosis, comprising
administering to said mammal a morphogen, wherein the morphogen:
(1) comprises a dimeric protein having an amino acid sequence with:
(a) at least 70% homology with the C-terminal seven-cysteine
skeleton of human OP-1, residues 330-431 of SEQ ID NO: 2; (b)
having greater than 60% amino acid sequence identity with said
C-terminal seven-cysteine skeleton of human OP-1; (c) defined by
Generic Sequence 7, SEQ ID NO: 4; (d) defined by Generic Sequence
8, SEQ ID NO: 5; (e) defined by Generic Sequence 9, SEQ ID NO: 6;
(f) defined by Generic Sequence 10, SEQ ID NO: 7; or (g) defined by
OPX, SEQ ID NO: 3; and (2) stimulates production of an N-CAM or L1
isoform by an NG108-15 cell in vitro; whereby motor function is
preserved in said mammal.
11. (Canceled)
12. A method of preserving motor function in a mammal with symptoms
of or at risk of a spinal cord injury, comprising administering to
said mammal a morphogen, wherein the morphogen: (1) comprises a
dimeric protein having an amino acid sequence with: (a) at least
70% homology with the C-terminal seven-cysteine skeleton of human
OP-1, residues 330-431 of SEQ ID NO: 2; (b) greater than 60% amino
acid sequence identity with said C-terminal seven-cysteine skeleton
of human OP-1; (c) defined by Generic Sequence 7, SEQ ID NO: 4; (d)
defined by Generic Sequence 8, SEQ ID NO: 5; (e) defined by Generic
Sequence 9, SEQ ID NO: 6; (f) defined by Generic Sequence 10, SEQ
ID NO: 7; or (g) defined by OPX, SEQ ID NO: 3; and (2) stimulates
production of an N-CAM or L1 isoform by an NG108-15 cell in vitro;
whereby motor function is preserved in said mammal.
13-18. (Canceled)
19. A method of preserving motor function in a mammal with symptoms
of or at risk of amyotrophic lateral sclerosis, comprising
administering to said mammal a morphogen selected from: human OP-1,
mouse OP-1, human OP-2, mouse OP-2,60A, GDF-1, BMP2A, BMP2B, DPP,
Vgl, Vgr-1, BMP3, BMP5, or BMP6, wherein said morphogen stimulates
production of an N-CAM or L1 isoform by an NG108-15 cell in vitro
whereby motor function is preserved in said mammal.
20. (Canceled)
21. A method of preserving motor function in a mammal with symptoms
of or at risk of a spinal cord injury, comprising administering a
morphogen selected from: human OP-1, mouse OP-1, human OP-2, mouse
OP-2, 60A, GDF-1, BMP2A, BMP2B, DPP, Vgl, Vgr-1, BMP3, BMP5, or
BMP6, wherein said morphogen stimulates production of an N-CAM or
L1 isoform by an NG108-15 cell in vitro whereby motor function is
preserved in said mammal.
22-23. (Canceled)
24. The method of claim 10, wherein the morphogen comprises a
dimeric protein having an amino acid sequence with at least 70%
homology with the C-terminal seven-cysteine skeleton of human OP-1,
residues 330-431 of SEQ ID NO: 2.
25. The method of claim 10, wherein the morphogen comprises a
dimeric protein having an amino acid sequence with greater than 60%
amino acid sequence identity with said C-terminal seven-cysteine
skeleton of human OP-1.
26. The method of claim 12, wherein the morphogen comprises a
dimeric protein having an amino acid sequence with at least 70%
homology with the C-terminal seven-cysteine skeleton of human OP-1,
residues 330-431 of SEQ ID NO: 2.
27. The method of claim 12, wherein the morphogen comprises a
dimeric protein having an amino acid sequence with greater than 60%
amino acid sequence identity with said C-terminal seven-cysteine
skeleton of human OP-1.
Description
RELATED APPLICATIONS
[0001] The present application is a continuation-in-part of U.S.
Ser. No. 08/260,675, filed Jun. 16, 1994, which is a file wrapper
continuation of U.S. Ser. No. 08/126,100, filed Sep. 23, 1993,
which is a file wrapper continuation of U.S. Ser. No. 07/922,813,
filed July 31, 1992 filed as a continuation-in-part of U.S. Ser.
No. 07/752,764 and copending U.S. Ser. No. 07/753,059, both filed
Aug. 30, 1991 as continuations-in-part of U.S. Ser. No. 07/667,274,
filed March 11, 1991. The above-mentioned applications are
incorporated by reference herein.
BACKGROUND OF THE INVENTION
[0002] 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 th e CNS, also provide a myelin sheath that
surrounds neural processes. The myelin sheath enables rapid
conduction along the neuron. In the peripheral nervous system,
axons of multiple neurons may bundle together in order to form a
nerve fiber. These, in turn, may be combined into fascicles or
bundles.
[0003] During development, differentiating neurons from the central
and peripheral nervous systems send out axons that grow and make
contact with specific target cells. In some cases, axons must cover
enormous distances; some grow into the periphery, whereas others
are confined within the central nervous system. In mammals, this
stage of neurogenesis is complete during the embryonic phase of
life and neuronal cells do not multiply once they have fully
differentiated.
[0004] A host of neuropathies have been identified that affect the
nervous system. The neuropathies, which may affect neurons
themselves or associated glial cells, may result from cellular
metabolic dysfunction, infection, exposure to toxic agents,
autoimmunity, malnutrition, or ischemia. In some cases, the
cellular neuropathy 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 immune
response to the initial injury then destroys neural pathways.
[0005] 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.
[0006] 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
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.
[0007] 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 cerbrospinal 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.
[0008] One type of morphoregulatory molecule associated with
neuronal cell growth, differentiation and development is the cell
adhesion molecule ("CAM"), most notably the nerve cell adhesion
molecule (N-CAM). The CAMs are members the immunoglobulin
super-family. They 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 have been identified. Of
these, the most thoroughly studied 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
demyelinating mutant. Bhat, Brain Res. 452: 373-377 (1988). Reduced
levels of N-CAM also have been associated with normal pressure
hydrocephalus, Werdelin, Acta Neurol. Scand. 79: 177-181 (1989),
and with type II schizophrenia. Lyons, et al., Biol. Psychiatry 23:
769-775 (1988). In addition, antibodies against N-CAM have been
shown to disrupt functional recovery in injured nerves. Remsen,
Exp. Neurobiol. 110: 268-273 (1990).
[0009] Currently no satisfactory method exists to repair the damage
caused by traumatic injuries of motor neurons and diseases of motor
neurons.
[0010] There are 15,000 to 18,000 new cases of spinal cord injury
each year in the United States. In addition, there are
approximately 200,000 survivors of spinal cord injury. The annual
cost of care for these patients exceeds $7 billion. The
pathophysiology following acute spinal cord trauma is a complex and
not fully understood mechanism. The primary tissue damage caused by
mechanical trauma occurs immediately and is irreversible. Allen, J.
Am. Med. Assoc. 57: 878-880 (1911). Experimental evidence indicates
that much of the post-traumatic tissue damage is the result of a
reactive process that begins within minutes after the injury and
continues for days or weeks. Janssen, et al., Spine 14: 23-32
(1989) and Panter, et al., (1992). This progressive,
self-destructive process includes pathophysiological mechanisms
such as hemorrhage, post-traumatic ischemia, edema, axonal and
neuronal necrosis, and demyelinization followed by cyst formation
and infarction. For review, see Tator, et al., J. Neurosurg, 75:
15-26 (199 i) and Faden, Crit. Rev. Neurobiol. 7: 175-186 (1993).
Proposed injurious factors include electrolyte changes whereby
increased intracellular calcium initiates a cascade of events
(Young, J. Neurotrauma 9, Suppl. 1: S9-S25 (1992) and Young, J.
Emerg. Med. 11: 13-22 (1993)), biochemical changes with
uncontrolled transmitter release (Liu, et al., Cell 66: 807-815
(1991) and Yanase, et al., J. Neurosurg. 83: 884-888 (1995),
arachidonic acid release, free-radical production, lipid
peroxidation (Braughler, et al, J. Neurotrauma 9, Suppl. 1: S1-S7
(1992), eicosanoid production (Demediuk, et al., J. Neurosci. Res.
20: 115-121 (1988), endogenous opioids (Faden, et al., Ann Neurol.
17: 386-390 (1985), metabolic changes including alterations in
oxygen and glucose (Faden, Crit. Rev. Neurobiol. 7: 175-186
(1993)), inflammatory-changes (Blight, J. Neurotrauma 9, Suppl. 1:
S83-S91 (1992), and astrocytic edema (Kinelberg, J. Neurotrauma 9,
Suppl. 1: S71-S81 (1992). For the past 400 years surgical
approaches including laminectomy and decompression, accompanied by
fusion, have been the most commonly practiced treatment strategies.
Hansebout, "Early Management of Acute Spinal Cord Injury", pp.
181-196 (1982) and Janssen, et al., Spine 14: 23-32 (1989).
However, these procedures have not involved the application of
techniques to augment the regenerative properties of spinal cord
tissue.
[0011] A host of diseases of motor neurons have been identified,
including demyelinating diseases, myelopathies, and diseases of
motor neurons such as amyotrophic lateral sclerosis (ALS). INTERNAL
MEDICINE, ch. 121-123 (4th ed., J. H. Stein, ed., Mosby, 1994).
Multiple sclerosis (MS) is the most common demyelinating disorder
of the central nervous system, causing patches of sclerosis (i.e.,
plaques) in the brain and spinal cord. MS has protean clinical
manifestations, depending upon the location and size of the plaque.
Typical symptoms include visual loss, diplopia, nystagmus,
dysarthria, weakness, paresthesias, bladder abnormalities, and mood
alterations. Myriad treatments have been proposed for this
long-term variable illness. The list of proposed treatments
encompasses everything from diet to electrical stimulation to
acupuncture, emotional support, and various forms of
immunosupressive therapy. None have proved to be satisfactory.
[0012] Progressive loss of lower and upper motor neurons occurs in
several diseases (e.g., primary lateral sclerosis, spinal muscular
atrophy, benign focal amyotrophy). However, ALS is the most common
form of motor neuron disease. Loss of both lower and upper motor
neurons occur in ALS. Symptoms include progressive skeletal muscle
wasting, weakness, gasciculations, and cramping. Some cases have
predominant involvement of brainstem motoneurons (progressive
bulbar palsy). Unfortunately, treatment of motor neuron and related
diseas is largely supportive at this time. INTERNAL MEDICINE, ch.
123 (4th ed., J. H. Stein, ed., Mosby, 1994).
[0013] Accordingly, there is a need in the art for treatments of
motor neurons disorders and injuries, and related deficits in
neural functions.
SUMMARY OF THE INVENTION
[0014] The present invention provides methods and compositions for
maintaining neural pathways in a mammal in vivo, including methods
for enhancing the survival of neural cells.
[0015] In a preferred embodiment, methods of the invention for
treating motor neuron defects, including amyotrophic lateral
sclerosis, multiple sclerosis, and spinal cord injury comprise
administering a morphogen comprising a dimeric protein having an
amino acid sequence selected from the group consisting of a
sequence have 70% homology with the C-terminal seven-cysteine
skeleton of human OP-1 (amino acids 330-341 of SEQ ID NO: 2), a
sequence having greater than 60% amino acid sequence identity with
human OP-1; generic sequence 7 (SEQ ID NO: 4); generic sequence 8
(SEQ ID NO: 6); generic sequence 10 (SEQ ID NO: 7); and OPX (SEQ ID
NO: 3); wherein the morphogen stimulates production of N-CAM or L1
isoforms by an NG108-15 cell in vivo. Spinal cord injuries include
injuries resulting from a tumor, mechanical trauma, and chemical
trauma. The same or similar methods are contemplated to restore
motor function in a mammal having amyotrophic lateral sclerosis,
multiple sclerosis, or a spinal cord injury. Administering one of
the aforementioned morphogens also provides a prophylactic
function. Such administration has the effect of preserving motor
function in a mammal having, or at risk of having, amyotrophic
lateral sclerosis, multiple sclerosis, or a spinal cord injury.
Also according to the invention, morphogen administration preserves
the integrity of the nigrostriatal pathway.
[0016] Specifically, methods of the invention for treating (pre- or
post-symptomatically) amyotrophic lateral sclerosis, multiple
sclerosis, or a spinal cord injury comprise administering a
morphogen selected from the group consisting of human OP-1, mouse
OP-1, human OP-2, mouse OP-2, 60A, GDF-1, BMP2A, BMP2B, DPP, Vgl,
Vgr-1, BMP3, BMP5, and BMP6. Such morphogens are capable of
stimulating production of N-CAM or L1 isoform by an NG108-15 cell
in vivo.
[0017] In a particularly-preferred embodiment, the morphogen is a
soluble complex, comprising at least one morphogen pro domain, or
fragment thereof, non-covalently attached to a mature
morphogen.
[0018] In one aspect, the invention features compositions and
therapeutic treatment methods comprising administering to a mammal
a therapeutically effective amount of a morphogenic protein
("morphogen"), as defined herein, upon injury to a neural pathway,
or in anticipation of such injury, for a time and at a
concentration sufficient to maintain the neural pathway, including
repairing damaged pathways, or inhibiting additional damage
thereto.
[0019] In another aspect, the invention features compositions and
therapeutic treatment methods for maintaining neural pathways. Such
treatment methods include administering to the mammal, upon injury
to a neural pathway or in anticipation of such injury, a compound
that stimulates a therapeutically effective concentration of an
endogenous morphogen. 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 capable of,
producing a morphogen and/or secreting a morphogen, and which cause
endogenous level of the morphogen to be altered.
[0020] In particular, the invention provides methods for protecting
neurons from the tissue destructive effects associated with the
body's immune and inflammatory response to nerve injury. The
invention also provides methods for stimulating neurons to maintain
their differentiated 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, particularly nerve cell adhesion
molecules (N-CAM). The invention also provides methods,
compositions and devices for stimulating cellular repair of damaged
neurons and neural pathways, including regenerating damaged
dendrites or axons. In addition, the invention also provides means
for evaluating the status of nerve tissue, and for detecting and
monitoring neuropathies by monitoring fluctuations in morphogen
levels.
[0021] In one aspect of the invention, the morphogens described
herein are useful in repairing damaged neural pathways of the
peripheral nervous system. In particular, morphogens are useful for
repairing damaged neural pathways, including transected or
otherwise damaged nerve fibers. Specifically, the morphogens
described herein are capable of stimulating complete axonal nerve
regeneration, including vascularization and reformation of the
myelin sheath. Preferably, the morphogen preferably is provided to
the site of injury in a biocompatible, bioresorbable carrier
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. 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. A preferred carrier comprises an extracellular matrix
composition derived, for example, from mouse sarcoma cells.
[0022] In a particularly preferred embodiment, a 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 neurite. Useful
channels comprise a biocompatible membrane, which may be tubular in
structure, having a dimension sufficient to span the gap in the
nerve to be repaired, and having openings adapted to receive
severed nerve ends. The 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 preferred
embodiment, the outer surface of the channel is substantially
impermeable.
[0023] 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.
[0024] Morphogens described herein are also useful in autologous
peripheral nerve segment implants, such as in the repair of damaged
or detached retinas, or other damage to the optic nerve.
[0025] In another aspect of the invention, morphogens described
herein are useful to protect against 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, 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 a morphogen,
either directly or systemically 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, may also 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.
[0026] Generally, morphogens useful in methods and compositions of
the invention are dimeric proteins that induce morphogenesis of one
or more eukaryotic (e.g., mammalian) cells, tissues or organs.
Tissue morphogenesis includes de novo or regenerative tissue
formation, such as occurs in a vertebrate embryo during
development. Of particular interest are morphogens that induce
tissue-specific morphogenesis at least of bone or neural tissue. As
defined herein, a morphogen comprises a pair of polypeptides that,
when folded, form a dimeric protein that elicits morphogenetic
responses in cells and tissues displaying morphogen-specific
receptors. That is, the morphogens generally induce a cascade of
events including all of the following 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.
"Progenitor" cells are uncommitted cells that are competent to
differentiate into one or more specific types of differentiated
cells, depend ing on their genomic repertoire and the tissue
specificity of the permissive environment in which morphogenesis is
induced. An exemplary progenitor cell is a hematopoeitic stem cell,
a mesenchymal stem cell, a basement epithelium cell, a neural crest
cell, or the like. Further, morphogens can delay or mitigate the
onset of senescence- or quiescence-associated loss of phenotype
and/or tissue function. Still further, morphogens can stimulate
phenotypic expression of a differentiated cell type, including
expression of metabolic and/or functional, e.g., secretory,
properties thereof. In addition, morphogens can induce
redifferentiation of committed cells (e.g., osteoblasts,
neuroblasts, or the like) under appropriate conditions. As noted
above, morphogens that induce proliferation and/or differentiation
at least of bone or neural tissue, and/or support the growth,
maintenance and/or functional properties of neural tissue, are of
particular interest herein. See, e.g., WO 92/15323, WO 93/04692, WO
94/03200 (providing more detailed disclosures as to the tissue
morphogenic properties of these proteins).
[0027] As used herein, the terms "morphogen," "bone morphogen,"
"bone morphogenic protein," "BMP," "morphogenic protein" and
"morphogenetic protein" all embrace the class of proteins typified
by human osteogenic protein 1 (hOP-1). Nucleotide and amino acid
sequences for hOP-1 are provided in SEQ ID NOS: 1 and 2,
respectively. For ease of description, hOP--I is considered a
representative morphogen. It will be appreciated that OP-1 is
merely representative of the TGF-.beta. subclass of true tissue
morphogens and is not intended to limit the description. Other
known and useful morphogens include, but are not limited to, BMP-2,
BMP-3, BMP-3b, BMP-4, BMP-5, BMP-6, BMP-8, BMP-9, BMP-10, BMP-11,
BMP-12, BMP-13, BMP-15, GDF-1, GDF-2, GDF-3, GDF-5, GDF-6, GDF-7,
GDF-8, GDF-9, GDF-10, GDF-11, GDF-12, 60A, NODAL, UNIVIN, SCREW,
ADMP, and NEURAL, and morphogenically-active amino acid variants of
any thereof.
[0028] In specific embodiments, useful morphogens include those
sharing the conserved seven cysteine skeleton, and sharing at least
70% amino acid sequence homology (similarity), within the
C-terminal seven-cysteine skeleton of human OP-1, residues 330-431
of SEQ ID NO: 2 (hereinafter referred to as the "reference
sequence"). In another embodiment, the invention encompasses use of
biologically active species (phylogenetic) variants of any of the
morphogenic proteins recited herein, including conservative amino
acid sequence variants, proteins encoded by degenerate nucleotide
sequence variants, and morphogenically-active proteins sharing the
conserved seven cysteine skeleton as defined herein and encoded by
a DNA competent to hybridize under standard stringency conditions
to a DNA encoding a morphogenic protein disclosed herein,
including, without limitation, OP-1 or BMP-2 or BMP-4. Presently,
however, the reference sequence is that of residues 330-431 of SEQ
ID NO: 2 (OP-1).
[0029] In still another embodiment, morphogens useful in methods
and compositions of the invention are defined as
morphogenically-active proteins having any one of the generic
sequences defined herein, including OPX (SEQ ID NO: 3) and Generic
Sequences 7 and 8 (SEQ ID NOS: 4 and 5, respectively), or Generic
Sequences 9 and 10 (SEQ ID NOS: 6 and 7, respectively). OPX
encompasses the observed variation between the known phylogenetic
counterparts of the osteogenic OP-1 and OP-2 proteins, and is
described by the amino acid sequence presented herein below and in
SEQ ID NO: 3. Generic Sequence 9 is a 96 amino acid sequence
containing the C-terminal six cysteine skeleton observed in hOP-1
(residues 335-431 of SEQ ID NO: 2) and wherein the remaining
residues encompass the observed variation among OP-1, OP-2, OP-3,
BMP-2, BMP-3, BMP-4, BMP-5, BMP-6, BMP-8, BMP-9, BMP-10, BMP-11,
BMP-15, GDF-1, GDF-3, GDF-5, GDF-6, GDF-7, GDF-8, GDF-9, GDF-10,
GDF-11, 60A, UNIVIN, NODAL, DORSALIN, NEURAL, SCREW and ADMP. That
is, each of the non-cysteine residues is independently selected
from the corresponding residue in this recited group of known,
naturally-sourced proteins. Generic Sequence 10 is a 102 amino acid
sequence which includes a five amino acid sequence added to the
N-terminus of the Generic Sequence 9 and defines the seven cysteine
skeleton observed in hOP-1 (330-431 SEQ ID NO: 2). Generic
Sequences 7 and 8 are 96 and 102 amino acid sequences,
respectively, containing either the six cysteine skeleton (Generic
Sequence 7) or the seven cysteine skeleton (Generic Sequence 8)
defined by hOP-1 and wherein the remaining non-cysteine residues
encompass the observed variation among OP-1, OP-2, OP-3, BMP-2,
BMP-3, BMP4, 60A, DPP, Vgl, BMP-5, BMP-6, Vgr-1, and GDF-1.
[0030] Of particular interest are morphogens which, when provided
to a specific tissue of a mammal, induce tissue-specific
morphogenesis or maintain the normal state of differentiation and
growth of that tissue. In preferred embodiments, the present
morphogens induce the formation of vertebrate (e.g., avian or
mammalian) body tissues, such as but not limited to nerve, eye,
bone, cartilage, bone marrow, ligament, tooth dentin, periodontium,
liver, kidney, lung, heart, or gastrointestinal lining. Preferred
methods may be carried out in the context of developing embryonic
tissue, or at an aseptic, unscarred wound site in post-embryonic
tissue. Methods of identifying such morphogens, or morphogen
receptor agonists, are known in the art and include assays for
compounds which induce morphogen-mediated responses (e.g.,
induction of endochondral bone formation, induction of
differentiation of metanephric mesenchyme, and the like). In a
preferred embodiment, morphogens of the invention, when implanted
in a mammal in conjunction with a matrix permissive of bone
morphogenesis, are capable of inducing a developmental cascade of
cellular and molecular events that culminates in endochondral bone
formation. See, U.S. Pat. No. 4,968,590; Sampath, et al., Proc.
Natl. Acad. Sci. USA 80: 6591-6595 (1983), the disclosures of which
are incorporated by reference herein.
[0031] In an alternative preferred embodiment, morphogens of the
invention are also capable of stimulating production of cell
adhesion molecules, including nerve cell adhesion molecules
(N-CAMs). In a preferred embodiment, the present morphogens are
capable of stimulating the production of N-CAM in vitro in NG108-15
cells, which are a preferred model system for assessing neuronal
differentiation, particularly motor neuron differentiation.
[0032] In still other embodiments, an agent which acts as an
agonist of a morphogen receptor may be administered instead of the
morphogen itself. An "agonist" of a receptor is a compound which
binds to the receptor, and for which the result of such binding is
similar to the result of binding the natural, endogenous ligand of
the receptor. That is, the compound must, upon interaction with the
receptor, produce the same or substantially similar transmembrane
and/or intracellular effects as the endogenous ligand. Thus, an
agonist of a morphogen receptor binds to the receptor and such
binding has the same or a functionally similar result as morphogen
binding (e.g., induction of morphogenesis). The activity or potency
of an agonist can be less than that of the natural ligand, in which
case the agonist is said to be a "partial agonist," or it can be
equal to or greater than that of the natural ligand, in which case
it is said to be a "full agonist." Thus, for example, a small
peptide or other molecule which can mimic the activity of a
morphogen in binding to and activating the morphogen's receptor may
be employed as an equivalent of the morphogen. Preferably the
agonist is a full agonist, but partial morphogen receptor agonists
may also be advantageously employed. Such an agonist may also be
referred to as a morphogen "mimic," "mimetic," or "analog."
[0033] Morphogen inducers and agonists can be identified by
mutation, site-specific mutagenesis, combinatorial chemistry, etc.
Such methods are well known in the art. For example, methods of
identifying morphogen inducers or agonists of morphogen receptors
may be found in U.S. Ser. No. 08/478,097 filed Jun. 7, 1995 and
U.S. Ser. No. 08/507,598 filed Jul. 26, 1995, the disclosures of
which are incorporated herein by reference. Candidate morphogen
inducers and agonists are then tested for their ability to induce
endochondral bone formation and preferably, to stimulate N-CAM
production in neurons or in a neuronal model system, such as
NG108-15 cells. Morphogen inducers and agonists identified
according to the present invention are capable of inducing
endochondral bone formation when implanted in a mammal in
conjunction with a matrix permissive of bone morphogenesis and are
capable of stimulating production of N-CAM in vitro.
[0034] The preferred methods, material, and examples that will now
be described are illustrative only and are not intended to be
limiting. Other features and advantages of the invention will be
apparent from the following detailed description, and from the
claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0035] FIG. 1 is a tabular presentation of the percent amino acid
sequence identity and percent amino acid sequence homology
("similarity") that various members of the family of morphogenic
proteins as defined herein share with hOP-1 in the C-terminal seven
cysteine skeleton;
[0036] FIG. 2 (Panels A and B) are photographs illustrating the
ability of morphogen (OP-1) to induce transformed
neuroblastoma.times.glioma cells (Panel 1A) to redifferentiate to a
morphology characteristic of untransformed neurons (Panel 1B);
[0037] FIG. 3A is a line graph depicting a dose response curve for
the induction of the 180 kDa and 140 kDa N-CAM isoforms in
morphogen-treated NG108-15 cells;
[0038] FIG. 3B is a photograph of a Western blot of whole cell
extracts from morphogen-treated NG108-15 cells with an
N-CAM-specific antibody; and
[0039] FIG. 4 is a line graph depicting the mean number of cell
aggregates counted in twenty (20) randomly selected magnified
viewing fields as a function of morphogen concentration.
[0040] FIG. 5 is a photograph of an immunoblot demonstrating the
presence of OP-1 in human serum.
[0041] FIG. 6 is a bar graph comparing the effects of OP-1 and
glial cells on axonal and dendritic length after one day in
vitro.
[0042] FIG. 7 is a bar graph comparing the effects of OP-1 and
glial cells on axonal and dendritic length after three days in
vitro.
[0043] FIG: 8 is a bar graph comparing the effects of OP-1 and
glial cells on dendritic branching after three days in vitro.
[0044] FIG. 9 (Panels A-C) are line graphs depicting 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: the percentage of cells
with dendrites (Panel A); the mean number of dendrites/cell (Panel
B); and the number of axons/cell (Panel C). The bars shown in Panel
B represent the SEM; where bars are not shown, the SEM was smaller
than the size of the symbol. Open symbols, control; filled symbols,
cells supplemented with 100 ng/ml OP-1 during the time course
study.
[0045] FIG. 10 is a line graph depicting the effects of varying
concentrations of OP-1 on dendritic growth. Sympathetic neurons
were exposed to OP-1 in culture for three 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.
[0046] FIG. 11 is a line graph depicting the effects of varying
concentrations of different morphogens on dendritic growth.
Sympathetic neurons were exposed to various concentrations of
BMP-2, OP-1, 60A, BMP-3 or CDMP-2 beginning on the fifth day in
vitro and then immunostained on day 10 with a dendrite-specific
antibody (SMI 32). Data are presented as the mean.+-.SEM. N=30.
[0047] FIG. 12 is a bar graph comparing the effects of OP-1 and
glial cells on synapse formation after three and four days in
vitro.
[0048] FIG. 13 is a bar graph depicting the effects of OP-1
treatment on the size of spinal cord neurons transplanted
intra-ocularly in vivo over a period of four weeks.
[0049] FIG. 14 is a photograph of the neurofilament staining of
intra-ocular cultures. Spinal cord transplant cultures were stained
four weeks post-grafting. Cultures were treated with weekly
injections of vehicle or OP-1.
[0050] FIG. 15 is a photograph of the choline acetyltransferase
staining of intra-ocular cultures. Spinal cord transplant cultures
were stained four weeks post-grafting. Cultures were treated with
weekly injections of vehicle or OP-1.
[0051] FIG. 16 is a bar graph depicting injury severity scores in
the forelimb placing task of sham animals (N=7; black bars),
vehicle-treated animals with traumatic brain injury (N=8; grey
bars), and OP-1-treated animals with traumatic brain injury (10
.mu.g/intracistemal injection; total OP-1 delivered in 2
injections=20 .mu.g/animal; N=7; white bars).
[0052] FIG. 17 is a bar graph depicting failure scores in the beam
walk task of sham animals (N=5; black bars), vehicle-treated
animals with traumatic brain injury (N=5; grey bars), and
OP-1-treated animals with traumatic brain injury (10
.mu.g/intracisternal injection; total OP-1 delivered in 2
injections=20 .mu.g/animal; N=4; white bars).
[0053] FIG. 18 is a bar graph depicting beam latency scores of sham
animals (N=5; black bars), vehicle-treated animals with traumatic
brain injury (N=5; grey bars), and OP-1-treated animals with
traumatic brain injury (10 .mu.g/intracistemal injection; total
OP-1 delivered in 2 injections=20 .mu.g/animal; N=4; white
bars).
DETAILED DESCRIPTION OF THE INVENTION
[0054] It has now been discovered that morphogens enhance survival
of neurons, and maintain neural pathways. As described herein,
morphogens are capable of enhancing survival of neurons,
stimulating neuronal CAM expression, maintaining the phenotypic
expression of differentiated neurons, inducing the
redifferentiation of transformed cells of neural origin, and
stimulating axonal growth over breaks in neural processes,
particularly large gaps in axons. Morphogens also protect against
tissue destruction associated with immunologically-related nerve
tissue damage. Finally, morphogens may be used as part of a method
for monitoring the viability of nerve tissue in a mammal.
[0055] A. Biochemical, Structural and Functional Properties of
Useful Morphogenic Proteins
[0056] As noted above, a protein is morphogenic as defined herein
if it induces the developmental cascade of cellular and molecular
events that culminate in the formation of new, organ-specific
tissue. In a preferred embodiment, a morphogen is a dimeric
protein, each polypeptide component of which has a sequence that
corresponds to, or is functionally equivalent to, at least the
conserved C-terminal six or seven cysteine skeleton of human OP-1,
included in SEQ ID NO: 2, and/or which shares 70% amino acid
sequence homology with OP-1 in this region. The morphogens are
generally competent to induce a cascade of events including the
following, 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. Under appropriate conditions morphogens are
also competent to induce redifferentiation of cells that have
undergone abnormal differentiation. Details of how the morphogens
useful in this invention were identified, as well as a description
on how to make, use and test them for morphogenic activity are
disclosed in numerous publications, including U.S. Pat. Nos.
5,011,691 and 5,266,683, and the international patent application
publications WO 92/15323; WO 93/04692; and WO 94/03200, each of
which are incorporated by reference herein. As disclosed therein,
the morphogens can be purified from naturally-sourced material or
recombinantly produced from prokaryotic or eukaryotic host cells,
using the genetic sequences disclosed therein. Alternatively, novel
morphogenic sequences can be identified following the procedures
disclosed therein.
[0057] The naturally-occurring morphogens share substantial amino
acid sequence homology in their C-terminal sequences (sharing e.g.,
a six or seven cysteine skeleton sequence). Typically, a
naturally-occurring morphogen is translated as a precursor, having
an N-terminal signal peptide sequence, typically less than about 35
residues in length, followed by a "pro" domain that is cleaved to
yield the mature polypeptide, which includes the biologically
active C-terminal skeleton sequence. 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, Nucleic Acids
Research 14: 4683-4691 (1986). The pro polypeptide typically is
about three times larger than the fully processed, mature
C-terminal polypeptide. Under native conditions, the protein is
secreted as a mature dimer and the cleaved pro polypeptide is
thought to remain associated therewith to form a protein complex,
presumably to improve the solubility of the mature dimeric protein.
The complexed form of a morphogen is generally observed to be more
soluble than the mature form under physiological conditions.
[0058] Natural-sourced morphogenic protein in its mature, native
form, is typically a glycosylated dimer, having an apparent
molecular weight of about 30-36 kDa as determined by SDS-PAGE. When
reduced, the 30 kDa protein gives rise to two glycosylated
polypeptide subunits having apparent molecular weights in the range
of about 16 kDa and about 18 kDa. The unglycosylated dimeric
protein, which also has morphogenic activity, typically has an
apparent molecular weight in the range of about 27 kDa. When
reduced, the 27 kDa protein gives rise to two unglycosylated
polypeptides having molecular weights typically in the range of
about 14 kDa to about 16 kDa.
[0059] In preferred embodiments, each of the polypeptide subunits
of a dimeric morphogenic protein as defined herein comprises an
amino acid sequence sharing a defined relationship with an amino
acid sequence of a reference morphogen. In one embodiment,
preferred morphogenic polypeptide chains share a defined
relationship with a sequence present in morphogenically-active
human OP-1, SEQ ID NO: 2. However, any one or more of the
naturally-occurring or biosynthetic morphogenic proteins disclosed
herein similarly could be used as a reference sequence. Preferred
morphogenic polypeptide chains share a defined relationship with at
least the C-terminal six cysteine skeleton of human OP-1, residues
335-431 of SEQ ID NO: 2. Preferably, morphogenic proteins share a
defined relationship with at least the C-terminal seven cysteine
skeleton of human OP-1, residues 330-431 of SEQ ID NO: 2.
[0060] Functionally equivalent sequences include functionally
equivalent arrangements of cysteine residues disposed within the
reference sequence, including amino acid insertions or deletions
which alter the linear arrangement of these cysteines, but do not
materially impair their relationship in the folded structure of the
dimeric morphogen protein, including their ability to form such
intra- or inter-chain disulfide bonds as may be necessary for
morphogenic activity. For example naturally-occurring morphogens
have been described in which at least one internal deletion (of one
residue; BMP2) or insertion (of four residues; GDF-1) is present
but does not abrogate biological activity. Functionally equivalent
sequences further include those wherein one or more amino acid
residues differ from the corresponding residue of a reference
sequence, e.g., the C-terminal seven cysteine skeleton of human
OP-1, provided that this difference does not destroy tissue
morphogenic activity. Accordingly, conservative substitutions of
corresponding amino acids in the reference sequence are preferred.
Amino acid residues that are "conservative substitutions" for
corresponding residues in a reference sequence are those that are
physically or functionally similar to the corresponding reference
residues, e.g., that have similar size, shape, electric charge,
chemical properties including the ability to form covalent or
hydrogen bonds, or the like. Particularly preferred conservative
substitutions are those fulfilling the criteria defined for an
accepted point mutation in Dayhoff, et al., 5 ATLAS OF PROTEIN
SEQUENCE AND STRUCTURE, Suppl. 3, ch. 22 pp. 354-352 (1978), Natl.
Biomed. Res. Found., Washington, D.C. 20007, the teachings of which
are incorporated by reference herein. Examples of conservative
substitutions include the substitution of one amino acid for
another with similar characteristics, e.g., substitutions within
the following groups: valine, glycine; glycine, alanine; valine,
isoleucine, leucine; aspartic acid, glutamic acid; asparagine,
glutamine; serine, threonine; lysine, arginine; and phenylalanine,
tyrosine. The term "conservative substitution" also includes the
use of a synthetic or derivatized amino acid in place of the
corresponding natural parent amino acid, provided that antibodies
raised to the resulting variant polypeptide also immunoreact with
the corresponding naturally sourced morphogen polypeptide.
[0061] The following publications disclose publications morphogen
polypeptide sequences, as well as relevant chemical and physical
properties, of naturally-occurring and/or synthetic morphogens:
OP-1 and OP-2: U.S. Pat. No. 5,011,691, U.S. Pat. No. 5,266,683,
Ozkaynak, et al., EMBO J. 9: 2085-2093 (1990); OP-3: WO 94/10203
(PCT US93/10520); BMP-2, BMP-3, and BMP-4: WO 88/00205, Wozney, et
al., Science 242: 1528-1534 (1988); BMP-5 and BMP-6: Celeste, et
al., PNAS 87: 9843-9847 (1991); Vgr-1: Lyons, et al, PNAS 86:
4554-4558 (1989); DPP: Padgett, et al., Nature 325: 81-84 (1987);
Vg-1: Weeks Cell 51: 861-867 (1987); BMP-9: WO 95/33830
(PCT/US95/07084); BMP-10: WO 94/26893 (PCT/US94/05290); BMP-11: WO
94/26892 (PCT/US94/05288); BMP-12: WO 95/16035 (PCT/US94/14030);
BMP-13: WO 95/16035 (PCT/US94/14030); GDF-1: WO 92/00382
(PCT/US91/04096) and Lee, et al., PNAS 88: 42504254 (1991); GDF-8:
WO 94/21681 (PCT/US94/03019); GDF-9: WO 94/15966 (PCT/US94/00685);
GDF-10: WO 95/10539 (PCT/US94/11440); GDF-11: WO 96/01845
(PCT/US95/08543); BMP-15: WO 96/36710 (PCT/US96/06540); MP121: WO
96/01316 (PCT/EP95/02552); GDF-5 (CDMP-1, MP52): WO 94/15949
(PCT/US94/00657) and WO 96/14335 (PCT/US94/12814) and WO 93/16099
(PCT/EP93/00350); GDF-6 (CDMP-2, BMP-13): WO 95/01801
(PCT/US94/07762) and WO 96/14335 and WO 95/10635 (PCT/US94/14030);
GDF-7 (CDMP-3, BMP-12): WO 95/10802 (PCT/US94/07799) and WO
95/10635 (PCT/US94/14030). In another embodiment, useful proteins
include biologically active biosynthetic constructs, including
novel biosynthetic morphogenic proteins and chimeric proteins
designed using sequences from two or more known morphogens. See
also the biosynthetic constructs 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).
[0062] In certain preferred embodiments, useful morphogenic
proteins include those in which the amino acid sequences comprise a
sequence sharing at least 70% amino acid sequence homology or
"similarity", and preferably 80% homology or similarity, with a
reference morphogenic protein selected from the exemplary,
naturally-occurring morphogenic proteins listed herein. Preferably,
the reference protein is human OP-1, and the reference sequence
thereof is the C-terminal seven cysteine skeleton present in
osteogenically active forms of human OP-1, residues 330-431 of SEQ
ID NO: 2. Useful morphogenic proteins accordingly include allelic,
phylogenetic counterpart and other variants of the preferred
reference sequence, whether naturally-occurring or biosynthetically
produced (e.g., including "muteins" or "mutant proteins"), as well
as novel members of the general morphogenic family of proteins
including those set forth and identified above. Certain
particularly preferred morphogenic polypeptides share at least 60%
amino acid identity with the preferred reference sequence of human
OP-1, still more preferably at least 65% amino acid identity
therewith.
[0063] In certain embodiments, a poly eptide suspected of being
functionally equivalent to a reference morphogen polypeptide is
aligned therewith using the method of Needleman, et al., J. Mol.
Biol. 48: 443-453 (1970), implemented conveniently by computer
programs such as the Align program (DNAstar, Inc.). As noted above,
internal gaps and amino acid insertions in the candidate sequence
are ignored for purposes of calculating the defined relationship,
conventionally expressed as a level of amino acid sequence
homology, or identity, between the candidate and reference
sequences. "Amino acid sequence homology" is understood herein to
include both amino acid sequence identity and similarity.
Homologous sequences share identical and/or similar amino acid
residues, where similar residues are conservation substitutions
for, or "allowed point mutations" of, corresponding amino acid
residues in an aligned reference sequence. Thus, a candidate
polypeptide sequence that shares 70% amino acid homology with a
reference sequence is one in which any 70% of the aligned residues
are either identical to, or are conservative substitutions of, the
corresponding residues in a reference sequence. In a preferred
embodiment, the reference sequence is the C-terminal seven cysteine
skeleton sequence of human OP-1.
[0064] FIG. 1 recites the percent amino acid sequence homology
(similarity) and percent identity within the C-terminal seven
cysteine skeletons of various representative members of the
TGF-.beta. family, using OP-1 as the reference sequence. The
percent homologies recited in the figure are determined by aligning
the sequences essentially following the method of Needleman, et
al., J. Mol. Biol., 48: 443-453 (1970), and using the Align Program
(DNAstar, Inc.). Insertions and deletions from the reference
morphogen sequence (the C-terminal, biologically active
seven-cysteine skeleton of hOP-1) are ignored for purposes of
calculation.
[0065] As is apparent to one of ordinary skill in the art reviewing
the sequences for the proteins listed in FIG. 1, significant amino
acid changes can be made from the reference sequence while
retaining substantial morphogenic activity. For example, while the
GDF-1 protein sequence shares only about 50% amino acid identity
with the hOP-1 sequence described herein, the GDF-1 sequence shares
greater than 70% amino acid sequence homology with the hOP-1
sequence, where "homology" is as defined above. Moreover, GDF-1
contains a four amino acid insert (Gly-Gly-Pro-Pro) between the two
residues corresponding to residue 372 and 373 of OP-1 (SEQ ID NO:
2). Similarly, BMP-3 has a "extra" residue, a valine, inserted
between the two residues corresponding to residues 385 and 386 of
hOP-1 (SEQ ID NO: 2). Also, BMP-2 and BMP-4 are both "missing" the
amino acid residue corresponding to residue 389 of OP-1 (SEQ ID NO:
2). None of these "deviations" from the reference sequence appear
to interfere substantially with biological activity.
[0066] In other preferred embodiments, the family of morphogenic
polypeptides useful in the present invention, and members thereof,
are defined by a generic amino acid sequence. For example, Generic
Sequence 7 (SEQ ID NO: 4) and Generic Sequence 8 (SEQ ID NO: 5)
disclosed below, encompass the observed variations between
preferred protein family members identified to date, including at
least OP-1, OP-2, OP-3, CBMP-2A, CBMP-2B, BMP-3, 60A, DPP, Vg1,
BMP-5, BMP-6, Vgr-1, and GDF-1. The amino acid sequences for these
proteins are described herein and/or in the art, as summarized
above. The generic sequences include both the amino acid identity
shared by these sequences in the C-terminal skeleton, defined by
the six and seven cysteine skeletons (Generic Sequences 7 and 8,
respectively), as well as alternative residues for the variable
positions within the sequence. The generic sequences provide an
appropriate cysteine skeleton where inter- or intramolecular
disulfide bonds can form, and contain certain critical amino acids
likely to influence the tertiary structure of the folded proteins.
In addition, the generic sequences allow for an additional cysteine
at position 36 (Generic Sequence 7) or position 41 (Generic
Sequence 8), thereby encompassing the morphogenically-active
sequences of OP-2 and OP-3.
1 Generic Sequence 7 (SEQ ID NO: 4) Leu Xaa Xaa Xaa Phe Xaa Xaa 1 5
Xaa Gly Trp Xaa Xaa Xaa Xaa Xaa Xaa Pro 10 15 Xaa Xaa Xaa Xaa Ala
Xaa Tyr Cys Xaa Gly 20 25 Xaa Cys Xaa Xaa Pro Xaa Xaa Xaa Xaa Xaa
30 35 Xaa Xaa Xaa Asn His Ala Xaa Xaa Xaa Xaa 40 45 Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa 50 55 Xaa Xaa Xaa Cys Cys Xaa Pro Xaa Xaa
Xaa 60 65 Xaa Xaa Xaa Xaa Xaa Leu Xaa Xaa Xaa Xaa 70 75 Xaa Xaa Xaa
Val Xaa Leu Xaa Xaa Xaa Xaa 80 85 Xaa Met Xaa Val Xaa Xaa Cys Xaa
Cys Xaa 90 95
[0067] wherein each Xaa independently is 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, Gin,
Ser, Lys or Ala); Xaa at res. 7=(Asp or Glu); 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. 13=(Trp or Ser); 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, Gin, 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, Ala,
or Ser); Xaa at res. 28=(Glu, Lys, Asp, Gin or Ala); Xaa at res.
30=(Ala, Ser, Pro, Gln, Ile or Asn); Xaa at res. 31=(Phe, Leu or
Tyr); Xaa at res. 33=(Leu, Val or Met); a, 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, Met 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, Gly or Leu); Xaa at res. 0.53=(Asn, Lys, Ala, Glu, Gly or
Phe); Xaa at res. 54=(Pro, Ser or Val); Xaa at res. 55=(Glu, Asp,
Asn, Gly, Val, Pro or Lys); Xaa at res. 56=(Thr, Ala, Val, Lys,
Asp, Tyr, Ser, Gly, Ile 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, Val 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,
Leu, 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, Arg 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, Arg or Val);
Xaa at res. 86=(Tyr, Glu or His); Xaa at res. 87=(Arg, Gln, Glu or
Pro); Xaa at res. 88=(Asn, Glu, Trp or Asp); Xaa at res. 90=(Val,
Thr, Ala or Ile); Xaa at res. 92=(Arg, Lys, Val, Asp, Gln 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).
[0068] Generic Sequence 8 (SEQ ID NO: 5) includes all of Generic
Sequence 7 (SEQ ID NO: 4) and in addition includes the following
sequence (SEQ ID NO: 8) at its N-terminus:
2 SEQ ID NO:8 Cys Xaa Xaa Xaa Xaa 1 5
[0069] Accordingly, beginning with residue 7, each "Xaa" in Generic
Sequence 8 is a specified amino acid defined as for Generic
Sequence 7, with the distinction that each residue number described
for Generic Sequence 7 is shifted by five in Generic Sequence 8.
Thus, "Xaa at res. 2=(Tyr or Lys)" in Generic Sequence 7 refers to
Xaa at res. 7 in Generic Sequence 8. In Generic Sequence 8, 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); and Xaa at res. 5=(Glu, Ser, His, Gly,
Arg, Pro, Thr, or Tyr).
[0070] In another embodiment, useful osteogenic proteins include
those defined by Generic Sequences 9 and 10 (SEQ ID NOS: 6 and 7,
respectively), described herein above. Specifically, Generic
Sequences 9 and 10 are composite amino acid sequences of the
following proteins: human OP-1, human OP-2, human OP-3, human
BMP-2, human BMP-3, human BMP-4, human BMP-5, human BMP-6, human
BMP-8, human BMP-9, human BMP-10, human BMP-11, Drosophila 60A,
Xenopus Vg-1, sea urchin UNIVIN, human CDMP-1 (mouse GDF-5), human
CDMP-2 (mouse GDF-6, human BMP-13), human CDMP-3 (mouse GDF-7,
human BMP-12), mouse GDF-3, human GDF-1, mouse GDF-1, chicken
DORSALIN, Drosophila dpp, Drosophila SCREW, mouse NODAL, mouse
GDF-8, human GDF-8, mouse GDF-9, mouse GDF-10, human GDF-11, mouse
GDF-11, human BMP-15, and rat BMP-3b. Like Generic Sequence 7,
Generic Sequence 9 accommodates the C-terminal six cysteine
skeleton and, like Generic Sequence 8, Generic Sequence 10
accommodates the seven cysteine skeleton.
3 Generic Sequence 9 (SEQ ID NO: 6) Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa 1 5 10 Xaa Xaa Xaa Xaa Xaa Xaa Pro Xaa Xaa Xaa 15 20 Xaa
Xaa Xaa Xaa Cys Xaa Gly Xaa Cys Xaa 25 30 Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa 35 40 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 45 50
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 55 60 Xaa Cys Xaa Pro Xaa
Xaa Xaa Xaa Xaa Xaa 65 70 Xaa Xaa Leu Xaa Xaa Xaa Xaa Xaa Xaa Xaa
75 80 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 85 90 Xaa Xaa Xaa Cys
Xaa Cys Xaa 95
[0071] 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. 1=(Phe, Leu or Glu); Xaa at res. 2=(Tyr,
Phe, His, Arg, Thr, Gln, Val or Glu); Xaa at res. 3=(Val, Ile, Leu
or Asp); Xaa at res. 4=(Ser, Asp, Glu, Asn or Phe); Xaa at res.
5=(Phe or Glu); Xaa at res. 6=(Arg, Gln, Lys, Ser, Glu, Ala or
Asn); Xaa at res. 7=(Asp, Glu, Leu, Ala or Gln); Xaa at res.
8=(leu, Val, Met, Ile or Phe); Xaa at res. 9=(Gly, His or Lys); Xaa
at res. 10=(Trp or Met); Xaa at res. 11=(Gln, Leu, His, Glu, Asn,
Asp, Ser or Gly); Xaa at res. 12=(Asp, Asn, Ser, Lys, Arg, Glu or
His); Xaa at res. 13=(Trp or Ser); Xaa at res. 14=(Ile or Val); Xaa
at res. 15=(Ile or Val); Xaa at res. 16=(Ala, Ser, Tyr or Trp); Xaa
at res. 18=(Glu, Lys, Gln, Met, Pro, Leu, Arg, His or Lys); Xaa at
res. 19=(Gly, Glu, Asp, Lys, Ser, Gln, Arg or Phe); Xaa at res.
20=(Tyr or Phe); Xaa at res. 21=(Ala, Ser, Gly, Met, Gln, His, Glu,
Asp, Leu, Asn, Lys or Thr); Xaa at res. 22=(Ala or Pro); Xaa at
res. 23=(Tyr, Phe, Asn, Ala or Arg); Xaa at res. 24=(Tyr, His, Glu,
Phe or Arg); Xaa at res. 26=(Glu, Asp, Ala, Ser, Tyr, His, Lys,
Arg, Gln or Gly); Xaa at res. 28=(Glu, Asp, Leu, Val, Lys, Gly,
Thr, Ala or Gin); Xaa at res. 30=(Ala, Ser, Ile, Asn, Pro, Glu,
Asp, Phe, Gin or Leu); Xaa at res. 31=(Phe, Tyr, Leu, Asn, Gly or
Arg); Xaa at res. 32=(Pro, Ser, Ala or Val); Xaa at res. 33=(Leu,
Met, Glu, Phe or Val); Xaa at res. 34=(Asn, Asp, Thr, Gly, Ala,
Arg, Leu or Pro); Xaa at res. 35=(Ser, Ala, Glu, Asp, Thr, Leu,
Lys, Gin or His); Xaa at res. 36=(Tyr, His, Cys, Ile, Arg, Asp,
Asn, Lys, Ser, Glu or Gly); Xaa at res. 37=(Met, Leu, Phe, Val, Gly
or Tyr); Xaa at res. 38=(Asn, Glu, Thr, Pro, Lys, His, Gly, Met,
Val or Arg); Xaa at res. 39=(Ala, Ser, Gly, Pro or Phe); Xaa at
res. 40=(Thr, Ser, Leu, Pro, His or Met); Xaa at res. 41=(Asn, Lys,
Val, Thr or Gin); Xaa at res. 42=(His, Tyr or Lys); Xaa at res.
43=(Ala, Thr, Leu or Tyr); Xaa at res. 44=(Ile, Thr, Val, Phe, Tyr,
Met or Pro); Xaa at res. 45=(Val, Leu, Met, Ile or His); Xaa at
res. 46=(Gin, Arg or Thr); Xaa at res. 47=(Thr, Ser, Ala, Asn or
His); Xaa at res. 48=(Leu, Asn or Ile); Xaa at res. 49=(Val, Met,
Leu, Pro or Ile); Xaa at res. 50=(His, Asn, Arg, Lys, Tyr or Gin);
Xaa at res. 51=(Phe, Leu, Ser, Asn, Met, Ala, Arg, Glu, Gly or
Gin); Xaa at res. 52=(Ile, Met, Leu, Val, Lys, Gln, Ala or Tyr);
Xaa at res. 53=(Asn, Phe, Lys, Glu, Asp, Ala, Gin, Gly, Leu or
Val); Xaa at res. 54=(Pro, Asn, Ser, Val or Asp); Xaa at res.
55=(Glu, Asp, Asn, Lys, Arg, Ser, Gly, Thr, Gin, Pro or His); Xaa
at res. 56=(Thr, His, Tyr, Ala, Ile, Lys, Asp, Ser, Gly or Arg);
Xaa at res. 57=(Val, Ile, Thr, Ala, Leu or Ser); Xaa at res.
58=(Pro, Gly, Ser, Asp or Ala); Xaa at res. 59=(Lys, Leu, Pro, Ala,
Ser, Glu, Arg or Gly); Xaa at res. 60=(Pro, Ala, Val, Thr or Ser);
Xaa at res. 61=(Cys, Val or Ser); Xaa at res. 63=(Ala, Val or Thr);
Xaa at res. 65=(Thr, Ala, Glu, Val, Gly, Asp or Tyr); Xaa at res.
66=(Gin, Lys, Glu, Arg or Val); Xaa at res. 67=(Leu, Met, Thr or
Tyr); Xaa at res. 68=(Asn, Ser, Gly, Thr, Asp, Glu, Lys or Val);
Xaa at res. 69=(Ala, Pro, Gly or Ser); Xaa at res. 70=(Ile, Thr,
Leu or Val); Xaa at res. 71=(Ser, Pro, Ala, Thr, Asn or Gly); Xaa
at res. 2=(Val, Ile, Leu or Met); Xaa at res. 74=(Tyr, Phe, Arg,
Thr, Tyr or Met); Xaa at res. 75=(Phe, Tyr, His, Leu, Ile, Lys, Gin
or Val); Xaa at res. 76=(Asp, Leu, Asn or Glu); Xaa at res.
77=(Asp, Ser, Arg, Asn, Glu, Ala, Lys, Gly or Pro); Xaa at res.
78=(Ser, Asn, Asp, Tyr, Ala, Gly, Gin, Met, Glu, Asn or Lys); Xaa
at res. 79=(Ser, Asn, Glu, Asp, Val, Lys, Gly, Gln or Arg); Xaa at
res. 80=(Asn, Lys, Thr, Pro, Val, Ile, Arg, Ser or Gln); Xaa at
res. 81=(Val, Ile, Thr or Ala); Xaa at res. 82=(Ile, Asn, Val, Leu,
Tyr, Asp or Ala); Xaa at res. 83=(Leu, Tyr, Lys or Ile); Xaa at
res. 84=(Lys, Arg, Asn, Tyr, Phe, Thr, Glu or Gly); Xaa at res.
85=(Lys, Arg, His, Gln, Asn, Glu or Val); Xaa at res. 86=(Tyr, His,
Glu or Ile); Xaa at res. 87=(Arg, Glu, Gln, Pro or Lys); Xaa at
res. 88=(Asn, Asp, Ala, Glu, Gly or Lys); Xaa at res. 89=(Met or
Ala); Xaa at res. 90=(Val, Ile, Ala, Thr, Ser or Lys); Xaa at res.
91=(Val or Ala); Xaa at res. 92=(Arg, Lys, Gln, Asp, Glu, Val, Ala,
Ser or Thr); Xaa at res. 93=(Ala, Ser, Glu, Gly, Arg or Thr); Xaa
at res. 95=(Gly, Ala or Thr); Xaa at res. 97=(His, Arg, Gly, Leu or
Ser). Further, after res. 53 in rBMP-3b and mGDF-10 there is an
Ile; after res. 54 in GDF-1 there is a T; after res. 54 in BMP-3
there is a V; after res. 78 in BMP-8 and Dorsalin there is a G;
after res. 37 in hGDF-1 there is Pro, Gly, Gly, Pro.
[0072] Generic Sequence 10 (SEQ ID NO: 7) includes all of Generic
Sequence 9 (SEQ ID NO: 6) and in addition includes the following
sequence (SEQ ID NO: 9) at its N-terminus:
4 SEQ ID NO: 9 Cys Xaa Xaa Xaa Xaa 1 5
[0073] Accordingly, beginning with residue 6, each "Xaa" in Generic
Sequence 10 is a specified amino acid defined as for Generic
Sequence 9, with the distinction that each residue number described
for Generic Sequence 9 is shifted by five in Generic Sequence 10.
Thus, "Xaa at res. 1=(Tyr, Phe, His, Arg, Thr, Lys, Gln, Val or
Glu)" in Generic Sequence 9 refers to Xaa at res. 6 in Generic
Sequence 10. In Generic Sequence 10, Xaa at res. 2=(Lys, Arg, Gln,
Ser, His, Glu, Ala, or Cys); Xaa at res. 3=(Lys, Arg, Met, Lys,
Thr, Leu, Tyr, or Ala); Xaa at res. 4=(His, Gln, Arg, Lys, Thr,
Leu, Val, Pro, or Tyr); and Xaa at res. 5=(Gln, Thr, His, Arg, Pro,
Ser, Ala, Gln, Asn, Tyr, Lys, Asp, or Leu).
[0074] Based upon alignment of the naturally-occurring morphogens
within the definition of Generic Sequence 10, it should be clear
that gaps and/or insertions of one or more amino acid residues can
be tolerated (without abrogating or substantially impairing
biological activity) at least between or involving residues 11-12,
42-43, 59-60, 68-69 and 83-84.
[0075] As noted above, certain preferred morphogenic polypeptide
sequences useful in this invention have greater than 60% identity,
preferably greater than 65% identity, with the amino acid sequence
defining the preferred reference sequence of hOP-1. These
particularly preferred sequences include allelic and phylogenetic
counterpart variants of the OP-1 and OP-2 proteins, including the
Drosophila 60A protein, as well as the closely related proteins
BMP-5, BMP-6 and Vgr-1. Accordingly, in certain particularly
preferred embodiments, useful morphogenic proteins include active
proteins comprising pairs of polypeptide chains within the generic
amino acid sequence herein referred to as "OPX" (SEQ ID NO: 3),
which defines the seven cysteine skeleton and accommodates the
homologies between several identified variants of OP-1 and OP-2.
Accordingly, each "Xaa" at a given position in OPX independently is
selected from the residues occurring at the corresponding position
in the C-terminal sequence of mouse or human OP-1 or OP-2.
Specifically, each "Xaa" is independently selected from a group of
one or more specified amino acids as defined below:
5 Cys Xaa Xaa His Glu Leu Tyr Val Ser Phe Xaa Asp Leu Gly Trp 1 5
10 15 Xaa Asp Trp Xaa Ile Ala Pro Xaa Gly Tyr Xaa Ala Tyr Tyr Cys
20 25 30 Glu Gly Glu Cys Xaa Phe Pro Leu Xaa Ser Xaa Met Asn Ala
Thr 35 40 45 Asn His Ala Ile Xaa Gln Xaa Leu Val His Xaa Xaa Xaa
Pro Xaa 50 55 60 Xaa Val Pro Lys Xaa Cys Cys Ala Pro Thr Xaa Leu
Xaa Ala Xaa 65 70 75 Ser Val Leu Tyr Xaa Asp Xaa Ser Xaa Asn Val
Ile Leu Xaa Lys 80 85 90 Xaa Arg Asn Met Val Val Xaa Ala Cys Gly
Cys His 95 100
[0076] wherein Xaa at res. 2=(Lys or Arg); Xaa at res. 3 (Lys or
Arg); Xaa at res. 11=(Arg or Gln); Xaa at res. 16=(Gin or Leu); Xaa
at res. 19=(Ile or Val); Xaa at res. 23=(Glu or Gln); Xaa at res.
26=(Ala or Ser); Xaa at res. 35=(Ala or Ser); Xaa at res. 39=(Asn
or Asp); Xaa at res. 41=(Tyr or Cys); Xaa at res. 50=(Val or Leu);
Xaa at res. 52=(Ser or Thr); Xaa at res. 56=(Phe or Leu); Xaa at
res. 57=(Ile or Met); Xaa at res. 58=(Asn or Lys); Xaa at res.
60=(Glu, Asp or Asn); Xaa at res. 61=(Thr, Ala or Val); Xaa at res.
65=(Pro or Ala); Xaa at res. 71=(Gln or Lys); Xaa at res. 73=(Asn
or Ser); Xaa at res. 75=(Ile or Thr); Xaa at res. 80=(Phe or Tyr);
Xaa at res. 82=(Asp or Ser); Xaa at res. 84=(Ser or Asn); Xaa at
res. 89=(Lys or Arg); Xaa at res. 91=(Tyr or His); and Xaa at res.
97=(Arg or Lys).
[0077] In still another preferred embodiment, useful
morphogenically-active proteins have polypeptide chains with amino
acid sequences comprising a sequence encoded by a nucleic acid that
hybridizes with DNA or RNA encoding reference morphogen sequences,
e.g., C-terminal sequences defining the conserved seven cysteine
skeletons of OP-1, OP-2, BMP-2, BMP-4, BMP-5, BMP-6, 60A, GDF-3,
GDF-5, GDF-6, GDF-7 and the like. As used herein, high stringency
hybridization conditions are defined as hybridization according to
known techniques 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. Standard
stringency conditions are well characterized in standard molecular
biology cloning texts. See, for example, MOLECULAR CLONING A
LABORATORY MANUAL, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis
(Cold Spring Harbor Laboratory Press: 1989); DNA CLONING, Volumes I
and II (D. N. Glover ed., 1985); OLIGONUCLEOTIDE SYNTHESIS (M. J.
Gait ed., 1984); NUCLEIC ACID HYBRIDIZATION (B. D. Hames & S.
J. Higgins eds. 1984); and B. Perbal, A PRACTICAL GUIDE TO
MOLECULAR CLONING (1984).
[0078] In other embodiments, as an alternative to the
administration of a morphogenic protein, an effective amount of an
agent competent to stimulate or induce increased endogenous
morphogen expression in a mammal may be administered by any of the
routes described herein. Such a morphogen inducer may be provided
to a mammal, e.g., by systemic administration to the mammal or by
direct administration to the neural tissue. A method for
identifying and testing inducers (stimulating agents) competent to
modulate the levels of endogenous morphogens in a given tissue is
described in published applications WO93/05172 and WO93/05751, each
of which is incorporated by reference herein. Briefly, candidate
compounds are identified and tested by incubation in vitro with
test tissue or cells, or a cultured cell line derived therefrom,
for a time sufficient to allow the compound to affect the
production, i.e., cause the expression and/or secretion, of a
morphogen produced by the cells of that tissue. Suitable tissue, or
cultured cells of a suitable tissue, are preferably selected from
renal epithelium, ovarian tissue, fibroblasts, and osteoblasts.
[0079] In yet other embodiments, an agent which acts as an agonist
of a morphogen receptor may be administered instead of the
morphogen itself. Such an agent may also be referred to an a
morphogen "mimic," "mimetic," or "analog." Thus, for example, a
small peptide or other molecule which can mimic the activity of a
morphogen in binding to and activating the morphogen's receptor may
be employed as an equivalent of the morphogen. Preferably the
agonist is a full agonist, but partial morphogen receptor agonists
may also be advantageously employed. Methods of identifying such
agonists are known in the art and include assays for compounds
which induce morphogen-mediated responses (e.g., induction of
differentiation of metanephric mesenchyme, induction of
endochondral bone formation). For example, methods of identifying
morphogen inducers or agonists of morphogen receptors may be found
in U.S. Ser. No. 08/478,097 filed Jun. 7, 1995 and U.S. Ser. No.
08/507,598 filed Jul. 26, 1995, disclosures of which are
incorporated herein by reference.
[0080] As a general matter, methods of the present invention may be
applied to the treatment of any mammalian subject at risk of or
afflicted with a neural tissue insult or neuropathy. The invention
is suitable for the treatment of any primate, preferably a higher
primate such as a human. In addition, however, the invention may be
employed in the treatment of domesticated mammals which are
maintained as human companions (e.g., dogs, cats, horses), which
have significant commercial value (e.g., goats, pigs, sheep,
cattle, sporting or draft animals), which have significant
scientific value (e.g., captive or free specimens of endangered
species, or inbred or engineered animal strains), or which
otherwise have value.
[0081] C. Formulations and Methods of Treatment
[0082] Morphogens, morphogen inducers, or agonists of morphogen
receptors of the present invention may be administered by any route
which is compatible with the particular morphogen, inducer, or
agonist employed. Thus, as appropriate, administration may be oral
or parenteral, including intravenous and intraperitoneal routes of
administration. In addition, administration may be by periodic
injections of a bolus of the morphogen, inducer or agonist, or may
be made more continuous by intravenous or intraperitoneal
administration from a reservoir which is external (e.g., an i.v.
bag) or internal (e.g., a bioerodable implant, or a colony of
implanted, morphogen-producing cells).
[0083] Therapeutic agents of the invention (i.e., morphogens,
morphogen inducers or agonists of morphogen receptors) may be
provided to an individual by any suitable means, directly (e.g.,
locally, as by injection, implantation or topical administration to
a tissue locus) or systemically (e.g., parenterally or orally).
Where the agent is to be provided parenterally, such as by
intravenous, subcutaneous, intramolecular, ophthalmic,
intraperitoneal, intramuscular, buccal, rectal, vaginal,
intraorbital, intracerebral, intracranial, intraspinal,
intraventricular, intrathecal, intracisternal, intracapsular,
intranasal or by aerosol administration, the agent preferably
comprises part of an aqueous or physiologically compatible fluid
suspension or solution. Thus, the morphogen carrier or vehicle is
physiologically acceptable so that in addition to delivery of the
desired agent to the patient, it does not otherwise adversely
affect the patient's electrolyte and/or volume balance. The fluid
medium for the agent thus can comprise normal physiologic saline
(e.g., 9.85% aqueous NaCl, 0.15M, pH 7-7.4).
[0084] Association of the mature morphogen dimer with a morphogen
pro domain results in the pro form of the morphogen which typically
is more soluble in physiological solutions than the corresponding
mature form. In fact, endogenous morphogens are thought to be
transported (e.g., secreted and circulated) in the mammalian body
in this form. This soluble form of the protein can be obtained from
culture medium of morphogen-secreting mammalian cells, e.g., cells
transfected with nucleic acid encoding and competent to express the
morphogen. Alternatively, a soluble species can be formulated by
complexing the mature, morphogenically-active polypeptide dimer (or
an active fragment thereof) with a morphogen pro domain polypeptide
or a solubility-enhancing fragment thereof. Solubility-enhancing
pro domain fragments can be any N-terminal, C-terminal or internal
fragment of the pro region of a member of the morphogen family that
complexes with the mature polypeptide dimer to enhance stability
and/or dissolubility of the resulting noncovalent or convalent
complex. Typically, useful fragments are those cleaved at the
proteolytic site Arg-Xaa-Xaa-Arg. A detailed description of soluble
complex forms of morphogenic proteins, including how to make, test
and use them, is described in WO 94/03600 (PCT US 93/07189). In the
case of OP-1, useful pro domain polypeptide fragments include the
intact pro domain polypeptide (residues 30-292) and fragments
48-292 and 158-292, all of SEQ ID NO: 2. Another molecule capable
of 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 may
also be useful.
[0085] Useful solutions for parenteral administration may be
prepared by any of the methods well known in the pharmaceutical
art, described, for example, in REMINGTON'S PHARMACEUTICAL SCIENCES
(Gennaro, A., ed.), Mack Pub., 1990. Formulations of the
therapeutic agents of the invention may include, for example,
polyalkylene glycols such as polyethylene glycol, oils of vegetable
origin, hydrogenated naphthalenes, and the like. Formulations for
direct administration, in particular, may include glycerol and
other compositions of high viscosity to help maintain the agent at
the desired locus. Biocompatible, preferably bioresorbable,
polymers, including, for example, hyaluronic acid, collagen,
tricalcium phosphate, polybutyrate, lactide, and glycolide polymers
and lactide/glycolide copolymers, may be useful excipients to
control the release of the agent in vivo. Other potentially useful
parenteral delivery systems for these agents include ethylene-vinyl
acetate copolymer particles, osmotic pumps, implantable infusion
systems, and liposomes. Formulations for inhalation administration
contain as excipients, for example, lactose, or may 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 may also include
glycocholate for buccal administration, methoxysalicylate for
rectal administration, or cutric acid for vaginal administration.
Suppositories for rectal administration may also be prepared by
mixing the morphogen, inducer or agonist with a non-irritating
excipient such as cocoa butter or other compositions which are
solid at room temperature and liquid at body temperatures.
[0086] Formulations for topical administration to the skin surface
may be prepared by dispersing the morphogen, inducer or agonist
with a dermatologically acceptable carrier such as a lotion, cream,
ointment or soap. Particularly useful are carriers capable of
forming a film or layer over the skin to localize application and
inhibit removal. For topical administration to internal tissue
surfaces, the agent may be dispersed in a liquid tissue adhesive or
other substance known to enhance adsorption to a tissue surface.
For example, hydroxypropylcellulose or fibrinogen/thrombin
solutions may be used to advantage. Alternatively, tissue-coating
solutions, such as pectin-containing formulations may be used.
[0087] Alternatively, the agents described herein may 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
and is also detected in the bloodstream. Maternal administration,
via ingested milk, may be a natural delivery route of TGF-.beta.
superfamily proteins. Letterio, et al., Science 264: 1936-1938
(1994), report that TGF-.beta. is present in murine milk, and that
radiolabelled TGF-.beta. is absorbed by gastrointestinal mucosa of
suckling juveniles. Labeled, ingested TGF-.beta. appears rapidly in
intact form in the juveniles' body tissues, including lung, heart
and liver. Finally, soluble form morphogen, e.g., mature morphogen
associated with the pro domain, is morphogenically-active. These
findings, as well as those disclosed in the examples below,
indicate that oral and parenteral administration are viable means
for administering TGF-.beta. superfamily proteins, including the
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 expressed, full length polypeptide sequence
and/or by association with one or more milk components.
Accordingly, the compounds provided herein may also be associated
with molecules capable of enhancing their solubility in vitro or in
vivo.
[0088] Where the morphogen is intended for use as a therapeutic for
disorders of the CNS, an additional problem must be addressed:
overcoming the blood-brain barrier, the brain capillary wall
structure that effectively screens out all but selected categories
of substances 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, or by intranasal administration or inhalation of
formulations suitable for uptake and retrograde transport by
olfactory neurons. 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 morphogens, inducers or agonists
provided herein can be derivatized or conjugated to a lipophilic
moiety or to a substance that is actively transported across the
blood-brain barrier, using standard means known to those skilled in
the art. See, for example, Pardridge, Endocrine Reviews 7: 314-330
(1986) and U.S. Pat. No. 4,801,575.
[0089] The compounds provided herein may also be associated with
molecules capable of targeting the morphogen, inducer or agonist to
the desired tissue. For example, an antibody, antibody fragment, or
other binding protein that interacts specifically with a surface
molecule on cells of the desired tissue, may be used. Useful
targeting molecules may be designed, for example, using the single
chain binding site technology disclosed in U.S. Pat. No. 5,091,513.
Targeting molecules can be covalently or non-covalently associated
with the morphogen, inducer or agonist.
[0090] As will be appreciated by one of ordinary skill in the art,
the formulated compositions contain therapeutically-effective
amounts of the morphogen, morphogen inducers or agonists of
morphogen receptors. That is, they contain an amount which provides
appropriate concentrations of the agent to the affected nervous
system tissue for a time sufficient to stimulate a detectable
restoration of impaired central or peripheral nervous system
function, up to and including a complete restoration thereof. As
will be appreciated by those skilled in the art, these
concentrations will vary depending upon a number of factors,
including the biological efficacy of the selected agent, the
chemical characteristics (e.g., hydrophobicity) of the specific
agent, the formulation thereof, including a mixture with one or
more excipients, the administration route, and the treatment
envisioned, including whether the active ingredient will be
administered directly into a tissue site, or whether it will be
administered systemically. The preferred dosage to be administered
is also likely to depend on variables such as the condition of the
diseased or damaged tissues, and the overall health status of the
particular mammal. As a general matter, single, daily, biweekly or
weekly dosages of 0.00001-1000 mg of a morphogen are sufficient,
with 0.0001-100 mg being preferable, and 0.001 to 10 mg being even
more preferable. Alternatively, a single, daily, biweekly or weekly
dosage of 0.01-1000 .mu.g/kg body weight, more preferably 0.01-10
mg/kg body weight, may be advantageously employed. The present
effective dose can be administered in a single dose or in a
plurality (two or more) of installment doses, as desired or
considered appropriate under the specific circumstances. A bolus
injection or diffusable infusion formulation can be used. If
desired to facilitate repeated or frequent infusions, implantation
of a semi-permanent stent (e.g., intravenous, intraperitoneal,
intracisternal or intracapsular) may be advisable.
[0091] The morphogens, inducers or agonists of the invention may,
of course, be administered alone or in combination with other
molecules known to be beneficial in the treatment of the conditions
described herein. For example, various well-known growth factors,
hormones, enzymes, therapeutic compositions, antibiotics, or other
bioactive agents can also be administered with the morphogen. Thus,
various known growth factors such as NGF, EGF, PDGF, IGF, FGF,
TGF-.alpha., and TGF-.beta., as well as enzymes, enzyme inhibitors,
antioxidants, anti-inflammatory agents, free radical scavenging
agents, antibiotics and/or chemoattractant/chemotactic factors, can
be included in the present morphogen formulation.
EXAMPLE 1
Preparation of Soluble Morphogen Protein Solutions for In vivo
Administration
[0092] A. Aqueous Solutions
[0093] While the mature dimeric morphogenic proteins defined herein
are typically sparingly soluble in physiological buffers, they can
be solubilized to form injectable suspensions or solutions. One
exemplary aqueous formulation containing a morphogen is made, for
example, by dispersing the morphogen in 50% ethanol containing
acetonitrile in 0.1% trifluoroacetic acid (TFA) or 0.1% HCl, or in
an equivalent solvent. 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)
or a similar carrier protein. The resultant solution is preferably
vortexed extensively to produce a physiologically acceptable
morphogen formulation.
[0094] In another embodiment, the morphogen, including OP-1, is
solubilized by reducing the pH of the solution. In one preferred
formulation, the protein is solubilized in 0.2 mM acetate buffer,
pH 4.5, containing 5% mannitol, to render the solution more
isotonic. Other standard means for creating physiologically
acceptable formulations are within the skill of the art.
[0095] B. Soluble Complex Formulations
[0096] Another preferred form is a dimeric morphogenic protein
comprising at least the C-terminal seven cysteine skeleton
characteristic of the morphogen family, complexed with a peptide
comprising a pro region of a member of the morphogen family, or a
solubility-enhancing fragment thereof, or an allelic, phylogenetic
or other sequence variant thereof. The solubility-enhancing
fragment is any N-terminal or C-terminal fragment of the pro domain
polypeptide of a member of the morphogen family that complexes with
the mature polypeptide dimer to enhance the stability of the
resulting soluble complex. Preferably, the dimeric morphogenic
protein is complexed with two such pro domain peptides.
[0097] As described above and in published application WO 94/03600,
incorporated by reference herein, the soluble complex form is
isolated from the cell culture media (or a body fluid) under
appropriate conditions. Alternatively, the complex is formulated in
vitro.
[0098] Soluble morphogen complexes are 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 generally as described
in WO 94/03600. The affinity column described below is a Zn-IMAC
column. The present example uses human OP-1, and is not intended to
be limiting. 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 includes 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
thereof).
[0099] In this example, OP-1 was expressed in mammalian (CHO,
Chinese hamster ovary) cells as described in the art (see, for
example, international application US90/05903 (WO 91/05802). The
CHO cell conditioned media containing 0.5% FBS is 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 purified soluble OP-1 is
next applied to an S-Sepharose action-exchange column equilibrated
in 20 mM NaPO.sub.4 (pH 7.0) with 50 mM NaCl. The protein then is
applied to a Sephacryl S-200HR column equilibrated in TBS. Using
substantially the same protocol, soluble morphogens can also be
isolated from one or more body fluids, including milk, serum,
cerebrospinal fluid or peritoneal fluid.
[0100] 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.
[0101] 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
polypeptide has an opportunity to associate with the mature dimeric
protein 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 domain peptides, while maintaining the association of
the pro domain peptides with the mature dimer. Useful denaturants
include 4-6 M 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 or 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 on the subject is GUIDE
TO PROTEIN PURIFICATION, M. Deutscher, ed., Academic Press, San
Diego, 1990, particularly section V. Complex formation may also be
aided by addition of one or more chaperone proteins.
[0102] 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, including any one or more of 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.
EXAMPLE 2
Identification of Morphogen-Expressing Tissue
[0103] Determining the tissue distribution of morphogens may be
used to identify different morphogens expressed in a given tissue,
as well as to identify new, related morphogens. Tissue distribution
also may 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 may be low.
For example, protein distribution may 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 may be
determined using standard Northern hybridization protocols and
transcript-specific probes.
[0104] Any probe capable of hybridizing specifically to a
transcript, and distinguishing the transcript of interest from
other, related transcripts may 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 may best be determined using a probe specific for the
pro region of the immature 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 untranslated pro region and the N-terminus of the mature
sequence. See Lyons, et al., PNAS 86: 4554-4558 (1989) 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 Earl-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 may be used,
for example, with hOP-1 (SEQ ID NO: 16) or human or mouse OP-2 (SEQ
ID NOS: 20 and 22.)
[0105] Using these morphogen-specific probes, which may 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., Anal. Biochem 162: 156-159 (1987) 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 mg) 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.
[0106] 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 co-pending
U.S. Ser. No. 752,764, and in Ozkaynak, et al., Biochem. Biophys.
Res. Comm. 179: 116-123 (1991), and Ozkaynak, et al., (JBC, in
press) (1992), the disclosures of which are incorporated herein by
reference. 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 3
Morphogen Localization in the Nervous System
[0107] 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 2, 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 (U.S. Ser. No. 752,764, and
Ozkaynak, et al., Biochem. Biophys. Res. Comm., 179: 11623 (1991)
and Ozkaynak, et al., JBC, in press (1992)). 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., PNAS 88: 4250-4254
(1991). 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., Development 111: 531-542 (1991)), although the
nerve tissue does not appear to be the primary expression tissue
for these genes. Ozkaynak, et al., JBC in press (1992).
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.
[0108] Immunolocalization studies, performed using standard
methodologies known in the art and disclosed in U.S. Ser. No.
752,764, filed Aug. 30, 1991, 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-OP-1 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.
[0109] As can be seen in FIG. 2, 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 (Panel 1A) and spinal cord (Panel 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.
[0110] 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.
[0111] OP-1 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.
[0112] 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 14. 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.
[0113] The endogenous morphogens which act on neuronal cells may be
expressed and secreted by nerve tissue cells, e.g., by neurons
and/or glial cells associated with the neurons, and/or they may be
transported to the neurons by the cerebrospinal fluid and/or
bloodstream. Recently, OP-1 has been identified in the human blood
(See Example 10, 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, Exp. Neurology 114: 254-257 (1991). The regenerative
property of these cells may be mediated by the secretion of a
morphogen by the Schwann cells.
EXAMPLE 4
Morphogen Enhancement of Neuronal Cell Survival
[0114] 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, J. Biol. Chem. 26: 3164-3169 (1986) and Freese,
et al., Brain Res. 521: 254-264 (1990). 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 substantia 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.
[0115] In this example, the cultured basal ganglia 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.
[0116] Dysfunctions in the basal ganglia of the substantia 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 may be administered.
EXAMPLE 5
Morphogen-Induced Redifferentiation of Transformed Cells
[0117] 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 cell line of neuronal
origin, NG105-115. Morphogen-induced redifferentiation of
transformed cells also has been shown in mouse neuroblastoma cells
(N1E-115) and in human embryo carcinoma cells (see copending U.S.
Ser. No. 752,764, incorporated herein by reference.)
[0118] 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. 2A). 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.
[0119] 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
ml 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. 2B). FIG. 4 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.
[0120] 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, retinoic 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..
[0121] The experiments also have been performed with highly
purified soluble morphogen (e.g., mature OP-1 associated with its
pro domain) which also specifically induced redifferentiation of
NG108-15 cells.
[0122] 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 may be administered or, alternatively, a
morphogen-stimulating agent may be administered.
EXAMPLE 6
Nerve Tissue Protection from Chemical Trauma
[0123] 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 may protect neurons
from the cytotoxic effects associated with excitatory amino acids
such as glutamate.
[0124] 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, may 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 7
Morphogen-Induced CAM Expression
[0125] 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.
[0126] N-CAMs are implicated in appropriate neural development,
including appropriate neurulation, 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
synapses 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.
[0127] The morphogens described herein 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.
[0128] In this example, NG108-15 cells were cultured for four days
in the presence of increasing concentrations of OP-1 and standard
Western blots performed on whole cell extracts. N-CAM isoforms were
detected with an antibody which cross-reacts 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 mg 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 FIG. 3. FIG. 3B is a Western blot of OP-1-treated NG108-15 cell
extracts, probed with mAb H28.123, showing the induction of all
three isoforms. FIG. 3A 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. 3B, N-CAM-120 is induced in response to morphogen
treatment. The increase in N-CAM expression corresponded in a
dose-dependent manner with the morphogen induction of multicellular
aggregates. Compare FIG. 3A and FIG. 4.
[0129] FIG. 4 graphs the mean number of multilayered aggregates
(clumps) counted per 20 randomly selected, microscopic viewing
fields in six 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.
[0130] 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 oligonucleotides sufficient to
inhibit N-CAM induction also inhibited formation of multilayered
cell aggregates. Specifically, incubation of morphogen-treated
NG108-15 cells with 0.3-3 mM N-CAM antisense S-oligos, 5-500 mM
unmodified N-CAM antisense oligos, or 10 mg/ml mAb H28.123
significantly inhibits cell aggregation. It is likely that
morphogen treatment also stimulates other CAMs, as inhibition is
not complete.
[0131] Finally, the above-described experiments have also been
performed with soluble morphogen (e.g., mature OP-1 dimer,
associated with its pro domain polypeptides as described in Example
1). The soluble form of morphogen also specifically induced CAM
expression.
[0132] 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 the
morphogens described herein to affect N-CAM expression can be
assessed in vitro using a suitable cell line, such as NG108-15 and
the methods described herein.
[0133] As described above, preferred morphogens, inducers, or
agonists of the present invention can induce both N-CAM expression
in vitro and endochondral bone formation when implanted in vivo in
a mammal in conjunction with a matrix permissive of bone
morphogenesis. Thus, the methods described herein can be used to
assess novel candidate morphogens, inducers, or agonists.
[0134] 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.
[0135] 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
Alzheimer's' disease, and the like. In clinical applications, the
morphogens themselves may be administered or, alternatively, a
morphogen-stimulating agent may be administered.
[0136] The efficacy of the morphogens described herein to affect
N-CAM expression may 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 may be evaluated by tissue biopsy as described
in Example 10, 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 12.
[0137] Alternatively, the level of N-CAM proteins or protein
fragments present in cerebrospinal fluid or serum also may 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 may be detected following the procedure described in
Example 10 and using an N-CAM specific antibody, such as mAb
H28.123.
EXAMPLE 8
Morphogen-Induced Nerve Gap Repair (PNS)
[0138] 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.
[0139] 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.
[0140] 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 0.12 mm of the tube consisted of an OP-1
gel prepared by mixing 1 to 5 mg of substantially pure CHO-produced
recombinant OP-1 with approximately 100 ml 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
sheath.
[0141] 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 180o 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).
[0142] 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 9
Morphogen-Induced Nerve Gap Repair (CNS)
[0143] 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, may be
evaluated using a rat crushed optic nerve model such as the one
described by Bignami, et al., Exp. Eye Res. 28: 63-69 (1979), 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 anesthetized 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 ml solution, and 25 mg morphogen) to the optic nerve,
e.g., just prior to the operation, concomitant with the operation,
or at specific times after the operation.
[0144] In the absence of therapy, the surgery induces glial
scarring of the crushed nerve, as determine d by immunofluorescence
staining for glial fibrillary acidic protein (GFA), a marker
protein for glial scarring, and by histology. Indirect
immunofluorescence on air-dried cryostat sections as described in
Bignami, et al., J. Comp. Neur. 153: 27-38 (1974), 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 10
Nerve Tissue Diagnostics
[0145] Morphogen localization in nerve tissue can be used as part
of a method for diagnosing a neurological disorder or neuropathy.
The method may 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 may be used as an indicator of
tissue dysfunction. Alternatively, fluctuation in morphogen levels
may 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.
[0146] Fluctuations in morphogen levels present in the
cerebrospinal fluid or bloodstream also may be used to evaluate
nerve tissue viability. For example, morphogens are detected
associated 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 may be released from dying cells into cerebrospinal
fluid. In addition, OP-1 recently has been identified in human
blood, which also may be a means of morphogen transport, and/or a
repository for the contents of dying cells.
[0147] Spinal fluid may be obtained from an individual by a
standard lumbar puncture, using standard methodologies known in the
medical art. Similarly, serum samples may 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 may be assessed by standard Western blot
(immunoblot), ELISA or RIA procedures. Briefly, for example, with
the ELISA, samples may be diluted in an appropriate buffer, such as
phosphate-buffered saline, and 50 ml 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 may be washed with a standard buffer
and incubated with 50 ml 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 may be detected using standard
procedures.
[0148] Alternatively, a morphogen-specific affinity column may 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 may 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 may be determined using standard protein
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.
[0149] 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 14, 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. 5 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.
[0150] Morphogens may 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 may 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 may be used
as indicators of a change in tissue status.
EXAMPLE 11
Alleviation of Immune Response-Mediated Nerve Tissue Damage
[0151] The morphogens described herein may 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 copending U.S. Ser. No. 753,059, filed Aug. 30, 1991,
the disclosure of which is incorporated herein. A primary source of
such damage to nerve tissue follows hypoxia or ischemia-reperfusion
of a blood supply to a neural pathway, such as may result from an
embolic stroke, or may be induced during a surgical procedure. As
described in U.S. Ser. No. 753,059, 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 may be assessed using methodologies and models known
to those skilled in the art and described below.
[0152] 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., Annals of
Neurology 25: 281-285 (1989), 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).
[0153] The effect of morphogen on cerebral infarcts can be assessed
by administering varying concentrations of morphogens, e.g., OP-1,
at different times 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 non-treated animals.
EXAMPLE 12
Animal Model for Assessing Morphogen Efficacy In Vivo
[0154] 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 may be determined by comparison with established animal
models. Presented below is an exemplary protocol for a rat brain
stab model.
[0155] Briefly, male Long Evans rats, obtained from standard
commercial sources, are anesthetized 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 ml solutions
containing either morphogen (e.g., OP-1, 25 mg) 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.
[0156] Three days post surgery, rats are sacrificed by decapitation
and their brains processed for sectioning. Scar tissue formation is
evaluated by immunofluorescence 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 13
In Vitro Model for Evaluating Morphogen Species Transport Across
the Blood-Brain Barrier
[0157] Described below is an in vitro method for evaluating the
facility with which selected morphogen species likely will pass
across the blood-brain barrier. A detailed description of the model
and protocol are provided by Audus, et al., Ann. N. Acad. Sci. 507:
9-18 (1987), the disclosure of which is incorporated herein by
reference.
[0158] 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.
[0159] After isolation, approximately 5.times.10.sup.5
cells/cm.sup.2 are plated on culture dishes or 5-12 mm 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.
[0160] 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.
[0161] 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 mm) 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.
[0162] 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 14
Screening Assay for Candidate. Compounds which Alter Endogenous
Morphogen Levels
[0163] Candidate compound(s) which may be administered to affect
the level of a given morphogen may be found using the following
screening assay, in which the level of morphogen production by a
cell type which produces 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. A more detailed description also may
be found in U.S. Ser. No. 752,861, incorporated hereinabove by
reference.
[0164] 14.1 Growth of Cells in Culture
[0165] Cell cultures of kidney, adrenals, urinary bladder, brain,
or other organs, may be prepared as described widely in the
literature. For example, kidneys may 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 may 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
may 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).
[0166] Samples for testing the level of morphogen production
includes culture supernatants or cell lysates, collected
periodically and evaluated for OP-1 production by immunoblot
analysis (Sambrook et al., eds., 1989, Molecular Cloning, Cold
Spring Harbor Press, Cold Spring Harbor, N.Y.), or a portion of the
cell culture itself, collected periodically and used to prepare
polyA+ RNA for RNA analysis. To monitor de novo OP-1 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.
[0167] 14.2 Determination of Level of Morphogenic Protein
[0168] In order to quantitate the production of a morphogenic
protein by a cell type, an immunoassay may be performed to detect
the morphogen using a polyclonal or monoclonal antibody specific
for that protein. For example, OP-1 may be detected using a
polyclonal-antibody specific for OP-1 in an ELISA, as follows.
[0169] 1 mg/100 ml 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 ml 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 ml 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 ml 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 ml substrate
(ELISA Amplification System Kit, Life Technologies, Inc., Bethesda,
Md.) is added to each well incubated at room temperature for 15
min. Then, 50 ml 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 ml 0.3 M sulfuric 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.
[0170] Polyclonal antibody may be prepared as follows. Each rabbit
is given a primary immunization of 100 .mu.g/500 ml E. coli
produced OP-1 monomer (amino acids 328-431 in SEQ ID NO:5) in 0.1%
SDS mixed with 500 .mu.l E. coli produced OP-1 monomer (amino acids
328-431 in SEQ ID NO: 5) in 0.1% SDS mixed with 50011 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
mg of antigen and bled (15 ml per bleed) at days seven and ten
after boosting.
[0171] Monoclonal antibody specific for a given morphogen may be
prepared as follows. A mouse is given two injections of E. coli
produced OP-1 monomer. The first injection contains 100 mg of OP-1
in complete Freund's adjuvant and is given subcutaneously. The
second injection contains 50 mg of OP-1 in incomplete adjuvant and
is given intraperitoneally. The mouse then receives a total of 230
mg 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 mg of OP-1 (307-431) and 30 mg 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.
EXAMPLE 15
Morphogen-Induced Dendritic Growth in Spinal Motor Neurons In
Vitro
[0172] In order to evaluate the effects of various neurotrophic
proteins on neurite outgrowth, dissociated motor neurons from the
spinal cord were exposed OP-1, BDNF, LIF, or GDNF in vitro.
[0173] Suspensions of motor neurons dissociated from the spinal
cord of rat fetuses (E14 day) were prepared and plated essentially
according to the method of Higgins, et al., CULTURING NERVE CELLS,
Banker and Goslin, eds., MIT Press, pp. 177-205 (1991),
incorporated by reference herein. Neurons were plated at low
density (about 15 cells/mm.sup.2) onto poly-D-lysine coated
coverslips and maintained in a serum-free medium, Higgins, et al.,
CULTURING NERVE CELLS (1991). Cytosine-b-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 the cultures virtually free
of normeuronal cells for 30 days. Exposure to vehicle, OP-1, BDNF,
LIF, or GDNF was initiated after the elimination of normeuronal
cells.
[0174] Cellular morphology was routinely assessed by intracellular
injection of fluorescent dyes (4% Lucifer Yellow or 5% 5,6
dicarboxyfluorescein; Bruckenstein and Higgins, Dev. Biol. 128:
924-936 (1988). Only neurons whose cell bodies were at least 150 mm
from their nearest neighbor were injected, because
density-dependent changes in morphology occur when somata of motor
neurons are separated by lesser distances. Highly purified
recombinant human 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. See
Sampath, et al., J. Biol. Chem. 267: 20352-20362 (1992).
[0175] Cultures were immunostained with antibodies previously shown
to react selectively with either axons or dendrites. Lein and
Higgins, Dev. Biol. 136: 330-345 (1989). Dendritic probes included
mAb to MAP2, to nonphosphorylated forms of the M and H
neurofilaments, and to the transferrin receptor. Axonal probes
included monoclonal antibodies against to synaptophysin, tau, and
phosphorylated forms of the H and the M and H neurofilament
subunits. All antigens were localized by indirect
immunofluorescence using previously described procedures. Lein and
Higgins, Dev. Biol. 136: 330-345 (1989). Image 1 Software
(Universal Imaging) was used for the morphometric analyses of
dendritic growth in immunostained cultures.
[0176] Addition of OP-1 enhanced dendritic growth of spinal motor
neurons. The dendrites that formed in the presence of OP-1 were
significantly longer, had a larger number of branchpoints, and had
a larger diameter than control spinal motor neurons. The effects of
OP-1 appeared to be specific to dendrites OP-1 did not
significantly affect the total length of axons. However, axon
length was significantly increased by LIF and GDNF. These
observations are summarized in Table I.
6TABLE I COMPARISON OF THE EFFECTS OF VARIOUS GROWTH HORMONES ON
DENDRITIC GROWTH OF SPINAL MOTOR NEURONS Total dendritic # of
dendritic Condition length branchpoints Least somal diameter Total
axonal length Control 65.09 .+-. 6.7 0.65 .+-. 0.17 8.81 .+-. 0.32
180.95 .+-. 14.5 OP-1 133.38 .+-. 10.0* 1.40 .+-. 0.22* 10.10 .+-.
0.44* 200.20 .+-. 11.9 BDNF 56.58 .+-. 8.6 0.45 .+-. 0.78 9.56 .+-.
0.32 158.10 .+-. 9.8 LIF 59.64 .+-. 8.3 0.58 .+-. 0.20 8.87 .+-.
0.29 272.00 .+-. 24.2* GDNF 52.06 .+-. 12.6 0.76 .+-. 0.39 8.66
.+-. 0.24 288.89 .+-. 23.5* All measurements are in micrometers.
Data are expressed as the mean .+-. SEM. N = 40. *Significantly
different from Control group.
EXAMPLE 16
Morphogen-Induced Dendritic Growth in Various Neurons In Vitro
[0177] In order to further evaluate the effects of morphogens on
neurite outgrowth, various neuronal populations were exposed OP-1
in vitro.
[0178] 16.1 Cortical Neurons
[0179] The effects of morphogens on neurite outgrowth were
evaluated in cortical neurons. Pregnant Balb/c mice (E8) were
euthanised by decapitation following CO.sub.2 anesthesia and the
embryos removed under sterile conditions. After carefully removing
the meninges, the frontal cerebral cortex was dissected in sterile
Hank's balanced salt solution (HBSS) without Ca.sup.+2/Mg.sup.+2
(Biowhittaker) containing 0.6% glucose and 0.5% HEPES (Sigma). The
cortex was minced to 1 mm thick pieces and dissociated into a
single-cell suspension using the following protocol. Pieces of
frontal cortex were placed in 4.5 ml of Ca.sup.+2/Mg.sup.+2-fre- e
HBSS in a 50 ml conical culture tube and incubated in a water bath
for 5 minutes at 37.degree. C. Then, 0.5 ml of 2.5% trypsin
solution (Gibco) was added and the tissue was then incubated for 10
minutes on a shaking device at 37.degree. C. The supernatant was
then removed and placed into another tube containing 0.5 ml fetal
bovine serum (FBS; Gibco). Five ml of 0.025% Deoxyribonuclease I
(Dnase; Calbiochem Corp.) in Ca.sup.+2/Mg.sup.+2-free HBSS was then
added to the pellet and the incubation was continued for another 5
min on a shaking device at 37.degree. C. At the end of incubation,
the trypsin was inactivated by adding 0.5 ml FBS. The supernatant
collected earlier was combined with the tissue and the cells were
then concentrated by centrifugation (1000 rpm, 5 min.) and the
supernatant was decanted. Fresh medium (2 ml) was added to
resuspend the pellet which was further dissociated into a
single-cell suspension by trituration using a pipet-tip.
[0180] The cells were plated in Neurobasal Medium (GIBCO), and
supplemented with B-27 Supplement (GIBCO), 1 mM glutamine and
penicillin/streptomycin. For all experiments, the cortical neurons
were plated at low density (1.times.10.sup.4 cells/0.5 ml) on
poly-D-lysine (50-100 .mu.g/ml; Sigma) coated coverslips inserted
into 24-well culture plate (Falcon). Cells were grown for two days
in vitro at 37.degree. C. in an atmosphere of 5% CO.sub.2. OP-1 (1,
10, 30, or 100 ng/ml) was added either three hours or 24 hours
after plating the cortical neurons. BSA was added to all wells at a
final concentration of 500 .mu.g/ml prior to adding OP-1. Control
cultures consisted of culture medium and medium with BSA 500
.mu.g/ml.
[0181] Mouse neurites were immunostained with M6, a mouse
neuron-specific monoclonal antibody. Cells were first incubated in
0.1 M phosphate buffered saline (PBS) containing 1% BSA for 30 min
at 25.degree. C. and then exposed to M6 in PBS (1:10) for 24 hrs at
4.degree. C. Immunofluorescent labeling for M6 was carried out
using biotinylated secondary antibodies anti-rat IgG (Sigma; 3
.mu.g/ml, 1:200, 1 hr at 37.degree. C.) followed by avidin-TRITC
conjugate (Sigma; 6.5 .mu.g/ml, 1:400, 1 hr at 37.degree. C., in
the dark).
[0182] Axons were identified with a rabbit polyclonal antiserum to
the 200 kDa neurofilament protein (NF-H; Sigma; 1:100, 91
.mu.g/ml). A monoclonal antibody to microtubule-associated protein
2 (MAP2; Boehringer-Mannheim; 1:100, 20 .mu.g/ml) was used as a
specific marker of dendrites. To visualize these intracellular
antigens, cells were permeabilized with 0.5% triton X-100 (TX) in
0.1 M PBS containing 1% BSA and 4% goat serum (GS; Sigma) for 1 hr
at 25.degree. C. Primary antibodies were diluted in 0.1M PBS, 1%
BSA, 4% GS with 0.5% TX and incubated for 1 hr at 37.degree. C.
After the primary incubation, the cells were washed three times in
PBS containing 4% GS. Labeling was detected with fluorescein- or
rhodamine-conjugated antibodies (1:400 in PBS, BSA, GS, and TX, 1
hr at 37.degree. C., in the dark). Mouse antibodies were visualized
with fluorescein-coupled goat anti-mouse Ig (Boehringer-Mannheim).
Rabbit or rat antibodies were visualized using indirect
immunofluorescence with rhodamine-conjugated goat anti-rat or
anti-rabbit Ig. Cultures were additionally stained with a nuclear
stain, 4',6-Diamidino-2-phenylindole dihydrochloride hydrate (DAPI;
Sigma; 0.1 .mu.g/ml, 5 min at room temperature). Coverslips were
washed once in sterile water and let dry for 10 minutes before
mounting onto glass slides in aqueous mounting solution
(Fluoromount; Southern Biotechnology). Slides were kept
refrigerated in the dark until examined.
[0183] Immunoreactive cells were examined in six different
microscopic fields selected at random on a minimum of five
coverslips for each experiment. Three experiments for each
condition were carried out. Only isolated neurons whose cell bodies
or processes that were not in contact with other neurons were
analyzed. A total of 100 neurons were examined for each
experimental condition.
[0184] For measurements of neurite length, neurons were examined at
a final image magnification of 400.times.. Fluorescent images of
the neurons were recorded with a CCD video camera (Dage) and
analyzed with a Macintosh PowerMac (9500/200) and image processing
program (NIH Image 1.59). Neurite lengths were measured by tracing
the total length of any neurite extending from a neuron cell body.
Recorded lengths were calibrated at the same magnification using a
ciroscope slide micrometer. The number of primary dendrites per
cell, the length of major neurites or axons, the length of primary
dendrites, the length and number of secondary dendrites, the total
length of primary or secondary dendrites, and the total neurite and
dendrite length per cell were calculated. Analysis of statistical
significance of any observed differences between monolayers was
performed using Student's t-test or ANOVA (SPSS/Mac, version 6.1,
SPSS Inc., Chicago, Ill.).
[0185] OP-1 enhanced dendritic growth of cortical neurons. The
length of primary dendrites and the number and length of secondary
dendrites were significantly increased in the presence of OP-1. The
increase in dendritic growth was dose-dependent. Maximal growth was
observed in the presence of 30-100 .mu.g/ml of OP-1. The effects of
OP-1 appeared to be dendrite-specific; OP-1 did not significantly
affect the cell morphology, body size, and axon length of cortical
neurons. These observations are summarized in Table II.
7TABLE II EFFECTS OF OP-1 ON DENDRITIC GROWTH OF CORTICAL NEURONS
Cell Body Condition Diameter Axon Length # Primary # Secondary BSA
3 hrs 15.9 .+-. 0.5 153.2 .+-. 9.1 3.5 .+-. 0.2 0.4 .+-. 0.14 OP-1,
1 .mu.g, 3 hrs 15.98 .+-. 0.5 151.1 .+-. 6.2 3.5 .+-. 0.2 0.55 .+-.
0.13 OP-1, 10 .mu.g, 3 hrs 17.8 .+-. 0.49 139.3 .+-. 8.5 4.0 .+-.
0.14 0.6 .+-. 0.13 OP-1, 30 .mu.g, 3 hrs 17.74 .+-. 0.5 134.5 .+-.
6.9 4.0 .+-. 0.15 0.96 .+-. 0.14 OP-1, 100 .mu.g, 3 hrs 17.3 .+-.
0.4 138.8 .+-. 6.6 3.6 .+-. 0.2 1.3 .+-. 0.2 BSA 24 hrs 16.3 .+-.
0.45 144.8 .+-. 5.9 3.5 .+-. 0.17 0.48 .+-. 0.11 Op-1, 1 .mu.g, 24
hrs 15.6 .+-. 0.5 142.6 .+-. 10.9 3.4 .+-. 0.2 0.4 .+-. 0.1 OP-1,
10 .mu.g, 24 hrs 16.7 .+-. 0.4 144.2 .+-. 8.16 3.2 .+-. 0.14 0.6
.+-. 0.1 OP-1, 30 .mu.g, 24 hrs 16.5 .+-. 0.4 139.4 .+-. 7.8 3.5
.+-. 0.2 0.86 .+-. 0.12 OP-1, 100 .mu.g, 24 hrs 17.3 .+-. 0.4 156.5
.+-. 11.1 3.0 .+-. 0.19 0.86 .+-. 0.17 Length Length Total Total
Total Condition Primary Secondary Primary Secondary Dendrite BSA 3
hrs 15.4 .+-. 0.6 3.4 .+-. 0.8 51.4 .+-. 4.5 3.6 .+-. 1.5 54.9 .+-.
5.1 OP-1, 1 .mu.g, 3 hrs 14.2 .+-. 0.4 4.7 .+-. 0.7 49.1 .+-. 3.5
5.7 .+-. 1.5 54.8 .+-. 4.6 OP-1, 10 .mu.g, 3 hrs 20.5 .+-. 0.6 4.6
.+-. 0.6 81.6 .+-. 3.5 5.8 .+-. 1.2 87.5 .+-. 4.3 OP-1, 30 .mu.g, 3
hrs 22.9 .+-. 0.5 7.6 .+-. 0.7 89.9 .+-. 4.6 11.1 .+-. 1.9 99.6
.+-. 5.7 OP-1, 100 .mu.g, 3 hrs 23.7 .+-. 0.6 9.9 .+-. 0.75 87.7
.+-. 5.3 16.2 .+-. 2.9 103.9 .+-. 7.6 BSA 24 hrs 16.7 .+-. 0.5 4.6
.+-. 0.6 55.9 .+-. 3.5 4.7 .+-. 1.1 60.6 .+-. 4.1 OP-1, 1 .mu.g, 24
hrs 13.7 .+-. 0.5 3.7 .+-. 0.7 46.3 .+-. 3.6 4.1 .+-. 1.0 50.4 .+-.
4.0 OP-1, 10 .mu.g, 24 hrs 16.3 .+-. 0.6 4.8 .+-. 0.7 50.8 .+-. 3.1
5.3 .+-. 7.7 56.1 .+-. 3.6 OP-1, 30 .mu.g, 24 hrs 18.9 .+-. 0.5 7.4
.+-. 0.8 65.6 .+-. 4.5 9.6 .+-. 1.6 75.5 .+-. 5.1 OP-1, 100 .mu.g,
24 hrs 22.1 .+-. 0.8 7.6 .+-. 1.0 64.0 .+-. 6.4 10.5 .+-. 2.2 74.8
.+-. 7.8
[0186] Three separate experiments were performed for each
condition. At least 30 isolated cells, identified by MAP2 and NF-H
immunolabeling and not touching other neurons were analyzed for
each experiment after 2DIV using computer assisted image analysis.
Data are expressed as means.+-.standard error.
[0187] 16.2 Hippocampal Neurons
[0188] The effects of morphogens on neurite outgrowth were
evaluated in hippocampal neurons. Primary hippocampal cultures were
prepared according to the method of Banker, et al. See Banker &
Cowan Brain Res. 126: 397-425 (1977); Banker & Goslin,
CULTURING NERVE CELLS (1991). A very low density of neurons was
plated over a monolayer of glial cells plated on poly-D-lysine
coated coverslips and maintained in a serum-free medium. Cellular
morphology was assessed by immunostaining for MAP2. Dendritic
length and branching was quantified using the Shoil concentric ring
analysis. Under these control conditions, hippocampal neurons
produce 4-6 minor processes. Over the first 24-48 hours, one of the
processes grows rapidly and becomes an axon. The other processes
extend very slowly and develop into mature dendrites after 6-10
days.
[0189] Because glial cells secrete trophic factors into the medium
that are critical for the development of hippocampal neurons, this
culture method was modified to assess the effects of OP-1.
Hippocampal neurons were plated in a serum-free medium without
glial cells. In the absence of glial cells, OP-1 markedly enhanced
the rate and extent of dendritic development of hippocampal neurons
cultured in serum-free medium. OP-1-treated neurons had
significantly increased number of Shoil ring intersections (39.6
vs. 16.02), dendritic length (FIGS. 6 and 7) and number of terminal
branches (13.05 vs. 6.64; FIG. 8). There were no significant
differences in the number of primary dendrites. The effects of OP-1
appeared to be dendrite-specific in this cell type. As illustrated
in FIG. 6; OP-1 did not significantly affect the total length of
axons.
[0190] 16.3 Sympathetic Neurons
[0191] (i) Effects of OP-1 on Dendritic Growth
[0192] In order to assess the effects of morphogens on sympathetic
neurons, 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 exposed to OP-1. The suspension were
prepared according to the method of Higgins, et al., CULTURING
NERVE CELLS, Banker and Goslin, eds., MIT Press, pp. 177-205
(1991), the teachings of which herein incorporated by reference.
Equivalent results were obtained with pre- and postnatal animals.
Neurons were plated at low density (about 15 cells/mm.sup.2) onto
poly-D-lysine coated coverslips and maintained in a serum-free
medium (Higgins, et al., CULTURING NERVE CELLS (1991)) containing
NGF (100 ng/ml). Cytosine-b-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 the cultures virtually free of normeuronal
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 mCi/ml, ICN) before
being dissociated. Because NT3 (50 ng/ml) enhances the survival of
immature sympathetic neurons, Birren, et al., Develop. 119: 597-610
(1993), 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 normeuronal cells.
[0193] Cellular morphology was routinely assessed by intracellular
injection of fluorescent dyes (4% Lucifer Yellow or 5% 5,6
dicarboxyfluorescein; Bruckenstein and Higgins, Dev. Biol. 128:
924-936 (1988)). Only neurons whose cell bodies were at least 150
mm from their nearest neighbor were injected, because
density-dependent changes in morphology occur when somata of
sympathetic neurons are separated by lesser distances. Highly
purified recombinant human 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. See Sampath, et al., J. Biol. Chem. 267: 20352-20362
(1992).
[0194] 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 one month in vitro. Most of the
remainder had either two 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.
[0195] 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 three days. The processes
that formed in the presence of OP-1 had the appearance of
dendrites. These processes 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 mm from the soma. The mean number of dendrites/cell continued
to increase during a four week exposure to OP-1, with most of the
change occurring during the first 10 days of treatment. After four
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. 9.
[0196] The effects of OP-1 appeared to be dendrite-specific in this
cell type. The effects of OP-1 on initial axon growth during the
first 48 hrs in culture were examined. OP-1 did not affect 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 the morphogen was not acting by
enhancing the survival of a subpopulation of neurons, as shown in
panel C of FIG. 9. The effects of OP-1 were also examined in the
delayed introduction paradigm, and no increase in axon number was
observed.
[0197] (ii) Effects of Various Concentrations of OP-1 on Dendritic
Growth
[0198] In order to assess whether the effects of morphogens on
dendritic growth were concentration-dependent, suspensions of
neurons dissociated from the superior cervical ganglia were exposed
to various concentrations of OP-1.
[0199] The effects of OP-1 were concentration-dependent (FIG. 10).
Significant changes in dendritic growth could be detected with
concentrations as low as 300 pg/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.
[0200] 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. In addition, three other changes were
observed in cellular morphology in this morphogen concentration
range. 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.
[0201] (iii) Effects of Various Morphogens on Dendritic Growth
[0202] Using the methods described above, other morphogens were
tested for their capacity to induce dendritic growth of sympathetic
neurons and their effects on the expression of cytoskeletal
proteins. Sympathetic neurons were dissociated from the superior
cervical ganglia of Holtzman (Harlan Sprague-Dawley) rat fetuses
(E21) or pups (1 day postnatal) and plated onto
poly-D-lysine-coated coverslips according to the method of Higgins,
et al., CULTURING NERVE CELLS, Banker and Goslin, eds., MIT Press,
pp.177-205 (1991). The cells were maintained in a serum-free medium
that contains nerve growth factor (100 ng/ml), and normeuronal
cells were eliminated by exposure to
cytosine-.beta.-D-arabinofuranoside (1 .mu.M) for 48-72 h beginning
on the second day after plating. The morphology of the neurons was
routinely assessed by intracellular injection of the fluorescent
dye Lucifer yellow (4%) and by immunostaining with
dendrite-specific antibodies. These included monoclonal antibody
SMI 32 (Stemberger Monocionals, Inc.) which recognizes
nonphosphorylated epitopes on the H and M neurofilament subunits
and AP20 (Sigma) and SMI 52 (Stemberger Monocionals, Inc.) which
both react with microtubule-associated protein-2 (MAP2). Highly
purified recombinant human proteins (BMP-2, BMP-3 and CDMP-2) and
Drosophila 60A were prepared as described previously for OP-1.
[0203] For Western blot analysis of cytoskeletal proteins,
sympathetic neurons were plated onto poly-D-lysine coated 35 mm
dishes and treated with 50 ng/ml of BMP-2, OP-1, 60A or CDMP-2 for
five days. Cells were then scraped off dishes in 50 mM Tris buffer
(pH 7.4) containing 0.1% SDS, 2% 2-mecaptoenthanol and 1 mM EDTA
and homogenized by passing through a 23 gauge needle at 4.degree.
C. Cell extracts were centrifuged at 12000.times.g for 15 minutes
and the protein concentrations of the supernatants were determined
using the Bradford dye reagent (Bio-Rad). Equal amounts of proteins
were resolved by SDS-PAGE, electrophoretically transferred onto a
nitrocellulose membrane, and probed with antibodies to MAP2 or an
antibody SMI 31 (Stemberger Monocionals, Inc.) to the
phosphorylated forms of the H and M neurofilament subunits.
Detection was accomplished using Chemiluminescent Substrate (Pierce
Chemical Co.) after sequential treatment with biotinylated goat
antimouse IgG (HyClone Laboratories, Inc.) and with horseradish
peroxidase-conjugated streptavidin (Amersham).
[0204] As illustrated in FIG. 11, all the morphogens tested (i.e.,
OP-1, BMP-2, BMP-3, CDMP-2, and 60A) induced significant dendritic
growth in sympathetic neurons. However, significant variations in
efficacy were observed. Treatment with maximally effective
concentrations (50 ng/ml) of BMP-2 or OP-1 for five days caused
virtually all of the neurons to form dendrites (Table III). These
processes exhibited a distinct taper, branched at "Y" shaped
angles, and extended approximately 100 .mu.m from the cell bodies
after five days of treatment. Examination of concentration effect
relationships (FIG. 1) revealed that the EC.sub.50 for BMP-2 (1.7
ng/ml) was similar to that for OP-1 (1.8 ng/ml) and that maximally
effective concentrations of these two growth factors had equivalent
effects on the cells, as assessed by both the number of dendrites
per cell and the length of the longest dendrite (Table III).
Moreover, the effects of OP-1 and BMP-2 were not additive (data not
shown) suggesting that the two ligands may share aspects of a
common signaling pathway. The Drosophila 60A protein also
stimulated dendritic growth and the EC.sub.50 (2.7 ng/ml) for this
activity was similar to that for OP-1 and BMP-2. However, 60A was
less efficacious, and at maximally effective concentrations caused
cells to form fewer dendrites (1.2/cell) than either OP-1 or BMP-2
(4.6 or 4.9/cell, respectively). BMP-3 and CDMP-2 only produced a
statistically significant increase in dendritic growth at the
highest concentration tested (100 ng/ml). The different efficacies
for promoting dendrite growth may indicate relatively stringent
structural requirements for this biological activity. OP-1, BMP-2
and 60A, which share a high sequence homology (89-90%) in the
conserved seven-cysteine skeleton sequence, had much higher
efficacies than BMP-3 and CDMP-2, which share 78% and 82% homology,
respectively, with the reference sequence of OP-1. See FIG. 1.
8TABLE III COMPARISON OF THE EFFECTS OF VARIOUS MORPHOGENS ON
DENDRITIC GROWTH OF SYMPATHETIC NEURONS Length of Growth Dendrites
% Cells with the Longest EC.sub.50 Factor per Cell Dendrites
Dendrite (pm) (ng/ml) Control 0.20 .+-. 0.14 13.3 6.0 .+-. 4.3 OP-1
4.62 .+-. 0.49* 100.0 112.3 .+-. 5.6 1.84 BMP-2 4.93 .+-. 0.46*
100.0 107.1 .+-. 5.6 1.66 60A 1.20 .+-. 0.26* 73.3 50.7 .+-. 9.0
2.70 BMP-3 0.44 .+-. 0.22 25.0 16.3 .+-. 7.6 26.05 CDMP-2 0.25 .+-.
0.14 18.8 12.5 .+-. 6.7 98.38 Sympathetic neurons were exposed to
50 ng/ml of each of the growth factors for five days and then
immunostained with a dendrite specific antibody (SMI 32). Cellular
morphology was analyzed by fluorescence microscopy using Metamorph
software Universal Imaging). Data are presented as the mean .+-.
SEM; N = 20-30. *p < vs control (Student's t-test).
[0205] The effects of various morphogens on the expression of
cytoskeletal proteins were also assessed using methods described
above. After normeuronal cells had been eliminated, sympathetic
neurons were treated with control medium or with 50 ng/ml of OP-1,
BMP-2, 60A or CDMP-2, for five days. Cultures were then solubilized
and subjected to Western blot analysis for MAP2 (primarily located
in dendrites) and for phosphorylated forms of the H and M
neurofilament subunits (primarily located in axons). The efficacy
of the various morphogens in increasing MAP2 expression correlated
with their ability to induce dendritic growth. Cultures exposed to
BMP-2, OP-1 or 60A exhibited significant increases (3.0.+-.0.4,
2.3.+-.0.4, and 1.8.+-.0.3 fold, respectively) in the expression of
high molecular weight forms of MAP2 when compared to control
cultures. The level of expression of MAP2 was not significantly
increased in cultures exposed to CDMP-2. None of the morphogens
tested affected the expression of the phosphorylated forms of the H
and M neurofilament subunits. These results show that morphogens
enhance the expression of a microtubule-associated protein which is
found in dendrites and which is required for the growth of these
processes. These observations suggest that regulation of MAP2
expression may be one of the mechanisms by which morphogens
regulate the morphological development of sympathetic neurons.
[0206] (iv) Comparison of OP-1 to Other Growth Factors
[0207] In order to evaluate whether the effects on dendritic
outgrowth are specific to morphogens, the effects of other growth
factors on dendritic growth were compared to those of OP-1.
[0208] 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., Brain Res. 367: 282-286 (1986), 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., IL-3, IL-4, IL-6, IL-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); R&D Systems (BDNF, NT3, NT4, bFGF).
[0209] As summarized in 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 and functionally distinct morphogen
sub-family. In addition, negative results were obtained with most
neurotrophins and nine other growth factors known to affect
neuronal survival or differentiation (Table IV). In other
experiments, negative results were also obtained with: TGF-.beta.2,
interleukins 1.beta., 2, 3, 4, 6, 7, and 8, PDGF, HGF, GM-CSF,
MCAF, RANTES, TGF-.alpha. and gamma interferon. Thus, it would
appear that the dendrite-promoting effect of morphogens is a highly
specific response that is observed with a very limited subset of
growth factors.
9TABLE IV COMPARISON OF THE EFFECTS OF OP-1 AND OTHER GROWTH
FACTORS ON DENDRITIC GROWTH GROWTH MEAN NUMBER OF FACTOR
DENDRITES/CELL NONE 0.8 .+-. 0.04 OP-1 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
[0210] 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. Only the results obtained with the
highest concentration tested (100 ng/ml) are shown in this table,
but lower concentrations yielded similar results. N>30 cells for
each condition
[0211] (v) Role of Morphogens in Glial-Induced Dendritic Growth
[0212] Sympathetic neurons extend only a single axon when grown in
the absence of serum or normeuronal cells. In contrast,
co-culturing sympathetic neurons with glial cells causes these
neurons to form dendrites. In order to assess the potential role of
morphogens in glial-induced dendritic growth, neuron and glial
cells were co-cultured in the presence of a monoclonal antibody
(mAb) raised against hOP-1.
[0213] Dendritic growth in sympathetic neurons grown with
astrocytes or Schwann cells was inhibited by 40-60% in the presence
of a hOP-1 mAb. SDS-PAGE analyses by hOP-1 mAb of proteins
immunoprecipitated from neuron-glia co-cultures revealed several
bands, the molecular weights of which corresponded to the cellular
and secreted forms of hOP-1. Immunocytochemical analyses of
co-cultures indicate that both neurons and glia express cytoplasmic
and surface staining for OP-1 and BMP-6. Similar patterns of
immunoreactivity were observed in glia grown in the absence of
neurons. However, neurons cultured in the absence of glia expressed
cytoplasmic but not surface staining for OP-1 or BMP-6. These data
are consistent with a role for morphogens in glia-induced dendritic
growth.
EXAMPLE 17
Morphogen-Induced Synaptic Formation
[0214] As described in Examples 15 and 16, OP-1 induces dendritic
growth in various populations of cultured neurons. To determine if
these dendrites are receptive to innervation, OP-1-treated cultured
bippocampal neurons were immunostained with antibodies to MAP2 and
synapsin. Sites of presynaptic contact were defined by puncta of
synapsin immunoreactivity. Given the poor growth of axons in
cultured hippocampal neurons maintained in a serum-free medium, a
heterochronic culture technique was used to assess the ability of
the OP-1-extended dendrites to receive axonal contacts. Cultured
neurons were grown in the presence of OP-1 for three days. New
neurons were plated on top of these more mature neurons and fixed
one day later. Previous work has shown that axonal contacts will
form within 24 hours of plating if more mature dendrites are
present within the culture. Fletcher, et al., J. Neurosci 14:
6695-6706 (1994). Using this heterochronic culture technique,
synapsin positive aggregates were found surrounding OP-1-induced
dendrites. As illustrated in FIG. 12, OP-1-treated cultured
hippocampal neurons had a significantly higher number of synapses
per neuron than untreated neurons or neurons co-cultured with glial
cells. These observations suggest that the OP-1 induced dendritic
outgrowth produces dendrites that are receptive to innervation.
EXAMPLE 18
Morphogen-Induced Dendritic Growth and Synaptogenesis In Vivo
[0215] In order to assess the effects of morphogen on dendritic
growth in vivo, rats are injected intraperitoneally once per day
with OP-1 at dose of 2 mg/kg. The control group consists of rats
injected intraperitoneally with the vehicle (20 mM arginine (pH
9.0), 150 mM NaCl with 0.1% Tween 80). After seven days, rats are
anesthetized with ether and the superior cervical ganglia,
hippocampus, and hypoglossal nucleus are removed. Subsequently,
rats are perfused with paraformaldehyde and the kidney and retina
are removed.
[0216] Superior cervical ganglia are desheathed and pinned in a
chamber superfused with an oxygenated physiological saline. For
intracellular staining, neurons are impaled with triangular glass
electrodes filled with a 4% solution of horseradish peroxidase
(HRP). HRP is introduced into the cell by iontophoresis and the
reaction product is visualized by the pyrocathecol-phenylenediamine
method. Hanker, et al., Histochem. J 9: 789-792 (1977); for details
see Purves and Hume, J. Neurosci. 1: 441-452 (1981); Forehand and
Purves, J. Neurosci. 4: 1-12 (1984). Five to ten cells/ganglion are
injected. After allowing two hours for dye diffusion, the ganglia
are fixed in 4% formaldehyde overnight. After dehydration, stained
neurons are viewed at 300.times.in whole-mount preparations and
traced with the aid of a camera lucida.
[0217] To confirm the light microscopic identification of processes
and to assess the state of differentiation of the dendrites formed
in the presence of OP-1, superior cervical ganglia are
immunostained with antibodies previously shown to react selectively
with either axons or dendrites. Lein and Higgins, Dev. Biol. 136:
330-345 (1989). Monoclonal antibodies (mAb) to MAP2 (e.g., AP14),
to nonphosphorylated forms of the M and H neurofilaments (SMI 32,
Stembery-Meyer Immunocytochemicals), and to the transferrin
receptor (MRC OX-26, Serotech) are used as dendritic markers and
mAb to synaptophysin (SY-38, Boehringer Mannheim), tau (e.g., Tau
1), and phosphorylated forms of the H (NE14, Boehringer Mannheim)
and the M and H (SMI 31, Sternbery-Meyer Immunocytochemicals)
neurofilament subunits are used as axonal markers. All antigens are
localized by indirect immunofluorescence using previously described
procedures. Lein and Higgins, Dev. Biol. 136: 330-345 (1989). Image
1 Software (Universal Imaging) is used for the morphometric
analyses of dendritic growth in immunostained cultures. In
addition, in order to determine the effects of OP-1 on
synaptogenesis in superior cervical ganglia in vivo, neurons are
immunostained with antibodies to synapsin. Sites of presynaptic
contact are defined by puncta of synapsin immunoreactivity.
[0218] Hippocampal or hypoglossal tissue is impregnated with
GolgiCox solution. Following dehydration, the tissue is embedded in
celloidin and sectioned at 160 lm on a microtome. Sections are then
developed in 5% sodium sulphite and mounted on a glass slide with
permount. Kidney and retinal tissue is removed from animals that
have been perfused with formaldehyde. The fixed tissue is embedded
in paraffin and sectioned at 160 .mu.m on a microtome. Sections are
then developed in 5% sodium sulphite and mounted on a glass slide
with permount. Sections of hippocampal, hypoglossal, kidney, and
retinal tissue are immunostained with antibodies previously shown
to react selectively with axons, dendrites, or synapsin. Antigens
are localized by indirect immunofluorescence, as described
above.
[0219] Dendritic and axonal processes are distinguished using
established criteria. Purves and Hume, J. Neurosci. 1: 441-452
(1981). Dendrites have numerous short processes arising from the
main shaft and branched into secondary and tertiary segments
relatively close to the cell soma. The axon is readily identified
as a smooth, thick process that usually could be followed for at
least several hundred microns and frequently can be seen exiting
the ganglion via a postganglionic nerve. The arbor of each neuron
is assessed by four measures of dendritic complexity. The number of
primary dendrites is determined by viewing the cells at
480.times.in multiple focal planes. A primary dendrite is defined
as any process extending from the soma a distance greater than one
cell diameter. Total dendritic lengths are measured from the camera
lucida tracings with the aid of a digitizing tablet and a general
purpose program for neural imaging. Voyvodic, Soc. Neurosci. Abstr.
12: 390 (1986). The radius of a circle incorporating the entire
arbor is measured as an indicator of the process length. Finally,
the extent of branching is determined by counting the number of
branches crossing a 50% circle. Scholl, J. Comp. Neurol. 244:
245-253 (1953). Sites of presynaptic contact are defined by puncta
of synapsin immunoreactivity.
[0220] Animals treated with OP-1 are expected to have significantly
enhanced dendritic growth when compared to control animals,
reflected in increased length, diameter, and number of processes.
Further, animals treated with OP-1 are expected to have
significantly increased number of synaptic contact when compared to
control animals.
EXAMPLE 19
Intra-Ocular Transplants
[0221] Intra-ocular grafting is a well established model which
offers an isolated environment in which CNS synaptic contact can be
selectively activated and pharmacologically characterized using
drug superfision techniques in vivo. See, for example, Olson, et
al., ADVANCES IN CELLULAR NEUROBIOLOGY, (Academic Press, 1983)
Grafts of identified CNS areas are placed into the anterior eye
chamber of syngeneic and allogeneic host rats. The development and
overall structural organization of the graft is relatively
organotypic in nature and the mature transplant usually provides an
in vivo replica of the grated area. Hoffer, et al., Brain Res.
79:165-184 (1974). After maturation of the transplant, host animals
can be anesthetized and pre and postsynaptic activity can be
examined using in vivo electrochemical and electrophysiological
techniques. Eriksdotter-Nilsson et al., Brain Res. 478: 269-280
(1989); Eriksdotter-Nilsson, et al., Exp. Brain Res. 74: 89-98
(1989); Hoffer, et al., Brain Res. 79: 165-184 (1974); Johansson,
et al., Exp. Neurol. 134: 25-34 (1995). After sacrifice of the host
animal, grafts and underlying irides can be processed for
histochemical evaluations of neural and glial elements, and for
localization of various transmitter-specific structures and
receptors. Bjorklund, et al., Dev. Brain Res. 6: 130-140 (1983);
Bergman, et al., Hippocampus 2: 339-348 (1992); Henschen, et al.,
Prog. Brain Res. 78: 187-191 (1988); Henschen, et al., Neuroscience
26: 193-213 (1988); and Henschen, et al., Brain Res. 36: 237-247
(1988). Using sequential grafting of fetal brain tissue pieces to
the anterior chamber of the eye, it is possible to study the
conditions under which mature brain and spinal cord tissue (grafts
which have resided in oculo for one or more months and which no
longer show morphological signs of growth or development) will
accept ingrowth of nerve fibers. Olson, et al., Brain Res. Bull. 9:
519-537 (1982). Thus, using the in oculo technique, isolated
replicas of defined pathways suitable for structural and functional
studies of CNS connectivity can be obtained.
[0222] In particular, it has been previously shown that grafted
spinal cord will survive and grow in oculo in a manner suggesting
that is possesses a considerable intrinsic determination of its
normal development. Henschen, et al., Exp. Brain Res. 60:
38-47(1985). Further, it has been previously demonstrated that
models of the descending coeruleo-spinal noradrenergic and
bulbospinal serotonergic pathways to spinal cord can be generated
when these CNS areas are co-grafted in oculo. Henschen, et al.,
Brain Res. Bull. 15: 335-342 (1985); Henschen, et al., Brain Res.
Bull 17: 801-808 (1986); Henschen, et al., Dev. Brain Res. 36:
237-247 (1987); Henschen, et al., Exp. Brain Res. 75: 317-326
(1989). In a similar vein, corticospinal and sensory and motor
pathways can be constructed in oculo, using co-grafts of cerebral
cortex (Palmer, et al., Exp. Brain Res. 87: 96-107 (1991); dorsal
root ganglia (Trok, et al., 1997), and muscle (Trok, et al., Brain
Res. 659: 138-146 (1994), with subsequent histological and
electrophysiological analysis. Thus, in oculo grafts provide a
unique isolated system of a much more "in vivo" than "in vitro"
nature, to study spinal cord connectivity and response to
injury.
[0223] In oculo transplants of specific CNS areas have been
employed to evaluate of various putative neurotrophic effects: NGF
in hippocampus (Eriksdotter-Nilsson et al., Brain Res. 478: 269-280
(1989) and Eriksdotter-Nilsson, et al., Exp. Brain Res. 74: 89-98
(1989)), FGF in cortical areas (Giacobini, et al., Exp. Brain Res.
86: 73-81 (1991)), IGF-1 in olfactory bulb (Giacobini, et al.,
1995)), GDNF in midbrain dopaminergic nucleus and spinal cord
(Johansson, et al., Exp. Neurol. 134: 25-34 (1995); Trok, et al.,
Neuroscience 71: 231-241 (1996); and Trok, et al., (1996)), and
BDNF, NT-3, NT-4, and CNTF in spinal cord (Trok, et al., 1997).
[0224] 20.1 Effects of OP-1 on Intra-Ocular Spinal Cord
Transplants
[0225] In oculo transplants Were employed to evaluate the effects
of morphogens on motor neurons. Fisher 344 rats were implanted with
syngeneic E18 spinal cord grafts, essentially as previously
described by Henschen, et al., Prog Brain Res. 78: 187-191 (1988).
OP-1 (0.5 .mu.g) or vehicle was injected into the anterior chamber
at weekly intervals. Each rat had an OP-1-treated graft in one eye
and a control graft in the contralateral eye. Survival and growth
of the graft was followed noninvasively by observation through the
cornea over a four week period. After sacrifice of the host
animals, grafts were evaluated by histological and
immunocytochemical techniques. Olson, L., et al., ADVANCES IN
CELLULAR NEUROBIOLOGY, pp. 407-442 (Academic Press, 1983);
Granholm, et al., Exp. Neurol. 118: 7-17 (1992).
[0226] As shown in FIG. 13, OP-1-treated grafts maintained a
significantly larger size over the four week observation period,
compared to control grafts. Transplants treated with weekly
injections of vehicle had minimal survival but manifested a marked
reduction in size, similar to what has been previously described
for E18 donors. Henschen, et al., Prog. Brain Res. 78: 187-191
(1988); Henschen, et al., Neuroscience 26: 193-213 (1988). In
contrast, grafts treated with weekly injections of 0.5 .mu.g of
OP-1 maintained a much greater size and, at the end of four weeks,
had a size equal to, or only slightly less than, the initial size
at grafting.
[0227] This positive effect of OP-1 was confirmed using
immunocytochemical techniques. Overall neuron density was evaluated
using MAP2 (i.e., neurofilament immunoreactivity). As seen in FIG.
14, the number of neurofilament-positive neuronal structures was
significantly higher in OP-1-treated grafts compared to
vehicle-treated transplants. In order to assess more specifically
the effects of OP-1 on motor neurons, immunocytochemical studies
were carried out using choline acetyltransferase (CHAT)
immunocytochemistry. The number of cholinergic cell bodies and
fibers was also significantly higher in OP-1-treated grafts than in
vehicle-treated transplants. See FIG. 15.
EXAMPLE 20
Traumatic Injury Model
[0228] The fluid percussion brain injury model was used to assess
the ability of morphogens to restore central nervous system
functions following significant traumatic brain injury.
[0229] I. Fluid Percussion Brain Injury Procedure
[0230] The animals used in this study were male Sprague-Dawley rats
weighing 250-300 grams (Charles River). The basic surgical
preparation for the fluid-percussion brain injury has been
previously described. Dietrich, et al., Acta Neuropathol. 87:
250-258 (1994) incorporated by reference herein. Briefly, rats were
anesthetized with 3% halothane, 30% oxygen, and a balance of
nitrous oxide. Tracheal intubation was performed and rats were
placed in a stereotaxic frame. A 4.8-mm craniotomy was then made
overlying the right parietal cortex, 3.8 mm posterior to bregma and
2.5 mm lateral to the midline. An injury tube was placed over the
exposed dura and bonded by adhesive. Dental acrylic was then poured
around the injury tube and the injury tube was then plugged with a
gelfoam sponge. The scalp was sutured closed and the animal
returned to its home case and allowed to recover overnight.
[0231] On the next day, fluid-percussion brain injury was produced
essentially as described by Dixon, et al., J. Neurosurg 67: 110-119
(1987) and Clifton, et al., J Cereb. Blood Flow Metab. 11: 114-121
(1991). The fluid percussion device consisted of a saline-filled
Plexiglas cylinder that is fitted with a transducer housing and
injury screw adapted for the rat's skull. The metal screw was
firmly connected to the plastic injury tube of the intubated
anesthetized rat (70% nitrous oxide, 1.5% halothane, and 30%
oxygen), and the injury was induced by the descent of a pendulum
that strikes the piston. Rats underwent mild-to-moderate head
injury, ranging from 1.6 to 1.9 atm. Brain temperature was
indirectly monitored with a thermistor probe inserted into the
right temporalis muscle and maintained at 37-37.5.degree. C. Rectal
temperature was also measured and maintained at 37.degree. C. prior
to and throughout the monitoring period.
[0232] II. Administration of Morphogen
[0233] Animals in the treatment group received OP-1
intracisternally at a dose of 10 .mu.g/injection. Control animals
received vehicle solutions lacking OP-1 but with all other
components at equivalent final concentrations. Both OP-1 and
vehicle-treated animals received two injections, one day and four
days following the fluid percussion injury.
[0234] To administer the injection, the animals were anesthetized
with halothane in 70% NO.sub.2/30% O.sub.2 and placed in a
stereotaxic frame. The procedure for intracisternal injection of
OP-1 containing solutions or vehicle-only solutions was identical.
Using aseptic technique, OP-1(1 or 10 .mu.g/injection) or an
equivalent volume of vehicle were introduced by percutaneous
injection (10 .mu.l/injection) into the cisterna magna using a
Hamilton syringe fitted with a 26 gauge needle (Yamada, et al.,
(1991) J. Cereb. Blood Flow Metab. 11: 472-478). Before each
injection, 1-2 .mu.l of cerebrospinal fluid (CSF) was drawn back
through the Hamilton syringe to verify needle placement in the
subarachnoid space. Preliminary studies demonstrated that a dye, 1%
Evans blue, delivered in this fashion diffused freely through the
basal cisterns and over the cerebral cortex within one hour of
injection. Animals were randomly assigned to either of the OP-1
treatment groups or to the vehicle treatment group. Animals
received two intracistemal injections (2.times.10 .mu.g/injection
OP-1 or 2.times.vehicle); the first injection was administered 24
hours after the brain injury and the second injection was
administered 4 days after the brain injury.
[0235] III. Behavioral Testing
[0236] Three standard functional/behavioral tests were used to
assess sensorimotor and reflex function after brain injury. The
tests have been fully described in the literature, including
Bederson, et al., (1986) Stroke 17: 472476; DeRyck, et al., (1992)
Brain Res. 573: 44-60; Markgraf, et al., (1992) Brain Res. 575:
238-246; and Alexis, et al., (1995) Stroke 26: 2338-2346.
[0237] A. The Forelimb Placing Test
[0238] Forelimb placing to three separate stimuli (visual, tactile,
and proprioceptive) was measured to assess sensorimotor
integration. DeRyck, et al., Brain Res. 573:44-60 (1992). For the
visual placing subtest, the animal is held upright by the
researcher and brought close to a table top. Normal placing of the
limb on the table is scored as "0," delayed placing (<2 sec) is
scored as "1," and no or very delayed placing (>2 sec) is scored
as "2." Separate scores are obtained first as the animal is brought
forward and then again as the animal is brought sideways to the
table (maximum score per limb=4; in each case higher numbers denote
greater deficits). For the tactile placing subtest, the animal is
held so that it cannot see or touch the table top with its
whiskers. The dorsal forepaw is touched lightly to the table top as
the animal is first brought forward and then brought sideways to
the table. Placing each time is scored as above (maximum score per
limb=4). For the proprioceptive placing subtest, the animal is
brought forward only and greater pressure is applied to the dorsal
forepaw; placing is scored as above (maximum score per limb=2).
Finally, the ability of animals to place the forelimb in response
to whisker stimulation by the tabletop was tested (maximum score
per limb 2). Then subscores were added to give the total forelimb
placing score per limb (range=0-12).
[0239] B. The Beam Balance Test
[0240] Beam balance is sensitive to motor cortical insults. This
task was used to assess gross vestibulomotor function by requiring
a rat to balance steadily on a narrow beam. Feeney, et al.,
Science, 217: 855-857 (1982); Goldstein, et al., Behav. Neurosci.
104: 318-325 (1990). The test involved three 60-second training
trials 24 hours before surgery to acquire baseline data. The
apparatus consisted of a 3/4-inch-wide beam, 10 inches in length,
suspended 1 ft. above a table top. The rat was positioned on the
beam and had to maintain steady posture with all limbs on top of
the beam for 60 seconds. The animals' performance was rated with
the scale of Clifton, et al., J. Cereb Blood Flow Metab. 11:
114-121(1991), which ranges from 1 to 6, with a score of 1 being
normal and a score of 6 indicating that the animal was unable to
support itself on the beam.
[0241] C. The Beam Walking Test
[0242] This was a test of sensorimotor integration specifically
examining hindlimb function. The testing apparatus and rating
procedures were adapted from Feeney, et al., Science, 217: 855-857
(1982). A 1-inch-wide beam, 4 ft. in length, was suspended 3 ft.
above the floor in a dimly lit room. At the far end of the beam was
a darkened goal box with a narrow entryway. At equal distances
along the beam, four 3-inch metal screws were positioned, angling
away from the beam's center. A white noise generator and bright
light source at the start of the beam motivated the animal to
traverse the beam and enter the goal box. Once inside the goal box,
the stimuli were terminated. The rat's latency to reach the goal
box (in seconds) and hindlimb performance as it traversed the beam
(based on a 1 to 7 rating scale) were recorded. A score of 7
indicates normal beam walking with less than 2 foot slips, and a
score of 1 indicates that the rat was unable to traverse the beam
in less than 80 seconds. Each rat was trained for three days before
surgery to acquire the task and to achieve normal performance (a
score of 7) on three consecutive trials. Three baseline trials were
collected 24 hours before surgery, and three testing trials were
recorded daily thereafter. Mean values of latency and score for
each day were computed.
[0243] IV. Results
[0244] As illustrated in FIGS. 16-18, OP-1 enhanced the recovery
from traumatic brain injury in all three behavioral measures. In
the forelimb placing tests, OP-1-treated animals showed a gradual
decrease in injury severity scores which attained statistical
significance by day 9. See FIG. 16. In the beam balance and beam
walking tests, OP-1-treated animals had performance scores that
were essentially identical to sham-control animals. See FIGS. 17
and 18. These observations suggests that morphogens are capable of
restoring impaired or lost sensory-motor functions following a
traumatic brain injury, including visual, tactile, and
proprioceptive placement, gross vestibulomotor function, and
sensorimotor integration.
[0245] Similar routine modifications can be made in other accepted
models of traumatic central nervous system injury, to confirm
efficacy of morphogen treatment to restore impaired or lost
sensory-motor functions.
Sequence CWU 1
1
* * * * *