U.S. patent application number 12/348077 was filed with the patent office on 2009-07-30 for use of compounds that interfere with the hedgehog signaling pathway for the manufacture of a medicament for preventing, inhibiting, and/or reversing ocular diseases related with ocular neovascularization.
This patent application is currently assigned to FONDAZIONE TELETHON. Invention is credited to Alberto Auricchio, Markus Hildinger, Enrico Maria Surace.
Application Number | 20090192115 12/348077 |
Document ID | / |
Family ID | 36282808 |
Filed Date | 2009-07-30 |
United States Patent
Application |
20090192115 |
Kind Code |
A1 |
Auricchio; Alberto ; et
al. |
July 30, 2009 |
USE OF COMPOUNDS THAT INTERFERE WITH THE HEDGEHOG SIGNALING PATHWAY
FOR THE MANUFACTURE OF A MEDICAMENT FOR PREVENTING, INHIBITING,
AND/OR REVERSING OCULAR DISEASES RELATED WITH OCULAR
NEOVASCULARIZATION
Abstract
The use of compounds that interfere with the hedgehog signaling
pathway for the manufacture of a medicament for preventing,
inhibiting, and/or reversing ocular diseases related with ocular
neovascularization. Particularly, the above-mentioned diseases are
(wet) age-related macular degeneration, (proliferative) diabetic
retinopathy, neovascular glaucoma, retinal vein occlusion, or
retinopathy of prematurity (ROP).
Inventors: |
Auricchio; Alberto; (Naples,
IT) ; Hildinger; Markus; (Naples, IT) ;
Surace; Enrico Maria; (Naples, IT) |
Correspondence
Address: |
YOUNG & THOMPSON
209 Madison Street, Suite 500
ALEXANDRIA
VA
22314
US
|
Assignee: |
FONDAZIONE TELETHON
|
Family ID: |
36282808 |
Appl. No.: |
12/348077 |
Filed: |
January 2, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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11002882 |
Dec 3, 2004 |
7517870 |
|
|
12348077 |
|
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Current U.S.
Class: |
514/44R |
Current CPC
Class: |
C12N 15/86 20130101;
C07K 16/18 20130101; A61K 31/58 20130101; A61P 27/02 20180101; A61K
31/00 20130101; A61K 31/56 20130101; A61K 31/445 20130101; C12N
15/113 20130101; C12N 2310/14 20130101; A61K 31/4355 20130101; A61K
38/1709 20130101; C12N 2750/14143 20130101 |
Class at
Publication: |
514/44 |
International
Class: |
A61K 31/711 20060101
A61K031/711; A61K 31/7105 20060101 A61K031/7105 |
Claims
1. A method of inhibiting, and/or reversing disease related with
ocular neovascularization in a mammalian subject comprising:
administering to the subject a therapeutically effective amount of
a substance that interferes with the Hedgehog signaling pathway,
wherein said substance is one of: (i) a soluble Hip1 protein
encoded in a transgene cassette of a gene transfer vector, wherein
the administration of said gene transfer vector to a mammalian
subject results in the expression and secretion of soluble Hip 1,
and (ii) a small interfering (ribo)nucleic acid.
2. The method of claim 1, wherein, the substance is said soluble
Hip1 protein encoded in a transgene cassette of a gene transfer
vector, and said soluble Hip1 protein is SEQ ID NO 6 or a
homologous protein.
3. The method of claim 1, wherein, the substance is said soluble
Hip1 protein encoded in a transgene cassette of a gene transfer
vector, and said soluble Hip1 protein is obtained from RIP1 by
deletion of the transmembrane segment located at the C-terminus,
and optionally containing a signal peptide.
4. The method of claim 1, wherein, the substance is said soluble
Hip1 protein encoded in a transgene cassette of a gene transfer
vector, and said soluble Hip1 protein is SEQ ID NO 4.
5. The method of claim 1, wherein, the substance is said soluble
Hip1 protein encoded in a transgene cassette of a gene transfer
vector, and said gene transfer vector is a recombinant
adeno-associated viral vector.
6. The method of claim 1, wherein, the substance is an isolated
small interfering RNA which comprises a sense RNA strand and an
antisense RNA strand, the sense and antisense RNA strands form an
RNA duplex, the sense RNA strand comprises nucleotide sequence SEQ
ID NO: 16, and the antisense RNA strand comprises the nucleotide
sequence SEQ ID NO: 15.
7. The method of claim 1, wherein, the substance is an isolated
small interfering RNA which comprises a sense RNA strand and an
antisense RNA strand, the sense and antisense RNA strands form an
RNA duplex, the sense RNA strand comprises a nucleotide sequence
SEQ ID NO: 17, and the antisense RNA strand comprises the
nucleotide sequence SEQ ID NO:18.
8. The method of claim 1, wherein, the substance is an isolated
small interfering RNA which comprises a sense RNA strand and an
antisense RNA strand, the sense and antisense RNA strands form an
RNA duplex, the sense RNA strand comprises the nucleotide sequence
SEQ ID NO: 19, and the antisense RNA strand comprises the
nucleotide sequence SEQ ID NO: 20.
9. The method of claim 1, wherein, the substance is a DNA sequence
corresponding to said small interfering (ribo)nucleic acid, and
said DNA sequence is comprised in a transgene cassette of a
recombinant adeno-associated viral vector.
10. The method of claim 1, wherein the substance is comprised in a
preparation suitable for and/or administered by local
administration.
11. The method of claim 1, wherein the substance is comprised in a
preparation suitable for and/or administered by topical
administration.
12. The method of claim 1, wherein the substance is comprised in a
preparation suitable for and/or administered by systemic
administration.
13. The method of claim 1, wherein the substance is comprised in a
preparation suitable for and/or administered by intravitreal,
injection.
14. The method of claim 1, wherein the substance is comprised in a
preparation suitable for and/or administered by subtretinal
injection.
15. The method of claim 1, wherein the substance is comprised in a
preparation suitable for and/or administered by intravitreal
administration.
16. The method of claim 1, wherein the substance is comprised in a
preparation suitable for and/or administered by intracavity
injection.
17. The method of claim 1, wherein the substance is comprised in a
preparation suitable for and/or administered by intraarterial
administration.
18. The method of claim 1, wherein the substance is comprised in a
preparation suitable for and/or administered by intravenous
administration.
19. The method of claim 1, wherein the substance is comprised in a
preparation suitable for and/or administered by intramuscular
administration.
20. The method of claim 1, wherein the substance is comprised in a
preparation suitable for and/or administered by injection into
tissue.
21. The method of claim 1, wherein the substance is comprised in a
preparation suitable for and/or administered by injection into gaps
in tissue.
22. The method of claim 1, wherein the substance is comprised in a
preparation suitable for and/or administered by inhalation anal or
nasal instillation.
23. The method of claim 1, wherein the disease related with ocular
neovascularization is selected from the group consisting of
age-related macular degeneration, diabetic retinopathy, retinopathy
of prematurity (ROP), neovascular glaucoma or retinal vein
occlusion.
Description
[0001] This application is a divisional application of Ser. No.
11/002,882, filed Dec. 3, 2004, currently pending. The teachings of
the above application is hereby incorporated by reference. Any
disclaimer that may have occurred during prosecution of the above
referenced application is hereby expressly disclaimed.
1. BACKGROUND OF THE INVENTION
[0002] In the following sections, the inventors include passages
and cite publications that are available in the public domain. The
author of the patent would like to acknowledge in particular "The
Merck Manual of Diagnosis and Therapy", and U.S. patent application
Ser. Nos. 10/652,298, 09/977,864, 09/88384. The author of the
patent can be contacted at hildinger@gmx.net.
[0003] It must be noted that as used herein and in the appended
claims, the singular forms "a" and "the" include plural referents
unless the context clearly dictates otherwise. Thus, for example,
reference to "a cell" or "the cell" includes a plurality ("cells"
or "the cells"), and so forth. Moreover, the word "or" can either
be exclusive in nature (i.e., either A or B, but not A and B
together), or inclusive in nature (A or B, including A alone, B
alone, but also A and B together). One of skill in the art will
realize which interpretation is the most appropriate--unless it is
detailed by reference in the text as "either A or B" (exclusive
"or") or "and/or" (inclusive "or").
1.1 Field of the Invention
[0004] The present invention relates to methods of preventing,
inhibiting, and/or reversing ocular neovascularization in a
mammalian subject comprising administering to the subject a
therapeutically effective amount of a substance that interferes
with the Hedgehog signaling pathway. Ocular neovascularization is a
major cause of blindness in developed countries. It is causally
involved in many ocular diseases including age-related macular
degeneration, (proliferative) diabetic retinopathy, neovascular
glaucoma, retinal vein occlusion, or retinopathy of prematurity
(ROP). Current treatments are of limited efficacy and associated
with significant adverse effects, reflecting the high unmet need in
those disease areas.
[0005] More specifically, the invention relates to the use of a
small molecule such as cyclopamine, or a protein such as a
(humanized) monoclonal antibody to interfere with Hedgehog
signaling. The inventors demonstrate in the present invention that
interference with Hedgehog signaling by means of e.g., cyclopamine,
is able to prevent, inhibit and/or reverse ocular
neovascularization in a mammalian subject, which will have a
therapeutic benefit on said subject.
1.2 Background of the Invention
[0006] Ocular neovascularization is involved in multiple
pathologies of the eye such as (wet) age-related macular
degeneration, (proliferative) diabetic retinopathy, neovascular
glaucoma, retinal vein occlusion, or retinopathy of prematurity
(ROP). Yet, there is still a high degree of unmet need in the
prevention, treatment and/or cure of diseases caused by ocular
neovascularization: Many patients affected by one of those diseases
will face (legal) blindness in the end.
[0007] The present invention relates to methods for preventing,
inhibiting, and/or reversing ocular neovascularization by
interfering with the Hedgehog signaling pathway in general, and by
inhibiting Smoothened (Smo) function in particular. The present
invention also comprises administering to a mammalian subject a
substance that interferes with the Hedgehog signaling pathway in
general, and that inhibits Smoothened (Smo) function in particular
such as (for example) cyclopamine.
[0008] The inventors are the first to show (1) the involvement of
Hedgehog signaling in general, and the requirement for Smoothened
activity in particular in ocular neovascularization, and (2) that
interfering with Hedgehog signaling in general, and inhibition of
Smoothened activation in particular can prevent, inhibit and/or
reverse ocular neovascularization. In the preferred embodiment, the
inventors prove that administering cyclopamine to a mammalian
subject can inhibit Smoothen activation and thus prevent and/or
inhibit ocular neovascularization. Generally speaking, upon
administration of a Hedgehog-interference substance (such as
cyclopamine), ocular neovascularization will be prevented,
inhibited and/or reversed, thus preventing and/or ameliorating the
pathologies associated with ocular neovascularization. Also
provided are pharmaceutical kits containing the Hedgehog-signaling
interfering substance in a suitable pharmaceutical suspension for
administration.
[0009] The present invention offers a novel, useful and non-obvious
method for treating, inhibiting and/or preventing ocular diseases
that are caused (in part) by ocular neovascularization by
administering to a mammalian subject a substance that interferes
with the Hedgehog signaling pathway. Also provided are
pharmaceutical kits containing the Hedgehog pathway interfering
substance in a suitable pharmaceutical suspension for
administration.
1.3 Description of Prior Art
1.3.1 Hedgehog Signaling
[0010] Analysis of the Drosophila melanogaster Hedgehog mutant,
named after its prominent phenotype--epidermal spikes in larval
segments that normally are devoid of these extensions--led to the
cloning of the original Hedgehog gene.
[0011] In vertebrates, the Hedgehog (Hh) protein family of secreted
glycoproteins comprises at least four members: Three of these
members, Sonic Hedgehog (Shh), Desert Hedgehog (Dhh), and India
Hedgehog (Ihh), seem to be expressed in all vertebrates, including
mammals. They are also highly conserved between species. A fourth
member, tiggie-winkle Hedgehog (Thh), appears specific to fish.
Exemplary Hedgehog genes and proteins are described in PCT
publications WO 95/18856 and WO 96/17924.
[0012] Hedgehog (Hh) proteins act as morphogens in many tissues
during embryonic development as mediators of intercellular
signaling. The Hedgehog pathway is important in regulating cell
patterning, differentiation, proliferation, survival and growth in
the embryo and the adult ([1]; [2]; [3]). Vertebrate Hedgehog
proteins are crucial to a number of epithelial-mesenchymal
inductive interactions during neuronal development, limb
development, lung, bone, hair follicle and gut formation ([4, 5]
[6][7] [8] [9]). More recently, their role in tumorigenesis and
angiogenesis has been described in adult organisms.
[0013] Shh, the most widely expressed Hedgehog protein, apparently
is also the most potent member of its family. It is broadly
expressed during development, and sonic null mice are embryonic
lethal ([10]; [11] [12]; [13]) Shh is primarily involved in
morphogenic and neuroinductive activities such as neural tube,
craniofacial, limb, and kidney development. Indian Hedgehog (Ihh)
is involved in bone development during embryogenesis and in bone
formation in the adult ([14]; [15]). Desert Hedgehog (Dhh) is the
most restricted in terms of expression, and Dhh null mice are
viable; it is expressed primarily in the testes, both in mouse
embryonic development and in the adult rodent and human ([16];
[17]).
[0014] Hedgehog proteins exert their function as part of the
Hedgehog signaling pathway that is tightly regulated by many
inhibitors at different levels. The Hedgehog receptor, Patched
(Ptc) is a 12 transmembrane protein with a sterol sensing domain.
The mammalian genome contains 2 patched genes, ptc1 and ptc2. In
the absence of Hedgehog, Ptc binds and inhibits the function of
Smoothened (Smo), a 7 transmembrane protein that functions as a
co-receptor. Once Hh binds to Ptc, Ptc does not further inhibit
Smo. Smo's activation in turn leads to the activation of fused
(Fu), a serine-threonine kinase, and the disassociation of a zinc
finger transcription factor of the mammalian Gli family
(corresponding to Ci in Drosophila), from the
microtubule-associated Fu-Gli-Su(fu) complex [Su(fu): Suppressor of
Fused]. Gli proteins are large, multifunctional transcription
factors, and their activities are intrinsically regulated. The
uncomplexed Gli protein is then transported into the nucleus where
it activates downstream target genes of the Hedgehog pathway
including genes associated with cell cycle progression, the ptc1
and gli1 gene ([18]; [19, 20]; [21]; [22]; [23]).
[0015] The various Hedgehog proteins consist of a signal peptide, a
highly conserved N-terminal region, and a more divergent C-terminal
domain. During their passage through the secretory pathway,
Hedgehog proteins are extensively modified: They undergo an
internal autoproteolytic cleavage yielding a 19 kD N-terminal
peptide and a 26-28 kD C-terminal peptide; the 19 kD peptide is
then further modified at its N--and C-termini by palmitoyl and
cholesteryl adducts, respectively. These lipid modifications lead
to a tight association of the 19 kD fragment with membranes. In
addition, the diffusion of the 19 kD N-terminal peptide is further
limited by binding to the Hip1, Patched1 (Ptc1) and Patched2 (Ptc2)
transmembrane receptors, all of which are expressed on Hh
responsive cells, and by interaction with heparin through an
N-terminal basic domain. The C-terminal peptide on the contrary is
freely diffusible. Yet, it is the 19 kD peptide that acts directly
on distant cells in developing tissues. This remote action requires
the transmembrane transporter-like protein Dispatched (Disp) for
release of Hh from secreting cells and other proteins and
enzymes.
[0016] Three Gli proteins have been identified in mammals (Gli1,
Gli2, Gli3). Proteolysis of Gli2 and Gli3 results in the generation
of strong repressors of gene transcription, whereas the Gli1
repressor seems to be weak. Shh inhibits repressor formation by
Gli3, but not by Gli2, and the formation of potent activators of
Gli2, and perhaps of other Gli proteins, depends on Hh signaling
([24-28]
[0017] Gli proteins can form Hedgehog signaling complexes with
several other signaling components, including the kinesin-like
protein Costal-2, Fu, and Su(fu) [29-33]. In these complexes Gli
proteins are regulated by cytoplasmic sequestration,
phosphorylation, and proteolysis. They can be found in both the
nucleus and cytoplasm. In the cytoplasm, they are part of a
multimolecular complex that is tethered to the cytoskeleton. In the
absence of Hh, Gli proteins are cleaved by the proteasome, and
C-terminally truncated forms termed "Gli repressors" translocate to
the nucleus, where they act as dominant transcriptional repressors.
Upon Hh-induced Smo activation, Gli repressor formation is
inhibited and full-length labile activators of transcription are
made instead.
1.3.2 Involvement of Hedgehog in Diseases
[0018] Prior art describes involvement of Hedgehog in several
diseases. When the Hedgehog-pathway is activated or maintained
inappropriately, cell proliferation can become misregulated. Some
examples are cancer [34-36] [37] [38] and skin-related disorders
such as psoriasis [39, 40].
[0019] Hedgehog misregulation has been associated with a whole
range of cancers including BCC, medulloblastoma, rhabdomyosarcoma,
cutaneous epithelial tumors, oral squamous cell carcinoma,
pancreatic cancer, digestive tract tumors, glioma, pancreatic
adenosarcoma, esophageal and stomach cancer, small cell lung
cancer. In addition to controlling cell differentiation and tissue
patterning, Hh signalling also regulates the proliferation of
distinct cell types via direct activation of cell-cycle control
genes such as cyclin D1 and cyclin D2 in mammalian cells [41],
which could explain its involvement in a vast variety of
tumors.
1.3.3 Vasculogenesis, Angiogenesis and Neovascularization
[0020] Vascular development involves vasculogenesis, where
endothelial cells form a primary tubular network, as well as
angiogenesis, in which vessel size and structure are modified, and
branching occurs to insure that all cells receive adequate oxygen
delivery. In adults, angiogenesis occurs in response to tissue
hypoxia/ischemia and plays an important role in determining the
progression of ischemic heart disease and cancer.
[0021] For purposes of this invention, vasculogenesis is a
physiological process, and pathological (abnormal) vasculogenesis
is referred to as neovascularization. Neovascularization--in a more
narrow definition--is a form of pathologic, abnormal vasculogenesis
that causes visual loss. Neovascularization can be caused by a
multitude of molecular mechanisms, and the basic pathological
mechanisms that trigger the cascade of events are many such as
ischemia, inflammatory, or metabolic events. One hypothesis for the
process of neovascularization suggests that increased vascular
permeability leads to retinal oedema and vascular fragility
resulting in haemorrhage, or fibro-vascular proliferation with
tractional and rhegmatogenous retinal detachment. Furthermore, for
purposes of this invention, angiogenesis can be either
physiological or pathological.
[0022] The discovery of the molecular mechanisms of physiological
vasculogenesis and angiogenesis vs. neovascularization and
pathological angiogenesis helped to recognize two classes of
diseases: One where the therapeutic angiogenesis can repair the
tissue damages (arteriosclerosis, ischemia-induced pathologies such
as myocardial infarction, coronary artery disease, peripheral
vascular disease, limb ischemia and stroke) and the other one where
inhibition of neovascularization and/or pathological angiogenesis
can cure the disease or delay its progression (age-related macular
degeneration, (proliferative) diabetic retinopathy, neovascular
glaucoma, retinal vein occlusion, or retinopathy of prematurity
(ROP), benign and malignant angiogenic tumors, progression of
malignant tumors, psoriasis, rheumatoid arthritis).
[0023] Thus, the induction of angiogenesis and vascular growth is
beneficial for tissue repair and would healing whereas inhibition
of angiogenic growth factors can prevent angiogenesis driven
pathologies.
[0024] Neovascularization in the retina is initiated by the
ischemic injury, hyperglycaemia induced PKC activation, and or
hypoxia. Hypoxia plays a causal role in the pathological events
that lead to the formation of new blood vessels, which ultimately
results in blindness. Under hypoxic stimuli HIF-1.alpha. triggers
the expression of a number of proangiogenic genes, including
Vascular Endothelial Growth Factor (VEGF). Glial cells in the
hypoxic retina not only produce VEGF, but also IL-8 and HGF as well
and pericytes start to express bFGF all responsible for the
neoangiogenesis. Yet, as the inventors demonstrate, the
Hedgehog-signaling pathway is also able of inducing VEGF
expression. VEGF is one of the most important growth factors that
have been extensively associated with neovascular diseases of the
eye.
[0025] As mentioned before, angiogenesis is the process by which
new capillaries are formed by sprouting from pre-existing vessels.
It is a highly attractive target for therapeutic intervention since
it represents a final common pathway in processes that are
multifactorial in aetiology. Angiogenesis is regulated through the
combined action of pro-angiogenic and anti-angiogenic factors.
[0026] Pro-angiogenic factors favor angiogenesis. They can be
classified into (a) Cytokines and growth factors such as Fibroblast
Growth Factor (FGF), Vascular Endothelial Growth Factor (VEGF),
TGF.alpha. and TGF.beta., Angiopoietins, Interleukins (IL-1, IL-6,
IL-8), GM-CSF, PDGF, PDECGF, EGF, TNF.alpha., HGF; (b) Cell surface
receptors such as Flk1 (=VEGFR2; =KDR; VEGF receptor), Flt1
(=VEGFR1; VEGF receptor; [8]), Neurophilin; (c) Integrins such as
.alpha.sub.v.beta.sub.3 (whose expression is induced by, e.g.,
TNF.alpha., FGF-2, GM-CSF etc.), alpha.sub.v.beta.sub.5; (d)
Proteases such as matrix metalloproteases (MMPs); (e) Small
molecules (e.g., retinoic acid; Cu).
[0027] Anti-angiogenic factors inhibit angiogenesis. They can be
classified into (a) Cytokines and growth factors such as Troponin,
Platelet Factor 4, TGF.beta. 1, Interferon alpha. and Interferon
.beta., Somatostatin; (b) Proteolytic fragments such as
Angiostatin, Endostatin (collagen XVIII fragment), Thrombospondin
fragment, Fibronectin fragments, Vasostatin (kallikrein fragment);
(c) Tissue inhibitors of metalloproteases (TIMPs); (d) Small
molecules such as some retinoids, zinc or 2-methoxyestradiol; (e)
Other proteins such as laminin or CM101.
1.3.4 Hedgehog Signaling in Angiogenesis and Neovascularization
[0028] Pola et al. [42] have shown that cells in the adult
vasculature both express ptc1 and can respond to exogenous
Hedgehog. They also were able to prove that Hedgehog is able to
induce robust neovascularization in the corneal pocket model of
angiogenesis. In the same publication, the group found that the
Hedgehog-signaling pathway is present in adult cardiovascular
tissues and can be activated in vivo. Shh was able to induce robust
angiogenesis, characterized by distinct large-diameter vessels. Shh
also augmented blood-flow recovery and limb salvage following
operatively induced hind-limb ischemia in aged mice. In vitro, Shh
had no effect on endothelial-cell migration or proliferation;
instead, it induced expression of two families of angiogenic
cytokines, including all three vascular endothelial growth factor-1
isoforms and angiopoietins-1 and -2 from interstitial mesenchymal
cells. In summary, the group discovered a novel role for Shh as an
indirect angiogenic factor that regulates expression of multiple
angiogenic cytokines--indicating a potential therapeutic use of Shh
for ischemic disorders.
[0029] Whereas this group has shown a role for Shh in ischemic
diseases, there has not yet been reports of the involvement of
Hedgehog in general and Shh in particular in ocular
neovascularization. Moreover, there have not yet been reports that
interfering with Hedgehog signaling in general and Shh signaling in
particular, and interference by inhibiting Smo activation in
detail, will inhibit, prevent, and/or revert ocular
neovascularization.
1.3.5 Eye Diseases
[0030] Ocular neovascularization is causatively involved in many
eye diseases such as (proliferative) diabetic retinopathy,
neovascular glaucoma, retinal vein occlusion an/or retinopathy of
prematurity (ROP).
1.3.5.1 Age-Related Macular Degeneration (AMD)
[0031] Age related macular degeneration (AMD) is the leading cause
of legal blindness in the elderly Caucasian population, but is
relatively rare in other races. The degenerative condition of the
central retina (macula) only affects central vision, leaving
peripheral vision intact. AMD affects approximately 30% or more of
the Caucasian population age 75 and greater. The molecular
mechanism underling AMD is not fully understood and is the major
obstacle in the development of aetiologic and prophylactic
treatments. The primary lesion appears to occur deep to the central
retina with deposits known as drusen. Drusen are thought to be
metabolic by-products, the increasing deposition of which may
further interfere with the high metabolic activity of the
macula.
[0032] There is no medical treatment for AMD and only a very small
number of subjects with late stage disease are amenable to a
palliative and minimally effective laser photocoagulation
therapy.
[0033] There are two different forms of AMD: In atrophic macular
degeneration (dry form), there is pigmentary disturbance in the
macular region, but no elevated macular scar and no hemorrhage or
exudation in the region of the macula; in exudative macular
degeneration (wet form), which is much less common (approximately
10% of all cases of AMD), there is formation of a subretinal
network of choroidal neovascularization often associated with
intraretinal hemorrhage, subretinal fluid, pigment epithelial
detachment, and hyperpigmentation. Eventually, this complex
contracts and leaves a distinct elevated scar at the posterior
pole. In that respect, "wet" AMD is more visually debilitating
compared to the dry form. Both forms of age-related macular
degeneration are often bilateral and are preceded by drusen in the
macular region.
[0034] For the present invention, the "wet" form of AMD is of
primary interest as it involves ocular neovascularization. This
form of the disease occurs when a tiny frond of vessels
(capillaries) breaks through a layer of the retina known as Bruch's
membrane, and grows beneath the macula. This is known as a
choroidal neovascular membrane (CNVM). The abnormal vessels of the
CNVM may leak fluid causing a localized swelling, or worse, result
in a localized bleed. This is the condition most likely to result
in legal blindness. It is important to realize, however, that even
"wet" AMD doesn't lead to "cane blindness", given that peripheral
vision remains intact, although often legally blind (<20/200
vision). Although most patients will not lose all vision, even the
reduction is a great burden on the affected individual. Current
therapy involves appropriate laser photocoagulation, if fluorescein
angiography demonstrates a neovascular network outside the fovea.
Yet, there still remains a high degree of unmet need in treating
and preventing "wet" AMD.
1.3.5.2 (Proliferative) Diabetic Retinopathy
[0035] Diabetic retinopathy is the leading cause of acquired
blindness among Americans under the age of 65. It can be
particularly severe in persons with insulin-dependent diabetes
mellitus (IDDM; type I diabetes mellitus); it also occurs
frequently in persons with chronic non-insulin-dependent diabetes
mellitus (NIDDM; type II diabetes mellitus). The degree of
retinopathy is highly correlated with the duration of the
diabetes.
[0036] Proliferative retinopathy is characterized by abnormal new
vessel formation (neovascularization), which grows on the vitreous
surface or extends into the vitreous cavity. In advanced disease,
neovascular membranes can occur, resulting in a traction retinal
detachment. Vitreous hemorrhages may result from
neovascularization. Visual symptoms vary, depending on pathologic
events. For example, a sudden severe loss of vision can occur when
there is intravitreal hemorrhage. Visual prognosis with
proliferative retinopathy is more guarded if associated with severe
retinal ischemia, extensive neovascularization, or extensive
fibrous tissue formation.
[0037] Proliferative diabetic retinopathy (PDR) carries the
greatest risk of visual loss. The condition is characterized by the
development of neovascularization on or adjacent to the optic nerve
and vitreous or pre-retinal hemorrhage (hemorrhage in the vitreous
humor or in front of the retina). PDR usually occurs in eyes with
advanced background diabetic retinopathy and is thought to be
secondary to ischemia (lack of oxygen or blood flow) of the retina.
The neovascular vessels are abnormal and have a tendency to break
and bleed into the vitreous humor of the eye. In addition to sudden
vision loss, this may lead to more permanent complications, such as
tractional retinal detachment and neovascular glaucoma.
[0038] Patients with PDR should receive scatter laser
photocoagulation (also known as PRP, or pan-retinal
photocoagulation, a laser treatment of the ischemic peripheral
retina) as soon as possible following diagnosis of the condition.
This treatment is also known as pan-retinal laser photocoagulation.
By causing regression of the neovascular tissues, the risk of
severe vision loss is substantially reduced. PRP is an in-office or
out-patient procedure done with or without an anesthetic injection
adjacent to the eye. The laser treatment usually takes less than 30
to 45 minutes per session. A complete laser treatment, however, may
require up to 3 or 4 different sessions, with a total of one to two
thousand laser applications ("spots").
[0039] In some patients with PDR, the vitreous hemorrhage prevents
the ophthalmologist from performing the laser treatment. Simply
put, the blood is in the way of the laser beam. If the vitreous
hemorrhage fails to clear within a few weeks or months, a
vitrectomy surgery may be performed to mechanically clear the
hemorrhage and laser photocoagulation is then applied, either at
the time of the vitrectomy or shortly thereafter. Patients who have
tractional retinal detachment are usually scheduled for vitrectomy
surgery promptly.
[0040] As comparison: Nonproliferative retinopathy (formerly known
as background retinopathy) is characterized by increased capillary
permeability, microaneurysms, hemorrhages, exudates, and edema.
Visual symptoms generally do not occur in the early stages of
nonproliferative retinopathy. However, significant early visual
changes can occur in a small number of patients, especially in
those with NIDDM.
[0041] There is no cure for diabetic retinopathy, and patients
still show high unmet need: Apart from diabetes and blood pressure
control, panretinal photocoagulation may diminish or eliminate
proliferative retinopathy and neovascularization of the iris. Early
photocoagulation decreases the risk of neovascular glaucoma
development. Vitrectomy may be useful in cases of vitreous
hemorrhage.
1.3.5.3 Retinopathy of Prematurity (ROP, Also Retrolental
Fibroplasia)
[0042] ROP is a bilateral ocular disorder of abnormal retinal
vascularization in premature infants, especially those of lowest
birth weight, with outcomes ranging from normal vision to
blindness.
[0043] Retinopathy of prematurity (ROP) represents a proliferation
of abnormal retinal blood vessels that occurs almost exclusively in
prematurely delivered infants. More than 80% of infants weighing
<1 kg at birth develop ROP. The risk of ROP rises with
decreasing birth weights, and advancing neonatology now frequently
allows infants weighing under two pounds to survive. The belief
that oxygen supplementation for premature infants causes ROP is
still widely held, at least amongst the general public.
Historically, ROP was recognized to occur primarily in infants who
required high oxygen supplementation due to their immature lungs.
Once this association was made, the delivery of oxygen was
curtailed in attempt to reduce ROP. This was somewhat effective in
reducing the incidence of ROP, but the incidence of cerebral palsy
and hyaline membrane (lung) disease increased. Other risk factors
for the development of ROP include extended duration of time in
oxygen, the need for respiratory stimulants such as aminophylline
or theophylline, and maternal bleeding from the conditions known as
abruptio placentae or placenta previa.
[0044] Neonatologists now give enough oxygen to sustain life and
prevent neurological disorders, yet limit the risk of ROP. The risk
of some degree of ROP in these infants is extremely high. Because
the inner retinal blood vessels start growing about midpregnancy
and have fully vascularized the retina by full term, their growth
is incomplete in premature infants. Susceptibility to ROP varies
but correlates with the proportion of retina that remains avascular
at birth. ROP occurs when normal blood vessels in the retina fail
to reach the retinal periphery, leaving retinal tissue ischemic
(oxygen deprived). When this occurs, neovascularization may occur
at the border of the ischemic (oxygen starved) and non-ischemic
retina. The neovascular tissue may grow into the vitreous humor
resulting in tractional membranes, and cause tractional retinal
detachment if not treated. In addition, an abnormal ridge of tissue
forms between the vascularized central retina and the
nonvascularized peripheral retina. In severe ROP, these new vessels
invade the vitreous, and sometimes the entire vasculature of the
eye becomes engorged ("plus" disease). The abnormal vessel growth
often subsides spontaneously but, in about 4% of survivors weighing
<1 kg at birth, progresses to produce retinal detachments and
vision loss within 2 to 12 mo postpartum.
[0045] Children with healed ROP have a higher incidence of myopia,
strabismus, and amblyopia. A few children with moderate, healed ROP
are left with cicatricial scars (e.g., dragged retina or retinal
folds) but no initial retinal detachments and are at risk for
retinal detachments later in life.
[0046] Prevention of premature birth is the best preventive of ROP.
After a preterm birth, oxygen should be used only in amounts
sufficient to avoid hypoxia. Improved ROP prevention remains a
focus of intensive investigation worldwide. In severe ROP,
cryotherapy or laser photocoagulation to ablate the peripheral
avascular retina can halve the incidence of retinal fold or
detachment. Retinal vascularization must be closely followed at
1-to 2-week intervals until the vessels have matured sufficiently
without reaching the preconditions for cryotherapy or laser
photocoagulation to the ischemic retina. If retinal detachments
occur in infancy, scleral buckling surgery or vitrectomy with
lensectomy may be considered, but these procedures are late rescue
efforts with low benefit.
1.3.5.4 Neovascular Glaucoma
[0047] Neovascular glaucoma is a form of glaucoma that most
commonly is associated with proliferative diabetic retinopathy
(PDR) and central retinal vein occlusion (CRVO). Other causative
factors include carotid occlusive disease (carotid artery plaques
resulting in significant lumen narrowing or occlusion), central
retinal artery occlusion (CRAO), temporal arteritis, and many other
conditions that result in ischemia (reduced blood flow) of the
retina or ciliary body.
[0048] The mechanism of neovascular glaucoma is the development of
neovascularization in the angle of the eye causing obstruction to
fluid egress via the trabecular meshwork, the primary outflow
pathway of the eye. When the retina is ischemic, it is theorized
that an angiogenic factor, acting as a local hormone, is released
from the ischemic tissue and this results in the development of
neovascularization. Neovascularization occurs primarily in the
following locations: optic nerve, retina, iris, and angle of the
eye.
[0049] Ablation of the ischemic tissue via pan-retinal laser
photocoagulation (PRP), therefore, is believed to minimize the
angiogenic factor production, and the neovascular tissue
consequently regresses. Medical management, including the use of
topical eye-drop medications and steroids, is usually used until
the PRP takes effect (vessels regress). Laser ablation of the
vessels in the angle of the eye is only occasionally effective and
may be used as a temporary measure to help keep the angle open.
[0050] For those patients in whom laser photocoagulation (PRP) is
not effective, glaucoma filtration surgery, implantation of a
glaucoma drainage device (tube shunt), or cyclocryotherapy
(freezing therapy of the ciliary body, which produces aqueous
fluid) may all be effective. Laser cyclophotocoagulation, which
entails use of a laser to destroy part of the ciliary body, may
also be used.
[0051] The prevention of neovascular glaucoma is extraordinarily
important since treatment is difficult and the prognosis is
generally poor. Prophylaxis includes the use of PRP laser for
patients with both proliferative diabetic retinopathy (PDR) and
central retinal vein occlusion (CRVO) in which extensive ischemia
(reduced blood flow) is present. Unfortunately, it is not always
possible to determine which patients with these underlying
conditions will develop neovascular glaucoma, even with the most
meticulous care.
1.3.5.5 Retinal Vein Occlusion: Central Retinal Vein Occlusion
[0052] Central retinal vein occlusion (CRVO) presents with mild to
severe, sudden, painless, visual loss. The majority of patients
will either have systemic hypertension, chronic open-angle
glaucoma, or significant atherosclerosis. The final insult of these
disorders causing the CRVO is generally a thrombus of the central
retinal vein just as it enters the eye. The ophthalmologist may
find mild to severe hemorrhages and cotton-wool spots in the
retina, the latter of which indicate ischemia.
[0053] The initial vision on presentation is a good predictor of
the final visual outcome. That is, the worse the vision initially,
the worse the final visual acuity. In fact, in half of patients,
final visual acuity remains within 3 lines on the eye chart. There
are two basic classes that ophthalmologists will use to classify
CRVO's upon presentation. These are the ischemic (poor blood flow)
and non-ischemic types. The latter of these types generally has
much better vision upon presentation and fewer clinical findings on
exam.
[0054] The prognosis for the non-ischemic type of CRVO is good. The
ischemic type almost always has vision of 20/100 or worse on
initial presentation, and has a much higher risk of developing
complications. These patients must be following carefully, perhaps
every few weeks, to evaluate for signs of neovascularization both
in the retina and on the iris. Neovascularization of the retina or
optic nerve may result in bleeding (vitreous hemorrhage) and
neovascularization of the iris may result in intractable glaucoma
(high pressure failing to respond to all conventional therapy).
Both conditions, if they develop, are typically treated with laser
to the retina (pan-retinal photocoagulation) in attempt to cause
regression of the neovascularization. Eyes considered to be in the
ischemic CRVO category, may also be treated with laser
photocoagulation of the retina to prevent these dreaded
complications.
1.3.5.6 Retinal Vein Occlusion: Branch Retinal Vein Occlusion
[0055] A branch retinal vein occlusion (BRVO) may present with
decreased vision, peripheral vision loss, distortion of vision, or
"blind spots." The condition is unilateral and usually develops in
a patient with hypertension or diabetes mellitus. The cause of the
condition is a localized thrombus development in a branch retinal
vein due to arteriosclerosis (hardening of the arteries) in an
adjacent branch retinal arteriole.
[0056] The ophthalmologist will see retinal hemorrhages along the
involved retinal vein, the pattern of which nearly always leads to
the correct diagnosis. Many ophthalmologists will obtain a
fluorescein angiogram during the recovery period if
neovascularization is suspected. A fluorescein angiogram is an
extraordinarily safe, in-office diagnostic procedure, in which
fluorescein dye is administered by IV or sometimes orally, and
retinal photography is subsequently completed.
[0057] Patients are typically re-evaluated every one to two months
to evaluate for chronic macular edema (swelling) and/or
neovascularization. If macular edema persists beyond 3 to 6 months
and visual acuity is reduced below 20/40, the patient may be
treated with focal laser. For those patients who meet the
guidelines for treatment, laser photocoagulation has been shown to
improve vision and to increase the chances that final visual acuity
will be 20/40 or better. If neovascularization develops, or if the
BRVO involves a significantly large area of retina, which may
predispose to the development of neovascularization, the patient
may undergo pan-retinal laser photocoagulation. Many patients will
have resolution of the retinal hemorrhages and macular swelling,
over a several month period, with retention of good vision. For
those requiring laser treatment, the ophthalmologist will use
rather strict criteria to determine which patients will benefit
from laser treatment.
[0058] According to the above, there is still a high degree of
unmet need in the prevention, treatment and/or cure of diseases
caused by ocular neovascularization. Particularly, current
treatments are limited efficacy and associated with significant
adverse effects.
[0059] The inventors of the present invention have now found that
the Hedgehog signaling is involved in ocular neovascularization and
that interfering with Hedgehog signaling upon administration of a
Hedgehog-interference substance can prevent, inhibit and/or reverse
ocular neovascularization.
1.3.6 Hedgehog Inhibitors
[0060] The Hedgehog (Hh) pathway can be blocked at different
levels, and many Hh inhibitors are in the public domain [43-47].
They are primarily investigated as potential anti-cancer agents.
The actual nature of the Hh inhibitor used does not limit the scope
of the invention. This invention primarily addresses the
therapeutic use of substances that interfere with Hedgehog
signaling in general, and Shh signaling in particular, to prevent,
inhibit and/or revert ocular neovascularization and the
pathological consequences resulting from ocular neovascularization.
Thus, the nature of the Hh inhibitor, and the biological target of
the Hh inhibitor should not be considered as limitations of the
present invention.
[0061] Inhibition of Hh signaling has been reported with
antibodies, such as (for example) antibodies directed against Sonic
Hedgehog (Shh). Moreover, several specific Smoothened (Smo)
inhibitors have been identified such as e.g., Cyclopamine [46, 48,
49]. Some other inhibitors of Smo have been identified in two
large-scale screens for small-molecule inhibitors. These inhibitors
include several that potently block a constitutively activated form
of Smo that is known to cause BCCs (SANT1-4, Cur61414) [47]
1.3.6.1 Cyclopamine
[0062] Cyclopamine, a natural alkaloid derivative that is isolated
from a plant of the lily family Veratum californicum, represents
the first member of a class of small chemical compounds that
specifically inhibit the Hh pathway. In its preferred embodiment,
administration of cyclopamine to a mammalian subject was used to
prevent ocular neovascularization. Cyclopamine is a potent
teratogen that specifically inhibits Smo activity by binding to its
heptahelical bundle. Treatment of mice that carry Hh-dependent
tumors with cyclopamine results in growth inhibition and regression
of cancerous tissue, but does not affect the health of treated
animals. Thus it seems that Hh inhibition by cyclopamine causes
little, if any, toxic effects on cells that do not depend on Hh
signalling. Cyclopamine, however, is difficult to synthesize in
large quantities and therefore is not applicable as a therapeutic
agent for diseases where a large quantity of drug is needed (e.g.,
cancer)--a factor that might also apply to a modified and more
effective version of this compound, KAADcyclopamine. Yet, for
ocular diseases, a smaller quantity of cyclopamine is sufficient to
be therapeutically effective. Moreover, as many ocular diseases
that involve ocular neovascularization affect primarily older
humans, teratogenicity might not be a safety limitation for the
majority of patients.
[0063] Apart from its use in cancer treatment, cyclopamine has also
been applied successfully to the treatment of skin-related
disorders such as psoriasis ([39, 40]).
[0064] Cyclopamine had been in-licensed by Curis from Johns Hopkins
University School of Medicine in September 2000. In August 2004,
data were published that showed the Hh signalling pathway played an
essential role in endothelial tube formation during vasculogenesis
[50, 51]. One month later, a study published in Cancer Research
showed that cyclopamine blocks the growth of breast cancer cells
via the Hh signalling pathway [52].
1.3.6.2 Hedgehog interacting protein 1 (Hip1)
[0065] As mentioned, in response to Hh, at least two proteins that
are up-regulated: Patched1 (Ptc1), the Hh receptor, a general
target in both invertebrate and vertebrate organisms, and Hip1, a
Hh-binding protein that is vertebrate specific [53]. Hip1 stands
for Hedgehog interacting protein 1 (Hip1). It is a negative
regulator of Hh signaling. It has been shown that loss of Hip1
function results in specific defects in two Hh target issues, the
lung, a target of Sonic hedgehog (Shh) signaling, and the
endochondral skeleton, a target of Indian hedgehog (Ihh)
signaling.
[0066] Human Hip1 (SEQ ID NO:4) is encoded in humans by the HHIP
gene (GeneID: 64399; SEQ ID NO:3). HHIP comprises 13 exons and
spans >91 kb encoding a protein of .about.700 amino acids which
shares 94% sequence identity with mouse Hip1 (SEQ ID NO:2). The
sequence of the mouse cDNA for Hip1 is listed in SEQ ID NO:1. HHIP
maps to chromosome 4q31.21.fwdarw. q31.3. It is a type I
transmembrane protein [54] that binds all mammalian Hh proteins
with an affinity similar to that of Ptc1, but this binding most
likely regulates the availability of ligand, thereby attenuating
signaling rather than activating a novel pathway. Like Ptc,
expression of Hip1 is up-regulated in response to Hh signaling
[54], but unlike Ptc, there is no evidence that it acts by directly
regulating Smo. Therefore, Hip1 adds a second layer of control to
the Hh negative feedback mechanism, a layer that is exclusive to
vertebrates.
[0067] By deleting the transmembrane domain and retaining and/or
inserting a secretion signal peptide, one can create a secreted
version of Hip1 (sHip1) (SEQ ID NO:6), which is encoded by the
correspondingly modified cDNA, sHIP1 (SEQ ID NO:5). This secreted
version is still able to bind and sequester Hedgehog, thus
interfering with Hedgehog signaling. A secreted version of Hip1 has
been published in prior art [55] [56].
1.3.7 Gene Transfer
[0068] Gene transfer systems can be classified along different
dimensions [0068] (A) Nature or origin of the system [0069] (B)
Delivery mechanism; [0070] (C) Site of gene transfer (e.g., ex
vivo, in vitro, in vivo).
[0069] (A) Based on the nature or origin of the gene transfer
system, existing delivery systems for nucleic acid compositions can
be subdivided into three groups: (1) viral vectors, (2) non-viral
vectors, and (3) naked nucleic acids. Regarding vector targeting
(specificity) and efficiency, viral vector systems are superior to
conventional non-viral vectors and naked nucleic acids. On the
other hand, non-viral vectors and naked nucleic acids are safer,
easier to upscale in production and allow for the delivery of
modified nucleic acids compared to viral vectors.
[0070] (B) Alternatively, based on the delivery mechanism, gene
transfer methods fall into the following three broad categories:
(1) physical (e.g., electroporation, direct gene transfer and
particle bombardment), (2) chemical (e.g. lipid-based carriers and
other non-viral vectors) and (3) biological (e.g. virus or
bacterium derived vectors).
[0071] (C) Gene therapy methodologies can also be described by
delivery site. Fundamental ways to deliver genes include ex vivo
gene transfer, in vivo gene transfer, and in vitro gene transfer.
In ex vivo gene transfer, cells are taken from the subject and
grown in cell culture. The nucleic acid composition is introduced
into the cells, the transduced or transfected cells are (in some
instances) expanded in number and then reimplanted in the subject.
In in vitro gene transfer, the transformed cells are cells growing
in culture, such as tissue culture cells, and not particular cells
from a particular subject. These "laboratory cells" are transfected
or transduced; the transfected or transduced cells are then in some
instances selected and/or expanded for either implantation into a
subject or for other uses. In vivo gene transfer involves
introducing the nucleic acid composition into the cells of the
subject when the cells are within the subject.
[0072] Several delivery mechanisms may be used to achieve gene
transfer in vivo, ex vivo, and/or in vitro:
[0073] Mechanical (i.e. physical) methods of DNA delivery can be
achieved by direct injection of DNA, such as microinjection of DNA
into germ or somatic cells, pneumatically delivered DNA-coated
particles, such as the gold particles used in a "gene gun," and
inorganic chemical approaches such as calcium phosphate
transfection. It has been found that physical injection of plasmid
DNA into muscle cells yields a high percentage of cells that are
transfected and have a sustained expression of marker genes. The
plasmid DNA may or may not integrate into the genome of the cells.
Non-integration of the transfected DNA would allow the transfection
and expression of gene product proteins in terminally
differentiated, non-proliferative tissues for a prolonged period of
time without fear of mutational insertions, deletions, or
alterations in the cellular or mitochondrial genome. Long-term, but
not necessarily permanent, transfer of therapeutic genes into
specific cells may provide treatments for genetic diseases or for
prophylactic use. The DNA could be reinjected periodically to
maintain the gene product level without mutations occurring in the
genomes of the recipient cells. Non-integration of exogenous DNAs
may allow for the presence of several different exogenous DNA
constructs within one cell with all of the constructs expressing
various gene products. Particle-mediated gene transfer may also be
employed for injecting DNA into cells, tissues and organs. With a
particle bombardment device, or "gene gun," a motive force is
generated to accelerate DNA coated high-density particles (such as
gold or tungsten) to a high velocity that allows penetration of the
target organs, tissues or cells. Electroporation for gene transfer
uses an electrical current to make cells or tissues susceptible to
electroporation-mediated gene transfer. A brief electric impulse
with a given field strength is used to increase the permeability of
a membrane in such a way that DNA molecules can penetrate into the
cells. The techniques of particle-mediated gene transfer and
electroporation are well known to those of ordinary skill in the
art.
[0074] Chemical methods of gene therapy involve carrier mediated
gene transfer through the use of fusogenic lipid vesicles such as
liposomes or other vesicles for membrane fusion. A carrier
harboring a DNA of interest can be conveniently introduced into
body fluids or the bloodstream and then site specifically directed
to the target organ or tissue in the body. Liposomes, for example,
can be developed which are cell specific or organ specific. The
foreign DNA carried by the liposome thus will be taken up by those
specific cells. Injection of immunoliposomes that are targeted to a
specific receptor on certain cells can be used as a convenient
method of inserting the DNA into the cells bearing the receptor.
Another carrier system that has been used is the
asialoglycoprotein/polylysine conjugate system for carrying DNA to
hepatocytes for in vivo gene transfer. Transfected DNA may also be
complexed with other kinds of carriers so that the DNA is carried
to the recipient cell and then resides in the cytoplasm or in the
nucleoplasm of the recipient cell. DNA can be coupled to carrier
nuclear proteins in specifically engineered vesicle complexes and
carried directly into the nucleus. Carrier mediated gene transfer
may also involve the use of lipid-based proteins which are not
liposomes. For example, lipofectins and cytofectins are lipid-based
positive ions that bind to negatively charged DNA, forming a
complex that can ferry the DNA across a cell membrane. Another
method of carrier mediated gene transfer involves receptor-based
endocytosis. In this method, a ligand (specific to a cell surface
receptor) is made to form a complex with a gene of interest and
then injected into the bloodstream; target cells that have the cell
surface receptor will specifically bind the ligand and transport
the ligand-DNA complex into the cell.
[0075] Biological gene therapy methodologies usually employ viral
vectors to insert genes into cells. The transduced cells may be
cells derived from the patient's normal tissue, the patient's
diseased tissue, or may be non-patient cells. Viral vectors that
have been used for gene therapy protocols include but are not
limited to, retroviruses, lentivruses, other RNA viruses such as
pollovirus or Sindbis virus, adenovirus, adeno-associated virus,
herpes viruses, simian virus 40, vaccinia and other DNA
viruses.
[0076] Replication-defective murine retroviral vectors are commonly
utilized gene transfer vectors. Murine leukemia retroviruses are
composed of a single strand RNA complexed with a nuclear core
protein and polymerase (pol) enzymes, encased by a protein core
(gag) and surrounded by a glycoprotein envelope (env) that
determines host range. The genomic structure of retroviruses
include the gag, pol, and env genes flanked by 5' and 3' long
terminal repeats (LTR). Retroviral vector systems exploit the fact
that a minimal vector containing the 5' and 3' LTRs and the
packaging signal are sufficient to allow vector packaging,
infection and integration into target cells providing that the
viral structural proteins are supplied in trans in the packaging
cell line. Fundamental advantages of retroviral vectors for gene
transfer include efficient infection and gene expression in most
dividing cell types, precise single copy vector integration into
target cell chromosomal DNA, and ease of manipulation of the
retroviral genome. For example, altered retrovirus vectors have
been used in ex vivo and in vitro methods to introduce genes into
peripheral and tumor-infiltrating lymphocytes, hepatocytes,
epidermal cells, myocytes, or other somatic cells (which may then
be introduced into the patient to provide the gene product from the
inserted DNA). For descriptions of various retroviral systems, see,
e.g., U.S. Pat. No. 5,219,740; [49-53]. The main disadvantage of
retroviral systems is that retroviral vectors can only infect
dividing cells. Lentiviral vectors overcome this limitation.
Nevertheless, production of retro-and lentiviral vectors is
complex, and the virions are not very stable compared to other
viruses. More recently, the danger of inducing cancer through
insertional mutagenesis has been raised as a major safety concern
[54] [55].
[0077] A number of adenovirus based gene delivery systems have also
been developed. Human adenoviruses are double stranded, linear DNA
viruses with a protein capsid that enter cells by receptor-mediated
endocytosis. Adenoviral vectors have a broad host range and are
highly infectious, even at low virus titers. Moreover, adenoviral
vectors can accommodate relatively long transgenes compared to
other systems. A number of adenovirus based gene delivery systems
have also been described [56-62]. The main limitation of adenoviral
vectors is their high degree of immunogenicity, which limits their
use in respect to applications that require long-term gene
expression.
[0078] For many applications, long-term gene expression (over
several years) will have to be achieved. This is also the case for
the present invention. So far, primarily adeno-associated virus
based vectors allow for this. Most other viral vectors are limited
by expression of viral genes so that transduced cells will be
eliminated by the immune system (e.g., adenoviral vectors), gene
silencing (retroviral vectors or lentiviral vectors) or
questionable safety profile (e.g., retroviral vectors or adenoviral
vectors).
1.3.7.1 Adeno-Associated Viral Vectors
[0079] The present invention uses adeno-associated virus-based
vectors [63] [64] [65] for the transfer of an RNAi expression
cassette into the appropriate target cells of a mammalian subject
in vivo.
[0080] Adeno associated virus (AAV) is a small nonpathogenic virus
of the parvoviridae family. AAV is distinct from the other members
of this family by its dependence on a helper virus for replication.
The approximately 5 kb genome of AAV consists of single stranded
DNA of either plus or minus polarity. The ends of the genome are
short inverted terminal repeats (ITRs), which can fold into hairpin
structures and serve as the origin of viral DNA replication.
Physically, the parvovirus virion is non-enveloped and its
icosohedral capsid is approximately 20 nm in diameter. To date, at
least 8 serologically distinct AAVs have been identified and
isolated from humans or primates and are referred to as AAV types
1-8. The most extensively studied of these isolates are AAV type 2
(AAV2) and AAV type 5 (AAV5).
[0081] The genome of AAV2 is 4680 nucleotides in length and
contains two open reading frames (ORFs). The left ORF encodes the
non-structural Rep proteins, Rep40, Rep52, Rep68 and Rep78, which
are involved in regulation of replication and transcription in
addition to the production of single-stranded progeny genomes.
Furthermore, two of the Rep proteins have been associated with the
preferential integration of AAV2 genomes into a region of the q arm
of human chromosome 19. Rep68/78 have also been shown to possess
NTP binding activity as well as DNA and RNA helicase activities.
The Rep proteins possess a nuclear localization signal as well as
several potential phosphorylation sites. Mutation of one of these
kinase sites resulted in a loss of replication activity. The ends
of the genome are short inverted terminal repeats, which have the
potential to fold into T-shaped hairpin structures that serve as
the origin of viral DNA replication. Within the ITR region two
elements have been described which are central to the function of
the ITR, a GAGC repeat motif and the terminal resolution site
(trs). The repeat motif has been shown to bind Rep when the ITR is
in either a linear or hairpin conformation. This binding serves to
position Rep68/78 for cleavage at the trs, which occurs in a site-
and strand-specific manner. In addition to their role in
replication, these two elements appear to be central to viral
integration. Contained within the chromosome 19 integration locus
is a Rep binding site with an adjacent trs. These elements have
been shown to be functional and necessary for locus specific
integration.
[0082] The right ORF of AAV2 encodes related capsid proteins
referred to as VP1, 2 and 3. These capsid proteins form the
icosahedral, non-enveloped virion particle of .about.20 nm
diameter. VP1, 2 and 3 are found in a ratio of 1:1:10. The capsid
proteins differ from each other by the use of alternative splicing
and an unusual start codon. Deletion analysis has shown that
removal or alteration of VP1, which is translated from an
alternatively spliced message, results in a reduced yield of
infectious particles. Mutations within the VP3 coding region result
in the failure to produce any single-stranded progeny DNA or
infectious particles.
[0083] The findings described in the context of AAV2 are generally
applicable to other AAV serotypes as well.
[0084] The following features of AAV have made it an attractive
vector for gene transfer. AAV vectors possess a broad host range
[66], transduce both dividing and non dividing cells in vitro and
in vivo and maintain high levels of expression of the transduced
genes in the absence of a significant immune response to the
transgene product in general. Moreover, as wild-type AAV is
non-pathogenic, AAV vector particles are assumed to be
non-pathogenic as well (in contrast to adenoviral vectors). Viral
particles are heat stable, resistant to solvents, detergents,
changes in pH and temperature. The ITRs have been shown to be the
only cis elements required for replication and packaging and may
contain some promoter activities. Thus, AAV vectors encode no viral
genes.
2. SUMMARY OF THE INVENTION
[0085] The following paragraphs will provide a summary of the
invention.
2.1 Substance or General Idea of the Claimed Invention
[0086] The present invention relates to methods for preventing,
inhibiting, and/or reversing ocular neovascularization by
interfering with the Hedgehog signaling pathway. Ocular
neovascularization is the leading cause of blindness in developed
countries. It is involved in many pathologies such as (wet)
age-related macular degeneration, (proliferative) diabetic
retinopathy, neovascular glaucoma, retinal vein occlusion or
retinopathy of prematurity (ROP). Yet, there is still a high degree
of unmet need in the prevention, treatment and/or cure of diseases
caused by ocular neovascularization.
[0087] Interference with the Hedgehog signaling pathway is achieved
by administering to a mammalian subject a substance that interferes
with the Hedgehog signaling pathway. In one embodiment, cyclopamine
interferes with the Hedgehog signaling pathway by inhibiting Smo
function, thus preventing ocular neovascularization in a mammalian
subject. In another embodiment, In another embodiment, a modified,
secreted form of Hip1 is either injected in the eye of a mammalian
subject, or is expressed by a gene transfer vector in the eye of a
mammalian subject. Said modified, secreted form of Hip1 sequesters
Hedgehog outside the cell, thus inhibiting binding of Hedgehog to
its receptor, Ptc1, and interfering with Hedgehog signaling.
[0088] The inventors are the first to show [0091] (1) The
involvement of Hedgehog signaling in ocular neovascularization, and
[0092] (2) That interfering with Hedgehog signaling can prevent,
inhibit and/or reverse ocular neovascularization. Upon
administration of a Hedgehog-interference substance, ocular
neovascularization will be prevented, inhibited and/or reversed,
thus preventing and/or ameliorating the pathologies associated with
ocular neovascularization. In one embodiment, the inventors prove
interference with the Hedgehog signaling pathway by administration
of cyclopamine to a mammalian subject in a model of choroidal
neovascularization.
[0089] Also provided are pharmaceutical kits containing the
Hedgehog-signaling interfering substance in a suitable
pharmaceutical suspension for administration.
[0090] We disclose that the method of the present invention [0095]
(1) Is effective in preventing, inhibiting, and/or reversing ocular
neovascularization in a mammalian subject in vivo [0096] (2) Will
lead to a therapeutic benefit for a mammalian subject suffering
from a disease caused by ocular neovascularization such as (wet)
age-related macular degeneration, (proliferative) diabetic
retinopathy, neovascular glaucoma, retinal vein occlusion or
retinopathy of prematurity (ROP). [0097] (3) Will achieve the
effects listed in (1) and (2) by interfering with the Hedgehog
signaling pathway.
[0091] A significant aspect of the present invention relates to the
demonstration that interfering with Hedgehog signaling in vivo in
mammalian subjects by administering a substance that interferes
with Hedgehog signaling is able to prevent, inhibit, and/or reverse
ocular neovascularization. This had not been previously described
in the art. Thus, the present invention provides, for the first
time, a demonstration of the importance of interfering with the
Hedgehog signaling pathway to prevent, inhibit, and/or reverse
ocular neovascularization. In the preferred embodiment, the
inventors prevent ocular neovascularization by administering
cyclopamine to the eye of a mammalian subject in vivo. Cyclopamine
is an inhibitor of Smo, and thus interferes with Hedgehog
signaling, particularly Sonic Hedgehog signaling.
[0092] Also disclosed are pharmaceutical kits containing a
substance that interferes with Hedgehog signaling in a suitable
pharmaceutical suspension for administration. In this aspect, the
invention provides a pharmaceutical kit for delivery of said
substance. The kit may contain a container for administration of a
predetermined dose. The kit further may contain a suspension
containing the substance that interferes with Hedgehog signaling
for delivery of a predetermined dose, said suspension comprising
[0100] (a) the substance that interferes with Hedgehog signaling,
and [0101] (b) a physiologically compatible carrier.
[0093] In one embodiment, the Hedgehog-signaling interfering
substance will be administered to the eye by intravitreal
injection. In another embodiment, the Hedgehog-signaling
interfering substance will be administered to the eye by subretinal
injection. In yet another embodiment, the Hedgehog-signaling
interfering substance is formulated in a suspension that can be
topically applied to the eye, either in the form of a creme or in
the form of eye drops. Many different formulations and ways of
administrations can be envisioned, and should not be considered
limiting on the scope of the present invention.
[0094] Practice of the present invention will provide useful
medical applications as described below under "Utility Of The
Present Invention". In summary, the invention provides a method for
treating a mammalian subject with a disease caused in total or in
part by ocular neovascularization by administering to the mammalian
subject in vivo a substance that interferes with the Hedgehog
signaling pathway in general, and the Shh signaling pathway in
particular. In its preferred embodiment, the mammalian subject is a
human patient, and the substance that interferes with Shh signaling
is cyclopamine, which inhibits Shh signaling by inhibiting Smo
function.
[0095] This invention also provides a method of treating a subject
having an eye disorder ameliorated by interfering with the Hedgehog
signaling pathway, comprising administering to the subject a
therapeutically effective amount of the instant pharmaceutical
composition comprising a substance that interferes with Hedgehog
signaling.
[0096] Moreover, this invention also provides a method of
inhibiting in a subject the onset of an eye disorder ameliorated by
interfering with the Hedgehog signaling pathway, comprising
administering to the subject a prophylactically effective amount of
the instant pharmaceutical composition comprising a substance that
interferes with Hedgehog signaling.
[0097] The exact nature of the substance that interferes with
Hedgehog signaling does not limit the scope of the invention: Said
substance can be a small molecule (such as cyclopamine), an
antibody (such as 5E1), a protein or protein fragment, an aptamer,
a small interfering RNA, a ribozyme, an antisense nucleic acid
molecule, a soluble receptor, a modified version of a Hedgehog
protein, a peptoid, or any other substance that interferes with
Hedgehog signaling. In case of proteins and nucleic acids: These
substances might be either directly administered to the patient, or
expressed within the patient by using a recombinant gene transfer
vector such as an AAV vector.
[0098] Moreover, the therapeutic of the present invention can be
administered to the subject by many means. The exact method chosen
should not limit the scope of the present invention. For example,
the therapeutic of the present invention can be injected
intravitreally or subretinally into the eye, it could be applied
topically (e.g., as a creme), or it can be administered in the form
of eye drops. In preferred embodiments, the therapeutic of the
invention is applied locally to the eye.
[0099] Similarly, the therapeutic of the present invention can be
formulated in different forms and suspended in a multitude of
pharmaceutically acceptable carriers. The choice of formulation and
pharmaceutical carrier should not limit the scope of the present
invention.
[0100] To summarize: The present invention describes for the first
time that interference with Hedgehog signaling in general, and Shh
signaling in particular, prevents, inhibits, and/or reverses ocular
neovascularization, a process involved in many eye-related
pathologies. Interference with Hedgehog signaling in general, and
sonic Hedgehog signaling in particular will exert a therapeutic
benefit on the individual treated with a Hedgehog-signaling
interference substance. The exact nature or composition of said
substance, its formulation and suspension, its way of
administration should not limit the scope of this invention: As
long as a substance--including a combination of
substances--interferes with Hedgehog signaling in general, and
Sonic Hedgehog signaling in particular, to inhibit, prevent, and/or
reverse ocular neovascularization in a mammalian subject, it should
be considered claimed by the present invention. Whereas the
inventors describe the usefulness of the present invention for a
range of eye diseases with high unmet need--such as (wet)
age-related macular degeneration, (proliferative) diabetic
retinopathy, neovascular glaucoma, retinal vein occlusion, or
retinopathy of prematurity (ROP)--it should be understood that the
artisan will be able to identify further eye diseases whose
pathology involve ocular neovascularization. The exact nature of
the eye disease should not be considered a limitation of the
present invention: All eye pathologies that involve ocular
neovascularization in their aetiology should be considered claimed
in the present invention.
2.2 Advantages of the Invention Over Prior Approaches
[0101] Ocular neovascularization is the leading cause of blindness
in developed countries due to its involvement in multiple
pathologies such as (wet) age-related macular degeneration,
(proliferative) diabetic retinopathy, neovascular glaucoma, retinal
vein occlusion, or retinopathy of prematurity (ROP). Yet, there is
still a high degree of unmet need in the prevention, treatment
and/or cure of diseases caused by ocular neovascularization.
[0102] The present invention relates to methods for preventing,
inhibiting, and/or reversing ocular neovascularization by
interfering with the Hedgehog signaling pathway by administering to
a mammalian subject a substance that interferes with the Hedgehog
signaling pathway. In the preferred embodiments, the
Hedgehog-signaling interfering substance is formulated in an
acceptable pharmaceutical carrier and/or suspension and provided in
a pharmaceutical kit.
[0103] Thus, the present invention is of great medical use to
address the high unmet need in eye disorders whose aetiology
involve ocular neovascularization. By making use of the present
invention, a therapeutic benefit can be achieved in a mammalian
individual (including a human being) suffering from or being at
risk of an eye disease that involves ocular neovascularization in
its pathological process.
[0104] The present invention is also novel in the respect that the
inventors are the first to show [0114] (1) The involvement of
Hedgehog signaling in general, and Shh signaling in particular, and
Smo activity in detail, in ocular neovascularization, and [0115]
(2) That interfering with Hedgehog signaling in general, Shh
signaling in particular, and Smo activity in detail, can prevent,
inhibit and/or reverse ocular neovascularization: Upon
administration of a Hedgehog-signaling-interference substance,
ocular neovascularization will be prevented, inhibited and/or
reversed, thus preventing and/or ameliorating the pathologies
associated with ocular neovascularization. In one embodiment,
cyclopamine is used as to interfere with Hedgehog signaling in
general, and Shh signaling in particular. Cyclopamine interferes
with Shh signaling by inhibiting Smo function, thus preventing
and/or inhibiting ocular neovascularization, e.g., in an animal
model of choroidal neovascularization.
[0105] It has been shown in prior art (Pola et al.) that cells in
the adult vasculature both express ptc1 and can respond to
exogenous Hedgehog. Pola et al. also were able to prove that
Hedgehog is able to induce robust neovascularization in the corneal
pocket model of angiogenesis. In the same publication, the group
found that the Hedgehog-signaling pathway is present in adult
cardiovascular tissues and can be activated in vivo. Shh was able
to induce robust angiogenesis, characterized by distinct
large-diameter vessels. Shh also augmented blood-flow recovery and
limb salvage following operatively induced hind-limb ischemia in
aged mice. In summary, the group discovered a novel role for Shh as
an indirect angiogenic factor that regulates expression of multiple
angiogenic cytokines--indicating a potential therapeutic use of Shh
for ischemic disorders.
[0106] However, whereas this group has shown a role for Shh in
ischemic diseases, there has not yet been reports of the
involvement of Hedgehog in general and Shh in particular in ocular
neovascularization. Moreover, there have not yet been reports that
interfering with Hedgehog signaling in general and Shh signaling in
particular, and interference by inhibiting Smo activation in
detail, will inhibit, prevent, and/or revert ocular
neovascularization and the pathological processes involved with it.
The inventors are the first to show that particular effect, thus
providing an advancement and improvement in the art over prior
knowledge.
[0107] Prior art also describes a role for inhibiting Hedgehog in
cancer and skin-related diseases such as Psoriasis. It has also
been published that cyclopamine is able of inhibiting Smo function,
and thus inhibiting Shh signaling. However, it has not yet been
published that interfering with the Hedgehog signaling pathway is
able to prevent, inhibit, and/or revert ocular neovascularization.
The inventors are the first to show that particular effect, thus
providing an advancement and improvement in the art over prior
knowledge.
[0108] Furthermore, the inventors are the first to show a
therapeutic benefit of using a Hedgehog-signaling-interfering
substance in a mammalian subject to prevent, inhibit and/or reverse
ocular neovascularization in vivo in an animal model of choroidal
neovascularization (CNV). Prior art has not yet described the
involvement of Shh signaling in ocular neovascularization, and that
cyclopamine can be used to modulate ocular neovascularization.
3. DETAILED DESCRIPTION OF THE INVENTION
[0109] The present invention relates to methods of preventing,
inhibiting, and/or reversing ocular neovascularization in a
mammalian subject comprising administering to the subject a
therapeutically effective amount of a substance that interferes
with the Hedgehog signaling pathway. Ocular neovascularization is a
major cause of blindness in developed countries. It is causally
involved in many ocular diseases including age-related macular
degeneration, (proliferative) diabetic retinopathy, neovascular
glaucoma, retinal vein occlusion or retinopathy of prematurity
(ROP). Current treatments are of limited efficacy and associated
with significant adverse effects, reflecting the high unmet need in
those disease areas.
[0110] More specifically, the invention relates to the use of a
small molecule such as cyclopamine, or a protein such as a
(humanized) monoclonal antibody to interfere with Hedgehog
signaling in general, and Shh signaling in particular. The
inventors demonstrate in the present invention that interference
with Hedgehog signaling by means of e.g., cyclopamine, is able to
prevent, inhibit and/or reverse ocular neovascularization in a
mammalian subject, which will have a therapeutic benefit on said
subject.
[0111] Also provided are pharmaceutical kits containing the
substance that interferes with Hedgehog signaling in a suitable
pharmaceutical suspension for administration.
[0112] The practice of the present invention will employ, unless
otherwise indicated, conventional methods of virology, chemistry,
microbiology, molecular biology and recombinant DNA techniques
within the skill of the art. Such techniques are explained fully in
the literature; see, e.g., Sambrook, et al. Molecular Cloning: A
Laboratory Manual (Current Edition); DNA Cloning: A Practical
Approach, vol. I & II (D. Glover, ed.); Oligonucleotide
Synthesis (N. Gait, ed., Current Edition); Nucleic Acid
Hybridization (B. Hames & S. Higgins, eds., Current Edition);
Transcription and Translation (B. Hames & S. Higgins, eds.,
Current Edition); CRC Handbook of Parvoviruses, vol. I & II (P.
Tijessen, ed.); Fundamental Virology, 2nd Edition, vol. I & II
(B. N. Fields and D. M. Knipe, eds.)
[0113] It is specific object of the present invention the use of
compounds that interfere with the hedgehog signaling pathway for
the manufacture of a medicament for preventing, inhibiting, and/or
reversing ocular diseases related with the ocular
neovascularization.
[0114] The compounds that interfere with the Hedgehog signaling
pathway can be selected from the group comprising a small molecule,
for example with a molecular weight of less than 2000 Daltons,
protein, peptoid, and/or nucleic acid.
[0115] Small molecules, according to the present invention can be
selected from the group consisting of steroidal alkaloids, compound
A, SANT1, SANT2, SANT3, SANT4, Cur61414, Forskolin, tomatidine,
AY9944, triparanol, compound B and functionally effective
derivative thereof.
[0116] Steroidal alkaloids can be selected from the group
consisting of cyclopamine or a functionally effective derivative
thereof, KAAD-cyclopamine, jervine, aldosterone, androstane,
androstene, androstenedione, androsterone, cholecalciferol,
cholestane, cholic acid, corticosterone, cortisol, cortisol
acetate, cortisone, cortisone acetate, deoxycorticosterone,
digitoxigenin, ergocalciferol, ergosterol, estradiol-17-a,
estradiol-17-.beta., estriol, estrane, estrone, hydrocortisone,
lanosterol, lithocholic acid, mestranol, .beta.-methasone,
prednisone, pregnane, pregnenolone, progesterone, spironolactone,
testosterone, triamcinolone and their derivatives.
[0117] In addition, small molecule can be represented by general
formula (I) (US patent application N. 20030022819)
##STR00001##
wherein, as valence and stability permit, R.sub.1 and R.sub.2,
independently for each occurrence, represent H, lower alkyl, aryl
(e.g., substituted or unsubstituted), aralkyl (e.g., substituted or
unsubstituted, e.g., --(CH.sub.2)naryl), or heteroaryl (e.g.,
substituted or unsubstituted), or heteroaralkyl (e.g., substituted
or unsubstituted, e.g., --(CH.sub.2).sub.nheteroaralkyl-); L,
independently for each occurrence, is absent or
represents--(CH.sub.2).sub.n-alkyl, -alkenyl-, -alkynyl-,
--(CH.sub.2).sub.nalkenyl-, --(CH.sub.2).sub.nalkynyl-,
--(CH.sub.2).sub.nO(CH.sub.2).sub.p--,
--(CH.sub.2).sub.nNR.sub.2(CH2).sub.p--, --(CH.sub.2).sub.n
S(CH.sub.2).sub.p--, --(CH.sub.2).sub.n alkenyl(CH.sub.2).sub.p--,
--(CH.sub.2).sub.n alkynyl(CH.sub.2).sub.p--,
--O(CH.sub.2).sub.n--, --NR.sub.2(CH.sub.2).sub.n--, or
--S(CH.sub.2).sub.n--; X.sub.1 and X.sub.2 can be selected,
independently, from --N(R.sub.8)--, --O--, --S--, --Se--, --N--N--,
--ON--CH--, --(R.sub.8)N--N(R.sub.8)--, --ON(R.sub.8)--, a
heterocycle, or a direct bond between L and Y.sub.1 or Y.sub.2,
respectively; Y.sub.1 and Y.sub.2 can be selected, independently,
from --C(--O)--, --C(--S)--, --S(O.sub.2)--, --S(O)--,
--C(--NCN)--, --P(--O)(OR.sub.2)--, a heteroaromatic group, or a
direct bond between X.sub.1 and Z.sub.1 or X.sub.2 and Z.sub.2,
respectively; Z.sub.1 and Z.sub.2 can be selected, independently,
from --N(R.sub.8)--, --O--, --S--, Se--, --N--N--, --ON--CH--,
--R.sub.8N--NR.sub.8--, --ONR.sub.8--, a heterocycle, or a direct
bond between Y.sub.1 or Y.sub.2, respectively, and L; R.sub.8,
independently for each occurrence, represents H, lower alkyl,
--(CH.sub.2)naryl (e.g., substituted or unsubstituted),
--(CH.sub.2)nheteroaryl (e.g., substituted or unsubstituted), or
two R.sub.8 taken together may form a 4-to 8-membered ring, e.g.,
with X.sub.1 and Z.sub.1 or X.sub.2 and Z.sub.1, which ring may
include one or more carbonyls; prepresents, independently for each
occurrence, an integer from 0 to 10, preferably from 0 to 3; and n,
individually for each occurence, represents an integer from 0 to
10, preferably from 0 to 5.
[0118] Small molecule can be also represented by general formula
(II) (US patent application N. 20030022819):
##STR00002##
wherein, as valence and stability permit, R.sub.1, R.sub.2,
R.sub.3, and R.sub.4, independently for each occurrence, represent
H, lower alkyl, --(CH.sub.2).sub.naryl (e.g., substituted or
unsubstituted), or --(CH.sub.2).sub.nheteroaryl (e.g., substituted
or unsubstituted); L, independently for each occurrence, is absent
or represents--(CH.sub.2).sub.n--, -alkenyl-, -alkynyl-,
--(CH.sub.2).sub.nalkenyl-, --(CH.sub.2).sub.n alkynyl-,
--(CH.sub.2).sub.nO(CH.sub.2).sub.p--,
--(CH.sub.2).sub.nNR.sub.8(CH.sub.2).sub.p--,
--(CH.sub.2).sub.nS(CH.sub.2).sub.p--,
--(CH.sub.2).sub.nalkenyl(CH.sub.2).sub.p--,
--(CH.sub.2).sub.nalkynyl(CH.sub.2).sub.p--, --O(CH.sub.2).sub.n--,
--NR.sub.8(CH.sub.2).sub.n--, or --S(CH.sub.2).sub.n; X and D,
independently, can be selected from --N(R.sub.8)--, --O--, --S--,
--(R.sub.8)N--N(R.sub.8)--, --ON(R.sub.8)--, or a direct bond; Y
and Z, independently, can be selected from O or S; E represents O,
S, or NR.sub.5, wherein R.sub.5 represents LR.sub.8 or --(C--O)
LR.sub.8. R.sub.8, independently for each occurrence, represents H,
lower alkyl, --(CH.sub.2).sub.naryl (e.g., substituted or
unsubstituted), --(CH.sub.2).sub.nheteroaryl (e.g., substituted or
unsubstituted), or two R.sub.8 taken together may form a 4-to
8-membered ring; p represents, independently for each occurrence,
an integer from 0 to 10, preferably from 0 to 3; n, individually
for each occurrence, represents an integer from 0 to 10, preferably
from 0 to 5; and q and r represent, independently for each
occurrence, an integer from 0-2.
[0119] The nucleic acid is selected from the group consisting of
(antisense) oligonucleotides, ribozymes, aptamers, and/or small
interfering (ribo)nucleic acids.
[0120] Particularly, the nucleic acid can encode a polypeptide and
is introduced into the eye by means of a viral vector, a non-viral
vector and/or naked DNA.
[0121] The nucleic acid can be an isolated small interfering RNA
comprising a sense RNA strand and an antisense RNA strand, wherein
the sense and antisense RNA strands form an RNA duplex, and wherein
the sense RNA strand comprises the nucleotide sequence SEQ ID NO:16
and the antisense RNA strand comprises the nucleotide sequence SEQ
ID NO:15.
[0122] In addition, the nucleic acid can be an isolated small
interfering RNA comprising a sense RNA strand and an antisense RNA
strand, wherein the sense and antisense RNA strands form an RNA
duplex, and wherein the sense RNA strand comprises the nucleotide
sequence SEQ ID NO:17 and the antisense RNA strand comprises the
nucleotide sequence SEQ ID NO:18.
[0123] The nucleic acid can be also an isolated small interfering
RNA comprising a sense RNA strand and an antisense RNA strand,
wherein the sense and antisense RNA strands form an RNA duplex, and
wherein the sense RNA strand comprises the nucleotide sequence SEQ
ID NO:19 and the antisense RNA strand comprises the nucleotide
sequence SEQ ID NO:20.
[0124] Typically, the siRNA of the invention is administered to a
mammalian subject at a concentration of about 10 to 200 mg/ml, or
about 100 to 1,000 nM. Application can be performed either
topically to the eye (topical instillation) in a volume from about
5 microliters to about 75 microliters, for example from about 7
microliters to about 50 microliters (in case of a mouse), or in a
volume of about 50 microliters to 500 microliters, for example from
about 100 microliters to about 250 microliters. Alternatively, the
siRNA of the invention can be administered by intravitreal or
subretinal injection in a volume of 1 microliter to about 5
microliters (in case of a mouse), or in a volume of about 300
microliters to 1,000 microliters (in case of a human). One of
ordinary skill in the art will be able to determine the appropriate
volume as a function of the subject to be treated and the route of
administration.
[0125] Substance that interferes with the Hedgehog signaling
pathway can be also a decoy protein which can be in a secreted form
of the Hedgehog interacting protein 1 (Hip1) or a homologous
protein.
[0126] The soluble Hip1 protein can be encoded in a transgene
cassette of a gene transfer vector with the administration of said
gene transfer vector to a mammalian subject resulting in the
expression and secretion of soluble Hip1. Said gene transfer vector
can be a recombinant adeno-associated viral vector.
[0127] The protein can be a mutant Hedgehog protein, an antibody
homolog directed against patched and/or smoothened. Said antibody
homolog can be a monoclonal antibody 5E1 or a monoclonal antibody
that binds the same epitope as 5E1 (5E1, Developmental Studies
Hybridoma Bank, Karen Jensen, Department of Biological Sciences,
The University of Iowa, 007 Biology Building East, Iowa City, Iowa
52242, tel: (319)335-3826, fax: (319)335-2077, 5 .mu.l available
for order on website: www.uiowa.edu/-dshbwww/l*ndex.html, e-mail:
dshb@uiowa.edu)
[0128] The above-mentioned compounds can interfere with the
different Hedgehog signaling, i.e. Sonic (Shh), Desert (Dhh) or
Indian (Ihh) Hedgehog signaling and can interact by means of
different mechanisms. Particularly, the compounds according to the
present invention can interact with Hedgehog receptor, patched
(Ptc), more particularly with the mammalian Hedgehog receptors Ptc1
and/or Ptc2, with the Hedgehog co-receptor smoothened (Smo), with
the serine-threonine kinase fused (Fu) or with the transcription
factor Gli1, Gli2 and/or Gli3.
[0129] Ocular diseases related with the ocular neovascularization
that can be treated according to the present invention are
represented by, for example, age-related macular degeneration,
diabetic retinopathy, retinopathy of prematurity (ROP), neovascular
glaucoma, retinal vein occlusion.
[0130] The medicament that can be used according to the present
invention can be suitable for and/or administered by local
administration, topical administration, systemic administration,
intravitreal injection, subtretinal injection, intravitreal
administration, intracavity injection, intra-arterial
administration, intravenous administration, intramuscular
administration, injection into tissue, injection into gaps in
tissue or inhalation and/or nasal instillation. In addition, the
compounds that interfere with the hedgehog signaling pathway can be
associated with agents modulating cAMP level.
[0131] The practice of the present invention will employ, unless
otherwise indicated, conventional methods of virology, chemistry,
microbiology, molecular biology and recombinant DNA techniques
within the skill of the art. Such techniques are explained fully in
the literature; see, e.g., Sambrook, et al. Molecular Cloning: A
Laboratory Manual (Current Edition); DNA Cloning: A Practical
Approach, vol. I & II (D. Glover, ed.); Oligonucleotide
Synthesis (N. Gait, ed., Current Edition); Nucleic Acid
Hybridization (B. Hames & S. Higgins, eds., Current Edition);
Transcription and Translation (B. Hames & S. Higgins, eds.,
Current Edition); CRC Handbook of Parvoviruses, vol. I & II (P.
Tijessen, ed.); Fundamental Virology, 2nd Edition, vol. I & II
(B. N. Fields and D. M. Knipe, eds.)
3.1 DEFINITIONS
[0132] In describing the present invention, the following terms
will be employed, and are intended to be defined as indicated
below.
[0133] For purposes of this invention, the "vascular development"
comprises at least two steps: "vasculogenesis" and physiological
angiogenesis. The term "vasculogenesis" refers to process, where
endothelial cells form a primary tubular network, whereas
"angiogenesis" refers to a process, in which vessel size and
structure are modified, and branching occurs to insure that all
cells are supplied with sufficient nutrients. Thus, "angiogenesis"
is defined as any alteration of an existing vascular bed or the
extension of existing vasculature which benefits tissue
perfusion.
[0134] For purposes of this invention, pathological (abnormal)
vasculogenesis is referred to as "neovascularization".
Neovascularization--in a more narrow definition--is a form of
pathologic, abnormal vasculogenesis in the eye ("ocular
neovascularization"). It leads to visual loss through increased
vascular permeability leading to retinal oedema, (b) vascular
fragility resulting in haemorrhage, or fibro-vascular proliferation
with tractional and rhegmatogenous retinal detachment. Choroidal
neovascularization is used synonymously with ocular
neovascularization. Furthermore, for purposes of this invention,
angiogenesis can be either physiological or pathological.
Furthermore, for purposes of this invention, "angiogenesis" can be
either physiological or pathological.
[0135] For purposes of this invention, the term "Hedgehog" is used
to refer generically to any member of the Hedgehog family,
including sonic, indian, desert and tiggy winkle. The term may be
used to indicate a protein or gene.
[0136] For purposes of this invention, the term "Hedgehog
transduction pathway" are all used to refer to the chain of events
normally mediated by Hedgehog, smoothened, ptc, and gli, among
others, and resulting in a changes in gene expression and other
phenotypic changes typical of Hedgehog activity. The Hedgehog
pathway can be activated even in the absence of a Hedgehog protein
by activating a downstream component. For example, overexpression
of smoothened will activate the pathway in the absence of Hedgehog.
gli and ptc gene expression are indicators of an active Hedgehog
signaling pathway.
[0137] For purposes of this invention, the term "Hedgehog
antagonist" refers to an agent that potentiates or recapitulates
the bioactivity of patched, such as to repress transcription of
target genes. In other words: A hedgehog antagonist is a substance
that interferes with the Hedgehog signaling pathway. The term
"Hedgehog antagonist" as used herein refers not only to any agent
that may act by directly inhibiting the normal function of the
Hedgehog protein, but also to any agent that inhibits the Hedgehog
signalling pathway, and thus recapitulates the function of ptc. A
Hedgehog antagonist may be a small molecule, an antibody (including
but not restricted to: a diabody, single chain antibody, monoclonal
antibody, IgG, IgM, IgA, IgD, IgE, or an antibody fragment
comprising at least one pair of variable regions), an antisense
nucleic acid, PNA or ribozyme, RNAi construct, aptamer, peptoid, or
a mutant Hedgehog protein that can disrupt or inhibit Hedgehog
signaling. An antibody may be directed to a protein encoded by any
of the genes in the Hedgehog pathway, including sonic, indian or
desert Hedgehog, smoothened, ptc-1, ptc-2, gli-1, gli-2, gli-3,
etc. In most cases, the antibody would inhibit the activity of the
target protein, but in the case of patched, such an antibody would
be an activator of patched. An antisense nucleic acid would
likewise decrease production of a protein encoded by any of the
genes in the Hedgehog pathway, with the exception of patched or
other genes encoding negative regulators of the Hedgehog signaling
pathway.
[0138] A preferred antagonist has at least the following
properties: (i) the isolated protein binds the receptor patched-1
with an affinity that may be less than, but is preferably at least
the same as, the binding of mature Hedgehog protein to patched-1;
and (ii) the isolated protein blocks alkaline phosphatase (AP)
induction by mature Hedgehog protein when tested in an in vitro
CH310T 1/2 cell-based AP induction assay. Antagonists of the
invention may also have the additional properties of being (iii)
unable to induce ptc-1 and gli-lexpression. Persons having ordinary
skill in the art can easily test any putative Hedgehog antagonist
for these properties. In particular, the mouse embryonic fibroblast
line C3HlOT I/2 is a mesenchymal stem cell line that is Hedgehog
responsive. Hedgehog treatment of the cells causes an upregulation
of gli-1 and patched-1 (known indicators of Hedgehog dependent
signaling) and also causes induction of alkaline phosphatase
activity, an indicator that the cells have differentiated down the
chondrocyte/bone osteoblast lineage.
[0139] For purposes of this invention, the term "protein" means a
polypeptide (native (i.e., naturally-occurring) or mutant),
oligopeptide, peptide, or other amino acid sequence. As used
herein, "protein" is not limited to native or full-length proteins,
but is meant to encompass protein fragments having a desired
activity or other desirable biological characteristics, as well as
mutants or derivatives of such proteins or protein fragments that
retain a desired activity or other biological
characteristic--including peptoids with a nitrogen based backbone.
Mutant proteins encompass proteins having an amino acid sequence
that is altered relative to the native protein from which it is
derived, where the alterations can include amino acid substitutions
(conservative or non-conservative), deletions, or additions (e.g.,
as in a fusion protein). "Protein" and "polypeptide" are used
interchangeably herein without intending to limit the scope of
either term.
[0140] For purposes of this invention, "amino acid" refers to a
monomeric unit of a peptide, polypeptide, or protein. There are
twenty amino acids found in naturally occurring peptides,
polypeptides and proteins, all of which are L-isomers. The term
also includes analogs of the amino acids and D-isomers of the
protein amino acids and their analogs.
[0141] For purposes of the present invention, "soluble" is intended
to mean a chimeric and/or modified receptor protein that is not
fixed to the surface of cells via a transmembrane domain. As such,
soluble forms of the chimeric binding proteins of the present
invention, while capable of binding to and inactivating its ligand,
do not comprise a transmembrane domain and thus generally do not
become associated with the cell membrane of cells in which the
molecule is expressed. A soluble form of the receptor exerts an
inhibitory effect on the biological activity of the ligand protein
by binding to its ligand, thereby preventing it from binding to its
natural receptors present on the surface of target cells.
[0142] For purposes of this invention, by "DNA" is meant a
polymeric form of desoxyribonucleotides (adenine, guanine, thymine,
or cytosine) in double-stranded or single-stranded form, either
relaxed and supercoiled, either linear circular. This term refers
only to the primary and secondary structure of the molecule, and
does not limit it to any particular tertiary forms. Thus, this term
includes single- and double-stranded DNA found, inter alia, in
linear DNA molecules (e.g., restriction fragments), viruses,
plasmids, and chromosomes. In discussing the structure of
particular DNA molecules, sequences may be described herein
according to the normal convention of giving only the sequence in
the 5' to 3' direction along the non-transcribed strand of DNA
(i.e., the strand having the sequence homologous to the mRNA). The
term captures molecules that include the four bases adenine,
guanine, thymine, or cytosine, as well as molecules that include
base analogues which are known in the art.
[0143] For purposes of this invention, "polynucleotide" as used
herein means a polymeric form of nucleotides of any length, either
ribonucleotides or desoxyribonucleotides. This term refers only to
the primary structure of the molecule. Thus, the term includes
double- and single-stranded DNA, as well as, double- and
single-stranded RNA. It also includes modifications, such as
methylation or capping, and unmodified forms of the
polynucleotide.
[0144] For the purpose of describing the relative position of
nucleotide sequences in a particular nucleic acid molecule
throughout the instant application, such as when a particular
nucleotide sequence is described as being situated "upstream,"
"downstream," "5'," or "3'" relative to another sequence, it is to
be understood that it is the position of the sequences in the
non-transcribed strand of a DNA molecule that is being referred to
as is conventional in the art.
[0145] For purposes of this invention, a "gene sequence" or "coding
sequence" or "protein coding sequence" or "open reading frame" or a
sequence which "encodes" a particular protein, is a nucleic acid
composition which is transcribed into RNA (in the case of DNA) and
potentially translated (in the case of mRNA) into a polypeptide in
vitro or in vivo when placed under the control of appropriate
regulatory sequences. The boundaries of the gene are determined by
a start codon at the 5' (amino) terminus and potentially a
translation stop codon at the 3' (carboxy) terminus. A gene
sequence can include, but is not limited to, cDNA from prokaryotic
or eukaryotic mRNA, genomic DNA sequences from prokaryotic or
eukaryotic DNA, and even synthetic DNA sequences. A transcription
termination sequence will usually be located 3' to the protein
coding sequence.
[0146] For purposes of this invention, by the term "transgene" is
meant a nucleic acid composition made out of DNA, which encodes a
peptide, oligopeptide or protein. The transgene may be operatively
linked to regulatory components in a manner which permits transgene
transcription, translation and/or ultimately directs expression of
a product encoded by the nucleic acid composition in the host cell,
e.g., the transgene is placed into operative association with a
promoter and enhancer elements, as well as other regulatory
sequences, such as introns or polyA sequences, useful for its
regulation. The composite association of the transgene with its
regulatory sequences is referred to herein as a "minicassette" or
"minigene". Minicasssettes or minigenes in their entirety are also
nucleic acid compositions. The exact nucleic acid composition will
depend upon the use to which the resulting nucleic acid transfer
vector will be put and is known to the artisan (Sambrook 1989,
Lodish et al. 2000). When taken up by a target cell, the nucleic
acid composition may remain present in the cell as a functioning
extrachromosomal molecule, or it may integrate into the cell's
chromosomal DNA, depending on the kind of transfer vector used.
[0147] For purposes of this invention, "heterologous" as it relates
to nucleic acid compositions denotes sequences that are not
normally joined together. Thus, a "heterologous" region of a
nucleic acid composition is a segment of nucleic acid within or
attached to another nucleic acid composition that is not found in
association with the other molecule in nature. For example, a
heterologous region of a nucleic acid composition could include a
coding sequence flanked by sequences not found in association with
the coding sequence in nature. Another example of a heterologous
coding sequence is a construct where the coding sequence itself is
not found in nature (e.g., synthetic sequences having codons
different from the native gene). Allelic variation or naturally
occurring mutational events do not give rise to heterologous DNA,
as used herein.
[0148] For purposes of this invention, "homology" or "homologous"
refers to the percent homology between two polynucleotide or two
polypeptide moiety. The correspondence between the sequence from
one moiety to another can be determined by techniques known in the
art. Two DNA or two polypeptide sequences are "substantially
homologous" to each other when at least about 80%, preferably at
least about 90%, and most preferably at least about 95% of the
nucleotides or amino acids match over a defined length of the
molecules, as determined using methods in the art.
[0149] The techniques for determining amino acid sequence homology
are well-known in the art. In general, "homology" means the exact
amino acid to amino acid comparison of two or more polypeptides at
the appropriate place, where amino acids are identical or possess
similar chemical and/or physical properties such as charge or
hydrophobicity. A so-termed "percent homology" then can be
determined between the compared polypeptide sequences. The programs
available in the Wisconsin Sequence Analysis Package (available
from Genetics Computer Group, Madison, Wis.), for example, the GAP
program, are capable of calculating homologies between two
polypeptide sequences. Other programs for determining homology
between polypeptide sequences are known in the art.
[0150] Homology for polynucleotides is determined essentially as
follows: Two polynucleotides are considered to be "substantially
homologous" to each other when at least about 80%, preferably at
least about 90%, and most preferably at least about 95% of the
nucleotides match over a defined length of the molecules, when
aligned using the default parameters of the search algorithm BLAST
2.0. The BLAST 2.0 program is publicly available.
[0151] Alternatively, homology for polynucleotides can be
determined by hybridization experiments. As used herein, a nucleic
acid sequence or fragment (such as for example, primers or probes),
is considered to selectively hybridize to a sequence 1, thus
indicating "substantial homology", if such a sequence is capable of
specifically hybridizing to the sequence 1 or a variant thereof or
specifically priming a polymerase chain reaction: (i) under typical
hybridization and wash conditions, such as those described, for
example, in Maniatis, (Molecular Cloning: A Laboratory Manual, 2nd
Edition, 1989) where preferred hybridization conditions are those
of lesser stringency and more preferred, higher stringency; or (ii)
using reduced stringency wash conditions that allow at most about
25-30% basepair mismatches, for example, 2.times.SSC, 0.1% SDS, at
room temperature twice, for 30 minutes each; then 2.times.SSC, 0.1%
SDS, 37 C, once for 30 minutes; the 2.times.SSC at room temperature
twice, 10 minutes each or (iii) under standard PCR conditions or
under "touch-down" PCR conditions such as described by [98]).
[0152] For purposes of this invention, the term "cell" means any
prokaryotic or eukaryotic cell, either ex vivo, in vitro or in
vivo, either separate (in suspension) or as part of a higher
structure such as--but not limited to--organs or tissues.
[0153] For purposes of this invention, the term "host cell" means a
cell that can be transduced and/or transfected by an appropriate
gene transfer vector. The nature of the host cell may vary from
gene transfer vector to gene transfer vector.
[0154] For purposes of this invention, by the term "a Therapeutic
of this invention" is meant a substance that interferes with
Hedgehog signaling in general, and Shh signaling in particular,
resulting in prevention, inhibition, and/or reversion of ocular
neovascularization.
[0155] For purposes of this invention, "treatment" refers to
prophylaxis and/or therapy.
[0156] "Pharmaceutically effective" levels are levels sufficient to
achieve a physiologic effect in a human or veterinary subject,
which effect may be therapeutic or prophylactic.
[0157] For purposes of this invention, by "mammalian subject" is
meant any member of the class Mammalia including, without
limitation, humans and nonhuman primates such as chimpanzees and
other apes and monkey species; farm animals such as cattle, sheep,
pigs, goats and horses; domestic mammals such as dogs and cats;
laboratory animals including rodents such as mice, rats and guinea
pigs, and the like. The term does not denote a particular age or
sex. Thus, adult and newborn subjects, as well as fetuses, whether
male or female, are intended to be covered.
[0158] For purposes of this invention, the terms "individual" or
"subject" or "patient" as used herein refer to vertebrates,
particularly members of the mammalian species and include but are
not limited to domestic animals, sports animals, primates and
humans; more particularly the term refer to humans.
[0159] For purposes of this invention, "mesenchymal cells" are
defined as cells of mesenchymal origin including fibroblasts,
stromal cells, smooth muscle cells, skeletal muscle cells, cells of
osteogenic origin such as chondrocytes, cells of hemaeopoietic
origin such as monocytes, macrophages, lymphocytes, granulocytes
and cells of adipose origin such as adipocytes.
[0160] For purpose of this invention, a "Hedgehog therapeutic",
whether it is a Hedgehog agonist or Hedgehog antagonist is said to
have "therapeutic efficacy" in modulating neovascularization, and
an amount of the therapeutic is said to be a "angiogenic modulatory
amount", if administration of that amount of the therapeutic is
sufficient to cause a significant modulation (i.e., increase or
decrease) in angiogenic activity when administered to a subject
(e.g., an animal model or human patient) needing modulation of
angiogenesis.
[0161] For purposes of this invention, a "substance that interferes
with the Hedgehog signaling pathway" or a "Hedgehog signaling
interfering substance" is a Hedgehog therapeutic with
Hedgehog-antagonistic function. Interference can be accomplished in
different ways, whereas one substance can exert one or more
interfering mechanisms: The substance can interfere with the
Hedgehog signaling pathway in general, and the Shh pathway in
particular by [0173] (1) De-activating the Hedgehog receptor or
inhibiting its activity; [0174] (2) Inhibiting activity of the
Hedgehog protein; [0175](3) Coating, or binding to, a Hedgehog
protein on the surface of a Hedgehog bearing or secreting cell with
sufficient specificity to de-activate or inhibit a
Hedgehog-ligand/Hedgehog interaction, e.g., the Hedgehog/patched
interaction; [0176](4) Coating, or binding to, a Hedgehog protein
on the surface of a Hedgehog-bearing or secreting cell with
sufficient specificity to modify, and preferably to de-activate or
inhibit, transduction of a Hedgehog-mediated signal e.g.,
Hedgehog/patched, smoothened, fused, or gli-mediated signaling;
[0177] (5) Coating, or binding to, a Hedgehog receptor or
coreceptor (e.g., patched or smoothened) in or on cells with
sufficient specificity to de-activate or inhibit the
Hedgehog/patched interaction; [0178] (6) Coating, or binding to, a
Hedgehog receptor or co-receptor (e.g., patched or smoothened) in
or on cells with sufficient specificity to modify, and preferably
to de-activate or inhibit transduction of Hedgehog receptor
mediated Hedgehog signaling, e.g., patched-mediated Hedgehog
signaling; [0179] (7) Exerting RNA interference with an RNA that
encodes a component of the Hedgehog signaling pathway
[0162] Moreover, more than one a substance that interferes with the
Hedgehog signaling pathway can be administered to a patient, e.g.,
an agent that binds to Hedgehog can be combined with an agent that
binds to Patched. Moreover, a Hedgehog therapeutic is an
"antagonist" if it modulates ocular neovascularization in such a
way as to inhibit, decelerate, reverse or otherwise slow ocular
neovascularization, regardless of the mode of action of such
therapeutic. For example, antagonist molecules may be antibody
homologs, certain fragments of Hedgehog, small interfering RNAs,
peptoids, aptamers, or small organic molecules that may be
administered and modulate Hedgehog binding sites on cells.
[0163] For purposes of this invention, the term "antibody homolog"
includes intact antibodies consisting of immunoglobulin light and
heavy chains linked via disulfide bonds. The term "antibody
homolog" is also intended to encompass a Hedgehog therapeutic
comprising one or more polypeptides selected from immunoglobulin
light chains, immunoglobulin heavy chains and antigen-binding
fragments thereof which are capable of binding to one or more
antigens (i.e., Hedgehog, Patched, and/or Smo). The component
polypeptides of an antibody homolog composed of more than one
polypeptide may optionally be disulfide-bound or otherwise
covalently crosslinked. Accordingly, therefore, "antibody homologs"
include intact immunoglobulins of types IgA, IgG, IgE, IgD, IgM (as
well as subtypes thereof), wherein the light chains of the
immunoglobulin may be of types kappa or lambda or portions of
intact antibodies that retain antigen-binding specificity, for
example, Fab fragments, Fab' fragments, F(ab')2 fragments, F(v)
fragments, heavy chain monomers or dimers, light chain monomers or
dimers, dimers consisting of one heavy and one light chain, and the
like. An "antibody homolog" also refers to monoclonal antibodies,
chimeric antibodies, single-chain antibodies, intrabodies,
humanized monoclonal antibodies and antibodies linked to other
molecules.
[0164] For purposes of this invention, a "humanized antibody
homolog" is an antibody homolog, produced by recombinant DNA
technology, in which some or all of the amino acids of a human
immunoglobulin light or heavy chain that are not required for
antigen binding have been substituted for the corresponding amino
acids from a non-human mammalian immunoglobulin light or heavy
chain.
[0165] For purposes of this invention, a "human antibody homolog"
is an antibody homolog in which all the amino acids of an
immunoglobulin light or heavy chain (regardless of whether or not
they are required for antigen binding) are derived from a human
source.
[0166] By "not substantially cross react" is meant that the
antibody has a binding affinity for a non-homologous protein which
is at least one order of magnitude, more preferably at least 2
orders of magnitude, and even more preferably at least 3 orders of
magnitude less than the binding affinity of the antibody for the
protein or proteins for which the antibody is immunospecific.
[0167] For purposes of this invention, the term "gene therapy"
means the transfer of nucleic acid compositions into cells of a
multicellular eukaryotic organism, be it in vivo, ex vivo or in
vitro (see also [57] [58]). The term "gene therapy" should not be
limited to the purpose of correcting metabolic disorders, but be
interpreted more as a technical term for the transfer of nucleic
acid compositions for therapeutic purposes in general, independent
of a specific therapeutic purpose. Therefore, the term "gene
therapy" would include--without limitation--correction of metabolic
disorders, cancer therapy, vaccination, monitoring of cell
populations, cell expansion, stem cell manipulation etc. by means
of transfer of nucleic acid compositions.
[0168] For purposes of this invention, "transfection" is used to
refer to the uptake of nucleic acid compositions by a cell. A cell
has been "transfected" when an exogenous nucleic acid composition
has crossed the cell membrane. A number of transfection techniques
are generally known in the art. See, e.g., [59, 60], Sambrook et
al. (1989) Molecular Cloning, a laboratory manual, Cold Spring
Harbor Laboratories, New York, Davis et al. (1986) Basic Methods in
Molecular Biology, Elsevier, and [61]. Such techniques can be used
to introduce one or more nucleic acid compositions, such as a
plasmid vector and other nucleic acid molecules, into suitable host
cells. The term refers to both stable and transient uptake of the
genetic material. For purposes of this invention, "transduction" is
a special form of "transfection" via a viral vector.
[0169] For purposes of this invention, "transduction" denotes the
delivery of a nucleic acid composition to, into or within a
recipient cell either in vivo, in vitro or ex vivo, via a virus or
viral vector, such as via a recombinant AAV virion. Transduction is
a special form of transfection, i.e., the term transfection
includes the term transduction.
[0170] For purposes of this invention, by "vector", "transfer
vector", "gene transfer vector" or "nucleic acid composition
transfer vector" is meant any element, such as a plasmid, phage,
transposon, cosmid, chromosome, virus, virion, etc., which is
capable of transferring and/or transporting a nucleic acid
composition to a host cell, into a host cell and/or to a specific
location and/or compartment within a host cell. Thus, the term
includes cloning and expression vehicles, as well as viral and
non-viral vectors and potentially naked or complexed DNA. However,
the term does not include cells that produce gene transfer vectors
such as retroviral packaging cell lines.
[0171] For purposes of this invention, by "recombinant virus",
"recombinant virion", "recombinant vector" or "recombinant viral
vector" is meant a virus that has been genetically altered, e.g.,
by the addition or insertion of a heterologous nucleic acid
composition into the particle.
[0172] For purposes of this invention, the term "RNA interference"
or "RNAi" is broadly defined and includes all posttranscriptional
and transcriptional mechanisms of RNA mediated inhibition of gene
expression, such as those described in P. D. Zamore Science 296,
1265 (2002). RNA interference is mediated by double-stranded RNA
(dsRNA), which can induce many different epigenetic gene-silencing
processes in eukaryotes, including the degradation of homologous
mRNAs--a process called RNA interference (RNAi) in animals and
post-transcriptional gene silencing (PTGS) in plants. RNA
interference (RNAi) has first been discovered in 1998 by Andrew
Fire and Craig Mello in C. elegans, confirming former studies of
PGTS in plants [62]. It now seems to be a ubiquitous
mechanism--also applicable to humans [63-73]. Double stranded RNA
has been shown to inhibit gene expression of genes having a
complementary sequence through a process termed RNA interference
(see, for example, Hammond et al. Nat. Rev. Genet. 2:110-119
(2001)).
[0173] For purposes of this invention, the term "small interfering
RNA" or "siRNA" as used herein means short interfering RNA which is
a double-stranded RNA complex that is less than 30 base pairs
(i.e., 60 nucleotides or bases) and preferably 21-25 base pairs
(i.e., 42-50 bases or nucleotides) in length. More generally,
double-stranded RNA that is responsible for inducing RNAi is termed
interfering RNA. Thus, a "small interfering RNA" or "siRNA" is a
double-stranded RNA complex that is capable of decreasing the
expression of a gene with which it shares homology. The region of
the gene or other nucleotide sequence over which there is homology
is known as the "RNAi target region", "target region", "RNAi target
sequence" or "target sequence".
[0174] In one embodiment the siRNA may be a "hairpin" or stem-loop
RNA molecule, comprising a sense region, a loop region and an
antisense region complementary to the sense region and thus capable
of forming an RNAi inducing dsRNA complex. In other embodiments the
siRNA comprises two distinct RNA molecules that are non-covalently
associated to form a dsRNA complex.
[0175] For purposes of this invention, the term "RNAi expression
cassette" as used herein means a nucleic acid composition which
encodes one or more RNA molecules which are capable of forming a
double-stranded RNA complex and thus are capable of inducing RNA
interference.
[0176] The design of the RNAi expression cassette does not limit
the scope of the invention. Different strategies to design an RNAi
expression cassette can be applied, and RNAi expression cassettes
based on different designs will be able to induce RNA interference
in vivo. (Although the design of the RNAi expression cassette does
not limit the scope of the invention, some RNAi expression cassette
designs are included in the detailed description of this invention
and below.) One of skill in the art will be able to choose among
different designs without undue effort.
[0177] Features common to all RNAi expression cassettes are that
they comprise an RNA coding region which encodes one or more RNA
molecules. After or during RNA expression from the RNAi expression
cassette, a double-stranded RNA complex may be formed by either a
single, self-complementary RNA molecule (intramolecular formation)
or two complementary RNA molecules (intermolecular formation).
Formation of the dsRNA complex may be initiated either inside or
outside the nucleus. The dsRNA complex will be capable of inducing
RNA interference either directly or indirectly.
[0178] In some embodiments, the RNAi inducing double-stranded RNA
complex (encoded by the RNAi expression cassette(s)) comprises a
first RNA portion capable of hybridizing under physiological
conditions to at least a portion of an mRNA molecule (the RNAi
target sequence of the RNAi target mRNA of the RNAi target gene),
and a second RNA portion wherein at least a part of the second RNA
portion is capable of hybridizing under physiological conditions to
the first portion. Preferably the first and second portions are
part of the same RNA molecule and are capable of hybridization at
physiological conditions, such as those existing within a cell and
upon hybridization the first and second portions form a
double-stranded RNA complex. For example, the RNAi inducing
double-stranded RNA complex (encoded by the RNAi expression
cassette(s)) is formed by a linear RNA molecule, which RNA
comprises a first portion capable of hybridizing to at least a
portion of an mRNA molecule and a second portion wherein at least
part of the second portion is capable of hybridizing to the first
portion to form a hairpin dsRNA complex. Thus, in some embodiments,
when introduced into a cell via rAAV gene transfer, expression of
the RNAi expression cassette gives rise to a single RNA molecule
capable of forming intramolecularly an RNAi inducing dsRNA complex.
However, it will be understood from the following description that
more than one rAAV genome or rAAV vector or RNAi expression
cassette or RNA coding region may be introduced into a cell, either
simultaneously or sequentially via rAAV mediated gene transfer, to
give rise to two or more RNA molecules capable of forming
intermolecularly an RNAi-inducing dsRNA complex. Typically, the two
RNA sequences capable of forming a dsRNA complex, whether intra- or
intermolecularly, are at least in part sense and at least in part
antisense sequences of a gene or nucleic acid sequence whose
expression is to be down-regulated or decreased.
[0179] In the preferred embodiment the RNAi expression cassette
comprises at least one RNA coding region. In other embodiments, the
RNAi expression cassette comprises two or more RNA coding regions.
The RNAi expression cassette also preferably comprises at least one
RNA Polymerase III promoter. The RNA Polymerase III promoter is
operably linked to the RNA coding region, and the RNA coding region
can also be linked to a termination sequence (terminator). In
addition, more than one RNA Polymerase III promoters may be
incorporated.
[0180] In certain embodiments the invention employs
ribozyme--containing RNA molecules--encoded by the RNAi expression
cassette--to generate dsRNA complexes, thereby overcoming certain
known difficulties associated with generating dsRNA such as the
removal of polyadenylation signals. In other embodiments the
invention is based on the ability of a portion of the RNA molecule
to encode an RNA or protein that enhances specific activity of
dsRNA. One example of this specific activity enhancing portion of
the RNA molecule is a portion of the molecule encoding the HIV Tat
protein to inhibit the cellular breakdown of dsRNA complexes. Such
a portion is additionally useful in treating disorders such as HIV
infection.
[0181] For purposes of this invention, the term "RNA expression
product" or "RNA product" refers to the RNA molecule or RNA
transcript transcribed (synthesized) from an RNAi expression
cassette.
[0182] The term "target mRNA" or "RNAi target mRNA" refers to any
mRNA whose expression in the host is to be reduced. The RNAi target
mRNA is the RNA transcript of the (RNAi) target gene.
[0183] The terms "double-stranded RNA complex" or "dsRNA complex"
as used herein are equivalent, and each shall mean a complex formed
either (a) by two linear molecules of RNA, wherein at least a
portion of the sequence of one molecule is complementary to, and is
capable of or has hybridized to, at least a portion of the sequence
of the other RNA molecule, or (b) by two portions of a linear RNA
molecule which are complementary to, and are capable of or have
therefore hybridized to, each other. The dsRNA complex is generated
by the RNA expression product(s) of the RNAi expression cassette(s)
and is able to mediate either directly or indirectly RNA
interference, thus mediating down-regulation of the expression of
the RNAi target gene.
[0184] In certain embodiments, said double-stranded RNA complex for
down-regulating expression of a mammalian gene comprises (i) a
first nucleotide sequence that hybridizes under stringent
conditions to a nucleotide sequence of at least one mammalian gene
and (ii) a second nucleotide sequence which is complementary to
said first nucleotide sequence. In a subgroup of those embodiments,
an RNA loop connects the first with the second nucleotide
sequence.
[0185] A dsRNA complex comprising a nucleotide sequence identical
to a portion of the RNAi target gene is preferred for inhibition.
RNA sequences with insertions, deletions, and single point
mutations relative to the RNAi target sequence have also been found
to be effective for inhibition. Thus, sequence identity may be
optimized by alignment algorithms known in the art and calculating
the percent difference between the nucleotide sequences.
Alternatively, the RNA duplex region of the dsRNA complex may be
defined functionally as a nucleotide sequence that is capable of
hybridizing with a portion of the target gene transcript.
[0186] An example for a dsRNA complex are siRNAs. RNAi-inducing
dsRNA complexes based on siRNAs are described [74] [75] [76]. The
dsRNA complex is generally at least about 15 base pairs in length
and is preferably about 15 to about 30 base pairs in length.
However, a significantly longer dsRNA complex can be used
effectively in some organisms. In a more preferred embodiment, the
dsRNA complex is between about 19 and 22 base pairs in length. The
dsRNA complex is preferably identical to the target nucleotide
sequence over this region. When the gene to be down-regulated is in
a family of highly conserved genes, the sequence of the duplex
region can be chosen with the aid of sequence comparison to target
only the desired gene. On the other hand, if there is sufficient
identity among a family of homologous genes within an organism, a
duplex region can be designed that would down regulate a plurality
of genes simultaneously. The RNA duplexes may be flanked by single
stranded regions on one or both sides of the duplex. For example,
in the case of the hairpin, the single stranded loop region would
connect the duplex region at one end.
[0187] For purposes of this invention, the term "RNA duplex" or
"RNA duplex region" means the part of the dsRNA complex that is
homologous and/or complementary to the RNAi target region. In
certain embodiments, the RNA duplex might comprise the whole dsRNA
complex. The RNA duplex is substantially homologous and/or
complementary (typically at least about 80% identical, more
preferably at least about 90% identical) in sequence to the RNAi
target sequence of the gene targeted for down regulation via RNA
interference.
[0188] For purposes of this invention, the term "Nucleic Acid
Ligand" as used herein is a non-naturally occurring nucleic acid
having a desirable action on a target. A nucleic acid ligand is
also referred to as an "aptamer" herein. A desirable action
includes, but is not limited to, binding of the target,
catalytically changing the target, reacting with the target in a
way which modifies/alters the target or the functional activity of
the target, covalently attaching to the target as in a suicide
inhibitor, and facilitating the reaction between the target and
another molecule. In the preferred embodiment, the desirable action
is specific binding to a target molecule, such target molecule
being a three dimensional chemical structure other than a
polynucleotide that binds to the nucleic acid ligand through a
mechanism which predominantly depends on Watson/Crick base pairing
or triple helix binding, wherein the nucleic acid ligand is not a
nucleic acid having the known physiological function of being bound
by the target molecule. Nucleic acid ligands include nucleic acids
that are identified from a candidate mixture of nucleic acids, said
nucleic acid ligand being a ligand of a given target by the method
comprising: a) contacting the candidate mixture with the target,
wherein relative to the candidate mixture may be partitioned from
the remainder of the candidate mixture; b) partitioning the
increased affinity nucleic acids from the remainder of the
candidate mixture; and c) amplifying the increased affinity nucleic
acids to yield a ligand-enriched mixture of nucleic acids.
[0189] For purposes of this invention, the term "SELEX" refers to a
methodology involving the combination of selection of nucleic acid
ligands which interact with a target in a desirable manner, for
example binding to a protein, with amplification of those selected
nucleic acids [77-79]. Iterative cycling of the
selection/amplification steps allows selection of one or a small
number of nucleic acids which interact most strongly with the
target from a pool which contains a very large number of nucleic
acids. Cycling of the selection/amplification procedure is
continued until a selected goal is achieved. In the present
invention, the SELEX methodology is employed to obtain nucleic acid
ligands to components of the Hedgehog signaling pathway.
[0190] For purposes of this invention, the term "aliphatic group"
refers to a straight-chain, branched-chain, or cyclic
aliphatichydrocarbon group and includes saturated and unsaturated
aliphatic groups, such as an alkyl group, analkenyl group, and an
alkynyl group.
[0191] For purposes of this invention, the terms "alkenyl" and
"alkynyl" refer to unsaturated aliphatic groups analogous in length
and possible substitution to the alkyls described above, but that
contain at least one double or triple bond, respectively.
[0192] For purposes of this invention, the terms "alkoxyl" or
"alkoxy" as used herein refers to an alkyl group, as defined above,
having anoxygen radical attached thereto. Representative alkoxyl
groups include methoxy, ethoxy, propyloxy, tert-butoxy and the
like. An "ether" is two hydrocarbons covalently linked by an
oxygen. Accordingly, the substituent of an alkyl that renders that
alkyl an ether is or resembles an alkoxyl, such as can be
represented by one of --O-alkyl, --O-alkenyl, --O-alkynyl,
--O--(CH.sub.2).sub.m--R8, where m and R8 are described above.
[0193] For purposes of this invention, the term "alkyl" refers to
the radical of saturated aliphatic groups, including straight-chain
alkyl groups, branched-chain alkyl groups, cycloalkyl (alicyclic)
groups, alkyl-substituted cycloalkyl groups, and
cycloalkyl-substituted alkyl groups. In preferred embodiments, a
straight chain or branched chain alkyl has 30 or fewer carbon atoms
in its backbone (e.g., C1-C30 for straight chains, C3-C30 for
branched chains), and more preferably 20 or fewer. Likewise,
preferred cycloalkyls have from 3-10 carbon atoms in their ring
structure, and more preferably have 5, 6 or 7 carbons in the ring
structure.
[0194] Moreover, the term "alkyl" (or "lower alkyl") as used
throughout the specification, examples, and claims is intended to
include both "unsubstituted alkyls" and "substituted alkyls", the
latter of which refers to alkyl moieties having substituents
replacing a hydrogen on one or more carbons of the hydrocarbon
backbone.
[0195] Such substituents can include, for example, a halogen, a
hydroxyl, a carbonyl (such as a carboxyl, an alkoxycarbonyl, a
formyl, or an acyl), a thiocarbonyl (such as a thioester, a
thioacetate, or a thioformate), an alkoxyl, a phosphoryl, a
phosphate, a phosphonate, a phosphinate, an amino, an amido, an
amidine, animine, a cyano, a nitro, an azido, a sulfhydryl, an
alkylthio, a sulfate, a sulfonate, a sulfamoyl, asulfonamido, a
sulfonyl, a heterocyclyl, an aralkyl, or an aromatic or
heteroaromatic moiety. It will be understood by those skilled in
the art that the moieties substituted on the hydrocarbon chain can
themselves be substituted, if appropriate. For instance, the
substituents of a substituted alkyl may include substituted and
unsubstituted forms of amino, azido, imino, amido, phosphoryl
(including phosphonate and phosphinate), sulfonyl (including
sulfate, sulfonamido, sulfamoyl and sulfonate), and silyl groups,
as well asethers, alkylthios, carbonyls (including ketones,
aldehydes, carboxylates, and esters), --CF.sub.3, --CN and the
like. Exemplary substituted alkyls are described below. Cycloalkyls
can be further substituted with alkyls, alkenyls, alkoxys,
alkylthios, aminoalkyls, carbonyl-substituted alkyls, --CF.sub.3,
--CN, and the like.
[0196] Unless the number of carbons is otherwise specified, "lower
alkyl" as used herein means an alkyl group, as defined above, but
having from one to ten carbons, more preferably from one to six
carbon atoms in its backbone structure. Likewise, "lower alkenyl"
and "lower alkynyl" have similar chain lengths. Throughout the
application, preferred alkyl groups are lower alkyls. In preferred
embodiments, a substituent designated herein as alkyl is a lower
alkyl.
[0197] For purposes of this invention, the term "alkylthio" refers
to an alkyl group, as defined above, having a sulfur radical
attached thereto. In preferred embodiments, the "alkylthio" moiety
is represented by one of --S-alkyl, --S-alkenyl, --S-alkynyl, and
--S--(CH.sub.2).sub.m--R8, wherein m and R8 are defined above.
Representative alkylthio groups include methylthio, ethylthio, and
the like.
[0198] For purposes of this invention, the term "aralkyl", as used
herein, refers to an alkyl group substituted with an aryl group
(e.g., anaromatic or heteroaromatic group).
[0199] For purposes of this invention, the term "aryl" as used
herein includes 5-, 6-, and 7-membered single-ring aromatic groups
that may include from zero to four heteroatoms, for example,
benzene, pyrrole, furan, thiophene, imidazole, oxazole, thiazole,
triazole, pyrazole, pyridine, pyrazine, pyridazine and pyrimidine,
and the like. Those aryl groups having heteroatoms in the ring
structure may also be referred to as "aryl heterocycles" or
"heteroaromatics." The aromatic ring can be substituted at one or
more ring positions with such substituents as described above, for
example, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl,
cycloalkyl, hydroxyl, alkoxyl, amino, nitro, sulfhydryl, imino,
amido, phosphate, phosphonate, phosphinate, carbonyl, carboxyl,
silyl, ether, alkylthio, sulfonyl, sulfonamido, ketone, aldehyde,
ester, heterocyclyl, aromatic or heteroaromatic moieties,
--CF.sub.3, --CN, or the like. The term "aryl" also includes
polycyclic ring systems having two or more cyclic rings in which
two or more carbons are common to two adjoining rings (the rings
are "fused rings") wherein at least one of the rings is aromatic,
e.g., the other cyclic rings can be cycloalkyls, cycloalkenyls,
cycloalkynyls, aryls and/or heterocyclyls.
[0200] For purposes of this invention, the term "heteroatom" as
used herein means an atom of any element other than carbon or
hydrogen. Preferred heteroatoms are boron, nitrogen, oxygen,
phosphorus, sulfur and selenium.
[0201] For purposes of this invention, the terms "heterocyclyl" or
"heterocyclic group" refer to 3-to 10-membered ring structures,
more preferably 3-to 7-membered rings, whose ring structures
include one to four heteroatoms. Heterocycles can also be
polycyclic. Heterocyclyl groups include, for example, thiophene,
thianthrene, furan, pyran, isobenzofuran, chromene, xanthene,
phenoxathiin, pyrrole, imidazole, pyrazole, isothiazole, isoxazole,
pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole,
indole, indazole, purine, quinolizine, isoquinoline, quinoline,
phthalazine, naphthyridine, quinoxaline, quinazoline, cinnoline,
pteridine, carbazole, carboline, phenanthridine, acridine,
pyrimidine, phenanthroline, phenazine, phenarsazine, phenothiazine,
furazan, phenoxazine, pyrrolidine, oxolane, thiolane, oxazole,
piperidine, piperazine, morpholine, lactones, lactams such as
azetidinones and pyrrolidinones, sultams, sultones, and the like.
The heterocyclic ring can be substituted at one or more positions
with such substituents as described above, as for example, halogen,
alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, amino,
nitro, sulfhydryl, imino, amido, phosphate, phosphonate,
phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl,
ketone, aldehyde, ester, a heterocyclyl, an aromatic or
heteroaromatic moiety, --CF.sub.3, --CN, or the like.
[0202] For purposes of this invention, the term, the term "nitro"
means --NO.sub.2; the term "halogen" designates --F, --Cl, --Br or
--I; the term "sulfhydryl" means --SH; the term "hydroxyl" means
--OH; and the term "sulfonyl" means --SO.sub.2--.
[0203] For purposes of this invention, the terms "polycyclyl" or
"polycyclic group" refer to two or more rings (e.g., cycloalkyls,
cycloalkenyls, cycloalkynyls, aryls and/or heterocyclyls) in which
two or more carbons are common to two adjoining rings, e.g., the
rings are "fused rings". Rings that are joined through non-adjacent
atoms are termed "bridged" rings. Each of the rings of the
polycycle can be substituted with such substituents as described
above, as for example, halogen, alkyl, aralkyl, alkenyl, alkynyl,
cycloalkyl, hydroxyl, amino, nitro, sulfhydryl, imino, amido,
phosphate, phosphonate, phosphinate, carbonyl, carboxyl, silyl,
ether, alkylthio, sulfonyl, ketone, aldehyde, ester, a
heterocyclyl, an aromatic or heteroaromatic moiety, --CF.sub.3,
--CN, or the like.
[0204] A "selenoalkyl" refers to an alkyl group having a
substituted seleno group attached thereto. Exemplary "selenoethers"
which may be substituted on the alkyl are selected from one of
--Se-alkyl, --Se-alkenyl, --Se-alkynyl, and
--Se--(CH.sub.2).sub.m--R8, m and R8 being defined above.
[0205] For purposes of this invention, the term "substituted" is
contemplated to include all permissible substituents of organic
compounds. In a broad aspect, the permissible substituents include
acyclic and cyclic, branched and unbranched, carbocyclic and
heterocyclic, aromatic and nonaromatic substituents of organic
compounds. Illustrative substituents include, for example, those
described herein above. The permissible substituents can be one or
more and the same or different for appropriate organic compounds.
For purposes of this invention, the heteroatoms such as nitrogen
may have hydrogen substituents and/or any permissible substituents
of organic compounds described herein which satisfy the valences of
the heteroatoms. This invention is not intended to be limited in
any manner by the permissible substituents of organic
compounds.
[0206] It will be understood that "substitution" or "substituted
with" includes the implicit proviso that such substitution is in
accordance with permitted valence of the substituted atom and the
substituent, and that the substitution results in a stable
compound, e.g., which does not spontaneously undergo transformation
such as by rearrangement, cyclization, elimination, etc.
[0207] For purposes of this invention, the definition of each
expression, e.g., alkyl, m, n, etc. when it occurs more than once
in any structure, is intended to be independent of its definition
elsewhere in the same structure.
[0208] The terms triflyl, tosyl, mesyl, and nonaflyl are
art-recognized and refer to trifluoromethanesulfonyl,
p-toluenesulfonyl, methanesulfonyl, and nonafluorobutanesulfonyl
groups, respectively. The terms triflate, tosylate, mesylate, and
nonaflate are art-recognized and refer to trifluoromethanesulfonate
ester, ptoluenesulfonateester, methanesulfonate ester, and
nonafluorobutanesulfonate ester functional groups and molecules
that contain said groups, respectively.
[0209] The abbreviations Me, Et, Ph, Tf, Nf, Ts, Ms represent
methyl, ethyl, phenyl, trifluoromethanesulfonyl,
nonafluorobutanesulfonyl, p-toluenesulfonyl and methanesulfonyl,
respectively. A more comprehensive list of the abbreviations
utilized by organic chemists of ordinary skill in the art appears
in the first issue of each volume of the Journal of Organic
Chemistry; this list is typically presented in a table entitled
Standard List of Abbreviations. The abbreviations contained in said
list, and all abbreviations utilized by organic chemists of
ordinary skill in the art are hereby incorporated by reference.
[0210] For purposes of this invention, the chemical of the
Elements, CAS version, Handbook of Chemistry and Physics, 67th Ed.,
1986-87, inside cover. Also for purposes of this invention, the
term "hydrocarbon" is contemplated to include all permissible
compounds having at least one hydrogen and one carbon atom. In a
broad aspect, the permissible hydrocarbons include acyclic and
cyclic, branched and unbranched, carbocyclic and heterocyclic,
aromatic and nonaromatic organic compounds that can be substituted
or unsubstituted.
3.2 GENERAL METHODS
[0211] The present invention relates to methods of preventing,
inhibiting, and/or reversing ocular neovascularization in a
mammalian subject comprising administering to the subject a
therapeutically effective amount of a substance that interferes
with the Hedgehog signaling pathway. Ocular neovascularization is a
major cause of blindness in developed countries. It is causally
involved in many ocular diseases including age-related macular
degeneration, (proliferative) diabetic retinopathy, neovascular
glaucoma, retinal vein occlusion or retinopathy of prematurity
(ROP). Current treatments are of limited efficacy and associated
with significant adverse effects, reflecting the high unmet need in
those disease areas.
[0212] The practice of the present invention will employ, unless
otherwise indicated, conventional methods of virology, chemistry,
microbiology, molecular biology and recombinant DNA techniques
within the skill of the art. Such techniques are explained fully in
the literature; see, e.g., Sambrook, et al. Molecular Cloning: A
Laboratory Manual (Current Edition); DNA Cloning: A Practical
Approach, vol. I & II (D. Glover, ed.); Oligonucleotide
Synthesis (N. Gait, ed., Current Edition); Nucleic Acid
Hybridization (B. Hames & S. Higgins, eds., Current Edition);
Transcription and Translation (B. Hames & S. Higgins, eds.,
Current Edition); CRC Handbook of Parvoviruses, vol. I & II (P.
Tijessen, ed.); Fundamental Virology, 2nd Edition, vol. I & II
(B. N. Fields and D. M. Knipe, eds.)
[0213] Numerous experimental methods are relevant to this invention
or experiments leading thereto, which are within routine skill in
the art. These include: methods for isolating nucleic acid
molecules, including, for example, phenol chloroform extraction,
quick lysis and capture on columns ([80]; U.S. Pat. No. 5,582,988);
methods of detecting and quantitating nucleic acid molecules;
methods of detecting and quantitating catalytic nucleic acid
activity; methods of amplifying a nucleic acid sequence including,
for example, PCR, SDA and TMA (also known as (SSR) [U.S. Pat. Nos.
4,683,202; 4,683,195; 4,000,159; 4,965,188; 5,176,995]; and methods
of determining whether a catalytic nucleic acid molecule cleaves an
amplified nucleic acid segment including, by way of example,
polyacrylamide gel electrophoresis and fluorescence resonance
energy transfer (FRET) [181]; PCT International Publication No. WO
94/29481].
[0214] Certain compounds of the present invention may exist in
particular geometric or stereoisomeric forms. The present invention
contemplates all such compounds, including cis- and trans-isomers,
R- and S enantiomers, diastereomers, (D)-isomers, (L)-isomers, the
racemic mixtures thereof, and other mixtures thereof, as falling
within the scope of the invention. Additional asymmetric carbon
atoms may be present in a substituent such as an alkyl group. All
such isomers, as well as mixtures thereof, are intended to be
included in this invention.
[0215] If, for instance, a particular enantiomer of a compound of
the present invention is desired, it may be prepared by asymmetric
synthesis, or by derivation with a chiral auxiliary, where the
resulting diastereomeric mixture is separated and the auxiliary
group cleaved to provide the pure desired enantiomers.
Alternatively, where the molecule contains a basic functional
group, such as amino, or an acidic functional group, such as
carboxyl, diastereomeric salts may be formed with an appropriate
optically active acid or base, followed by resolution of the
diastereomers thus formed by fractional crystallization or
chromatographic means well known in the art, and subsequent
recovery of the pure enantiomers.
[0216] Contemplated equivalents of the compounds described above
include compounds which otherwise correspond thereto, and which
have the same general properties thereof (e.g., the ability to
inhibit Hedgehog signaling), wherein one or more simple variations
of substituents are made which do not adversely affect the efficacy
of the compound. In general, the compounds of the present invention
may be prepared by the methods illustrated in the general reaction
schemes as, for example, described below, or by modifications
thereof, using readily available starting materials, reagents and
conventional synthesis procedures. In these reactions, it is also
possible to make use of variants that are in themselves known, but
are not mentioned here.
3.2.1 Small Molecule Inhibitors
[0217] Small molecule antagonists of Hedgehog signaling have been
described extensively in prior art. Formulas and methods of making
the compositions are described in detail in the following U.S.
patents and U.S. patent applications the specifications of which
are expressly incorporated by reference herein: U.S. patent
applications Ser. No. 20040110663 Dudek et al.: Hedgehog
antagonists, methods and uses related thereto 20040060568 Dudek et
al.: Hedgehog antagonists, methods and uses related thereto
20030022819 Ling et al.: Angiogenesis-modulating compositions and
uses.
[0218] To summarize: In some embodiments of the present invention,
a Hedgehog antagonist may be a small organic molecule. Such a small
organic molecule may antagonize Hedgehog signal transduction via an
interaction with but not limited to Hedgehog, patched (ptc), gli,
and/or smoothened. It is, therefore, specifically contemplated that
these small molecules which interfere with aspects of Hedgehog,
ptc, or smoothened signal transduction activity will likewise be
capable of inhibiting ocular neovascularization.
[0219] Thus, it is contemplated that in certain embodiments, these
compounds may be useful for inhibiting Hedgehog activity. In
preferred embodiments, the subject inhibitors are organic molecules
having a molecular weight less than 2500 kD, more preferably less
than 1500 kD, and even more preferably less than 750 kD, and are
capable of antagonizing Hedgehog signaling, preferably specifically
in target cells.
[0220] In certain preferred embodiments, the subject inhibitors
inhibit Hedgehog-mediated signal transduction with an IC50 of 1 mM
or less, more preferably of 1 .mu.M or less, and even more
preferably of 1 nM or less. Moreover, the subject methods can be
performed on cells that are provided in culture (in vitro), or on
cells in a whole animal (in vivo).
[0221] Hedgehog antagonists include AY9944, triparanol, jervine,
cyclopamine and tomatidine, compound A and compound B (U.S. patent
application Ser. No. 09/977,864).
[0222] In some embodiments, steroidal alkaloids are used to
interfere with Hedgehog signaling. Steroidal alkaloids have a
fairly complex nitrogen-containing nucleus. Two exemplary classes
of steroidal alkaloids for use in the subject methods are the
Solanum type and the Veratrum type. The above notwithstanding, in a
preferred embodiment, the methods and compositions of the present
invention make use of compounds having a steroidal alkaloid ring
system of cyclopamine.
[0223] There are more than 50 naturally occurring veratrum
alkaloids including veratramine, cyclopamine, cycloposine, jervine,
and muldamine occurring in plants of the Veratrum spp. The
Zigadenus spp., deathcamas, also produces several veratrum-type of
steroidal alkaloids including zygacine. In general, many of the
veratrum alkaloids (e.g., jervine, cyclopamine and cycloposine)
consist of a modified steroid skeleton attached spiro to a
furanopiperidine. Some veratrum-type alkaloids are depicted in FIG.
1.
[0224] An example of the Solanum type is solanidine. This steroidal
alkaloid is the nucleus (i.e., aglycone) for two important
glycoalkaloids, solanine and chaconine, found in potatoes. Other
plants in the Solanum family including various nightshades,
Jerusalem cherries, and tomatoes also contain solanum-type
glycoalkaloids. Glycoalkaloids are glycosides of alkaloids. A
typical solanum-type alkaloid is depicted in FIG. 2
(Solanidine).
[0225] Another class of smoothened antagonists can be based on the
veratrum-type steroidal alkaloids resembling verticine and
zygacine, or unsaturated forms thereof and/or seco-, nor- or
homo-derivatives thereof.
[0226] Based on these structures, and the possibility that certain
unwanted side effects can be reduced by some manipulation of the
structure, a wide range of steroidal alkaloids is contemplated as
potential smoothened antagonists for use in the subject method.
[0227] In certain embodiments, the subject antagonists can be
chosen on the basis of their selectively for the smoothened
pathway. This selectivity can be for the smoothened pathway versus
other steroid-mediated pathways (such as testosterone or estrogen
mediated activities), as well as selectivity for particular
Hedgehog/ptc/smoothened pathways, e.g., which isotype specific for
ptc (e.g., ptc-1, ptc-2) or Hedgehog (e.g., Shh, Ihh, Dhh, etc.).
For instance, the subject method may employ steroidal alkaloids
which do not substantially interfere with the biological activity
of such steroids as aldosterone, androstane, androstene,
androstenedione, androsterone, cholecalciferol, cholestane, cholic
acid, corticosterone, cortisol, cortisol acetate, cortisone,
cortisone acetate, deoxycorticosterone, digitoxigenin,
ergocalciferol, ergosterol, estradiol-17-a, estradiol-17-.beta.,
estriol, estrane, estrone, hydrocortisone, lanosterol, lithocholic
acid, mestranol, .beta.-methasone, prednisone, pregnane,
pregnenolone, progesterone, spironolactone, testosterone,
triamcinolone and their derivatives, at least so far as those
activities are unrelated to ptc related signaling.
[0228] In one embodiment, the subject steroidal alkaloid for use in
the present method has a kd for members of the nuclear hormone
receptor superfamily of greater than 1 .mu.M, and more preferably
greater than 1 mM, e.g., it does not bind estrogen, testosterone
receptors or the like. Preferably, the subject smoothened
antagonist has no estrogenic activity at physiological
concentrations (e.g., in the range of 1 ng-1 mg/kg).
[0229] In this manner, untoward side effects which may be
associated certain members of the steroidal alkaloid class can be
reduced. For example, using drug screening assays, the application
of combinatorial and medicinal chemistry techniques to the
steroidal alkaloids provides a means for reducing such unwanted
negative side effects including personality changes, shortened life
spans, cardiovascular diseases and vascular occlusion, organ
toxicity, hyperglycemia and diabetes, Cushnoid features, "wasting"
syndrome, steroidal glaucoma, hypertension, peptic ulcers, and
increased susceptibility to infections. For certain embodiments, it
will be beneficial to reduce the teratogenic activity relative
tojervine, as for example, in the use of the subject method to
selectively inhibit spermatogenesis.
[0230] In a preferred embodiment, the subject antagonists are
steroidal alkaloids other than spirosolane, tomatidine, jervine,
etc. In particular embodiments, the steroidal alkaloid is chosen
for use because it is more selective for one patched isoform over
the next, e.g., 10-fold, and more preferably at least 100-or even
1000-fold more selective for one patched pathway (ptc-1, ptc-2)
over another. Likewise, the steroidal alkaloid may be chosen for
use because it is more selective for one smoothened isoform over
the next, e.g., 10-fold, and more preferably at least 100-or even
1000-fold more selective for one wild-type smoothened protein
(should various isoforms exist) or for activated smoothened mutants
relative to wild-type smoothened.
[0231] In certain embodiments, the subject method can be carried
out conjointly with the administration of growth and/or trophic
factors, or compositions that also act on other parts of the
Hedgehog/smoothened pathway. For instance, it is contemplated that
the subject methods can include treatment with an agent that
modulates cAMP levels, e.g., increasing or decreasing intracellular
levels of cAMP.
[0232] In one embodiment, the subject method utilizes a smoothened
antagonist, and the conjoint agent elevates cAMP levels in order to
enhance the efficacy of the smoothened antagonist. For example,
compounds that may activate adenylate cyclase include forskolin
(FK), cholera toxin (CT), pertussis toxin (PT), prostaglandins
(e.g., PGE-1 and PGE-2), colforsin and .beta.-adrenergic receptor
agonists. .beta.-Adrenergic receptor agonists (sometimes referred
to herein as ".beta.-adrenergic agonists") include albuterol,
bambuterol, bitolterol, carbuterol, clenbuterol, clorprenaline,
denopamine, dioxethedrine, dopexamine, ephedrine, epinephrine,
etafedrine, ethylnorepinephrine, fenoterol, formoterol,
hexoprenaline, ibopamine, isoetharine, isoproterenol, mabuterol,
metaproterenol, methoxyphenamine, norepinephrine, oxyfedrine,
pirbuterol, prenalterol, procaterol, propranolol, protokylol,
quinterenol, reproterol, rimiterol, ritodrine, salmefamol,
soterenol, salmeterol, terbutaline, tretoquinol, tulobuterol, and
xamoterol.
[0233] Compounds which may inhibit a cAMP phosphodiesterase include
anrinone, milrinone, xanthine, methylxanthine, anagrelide,
cilostamide, medorinone, indolidan, rolipram,
3-isobutyl-1-methylxanthine(IBMX), chelerythrine, cilostazol,
glucocorticoids, griseolic acid, etazolate, caffeine, indomethacin,
papverine, MDL 12330A, SQ 22536, GDPssS, clonidine, type III and
type IV phosphodiesterase inhibitors, methylxanthines such as
pentoxifylline, theophylline, theobromine, pyrrolidinones and
phenyl cycloalkane and cycloalkene derivatives (described in PCT
publications Nos. WO 92/19594 and WO 92/10190), lisophylline, and
fenoxamine.
[0234] Analogs of cAMP which may be useful in the present method
include dibutyryl-cAMP (db-cAMP),(8-(4)-chlorophenylthio)-cAMP
(cpt-cAMP), 8-[(4-bromo-2,3-dioxobutyl)thio]-cAMP,
2-[(4-bromo-2,3-dioxobutyl)thio]-cAMP, 8-bromo-cAMP,
dioctanoyl-cAMP, Spadenosine 3':5'-cyclic phosphorothioate,
8-piperidino-cAMP, N6-phenyl-cAMP, 8-methylamino-cAMP,
8-(6-aminohexyl)amino-cAMP, 2'-deoxycAMP, N6,2'-O-dibutryl-cAMP,
N6,2'-O-disuccinyl-cAMP, N6-monobutyryl-cAMP, 2'-O-monobutyrylcAMP,
2'-O-monobutryl-8-bromo-cAMP, N6-monobutryl-2'-deoxy-cAMP, and
2'-O-monosuccinylcAMP.
[0235] Compounds which may reduce the levels or activity of cAMP
include prostaglandylinositol cyclicphosphate (cyclic PIP),
endothelins (ET)-1 and -3, norepinepurine, K252a, dideoxyadenosine,
dynorphins, melatonin, pertussis toxin, staurosporine, Gi 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.
[0236] In certain embodiments, a compound which is an antagonist of
the Hedgehog pathway is chosen to selectively antagonize Hedgehog
activity over protein kinases other than PKA, such as PKC, e.g.,
the compound modulates the activity of the Hedgehog pathway 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 the Hedgehog pathway may inhibit Hedgehog 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.
3.2.2 Protein Therapeutics
[0237] In some embodiments of the present invention, polypeptides
(including proteins) and/or modified polypeptides/proteins are used
as therapeutics.
[0238] There are several variants that are able to function as
antagonists. At least five different techniques can be envisioned:
Antibody antagonists, Dominant-negative versions of Hedgehog,
soluble receptors ("interceptors"), randomly selected peptide
antagonists, or randomly selected peptoid antagonists.
3.2.2.1 General Methods
[0239] There are two general ways of administering a polypeptide to
a subject: Either one administers the isolated polypeptide, or one
administers a gene transfer vector that encodes the desired
polypeptide. In the first case, the isolated polypeptide is
synthesized outside the mammalian subject to be treated, in the
second case, the polypeptide is synthesized within the mammalian
subject, using the cellular machinery of the transduced cell(s)
within the mammalian subject.
[0240] Isolated polypeptides can be produced by any suitable method
known in the art. Such methods range from direct protein synthetic
methods to constructing a DNA sequence encoding isolated
polypeptide sequences and expressing those sequences in a suitable
transformed host.
[0241] Standard methods may be applied to synthesize an isolated
polypeptide sequence of interest using standard methods of in vitro
protein synthesis.
[0242] In one embodiment of a recombinant method, a DNA sequence is
constructed by isolating or synthesizing a DNA sequence encoding a
wild type protein of interest. Optionally, the sequence may be
mutagenized by site-specific mutagenesis to provide functional
analogs thereof, or modified by any other means, e.g., by fusing to
another gene sequence, thus generating fusion proteins, or by
deleting specific parts of the gene sequence, resulting in the
expression of a protein that lacks specific parts compared to the
wild-type form. For example, a transmembrane domain can be deleted,
thus creating a secreted version of a protein that--in its original
state--is membrane anchored.
[0243] Another method of constructing a DNA sequence encoding a
polypeptide of interest would be by chemical synthesis using an
oligonucleotide synthesizer. Such oligonucleotides may be
preferably designed based on the amino acid sequence of the desired
polypeptide, and preferably selecting those codons that are favored
in the host cell in which the recombinant polypeptide of interest
will be produced. For example, a DNA oligomer containing a
nucleotide sequence coding for the particular isolated polypeptide
may be synthesized. In one embodiment, several small
oligonucleotides coding for portions of the desired polypeptide may
be synthesized and then ligated. The individual oligonucleotides
typically contain 5' or 3' overhangs for complementary assembly. A
complete amino acid sequence may be used to construct a
back-translated gene.
[0244] Once assembled (by synthesis, polymerase chain reaction,
site-directed mutagenesis, or by any other method), the mutant DNA
sequences encoding a particular isolated polypeptide of interest
will be inserted into an expression vector and operatively linked
to an expression control sequence appropriate for expression of the
protein in a desired host. Proper assembly may be confirmed by
nucleotide sequencing, restriction mapping, and expression of a
biologically active polypeptide in a suitable host. As is well
known in the art, in order to obtain high expression levels of a
transfected gene in a host, the gene must be operatively linked to
transcriptional and translational expression control sequences that
are functional in the chosen expression host.
[0245] The choice of expression control sequence and expression
vector will depend upon the choice of the corresponding host. A
wide variety of expression host/vector combinations may be
employed. Useful expression vectors for eukaryotic hosts, include,
for example, vectors comprising expression control sequences from
SV40, bovine papilloma virus, retrovirus, adenovirus and
cytomegalovirus. Useful expression vectors for bacterial hosts
include known bacterial plasmids, such as plasmids from Escherichia
coli, including pCR1, pBR322, pMB9 and their derivatives, wider
host range plasmids, such as M13 and filamentous single-stranded
DNA phages. Preferred E. coli vectors include pL vectors containing
the lambda phage pL promoter (U.S. Pat. No. 4,874,702), pET vectors
containing the T7 polymerase promoter [82] and the pSP72 vector.
Useful expression vectors for yeast cells, for example, include the
2 g and centromere plasmids.
[0246] Further, within each specific expression vector, various
sites may be selected for insertion of these DNA sequences. These
sites are usually designated by the restriction endonuclease which
cuts them. They are well-recognized by those of skill in the art.
It will be appreciated that a given expression vector useful in
this invention need not have a restriction endonuclease site for
insertion of the chosen DNA fragment. Instead, the vector may be
joined by the fragment by alternate means.
[0247] The expression vector, and the site chosen for insertion of
a selected DNA fragment and operative linking to an expression
control sequence, is determined by a variety of factors such as:
the number of sites susceptible to a particular restriction enzyme,
the size of the polypeptide, how easily the polypeptide is
proteolytically degraded, and the like. The choice of a vector and
insertion site for a given DNA is determined by a balance of these
factors.
[0248] To provide for adequate transcription of the recombinant
constructs of the invention, a suitable promoter/enhancer sequence
may preferably be incorporated into the recombinant vector,
provided that the promoter/expression control sequence is capable
of driving transcription of a nucleotide sequence encoding the
polypeptide of interest. Any of a wide variety of expression
control sequences may be used in these vectors. Such useful
expression control sequences include the expression control
sequences associated with structural genes of the foregoing
expression vectors. Examples of useful expression control sequences
include, for example, the-early and late promoters of SV40 or
adenovirus, the lac system, the trp system, the TAC or TRC system,
the major operator and promoter regions of phage lambda, for
example pL, the control regions of 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 .alpha.-mating system and other sequences known to control
the expression of genes of prokaryotic or eukaryotic cells and
their viruses, and various combinations thereof. Many of the
vectors mentioned are commercially available. One supplier is
Invitrogen, Carlsbad, USA, www.invitrogen.com. The company also
offers detailed information on the sequence of the vectors it
sells.
[0249] Any suitable host may be used to produce in quantity the
isolated polypeptides of the invention, including bacteria, fungi
(including yeasts), plants, insects, mammals, or other appropriate
animal cells or cell lines, as well as transgenic animals or
plants. More particularly, these hosts may include well known
eukaryotic and prokaryotic hosts, such as strains of E. coli,
Pseudomonas, Bacillus, Streptomyces, fungi, yeast (e.g.,
Hansenula), insect cells such as Spodoptera firugiperda (SF9), and
High Five TM, animal cells such as Chinese hamster ovary (CHO),
mouse cells such as NS/O cells, African green monkey cells, COS 1,
COS 7, BSC 1, BSC 40, and BMT 10, and human cells, as well as plant
cells.
[0250] Promoters which may be used to control the expression of
polypeptides in eukaryotic cells include, but are not limited to,
the SV40 early promoter region [83], the promoter contained in the
3' long terminal repeat of Rous sarcoma virus [84], the herpes
thymidine kinase promoter [85], the regulatory sequences of the
metallothionine gene [86].
[0251] In case the polypeptide is expressed in plants, plant
expression vectors should be used comprising the nopaline
synthetase promoter region [87] or the cauliflower mosaic virus 35S
RNA promoter [88], and the promoter for the photosynthetic enzyme
ribulose biphosphatecarboxylase [87].
[0252] In case the polypeptide is expressed in yeast or other
fungi, promoter elements should be chosen such as the Gal 4
promoter, the ADC (alcohol dehydrogenase) promoter, PGK
(phosphoglycerolkinase) promoter, alkaline phosphatase
promoter.
[0253] In case the polypeptide is expressed in transgenic animals,
the following animal transcriptional control regions can be used,
which exhibit tissue specificity and have been utilized in
transgenic animals: elastase I gene control region which is active
in pancreatic cells ([89]; [90]); insulin gene enhancers for
promoters which are active in pancreatic cells ([91]);
immunoglobulin gene enhancers or promoters which are active in
lymphoid cells ([92]; [93]; [94]); the cytomegalovirus early
promoter and enhancer regions ([95]); mouse mammary tumor virus
control region which is active in testicular, breast, lymphoid and
mast cells ([96]); albumin gene control region which is active in
liver [97]; .alpha.-fetoprotein gene control region which is active
in liver ([98]; [99]); .alpha.-antitrypsin gene control region
which is active in the liver ([100]); beta.-globin gene control
region which is active in myeloid cells ([101]), myelin basic
protein gene control region which is active in oligodendrocyte
cells in the brain [102]; myosin light chain-2 gene control region
which is active in skeletal muscle [103]; and gonadotropic
releasing hormone gene control region which is active in the
hypothalamus ([104]).
[0254] Operative linking of a DNA sequence to an expression control
sequence includes the provision of a translation start signal in
the correct reading frame upstream of the DNA sequence. If the
particular DNA sequence being expressed does not begin with a
methionine, the start signal will result in an additional amino
acid (methionine) being located at the N-terminus of the product.
If a hydrophobic moiety is to be linked to the N-terminal
methionyl-containing protein, the protein may be employed directly
in the compositions of the invention. Yet, methods are available in
the art to remove N-terminal methionines from polypeptides
expressed with them. For example, certain hosts and fermentation
conditions permit removal of substantially all of the N-terminal
methionine in vivo.
[0255] It should be understood that not all vectors and expression
control sequences will function equally well to express a given
isolated polypeptide. Neither will all hosts function equally well
with the same expression system. However, one of skill in the art
may make a selection among these vectors, expression control
systems and hosts without undue experimentation.
[0256] Successful incorporation of these polynucleotide constructs
into a given expression vector may be identified by three general
approaches: (a) DNA-DNA hybridization, (b) presence or absence of
"marker" gene functions, and (c) expression of inserted sequences.
In the first approach, the presence of the gene inserted in an
expression vector can be detected by DNA-DNA hybridization using
probes comprising sequences that are homologous to the inserted
gene. In the second approach, the recombinant vector/host system
can be identified and selected based upon the presence or absence
of certain "marker" gene functions (e.g., thymidine kinase
activity, resistance to antibiotics such as G418, transformation
phenotype, occlusion body formation in baculovirus, etc.) caused by
the insertion of foreign genes in the vector. For example, if the
polynucleotide is inserted so as to interrupt a marker gene
sequence of the vector, recombinants containing the insert can be
identified by the absence of the marker gene function. In the third
approach, recombinant expression vectors can be identified by
assaying the foreign gene product expressed by the recombinant
vector. Such assays can be based, for example, on the physical or
functional properties of the gene product in bioassay systems.
[0257] Recombinant nucleic acid molecules which encode modified
protein therapeutics may be obtained by any method known in the art
(Maniatis et al., 1982, Molecular Cloning; A Laboratory Manual,
Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.) or
obtained from publicly available clones. Modifications
comprise--but are not limited to--deletions, insertions, point
mutations, fusions to other polypeptides. In some embodiments of
the invention, a recombinant vector system may be created to
accommodate sequences encoding the therapeutic of interest in the
correct reading frame with a synthetic hinge region. Additionally,
it may be desirable to include, as part of the recombinant vector
system, nucleic acids corresponding to the 3' flanking region of an
immunoglobulin gene including RNA cleavage/polyadenylation sites
and downstream sequences. Furthermore, it may be desirable to
engineer a signal sequence upstream of the modified protein
therapeutic to facilitate the secretion of the protein therapeutic
from a cell transformed with the recombinant vector. This is
particularly of interest in embodiments, where a normally
membrane-bound protein is modified in a way so that it will be
secreted instead.
[0258] There are also commercial services available that will
synthesize any desired gene or genetic sequence based upon an
electronically submitted gene sequence (e.g., Medigenomix, Munich,
Germany; Geneart, Regensburg, Germany). They will also provide the
insertion of the gene sequence into a gene expression vector of
choice. These commercial services not only enable the artisan, but
everyone to gain access to any genetic vector they desire. This
actually should be considered the optimal method for obtaining a
gene transfer vector.
[0259] The proteins produced by a transformed host can be purified
according to any suitable method. Such standard methods include
chromatography (e.g., ion exchange, affinity, and sizing column
chromatography), centrifugation, differential solubility, or by any
other standard technique for protein purification. For
immunoaffinity chromatography, the protein of interest may be
isolated by binding it to an affinity column comprising antibodies
that were raised against said protein or a cross-reactive protein
and were affixed to a stationary support. to give a substantially
pure protein. By the term "substantially pure" is intended that the
protein is free of the impurities that are naturally associated
therewith. Substantial purity may be evidenced by a single band by
electrophoresis. Isolated proteins can also be characterized
physically using such techniques as proteolysis, nuclear magnetic
resonance, and X-ray crystallography.
[0260] In one embodiment, a modified, secreted version of Hip1 is
purified by passing a supernatant containing said secreted version
of Hip1 through an affinity column that has Shh bound to it. The
bound target protein may then be eluted by treatment with a
chaotropic salt or by elution with aqueous acetic acid. In one
embodiment, specific immunoglobulin fusion proteins may be purified
by passing a solution containing the fusion protein through a
column which contains immobilized protein A or protein G which
selectively binds the Fc portion of the fusion protein (PCT
Application, Publication No. WO87/00329).
[0261] Alternatively, affinity tags such as hexa-histidine, maltose
binding domain, influenza coat sequence, and
glutathione-S-transferase can be attached to the protein to allow
easy purification by passage over an appropriate affinity
column.
3.2.2.2 Soluble Receptors (Interceptors)
[0262] In certain embodiments, soluble receptors are used to
interfere with Hedgehog signaling. Many receptors are normally
membrane-bound proteins and can be "solubilized" by removing their
transmembrane domain. In instances where removing the transmembrane
domain leaves the remaining domains functional, particularly the
domain that interacts with the ligand, a soluble receptor can be
used as an antagonist: Said soluble receptor ("interceptor") is
able to bind and thus sequester its ligand so that the ligand
cannot bind any more to the functional, membrane-bound receptor. In
other words, the soluble receptor competes with the membrane-bound
receptor for ligand binding. By providing the soluble receptor in
excess, one can prevent binding of the ligand (e.g., Hedgehog) to
its receptor, thus preventing activation of the corresponding
signaling pathway.
[0263] In one embodiment, Hip1 is modified in a way so that it
lacks its transmembrane domain. The transmembrane domain is removed
from the genetic sequence of the HHIP gene by standard
recombination technologies, preferentially by PCR mutagenesis. The
modified HHIP gene can then be expressed in appropriate host cells.
The synthesized, modified Hip1 is then secreted from its host
cells, but is still able to bind and thus sequester Hedgehog.
3.2.2.3 Dominant-Negative Hedgehog Variants
[0264] Several Hedgehog variants are unable to elicit a
Hedgehog-dependent response on C3H10T1/2 cells, but they competed
with mature Hedgehog for function and therefore serve as functional
antagonists. The synthesis and use of such Hedgehog antagonist
moieties are described in prior art.
[0265] To summarize: Certain Hedgehog variants that contain
N-terminal modifications can block Hedgehog function because they
lack the ability to elicit a Hedgehog-dependent response but retain
the ability to bind to the Hedgehog receptor, patched-1. The
critical primary amino acid sequence that defines whether a
Hedgehog polypeptide (i.e., a Sonic, Indian or Desert Hedgehog) is
a functional Hedgehog antagonist is the N-terminal cysteine residue
that corresponds to Cys-1 of the mature Hedgehog. So long as the
Hedgehog polypeptide either lacks this N-terminal cysteine
completely or contains this N-terminal cysteine in a modified form
(e.g. chemically modified or included as part of an N-terminal
extension moiety), the resulting polypeptide can act as a
functional Hedgehog antagonist.
[0266] Provided that, for example, a Sonic Hedgehog has an
N-terminal cysteine corresponding to Cys-1 that is altered or
otherwise modified, it can antagonize the action of any other
member of the Hedgehog family. One skilled in the art can alter the
structure of the antagonist, e.g., by producing fragments or
analogs, and test the newly produced structures for antagonist
activity. These, or analogous methods, can be used to make and
screen fragments and analogs of a antagonist polypeptides.
[0267] Antagonist forms of Hedgehog may be identified by using a
Hedgehog sensitive screening system. For example, a cell line
transfected with a gli-1-lacz reporter gene construct could be
monitored for .beta.-galactosidase activity. Gli-1 is a reporter
for activation of the Hedgehog signaling pathway and Hedgehog
mutants that inhibit gli-1-driven reporter gene expression would be
Hedgehog antagonists. Any number of reporter genes may be used,
including luciferase, green fluorescent protein (and variants
including yellow, red, blue and cyan), GUS, and other fluorescent
or chromogenic proteins.
3.2.2.4 Antibody Antagonists
[0268] It is anticipated that antibodies can act as hedgehog
antagonists. Antibodies can have extraordinary affinity and
specificity for particular epitopes. Antibodies that bind to any
protein in the hedgehog signaling pathway may have the capacity to
act as antagonists. Antibodies that bind to hedgehog or smoothened
may act by simply sterically hindering the proper protein-protein
interactions or occupying active sites. Antibodies that bind to
patched proteins may act as antagonists if they cause
hyperactivation of the patched protein, for example stimulating
patched association with smoothened. Proteins with extracellular
domains are readily bound by exogenously supplied antibodies.
[0269] Several anti-hedgehog or patched monoclonal antibodies have
been previously described. These anti-hedgehog or patched
monoclonal antibodies and others will be useful in the methods of
treatment according to the present invention.
[0270] In one embodiment of the present invention, hedgehog
antibodies are used to interfere with hedgehog signaling: By
binding to secreted Hedgehog in general, and Shh in particular,
preferentially the N-terminal fragment, antibodies can prevent Shh
to interact with its receptor Patch proteins. Preferred antibodies
are specifically immunoreactive with a vertebrate hedgehog protein.
For example, by using immunogens derived from hedgehog protein,
monoclonal or polyclonal antibodies can be made using standard
protocols (See, for example, Antibodies: A laboratory manual ed. by
Harlow and Lane (Cold Spring Harbor Press: 1988)). A mammal, such
as a mouse, a hamster or rabbit can be immunized with an
immunogenic form of the peptide (e.g., a vertebrate hedgehog
polypeptide or an antigenic fragment which is capable of eliciting
an antibody response). Techniques for conferring immunogenicity on
a protein or peptide include conjugation to carriers or other
techniques well known in the art. An immunogenic portion of a
hedgehog protein can be administered in the presence of adjuvant.
The progress of immunization can be monitored by detection of
antibody titers in plasma or serum. Standard ELISA or other
immunoassays can be used with the immunogen as antigen to assess
the levels of antibodies. In a preferred embodiment, the subject
antibodies are immunospecific for antigenic determinants of a
hedgehog protein of a vertebrate organism. In yet a further
preferred embodiment the present invention provides, for example,
antibodies which are immunospecific for discrete hedgehog family
member, e.g. Shh versus Dhh versus Ihh. Antibodies which are
immunospecific for hedgehog, or for a specific hedgehog family
member do not substantially cross-react with non-homologous
proteins. In one embodiment, the antibody does not substantially
cross-react with an invertebrate hedgehog protein.
[0271] The term antibody as used herein is intended to include
fragments thereof that are also specifically reactive with one or
more of the vertebrate hedgehog polypeptides. Antibodies can be
fragmented using conventional techniques, and the fragments are
screened for utility in the same manner as described above for
whole antibodies. For example, F(ab).sub.2 fragments can be
generated by treating antibody with pepsin. The resulting
F(ab).sub.2 fragment can be treated to reduce disulfide bridges to
produce Fab fragments. The antibody of the present invention is
further intended to include bispecific and chimeric molecules
having affinity for a hedgehog protein conferred by at least one
CDR region of the antibody.
[0272] Both monoclonal and polyclonal antibodies immunoreactive
with hedgehog polypeptides can be used as hedgehog antagonists.
Although not all hedgehog antibodies function as hedgehog
antagonists, antibodies with hedgehog antagonist activity can be
identified in much the same way as other hedgehog antagonists. For
example, candidate antibodies can be administered to cells
expressing a reporter protein under the control of a
Hedgehog-controlled promoter, and antibodies that cause decreased
reporter gene expression are antagonists.
[0273] In one variation, antibodies of the invention can be single
chain antibodies (scFv), comprising variable antigen binding
domains linked by a polypeptide linker. Single chain antibodies are
expressed as a single polypeptide chain and can be expressed in
bacteria and as part of a phage display library. In this way,
phages that express the appropriate scFv will have hedgehog
antagonist activity. The nucleic acid encoding the single chain
antibody can then be recovered from the phage and used to produce
large quantities of the scFv. Construction and screening of scFv
libraries is extensively described in various publications (U.S.
Pat. Nos. 5,258,498; 5,482,858; 5,091,513; 4,946,778; 5,969,108;
5,871,907; 5,223,409; 5,225,539).
[0274] An illustrative example of a hedgehog antibody which
functions as a hedgehog antagonist is 5E1. As noted in the Examples
provided herein, 5E1 functions in vitro and in vivo as a hedgehog
antagonist. The invention specifically contemplates the use of 5E1,
or an antibody which recognizes the same epitope as 5E1 in the
subject methods.
[0275] The technology for producing monoclonal antibodies is well
known. See, for example, WO93/09229, or U.S. Pat. No.
5,411,941,
[0276] Fully human monoclonal antibody homologs against hedgehog or
patched are another preferred binding agent that may block or coat
hedgehog ligands in the method of the invention. In their intact
form these may be prepared using in vitro-primed human splenocytes,
as described in [105]. Alternatively, they may be prepared by
repertoire cloning as described in [106] or in [107] or in U.S.
Pat. No. 5,798,230 that describes preparation of human monoclonal
antibodies from human B cells. According to this process, human
antibody-producing B cells are immortalized by infection with an
Epstein-Barr virus, or a derivative thereof, that expresses
Epstein-Barr virus nuclear antigen 2 (EBNA2). EBNA2 function, which
is required for immortalization, is subsequently shut off, which
results in an increase in antibody production.
[0277] In yet another method for producing fully human antibodies,
U.S. Pat. No. 5,789,650 describes-transgenic non-human animals
capable of producing heterologous antibodies and transgenic
non-human animals having inactivated endogenous immunoglobulin
genes.
[0278] Large nonimmunized human phage display libraries may also be
used to isolate high affinity antibodies that can be developed as
human therapeutics using standard phage technology [108-110].
[0279] Yet another preferred binding agent that may block or coat
hedgehog ligands in the method of the invention is a humanized
recombinant antibody homolog having anti-hedgehog, anti-Smo or
patched specificity. Following the early methods for the
preparation of true "chimeric antibodies" (where the entire
constant and entire variable regions are derived from different
sources), a new approach was described in EP 0239400 (Winter et
al.) whereby antibodies are altered by substitution (within a given
variable region) of their complementarity determining regions
(CDRs) for one species with those from another. The process for
humanizing monoclonal antibodies via CDR "grafting" has been termed
"reshaping" [111-113]. See also U.S. Pat. Nos. 5,693,762;
5,693,761; 5,585,089; and 5,530,101 (Protein Design Labs).
3.2.2.5 Peptoids
[0280] Peptoids can be developed for any given protein,
particularly for secreted proteins such as proteins of the Hedgehog
family. How to generate and isolate peptoids has been described in
prior art. U.S. Patent application 20040161798 is included hereby
by reference. One of ordinary skill in the art will be able to
generate and isolate peptoids that interfere with Hedgehog
signaling function. Peptoids against Hedgehog proteins, Gli-1, or
Fu are of particular interest. One strength of peptoids is that
they can also pass through plasma membranes and thus, they can be
targeted against intracellular proteins such as, e.g., Gli-1.
3.2.3 Antisense, Ribozyme, Triple Helix RNA Interference and
Aptamer Techniques
[0281] Another aspect of the invention relates to the use of
nucleic acids and/or modified nucleic acids as therapeutics. In
some embodiments, these nucleic acids are produced inside cells via
means of gene transfer vectors. In other embodiments, these nucleic
acids are directly administered to the mammalian subject in vivo.
At least four different techniques have been described in prior
are: Antisense, ribozyme, RNA interference and aptamers.
[0282] Antisense RNA and DNA, ribozyme, and triple helix molecules
of the invention may be prepared by any method known in the art for
the synthesis of DNA and RNA molecules. These include techniques
for chemically synthesizing oligodeoxyribonucleotides and
oligoribonucleotides well known in the art such as for example
solid phase phosphoramidite chemical synthesis. Alternatively, RNA
molecules may be generated by in vitro and in vivo transcription of
DNA sequences encoding the antisense RNA molecule. Such DNA
sequences may be incorporated into a wide variety of vectors that
incorporate suitable RNA polymerase promoters such as the T7 or SP6
polymerase promoters. Alternatively, antisense cDNA constructs that
synthesize antisense RNA constitutively or inducibly, depending on
the promoter used, can be introduced stably into cell lines.
[0283] Moreover, various well-known modifications to nucleic acid
molecules may be introduced as a means of increasing intracellular
stability and half-life. Possible modifications include but are not
limited to the addition of flanking sequences of ribonucleotides or
deoxyribonucleotides to the 5' and/or 3' ends of the molecule or
the use of phosphorothioate or 2' O-methyl rather than
phosphodiesterase linkages within the oligodeoxyribonucleotide
backbone.
3.2.3.1 Antisense
[0284] As used herein, "antisense" therapy refers to administration
or in situ generation of oligonucleotide molecules or their
derivatives which specifically hybridize (e.g., bind) under
cellular conditions, with the cellular mRNA and/or genomic DNA
encoding one or more of the subject hedgehog pathway proteins so as
to 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 that relies on specific binding to
oligonucleotide sequences.
[0285] 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 cellular mRNA which encodes a
hedgehog signaling protein. Alternatively, the antisense construct
is an oligonucleotide probe that 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 hedgehog
signaling gene. Such oligonucleotide probes are preferably modified
oligonucleotides that are resistant to endogenous nucleases, e.g.,
exonucleases and/or endonucleases, and are therefore stable in
vivo. Exemplary nucleic acid molecules for use as antisense
oligonucleotides are phosphoramidate, 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, in [114]; or [115]. With respect
to antisense DNA, oligodeoxyribonucleotides derived from the
translation initiation site, e.g., between the -10 and +10 regions
of the hedgehog signaling gene nucleotide sequence of interest, are
preferred.
[0286] Antisense approaches involve the design of oligonucleotides
(either DNA or RNA) that are complementary to mRNA encoding a
hedgehog signaling protein. The antisense oligonucleotides will
bind to the mRNA transcripts and prevent translation. Absolute
complementarity, although preferred, is not required. In the case
of double-stranded antisense nucleic acids, a single strand of the
duplex DNA may thus be tested, or triplex formation may be assayed.
The ability to hybridize will depend on both the degree of
complementarity and the length of the antisense nucleic acid.
Generally, the longer the hybridizing nucleic acid, the more base
mismatches with an RNA it may contain and still form a stable
duplex (or triplex, as the case may be). One skilled in the art can
ascertain a tolerable degree of mismatch by use of standard
procedures to determine the melting point of the hybridized
complex.
[0287] Oligonucleotides that are complementary to the 5' end of the
mRNA, e.g., the 5' untranslated sequence up to and including the
AUG initiation codon, should work most efficiently at inhibiting
translation. However, sequences complementary to the 3'
untranslated sequences of mRNAs have recently been shown to be
effective at inhibiting translation of mRNAs as well [116].
Therefore, oligonucleotides complementary to either the 5' or 3'
untranslated, non-coding regions of a gene could be used in an
antisense approach to inhibit translation of that mRNA.
Oligonucleotides complementary to the 5' untranslated region of the
mRNA should include the complement of the AUG start codon.
Antisense oligonucleotides complementary to mRNA coding regions are
less efficient inhibitors of translation but could also be used in
accordance with the invention. Whether designed to hybridize to the
5', 3' or coding region of mRNA, antisense nucleic acids should be
at least six nucleotides in length, and are preferably less that
about 100 and more preferably less than about 50, 25, 17 or 10
nucleotides in length.
[0288] Regardless of the choice of target sequence, it is preferred
that in vitro studies are first performed to quantitate the ability
of the antisense oligonucleotide to quantitate the ability of the
antisense oligonucleotide to inhibit gene expression. It is
preferred that these studies utilize controls that distinguish
between antisense gene inhibition and nonspecific biological
effects of oligonucleotides. It is also preferred that these
studies compare levels of the target RNA or protein with that of an
internal control RNA or protein. Additionally, it is envisioned
that results obtained using the antisense oligonucleotide are
compared with those obtained using a control oligonucleotide. It is
preferred that the control oligonucleotide is of approximately the
same length as the test oligonucleotide and that the nucleotide
sequence of the oligonucleotide differs from the antisense sequence
no more than is necessary to prevent specific hybridization to the
target sequence.
[0289] The oligonucleotides can be DNA or RNA or chimeric mixtures
or derivatives or modified versions thereof, single-stranded or
double-stranded. The oligonucleotide can be modified at the base
moiety, sugar moiety, or phosphate backbone, for example, to
improve stability of the molecule, hybridization, etc. The
oligonucleotide may include other appended groups such as peptides
(e.g., for targeting host cell receptors), or agents facilitating
transport across the cell membrane (see, e.g., [117]; [118]; PCT
Publication No. WO88/09810) or the blood-brain barrier (see, e.g.,
PCT Publication No. WO89/10134), hybridization-triggered cleavage
agents [114] or intercalating agents [119]. To this end, the
oligonucleotide may be conjugated to another molecule, e.g., a
peptide, hybridization triggered cross-linking agent, transport
agent, hybridization-triggered cleavage agent, etc.
[0290] The antisense oligonucleotide may comprise at least one
modified base moiety which is selected from the group including but
not limited to 5-fluorouracil, 5-bromouracil, 5-chlorouracil,
5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine,
5-(carboxyhydroxytriethyl) uracil,
5-carboxymethylaminomethyl-2-thiouridine,
5-carboxymethylaminomethyluracil, dihydrouracil,
.beta.-D-galactosylqueosine, inosine, N6-isopentenyladenine,
1-methylguanine, 1-methylinosine, 2,2-dimethylguanine,
2-methyladenine, 2-methylguanine, 3-methylcytosine,
5-methylcytosine, N6-adenine, 7-methylguanine,
5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil;
.beta.-D-mannosylqueosine, 5'-methoxycarboxymethyluracil,
5-methoxyuracil, 2-methylthio-N6-isopentenyladenine,
uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine,
2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil,
5-methyluracil, uracil-5-oxyacetic acid methyl ester,
uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil,
3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and
2,6-diaminopurine.
[0291] The antisense oligonucleotide may also comprise at least one
modified sugar moiety selected from the group including but not
limited to arabinose, 2-fluoroarabinose, xylulose, and hexose.
[0292] The antisense oligonucleotide can also contain a neutral
peptide-like backbone. Such molecules are termed peptide nucleic
acid (PNA)-oligomers and are described, e.g., in [120] and [121].
One advantage of PNA oligomers is their capability to bind to
complementary DNA essentially independently from the ionic strength
of the medium due to the neutral backbone of the DNA. In yet
another embodiment, the antisense oligonucleotide comprises at
least one modified phosphate backbone selected from the group
consisting of a phosphorothioate, a phosphorodithioate, a
phosphoramidothioate, a phosphoramidate, a phosphordiamidate, a
methylphosphonate, an alkyl phosphotriester, and a formacetal or
analog thereof.
[0293] In yet a further embodiment, the antisense oligonucleotide
is an--anomeric oligonucleotide. An--anomeric oligonucleotide forms
specific double-stranded hybrids with complementary RNA in which,
contrary to the usual units, the strands run parallel to each other
[122]. The oligonucleotide is a 2'-O-methylribonucleotide [123], or
a chimeric RNA-DNA analogue.
[0294] Oligonucleotides of the invention may be synthesized by
standard methods known in the art, e.g., by use of an automated DNA
synthesizer (such as are commercially available from Biosearch,
Applied Biosystems, etc.). As examples, phosphorothioate
oligonucleotides may be synthesized by the method of Stein et al.
[124-127], methylphosphonate oligonucleotides can be prepared by
use of controlled pore glass polymer supports [128].
[0295] While antisense nucleotides complementary to the coding
region of an mRNA sequence can be used, those complementary to the
transcribed untranslated region and to the region comprising the
initiating methionine are most preferred.
[0296] The antisense molecules can be delivered to cells that
express hedgehog signaling genes in vivo. A number of methods have
been developed for delivering antisense DNA or RNA to cells; e.g.,
antisense molecules can be injected directly into the tissue site,
or modified antisense molecules, designed to target the desired
cells (e.g., antisense linked to peptides or antibodies that
specifically bind receptors or antigen expressed on the target cell
surface) can be administered systematically.
[0297] However, it may be difficult to achieve intracellular
concentrations of the antisense sufficient to suppress translation
on endogenous mRNAs in certain instances. Therefore a preferred
approach utilizes a recombinant DNA construct in which the
antisense oligonucleotide is placed under the control of a strong
pol m or pol II promoter. The use of such a construct to transfect
target cells in the patient will result in the transcription of
sufficient amounts of single stranded RNAs that will form
complementary base pairs with the endogenous hedgehog signaling
transcripts and thereby prevent translation. For example, a vector
can be introduced in vivo such that it is taken up by a cell and
directs the transcription of an antisense RNA. Such a vector can
remain episomal or become chromosomally integrated, as long as it
can be transcribed to produce the desired antisense RNA. Such
vectors can be constructed by recombinant DNA technology methods
standard in the art. Vectors can be plasmid, viral, or others known
in the art, used for replication and expression in mammalian cells.
Expression of the sequence encoding the antisense RNA can be by any
promoter known in the art to act in mammalian, preferably human
cells. Such promoters can be inducible or constitutive. Such
promoters include but are not limited to: the SV40 early promoter
region [83], the promoter contained in the 3' long terminal repeat
of Rous sarcoma virus [84], the herpes thymidine kinase promoter
[85], the regulatory sequences of the metallothionein gene
(Brinster et al, 1982, Nature 296:3942), etc. Any type of plasmid,
cosmid, YAC or viral vector can be used to prepare the recombinant
DNA construct that can be introduced directly into the tissue site.
Alternatively, viral vectors can be used which selectively infect
the desired tissue, in which case administration may be
accomplished by another route (e.g., systematically).
3.2.3.2 Ribozymes
[0298] Ribozyme molecules designed to catalytically cleave hedgehog
signaling mRNA transcripts can also be used to prevent translation
of mRNA (See, e.g., PCT International Publication WO90/11364,
published Oct. 4, 1990; [129], U.S. Pat. No. 5,093,246). While
ribozymes that cleave mRNA at site-specific recognition sequences
can be used to destroy particular mRNAs, the use of hammerhead
ribozymes is preferred. Hammerhead ribozymes cleave mRNAs at
locations dictated by flanking regions that form complementary base
pairs with the target mRNA. The sole requirement is that the target
mRNA have the following sequence of two bases: 5'-UG-3'. The
construction and production of hammerhead ribozymes is well known
in the art and is described more fully in [130].
[0299] The ribozymes of the present invention also include RNA
endoribonucleases (hereinafter "Cech-type ribozymes") such as the
one which occurs naturally in Tetrahymena thermophila (known as the
IVS, or L-19 IVS RNA) and which has been extensively described by
Thomas Cech and collaborators [113-135], published International
patent application No. WO88/04300; [136]. The Cech-type ribozymes
have an eight base pair active site that hybridizes to a target RNA
sequence whereafter cleavage of the target RNA takes place. The
invention encompasses those Cech-type ribozymes that target eight
base-pair active site sequences.
[0300] As in the antisense approach, the ribozymes can be composed
of modified oligonucleotides (e.g., for improved stability,
targeting, etc.) and should be delivered to cells that express
hedgehog signaling genes in vivo. A preferred method of delivery
involves using a DNA construct "encoding" the ribozyme under the
control of a strong constitutive pol III or pol II promoter, so
that transfected cells will produce sufficient quantities of the
ribozyme to destroy targeted messages and inhibit translation.
Because ribozymes unlike antisense molecules, are catalytic, a
lower intracellular concentration is required for efficiency.
3.2.3.3 Triple Helix Formation
[0301] Alternatively, endogenous hedgehog signaling gene expression
can be reduced by targeting deoxyribonucleotide sequences
complementary to the regulatory region of the gene (i.e., the
promoter and/or enhancers) to form triple helical structures that
prevent transcription of the gene in target cells in the body. (See
generally, [137]; [138]).
[0302] Nucleic acid molecules to be used in triple helix formation
for the inhibition of transcription are preferably single stranded
and composed of deoxyribonucleotides. The base composition of these
oligonucleotides should promote triple helix formation via
Hoogsteen base pairing rules, which generally require sizable
stretches of either purines or pyrimidines to be present on one
strand of a duplex. Nucleotide sequences may be pyrimidine-based,
which will result in TAT and CGC triplets across the three
associated strands of the resulting triple helix. The
pyrimidine-rich molecules provide base complementarity to a
purine-rich region of a single strand of the duplex in a parallel
orientation to that strand. In addition, nucleic acid molecules may
be chosen that are purine-rich, for example, containing a stretch
of G residues. These molecules will form a triple helix with a DNA
duplex that is rich in GC pairs, in which the majority of the
purine residues are located on a single strand of the targeted
duplex, resulting in CGC triplets across the three strands in the
triplex.
[0303] Alternatively, the potential sequences that can be targeted
for triple helix formation may be increased by creating a so-called
"switchback" nucleic acid molecule. Switchback molecules are
synthesized in an alternating 5'-3',3'-5' manner, such that they
base pair with first one strand of a duplex and then the other,
eliminating the necessity for a sizable stretch of either purines
or pyrimidines to be present on one strand of a duplex.
3.2.3.4 RNA Interference
[0304] The discovery that RNA interference (RNAi) seems to be a
ubiquitous mechanism to silence genes suggests an alternative,
novel approach to decrease gene expression, which is able to
overcome the limitations of the other approaches outlined above.
Short interfering RNAs (siRNAs) are at the heart of RNAi. The
antisense strand of the siRNA is used by an RNAi silencing complex
to guide cleavage of complementary mRNA molecules, thus silencing
expression of the corresponding gene [6-10].
[0305] The present invention--leveraging RNAi--thus differs from
other nucleic acid based strategies (antisense and ribozyme
methods) in both approach and effectiveness:
[0306] (a) Compared to antisense strategies, RNAi leverages a
catalytic process, i.e., a small amount of siRNA is capable of
decreasing the concentration of the target gene mRNA within the
target cell. As antisense is based on a stoichiometric process, a
much larger concentration of effector molecules is required within
the target cell, i.e., a concentration is required that is equal to
or greater than the concentration of endogenous mRNA. Thus, as RNAi
is a catalytic process, a lower amount of effector molecules (i.e.,
siRNAs) is sufficient to mediate a therapeutic effect.
[0307] (b) Compared to ribozymes (which have a catalytic function
as well), RNAi seems to be a more flexible strategy, which allows
targeting a higher variety of target sequences and thus offers more
flexibility in construct design. Moreover, design of RNAi
constructs is fast and convenient as the artisan can design those
constructs based on the sequence information of the RNAi target
gene. With ribozymes, more trial-and-error experiments and more
sophisticated design algorithms are required as ribozymes are more
complex in nature. Last, RNAi is more efficacious in vivo compared
to ribozymes as RNAi leverages ubiquitous, endogenous cell
machinery.
[0308] The present invention also differs from protein-based
strategies, as RNAi does not require the expression of
non-endogenous proteins (such as artificial transcription factors),
thus lowering the risk of an unintended immune response.
[0309] In summary, RNAi-mediated down-regulation of gene expression
is a novel mechanism with clear advantages over existing gene
expression down-regulation approaches.
[0310] RNAi constructs comprise double stranded RNA that can
specifically block expression of a target gene. Accordingly, RNAi
constructs can act as antagonists by specifically blocking
expression of a particular gene. "RNA interference" or "RNAi" is a
term initially applied to a phenomenon observed in plants and worms
where double-stranded RNA (dsRNA) blocks gene expression in a
specific and post-transcriptional manner. Without being bound by
theory, RNAi appears to involve mRNA degradation, however the
biochemical mechanisms are currently an active area of research.
Despite some mystery regarding the mechanism of action, RNAi
provides a useful method of inhibiting gene expression in vitro or
in vivo.
[0311] As used herein, the term "dsRNA" refers to siRNA molecules,
or other RNA molecules including a double stranded feature and able
to be processed to siRNA in cells, such as hairpin RNA
moieties.
[0312] The term "loss-of-function," as it refers to genes inhibited
by the subject RNAi method, refers to a diminishment in the level
of expression of a gene when compared to the level in the absence
of RNAi constructs.
[0313] As used herein, the phrase "mediates RNAi" refers to
(indicates) the ability to distinguish which RNAs are to be
degraded by the RNAi process, e.g., degradation occurs in a
sequence-specific manner rather than by a sequence-independent
dsRNA response, e.g., a PKR response.
[0314] As used herein, the term "RNAi construct" is a generic term
used throughout the specification to include small interfering RNAs
(siRNAs), hairpin RNAs, and other RNA species which can be cleaved
in vivo to form siRNAs. RNAi constructs herein also include
expression vectors (also referred to as RNAi expression vectors)
capable of giving rise to transcripts which form dsRNAs or hairpin
RNAs in cells, and/or transcripts which can produce siRNAs in
vivo.
[0315] "RNAi expression vector" (also referred to herein as a
"dsRNA-encoding plasmid") refers to replicable nucleic acid
constructs used to express (transcribe) RNA which produces siRNA
moieties in the cell in which the construct is expressed. Such
vectors include a transcriptional unit comprising an assembly of
(1) genetic element(s) having a regulatory role in gene expression,
for example, promoters, operators, or enhancers, operatively linked
to (2) a "coding" sequence which is transcribed to produce a
double-stranded RNA (two RNA moieties that anneal in the cell to
form an siRNA, or a single hairpin RNA which can be processed to an
siRNA), and (3) appropriate transcription initiation and
termination sequences. The choice of promoter and other regulatory
elements generally varies according to the intended host cell. In
general, expression vectors of utility in recombinant DNA
techniques are often in the form of "plasmids" which refer to
circular double stranded DNA loops which, in their vector form are
not bound to the chromosome. In the present specification,
"plasmid" and "vector" are used interchangeably as the plasmid is
the most commonly used form of vector. However, 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.
[0316] The RNAi constructs contain a nucleotide sequence that
hybridizes under physiologic conditions of the cell to the
nucleotide sequence of at least a portion of the mRNA transcript
for the gene to be inhibited (i.e., the "target" gene). The
double-stranded RNA need only be sufficiently similar to natural
RNA that it has the ability to mediate RNAi. Thus, the invention
has the advantage of being able to tolerate sequence variations
that might be expected due to genetic mutation, strain polymorphism
or evolutionary divergence. The number of tolerated nucleotide
mismatches between the target sequence and the RNAi construct
sequence is no more than 1 in 5 basepairs, or 1 in 10 basepairs, or
1 in 20 basepairs, or 1 in 50 basepairs.
[0317] Mismatches in the center of the siRNA duplex are most
critical and may essentially abolish cleavage of the target RNA. In
contrast, nucleotides at the 3' end of the siRNA strand that is
complementary to the target RNA do not significantly contribute to
specificity of the target recognition.
[0318] Sequence identity may be optimized by sequence comparison
and alignment algorithms known in the art (see Gribskov and
Devereux, Sequence Analysis Primer, Stockton Press, 1991, and
references cited therein) and calculating the percent difference
between the nucleotide sequences by, for example, the
Smith-Waterman algorithm as implemented in the BESTFIT software
program using default parameters (e.g., University of Wisconsin
Genetic Computing Group). Greater than 90% sequence identity, or
even 100% sequence identity, between the inhibitory RNA and the
portion of the target gene is preferred. Alternatively, the duplex
region of the RNA may be defined functionally as a nucleotide
sequence that is capable of hybridizing with a portion of the
target gene transcript (e.g., 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM
EDTA, 50.degree. C. or 70.degree. C. hybridization for 12-16 hours;
followed by washing).
[0319] Production of RNAi constructs can be carried out by chemical
synthetic methods or by recombinant nucleic acid techniques.
Endogenous RNA polymerase of the treated cell may mediate
transcription in vivo, or cloned RNA polymerase can be used for
transcription in vitro. The RNAi constructs may include
modifications to either the phosphate-sugar backbone or the
nucleoside, e.g., to reduce susceptibility to cellular nucleases,
improve bioavailability, improve formulation characteristics,
and/or change other pharmacokinetic properties. For example, the
phosphodiester linkages of natural RNA may be modified to
[0320] include at least one of an nitrogen or sulfur heteroatom.
Modifications in RNA structure may be tailored to allow specific
genetic inhibition while avoiding a general response to dsRNA.
Likewise, bases may be modified to block the activity of adenosine
deaminase. The RNAi construct may be produced enzymatically or by
partial/total organic synthesis, any modified ribonucleotide can be
introduced by in vitro enzymatic or organic synthesis.
[0321] Methods of chemically modifying RNA molecules can be adapted
for modifying RNAi constructs (see, for example, [139]; [140];
[141]). Merely to illustrate, the backbone of an RNAi construct can
be modified with phosphorothioates, phosphoramidate,
phosphodithioates, chimeric methylphosphonatephosphodiesters,
peptide nucleic acids, 5-propynyl-pyrimidine containing oligomers
or sugar modifications (e.g., 2'-substituted ribonucleosides,
a-configuration).
[0322] The double-stranded structure may be formed by a single
self-complementary RNA strand or two complementary RNA strands. RNA
duplex formation may be initiated either inside or outside the
cell. The RNA may be introduced in an amount which allows delivery
of at least one copy per cell. Higher doses (e.g., at least 5, 10,
100, 500 or 1000 copies per cell) of double-stranded material may
yield more effective
[0323] inhibition, while lower doses may also be useful for
specific applications. Inhibition is sequence-specific in that
nucleotide sequences corresponding to the duplex region of the RNA
are targeted for genetic inhibition.
[0324] In certain embodiments, the subject RNAi constructs are
"small interfering RNAs" or "siRNAs." These nucleic acids are
around 19-30 nucleotides in length, and even more preferably 21-23
nucleotides in length, e.g., corresponding in length to the
fragments generated by nuclease "dicing" of longer doublestranded
RNAs. The siRNAs are understood to recruit nuclease complexes and
guide the complexes to the target mRNA by pairing to the specific
sequences. As a result, the target mRNA is degraded by the
nucleases in the protein complex. In a particular embodiment, the
21-23 nucleotides siRNA molecules comprise a 3' hydroxyl group.
[0325] The siRNA molecules of the present invention can be obtained
using a number of techniques known to those of skill in the art.
For example, the siRNA can be chemically synthesized or
recombinantly produced using methods known in the art. For example,
short sense and antisense RNA oligomers can be synthesized and
annealed to form double-stranded RNA structures with 2-nucleotide
overhangs at each end ([75], [142]). These double-stranded siRNA
structures can then be directly introduced to cells, either by
passive uptake or a delivery system of choice, such as described
below.
[0326] In certain embodiments, the siRNA constructs can be
generated by processing of longer doublestranded RNAs, for example,
in the presence of the enzyme dicer. In one embodiment, the
Drosophila in vitro system is used. In this embodiment, dsRNA is
combined with a soluble extract derived from Drosophila embryo,
thereby producing a combination. The combination is maintained
under conditions in which the dsRNA is processed to RNA molecules
of about 21 to about 23 nucleotides.
[0327] The siRNA molecules can be purified using a number of
techniques known to those of skill in the art. For example, gel
electrophoresis can be used to purify siRNAs. Alternatively,
non-denaturing methods, such as non-denaturing column
chromatography, can be used to purify the siRNA. In addition,
chromatography (e.g., size exclusion chromatography), glycerol
gradient centrifugation, affinity purification with antibody can be
used to purify siRNAs.
[0328] In certain preferred embodiments, at least one strand of the
siRNA molecules has a 3' overhang from about 1 to about 6
nucleotides in length, though may be from 2 to 4 nucleotides in
length. More preferably, the 3' overhangs are 1-3 nucleotides in
length. In certain embodiments, one strand having a 3' overhang and
the other strand being blunt-ended or also having an overhang. The
length of the overhangs may be the same or different for each
strand. In order to further enhance the stability of the siRNA, the
3' overhangs can be stabilized against degradation. In one
embodiment, the RNA is stabilized by including purine nucleotides,
such as adenosine or guanosine nucleotides. Alternatively,
substitution of pyrimidine nucleotides by modified analogues, e.g.,
substitution of uridine nucleotide 3' overhangs by
2'-deoxythyinidine is tolerated and does not affect the efficiency
of RNAi. The absence of a 2' hydroxyl significantly enhances the
nuclease resistance of the overhang in tissue culture medium and
may be beneficial in vivo.
[0329] In other embodiments, the RNAi construct is in the form of a
long double-stranded RNA. In certain embodiments, the RNAi
construct is at least 25, 50, 100, 200, 300 or 400 bases. In
certain embodiments, the RNAi construct is 400-800 bases in length.
The double-stranded RNAs are digested intracellularly, e.g., to
produce siRNA sequences in the cell. However, use of long
double-stranded RNAs in vivo is not always practical, presumably
because of deleterious effects that may be caused by the
sequence-independent dsRNA response. In such embodiments, the use
of local delivery systems and/or agents which reduce the effects of
interferon or PKR are preferred.
[0330] In certain embodiments, the RNAi construct is in the form of
a hairpin structure (named as hairpin RNA). The hairpin RNAs can be
synthesized exogenously or can be formed by transcribing from RNA
polymerase III promoters in vivo. Examples of making and using such
hairpin RNAs for gene silencing in mammalian cells are described
in, for example, [76]; [143]; [144]; [145]). Preferably, such
hairpin RNAs are engineered in cells or in an animal to ensure
continuous and stable suppression of a desired gene. It is known in
the art that siRNAs can be produced by processing a hairpin RNA in
the cell.
[0331] In yet other embodiments, a plasmid is used to deliver the
double-stranded RNA, e.g., as a transcriptional product. In such
embodiments, the plasmid is designed to include a "coding sequence"
for each of the sense and antisense strands of the RNAi construct.
The coding sequences can be the same sequence, e.g., flanked by
inverted promoters, or can be two separate sequences each under
transcriptional control of separate promoters. After the coding
sequence is transcribed, the complementary RNA transcripts
base-pair to form the double-stranded RNA.
[0332] PCT application WO01/77350 describes an exemplary vector for
bi-directional transcription of a transgene to yield both sense and
antisense RNA transcripts of the same transgene in a eukaryotic
cell. Accordingly, in certain embodiments, the present invention
provides a recombinant vector having the following unique
characteristics: it comprises a viral replicon having two
overlapping transcription units arranged in an opposing orientation
and flanking a transgene for an RNAi construct of interest, wherein
the two overlapping transcription units yield both sense and
antisense RNA transcripts from the same transgene fragment in a
host cell.
[0333] RNAi constructs can comprise either long stretches of double
stranded RNA identical or substantially identical to the target
nucleic acid sequence or short stretches of double stranded RNA
identical to substantially identical to only a region of the target
nucleic acid sequence. Exemplary methods of making and delivering
either long or short RNAi constructs can be found, for example, in
WO01/68836 and WO01/75164.
[0334] Exemplary RNAi constructs that specifically recognize a
particular gene, or a particular family of genes can be selected
using methodology outlined in detail above with respect to the
selection of antisense oligonucleotide. Similarly, methods of
delivery RNAi constructs include the methods for delivery antisense
oligonucleotides outlined in detail above.
[0335] In general, it is anticipated that any of the foregoing
methods that decrease the presence or translation of hedgehog,
smoothened or gli-1 mRNA will act as hedgehog antagonists, while
methods that decrease the production of patched will have an
agonist effect.
[0336] In certain embodiments, the subject antagonists can be
chosen on the basis of their selectively for the hedgehog pathway.
This selectivity can be for the hedgehog pathway versus other
pathways, or for selectivity between particular hedgehog pathways,
e.g., e.g., ptc-1, ptc-2, etc. In certain preferred embodiments,
the subject inhibitors inhibit hedgehog-mediated signal
transduction with an ED 50 of 1 mM or less, more preferably of 1
.mu.M or less, and even more preferably of 1 nM or less.
[0337] In particular embodiments, the small molecule is chosen for
use because it is more selective for one patched isoform over the
next, e.g., 10 fold, and more preferably at least 100 or even 1000
fold more selective for one patched pathway (ptc-1, ptc-2) over
another.
[0338] In certain embodiments, a compound which is an antagonist of
the hedgehog pathway is chosen to selectively antagonize hedgehog
activity over protein kinases other than PKA, such as PKC, e.g.,
the compound modulates the activity of the hedgehog pathway 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 the hedgehog pathway may inhibit hedgehog activity
with a Ki at least an order of magnitude lower than its Ki 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, the Ki for PKA inhibition is less
than 10 nM, preferably less than 1 nM, even more preferably less
than 0.1 nM.
[0339] The design of the RNAi expression cassette does not limit
the scope of the invention. Different strategies to design an RNAi
expression cassette can be applied, and RNAi expression cassettes
based on different designs will be able to induce RNA interference
in vivo. (Although the design of the RNAi expression cassette does
not limit the scope of the invention, some RNAi expression cassette
designs are included in the detailed description of this invention
and below.)
[0340] Features common to all RNAi expression cassettes are that
they comprise an RNA coding region which encodes an RNA molecule
which is capable of inducing RNA interference either alone or in
combination with another RNA molecule by forming a double-stranded
RNA complex either intramolecularly or intermolecularly.
[0341] Different design principles can be used to achieve that same
goal and are known to those of skill in the art. For example, the
RNAi expression cassette may encode one or more RNA molecules.
After or during RNA expression from the RNAi expression cassette, a
double-stranded RNA complex may be formed by either a single,
self-complementary RNA molecule or two complementary RNA molecules.
Formation of the dsRNA complex may be initiated either inside or
outside the nucleus.
[0342] The RNAi target gene does not limit the scope of this
invention and may be any gene that participates in the hedgehog
signaling pathway. Thus, the choice of the RNAi target gene is not
limiting for the present invention: The artisan will know how to
design an RNAi expression cassette to down-regulate the gene
expression of any RNAi target gene of interest. Depending on the
particular RNAi target gene and method of delivery, the procedure
may provide partial or complete loss of function for the RNAi
target gene.
3.2.3.5 Aptamers
[0343] Aptamers are a non-naturally occurring nucleic acid having a
desirable action on a target. A desirable action includes, but is
not limited to, binding of the target, catalytically changing the
target, reacting with the target in a way which modifies/alters the
target or the functional activity of the target, covalently
attaching to the target as in a suicide inhibitor, facilitating the
reaction between the target and another molecule. The target in
case of the, present invention is a component of the Hedgehog
signaling pathway.
[0344] Aptamers are identified based on the SELEX process
[146-148]. In its most basic form, the SELEX process may be defined
by the following series of steps:
[0345] 1) A candidate mixture of nucleic acids of differing
sequence is prepared. The candidate mixture generally includes
regions of fixed sequences (i.e., each of the members of the
candidate mixture contains the same sequences in the same location)
and regions of randomized sequences. The fixed sequence regions are
selected either: (a) to assist in the amplification steps described
below, (b) to mimic a sequence known to bind to the target, or (c)
to enhance the concentration of a given structural arrangement of
the nucleic acids in the candidate mixture. The randomized
sequences can be totally randomized (i.e., the probability of
finding a base at any position being one in four) or only partially
randomized (e.g., the probability of finding a base at any location
can be selected at any level between 0 and 100 percent).
[0346] 2) The candidate mixture is contacted with the selected
target under conditions favorable for binding between the target
and members of the candidate mixture. Under these circumstances,
the interaction between the target and the nucleic acids of the
candidate mixture can be considered as forming nucleic acid-target
pairs between the target and those nucleic acids having the
strongest affinity for the target.
[0347] 3) The nucleic acids with the highest affinity for the
target are partitioned from those nucleic acids with lesser
affinity to the target. Because only an extremely small number of
sequences (and possibly only one molecule of nucleic acid)
corresponding to the highest affinity nucleic acids exist in the
candidate mixture, it is generally desirable to set the
partitioning criteria so that a significant amount of the nucleic
acids in the candidate mixture (approximately 5-50%) are retained
during partitioning.
[0348] 4) Those nucleic acids selected during partitioning as
having the relatively higher affinity to the target are then
amplified to create a new candidate mixture that is enriched in
nucleic acids having a relatively higher affinity for the
target.
[0349] 5) By repeating the partitioning and amplifying steps above,
the newly formed candidate mixture contains fewer and fewer weakly
binding sequences, and the average degree of affinity of the
nucleic acids to the target will generally increase. Taken to its
extreme, the SELEX process will yield a candidate mixture
containing one or a small number of unique nucleic acids
representing those nucleic acids from the original candidate
mixture having the highest affinity to the target molecule.
[0350] In order to produce nucleic acids desirable for use as a
pharmaceutical, it is preferred that the nucleic acid ligand (1)
binds to the target in a manner capable of achieving the desired
effect on the target; (2) be as small as possible to obtain the
desired effect; (3) be as stable as possible; and (4) be a specific
ligand to the chosen target. In most situations, it is preferred
that the nucleic acid ligand have the highest possible affinity to
the target.
[0351] The SELEX patent applications describe and elaborate on this
process in great detail. Included are targets that can be used in
the process; methods for partitioning nucleic acids within a
candidate mixture; and methods for amplifying partitioned nucleic
acids to generate enriched candidate mixture. The SELEX patent
applications also describe ligands obtained to a number of target
species, including both protein targets where the protein is and is
not a nucleic acid binding protein. The SELEX method further
encompasses combining selected nucleic acid ligands with lipophilic
or non-immunogenic, high molecular weight compounds in a diagnostic
or therapeutic complex as described in U.S. patent application Ser.
No. 08/434,465, filed May 4, 1995, entitled "Nucleic Acid Ligand
Complexes".
[0352] In certain embodiments of the present invention it is
desirable to provide a complex comprising one or more nucleic acid
ligands to components of the Hedgehog signaling pathway covalently
linked with a non-immunogenic, high molecular weight compound or
lipophilic compound. A non-immunogenic, high molecular weight
compound is a compound between approximately 100 Da to 1,000,000
Da, more preferably approximately 1000 Da to 500,000 Da, and most
preferably approximately 1000 Da to 200,000 Da, that typically does
not generate an immunogenic response. For the purposes of this
invention, an immunogenic response is one that causes the organism
to make antibody proteins. In one preferred embodiment of the
invention, the non-immunogenic, high molecular weight compound is a
polyalkylene glycol. In the most preferred embodiment, the
polyalkylene glycol is polyethylene glycol (PEG). More preferably,
the PEG has a molecular weight of about 10-80K. Most preferably,
the PEG has a molecular weight of about 20-45K. In certain
embodiments of the invention, the non-immunogenic, high molecular
weight compound can also be a nucleic acid ligand.
3.2.4 Gene Transfer
[0353] In some embodiments, gene transfer to the eye is used
instead of administering an exogenously synthesized protein. Upon
transduction of corresponding host cells within the eye, said cells
will start producing the protein encoded by the gene transfer
vector. One vector system of particular importance for ocular gene
transfer is a system based on recombinant AAV virions.
3.2.4.1 Recombinant AAV Virions
[0354] The recombinant AAV virions of the preferred embodiment,
comprising an RNAi expression cassette, can be produced using
standard methodology, known to the artisan. The Methods generally
involve the steps of
[0355] (1) introducing an AAV vector construct into a host cell
(e.g., 293 cells);
[0356] (2) introducing an AAV packaging construct into the host
cell, where the packaging construct includes AAV coding regions
(e.g., rep and cap sequences) capable of being expressed in the
host cell to complement AAV packaging functions missing from the
AAV vector construct;
[0357] (3) introducing one or more helper viruses and/or accessory
function vector constructs into the host cell, wherein the helper
virus and/or accessory function vector constructs provide accessory
functions capable of supporting efficient recombinant AAV ("rAAV")
virion production in the host cell; and
[0358] (4) culturing the host cell to produce rAAV virions.
[0359] The AAV vector construct, AAV packaging construct and the
helper virus or accessory function vector constructs can be
introduced into the host cell either simultaneously or serially,
using standard transfection techniques.
[0360] In one embodiment, pseudotyped rAAV virions are produced, in
which a non-AAV5 serotype ITR based RNAi expression cassette is
packaged in an AAV5 capsid. The inventors have previously found
that this pseudotyping can be achieved by utilizing a Rep protein
(or a functional portion thereof) of the same serotype or a
cross-reactive serotype as that of the ITRs found in the minigene
in the presence of sufficient packaging and accessory functions to
permit packaging [83]. Thus, an AAV2 minigene (harboring an RNAi
expression cassette) can be pseudotyped in an AAV5 capsid by use of
a rep protein from AAV2 or a cross-reactive serotype, e.g., AAV1,
AAV3, AAV4 or AAV6. Similarly, an AAV minigene containing AAV1 5'
ITRs and AAV2 3' ITRs may be pseudotyped in an AAV5 capsid by use
of a Rep protein from AAV 1, AAV2, or another cross-reactive
serotype. However, because AAV5 is not cross-reactive with the
other AAV serotypes, an AAV5 minigene can be pseudotyped in a
heterologous AAV capsid only by use of an AAV5 Rep protein.
[0361] In certain embodiments, the invention provides an rAAV
virion, in which both the AAV ITRs and capsid protein are
independently selected from among AAV serotypes, including, without
limitation, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8. For
example, the invention may utilize a rAAV1 vector, a rAAV2 vector,
a rAAV2/1 vector, and rAAV1/2 vector and/or a rAAV2/5 vector, as
desired--following the nomenclature rAAVx/y with x: serotype source
of ITRs, y: serotype source of capsid; rAAVz with z as serotype
source of ITRs and capsid.
[0362] In another embodiment of this method, the delivery of vector
with an AAV capsid protein may precede or follow delivery of a
heterologous molecule (e.g., gene) via a vector with a different
serotype AAV capsid protein. Thus, delivery via multiple rAAV
vectors may be used for repeat delivery of a desired molecule to a
selected host cell. Desirably, subsequently administered rAAV carry
the same minigene as the first rAAV vector, but the subsequently
administered vectors contain capsid proteins of serotypes which
differ from the first vector. For example, if a first rAAV has an
AAV5 capsid protein, subsequently administered rAAV may have capsid
proteins selected from among the other serotypes, including AAV2,
AAV1, AAV3A, AAV3B, AAV4 and AAV6. Alternatively, if a first rAAV
has an AAV2 capsid protein, subsequently administered rAAV may have
an AAV5 cansid. Still other suitable combinations will be readily
apparent to one of skill in the art.
[0363] The host cell for rAAV virion production itself may be
selected from any biological organism, including prokaryotic (e.g.,
bacterial) cells, and eukaryotic cells, including, insect cells,
yeast cells and mammalian cells. Particularly desirable host cells
are selected from among any mammalian species, including, without
limitation, cells such as A549, WEHI, 3T3, 10T1/2, BHK, MDCK, COS
1, COS 7, BSC 1, BSC 40, BMT 10, VERO, W138, HeLa, 293 cells (which
express functional adenoviral E1), Saos, C2C12, L cells, HT1080,
HepG2 and primary fibroblast, hepatocyte and myoblast cells derived
from mammals including human, monkey, mouse, rat, rabbit, and
hamster. The selection of the mammalian species providing the cells
is not a limitation of this invention; nor is the type of mammalian
cell, i.e., fibroblast, hepatocyte, tumor cell, etc. The
requirements for the cell used is that it not carry any adenovirus
gene other than E1, E2a and/or E4 ORF6; it not contain any other
virus gene which could result in homologous recombination of a
contaminating virus during the production of rAAV; and it is
capable of infection or transfection of DNA and expression of the
transfected DNA.
[0364] One host cell useful in the present invention is a host cell
stably transformed with the sequences encoding rep and cap, and
which is transfected with the adenovirus E1, E2a, and E40RF6 DNA
and a construct carrying the minigene as described above. Stable
rep and/or cap expressing cell lines, such as B-50
(PCT/US98/19463), or those described in U.S. Pat. No. 5,658,785,
may also be similarly employed. Another desirable host cell
contains the minimum adenoviral DNA which is sufficient to express
E4 ORF6.
[0365] The preparation of a host cell according to this invention
involves techniques such as assembly of selected DNA sequences.
This assembly may be accomplished utilizing conventional
techniques. Such techniques include cDNA and genomic cloning, which
are well known and are described in Sambrook et al., cited above,
use of overlapping oligonucleotide sequences of the adenovirus and
AAV genomes, combined with polymerase chain reaction, synthetic
methods, and any other suitable methods which provide the desired
nucleotide sequence.
[0366] Introduction of the molecules (as plasmids or viruses) into
the host cell may also be accomplished using techniques known to
the skilled artisan and as discussed throughout the specification.
In the preferred embodiment, standard transfection techniques are
used, e.g., CaPO.sub.4 transfection or electroporation, and/or
infection by hybrid adenovirus/AAV vectors into cell lines such as
the human embryonic kidney cell line HEK 293 (a human kidney cell
line containing functional adenovirus E1 genes which provides
trans-acting E1 proteins). Thus produced, the rAAV may be used to
prepare the compositions and kits described herein, and used in the
method of the invention.
3.2.4.2 AAV Vector Constructs
[0367] AAv vector constructs are constructed using known techniques
to at least provide, as operatively linked components in the
direction of transcription, (a) control elements including a
transcriptional initiation region, (b) the DNA of interest (here:
at least an RNAi expression cassette), and (c) a transcriptional
termination region. The control elements are selected to be
functional in the targeted cell. The resulting construct, which
contains the operatively linked components, is bounded (5' and 3')
with functional AAV ITR sequences. The nucleotide sequences of AAV
ITR regions are known. See, e.g., [84]; Berns, K. I. "Parvoviridae
and their Replication" in Fundamental Virology, 2nd Edition, (B. N.
Fields and D. M. Knipe, eds.) for the AAV-2 sequence. AAV ITRs used
in the vectors of the invention need not have a wild-type
nucleotide sequence, and may be altered, e.g., by the insertion,
deletion or substitution of nucleotides.
[0368] Additionally, AAV ITRs may be derived from any of several
AAV serotypes, including AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6,
AAV-7, AAV-8, etc. The 5 and 3' ITRs which flank a selected
transgene expression cassette in an AAV vector plasmid need not
necessarily be identical or derived from the same AAV serotype, as
long as they function as intended, i.e., to allow for excision and
replication of the bounded nucleotide sequence of interest when AAV
rep gene products are present in the cell. Thus, rAAV vector design
and production allows for exchanging of the capsid proteins between
different AAV serotypes: Homologous vectors comprising an
expression cassette flanked by e.g., AAV2-ITRs and packaged in an
AAV2 capsid, can be produced as well as heterologous, hybrid
vectors where the transgene expression cassette is flanked by e.g.,
AAV2 ITRs, but the capsid originates from another AAV serotype: The
following combinations are feasible: rAAV2/1-8, where the first
number defines the genome and the second the capsid of the AAV of
origin. In its preferred embodiment, the gene transfer vector is
produced using a rAAV2/5 design.
[0369] Suitable minigenes for use in AAV vectors will generally be
less than about 5 kilobases (kb) in size, which is the case for
RNAi expression cassettes. Given the size of most RNAi expression
cassettes, other minigenes might be included in the same AAV vector
comprising another gene of interest.
[0370] The AAV sequences used in generating the minigenes, vectors,
and capsids, and other constructs used in the present invention may
be obtained from a variety of sources. For example, the sequences
may be provided by AAV type 5, AAV type 2, AAV type 1, AAV type 3,
AAV type 4, AAV type 6, or other AAV serotypes or other
densoviruses. A variety of these viral serotypes and strains are
available from the American Type Culture Collection, Manassas, Va.,
or are available from a variety of academic or commercial sources.
Alternatively, it may be desirable to synthesize sequences used in
preparing the vectors and viruses of the invention using known
techniques, which may utilize AAV sequences which are published
and/or available from a variety of databases. The source of the
sequences utilized in preparation of the constructs of the
invention is not a limitation of the present invention.
3.2.4.3 rAAV Virion Production
[0371] In order to produce rAAV virions, an AAV vector construct
that has been constructed as described above is introduced into a
suitable host cell using known techniques, such as by transfection.
A number of transfection techniques are generally known in the art.
See, e.g., [69,70], Sambrook et al. (1989) Molecular Cloning, a
laboratory manual, Cold Spring Harbor Laboratories, New York, Davis
et al. (1986) Basic Methods in Molecular Biology, Elsevier, and
[71]. Particularly suitable transfection methods include calcium
phosphate co-precipitation [69], direct micro-injection into
cultured cells [85], electroporation [86], liposome mediated gene
transfer [87], lipid-mediated transduction [88], and nucleic acid
delivery using high-velocity microprojectiles.
[0372] The AAV vector construct harboring the AAV minigene is
preferably carried on a plasmid which is delivered to a host cell
by transfection. The plasmids useful in this invention may be
engineered such that they are suitable for replication and,
optionally, integration in prokaryotic cells, mammalian cells, or
both. These plasmids (or other vectors carrying the 5' AAV
ITR-heterologous molecule-3' AAV ITR) may contain sequences
permitting replication of the AAV minigene in eukaryotes and/or
prokaryotes and selection markers for these systems. Selectable
markers or reporter genes may include sequences encoding geneticin,
hygromicin or purimycin resistance, among others. The plasmids may
also contain certain selectable reporters or marker genes that can
be used to signal the presence of the vector in bacterial cells,
such as ampicillin resistance. Other components of the plasmid may
include an origin of replication and an amplicon, such as the
amplicon system employing the Epstein Barr virus nuclear antigen.
This amplicon system, or other similar amplicon components permit
high copy episomal replication in the cells. Preferably, the
molecule carrying the AAV minigene is transfected into the cell,
where it may exist transiently or as an episome. Alternatively, the
AAV minigene (carrying the 5' AAV ITR-heterologous molecule-3' AAV
ITR) may be stably integrated into a chromosome of the host cell.
Suitable transfection techniques are known and may readily be
utilized to deliver the AAV minigene to the host cell.
[0373] Generally, when delivering the AAV vector construct
comprising the AAV minigene by transfection, the vector is
delivered in an amount from about 5 .mu.g to about 100 .mu.g DNA,
and preferably about 10 to about 50 .mu.g DNA to about 10.sup.4
cells to about 10.sup.13 cells, and preferably about 10.sup.5
cells. However, the relative amounts of vector DNA to host cells
may be adjusted, taking into consideration such factors as the
selected vector, the delivery method and the host cells
selected.
[0374] For the purposes of the invention, suitable host cells for
producing rAAV virions include microorganisms, yeast cells, insect
cells, and mammalian cells, that can be, or have been, used for
transfection. The term includes the progeny of the original cell
which has been transfected. Thus, a "host cell" as used herein
generally refers to a cell which has been transfected with an
exogenous DNA sequence. Cells from the stable human cell line, 293
(readily available through, e.g., the ATCC under Accession No. ATCC
CRL1573) are preferred in the practice of the present invention.
Particularly, the human cell line 293 is a human embryonic kidney
cell line that has been transformed with adenovirus type-5 DNA
fragments [89], and expresses the adenoviral E1a and E1b genes
[90]. The 293 cell line is readily transfected, and provides a
particularly convenient platform in which to produce rAAV
virions.
[0375] The components required to be cultured in the host cell to
package the AAV minigene in the AAV capsid may be provided to the
host cell in trans. Alternatively, any one or more of the required
components (e.g., minigene, rep sequences, cap sequences, and/or
accessory functions) may be provided by a stable host cell which
has been engineered to contain one or more of the required
components using methods known to those of skill in the art.
[0376] The minigene, rep sequences, cap sequences, and accessory
(helper) functions required for producing the rAAV of the invention
may be delivered to the packaging-host cell in the form of any
genetic element, e.g., naked DNA, a plasmid, phage, transposon,
cosmid, virus, etc. which transfer the sequences carried thereon.
The selected-genetic element may be delivered by any suitable
method, including transfection, electroporation, liposome delivery,
membrane fusion techniques, high velocity DNA-coated pellets, viral
infection and protoplast fusion.
3.2.4.4 AAV Packaging Functions
[0377] Host cells containing the above described AAV vector
constructs must be rendered capable of providing AAV packaging
functions in order to replicate and encapsidate the nucleotide
sequences flanked by the AAV ITRs to produce rAAV virions. AAV
packaging functions are generally AAV-derived coding sequences
which can be expressed to provide AAV gene products that, in turn,
function in trans for productive AAV replication and genome
encapsidation. AAV packaging functions are used herein to
complement necessary AAV functions that are missing from the AAV
vectors. Thus, AAV packaging functions include one, or both of the
major AAV ORFs, namely the rep and cap coding regions, or
functional homologues thereof.
[0378] By "AAV rep coding region" is meant the art-recognized
region of the AAV genome which encodes the replication proteins
Rep78; Rep68; Rep52 and Rep40. These Rep expression products have
been shown to possess many functions, including recognition,
binding and nicking of the AAV origin of DNA replication, DNA
helicase activity and modulation of transcription from AAV (or
other heterologous) promoters. The Rep expression products are
collectively required for replicating the AAV genome. For a
description of the AAV rep coding region, see, e.g., [84, 91].
Suitable homologues of the AAV rep coding region include the human
herpesvirus 6 (HHV-6) rep gene which is also known to mediate AAV-2
DNA replication [92].
[0379] By "AAV cap coding region" is meant the art-recognized
region of the AAV genome which encodes the capsid proteins VP1,
VP2, and VP3, or functional homologues thereof. These cap
expression products supply the packaging functions which are
collectively required for packaging the viral genome. For a
description of the AAV cap coding region, see, e.g., [84, 91].
[0380] AAv packaging functions are introduced into the host cell by
transfecting the host cell with an AAV packaging construct either
prior to, or concurrently with, the transfection of the AAV vector
construct. AAV packaging constructs are thus used to provide at
least transient expression of AAV rep and/or cap genes to
complement missing AAV functions that are necessary for productive
AAV infection. AAV packaging constructs lack AAV ITRs and can
neither replicate nor package themselves. These constructs can be
in the form of a plasmid, phage, transposon, cosmid, virus, or
virion. A number of AAV packaging constructs have been described,
such as the commonly used plasmids pAAV/Ad and pIM29+45 which
encode both Rep and Cap expression products. See, e.g., [93, 94]. A
number of other vectors have been described which encode Rep and/or
Cap expression products. See, e.g., U.S. Pat. No. 5,139,941.
[0381] Additionally, when pseudotyping an AAV vector in an AAV5
capsid, the sequences encoding each of the essential Rep proteins
may be supplied by the same AAV serotype as the ITRs, or the
sequences encoding the Rep proteins may be supplied by different,
but cross-reactive, AAV serotypes (e.g., AAV1, AAV2, AAV3, AAV4 and
AAV6). For example, the Rep78/68 sequences may be from AAV2,
whereas the Rep52/40 sequences may from AAV1.
[0382] In one embodiment, the host cell stably contains the capsid
ORF under the control of a suitable promoter, such as those
described above. Most desirably, in this embodiment, the capsid ORF
is expressed under the control of an inducible promoter. In another
embodiment, the capsid ORF is supplied to the host cell in trans.
When delivered to the host cell in trans, the capsid ORF may be
delivered via a plasmid that contains the sequences necessary to
direct expression of the selected capsid ORF in the host cell. Most
desirably, when delivered to the host cell in trans, the plasmid
carrying the capsid ORF also carries other sequences required for
packaging the rAAV, e.g., the rep sequences.
[0383] In another embodiment, the host cell stably contains the rep
sequences under the control of a suitable promoter, such as those
described above. Most desirably, in this embodiment, the essential
Rep proteins are expressed under the control of an inducible
promoter. In another embodiment, the rep ORF is supplied to the
host cell in trans. When delivered to the host cell in trans, the
rep ORF may be delivered via a plasmid which contains the sequences
necessary to direct expression of the selected rep ORF in the host
cell. Most desirably, when delivered to the host cell in trans, the
plasmid carrying the rep ORF also carries other sequences required
for packaging the rAAV, e.g., the cap sequences.
[0384] Thus, in one embodiment, the rep and cap sequences may be
transfected into the host cell on a single nucleic acid molecule
and exist in the cell as an episome. In another embodiment, the rep
and cap sequences are stably integrated into the genome of the
cell. Another embodiment has the rep and cap sequences transiently
expressed in the host cell. For example, a useful nucleic acid
molecule for such transfection comprises, from 5' to 3', a
promoter, an optional spacer interposed between the promoter and
the start site of the rep gene sequence, an AAV rep gene sequence,
and an AAV cap gene. sequence.
[0385] Optionally, the rep and/or cap sequences may be supplied on
a vector that contains other DNA sequences that are to be
introduced into the host cells. For instance, the vector may
contain the rAAV vector construct comprising the AAV minigene. The
vector may comprise one or more of the genes encoding the helper
functions, e.g., the adenoviral proteins E1, E2a, and E40RF6, and
the gene for VAI RNA.
[0386] In another embodiment, the promoter for rep is an inducible
promoter, as discussed above in connection with regulatory
sequences and promoters. One preferred promoter for rep expression
is the T7 promoter. The vector comprising the rep gene regulated by
the T7 promoter and the cap gene, is transfected or transduced into
a cell which either constitutively or inducibly expresses the T7
polymerase. See WO 98/10088, published Mar. 12, 1998.
[0387] Preferably, the promoter used in the AAV packaging construct
may be any of the constitutive, inducible or native promoters known
to one of skill in the art or as discussed above. In one
embodiment, an AAV p5 promoter sequence is employed. The selection
of the AAV to provide any of these sequences does not limit the
invention.
[0388] The spacer is an optional element in the design of the AAV
packaging construct. The spacer is a DNA sequence interposed
between the promoter and the rep gene ATG start site. The spacer
may have any desired design; that is, it may be a random sequence
of nucleotides, or alternatively, it may encode a gene product,
such as a marker gene. The spacer may contain genes that typically
incorporate start/stop and polyA sites. The spacer may be a
non-coding DNA sequence from a prokaryote or eukaryote, a
repetitive non-coding sequence, a coding sequence without
transcriptional controls or a coding sequence with transcriptional
controls. Two exemplary sources of spacer sequences are the X phage
ladder sequences or yeast ladder sequences, which are available
commercially, e.g., from Gibco or Invitrogen, among others. The
spacer may be of any size sufficient to reduce expression of the
rep78 and rep68 gene products, leaving the rep52, rep40 and cap
gene products expressed at normal levels. The length of the spacer
may therefore range from about 10 bp to about 10.0 kbp, preferably
in the range of about 100 bp to about 8.0 kbp. To reduce the
possibility of recombination, the spacer is preferably less than 2
kbp in length; however, the invention is not so limited.
[0389] Although the molecule(s) providing rep and cap may exist in
the host cell transiently (i.e., through transfection), it is
preferred that one or both of the rep and cap proteins and the
promoter(s) controlling their expression be stably expressed in the
host cell, e.g., as an episome or by integration into the
chromosome of the host cell. The methods employed for constructing
embodiments of this invention are conventional genetic engineering
or recombinant engineering techniques such as those described in
the references above. While this specification provides
illustrative examples of specific constructs, using the information
provided herein, one of skill in the art may select and design
other suitable constructs, using a choice of spacers, promoters,
and other elements, including at least one translational start and
stop signal, and the optional addition of polyadenylation
sites.
3.2.4.5 AAV Accessory Functions
[0390] The host cell (or packaging cell) must also be rendered
capable of providing non-AAV derived functions, or "accessory
functions", in order to produce rAAV virions. Accessory functions
are non-AAV derived viral and/or cellular functions upon which AAV
is dependent for its replication. Thus, accessory functions include
at least those non AAV proteins and RNAs that are required in AAV
replication, including those involved in activation of AAV gene
transcription, stage specific AAV mRNA splicing, AAV DNA
replication, synthesis of rep and cap expression products and AAV
capsid assembly. Viral-based accessory functions can be derived
from any of the known helper viruses.
[0391] Particularly, accessory functions can be introduced into and
then expressed in host cells using methods known to those of skill
in the art. Commonly, accessory functions are provided by infection
of the host cells with an unrelated helper virus. A number of
suitable helper viruses are known, including adenoviruses, Herpes
viruses such as Herpes Simplex Virus types 1 and 2, and vaccinia
viruses. Non-viral accessory functions will also find use herein,
such as those provided by cell synchronization using any of various
known agents [95-97]. Alternatively and preferentially, accessory
functions can be provided using an accessory function vector
construct. Accessory function vector constructs include nucleotide
sequences that provide one or more accessory functions. An
accessory function vector is capable of being introduced into a
suitable host cell in order to support efficient AAV virion
production in the host cell. Accessory function vectors can be in
the form of a plasmid, phage, virus, transposon or cosmid.
Accessory vector constructs can also be in the form of one or more
linearized DNA or RNA fragments which, when associated with the
appropriate control elements and enzymes, can be transcribed or
expressed in a host cell to provide accessory functions.
[0392] Nucleic acid sequences providing the accessory functions can
be obtained from natural sources, such as from the genome of
adenovirus (especially Adenovirus serotype 5), or constructed using
recombinant or synthetic methods known in the art. In this regard,
adenovirus-derived accessory functions have been widely studied,
and a number of adenovirus genes involved in accessory functions
have been identified and partially characterized. See, e.g.,
Carter, B. J. (1990) "Adeno-Associated Virus Helper Functions," in
CRC Handbook of Parvoviruses, vol. I (P. Tijssen, ed.), and [91].
Specifically, early adenoviral gene regions E1a, E2a, E4, VAI RNA
and, possibly, E1b are thought to participate in the accessory
process [98]. Herpes Virus-derived accessory functions have been
described as well [99]. Vaccinia virus-derived accessory functions
have also been described [95].
[0393] Most desirably, the necessary accessory functions are
provided from an adenovirus source. In one embodiment, the host
cell is provided with and/or contains an E1a gene product, an E1b
gene product, an E2a gene product, and/or an E4 ORF6 gene product.
The host cell may contain other adenoviral genes such as VAI RNA,
but these genes are not required. In a preferred embodiment, no
other adenovirus genes or gene functions are present in the host
cell. The DNA sequences encoding the adenovirus E4 ORF6 genes and
the E1 genes and/or E2a genes useful in this invention may be
selected from among any known adenovirus type, including the
presently identified 46 human types [see, e.g., American Type
Culture Collection]. Similarly, adenoviruses known to infect other
animals may supply the gene sequences. The selection of the
adenovirus type for each E1, E2a, and E4 ORF6 gene sequence does
not limit this invention. The sequences for a number of adenovirus
serotypes, including that of serotype Ad5, are available from
Genbank. A variety of adenovirus strains are available from the
American Type Culture Collection (ATCC), Manassas, Va., or are
available by request from a variety of commercial and institutional
sources. Any one or more of human adenoviruses Types 1 to 46 may
supply any of the adenoviral sequences, including E1, E2a, and/or
E4 ORF6.
[0394] The adenovirus E1a, E1b, E2a, and/or E40RF6 gene products,
as well as any other desired accessory functions, can be provided
using any means that allows their expression in a cell. Each of the
sequences encoding these products may be on a separate vector, or
one or more genes may be on the same vector. The vector may be any
vector known in the art or disclosed above, including plasmids,
cosmids and viruses.
Introduction into the Host Cell
[0395] of the vector may be achieved by any means known in the art
or as disclosed above, including transfection, infection,
electroporation, liposome delivery, membrane fusion techniques,
high velocity DNA-coated pellets, viral infection and protoplast
fusion, among others. One or more of the adenoviral genes may be
stably integrated into the genome of the host cell, stably
expressed as episomes, or expressed transiently. The gene products
may all be expressed transiently, on an episome or stably
integrated, or some of the gene products may be expressed stably
while others are expressed transiently. Furthermore, the promoters
for each of the adenoviral genes may be selected independently from
a constitutive promoter, an inducible promoter or a native
adenoviral promoter. The promoters may be regulated by a specific
physiological state of the organism or cell (i.e., by the
differentiation state or in replicating or quiescent cells) or by
exogenously-added factors, for example.
[0396] As a consequence of the infection of the host cell with a
helper virus, or transfection of the host cell with an accessory
function vector construct, accessory functions are expressed which
transactivate the AAV packaging construct to produce AAV Rep and/or
Cap proteins. The Rep expression products direct excision of the
recombinant DNA (including the DNA of interest) from the AAV vector
construct. The Rep proteins also serve to replicate the AAV genome.
The expressed Cap proteins assemble into capsids, and the
recombinant AAV genome is packaged into the capsids. Thus,
productive AAV replication ensues, and the DNA is packaged into
rAAV virions.
[0397] Following recombinant AAV replication, rAAV virions can be
purified from the host cell using a variety of conventional
purification methods, such as CsCl gradients or column
purification. Further, if helper virus infection is employed to
express the accessory functions, residual helper virus can be
inactivated, using known methods. For example, adenovirus can be
inactivated by heating to temperatures of approximately 60.degree.
C. for, e.g., 20 minutes or more. This treatment selectively
inactivates the helper virus which is heat labile, while preserving
the rAAV which is heat stable. The resulting rAAV virions are then
ready for use for DNA delivery to a variety of target cells.
3.2.4.6 In Vivo Delivery rAAV Virions and Pharmaceutical
Compositions
[0398] The present invention relates to a method for the transfer
of nucleic acid compositions to the cells of an individual in
general and to the transfer of RNAi expression cassettes in
particular. The method comprises the step of contacting cells of
said individual with rAAV-based gene transfer vectors which include
at least one RNAi expression cassette, thereby delivering said RNAi
expression cassette to the nucleus within said cells. The rAAV
vectors are administered to the cells of said individual on an in
vivo basis, i.e., the contact with the cells of the individual
takes place within the body of the individual in accordance with
the procedures which are most typically employed.
[0399] The rAAV virion is preferably suspended in a
pharmaceutically acceptable delivery vehicle (i.e., physiologically
compatible carrier), for administration to a human or non-human
mammalian patient. Suitable carriers may be readily selected by one
of skill in the art and may depend on the nature of the nucleic
acid transfer vector chosen. Pharmaceutical compositions will
comprise sufficient genetic material to produce a therapeutically
effective amount of dsRNA complexes. The pharmaceutical
compositions will also contain a pharmaceutically acceptable
excipient. Such excipients include any pharmaceutical agent that
does not itself induce an immune response harmful to the individual
receiving the composition, and which may be administered without
undue toxicity. Pharmaceutically acceptable excipients include, but
are not limited to, liquids such as water, saline, glycerol and
ethanol. Pharmaceutically acceptable salts can be included therein,
for example, mineral acid salts such as hydrochlorides,
hydrobromides, phosphates, sulfates, and the like; and the salts of
organic acids such as acetates, propionates, malonates, benzoates,
and the like. Additionally, auxiliary substances, such as wetting
or emulsifying agents, pH buffering substances, and the like, may
be present in such vehicles. Other exemplary carriers include
lactose, sucrose, calcium phosphate, gelatin, dextran, agar,
pectin, peanut oil, sesame oil, and water. The selection of the
carrier is not a limitation of the present invention. Optionally,
the compositions of the invention may contain, in addition to the
rAAV virions and carrier(s), other conventional pharmaceutical
ingredients, such as preservatives, or chemical stabilizers.
Suitable exemplary ingredients include microcrystalline cellulose,
carboxymethylcellulose sodium, polysorbate 80, phenylethyl alcohol,
chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide,
propyl gallate, the parabens, ethyl vanillin, glycerin, phenol,
parachlorophenol, gelatin and albumin. A thorough discussion of
pharmaceutically acceptable excipients is available in REMINGTON'S
PHARMACEUTICAL SCIENCES (Mack Pub. Co., N.J. 1991).
[0400] Solutions, suspensions and powders for reconstitutable
delivery systems include vehicles such as suspending agents (e.g.,
gums, zanthans, cellulosics and sugars), humectants (e.g.,
sorbitol), solubilizers (e.g., ethanol, water, PEG and propylene
glycol), surfactants (e.g., sodium lauryl sulfate, Spans, Tweens,
and cetyl pyridine), preservatives and antioxidants (e.g.,
parabens, vitamins E and C, and ascorbic acid), anti-caking agents,
coating agents, and chelating agents (e.g., EDTA).
[0401] In this invention, administering the instant pharmaceutical
composition can be effected or performed using any of the various
methods and delivery systems known to those skilled in the art. The
administering can be performed, for example, intravenously, orally,
via implant, transmucosally, transdermally, intramuscularly, and
subcutaneously. In addition, the instant pharmaceutical
compositions ideally contain one or more routinely used
pharmaceutically acceptable carriers. Such carriers are well known
to those skilled in the art. The following delivery systems, which
employ a number of routinely used carriers, are only representative
of the many embodiments envisioned for administering the instant
composition.
[0402] Injectable drug delivery systems include solutions,
suspensions, gels, microspheres and polymeric injectables, and can
comprise excipients such as solubility-altering agents (e.g.,
ethanol, propylene glycol and sucrose) and polymers (e.g.,
polycaprylactones and PLGA's). Implantable systems include rods and
discs, and can contain excipients such as PLGA and
polycaprylactone.
[0403] Determining a therapeutically or prophylactically effective
amount of the instant pharmaceutical composition can be done based
on animal data using routine computational methods. Appropriate
doses will depend, among other factors, on the specifics of the
transfer vector chosen, on the route of administration, on the
mammal being treated (e.g., human or non-human primate or other
mammal), age, weight, and general condition of the subject to be
treated, the severity of the cancer being treated, the location of
the cancer being treated and the mode of administration. Thus, the
appropriate dosage may vary from patient to patient. An appropriate
effective amount can be readily determined by one of skill in the
art. In one specific embodiment, the nucleic acid transfer vector
is an AAV2/5 hybrid vector. A therapeutically effective human
dosage for in vivo delivery of said vector according to the present
invention is believed to be in the range of from about 20 to about
50 ml of saline solution containing concentrations of from about
10.sup. 10 to 10.sup. 14 functional vector/ml solution. The dosage
will be adjusted to balance the therapeutic benefit against any
side effects. In yet another embodiment, pharmaceutically effective
dose of the rAAV is generally in the range of concentrations of
from about 1.times.10.sup.5 to 1.times.10.sup.50 genomes rAAV,
about 10.sup.8 to 10.sup.20 genomes rAAV, about 10.sup.10 to about
10.sup.16 genomes, or about 10.sup.11 to 10.sup.16 genomes rAAV. A
preferred human dosage may be about 1.times.10.sup.13 AAV genomes
rAAV. Such concentrations may be delivered in about 0.001 ml to 100
ml, 0.05 to 50 ml, or 10 to 25 ml of a carrier solution. Other
effective dosages can be readily established by one of ordinary
skill in the art through routine trials establishing dose response
curves.
[0404] Dosage treatment may be a single dose schedule or a multiple
dose schedule. Moreover, the subject may be administered as many
doses as appropriate. One of skill in the art can readily determine
an appropriate number of doses. However, the dosage may need to be
adjusted to take into consideration an alternative route of
administration, or balance the therapeutic benefit against any side
effects. Such dosages may vary depending upon the therapeutic
application for which the recombinant vector is employed.
[0405] The vector particles are administered in sufficient amounts
to enter the desired cells and to guarantee sufficient levels of
functionality of the transferred nucleic acid composition to
provide a therapeutic benefit without undue adverse, or with
medically acceptable, physiological effects which can be determined
by those skilled in the medical arts.
[0406] In some embodiments, conventional pharmaceutically
acceptable routes of administration of rAAV may be combined. These
routes include, but are not limited to, direct delivery to the
liver, intravenous, intramuscular, subcutaneous, intradermal, oral
and other parental routes of administration.
[0407] Optionally, in specific embodiments, rAAV-mediated delivery
according to the invention may be combined with delivery by other
viral and non-viral vectors. Such other viral vectors including,
without limitation, adenoviral vectors, retroviral vectors,
lentiviral vectors. herpes simplex virus (HSV) vectors, and
baculovirus vectors may be readily selected and generated according
to methods known in the art. Similarly, non-viral vectors,
including, without limitation, liposomes, lipid-based vectors,
polyplex vectors, molecular conjugates, polyamines and polycation
vectors, may be readily selected and generated according to methods
known in the art. When administered by these alternative routes,
the dosage is desirable in the range described above.
[0408] In one embodiment, the route of administration is inhalation
with lung cells as RNAi target cells. In that instance, when
prepared for use as an inhalant: the pharmaceutical compositions
are prepared as fluid unit doses using the rAAV and a suitable
pharmaceutical vehicle for delivery by an atomizing spray pump, or
by dry powder for insufflation. For use as aerosols, the rAAV can
be packaged in a pressurized aerosol container together with a
gaseous or liquefied propellant, for example,
dichlorodifluormethane, carbon dioxide, nitrogen, propane, and the
like, with the usual components such as cosolvents and wetting
agents, as may be necessary or desirable. A pharmaceutical kit of
said embodiment, desirably contains a container for oral or
intranasal inhalation, which delivers a metered dose in one, two,
or more actuations. Suitably, the kit also contains instructions
for use of the spray pump or other delivery device, instructions on
dosing, and an insert regarding the active agent (i.e., the
transgene and/or rAAV). A single actuation of a pump spray or
inhaler generally delivers contains in the range of about 10.sup.5
to about 10.sup.15 genome copies (GC), about 10.sup.8 to about
10.sup.12, and/or about 10.sup.10 GC, in a liquid containing 10
.mu.g to 250 .mu.g carrier, 25 .mu.g to 100 .mu.g, or 40 .mu.g to
50 .mu.g, carrier. Suitably, a dose is delivered in one or two
actuations. However, other suitable delivery methods may be readily
determined. The doses may be repeated daily, weekly, or monthly,
for a predetermined length of time or as prescribed.
3.2.5 Pharmaceutical Preparations
[0409] In another aspect, the present invention provides
pharmaceutical preparations comprising hedgehog antagonists. The
hedgehog antagonists for use in the subject method may be
conveniently formulated for administration with a biologically
acceptable medium, such as water, buffered saline, polyol (for
example, glycerol, propylene glycol, liquid polyethylene glycol and
the like) or suitable mixtures thereof. The optimum concentration
of the active ingredient(s) in the chosen medium can be determined
empirically, according to procedures well known to medicinal
chemists. As used herein, "biologically acceptable medium" includes
any and all solvents, dispersion media, and the like which may be
appropriate for the desired route of administration of the
pharmaceutical preparation. The use of such media for
pharmaceutically active substances is known in the art. Except
insofar as any conventional media or agent is incompatible with the
activity of the hedgehog antagonist, its use in the pharmaceutical
preparation of the invention is contemplated. Suitable vehicles and
their formulation inclusive of other proteins are described, for
example, in the book Remington's Pharmaceutical Sciences
(Remington's Pharmaceutical Sciences. Mack Publishing Company,
Easton, Pa., USA 1985). These vehicles include injectable "deposit
formulations".
[0410] Pharmaceutical formulations of the present invention can
also include veterinary compositions, e.g., pharmaceutical
preparations of the hedgehog antagonists suitable for veterinary
uses, e.g., for the treatment of livestock or domestic animals,
e.g., dogs.
[0411] 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 proteinaceous
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 a hedgehog antagonist at a particular target site.
[0412] The preparations of the present invention may be given
orally, parenterally, topically, or rectally. They are, of course,
given by forms suitable for each administration route. For example,
they are administered in tablets or capsule form, by injection,
inhalation, eye lotion, ointment, suppository, controlled release
patch, etc. administration by injection, infusion or inhalation;
topical by lotion or ointment; and rectal by suppositories. Oral
and topical administrations are preferred.
[0413] The phrases "parenteral administration" and "administered
parenterally" as used herein means modes of administration other
than enteral and topical administration, usually by injection, and
includes, without limitation, intravenous, intramuscular,
intraarterial, intrathecal, intracapsular, intraorbital,
intracardiac, intradermal, intraperitoneal, transtracheal,
subcutaneous, subcuticular, intraarticular, subcapsular,
subarachnoid, intraspinal and intrasternal injection and
infusion.
[0414] The phrases "systemic administration," "administered
systemically," "peripheral administration" and "administered
peripherally" as used herein mean the administration of a compound,
drug or other material other than directly into the central nervous
system, such that it enters the patient's system and, thus, is
subject to metabolism and other like processes, for example,
subcutaneous administration.
[0415] These compounds may be administered to humans and other
animals for therapy by any suitable route of administration,
including orally, nasally, as by, for example, a spray, rectally,
intravaginally, parenterally, intracistemally and topically, as by
powders, ointments or drops, including buccally and
sublingually.
[0416] Regardless of the route of administration selected, the
compounds of the present invention, which may be used in a suitable
hydrated form, and/or the pharmaceutical compositions of the
present invention, are formulated into pharmaceutically acceptable
dosage forms such as described below or by other conventional
methods known to those of skill in the art.
[0417] Actual dosage levels of the active ingredients in the
pharmaceutical compositions of this invention may be varied so as
to obtain an amount of the active ingredient that is effective to
achieve the desired therapeutic response for a particular patient,
composition, and mode of administration, without being toxic to the
patient.
[0418] The selected dosage level will depend upon a variety of
factors including the activity of the particular compound of the
present invention employed, or the ester, salt or amide thereof,
the route of administration, the time of administration, the rate
of excretion of the particular compound being employed, the
duration of the treatment, other drugs, compounds and/or materials
used in combination with the particular hedgehog antagonist
employed, the age, sex, weight, condition, general health and prior
medical history of the patient being treated, and like factors well
known in the medical arts.
[0419] A physician or veterinarian having ordinary skill in the art
can readily determine and prescribe the effective amount of the
pharmaceutical composition required. For example, the physician or
veterinarian could start doses of the compounds of the invention
employed in the pharmaceutical composition at levels lower than
that required in order to achieve the desired therapeutic effect
and gradually increase the dosage until the desired effect is
achieved.
[0420] In general, a suitable daily dose of a compound of the
invention will be that amount of the compound that is the lowest
dose effective to produce a therapeutic effect. Such an effective
dose will generally depend upon the factors described above.
Generally, intravenous, intracerebroventricular and subcutaneous
doses of the compounds of this invention for a patient will range
from about 0.0001 to about 100 mg per kilogram of body weight per
day.
[0421] If desired, the effective daily dose of the active compound
may be administered as two, three, four, five, six or more
sub-doses administered separately at appropriate intervals
throughout the day, optionally, in unit dosage forms.
[0422] The term "treatment" is intended to encompass also
prophylaxis, therapy and cure.
[0423] The patient receiving this treatment is any animal in need,
including primates, in particular humans, and other mammals such as
equines, cattle, swine and sheep; and poultry and pets in
general.
[0424] The compound of the invention can be administered as such or
in admixtures with pharmaceutically acceptable and/or sterile
carriers and can also be administered in conjunction with other
antimicrobial agents such as penicillins, cephalosporins,
aminoglycosides and glycopeptLdes. Conjunctive therapy thus
includes sequential, simultaneous and separate administration of
the active compound in a way that the therapeutic effects of the
first administered one is not entirely disappeared when the
subsequent is administered.
3.2.6 Pharmaceutical Composition
[0425] While it is possible for a therapeutic of the present
invention to be administered alone, it is preferable to administer
the therapeutic as a pharmaceutical formulation (composition). The
hedgehog antagonists according to the invention may be formulated
for administration in any convenient way for use in human or
veterinary medicine. In certain embodiments, the compound included
in the pharmaceutical preparation may be active itself, or may be a
prodrug, e.g., capable of being converted to an active compound in
a physiological setting.
[0426] Thus, another aspect of the present invention provides
pharmaceutically acceptable compositions comprising a
therapeutically effective amount of one or more of the compounds
described above, formulated together with one or more
pharmaceutically acceptable carriers (additives) and/or diluents.
As described in detail below, the pharmaceutical compositions of
the present invention may be specially formulated for
administration in solid or liquid form, including those adapted for
the following: (1) oral administration, for example, drenches
(aqueous or non-aqueous solutions or suspensions), tablets,
boluses, powders, granules, pastes for application to the tongue;
(2) parenteral administration, for example, by subcutaneous,
intramuscular or intravenous injection as, for example, a sterile
solution or suspension; (3) topical application, for example, as a
cream, ointment or spray applied to the skin; or (4) intravaginally
or intrarectally, for example, as a pessary, cream or foam.
However, in certain embodiments the subject compounds may be simply
dissolved or suspended in sterile water. In certain embodiments,
the pharmaceutical preparation is non-pyrogenic, i.e., does not
elevate the body temperature of a patient.
[0427] The phrase "therapeutically effective amount" as used herein
means that amount of a compound, material, or composition
comprising a compound of the present invention which is effective
for producing some desired therapeutic effect by overcoming a
hedgehog gain-of-function phenotype in at least a subpopulation of
cells in an animal and thereby blocking the biological consequences
of that pathway in the treated cells, at a reasonable benefit/risk
ratio applicable to any medical treatment.
[0428] The phrase "pharmaceutically acceptable" is employed herein
to refer to those compounds, materials, compositions, and/or dosage
forms which are, within the scope of sound medical judgment,
suitable for use in contact with the tissues of human beings and
animals without excessive toxicity, irritation, allergic response,
or other problem or complication, commensurate with a reasonable
benefit/risk ratio.
[0429] The phrase "pharmaceutically acceptable carrier" as used
herein means a pharmaceutically acceptable material, composition or
vehicle, such as a liquid or solid filler, diluent, excipient,
solvent or encapsulating material, involved in carrying or
transporting the subject antagonists from one organ, or portion of
the body, to another organ, or portion of the body. Each carrier
must be "acceptable" in the sense of being compatible with the
other ingredients of the formulation and not injurious to the
patient. Some examples of materials which can serve as
pharmaceutically acceptable carriers include: (1) sugars, such as
lactose, glucose and sucrose; (2) starches, such as corn starch and
potato starch; (3) cellulose, and its derivatives, such as sodium
carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4)
powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8)
excipients, such as cocoa butter and suppository waxes; (9) oils,
such as peanut oil, cottonseed oil, safflower oil, sesame oil,
olive oil, corn oil and soybean oil; (10) glycols, such as
propylene glycol; (11) polyols, such as glycerin, sorbitol,
mannitol and polyethylene glycol; (12) esters, such as ethyl oleate
and ethyl laurate; (13) agar; (14) buffering agents, such as
magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16)
pyrogen-free water; (17) isotonic saline; (18) Ringer's solution;
(19) ethyl alcohol; (20) phosphate buffer solutions; and (21) other
non-toxic compatible substances employed in pharmaceutical
formulations.
[0430] As set out above, certain embodiments of the present
hedgehog antagonists may contain a basic functional group, such as
amino or alkylamino, and are, thus, capable of forming
pharmaceutically acceptable salts with pharmaceutically acceptable
acids. The term "pharmaceutically acceptable salts" in this
respect, refers to the relatively non-toxic, inorganic and organic
acid addition salts of compounds of the present invention. These
salts can be prepared in situ during the final isolation and
purification of the compounds of the invention, or by separately
reacting a purified compound of the invention in its free base
[0431] form with a suitable organic or inorganic acid, and
isolating the salt thus formed. Representative salts include the
hydrobromide, hydrochloride, sulfate, bisulfate, phosphate,
nitrate, acetate, valerate, oleate, palmitate, stearate, laurate,
benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate,
succinate, tartrate, naphthylate, mesylate, glucoheptonate,
lactobionate, and laurylsulphonate salts and the like [149].
[0432] The pharmaceutically acceptable salts of the subject
compounds include the conventional nontoxic salts or quaternary
ammonium salts of the compounds, e.g., from non-toxic organic or
inorganic acids. For example, such conventional nontoxic salts
include those derived from inorganic acids such as hydrochloride,
hydrobromic, sulfuric, sulfamic, phosphoric, nitric, and the like;
and the salts prepared from organic acids such as acetic,
propionic, succinic, glycolic, stearic, lactic, malic, tartaric,
citric, ascorbic, palmitic, maleic, hydroxymaleic, phenylacetic,
glutamic, benzoic, salicyclic, sulfanilic, 2-acetoxybenzoic,
fumaric, toluenesulfonic, methanesulfonic, ethane disulfonic,
oxalic, isothionic, and the like.
[0433] In other cases, the compounds of the present invention may
contain one or more acidic functional groups and, thus, are capable
of forming pharmaceutically acceptable salts with pharmaceutically
acceptable bases. The term "pharmaceutically acceptable salts" in
these instances refers to the relatively non-toxic, inorganic and
organic base addition salts of compounds of the present invention.
These salts can likewise be prepared in situ during the final
isolation and purification of the compounds, or by separately
reacting the purified compound in its free acid form with a
suitable base, such as the hydroxide, carbonate or bicarbonate of a
pharmaceutically acceptable metal cation; with ammonia, or with a
pharmaceutically acceptable organic primary, secondary or tertiary
amine. Representative alkali or alkaline earth salts include the
lithium, sodium, potassium, calcium, magnesium, and aluminum salts
and the like. Representative organic amines useful for the
formation of base addition salts include ethylamine, diethylamine,
ethylenediamine, ethanolamine, diethanolamine, piperazine and the
like. (See, for example, [149]). Wetting agents, emulsifiers and
lubricants, such as sodium lauryl sulfate and magnesium stearate,
as well as coloring agents, release agents, coating agents,
sweetening, flavoring and perfuming agents, preservatives and
antioxidants can also be present in the compositions.
[0434] Examples of pharmaceutically acceptable antioxidants
include: (1) water soluble antioxidants, such as ascorbic acid,
cysteine hydrochloride, sodium bisulfate, sodium metabisulfite,
sodium sulfite and the like; (2) oil-soluble antioxidants, such as
ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated
hydroxytoluene (BHT), lecithin, propyl gallate, alpha.-tocopherol,
and the like; and (3) metal chelating agents, such as citric acid,
ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid,
phosphoric acid, and the like.
[0435] Formulations of the present invention include those suitable
for oral, nasal, topical (including buccal and sublingual), rectal,
vaginal and/or parenteral administration. The formulations may
conveniently be presented in unit dosage form and may be prepared
by any methods well known in the art of pharmacy. The amount of
active ingredient which can be combined with a carrier material to
produce a single dosage form will vary depending upon the host
being treated, the particular mode of administration. The amount of
active ingredient that can be combined with a carrier material to
produce a single dosage form will generally be that amount of the
compound that produces a therapeutic effect. Generally, out of one
hundred percent, this amount will range from about 1 percent to
about ninety-nine percent of active ingredient, preferably from
about 5 percent to about 70 percent, most preferably from about 10
percent to about 30 percent.
[0436] Methods of preparing these formulations or compositions
include the step of bringing into association a compound of the
present invention with the carrier and, optionally, one or more
accessory ingredients. In general, the formulations are prepared by
uniformly and intimately bringing into association a compound of
the present invention with liquid carriers, or finely divided solid
carriers, or both, and then, if necessary, shaping the product.
[0437] Formulations of the invention suitable for oral
administration may be in the form of capsules, cachets, pills,
tablets, lozenges (using a flavored basis, usually sucrose and
acacia or tragacanth), powders, granules, or as a solution or a
suspension in an aqueous or non-aqueous liquid, or as an
oil-in-water or water-in-oil liquid emulsion, or as an elixir or
syrup, or as pastilles (using an inert base, such as gelatin and
glycerin, or sucrose and acacia) and/or as mouth washes and the
like, each containing a predetermined amount of a compound of the
present invention as an active ingredient. A compound of the
present invention may also be administered as a bolus, electuary or
paste.
[0438] In solid dosage forms of the invention for oral
administration (capsules, tablets, pills, dragees, powders,
granules and the like), the active ingredient is mixed with one or
more pharmaceutically acceptable carriers, such as sodium citrate
or dicalcium phosphate, and/or any of the following: (1) fillers or
extenders, such as starches, lactose, sucrose, glucose, mannitol,
and/or silicic acid; (2) binders, such as, for example,
carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone,
sucrose and/or acacia; (3) humectants, such as glycerol; (4)
disintegrating agents, such as agar-agar, calcium carbonate, potato
or tapioca starch, alginic acid, certain silicates, and sodium
carbonate; (5) solution retarding agents, such as paraffin; (6)
absorption accelerators, such as quaternary ammonium compounds; (7)
wetting agents, such as, for example, cetyl alcohol and glycerol
monostearate; (8) absorbents, such as kaolin and bentonite clay;
(9) lubricants, such a talc, calcium stearate, magnesium stearate,
solid polyethylene glycols, sodium lauryl sulfate, and mixtures
thereof; and (10) coloring agents. In the case of capsules, tablets
and pills, the pharmaceutical compositions may also comprise
buffering agents. Solid compositions of a similar type may also be
employed as fillers in soft and hard-filled gelatin capsules using
such excipients as lactose or milk sugars, as well as high
molecular weight polyethylene glycols and the like.
[0439] A tablet may be made by compression or molding, optionally
with one or more accessory ingredients. Compressed tablets may be
prepared using binder (for example, gelatin or hydroxypropylmethyl
cellulose), lubricant, inert diluent, preservative, disintegrant
(for example, sodium starch glycolate or cross-linked sodium
carboxymethyl cellulose), surface-active or dispersing agent.
Molded tablets may be made by molding in a suitable machine a
mixture of the powdered compound moistened with an inert liquid
diluent.
[0440] The tablets, and other solid dosage forms of the
pharmaceutical compositions of the present invention, such as
dragees, capsules, pills and granules, may optionally be scored or
prepared with coatings and shells, such as enteric coatings and
other coatings well known in the pharmaceutical-formulating art.
They may also be formulated so as to provide slow or controlled
release of the active ingredient therein using, for example,
hydroxypropylmethyl cellulose in varying proportions to provide the
desired release profile, other polymer matrices, liposomes and/or
microspheres. They may be sterilized by, for example, filtration
through a bacteria-retaining filter, or by incorporating
sterilizing agents in the form of sterile solid compositions that
can be dissolved in sterile water, or some other sterile injectable
medium immediately before use. These compositions may also
optionally contain opacifying agents and may be of a composition
that they release the active ingredient(s) only, or preferentially,
in a certain portion of the gastrointestinal tract, optionally, in
a delayed manner. Examples of embedding compositions that can be
used include polymeric substances and waxes. The active ingredient
can also be in micro-encapsulated form, if appropriate, with one or
more of the above-described excipients.
[0441] Liquid dosage forms for oral administration of the compounds
of the invention include pharmaceutically acceptable emulsions,
microemulsions, solutions, suspensions, syrups and elixirs. In
addition to the active ingredient, the liquid dosage forms may
contain inert diluents commonly used in the art, such as, for
example, water or other solvents, solubilizing agents and
emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl
carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate,
propylene glycol, 1,3-butylene glycol, oils (in particular,
cottonseed, groundnut, corn, germ, olive, castor and sesame oils),
glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty
acid esters of sorbitan, and mixtures thereof.
[0442] Besides inert diluents, the oral compositions can also
include adjuvants such as wetting agents, emulsifying and
suspending agents, sweetening, flavoring, coloring, perfuming and
preservative agents.
[0443] Suspensions, in addition to the active compounds, may
contain suspending agents as, for example, ethoxylated isostearyl
alcohols, polyoxyethylene sorbitol and sorbitan esters,
microcrystalline cellulose, aluminum metahydroxide, bentonite,
agar-agar and tragacanth, and mixtures thereof.
[0444] It is known that sterols, such as cholesterol, will form
complexes with cyclodextrins. Thus, in preferred embodiments, where
the inhibitor is a steroidal alkaloid, it may be formulated with
cyclodextrins, such as a-, .beta.-and ?-cyclodextrin,
dimethyl-.beta. cyclodextrin and
2-hydroxypropyl-.beta.-cyclodextrin.
[0445] Formulations of the pharmaceutical compositions of the
invention for rectal or vaginal administration may be presented as
a suppository, which may be prepared by mixing one or more
compounds of the invention with one or more suitable nonirritating
excipients or carriers comprising, for example, cocoa butter,
polyethylene glycol, a suppository wax or a salicylate, and which
is solid at room temperature, but liquid at body temperature and,
therefore, will melt in the rectum or vaginal cavity and release
the active hedgehog antagonist.
[0446] Formulations of the present invention which are suitable for
vaginal administration also include pessaries, tampons, creams,
gels, pastes, foams or spray formulations containing such carriers
as are known in the art to be appropriate.
[0447] Dosage forms for the topical or transdermal administration
of a compound of this invention include powders, sprays, ointments,
pastes, creams, lotions, gels, solutions, patches and inhalants.
The active compound may be mixed under sterile conditions with a
pharmaceutically acceptable carrier, and with any preservatives,
buffers, or propellants that may be required.
[0448] The ointments, pastes, creams and gels may contain, in
addition to an active compound of this invention, excipients, such
as animal and vegetable fats, oils, waxes, paraffins, starch,
tragacanth, cellulose derivatives, polyethylene glycols, silicones,
bentonites, silicic acid, talc and zinc oxide, or mixtures
thereof.
[0449] Powders and sprays can contain, in addition to a compound of
this invention, excipients such as lactose, talc, silicic acid,
aluminum hydroxide, calcium silicates and polyamide powder, or
mixtures of these substances. Sprays can additionally contain
customary propellants, such as chlorofluorohydrocarbons and
volatile, unsubstituted hydrocarbons, such as butane and
propane.
[0450] Transdermal patches have the added advantage of providing
controlled delivery of a compound of the present invention to the
body. Such dosage forms can be made by dissolving or dispersing the
hedgehog antagonists in the proper medium. Absorption enhancers can
also be used to increase the flux of the hedgehog antagonists
across the skin. The rate of such flux can be controlled by either
providing a rate controlling membrane or dispersing the compound in
a polymer matrix or gel.
[0451] Ophthalmic formulations, eye ointments, powders, solutions
and the like, are also contemplated as being within the scope of
this invention.
[0452] Pharmaceutical compositions of this invention suitable for
parenteral administration comprise one or more compounds of the
invention in combination with one or more pharmaceutically
acceptable sterile isotonic aqueous or nonaqueous solutions,
dispersions, suspensions or emulsions, or sterile powders which may
be reconstituted into sterile injectable solutions or dispersions
just prior to use, which may contain antioxidants, buffers,
bacteriostats, solutes which render the formulation isotonic with
the blood of the intended recipient or suspending or thickening
agents.
[0453] Examples of suitable aqueous and nonaqueous carriers that
may be employed in the pharmaceutical compositions of the invention
include water, ethanol, polyols (such as glycerol, propylene
glycol, polyethylene glycol, and the like), and suitable mixtures
thereof, vegetable oils, such as olive oil, and injectable organic
esters, such as ethyl oleate. Proper fluidity can be maintained,
for example, by the use of coating materials, such as lecithin, by
the maintenance of the required particle size in the case of
dispersions, and by the use of surfactants.
[0454] These compositions may also contain adjuvants such as
preservatives, wetting agents, emulsifying agents and dispersing
agents. Prevention of the action of microorganisms may be ensured
by the inclusion of various antibacterial and antifungal agents,
for example, paraben, chlorobutanol, phenol sorbic acid, and the
like. It may also be desirable to include isotonic agents, such as
sugars, sodium chloride, and the like into the compositions. In
addition, prolonged absorption of the injectable pharmaceutical
form may be brought about by the inclusion of agents that delay
absorption such as aluminum monostearate and gelatin.
[0455] In some cases, in order to prolong the effect of a drug, it
is desirable to slow the absorption of the drug from subcutaneous
or intramuscular injection. This may be accomplished by the use of
a liquid suspension of crystalline or amorphous material having
poor water solubility. The rate of absorption of the drug then
depends upon its rate of dissolution, which, in turn, may depend
upon crystal size and crystalline form. Alternatively, delayed
absorption of a parenterally administered drug form is accomplished
by dissolving or suspending the drug in an oil vehicle.
[0456] Injectable depot forms are made by forming microencapsule
matrices of the subject compounds in biodegradable polymers such as
polylactide-polyglycolide. Depending on the ratio of drug to
polymer, and
[0457] the nature of the particular polymer employed, the rate of
drug release can be controlled. Examples of other biodegradable
polymers include poly(orthoesters) and poly(anhydrides). Depot
injectable formulations are also prepared by entrapping the drug in
liposomes or microemulsions that are compatible with body
tissue.
[0458] When the compounds of the present invention are administered
as pharmaceuticals, to humans and animals, they can be given per se
or as a pharmaceutical composition containing, for example, 0.1 to
99.5% (more preferably, 0.5 to 90%) of active ingredient in
combination with a pharmaceutically acceptable carrier.
[0459] The addition of the active compound of the invention to
animal feed is preferably accomplished by preparing an appropriate
feed premix containing the active compound in an effective amount
and incorporating the premix into the complete ration.
[0460] Alternatively, an intermediate concentrate or feed
supplement containing the active ingredient can be blended into the
feed. The way in which such feed premixes and complete rations can
be prepared and administered are described in reference books (such
as "Applied Animal Nutrition", W.H. Freedman and CO., San
Francisco, U.S.A., 1969 or "Livestock Feeds and Feeding" 0 and B
books, Corvallis, Oreg., U.S.A., 1977).
[0461] In any of the foregoing embodiment, the invention
contemplates that the pharmaceutical preparations may be
non-pyrogenic.
[0462] The pharmaceutical preparations for use in the methods of
the present invention may comprise combinations of two or more
hedgehog antagonists. For example, two hedgehog antibodies may be
combined with a pharmaceutically acceptable carrier or excipient.
The two antibodies may act additively or synergistically. In
another example, one or more hedgehog antibodies may be combined
with one or more non-antibody hedgehog antagonists (e.g., one or
more small organic molecules), and with a pharmaceutically
acceptable carrier or excipients. Said combination of hedgehog
antagonists may act additively or synergistically.
3.3 DESCRIPTIONS OF FIGURES
[0463] The present invention now will be described by way of
illustrating but not limiting way, according to preferred
embodiments thereof, with particular reference to the figures of
the enclosed drawings, wherein:
[0464] FIG. 1 shows FIG. 1 shows some representative examples of
the chemical/structural formulas of steroidal alkaloids of the
Veratrum type.
[0465] FIG. 2 shows a representative example of the
chemical/structural formulas of steroidal alkaloids of the Solanum
type.
[0466] FIG. 3 shows the result of an experiment where laser-induced
CNV triggers the expression of Shh and Ptc. One can clearly see the
upregulation of Shh (Shh CNV), Ptch (Ptch CNV), and VEGF (VEGF CNV)
in laser-treated eyes (CNV) in comparison to control eyes (i.e.,
VEGF, Shh, Ptch). Actin functioned as an internal control.
[0467] FIG. 4 shows the result of an experiment where hypoxia was
induced in 293 cells (using desferroxamine), and the supernatant
was tested twenty-four hours after induction for the presence of
VEGF and Shh.
[0468] FIG. 5 shows the result of an angiography after
fluorescein-dextran perfusion, where one group of "ROP" mice, a
model of ocular neovascularization, was treated with cyclopamine
("Cyclopamine") and the other group ("ROP") was untreated. Robust
inhibition of ocular neovascularization (decreased neovascular
tufts and vessel leakage) was observed in all eyes injected with
cyclopamine ("Cyclopamine"; FIG. 5 left) when compared with the
control eyes ("ROP"; FIG. 5 right).
3.4 DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0469] The following paragraphs will provide a detailed description
of the preferred embodiments.
3.4.1 Example 1
Laser-Induced CNV triggers the Expression of Shh and Ptc
[0470] To determine whether the Shh pathway is activated in
experimental choroid neovascularization (CNV model), we performed
RT-PCR analyses on laser treated, and control eyes.
[0471] We performed two independent experiments using two groups of
animals of the same age (n=3). Choroidal neovascularization was
induced in one eye by 20 burns around the entire surface of the
retina using a diode laser. The contralateral eye was used as a
control.
[0472] Three days after treatment retinas were harvested and
pulled. Following RNA extraction, cDNA was synthesized and used as
a template for semi-quantitative RT-PCR analysis. Shh was
upregulated in laser-treated eyes in comparison to control eyes
(FIG. 3: Laser-induced CNV triggers the expression of Shh and Ptc).
Interestingly Ptc expression was also induced, implying that Ptc is
a transcriptional target of Shh.
[0473] Semi-quantitative RT-PCR analysis was performed using Actin
as a control. First-strand cDNA was synthesized from total RNA by
reverse transcriptase (Stratagene, La Jolla, Calif., USA) using
random primers. The cDNAs were amplified by PCR (94.degree. C. for
1 minute, 58.degree. C. for 1 minute 20 seconds, and 72.degree. C.
for 1 minute 15 seconds) using Taq DNA polymerase (Promega Corp.,
Madison, Wis., USA).
[0474] The reactions were carried out in the presence of the
following pairs of specific primers: TABLE-US-00001 SEQ ID NO: 9
(Shh forward primer): 5'GTG AGG CTGCGA GTG ACC G-3'; SEQ ID NO: 10
(Shh reverse primer): 5'-CCT GGT CGT CAG CCG CCA GCA CGC-3'; SEQ ID
NO: 11 (Ptc forward primer): 5'-CTG CTG CTA TCC ATC AGC GT-3'; SEQ
ID NO: 12 (Ptc reverse primer): 5'-AAG AAG GAT AAG AGG ACA
GG-3'.
[0475] PCR products were electrophoresed on a 1.5% agarose gel and
visualized by ethidium bromide.
[0476] As can be seen in FIG. 3, laser-induced CNV triggers the
expression of Shh and Ptc. One can clearly see the upregulation of
Shh (Shh CNV), Ptch (Ptch CNV), and VEGF (VEGF CNV) in
laser-treated eyes (CNV) in comparison to control eyes (i.e., VEGF,
Shh, Ptch). Actin functioned as an internal control.
3.4.2 Example 2
[0477] Cyclopamine is Able to Inhibit Hypoxia-Induced VEGF
Expression in HEK-293 Cells
[0478] To assess the ability of cyclopamine (purchased from Toronto
Research Chemicals Inc. North York, ON, Canada) to inhibit VEGF
protein expression induced in hypoxia, we added to the cell medium
(10% Fetal Calf Serum FCS in DMEM) of 293 cells both desferroxamine
(Sigma, St. Louis, Mo.) to a final concentration of 100 .mu.M (to
induce hypoxia) and Cyclopamine to a final concentration of 10
.mu.M dissolved in 5 .mu.l of 95% ethanol. Five pl of 95% ethanol
was added to untreated control.
[0479] Twenty-four hours after induction of hypoxia the supernatant
of the 293 cells was removed from wells and a human VEGF ELISA (R
& D Systems, Minneapolis, Minn.) was performed on the cell
supernatants (FIG. 4) as described in the Quantikine human VEGF
ELISA protocol using a dilution of the medium of 1:10. ELISA
results were read on a Bio-Rad Reader 550 ELISA plate reader
(Bio-Rad Laboratories, Inc, CA).
[0480] As can be seen in FIG. 4, hypoxia induction resulted in the
expression and secretion of both VEGF and Sonic Hedgehog in 293
cells.
3.4.3 Example 3
Cyclopamine Administration to ROP Mice results in Inhibition of
Ocular Neovascularization
[0481] To determine whether inhibition of the Shh signaling pathway
is able to reduce retinal (ocular) neovascularization, Cyclopamine
was administrated to a mouse model of Retinopathy of Prematurity
(ROP).
[0482] C57/BL6 mice were used in this study. Mice from postnatal
day 7 (P7) to P12 were exposed to hyperoxia (25% nitrogen, 75%
oxygen). At P12 mice returned to room air until P17. The group of
mice treated with cyclopamine received a daily subcutaneous
injection of 50 mg/kg of the drug dissolved in 95% of ethanol and
then mixed 1:4 in triolein (Sigma, St. Louis, Mo.) in a final
volume of 50 .mu.l, from P12 to P17, controls received the vehicle
alone.
[0483] On P17 angiography was performed: Mice were deeply
anesthetized with Avertin and perfused with a solution containing
fluorescein-dextran (50 mg/ml; 2 million molecular weight, Sigma,
St. Louis, Mo.) in phosphate-buffered saline (PBS). Then, eyes were
enucleated and fixed in paraformaldehyde, and the retinas were
dissected, flat-mounted and viewed by fluorescence microscopy.
Robust inhibition of ocular neovascularization (decreased
neovascular tufts and vessel leakage) was observed in all eyes
injected with cyclopamine when compared with the control eyes (FIG.
5).
3.4.4 Example 4
AAV-Mediated Gene Transfer of Soluble Hip1 to ROP Mice Results in
Inhibition of Ocular Neovascularization
[0484] This example provides a rAAV virion comprising a nucleic
acid composition that comprises a soluble Hip1 (sHIP1) minigene and
AAV2 ITRs. Said rAAV virion is pseudotyped in a capsid of AAV
serotype 2 (AAV2)--resulting in an AAV2/2 virion, referred to as
AAV2/2 CMV sHIP1, encoded by AAV2 CMV sHIP1 (SEQ ID NO:7). The use
of rAAV is particularly desirable, as it allows for long-term and
high-level gene expression in the eye. The artisan will be able to
reconstruct AAV2 CMV sHIP1 from the sequence information provided.
Alternatively, the artisan can order a plasmid comprising the AAV2
CMV sHIP1 sequence from a commercial service such as--for
example--Geneart (www.geneart.de; Regensburg, Germany).
Alternatively, the inventors will enable the practice of the
invention by providing the AAV2 CMV sHIP1 plasmid or any other
reagent that is not commercially available within 4 weeks upon
request (the inventors can be contacted by e-mailing a
corresponding request to: hildinger@gmx.net).
[0485] The sHIP1 sequence was cloned using PCR with mouse cDNA
retina as a template. The primers were designed to amplify the
sequence from the Hip1 start codon up to the deletion of the last
22 amino acids at the C-terminus. The primers include the cloning
sites (NotI on the 5' site, BamHI on the 3' site); their sequence
is listed in SEQ ID NO:13 (forward primer) and SEQ ID NO:14
(reverse primer).
[0486] sHip1 secretion from the transduced target cells is achieved
by transducing said target cells with a gene transfer vector
comprising a nucleic acid composition which comprises the sHip1
coding sequence and all the regulatory sequences necessary for its
transcription and translation in said target cells with said target
cells being eye cells such as--for example--retinal pigment
epithelium cells. In this embodiment, expression is driven by a CMV
promoter/enhancer, and a bovine growth hormone polyadenylation
signal is used to terminate transcription.
[0487] Vector is administered via subretinal injection at a dose of
10.sup.10 genomic particles. Alternatively, vector can also be
administered via intravitreal injection.
[0488] As a control, a rAAV virion expressing lacZ as transgene
(AAV 2/2 CMV lacZ) is used as lacZ does not possess anti-angiogenic
activity. The sequence and cloning of AAV2 CMV lacZ has been
described in prior art [71].
[0489] Both AAV2/2 CMV sHIP1 and AAV2/2 CMV lacZ are prepared by
triple transfection and purified by CsCl.sub.2 gradients as
described herein and in prior art [71]. Physical titers are
assessed by Real Time PCR.
[0490] To determine whether inhibition of the Shh signaling pathway
through expression and secretion of soluble Hip1 is able to reduce
retinal (ocular) neovascularization, AAV2/2 CMV sHIP1 and AAV2/2
CMV lacZ are administrated to a mouse model of Retinopathy of
Prematurity (ROP), respectively.
[0491] 10 C57/BL6 mice are used in this study. Mice from postnatal
day 7 (P7) to P12 are exposed to hyperoxia (25% nitrogen, 75%
oxygen). At P12 mice return to room air until P17. The group of
mice to be treated with AAV 2/2 CMV sHIP1 (5 mice) receives
10.sup.10 genomic particles of AAV 2/2 CMV sHIP1 in a final volume
of 50 .mu.l at day P3, the control group of mice (5 mice) receives
the same amount of genomic particles of AAV 2/2 CMV lacZ in a final
volume of f 50 .mu.l at day P3. Vector is administered by
subretinal injection.
[0492] On P17 angiography are performed: Mice are deeply
anesthetized with Avertin and perfused with a solution containing
fluorescein-dextran (50 mg/ml; 2 million molecular weight, Sigma,
St. Louis, Mo.) in phosphate-buffered saline (PBS). Then, eyes are
enucleated and fixed in paraformaldehyde, and the retinas are
dissected, flat-mounted and viewed by fluorescence microscopy.
Robust inhibition of ocular neovascularization (decreased
neovascular tufts and vessel leakage) is observed in all eyes
injected with AAV 2/2 CMV sHIP1 when compared with the control
("lacZ") eyes.
3.4.5 Example 5
Administration of Soluble Hip1 to ROP Mice results in Inhibition of
Ocular Neovascularization
[0493] Soluble Hip1 is produced as follows: 293 cells are
transfected with AAV2 CMV sHIP1 using the CsCl.sub.2 method. Three
days upon transfection, supernatant is harvested and filtered
through a 22-.mu.m filter to remove cell debris. To purify soluble
Hip1, the supernatant is passed through an affinity column with
covalently attached polyclonal rabbit-anti-Hip1 antibodies. Hip1 is
eluted with 15 ml of PBS, pH7.4, plus 0.4M NaCl. The eluate is
concentrated to about 0.5 ml with a Millipore Biomax-100K NMWL
filter device (UFV2BHK40) by centrifugation. To adjust the NaCl
concentration to physiological levels, the filter device is
refilled with PBS, pH 7.4, and the soluble Hip1 is concentrated to
0.5 ml again. After removal of the target-protein-containing
solution, the membrane of the filter device is washed three times
with 100 .mu.l of PBS, pH 7.4, which is then added to the other
part.
[0494] To determine whether inhibition of the Shh signaling pathway
by administration of soluble Hip1 is able to reduce retinal
(ocular) neovascularization, soluble Hip1 is administrated to a
mouse model of Retinopathy of Prematurity (ROP).
[0495] C57/BL6 mice are used in this study. Mice from postnatal day
7 (P7) to P12 are exposed to hyperoxia (25% nitrogen, 75% oxygen).
At P12 mice return to room air until P17. The group of five mice
treated with soluble Hip1 received a daily subretinal injection of
50 mg/kg of soluble Hip1 dissolved in Phosphate-Buffered Saline
(PBS) from P12 to P17, controls receive the vehicle alone.
[0496] On P17 angiography is performed: Mice are deeply
anesthetized with Avertin and perfused with a solution containing
fluorescein-dextran (50 mg/ml; 2 million molecular weight, Sigma,
St. Louis, Mo.) in Phosphate-Buffered Saline (PBS). Then, eyes are
enucleated and fixed in paraformaldehyde, and the retinas are
dissected, flat-mounted and viewed by fluorescence microscopy.
Robust inhibition of ocular neovascularization (decreased
neovascular tufts and vessel leakage) is observed in all eyes
injected with soluble Hip1 when compared with the control eyes.
3.4.6 Example 6
Administration of an siRNA targeting Sonic Hedgehog to ROP Mice
Results in Inhibition of Ocular Neovascularization
[0497] The siRNA targeting Sonic Hedgehog is ordered from a
commercial vendor (Qiagen GmbH, Hilden, Germany) according to our
specifications: The siRNA duplex is represented in SEQ ID NO:16
(sense strand) and. SEQ ID NO:15 (antisense strand), wherein the
sense and antisense RNA strands form an RNA duplex. The sense and
antisense strand are ordered pre-annealed.
[0498] To determine whether inhibition of the Shh signaling pathway
by administration of an siRNA targeting Shh is able to reduce
retinal (ocular) neovascularization, siRNA targeting Shh is
administrated to a mouse model of Retinopathy of Prematurity
(ROP).
[0499] C57/BL6 mice are used in this study. Mice from postnatal day
7 (P7) to P12 are exposed to hyperoxia (25% nitrogen, 75% oxygen).
At P12 mice return to room air until P17. The group of five mice
treated with siRNA targeting Shh receive a daily subretinal
injection (1 .mu.l) of 100 mg/ml siRNA targeting Shh dissolved in
Phosphate-Buffered Saline (PBS) from P12 to P17, controls receive
the vehicle alone.
[0500] On P17 angiography is performed: Mice are deeply
anesthetized with Avertin and perfused with a solution containing
fluorescein-dextran (50 mg/ml; 2 million molecular weight, Sigma,
St. Louis, Mo.) in Phosphate-Buffered Saline (PBS). Then, eyes are
enucleated and fixed in paraformaldehyde, and the retinas are
dissected, flat-mounted and viewed by fluorescence microscopy.
Robust inhibition of ocular neovascularization (decreased
neovascular tufts and vessel leakage) is observed in all eyes
injected with siRNA targeting Shh when compared with the control
eyes.
[0501] To summarize: A small interfering RNA targeting Sonic
Hedgehog is injected into the eye of a mammalian subject. One
variant of an siRNA targeting human and mouse Sonic Hedgehog is
represented in SEQ ID NO:16 (sense strand) and SEQ ID NO:15
(antisense strand), wherein the sense and antisense RNA strands
form an RNA duplex. The sense and antisense strand can be purchased
already annealed (i.e., in duplex form) from commercial vendors
such as Qiagen GmbH, Hilden, Germany. Many different siRNAs can be
designed to target Sonic Hedgehog. Theoretically, each 19 to 25
nucleotide stretch within the Sonic Hedgehog gene can be used as
design template for an siRNA. The exact method, design (e.g.,
stem-loop vs. annealed duplex) and sequence of the siRNA targeting
Sonic Hedgehog should not limit the scope of the present invention.
When administering the siRNA targeting Sonic Hedgehog in a
sufficient amount, Shh translation and secretion will be inhibited,
thus preventing the activation of the Hedgehog signaling pathway to
the extent that ocular neovascularization will be prevented.
[0502] Preferably, the siRNA of the invention is ribonucleoside
phosphoramidites and a conventional DNA/RNA synthesizer. The siRNA
can be synthesized as two separate, complementary RNA molecules, or
as a single RNA molecule with two complementary regions. Commercial
suppliers of synthetic RNA molecules or synthesis reagents include
Proligo (Hamburg, Germany), Dharmacon Research (Lafayette, Colo.,
USA), Pierce Chemical (part of Perbio Science, Rockford, Ill.,
USA), Glen Research (Sterling, Va., USA), ChemGenes (Ashland,
Mass., USA) and Cruachem (Glasgow, UK).
3.4.7 Example 7
Administration of an siRNA targeting Smoothened to ROP Mice results
in Inhibition of Ocular Neovascularization
[0503] The siRNA targeting Smoothened is ordered from a commercial
vendor (Qiagen GmbH, Hilden, Germany) according to our
specifications: The siRNA duplex is represented in SEQ ID NO:17
(sense strand) and SEQ ID NO:18 (antisense strand), wherein the
sense and antisense RNA strands form an RNA duplex. The sense and
antisense strand are ordered pre-annealed.
[0504] To determine whether inhibition of the Shh signaling pathway
by administration of an siRNA targeting Smoothened is able to
reduce retinal (ocular) neovascularization, siRNA targeting Shh is
administrated to a mouse model of Retinopathy of Prematurity
(ROP).
[0505] C57/BL6 mice are used in this study. Mice from postnatal day
7 (P7) to P12 are exposed to hyperoxia (25% nitrogen, 75% oxygen).
At P12 mice return to room air until P17. The group of five mice
treated with siRNA targeting Smoothened receive a daily subretinal
injection (1 .mu.l) 100 mg/ml of siRNA targeting Smoothened
dissolved in Phosphate-Buffered Saline (PBS) from P12 to P17,
controls receive the vehicle alone.
[0506] On P17 angiography is performed: Mice are deeply
anesthetized with Avertin and perfused with a solution containing
fluorescein-dextran (50 mg/ml; 2 million molecular weight, Sigma,
St. Louis, Mo.) in Phosphate-Buffered Saline (PBS). Then, eyes are
enucleated and fixed in paraformaldehyde, and the retinas are
dissected, flat-mounted and viewed by fluorescence microscopy.
Robust inhibition of ocular neovascularization (decreased
neovascular tufts and vessel leakage) is observed in all eyes
injected with siRNA targeting Smoothened when compared with the
control eyes.
[0507] To summarize: A small interfering RNA targeting Smoothened
is injected into the eye of a mammalian subject. One variant of an
siRNA targeting human and mouse Smoothened is represented in SEQ ID
NO:17 (sense strand) and SEQ ID NO:18 (antisense strand), wherein
the sense and antisense RNA strands form an RNA duplex. The sense
and antisense strand can be purchased already annealed (i.e., in
duplex form) from commercial vendors such as Qiagen GmbH, Hilden,
Germany. Many different siRNAs can be designed to target
Smoothened. Theoretically, each 19 to 25 nucleotide stretch within
the Smoothened gene can be used as design template for an siRNA.
The exact method, design (e.g., stem-loop vs. annealed duplex) and
sequence of the siRNA targeting Smoothened should not limit the
scope of the present invention. When administering the siRNA
targeting Smoothened in a sufficient amount, Smoothened translation
and expression will be inhibited, thus preventing the activation of
the Hedgehog signaling pathway to the extent that ocular
neovascularization will be prevented.
[0508] Preferably, the siRNA of the invention is chemically
synthesized using appropriately protected ribonucleoside
phosphoramidites and a conventional DNA/RNA synthesizer. The siRNA
can be synthesized as two separate, complementary RNA molecules, or
as a single RNA molecule with two complementary regions. Commercial
suppliers of synthetic RNA molecules or synthesis reagents include
Proligo (Hamburg, Germany), Dharmacon Research (Lafayette, Colo.,
USA), Pierce Chemical (part of Perbio Science, Rockford, Ill.,
USA), Glen Research (Sterling, Va., USA), ChemGenes (Ashland,
Mass., USA) and Cruachem (Glasgow, UK).
3.4.8 Example 8
Administration of an siRNA targeting Gli1 to ROP Mice results in
Inhibition of Ocular Neovascularization
[0509] The siRNA targeting Gli1 is ordered from a commercial vendor
(Qiagen GmbH, Hilden, Germany) according to our specifications: The
siRNA duplex is represented in SEQ ID NO:19 (sense strand) and SEQ
ID NO:20 (antisense strand), wherein the sense and antisense RNA
strands form an RNA duplex. The sense and antisense strand are
ordered pre-annealed.
[0510] To determine whether inhibition of the Shh signaling pathway
by administration of an siRNA targeting Gli1 is able to reduce
retinal (ocular) neovascularization, siRNA targeting Shh is
administrated to a mouse model of Retinopathy of Prematurity
(ROP).
[0511] C57/BL6 mice are used in this study. Mice from postnatal day
7 (P7) to P12 are exposed to hyperoxia (25% nitrogen, 75% oxygen).
At P12 mice return to room air until P17. The group of five mice
treated with siRNA targeting Gli1 receive a daily subretinal
injection (1 .mu.l) of 100 mg/ml siRNA targeting Gli1 dissolved in
Phosphate-Buffered Saline (PBS) from P12 to P17, controls receive
the vehicle alone.
[0512] On P17 angiography is performed: Mice are deeply
anesthetized with Avertin and perfused with a solution containing
fluorescein-dextran (50 mg/ml; 2 million molecular weight, Sigma,
St. Louis, Mo.) in Phosphate-Buffered Saline (PBS). Then, eyes are
enucleated and fixed in paraformaldehyde, and the retinas are
dissected, flat-mounted and viewed by fluorescence microscopy.
Robust inhibition of ocular neovascularization (decreased
neovascular tufts and vessel leakage) is observed in all eyes
injected with siRNA targeting Gli1 when compared with the control
eyes.
[0513] To summarize: A small interfering RNA targeting Gli1 is
injected into the eye of a mammalian subject. One variant of an
siRNA targeting human and mouse Gli1 is represented in SEQ ID NO:19
(sense strand) and SEQ ID NO:20 (antisense strand), wherein the
sense and antisense RNA strands form an RNA duplex. The sense and
antisense strand can be purchased already annealed (i.e., in duplex
form) from commercial vendors such as Qiagen GmbH, Hilden, Germany.
Many different siRNAs can be designed to target Gli1.
Theoretically, each 19 to 25 nucleotide stretch within the Gli1
gene can be used as design template for an siRNA. The exact method,
design (e.g., stem-loop vs. annealed duplex) and sequence of the
siRNA targeting Gli1 should not limit the scope of the present
invention. When administering the siRNA targeting Gli1 in a
sufficient amount, Gli1 translation and expression will be
inhibited, thus preventing the activation of the Hedgehog signaling
pathway to the extent that ocular neovascularization will be
prevented.
[0514] Preferably, the siRNA of the invention are chemically
synthesized using appropriately protected ribonucleoside
phosphoramidites and a conventional DNA/RNA synthesizer. The siRNA
can be synthesized as two separate, complementary RNA molecules, or
as a single RNA molecule with two complementary regions. Commercial
suppliers of synthetic RNA molecules or synthesis reagents include
Proligo (Hamburg, Germany), Dharmacon Research (Lafayette, Colo.,
USA), Pierce Chemical (part of Perbio Science, Rockford, Ill.,
USA), Glen Research (Sterling, Va., USA), ChemGenes (Ashland,
Mass., USA) and Cruachem (Glasgow, UK).
3.4.9 Other Embodiments
[0515] In some embodiments, a monoclonal antibody against Hedgehog
is injected into the eye of a mammalian subject. In one subset of
said embodiments, the 5 .mu.l monoclonal antibody is injected in
the eye of a mouse to prevent choroidal neovascularization in the
CNV mouse model. Said antibody binds to Hedgehog and thus prevents
binding of Hedgehog to its receptor, Ptc-1. This in turn prevents
ocular neovascularization.
[0516] In yet other embodiments, a soluble form of Hip1 is injected
into the eye of a mammalian subject. One variant of a soluble form
of Hip1 is represented in SEQ ID NO:6. There are many different
ways of creating a soluble version of Hip1. All ways have in common
that the transmembrane domain of Hip1 is removed, which enables
secretion of Hip1. The exact method and sequence of the soluble
Hip1 variant should not limit the scope of the present invention.
Moreover, any homologous protein to the soluble form of Hip1 (SEQ
ID NO:6) should also fall within the scope of the present
invention. The soluble Hip1 variant is still able to bind and thus
sequester Hedgehog. In other words: The soluble Hip1 variant
competes with the Hedgehog receptor Ptc1 for Hedgehog binding. When
administering the soluble Hip1 variant in excess, the soluble Hip1
variants sequester a sufficient amount of free Hedgehog to prevent
activation of the Hedgehog signaling pathway to the extent that
ocular neovascularization will be prevented. In one subset of said
embodiments, the soluble variant of Hip1 is pegylated.
[0517] In yet other embodiments, a soluble variant of Hip1 is
expressed within the mammalian subject. This is achieved by
administering to said subject a gene transfer vector encoding said
soluble Hip1 variant. Thus, the soluble Hip1 variant is expressed
and secreted from cells of said mammalian subject. The secreted
Hip1 variants are then able to locally sequester Hedgehog, thus
preventing activation of the Hedgehog signaling pathway, which--in
the end--will prevent, inhibit, and/or reverse ocular
neovascularization.
3.4.10 Preferred Embodiment
[0518] In its preferred embodiment, cyclopamine is administered to
a mammalian subject in general, and to a human being in particular,
by subcutaneous injection at a concentration of 50 mg/kg. The drug
is dissolved in 95% of ethanol and then mixed 1:4 in triolein.
Alternatively, one of skill in the art will be able to formulate
cyclopamine also in eye drops.
[0519] Furthermore, in its preferred embodiment, the
Hedgehog-signaling interfering substance is administered to a
mammalian subject affected by the wet form of AMD.
[0520] Cyclopamine binds to and thus inhibits Smo activation, thus
interfering with the Hedgehog-signaling pathway. This in turn
inhibits, prevents and/or reverses ocular neovascularization, which
is involved in the pathology of wet AMD.
[0521] Although the present invention has been described with
reference to specific embodiments and examples, numerous
modifications and variations can be made and still the result will
come within the scope of the invention. No limitation with respect
to the specific embodiments disclosed herein is intended or should
be inferred.
3. PRIOR ART
[0522] U.S. Patent Applications TABLE-US-00002 U.S. patent
application Inventor: Title 20040110663 Dudek et al.: Hedgehog
antagonists, methods and uses related thereto 20040060568 Dudek et
al.: Hedgehog antagonists, methods and uses related thereto
20040048282 Smolyar: Regulation of human patched-like protein
20040030099 Smolyar: Regulation of human patched-like protein
20040023949 Baxter et al.: Mediators of hedgehog signaling
pathways, compositions and uses related thereto 20030022819 Ling et
al.: Angiogenesis-modulating compositions and use 20040161798
xxx
[0523] U.S. patents TABLE-US-00003 U.S. patent Inventor: Title U.S.
Pat. No. 6,713,065 Baron et al.: Methods of using hedgehog proteins
to modulate hematopoiesis and vascular growth U.S. Pat. No.
6,552,016 Baxter et al.: Mediators of hedgehog signaling pathways,
compositions and uses related thereto U.S. Pat. No. 6,686,388 Dudek
et al.: Regulators of the hedgehog pathway, compositions and uses
related thereto U.S. Pat. No. 6,683,108 Baxter et al.: Agonists of
hedgehog signaling pathways and uses related thereto U.S. Pat. No.
6,639,051 Wang et al.: Regulation of epithelial tissue by
hedgehog-like polypeptides, and formulations and uses related
thereto U.S. Pat. No. 6,605,700 Bumcrot: Human patched genes and
proteins, and uses related thereto U.S. Pat. No. 6,552,016 Baxter
et al.: Mediators of hedgehog signaling pathways, compositions and
uses related thereto U.S. Pat. No. 6,468,978 Esswein et al.: Active
hedgehog protein conjugate U.S. Pat. No. 6,309,879 Bumcrot: Human
patched genes and proteins, and uses related thereto U.S. Pat. No.
6,291,516 Dudek et al.: Regulators of the hedgehog pathway,
compositions and uses related thereto U.S. Pat. No. 6,432,970
Beachy et al.: Inhibitors of hedgehog signaling pathways,
compositions and uses related thereto U.S. Pat. No. 6,291,516 Dudek
et al.: Regulators of the hedgehog pathway, compositions and uses
related thereto U.S. Pat. No. 6,288,048 Beachy et al.: Cholesterol
and hedgehog signaling U.S. Pat. No. 6,057,091 Beachy et al.:
Method of identifying compounds affecting hedgehog cholesterol
transfer U.S. Pat. No. 5,891,875 Hipskind et al.: Morpholinyl
tachykinin receptor antagonists U.S. Pat. No. 5,260,210 Rubin et
al.: Blood-brain barrier model U.S. Pat. No. 5,795,756 Johnson et
al.: Method and compounds for the inhibition of adenylyl cyclase
U.S. Pat. No. 4,874,702 Fiers et al.: Vectors and methods for
making such vectors and for expressive cloned genes U.S. Pat. No.
5,258,498 Huston et al.: Polypeptide linkers for production of
biosynthetic proteins U.S. Pat. No. 5,482,858 Huston et al.:
Polypeptide linkers for production of biosynthetic proteins U.S.
Pat. No. 5,091,513 Huston et al.: Biosynthetic antibody binding
sites U.S. Pat. No. 4,946,778 Ladner et al.: Single polypeptide
chain binding molecules U.S. Pat. No. 5,969,108 McCafferty et al.:
Methods for producing members of specific binding pairs U.S. Pat.
No. 5,871,907 Winter et al.: Methods for producing members of
specific binding pairs U.S. Pat. No. 5,223,409 Ladner et al.:
Directed evolution of novel binding proteins U.S. Pat. No.
5,225,539 Winter: Recombinant altered antibodies and methods of
making altered antibodies U.S. Pat. No. 5,411,941 Grinna et al.:
Heterodimeric osteogenic factor U.S. Pat. No. 5,798,230 Bornkamm et
al.: Process for the preparation of human monoclonal antibodies and
their use U.S. Pat. No. 5,789,650 Lonberg et al.: Transgenic
non-human animals for producing heterologous antibodies U.S. Pat.
No. 5,693,762 Queen et al.: Humanized immunoglobulins U.S. Pat. No.
5,693,761 Queen et al.: Polynucleotides encoding improved humanized
immunoglobulins U.S. Pat. No. 5,585,089 Queen et al.: Humanized
immunoglobulins U.S. Pat. No. 5,530,101 Queen et al.: Humanized
immunoglobulins U.S. Pat. No. 5,176,996 Hogan et al.: Method for
making synthetic oligonucleotides which bind specifically to target
sites on duplex DNA molecules, by forming a colinear triplex, the
synthetic oligonucleotides and methods of use U.S. Pat. No.
5,264,564 Matteucci: Oligonucleotide analogs with novel linkages
U.S. Pat. No. 5,256,775 Froehler: Exonuclease-resistant
oligonucleotides U.S. Pat. No. 5,093,246 Cech et al.: RNA ribozyme
polymerases, dephosphorylases, restriction endoribonucleases and
methods U.S. Pat. No. 5,658,785 Johnson: Adeno-associated virus
materials and methods U.S. Pat. No. 5,139,941 Muzyczka et al.: AAV
transduction vectors
[0524] Other patents TABLE-US-00004 Patent Title WO9952534A1 Use of
steroidal alkaloid derivatives as inhibitors of hedgehog signaling
pathways
PRIOR ART PUBLICATIONS
Prior Art Publications
[0525] 1. Ingham, P. W., Signalling by hedgehog family proteins in
Drosophila and vertebrate development. Curr Opin Genet Dev, 1995.
5(4): p. 492-8. [0526] 2. Perrimon, N., Hedgehog and beyond. Cell,
1995. 80(4): p. 517-20. [0527] 3. Hammerschmidt, M., Brook, A., and
McMahon, A. P., The world according to hedgehog. Trends Genet,
1997. 13(1): p. 14-21. [0528] 4. Ericson, J., Muhr, J., Jessell, T.
M., and Edlund, T., Sonic hedgehog: a common signal for ventral
patterning along the rostrocaudal axis of the neural tube. Int J
Dev Biol, 1995. 39(5): p. 809-16. [0529] 5. Ericson, J., Muhr, J.,
Placzek, M., Lints, T., Jessell, T. M., and Edlund, T., Sonic
hedgehog induces the differentiation of ventral forebrain neurons:
a common signal for ventral patterning within the neural tube.
Cell, 1995. 81(5): p. 747-56. [0530] 6. Roberts, D. J., Johnson, R.
L., Burke, A. C., Nelson, C. E., Morgan, B. A., and Tabin, C.,
Sonic hedgehog is an endodermal signal inducing Bmp-4 and Hox genes
during induction and regionalization of the chick hindgut.
Development, 1995. 121(10): p. 3163-74. [0531] 7. Apelqvist, A.,
Ahlgren, U., and Edlund, H., Sonic hedgehog directs specialised
mesoderm differentiation in the intestine and pancreas. Curr Biol,
1997. 7(10): p. 801-4. [0532] 8. Dodd, J., Jessell, T. M., and
Placzek, M., The when and where of floor plate induction. Science,
1998. 282(5394): p. 1654-7. [0533] 9. Dockter, J. L., Sclerotome
induction and differentiation. Curr Top Dev Biol, 2000. 48: p.
77-127. [0534] 10. Bitgood, M. J. and McMahon, A. P., Hedgehog and
Bmp genes are coexpressed at many diverse sites of cell-cell
interaction in the mouse embryo. Dev Biol, 1995. 172(1): p. 126-38.
[0535] 11. Chiang, C., Litingtung, Y., Lee, E., Young, K. E.,
Corden, J. L., Westphal, H., and Beachy, P. A., Cyclopia and
defective axial patterning in mice lacking Sonic hedgehog gene
function. Nature, 1996. 383(6599): p. 407-13. [0536] 12.
Litingtung, Y., Lei, L., Westphal, H., and Chiang, C., Sonic
hedgehog is essential to foregut development. Nat Genet, 1998.
20(1): p. 58-61. [0537] 13. St-Jacques, B., Dassule, H. R.,
Karavanova, I., Botchkarev, V. A., Li, J., Danielian, P. S.,
McMahon, J. A., Lewis, P. M., Paus, R., and McMahon, A. P., Sonic
hedgehog signaling is essential for hair development. Curr Biol,
1998. 8(19): p. 1058-68. [0538] 14. St-Jacques, B., Hammerschmidt,
M., and McMahon, A. P., Indian hedgehog signaling regulates
proliferation and differentiation of chondrocytes and is essential
for bone formation. Genes Dev, 1999. 13(16): p. 2072-86. [0539] 15.
Karp, S. J., Schipani, E., St-Jacques, B., Hunzelman, J.,
Kronenberg, H., and McMahon, A. P., Indian hedgehog coordinates
endochondral bone growth and morphogenesis via parathyroid hormone
related-protein-dependent and-independent pathways. Development,
2000. 127(3): p. 543-8. [0540] 16. Bitgood, M. J., Shen, L., and
McMahon, A. P., Sertoli cell signaling by Desert hedgehog regulates
the male germline. Curr Biol, 1996. 6(3): p. 298-304. [0541] 17.
Parmantier, E., Lynn, B., Lawson, D., Turmaine, M., Namini, S. S.,
Chakrabarti, L., McMahon, A. P., Jessen, K. R., and Mirsky, R.,
Schwann cell-derived Desert hedgehog controls the development of
peripheral nerve sheaths. Neuron, 1999. 23(4): p. 713-24. [0542]
18. Ding, Q., Fukami, S., Meng, X., Nishizaki, Y., Zhang, X.,
Sasaki, H., Dlugosz, A., Nakafuku, M., and Hui, C., Mouse
suppressor of fused is a negative regulator of sonic hedgehog
signaling and alters the subcellular distribution of Gli1. Curr
Biol, 1999. 9(19): p. 1119-22. [0543] 19. Murone, M., Rosenthal,
A., and de Sauvage, F. J., Hedgehog signal transduction: from flies
to vertebrates. Exp Cell Res, 1999. 253(1): p. 25-33. [0544] 20.
Murone, M., Rosenthal, A., and de Sauvage, F. J., Sonic hedgehog
signaling by the patched-smoothened receptor complex. Curr Biol,
1999. 9(2): p. 76-84. [0545] 21. Pearse, R. V., 2nd, Collier, L.
S., Scott, M. P., and Tabin, C. J., Vertebrate homologs of
Drosophila suppressor of fused interact with the gli family of
transcriptional regulators. Dev Biol, 1999. 212(2): p. 323-36.
[0546] 22. Stone, D. M., Murone, M., Luoh, S., Ye, W., Armanini, M.
P., Gurney, A., Phillips, H., Brush, J., Goddard, A., de Sauvage,
F. J., and Rosenthal, A., Characterization of the human suppressor
of fused, a negative regulator of the zinc-finger transcription
factor Gli. J Cell Sci, 1999. 112 (Pt 23): p. 4437-48. [0547] 23.
Hynes, M., Ye, W., Wang, K., Stone, D., Murone, M., Sauvage, F.,
and Rosenthal, A., The seven-transmembrane receptor smoothened
cell-autonomously induces multiple ventral cell types. Nat
Neurosci, 2000. 3(1): p. 41-6. [0548] 24. Bai, C. B., Auerbach, W.,
Lee, J. S., Stephen, D., and Joyner, A. L., Gli2, but not Gli1, is
required for initial Shh signaling and ectopic activation of the
Shh pathway. Development, 2002. 129(20): p. 4753-61. [0549] 25.
Bai, C. B. and Joyner, A. L., Gli1 can rescue the in vivo function
of Gli2. Development, 2001. 128(24): p. 5161-72. [0550] 26.
Brewster, R., Lee, J., and Ruiz i Altaba, A., Gli/Zic factors
pattern the neural plate by defining domains of cell
differentiation. Nature, 1998. 393(6685): p. 579-83. [0551] 27.
Brewster, R., Mullor, J. L., and Ruiz i Altaba, A., Gli2 functions
in FGF signaling during antero-posterior patterning. Development,
2000. 127(20): p. 4395-405. [0552] 28. Buttitta, L., Mo, R., Hui,
C. C., and Fan, C. M., Interplays of Gli2 and Gli3 and their
requirement in mediating Shh-dependent sclerotome induction.
Development, 2003. 130(25): p. 6233-43. [0553] 29. Dunaeva, M.,
Michelson, P., Kogerman, P., and Toftgard, R., Characterization of
the physical interaction of Gli proteins with SUFU proteins. J Biol
Chem, 2003. 278(7): p. 5116-22. [0554] 30. Kalderon, D., Hedgehog
signaling: Costal-2 bridges the transduction gap. Curr Biol, 2004.
14(2): p. R67-9. [0555] 31; Lum, L., Zhang, C., Oh, S., Mann, R.
K., von Kessler, D. P., Taipale, J., Weis-Garcia, F., Gong, R.,
Wang, B., and Beachy, P. A., Hedgehog signal transduction via
Smoothened association with a cytoplasmic complex scaffolded by the
atypical kinesin, Costal-2. Mol Cell, 2003. 12(5): p. 1261-74.
[0556] 32. Merchant, M., Vajdos, F. F., Ultsch, M., Maun, H. R.,
Wendt, U., Cannon, J., Desmarais, W., Lazarus, R. A., de Vos, A.
M., and de Sauvage, F. J., Suppressor of fused regulates Gli
activity through a dual binding mechanism. Mol Cell Biol, 2004.
24(19): p. 8627-41. [0557] 33. Monnier, V., Dussillol, F., Alves,
G., Lamour-Isnard, C., and Plessis, A., Suppressor of fused links
fused and Cubitus interruptus on the hedgehog signalling pathway.
Curr Biol, 1998. 8(10): p. 583-6. [0558] 34. Bak, M., Hansen, C.,
Tommerup, N., and Larsen, L. A., The Hedgehog signaling
pathway--implications for drug targets in cancer and
neurodegenerative disorders. Pharmacogenomics, 2003. 4(4): p.
411-29. [0559] 35. Bale, A. E., Hedgehog signaling and human
disease. Annu Rev Genomics Hum Genet, 2002. 3: p. 47-65. [0560] 36.
Matise, M. P. and Joyner, A. L., Gli genes in development and
cancer. Oncogene, 1999. 18(55): p. 7852-9. [0561] 37. Taipale, J.
and Beachy, P. A., The Hedgehog and Wnt signalling pathways in
cancer. Nature, 2001. 411(6835): p. 349-54. [0562] 38. Hebrok, M.,
Hedgehog signaling in pancreas development. Mech Dev, 2003. 120(1):
p. 45-57. [0563] 39. Kuenzli, S., Sorg, O., and Saurat, J. H.,
Cyclopamine, hedgehog and psoriasis. Dermatology, 2004. 209(2): p.
81-3. [0564] 40. Tas, S, and Avci, O., Rapid clearance of psoriatic
skin lesions induced by topical cyclopamine. A preliminary proof of
concept study. Dermatology, 2004. 209(2): p. 126-31. [0565] 41.
Duman-Scheel, M., Weng, L., Xin, S., and Du, W., Hedgehog regulates
cell growth and proliferation by inducing Cyclin D and Cyclin E.
Nature, 2002. 417(6886): p. 299-304. [0566] 42. Pola, R., Ling, L.
E., Silver, M., Corbley, M. J., Kearney, M., Blake Pepinsky, R.,
Shapiro, R., Taylor, F. R., Baker, D. P., Asahara, T., and Isner,
J. M., The morphogen Sonic hedgehog is an indirect angiogenic agent
upregulating two families of angiogenic growth factors. Nat Med,
2001. 7(6): p. 706-11. [0567] 43. Chen, J. K., Taipale, J., Young,
K. E., Maiti, T., and Beachy, P. A., Small molecule modulation of
Smoothened activity. Proc Natl Acad Sci USA, 2002. 99(22): p.
14071-6. [0568] 44. Frank-Kamenetsky, M., Zhang, X. M., Bottega,
S., Guicherit, O., Wichterle, H., Dudek, H., Bumcrot, D., Wang, F.
Y., Jones, S., Shulok, J., Rubin, L. L., and Porter, J. A.,
Small-molecule modulators of Hedgehog signaling: identification and
characterization of Smoothened agonists and antagonists. J Biol,
2002. 1(2): p. 10. [0569] 45. Bijlsma, M. F., Spek, C. A., and
Peppelenbosch, M. P., Hedgehog: an unusual signal transducer.
Bioessays, 2004. 26(4): p. 387-94. [0570] 46. Gaffield, W.,
Incardona, J. P., Kapur, R. P., and Roelink, H., A looking glass
perspective: thalidomide and cyclopamine. Cell Mol Biol
(Noisy-le-grand), 1999. 45(5): p. 579-88. [0571] 47. Pasca di
Magliano, M. and Hebrok, M., Hedgehog signalling in cancer
formation and maintenance. Nat Rev Cancer, 2003. 3(12): p. 903-11.
[0572] 48. Kim, S. K. and Melton, D. A., Pancreas development is
promoted by cyclopamine, a hedgehog signaling inhibitor. Proc Natl
Acad Sci USA, 1998. 95(22): p. 13036-41. [0573] 49. Incardona, J.
P., Gaffield, W., Kapur, R. P., and Roelink, H., The teratogenic
Veratrum alkaloid cyclopamine inhibits sonic hedgehog signal
transduction. Development, 1998. 125(18): p. 3553-62. [0574] 50.
Vokes, S. A., Yatskievych, T. A., Heimark, R. L., McMahon, J.,
McMahon, A. P., Antin, P. B., and Krieg, P. A., Hedgehog signaling
is essential for endothelial tube formation during vasculogenesis.
Development, 2004. 131(17): p. 4371-80. [0575] 51. Olsen, C. L.,
Hsu, P. P., Glienke, J., Rubanyi, G. M., and Brooks, A. R.,
Hedgehog-interacting protein is highly expressed in endothelial
cells but down-regulated during angiogenesis and in several human
tumors. BMC Cancer, 2004. 4(1): p. 43. [0576] 52. Kubo, M.,
Nakamura, M., Tasaki, A., Yamanaka, N., Nakashima, H., Nomura, M.,
Kuroki, S., and Katano, M., Hedgehog signaling pathway is a new
therapeutic target for patients with breast cancer. Cancer Res,
2004. 64(17): p. 6071-4. [0577] 53. Chuang, P. T., Kawcak, T., and
McMahon, A. P., Feedback control of mammalian Hedgehog signaling by
the Hedgehog-binding protein, Hip1, modulates Fgf signaling during
branching morphogenesis of the lung. Genes Dev, 2003. 17(3): p.
342-7. [0578] 54. Chuang, P. T. and McMahon, A. P., Vertebrate
Hedgehog signalling modulated by induction of a Hedgehog-binding
protein. Nature, 1999. 397(6720): p. 617-21. [0579] 55. Zeng, X.,
Goetz, J. A., Suber, L. M., Scott, W. J., Jr., Schreiner, C. M.,
and Robbins, D. J., A freely diffusible form of Sonic hedgehog
mediates long-range signalling. Nature, 2001. 411(6838): p. 716-20.
[0580] 56. Coulombe, J., Traiffort, E., Loulier, K., Faure, H., and
Ruat, M., Hedgehog interacting protein in the mature brain:
membrane-associated and soluble forms. Mol Cell Neurosci, 2004.
25(2): p. 323-33. [0581] 57. Izraeli, S, and Rechavi, G., Molecular
medicine--an overview. Isr Med Assoc J, 2002. 4(8): p. 638-40.
[0582] 58. Mohr, L. and Geissler, M., [Gene therapy: new
developments]. Schweiz Rundsch Med Prax, 2002. 91(51-52): p.
2227-35 [0583] 59. Graham, F. L. and van der Eb, A. J., A new
technique for the assay of infectivity of human adenovirus 5 DNA.
Virology, 1973. 52(2): p. 456-67. [0584] 60. Graham, F. L. and van
der Eb, A. J., Transformation of rat cells by DNA of human
adenovirus 5. Virology, 1973. 54(2): p. 536-9. [0585] 61. Chu, G.
and Sharp, P. A., SV40 DNA transfection of cells in suspension:
analysis of efficiency of transcription and translation of
T-antigen. Gene, 1981. 13(2): p. 197-202. [0586] 62. Fire, A., Xu,
S., Montgomery, M. K., Kostas, S. A., Driver, S. E., and Mello, C.
C., Potent and specific genetic interference by double-stranded RNA
in Caenorhabditis elegans. Nature, 1998. 391(6669): p. 806-11.
[0587] 63. Bosher, J. M. and Labouesse, M., RNA interference:
genetic wand and genetic watchdog. Nat Cell Biol, 2000. 2(2): p.
E31-6. [0588] 64. Sharp, P. A., RNA interference--2001. Genes Dev,
2001. 15(5): p. 485-90. [0589] 65. Hammond, S. M., Caudy, A. A.,
and Hannon, G. J., Post-transcriptional gene silencing by
double-stranded RNA. Nat Rev Genet, 2001. 2(2): p. 110-9. [0590]
66. Zamore, P. D., RNA interference: listening to the sound of
silence. Nat Struct Biol, 2001. 8(9): p. 746-50. [0591] 67. Moss,
E. G., RNA interference: it's a small RNA world. Curr Biol, 2001.
11(19): p. R772-5. [0592] 68. Fjose, A., Ellingsen, S., Wargelius,
A., and Seo, H. C., RNA interference: mechanisms and applications.
Biotechnol Annu Rev, 2001. 7: p. 31-57. [0593] 69. Tuschl, T., RNA
interference and small interfering RNAs. Chembiochem, 2001. 2(4):
p. 239-45. [0594] 70. Cullen, B. R., RNA interference: antiviral
defense and genetic tool. Nat Immunol, 2002. 3(7): p. 597-9. [0595]
71. Hannon, G. J., RNA interference. Nature, 2002. 418(6894): p.
244-51. [0596] 72. Kitabwalla, M. and Ruprecht, R. M., RNA
interference--a new weapon against HIV and beyond. N Engl J Med,
2002. 347(17): p. 1364-7. [0597] 73. McManus, M. T. and Sharp, P.
A., Gene silencing in mammals by small interfering RNAs. Nat Rev
Genet, 2002. 3(10): p. 737-47. [0598] 74. Elbashir, S. M.,
Harborth, J., Lendeckel, W., Yalcin, A., Weber, K., and Tuschl, T.,
Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured
mammalian cells. Nature, 2001. 411(6836): p. 494-8. [0599] 75.
Caplen, N. J., Parrish, S., Imani, F., Fire, A., and Morgan, R. A.,
Specific inhibition of gene expression by small double-stranded
RNAs in invertebrate and vertebrate systems. Proc Natl Acad Sci
USA, 2001. 98(17): p. 9742-7. [0600] 76. Paddison, P. J., Caudy, A.
A., Bernstein, E., Hannon, G. J., and Conklin, D. S., Short hairpin
RNAs (shRNAs) induce sequence-specific silencing in mammalian
cells. Genes Dev, 2002. 16(8): p. 948-58. [0601] 77. Kusser, W.,
Chemically modified nucleic acid aptamers for in vitro selections:
evolving evolution. J Biotechnol, 2000. 74(1): p. 27-38. [0602] 78.
Sun, S., Technology evaluation: SELEX, Gilead Sciences Inc. Curr
Opin Mol Ther, 2000. 2(1): p. 100-5. [0603] 79. Brody, E. N. and
Gold, L., Aptamers as therapeutic and diagnostic agents. J
Biotechnol, 2000. 74(1): p. 5-13. [0604] 80. Kramvis, A., Bukofzer,
S., and Kew, M. C., Comparison of hepatitis B virus DNA extractions
from serum by the QIAamp blood kit, GeneReleaser, and the
phenol-chloroform method. J Clin Microbiol, 1996. 34(11): p.
2731-3. [0605] 81. Cuenoud, B. and Szostak, J. W., A DNA
metalloenzyme with DNA ligase activity. Nature, 1995. 375(6532): p.
611-4. [0606] 82. Studier, F. W., Rosenberg, A. H., Dunn, J. J.,
and Dubendorff, J. W., Use of T7 RNA polymerase to direct
expression of cloned genes. Methods Enzymol, 1990. 185: p. 60-89.
[0607] 83. Benoist, C. and Chambon, P., In vivo sequence
requirements of the SV40 early promotor region. Nature, 1981.
290(5804): p. 304-10.
[0608] 84. Yamamoto, T., de Crombrugghe, B., and Pastan, I.,
Identification of a functional promoter in the long terminal repeat
of Rous sarcoma virus. Cell, 1980. 22(3): p. 787-97. [0609] 85.
Wagner, E. F., Stewart, T. A., and Mintz, B., The human beta-globin
gene and a functional viral thymidine kinase gene in developing
mice. Proc Natl Acad Sci USA, 1981. 78(8): p. 5016-20. [0610] 86.
Brinster, R. L., Chen, H. Y., Warren, R., Sarthy, A., and Palmiter,
R. D., Regulation of metallothionein--thymidine kinase fusion
plasmids injected into mouse eggs. Nature, 1982. 296(5852): p.
39-42. [0611] 87. Herrera-Estrella, L., Van den Broeck, G.,
Maenhaut, R., Van Montagu, M., Schell, J., Timko, M., and Cashmore,
A., Light-inducible and chloroplast-associated expression of a
chimaeric gene introduced into Nicotiana tabacum using a Ti plasmid
vector. Nature, 1984. 310(5973): p. 115-20. [0612] 88. Gardner, R.
C., Howarth, A. J., Hahn, P., Brown-Luedi, M., Shepherd, R. J., and
Messing, J., The complete nucleotide sequence of an infectious
clone of cauliflower mosaic virus by M13 mp 7 shotgun sequencing.
Nucleic Acids Res, 1981. 9(12): p. 2871-88. [0613] 89. Swift, G.
H., Hammer, R. E., MacDonald, R. J., and Brinster, R. L.,
Tissue-specific expression of the rat pancreatic elastase I gene in
transgenic mice. Cell, 1984. 38(3): p. 639-46. [0614] 90.
MacDonald, R. J., Hammer, R. E., Swift, G. H., Ornitz, D. M.,
Davis, B. P., Palmiter, R. D., and Brinster, R. L., Tissue-specific
expression of pancreatic genes in transgenic mice. Ann NY Acad Sci,
1986. 478: p. 131-46. [0615] 91. Hanahan, D., Heritable formation
of pancreatic beta-cell tumours in transgenic mice expressing
recombinant insulin/simian virus 40 oncogenes. Nature, 1985.
315(6015): p. 115-22. [0616] 92. Grosschedl, R., Weaver, D.,
Baltimore, D., and Costantini, F., Introduction of a mu
immunoglobulin gene into the mouse germ line: specific expression
in lymphoid cells and synthesis of functional antibody. Cell, 1984.
38(3): p. 647-58. [0617] 93. Adams, J. M., Harris, A. W., Pinkert,
C. A., Corcoran, L. M., Alexander, W. S., Cory, S., Palmiter, R.
D., and Brinster, R. L., The c-myc oncogene driven by
immunoglobulin enhancers induces lymphoid malignancy in transgenic
mice. Nature, 1985. 318(6046): p. 533-8. [0618] 94. Alexander, W.
S., Schrader, J. W., and Adams, J. M., Expression of the c-myc
oncogene under control of an immunoglobulin enhancer in E mu-myc
transgenic mice. Mol Cell Biol, 1987. 7(4): p. 1436-44. [0619] 95.
Boshart, M., Weber, F., Jahn, G., Dorsch-Hasler, K., Fleckenstein,
B., and Schaffner, W., A very strong enhancer is located upstream
of an immediate early gene of human cytomegalovirus. Cell, 1985.
41(2): p. 521-30. [0620] 96. Leder, A., Pattengale, P. K., Kuo, A.,
Stewart, T. A., and Leder, P., Consequences of widespread
deregulation of the c-myc gene in transgenic mice: multiple
neoplasms and normal development. Cell, 1986. 45(4): p. 485-95.
[0621] 97. Pinkert, C. A., Ornitz, D. M., Brinster, R. L., and
Palmiter, R. D., An albumin enhancer located 10 kb upstream
functions along with its promoter to direct efficient,
liver-specific expression in transgenic mice. Genes Dev, 1987.
1(3): p. 268-76. [0622] 98. Krumlauf, R., Hammer, R. E., Tilghman,
S. M., and Brinster, R. L., Developmental regulation of
.alpha.-fetoprotein genes in transgenic mice. Mol Cell Biol, 1985.
5(7): p. 1639-48. [0623] 99. Hammer, R. E., Krumlauf, R., Camper,
S. A., Brinster, R. L., and Tilghman, S. M., Diversity of
.alpha.-fetoprotein gene expression in mice is generated by a
combination of separate enhancer elements. Science, 1987.
235(4784): p. 53-8. [0624] 100. Kelsey, G. D., Povey, S., Bygrave,
A. E., and Lovell-Badge, R. H., Species- and tissue-specific
expression of human alpha. 1-antitrypsin in transgenic mice. Genes
Dev, 1987. 1(2): p. 161-71. [0625] 101. Kollias, G., Wrighton, N.,
Hurst, J., and Grosveld, F., Regulated expression of human A
gamma-, beta-, and hybrid gamma beta-globin genes in transgenic
mice: manipulation of the developmental expression patterns. Cell,
1986. 46(1): p. 89-94. [0626] 102. Readhead, C., Popko, B.,
Takahashi, N., Shine, H. D., Saavedra, R. A., Sidman, R. L., and
Hood, L., Expression of a myelin basic protein gene in transgenic
shiverer mice: correction of the dysmyelinating phenotype. Cell,
1987. 48(4): p. 703-12. [0627] 103. Shani, M., Tissue-specific
expression of rat myosin light-chain 2 gene in transgenic mice.
Nature, 1985. 314(6008): p. 283-6. [0628] 104. Mason, A. J.,
Hayflick, J. S., Zoeller, R. T., Young, W. S., 3rd, Phillips, H.
S., Nikolics, K., and Seeburg, P. H., A deletion truncating the
gonadotropin-releasing hormone gene is responsible for hypogonadism
in the hpg mouse. Science, 1986. 234(4782): p. 1366-71. [0629] 105.
Boerner, P., Lafond, R., Lu, W. Z., Brams, P., and Royston, I.,
Production of antigen-specific human monoclonal antibodies from in
vitro-primed human splenocytes. J Immunol, 1991. 147(1): p. 86-95.
[0630] 106. Persson, M. A., Caothien, R. H., and Burton, D. R.,
Generation of diverse high-affinity human monoclonal antibodies by
repertoire cloning. Proc Natl Acad Sci USA, 1991. 88(6): p. 2432-6.
[0631] 107. Huang, C. and Stollar, B. D., Construction of
representative immunoglobulin variable region cDNA libraries from
human peripheral blood lymphocytes without in vitro stimulation. J
Immunol Methods, 1991. 141(2): p. 227-36. [0632] 108. Vaughan, T.
J., Osbourn, J. K., and Tempest, P. R., Human antibodies by design.
Nat Biotechnol, 1998. 16(6): p. 535-9. [0633] 109. Vaughan, T. J.,
Williams, A. J., Pritchard, K., Osbourn, J. K., Pope, A. R.,
Earnshaw, J. C., McCafferty, J., Hodits, R. A., Wilton, J., and
Johnson, K. S., Human antibodies with sub-nanomolar affinities
isolated from a large non-immunized phage display library. Nat
Biotechnol, 1996. 14(3): p. 309-14. [0634] 110. Vaughan, C. K. and
Sollazzo, M., Of minibody, camel and bacteriophage. Comb Chem High
Throughput Screen, 2001. 4(5): p. 417-30. [0635] 111. Riechmann,
L., Foote, J., and Winter, G., Expression of an antibody Fv
fragment in myeloma cells. J Mol Biol, 1988. 203(3): p. 825-8.
[0636] 112. Riechmann, L., Clark, M., Waldmann, H., and Winter, G.,
Reshaping human antibodies for therapy. Nature, 1988. 332(6162): p.
323-7. [0637] 113. Verhoeyen, M. and Riechmann, L., Engineering of
antibodies. Bioessays, 1988. 8(2): p. 74-8. [0638] 114. van der
Krol, A. R., Mol, J. N., and Stuitje, A. R., Modulation of
eukaryotic gene expression by complementary RNA or DNA sequences.
Biotechniques, 1988. 6(10): p. 958-76. [0639] 115. Stein, C. A. and
Cohen, J. S., Oligodeoxynucleotides as inhibitors of gene
expression: a review. Cancer Res, 1988. 48(10): p. 2659-68. [0640]
116. Wagner, R. W., Gene inhibition using antisense
oligodeoxynucleotides. Nature, 1994. 372(6504): p. 333-5. [0641]
117. Letsinger, R. L., Zhang, G. R., Sun, D. K., Ikeuchi, T., and
Sarin, P. S., Cholesteryl-conjugated oligonucleotides: synthesis,
properties, and activity as inhibitors of replication of human
immunodeficiency virus in cell culture. Proc Natl Acad Sci USA,
1989. 86(17): p. 6553-6. [0642] 118. Lemaitre, M., Bayard, B., and
Lebleu, B., Specific antiviral activity of a
poly(L-lysine)-conjugated oligodeoxyribonucleotide sequence
complementary to vesicular stomatitis virus N protein mRNA
initiation site. Proc Natl Acad Sci USA, 1987. 84(3): p. 648-52.
[0643] 119. Zon, G., Oligonucleotide analogues as potential
chemotherapeutic agents. Pharm Res, 1988. 5(9): p. 539-49. [0644]
120. Perry-O'Keefe, H., Yao, X. W., Coull, J. M., Fuchs, M., and
Egholm, M., Peptide nucleic acid pre-gel hybridization: an
alternative to southern hybridization. Proc Natl Acad Sci USA,
1996. 93(25): p. 14670-5. [0645] 121. Egholm, M., Buchardt, O.,
Christensen, L., Behrens, C., Freier, S. M., Driver, D. A., Berg,
R. H., Kim, S. K., Norden, B., and Nielsen, P. E., PNA hybridizes
to complementary oligonucleotides obeying the Watson-Crick
hydrogen-bonding rules. Nature, 1993. 365(6446): p. 566-8. [0646]
122. Gautier, C., Morvan, F., Rayner, B., Huynh-Dinh, T., Igolen,
J., Imbach, J. L., Paoletti, C., and Paoletti, J., Alpha-DNA. IV:
Alpha-anomeric and beta-anomeric tetrathymidylates covalently
linked to intercalating oxazolopyridocarbazole. Synthesis,
physicochemical properties and poly (rA) binding. Nucleic Acids
Res, 1987. 15(16): p. 6625-41. [0647] 123. Inoue, H., Hayase, Y.,
Imura, A., Iwai, S., Miura, K., and Ohtsuka, E., Synthesis and
hybridization studies on two complementary
nona(2'-O-methyl)ribonucleotides. Nucleic Acids Res, 1987. 15(15):
p. 6131-48. [0648] 124. Loke, S. L., Stein, C., Zhang, X., Avigan,
M., Cohen, J., and Neckers, L. M., Delivery of c-myc antisense
phosphorothioate oligodeoxynucleotides to hematopoietic cells in
culture by liposome fusion: specific reduction in c-myc protein
expression correlates with inhibition of cell growth and DNA
synthesis. Curr Top Microbiol Immunol, 1988. 141: p. 282-9. [0649]
125. Stein, C. A., Mori, K., Loke, S. L., Subasinghe, C.,
Shinozuka, K., Cohen, J. S., and Neckers, L. M., Phosphorothioate
and normal oligodeoxyribonucleotides with 5'-linked acridine:
characterization and preliminary kinetics of cellular uptake. Gene,
1988. 72(1-2): p. 333-41. [0650] 126. Stein, C. A., Subasinghe, C.,
Shinozuka, K., and Cohen, J. S., Physicochemical properties of
phosphorothioate oligodeoxynucleotides. Nucleic Acids Res, 1988.
16(8): p. 3209-21. [0651] 127. Matsukura, M., Zon, G., Shinozuka,
K., Stein, C. A., Mitsuya, H., Cohen, J. S., and Broder, S.,
Synthesis of phosphorothioate analogues of
oligodeoxyribonucleotides and their antiviral activity against
human immunodeficiency virus (HIV). Gene, 1988. 72(1-2): p. 343-7.
[0652] 128. Sarin, P. S., Agrawal, S., Civeira, M. P., Goodchild,
J., Ikeuchi, T., and Zamecnik, P. C., Inhibition of acquired
immunodeficiency syndrome virus by oligodeoxynucleoside
methylphosphonates. Proc Natl Acad Sci USA, 1988. 85(20): p.
7448-51. [0653] 129. Sarver, N., Cantin, E. M., Chang, P. S., Zaia,
J. A., Ladne, P. A., Stephens, D. A., and Rossi, J. J., Ribozymes
as potential anti-HIV-1 therapeutic agents. Science, 1990.
247(4947): p. 1222-5. [0654] 130. Haseloff, J. and Gerlach, W. L.,
Simple RNA enzymes with new and highly specific endoribonuclease
activities. Nature, 1988. 334(6183): p. 585-91. [0655] 131. Zaug,
A. J. and Cech, T. R., The intervening sequence RNA of Tetrahymena
is an enzyme. Science, 1986. 231(4737): p. 470-5. [0656] 132. Zaug,
A. J., Kent, J. R., and Cech, T. R., Reactions of the intervening
sequence of the Tetrahymena ribosomal ribonucleic acid precursor:
pH dependence of cyclization and site-specific hydrolysis.
Biochemistry, 1985. 24(22): p. 6211-8. [0657] 133. Zaug, A. J.,
Grabowski, P. J., and Cech, T. R., Autocatalytic cyclization of an
excised intervening sequence RNA is a cleavage-ligation reaction.
Nature, 1983. 301(5901): p. 578-83. [0658] 134. Cech, T. R., Zaug,
A. J., and Grabowski, P. J., In vitro splicing of the ribosomal RNA
precursor of Tetrahymena: involvement of a guanosine nucleotide in
the excision of the intervening sequence. Cell, 1981. 27(3 Pt 2):
p. 487-96. [0659] 135. Zaug, A. J. and Cech, T. R., The intervening
sequence excised from the ribosomal RNA precursor of Tetrahymena
contains a 5-terminal guanosine residue not encoded by the DNA.
Nucleic Acids Res, 1982. 10(9): p. 2823-38. [0660] 136. Been, M. D.
and Cech, T. R., One binding site determines sequence specificity
of Tetrahymena pre-rRNA self-splicing, trans-splicing, and RNA
enzyme activity. Cell, 1986 47 (2): p. 207-16. [0661] 137. Helene,
C., The anti-gene strategy: control of gene expression by
triplex-forming-oligonucleotides. Anticancer Drug Des, 1991. 6(6):
p. 569-84. [0662] 138. Maher, L. J., 3rd, DNA triple-helix
formation: an approach to artificial gene repressors? Bioessays,
1992. 14(12): p. 807-15. [0663] 139. Heidenreich, O., Gryaznov, S.,
and Nerenberg, M., RNase H-independent antisense activity of
oligonucleotide N3'.fwdarw.P5' phosphoramidates. Nucleic Acids Res,
1997. 25(4): p. 776-80. [0664] 140. Wilson, W. D., Mizan, S.,
Tanious, F. A., Yao, S., and Zon, G., The interaction of
intercalators and groove-binding agents with DNA triple-helical
structures: the influence of ligand structure, DNA backbone
modifications and sequence. J Mol Recognit, 1994. 7(2): p. 89-98.
[0665] 141. Chen, J. K., Schultz, R. G., Lloyd, D. H., and
Gryaznov, S. M., Synthesis of oligodeoxyribonucleotide
N3'.fwdarw.P5' phosphoramidates. Nucleic Acids Res, 1995. 23(14):
p. 2661-8. [0666] 142. Elbashir, S. M., Martinez, J., Patkaniowska,
A., Lendeckel, W., and Tuschl, T., Functional anatomy of siRNAs for
mediating efficient RNAi in Drosophila melanogaster embryo lysate.
Embo J, 2001. 20(23): p. 6877-88. [0667] 143. McCaffrey, A. P.,
Meuse, L., Pham, T. T., Conklin, D. S., Hannon, G. J., and Kay, M.
A., RNA interference in adult mice. Nature, 2002. 418(6893): p.
38-9. [0668] 144. McManus, M. T., Petersen, C. P., Haines, B. B.,
Chen, J., and Sharp, P. A., Gene silencing using micro-RNA designed
hairpins. Rna, 2002. 8(6): p. 842-50. [0669] 145. Yu, J. Y.,
DeRuiter, S. L., and Turner, D. L., RNA interference by expression
of short-interfering RNAs and hairpin RNAs in mammalian cells. Proc
Natl Acad Sci USA, 2002. 99(9): p. 6047-52. [0670] 146. Gold, L.,
Brown, D., He, Y., Shtatland, T., Singer, B. S., and Wu, Y., From
oligonucleotide shapes to genomic SELEX: novel biological
regulatory loops. Proc Natl Acad Sci USA, 1997. 94(1): p. 59-64.
[0671] 147. Gold, L., The SELEX process: a surprising source of
therapeutic and diagnostic compounds. Harvey Lect, 1995. 91: p.
47-57. [0672] 148. Klug, S. J. and Famulok, M., All you wanted to
know about SELEX. Mol Biol Rep, 1994. 20(2): p. 97-107. [0673] 149.
Berge, S. M., Bighley, L. D., and Monkhouse, D. C., Pharmaceutical
salts. J Pharm Sci, 1977. 66(1): p. 1-19.
Sequence CWU 1
1
2012103DNAMus musculusCDS(1)..(2103)Hip1 cDNA (murine) 1atg ctg aag
atg ctc tcg ttt aag ctg cta ctg ctg gcc gtg gct ctg 48Met Leu Lys
Met Leu Ser Phe Lys Leu Leu Leu Leu Ala Val Ala Leu1 5 10 15ggc ttc
ttt gaa gga gat gcg aag ttt ggg gaa agg aac gag ggg agc 96Gly Phe
Phe Glu Gly Asp Ala Lys Phe Gly Glu Arg Asn Glu Gly Ser20 25 30gga
gcg aga agg aga cgg tgc ctg aat ggg aac ccc cca aag cgc cta 144Gly
Ala Arg Arg Arg Arg Cys Leu Asn Gly Asn Pro Pro Lys Arg Leu35 40
45aag aga agg gac agg cgg gtg atg tcc cag ctg gag ctg ctc agt gga
192Lys Arg Arg Asp Arg Arg Val Met Ser Gln Leu Glu Leu Leu Ser
Gly50 55 60gga gag atc ctg tgt ggt ggc ttc tac cca cga gta tct tgc
tgc ctg 240Gly Glu Ile Leu Cys Gly Gly Phe Tyr Pro Arg Val Ser Cys
Cys Leu65 70 75 80cag agt gac agc cct gga ttg ggg cgt ctg gag aac
aag atc ttt tct 288Gln Ser Asp Ser Pro Gly Leu Gly Arg Leu Glu Asn
Lys Ile Phe Ser85 90 95gcc acc aac aac tca gaa tgc agc agg ctg ctg
gag gag atc caa tgt 336Ala Thr Asn Asn Ser Glu Cys Ser Arg Leu Leu
Glu Glu Ile Gln Cys100 105 110gct ccc tgc tcc ccg cat tcc cag agc
ctc ttc tac aca cct gaa aga 384Ala Pro Cys Ser Pro His Ser Gln Ser
Leu Phe Tyr Thr Pro Glu Arg115 120 125gat gtc ctg gat ggg gac cta
gca ctt cca ctc ctc tgc aaa gac tac 432Asp Val Leu Asp Gly Asp Leu
Ala Leu Pro Leu Leu Cys Lys Asp Tyr130 135 140tgc aaa gaa ttc ttt
tat act tgc cga ggc cat att cca ggt ctt ctt 480Cys Lys Glu Phe Phe
Tyr Thr Cys Arg Gly His Ile Pro Gly Leu Leu145 150 155 160caa aca
act gct gat gaa ttt tgc ttt tac tat gca aga aaa gat gct 528Gln Thr
Thr Ala Asp Glu Phe Cys Phe Tyr Tyr Ala Arg Lys Asp Ala165 170
175ggg tta tgc ttt cca gac ttc ccg aga aag caa gtc aga gga cca gca
576Gly Leu Cys Phe Pro Asp Phe Pro Arg Lys Gln Val Arg Gly Pro
Ala180 185 190tct aac tac ttg ggc cag atg gaa gac tac gag aaa gtg
ggg ggg atc 624Ser Asn Tyr Leu Gly Gln Met Glu Asp Tyr Glu Lys Val
Gly Gly Ile195 200 205agc aga aaa cac aaa cac aac tgc ctc tgt gtc
cag gag gtc atg agt 672Ser Arg Lys His Lys His Asn Cys Leu Cys Val
Gln Glu Val Met Ser210 215 220ggg ctg cgg cag cct gtg agc gct gtg
cac agc ggg gat ggc tcc cat 720Gly Leu Arg Gln Pro Val Ser Ala Val
His Ser Gly Asp Gly Ser His225 230 235 240cgg ctc ttc att cta gag
aag gaa ggc tac gtg aag att cta acc cca 768Arg Leu Phe Ile Leu Glu
Lys Glu Gly Tyr Val Lys Ile Leu Thr Pro245 250 255gaa gga gaa ctg
ttc aag gag cct tac ttg gac att cac aaa ctt gtt 816Glu Gly Glu Leu
Phe Lys Glu Pro Tyr Leu Asp Ile His Lys Leu Val260 265 270caa agt
gga ata aag gga gga gac gaa agg ggc ctg cta agc ctg gca 864Gln Ser
Gly Ile Lys Gly Gly Asp Glu Arg Gly Leu Leu Ser Leu Ala275 280
285ttc cat ccc aat tac aag aaa aat gga aag ctg tat gtg tct tat acc
912Phe His Pro Asn Tyr Lys Lys Asn Gly Lys Leu Tyr Val Ser Tyr
Thr290 295 300acc aac cag gaa cgg tgg gct att ggg cct cac gac cac
att ctt cgg 960Thr Asn Gln Glu Arg Trp Ala Ile Gly Pro His Asp His
Ile Leu Arg305 310 315 320gtt gtg gaa tac aca gta tcc agg aaa aac
ccc cat caa gtt gat gtg 1008Val Val Glu Tyr Thr Val Ser Arg Lys Asn
Pro His Gln Val Asp Val325 330 335aga aca gcc agg gtg ttt ctg gaa
gtc gca gag ctc cac cga aag cat 1056Arg Thr Ala Arg Val Phe Leu Glu
Val Ala Glu Leu His Arg Lys His340 345 350ctt ggg gga cag ctg ctc
ttt ggt cct gat ggc ttt ttg tac atc atc 1104Leu Gly Gly Gln Leu Leu
Phe Gly Pro Asp Gly Phe Leu Tyr Ile Ile355 360 365ctt ggg gat ggt
atg atc aca ttg gat gac atg gaa gag atg gat ggg 1152Leu Gly Asp Gly
Met Ile Thr Leu Asp Asp Met Glu Glu Met Asp Gly370 375 380tta agt
gac ttc aca ggc tct gtg ctg agg ctg gac gtg gac acc gac 1200Leu Ser
Asp Phe Thr Gly Ser Val Leu Arg Leu Asp Val Asp Thr Asp385 390 395
400atg tgc aat gtg cct tat tcc ata cct cgg agt aac cct cac ttc aac
1248Met Cys Asn Val Pro Tyr Ser Ile Pro Arg Ser Asn Pro His Phe
Asn405 410 415agc acc aac cag ccc cca gaa gta ttt gcc cac ggc ctc
cat gat cca 1296Ser Thr Asn Gln Pro Pro Glu Val Phe Ala His Gly Leu
His Asp Pro420 425 430ggc aga tgt gcc gtg gat cga cat cct act gat
ata aac atc aat tta 1344Gly Arg Cys Ala Val Asp Arg His Pro Thr Asp
Ile Asn Ile Asn Leu435 440 445aca ata ctt tgc tca gat tcc aac ggg
aaa aac agg tca tca gcc aga 1392Thr Ile Leu Cys Ser Asp Ser Asn Gly
Lys Asn Arg Ser Ser Ala Arg450 455 460atc cta cag ata ata aag gga
aga ggt tat gaa agt gag cca tct ctt 1440Ile Leu Gln Ile Ile Lys Gly
Arg Gly Tyr Glu Ser Glu Pro Ser Leu465 470 475 480ctt gaa ttc aag
cca ttc agt aac ggc cct ttg gtt ggt gga ttt gtt 1488Leu Glu Phe Lys
Pro Phe Ser Asn Gly Pro Leu Val Gly Gly Phe Val485 490 495tac aga
ggc tgt cag tct gaa aga ttg tac gga agc tat gtg ttc gga 1536Tyr Arg
Gly Cys Gln Ser Glu Arg Leu Tyr Gly Ser Tyr Val Phe Gly500 505
510gat cgc aat ggg aat ttc tta acc ctc cag caa agc cca gtg acc aag
1584Asp Arg Asn Gly Asn Phe Leu Thr Leu Gln Gln Ser Pro Val Thr
Lys515 520 525caa tgg caa gaa aag ccg ctc tgc ctg ggt gcc agc agc
tcc tgt cga 1632Gln Trp Gln Glu Lys Pro Leu Cys Leu Gly Ala Ser Ser
Ser Cys Arg530 535 540ggc tac ttt tcg ggt cac atc ttg gga ttt gga
gaa gat gaa tta gga 1680Gly Tyr Phe Ser Gly His Ile Leu Gly Phe Gly
Glu Asp Glu Leu Gly545 550 555 560gag gtt tac att cta tca agc agt
aag agt atg acc cag act cac aat 1728Glu Val Tyr Ile Leu Ser Ser Ser
Lys Ser Met Thr Gln Thr His Asn565 570 575gga aaa ctc tac aag atc
gta gac ccc aaa aga cct tta atg cct gag 1776Gly Lys Leu Tyr Lys Ile
Val Asp Pro Lys Arg Pro Leu Met Pro Glu580 585 590gaa tgc aga gtc
aca gtt caa cct gcc cag cca ctg acc tcc gat tgc 1824Glu Cys Arg Val
Thr Val Gln Pro Ala Gln Pro Leu Thr Ser Asp Cys595 600 605tcc cgg
ctc tgt cga aac ggc tac tac acc ccc act ggc aag tgc tgc 1872Ser Arg
Leu Cys Arg Asn Gly Tyr Tyr Thr Pro Thr Gly Lys Cys Cys610 615
620tgc agt ccc ggc tgg gag gga gac ttc tgc aga att gcc aag tgt gag
1920Cys Ser Pro Gly Trp Glu Gly Asp Phe Cys Arg Ile Ala Lys Cys
Glu625 630 635 640cca gcg tgc cgt cat gga ggt gtc tgt gtc aga ccg
aac aag tgc ctc 1968Pro Ala Cys Arg His Gly Gly Val Cys Val Arg Pro
Asn Lys Cys Leu645 650 655tgt aaa aag ggc tat ctt ggt cct caa tgt
gaa caa gtg gac agg aac 2016Cys Lys Lys Gly Tyr Leu Gly Pro Gln Cys
Glu Gln Val Asp Arg Asn660 665 670gtc cgc aga gtg acc agg gca ggt
atc ctt gat cag atc att gac atg 2064Val Arg Arg Val Thr Arg Ala Gly
Ile Leu Asp Gln Ile Ile Asp Met675 680 685acg tct tac ttg ctg gat
ctc aca agt tac att gta tag 2103Thr Ser Tyr Leu Leu Asp Leu Thr Ser
Tyr Ile Val690 695 7002700PRTMus musculus 2Met Leu Lys Met Leu Ser
Phe Lys Leu Leu Leu Leu Ala Val Ala Leu1 5 10 15Gly Phe Phe Glu Gly
Asp Ala Lys Phe Gly Glu Arg Asn Glu Gly Ser20 25 30Gly Ala Arg Arg
Arg Arg Cys Leu Asn Gly Asn Pro Pro Lys Arg Leu35 40 45Lys Arg Arg
Asp Arg Arg Val Met Ser Gln Leu Glu Leu Leu Ser Gly50 55 60Gly Glu
Ile Leu Cys Gly Gly Phe Tyr Pro Arg Val Ser Cys Cys Leu65 70 75
80Gln Ser Asp Ser Pro Gly Leu Gly Arg Leu Glu Asn Lys Ile Phe Ser85
90 95Ala Thr Asn Asn Ser Glu Cys Ser Arg Leu Leu Glu Glu Ile Gln
Cys100 105 110Ala Pro Cys Ser Pro His Ser Gln Ser Leu Phe Tyr Thr
Pro Glu Arg115 120 125Asp Val Leu Asp Gly Asp Leu Ala Leu Pro Leu
Leu Cys Lys Asp Tyr130 135 140Cys Lys Glu Phe Phe Tyr Thr Cys Arg
Gly His Ile Pro Gly Leu Leu145 150 155 160Gln Thr Thr Ala Asp Glu
Phe Cys Phe Tyr Tyr Ala Arg Lys Asp Ala165 170 175Gly Leu Cys Phe
Pro Asp Phe Pro Arg Lys Gln Val Arg Gly Pro Ala180 185 190Ser Asn
Tyr Leu Gly Gln Met Glu Asp Tyr Glu Lys Val Gly Gly Ile195 200
205Ser Arg Lys His Lys His Asn Cys Leu Cys Val Gln Glu Val Met
Ser210 215 220Gly Leu Arg Gln Pro Val Ser Ala Val His Ser Gly Asp
Gly Ser His225 230 235 240Arg Leu Phe Ile Leu Glu Lys Glu Gly Tyr
Val Lys Ile Leu Thr Pro245 250 255Glu Gly Glu Leu Phe Lys Glu Pro
Tyr Leu Asp Ile His Lys Leu Val260 265 270Gln Ser Gly Ile Lys Gly
Gly Asp Glu Arg Gly Leu Leu Ser Leu Ala275 280 285Phe His Pro Asn
Tyr Lys Lys Asn Gly Lys Leu Tyr Val Ser Tyr Thr290 295 300Thr Asn
Gln Glu Arg Trp Ala Ile Gly Pro His Asp His Ile Leu Arg305 310 315
320Val Val Glu Tyr Thr Val Ser Arg Lys Asn Pro His Gln Val Asp
Val325 330 335Arg Thr Ala Arg Val Phe Leu Glu Val Ala Glu Leu His
Arg Lys His340 345 350Leu Gly Gly Gln Leu Leu Phe Gly Pro Asp Gly
Phe Leu Tyr Ile Ile355 360 365Leu Gly Asp Gly Met Ile Thr Leu Asp
Asp Met Glu Glu Met Asp Gly370 375 380Leu Ser Asp Phe Thr Gly Ser
Val Leu Arg Leu Asp Val Asp Thr Asp385 390 395 400Met Cys Asn Val
Pro Tyr Ser Ile Pro Arg Ser Asn Pro His Phe Asn405 410 415Ser Thr
Asn Gln Pro Pro Glu Val Phe Ala His Gly Leu His Asp Pro420 425
430Gly Arg Cys Ala Val Asp Arg His Pro Thr Asp Ile Asn Ile Asn
Leu435 440 445Thr Ile Leu Cys Ser Asp Ser Asn Gly Lys Asn Arg Ser
Ser Ala Arg450 455 460Ile Leu Gln Ile Ile Lys Gly Arg Gly Tyr Glu
Ser Glu Pro Ser Leu465 470 475 480Leu Glu Phe Lys Pro Phe Ser Asn
Gly Pro Leu Val Gly Gly Phe Val485 490 495Tyr Arg Gly Cys Gln Ser
Glu Arg Leu Tyr Gly Ser Tyr Val Phe Gly500 505 510Asp Arg Asn Gly
Asn Phe Leu Thr Leu Gln Gln Ser Pro Val Thr Lys515 520 525Gln Trp
Gln Glu Lys Pro Leu Cys Leu Gly Ala Ser Ser Ser Cys Arg530 535
540Gly Tyr Phe Ser Gly His Ile Leu Gly Phe Gly Glu Asp Glu Leu
Gly545 550 555 560Glu Val Tyr Ile Leu Ser Ser Ser Lys Ser Met Thr
Gln Thr His Asn565 570 575Gly Lys Leu Tyr Lys Ile Val Asp Pro Lys
Arg Pro Leu Met Pro Glu580 585 590Glu Cys Arg Val Thr Val Gln Pro
Ala Gln Pro Leu Thr Ser Asp Cys595 600 605Ser Arg Leu Cys Arg Asn
Gly Tyr Tyr Thr Pro Thr Gly Lys Cys Cys610 615 620Cys Ser Pro Gly
Trp Glu Gly Asp Phe Cys Arg Ile Ala Lys Cys Glu625 630 635 640Pro
Ala Cys Arg His Gly Gly Val Cys Val Arg Pro Asn Lys Cys Leu645 650
655Cys Lys Lys Gly Tyr Leu Gly Pro Gln Cys Glu Gln Val Asp Arg
Asn660 665 670Val Arg Arg Val Thr Arg Ala Gly Ile Leu Asp Gln Ile
Ile Asp Met675 680 685Thr Ser Tyr Leu Leu Asp Leu Thr Ser Tyr Ile
Val690 695 70032103DNAHomo sapiensCDS(1)..(2103)Hip1 cDNA (human)
3atg ctg aag atg ctc tcc ttt aag ctg ctg ctg ctg gcc gtg gct ctg
48Met Leu Lys Met Leu Ser Phe Lys Leu Leu Leu Leu Ala Val Ala Leu1
5 10 15ggc ttc ttt gaa gga gat gct aag ttt ggg gaa aga aac gaa ggg
agc 96Gly Phe Phe Glu Gly Asp Ala Lys Phe Gly Glu Arg Asn Glu Gly
Ser20 25 30gga gca agg agg aga agg tgc ctg aat ggg aac ccc ccg aag
cgc ctg 144Gly Ala Arg Arg Arg Arg Cys Leu Asn Gly Asn Pro Pro Lys
Arg Leu35 40 45aaa agg aga gac agg agg atg atg tcc cag ctg gag ctg
ctg agt ggg 192Lys Arg Arg Asp Arg Arg Met Met Ser Gln Leu Glu Leu
Leu Ser Gly50 55 60gga gag atg ctg tgc ggt ggc ttc tac cct cgg ctg
tcc tgc tgc ctg 240Gly Glu Met Leu Cys Gly Gly Phe Tyr Pro Arg Leu
Ser Cys Cys Leu65 70 75 80cgg agt gac agc ccg ggg cta ggg cgc ctg
gag aat aag ata ttt tct 288Arg Ser Asp Ser Pro Gly Leu Gly Arg Leu
Glu Asn Lys Ile Phe Ser85 90 95gtt acc aac aac aca gaa tgt ggg aag
tta ctg gag gaa atc aaa tgt 336Val Thr Asn Asn Thr Glu Cys Gly Lys
Leu Leu Glu Glu Ile Lys Cys100 105 110gca ctt tgc tct cca cat tct
caa agc ctg ttc cac tca cct gag aga 384Ala Leu Cys Ser Pro His Ser
Gln Ser Leu Phe His Ser Pro Glu Arg115 120 125gaa gtc ttg gaa aga
gac cta gta ctt cct ctg ctc tgc aaa gac tat 432Glu Val Leu Glu Arg
Asp Leu Val Leu Pro Leu Leu Cys Lys Asp Tyr130 135 140tgc aaa gaa
ttc ttt tac act tgc cga ggc cat att cca ggt ttc ctt 480Cys Lys Glu
Phe Phe Tyr Thr Cys Arg Gly His Ile Pro Gly Phe Leu145 150 155
160caa aca act gcg gat gag ttt tgc ttt tac tat gca aga aaa gat ggt
528Gln Thr Thr Ala Asp Glu Phe Cys Phe Tyr Tyr Ala Arg Lys Asp
Gly165 170 175ggg ttg tgc ttt cca gat ttt cca aga aaa caa gtc aga
gga cca gca 576Gly Leu Cys Phe Pro Asp Phe Pro Arg Lys Gln Val Arg
Gly Pro Ala180 185 190tct aac tac ttg gac cag atg gaa gaa tat gac
aaa gtg gaa gag atc 624Ser Asn Tyr Leu Asp Gln Met Glu Glu Tyr Asp
Lys Val Glu Glu Ile195 200 205agc aga aag cac aaa cac aac tgc ttc
tgt att cag gag gtt gtg agt 672Ser Arg Lys His Lys His Asn Cys Phe
Cys Ile Gln Glu Val Val Ser210 215 220ggg ctg cgg cag ccc gtt ggt
gcc ctg cat agt ggg gat ggc tcg caa 720Gly Leu Arg Gln Pro Val Gly
Ala Leu His Ser Gly Asp Gly Ser Gln225 230 235 240cgt ctc ttc att
ctg gaa aaa gaa ggt tat gtg aag ata ctt acc cct 768Arg Leu Phe Ile
Leu Glu Lys Glu Gly Tyr Val Lys Ile Leu Thr Pro245 250 255gaa gga
gaa att ttc aag gag cct tat ttg gac att cac aaa ctt gtt 816Glu Gly
Glu Ile Phe Lys Glu Pro Tyr Leu Asp Ile His Lys Leu Val260 265
270caa agt gga ata aag gga gga gat gaa aga gga ctg cta agc ctc gca
864Gln Ser Gly Ile Lys Gly Gly Asp Glu Arg Gly Leu Leu Ser Leu
Ala275 280 285ttc cat ccc aat tac aag aaa aat gga aag ttg tat gtg
tcc tat acc 912Phe His Pro Asn Tyr Lys Lys Asn Gly Lys Leu Tyr Val
Ser Tyr Thr290 295 300acc aac caa gaa cgg tgg gct atc ggg cct cat
gac cac att ctt agg 960Thr Asn Gln Glu Arg Trp Ala Ile Gly Pro His
Asp His Ile Leu Arg305 310 315 320gtt gtg gaa tac aca gta tcc aga
aaa aat cca cac caa gtt gat ttg 1008Val Val Glu Tyr Thr Val Ser Arg
Lys Asn Pro His Gln Val Asp Leu325 330 335aga aca gcc aga gtc ttt
ctt gaa gtt gca gaa ctc cac aga aag cat 1056Arg Thr Ala Arg Val Phe
Leu Glu Val Ala Glu Leu His Arg Lys His340 345 350ctg gga gga caa
ctg ctc ttt ggc cct gac ggc ttt ttg tac atc att 1104Leu Gly Gly Gln
Leu Leu Phe Gly Pro Asp Gly Phe Leu Tyr Ile Ile355 360 365ctt ggt
gat ggg atg att aca ctg gat gat atg gaa gaa atg gat ggg 1152Leu Gly
Asp Gly Met Ile Thr Leu Asp Asp Met Glu Glu Met Asp Gly370 375
380tta agt gat ttc aca ggc tca gtg cta cgg ctg gat gtg gac aca gac
1200Leu Ser Asp Phe Thr Gly Ser Val Leu Arg Leu Asp Val Asp Thr
Asp385 390 395 400atg tgc aac gtg cct tat tcc ata cca agg agc aac
cca cac ttc aac 1248Met Cys Asn Val Pro Tyr Ser Ile Pro Arg Ser Asn
Pro His Phe Asn405 410 415agc acc aac cag ccc ccc gaa gtg ttt gct
cat ggg ctc cac gat cca 1296Ser Thr Asn Gln Pro Pro Glu Val Phe Ala
His Gly Leu His Asp Pro420 425 430ggc aga tgt gct gtg gat aga cat
ccc act gat ata aac atc aat tta 1344Gly Arg Cys Ala Val Asp Arg His
Pro Thr Asp Ile Asn Ile Asn Leu435 440 445acg ata ctg tgt tca gac
tcc aat gga aaa aac aga tca tca gcc aga 1392Thr Ile Leu Cys Ser Asp
Ser Asn Gly Lys Asn Arg Ser Ser Ala Arg450 455 460att cta cag ata
ata aag ggg aaa gat tat gaa
agt gag cca tca ctt 1440Ile Leu Gln Ile Ile Lys Gly Lys Asp Tyr Glu
Ser Glu Pro Ser Leu465 470 475 480tta gaa ttc aag cca ttc agt aat
ggt cct ttg gtt ggt gga ttt gta 1488Leu Glu Phe Lys Pro Phe Ser Asn
Gly Pro Leu Val Gly Gly Phe Val485 490 495tac cgg ggc tgc cag tca
gaa aga ttg tat gga agc tac gtg ttt gga 1536Tyr Arg Gly Cys Gln Ser
Glu Arg Leu Tyr Gly Ser Tyr Val Phe Gly500 505 510gat cgt aat ggg
aat ttc cta act ctc cag caa agt cct gtg aca aag 1584Asp Arg Asn Gly
Asn Phe Leu Thr Leu Gln Gln Ser Pro Val Thr Lys515 520 525cag tgg
caa gaa aaa cca ctc tgt ctc ggc act agt ggg tcc tgt aga 1632Gln Trp
Gln Glu Lys Pro Leu Cys Leu Gly Thr Ser Gly Ser Cys Arg530 535
540ggc tac ttt tcc ggt cac atc ttg gga ttt gga gaa gat gaa cta ggt
1680Gly Tyr Phe Ser Gly His Ile Leu Gly Phe Gly Glu Asp Glu Leu
Gly545 550 555 560gaa gtt tac att tta tca agc agt aaa agt atg acc
cag act cac aat 1728Glu Val Tyr Ile Leu Ser Ser Ser Lys Ser Met Thr
Gln Thr His Asn565 570 575gga aaa ctc tac aaa att gta gat ccc aaa
aga cct tta atg cct gag 1776Gly Lys Leu Tyr Lys Ile Val Asp Pro Lys
Arg Pro Leu Met Pro Glu580 585 590gaa tgc aga gcc acg gta caa cct
gca cag aca ctg act tca gag tgc 1824Glu Cys Arg Ala Thr Val Gln Pro
Ala Gln Thr Leu Thr Ser Glu Cys595 600 605tcc agg ctc tgt cga aac
ggc tac tgc acc ccc acg gga aag tgc tgc 1872Ser Arg Leu Cys Arg Asn
Gly Tyr Cys Thr Pro Thr Gly Lys Cys Cys610 615 620tgc agt cca ggc
tgg gag ggg gac ttc tgc aga act gca aaa tgt gag 1920Cys Ser Pro Gly
Trp Glu Gly Asp Phe Cys Arg Thr Ala Lys Cys Glu625 630 635 640cca
gca tgt cgt cat gga ggt gtc tgt gtt aga ccg aac aag tgc ctc 1968Pro
Ala Cys Arg His Gly Gly Val Cys Val Arg Pro Asn Lys Cys Leu645 650
655tgt aaa aaa gga tat ctt ggt cct caa tgt gaa caa gtg gac aga aac
2016Cys Lys Lys Gly Tyr Leu Gly Pro Gln Cys Glu Gln Val Asp Arg
Asn660 665 670atc cgc aga gtg acc agg gca ggt att ctt gat cag atc
att gac atg 2064Ile Arg Arg Val Thr Arg Ala Gly Ile Leu Asp Gln Ile
Ile Asp Met675 680 685aca tct tac ttg ctg gat cta aca agt tac att
gta tag 2103Thr Ser Tyr Leu Leu Asp Leu Thr Ser Tyr Ile Val690 695
7004700PRTHomo sapiens 4Met Leu Lys Met Leu Ser Phe Lys Leu Leu Leu
Leu Ala Val Ala Leu1 5 10 15Gly Phe Phe Glu Gly Asp Ala Lys Phe Gly
Glu Arg Asn Glu Gly Ser20 25 30Gly Ala Arg Arg Arg Arg Cys Leu Asn
Gly Asn Pro Pro Lys Arg Leu35 40 45Lys Arg Arg Asp Arg Arg Met Met
Ser Gln Leu Glu Leu Leu Ser Gly50 55 60Gly Glu Met Leu Cys Gly Gly
Phe Tyr Pro Arg Leu Ser Cys Cys Leu65 70 75 80Arg Ser Asp Ser Pro
Gly Leu Gly Arg Leu Glu Asn Lys Ile Phe Ser85 90 95Val Thr Asn Asn
Thr Glu Cys Gly Lys Leu Leu Glu Glu Ile Lys Cys100 105 110Ala Leu
Cys Ser Pro His Ser Gln Ser Leu Phe His Ser Pro Glu Arg115 120
125Glu Val Leu Glu Arg Asp Leu Val Leu Pro Leu Leu Cys Lys Asp
Tyr130 135 140Cys Lys Glu Phe Phe Tyr Thr Cys Arg Gly His Ile Pro
Gly Phe Leu145 150 155 160Gln Thr Thr Ala Asp Glu Phe Cys Phe Tyr
Tyr Ala Arg Lys Asp Gly165 170 175Gly Leu Cys Phe Pro Asp Phe Pro
Arg Lys Gln Val Arg Gly Pro Ala180 185 190Ser Asn Tyr Leu Asp Gln
Met Glu Glu Tyr Asp Lys Val Glu Glu Ile195 200 205Ser Arg Lys His
Lys His Asn Cys Phe Cys Ile Gln Glu Val Val Ser210 215 220Gly Leu
Arg Gln Pro Val Gly Ala Leu His Ser Gly Asp Gly Ser Gln225 230 235
240Arg Leu Phe Ile Leu Glu Lys Glu Gly Tyr Val Lys Ile Leu Thr
Pro245 250 255Glu Gly Glu Ile Phe Lys Glu Pro Tyr Leu Asp Ile His
Lys Leu Val260 265 270Gln Ser Gly Ile Lys Gly Gly Asp Glu Arg Gly
Leu Leu Ser Leu Ala275 280 285Phe His Pro Asn Tyr Lys Lys Asn Gly
Lys Leu Tyr Val Ser Tyr Thr290 295 300Thr Asn Gln Glu Arg Trp Ala
Ile Gly Pro His Asp His Ile Leu Arg305 310 315 320Val Val Glu Tyr
Thr Val Ser Arg Lys Asn Pro His Gln Val Asp Leu325 330 335Arg Thr
Ala Arg Val Phe Leu Glu Val Ala Glu Leu His Arg Lys His340 345
350Leu Gly Gly Gln Leu Leu Phe Gly Pro Asp Gly Phe Leu Tyr Ile
Ile355 360 365Leu Gly Asp Gly Met Ile Thr Leu Asp Asp Met Glu Glu
Met Asp Gly370 375 380Leu Ser Asp Phe Thr Gly Ser Val Leu Arg Leu
Asp Val Asp Thr Asp385 390 395 400Met Cys Asn Val Pro Tyr Ser Ile
Pro Arg Ser Asn Pro His Phe Asn405 410 415Ser Thr Asn Gln Pro Pro
Glu Val Phe Ala His Gly Leu His Asp Pro420 425 430Gly Arg Cys Ala
Val Asp Arg His Pro Thr Asp Ile Asn Ile Asn Leu435 440 445Thr Ile
Leu Cys Ser Asp Ser Asn Gly Lys Asn Arg Ser Ser Ala Arg450 455
460Ile Leu Gln Ile Ile Lys Gly Lys Asp Tyr Glu Ser Glu Pro Ser
Leu465 470 475 480Leu Glu Phe Lys Pro Phe Ser Asn Gly Pro Leu Val
Gly Gly Phe Val485 490 495Tyr Arg Gly Cys Gln Ser Glu Arg Leu Tyr
Gly Ser Tyr Val Phe Gly500 505 510Asp Arg Asn Gly Asn Phe Leu Thr
Leu Gln Gln Ser Pro Val Thr Lys515 520 525Gln Trp Gln Glu Lys Pro
Leu Cys Leu Gly Thr Ser Gly Ser Cys Arg530 535 540Gly Tyr Phe Ser
Gly His Ile Leu Gly Phe Gly Glu Asp Glu Leu Gly545 550 555 560Glu
Val Tyr Ile Leu Ser Ser Ser Lys Ser Met Thr Gln Thr His Asn565 570
575Gly Lys Leu Tyr Lys Ile Val Asp Pro Lys Arg Pro Leu Met Pro
Glu580 585 590Glu Cys Arg Ala Thr Val Gln Pro Ala Gln Thr Leu Thr
Ser Glu Cys595 600 605Ser Arg Leu Cys Arg Asn Gly Tyr Cys Thr Pro
Thr Gly Lys Cys Cys610 615 620Cys Ser Pro Gly Trp Glu Gly Asp Phe
Cys Arg Thr Ala Lys Cys Glu625 630 635 640Pro Ala Cys Arg His Gly
Gly Val Cys Val Arg Pro Asn Lys Cys Leu645 650 655Cys Lys Lys Gly
Tyr Leu Gly Pro Gln Cys Glu Gln Val Asp Arg Asn660 665 670Ile Arg
Arg Val Thr Arg Ala Gly Ile Leu Asp Gln Ile Ile Asp Met675 680
685Thr Ser Tyr Leu Leu Asp Leu Thr Ser Tyr Ile Val690 695
70052037DNAMus musculusCDS(1)..(2037)soluble Hip1 cDNA (murine)
5atg ctg aag atg ctc tcg ttt aag ctg cta ctg ctg gcc gtg gct ctg
48Met Leu Lys Met Leu Ser Phe Lys Leu Leu Leu Leu Ala Val Ala Leu1
5 10 15ggc ttc ttt gaa gga gat gcg aag ttt ggg gaa agg aac gag ggg
agc 96Gly Phe Phe Glu Gly Asp Ala Lys Phe Gly Glu Arg Asn Glu Gly
Ser20 25 30gga gcg aga agg aga cgg tgc ctg aat ggg aac ccc cca aag
cgc cta 144Gly Ala Arg Arg Arg Arg Cys Leu Asn Gly Asn Pro Pro Lys
Arg Leu35 40 45aag aga agg gac agg cgg gtg atg tcc cag ctg gag ctg
ctc agt gga 192Lys Arg Arg Asp Arg Arg Val Met Ser Gln Leu Glu Leu
Leu Ser Gly50 55 60gga gag atc ctg tgt ggt ggc ttc tac cca cga gta
tct tgc tgc ctg 240Gly Glu Ile Leu Cys Gly Gly Phe Tyr Pro Arg Val
Ser Cys Cys Leu65 70 75 80cag agt gac agc cct gga ttg ggg cgt ctg
gag aac aag atc ttt tct 288Gln Ser Asp Ser Pro Gly Leu Gly Arg Leu
Glu Asn Lys Ile Phe Ser85 90 95gcc acc aac aac tca gaa tgc agc agg
ctg ctg gag gag atc caa tgt 336Ala Thr Asn Asn Ser Glu Cys Ser Arg
Leu Leu Glu Glu Ile Gln Cys100 105 110gct ccc tgc tcc ccg cat tcc
cag agc ctc ttc tac aca cct gaa aga 384Ala Pro Cys Ser Pro His Ser
Gln Ser Leu Phe Tyr Thr Pro Glu Arg115 120 125gat gtc ctg gat ggg
gac cta gca ctt cca ctc ctc tgc aaa gac tac 432Asp Val Leu Asp Gly
Asp Leu Ala Leu Pro Leu Leu Cys Lys Asp Tyr130 135 140tgc aaa gaa
ttc ttt tat act tgc cga ggc cat att cca ggt ctt ctt 480Cys Lys Glu
Phe Phe Tyr Thr Cys Arg Gly His Ile Pro Gly Leu Leu145 150 155
160caa aca act gct gat gaa ttt tgc ttt tac tat gca aga aaa gat gct
528Gln Thr Thr Ala Asp Glu Phe Cys Phe Tyr Tyr Ala Arg Lys Asp
Ala165 170 175ggg tta tgc ttt cca gac ttc ccg aga aag caa gtc aga
gga cca gca 576Gly Leu Cys Phe Pro Asp Phe Pro Arg Lys Gln Val Arg
Gly Pro Ala180 185 190tct aac tac ttg ggc cag atg gaa gac tac gag
aaa gtg ggg ggg atc 624Ser Asn Tyr Leu Gly Gln Met Glu Asp Tyr Glu
Lys Val Gly Gly Ile195 200 205agc aga aaa cac aaa cac aac tgc ctc
tgt gtc cag gag gtc atg agt 672Ser Arg Lys His Lys His Asn Cys Leu
Cys Val Gln Glu Val Met Ser210 215 220ggg ctg cgg cag cct gtg agc
gct gtg cac agc ggg gat ggc tcc cat 720Gly Leu Arg Gln Pro Val Ser
Ala Val His Ser Gly Asp Gly Ser His225 230 235 240cgg ctc ttc att
cta gag aag gaa ggc tac gtg aag att cta acc cca 768Arg Leu Phe Ile
Leu Glu Lys Glu Gly Tyr Val Lys Ile Leu Thr Pro245 250 255gaa gga
gaa ctg ttc aag gag cct tac ttg gac att cac aaa ctt gtt 816Glu Gly
Glu Leu Phe Lys Glu Pro Tyr Leu Asp Ile His Lys Leu Val260 265
270caa agt gga ata aag gga gga gac gaa agg ggc ctg cta agc ctg gca
864Gln Ser Gly Ile Lys Gly Gly Asp Glu Arg Gly Leu Leu Ser Leu
Ala275 280 285ttc cat ccc aat tac aag aaa aat gga aag ctg tat gtg
tct tat acc 912Phe His Pro Asn Tyr Lys Lys Asn Gly Lys Leu Tyr Val
Ser Tyr Thr290 295 300acc aac cag gaa cgg tgg gct att ggg cct cac
gac cac att ctt cgg 960Thr Asn Gln Glu Arg Trp Ala Ile Gly Pro His
Asp His Ile Leu Arg305 310 315 320gtt gtg gaa tac aca gta tcc agg
aaa aac ccc cat caa gtt gat gtg 1008Val Val Glu Tyr Thr Val Ser Arg
Lys Asn Pro His Gln Val Asp Val325 330 335aga aca gcc agg gtg ttt
ctg gaa gtc gca gag ctc cac cga aag cat 1056Arg Thr Ala Arg Val Phe
Leu Glu Val Ala Glu Leu His Arg Lys His340 345 350ctt ggg gga cag
ctg ctc ttt ggt cct gat ggc ttt ttg tac atc atc 1104Leu Gly Gly Gln
Leu Leu Phe Gly Pro Asp Gly Phe Leu Tyr Ile Ile355 360 365ctt ggg
gat ggt atg atc aca ttg gat gac atg gaa gag atg gat ggg 1152Leu Gly
Asp Gly Met Ile Thr Leu Asp Asp Met Glu Glu Met Asp Gly370 375
380tta agt gac ttc aca ggc tct gtg ctg agg ctg gac gtg gac acc gac
1200Leu Ser Asp Phe Thr Gly Ser Val Leu Arg Leu Asp Val Asp Thr
Asp385 390 395 400atg tgc aat gtg cct tat tcc ata cct cgg agt aac
cct cac ttc aac 1248Met Cys Asn Val Pro Tyr Ser Ile Pro Arg Ser Asn
Pro His Phe Asn405 410 415agc acc aac cag ccc cca gaa gta ttt gcc
cac ggc ctc cat gat cca 1296Ser Thr Asn Gln Pro Pro Glu Val Phe Ala
His Gly Leu His Asp Pro420 425 430ggc aga tgt gcc gtg gat cga cat
cct act gat ata aac atc aat tta 1344Gly Arg Cys Ala Val Asp Arg His
Pro Thr Asp Ile Asn Ile Asn Leu435 440 445aca ata ctt tgc tca gat
tcc aac ggg aaa aac agg tca tca gcc aga 1392Thr Ile Leu Cys Ser Asp
Ser Asn Gly Lys Asn Arg Ser Ser Ala Arg450 455 460atc cta cag ata
ata aag gga aga ggt tat gaa agt gag cca tct ctt 1440Ile Leu Gln Ile
Ile Lys Gly Arg Gly Tyr Glu Ser Glu Pro Ser Leu465 470 475 480ctt
gaa ttc aag cca ttc agt aac ggc cct ttg gtt ggt gga ttt gtt 1488Leu
Glu Phe Lys Pro Phe Ser Asn Gly Pro Leu Val Gly Gly Phe Val485 490
495tac aga ggc tgt cag tct gaa aga ttg tac gga agc tat gtg ttc gga
1536Tyr Arg Gly Cys Gln Ser Glu Arg Leu Tyr Gly Ser Tyr Val Phe
Gly500 505 510gat cgc aat ggg aat ttc tta acc ctc cag caa agc cca
gtg acc aag 1584Asp Arg Asn Gly Asn Phe Leu Thr Leu Gln Gln Ser Pro
Val Thr Lys515 520 525caa tgg caa gaa aag ccg ctc tgc ctg ggt gcc
agc agc tcc tgt cga 1632Gln Trp Gln Glu Lys Pro Leu Cys Leu Gly Ala
Ser Ser Ser Cys Arg530 535 540ggc tac ttt tcg ggt cac atc ttg gga
ttt gga gaa gat gaa tta gga 1680Gly Tyr Phe Ser Gly His Ile Leu Gly
Phe Gly Glu Asp Glu Leu Gly545 550 555 560gag gtt tac att cta tca
agc agt aag agt atg acc cag act cac aat 1728Glu Val Tyr Ile Leu Ser
Ser Ser Lys Ser Met Thr Gln Thr His Asn565 570 575gga aaa ctc tac
aag atc gta gac ccc aaa aga cct tta atg cct gag 1776Gly Lys Leu Tyr
Lys Ile Val Asp Pro Lys Arg Pro Leu Met Pro Glu580 585 590gaa tgc
aga gtc aca gtt caa cct gcc cag cca ctg acc tcc gat tgc 1824Glu Cys
Arg Val Thr Val Gln Pro Ala Gln Pro Leu Thr Ser Asp Cys595 600
605tcc cgg ctc tgt cga aac ggc tac tac acc ccc act ggc aag tgc tgc
1872Ser Arg Leu Cys Arg Asn Gly Tyr Tyr Thr Pro Thr Gly Lys Cys
Cys610 615 620tgc agt ccc ggc tgg gag gga gac ttc tgc aga att gcc
aag tgt gag 1920Cys Ser Pro Gly Trp Glu Gly Asp Phe Cys Arg Ile Ala
Lys Cys Glu625 630 635 640cca gcg tgc cgt cat gga ggt gtc tgt gtc
aga ccg aac aag tgc ctc 1968Pro Ala Cys Arg His Gly Gly Val Cys Val
Arg Pro Asn Lys Cys Leu645 650 655tgt aaa aag ggc tat ctt ggt cct
caa tgt gaa caa gtg gac agg aac 2016Cys Lys Lys Gly Tyr Leu Gly Pro
Gln Cys Glu Gln Val Asp Arg Asn660 665 670gtc cgc aga gtg acc agg
tga 2037Val Arg Arg Val Thr Arg6756678PRTMus musculus 6Met Leu Lys
Met Leu Ser Phe Lys Leu Leu Leu Leu Ala Val Ala Leu1 5 10 15Gly Phe
Phe Glu Gly Asp Ala Lys Phe Gly Glu Arg Asn Glu Gly Ser20 25 30Gly
Ala Arg Arg Arg Arg Cys Leu Asn Gly Asn Pro Pro Lys Arg Leu35 40
45Lys Arg Arg Asp Arg Arg Val Met Ser Gln Leu Glu Leu Leu Ser Gly50
55 60Gly Glu Ile Leu Cys Gly Gly Phe Tyr Pro Arg Val Ser Cys Cys
Leu65 70 75 80Gln Ser Asp Ser Pro Gly Leu Gly Arg Leu Glu Asn Lys
Ile Phe Ser85 90 95Ala Thr Asn Asn Ser Glu Cys Ser Arg Leu Leu Glu
Glu Ile Gln Cys100 105 110Ala Pro Cys Ser Pro His Ser Gln Ser Leu
Phe Tyr Thr Pro Glu Arg115 120 125Asp Val Leu Asp Gly Asp Leu Ala
Leu Pro Leu Leu Cys Lys Asp Tyr130 135 140Cys Lys Glu Phe Phe Tyr
Thr Cys Arg Gly His Ile Pro Gly Leu Leu145 150 155 160Gln Thr Thr
Ala Asp Glu Phe Cys Phe Tyr Tyr Ala Arg Lys Asp Ala165 170 175Gly
Leu Cys Phe Pro Asp Phe Pro Arg Lys Gln Val Arg Gly Pro Ala180 185
190Ser Asn Tyr Leu Gly Gln Met Glu Asp Tyr Glu Lys Val Gly Gly
Ile195 200 205Ser Arg Lys His Lys His Asn Cys Leu Cys Val Gln Glu
Val Met Ser210 215 220Gly Leu Arg Gln Pro Val Ser Ala Val His Ser
Gly Asp Gly Ser His225 230 235 240Arg Leu Phe Ile Leu Glu Lys Glu
Gly Tyr Val Lys Ile Leu Thr Pro245 250 255Glu Gly Glu Leu Phe Lys
Glu Pro Tyr Leu Asp Ile His Lys Leu Val260 265 270Gln Ser Gly Ile
Lys Gly Gly Asp Glu Arg Gly Leu Leu Ser Leu Ala275 280 285Phe His
Pro Asn Tyr Lys Lys Asn Gly Lys Leu Tyr Val Ser Tyr Thr290 295
300Thr Asn Gln Glu Arg Trp Ala Ile Gly Pro His Asp His Ile Leu
Arg305 310 315 320Val Val Glu Tyr Thr Val Ser Arg Lys Asn Pro His
Gln Val Asp Val325 330 335Arg Thr Ala Arg Val Phe Leu Glu Val Ala
Glu Leu His Arg Lys His340 345 350Leu Gly Gly Gln Leu Leu Phe Gly
Pro Asp Gly Phe Leu Tyr Ile Ile355 360 365Leu Gly Asp Gly Met Ile
Thr Leu Asp Asp Met Glu Glu Met Asp Gly370 375 380Leu Ser Asp Phe
Thr Gly Ser Val Leu Arg Leu Asp Val Asp Thr Asp385 390 395 400Met
Cys Asn Val Pro Tyr Ser Ile Pro Arg Ser Asn Pro His Phe Asn405 410
415Ser Thr Asn Gln Pro Pro Glu Val Phe Ala His Gly Leu His Asp
Pro420
425 430Gly Arg Cys Ala Val Asp Arg His Pro Thr Asp Ile Asn Ile Asn
Leu435 440 445Thr Ile Leu Cys Ser Asp Ser Asn Gly Lys Asn Arg Ser
Ser Ala Arg450 455 460Ile Leu Gln Ile Ile Lys Gly Arg Gly Tyr Glu
Ser Glu Pro Ser Leu465 470 475 480Leu Glu Phe Lys Pro Phe Ser Asn
Gly Pro Leu Val Gly Gly Phe Val485 490 495Tyr Arg Gly Cys Gln Ser
Glu Arg Leu Tyr Gly Ser Tyr Val Phe Gly500 505 510Asp Arg Asn Gly
Asn Phe Leu Thr Leu Gln Gln Ser Pro Val Thr Lys515 520 525Gln Trp
Gln Glu Lys Pro Leu Cys Leu Gly Ala Ser Ser Ser Cys Arg530 535
540Gly Tyr Phe Ser Gly His Ile Leu Gly Phe Gly Glu Asp Glu Leu
Gly545 550 555 560Glu Val Tyr Ile Leu Ser Ser Ser Lys Ser Met Thr
Gln Thr His Asn565 570 575Gly Lys Leu Tyr Lys Ile Val Asp Pro Lys
Arg Pro Leu Met Pro Glu580 585 590Glu Cys Arg Val Thr Val Gln Pro
Ala Gln Pro Leu Thr Ser Asp Cys595 600 605Ser Arg Leu Cys Arg Asn
Gly Tyr Tyr Thr Pro Thr Gly Lys Cys Cys610 615 620Cys Ser Pro Gly
Trp Glu Gly Asp Phe Cys Arg Ile Ala Lys Cys Glu625 630 635 640Pro
Ala Cys Arg His Gly Gly Val Cys Val Arg Pro Asn Lys Cys Leu645 650
655Cys Lys Lys Gly Tyr Leu Gly Pro Gln Cys Glu Gln Val Asp Arg
Asn660 665 670Val Arg Arg Val Thr Arg67576814DNAArtificialsynthetic
recombinant AAV vector (plasmid) 7agcgcccaat acgcaaaccg cctctccccg
cgcgttggcc gattcattaa tgcagctggc 60acgacaggtt tcccgactgg aaagcgggca
gtgagcgcaa cgcaattaat gtgagttagc 120tcactcatta ggcaccccag
gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa 180ttgtgagcgg
ataacaattt cacacaggaa acagctatga ccatgattac gccagattta
240attaaggctg cgcgctcgct cgctcactga ggccgcccgg gcaaagcccg
ggcgtcgggc 300gacctttggt cgcccggcct cagtgagcga gcgagcgcgc
agagagggag tggccaactc 360catcactagg ggttccttgt agttaatgat
taacccgcca tgctacttat ctacgtagcc 420atgctctagg aagatcggaa
ttcgccctta agctagctag ttattaatag taatcaatta 480cggggtcatt
agttcatagc ccatatatgg agttccgcgt tacataactt acggtaaatg
540gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaataatg
acgtatgttc 600ccatagtaac gccaataggg actttccatt gacgtcaatg
ggtggagtat ttacggtaaa 660ctgcccactt ggcagtacat caagtgtatc
atatgccaag tacgccccct attgacgtca 720atgacggtaa atggcccgcc
tggcattatg cccagtacat gaccttatgg gactttccta 780cttggcagta
catctacgta ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt
840acatcaatgg gcgtggatag cggtttgact cacggggatt tccaagtctc
caccccattg 900acgtcaatgg gagtttgttt tggcaccaaa atcaacggga
ctttccaaaa tgtcgtaaca 960actccgcccc attgacgcaa atgggcggta
ggcgtgtacg gtgggaggtc tatataagca 1020gagctggttt agtgaaccgt
cagatcctgc agaagttggt cgtgaggcac tgggcaggta 1080agtatcaagg
ttacaagaca ggtttaagga gaccaataga aactgggctt gtcgagacag
1140agaagactct tgcgtttctg ataggcacct attggtctta ctgacatcca
ctttgccttt 1200ctctccacag gtgtccaggc ggccgcc atg ctg aag atg ctc
tcg ttt aag ctg 1254Met Leu Lys Met Leu Ser Phe Lys Leu1 5cta ctg
ctg gcc gtg gct ctg ggc ttc ttt gaa gga gat gcg aag ttt 1302Leu Leu
Leu Ala Val Ala Leu Gly Phe Phe Glu Gly Asp Ala Lys Phe10 15 20
25ggg gaa agg aac gag ggg agc gga gcg aga agg aga cgg tgc ctg aat
1350Gly Glu Arg Asn Glu Gly Ser Gly Ala Arg Arg Arg Arg Cys Leu
Asn30 35 40ggg aac ccc cca aag cgc cta aag aga agg gac agg cgg gtg
atg tcc 1398Gly Asn Pro Pro Lys Arg Leu Lys Arg Arg Asp Arg Arg Val
Met Ser45 50 55cag ctg gag ctg ctc agt gga gga gag atc ctg tgt ggt
ggc ttc tac 1446Gln Leu Glu Leu Leu Ser Gly Gly Glu Ile Leu Cys Gly
Gly Phe Tyr60 65 70cca cga gta tct tgc tgc ctg cag agt gac agc cct
gga ttg ggg cgt 1494Pro Arg Val Ser Cys Cys Leu Gln Ser Asp Ser Pro
Gly Leu Gly Arg75 80 85ctg gag aac aag atc ttt tct gcc acc aac aac
tca gaa tgc agc agg 1542Leu Glu Asn Lys Ile Phe Ser Ala Thr Asn Asn
Ser Glu Cys Ser Arg90 95 100 105ctg ctg gag gag atc caa tgt gct ccc
tgc tcc ccg cat tcc cag agc 1590Leu Leu Glu Glu Ile Gln Cys Ala Pro
Cys Ser Pro His Ser Gln Ser110 115 120ctc ttc tac aca cct gaa aga
gat gtc ctg gat ggg gac cta gca ctt 1638Leu Phe Tyr Thr Pro Glu Arg
Asp Val Leu Asp Gly Asp Leu Ala Leu125 130 135cca ctc ctc tgc aaa
gac tac tgc aaa gaa ttc ttt tat act tgc cga 1686Pro Leu Leu Cys Lys
Asp Tyr Cys Lys Glu Phe Phe Tyr Thr Cys Arg140 145 150ggc cat att
cca ggt ctt ctt caa aca act gct gat gaa ttt tgc ttt 1734Gly His Ile
Pro Gly Leu Leu Gln Thr Thr Ala Asp Glu Phe Cys Phe155 160 165tac
tat gca aga aaa gat gct ggg tta tgc ttt cca gac ttc ccg aga 1782Tyr
Tyr Ala Arg Lys Asp Ala Gly Leu Cys Phe Pro Asp Phe Pro Arg170 175
180 185aag caa gtc aga gga cca gca tct aac tac ttg ggc cag atg gaa
gac 1830Lys Gln Val Arg Gly Pro Ala Ser Asn Tyr Leu Gly Gln Met Glu
Asp190 195 200tac gag aaa gtg ggg ggg atc agc aga aaa cac aaa cac
aac tgc ctc 1878Tyr Glu Lys Val Gly Gly Ile Ser Arg Lys His Lys His
Asn Cys Leu205 210 215tgt gtc cag gag gtc atg agt ggg ctg cgg cag
cct gtg agc gct gtg 1926Cys Val Gln Glu Val Met Ser Gly Leu Arg Gln
Pro Val Ser Ala Val220 225 230cac agc ggg gat ggc tcc cat cgg ctc
ttc att cta gag aag gaa ggc 1974His Ser Gly Asp Gly Ser His Arg Leu
Phe Ile Leu Glu Lys Glu Gly235 240 245tac gtg aag att cta acc cca
gaa gga gaa ctg ttc aag gag cct tac 2022Tyr Val Lys Ile Leu Thr Pro
Glu Gly Glu Leu Phe Lys Glu Pro Tyr250 255 260 265ttg gac att cac
aaa ctt gtt caa agt gga ata aag gga gga gac gaa 2070Leu Asp Ile His
Lys Leu Val Gln Ser Gly Ile Lys Gly Gly Asp Glu270 275 280agg ggc
ctg cta agc ctg gca ttc cat ccc aat tac aag aaa aat gga 2118Arg Gly
Leu Leu Ser Leu Ala Phe His Pro Asn Tyr Lys Lys Asn Gly285 290
295aag ctg tat gtg tct tat acc acc aac cag gaa cgg tgg gct att ggg
2166Lys Leu Tyr Val Ser Tyr Thr Thr Asn Gln Glu Arg Trp Ala Ile
Gly300 305 310cct cac gac cac att ctt cgg gtt gtg gaa tac aca gta
tcc agg aaa 2214Pro His Asp His Ile Leu Arg Val Val Glu Tyr Thr Val
Ser Arg Lys315 320 325aac ccc cat caa gtt gat gtg aga aca gcc agg
gtg ttt ctg gaa gtc 2262Asn Pro His Gln Val Asp Val Arg Thr Ala Arg
Val Phe Leu Glu Val330 335 340 345gca gag ctc cac cga aag cat ctt
ggg gga cag ctg ctc ttt ggt cct 2310Ala Glu Leu His Arg Lys His Leu
Gly Gly Gln Leu Leu Phe Gly Pro350 355 360gat ggc ttt ttg tac atc
atc ctt ggg gat ggt atg atc aca ttg gat 2358Asp Gly Phe Leu Tyr Ile
Ile Leu Gly Asp Gly Met Ile Thr Leu Asp365 370 375gac atg gaa gag
atg gat ggg tta agt gac ttc aca ggc tct gtg ctg 2406Asp Met Glu Glu
Met Asp Gly Leu Ser Asp Phe Thr Gly Ser Val Leu380 385 390agg ctg
gac gtg gac acc gac atg tgc aat gtg cct tat tcc ata cct 2454Arg Leu
Asp Val Asp Thr Asp Met Cys Asn Val Pro Tyr Ser Ile Pro395 400
405cgg agt aac cct cac ttc aac agc acc aac cag ccc cca gaa gta ttt
2502Arg Ser Asn Pro His Phe Asn Ser Thr Asn Gln Pro Pro Glu Val
Phe410 415 420 425gcc cac ggc ctc cat gat cca ggc aga tgt gcc gtg
gat cga cat cct 2550Ala His Gly Leu His Asp Pro Gly Arg Cys Ala Val
Asp Arg His Pro430 435 440act gat ata aac atc aat tta aca ata ctt
tgc tca gat tcc aac ggg 2598Thr Asp Ile Asn Ile Asn Leu Thr Ile Leu
Cys Ser Asp Ser Asn Gly445 450 455aaa aac agg tca tca gcc aga atc
cta cag ata ata aag gga aga ggt 2646Lys Asn Arg Ser Ser Ala Arg Ile
Leu Gln Ile Ile Lys Gly Arg Gly460 465 470tat gaa agt gag cca tct
ctt ctt gaa ttc aag cca ttc agt aac ggc 2694Tyr Glu Ser Glu Pro Ser
Leu Leu Glu Phe Lys Pro Phe Ser Asn Gly475 480 485cct ttg gtt ggt
gga ttt gtt tac aga ggc tgt cag tct gaa aga ttg 2742Pro Leu Val Gly
Gly Phe Val Tyr Arg Gly Cys Gln Ser Glu Arg Leu490 495 500 505tac
gga agc tat gtg ttc gga gat cgc aat ggg aat ttc tta acc ctc 2790Tyr
Gly Ser Tyr Val Phe Gly Asp Arg Asn Gly Asn Phe Leu Thr Leu510 515
520cag caa agc cca gtg acc aag caa tgg caa gaa aag ccg ctc tgc ctg
2838Gln Gln Ser Pro Val Thr Lys Gln Trp Gln Glu Lys Pro Leu Cys
Leu525 530 535ggt gcc agc agc tcc tgt cga ggc tac ttt tcg ggt cac
atc ttg gga 2886Gly Ala Ser Ser Ser Cys Arg Gly Tyr Phe Ser Gly His
Ile Leu Gly540 545 550ttt gga gaa gat gaa tta gga gag gtt tac att
cta tca agc agt aag 2934Phe Gly Glu Asp Glu Leu Gly Glu Val Tyr Ile
Leu Ser Ser Ser Lys555 560 565agt atg acc cag act cac aat gga aaa
ctc tac aag atc gta gac ccc 2982Ser Met Thr Gln Thr His Asn Gly Lys
Leu Tyr Lys Ile Val Asp Pro570 575 580 585aaa aga cct tta atg cct
gag gaa tgc aga gtc aca gtt caa cct gcc 3030Lys Arg Pro Leu Met Pro
Glu Glu Cys Arg Val Thr Val Gln Pro Ala590 595 600cag cca ctg acc
tcc gat tgc tcc cgg ctc tgt cga aac ggc tac tac 3078Gln Pro Leu Thr
Ser Asp Cys Ser Arg Leu Cys Arg Asn Gly Tyr Tyr605 610 615acc ccc
act ggc aag tgc tgc tgc agt ccc ggc tgg gag gga gac ttc 3126Thr Pro
Thr Gly Lys Cys Cys Cys Ser Pro Gly Trp Glu Gly Asp Phe620 625
630tgc aga att gcc aag tgt gag cca gcg tgc cgt cat gga ggt gtc tgt
3174Cys Arg Ile Ala Lys Cys Glu Pro Ala Cys Arg His Gly Gly Val
Cys635 640 645gtc aga ccg aac aag tgc ctc tgt aaa aag ggc tat ctt
ggt cct caa 3222Val Arg Pro Asn Lys Cys Leu Cys Lys Lys Gly Tyr Leu
Gly Pro Gln650 655 660 665tgt gaa caa gtg gac agg aac gtc cgc aga
gtg acc agg tga 3264Cys Glu Gln Val Asp Arg Asn Val Arg Arg Val Thr
Arg670 675ggatccaatc aacctctgga ttacaaaatt tgtgaaagat tgactggtat
tcttaactat 3324gttgctcctt ttacgctatg tggatacgct gctttaatgc
ctttgtatca tgctattgct 3384tcccgtatgg ctttcatttt ctcctccttg
tataaatcct ggttgctgtc tctttatgag 3444gagttgtggc ccgttgtcag
gcaacgtggc gtggtgtgca ctgtgtttgc tgacgcaacc 3504cccactggtt
ggggcattgc caccacctgt cagctccttt ccgggacttt cgctttcccc
3564ctccctattg ccacggcgga actcatcgcc gcctgccttg cccgctgctg
gacaggggct 3624cggctgttgg gcactgacaa ttccgtggtg ttgtcgggga
agctgacgtc ctttccatgg 3684ctgctcgcct gtgttgccac ctggattctg
cgcgggacgt ccttctgcta cgtcccttcg 3744gccctcaatc cagcggacct
tccttcccgc ggcctgctgc cggctctgcg gcctcttccg 3804cgtcttcgag
atctgcctcg actgtgcctt ctagttgcca gccatctgtt gtttgcccct
3864cccccgtgcc ttccttgacc ctggaaggtg ccactcccac tgtcctttcc
taataaaatg 3924aggaaattgc atcgcattgt ctgagtaggt gtcattctat
tctggggggt ggggtggggc 3984aggacagcaa gggggaggat tgggaagaca
atagcaggca tgctggggac tcgagttaag 4044ggcgaattcc cgattaggat
cttcctagag catggctacg tagataagta gcatggcggg 4104ttaatcatta
actacaagga acccctagtg atggagttgg ccactccctc tctgcgcgct
4164cgctcgctca ctgaggccgg gcgaccaaag gtcgcccgac gcccgggctt
tgcccgggcg 4224gcctcagtga gcgagcgagc gcgcagcctt aattaaccta
attcactggc cgtcgtttta 4284caacgtcgtg actgggaaaa ccctggcgtt
acccaactta atcgccttgc agcacatccc 4344cctttcgcca gctggcgtaa
tagcgaagag gcccgcaccg atcgcccttc ccaacagttg 4404cgcagcctga
atggcgaatg ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg
4464gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc
tcctttcgct 4524ttcttccctt cctttctcgc cacgttcgcc ggctttcccc
gtcaagctct aaatcggggg 4584ctccctttag ggttccgatt tagtgcttta
cggcacctcg accccaaaaa acttgattag 4644ggtgatggtt cacgtagtgg
gccatcgccc cgatagacgg tttttcgccc tttgacgctg 4704gagttcacgt
tcctcaatag tggactcttg ttccaaactg gaacaacact caaccctatc
4764tcggtctatt cttttgattt ataagggatt tttccgattt cggcctattg
gttaaaaaat 4824gagctgattt aacaaaaatt taacgcgaat tttaacaaaa
tattaacgtt tataatttca 4884ggtggcatct ttcggggaaa tgtgcgcgga
acccctattt gtttattttt ctaaatacat 4944tcaaatatgt atccgctcat
gagacaataa ccctgataaa tgcttcaata atattgaaaa 5004aggaagagta
tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt
5064tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc
tgaagatcag 5124ttgggtgcac gagtgggtta catcgaactg gatctcaata
gtggtaagat ccttgagagt 5184tttcgccccg aagaacgttt tccaatgatg
agcactttta aagttctgct atgtggcgcg 5244gtattatccc gtattgacgc
cgggcaagag caactcggtc gccgcataca ctattctcag 5304aatgacttgg
ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta
5364agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa
cttacttctg 5424acaacgatcg gaggaccgaa ggagctaacc gcttttttgc
acaacatggg ggatcatgta 5484actcgccttg atcgttggga accggagctg
aatgaagcca taccaaacga cgagcgtgac 5544accacgatgc ctgtagtaat
ggtaacaacg ttgcgcaaac tattaactgg cgaactactt 5604actctagctt
cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca
5664cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg
agccggtgag 5724cgtgggtctc gcggtatcat tgcagcactg gggccagatg
gtaagccctc ccgtatcgta 5784gttatctaca cgacggggag tcaggcaact
atggatgaac gaaatagaca gatcgctgag 5844ataggtgcct cactgattaa
gcattggtaa ctgtcagacc aagtttactc atatatactt 5904tagattgatt
taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat
5964aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc
agaccccgta 6024gaaaagatca aaggatcttc ttgagatcct ttttttctgc
gcgtaatctg ctgcttgcaa 6084acaaaaaaac caccgctacc agcggtggtt
tgtttgccgg atcaagagct accaactctt 6144tttccgaagg taactggctt
cagcagagcg cagataccaa atactgtcct tctagtgtag 6204ccgtagttag
gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta
6264atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg
gttggactca 6324agacgatagt taccggataa ggcgcagcgg tcgggctgaa
cggggggttc gtgcacacag 6384cccagcttgg agcgaacgac ctacaccgaa
ctgagatacc tacagcgtga gctatgagaa 6444agcgccacgc ttcccgaagg
gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga 6504acaggagagc
gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc
6564gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg
ggggcggagc 6624ctatggaaaa acgccagcaa cgcggccttt ttacggttcc
tggccttttg ctgcggtttt 6684gctcacatgt tctttcctgc gttatcccct
gattctgtgg ataaccgtat taccgccttt 6744gagtgagctg ataccgctcg
ccgcagccga acgaccgagc gcagcgagtc agtgagcgag 6804gaagcggaag
68148678PRTArtificialsynthetic recombinant AAV vector (plasmid)
8Met Leu Lys Met Leu Ser Phe Lys Leu Leu Leu Leu Ala Val Ala Leu1 5
10 15Gly Phe Phe Glu Gly Asp Ala Lys Phe Gly Glu Arg Asn Glu Gly
Ser20 25 30Gly Ala Arg Arg Arg Arg Cys Leu Asn Gly Asn Pro Pro Lys
Arg Leu35 40 45Lys Arg Arg Asp Arg Arg Val Met Ser Gln Leu Glu Leu
Leu Ser Gly50 55 60Gly Glu Ile Leu Cys Gly Gly Phe Tyr Pro Arg Val
Ser Cys Cys Leu65 70 75 80Gln Ser Asp Ser Pro Gly Leu Gly Arg Leu
Glu Asn Lys Ile Phe Ser85 90 95Ala Thr Asn Asn Ser Glu Cys Ser Arg
Leu Leu Glu Glu Ile Gln Cys100 105 110Ala Pro Cys Ser Pro His Ser
Gln Ser Leu Phe Tyr Thr Pro Glu Arg115 120 125Asp Val Leu Asp Gly
Asp Leu Ala Leu Pro Leu Leu Cys Lys Asp Tyr130 135 140Cys Lys Glu
Phe Phe Tyr Thr Cys Arg Gly His Ile Pro Gly Leu Leu145 150 155
160Gln Thr Thr Ala Asp Glu Phe Cys Phe Tyr Tyr Ala Arg Lys Asp
Ala165 170 175Gly Leu Cys Phe Pro Asp Phe Pro Arg Lys Gln Val Arg
Gly Pro Ala180 185 190Ser Asn Tyr Leu Gly Gln Met Glu Asp Tyr Glu
Lys Val Gly Gly Ile195 200 205Ser Arg Lys His Lys His Asn Cys Leu
Cys Val Gln Glu Val Met Ser210 215 220Gly Leu Arg Gln Pro Val Ser
Ala Val His Ser Gly Asp Gly Ser His225 230 235 240Arg Leu Phe Ile
Leu Glu Lys Glu Gly Tyr Val Lys Ile Leu Thr Pro245 250 255Glu Gly
Glu Leu Phe Lys Glu Pro Tyr Leu Asp Ile His Lys Leu Val260 265
270Gln Ser Gly Ile Lys Gly Gly Asp Glu Arg Gly Leu Leu Ser Leu
Ala275 280 285Phe His Pro Asn Tyr Lys Lys Asn Gly Lys Leu Tyr Val
Ser Tyr Thr290 295 300Thr Asn Gln Glu Arg Trp Ala Ile Gly Pro His
Asp His Ile Leu Arg305 310 315 320Val Val Glu Tyr Thr Val Ser Arg
Lys Asn Pro His Gln Val Asp Val325 330 335Arg Thr Ala Arg Val Phe
Leu Glu Val Ala Glu Leu His Arg Lys His340 345 350Leu Gly Gly Gln
Leu Leu Phe Gly Pro Asp Gly Phe Leu Tyr Ile Ile355 360 365Leu Gly
Asp Gly Met Ile Thr Leu Asp Asp Met Glu Glu Met Asp Gly370 375
380Leu Ser Asp Phe Thr Gly Ser Val Leu Arg Leu Asp Val Asp Thr
Asp385 390 395 400Met Cys Asn Val Pro Tyr Ser Ile Pro Arg Ser Asn
Pro His Phe Asn405 410
415Ser Thr Asn Gln Pro Pro Glu Val Phe Ala His Gly Leu His Asp
Pro420 425 430Gly Arg Cys Ala Val Asp Arg His Pro Thr Asp Ile Asn
Ile Asn Leu435 440 445Thr Ile Leu Cys Ser Asp Ser Asn Gly Lys Asn
Arg Ser Ser Ala Arg450 455 460Ile Leu Gln Ile Ile Lys Gly Arg Gly
Tyr Glu Ser Glu Pro Ser Leu465 470 475 480Leu Glu Phe Lys Pro Phe
Ser Asn Gly Pro Leu Val Gly Gly Phe Val485 490 495Tyr Arg Gly Cys
Gln Ser Glu Arg Leu Tyr Gly Ser Tyr Val Phe Gly500 505 510Asp Arg
Asn Gly Asn Phe Leu Thr Leu Gln Gln Ser Pro Val Thr Lys515 520
525Gln Trp Gln Glu Lys Pro Leu Cys Leu Gly Ala Ser Ser Ser Cys
Arg530 535 540Gly Tyr Phe Ser Gly His Ile Leu Gly Phe Gly Glu Asp
Glu Leu Gly545 550 555 560Glu Val Tyr Ile Leu Ser Ser Ser Lys Ser
Met Thr Gln Thr His Asn565 570 575Gly Lys Leu Tyr Lys Ile Val Asp
Pro Lys Arg Pro Leu Met Pro Glu580 585 590Glu Cys Arg Val Thr Val
Gln Pro Ala Gln Pro Leu Thr Ser Asp Cys595 600 605Ser Arg Leu Cys
Arg Asn Gly Tyr Tyr Thr Pro Thr Gly Lys Cys Cys610 615 620Cys Ser
Pro Gly Trp Glu Gly Asp Phe Cys Arg Ile Ala Lys Cys Glu625 630 635
640Pro Ala Cys Arg His Gly Gly Val Cys Val Arg Pro Asn Lys Cys
Leu645 650 655Cys Lys Lys Gly Tyr Leu Gly Pro Gln Cys Glu Gln Val
Asp Arg Asn660 665 670Val Arg Arg Val Thr
Arg675919DNAArtificialsynthetic oligonucleotide for PCR 9gtgaggctgc
gagtgaccg 191024DNAArtificialsynthetic PCR oligonucleotide
10cctggtcgtc agccgccagc acgc 241120DNAArtificialsynthetic PCR
oligonucleotide 11ctgctgctat ccatcagcgt
201220DNAArtificialsynthetic PCR oligonucleotide 12aagaaggata
agaggacagg 201340DNAArtificialsynthetic PCR oligonucleotide primer
13aagcggccgc atgctgaaga tgctctcgtt taagctgcta
401432DNAArtificialsynthetic PCR oligonucleotide primer
14aaggatccct acctggtcac tctgcggacg tt 321523RNAartificialsynthetic
Shh-siRNA-antisense 15uaugaugucg ggguuguaau uuu
231623RNAartificialsynthetic Shh-siRNA-sense 16aauuacaacc
ccgacaucau auu 231722RNAartificialsynthetic Smo-siRNA-sense
17aaggccuucu cuaagcggca uu 221822RNAartificialsynthetic
Smo-siRNA-antisense 18ugccgcuuag agaaggccuu uu
221921RNAartificialsynthetic Gli1-siRNA-sense 19acgccgcagc
agcagcuccu u 212021RNAartificialsynthetic Gli1-siRNA-antisense
20ggagcugcug cugcggcguu u 21
* * * * *
References