U.S. patent application number 10/849704 was filed with the patent office on 2004-11-25 for neuroprotective methods and reagents.
This patent application is currently assigned to Curis, Inc.. Invention is credited to Mahanthappa, Nagesh K..
Application Number | 20040235739 10/849704 |
Document ID | / |
Family ID | 25383052 |
Filed Date | 2004-11-25 |
United States Patent
Application |
20040235739 |
Kind Code |
A1 |
Mahanthappa, Nagesh K. |
November 25, 2004 |
Neuroprotective methods and reagents
Abstract
One aspect of the present application relates to a method for
limiting damage to neuronal cells by ischemic or epoxic conditions,
e.g., such as may be manifest by a reduction in brain infarct
volume, by administering to an individual a hedgehog therapeutic or
ptc therapeutic in an amount effective for reducing cerebral
infarct volume.
Inventors: |
Mahanthappa, Nagesh K.;
(Cambridge, MA) |
Correspondence
Address: |
ROPES & GRAY LLP
ONE INTERNATIONAL PLACE
BOSTON
MA
02110-2624
US
|
Assignee: |
Curis, Inc.
Cambridge
MA
|
Family ID: |
25383052 |
Appl. No.: |
10/849704 |
Filed: |
May 19, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10849704 |
May 19, 2004 |
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09418221 |
Oct 14, 1999 |
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6767888 |
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09418221 |
Oct 14, 1999 |
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08883656 |
Jun 27, 1997 |
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Current U.S.
Class: |
514/200 ;
514/13.8; 514/14.7; 514/14.9; 514/15.1; 514/17.7 |
Current CPC
Class: |
A61K 31/47 20130101;
A61P 9/10 20180101; A61K 31/496 20130101; A61K 31/711 20130101 |
Class at
Publication: |
514/012 |
International
Class: |
A61K 038/17 |
Claims
1. A method for limiting damage to neuronal cells by ischemic or
epoxic conditions, comprising administering to an individual a ptc
therapeutic in an amount effective for reducing cerebral infarct
volume relative to the absence of administration of the ptc
therapeutic, wherein the ptc therapeutic inhibits PKC with a
K.sub.i greater than 1 .mu.M.
2. A method for protecting cerebral tissue of a mammal against the
repercussions of ischemia which comprises administering to the
mammal in need thereof a therapeutically effective amount of a ptc
therapeutic therapeutic, wherein the ptc therapeutic inhibits PKC
with a K.sub.i greater than 1 .mu.M.
3. A method for the treatment of cerebral infarctions which
comprises administering to a patient in need thereof a
therapeutically effective amount of a ptc therapeutic therapeutic,
wherein the ptc therapeutic inhibits PKC with a K.sub.i greater
than 1 .mu.M.
4. A method for the treatment of cerebral ischemia which comprises
administering to a patient in need thereof a therapeutically
effective amount of a ptc therapeutic therapeutic, wherein the ptc
therapeutic inhibits PKC with a K.sub.i greater than 1 .mu.M.
5. A method for the treatment of stroke which comprises
administering to a patient in need thereof a therapeutically
effective amount of a ptc therapeutic therapeutic, wherein the ptc
therapeutic inhibits PKC with a K.sub.i greater than 1 .mu.M.
6. A method for the treatment of transient ischemia attack which
comprises administering to a patient in need thereof a
therapeutically effective amount of a ptc therapeutic therapeutic,
wherein the ptc therapeutic inhibits PKC with a K.sub.i greater
than 1 .mu.M.
7. The method of claim 1, wherein the ptc therapeutic binds to
patched and mimics hedgehog-mediated patched signal
transduction.
8. The method of claim 7, wherein the ptc therapeutic is a small
organic molecule.
9. The method of claim 7, wherein the binding of the ptc
therapeutic to patched results in upregulation of patched and/or
gli expression.
10. The method of claim 8, wherein the ptc therapeutic is a small
organic molecule which interacts with neuronal cells to mimic
hedgehog-mediated patched signal transduction.
11. The method of claim 1, wherein the ptc therapeutic mimics
hedgehog-mediated patched signal transduction by altering the
localization, protein-protein binding and/or enzymatic activity of
an intracellular protein involved in a patched signal pathway.
12. The method of claim 1, wherein the ptc therapeutic alters the
level of expression of a hedgehog protein, a patched protein or a
protein involved in the intracellular signal transduction pathway
of patched.
13. The method of claim 11, wherein the ptc therapeutic is a small
organic molecule which binds to patched and regulates
patched-dependent gene expression.
14. The method of claim 11, wherein the ptc therapeutic is an
inhibitor of protein kinase A (PKA).
15. The method of claim 14, wherein the PKA inhibitor is a
5-isoquinolinesulfonamide.
16. The method of claim 15, wherein the PKA inhibitor is
represented in the general formula: 6wherein, R.sub.1 and R.sub.2
each can independently represent hydrogen, and as valence and
stability permit a lower alkyl, a lower alkenyl, a lower alkynyl, a
carbonyl, a thiocarbonyl, an amino, an acylamino, an amido, a
cyano, a nitro, an azido, a sulfate, a sulfonate, a sulfonamido,
--(CH.sub.2).sub.m--R.sub.8- , --(CH.sub.2).sub.m--OH,
--(CH.sub.2).sub.m--O-lower alkyl, --(CH.sub.2).sub.m--O-lower
alkenyl, --(CH.sub.2).sub.n--O--(CH.sub.2).su- b.m--R.sub.8,
--(CH.sub.2).sub.m--SH, --(CH.sub.2).sub.m--S-lower alkyl,
--(CH.sub.2).sub.m--S-lower alkenyl,
--(CH.sub.2).sub.n--S--(CH.sub.2).su- b.m--R.sub.8, or R.sub.1 and
R.sub.2 taken together with N form a substituted or unsubstituted
heterocycle; R.sub.3 is absent or represents one or more
substitutions to the isoquinoline ring such as a lower alkyl, a
lower alkenyl, a lower alkynyl, a carbonyl (such as a carboxyl, an
ester, a formate, or a ketone), a thiocarbonyl (such as a
thioester, a thioacetate, or a thioformate), an amino, an
acylamino, an amido, a cyano, a nitro, an azido, a sulfate, a
sulfonate, a sulfonamido, --(CH.sub.2).sub.m--R.sub.8,
--(CH.sub.2).sub.m--OH, --(CH.sub.2).sub.m--O-lower alkyl,
--(CH.sub.2).sub.m--O-lower alkenyl,
--(CH.sub.2).sub.n--O--(CH.sub.2).sub.m--R.sub.8,
--(CH.sub.2).sub.m--SH, --(CH.sub.2).sub.m--S-lower alkyl,
--(CH.sub.2).sub.m--S-lower alkenyl,
--(CH.sub.2).sub.n--S--(CH.sub.2).sub.m--R.sub.8; R.sub.8
represents a substituted or unsubstituted aryl, aralkyl,
cycloalkyl, cycloalkenyl, or heterocycle; and n and m are
independently for each occurrence zero or an integer in the range
of 1 to 6.
17. The method of claim 14, wherein the PKA inhibitor is selected
from the group consisting of
N-[2-((p-bromocinnamyl)amino)ethyl]-5-isoquinolinesul- fonamide,
KT5720, and PKA Heat Stable Inhibitor isoform .alpha..
18. The method of claim 5, wherein the stroke is a thrombotic
stroke.
19. The method of claim 5, wherein the stroke is an embolic
stroke.
20. The method of claim 1, wherein the conditions result in
cerebral hypoxia.
21. The method of claim 1, wherein the conditions result in
progressive loss of neurons due to oxygen deprivation.
22. The method of claim 3, wherein the patient is treated
prophylactically.
23. The method of claim 1, wherein the individual is treated
prophylactically.
24. The method of claim 2, wherein the mammal is treated
prophylactically.
25. The method of claim 1, wherein the patient is hypotensive.
26. The method of claim 1, further comprising administering one or
more of an anticoagulant, an antiplatelet agent, a thrombin
inhibitor, and/or a thrombolytic agent.
27. The method of claim 1, further comprising performing vascular
surgery.
28. The method of claim 27, wherein the vascular surgery comprises
carotid endarterectomy.
29. The method of claim 1, wherein treatment of the patient with
the ptc therapeutic results in at least a 25% reduction in cerebral
infarct volumes relative to absence of treatment with the ptc
therapeutic.
30. The method of claim 29, wherein treatment of the patient with
the ptc therapeutic results in at least a 50% reduction in cerebral
infarct volumes relative to absence of treatment with the ptc
therapeutic.
31. The method of claim 29, wherein treatment of the patient with
the ptc therapeutic results in at least a 70% reduction in cerebral
infarct volumes relative to absence of treatment with the ptc
therapeutic.
32. The method of claim 1, wherein the ptc therapeutic inhibits the
activity of PKA, cAMP, or adenylate cyclase.
33. The method of claim 1, wherein the ptc therapeutc agonizes the
activity of cAMP phosphodiesterase.
34. A therapeutic preparation of a small molecule antagonist of
patched, which patched antagonist inhibits PKC with a K.sub.1
greater than 100 nM and is provided in a pharmaceutically
acceptable carrier and in an amount sufficient to provide
protection against neuronal cell death under ischemic and/or
hypoxic conditions.
35. The preparation of claim 34, which patched antagonist binds to
patched.
36. The preparation of claim 34, wherein the patched antagonist is
provided in an amount sufficient to produce, upon a dosage regimen
of 7 days, at least a 70% decrease in infarct volume in an MCAO
model relative to the absence of the patched antagonist.
37. The preparation of claim 34, wherein the patched antagonist is
provided in an amount sufficient to produce, upon a dosage regimen
of 3 days, at least a 70% decrease in infarct volume in an MCAO
model relative to the absence of the patched antagonist.
Description
[0001] This application is a continuation-in-part of U.S. patent
application Ser. No. 08/883,656, filed Jun. 27, 1997, incorporated
herein by reference in its entirety.
BACKGROUND OF THE INVENTION
[0002] Stroke kills more than 150,000 people annually and accounts
for about one of every 15 U.S. deaths. It is presently the third
largest cause of death, ranking behind diseases of the heart and
cancer, according to the National Center for Health Statistics.
[0003] On average, someone suffers a stroke in the United States
every minute; every 3.4 minutes someone dies of a stroke. Based on
the Framingham Heart Study, approximately 500,000 people suffer a
new or recurrent brain attack each year. Approximately 3,890,000
stroke survivors are alive today. From 1984 to 1994, the death rate
from stroke declined 19.8 percent, but the actual number of deaths
from brain attack rose slightly.
[0004] Stroke is the leading cause of serious, long-term disability
in the United States. Stroke accounts for half of all patients
hospitalized for acute neurological disease. In 1991-92 one million
Americans age 15 and older had disabilities resulting from stroke.
According to the Framingham Heart Study, 31 percent of brain attack
survivors needed help caring for themselves; 20 percent needed help
walking; and 71 percent had an impaired ability to work when
examined an average of seven years later. Sixteen percent had to be
institutionalized. About 31 percent of people who have an initial
stroke die within a year. This percentage is higher among people
older than age 65. About two-thirds of men and women who have a
brain attack die within 12 years; long-term survivorship is worse
in men than in women. 407,000 males and 478,000 females were
discharged from hospitals in 1994 after having a stroke. For
statistics, see for example the homepage for the American Heart
Association at http://www.amhrt.org/1997/stats/Stroke.html
[0005] Stroke is defined as a sudden impairment of body functions
caused by a disruption in, e.g., the supply of blood to the brain.
For instance, a stroke occurs when a blood vessel bringing oxygen
and nutrients to the brain is interrupted by any method including
low blood pressure, clogging by atherosclerotic plaque, a blood
clot, or some other particle, or when a blood vessel bursts.
[0006] Because of the blockage or rupture, part of the brain fails
to get the blood flow that it requires. Brain tissue that receives
an inadequate supply of blood is said to be ischemic. Deprived of
oxygen and nutrients, nerve cells and other cell types within the
brain begin to fail, creating an infarct (an area of cell death, or
necrosis). As nerve cells (neurons) fail and die, the part of the
body controlled by those neurons cannot function either. The
devastating effects of ischemia are often permanent because brain
tissue has very limited repair capabilities and lost neurons are
not usually replaced.
[0007] Cerebral ischemia may be incomplete (blood flow is reduced
but not entirely cut off), complete (total loss of tissue
perfusion), transient or permanent. If ischemia is incomplete and
persists for no more than ten to fifteen minutes, neural death
might not occur. More prolonged or complete ischemia results in
infarction. Depending on the site and extent of the infarction,
mild to severe neurological disability or death will follow. Thus,
the chain of causality leading to neurological deficit in stroke
has two principal components: loss of blood supply, and cell damage
and death.
[0008] Thrombosis is the blockage of an artery by a large deposit
that usually results from the combination of atherosclerosis and
blood clotting. Thrombotic stroke (also called cerebral thrombosis)
results when a deposit in a brain or neck artery reaches occlusive
proportions. Most strokes are of this type.
[0009] Embolism is the blockage of an artery or vein by an embolus.
Emboli are often small pieces of blood clot that break off from
larger clots. Embolic stroke (also called cerebral embolism) occurs
when an embolus is carried in the bloodstream to a brain or neck
artery. If the embolus reaches an artery that is too small for it
to pass through, it plugs the artery and cuts off the blood supply
to downstream tissues. Embolic stroke is the clinical expression of
this event.
[0010] To a modest extent, the brain is protected against cerebral
ischemia by compensatory mechanisms that include: collateral
circulation (overlapping local blood supplies), and arteriolar
auto-regulation (local smooth muscle control of blood flow in the
smallest arterial channels). If compensatory mechanisms operate
efficiently, slightly diminished cerebral blood flow produces
neither tissue ischemia nor abnormal signs and symptoms. Usually,
such mechanisms must act within minutes to restore blood flow if
permanent infarction damage is to be avoided or reduced. Arteriolar
auto-regulation works by shunting blood from noncritical regions to
infarct zones.
[0011] Even in the face of systemic hypotension, auto-regulation
may be sufficient to adjust the circulation and thereby preserve
the vitality and function of brain tissue. Alternatively, ischemia
may be sufficiently prolonged and compensatory mechanisms
sufficiently inadequate that a catastrophic stroke results. With
these as the extremes, the gradation of ischemic stroke are
described below.
[0012] A transient ischemic attack (TIA) is conventionally
described as a loss of neurologic function caused by ischemia,
abrupt in onset, persisting for less than 24 hours, and clearing
without residual signs. Most TIAs last only a few minutes. However,
neurologic disability may persist for more than 24 hours before
clearing. Such an event is called a reversible ischemic
neurological disability (RIND).
[0013] An ischemic event that is sufficiently severe to cause
persistent disability but that is short of a calamitous stroke, is
called a partial nonprogressing stroke (PNS). The penultimate
ischemic event, a completed stroke, produces major functional loss.
The ultimate ischemic insult is death.
[0014] Focal cerebral ischemia must be distinguished from global
cerebral hypoxia. In cerebral hypoxia the oxygen supply to the
brain is diminished even though blood flow and blood pressure may
be normal. Discriminating between diagnoses of patients with acute
neurological deficit is critical because patient management takes
disparate paths.
[0015] There are generally distinct clinical outcomes in stroke
versus cerebral hypoxia, although both sets of patients may suffer
death or permanent damage. Hypoxia patients who survive past an
acute life-threatening period usually show few immediate symptoms
of long term damage. Instead, clinical manifestations such as
mental deterioration, urinary and fecal incontinence, gait and
speech disturbances, tremor and weakness are delayed for periods
that may vary from days to weeks. However, as in stroke,
progressive loss of neurons due to oxygen deprivation is believed
to be a factor in such detrimental effects of hypoxia.
[0016] It is an objective of the present application to provide new
drugs for treatment and prophylaxis of cerebral ischemia, such as
stroke.
[0017] It is also an objective of the present application to
provide new drugs for treatment and prophylaxis of cerebral
hypoxia.
SUMMARY OF THE INVENTION
[0018] One aspect of the present application relates to a method
for limiting damage to neuronal cells by ischemic or epoxic
conditions, e.g., such as may be manifest by a reduction in brain
infarct volume, by administering to an individual a hedgehog
therapeutic or ptc therapeutic in an amount effective for reducing
cerebral infarct volume relative to the absence of administeration
of the hedgehog therapeutic or ptc therapeutic.
[0019] In other embodiments, the subject method can be used for
protecting cerebral tissue of a mammal against the repercussions of
ischemia; for treating cerebral infarctions; for treating cerebral
ischemia; for treatment of stroke; and/or for treating transient
ischemia attacks. In embodiments wherein the patient is treated
with a ptc therapeutic, such therapeutics are preferably small
organic molecules which mimic hedgehog effects onpatched-mediated
signals.
[0020] Wherein the subject method is carried out using a hedgehog
therapeutic, the hedgehog therapeutic preferably a polypeptide
including a hedgehog portion comprising at least a bioactive
extracellular portion of a hedgehog protein, e.g., the hedgehog
portion includes at least 50, 100 or 150 amino acid residues of an
N-terminal half of a hedgehog protein. In preferred embodiments,
the hedgehog portion includes at least a portion of the hedgehog
protein corresponding to a 19 kd fragment of the extracellular
domain of a hedgehog protein.
[0021] In preferred embodiments, the hedgehog portion has an amino
acid sequence at least 60, 75, 85, or 95 percent identical with a
hedgehog protein of any of SEQ ID Nos. 10-18, though sequences
identical to those sequence listing entries are also contemplated
as useful in the present method. The hedgehog portion can be
encoded by a nucleic acid which hybridizes under stringent
conditions to a nucleic acid sequence of any of SEQ ID Nos. 1-9,
e.g., the hedgehog portion can be encoded by a vertebrate hedgehog
gene, especially a human hedgehog gene.
[0022] In other embodiments, the subject method can be carried out
by administering a gene activation construct, wherein the gene
activation construct is designed to recombine with a genomic
hedgehog gene of the patient to provide a heterologous
transcriptional regulatory sequence operatively linked to a coding
sequence of the hedgehog gene.
[0023] In still other embodiments, the subject method can be
practiced with the administration of a gene therapy construct
encoding a hedgehog polypeptide. For instance, the gene therapy
construct can be provided in a composition selected from a group
consisting of a recombinant viral particle, a liposome, and a
poly-cationic nucleic acid binding agent.
[0024] Where the subject method is carried out using a ptc
therapeutic, the therapeutic can be, e.g., a molecule which binds
to patched and mimics hedgehog-mediated patched signal
transduction. For instance, the binding of the therapeutic to
patched may result in upregulation of patched and/or gli
expression.
[0025] In other embodiments, the ptc therapeutic mimics
hedgehog-mediated patched signal transduction by altering the
localization, protein-protein binding and/or enzymatic activity of
an intracellular protein involved in a patched signal pathway.
[0026] In a preferred embodiment, the ptc therapeutic is a small
organic molecule, e.g., less than 5 kd, more preferably less than
2.5 kd. For instance, the present invention contemplates the use of
small organic molecules which interact with neuronal cells to mimic
hedgehog-mediated patched signal transduction.
[0027] In a preferred embodiment, the ptc therapeutic is a PKA
inhibitor. A variety of PKA inhibitors are known in the art,
including both peptidyl and organic compounds. For instance, the
ptc therapeutic can be a 5-isoquinolinesulfonamide, such as
represented in the general formula: 1
[0028] wherein,
[0029] R.sub.1 and R.sub.2 each can independently represent
hydrogen, and as valence and stability permit a lower alkyl, a
lower alkenyl, a lower alkynyl, a carbonyl (such as a carboxyl, an
ester, a formate, or a ketone), a thiocarbonyl (such as a
thioester, a thioacetate, or a thioformate), an amino, an
acylamino, an amido, a cyano, a nitro, an azido, a sulfate, a
sulfonate, a sulfonamido, --(CH.sub.2).sub.m--R.sub.8- ,
--(CH.sub.2).sub.m--OH, --(CH.sub.2).sub.m--O-lower alkyl,
--(CH.sub.2).sub.m--O-lower alkenyl,
--(CH.sub.2).sub.n--O--(CH.sub.2).su- b.m--R.sub.8,
--(CH.sub.2).sub.m--SH, --(CH.sub.2).sub.m--S-lower alkyl,
--(CH.sub.2).sub.m--S-lower alkenyl,
--(CH.sub.2).sub.n--S--(CH.sub.2).su- b.m--R.sub.8, or
[0030] R.sub.1 and R.sub.2 taken together with N form a heterocycle
(substituted or unsubstituted);
[0031] R.sub.3 is absent or represents one or more substitutions to
the isoquinoline ring such as a lower alkyl, a lower alkenyl, a
lower alkynyl, a carbonyl (such as a carboxyl, an ester, a formate,
or a ketone), a thiocarbonyl (such as a thioester, a thioacetate,
or a thioformate), an amino, an acylamino, an amido, a cyano, a
nitro, an azido, a sulfate, a sulfonate, a sulfonamido,
--(CH.sub.2).sub.m--R.sub.8- , --(CH.sub.2).sub.m--OH,
--(CH.sub.2).sub.m--O-lower alkyl, --(CH.sub.2).sub.m--O-lower
alkenyl, --(CH.sub.2).sub.n--O--(CH.sub.2).su- b.m--R.sub.8,
--(CH.sub.2).sub.m--SH, --(CH.sub.2).sub.m--S-lower alkyl,
--(CH.sub.2).sub.m--S-lower alkenyl,
--(CH.sub.2).sub.n--S--(CH.sub.2).su- b.m--R.sub.8;
[0032] R.sub.8 represents a substituted or unsubstituted aryl,
aralkyl, cycloalkyl, cycloalkenyl, or heterocycle; and
[0033] n and m are independently for each occurrence zero or an
integer in the range of 1 to 6. Exemplary PKA inhibitors of this
class include
N-[2-((p-bromocinnamyl)amino)ethyl]-5-isoquinoline-sulfonamide and
1-(5-isoquinolinesulfonyl)-2-methylpiperazine. Other PKA inhibitors
which can be used in the subject method include KT5720; and PKA
Heat Stable Inhibitor (isoform .alpha.).
[0034] In yet other embodiments of the present invention, the ptc
therapeutic alters the level of expression of a hedgehog protein, a
patched protein or another protein involved in the intracellular
signal transduction pathway of patched. In this regard, the ptc
therapeutic can be an antisense construct which inhibits the
expression of a protein which is involved in the signal
transduction pathway of patched and the expression of which
antagonizes hedgehog-mediated signals. For example, the antisense
molecule can be one which hyridizes to a patched transcript or
genomic sequence, such as 5'-GTCCTGGCGCCGCCGCCGCCGTCGCC,
5'-TTCCGATG-ACCGGCCTTTCGCGGTGA or
5'-GTGCACGGAAAGGTGCAGGCCACACT.
[0035] In yet other embodiments, the subject method can be carried
out with a gene activation construct, which construct recombines
with a genomic hedgehog gene of the patient, e.g., to form a
chimeric gene, providing a heterologous transcriptional regulatory
sequence operatively linked to a coding sequence of the hedgehog
gene. The transcriptional regulatory sequence can provide for
constitutive or inducible expression of the hedgehog gene.
[0036] The subject method can be used as part of a treatment for
stroke, e.g., thrombotic stroke and/or embolic stroke.
[0037] The subject method can also be used to treat hypoxic
conditions which otherwise result in cerebral hypoxia.
[0038] The subject method can be used prophylactically or as an
ipso facto treatment. It can be used to treat patients who are
hypotensive.
[0039] The subject method can also be used as part of a therapy
including administering one or more of an anticoagulant, an
antiplatelet agent, a thrombin inhibitor, and/or a thrombolytic
agent, and/or in conjunction with vascular surgery, e.g., carotid
endarterectomy.
[0040] In preferred embodiments, the subject method results in at
least a 25%, 50%, 70%, 75%, or 90% reduction in cerebral infarct
volumes relative to the absence of treatment with the therapeutic,
e.g., as measured in a stroke model such as the MCAO model.
BRIEF DESCRIPTION OF THE DRAWINGS
[0041] FIG. 1 is a graph demonstrating the effect of systemic
hedgehog treatment on cerebral infarction volume in rat models of
middle cerebral artery occlusion.
DETAILED DESCRIPTION OF THE INVENTION
[0042] Stroke occurs when the flow of oxygen and nutrients to the
brain is inhibited/interrupted due to any cause. Thus, in certain
indications, stroke is a form interrupted of cardiovascular disease
that affects the arteries of the central nervous system. For
example, a stroke occurs when a blood vessel bringing oxygen and
nutrients to the brain bursts or is clogged by a blood clot or some
other particle. Because of this rupture or blockage, part of the
brain doesn't get the flow of blood it needs. Deprived of oxygen,
nerve cells in the affected area of the brain can't function and
die within minutes. Depending on the part of the brain affected by
the brain attack/stroke, there may be loss of normal function.
Strokes are the third most common cause of death in United States.
Stroke is the most common cause of disability of all conditions in
adults.
[0043] In terms of treatment, once a patient experiences symptoms
of a transient ischemic attack, the goal of therapy is prevention
of stroke. If a stroke occurs, the goal of therapy changes to the
limiting of damage. Preventing stroke and limiting the damage of
stroke are currently carried out in the art through medication or
surgery. In both cases, the treatment involves reducing or removing
blocks, building up in blood vessels and preventing further cell
death about neuronal populations. These treatments include the use
of (a) anticoagulants, (b) antiplatelet agents, and (c) vascular
surgery. For instance, anticoagulation drug therapy inhibits the
coagulation process. Heparin, which inhibits enzymes and platelets
that cause clots, is used in acute settings. For long term
prevention, warfarin offers anticoagulation by stopping production
of Vitamin K dependent coagulation factors. With both drugs, there
runs a risk of hemorrhage and is only used for ischemic strokes.
Strokes involving certain areas also do not warrant this therapy.
Another therapy known in the art, antiplatelet therapy with
aspirin, provides one of the most important preventive tools
available. At low daily doses, aspirin has been shown to reduce the
incidence of stroke. Specifically, low doses of aspirin block the
production of a chemical called thromboxane. Thromboxane's function
is to activate platelets to bind together and thus form blood
clots. Finally, carotid endarterectomy is the surgical procedure
where the plaque at the origin of the carotid artery is removed.
This is the treatment of choice of patients with TIA's caused by
embolism, low flow, and with minor strokes due to narrowing greater
than 70% of the internal carotid.
[0044] I. Overview
[0045] The present application is directed to compositions and
methods for the prevention and treatment of ischemic injury to the
brain, such as resulting from stroke. The invention derives, at
least in part, from the observation of a protective effect by the
so called "hedgehog" proteins on animal stroke models. Briefly, as
described in the appended examples, we investigated the
neuroprotective potential of hedgehog proteins in a rat model of
focal cerebral ischemia that used permanent occlusion of the middle
cerebral artery. Intravenous infusion of vehicle (control) or Shh
(sonic hedgehog) was administered for 3 hours beginning 30 minutes
after occlusion, and resulted in about a 70 percent reduction in
total infarct size (P=0.0039), relative to the control, when
examined 24 hours post-occlusion. Measurements of arterial blood
pressure, blood gases, glucose, hematocrit and osmolality revealed
no difference among vehicle- and Shh-treated animals. These results
show that the intravenous hedgehog protein reduces neuronal damage
due to stroke. There was no apparent cytotoxicity associated with
administration of the hedgehog polypeptide.
[0046] These results, in comparison to neuroprotective agents
described in the art, suggest an unexpectedly good neuroprotective
activity for hedgehog in the treatment of stroke. For example, the
non-competitive antagonist of the NMDA receptor, MK-801, was
typically reported to produce less than a 50% reduction in infarct
volume. Work on MK-801 was halted because of significant safety
concerns, mostly related to vacuolization seen in neurons of animal
models. Moreover, MK-801 has a relatively short therapeutic window
and must be given within a few hours of the ischemic attack.
[0047] Another neuroprotective agent presently being investigated
for use in the treatment of stroke is basic fibroblast growth
factor (bFGF). In one study, (Tatlisumak et al. (1996) Stroke
27:2292), bFGF (45 .mu.g/kg/hr) or vehicle was infused
intravenously for three hours beginning 30 minutes after permanent
middle cerebral artery occlusion by intraluminal suture in mature
Sprague-Dawley rats. After 24 hours, neurological deficit and
infract volume were significantly improved (approximately 50%
reduction in infarct volume) in the FGF group. Autoradiography
following intravenous administration of radiolabeled bFGF showed
that labeled FGF (confirmed by immunoprecipitation) crossed the
damaged blood brain barrier to enter the ischemic, but not the
non-ischemic hemisphere.
[0048] A second model (Jiang et al. (1995) Stroke 26:1-40),
utilized mature Wistar rates which underwent temporary occlusion of
the middle cerebral artery by intra-arterial suture for two hours.
At the time of reperfusion either bFGF (45 .mu.g/kg/hr) or vehicle
were infused intravenously over three hours. At seven days after
ischemia, infarct volume was significantly reduced in the bFGF
treated animals (approximately 40% reduction in infarct volume),
and only the bFGF treated animals regained their weight after
surgery.
[0049] In one aspect, the present invention provides pharmaceutical
preparations and methods for preventing/treating cerebral ischemia
and the like utilizing, as an active ingredient, a hedgehog
polypeptide or a mimetic thereof.
[0050] The subject hedgehog treatments are effective on both human
and animal subjects afflicted with these conditions. Animal
subjects to which the invention is applicable extend to both
domestic animals and livestock, raised either as pets or for
commercial purposes. Examples are dogs, cats, cattle, horses,
sheep, hogs and goats.
[0051] However, without wishing to be bound by any particular
theory, the reduction in infarct size in the present studies may be
due at least in part to the ability of hedgehog proteins to
antagonize (directly or indirectly) patched-mediated regulation of
gene expression and other physiological effects mediated by the
patched gene. The patched gene product, a cell surface protein, is
understood to signal through a pathway which regulates
transcription of a variety of genes involved in neuronal cell
development. In the CNS and other tissue, the introduction of
hedgehog relieves (derepresses) this inhibition conferred by
patched, allowing expression of particular gene programs.
[0052] Accordingly, the present invention contemplates the use of
other agents which are capable of mimicking the effect of the
hedgehog protein on patched signalling, e.g., as may be identified
from the drug screening assays described below.
[0053] II. Definitions
[0054] For convience, certain terms employed in the specfication,
examples, and appended claims are collected here.
[0055] A "stroke" is a sudden loss of function caused by a cutoff
in the blood supply to the brain. Stroke presents with different
levels of severity ranging from "transient ischemic attack" or
"TIA" (no permanent disability), to "partial nonprogressing stroke"
(persistent but no calamitous damage), to "complete stroke"
(permanent, calamitous neurological deficit). Ischemia (diminished
or stopped blood flow) and infarction (cell damage and death within
the zone of ischemia) are the pathologic processes in stroke that
lead to neurologic deficits.
[0056] "Ischemic stroke" is caused by an obstruction of blood
vessels supplying the brain. The primary subcategories of ischemic
stroke are thrombotic stroke, embolic stroke and lacunar
infarctions.
[0057] "Hemorrhagic stroke" is caused by the rupture of blood
vessels supplying the brain. The primary subcategories of
hemorrhagic stroke are subarachnoid hemorrhage (SAH) and
intracerebral hemorrhage (ICH).
[0058] The term "ischemic damage" refers to a reduction in the
biological capability of a neuronal cell, including cell death,
induced by a reduced blood flow, or an otherwise reduced level of
oxygen to the affected neuronal cells, whether it be the result of
ischemic stroke, hemmorrhagic stroke, hypoxia or the like.
[0059] The term "hedgehog therapeutic" refers to various forms of
hedgehog polypeptides, as well as peptidomimetics, which are
neuroprotective for neuronal cells, and in particular, enhance the
survival of neurons under ischemic and/or epoxic conditions. These
include naturally occurring forms of hedgehog proteins, as well as
modified or mutant forms generated by molecular biological
techniques, chemical synthesis, etc. While in preferred embodiments
the hedgehog polypeptide is derived from a vertebrate homolog,
cross-sepcies activity reported in the literature supports the use
of hedgehog peolypeptides from invertebrate organisms as well.
Naturally and non-naturally occurring hedgehog therapeutics
referred to herein as "agonists" mimic or potentiate (collectively
"agonize") the effects of a naturally-occurring hedgehog protein as
a neuroprotective agent. In addition, the term "hedgehog
therapeutic" includes molecules which can activate expression of an
endogenous hedgehog gene. The term also includes gene therapy
constructs for causing expression of hedgehog polypeptides in vivo,
as for example, expression constructs encoding recombinant hedgehog
polypeptides as well as trans-activation constructs for altering
the regulatory sequences of an endogenous hedgehog gene by
homologous recombination.
[0060] In particular, the term "hedgehog polypeptide" encompasses
hedgehog proteins and peptidyl fragments thereof.
[0061] As used herein the term "bioactive fragment", with reference
to portions of hedgehog proteins, refers to a fragment of a
full-length hedgehog protein, wherein the fragment specifically
agonizes neuroprotective events mediated by wild-type hedgehog
proteins. The hedgehog bioactive fragment preferably is a soluble
extracellular portion of a hedgehog protein, where solubility is
with reference to physiologically compatible solutions. Exemplary
bioactive fragments are described in PCT publications WO 95/18856
and WO 96/17924.
[0062] The term "ptc therapeutic" refers to agents which mimic the
effect of naturally occuning hedgehog proteins on patched
signalling. The ptc therapeutic can be, e.g., a peptide, a nucleic
acid, a carbohydrate, a small organic molecule, or natural product
extract (or fraction thereof).
[0063] A "patient" or "subject" to be treated by the subject method
is a mammals, including a human.
[0064] A "therapeutically effective amount" of, e.g., a hedgehog or
ptc therapeutic, with respect to the subject method of treatment,
refers to an amount of the therapeutic (in a preparation) which
when applied as part of a desired dosage regimen causes a decrease
in ischemia- and/or hypoxia-induced neuronal cell death (i.e., a
reduction in the volume/size of a cerebral infarct caused thereby)
according to clinically acceptable standards for the treatment or
prevention of those disorder.
[0065] By "protection from damage to neural tissue" it is meant
reduction in the total stroke volume and/or infarct volume
resulting from, e.g., ischemic or hypoxic conditions, preferably as
manifested by less neurological and/or cognitive deficits.
[0066] "Homology" and "identity" each refer to sequence similarity
between two polypeptide sequences, with identity being a more
strict comparison. Homology and identity can each be determined by
comparing a position in each sequence which, may be aligned for
purposes of comparison. When a position in the compared sequence is
occupied by the same amino acid residue, then the polypeptides can
be referred to as identical at that position; when the equivalent
site is occupied by the same amino acid (e.g., identical) or a
similar amino acid (e.g., similar in steric and/or electronic
nature), then the molecules can be referred to as homologous at
that position. A percentage of homology or identity between
sequences is a function of the number of matching or homologous
positions shared by the sequences. An "unrelated" or
"non-homologous" sequence shares less than 40 percent identity,
though preferably less than 25 percent identity, with an AR
sequence of the present invention.
[0067] The term "corresponds to", when referring to a particular
polypeptide or nucleic acid sequence is meant to indicate that the
sequence of interest is identical or homologous to the reference
sequence to which it is said to correspond.
[0068] The terms "recombinant protein", "heterologous protein" and
"exogenous protein" are used interchangeably throughout the
specification and refer to a polypeptide which is produced by
recombinant DNA techniques, wherein generally, DNA encoding the
polypeptide is inserted into a suitable expression construct which
is in turn used to transform a host cell to produce the
heterologous protein. That is, the polypeptide is expressed from a
heterologous nucleic acid.
[0069] A "chimeric protein" or "fusion protein" is a fusion of a
first amino acid sequence encoding a hedgehog polypeptide with a
second amino acid sequence defining a domain foreign to and not
substantially homologous with any domain of hh protein. A chimeric
protein may present a foreign domain which is found (albeit in a
different protein) in an organism which also expresses the first
protein, or it may be an "interspecies", "intergenic", etc. fusion
of protein structures expressed by different kinds of organisms. In
general, a fusion protein can be represented by the general formula
(X).sub.n--(hh).sub.m--(Y).sub.n, wherein hh represents all or a
portion of the hedgehog protein, X and Y each independently
represent an amino acid sequences which are not naturally found as
a polypeptide chain contiguous with the hedgehog sequence, m is an
integer greater than or equal to 1, and each occurrence of n is,
independently, 0 or an integer greater than or equal to 1 (n and m
are preferably no greater than 5 or 10).
[0070] As used herein, the term "vector" refers to a nucleic acid
molecule capable of transporting another nucleic acid to which it
has been linked. The term "expression vector" includes plasmids,
cosmids or phages capable of synthesizing, for example, the subject
hedgehog polypeptides encoded by the respective recombinant gene
carried by the vector. Preferred vectors are those capable of
autonomous replication and/expression of nucleic acids to which
they are linked. In the present specification, "plasmid" and
"vector" are used interchangeably as the plasmid is the most
commonly used form of vector. Moreover, the invention is intended
to include such other forms of expression vectors which serve
equivalent functions and which become known in the art subsequently
hereto.
[0071] "Transcriptional regulatory sequence" is a generic term used
throughout the specification to refer to DNA sequences, such as
initiation signals, enhancers, and promoters, as well as
polyadenylation sites, which induce or control transcription of
protein (or antisense) coding sequences with which they are
operably linked. In preferred embodiments, transcription of a
recombinant gene is under the control of a promoter sequence (or
other transcriptional regulatory sequence) which controls the
expression of the recombinant gene in a cell-type in which
expression is intended. It will also be understood that the
recombinant gene can be under the control of transcriptional
regulatory sequences which are the same or which are different from
those sequences which control transcription of the
naturally-occurring form of the regulatory protein.
[0072] The term "operably linked" refers to the arrangement of a
transcriptional regulatory element relative to another
transcribable nucleic acid sequence, such that the transcriptional
regulatory element can regulate the rate of transcription from the
transcribable sequence(s).
[0073] III. Exemplary Applications of Method and Compositions
[0074] Central nervous system tissue is particularly vulnerable to
damage caused by ischemic conditions. The subject method has wide
applicability to the treatment or prophylaxis of ischemic or
hypoxic damage marked by neuronal cell death. The instant treatment
can be used to treat or prevent injury or disease to brain tissue
resulting from ischemia, e.g., as caused from insufficient oxygen.
The types of ischemia for which the subject method can be used as
part of a treatment include, but are not limited to those which may
last for only transient periods of time to those which may last for
lengthy durations, as in stroke. In the regard, the subject method
is useful for treatment and prevention of injury to the brain and
spinal cord and edema due to head trauma, spinal trauma, stroke,
hypotension, arrested breathing, cardiac arrest, Rey's syndrome,
cerebral thrombosis, embolism, hemorrhage or tumors,
encephalomyelitis, hydroencephalitis, and operative and
postoperative brain injury.
[0075] In general, the method can be characterized as including a
step of administering to an animal an amount of a ptc or hedgehog
therapeutic effective to enhance the survival of neuronal cells
under such ischemic or hypoxic conditions. The mode of
administration and dosage regimens will vary depending on the
severity of the ischemic or hypoxic attack, e.g., the dosage may be
altered as between a transient ischemic attack, a partial
nonprogressing stroke, and a complete stroke. In preferred
embodiments, the ptc or hedeghog therapeutic is administered
systemically initially (i.e., while the blood brain barrier is
disrupted), then locally for medium to long term care.
[0076] When used to treat stroke, the clinician should not only
define the level of stroke severity, but also the "pace" or "tempo"
of the illness. This is because the pace of progression helps to
dictate the urgency for evaluation and treatment. A patient who
suffers a TIA in the morning has a higher risk for stroke in the
afternoon than a patient who suffered a single TIA a month earlier.
Where the risk of stroke remains high, the subject hedgehog and ptc
therapeutics can be used prophylatically in order to minimize
ischemic damage which may result from an eventual stroke. A patient
who is worsening under supervision requires more urgent management
than one who has been stable for a week or more.
[0077] The subject method may also find particular utility in
treating or preventing the adverse neurological consequences of
surgery. For example, coronary bypass surgery requires the use of
heart-lung machines, which tend to introduce air bubbles into the
circulatory system that may lodge in the brain. The presence of
such air bubbles robs neuronal tissue of oxygen, resulting in
anoxia and ischemia. Pre- or post-surgical administration of the
hedgehog and/or ptc therapeutics of the present invention will
treat or prevent the resulting ischemia. In a preferred embodiment,
the subject therapeutics are administered to patients undergoing
cardiopulmonary bypass surgery or carotid endarterectomy
surgery.
[0078] In still other embodiments, the subject method can be used
in the prevention and/or treatment of hypoxia, e.g., as a
neuroprotective agent. For instance, the subject method can be used
prophylactically to lessen the neuronal cell death caused by
altitude-induced hypoxia.
[0079] A method which is "neuroprotective", in the case of cerebral
ischemia, results in diminished infarct volume relative to that
which would occur in the absence of treatment with a hedgehog or
ptc therapeutic. That is a neuroprotective therapy is intended to
maintain or rescue damaged nerve cells, preventing their death.
[0080] The treatment methods of the present invention can be
combined with the use of (a) anticoagulants, (b) antiplatelet
agents, and/or (c) vascular surgery. Co-administered with suitable
anti-coagulant agents, antiplatelet agents, thrombin inhibitors,
and/or thrombolytic agents, may afford an efficacy advantage over
any of the agents alone, and may do so while permitting the use of
lower doses of each. A lower dosage minimizes the potential of side
effects, thereby providing an increased margin of safety. The two
(or more) agents are administered in combination according to the
invention. The term "in combination" in this context means that the
drugs are given substantially contemporaneously, either
simultaneously or sequentially. If given sequentially, at the onset
of administration of the second agent, the first of the two agents
is preferably still detectable at effective concentrations at the
site of treatment.
[0081] The term "anti-coagulant agents" (or coagulation inhibitory
agents), as used herein, denotes agents that inhibit blood
coagulation. Such agents include warfarin, heparin, or low
molecular weight heparin (LMWH), including pharmaceutically
acceptable salts or prodrugs thereof. For reasons of efficacy, the
preferable anti-coagulant agents are warfarin or heparin or LMWH.
The warfarin employed herein, may be, for exarnple, crystalline
warfarin or amorphous sodium warfarin. The heparin employed herein
may be, for example, the sodium or sulfate salts thereof.
[0082] The term "anti-platelet agents" (or platelet inhibitory
agents), as used herein, denotes agents that inhibit platelet
function such as by inhibiting the aggregation, adhesion or
granular secretion of platelets. Such agents include the various
known non-steroidal anti-inflammatory drugs (NSAIDS) such as
aspirin, ibuprofen, naproxen, sulindac, indomethacin, mefenamate,
droxicam, diclofenac, sulfinpyrazone, and piroxicam, including
pharmaceutically acceptable salts or prodrugs thereof. Of the
NSAIDS, aspirin (acetylsalicyclic acid or ASA), which has been well
researched and widely used with good results, and piroxicam, which
exerts its anti-platelet effect when dosed once daily, are
preferred compounds, especially aspirin. Piroxicam is commercially
available from Pfizer Inc. (New York, N.Y.), as FELDANE TM. Other
suitable anti-platelet agents include ticlopidine, including
pharmaceutically acceptable salts or prodrugs thereof Ticlopidine
is also a preferred compound since it is known to be gentle on the
gastro-intestinal tract in use. Still other suitable platelet
inhibitory agents include thromboxane-A2-receptor antagonists and
thromboxane-A2-synthetase inhibitors, as well as pharmaceutically
acceptable salts or prodrugs thereof.
[0083] The phrase "thrombin inhibitors" (or anti-thrombin agents),
as used herein, denotes inhibitors of the serine protease thrombin.
By inhibiting thrombin, various thrombin mediated processes, such
as thrombin-mediated platelet activation (that is, for example, the
aggregation of platelets, and/or the granular secretion of
plasminogen activator inhibitor-i and/or serotonin) and/or fibrin
formation are disrupted. Such inhibitors include boropeptides,
hirudin and argatroban, including pharmaceutically acceptable salts
and prodrugs thereof. Preferably the thrombin inhibitors are
boropeptides. By boropeptides, it is meant, N-acetyl and peptide
derivatives of boronic acid, such as C-terminal alpha -aminoboronic
acid derivatives of lysine, ornithine, arginine, homoarginine and
corresponding isothiouronium analogs thereof. The term hirudin, as
used herein, includes suitable derivatives or analogs of hirudin,
referred to herein as hirulogs, such as disulfatohirudin.
[0084] The phrase "thrombolytic agents" or "fibrinolytic agents" or
"thrombolytics" or "fibrinolytics", as used herein, denotes agents
that lyse blood clots (thrombi). Such agents include tissue
plasminogen activator, anistreplase, urokinase or streptokinase,
including pharmaceutically acceptable salts or prodrugs thereof.
Tissue plasminogen activator (tPA) is commercially available from
Genentech Inc., South San Francisco, Calif. The term anistreplase,
as used herein, refers to anisoylated plasminogen streptokinase
activator complex, as described, for example, in European Patent
Application No. 0 28 489, the disclosures of which are hereby
incorporated herein by reference herein, in their entirety.
Anistreplase is commercially available from the Beecham Group,
Middlesex, England, under the trademark EMINASE TM. The term
urokinase, as used herein, is intended to denote both dual and
single chain urokinase, the latter also being referred to herein as
prourokinase.
[0085] In yet other embodiments, the subject method can be carried
out conjointly with the administration of growth and/or trophic
factors. For instance, the trophic growth factor basic FGF has been
demonstrated in the art to be useful in the functional recovery
following experimental stroke. In experiments providing exogenous
administration of bFGF after infarction, the early administration
of bFGF was found to reduce infarct size. See, for example,
Kawamata et al. (1997) Adv Neurol 73: 377-82. Likewise,
progesterone has been shown to be neuroprotective after transient
middle cerebral artery occlusion in male rats. Jiang et al. (1996)
Brain Res 735:101-7. Other agents with which the subject hedgehog
and ptc therapeutics can be coadministered include
nitro-L-arginine, transforming growth factor-.beta.1 (TGF-beta 1)
has been shown to rescue cultured neurons from excitotoxic and
hypoxic cell death and to reduce infarct size after focal cerebral
ischemia in mice and rabbits. In other instances, the combinatorial
therapy can include a trophic factor such as nerve growth factor,
cilliary neurotrophic growth factor, schwanoma-derived growth
factor, glial growth factor, stiatal-derived neuronotrophic factor,
platelet-derived growth factor, and scatter factor (HGF-SF).
Antimitogenic agents can also be used, as for example, cytosine,
arabinoside, 5-fluorouracil, hydroxyurea, and methotrexate.
[0086] Determination of a therapeutically effective amount and a
prophylactically effective amount of a hedgehog or ptc therapeutic,
e.g., to be adequately neuroprotective, can be readily made by the
physician or veterinarian (the "attending clinician"), as one
skilled in the art, by the use of known techniques and by observing
results obtained under analogous circumstances. The dosages may be
varied depending upon the requirements of the patient in the
judgment of the attending clinician, the severity of the condition
being treated, the risk of further ischemic or hypoxic damage to
the CNS, and the particular agent being employed. In determining
the therapeutically effective neuroprotective amount or dose, and
the prophylactically effective amount or dose, a number of factors
are considered by the attending clinician, including, but not
limited to: the specific cause of the ischemic or hypoxic state and
its likelihood of recurring or worsening; pharmacodynamic
characteristics of the particular agent and its mode and route of
administration; the desired time course of treatment; the species
of mammal; its size, age, and general health; the response of the
individual patient; the particular compound administered; the
bioavailability characteristics of the preparation administered;
the dose regimen selected; the kind of concurrent treatment (i.e.,
the interaction of the hedgehog or ptc therapeutic with other
co-administered therapeutics); and other relevant
circumstances.
[0087] Treatment can be initiated with smaller dosages which are
less than the optimum dose of the agent. Thereafter, the dosage
should be increased by small increments until the optimum effect
under the circumstances is reached. For convenience, the total
daily dosage may be divided and administered in portions during the
day if desired. A therapeutically effective antineoplastic amount
and a prophylactically effective neuroprotective amount of a
hedgehog polypeptide, for instance, is expected to vary from
concentrations about 0.1 nanogram per kilogram of body weight per
day (kg/day) to about 100 kg/day.
[0088] Potential hedgehog and ptc therapeutics, such as described
below, can be tested by measuring the volume of cerebral infarction
in animals receiving systemic injections. For instance, selected
agents can be evaluated in the focal stroke model involving
permanent middle cerebral artery occlusion (MCAO) in the
spontaneously hypertensive rat. This procedure results in a
reliably large neocortical infarct volume that is measured by means
of vital dye exclusion in serial slices through the brain 24 hours
after MCAO. Tamura et al. (1981) J Cerebral Blood Flow and
Metabolism 1:53-60.
[0089] The middle cerebral artery is the cerebral blood vessel most
susceptible to stroke in humans. In animals, coagulation, permanent
ligation or permanent placement of an occluding thread in the
artery produces a permanent focal stroke affecting the MCA
territory. Transient ligation or occlusion results in transient
focal stroke. Both transient and permanent focal strokes result in
varying degrees of edema and infarction in the affected brain
regions. The ability of compounds to reduce the volumes of edema
and infarction is considered a measure of their potential as
anti-stroke treatment.
[0090] Compounds which are determined to be effective for the
prevention or treatment of cerebral infarction and the like in
animals, e.g., dogs, rodents, may also be useful in treatment of
tumors in humans. Those skilled in the art of treating such
disorders in humans will be guided, from the data obtained in
animal studies, to the correct dosage and route of administration
of the compound to humans. In general, the determination of dosage
and route of administration in humans is expected to be similar to
that used to determine administration in animals.
[0091] The identification of those patients who are in need of
prophylactic treatment for ischemic or hypoxic states is well
within the ability and knowledge of one skilled in the art. Certain
of the methods for identification of patients which are at risk of
cerebral infarction which can be treated by the subject method are
appreciated in the medical arts, such as family history of the
development of a particular disease state and the presence of risk
factors associated with the development of that disease state in
the subject patient. A clinician skilled in the art can readily
identify such candidate patients, by the use of, for example,
clinical tests, physical examination and medical/family
history.
[0092] IV. Exemplary Hedgehog Therapeutic Compounds.
[0093] The hedgehog therapeutic compositions of the subject method
can be generated by any of a variety of techniques, including
purification of naturally occurring proteins, recombinantly
produced proteins and synthetic chemistry. Polypeptide forms of the
hedgehog therapeutics are preferably derived from vertebrate
hedgehog proteins, e.g., have sequences corresponding to naturally
occurring hedgehog proteins, or fragments thereof, from vertebrate
organisms. However, it will be appreciated that the hedgehog
polypeptide can correspond to a hedgehog protein (or fragment
thereof) which occurs in any metazoan organism.
[0094] The various naturally-occurring hedgehog proteins from which
the subject therapeutics can be derived are characterized by a
signal peptide, a highly conserved N-terminal region, and a more
divergent C-terminal domain. In addition to signal sequence
cleavage in the secretory pathway (Lee, J. J. et al. (1992) Cell
71:33-50; Tabata, T. et al. (1992) Genes Dev. 2635-2645; Chang, D.
E. et al. (1994) Development 120:3339-3353), hedgehog precursor
proteins naturally undergo an internal autoproteolytic cleavage
which depends on conserved sequences in the C-terminal portion (Lee
et al. (1994) Science 266:1528-1537; Porter et al. (1995) Nature
374:363-366). This autocleavage leads to a 19 kD N-terrninal
peptide and a C-terminal peptide of 26-28 kD (Lee et al. (1992)
supra; Tabata et al. (1992) supra; Chang et al. (1994) supra; Lee
et al. (1994) supra; Bumcrot, D. A., et al. (1995) Mol. Cell. Biol.
15:2294-2303; Porter et al. (1995) supra; Ekker, S. C. et al.
(1995) Curr. Biol. 5:944-955; Lai, C. J. et al. (1995) Development
121:2349-2360). The N-terminal peptide stays tightly associated
with the surface of cells in, which it was synthesized, while the
C-terminal peptide is freely diffusible both in vitro and in vivo
(Lee et al. (1994) supra; Bumcrot et al. (1995) supra; Mart', E. et
al. (1995) Development 121:2537-2547; Roelink, H. et al. (1995)
Cell 81:445-455). Cell surface retention of the N-terminal peptide
is dependent on autocleavage, as a truncated form of hedgehog
encoded by an RNA which terminates precisely at the normal position
of internal cleavage is diffusible in vitro (Porter et al. (1995)
supra) and in vivo (Porter, J. A. et al. (1996) Cell 86, 21-34).
Biochemical studies have shown that the autoproteolytic cleavage of
the hedgehog precursor protein proceeds through an internal
thioester intermediate which subsequently is cleaved in a
nucleophilic substitution. It is suggested that the nucleophile is
a small lipophilic molecule, more particularly cholesterol, which
becomes covalently bound to the C-terminal end of the N-peptide
(Porter et al. (1996) supra), tethering it to the cell surface.
[0095] The vertebrate family of hedgehog genes includes at least
four members, e.g., paralogs of the single drosophila hedgehog gene
(SEQ ID No. 19). Three of these members, herein referred to as
Desert hedgehog (Dhh), Sonic hedgehog (Shh) and Indian hedgehog
(Ihh), apparently exist in all vertebrates, including fish, birds,
and mammals. A fourth member, herein referred to as tiggie-winkle
hedgehog (Thh), appears specific to fish. According to the appended
sequence listing, (see also Table 1) a chicken Shh polypeptide is
encoded by SEQ ID No:1; a mouse Dhh polypeptide is encoded by SEQ
ID No:2; a mouse lhh polypeptide is encoded by SEQ ID No:3; a mouse
Shh polypeptide is encoded by SEQ ID No:4 a zebrafish Shh
polypeptide is encoded by SEQ ID No:5; a human Shh polypeptide is
encoded by SEQID No:6; a human Ihh polypeptide is encoded by SEQ ID
No:7; and a zebrafish Thh is encoded by SEQ ID No.8.
1TABLE 1 Guide to hedgehog sequences in Sequence Listing Nucleotide
Amino Acid Chicken Shh SEQ ID No. 1 SEQ ID No. 10 Mouse Dhh SEQ ID
No. 2 SEQ ID No. 11 Mouse Ihh SEQ ID No. 3 SEQ ID No. 12 Mouse Shh
SEQ ID No. 4 SEQ ID No. 13 Zebrafish Shh SEQ ID No. 5 SEQ ID No. 14
Human Shh SEQ ID No. 6 SEQ ID No. 15 Human Ihh SEQ ID No. 7 SEQ ID
No. 16 Zebrafish Thh SEQ ID No. 8 SEQ ID No. 17 Drosophila HH SEQ
ID No. 9 SEQ ID No. 18
[0096] In addition to the sequence variation between the various
hedgehog homologs, the hedgehog proteins are apparently present
naturally in a number of different forms, including a pro-form, a
full-length mature form, and several processed fragments thereof.
The pro-form includes an N-terminal signal peptide for directed
secretion of the extracellular domain, while the full-length mature
form lacks this signal sequence.
[0097] As described above, further processing of the mature form
occurs in some instances to yield biologically active fragments of
the protein. For instance, sonic hedgehog undergoes additional
proteolytic processing to yield two peptides of approximately 19
kDa and 27 kDa, the 19 kDa fragment corresponding to an proteolytic
N-terminal portion of the mature protein. In addition to
proteolytic fragmentation, the vertebrate hedgehog proteins can
also be modified post-translationally, such as by glycosylation
and/or addition of cholesterol, though bacterially produced (e.g.,
unglycosylated/uncholesterolized) forms of the proteins still
maintain certain of the bioactivities of the native protein.
Bioactive fragments of hedgehog polypeptides of the present
invention have been generated and are described in great detail in,
e.g., PCT publications WO 95/18856 and WO 96/17924.
[0098] Moreover, mutagenesis can be used to create modified hh
polypeptides, e.g., for such purposes as enhancing therapeutic or
prophylactic efficacy, or stability (e.g., ex vivo shelf life and
resistance to proteolytic degradation in vivo). Such modified
peptides can be produced, for instance, by amino acid substitution,
deletion, or addition. Modified hedgehog polypeptides can also
include those with altered post-translational processing relative
to a naturally occurring hedgehog protein, e.g., altered
glycosylation, cholesterolization, prenylation and the like.
[0099] In one embodiment, the hedgehog therapeutic is a polypeptide
encodable by a nucleotide sequence that hybridizes under stringent
conditions to a hedgehog coding sequence represented in one or more
of SEQ ID Nos: 1-9. Appropriate stringency conditions which promote
DNA hybridization, for example, 6.0.times.sodium chloride/sodium
citrate (SSC) at about 45.degree. C., followed by a wash of
2.0.times.SSC at 50.degree. C., are known to those skilled in the
art or can be found in Current Protocols in Molecular Biology, John
Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. For example, the salt
concentration in the wash step can be selected from a low
stringency of about 2.0.times.SSC at 50.degree. C. to a high
stringency of about 0.2.times.SSC at 50.degree. C. In addition, the
temperature in the wash step can be increased from low stringency
conditions at room temperature, about 22.degree. C., to high
stringency conditions at about 65.degree. C.
[0100] As described in the literature, genes for other hedgehog
proteins, e.g., from other animals, can be obtained from MRNA or
genomic DNA samples using techniques well known in the art. For
example, a cDNA encoding a hedgehog protein can be obtained by
isolating total mRNA from a cell, e.g., a mammalian cell, e.g., a
human cell, including embryonic cells. Double stranded cDNAs can
then be prepared from the total MRNA, and subsequently inserted
into a suitable plasmid or bacteriophage vector using any one of a
number of known techniques. The gene encoding a hedgehog protein
can also be cloned using established polymerase chain reaction
techniques.
[0101] Preferred nucleic acids encode a hedgehog polypeptide
comprising an amino acid sequence at least 60% homologous, more
preferably 70% homologous and most preferably 80% homologous with
an amino acid sequence selected from the group consisting of SEQ ID
Nos: 10-18. Nucleic acids which encode polypeptides at least about
90%, more preferably at least about 95%, and most preferably at
least about 98-99% homology with an amino acid sequence represented
in one of SEQ ID Nos:10-18 are also within the scope of the
invention.
[0102] Hedgehog polypeptides preferred by the present invention, in
addition to native hedgehog proteins, are at least 60% homologous,
more preferably 70% homologous and most preferably 80% homologous
with an amino acid sequence represented by any of SEQ ID Nos:10-18.
Polypeptides which are at least 90%, more preferably at least 95%,
and most preferably at least about 98-99% homologous with a
sequence selected from the group consisting of SEQ ID Nos:10-18 are
also within the scope of the invention. The only prerequisite is
that the hedgehog polypeptide is capable of protecting neuronal
cells against ischemic damage.
[0103] The term "recombinant protein" refers to a polypeptide of
the present invention which is produced by recombinant DNA
techniques, wherein generally, DNA encoding a hedgehog polypeptide
is inserted into a suitable expression vector which is in turn used
to transform a host cell to produce the heterologous protein.
Moreover, the phrase "derived from", with respect to a recombinant
hedgehog gene, is meant to include within the meaning of
"recombinant protein" those proteins having an amino acid sequence
of a native nedgehog protein, or an amino acid sequence similar
thereto which is generated by mutations including substitutions and
deletions (including truncation) of a naturally occurring form of
the protein.
[0104] The method of the present invention can also be carried out
using variant forms of the naturally occurring hedgehog
polypeptides, e.g., mutational variants.
[0105] As is known in the art, hedgehog polypeptides can be
produced by standard biological techniques. For example, a host
cell transfected with a nucleic acid vector directing expression of
a nucleotide sequence encoding the subject polypeptides can be
cultured under appropriate conditions to allow expression of the
peptide to occur. The polypeptide hedgehog may be secreted and
isolated from a mixture of cells and medium containing the
recombinant hedgehog polypeptide. Alternatively, the peptide may be
retained cytoplasmically by removing the signal peptide sequence
from the recombinant hedgehog gene and the cells harvested, lysed
and the protein isolated. A cell culture includes host cells, media
and other byproducts. Suitable media for cell culture are well
known in the art. The recombinant hedgehog polypeptide can be
isolated from cell culture medium, host cells, or both using
techniques known in the art for purifying proteins including
ion-exchange chromatography, gel filtration chromatography,
ultrafiltration, electrophoresis, and immunoaffinity purification
with antibodies specific for such peptide. In a preferred
embodiment, the recombinant hedgehog polypeptide is a fusion
protein containing a domain which facilitates its purification,
such as an hedgehoglGST fusion protein. The host cell may be any
prokaryotic or eukaryotic cell.
[0106] Recombinant hedgehog genes can be produced by ligating
nucleic acid encoding an hedgehog protein, or a portion thereof,
into a vector suitable for expression in either prokaryotic cells,
eukaryotic cells, or both. Expression vectors for production of
recombinant forms of the subject hedgehog polypeptides include
plasmids and other vectors. For instance, suitable vectors for the
expression of a hedgehog polypeptide include plasmids of the types:
pBR322-derived plasmids, pEMBL-derived plasmids, pEX-derived
plasmids, pBTac-derived plasmids and pUC-derived plasmids for
expression in prokaryotic cells, such as E. coli.
[0107] A number of vectors exist for the expression of recombinant
proteins in yeast. For instance, YEP24, YIP5, YEP5 1, YEP52, pYES2,
and YRP 17 are cloning and expression vehicles useful in the
introduction of genetic constructs into S. cerevisiae (see, for
example, Broach et al. (1983) in Experimental Manipulation of Gene
Expression, ed. M. Inouye Academic Press, p. 83, incorporated by
reference herein). These vectors can replicate in E. coli due the
presence of the pBR322 ori, and in S. cerevisiae due to the
replication determinant of the yeast 2 micron plasmid. In addition,
drug resistance markers such as ampicillin can be used. In an
illustrative embodiment, an hedgehog polypeptide is produced
recombinantly utilizing an expression vector generated by
sub-cloning the coding sequence of one of the hedgehog genes
represented in SEQ ID Nos:1-9 or 19.
[0108] The preferred mammalian expression vectors contain both
prokaryotic sequences, to facilitate the propagation of the vector
in bacteria, and one or more eukaryotic transcription units that
are expressed in eukaryotic cells. The pcDNAI/amp, pcDNAI/neo,
pRc/CMV, pSV2gpt, pSV2neo, pSV2-dhfr, pTk2, pRSVneo, pMSG, pSVT7,
pko-neo and pHyg derived vectors are examples of mammalian
expression vectors suitable for transfection of eukaryotic cells.
Some of these vectors are modified with sequences from bacterial
plasmids, such as pBR322, to facilitate replication and drug
resistance selection in both prokaryotic and eukaryotic cells.
Alternatively, derivatives of viruses such as the bovine
papillomavirus (3PV-1), or Epstein-Barr virus (pHEBo, pREP-derived
and p205) can be used for transient expression of proteins in
eukaryotic cells. The various methods employed in the preparation
of the plasmids and transformation of host organisms are well known
in the art. For other suitable expression systems for both
prokaryotic and eukaryotic cells, as well as general recombinant
procedures, see Molecular Cloning A Laboratory Manual, 2nd Ed., ed.
by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory
Press: 1989) Chapters 16 and 17.
[0109] In some instances, it may be desirable to express the
recombinant hedgehog polypeptide by the use of a baculovirus
expression system. Examples of such baculovirus expression systems
include pVL-derived vectors (such as pVL1392, pVL1393 and pVL941),
pAcUW-derived vectors (such as pAcUW1), and pBlueBac-derived
vectors (such as the 13-gal containing pBlueBac III).
[0110] When it is desirable to express only a portion of a hedgehog
protein, such as a form lacking a portion of the N-terminus, i.e.,
a truncation mutant which lacks the signal peptide, it may be
necessary to add a start codon (ATG) to the oligonucleotide
fragment containing the desired sequence to be expressed. It is
well known in the art that a methionine at the N-terminal position
can be enzymatically cleaved by the use of the enzyme methionine
aminopeptidase (MAP). MAP has been cloned from E. coli (Ben-Bassat
et al. (1987) J. Bacteriol. 169:751-757) and Salmonella typhimurium
and its in vitro activity has been demonstrated on recombinant
proteins (Miller et al. (1987) PNAS 84:2718-1722). Therefore,
removal of an N-terminal methionine, if desired, can be achieved
either in vivo by expressing hedgehog-derived polypeptides in a
host which produces MAP (e.g., E. coli or CM89 or S. cerevisiae),
or in vitro by use of purified MAP (e.g., procedure of Miller et
al., supra).
[0111] Alternatively, the coding sequences for the polypeptide can
be incorporated as a part of a fusion gene including a nucleotide
sequence encoding a different polypeptide. It is widely appreciated
that fusion proteins can also facilitate the expression of
proteins, and accordingly, can be used in the expression of the
hedgehog polypeptides of the present invention. For example,
hedgehog polypeptides can be generated as glutathione-S-transferase
(GST-fusion) proteins. Such GST-fusion proteins can enable easy
purification of the hedgehog polypeptide, as for example by the use
of glutathione-derivatized matrices (see, for example, Current
Protocols in Molecular Biology, eds. Ausubel et al. (N.Y.: John
Wiley & Sons, 1991)). In another embodiment, a fusion gene
coding for a purification leader sequence, such as a
poly-(His)/enterokinase cleavage site sequence, can be used to
replace the signal sequence which naturally occurs at the
N-terminus of the hedgehog protein (e.g., of the pro-form, in order
to permit purification of the poly(His)-hedgehog protein by
affinity chromatography using a Ni.sup.2+ metal resin. The
purification leader sequence can then be subsequently removed by
treatment with enterokinase (e.g., see Hochuli et al. (1987) J.
Chromatography 411:177; and Janknecht et al. PNAS 88:8972).
[0112] Techniques for making fusion genes are known to those
skilled in the art. Essentially, the joining of various DNA
fragments coding for different polypeptide sequences is performed
in accordance with conventional techniques, employing blunt-ended
or stagger-ended termini for ligation, restriction enzyme digestion
to provide for appropriate termini, filling-in of cohesive ends as
appropriate, alkaline phosphatase treatment to avoid undesirable
joining, and enzymatic ligation. In another embodiment, the fusion
gene can be synthesized by conventional techniques including
automated DNA synthesizers. Alternatively, PCR amplification of
gene fragments can be carried out using anchor primers which give
rise to complementary overhangs between two consecutive gene
fragments which can subsequently be annealed to generate a chimeric
gene sequence (see, for example, Current Protocols in Molecular
Biology, eds. Ausubel et al. John Wiley & Sons: 1992).
[0113] Hedgehog polypeptides may also be chemically modified to
create hedgehog derivatives by forming covalent or aggregate
conjugates with other chemical moieties, such as glycosyl groups,
cholesterol, isoprenyl, myristyl, lipids, phosphate, acetyl groups
and the like. Covalent derivatives of hedgehog proteins can be
prepared by linking the chemical moieties to functional groups on
amino acid sidechains of the protein or at the N-terminus or at the
C-terminus of the polypeptide.
[0114] For instance, hedgehog proteins can be generated to include
a moiety, other than sequence naturally associated with the
protein, that binds a component of the extracellular matrix and
enhances localization of the analog to cell surfaces. For example,
sequences derived from the fibronectin "type-III repeat", such as a
tetrapeptide sequence R-G-D-S (Pierschbacher et al. (1984) Nature
309:30-3; and Kornblihtt et al. (1985) EMBO 4:1755-9) can be added
to the hedgehog polypeptide to support attachment of the chimeric
molecule to a cell through binding ECM components (Ruoslahti et al.
(1987) Science 238:491-497; Pierschbacheret al. (1987) J. Biol.
Chem. 262:17294-8.; Hynes (1987) Cell 48:549-54; and Hynes (1992)
Cell 69:11-25).
[0115] In a preferred embodiment, the hedgehog polypeptide is
isolated from, or is otherwise substantially free of, other
cellular proteins, especially other extracellular or cell surface
associated proteins which may normally be associated with the
hedgehog polypeptide. The term "substantially free of other
cellular or extracellular proteins" (also referred to herein as
"contaminating proteins") or "substantially pure or purified
preparations" are defined as encompassing preparations of hedgehog
polypeptides having less than 20% (by dry weight) contaminating
protein, and preferably having less than 5% contaminating protein.
By "purified", it is meant that the indicated molecule is present
in the substantial absence of other biological macromolecules, such
as other proteins. The term "purified" as used herein preferably
means at least 80% by dry weight, more preferably in the range of
95-99% by weight, and most preferably at least 99.8% by weight, of
biological macromolecules of the same type present (but water.
buffers, and other small molecules, especially molecules having a
molecular weight of less than 5000, can be present). The term
"pure" as used herein preferably has the same numerical limits as
"purified" immediately above.
[0116] As described above for recombinant polypeptides, isolated
hedgehog polypeptides can include all or a portion of the amino
acid sequences represented in any of SEQ ID Nos:10-18, or a
homologous sequence thereto. Preferred fragments of the subject
hedgehog proteins correspond to the N-terminal and C-terminal
proteolytic fragments of the mature protein. Bioactive fragments of
hedgehog polypeptides are described in great detail in PCT
publications WO 95/18856 and WO 96/17924.
[0117] With respect to bioctive fragments of hedgehog polypeptide,
preferred hedgehog therapeutics include at least 50 amino acid
residues of a hedgehog polypeptide, more preferably at least 100,
and even more preferably at least 150.
[0118] Another preferred hedgehog polypeptide which can be included
in the hedgehog therapeutic is an N-terminal fragment of the mature
protein having a molecular weight of approximately 19 kDa.
[0119] Preferred human hedgehog proteins include N-terminal
fragments corresponding approximately to residues 24-197 of SEQ ID
No. 15 and 28-202 of SEQ ID No. 16. By "corresponding
approximately" it is meant that the sequence of interest is at most
20 amino acid residues different in length to the reference
sequence, though more preferably at most 5, 10 or 15 amino acid
different in length.
[0120] Still other preferred hedgehog polypeptides include an amino
acid sequence represented by the formula A-B wherein: (i) A
represents all or the portion of the amino acid sequence designated
by residues 1-168 of SEQ ID No:19; and B represents at least one
amino acid residue of the amino acid sequence designated by
residues 169-221 of SEQ ID No: 19; (ii) A represents all or the
portion of the amino acid sequence designated by residues 24-193 of
SEQ ID No: 15; and B represents at least one amino acid residue of
the amino acid sequence designated by residues 194-250 of SEQ ID
No:15; (iii) A represents all or the portion of the amino acid
sequence designated by residues 25-193 of SEQ ID No:13; and B
represents at least one amino acid residue of the amino acid
sequence designated by residues 194-250 of SEQ ID No:13; (iv) A
represents all or the portion of the amino acid sequence designated
by residues 23-193 of SEQ ID No: 1; and B represents at least one
amino acid residue of the amino acid sequence designated by
residues 194-250 of SEQ ID No: 1; (v) A represents all or the
portion of the amino acid sequence designated by residues 28-197 of
SEQ ID No: 12; and B represents at least one amino acid residue of
the amino acid sequence designated by residues 198-250 of SEQ ID
No:12; or (vi) A represents all or the portion of the amino acid
sequence designated by residues 29-197 of SEQ ID No:16; and B
represents at least one amino acid residue of the amino acid
sequence designated by residues 198-250 of SEQ ID No:16. In certain
preferred embodiments, A and B together represent a contiguous
polypeptide sequence of the designated sequence, A represents at
least 25, 50, 75, 100, 125 or 150 amino acids of the designated
sequence, and B represents at least 5, 10, or 20 amino acid
residues of the amino acid sequence designated by corresponding
entry in the sequence listing, and A and B together preferably
represent a contiguous sequence corresponding to the sequence
listing entry. Similar fragments from other hedgehog proteins are
also contemplated, e.g., fragments which correspond to the
preferred fragments from the sequence listing entries which are
enumerated above.
[0121] Isolated peptidyl portions of hedgehog proteins can be
obtained by screening peptides recombinantly produced from the
corresponding fragment of the nucleic acid encoding such peptides.
In addition, fragments can be chemically synthesized using
techniques known in the art such as conventional Merrifield solid
phase f-Moc or t-Boc chemistry. For example, a hedgehog polypeptide
of the present invention may be arbitrarily divided into fragments
of desired length with no overlap of the fragments, or preferably
divided into overlapping fragments of a desired length. The
fragments can be produced (recombinantly or by chemical synthesis)
and tested to identify those peptidyl fragments which can function
as agonists of a wild-type (e.g., "authentic") hedgehog protein.
For example, Romn et al. (1994) Eur J Biochem 222:65-73 describe
the use of competitive-binding assays using short, overlapping
synthetic peptides from larger proteins to identify binding
domains.
[0122] The recombinant hedgehog polypeptides of the present
invention also include homologs of the authentic hedgehog proteins,
such as versions of those protein which are resistant to
proteolytic cleavage, as for example, due to mutations which alter
potential cleavage sequences or which inactivate an enzymatic
activity associated with the protein. Hedgehog homologs of the
present invention also include proteins which have been
post-translationally modified in a manner different than the
authentic protein. Exemplary derivatives of hedgehog proteins
include polypeptides which lack glycosylation sites (e.g., to
produce an unglycosylated protein), which lack sites for
cholesterolization, and/or which lack N-terminal and/or C-terminal
sequences.
[0123] Modification of the structure of the subject hedgehog
polypeptides can also be for such purposes as enhancing therapeutic
or prophylactic efficacy, or stability (e.g., ex vivo shelf life
and resistance to proteolytic degradation in vivo). Such modified
peptides, when designed to retain at least one activity of the
naturally-occurring form of the protein, are considered fuinctional
equivalents of the hedgehog polypeptides described in more detail
herein. Such modified peptides can be produced, for instance, by
amino acid substitution, deletion, or addition.
[0124] It is well known in the art that certain isolated
replacements of amino acids, e.g., replacement of an amino acid
residue with another related amino acid (i.e., isosteric and/or
isoelectric mutations), can be carried out without major effect on
the biological activity of the resulting molecule. Conservative
replacements are those that take place within a family of amino
acids that are related in their side chains. Genetically encoded
amino acids are can be divided into four families: (1)
acidic=aspartate, glutamate; (2) basic=lysine, arginine, histidine;
(3) nonpolar=alanine, valine, leucine, isoleucine, proline,
phenylalanine, methionine, tryptophan; and (4) uncharged
polar=glycine, asparagine, glutamine, cysteine, serine, threonine,
tyrosine. Phenylalanine, tryptophan, and tyrosine are sometimes
classified jointly as aromatic amino acids. In similar fashion, the
amino acid repertoire can be grouped as (1) acidic=aspartate,
glutamate; (2) basic=lysine, arginine histidine, (3)
aliphatic=glycine, alanine, valine, leucine, isoleucine, serine,
threonine, with serine and threonine optionally be grouped
separately as aliphatic-hydroxyl; (4) aromatic=phenylalanine,
tyrosine, tryptophan; (5) amide=asparagine, glutamine; and (6)
sulfur -containing=cysteine and methionine. (see, for example,
Biochemistry, 2nd ed., Ed. by L. Stryer, W H Freeman and Co.:
1981). Whether a change in the amino acid sequence of a peptide
results in a functional hedgehog homolog (e.g., functional in the
sense that it acts to mimic or antagonize the wild-type form) can
be readily determined by assessing the ability of the variant
peptide to produce a response in cells in a fashion similar to the
wild-type protein, or competitively inhibit such a response.
Polypeptides in which more than one replacement has taken place can
readily be tested in the same manner.
[0125] It is specifically contemplated that the methods of the
present invention can be carried out using homologs of naturally
occurring hedgehog proteins. In one embodiment, the invention
contemplates using hedgehog polypeptides generated by combinatorial
mutagenesis. Such methods, as are known in the art, are convenient
for generating both point and truncation mutants, and can be
especially useful for identifying potential variant sequences
(e.g., homologs) that are functional in binding to a receptor for
hedgehog proteins. The purpose of screening such combinatorial
libraries is to generate, for example, novel hedgehog homologs
which can act as neuroprotective agents. To illustrate, hedgehog
homologs can be engineered by the present method to provide more
efficient binding to a cognate receptor, such as patched, retaining
neuroprotective activity. Thus, combinatorially-derived homologs
can be generated to have an increased potency relative to a
naturally occurring form of the protein. Moreover, manipulation of
certain domains of hedgehog by the present method can provide
domains more suitable for use in fusion proteins, such as one that
incorporates portions of other proteins which are derived from the
extracellular matrix and/or which bind extracellular matrix
components.
[0126] To further illustrate the state of the art of combinatorial
mutagenesis, it is noted that the review article of Gallop et al.
(1994) J Med Chem 37:1233 describes the general state of the art of
combinatorial libraries as of the earlier 1990's. In particular,
Gallop et al. state at page 1239 "[s]creening the analog libraries
aids in determining the minimum size of the active sequence and in
identifying those residues critical for binding and intolerant of
substitution". In addition, the Ladner et al. PCT publication
WO90/02809, the Goeddel et al. U.S. Pat. No. 5,223,408, and the
Markland et al. PCT publication WO92/15679 illustrate specific
techniques which one skilled in the art could utilize to generate
libraries of hedgehog variants which can be rapidly screened to
identify variants/fragments which retained a particular activity of
the hedgehog polypeptides. These techniques are exemplary of the
art and demonstrate that large libraries of related
variants/truncants can be generated and assayed to isolate
particular variants without undue experimentation. Gustin et al.
(1993) Virology 193:653, and Bass et al. (1990) Proteins:Structure,
Function and Genetics 8:309-314 also describe other exemplary
techniques from the art which can be adapted as means for
generating mutagenic variants of hedgehog polypeptides.
[0127] Indeed, it is plain from the combinatorial mutagenesis art
that large scale mutagenesis of hedgehog proteins, without any
preconceived ideas of which residues were critical to the
biological fuiction, and generate wide arrays of variants having
equivalent biological activity. Indeed, it is the ability of
combinatorial techniques to screen billions of different
variants-by high throughout analysis that removes any requirement
of a priori understanding or knowledge of critical residues.
[0128] To illustrate, the amino acid sequences for a population of
hedgehog homologs or other related proteins are aligned, preferably
to promote the highest homology possible. Such a population of
variants can include, for example, hedgehog homologs from one or
more species. Amino acids which appear at each position of the
aligned sequences are selected to create a degenerate set of
combinatorial sequences. In a preferred embodiment, the variegated
library of hedgehog variants is generated by combinatorial
mutagenesis at the nucleic acid level, and is encoded by
a.variegated gene library. For instance, a mixture of synthetic
oligonucleotides can be enzymatically ligated into gene sequences
such that the degenerate set of potential hedgehog sequences are
expressible as individual polypeptides, or alternatively, as a set
of larger fusion proteins (e.g., for phage display) containing the
set of hedgehog sequences therein.
[0129] As illustrated in PCT publication WO 95/18856, to analyze
the sequences of a population of variants, the amino acid sequences
of interest can be aligned relative to sequence homology. The
presence or absence of amino acids from an aligned sequence of a
particular variant is relative to a chosen consensus length of a
reference sequence, which can be real or artificial.
[0130] In an illustrative embodiment, alignment of exons 1, 2 and a
portion of exon 3 encoded sequences (e.g., the N-terminal
approximately 221 residues of the mature protein) of each of the
Shh clones produces a degenerate set of Shh polypeptides
represented by the general formula:
2 C-G-P-G-R-G-X(1)-G-X(2)-R-R-H-P-K- (SEQ ID No: 19)
K-L-T-P-L-A-Y-K-Q-F-I-P-N-V-A-E-K-
T-L-G-A-S-G-R-Y-E-G-K-I-X(3)-R-N- S-E-R-F-K-E-L-T-P-N-Y-N-
-P-D-I-I-F- K-D-E-E-N-T-G-A-D-R-L-M-T-Q-R-C-K-
D-K-L-N-X(4)-L-A-I-S-V-M-N-X(5)-W-
P-G-V-X(6)-L-R-V-T-E-G-W-D-E-D-G- H-H-X(7)-E-E-S-L-H-Y-E--
G-R-A-V-D- I-T-T-S-D-R-D-X(8)-S-K-Y-G-X(9)-L-
X(10)-R-L-A-V-E-A-G-F-D-W-V-Y-Y-E-
S-K-A-H-I-H-C-S-V-K-A-E-N-S-V-A-A-
K-S-G-G-C-F-P-G-S-A-X(11)-V-X(12)-
L-X(13)-X(14)-G-G-X(15)-K-X-(16)- V-K-D-L-X(17)-P-G-D-X(1-
8)-V-L-A-A- D-X(19)-X(20)-G-X(21)-L-X(22)-
X(23)-S-D-F-X(24)-X(25)-F-X(26)-D- R,
[0131] wherein each of the degenerate positions "X" can be an amino
acid which occurs in that position in one of the human, mouse,
chicken or zebrafish Shh clones, or, to expand the library, each X
can also be selected from amongst amino acid residue which would be
conservative substitutions for the amino acids which appear
naturally in each of those positions. For instance, Xaa(1)
represents Gly, Ala, Val, Leu, Ile, Phe, Tyr or Trp ; Xaa(2)
represents Arg, His or Lys; Xaa(3) represents Gly, Ala, Val, Leu,
Ile, Ser or Thr; Xaa(4) represents Gly, Ala, Val, Leu, Ile, Ser or
Thr; Xaa(5) represents Lys, Arg, His, Asn or Gln; Xaa(6) represents
Lys, Arg or His; Xaa(7) represents Ser, Thr, Tyr, Trp or Phe;
Xaa(8) represents Lys, Arg or His; Xaa(9) represents Met, Cys, Ser
or Thr; Xaa(10) represents Gly, Ala, Val, Leu, Ile, Ser or Thr;
Xaa(l 1) represents Leu, Val, Met, Thr or Ser; Xaa(12) represents
His, Phe, Tyr, Ser, Thr, Met or Cys; Xaa(13) represents Gln, Asn,
Glu, or Asp; Xaa(14) represents His, Phe, Tyr, Thr, Gln, Asn, Glu
or Asp; Xaa(15) represents Gln, Asn, Glu, Asp, Thr, Ser, Met or
Cys; Xaa(16) represents Ala, Gly, Cys, Leu, Val or Met; Xaa(17)
represents Arg, Lys, Met, Ile, Asn, Asp, Glu, Gln, Ser, Thr or Cys;
Xaa(18) represents Arg, Lys, Met or Ile; Xaa(19) represents Ala,
Gly, Cys, Asp, Glu, Gln, Asn, Ser, Thr or Met; Xaa(20) represents
Ala, Gly, Cys, Asp, Asn, Glu or Gln; Xaa(21) represents Arg, Lys,
Met, Ile, Asn, Asp, Glu or Gln; Xaa(22) represent Leu, Val, Met or
Ile; Xaa(23) represents Phe, Tyr, Thr, His or Trp; Xaa(24)
represents Ile, Val, Leu or Met; Xaa(25) represents Met, Cys, Ile,
Leu, Val, Thr or Ser; Xaa(26) represents Leu, Val, Met, Thr or Ser.
In an even more expansive library, each X can be selected from any
amino acid.
[0132] In similar fashion, alignment of each of the human, mouse,
chicken and zebrafish hedgehog clones, can provide a degenerate
polypeptide sequence represented by the general formula:
3 C-G-P-G-R-G-X(1)-X(2)-X(3)-R-R- (SEQ ID No: 20)
X(4)-X(5)-X(6)-P-K-X(7)-L-X(8)-P- L-X(9)-Y-K-Q-F-X(10)-P-- X(11)-
X(12)-X(13)-E-X(14)-T-L-G-A-S-G- X(15)-X(16)-E-G-X(17)-X(18)-X(19)-
R-X(20)-S-E-R-F-X(21)-X(22)-L-T-P-
N-Y-N-P-D-I-I-F-K-D-E-E-N-X(23)-G- A-D-R-L-M-T-X(24)-R-C-K-X(25)-
X(26)-X(27)-N-X(28)-L-A-I-- S-V-M-N-
X(29)-W-P-G-V-X(30)-L-R-V-T-E-G- X(31)-D-E-D-G-H-H-X(32)-X(33)-
X(34)-S-L-H-Y-E-G-R-A-X(35- )-D-I-T-
T-S-D-R-D-X(36)-X(37)-K-Y-G-X(38)-
L-X(39)-R-L-A-V-E-A-G-F-D-W-V-Y-Y-
E-S-X(40)-X(41)-H-X(42)-H-X(43)-S- V-K-X(44)-X(45),
[0133] wherein, as above, each of the degenerate positions "X" can
be an amino acid which occurs in a corresponding position in one of
the wild-type clones, and may also include amino acid residue which
would be conservative substitutions, or each X can be any amino
acid residue. In an exemplary embodiment, Xaa(1) represents Gly,
Ala, Val, Leu, Ile, Pro, Phe or Tyr; Xaa(2) represents Gly, Ala,
Val, Leu or Ile; Xaa(3) represents Gly, Ala, Val, Leu, Ile, Lys,
His or Arg; Xaa(4) represents Lys, Arg or His; Xaa(5) represents
Phe, Trp, Tyr or an amino acid gap; Xaa(6) represents Gly, Ala,
Val, Leu, Ile or an amino acid gap; Xaa(7) represents Asn, Gln,
His, Arg or Lys; Xaa(8) represents Gly, Ala, Val, Leu, Ile, Ser or
Thr; Xaa(9) represents Gly, Ala, Val, Leu, Ile, Ser or Thr; Xaa(10)
represents Gly, Ala, Val, Leu, Ile, Ser or Thr; Xaa(l 1) represents
Ser, Thr, Gln or Asn; Xaa(12) represents Met, Cys, Gly, Ala, Val,
Leu, Ile, Ser or Thr; Xaa(13) represents Gly, Ala, Val, Leu, Ile or
Pro; Xaa(14) represents Arg, His or Lys; Xaa(15) represents Gly,
Ala, Val, Leu, Ile, Pro, Arg, His or Lys; Xaa(16) represents Gly,
Ala, Val, Leu, Ile, Phe or Tyr; Xaa(17) represents Arg, His or Lys;
Xaa(18) represents Gly, Ala, Val, Leu, Ile, Ser or Thr; Xaa(19)
represents Thr or Ser; Xaa(20) represents Gly, Ala, Val, Leu, Ile,
Asn or Gln; Xaa(21) represents Arg, His or Lys; Xaa(22) represents
Asp or Glu; Xaa(23) represents Ser or Thr; Xaa(24) represents Glu,
Asp, Gln or Asn; Xaa(25) represents Glu or Asp; Xaa(26) represents
Arg, His or Lys; Xaa(27) represents Gly, Ala, Val, Leu or Ile;
Xaa(28) represents Gly, Ala, Val, Leu, Ile, Thr or Ser; Xaa(29)
represents Met, Cys, Gln, Asn, Arg, Lys or His; Xaa(30) represents
Arg, His or Lys; Xaa(31) represents Trp, Phe, Tyr, Arg, His or Lys;
Xaa(32) represents Gly, Ala, Val, Leu, Ile, Ser, Thr, Tyr or Phe;
Xaa(33) represents Gin, Asn, Asp or Glu; Xaa(34) represents Asp or
Glu; Xaa(35) represents Gly, Ala, Val, Leu, or Ile; Xaa(36)
represents Arg, His or Lys; Xaa(37) represents Asn, Gln, Thr or
Ser; Xaa(38) represents Gly, Ala, Val, Leu, Ile, Ser, Thr, Met or
Cys; Xaa(39) represents Gly, Ala, Val, Leu, Ile, Thr or Ser;
Xaa(40) represents Arg, His or Lys; Xaa(4 1) represents Asn, Gln,
Gly, Ala, Val, Leu or Ile; Xaa(42) represents Gly, Ala, Val, Leu or
Ile; Xaa(43) represents Gly; Ala, Val, Leu, Ile, Ser, Thr or Cys;
Xaa(44) represents Gly, Ala, Val, Leu, Ile, Thr or Ser; and Xaa(45)
represents Asp or Glu.
[0134] There are many ways by which the library of potential
hedgehog homologs can be generated from a degenerate
oligonucleotide sequence. Chemical synthesis of a degenerate gene
sequence can be carried out in an automatic DNA synthesizer, and
the synthetic genes then ligated into an appropriate expression
vector. The purpose of a degenerate set of genes is to provide, in
one mixture, all of the sequences encoding the desired set of
potential hedgehog sequences. The synthesis of degenerate
oligonucleotides is well known in the art (see for example, Narang,
SA (1983) Tetrahedron 39:3; Itakura et al. (1981) Recombinant DATA,
Proc 3rd Cleveland Sympos. Macromolecules, ed. A G Walton,
Arnsterdam: Elsevier pp273-289; Itakura et al. (1984) Annu. Rev.
Biochem. 53:323; Itakura et al. (1984) Science 198:1056; Ike et al.
(1983) Nucleic Acid Res. 11:477. Such techniques have been employed
in the directed evolution of other proteins (see, for example,
Scott et al. (1990) Science 249:386-390; Roberts et al. (1992) PNAS
89:2429-2433; Devlin et al. (1990) Science 249: 404-406; Cwirla et
al. (1990) PNAS 87: 6378-6382; as well as U.S. Pat. Nos. 5,223,409,
5,198,346, and 5,096,815).
[0135] A wide range of techniques are known in the art for
screening gene products of combinatorial libraries made by point
mutations, and for screening cDNA libraries for gene products
having a certain property. Such techniques will be generally
adaptable for rapid screening of the gene libraries generated by
the combinatorial mutagenesis of hedgehog homologs. The most widely
used techniques for screening large gene libraries typically
comprises cloning the gene library into replicable expression
vectors, transforming appropriate cells with the resulting library
of vectors, and expressing the combinatorial genes under conditions
in which detection of a desired activity facilitates relatively
easy isolation of the vector encoding the gene whose product was
detected. Each of the illustrative assays described below are
amenable to high through-put analysis as necessary to screen large
numbers of degenerate hedgehog sequences created by combinatorial
mutagenesis techniques.
[0136] In one embodiment, the combinatorial library is designed to
be secreted (e.g., the polypeptides of the library all include a
signal sequence but no transmembrane or cytoplasmic domains), and
is used to transfect a eukaryotic cell that can be co-cultured with
neuronal cells. A functional hedgehog protein secreted by the cells
expressing the combinatorial library will diffuse to neighboring
neuronal cells and induce a particular biological response, such as
protection against cell death under oxygen-deprevation conditions
(e.g., high CO.sub.2 culture -conditions). The pattern of detection
of proliferation will resemble a gradient function, and will allow
the isolation (generally after several repetitive rounds of
selection) of cells producing hedgehog homologs active as
neuroprotective agents with respect to neuronal cells.
[0137] To illustrate, target neuronal cells are cultured in 24-well
microtitre plates. Other eukaryotic cells are transfected with the
combinatorial hedgehog gene library and cultured in cell culture
inserts (e.g., Collaborative Biomedical Products, Catalog #40446)
that are able to fit into the wells of the microtitre plate. The
cell culture inserts are placed in the wells such that recombinant
hedgehog homologs secreted by the cells in the insert can diffuse
through the porous bottom of the insert and contact the target
cells in the microtitre plate wells. After a period of time
sufficient for fimctional forms of a hedgehog protein to produce a
measurable response in the target cells, such as neuroprotection,
the inserts are removed and the effect of the variant hedgehog
proteins on the target cells determined. Cells from the inserts
corresponding to wells which score positive for activity can be
split and re-cultured on several inserts, the process being
repeated until the active clones are identified.
[0138] In yet another screening assay, the candidate hedgehog gene
products are displayed on the surface of a cell or viral particle,
and the ability of particular cells or viral particles to associate
with a hedgehog-binding moiety (such as the patched protein or
other hedgehog receptor) via this gene product is detected in a
"panning assay". Such panning steps can be carried out on cells
cultured from embryos. For instance, the gene library can be cloned
into the gene for a surface membrane protein of a bacterial cell,
and the resulting fusion protein detected by panning (Ladner et
al., WO 88/06630; Fuchs et al. (1991) Bio/Technology 9:1370-1371;
and Goward et al. (1992) TIBS 18:136-140). In a similar fashion,
fluorescently labeled molecules which bind hedgehog can be used to
score for potentially functional hedgehog homologs. Cells can be
visually inspected and separated under a fluorescence microscope,
or, where the morphology of the cell permits, separated by a
fluorescence-activated cell sorter.
[0139] In an alternate embodiment, the gene library is expressed as
a fusion protein on the surface of a viral particle. For instance,
in the filamentous phage system, foreign peptide sequences can be
expressed on the surface of infectious phage, thereby conferring
two significant benefits. First, since these phage can be applied
to affinity matrices at very high concentrations, large number of
phage can be screened at one time. Second, since each infectious
phage displays the combinatorial gene product on its surface, if a
particular phage is recovered from an affinity matrix in low yield,
the phage can be amplified by another round of infection. The group
of almost identical E. coli filamentous phages M13, fd, and fl are
most often used in phage display libraries, as either of the phage
gIII or gVIII coat proteins can be used to generate fusion proteins
without disrupting the ultimate packaging of the viral particle
(Ladner et al. PCT publication WO 90/02909; Garrard et al., PCT
publication WO 92/09690; Marks et al. (1992) J. Biol. Chem.
267:16007-16010; Griffths et al. (1993) EMBO J 12:725-734; Clackson
et al. (1991) Nature 352:624-628; and Barbas et al. (1992) PNAS
89:4457-4461).
[0140] In an illustrative embodiment, the recombinant phage
antibody system (RPAS, Pharamacia Catalog number 27-9400-01) can be
easily modified for use in expressing and screening hedgehog
combinatorial libraries. For instance, the pCANTAB 5 phagemid of
the RPAS kit contains the gene which encodes the phage gill coat
protein. The hedgehog combinatorial gene library can be cloned into
the phagemid adjacent to the gilI signal sequence such that it will
be expressed as a gIII fusion protein. After ligation, the phagemid
is used to transform competent E. coli TG1 cells. Transformed cells
are subsequently infected with M13KO7 helper phage to rescue the
phagemid and its candidate hedgehog gene insert. The resulting
recombinant phage contain phagemid DNA encoding a specific
candidate hedgehog, and display one or more copies of the
corresponding fusion coat protein. The phage-displayed candidate
hedgehog proteins which are capable of binding an hedgehog receptor
are selected or enriched by panning. For instance, the phage
library can be applied to cells which express the patched protein
and unbound phage washed away from the cells. The bound phage is
then isolated, and if the recombinant phage express at least one
copy of the wild type glll coat protein, they will retain their
ability to infect E. coli. Thus, successive rounds of reinfection
of E. coli, and panning will greatly enrich for hedgehog homologs,
which can then be screened for further biological activities in
order to differentiate agonists and antagonists.
[0141] Combinatorial mutagenesis has a potential to generate very
large libraries of mutant proteins, e.g., in the order of 10.sup.26
molecules. Combinatorial libraries of this size may be technically
challenging to screen even with high throughput screening assays
such as phage display. To overcome this problem, a new technique
has been developed recently, recrusive ensemble mutagenesis (REM),
which allows one to avoid the very high proportion of
non-functional proteins in a random library and simply enhances the
frequency of functional proteins, thus decreasing the complexity
required to achieve a useful sampling of sequence space. REM is an
algorithm which enhances the frequency of functional mutants in a
library when an appropriate selection or screening method is
employed (Arkin and Yourvan, 1992, PNAS USA 89:7811-7815; Yourvan
et al., 1992, Parallel Problem Solvingfrom Nature, 2., In Maenner
and Manderick, eds., Elsevir Publishing Co., Amsterdam, pp.
401-410; Delgrave et al., 1993, Protein Engineering 6(3):327-33
1).
[0142] The invention also provides for reduction of the hedgehog
protein to generate mimetics, e.g., peptide or non-peptide agents,
which are able to mimic the neuroprotective activity of a
naturally-occurring hedgehog polypeptide. Thus, such mutagenic
techniques as described above are also useful to map the
determinants of the hedgehog proteins which participate in
protein-protein interactions involved in, for example, binding of
the subject hedgehog polypeptide to other extracellular matrix
components such as its receptor(s). To illustrate, the critical
residues of a subject hedgehog polypeptide which are involved in
molecular recognition of an hedgehog receptor such as patched can
be determined and used to generate hedgehog-derived peptidomimetics
which competitively bind with that moiety. By employing, for
example, scanning mutagenesis to map the amino acid residues of
each of the subject hedgehog proteins which are involved in binding
other extracellular proteins, peptidomimetic compounds can be
generated which mimic those residues of the hedgehog protein which
facilitate the interaction. After distinguishing between agonist
and antagonists, such agonistic mimetics may be used to mimic the
normal function of a hedgehog protein in the treatment ischemia.
For instance, non-hydrolyzable peptide analogs of such residues can
be generated using benzodiazepine (e.g., see Freidinger et al. in
Peptides: Chemistry and Biology, G. R. Marshall ed., ESCOM
Publisher:Leiden, Netherlands, 1988), azepine (e.g., see Huffman et
al. in Peptides: Chemistry and Biology, G. R. Marshall ed., ESCOM
Publisher: Leiden, Netherlands, 1988), substituted gama lactam
rings (Garvey et al. in Peptides: Chemistry and Biology, G. R.
Marshall ed., ESCOM Publisher: Leiden, Netherlands, 1988),
keto-methylene pseudopeptides (Ewenson et al. (1986) J Med Chem
29:295; and Ewenson et al. in Peptides: Structure and Function
(Proceedings of the 9th American Peptide Symposium) Pierce Chemical
Co. Rockland, Ill., 1985), .beta.-turn dipeptide cores (Nagai et
al. (1985) Tetrahedron Lett 26:647; and Sato et al. (1986) J Chem
Soc Perkin Trans 1:1231), and .beta.-aminoalcohols (Gordon et al.
(1985) Biochem Biophys Res Communl 26:419; and Dann et al. (1986)
Biochem Biophys Res Commun 134:71).
[0143] Recombinantly produced forms of the hedgehog proteins can be
produced using, e.g, expression vectors containing a nucleic acid
encoding a hedgehog polypeptide, operably linked to at least one
transcriptional regulatory sequence. Operably linked is intended to
mean that the nucleotide sequence is linked to a regulatory
sequence in a manner which allows expression of the nucleotide
sequence. Regulatory sequences are art-recognized and are selected
to direct expression of a hedgehog polypeptide. Accordingly, the
term transcriptional regulatory sequence includes promoters,
enhancers and other expression control elements. Such regulatory
sequences are described in Goeddel; Gene Expression Technology:
Methods in Enzymology 185, Academic Press, San Diego, Calif.
(1990). For instance, any of a wide variety of expression control
sequences, sequences that control the expression of a DNA sequence
when operatively linked to it, may be used in these vectors to
express DNA sequences encoding hedgehog polypeptide. Such useful
expression control sequences, include, for example, a viral LTR,
such as the LTR of the Moloney murine leukemia virus, the early and
late promoters of SV40, adenovirus or cytomegalovirus immediate
early promoter, the lac system, the trp system, the TAC or TRC
system, T7 promoter whose expression is directed by T7 RNA
polymerase, the major operator and promoter regions of phage
.lambda., the control regions for fd coat protein, the promoter for
3-phosphoglycerate kinase or other glycolytic enzymes, the
promoters of acid phosphatase, e.g., Pho5, the promoters of the
yeast ax-mating factors, the polyhedron promoter of the baculovirus
system and other sequences known to. control the expression of
genes of prokaryotic or eukaryotic cells or their viruses, and
various combinations thereof. It should be understood that the
design of the expression vector may depend on such factors as the
choice of the host cell to be transformed and/or the type of
protein desired to be expressed. Moreover, the vector's copy
number, the ability to control that copy number and the expression
of any other proteins encoded by the vector, such as antibiotic
markers, should also be considered.
[0144] In addition to providing a ready source of hedgehog
polypeptides for purification, the gene constructs of the present
invention can also be used as a part of a gene therapy protocol to
deliver nucleic acids encoding either a neuroprotective form of a
hedgehog polypeptide. Thus, another aspect of the invention
features expression vectors for in vivo transfection of a hedgehog
polypeptide in particular cell types so as to cause ectopic
expression of a hedgehog polypeptide in neuronal tissue.
[0145] Formulations of such expression constructs may be
administered in any biologically effective carrier, e.g., any
formulation or composition capable of effectively delivering the
recombinant gene to cells in vivo. Approaches include insertion of
the hedgehog coding sequence in viral vectors including recombinant
retroviruses, adenovirus, adeno-associated virus, and herpes
simplex virus-1, or recombinant bacterial or eukaryotic plasmids.
Viral vectors transfect cells directly; plasmid DNA can be
delivered with the help of, for example, cationic liposomes
(lipofectin) or derivatized (e.g., antibody conjugated), polylysine
conjugates, gramacidin S, artificial viral envelopes or other such
intracellular carriers, as well as direct injection of the gene
construct or CaPO.sub.4 precipitation carried out in vivo. It will
be appreciated that because transduction of appropriate target
cells represents the critical first step in gene therapy, choice of
the particular gene delivery system will depend on such factors as
the phenotype of the intended target and the route of
administration, e.g., locally or systemically. Furthermore, it will
be recognized that the particular gene construct provided for in
vivo transduction of hedgehog expression are also useful for in
vitro transduction of cells, such as-for use in the ex vivo tissue
culture systems described below.
[0146] A preferred approach for in vivo introduction of nucleic
acid into a cell is by use of a viral vector containing nucleic
acid, e.g., a cDNA, encoding the particular form of the hedgehog
polypeptide desired. Infection of cells with a viral vector has the
advantage that a large proportion of the targeted cells can receive
the nucleic acid. Additionally, molecules encoded within the viral
vector, e.g., by a cDNA contained in the viral vector, are
expressed efficiently in cells which have taken up viral vector
nucleic acid.
[0147] Retrovirus vectors and adeno-associated virus vectors are
generally understood to be the recombinant gene delivery system of
choice for the transfer of exogenous genes in vivo, particularly
into humans. These vectors provide efficient delivery of genes into
cells, and the transferred nucleic acids are stably integrated into
the chromosomal DNA of the host. A major prerequisite for the use
of retroviruses is to ensure the safety of their use, particularly
with regard to the possibility of the spread of wild-type virus in
the cell population. The development of specialized cell lines
(termed "packaging cells") which produce only replication-defective
retroviruses has increased the utility of retroviruses for gene
therapy, and defective retroviruses are well characterized for use
in gene transfer for gene therapy purposes (for a review see
Miller, A. D. (1990) Blood 76:271). Thus, recombinant retrovirus
can be constructed in which part of the retroviral coding sequence
(gag, pol, env) has been replaced by nucleic acid encoding a
hedgehog polypeptide and renders the retrovirus replication
defective. The replication defective retrovirus is then packaged
into virions which can be used to infect a target cell through the
use of a helper virus by standard techniques. Protocols for
producing recombinant retroviruses and for infecting cells in vitro
or in vivo with such viruses can be found in Current Protocols in
Molecular Biology, Ausubel, F. M. et al. (eds.) Greene Publishing
Associates, (1989), Sections 9.10-9.14 and other standard
laboratory manuals. Examples of suitable retroviruses include pLJ,
pZIP, pWE and pEM which are well known to those skilled in the art.
Examples of suitable packaging virus lines for preparing both
ecotropic and amphotropic retroviral systems include Crip, Cre, 2
and Am. Retroviruses have been used to introduce a variety of genes
into many different cell types, including neuronal cells, in vitro
and/or in vivo (see, for example, Eglitis, et al. (1985) Science
230:1395-1398; Danos and Mulligan (1988) Proc. Natl. Acad. Sci. USA
85:6460-6464; Wilson et al. (1988) Proc. Natl. Acad. Sci. USA
85:3014-3018; Armentano et al. (1990) Proc. Natl. Acad. Sci. USA
87:6141-6145; Huber et al. (1991) Proc. Natl. Acad. Sci. USA
88:8039-8043; Ferry et al. (1991) Proc. Natl. Acad. Sci. USA
88:8377-8381; Chowdhury et al. (1991) Science 254:1802-1805; van
Beusechem et al. (1992) Proc. Natl. Acad. Sci. USA 89:7640-7644;
Kay et al. (1992) Human Gene Therapy 3:641-647; Dai et al. (1992)
Proc. Natl. Acad. Sci. USA 89:10892-10895; Hwu et al. (1993) J.
Immunol. 150:4104-4115; U.S. Pat. No. 4,868,116; U.S. Pat. No.
4,980,286; PCT Application WO 89/07136; PCT Application WO
89/02468; PCT Application WO 89/05345; and PCT Application WO
92/07573).
[0148] Furthermore, it has been shown that it is possible to limit
the infection spectrum of retroviruses and consequently of
retroviral-based vectors, by modifying the viral packaging proteins
on the surface of the viral particle (see, for example PCT
publications WO93/25234 and WO94/06920). For instance, strategies
for the modification of the infection spectrum of retroviral
vectors include: coupling antibodies specific for cell surface
antigens to the viral env protein (Roux et al. (1989) PNAS
86:9079-9083; Julan et al. (1992) J. Gen Virol 73:3251-3255; and
Goud et al. (1983) Virology 163:251-254); or coupling cell surface
receptor ligands to the viral env proteins (Neda et al. (1991) J
Biol Chem 266:14143-14146). Coupling can be in the form of the
chemical cross-linking with a protein or other variety (e.g.,
lactose to convert the env protein to an asialoglycoprotein), as
well as by generating fusion proteins (e.g., single-chain
antibody/env fusion proteins). This technique, while useful to
limit or otherwise direct the infection to certain tissue types,
can also be used to convert an ecotropic vector into an amphotropic
vector.
[0149] Moreover, use of retroviral gene delivery can be further
enhanced by the use of tissue- or cell-specific transcriptional
regulatory sequences which control expression of the hedgehog gene
of the retroviral vector.
[0150] Another viral gene delivery system useful in the present
method utilizes adenovirus-derived vectors. The genome of an
adenovirus can be manipulated such that it encodes and expresses a
gene product of interest but is inactivated in terms of its ability
to replicate in a normal lytic viral life cycle. See for example
Berkner et al. (1988) BioTechniques 6:616; Rosenfeld et al. (1991)
Science 252:431-434; and Rosenfeld et al. (1992) Cell 68:143-155.
Suitable adenoviral vectors derived from the adenovirus strain Ad
type 5 dl324 or other strains of adenovirus (e.g., Ad2, Ad3, Ad7
etc.) are well known to those skilled in the art. Recombinant
adenoviruses can be advantageous in certain circumstances in that
they can be used to infect a wide variety of cell types, including
neuronal cells (Rosenfeld et-al. (1992) cited supra).
[0151] Furthermore, the virus particle is relatively stable and
amenable to purification and concentration, and as above, can be
modified so as to affect the spectrum of infectivity. Additionally,
introduced adenoviral DNA (and foreign DNA contained therein) is
not integrated into the genome of a host cell but remains episomal,
thereby avoiding potential problems that can occur as a result of
insertional mutagenesis in situations where introduced DNA becomes
integrated into the host genome (e.g., retroviral DNA). Moreover,
the carrying capacity of the adenoviral genome for foreign DNA is
large (up to 8 kilobases) relative to other gene delivery vectors
(Berkner et al. cited supra; Haj-Ahmand and Graham (1986) J. Virol.
57:267). Most replication-defective adenoviral vectors currently in
use and therefore favored by the present invention are deleted for
all or parts of the viral E1 and E3 genes but retain as much as 80%
of the adenoviral genetic material (see, e.g., Jones et al. (1979)
Cell 16:683; Berkner et al., supra; and Graham et al. in Methods in
Molecular Biology, E. J. Murray, Ed. (Humana, Clifton, N.J., 1991)
vol. 7. pp. 109-127). Expression of the inserted hedgehog gene can
be under control of, for example, the EIA promoter, the major late
promoter (MLP) and associated leader sequences, the E3 promoter, or
exogenously added promoter sequences.
[0152] In addition to viral transfer methods, such as those
illustrated above, non-viral methods can also be employed to cause
expression of a hedgehog polypeptide in the tissue of an animal.
Most nonviral methods of gene transfer rely on normal mechanisms
used by mammalian cells for the uptake and intracellular transport
of macromolecules. In preferred embodiments, non-viral gene
delivery systems of the present invention rely on endocytic
pathways for the uptake of the hedgehog polypeptide gene by the
targeted cell. Exemplary gene delivery systems of this type include
liposomal derived systems, poly-lysine conjugates, and artificial
viral envelopes.
[0153] In clinical settings, the gene delivery systems for the
therapeutic hedgehog gene can be introduced into a patient by any
of a number of methods, each of which is familiar in the art. For
instance, a pharmaceutical preparation of the gene delivery system
can be introduced systemically, e.g., by intravenous injection, and
specific transduction of the protein in the target cells occurs
predominantly from specificity of transfection provided by the gene
delivery vehicle, cell-type or tissue-type expression due to the
transcriptional regulatory sequences controlling expression of the
receptor gene, or a combination thereof. In other embodiments,
initial delivery of the recombinant gene is more limited with
introduction into the animal being quite localized. For example,
the gene delivery vehicle can be introduced by catheter (see U.S.
Pat. No. 5,328,470) or by stereotactic injection (e.g., Chen et al.
(1994) PNAS 91: 3054-3057). A hedgehog expression construct can be
delivered in a gene therapy construct to dermal cells by, e.g.,
electroporation using techniques described, for example, by Dev et
al. ((1994) Cancer Treat Rev 20:105-115).
[0154] The pharmaceutical preparation of the gene therapy construct
can consist essentially of the gene delivery system in an
acceptable diluent, or can comprise a slow release matrix in which
the gene delivery vehicle is imbedded. Alternatively, where the
complete gene delivery system can be produced intact from
recombinant cells, e.g., retroviral vectors, the pharmaceutical
preparation can comprise one or more cells which produce the gene
delivery system.
[0155] In yet another embodiment, the hedgehog or ptc therapeutic
can be a "gene activation" construct which, by homologous
recombination with a genomic DNA, alters the transcriptional
regulatory sequences of an endogenous gene. For instance, the gene
activation construct can replace the endogenous promoter of a
hedgehog gene with a heterologous promoter, e.g., one which causes
constitutive expression of the hedgehog gene or which causes
inducible expression of the gene under conditions different from
the normal expression pattern of the gene. Other genes in the
patched signaling pathway can be similarly targeted. A variety of
different formats for the gene activation constructs are available.
See, for example, the Transkaryotic Therapies, Inc PCT publications
WO93/09222, WO95/31560, WO96/29411, WO95/31560 and WO94/12650.
[0156] In preferred embodiments, the nucleotide sequence used as
the gene activation construct can be comprised of (1) DNA from some
portion of the endogenous hedgehog gene (exon sequence, intron
sequence, promoter sequences, etc.) which direct recombination and
(2) heterologous transcriptional regulatory sequence(s) which is to
be operably linked to the coding sequence for the genomic hedgehog
gene upon recombination of the gene activation construct. For use
in generating cultures of hedgehog producing cells, the construct
may further include a reporter gene to detect the presence of the
knockout construct in the cell.
[0157] The gene activation construct is inserted into a cell, and
integrates with the genomic DNA of the cell in such a position so
as to provide the heterologous regulatory sequences in operative
association with the native hedgehog gene. Such insertion occurs by
homologous recombination, i.e., recombination regions of the
activation construct that are homologous to the endogenous hedgehog
gene sequence hybridize to the genomic DNA and recombine with the
genomic sequences so that the construct is incorporated into the
corresponding position of the genomic DNA.
[0158] The terms "recombination region" or "targeting sequence"
refer to a segment (i.e., a portion) of a gene activation construct
having a sequence that is substantially identical to or
substantially complementary to a genomic gene sequence, e.g.,
including 5' flanking sequences of the genomic gene, and can
facilitate homologous recombination between the genomic sequence
and the targeting transgene construct.
[0159] As used herein, the term "replacement region" refers to a
portion of an activation construct which becomes integrated into an
endogenous chromosomal location following homologous recombination
between a recombination region and a genomic sequence.
[0160] The heterologous regulatory sequences, e.g., which are
provided in the replacement region, can include one or more of a
variety elements, including: promoters (such as constitutive or
inducible promoters), enhancers, negative regulatory elements,
locus control regions, transcription factor binding sites, or
combinations thereof. Promoters/enhancers which may be used to
control the expression of the targeted gene in vivo include, but
are not limited to, the cytomegalovirus (CMV) promoter/enhancer
(Karasuyama et al., 1989, J. Exp. Med, 169:13), the human
.beta.-actin promoter (Gunning et al. (1987) PNAS 84:4831-4835),
the glucocorticoid-inducible promoter present in the mouse mammary
tumor virus long terminal repeat (MMTV LTR) (Klessig et al. (1984)
Mol. Cell Biol. 4:1354-1362), the long terminal repeat sequences of
Moloney murine leukemia virus (MuLV LTR) (Weiss et al. (1985) RNA
Tumor Viruses, Cold Spring Harbor Laboratory, Cold Spring Harbor,
N.Y.), the SV40 early or late region promoter (Bemoist et al.
(1981) Nature 290:304-310; Templeton et al. (1984) Mol. Cell Biol.,
4:817; and Sprague et al. (1983) J. Virol., 45:773), the promoter
contained in the 3' long terminal repeat of Rous sarcoma virus
(RSV) (Yamamoto et al., 1980, Cell, 22:787-797), the herpes simplex
virus (HSV) thymidine kinase promoter/enhancer (Wagner et al.
(1981) PNAS 82:3567-71), and the herpes simplex virus LAT promoter
(Wolfe et al. (1992) Nature Genetics, 1:379-384).
[0161] In an exemplary embodiment, portions of the 5' flanking
region of the human Shh gene are amplified using primers which add
restriction sites, to generate the following fragments
4 5'-gcgcgcttcgaaGCGAGGCAGCCAGCGAGGGAGAGAGCGAGCGGGCG
AGCCGGAGCGAGGAAatcgatgcgcgc (primer 1)
5'-gcgcgcagatctGGGAAAGCGCAAGAGAGAGCGCACACGCACACACC
CGCCGCGCGCACTCGggatccgcgcgc (primer 2)
[0162] As illustrated, primer 1 includes a 5' non-coding region of
the human Shh gene and is flanked by an AsuII and ClaI restriction
sites. Primer 2 includes a portion of the 5' non-coding region
immediately 3' to that present in primer 1. The hedgehog gene
sequence is flanked by XhoII and BamHI restriction sites. The
purified amplimers are cut with each of the enzymes as
appropriate.
[0163] The vector pCDNA1.1 (Invitrogen) includes a CMV promoter.
The plasmid is cut with with AsuII, which cleaves just 3' to the
CMV promoter sequence. The AsuII/ClaI fragment of primer 1 is
ligated to the AsuII cleavage site of the pcDNA vector. The
ClaI/AsuII ligation destroys the AsuII site at the 3' end of a
properly inserted primer 1.
[0164] The vector is then cut with BamHI, and an XhoII/BamHI
fragment of primer 2 is ligated to the BamHI cleavage site. As
above, the BamHI/XhoII ligation destroys the BamHI site at the 5'
end of a properly inserted primer 2.
[0165] Individual colonies are selected, cut with AsuIl and BamHI,
and the size of the AsuII/BamHI fragment determined. Colonies in
which both the primer 1 and primer 2 sequences are correctly
inserted are further amplified, and cut with AsuIl and BamHI to
produce the gene activation construct:
5 cgaagcgaggcagccagcgagggagagagcgagcgggcgagccggagcga
ggaaATCGAAGGTTCGAATCCTTCCCCCACCACCATCACTTTCAAAAGTC
CGAAAGAATCTGCTCCCTGCTTGTGTGTTGGAGGTCGCTGAGTAGTGCGC
GAGTAAAATTTAAGCTACAACAAGGCAAGGCTTGACCGACAATTGCATGA
AGAATCTGCTTAGGGTTAGGCGTTTTGCGCTGCTTCGCGATGTACGGGCC
AGATATACGCGTTGACATTGATTATTGACTAGTTATTAATAGTAATCAAT
TACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAAC
TTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTG
ACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCA
TTGACGTCAATGGGTGGACTATTTACGGTAAACTGCCCACTTGGCAGTAC
ATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGT
AAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCC
TACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGC
GGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGA
TTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCA
AAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGC
AAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCTC
TGGCTAACTAGAGAACCCACTGCTTACTGGCTTATCGAAATTAATACGAC
TCACTATAGGGAGACCCAAGCTTGGTACCGAGCTCGGATCgatctgggaa
agcgcaagagagagcgcacacgcacacacccgccgcgcgcactcgg
[0166] In this construct, the flanking primer 1 and primer 2
sequences provide the recombination region which permits the
insertion of the CMV promoter in front of the coding sequence for
the human Shh gene. Other heterologous promoters (or other
transcriptional regulatory sequences) can be inserted in a genomic
hedgehog gene by a similar method.
[0167] In still other embodiments, the replacement region merely
deletes a negative transcriptional control element of the native
gene, e.g., to activate expression, or ablates a positive control
element, e.g., to inhibit expression of the targeted gene.
[0168] V. Exemplary ptc Therapeutic Compounds.
[0169] In another embodiment, the subject method is carried out
using a ptc therapeutic composition. Such compositions can be
generated with, for example, compounds which bind to patched and
alter its signal transduction activity, compounds which alter the
binding and/or enzymatic activity of a protein (e.g.,
intracellular) involved in patched signal pathway, and compounds
which alter the level of expression of a hedgehog protein, a
patched protein or a protein involved in the intracellular signal
transduction pathway of patched.
[0170] The availability of purified and recombinant hedgehog
polypeptides facilitates the generation of assay systems which can
be used to screen for drugs, such as small organic molecules, which
are either agonists or antagonists of the normal cellular function
of a hedgehog and/or patched protein, particularly in their role in
the pathogenesis of neuronal cell death. In one embodiment, the
assay evaluates the ability of a compound to modulate binding
between a hedgehog polypeptide and a hedgehog receptor such as
patched. In other embodiments, the assay merely scores for the
ability of a test compound to alter the signal transduction
activity of the patched protein. In this manner, a variety of
hedgehog and/or ptc therapeutics, which will include ones with
neuroprotective activity, can be identified. A variety of assay
formats will suffice and, in light of the present disclosure, will
be comprehended by a skilled artisan.
[0171] In many drug screening programs which test libraries of
compounds and natural extracts, high throughput assays are
desirable in order to maximize the number of compounds surveyed in
a given period of time. Assays which are performed in cell-free
systems, such as may be derived with purified or semi-purified
proteins, are often preferred as "primary" screens in that they can
be generated to permit rapid development and relatively easy
detection of an alteration in a molecular target which is mediated
by a test compound. Moreover, the effects of cellular toxicity
and/or bioavailability of the test compound can be generally
ignored in the in vitro system, the assay instead being focused
primarily on the effect of the drug on the molecular target as may
be manifest in an alteration of binding affinity with receptor
proteins.
[0172] Accordingly, in an exemplary screening assay for ptc
therapeutics, the compound of interest is contacted with a mixture
including a hedgehog receptor protein (e.g., a cell expressing the
patched receptor) and a hedgehog protein under conditions in which
it is ordinarily capable of binding the hedgehog protein. To the
mixture is then added a composition containing a test compound.
Detection and quantification of receptor/hedgehog complexes
provides a means for determining the test compound's efficacy at
inhibiting (or potentiating) complex formation between the receptor
protein and the hedgehog polypeptide. Moreover, a control assay can
also be performed to provide a baseline for comparison. In the
control assay, isolated and purified hedgehog polypeptide is added
to the receptor protein, and the formation of receptor/hedgehog
complex is quantitated in the absence of the test compound.
[0173] Agonist and antagonists of neuroprotection can be
distinguished, and the efficacy of the compound can be assessed, by
subsequent testing with neuronal cells.
[0174] In an illustrative embodiment, the polypeptide utilized as a
hedgehog receptor can be generated from the patched protein.
Accordingly, an exemplary screening assay includes all or a
suitable portion of the patched protein which can be obtained from,
for example, the human patched gene (GenBank U43148) or other
vertebrate sources (see GenBank Accession numbers U40074 for
chicken patched and U46155 for mouse patched), as well as from
drosophila (GenBank Accession number M28999) or other invertebrate
sources. The patched protein can be provided in the screening assay
as a whole protein (preferably expressed on the surface of a cell),
or alternatively as a fragment of the full length protein which
binds to hedgehog polypeptides, e.g., as one or both of the
substantial extracellular domains (e.g., corresponding to residues
Asnl20-Ser438 and/or Arg770-Trp1027 of the human patched protein).
For instance, the patched protein can be provided in soluble form,
as for example a preparation of one of the extracellular domains,
or a preparation of both of the extracellular domains which are
covalently connected by an unstructured linker (see, for example,
Huston et al. (1988) PNAS 85:4879; and U.S. Pat. No. 5,091,513). In
other embodiments, the protein can be provided as part of a
liposomal preparation or expressed on the surface of a cell. The
patched protein can derived from a recombinant gene, e.g., being
ectopically expressed in a heterologous cell. For instance, the
protein can be expressed on oocytes, mammalian cells (e.g., COS,
CHO, 3T3 or the like), or yeast cells by standard recombinant DNA
techniques. These recombinant cells can be used for receptor
binding, signal transduction or gene expression assays. Marigo et
al. (1996) Development 122:1225-1233 illustrates a binding assay of
human hedgehog to chick patched protein ectopically expressed in
Xenopus laevis oocytes. The assay system of Marigo et al. can be
adapted to the present drug screening assays. As illustrated in
that reference, Shh binds to the patched protein in a selective,
saturable, dose-dependent manner, thus demonstrating that patched
is a receptor for Shh.
[0175] Complex formation between the hedgehog polypeptide and a
hedgehog receptor may be detected by a variety of techniques. For
instance, modulation of the formation of complexes can be
quantitated using, for example, detectably labelled proteins such
as radiolabelled, fluorescently labelled, or enzymatically labelled
hedgehog polypeptides, by immunoassay, or by chromatographic
detection.
[0176] Typically, for cell-free assays, it will be desirable to
immobilize either the hedgehog receptor or the hedgehog polypeptide
to facilitate separation of receptor/hedgehog complexes from
uncomplexed forms of one of the proteins, as well as to accommodate
automation of the assay. In one embodiment, a fusion protein can be
provided which adds a domain that allows the protein to be bound to
a matrix. For example, glutathione-S-transferase/receptor
(GST/receptor) fusion proteins can be adsorbed onto glutathione
sepharose beads (Sigma Chemical, St. Louis, Mo.) or glutathione
derivatized microtitre plates, which are then combined with the
hedgehog polypeptide, e.g., an .sup.35S-labeled hedgehog
polypeptide, and the test compound and incubated under conditions
conducive to complex formation, e.g., at physiological conditions
for salt and pH, though slightly more stringent conditions may be
desired. Following incubation, the beads are washed to remove any
unbound hedgehog polypeptide, and the matrix bead-bound radiolabel
determined directly (e.g., beads placed in scintillant), or in the
supernatant after the receptor/hedgehog complexes are dissociated.
Alternatively, the complexes can be dissociated from the bead,
separated by SDS-PAGE gel, and the level of hedgehog polypeptide
found in the bead fraction quantitated from the gel using standard
electrophoretic techniques.
[0177] Other techniques for immobilizing proteins on matrices are
also available for use in the subject assay. For instance, soluble
portions of the hedgehog receptor protein can be immobilized
utilizing conjugation of biotin and stireptavidin. For instance,
biotinylated receptor molecules can be prepared from biotin-NHS
(N-hydroxy-succinimide) using techniques well known in the art
(e.g., biotinylation kit, Pierce Chemicals, Rockford, Ill.), and
immobilized in the wells of streptavidin-coated 96 well plates
(Pierce Chemical). Alternatively, antibodies reactive with the
hedgehog receptor but which do not interfere with hedgehog binding
can be derivatized to the wells of the plate, and the receptor
trapped in the wells by antibody conjugation. As above,
preparations of a hedgehog polypeptide and a test compound are
incubated in the receptor-presenting wells of the plate, and the
amount of receptor/hedgehog complex trapped in the well can be
quantitated. Exemplary methods for detecting such complexes, in
addition to those described above for the GST-immobilized
complexes, include immunodetection of complexes using antibodies
reactive with the hedgehog polypeptide, or which are reactive with
the receptor protein and compete for binding with the hedgehog
polypeptide; as well as enzyme-linked assays which rely on
detecting an enzymatic activity associated with the hedgehog
polypeptide. In the instance of the latter, the enzyme can be
chemically conjugated or provided as a fusion protein with the
hedgehog polypeptide. To illustrate, the hedgehog polypeptide can
be chemically cross-linked or genetically fused with alkaline
phosphatase, and the amount of hedgehog polypeptide trapped in the
complex can be assessed with a chromogenic substrate of the enzyme,
e.g., paranitrophenylphosphate. Likewise, a fusion protein
comprising the hedgehog polypeptide and glutathione-S-transferase
can be provided, and complex formation quantitated by detecting the
GST activity using 1-chloro-2,4-dinitrobenzene (Habig et al (1974)
J Biol Chem 249:7130).
[0178] For processes which rely on immunodetection for quantitating
one of the proteins trapped in the complex, antibodies against the
protein, such as the anti-hedgehog antibodies described herein, can
be used. Alternatively, the protein to be detected in the complex
can be "epitope tagged" in the form of a fusion protein which
includes, in addition to the hedgehog polypeptide or hedgehog
receptor sequence, a second polypeptide for which antibodies are
readily available (e.g., from commercial sources). For instance,
the GST fusion proteins described above can also be used for
quantification of binding using antibodies against the GST moiety.
Other useful epitope tags include myc-epitopes (e.g., see Ellison
et al. (1991) J Biol Chem 266:21150-21157) which includes a
10-residue sequence from c-myc, as well as the pFLAG system
(International Biotechnologies, Inc.) or the pEZZ-protein A system
(Pharamacia, N.J.).
[0179] Where the desired portion of the hedgehog receptor (or other
hedgehog binding molecule) cannot be provided in soluble form,
liposomal vesicles can be used to provide manipulatable and
isolatable sources of the receptor. For example, both authentic and
recombinant forms of the patched protein can be reconstituted in
artificial lipid vesicles (e.g., phosphatidylcholine liposomes) or
in cell membrane-derived vesicles (see, for example, Bear et al.
(1992) Cell 68:809-818; Newton et al. (1983) Biochemistry
22:6110-6117; and Reber et al. (1987) J Biol Chem
262:11369-11374).
[0180] In addition to cell-free assays, such as described above,
the readily available source of hedgehog proteins provided by the
art also facilitates the generation of cell-based assays for
identifying small molecule agonists of the neuroprotective activity
of wild-type hedgehog proteins. Analogous to the cell-based assays
described above for screening combinatorial libraries, neuronal
cells which are sensitive to hedgehog-dependent protection against
ischemic damage can be contacted with a hedgehog protein and a test
agent of interest, with the assay scoring for anything from simple
binding to the cell to modulation in hedgehog inductive responses
by the target cell in the presence and absence of the test agent.
As with the cell-free assays, agents which produce a statistically
significant change in hedgehog activities (either inhibition or
potentiation) can be identified.
[0181] In addition to characterizing cells that naturally express
the patched protein, cells which have been genetically engineered
to ectopically express patched can be utilized for drug screening
assays. As an example, cells which either express low levels or
lack expression of the patched protein, e.g., Xenopus laevis
oocytes, COS cells or yeast cells, can be genetically modified
using standard techniques to ectopically express the patched
protein. (see Marigo et al., supra).
[0182] The resulting recombinant cells, e.g., which express a
functional patched receptor, can be utilized in receptor binding
assays to identify agonist or anatagonists of hedgehog binding.
Binding assays can be performed using whole cells. Furthermore, the
recombinant cells of the present invention can be engineered to
include other heterologous genes encoding proteins involved in
hedgehog-dependent signal pathways. For example, the gene products
of one or more of smoothened, costal-2 and/orfused can be
co-expressed with patched in the reagent cell, with assays being
sensitive to the functional reconstituion of the hedgehog signal
transduction cascade.
[0183] Alternatively, liposomal preparations using reconstituted
patched protein can be utilized. Patched protein purified from
detergent extracts from both authentic and recombinant origins can
be reconstituted in artificial lipid vesicles (e.g.,
phosphatidylcholine liposomes) or in cell membrane-derived vesicles
(see, for example; Bear et al. (1992) Cell 68:809-818; Newton et
al. (1983) Biochemistry 22:6110-6117; and Reber et al. (1987) J
Biol Chem 262:11369-11374). The lamellar structure and size of the
resulting liposomes can be characterized using electron microscopy.
External orientation of the patched protein in the reconstituted
membranes can be demonstrated, for example, by immunoelectron
microscopy. The hedgehog protein binding activity of liposomes
containing patched and liposomes without the protein in the
presence of candidate agents can be compared in order to identify
potential modulators of the hedgehog-patched interaction.
[0184] The hedgehog protein used in these cell-based assays can be
provided as a purified source (natural or recombinant in origin),
or in the form of cells/tissue which express the protein and which
are co-cultured with the target cells. As in the cell-free assays,
where simple binding (rather than induction) is the hedgehog
activity scored for in the assay, the protein can be labelled by
any of the above-mentioned techniques, e.g., fluorescently,
enzymatically or radioactively, or detected by immunoassay.
[0185] In addition to binding studies, functional assays can be
used to identified modulators, i.e., agonists of hedgehog or
patched activities. By detecting changes in intracellular signals,
such as alterations in second messengers or gene expression in
patched-expressing cells contacted with a test agent, candidate
antagonists to patched signaling can be identified (e.g., having a
hedgehog-like activity).
[0186] A number of gene products have been implicated in
patched-mediated signal transduction, including patched, the
transcription factor cubitus interruptus (ci), the serine/threonine
kinase fused (fu) and the gene products of costal-2, smoothened and
suppressor of fused.
[0187] The interaction of a hedgehog protein with patched sets in
motion a cascade involving the activation and inhibition of
downstream effectors, the ultimate consequence of which is, in some
instances, a detectable change in the transcription or translation
of a gene. Potential transcriptional targets of patched signaling
are the patched gene itself (Hidalgo and Ingham, 1990 Development
110, 291-301; Marigo et al., 1996 ) and the vertebrate homologs of
the drosophila cubitus interruptus gene, the GLI genes (Hui et al.
(1994) Dev Biol 162:402-413). Patched gene expression has been
shown to be induced in cells of the limb bud and the neural plate
that are responsive to Shh. (Marigo et al. (1996) PNAS, in press;
Marigo et al. (1996) Development 122:1225-1233). The GLI genes
encode putative transcription factors having zinc finger DNA
binding domains (Orenic et al. (1990) Genes & Dev 4:1053-1067;
Kinzler et al. (1990) Mol Cell Biol 10:634-642). Transcription of
the GLI gene has been reported to be upregulated in response to
hedgehog in limb buds, while transcription of the GLI3 gene is
downregulated in response to hedgehog induction (Marigo et al.
(1996) Development 122:1225-1233). By selecting transcriptional
regulatory sequences from such target genes, e.g., from patched or
GLI genes, that are responsible for the up- or down-regulation of
these genes in response to patched signalling, and operatively
linking such promoters to a reporter gene, one can derive a
transcription based assay which is sensitive to the ability of a
specific test compound to modify patched signalling pathways.
Expression of the reporter gene, thus, provides a valuable
screening tool for the development of compounds that act as
antagonists of ptc, e.g., which may be useful as neuroprotective
agents.
[0188] Reporter gene based assays of this invention measure the end
stage of the above described cascade of events, e.g.,
transcriptional modulation. Accordingly, in practicing one
embodiment of the assay, a reporter gene construct is inserted into
the reagent cell in order to generate a detection signal dependent
on ptc signaling. To identify potential regulatory elements
responsive to ptc signaling present in the transcriptional
regulatory sequence of a target gene, nested deletions of genomic
clones of the target gene can be constructed using standard
techniques. See, for example, Current Protocols in Molecular
Biology, Ausubel, F. M. et al. (eds.) Greene Publishing Associates,
(1989); U.S. Pat. No. 5,266,488; Sato et al. (1995) J Biol Chem
270:10314-10322; and Kube et al. (1995) Cytokine 7:1-7. A nested
set of DNA fragments from the gene's 5'-flanking region are placed
upstream of a reporter gene, such as the luciferase gene, and
assayed for their ability to direct reporter gene expression in
patched expressing cells. Host cells transiently transfected with
reporter gene constructs can be scored for the induction of
expression of the reporter gene in the presence and absence of
hedgehog to determine regulatory sequences which are responsive to
patched-dependent signalling.
[0189] In practicing one embodiment of the assay, a reporter gene
construct is inserted into the reagent cell in order to generate a
detection signal dependent on second messengers generated by
induction with hedgehog protein. Typically, the reporter gene
construct will include a reporter gene in operative linkage with
one or more transcriptional regulatory elements responsive to the
hedgehog activity, with the level of expression of the reporter
gene providing the hedgehog-dependent detection signal. The amount
of transcription from the reporter gene may be measured using any
method known to those of skill in the art to be suitable. For
example, mRNA expression from the reporter gene may be detected
using RNAse protection or RNA-based PCR, or the protein product of
the reporter gene may be identified by a characteristic stain or an
intrinsic activity. The amount of expression from the reporter gene
is then compared to the amount of expression in either the same
cell in the absence of the test compound (or hedgehog) or it may be
compared with the amount of transcription in a substantially
identical cell that lacks the target receptor protein. Any
statistically or otherwise significant difference in the amount of
transcription indicates that the test compound has in some manner
altered the signal transduction of the patched protein, e.g., the
test compound is a potential ptc therapeutic.
[0190] As described in firther detail below, in preferred
embodiments the gene product of the reporter is detected by an
intrinsic activity associated with that product. For instance, the
reporter gene may encode a gene product that, by enzymatic
activity, gives rise to a detection signal based on color,
fluorescence, or luminescence. In other preferred embodiments, the
reporter or marker gene provides a selective growth advantage,
e.g., the reporter gene may enhance cell viability, relieve a cell
nutritional requirement, and/or provide resistance to a drug.
[0191] Preferred reporter genes are those that are readily
detectable. The reporter gene may also be included in the construct
in the form of a fusion gene with a gene that includes desired
transcriptional regulatory sequences or exhibits other desirable
properties. Examples of reporter genes include, but are not limited
to CAT (chloramphenicol acetyl transferase) (Alton and Vapnek
(1979), Nature 282: 864-869) luciferase, and other enzyme detection
systems, such as beta-galactosidase; firefly luciferase (deWet et
al. (1987), Mol. Cell. Biol. 7:725-737); bacterial luciferase
(Engebrecht and Silverman (1984), PNAS 1: 4154-4158; Baldwin et al.
(1984), Biochemistry 23: 3663-3667); alkaline phosphatase (Toh et
al. (1989) Eur. J. Biochem. 182: 231-238, Hall et al. (1983) J.
Mol. Appl. Gen. 2: 101), human placental secreted alkaline
phosphatase (Cullen and Malim (1992) Methods in Enzymol.
216:362-368).
[0192] Transcriptional control elements which may be included in a
reporter gene construct include, but are not limited to, promoters,
enhancers, and repressor and activator binding sites. Suitable
transcriptional regulatory elements may be derived from the
transcriptional regulatory regions of genes whose expression is
induced after modulation of a patched signal transduction pathway.
The characteristics of preferred genes from which the
transcriptional control elements are derived include, but are not
limited to, low or undetectable expression in quiescent cells,
rapid induction at the transcriptional level within minutes of
extracellular stimulation, induction that is transient and
independent of new protein synthesis, subsequent shut-off of
transcription requires new protein synthesis, and mRNAs transcribed
from these genes have a short half-life. It is not necessary for
all of these properties to be present.
[0193] In yet other embodiments, second messenger generation can be
measured directly in the detection step, such as mobilization of
intracellular calcium, phospholipid metabolism or adenylate cyclase
activity are quantitated, for instance, the products of
phospholipid hydrolysis IP.sub.3, DAG or cAMP could be measured For
example, recent studies have implicated protein kinase A (PKA) as a
possible component of hedgehog/patched signaling (Hammerschmidt et
al. (1996) Genes & Dev 10:647). High PKA activity has been
shown to antagonize hedgehog signaling in these systems.
Conversely, inhibitors of PKA will mimic and/or potentiate the
action of hedgehog. Although it is unclear whether PKA acts
directly downstream or in parallel with hedgehog signaling, it is
possible that hedgehog signalling occurs via inhibition of PKA
activity. Thus, detection of PKA activity provides a potential
readout for the instant assays. In certain embodiments, a preferred
ptc therapeutic inhibits PKA with a K.sub.i less than 10 nM,
preferably less than 1 nM, even more preferably less than 0.1
InM.
[0194] In a preferred embodiment, the ptc therapeutic is a PKA
inhibitor. A variety of PKA inhibitors are known in the art,
including both peptidyl and organic compounds. For instance, the
ptc therapeutic can be a 5-isoquinolinesulfonamide, such as
represented in the general formula: 2
[0195] wherein,
[0196] R.sub.1 and R.sub.2 each can independently represent
hydrogen, and as valence and stability permit a lower alkyl, a
lower alkenyl, a lower alkynyl, a carbonyl (such as a carboxyl, an
ester, a formate, or a ketone), a thiocarbonyl (such as a
thioester, a thioacetate, or a thioformate), an amino, an
acylamino, an amido, a cyano, a nitro, an azido, a sulfate, a
sulfonate, a sulfonamido, --(CH.sub.2).sub.m--R.sub.8- ,
--(CH.sub.2).sub.m--OH, --(CH.sub.2).sub.m--O-lower alkyl,
--(CH.sub.2).sub.m--O-lower alkenyl,
--(CH.sub.2).sub.n--O--(CH.sub.2).su- b.m--R.sub.8,
--(CH.sub.2).sub.m--SH, --(CH.sub.2).sub.m--S-lower alkyl,
--(CH.sub.2).sub.m--S-lower alkenyl,
--(CH.sub.2).sub.n--S--(CH.sub.2).su- b.m--R.sub.8, or
[0197] R.sub.1 and R.sub.2 taken together with N form a heterocycle
(substituted or unsubstituted);
[0198] R.sub.3 is absent or represents one or more substitutions to
the isoquinoline ring such as a lower alkyl, a lower alkenyl, a
lower alkynyl, a carbonyl (such as a carboxyl, an ester, a formate,
or a ketone), a thiocarbonyl (such as a thioester, a thioacetate,
or a thioformate), an amino, an acylamino, an amido, a cyano, a
nitro, an azido, a sulfate, a sulfonate, a sulfonamido,
--(CH2).sub.m--R.sub.8, --(CH.sub.2).sub.m--OH,
--(CH.sub.2).sub.m--O-lower alkyl, --(CH.sub.2).sub.m--O-lower
alkenyl, --(CH.sub.2).sub.n--O--(CH.sub.2).su- b.m--R.sub.8,
--(CH.sub.2).sub.m--SH, --(CH.sub.2).sub.m--S-lower alkyl,
--(CH.sub.2).sub.m--S-lower alkenyl,
--(CH.sub.2).sub.n--S--(CH.sub.2).su- b.m--R.sub.8;
[0199] R.sub.8 represents a substituted or unsubstituted aryl,
aralkyl, cycloalkyl, cycloalkenyl, or heterocycle; and
[0200] n and m are independently for each occurrence zero or an
integer in the range of 1 to 6. In a preferred embodiment, the PKA
inhibitor is
N-[2-((p-bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide
(H-89; Calbiochem Cat. No. 371963), e.g., having the formula: 3
[0201] In another embodiment, the PKA inhibitor is
l-(5-isoquitiolinesulfo- nyl)-2-methylpiperazine (H-7; Calbiochem
Cat. No. 371955), e.g., having the formula: 4
[0202] In still other embodiments, the PKA inhibitor is KT5720
(Calbiochem Cat. No. 420315), having the structure 5
[0203] The hedgehog pathway can be agonized by antagonizing the
cAMP pathway, e.g., by using an agonist of of cAMP
phosphodiesterase, or by using an antagonist of adenylate cyclase,
cAMP or protein kinase A (PKA). Compounds which may reduce the
levels or activity of cAMP include prostaglandylinositol cyclic
phosphate (cyclic PIP), endothelins (ET)-1 and -3, norepinepurine,
K252a, dideoxyadenosine, dynorphins, melatonin, pertussis toxin,
staurosporine, G.sub.i agonists, MDL 12330A, SQ 22536, GDPssS and
clonidine, beta-blockers, and ligands of G-protein coupled
receptors. Additional compounds are disclosed in U.S. Pat. Nos.
5,891,875, 5,260,210, and 5,795,756.
[0204] Exemplary peptidyl inhibitors of PKA activity include the
PKA Heat Stable Inhibitor (isoform .alpha.; see, for example,
Calbiochem Cat. No. 539488, and Wen et al. (1995) J Biol Chem
270:2041).
[0205] In certain embodiments, a compound which is an agonist or
antagonist of PKA is chosen to be selective for PKA over other
protein kinases, such as PKC, e.g., the compound modulates the
activity of PKA at least an order of magnitude more strongly than
it modulates the activity of another protein kinase, preferably at
least two orders of magnitude more strongly, even more preferably
at least three orders of magnitude more strongly. Thus, for
example, a preferred inhibitor of PKA may inhibit PKA activity with
a K.sub.i at least an order of magnitude lower than its K.sub.i for
inhibition of PKC, preferably at least two orders of magnitude
lower, even more preferably at least three orders of magnitude
lower. In certain embodiments, a ptc therapeutic inhibits PKC with
a K.sub.i greater than 1 .mu.M, greater than 100 nM, preferably
greater than 10 nM.
[0206] Certain hedgehog receptors may stimulate the activity of
phospholipases. Inositol lipids can be extracted and analyzed using
standard lipid extraction techniques. Water soluble derivatives of
all three inositol lipids (IP.sub.1, IP.sub.2, IP.sub.3) can also
be quantitated using radiolabelling techniques or HPLC.
[0207] The mobilization of intracellular calcium or the influx of
calcium from outside the cell may be a response to hedgehog
stimulation or lack there of. Calcium flux in the reagent cell can
be measured using standard techniques. The choice of the
appropriate calcium indicator, fluorescent, bioluminescent,
metallochromic, or Ca.sup.++-sensitive microelectrodes depends on
the cell type and the magnitude and time constant of the event
under study (Borle (1990) Environ Health Perspect 84:45-56). As an
exemplary method of Ca.sup.++ detection, cells could be loaded with
the Ca.sup.++ sensitive fluorescent dye fura-2 or indo-1, using
standard methods, and any change in Ca.sup.++ measured using a
fluorometer.
[0208] In certain embodiments of the assay, it may be desirable to
screen for changes in cellular phosphorylation. As an example, the
drosophila gene fused (fu) which encodes a serine/threonine kinase
has been identified as a potential downstream target in hedgehog
signaling. (Preat et al., 1990 Nature 347, 87-89; Therond et al.
1993, Mech. Dev. 44. 65-80). The ability of compounds to modulate
serine/threonine kinase activation could be screened using colony
immunoblotting (Lyons and Nelson (1984) Proc. Natl. Acad Sci. USA
81:7426-7430) using antibodies against phosphorylated serine or
threonine residues. Reagents for performing such assays are
commercially available, for example, phosphoserine and
phosphothreonine specific antibodies which measure increases in
phosphorylation of those residues can be purchased from commercial
sources.
[0209] In yet another embodiment, the ptc therapeutic is an
antisense molecule which inhibits expression of a protein involved
in a patched-mediated signal transduction pathway. To illustrate,
by inhibiting the expression of a protein involved in patched
signals, such as fused, costail-2, smoothened and/or Gli genes, or
patched itself, the ability of the patched signal pathway(s) to
alter the ability of a cell to withstand ischemic conditions can be
altered, e.g., potentiated or repressed.
[0210] As used herein, "antisense" therapy refers to administration
or in situ generation of oligonucleotide probes or their
derivatives which specifically hybridize (e.g., bind) under
cellular conditions with cellular mRNA and/or genomic DNA encoding,
a hedgehog protein, patched, or a protein involved in
patched-mediated signal transduction. The hybridization should
inhibit expression of that protein, e.g., by inhibiting
transcription and/or translation. The binding may be by
conventional base pair complementarity, or, for example, in the
case of binding to DNA duplexes, through specific interactions in
the major groove of the double helix. In general, "antisense"
therapy refers to the range of techniques generally employed in the
art, and includes any therapy which relies on specific binding to
oligonucleotide sequences.
[0211] An antisense construct of the present invention can be
delivered, for example, as an expression plasmid which, when
transcribed in the cell, produces RNA which is complementary to at
least a unique portion of the target cellular mRNA. Alternatively,
the antisense construct is an oligonucleotide probe which is
generated ex vivo and which, when introduced into the cell causes
inhibition of expression by hybridizing with the mRNA and/or
genomic sequences of a target gene. Such oligonucleotide probes are
preferably modified oligonucleotide which are resistant to
endogenous nucleases, e.g., exonucleases and/or endonucleases, and
is therefore stable in vivo. Exemplary nucleic acid molecules for
use as antisense oligonucleotides are phosphbramidate,
phosphothioate and methylphosphonate analogs of DNA (see also U.S.
Pat. Nos. 5,176,996; 5,264,564; and 5,256,775). Additionally,
general approaches to constructing oligomers useful in antisense
therapy have been reviewed, for example, by Van der Krol et al.
(1988) Biotechniques 6:958-976; and Stein et al. (1988) Cancer Res
48:2659-2668.
[0212] Several considerations should be taken into account when
constructing antisense oligonucleotides for the use in the methods
of the invention: (1) oligos should have a GC content of 50% or
more; (2) avoid sequences with stretches of 3 or more G's; and (3)
oligonucleotides should not be longer than 25-26 mers. When testing
an antisense oligonucleotide, a mismatched control can be
constructed. The controls can be generated by reversing the
sequence order of the corresponding antisense oligonucleotide in
order to conserve the same ratio of bases.
[0213] In an illustrative embodiment, the ptc therapeutic can be an
antisense construct for inhibiting the expression of patched, e.g.,
to mimic the inhibition of patched by hedgehog. Exemplary antisense
constructs include:
6 5'-GTCCTGGCGCCGCCGCCGCCGTCGCC 5'-TTCCGATGACCGGCCTTTCGCGGTGA
5'-GTGCACGGAAAGGTGCAGGCCA- CACT
[0214] VI. Exemplary Pharmaceutical Preparations of Hedgehog and
ptc Therapeutics
[0215] The source of the hedgehog and ptc therapeutics to be
formulated will depend on the particular form of the agent. Small
organic molecules and peptidyl fragments can be chemically
synthesized and provided in a pure form suitable for
pharmaceutical/cosmetic usage. Products of natural extracts can be
purified according to techniques known in the art. For example, the
Cox et al. U.S. Pat. No. 5,286,654 describes a method for purifying
naturally occurring forms of a secreted protein and can be adapted
for purification of hedgehog polypeptides. Recombinant sources of
hedgehog polypeptides are also available. For example, the gene
encoding hedgehog polypeptides, are known, inter alia, from PCT
publications WO 95/18856 and WO 96/17924.
[0216] Those of skill in treating neural tissues can determine the
effective amount of an hedgehog or ptc therapeutic to be formulated
in a pharmaceutical or cosmetic preparation.
[0217] The hedgehog or ptc therapeutic formulations used in the
method of the invention are most preferably applied in the form of
appropriate compositions. As appropriate compositions there may be
cited all compositions usually employed for systemically or locally
(such as intrathecal) administering drugs. The pharmaceutically
acceptable carrier should be substantially inert, so as not to act
with the active component. Suitable inert carriers include water,
alcohol polyethylene glycol, mineral oil or petroleum gel,
propylene glycol and the like.
[0218] To prepare the pharmaceutical compositions of this
invention, an effective amount of the particular hedgehog or ptc
therapeutic as the active ingredient is combined in intimate
admixture with a pharmaceutically acceptable carrier, which carrier
may take a wide variety of forms depending on the form of
preparation desired for administration. These pharmaceutical
compositions are desirable in unitary dosage form suitable,
particularly, for administration orally, rectally, percutaneously,
or by parenteral injection. For example, in preparing the
compositions in oral dosage form, any of the usual pharmaceutical
media may be employed such as, for example, water, glycols, oils,
alcohols and the like in the case of oral liquid preparations such
as suspensions, syrups, elixirs and solutions; or solid carriers
such as starches, sugars, kaolin, lubricants, binders,
disintegrating agents and the like in the case of powders, pills,
capsules, and tablets. Because of their ease in administration,
tablets and capsules represents the most advantageous oral dosage
unit form, in which case solid pharmaceutical carriers are
obviously employed. For parenteral compositions, the carrier will
usually comprise sterile water, at least in large part, though
other ingredients, for example, to aid solubility, may be included.
Injectable solutions, for example, may be prepared in which the
carrier comprises saline solution, glucose solution or a mixture of
saline and glucose solution. Injectable suspensions may also be
prepared in which case appropriate liquid carriers, suspending
agents and the like may be employed. Also included are solid form
preparations which are intended to be converted, shortly before
use, to liquid form preparations. In the compositions suitable for
percutaneous administration, the carrier optionally comprises a
penetration enhancing agent and/or a suitable wetting agent,
optionally combined with suitable additives of any nature in minor
proportions, which additives do not introduce a significant
deleterious effect on the skin.
[0219] It is especially advantageous to formulate the subject
compositions in dosage unit form for ease of administration and
uniformity of dosage. Dosage unit form as used in the specification
and claims herein refers to physically discrete units suitable as
unitary dosages, each unit containing a predetermined quantity of
active ingredient calculated to produce the desired therapeutic
effect in association with the required pharmaceutical carrier.
Examples of such dosage unit forms are tablets (including scored or
coated tablets), capsules, pills, powders packets, wafers,
injectable solutions or suspensions, teaspoonfuls, tablespoonfuls
and the like, and segregated multiples thereof.
[0220] The pharmaceutical preparations of the present invention can
be used, as stated above, for the many applications which can be
considered cosmetic uses. Cosmetic compositions known in the art,
preferably hypoallergic and pH controlled are especially preferred,
and include toilet waters, packs, lotions, skin milks or milky
lotions. The preparations contain, besides the hedgehog or ptc
therapeutic, components usually employed in such preparations.
Examples of such components are oils, fats, waxes, surfactants,
humectants, thickening agents, antioxidants, viscosity stabilizers,
chelating agents, buffers, preservatives, perfumes, dyestuffs,
lower alkanols, and the like. If desired, further ingredients may
be incorporated in the compositions, e.g., antiinflammatory agents,
antibacterials, antifungals, disinfectants, vitamins, sunscreens,
antibiotics, or other anti-acne agents.
[0221] Examples of oils comprise fats and oils such as olive oil
and hydrogenated oils; waxes such as beeswax and lanolin;
hydrocarbons such as liquid paraffin, ceresin, and squalane; fatty
acids such as stearic acid and oleic acid; alcohols such as cetyl
alcohol, stearyl alcohol, lanolin alcohol, and hexadecanol; and
esters such as isopropyl myristate, isopropyl palmitate and butyl
stearate. As examples of surfactants there may be cited anionic
surfactants such as sodium stearate, sodium cetylsulfate,
polyoxyethylene laurylether phosphate, sodium N-acyl glutamate;
cationic surfactants such as stearyldimethylbenzylammonium chloride
and stearyltrimethylammonium chloride; ampholytic surfactants such
as alkylaminoethylglycine hydrocloride solutions and lecithin; and
nonionic surfactants such as glycerin monostearate, sorbitan
monostearate, sucrose fatty acid esters, propylene glycol
monostearate, polyoxyethylene oleylether, polyethylene glycol
monostearate, polyoxyethylene sorbitan monopalmitate,
polyoxyethylene coconut fatty acid monoethanolamide,
polyoxypropylene glycol (e.g., the materials sold under the
trademark "Pluronic"), polyoxyethylene castor oil, and
polyoxyethylene lanolin. Examples of humectants include glycerin,
1,3-butylene glycol, and propylene glycol; examples of lower
alcohols include ethanol and isopropanol; examples of thickening
agents include xanthan gum, hydroxypropyl cellulose, hydroxypropyl
methyl cellulose, polyethylene glycol and sodium carboxymethyl
cellulose; examples of antioxidants comprise butylated
hydroxytoluene, butylated hydroxyanisole, propyl gallate, citric
acid and ethoxyquin; examples of chelating agents include disodium
edetate and ethanehydroxy diphosphate; examples- of buffers
comprise citric acid, sodium citrate, boric acid, borax, and
disodium hydrogen phosphate; and examples of preservatives are
methyl parahydroxybenzoate, ethyl parahydroxybenzoate,
dehydroacetic acid, salicylic acid and benzoic acid.
[0222] For preparing ointments, creams, toilet waters, skin milks,
and the like, typically from 0.01 to 10% in particular from 0.1 to
5% and more in particular from 0.2 to 2.5% of the active
ingredient, e.g., of the hedgehog or ptc therapeutic, will be
incorporated in the compositions. In ointments or creams, the
carrier for example consists of 1 to 20%, in particular 5 to 15% of
a humectant, 0.1 to 10% in particular from 0.5 to 5% of a thickener
and water; or said carrier may consist of 70 to 99%, in particular
20 to 95% of a surfactant, and 0 to 20%, in particular 2.5 to 15%
of a fat; or 80 to 99.9% in particular 90 to 99% of a thickener; or
5 to 15% of a surfactant, 2-15% of a humectant, 0 to 80% of an oil,
very small ( <2%) amounts of preservative, coloring agent and/or
perfume, and water. In a toilet water, the carrier for example
consists of 2 to 10% of a lower alcohol, 0.1 to 10% or in
particular 0.5 to 1% of a surfactant, 1 to 20%, in particular 3 to
7% of a humectant, 0 to 5% of a buffer, water and small amounts (
<2%) of preservative, dyestuff and/or perfume. In a skin milk,
the carrier typically consists of 10-50% of oil, 1 to 10% of
surfactant, 50-80% of water and 0 to 3% of preservative and/or
perfume. In the aforementioned preparations, all % symbols refer to
weight by weight percentage.
[0223] Particular compositions for use in the method of the present
invention are those wherein the hedgehog or ptc therapeutic is
formulated in liposome-containing compositions. Liposomes are
artificial vesicles formed by amphiphatic molecules such as polar
lipids, for example, phosphatidyl cholines, ethanolamines and
serines, sphingomyelins, cardiolipins, plasmalogens, phosphatidic
acids and cerebiosides. Liposomes are formed when suitable
amphiphathic molecules are allowed to swell in water or aqueous
solutions to form liquid crystals usually of multilayer structure
comprised of many bilayers separated from each other by aqueous
material (also referred to as coarse liposomes). Another type of
liposome known to be consisting of a single bilayer encapsulating
aqueous material is referred to as a unilamellar vesicle. If
water-soluble materials are included in the aqueous phase during
the swelling of the lipids they become entrapped in the aqueous
layer between the lipid bilayers.
[0224] Water-soluble active ingredients such as, for example,
various salt forms of a hedgehog polypeptide, are encapsulated in
the aqueous spaces between the molecular layers. The lipid soluble
active ingredient of hedgehog or ptc therapeutic, such as an
organic mimetic, is predominantly incorporated into the lipid
layers, although polar head groups may protude from the layer into
the aqueous space. The encapsulation of:these compounds can be
achieved by a number of methods. The method most commonly used
involves casting a thin film of phospholipid onto the walls of a
flask by evaporation from an organic solvent. When this film is
dispersed in a suitable aqueous medium, multilamellar liposomes are
formed. Upon suitable sonication, the coarse liposomes form smaller
similarly closed vesicles.
[0225] Water-soluble active ingredients are usually incorporated by
dispersing the cast film with an aqueous solution of the compound.
The unencapsulated compound is then removed by centrifugation,
chromatography, dialysis or other art-known suitable procedures.
The lipid-soluble active ingredient is usually incorporated by
dissolving it in the organic solvent with the phospholipid prior to
casting the film. If the solubility of the material in the lipid
phase is not exceeded or the amount present is not in excess of
that which can be bound to the lipid, liposomes prepared by the
above method usually contain most of the material bound in the
lipid bilayers; separation of the liposomes from unencapsulated
material is not required.
[0226] A particularly convenient method for preparing liposome
formulated forms of hedgehog and ptc therapeutics is the method
described in EP-A-253,619, incorporated herein by reference. In
this method, single bilayered liposomes containing encapsulated
active ingredients are prepared by dissolving the lipid component
in an organic medium, injecting the organic solution of the lipid
component under pressure into an aqueous component while
simultaneously mixing the organic and aqueous components with a
high speed homogenizer or mixing means, whereupon the liposomes are
formed spontaneously.
[0227] The single bilayered liposomes containing the encapsulated
hedgehog or ptc therapeutic can be employed directly or they can be
employed in a suitable pharmaceutically acceptable carrier for
localized administration. The viscosity of the liposomes can be
increased by the addition of one or more suitable thickening agents
such as, for example xanthan gum, hydroxypropyl cellulose,
hydroxypropyl methylcellulose and mixtures thereof. The aqueous
component may consist of water alone or it may contain
electrolytes, buffered systems and other ingredients, such as, for
example, preservatives. Suitable electrolytes which can be employed
include metal salts such as alkali metal and alkaline earth metal
salts. The preferred metal salts are calcium chloride, sodium
chloride and potassium chloride. The concentration of the
electrolyte may vary from zero to 260 mM, preferably from 5 mM to
160 mM. The aqueous component is placed in a suitable vessel which
can be adapted to effect homogenization by effecting great
turbulence during the injection of the organic component.
Homogenization of the two components can be accomplished within the
vessel, or, alternatively, the aqueous and organic components may
be injected separately into a mixing means which is located outside
the vessel. In the latter case, the liposomes are formed in the
mixing means and then transferred to another vessel for collection
purpose.
[0228] The organic component consists of a suitable non-toxic,
pharmaceutically acceptable solvent such as, for example ethanol,
glycerol, propylene glycol and polyethylene glycol, and a suitable
phospholipid which is soluble in the solvent. Suitable
phospholipids which can be employed include lecithin,
phosphatidylcholine, phosphatydylserine, phosphatidylethanol-amine,
phosphatidylinositol, lysophosphatidylcholine and phospha-tidyl
glycerol, for example. Other lipophilic additives may be employed
in order to selectively modify the characteristics of the
liposomes. Examples of such other additives include stearylamine,
phosphatidic acid, tocopherol, cholesterol and lanolin
extracts.
[0229] In addition, other ingredients which can prevent oxidation
of the phospholipids may be added to the organic component.
Examples of such other ingredients include tocopherol, butylated
hydroxyanisole, butylated hydroxytoluene, ascorbyl palmitate and
ascorbyl oleate. Preservatives such a benzoic acid, methyl paraben
and propyl paraben may also be added.
[0230] Methods of introduction may also be provided by rechargeable
or biodegradable devices. Various slow release polymeric devices
have been developed and tested in vivo in recent years for the
controlled delivery of drugs, including proteinacious
biopharmaceuticals. A variety of biocompatible polymers (including
hydrogels), including both biodegradable and non-degradable
polymers, can be used to form an implant for the sustained release
of an hh at a particular target site. Such embodiments of the
present invention can be used for the delivery of an exogenously
purified hedgehog protein, which has been incorporated in the
polymeric device, or for the delivery of hedgehog produced by a
cell encapsulated in the polymeric device.
[0231] An essential feature of certain embodiments of the implant
can be the linear release of the therapeutic, which can be achieved
through the manipulation of the polymer composition and form. By
choice of monomer composition or polymerization technique, the
amount of water, porosity and consequent permeability
characteristics can be controlled. The selection of the shape,
size, polymer, and method for implantation can be determined on an
individual basis according to the disorder to be treated and the
individual patient response. The generation of such implants is
generally known in the art. See, for example, Concise Encylopedia
of Medical & Dental Materials, ed. by David Williams (MIT
Press: Cambridge, Mass., 1990); and the Sabel et al. U.S. Pat. No.
4,883,666.
[0232] In another embodiment of an implant, a source of cells
producing the therapeutic, e.g., secreting a soluble form of a
hedgehog protein, is encapsulated in implantable hollow fibers or
the like. Such fibers can be pre-spun and subsequently loaded with
the cell source (Aebischer et al. U.S. Pat. No. 4,892,538;
Aebischer et al. U.S. Pat. No. 5,106,627; Hoffman et al. (1990)
Expt. Neurobiol. 110:39-44; Jaeger et al. (1990) Prog. Brain Res.
82:41-46; and Aebischer et al. (1991) J. Biomech. Eng.
113:178-183), or can be co-extruded with a polymer which acts to
form a polymeric coat about the cells (Lim U.S. Pat. No. 4,391,909;
Sefton U.S. Pat. No. 4,353,888; Sugamori et al. (1989) Trans. Am.
Artif. Intern. Organs 35:791-799; Sefton et al. (1987) Biotehnol.
Bioeng. 29:1135-1143; and Aebischeretal. (1991) Biomaterials
12:50-55).
[0233] Exemplification
[0234] The invention now being generally described, it will be more
readily understood by reference to the following examples which are
included merely for purposes of illustration of certain aspects and
embodiments of the present invention, and are not intended to limit
the invention.
[0235] Sonic Hedgehog (Shh) was evaluated in the focal stroke model
involving permanent middle cerebral artery occlusion (MCAO) in the
spontaneously hypertensive rat. Samples of the proteins were tested
as a neuroprotective agent by measuring the volume of cerebral
infarction, by means of vital dye exclusion, in animals receiving
systemic injections. For review of the MCAO, see Tamura et al.
(1981) J Cerebral Blood Flow and Metabolism 1:53-60.
[0236] Briefly, male Wistar rats, weighing about 270-300g were
treated systemically with Shh at 500 .mu.g/kg/hr for 3 hrs at 0.5
ml/hr. Control animals received buffer at same dilution as Shh
stock for the same period of time and volumes.
[0237] Prior to administration of the Shh or control stocks, the
MCAO animals were generated as follows: the rats were anesthesized,
with 400 mg/ml chloral hydrate, and their femoral vein and artery
were cannulated. Mean arterial blood pressure was monitered and
blood samples taken for blood gas measurements. A half-hour later,
the middle cereberal artery was occluded with a nylon monofilament
suture inserted via carotid artery. Half-hour after onset of
occlusion, having allowed animal to awake, infusion of Shh or
buffer/vehicle was started. The catheters were removed, and the
animals were returned to their cages. At 24 hours post-surgery, the
animals sacrificed by decapitation. Their brains were removed and
cut into 2 mm serial, coronal sections. The sections stained with
TTC stain and then fixed in neutral buffered formalin. Infarct
volumes measured by quantitative morphometry and expressed as a
percentage of the total hemispheric volume (normalized against the
contralateral hemisphere to correct for edema-associated
swelling).
[0238] FIG. 1 illustrates the results of the above-referenced
experiments. A substantial decrease in the volume of the cerebral
infarct was observed in the hedgehog treated rats relative to the
control rats. While not shown in FIG. 1, its was further observed
that there was no statistically significant effect of hedgehog on
blood pressure, pH, pO.sub.2, or pCO.sub.2.
[0239] All of the above-cited references and publications are
hereby incorporated by reference.
[0240] Equivalents
[0241] Those skilled-in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein. Such equivalents are intended to be encompassed by the
following claims.
Sequence CWU 1
1
26 1 1277 DNA chicken Shh CDS (1)..(1275) 1 atg gtc gaa atg ctg ctg
ttg aca aga att ctc ttg gtg ggc ttc atc 48 Met Val Glu Met Leu Leu
Leu Thr Arg Ile Leu Leu Val Gly Phe Ile 1 5 10 15 tgc gct ctt tta
gtc tcc tct ggg ctg act tgt gga cca ggc agg ggc 96 Cys Ala Leu Leu
Val Ser Ser Gly Leu Thr Cys Gly Pro Gly Arg Gly 20 25 30 att gga
aaa agg agg cac ccc aaa aag ctg acc ccg tta gcc tat aag 144 Ile Gly
Lys Arg Arg His Pro Lys Lys Leu Thr Pro Leu Ala Tyr Lys 35 40 45
cag ttt att ccc aat gtg gca gag aag acc cta ggg gcc agt gga aga 192
Gln Phe Ile Pro Asn Val Ala Glu Lys Thr Leu Gly Ala Ser Gly Arg 50
55 60 tat gaa ggg aag atc aca aga aac tcc gag aga ttt aaa gaa cta
acc 240 Tyr Glu Gly Lys Ile Thr Arg Asn Ser Glu Arg Phe Lys Glu Leu
Thr 65 70 75 80 cca aat tac aac cct gac att att ttt aag gat gaa gag
aac acg gga 288 Pro Asn Tyr Asn Pro Asp Ile Ile Phe Lys Asp Glu Glu
Asn Thr Gly 85 90 95 gct gac aga ctg atg act cag cgc tgc aag gac
aag ctg aat gcc ctg 336 Ala Asp Arg Leu Met Thr Gln Arg Cys Lys Asp
Lys Leu Asn Ala Leu 100 105 110 gcg atc tcg gtg atg aac cag tgg ccc
ggg gtg aag ctg cgg gtg acc 384 Ala Ile Ser Val Met Asn Gln Trp Pro
Gly Val Lys Leu Arg Val Thr 115 120 125 gag ggc tgg gac gag gat ggc
cat cac tcc gag gaa tcg ctg cac tac 432 Glu Gly Trp Asp Glu Asp Gly
His His Ser Glu Glu Ser Leu His Tyr 130 135 140 gag ggt cgc gcc gtg
gac atc acc acg tcg gat cgg gac cgc agc aag 480 Glu Gly Arg Ala Val
Asp Ile Thr Thr Ser Asp Arg Asp Arg Ser Lys 145 150 155 160 tac gga
atg ctg gcc cgc ctc gcc gtc gag gcc ggc ttc gac tgg gtc 528 Tyr Gly
Met Leu Ala Arg Leu Ala Val Glu Ala Gly Phe Asp Trp Val 165 170 175
tac tac gag tcc aag gcg cac atc cac tgc tcc gtc aaa gca gaa aac 576
Tyr Tyr Glu Ser Lys Ala His Ile His Cys Ser Val Lys Ala Glu Asn 180
185 190 tca gtg gca gcg aaa tca gga ggc tgc ttc cct ggc tca gcc aca
gtg 624 Ser Val Ala Ala Lys Ser Gly Gly Cys Phe Pro Gly Ser Ala Thr
Val 195 200 205 cac ctg gag cat gga ggc acc aag ctg gtg aag gac ctg
agc cct ggg 672 His Leu Glu His Gly Gly Thr Lys Leu Val Lys Asp Leu
Ser Pro Gly 210 215 220 gac cgc gtg ctg gct gct gac gcg gac ggc cgg
ctg ctc tac agt gac 720 Asp Arg Val Leu Ala Ala Asp Ala Asp Gly Arg
Leu Leu Tyr Ser Asp 225 230 235 240 ttc ctc acc ttc ctc gac cgg atg
gac agc tcc cga aag ctc ttc tac 768 Phe Leu Thr Phe Leu Asp Arg Met
Asp Ser Ser Arg Lys Leu Phe Tyr 245 250 255 gtc atc gag acg cgg cag
ccc cgg gcc cgg ctg cta ctg acg gcg gcc 816 Val Ile Glu Thr Arg Gln
Pro Arg Ala Arg Leu Leu Leu Thr Ala Ala 260 265 270 cac ctg ctc ttt
gtg gcc ccc cag cac aac cag tcg gag gcc aca ggg 864 His Leu Leu Phe
Val Ala Pro Gln His Asn Gln Ser Glu Ala Thr Gly 275 280 285 tcc acc
agt ggc cag gcg ctc ttc gcc agc aac gtg aag cct ggc caa 912 Ser Thr
Ser Gly Gln Ala Leu Phe Ala Ser Asn Val Lys Pro Gly Gln 290 295 300
cgt gtc tat gtg ctg ggc gag ggc ggg cag cag ctg ctg ccg gcg tct 960
Arg Val Tyr Val Leu Gly Glu Gly Gly Gln Gln Leu Leu Pro Ala Ser 305
310 315 320 gtc cac agc gtc tca ttg cgg gag gag gcg tcc gga gcc tac
gcc cca 1008 Val His Ser Val Ser Leu Arg Glu Glu Ala Ser Gly Ala
Tyr Ala Pro 325 330 335 ctc acc gcc cag ggc acc atc ctc atc aac cgg
gtg ttg gcc tcc tgc 1056 Leu Thr Ala Gln Gly Thr Ile Leu Ile Asn
Arg Val Leu Ala Ser Cys 340 345 350 tac gcc gtc atc gag gag cac agt
tgg gcc cat tgg gcc ttc gca cca 1104 Tyr Ala Val Ile Glu Glu His
Ser Trp Ala His Trp Ala Phe Ala Pro 355 360 365 ttc cgc ttg gct cag
ggg ctg ctg gcc gcc ctc tgc cca gat ggg gcc 1152 Phe Arg Leu Ala
Gln Gly Leu Leu Ala Ala Leu Cys Pro Asp Gly Ala 370 375 380 atc cct
act gcc gcc acc acc acc act ggc atc cat tgg tac tca cgg 1200 Ile
Pro Thr Ala Ala Thr Thr Thr Thr Gly Ile His Trp Tyr Ser Arg 385 390
395 400 ctc ctc tac cgc atc ggc agc tgg gtg ctg gat ggt gac gcg ctg
cat 1248 Leu Leu Tyr Arg Ile Gly Ser Trp Val Leu Asp Gly Asp Ala
Leu His 405 410 415 ccg ctg ggc atg gtg gca ccg gcc agc tg 1277 Pro
Leu Gly Met Val Ala Pro Ala Ser 420 425 2 1191 DNA murine Dhh CDS
(1)..(1188) 2 atg gct ctg ccg gcc agt ctg ttg ccc ctg tgc tgc ttg
gca ctc ttg 48 Met Ala Leu Pro Ala Ser Leu Leu Pro Leu Cys Cys Leu
Ala Leu Leu 1 5 10 15 gca cta tct gcc cag agc tgc ggg ccg ggc cga
gga ccg gtt ggc cgg 96 Ala Leu Ser Ala Gln Ser Cys Gly Pro Gly Arg
Gly Pro Val Gly Arg 20 25 30 cgg cgt tat gtg cgc aag caa ctt gtg
cct ctg cta tac aag cag ttt 144 Arg Arg Tyr Val Arg Lys Gln Leu Val
Pro Leu Leu Tyr Lys Gln Phe 35 40 45 gtg ccc agt atg ccc gag cgg
acc ctg ggc gcg agt ggg cca gcg gag 192 Val Pro Ser Met Pro Glu Arg
Thr Leu Gly Ala Ser Gly Pro Ala Glu 50 55 60 ggg agg gta aca agg
ggg tcg gag cgc ttc cgg gac ctc gta ccc aac 240 Gly Arg Val Thr Arg
Gly Ser Glu Arg Phe Arg Asp Leu Val Pro Asn 65 70 75 80 tac aac ccc
gac ata atc ttc aag gat gag gag aac agc ggc gca gac 288 Tyr Asn Pro
Asp Ile Ile Phe Lys Asp Glu Glu Asn Ser Gly Ala Asp 85 90 95 cgc
ctg atg aca gag cgt tgc aaa gag cgg gtg aac gct cta gcc atc 336 Arg
Leu Met Thr Glu Arg Cys Lys Glu Arg Val Asn Ala Leu Ala Ile 100 105
110 gcg gtg atg aac atg tgg ccc gga gta cgc cta cgt gtg act gaa ggc
384 Ala Val Met Asn Met Trp Pro Gly Val Arg Leu Arg Val Thr Glu Gly
115 120 125 tgg gac gag gac ggc cac cac gca cag gat tca ctc cac tac
gaa ggc 432 Trp Asp Glu Asp Gly His His Ala Gln Asp Ser Leu His Tyr
Glu Gly 130 135 140 cgt gcc ttg gac atc acc acg tct gac cgt gac cgt
aat aag tat ggt 480 Arg Ala Leu Asp Ile Thr Thr Ser Asp Arg Asp Arg
Asn Lys Tyr Gly 145 150 155 160 ttg ttg gcg cgc cta gct gtg gaa gcc
gga ttc gac tgg gtc tac tac 528 Leu Leu Ala Arg Leu Ala Val Glu Ala
Gly Phe Asp Trp Val Tyr Tyr 165 170 175 gag tcc cgc aac cac atc cac
gta tcg gtc aaa gct gat aac tca ctg 576 Glu Ser Arg Asn His Ile His
Val Ser Val Lys Ala Asp Asn Ser Leu 180 185 190 gcg gtc cga gcc gga
ggc tgc ttt ccg gga aat gcc acg gtg cgc ttg 624 Ala Val Arg Ala Gly
Gly Cys Phe Pro Gly Asn Ala Thr Val Arg Leu 195 200 205 cgg agc ggc
gaa cgg aag ggg ctg agg gaa cta cat cgt ggt gac tgg 672 Arg Ser Gly
Glu Arg Lys Gly Leu Arg Glu Leu His Arg Gly Asp Trp 210 215 220 gta
ctg gcc gct gat gca gcg ggc cga gtg gta ccc acg cca gtg ctg 720 Val
Leu Ala Ala Asp Ala Ala Gly Arg Val Val Pro Thr Pro Val Leu 225 230
235 240 ctc ttc ctg gac cgg gat ctg cag cgc cgc gcc tcg ttc gtg gct
gtg 768 Leu Phe Leu Asp Arg Asp Leu Gln Arg Arg Ala Ser Phe Val Ala
Val 245 250 255 gag acc gag cgg cct ccg cgc aaa ctg ttg ctc aca ccc
tgg cat ctg 816 Glu Thr Glu Arg Pro Pro Arg Lys Leu Leu Leu Thr Pro
Trp His Leu 260 265 270 gtg ttc gct gct cgc ggg cca gcg cct gct cca
ggt gac ttt gca ccg 864 Val Phe Ala Ala Arg Gly Pro Ala Pro Ala Pro
Gly Asp Phe Ala Pro 275 280 285 gtg ttc gcg cgc cgc tta cgt gct ggc
gac tcg gtg ctg gct ccc ggc 912 Val Phe Ala Arg Arg Leu Arg Ala Gly
Asp Ser Val Leu Ala Pro Gly 290 295 300 ggg gac gcg ctc cag ccg gcg
cgc gta gcc cgc gtg gcg cgc gag gaa 960 Gly Asp Ala Leu Gln Pro Ala
Arg Val Ala Arg Val Ala Arg Glu Glu 305 310 315 320 gcc gtg ggc gtg
ttc gca ccg ctc act gcg cac ggg acg ctg ctg gtc 1008 Ala Val Gly
Val Phe Ala Pro Leu Thr Ala His Gly Thr Leu Leu Val 325 330 335 aac
gac gtc ctc gcc tcc tgc tac gcg gtt cta gag agt cac cag tgg 1056
Asn Asp Val Leu Ala Ser Cys Tyr Ala Val Leu Glu Ser His Gln Trp 340
345 350 gcc cac cgc gcc ttc gcc cct ttg cgg ctg ctg cac gcg ctc ggg
gct 1104 Ala His Arg Ala Phe Ala Pro Leu Arg Leu Leu His Ala Leu
Gly Ala 355 360 365 ctg ctc cct ggg ggt gca gtc cag ccg act ggc atg
cat tgg tac tct 1152 Leu Leu Pro Gly Gly Ala Val Gln Pro Thr Gly
Met His Trp Tyr Ser 370 375 380 cgc ctc ctt tac cgc ttg gcc gag gag
tta atg ggc tga 1191 Arg Leu Leu Tyr Arg Leu Ala Glu Glu Leu Met
Gly 385 390 395 3 1281 DNA murine Ihh CDS (1)..(1233) 3 atg tct ccc
gcc tgg ctc cgg ccc cga ctg cgg ttc tgt ctg ttc ctg 48 Met Ser Pro
Ala Trp Leu Arg Pro Arg Leu Arg Phe Cys Leu Phe Leu 1 5 10 15 ctg
ctg ctg ctt ctg gtg ccg gcg gcg cgg ggc tgc ggg ccg ggc cgg 96 Leu
Leu Leu Leu Leu Val Pro Ala Ala Arg Gly Cys Gly Pro Gly Arg 20 25
30 gtg gtg ggc agc cgc cgg agg ccg cct cgc aag ctc gtg cct ctt gcc
144 Val Val Gly Ser Arg Arg Arg Pro Pro Arg Lys Leu Val Pro Leu Ala
35 40 45 tac aag cag ttc agc ccc aac gtg ccg gag aag acc ctg ggc
gcc agc 192 Tyr Lys Gln Phe Ser Pro Asn Val Pro Glu Lys Thr Leu Gly
Ala Ser 50 55 60 ggg cgc tac gaa ggc aag atc gcg cgc agc tct gag
cgc ttc aaa gag 240 Gly Arg Tyr Glu Gly Lys Ile Ala Arg Ser Ser Glu
Arg Phe Lys Glu 65 70 75 80 ctc acc ccc aac tac aat ccc gac atc atc
ttc aag gac gag gag aac 288 Leu Thr Pro Asn Tyr Asn Pro Asp Ile Ile
Phe Lys Asp Glu Glu Asn 85 90 95 acg ggt gcc gac cgc ctc atg acc
cag cgc tgc aag gac cgt ctg aac 336 Thr Gly Ala Asp Arg Leu Met Thr
Gln Arg Cys Lys Asp Arg Leu Asn 100 105 110 tca ctg gcc atc tct gtc
atg aac cag tgg cct ggt gtg aaa ctg cgg 384 Ser Leu Ala Ile Ser Val
Met Asn Gln Trp Pro Gly Val Lys Leu Arg 115 120 125 gtg acc gaa ggc
cgg gat gaa gat ggc cat cac tca gag gag tct tta 432 Val Thr Glu Gly
Arg Asp Glu Asp Gly His His Ser Glu Glu Ser Leu 130 135 140 cac tat
gag ggc cgc gcg gtg gat atc acc acc tca gac cgt gac cga 480 His Tyr
Glu Gly Arg Ala Val Asp Ile Thr Thr Ser Asp Arg Asp Arg 145 150 155
160 aat aag tat gga ctg ctg gcg cgc tta gca gtg gag gcc ggc ttc gac
528 Asn Lys Tyr Gly Leu Leu Ala Arg Leu Ala Val Glu Ala Gly Phe Asp
165 170 175 tgg gtg tat tac gag tcc aag gcc cac gtg cat tgc tct gtc
aag tct 576 Trp Val Tyr Tyr Glu Ser Lys Ala His Val His Cys Ser Val
Lys Ser 180 185 190 gag cat tcg gcc gct gcc aag aca ggt ggc tgc ttt
cct gcc gga gcc 624 Glu His Ser Ala Ala Ala Lys Thr Gly Gly Cys Phe
Pro Ala Gly Ala 195 200 205 cag gtg cgc cta gag aac ggg gag cgt gtg
gcc ctg tca gct gta aag 672 Gln Val Arg Leu Glu Asn Gly Glu Arg Val
Ala Leu Ser Ala Val Lys 210 215 220 cca gga gac cgg gtg ctg gcc atg
ggg gag gat ggg acc ccc acc ttc 720 Pro Gly Asp Arg Val Leu Ala Met
Gly Glu Asp Gly Thr Pro Thr Phe 225 230 235 240 agt gat gtg ctt att
ttc ctg gac cgc gag cca aac cgg ctg aga gct 768 Ser Asp Val Leu Ile
Phe Leu Asp Arg Glu Pro Asn Arg Leu Arg Ala 245 250 255 ttc cag gtc
atc gag act cag gat cct ccg cgt cgg ctg gcg ctc acg 816 Phe Gln Val
Ile Glu Thr Gln Asp Pro Pro Arg Arg Leu Ala Leu Thr 260 265 270 cct
gcc cac ctg ctc ttc att gcg gac aat cat aca gaa cca gca gcc 864 Pro
Ala His Leu Leu Phe Ile Ala Asp Asn His Thr Glu Pro Ala Ala 275 280
285 cac ttc cgg gcc aca ttt gcc agc cat gtg caa cca ggc caa tat gtg
912 His Phe Arg Ala Thr Phe Ala Ser His Val Gln Pro Gly Gln Tyr Val
290 295 300 ctg gta tca ggg gta cca ggc ctc cag cct gct cgg gtg gca
gct gtc 960 Leu Val Ser Gly Val Pro Gly Leu Gln Pro Ala Arg Val Ala
Ala Val 305 310 315 320 tcc acc cac gtg gcc ctt ggg tcc tat gct cct
ctc aca agg cat ggg 1008 Ser Thr His Val Ala Leu Gly Ser Tyr Ala
Pro Leu Thr Arg His Gly 325 330 335 aca ctt gtg gtg gag gat gtg gtg
gcc tcc tgc ttt gca gct gtg gct 1056 Thr Leu Val Val Glu Asp Val
Val Ala Ser Cys Phe Ala Ala Val Ala 340 345 350 gac cac cat ctg gct
cag ttg gcc ttc tgg ccc ctg cga ctg ttt ccc 1104 Asp His His Leu
Ala Gln Leu Ala Phe Trp Pro Leu Arg Leu Phe Pro 355 360 365 agt ttg
gca tgg ggc agc tgg acc cca agt gag ggt gtt cac tcc tac 1152 Ser
Leu Ala Trp Gly Ser Trp Thr Pro Ser Glu Gly Val His Ser Tyr 370 375
380 cct cag atg ctc tac cgc ctg ggg cgt ctc ttg cta gaa gag agc acc
1200 Pro Gln Met Leu Tyr Arg Leu Gly Arg Leu Leu Leu Glu Glu Ser
Thr 385 390 395 400 ttc cat cca ctg ggc atg tct ggg gca gga agc
tgaagggact ctaaccactg 1253 Phe His Pro Leu Gly Met Ser Gly Ala Gly
Ser 405 410 ccctcctgga actgctgtgc gtggatcc 1281 4 1313 DNA murine
Shh CDS (1)..(1311) 4 atg ctg ctg ctg ctg gcc aga tgt ttt ctg gtg
atc ctt gct tcc tcg 48 Met Leu Leu Leu Leu Ala Arg Cys Phe Leu Val
Ile Leu Ala Ser Ser 1 5 10 15 ctg ctg gtg tgc ccc ggg ctg gcc tgt
ggg ccc ggc agg ggg ttt gga 96 Leu Leu Val Cys Pro Gly Leu Ala Cys
Gly Pro Gly Arg Gly Phe Gly 20 25 30 aag agg cgg cac ccc aaa aag
ctg acc cct tta gcc tac aag cag ttt 144 Lys Arg Arg His Pro Lys Lys
Leu Thr Pro Leu Ala Tyr Lys Gln Phe 35 40 45 att ccc aac gta gcc
gag aag acc cta ggg gcc agc ggc aga tat gaa 192 Ile Pro Asn Val Ala
Glu Lys Thr Leu Gly Ala Ser Gly Arg Tyr Glu 50 55 60 ggg aag atc
aca aga aac tcc gaa cga ttt aag gaa ctc acc ccc aat 240 Gly Lys Ile
Thr Arg Asn Ser Glu Arg Phe Lys Glu Leu Thr Pro Asn 65 70 75 80 tac
aac ccc gac atc ata ttt aag gat gag gaa aac acg gga gca gac 288 Tyr
Asn Pro Asp Ile Ile Phe Lys Asp Glu Glu Asn Thr Gly Ala Asp 85 90
95 cgg ctg atg act cag agg tgc aaa gac aag tta aat gcc ttg gcc atc
336 Arg Leu Met Thr Gln Arg Cys Lys Asp Lys Leu Asn Ala Leu Ala Ile
100 105 110 tct gtg atg aac cag tgg cct gga gtg agg ctg cga gtg acc
gag ggc 384 Ser Val Met Asn Gln Trp Pro Gly Val Arg Leu Arg Val Thr
Glu Gly 115 120 125 tgg gat gag gac ggc cat cat tca gag gag tct cta
cac tat gag ggt 432 Trp Asp Glu Asp Gly His His Ser Glu Glu Ser Leu
His Tyr Glu Gly 130 135 140 cga gca gtg gac atc acc acg tcc gac cgg
gac cgc agc aag tac ggc 480 Arg Ala Val Asp Ile Thr Thr Ser Asp Arg
Asp Arg Ser Lys Tyr Gly 145 150 155 160 atg ctg gct cgc ctg gct gtg
gaa gca ggt ttc gac tgg gtc tac tat 528 Met Leu Ala Arg Leu Ala Val
Glu Ala Gly Phe Asp Trp Val Tyr Tyr 165 170 175 gaa tcc aaa gct cac
atc cac tgt tct gtg aaa gca gag aac tcc gtg 576 Glu Ser Lys Ala His
Ile His Cys Ser Val Lys Ala Glu Asn Ser Val 180 185 190 gcg gcc aaa
tcc ggc ggc tgt ttc ccg gga tcc gcc acc gtg cac ctg 624 Ala Ala Lys
Ser Gly Gly Cys Phe Pro Gly Ser Ala Thr Val His Leu 195 200 205 gag
cag ggc ggc acc aag ctg gtg aag gac tta cgt ccc gga gac cgc 672 Glu
Gln Gly Gly Thr Lys Leu Val Lys Asp Leu Arg Pro Gly Asp Arg 210 215
220 gtg ctg gcg gct gac gac cag ggc cgg ctg ctg tac agc gac ttc ctc
720 Val Leu Ala Ala Asp Asp Gln Gly Arg Leu Leu Tyr Ser Asp Phe Leu
225 230 235 240 acc ttc ctg gac cgc gac gaa ggc gcc aag aag gtc ttc
tac gtg atc 768 Thr Phe Leu Asp Arg Asp Glu Gly Ala Lys Lys Val Phe
Tyr Val Ile 245 250 255 gag acg ctg gag ccg cgc gag cgc ctg ctg ctc
acc gcc gcg cac ctg 816 Glu
Thr Leu Glu Pro Arg Glu Arg Leu Leu Leu Thr Ala Ala His Leu 260 265
270 ctc ttc gtg gcg ccg cac aac gac tcg ggg ccc acg ccc ggg cca agc
864 Leu Phe Val Ala Pro His Asn Asp Ser Gly Pro Thr Pro Gly Pro Ser
275 280 285 gcg ctc ttt gcc agc cgc gtg cgc ccc ggg cag cgc gtg tac
gtg gtg 912 Ala Leu Phe Ala Ser Arg Val Arg Pro Gly Gln Arg Val Tyr
Val Val 290 295 300 gct gaa cgc ggc ggg gac cgc cgg ctg ctg ccc gcc
gcg gtg cac agc 960 Ala Glu Arg Gly Gly Asp Arg Arg Leu Leu Pro Ala
Ala Val His Ser 305 310 315 320 gtg acg ctg cga gag gag gag gcg ggc
gcg tac gcg ccg ctc acg gcg 1008 Val Thr Leu Arg Glu Glu Glu Ala
Gly Ala Tyr Ala Pro Leu Thr Ala 325 330 335 cac ggc acc att ctc atc
aac cgg gtg ctc gcc tcg tgc tac gct gtc 1056 His Gly Thr Ile Leu
Ile Asn Arg Val Leu Ala Ser Cys Tyr Ala Val 340 345 350 atc gag gag
cac agc tgg gca cac cgg gcc ttc gcg cct ttc cgc ctg 1104 Ile Glu
Glu His Ser Trp Ala His Arg Ala Phe Ala Pro Phe Arg Leu 355 360 365
gcg cac gcg ctg ctg gcc gcg ctg gca ccc gcc cgc acg gac ggc ggg
1152 Ala His Ala Leu Leu Ala Ala Leu Ala Pro Ala Arg Thr Asp Gly
Gly 370 375 380 ggc ggg ggc agc atc cct gca gcg caa tct gca acg gaa
gcg agg ggc 1200 Gly Gly Gly Ser Ile Pro Ala Ala Gln Ser Ala Thr
Glu Ala Arg Gly 385 390 395 400 gcg gag ccg act gcg ggc atc cac tgg
tac tcg cag ctg ctc tac cac 1248 Ala Glu Pro Thr Ala Gly Ile His
Trp Tyr Ser Gln Leu Leu Tyr His 405 410 415 att ggc acc tgg ctg ttg
gac agc gag acc atg cat ccc ttg gga atg 1296 Ile Gly Thr Trp Leu
Leu Asp Ser Glu Thr Met His Pro Leu Gly Met 420 425 430 gcg gtc aag
tcc agc tg 1313 Ala Val Lys Ser Ser 435 5 1256 DNA zebrafish Shh
CDS (1)..(1254) 5 atg cgg ctt ttg acg aga gtg ctg ctg gtg tct ctt
ctc act ctg tcc 48 Met Arg Leu Leu Thr Arg Val Leu Leu Val Ser Leu
Leu Thr Leu Ser 1 5 10 15 ttg gtg gtg tcc gga ctg gcc tgc ggt cct
ggc aga ggc tac ggc aga 96 Leu Val Val Ser Gly Leu Ala Cys Gly Pro
Gly Arg Gly Tyr Gly Arg 20 25 30 aga aga cat ccg aag aag ctg aca
cct ctc gcc tac aag cag ttc ata 144 Arg Arg His Pro Lys Lys Leu Thr
Pro Leu Ala Tyr Lys Gln Phe Ile 35 40 45 cct aat gtc gcg gag aag
acc tta ggg gcc agc ggc aga tac gag ggc 192 Pro Asn Val Ala Glu Lys
Thr Leu Gly Ala Ser Gly Arg Tyr Glu Gly 50 55 60 aag ata acg cgc
aat tcg gag aga ttt aaa gaa ctt act cca aat tac 240 Lys Ile Thr Arg
Asn Ser Glu Arg Phe Lys Glu Leu Thr Pro Asn Tyr 65 70 75 80 aat ccc
gac att atc ttt aag gat gag gag aac acg gga gcg gac agg 288 Asn Pro
Asp Ile Ile Phe Lys Asp Glu Glu Asn Thr Gly Ala Asp Arg 85 90 95
ctc atg aca cag aga tgc aaa gac aag ctg aac tcg ctg gcc atc tct 336
Leu Met Thr Gln Arg Cys Lys Asp Lys Leu Asn Ser Leu Ala Ile Ser 100
105 110 gta atg aac cac tgg cca ggg gtt aag ctg cgt gtg aca gag ggc
tgg 384 Val Met Asn His Trp Pro Gly Val Lys Leu Arg Val Thr Glu Gly
Trp 115 120 125 gat gag gac ggt cac cat ttt gaa gaa tca ctc cac tac
gag gga aga 432 Asp Glu Asp Gly His His Phe Glu Glu Ser Leu His Tyr
Glu Gly Arg 130 135 140 gct gtt gat att acc acc tct gac cga gac aag
agc aaa tac ggg aca 480 Ala Val Asp Ile Thr Thr Ser Asp Arg Asp Lys
Ser Lys Tyr Gly Thr 145 150 155 160 ctg tct cgc cta gct gtg gag gct
gga ttt gac tgg gtc tat tac gag 528 Leu Ser Arg Leu Ala Val Glu Ala
Gly Phe Asp Trp Val Tyr Tyr Glu 165 170 175 tcc aaa gcc cac att cat
tgc tct gtc aaa gca gaa aat tcg gtt gct 576 Ser Lys Ala His Ile His
Cys Ser Val Lys Ala Glu Asn Ser Val Ala 180 185 190 gcg aaa tct ggg
ggc tgt ttc cca ggt tcg gct ctg gtc tcg ctc cag 624 Ala Lys Ser Gly
Gly Cys Phe Pro Gly Ser Ala Leu Val Ser Leu Gln 195 200 205 gac gga
gga cag aag gcc gtg aag gac ctg aac ccc gga gac aag gtg 672 Asp Gly
Gly Gln Lys Ala Val Lys Asp Leu Asn Pro Gly Asp Lys Val 210 215 220
ctg gcg gca gac agc gcg gga aac ctg gtg ttc agc gac ttc atc atg 720
Leu Ala Ala Asp Ser Ala Gly Asn Leu Val Phe Ser Asp Phe Ile Met 225
230 235 240 ttc aca gac cga gac tcc acg acg cga cgt gtg ttt tac gtc
ata gaa 768 Phe Thr Asp Arg Asp Ser Thr Thr Arg Arg Val Phe Tyr Val
Ile Glu 245 250 255 acg caa gaa ccc gtt gaa aag atc acc ctc acc gcc
gct cac ctc ctt 816 Thr Gln Glu Pro Val Glu Lys Ile Thr Leu Thr Ala
Ala His Leu Leu 260 265 270 ttt gtc ctc gac aac tca acg gaa gat ctc
cac acc atg acc gcc gcg 864 Phe Val Leu Asp Asn Ser Thr Glu Asp Leu
His Thr Met Thr Ala Ala 275 280 285 tat gcc agc agt gtc aga gcc gga
caa aag gtg atg gtt gtt gat gat 912 Tyr Ala Ser Ser Val Arg Ala Gly
Gln Lys Val Met Val Val Asp Asp 290 295 300 agc ggt cag ctt aaa tct
gtc atc gtg cag cgg ata tac acg gag gag 960 Ser Gly Gln Leu Lys Ser
Val Ile Val Gln Arg Ile Tyr Thr Glu Glu 305 310 315 320 cag cgg ggc
tcg ttc gca cca gtg act gca cat ggg acc att gtg gtc 1008 Gln Arg
Gly Ser Phe Ala Pro Val Thr Ala His Gly Thr Ile Val Val 325 330 335
gac aga ata ctg gcg tcc tgt tac gcc gta ata gag gac cag ggg ctt
1056 Asp Arg Ile Leu Ala Ser Cys Tyr Ala Val Ile Glu Asp Gln Gly
Leu 340 345 350 gcg cat ttg gcc ttc gcg ccc gcc agg ctc tat tat tac
gtg tca tca 1104 Ala His Leu Ala Phe Ala Pro Ala Arg Leu Tyr Tyr
Tyr Val Ser Ser 355 360 365 ttc ctg tcc ccc aaa act cca gca gtc ggt
cca atg cga ctt tac aac 1152 Phe Leu Ser Pro Lys Thr Pro Ala Val
Gly Pro Met Arg Leu Tyr Asn 370 375 380 agg agg ggg tcc act ggt act
cca ggc tcc tgt cat caa atg gga acg 1200 Arg Arg Gly Ser Thr Gly
Thr Pro Gly Ser Cys His Gln Met Gly Thr 385 390 395 400 tgg ctt ttg
gac agc aac atg ctt cat cct ttg ggg atg tca gta aac 1248 Trp Leu
Leu Asp Ser Asn Met Leu His Pro Leu Gly Met Ser Val Asn 405 410 415
tca agc tg 1256 Ser Ser 6 1425 DNA Homo sapien Shh CDS (1)..(1425)
"nnn" encoding "Xaa" at position 1387-1389 may be a, t, c, g, other
or unknown 6 atg ctg ctg ctg gcg aga tgt ctg ctg cta gtc ctc gtc
tcc tcg ctg 48 Met Leu Leu Leu Ala Arg Cys Leu Leu Leu Val Leu Val
Ser Ser Leu 1 5 10 15 ctg gta tgc tcg gga ctg gcg tgc gga ccg ggc
agg ggg ttc ggg aag 96 Leu Val Cys Ser Gly Leu Ala Cys Gly Pro Gly
Arg Gly Phe Gly Lys 20 25 30 agg agg cac ccc aaa aag ctg acc cct
tta gcc tac aag cag ttt atc 144 Arg Arg His Pro Lys Lys Leu Thr Pro
Leu Ala Tyr Lys Gln Phe Ile 35 40 45 ccc aat gtg gcc gag aag acc
cta ggc gcc agc gga agg tat gaa ggg 192 Pro Asn Val Ala Glu Lys Thr
Leu Gly Ala Ser Gly Arg Tyr Glu Gly 50 55 60 aag atc tcc aga aac
tcc gag cga ttt aag gaa ctc acc ccc aat tac 240 Lys Ile Ser Arg Asn
Ser Glu Arg Phe Lys Glu Leu Thr Pro Asn Tyr 65 70 75 80 aac ccc gac
atc ata ttt aag gat gaa gaa aac acc gga gcg gac agg 288 Asn Pro Asp
Ile Ile Phe Lys Asp Glu Glu Asn Thr Gly Ala Asp Arg 85 90 95 ctg
atg act cag agg tgt aag gac aag ttg aac gct ttg gcc atc tcg 336 Leu
Met Thr Gln Arg Cys Lys Asp Lys Leu Asn Ala Leu Ala Ile Ser 100 105
110 gtg atg aac cag tgg cca gga gtg aaa ctg cgg gtg acc gag ggc tgg
384 Val Met Asn Gln Trp Pro Gly Val Lys Leu Arg Val Thr Glu Gly Trp
115 120 125 gac gaa gat ggc cac cac tca gag gag tct ctg cac tac gag
ggc cgc 432 Asp Glu Asp Gly His His Ser Glu Glu Ser Leu His Tyr Glu
Gly Arg 130 135 140 gca gtg gac atc acc acg tct gac cgc gac cgc agc
aag tac ggc atg 480 Ala Val Asp Ile Thr Thr Ser Asp Arg Asp Arg Ser
Lys Tyr Gly Met 145 150 155 160 ctg gcc cgc ctg gcg gtg gag gcc ggc
ttc gac tgg gtg tac tac gag 528 Leu Ala Arg Leu Ala Val Glu Ala Gly
Phe Asp Trp Val Tyr Tyr Glu 165 170 175 tcc aag gca cat atc cac tgc
tcg gtg aaa gca gag aac tcg gtg gcg 576 Ser Lys Ala His Ile His Cys
Ser Val Lys Ala Glu Asn Ser Val Ala 180 185 190 gcc aaa tcg gga ggc
tgc ttc ccg ggc tcg gcc acg gtg cac ctg gag 624 Ala Lys Ser Gly Gly
Cys Phe Pro Gly Ser Ala Thr Val His Leu Glu 195 200 205 cag ggc ggc
acc aag ctg gtg aag gac ctg agc ccc ggg gac cgc gtg 672 Gln Gly Gly
Thr Lys Leu Val Lys Asp Leu Ser Pro Gly Asp Arg Val 210 215 220 ctg
gcg gcg gac gac cag ggc cgg ctg ctc tac agc gac ttc ctc act 720 Leu
Ala Ala Asp Asp Gln Gly Arg Leu Leu Tyr Ser Asp Phe Leu Thr 225 230
235 240 ttc ctg gac cgc gac gac ggc gcc aag aag gtc ttc tac gtg atc
gag 768 Phe Leu Asp Arg Asp Asp Gly Ala Lys Lys Val Phe Tyr Val Ile
Glu 245 250 255 acg cgg gag ccg cgc gag cgc ctg ctg ctc acc gcc gcg
cac ctg ctc 816 Thr Arg Glu Pro Arg Glu Arg Leu Leu Leu Thr Ala Ala
His Leu Leu 260 265 270 ttt gtg gcg ccg cac aac gac tcg gcc acc ggg
gag ccc gag gcg tcc 864 Phe Val Ala Pro His Asn Asp Ser Ala Thr Gly
Glu Pro Glu Ala Ser 275 280 285 tcg ggc tcg ggg ccg cct tcc ggg ggc
gca ctg ggg cct cgg gcg ctg 912 Ser Gly Ser Gly Pro Pro Ser Gly Gly
Ala Leu Gly Pro Arg Ala Leu 290 295 300 ttc gcc agc cgc gtg cgc ccg
ggc cag cgc gtg tac gtg gtg gcc gag 960 Phe Ala Ser Arg Val Arg Pro
Gly Gln Arg Val Tyr Val Val Ala Glu 305 310 315 320 cgt gac ggg gac
cgc cgg ctc ctg ccc gcc gct gtg cac agc gtg acc 1008 Arg Asp Gly
Asp Arg Arg Leu Leu Pro Ala Ala Val His Ser Val Thr 325 330 335 cta
agc gag gag gcc gcg ggc gcc tac gcg ccg ctc acg gcc cag ggc 1056
Leu Ser Glu Glu Ala Ala Gly Ala Tyr Ala Pro Leu Thr Ala Gln Gly 340
345 350 acc att ctc atc aac cgg gtg ctg gcc tcg tgc tac gcg gtc atc
gag 1104 Thr Ile Leu Ile Asn Arg Val Leu Ala Ser Cys Tyr Ala Val
Ile Glu 355 360 365 gag cac agc tgg gcg cac cgg gcc ttc gcg ccc ttc
cgc ctg gcg cac 1152 Glu His Ser Trp Ala His Arg Ala Phe Ala Pro
Phe Arg Leu Ala His 370 375 380 gcg ctc ctg gct gca ctg gcg ccc gcg
cgc acg gac cgc ggc ggg gac 1200 Ala Leu Leu Ala Ala Leu Ala Pro
Ala Arg Thr Asp Arg Gly Gly Asp 385 390 395 400 agc ggc ggc ggg gac
cgc ggg ggc ggc ggc ggc aga gta gcc cta acc 1248 Ser Gly Gly Gly
Asp Arg Gly Gly Gly Gly Gly Arg Val Ala Leu Thr 405 410 415 gct cca
ggt gct gcc gac gct ccg ggt gcg ggg gcc acc gcg ggc atc 1296 Ala
Pro Gly Ala Ala Asp Ala Pro Gly Ala Gly Ala Thr Ala Gly Ile 420 425
430 cac tgg tac tcg cag ctg ctc tac caa ata ggc acc tgg ctc ctg gac
1344 His Trp Tyr Ser Gln Leu Leu Tyr Gln Ile Gly Thr Trp Leu Leu
Asp 435 440 445 agc gag gcc ctg cac ccg ctg ggc atg gcg gtc aag tcc
agc nnn agc 1392 Ser Glu Ala Leu His Pro Leu Gly Met Ala Val Lys
Ser Ser Xaa Ser 450 455 460 cgg ggg gcc ggg gga ggg gcg cgg gag ggg
gcc 1425 Arg Gly Ala Gly Gly Gly Ala Arg Glu Gly Ala 465 470 475 7
1622 DNA Homo sapien Ihh CDS (51)..(1283) 7 catcagccca ccaggagacc
tcgcccgccg ctcccccggg ctccccggcc atg tct 56 Met Ser 1 ccc gcc cgg
ctc cgg ccc cga ctg cac ttc tgc ctg gtc ctg ttg ctg 104 Pro Ala Arg
Leu Arg Pro Arg Leu His Phe Cys Leu Val Leu Leu Leu 5 10 15 ctg ctg
gtg gtg ccc gcg gca tgg ggc tgc ggg ccg ggt cgg gtg gtg 152 Leu Leu
Val Val Pro Ala Ala Trp Gly Cys Gly Pro Gly Arg Val Val 20 25 30
ggc agc cgc cgg cga ccg cca cgc aaa ctc gtg ccg ctc gcc tac aag 200
Gly Ser Arg Arg Arg Pro Pro Arg Lys Leu Val Pro Leu Ala Tyr Lys 35
40 45 50 cag ttc agc ccc aat gtg ccc gag aag acc ctg ggc gcc agc
gga cgc 248 Gln Phe Ser Pro Asn Val Pro Glu Lys Thr Leu Gly Ala Ser
Gly Arg 55 60 65 tat gaa ggc aag atc gct cgc agc tcc gag cgc ttc
aag gag ctc acc 296 Tyr Glu Gly Lys Ile Ala Arg Ser Ser Glu Arg Phe
Lys Glu Leu Thr 70 75 80 ccc aat tac aat cca gac atc atc ttc aag
gac gag gag aac aca ggc 344 Pro Asn Tyr Asn Pro Asp Ile Ile Phe Lys
Asp Glu Glu Asn Thr Gly 85 90 95 gcc gac cgc ctc atg acc cag cgc
tgc aag gac cgc ctg aac tcg ctg 392 Ala Asp Arg Leu Met Thr Gln Arg
Cys Lys Asp Arg Leu Asn Ser Leu 100 105 110 gct atc tcg gtg atg aac
cag tgg ccc ggt gtg aag ctg cgg gtg acc 440 Ala Ile Ser Val Met Asn
Gln Trp Pro Gly Val Lys Leu Arg Val Thr 115 120 125 130 gag ggc tgg
gac gag gac ggc cac cac tca gag gag tcc ctg cat tat 488 Glu Gly Trp
Asp Glu Asp Gly His His Ser Glu Glu Ser Leu His Tyr 135 140 145 gag
ggc cgc gcg gtg gac atc acc aca tca gac cgc gac cgc aat aag 536 Glu
Gly Arg Ala Val Asp Ile Thr Thr Ser Asp Arg Asp Arg Asn Lys 150 155
160 tat gga ctg ctg gcg cgc ttg gca gtg gag gcc ggc ttt gac tgg gtg
584 Tyr Gly Leu Leu Ala Arg Leu Ala Val Glu Ala Gly Phe Asp Trp Val
165 170 175 tat tac gag tca aag gcc cac gtg cat tgc tcc gtc aag tcc
gag cac 632 Tyr Tyr Glu Ser Lys Ala His Val His Cys Ser Val Lys Ser
Glu His 180 185 190 tcg gcc gca gcc aag acg ggc ggc tgc ttc cct gcc
gga gcc cag gta 680 Ser Ala Ala Ala Lys Thr Gly Gly Cys Phe Pro Ala
Gly Ala Gln Val 195 200 205 210 cgc ctg gag agt ggg gcg cgt gtg gcc
ttg tca gcc gtg agg ccg gga 728 Arg Leu Glu Ser Gly Ala Arg Val Ala
Leu Ser Ala Val Arg Pro Gly 215 220 225 gac cgt gtg ctg gcc atg ggg
gag gat ggg agc ccc acc ttc agc gat 776 Asp Arg Val Leu Ala Met Gly
Glu Asp Gly Ser Pro Thr Phe Ser Asp 230 235 240 gtg ctc att ttc ctg
gac cgc gag ccc cac agg ctg aga gcc ttc cag 824 Val Leu Ile Phe Leu
Asp Arg Glu Pro His Arg Leu Arg Ala Phe Gln 245 250 255 gtc atc gag
act cag gac ccc cca cgc cgc ctg gca ctc aca ccc gct 872 Val Ile Glu
Thr Gln Asp Pro Pro Arg Arg Leu Ala Leu Thr Pro Ala 260 265 270 cac
ctg ctc ttt acg gct gac aat cac acg gag ccg gca gcc cgc ttc 920 His
Leu Leu Phe Thr Ala Asp Asn His Thr Glu Pro Ala Ala Arg Phe 275 280
285 290 cgg gcc aca ttt gcc agc cac gtg cag cct ggc cag tac gtg ctg
gtg 968 Arg Ala Thr Phe Ala Ser His Val Gln Pro Gly Gln Tyr Val Leu
Val 295 300 305 gct ggg gtg cca ggc ctg cag cct gcc cgc gtg gca gct
gtc tct aca 1016 Ala Gly Val Pro Gly Leu Gln Pro Ala Arg Val Ala
Ala Val Ser Thr 310 315 320 cac gtg gcc ctc ggg gcc tac gcc ccg ctc
aca aag cat ggg aca ctg 1064 His Val Ala Leu Gly Ala Tyr Ala Pro
Leu Thr Lys His Gly Thr Leu 325 330 335 gtg gtg gag gat gtg gtg gca
tcc tgc ttc gcg gcc gtg gct gac cac 1112 Val Val Glu Asp Val Val
Ala Ser Cys Phe Ala Ala Val Ala Asp His 340 345 350 cac ctg gct cag
ttg gcc ttc tgg ccc ctg aga ctc ttt cac agc ttg 1160 His Leu Ala
Gln Leu Ala Phe Trp Pro Leu Arg Leu Phe His Ser Leu 355 360 365 370
gca tgg ggc agc tgg acc ccg ggg gag ggt gtg cat tgg tac ccc cag
1208 Ala Trp Gly Ser Trp Thr Pro Gly Glu Gly Val His Trp Tyr Pro
Gln 375 380 385 ctg ctc tac cgc ctg ggg cgt ctc ctg cta gaa gag ggc
agc ttc cac 1256 Leu Leu Tyr Arg Leu Gly Arg Leu Leu Leu Glu Glu
Gly Ser Phe His 390 395 400 cca ctg ggc atg tcc ggg gca ggg agc
tgaaaggact ccaccgctgc 1303 Pro Leu Gly Met Ser Gly Ala Gly Ser
405 410 cctcctggaa ctgctgtact gggtccagaa gcctctcagc caggagggag
ctggccctgg 1363 aagggacctg agctggggga cactggctcc tgccatctcc
tctgccatga agatacacca 1423 ttgagacttg actgggcaac accagcgtcc
cccacccgcg tcgtggtgta gtcatagagc 1483 tgcaagctga gctggcgagg
ggatggttgt tgacccctct ctcctagaga ccttgaggct 1543 ggcacggcga
ctcccaactc agcctgctct cactacgagt tttcatactc tgcctccccc 1603
attgggaggg cccattccc 1622 8 1251 DNA Zebrafish Thh CDS (1)..(1248)
8 atg gac gta agg ctg cat ctg aag caa ttt gct tta ctg tgt ttt atc
48 Met Asp Val Arg Leu His Leu Lys Gln Phe Ala Leu Leu Cys Phe Ile
1 5 10 15 agc ttg ctt ctg acg cct tgt gga tta gcc tgt ggt cct ggt
aga ggt 96 Ser Leu Leu Leu Thr Pro Cys Gly Leu Ala Cys Gly Pro Gly
Arg Gly 20 25 30 tat gga aaa cga aga cac cca aag aaa tta acc ccg
ttg gct tac aag 144 Tyr Gly Lys Arg Arg His Pro Lys Lys Leu Thr Pro
Leu Ala Tyr Lys 35 40 45 caa ttc atc ccc aac gtt gct gag aaa acg
ctt gga gcc agc ggc aaa 192 Gln Phe Ile Pro Asn Val Ala Glu Lys Thr
Leu Gly Ala Ser Gly Lys 50 55 60 tac gaa ggc aaa atc aca agg aat
tca gag aga ttt aaa gag ctg att 240 Tyr Glu Gly Lys Ile Thr Arg Asn
Ser Glu Arg Phe Lys Glu Leu Ile 65 70 75 80 ccg aat tat aat ccc gat
atc atc ttt aag gac gag gaa aac aca aac 288 Pro Asn Tyr Asn Pro Asp
Ile Ile Phe Lys Asp Glu Glu Asn Thr Asn 85 90 95 gct gac agg ctg
atg acc aag cgc tgt aag gac aag tta aat tcg ttg 336 Ala Asp Arg Leu
Met Thr Lys Arg Cys Lys Asp Lys Leu Asn Ser Leu 100 105 110 gcc ata
tcc gtc atg aac cac tgg ccc ggc gtg aaa ctg cgc gtc act 384 Ala Ile
Ser Val Met Asn His Trp Pro Gly Val Lys Leu Arg Val Thr 115 120 125
gaa ggc tgg gat gag gat ggt cac cat tta gaa gaa tct ttg cac tat 432
Glu Gly Trp Asp Glu Asp Gly His His Leu Glu Glu Ser Leu His Tyr 130
135 140 gag gga cgg gca gtg gac atc act acc tca gac agg gat aaa agc
aag 480 Glu Gly Arg Ala Val Asp Ile Thr Thr Ser Asp Arg Asp Lys Ser
Lys 145 150 155 160 tat ggg atg cta tcc agg ctt gca gtg gag gca gga
ttc gac tgg gtc 528 Tyr Gly Met Leu Ser Arg Leu Ala Val Glu Ala Gly
Phe Asp Trp Val 165 170 175 tat tat gaa tct aaa gcc cac ata cac tgc
tct gtc aaa gca gaa aat 576 Tyr Tyr Glu Ser Lys Ala His Ile His Cys
Ser Val Lys Ala Glu Asn 180 185 190 tca gtg gct gct aaa tca gga gga
tgt ttt cct ggg tct ggg acg gtg 624 Ser Val Ala Ala Lys Ser Gly Gly
Cys Phe Pro Gly Ser Gly Thr Val 195 200 205 aca ctt ggt gat ggg acg
agg aaa ccc atc aaa gat ctt aaa gtg ggc 672 Thr Leu Gly Asp Gly Thr
Arg Lys Pro Ile Lys Asp Leu Lys Val Gly 210 215 220 gac cgg gtt ttg
gct gca gac gag aag gga aat gtc tta ata agc gac 720 Asp Arg Val Leu
Ala Ala Asp Glu Lys Gly Asn Val Leu Ile Ser Asp 225 230 235 240 ttt
att atg ttt ata gac cac gat ccg aca acg aga agg caa ttc atc 768 Phe
Ile Met Phe Ile Asp His Asp Pro Thr Thr Arg Arg Gln Phe Ile 245 250
255 gtc atc gag acg tca gaa cct ttc acc aag ctc acc ctc act gcc gcg
816 Val Ile Glu Thr Ser Glu Pro Phe Thr Lys Leu Thr Leu Thr Ala Ala
260 265 270 cac cta gtt ttc gtt gga aac tct tca gca gct tcg ggt ata
aca gca 864 His Leu Val Phe Val Gly Asn Ser Ser Ala Ala Ser Gly Ile
Thr Ala 275 280 285 aca ttt gcc agc aac gtg aag cct gga gat aca gtt
tta gtg tgg gaa 912 Thr Phe Ala Ser Asn Val Lys Pro Gly Asp Thr Val
Leu Val Trp Glu 290 295 300 gac aca tgc gag agc ctc aag agc gtt aca
gtg aaa agg att tac act 960 Asp Thr Cys Glu Ser Leu Lys Ser Val Thr
Val Lys Arg Ile Tyr Thr 305 310 315 320 gag gag cac gag ggc tct ttt
gcg cca gtc acc gcg cac gga acc ata 1008 Glu Glu His Glu Gly Ser
Phe Ala Pro Val Thr Ala His Gly Thr Ile 325 330 335 ata gtg gat cag
gtg ttg gca tcg tgc tac gcg gtc att gag aac cac 1056 Ile Val Asp
Gln Val Leu Ala Ser Cys Tyr Ala Val Ile Glu Asn His 340 345 350 aaa
tgg gca cat tgg gct ttt gcg ccg gtc agg ttg tgt cac aag ctg 1104
Lys Trp Ala His Trp Ala Phe Ala Pro Val Arg Leu Cys His Lys Leu 355
360 365 atg acg tgg ctt ttt ccg gct cgt gaa tca aac gtc aat ttt cag
gag 1152 Met Thr Trp Leu Phe Pro Ala Arg Glu Ser Asn Val Asn Phe
Gln Glu 370 375 380 gat ggt atc cac tgg tac tca aat atg ctg ttt cac
atc ggc tct tgg 1200 Asp Gly Ile His Trp Tyr Ser Asn Met Leu Phe
His Ile Gly Ser Trp 385 390 395 400 ctg ctg gac aga gac tct ttc cat
cca ctc ggg att tta cac tta agt 1248 Leu Leu Asp Arg Asp Ser Phe
His Pro Leu Gly Ile Leu His Leu Ser 405 410 415 tga 1251 9 1416 DNA
Drosophila HH CDS (1)..(1413) 9 atg gat aac cac agc tca gtg cct tgg
gcc agt gcc gcc agt gtc acc 48 Met Asp Asn His Ser Ser Val Pro Trp
Ala Ser Ala Ala Ser Val Thr 1 5 10 15 tgt ctc tcc ctg gga tgc caa
atg cca cag ttc cag ttc cag ttc cag 96 Cys Leu Ser Leu Gly Cys Gln
Met Pro Gln Phe Gln Phe Gln Phe Gln 20 25 30 ctc caa atc cgc agc
gag ctc cat ctc cgc aag ccc gca aga aga acg 144 Leu Gln Ile Arg Ser
Glu Leu His Leu Arg Lys Pro Ala Arg Arg Thr 35 40 45 caa acg atg
cgc cac att gcg cat acg cag cgt tgc ctc agc agg ctg 192 Gln Thr Met
Arg His Ile Ala His Thr Gln Arg Cys Leu Ser Arg Leu 50 55 60 acc
tct ctg gtg gcc ctg ctg ctg atc gtc ttg ccg atg gtc ttt agc 240 Thr
Ser Leu Val Ala Leu Leu Leu Ile Val Leu Pro Met Val Phe Ser 65 70
75 80 ccg gct cac agc tgc ggt cct ggc cga gga ttg ggt cgt cat agg
gcg 288 Pro Ala His Ser Cys Gly Pro Gly Arg Gly Leu Gly Arg His Arg
Ala 85 90 95 cgc aac ctg tat ccg ctg gtc ctc aag cag aca att ccc
aat cta tcc 336 Arg Asn Leu Tyr Pro Leu Val Leu Lys Gln Thr Ile Pro
Asn Leu Ser 100 105 110 gag tac acg aac agc gcc tcc gga cct ctg gag
ggt gtg atc cgt cgg 384 Glu Tyr Thr Asn Ser Ala Ser Gly Pro Leu Glu
Gly Val Ile Arg Arg 115 120 125 gat tcg ccc aaa ttc aag gac ctc gtg
ccc aac tac aac agg gac atc 432 Asp Ser Pro Lys Phe Lys Asp Leu Val
Pro Asn Tyr Asn Arg Asp Ile 130 135 140 ctt ttc cgt gac gag gaa ggc
acc gga gcg gat ggc ttg atg agc aag 480 Leu Phe Arg Asp Glu Glu Gly
Thr Gly Ala Asp Gly Leu Met Ser Lys 145 150 155 160 cgc tgc aag gag
aag cta aac gtg ctg gcc tac tcg gtg atg aac gaa 528 Arg Cys Lys Glu
Lys Leu Asn Val Leu Ala Tyr Ser Val Met Asn Glu 165 170 175 tgg ccc
ggc atc cgg ctg ctg gtc acc gag agc tgg gac gag gac tac 576 Trp Pro
Gly Ile Arg Leu Leu Val Thr Glu Ser Trp Asp Glu Asp Tyr 180 185 190
cat cac ggc cag gag tcg ctc cac tac gag ggc cga gcg gtg acc att 624
His His Gly Gln Glu Ser Leu His Tyr Glu Gly Arg Ala Val Thr Ile 195
200 205 gcc acc tcc gat cgc gac cag tcc aaa tac ggc atg ctc gct cgc
ctg 672 Ala Thr Ser Asp Arg Asp Gln Ser Lys Tyr Gly Met Leu Ala Arg
Leu 210 215 220 gcc gtc gag gct gga ttc gat tgg gtc tcc tac gtc agc
agg cgc cac 720 Ala Val Glu Ala Gly Phe Asp Trp Val Ser Tyr Val Ser
Arg Arg His 225 230 235 240 atc tac tgc tcc gtc aag tca gat tcg tcg
atc agt tcc cac gtg cac 768 Ile Tyr Cys Ser Val Lys Ser Asp Ser Ser
Ile Ser Ser His Val His 245 250 255 ggc tgc ttc acg ccg gag agc aca
gcg ctg ctg gag agt gga gtc cgg 816 Gly Cys Phe Thr Pro Glu Ser Thr
Ala Leu Leu Glu Ser Gly Val Arg 260 265 270 aag ccg ctc ggc gag ctc
tct atc gga gat cgt gtt ttg agc atg acc 864 Lys Pro Leu Gly Glu Leu
Ser Ile Gly Asp Arg Val Leu Ser Met Thr 275 280 285 gcc aac gga cag
gcc gtc tac agc gaa gtg atc ctc ttc atg gac cgc 912 Ala Asn Gly Gln
Ala Val Tyr Ser Glu Val Ile Leu Phe Met Asp Arg 290 295 300 aac ctc
gag cag atg caa aac ttt gtg cag ctg cac acg gac ggt gga 960 Asn Leu
Glu Gln Met Gln Asn Phe Val Gln Leu His Thr Asp Gly Gly 305 310 315
320 gca gtg ctc acg gtg acg ccg gct cac ctg gtt agc gtt tgg cag ccg
1008 Ala Val Leu Thr Val Thr Pro Ala His Leu Val Ser Val Trp Gln
Pro 325 330 335 gag agc cag aag ctc acg ttt gtg ttt gcg cat cgc atc
gag gag aag 1056 Glu Ser Gln Lys Leu Thr Phe Val Phe Ala His Arg
Ile Glu Glu Lys 340 345 350 aac cag gtg ctc gta cgg gat gtg gag acg
ggc gag ctg agg ccc cag 1104 Asn Gln Val Leu Val Arg Asp Val Glu
Thr Gly Glu Leu Arg Pro Gln 355 360 365 cga gtg gtc aag ttg ggc agt
gtg cgc agt aag ggc gtg gtc gcg ccg 1152 Arg Val Val Lys Leu Gly
Ser Val Arg Ser Lys Gly Val Val Ala Pro 370 375 380 ctg acc cgc gag
ggc acc att gtg gtc aac tcg gtg gcc gcc agt tgc 1200 Leu Thr Arg
Glu Gly Thr Ile Val Val Asn Ser Val Ala Ala Ser Cys 385 390 395 400
tat gcg gtg atc aac agt cag tcg ctg gcc cac tgg gga ctg gct ccc
1248 Tyr Ala Val Ile Asn Ser Gln Ser Leu Ala His Trp Gly Leu Ala
Pro 405 410 415 atg cgc ctg ctg tcc acg ctg gag gcg tgg ctg ccc gcc
aag gag cag 1296 Met Arg Leu Leu Ser Thr Leu Glu Ala Trp Leu Pro
Ala Lys Glu Gln 420 425 430 ttg cac agt tcg ccg aag gtg gtg agc tcg
gcg cag cag cag aat ggc 1344 Leu His Ser Ser Pro Lys Val Val Ser
Ser Ala Gln Gln Gln Asn Gly 435 440 445 atc cat tgg tat gcc aat gcg
ctc tac aag gtc aag gac tac gtg ctg 1392 Ile His Trp Tyr Ala Asn
Ala Leu Tyr Lys Val Lys Asp Tyr Val Leu 450 455 460 ccg cag agc tgg
cgc cac gat tga 1416 Pro Gln Ser Trp Arg His Asp 465 470 10 425 PRT
chicken Shh 10 Met Val Glu Met Leu Leu Leu Thr Arg Ile Leu Leu Val
Gly Phe Ile 1 5 10 15 Cys Ala Leu Leu Val Ser Ser Gly Leu Thr Cys
Gly Pro Gly Arg Gly 20 25 30 Ile Gly Lys Arg Arg His Pro Lys Lys
Leu Thr Pro Leu Ala Tyr Lys 35 40 45 Gln Phe Ile Pro Asn Val Ala
Glu Lys Thr Leu Gly Ala Ser Gly Arg 50 55 60 Tyr Glu Gly Lys Ile
Thr Arg Asn Ser Glu Arg Phe Lys Glu Leu Thr 65 70 75 80 Pro Asn Tyr
Asn Pro Asp Ile Ile Phe Lys Asp Glu Glu Asn Thr Gly 85 90 95 Ala
Asp Arg Leu Met Thr Gln Arg Cys Lys Asp Lys Leu Asn Ala Leu 100 105
110 Ala Ile Ser Val Met Asn Gln Trp Pro Gly Val Lys Leu Arg Val Thr
115 120 125 Glu Gly Trp Asp Glu Asp Gly His His Ser Glu Glu Ser Leu
His Tyr 130 135 140 Glu Gly Arg Ala Val Asp Ile Thr Thr Ser Asp Arg
Asp Arg Ser Lys 145 150 155 160 Tyr Gly Met Leu Ala Arg Leu Ala Val
Glu Ala Gly Phe Asp Trp Val 165 170 175 Tyr Tyr Glu Ser Lys Ala His
Ile His Cys Ser Val Lys Ala Glu Asn 180 185 190 Ser Val Ala Ala Lys
Ser Gly Gly Cys Phe Pro Gly Ser Ala Thr Val 195 200 205 His Leu Glu
His Gly Gly Thr Lys Leu Val Lys Asp Leu Ser Pro Gly 210 215 220 Asp
Arg Val Leu Ala Ala Asp Ala Asp Gly Arg Leu Leu Tyr Ser Asp 225 230
235 240 Phe Leu Thr Phe Leu Asp Arg Met Asp Ser Ser Arg Lys Leu Phe
Tyr 245 250 255 Val Ile Glu Thr Arg Gln Pro Arg Ala Arg Leu Leu Leu
Thr Ala Ala 260 265 270 His Leu Leu Phe Val Ala Pro Gln His Asn Gln
Ser Glu Ala Thr Gly 275 280 285 Ser Thr Ser Gly Gln Ala Leu Phe Ala
Ser Asn Val Lys Pro Gly Gln 290 295 300 Arg Val Tyr Val Leu Gly Glu
Gly Gly Gln Gln Leu Leu Pro Ala Ser 305 310 315 320 Val His Ser Val
Ser Leu Arg Glu Glu Ala Ser Gly Ala Tyr Ala Pro 325 330 335 Leu Thr
Ala Gln Gly Thr Ile Leu Ile Asn Arg Val Leu Ala Ser Cys 340 345 350
Tyr Ala Val Ile Glu Glu His Ser Trp Ala His Trp Ala Phe Ala Pro 355
360 365 Phe Arg Leu Ala Gln Gly Leu Leu Ala Ala Leu Cys Pro Asp Gly
Ala 370 375 380 Ile Pro Thr Ala Ala Thr Thr Thr Thr Gly Ile His Trp
Tyr Ser Arg 385 390 395 400 Leu Leu Tyr Arg Ile Gly Ser Trp Val Leu
Asp Gly Asp Ala Leu His 405 410 415 Pro Leu Gly Met Val Ala Pro Ala
Ser 420 425 11 396 PRT murine Dhh 11 Met Ala Leu Pro Ala Ser Leu
Leu Pro Leu Cys Cys Leu Ala Leu Leu 1 5 10 15 Ala Leu Ser Ala Gln
Ser Cys Gly Pro Gly Arg Gly Pro Val Gly Arg 20 25 30 Arg Arg Tyr
Val Arg Lys Gln Leu Val Pro Leu Leu Tyr Lys Gln Phe 35 40 45 Val
Pro Ser Met Pro Glu Arg Thr Leu Gly Ala Ser Gly Pro Ala Glu 50 55
60 Gly Arg Val Thr Arg Gly Ser Glu Arg Phe Arg Asp Leu Val Pro Asn
65 70 75 80 Tyr Asn Pro Asp Ile Ile Phe Lys Asp Glu Glu Asn Ser Gly
Ala Asp 85 90 95 Arg Leu Met Thr Glu Arg Cys Lys Glu Arg Val Asn
Ala Leu Ala Ile 100 105 110 Ala Val Met Asn Met Trp Pro Gly Val Arg
Leu Arg Val Thr Glu Gly 115 120 125 Trp Asp Glu Asp Gly His His Ala
Gln Asp Ser Leu His Tyr Glu Gly 130 135 140 Arg Ala Leu Asp Ile Thr
Thr Ser Asp Arg Asp Arg Asn Lys Tyr Gly 145 150 155 160 Leu Leu Ala
Arg Leu Ala Val Glu Ala Gly Phe Asp Trp Val Tyr Tyr 165 170 175 Glu
Ser Arg Asn His Ile His Val Ser Val Lys Ala Asp Asn Ser Leu 180 185
190 Ala Val Arg Ala Gly Gly Cys Phe Pro Gly Asn Ala Thr Val Arg Leu
195 200 205 Arg Ser Gly Glu Arg Lys Gly Leu Arg Glu Leu His Arg Gly
Asp Trp 210 215 220 Val Leu Ala Ala Asp Ala Ala Gly Arg Val Val Pro
Thr Pro Val Leu 225 230 235 240 Leu Phe Leu Asp Arg Asp Leu Gln Arg
Arg Ala Ser Phe Val Ala Val 245 250 255 Glu Thr Glu Arg Pro Pro Arg
Lys Leu Leu Leu Thr Pro Trp His Leu 260 265 270 Val Phe Ala Ala Arg
Gly Pro Ala Pro Ala Pro Gly Asp Phe Ala Pro 275 280 285 Val Phe Ala
Arg Arg Leu Arg Ala Gly Asp Ser Val Leu Ala Pro Gly 290 295 300 Gly
Asp Ala Leu Gln Pro Ala Arg Val Ala Arg Val Ala Arg Glu Glu 305 310
315 320 Ala Val Gly Val Phe Ala Pro Leu Thr Ala His Gly Thr Leu Leu
Val 325 330 335 Asn Asp Val Leu Ala Ser Cys Tyr Ala Val Leu Glu Ser
His Gln Trp 340 345 350 Ala His Arg Ala Phe Ala Pro Leu Arg Leu Leu
His Ala Leu Gly Ala 355 360 365 Leu Leu Pro Gly Gly Ala Val Gln Pro
Thr Gly Met His Trp Tyr Ser 370 375 380 Arg Leu Leu Tyr Arg Leu Ala
Glu Glu Leu Met Gly 385 390 395 12 411 PRT murine Ihh 12 Met Ser
Pro Ala Trp Leu Arg Pro Arg Leu Arg Phe Cys Leu Phe Leu 1 5 10 15
Leu Leu Leu Leu Leu Val Pro Ala Ala Arg Gly Cys Gly Pro Gly Arg 20
25 30 Val Val Gly Ser Arg Arg Arg Pro Pro Arg Lys Leu Val Pro Leu
Ala 35 40 45 Tyr Lys Gln Phe Ser Pro Asn Val Pro Glu Lys Thr Leu
Gly Ala Ser 50 55 60 Gly Arg Tyr Glu Gly Lys Ile Ala Arg Ser Ser
Glu Arg Phe Lys Glu 65 70 75 80 Leu Thr Pro Asn Tyr Asn Pro Asp Ile
Ile Phe Lys Asp Glu Glu Asn 85 90 95 Thr Gly Ala Asp Arg Leu
Met
Thr Gln Arg Cys Lys Asp Arg Leu Asn 100 105 110 Ser Leu Ala Ile Ser
Val Met Asn Gln Trp Pro Gly Val Lys Leu Arg 115 120 125 Val Thr Glu
Gly Arg Asp Glu Asp Gly His His Ser Glu Glu Ser Leu 130 135 140 His
Tyr Glu Gly Arg Ala Val Asp Ile Thr Thr Ser Asp Arg Asp Arg 145 150
155 160 Asn Lys Tyr Gly Leu Leu Ala Arg Leu Ala Val Glu Ala Gly Phe
Asp 165 170 175 Trp Val Tyr Tyr Glu Ser Lys Ala His Val His Cys Ser
Val Lys Ser 180 185 190 Glu His Ser Ala Ala Ala Lys Thr Gly Gly Cys
Phe Pro Ala Gly Ala 195 200 205 Gln Val Arg Leu Glu Asn Gly Glu Arg
Val Ala Leu Ser Ala Val Lys 210 215 220 Pro Gly Asp Arg Val Leu Ala
Met Gly Glu Asp Gly Thr Pro Thr Phe 225 230 235 240 Ser Asp Val Leu
Ile Phe Leu Asp Arg Glu Pro Asn Arg Leu Arg Ala 245 250 255 Phe Gln
Val Ile Glu Thr Gln Asp Pro Pro Arg Arg Leu Ala Leu Thr 260 265 270
Pro Ala His Leu Leu Phe Ile Ala Asp Asn His Thr Glu Pro Ala Ala 275
280 285 His Phe Arg Ala Thr Phe Ala Ser His Val Gln Pro Gly Gln Tyr
Val 290 295 300 Leu Val Ser Gly Val Pro Gly Leu Gln Pro Ala Arg Val
Ala Ala Val 305 310 315 320 Ser Thr His Val Ala Leu Gly Ser Tyr Ala
Pro Leu Thr Arg His Gly 325 330 335 Thr Leu Val Val Glu Asp Val Val
Ala Ser Cys Phe Ala Ala Val Ala 340 345 350 Asp His His Leu Ala Gln
Leu Ala Phe Trp Pro Leu Arg Leu Phe Pro 355 360 365 Ser Leu Ala Trp
Gly Ser Trp Thr Pro Ser Glu Gly Val His Ser Tyr 370 375 380 Pro Gln
Met Leu Tyr Arg Leu Gly Arg Leu Leu Leu Glu Glu Ser Thr 385 390 395
400 Phe His Pro Leu Gly Met Ser Gly Ala Gly Ser 405 410 13 437 PRT
murine Shh 13 Met Leu Leu Leu Leu Ala Arg Cys Phe Leu Val Ile Leu
Ala Ser Ser 1 5 10 15 Leu Leu Val Cys Pro Gly Leu Ala Cys Gly Pro
Gly Arg Gly Phe Gly 20 25 30 Lys Arg Arg His Pro Lys Lys Leu Thr
Pro Leu Ala Tyr Lys Gln Phe 35 40 45 Ile Pro Asn Val Ala Glu Lys
Thr Leu Gly Ala Ser Gly Arg Tyr Glu 50 55 60 Gly Lys Ile Thr Arg
Asn Ser Glu Arg Phe Lys Glu Leu Thr Pro Asn 65 70 75 80 Tyr Asn Pro
Asp Ile Ile Phe Lys Asp Glu Glu Asn Thr Gly Ala Asp 85 90 95 Arg
Leu Met Thr Gln Arg Cys Lys Asp Lys Leu Asn Ala Leu Ala Ile 100 105
110 Ser Val Met Asn Gln Trp Pro Gly Val Arg Leu Arg Val Thr Glu Gly
115 120 125 Trp Asp Glu Asp Gly His His Ser Glu Glu Ser Leu His Tyr
Glu Gly 130 135 140 Arg Ala Val Asp Ile Thr Thr Ser Asp Arg Asp Arg
Ser Lys Tyr Gly 145 150 155 160 Met Leu Ala Arg Leu Ala Val Glu Ala
Gly Phe Asp Trp Val Tyr Tyr 165 170 175 Glu Ser Lys Ala His Ile His
Cys Ser Val Lys Ala Glu Asn Ser Val 180 185 190 Ala Ala Lys Ser Gly
Gly Cys Phe Pro Gly Ser Ala Thr Val His Leu 195 200 205 Glu Gln Gly
Gly Thr Lys Leu Val Lys Asp Leu Arg Pro Gly Asp Arg 210 215 220 Val
Leu Ala Ala Asp Asp Gln Gly Arg Leu Leu Tyr Ser Asp Phe Leu 225 230
235 240 Thr Phe Leu Asp Arg Asp Glu Gly Ala Lys Lys Val Phe Tyr Val
Ile 245 250 255 Glu Thr Leu Glu Pro Arg Glu Arg Leu Leu Leu Thr Ala
Ala His Leu 260 265 270 Leu Phe Val Ala Pro His Asn Asp Ser Gly Pro
Thr Pro Gly Pro Ser 275 280 285 Ala Leu Phe Ala Ser Arg Val Arg Pro
Gly Gln Arg Val Tyr Val Val 290 295 300 Ala Glu Arg Gly Gly Asp Arg
Arg Leu Leu Pro Ala Ala Val His Ser 305 310 315 320 Val Thr Leu Arg
Glu Glu Glu Ala Gly Ala Tyr Ala Pro Leu Thr Ala 325 330 335 His Gly
Thr Ile Leu Ile Asn Arg Val Leu Ala Ser Cys Tyr Ala Val 340 345 350
Ile Glu Glu His Ser Trp Ala His Arg Ala Phe Ala Pro Phe Arg Leu 355
360 365 Ala His Ala Leu Leu Ala Ala Leu Ala Pro Ala Arg Thr Asp Gly
Gly 370 375 380 Gly Gly Gly Ser Ile Pro Ala Ala Gln Ser Ala Thr Glu
Ala Arg Gly 385 390 395 400 Ala Glu Pro Thr Ala Gly Ile His Trp Tyr
Ser Gln Leu Leu Tyr His 405 410 415 Ile Gly Thr Trp Leu Leu Asp Ser
Glu Thr Met His Pro Leu Gly Met 420 425 430 Ala Val Lys Ser Ser 435
14 418 PRT zebrafish Shh 14 Met Arg Leu Leu Thr Arg Val Leu Leu Val
Ser Leu Leu Thr Leu Ser 1 5 10 15 Leu Val Val Ser Gly Leu Ala Cys
Gly Pro Gly Arg Gly Tyr Gly Arg 20 25 30 Arg Arg His Pro Lys Lys
Leu Thr Pro Leu Ala Tyr Lys Gln Phe Ile 35 40 45 Pro Asn Val Ala
Glu Lys Thr Leu Gly Ala Ser Gly Arg Tyr Glu Gly 50 55 60 Lys Ile
Thr Arg Asn Ser Glu Arg Phe Lys Glu Leu Thr Pro Asn Tyr 65 70 75 80
Asn Pro Asp Ile Ile Phe Lys Asp Glu Glu Asn Thr Gly Ala Asp Arg 85
90 95 Leu Met Thr Gln Arg Cys Lys Asp Lys Leu Asn Ser Leu Ala Ile
Ser 100 105 110 Val Met Asn His Trp Pro Gly Val Lys Leu Arg Val Thr
Glu Gly Trp 115 120 125 Asp Glu Asp Gly His His Phe Glu Glu Ser Leu
His Tyr Glu Gly Arg 130 135 140 Ala Val Asp Ile Thr Thr Ser Asp Arg
Asp Lys Ser Lys Tyr Gly Thr 145 150 155 160 Leu Ser Arg Leu Ala Val
Glu Ala Gly Phe Asp Trp Val Tyr Tyr Glu 165 170 175 Ser Lys Ala His
Ile His Cys Ser Val Lys Ala Glu Asn Ser Val Ala 180 185 190 Ala Lys
Ser Gly Gly Cys Phe Pro Gly Ser Ala Leu Val Ser Leu Gln 195 200 205
Asp Gly Gly Gln Lys Ala Val Lys Asp Leu Asn Pro Gly Asp Lys Val 210
215 220 Leu Ala Ala Asp Ser Ala Gly Asn Leu Val Phe Ser Asp Phe Ile
Met 225 230 235 240 Phe Thr Asp Arg Asp Ser Thr Thr Arg Arg Val Phe
Tyr Val Ile Glu 245 250 255 Thr Gln Glu Pro Val Glu Lys Ile Thr Leu
Thr Ala Ala His Leu Leu 260 265 270 Phe Val Leu Asp Asn Ser Thr Glu
Asp Leu His Thr Met Thr Ala Ala 275 280 285 Tyr Ala Ser Ser Val Arg
Ala Gly Gln Lys Val Met Val Val Asp Asp 290 295 300 Ser Gly Gln Leu
Lys Ser Val Ile Val Gln Arg Ile Tyr Thr Glu Glu 305 310 315 320 Gln
Arg Gly Ser Phe Ala Pro Val Thr Ala His Gly Thr Ile Val Val 325 330
335 Asp Arg Ile Leu Ala Ser Cys Tyr Ala Val Ile Glu Asp Gln Gly Leu
340 345 350 Ala His Leu Ala Phe Ala Pro Ala Arg Leu Tyr Tyr Tyr Val
Ser Ser 355 360 365 Phe Leu Ser Pro Lys Thr Pro Ala Val Gly Pro Met
Arg Leu Tyr Asn 370 375 380 Arg Arg Gly Ser Thr Gly Thr Pro Gly Ser
Cys His Gln Met Gly Thr 385 390 395 400 Trp Leu Leu Asp Ser Asn Met
Leu His Pro Leu Gly Met Ser Val Asn 405 410 415 Ser Ser 15 475 PRT
Homo sapien Shh Xaa at position 463 is any or unknown amino acid 15
Met Leu Leu Leu Ala Arg Cys Leu Leu Leu Val Leu Val Ser Ser Leu 1 5
10 15 Leu Val Cys Ser Gly Leu Ala Cys Gly Pro Gly Arg Gly Phe Gly
Lys 20 25 30 Arg Arg His Pro Lys Lys Leu Thr Pro Leu Ala Tyr Lys
Gln Phe Ile 35 40 45 Pro Asn Val Ala Glu Lys Thr Leu Gly Ala Ser
Gly Arg Tyr Glu Gly 50 55 60 Lys Ile Ser Arg Asn Ser Glu Arg Phe
Lys Glu Leu Thr Pro Asn Tyr 65 70 75 80 Asn Pro Asp Ile Ile Phe Lys
Asp Glu Glu Asn Thr Gly Ala Asp Arg 85 90 95 Leu Met Thr Gln Arg
Cys Lys Asp Lys Leu Asn Ala Leu Ala Ile Ser 100 105 110 Val Met Asn
Gln Trp Pro Gly Val Lys Leu Arg Val Thr Glu Gly Trp 115 120 125 Asp
Glu Asp Gly His His Ser Glu Glu Ser Leu His Tyr Glu Gly Arg 130 135
140 Ala Val Asp Ile Thr Thr Ser Asp Arg Asp Arg Ser Lys Tyr Gly Met
145 150 155 160 Leu Ala Arg Leu Ala Val Glu Ala Gly Phe Asp Trp Val
Tyr Tyr Glu 165 170 175 Ser Lys Ala His Ile His Cys Ser Val Lys Ala
Glu Asn Ser Val Ala 180 185 190 Ala Lys Ser Gly Gly Cys Phe Pro Gly
Ser Ala Thr Val His Leu Glu 195 200 205 Gln Gly Gly Thr Lys Leu Val
Lys Asp Leu Ser Pro Gly Asp Arg Val 210 215 220 Leu Ala Ala Asp Asp
Gln Gly Arg Leu Leu Tyr Ser Asp Phe Leu Thr 225 230 235 240 Phe Leu
Asp Arg Asp Asp Gly Ala Lys Lys Val Phe Tyr Val Ile Glu 245 250 255
Thr Arg Glu Pro Arg Glu Arg Leu Leu Leu Thr Ala Ala His Leu Leu 260
265 270 Phe Val Ala Pro His Asn Asp Ser Ala Thr Gly Glu Pro Glu Ala
Ser 275 280 285 Ser Gly Ser Gly Pro Pro Ser Gly Gly Ala Leu Gly Pro
Arg Ala Leu 290 295 300 Phe Ala Ser Arg Val Arg Pro Gly Gln Arg Val
Tyr Val Val Ala Glu 305 310 315 320 Arg Asp Gly Asp Arg Arg Leu Leu
Pro Ala Ala Val His Ser Val Thr 325 330 335 Leu Ser Glu Glu Ala Ala
Gly Ala Tyr Ala Pro Leu Thr Ala Gln Gly 340 345 350 Thr Ile Leu Ile
Asn Arg Val Leu Ala Ser Cys Tyr Ala Val Ile Glu 355 360 365 Glu His
Ser Trp Ala His Arg Ala Phe Ala Pro Phe Arg Leu Ala His 370 375 380
Ala Leu Leu Ala Ala Leu Ala Pro Ala Arg Thr Asp Arg Gly Gly Asp 385
390 395 400 Ser Gly Gly Gly Asp Arg Gly Gly Gly Gly Gly Arg Val Ala
Leu Thr 405 410 415 Ala Pro Gly Ala Ala Asp Ala Pro Gly Ala Gly Ala
Thr Ala Gly Ile 420 425 430 His Trp Tyr Ser Gln Leu Leu Tyr Gln Ile
Gly Thr Trp Leu Leu Asp 435 440 445 Ser Glu Ala Leu His Pro Leu Gly
Met Ala Val Lys Ser Ser Xaa Ser 450 455 460 Arg Gly Ala Gly Gly Gly
Ala Arg Glu Gly Ala 465 470 475 16 411 PRT Homo sapien Ihh 16 Met
Ser Pro Ala Arg Leu Arg Pro Arg Leu His Phe Cys Leu Val Leu 1 5 10
15 Leu Leu Leu Leu Val Val Pro Ala Ala Trp Gly Cys Gly Pro Gly Arg
20 25 30 Val Val Gly Ser Arg Arg Arg Pro Pro Arg Lys Leu Val Pro
Leu Ala 35 40 45 Tyr Lys Gln Phe Ser Pro Asn Val Pro Glu Lys Thr
Leu Gly Ala Ser 50 55 60 Gly Arg Tyr Glu Gly Lys Ile Ala Arg Ser
Ser Glu Arg Phe Lys Glu 65 70 75 80 Leu Thr Pro Asn Tyr Asn Pro Asp
Ile Ile Phe Lys Asp Glu Glu Asn 85 90 95 Thr Gly Ala Asp Arg Leu
Met Thr Gln Arg Cys Lys Asp Arg Leu Asn 100 105 110 Ser Leu Ala Ile
Ser Val Met Asn Gln Trp Pro Gly Val Lys Leu Arg 115 120 125 Val Thr
Glu Gly Trp Asp Glu Asp Gly His His Ser Glu Glu Ser Leu 130 135 140
His Tyr Glu Gly Arg Ala Val Asp Ile Thr Thr Ser Asp Arg Asp Arg 145
150 155 160 Asn Lys Tyr Gly Leu Leu Ala Arg Leu Ala Val Glu Ala Gly
Phe Asp 165 170 175 Trp Val Tyr Tyr Glu Ser Lys Ala His Val His Cys
Ser Val Lys Ser 180 185 190 Glu His Ser Ala Ala Ala Lys Thr Gly Gly
Cys Phe Pro Ala Gly Ala 195 200 205 Gln Val Arg Leu Glu Ser Gly Ala
Arg Val Ala Leu Ser Ala Val Arg 210 215 220 Pro Gly Asp Arg Val Leu
Ala Met Gly Glu Asp Gly Ser Pro Thr Phe 225 230 235 240 Ser Asp Val
Leu Ile Phe Leu Asp Arg Glu Pro His Arg Leu Arg Ala 245 250 255 Phe
Gln Val Ile Glu Thr Gln Asp Pro Pro Arg Arg Leu Ala Leu Thr 260 265
270 Pro Ala His Leu Leu Phe Thr Ala Asp Asn His Thr Glu Pro Ala Ala
275 280 285 Arg Phe Arg Ala Thr Phe Ala Ser His Val Gln Pro Gly Gln
Tyr Val 290 295 300 Leu Val Ala Gly Val Pro Gly Leu Gln Pro Ala Arg
Val Ala Ala Val 305 310 315 320 Ser Thr His Val Ala Leu Gly Ala Tyr
Ala Pro Leu Thr Lys His Gly 325 330 335 Thr Leu Val Val Glu Asp Val
Val Ala Ser Cys Phe Ala Ala Val Ala 340 345 350 Asp His His Leu Ala
Gln Leu Ala Phe Trp Pro Leu Arg Leu Phe His 355 360 365 Ser Leu Ala
Trp Gly Ser Trp Thr Pro Gly Glu Gly Val His Trp Tyr 370 375 380 Pro
Gln Leu Leu Tyr Arg Leu Gly Arg Leu Leu Leu Glu Glu Gly Ser 385 390
395 400 Phe His Pro Leu Gly Met Ser Gly Ala Gly Ser 405 410 17 416
PRT Zebrafish Thh 17 Met Asp Val Arg Leu His Leu Lys Gln Phe Ala
Leu Leu Cys Phe Ile 1 5 10 15 Ser Leu Leu Leu Thr Pro Cys Gly Leu
Ala Cys Gly Pro Gly Arg Gly 20 25 30 Tyr Gly Lys Arg Arg His Pro
Lys Lys Leu Thr Pro Leu Ala Tyr Lys 35 40 45 Gln Phe Ile Pro Asn
Val Ala Glu Lys Thr Leu Gly Ala Ser Gly Lys 50 55 60 Tyr Glu Gly
Lys Ile Thr Arg Asn Ser Glu Arg Phe Lys Glu Leu Ile 65 70 75 80 Pro
Asn Tyr Asn Pro Asp Ile Ile Phe Lys Asp Glu Glu Asn Thr Asn 85 90
95 Ala Asp Arg Leu Met Thr Lys Arg Cys Lys Asp Lys Leu Asn Ser Leu
100 105 110 Ala Ile Ser Val Met Asn His Trp Pro Gly Val Lys Leu Arg
Val Thr 115 120 125 Glu Gly Trp Asp Glu Asp Gly His His Leu Glu Glu
Ser Leu His Tyr 130 135 140 Glu Gly Arg Ala Val Asp Ile Thr Thr Ser
Asp Arg Asp Lys Ser Lys 145 150 155 160 Tyr Gly Met Leu Ser Arg Leu
Ala Val Glu Ala Gly Phe Asp Trp Val 165 170 175 Tyr Tyr Glu Ser Lys
Ala His Ile His Cys Ser Val Lys Ala Glu Asn 180 185 190 Ser Val Ala
Ala Lys Ser Gly Gly Cys Phe Pro Gly Ser Gly Thr Val 195 200 205 Thr
Leu Gly Asp Gly Thr Arg Lys Pro Ile Lys Asp Leu Lys Val Gly 210 215
220 Asp Arg Val Leu Ala Ala Asp Glu Lys Gly Asn Val Leu Ile Ser Asp
225 230 235 240 Phe Ile Met Phe Ile Asp His Asp Pro Thr Thr Arg Arg
Gln Phe Ile 245 250 255 Val Ile Glu Thr Ser Glu Pro Phe Thr Lys Leu
Thr Leu Thr Ala Ala 260 265 270 His Leu Val Phe Val Gly Asn Ser Ser
Ala Ala Ser Gly Ile Thr Ala 275 280 285 Thr Phe Ala Ser Asn Val Lys
Pro Gly Asp Thr Val Leu Val Trp Glu 290 295 300 Asp Thr Cys Glu Ser
Leu Lys Ser Val Thr Val Lys Arg Ile Tyr Thr 305 310 315 320 Glu Glu
His Glu Gly Ser Phe Ala Pro Val Thr Ala His Gly Thr Ile 325 330 335
Ile Val Asp Gln Val Leu Ala Ser Cys Tyr Ala Val Ile Glu Asn His 340
345 350 Lys Trp Ala His Trp Ala Phe Ala Pro Val Arg Leu Cys His Lys
Leu 355 360 365 Met Thr Trp Leu Phe Pro Ala
Arg Glu Ser Asn Val Asn Phe Gln Glu 370 375 380 Asp Gly Ile His Trp
Tyr Ser Asn Met Leu Phe His Ile Gly Ser Trp 385 390 395 400 Leu Leu
Asp Arg Asp Ser Phe His Pro Leu Gly Ile Leu His Leu Ser 405 410 415
18 471 PRT Drosophila HH 18 Met Asp Asn His Ser Ser Val Pro Trp Ala
Ser Ala Ala Ser Val Thr 1 5 10 15 Cys Leu Ser Leu Gly Cys Gln Met
Pro Gln Phe Gln Phe Gln Phe Gln 20 25 30 Leu Gln Ile Arg Ser Glu
Leu His Leu Arg Lys Pro Ala Arg Arg Thr 35 40 45 Gln Thr Met Arg
His Ile Ala His Thr Gln Arg Cys Leu Ser Arg Leu 50 55 60 Thr Ser
Leu Val Ala Leu Leu Leu Ile Val Leu Pro Met Val Phe Ser 65 70 75 80
Pro Ala His Ser Cys Gly Pro Gly Arg Gly Leu Gly Arg His Arg Ala 85
90 95 Arg Asn Leu Tyr Pro Leu Val Leu Lys Gln Thr Ile Pro Asn Leu
Ser 100 105 110 Glu Tyr Thr Asn Ser Ala Ser Gly Pro Leu Glu Gly Val
Ile Arg Arg 115 120 125 Asp Ser Pro Lys Phe Lys Asp Leu Val Pro Asn
Tyr Asn Arg Asp Ile 130 135 140 Leu Phe Arg Asp Glu Glu Gly Thr Gly
Ala Asp Gly Leu Met Ser Lys 145 150 155 160 Arg Cys Lys Glu Lys Leu
Asn Val Leu Ala Tyr Ser Val Met Asn Glu 165 170 175 Trp Pro Gly Ile
Arg Leu Leu Val Thr Glu Ser Trp Asp Glu Asp Tyr 180 185 190 His His
Gly Gln Glu Ser Leu His Tyr Glu Gly Arg Ala Val Thr Ile 195 200 205
Ala Thr Ser Asp Arg Asp Gln Ser Lys Tyr Gly Met Leu Ala Arg Leu 210
215 220 Ala Val Glu Ala Gly Phe Asp Trp Val Ser Tyr Val Ser Arg Arg
His 225 230 235 240 Ile Tyr Cys Ser Val Lys Ser Asp Ser Ser Ile Ser
Ser His Val His 245 250 255 Gly Cys Phe Thr Pro Glu Ser Thr Ala Leu
Leu Glu Ser Gly Val Arg 260 265 270 Lys Pro Leu Gly Glu Leu Ser Ile
Gly Asp Arg Val Leu Ser Met Thr 275 280 285 Ala Asn Gly Gln Ala Val
Tyr Ser Glu Val Ile Leu Phe Met Asp Arg 290 295 300 Asn Leu Glu Gln
Met Gln Asn Phe Val Gln Leu His Thr Asp Gly Gly 305 310 315 320 Ala
Val Leu Thr Val Thr Pro Ala His Leu Val Ser Val Trp Gln Pro 325 330
335 Glu Ser Gln Lys Leu Thr Phe Val Phe Ala His Arg Ile Glu Glu Lys
340 345 350 Asn Gln Val Leu Val Arg Asp Val Glu Thr Gly Glu Leu Arg
Pro Gln 355 360 365 Arg Val Val Lys Leu Gly Ser Val Arg Ser Lys Gly
Val Val Ala Pro 370 375 380 Leu Thr Arg Glu Gly Thr Ile Val Val Asn
Ser Val Ala Ala Ser Cys 385 390 395 400 Tyr Ala Val Ile Asn Ser Gln
Ser Leu Ala His Trp Gly Leu Ala Pro 405 410 415 Met Arg Leu Leu Ser
Thr Leu Glu Ala Trp Leu Pro Ala Lys Glu Gln 420 425 430 Leu His Ser
Ser Pro Lys Val Val Ser Ser Ala Gln Gln Gln Asn Gly 435 440 445 Ile
His Trp Tyr Ala Asn Ala Leu Tyr Lys Val Lys Asp Tyr Val Leu 450 455
460 Pro Gln Ser Trp Arg His Asp 465 470 19 221 PRT Artificial
Sequence Description of Artificial Sequence degenerate polypeptide
sequence 19 Cys Gly Pro Gly Arg Gly Xaa Gly Xaa Arg Arg His Pro Lys
Lys Leu 1 5 10 15 Thr Pro Leu Ala Tyr Lys Gln Phe Ile Pro Asn Val
Ala Glu Lys Thr 20 25 30 Leu Gly Ala Ser Gly Arg Tyr Glu Gly Lys
Ile Xaa Arg Asn Ser Glu 35 40 45 Arg Phe Lys Glu Leu Thr Pro Asn
Tyr Asn Pro Asp Ile Ile Phe Lys 50 55 60 Asp Glu Glu Asn Thr Gly
Ala Asp Arg Leu Met Thr Gln Arg Cys Lys 65 70 75 80 Asp Lys Leu Asn
Xaa Leu Ala Ile Ser Val Met Asn Xaa Trp Pro Gly 85 90 95 Val Xaa
Leu Arg Val Thr Glu Gly Trp Asp Glu Asp Gly His His Xaa 100 105 110
Glu Glu Ser Leu His Tyr Glu Gly Arg Ala Val Asp Ile Thr Thr Ser 115
120 125 Asp Arg Asp Xaa Ser Lys Tyr Gly Xaa Leu Xaa Arg Leu Ala Val
Glu 130 135 140 Ala Gly Phe Asp Trp Val Tyr Tyr Glu Ser Lys Ala His
Ile His Cys 145 150 155 160 Ser Val Lys Ala Glu Asn Ser Val Ala Ala
Lys Ser Gly Gly Cys Phe 165 170 175 Pro Gly Ser Ala Xaa Val Xaa Leu
Xaa Xaa Gly Gly Xaa Lys Xaa Val 180 185 190 Lys Asp Leu Xaa Pro Gly
Asp Xaa Val Leu Ala Ala Asp Xaa Xaa Gly 195 200 205 Xaa Leu Xaa Xaa
Ser Asp Phe Xaa Xaa Phe Xaa Asp Arg 210 215 220 20 167 PRT
Artificial Sequence Description of Artificial Sequence degenerate
polypeptide sequence 20 Cys Gly Pro Gly Arg Gly Xaa Xaa Xaa Arg Arg
Xaa Xaa Xaa Pro Lys 1 5 10 15 Xaa Leu Xaa Pro Leu Xaa Tyr Lys Gln
Phe Xaa Pro Xaa Xaa Xaa Glu 20 25 30 Xaa Thr Leu Gly Ala Ser Gly
Xaa Xaa Glu Gly Xaa Xaa Xaa Arg Xaa 35 40 45 Ser Glu Arg Phe Xaa
Xaa Leu Thr Pro Asn Tyr Asn Pro Asp Ile Ile 50 55 60 Phe Lys Asp
Glu Glu Asn Xaa Gly Ala Asp Arg Leu Met Thr Xaa Arg 65 70 75 80 Cys
Lys Xaa Xaa Xaa Asn Xaa Leu Ala Ile Ser Val Met Asn Xaa Trp 85 90
95 Pro Gly Val Xaa Leu Arg Val Thr Glu Gly Xaa Asp Glu Asp Gly His
100 105 110 His Xaa Xaa Xaa Ser Leu His Tyr Glu Gly Arg Ala Xaa Asp
Ile Thr 115 120 125 Thr Ser Asp Arg Asp Xaa Xaa Lys Tyr Gly Xaa Leu
Xaa Arg Leu Ala 130 135 140 Val Glu Ala Gly Phe Asp Trp Val Tyr Tyr
Glu Ser Xaa Xaa His Xaa 145 150 155 160 His Xaa Ser Val Lys Xaa Xaa
165 21 74 DNA Artificial Sequence Description of Artificial
Sequence primer 21 gcgcgcttcg aagcgaggca gccagcgagg gagagagcga
gcgggcgagc cggagcgagg 60 aaatcgatgc gcgc 74 22 74 DNA Artificial
Sequence Description of Artificial Sequence primer 22 gcgcgcagat
ctgggaaagc gcaagagaga gcgcacacgc acacacccgc cgcgcgcact 60
cgggatccgc gcgc 74 23 996 DNA Artificial Sequence Description of
Artificial Sequence gene activation construct 23 cgaagcgagg
cagccagcga gggagagagc gagcgggcga gccggagcga ggaaatcgaa 60
ggttcgaatc cttcccccac caccatcact ttcaaaagtc cgaaagaatc tgctccctgc
120 ttgtgtgttg gaggtcgctg agtagtgcgc gagtaaaatt taagctacaa
caaggcaagg 180 cttgaccgac aattgcatga agaatctgct tagggttagg
cgttttgcgc tgcttcgcga 240 tgtacgggcc agatatacgc gttgacattg
attattgact agttattaat agtaatcaat 300 tacggggtca ttagttcata
gcccatatat ggagttccgc gttacataac ttacggtaaa 360 tggcccgcct
ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt 420
tcccatagta acgccaatag ggactttcca ttgacgtcaa tgggtggact atttacggta
480 aactgcccac ttggcagtac atcaagtgta tcatatgcca agtacgcccc
ctattgacgt 540 caatgacggt aaatggcccg cctggcatta tgcccagtac
atgaccttat gggactttcc 600 tacttggcag tacatctacg tattagtcat
cgctattacc atggtgatgc ggttttggca 660 gtacatcaat gggcgtggat
agcggtttga ctcacgggga tttccaagtc tccaccccat 720 tgacgtcaat
gggagtttgt tttggcacca aaatcaacgg gactttccaa aatgtcgtaa 780
caactccgcc ccattgacgc aaatgggcgg taggcgtgta cggtgggagg tctatataag
840 cagagctctc tggctaacta gagaacccac tgcttactgg cttatcgaaa
ttaatacgac 900 tcactatagg gagacccaag cttggtaccg agctcggatc
gatctgggaa agcgcaagag 960 agagcgcaca cgcacacacc cgccgcgcgc actcgg
996 24 26 DNA Artificial Sequence Description of Artificial
Sequence antisense construct 24 gtcctggcgc cgccgccgcc gtcgcc 26 25
26 DNA Artificial Sequence Description of Artificial Sequence
antisense construct 25 ttccgatgac cggcctttcg cggtga 26 26 26 DNA
Artificial Sequence Description of Artificial Sequence antisense
construct 26 gtgcacggaa aggtgcaggc cacact 26
* * * * *
References